10Th European Cytogenetics Conference 2015 4–7 July 2015, Strasbourg

Home , Edwards syndrome, Marker chromosome, Patau syndrome, Tetrasomy, XXYY syndrome

Chromosome Res (2015) 23(Suppl 1):S1–S157 DOI 10.1007/s10577-015-9476-6

10th European Cytogenetics Conference 2015 4–7 July 2015, Strasbourg – France

Published online: 1 July 2015 # Springer Science+Business Media Dordrecht 2015

EDITORIAL Mendel’slaws.Twoexceptionalandwell-known speakers, Trude Schwarzacher and Marcus This Supplement of Chromosome Research contains Pembrey, will show us the relevance of Mendel’s abstracts of the invited lectures and submitted oral work to the genetics of today. and poster presentations at the 10th European In the Opening lecture Terry Hassold will give us Cytogenetics Conference held on 4–7July2015in new insights into the age-old problem of aneuploidy in Strasbourg. humans. An important topic at this conference is the It was 150 years ago, in 1865, that Mendel first impact of next generation sequencing (NGS) and whole presented his paper on Experiments on Plant genome sequencing. In recent years the massive Hybridization at the Natural History Society in Brno sequencing has changed the practice of clinical (now in Czech Republic); the paper was published the genetics and has implications for cytogenetics. In following year. Gregor Johann Mendel was a German- the session “Impact of Whole Genome Sequencing speaking Augustinian friar who carried out his now on Future Diagnostics”, Joris Veltman will talk about famous experiments on pea plants in the experimental NGS and cytogenetics and Claudia Haferlach will garden at his monastery. He studied seven characteris- address the impact of NGS on the genetic diagnosis of tics of pea plants: plant height, pod shape and colour, malignant haemopathies. The sessions on Chromosome seed shape and colour, and flower position and colour. Rearrangements include among others a lecture by He coined the terms ‘recessive’ and ‘dominant’ in Sandra Rodriguez Perales on engineering of human reference to certain traits and he demonstrated the tumour-associated chromosomal translocations by actions of invisible ‘factors’— now called genes— CRISPR-Cas9 system. that provided for visible traits in predictable ways. Another highlight of the programme is the session on He kept meticulous records of his observations and based Prenatal Diagnosis in which Charles Strom will talk on these he formulated the laws of segregation and of about the positive predictive value of non-invasive pre- independent assortment, which later came to be known as natal screening (NIPS), and Kamlesh Madan will speak Mendel’slawsofinheritance.However,itwasmorethan about the impact of prenatal technologies on sex ratio. three decades later, at the turn of the 20th century, that the Besides these highlights, there are two interesting profound significance of Mendel’sworkwasrecognized. topics in the session on Clinical Cytogenetics, one is His work was rediscovered and his experimental on the CNVs on the X chromosome by Damien findings were verified by a number of scientists, Sanlanville and the other is an intriguing question thus ushering in the era of modern genetics. At this “Can Down syndrome be treated?” posed by Renaud Conference we celebrate the 150-year anniversary of Touraine. In the session on Animal and Plant S2 10th European Cytogenetics Conference 2015

Cytogenetics, Jaroslav Dolezol will discuss polyploidy As always, it is our intention that this meeting should and interspecific hybridization in plants, and Elena provide the participants with an opportunity for not only Giulotto will talk about the functional organization of contributing their knowledge and experience, but also satellite-less equid centromeres. for interacting with each other. Strasbourg, being one of Finally, there are a number of interesting topics Europe’s most attractive cities is the ideal place for this; in sessions on Tumour Cytogenetics, Fragile sites the entire town centre has been listed as UNESCO and Chromosome Instability, Segmental Duplications World Heritage. We hope that the participants will take and CNVs and Accreditation and Quality control. the opportunity to visit the centre to discover its history, The programme ends with the Keynote Lecture on its cuisine and its unique cultural atmosphere. the 22q11.2 rearrangement disorders by Bernice Finally, on behalf of the ECA and the Scientific Morrow. Programme Committee, I wish all participants a stimu- Iwouldliketodrawyourattentiontothemany lating, useful and an enjoyable meeting. interesting submitted oral communications and posters, the abstracts of which also appear in this José M. García-Sagredo supplement. President 10th European Cytogenetics Conference 2015 S3

COMMITTEES

E.C.A. BOARD OF DIRECTORS SCIENTIFIC PROGRAMME COMMITTEE

Sevilhan ARTAN Mariano ROCCHI–Chair Juan Cruz CIGUDOSA Claudia HAFERLACH Jean-Michel DUPONT Damien SANLAVILLE José M. GARCIA-SAGREDO Eva KLOPOCKI J.S. (Pat) HESLOP-HARRISON Joris VERMEESCH Thierry LAVABRE-BERTRAND Orsetta ZUFFARDI Nicole de LEEUW Kamlesh MADAN Konstantin MILLER Felix MITELMAN Harald RIEDER Mariano ROCCHI Elisabeth SYK LUNDBERG Roberta VANNI

E.C.A. COMMITTEE

Jose M. GARCIA-SAGREDO President Elisabeth SYK LUNDBERG First Vice-President Kamlesh MADAN Second Vice-President Konstantin MILLER General Secretary Jean-Michel DUPONT Treasurer

Organizing Secretariat

Yıldız Posta Cad. Akın Sitesi 1. Blok No:6 Kat:1 D:3 Gayrettepe 34349 Beşiktaş/Istanbul, TURKEY

Email : [email protected] Phone : +90-212347-6300 Fax : +90-212347-6363

Visit www.dekon.com.tr for more information.

Editors of the abstract book: José M. Garcia-Sagredo, Kamlesh Madan, Konstantin Miller

The European Cytogeneticists Association (E.C.A.) is indebted to the following companies and institutes for their support: S4 10th European Cytogenetics Conference 2015 10th European Cytogenetics Conference 2015 S5

10TH EUROPEAN CYTOGENETICS CONFERENCE PROGRAM

SATURDAY, 4 JULY 2015

14:00–15:00 Room 12 Permanent Working Group–Clinical and Molecular Approaches to Cytogenetic Syndromes 15:00–16:00 Room 12 Permanent Working Group–Molecular Cytogenetics and Array-CGH 16:00–17:00 Room 12 Permanent Working Group–Marker Chromosomes 14:00–15:00 Room Boston Permanent Working Group–Cytogenetics of Haematological Malignancies 15:00–16:00 Room Boston Permanent Working Group–Prenatal Diagnosis 16:00–17:00 Room Boston Permanent Working Group–Animal, Plant and Comparative Cytogenetics 14:00–15:00 Room Dresden CEQAS Participants’ Meeting 15:00–16:00 Room Dresden Permanent Working Group–Quality Issues and Training in Cytogenetics 16:00–17:00 Room Dresden International Breakpoint Mapping Consortium, Ancillary Meeting 14:00–15:00 Room Leicester Permanent Working Group–Cancer Cytogenetics, Solid Tumor Studies 15:00–16:00 Room Leicester Permanent Working Group–Cytogenetics, Toxicology and Mutagenesis 16:00–17:00 Room Leicester Permanent Working Group–Meiotic Studies 17:00–17:50 Industry-sponsored Satellite Symposium

18:00–19:00 Opening Lecture Chairs: José M. Garcia-Sagredo, Damien Sanlaville Aneuploidy in humans: new insights into an age-old problem Terry Hassold (USA)

SUNDAY, 5 JULY 2015

08:30–10:30 Plenary Session: Clinical Cytogenetics Chairs: Elisabeth Syk Lundberg, Kamlesh Madan 08:30–09:05 CNV on the X Chromosome. Clinical interpretation and pitfalls Damien Sanlaville (F) 09:05–09:40 Can Down syndrome be treated? Renaud Touraine (F) 09:40–10:30 Selected Oral Presentations Detecting small Copy Number Variations, smaller than 400 kb, improves the diagnostic yield of CMA in intellectual disability Marie Faoucher (F) The power of social media for karyotype-phenotype analysis of rare chromosome disorders Conny van Ravenswaaij-Arts (NL) Reorganisation of chromosome 9 pericentromeric segmental duplications in inv(9) carriers and in evolution Stefan Müller (D) 10:30–11:00 Coffee Break 11:00–12:00 Plenary Session: 150 Years Since Mendel’s Laws (Brno, Natural History Society 1865, published 1866) Chairs: Pat Heslop-Harrison, Mariano Rocchi 11:00–11:30 Mendel, his time, and the rediscovery of his laws Trude Schwarzacher (GB) 11:30–12:00 From genetic to epigenetic inheritance S6 10th European Cytogenetics Conference 2015

Marcus Pembrey (GB) 12:00–14:40 Poster Session 12:40–13:30 Industry-sponsored Satellite Symposium 13:40–14:30 Industry-sponsored Satellite Symposium 14:40–15:45 Concurrent Session: Chromosomal Rearrangements I Chairs: Orsetta Zuffardi, Damien Sanlaville 14:40–15:05 Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9 system Sandra Rodriguez Perales (E) 15:05–15:30 Genome rearrangements in hereditary disorders Karl Heinimann (CH) 15:30–15:45 Selected Oral Presentation Unraveling the internal structure of neocentromeres in ring chromosomes harboring genomic amplification in soft tissue tumors Gemma Macchia (I) 14:40–15:45 Concurrent Session: Segmental Duplications and Copy Number Variants I Chairs: Joris Vermeesch, Serge Romana 14:40–15:05 Salivary amylase CNV and obesity Philippe Froguel (GB) 15:05–15:30 Polymorphism of the pseudoautosomal region I Martin A. Mensah (D) 15:30–15:45 Selected Oral Presentation Unbalanced de novo chromosome translocations and inversions: a two-step origin? Maria Clara Bonaglia (I) 15:45–16:15 Coffee Break 16:15–17:20 Concurrent Session: Fragile Sites and Chromosome Instability Chairs: Lidia Larizza, Jean-Michel Dupont 16:15–16:40 Fragile X Syndrome: an overview Jean-Louis Mandel (F) 16:40–17:05 CINister problems, modeling aneuploidy in the mouse Floris Foijer (NL) 17:05–17:20 Selected Oral Presentation The International Breakpoint Mapping Consortium (IBMC). Systematic mapping of chromosomal breakpoints in the context of phenotypes and nuclear genome organization Niels Tommerup (DK) 16:15–17:20 Concurrent Session: Segmental Duplications and Copy Number Variants II Chairs: Nicole de Leeuw, Juan Cigudosa 16:15–16:40 The growing importance of CNVs: new insights for detection and clinical interpretation Sebastien Jacquemont (CDN) 16:40–17:05 Copy number variation as a key to unlocking the enigma that is schizophrenia Aiden Corvin (IRL) 17:05–17:20 Selected Oral Presentation Direct visualisation of complex structural and copy-number variation by molecular combing and multicolour-FISH Fengtang Yang (GB) 17:20–18:30 Poster Session 17:30–18:20 Industry-sponsored Satellite Symposium 18:30 E.C.A. General Assembly 10th European Cytogenetics Conference 2015 S7

MONDAY, 6 JULY 2015

08:30–10:30 Plenary Session: Tumor cytogenetics I Chairs: Felix Mitelman, Claudia Haferlach 08:30–09:00 Molecular breakdown of AML with EVI1 Ruud Delwel (NL) 09:00–09:30 Myelodysplastic syndromes: from 5q- to somatic mutation of RNA splicing machinery Mario Cazzola (I) 09:30–10:30 Selected Oral Presentations Comparison of microarray-based genomic profiling and karyotyping in myelodysplastic syndromes (a HOVON89 study) Marian Stevens-Kroef (NL) Epigenetic regulation of UNCX, a novel homeobox transcription factor gene activated in acute myeloid leukemia Giulia Daniele (I) Rapid and comprehensive routine diagnostic approach for prognostic genetic markers in multiple myeloma Elodie David (CH) 10:30–11:00 Coffee Break 11:00–12:15 Concurrent Session: Chromosomal Rearrangements II Chairs: Nicole de Leeuw, Franck Pellestor 11:00–11:30 Retrotransposable elements, unbalanced translocations and chimerism Joris Vermeesch (B) 11:30–12:00 SD, CNV and complex regions of the human genome Francesca Antonacci (I) 12:00–12:15 Selected Oral Presentation Meiotic recombination and complex chromosome rearrangements: how whole-genome sequencing helps to resolve them Vincent Gatinois (F) 11:00–12:15 Concurrent Session: Animal and Plant Cytogenetics Chairs: Pat Heslop Harrison, Valerie Fillon 11:00–11:30 Chromosome-centric approaches unravel the impact of polyploidization and interspecific hybridization Jaroslav Dolezel (CZ) 11:30–12:00 Functional organization of satellite-less equid centromeres Elena Giulotto (I) 12:00–12:15 Selected Oral Presentation Comparative chromosome painting in lacertid lizards: highly conserved karyotypes but independent origins of sex chromosomes Veronica Rojo (E) 12:15–14:30 Poster Session 12:40–13:30 Industry-sponsored Satellite Symposium 13:40–14:30 Industry-sponsored Satellite Symposium 14:30–15:45 Plenary Session: Impact of Whole Genome Sequencing on Future Diagnostics Chairs: Eva Klopocki, Juan Cigudosa 14:30–15:00 NGS and cytogenetics Joris Veltman (NL) 15:00–15:30 Genetic diagnosis in malignant hemopathies Claudia Haferlach (D) S8 10th European Cytogenetics Conference 2015

15:30–15:45 Selected Oral Presentation Exome sequencing and SNP arrays as a tool to describe genetic diversity of premalignant and malignant cells: lesson from monoclonal gammopathies Aneta Mikulasova (CZ) 15:45–16:15 Coffee Break 16:15–17:30 Concurrent Session: Tumor Cytogenetics II Chairs: Roberta Vanni, Thierry Lavabre-Bertrand 16:15–16:45 Constitutional and somatic rearrangement of chromosome 21 in ALL Christine J. Harrison (GB) 16:45–17:15 Developmental disease and cancer: Biological and Clinical Overlaps Alfonso Bellacosa (USA) 17:15–17:30 Selected Oral Presentation Assessment of SNP genomic microarray analysis as an alternative to FISH analysis for the detection of copy number changes in pediatric solid tumors Mary Shago (CDN) 16:15–17:30 Concurrent Session: Accreditation and Quality Control Chairs: Harald Rieder, Martine Doco-Fency 16:15–16:40 European Standards for Clinical Laboratory Geneticists Thomas Liehr (D) 16:40–17:00 Changes in ISO 15189: practical implications Konstantin Miller (D) 17:00–17:30 Mandatory 15189 ISO accreditation for French medical laboratories: how to best reconcile regulatory and normative requirements for cytogenetics? Philippe Lochu (F) 17:30–18:30 Poster Session 17:30–18:20 Industry-sponsored Satellite Symposium

TUESDAY, 7 JULY 2015

09:00–10:30 Plenary Session: Prenatal Diagnosis Chairs: Konstantin Miller, Sevilhan Artan 09:00–09:30 The Positive Predictive Value of Noninvasive Prenatal Screening (NIPS) Charles Strom (USA) 09:30–10:00 Prenatal technologies and sex ratio Kamlesh Madan (NL) 10:00–10:30 Selected Oral Presentations Noninvasive prenatal testing: more caution is needed in high risk pregnancies Beatrice Oneda (CH) Digital PCR in an efficient alternative to massively parallel sequencing as a non–invasie, prenatal test for trisomy 21: the results of a pilot study Laila Allach El Khattabi (F) 10:30–10:50 Coffee Break 10:50–11:40 Industry-sponsored Satellite Symposium 11:50–12:20 Keynote Lecture Chairs: Mariano Rocchi, José M. Garcia-Sagredo The chromosome 22q11.2 rearrangement disorders Bernice E. Morrow (USA) 12:20 Closing Ceremony Chairs: José M. Garcia-Sagredo, Damien Sanlaville 10th European Cytogenetics Conference 2015 S9

Invited Lecture Abstracts Intellectual disability (ID) and multiple congenital anomalies (MCA) have an estimated prevalence of L1 3%–5 % in resource-rich countries. These conditions make significant demands on healthcare expenditure. Aneuploidy in humans: new insights into an age-old Standard karyotyping can identify about 10 % of chro- problem mosomal abnormalities in patients with ID/MCA. Genome-wide array comparative genomic hybridization Terry Hassold (array-CGH or SNP array) has become the first-line School of Molecular Biosciences, Washington State diagnostic tool in the clinical investigation of patients University, Pullman WA–USA presenting with ID/MCA. This approach detects pathogenic genomic imbalances with a mean diagnostic yield Aneuploidy is the most common genetic complication of 12.2 % in these patients (Miller et al. AJHG 2010). of pregnancy, with approximately 0.2–0.3 % of new- The X chromosome is often implicated in intellectual born infants being trisomic. However, this represents disability. More than 100 loci on the X chromosome are just the tip of a large iceberg, because most aneuploid associated with intellectual disability and copy number conceptions die in utero. Indeed, studies of preimplan- variations of the X chromosome are frequently detected tation embryos suggest that a large proportion, if not a in routine diagnostics. majority, of fertilized human eggs have extra or missing In this session, we will focus on the difficulties of chromosomes. Because the vast majority of errors result interpreting X chromosome CNV detected by array- from the fertilization of a chromosomally abnormal egg CGH/SNP array: by a normal sperm, attention has focused on why human female meiosis is so error-prone. 1) Potential sex mismatch may make results for the X In this presentation, we will summarize recent studies chromosome difficult to interpret. Moreover, abnormal indicating that there are multiple routes to female- log ratios differ between males and females. Some derived aneuploidy; e.g., studies of model organisms examples are presented. indicate the contribution of errors occurring during the 2) Females have two X chromosomes. One of these long meiotic arrest stage or as part of the meiotic cell chromosomes is inactivated. This inactivation is usually cycle checkpoint machinery. We will also discuss our random, but this is not always the case. We will discuss own work, which has focused on analyzing human the utility of X inactivation studies in females with meiosis “as it happens” in fetal oocytes and in spermato- X-chromosome CNV. cytes. These studies demonstrate remarkable differences 3) Chromosome inactivation does not involve the between human males and females in the way in which entire X chromosome. Some regions of the chromo- chromosomes find and synapse with one another, some, such as the pseudo-autosomal regions, escape in the packaging of chromatin, and in the control inactivation. Careful analysis of the log ratio is required of the meiotic recombination pathway. Further, for these regions. they suggest that errors in fetal oogenesis are 4) The interpretation of X-chromosome CNV may relatively common and are important contributors differ between male and female patients. For example, X to male:female differences in meiotic nondisjunc- chromosome duplication in males results in X functional tion, as well as to the maternal age-related increase disomy. in aneuploidy levels in our species. 5) Several recurrent CNVs have been identified by array-CGH/SNP array. We present the most frequent of L2 these CNVs, together with their associated clinical phenotypes. CNV on the X chromosome. Clinical interpretation 6) We discuss some difficult-to-interpret recurrent and pitfalls. CNVs found on the X chromosome, such as CNV at the STS locus. Indeed, the discovery of numerous copy num- Damien Sanlaville ber variants of benign or unknown clinical significance Genetics Department, Hospices Civils de Lyon, has rendered interpretation problematic. For example, the UCBL1, Lyon-France submicroscopic duplication of Xp22.31 has been S10 10th European Cytogenetics Conference 2015 reported as a possible cause of intellectual disability The very first question is the likelihood of effect on and/or developmental delay or as a benign variant. an adult brain: is it too late to expect any effect after birth 7) We present several cases with incidental findings or infancy? We can consider that the nature of brain and for which genetic counseling is difficult. Indeed, defect in DS is not only developmental but also most X-chromosome CNVs are inherited from the functional, making room for possible cognitive mother and carrier status might be revealed. improvement even in older children or adults. It is also very difficult to evaluate amelioration in Finally, we present our experience with a custom X cognitive functions since there are no gold standard array containing more than 100,000 X-chromosome tests because no trial in any intellectual disorder has probes, making it possible to detect CNVover less than been successful so far. Along these lines, what is the 1 kb, and we discuss the utility of such arrays for meaning in real life of a statistically significant diagnosis. modification of few points or seconds in a neuro- psychological test? L3 In addition, cognitive functions are a “work in progress” that depends on environmental experiences Can Down Syndrome be treated? and positive effects may require adequate stimulating environment and a longer period of time than the Renaud Touraine, Marie Ducrocq, Bénédicte de usual duration of clinical trials. Fréminville Nevertheless better knowledge of brain functioning Service de Génétique, Centre de Référence des and chromosome 21 genes content have allowed several Anomalies du Développement, CHU de Saint Etienne, teams to identify potential therapeutic approaches France in DS. These different approaches are not exclusive to each other and may not be specific to DS since Down Syndrome (DS or Trisomy 21) is still the most some deficient pathways may be affected in other frequently recognized cause of intellectual deficiency causes of ID as well. with an incidence at birth of 1/1500-1/2000 in most The two most promising molecules currently in trial European countries. A further decrease is likely to come in persons with DS are EGCG and RG1662. with the use of non-invasive prenatal diagnosis as a EGCG (epigallocatechinegallate) is an inhibitor of replacement for current prenatal screening strategies. DYR1A whose coding gene is on the chromosome 21. Eventually, giving birth to a child whatever his chromo- DYRK1A overexpression has clearly a role in brain somal status is parents’ choice, and life expectancy of dysfunction in DS persons and DS animal models. In people with DS is more than 55 years, making DS not a these animal models, EGCG administration reduced the “disappearing” birth defect negative impact of the presence of 3 copies of Dyrk1a. Persons with DS often have mild intellectual defi- EGCG is a polyphenol extracted from green tea leafs. ciency (ID), less frequently severe ID. They benefit The first trial of EGCG therapy versus placebo in adults from early intervention and continuous educative and with DS has been conducted in Barcelona, Spain re-educative supports. Self-determination is also a major (Prof de la Torre) and its results are expected soon. source of improvement. Therefore a majority of persons RG1662 is a selective GABAA alpha5 negative with DS are able to attain convenient autonomy to live allosteric modulator developed by ROCHE™. In simple in our societies, as long as some support is maintained. words, it can be viewed as an “anti-diazepam” drug. These limited results are not sufficient indeed and a RG1662 re-establishes a balance between excitatory better achievement for a larger number of those persons and inhibitory systems in brain (related to Glutamate is on hold. and GABA respectively). A phase II double-blind Up to 2014, no drug or supplement mixture has multicentric trial is currently in progress in adults and proven any efficacy on improving cognitive perfor- adolescents with DS (CLEMATIS®). mance in persons with DS. This year and the very next The hope of reducing the burden of DS has never may be the beginning of effective therapies. been so well founded, even if it will not “treat” DS but Doing therapeutic trials to increase cognitive functions its most handicapping consequence i.e. intellectual in people with ID raised several difficulties. deficiency. 10th European Cytogenetics Conference 2015 S11

L4 Dutch Hugo de Vries and the German Carl Correns, and to some extend the Austrian Erich von Tschermak. Mendel, his time, and the rediscovery of his laws. Following their publications (in 1900 within 2 months), Mendel’s results were replicated and genetic linkage Trude Schwarzacher formally described. Department of Biology, University of Leicester, LE1 I will be presenting Mendel’s discoveries in the con- 7RH, UK text of data from our own lab, studying chromosomes, inheritance, combination and recombination of genes, ‘Few publications have so enduringly and variously and in particular repetitive DNA elements in plants and influenced science as had the short monograph animals. We now know about mechanisms of [Versuche über Pflanzenhybriden, 1865] by the Mendelian inheritance of genes and alleles, directly Augustinian monk of Brünn [now Brno], Pater Gregor giving quantitative traits and linkage, and genetic maps. Mendel. Forgotten for decades, within a few years But we also understand and can ascribe mechanisms for after its rediscovery it gave a mighty impetus to non-Mendelian inheritance and many aspects of the doctrine of heredity; and as Mendelism, his LaMarkian inheritance: linkage drag, and the teaching had now become the central theme of ever-increasing fields of epigenetics, genomic biological research as well as the foundation of interactions and chromatin cytogenetics that are manifold practical application’— this is how Hugo largely dependent on the repetitive landscape of Iltis (1966) starts the preface of his book ‘The Life chromosomes. of Mendel’. More details will be available at our website, www. I will be looking at the scientific landscape of the molcyt.com. time of Mendel’s discovery in the mid 19th century, as well as the rediscovery 50 years later and describe the key findings. These involved his famous studies with L5 peas, introducing the terms dominant and recessive, but Mendel also worked with bees and mice and the plant From Genetic to Epigenetic Inheritance. Hieraceum. But his paper was seen as essentially about hybridization rather than inheritance and had little Marcus Pembrey impact and was cited only three times over the Genetics and Epigenetics in Health and Disease Section, next 35 years. At that time blended inheritance UCL Institute of Child Health, 30 Guilford was the accepted theory of inheritance where Street, London WC1N 1EH, UK traits from each parent are averaged together— Centre for Child and Adolescent Health, School of not so different from what we now know to be Social & 10 Community Medicine, Oakfield Grove, the case for multiple genes and quantitative traits— University of Bristol, Bristol BS8 2BN, UK while Mendelian discontinuous inheritance applies to single genes. By the time Mendel’s Laws were ‘rediscovered’ and By the turn of the 19th century, there were rapid integrated into the chromosomal theory of inheritance, advances in cytology, and especially in the knowledge August Weismann’s idea of a barrier between the soma of the nucleus. Cell division is always preceded by and germ plasm (proposed in the 1890’s) had taken nuclear division (mitosis) and chromosomes were hold. This, and the difficulty of demonstrating stained and named (Waldeyer 1888). Boveri, Lamarckian inheritance of acquired characteristics using Weismann and Flemming showed their integrity and inbred animals, helped define the favoured view of continuity and proved that the number of chromosomes biological inheritance in the 20th century. Inheritance is fixed for each species. Thus, it was assumed was either transmission of DNA and its sequence that ‘heredity factors’ are contained in the chromo- variations or social transmission through imitation and somes and that each sexually reproduced organism learning. By the 1960’s somatic nuclear transplantation has received a complete set of paternal and maternal experiments indicated that all cells had a full comple- chromosomes. Finally, Mendel’slawswere ment of DNA and therefore some form of epigenetic rediscovered independently of each other by the regulation of gene expression during development had S12 10th European Cytogenetics Conference 2015 to exist. Modification of DNA by methylation of cytosines L6 was shown in 1975 and it soon became clear that methylation patterns influenced gene expression and Engineering human tumour-associated chromosomal were stably transmitted though somatic cell division to translocations with the RNA-guided CRISPR-Cas9 create a cellular memory. Studies of early mouse devel- system. opment in the 1980s, whilst challenging Weismann, also showed genome wide erasure of DNA methylation in Sandra Rodriguez Perales the preimplantation embryo. However imprinted genes Molecular Cytogenetics Group, Centro Nacional escape this early methylation erasure. Discovered in the Investigaciones Oncológicas (CNIO) C/ Melchor 1980’s and 1990’s, mammalian genomic imprinting was Fernández Almagro, Madrid-Spain the starting point for a reconsideration of the idea that sperm might carry information about the ancestral Cancer is a multistep process that involves multiple environment— with transgenerational epigenetic changes in its genome, including genetic specific point inheritance as a candidate mechanism. The focus was mutations and other large genome rearrangements. A on male-line transgenerational effects because female- frequent and key genomic event in cancer initiation line studies are confounded by transplacental signals, and progression are chromosome translocations, an etc. There were animal experiments at this time showing exchange of large DNA fragments between two non- male-line transgenerational effects of drugs and hor- homologous chromosomes that in many cases leads mones, but no epigenetic studies. Human male-line stud- to the creation of new fusion genes. Those chromosomal ies really took off around 2000withthethree-generation translocations are generated through the illegitimate historic analysis of food supply in childhood and joining of two non-homologous chromosomes affected descendant longevity by Lars Olov Bygren and by double-strand breaks (DSB). Despite their importance, colleagues using his Overkalix cohorts in Northern it is difficult to generate models (either murine or human Sweden. It was also about this time that environ- cellular models) to study the complex scenario of cancer mental epigenomics became established as an idea. genetics; but the cancer-modelling field is now I will describe my collaboration with Bygren and my experiencing a conversion with the recent emergence work with Professor Jean Golding to demonstrate of the RNA-programmable CRISPR-Cas9 system, a transgenerational responses to early-life parental smoke flexible methodology to produce essentially any exposure using the Avon Longitudinal Study of Parents desired modification in the genome. and Children (ALSPAC). This characterisation of We have developed a new strategy for generating human transgenerational responses does suggest cancer-related human chromosomal translocations some form of non-genetic, non-social transmission based on the CRISPR system. Using this approach, we is occurring between generations [see Pembrey et al. J have induced chromosomal translocations resembling Med Genet 2014;51:563 and Golding et al. Am J Hum those described in acute myeloid leukaemia (AML) Biol 2014;26:731]. There are three main messages to and Ewing’s sarcoma (ES) with unprecedented high take from these initial (male-line) transgenerational frequencies in human cell lines and primary human stem responses to early-life parental or ancestral food supply cells. Cytogenetic and molecular analysis of the or tobacco exposures: (i) they are not usually engineered cells revealed the reliability and accurate- Lamarckian inheritance of acquired characteristics; ly of the CRISPR system approach, providing a power- (ii) there are exposure-sensitive periods during ful tool for cancer studies. Furthermore, we have also development; and (iii) both the transmission and used the CRISPR system to induce other cooperative offspring outcomes are often sex-specific. It looks as if hits found in Ewing sarcoma patients. Using this transgenerational responses contribute to developmental advance, it is now possible to easily and accurately variation in the population. Support for this conclusion generate cell and animal models harbouring the same comes from rodent experiments over the last alterations that define tumour cells in human patients, decade. They have started to provide direct evidence allowing researchers to experimentally recapitulate the of transgenerational epigenetic inheritance particu- genomic alterations needed to transform a healthy larly in terms of changes in sperm microRNAs or cell/organism or tissue. We have demonstrated that the DNA methylation. CRISPR system has been prove as a robust technology 10th European Cytogenetics Conference 2015 S13 that made possible to generate animal and human cell most frequent recombination-associated motifs. models that recapitulate tumour specific cooperative Finally, assuming the sequence-related vulnerability of alterations rapidly and at low cost, providing a new DNA to mutation(s) we wondered if the DNA motif- way to interrogate the development and progression of based logic derived from germ line GR sites could be cancers. These advances will in time lead to the devel- extrapolated to a genomic scale. We therefore opment of better therapeutic approaches to the treatment partitioned the human genome HG18 into 5 kb windows of cancer, thanks to the use of more accurate and faithful and calculated the ratio of nhGR to hGR motifs, referred models. to as recombination type ratio (RTR). As we could show for all CRC genes investigated as well as for 16 L7 additional, clinically important gene loci with adequate numbers (≥15) of precisely mapped germ line GRs, an Genome rearrangements in hereditary disorders. RTR cut-off of 6.6 correctly associated the predominant type of recombination for the majority (>85 %) of Michal B. Kovac and Karl Heinimann pathogenic rearrangements. Hence, our study lends Research Group Human Genomics, Dept. of further support to the sequence-directed nature of Biomedicine, University of Basel, Basel-Switzerland human mutations and provides a new approach to study the mechanisms underlying GRs on a genomic scale Several lines of evidence suggest that genomic rear- within and, possibly, between species. rangements (GRs) in humans occur in a non-random manner and correlate with local sequence composi- L8 tion. Whereas most of the pertinent data on the processes leading to this type of genetic instability Salivary amylase CNV and obesity have come from studies on somatic tissues, little is known about GRs in the germ line ofpatientswithhereditary Philippe Froguel disorders. In this study we used 112 pathogenic germ line Pasteur Institute of Lille and Department of Genomics, GRs to investigate how sequence architecture impacts on Imperial College London-UK the type and frequency of GRs in genes predisposing to hereditary colorectal cancer, namely APC, MLH1, Frequent Nucleotide Polymorphisms (SNP) only MSH2, MSH6 and PMS2. Of these, we fine- explain a small fraction of obesity heritability. mapped 17 GRs (from a discovery set of 31 Beyong point mutations, it has been proposed that GRs) at 1 bp resolution using custom-designed Structure Variants may contribute to complex traits. arrays and chromosome walking, whereas the We previously evidenced a potent gene dosage effect at breakpoint (BP) positions for the remainder (n=95) chromosome 16p11 where large Copy Number Variant were retrieved from public sources. To analyse in (CNV) cause highly severe obesity (when DNA is ahypothesis-freemannerthebreakpoint-flanking deleted) or medical leanness (when DNA is duplicated). sequences we used available and custom algo- Further, we developed system biology approaches in rithms to identify 4-mer sequence motifs for human, combining transcriptomics in adipose tissue homology-driven (hGR) and non-homology-driven and GWAS analysis in sib-pairs discordant for obesity, rearrangements (nhGR) as well as complexity analysis and we identified a gene dosage effect of a multi-allelic for higher order features of genome architecture. CNV linked to obesity on chromosome 1 at amylase Subsequently, all statistically significant 4-mer genes regions. This complex region is known to witness sequences were cross-referenced with motifs reported human evolution and natural selection associated with in the context of genome reorganisation to look for agriculture development and starch consumption. Using mechanisms underlying hGR and nhGR formation. qPCR and novel digital PCR analysis we showed that In hGR BPs the most common motifs consisted of high number of copies of salivary amylase (AMY1) mutational “super hotspots” (40 %) and DNA strongly protects against obesity, in particular in high polymerase arrest / slippage sites (25 %) compared starch diet populations. Our recent data also involves to nhGR BPs, where DNA topoisomerase sites (27 %) polymorphic pancreatic amylase (AMY2) as a putative and “super hotspots” (19 %) represented the player. Both plasma enzyme activities strongly S14 10th European Cytogenetics Conference 2015 associates with adiposity and metabolic phenotypes. Floris Foijer Metabonomics analysis in human and animal studies European Research Institute for the Biology of Ageing incriminates the gut microbiome as a link between diet, (ERIBA), University of Groningen, University Medical amylase CNVs and obesity. Salivary amylase is a potent Center Groningen, A. Deusinglaan 1, NL-9713 AV marker of response to stress and environment induced Groningen-The Netherlands stress is a well known risk factor for obesity and metabolic diseases. Two out of three tumors display abnormal chromosome content, a state defined as aneuploid. Paradoxically, L9 aneuploidy inhibits cell proliferation of non- transformed cells, suggesting that cancer cells have Polymorphism of the pseudoautosomal region I. acquired mutations that help them cope with the disad- vantages of aneuploidy. Together, these features make Martin A. Mensah, Hestand MS, Larmuseau MHD, aneuploidy a promising target for cancer therapy. To Isrie M, Vanderheyden N, et al. (2014) better understand the responses to aneuploidy in vivo, Pseudoautosomal Region 1 Length Polymorphism in we developed conditional knockout mouse models, in the Human Population. PLoS Genet 10(11): e1004578. which we can provoke aneuploidy in tissues of choice, doi:10.1371/journal.pgen.1004578.doi:10.1371/ at time points of choice. Our models have allowed us to journal.pgen.1004578 induce highly aneuploid cancers in various tissues, Institut fur Medizinische Genetik und Humangenetik, including liver and T-cells in a highly reproducible Charite— Universitatsmedizin Berlin-Germany fashion. This allowed us to extract common features of aneuploid cancer cells in vivo, and allows us to The human sex chromosomes differ in sequence, except further investigate how aneuploid cancer cells cope for the pseudoautosomal regions (PAR) at the terminus with the detrimental consequences of aneuploidy. In of the short and the long arms, denoted as PAR1 and my presentation, I will discuss how different tissues PAR2. The boundary between PAR1 and the unique X show sometimes quite opposing responses to and Y sequences was established during the divergence aneuploidy, suggesting lineage-specific aneuploidy-coping of the great apes. During a copy number variation strategies. Understanding these tissue-specific responses screen, we noted a paternally inherited chromosome X will be crucial for the future development of aneuploidy duplication in 15 independent families. Subsequent ge- targeting cancer therapies. Furthermore, I will discuss nomic analysis including BAC mediated targeted how we use our single cell-sequencing platform to paired-end and single molecule sequencing demonstrated quantify karyotype heterogeneity in aneuploid mouse that an insertional translocation of X chromosomal tumors and human tumors. This data is is revealing that sequence into the Y chromosome generates an extended human aneuploid tumors, similar to our aneuploid PAR. The insertion is generated by non-allelic mouse tumors, exhibit frequent missegregation events, homologous recombination between a 548 bp LTR6B especially when put under selective pressure, which repeat within the Y chromosome PAR1 and a second might impact therapeutic response. LTR6B repeat located 105 kb from the PAR boundary on the X chromosome. The identification of the reciprocal L12 deletion on the X chromosome in one family and the occurrence of the variant in different chromosome Y The growing importance of CNVs: new insights for haplogroups demonstrate this is a recurrent genomic detection and clinical interpretation. rearrangement in the human population. This finding represents a novel mechanism shaping sex chromosomal Sebastien Jacquemont evolution. CHU Saint Justine, University of Montréal, Montréal-Canada L11 Copy Number Variants (CNVs) have been recognized CINister problems, modeling aneuploidy in the as major contributors to neurodevelopemental disorders mouse (NDs) but the effects of these variants on cognition and 10th European Cytogenetics Conference 2015 S15 behavior and how they lead to neuropsychiatric The current state of play in our understanding of the disorders remain largely unknown. Because some CNVs genetic architecture of schizophrenia and how this may (such as those at the 16p11.2 locus) occur recurrently impact clinical care will be reviewed. In particular, this at a specific locus as either a loss or reciprocal gain of presentation will provide an update on the latest findings genomic copies, they allow beyond a simple case– from CNV discovery, how these findings overlap across control study, to investigate relationships between clinical boundaries and their likely diagnostic relevance. gene dosage and clinical as well as intermediate CNVs can arise through a number of different mecha- traits. Our group has studied the 16p11.2 genomic nisms and detecting risk variants may require different region and characterized the effect of gene dosage on research approaches. As illustration, we recently cognitive, behavioral and structural neuroimaging identified a novel risk duplication at chr20p12.2 phenotypes as well as medical comorbidities (obesity identified in the Irish and UK populations that is likely in particular). We are extending this systematic study to to have been inherited as a single ancestral event. all major recurrent genomic variants associated with Finally, we consider where the field is moving and autism and schizophrenia to understand how multiple how genomics may reshape how we diagnose and treat genomic loci may converge on key mechanisms leading schizophrenia in the future. to these 2 neuropsychiatric disorders. L14 L13 Molecular breakdown of AML with EVI1. Copy number variation as a key to unlocking the enigma that is schizophrenia Ruud Delwel Department of Hematology, Erasmus University Aiden Corvin Medical Center, Rotterdam-The Netherlands Department of Psychiatry, Psychosis Research Group, Trinity College Dublin, Dublin-Ireland Acute myeloid leukemia (AML) is the most common form of acute leukemia. Understanding of the Schizophrenia is a complex, heritable brain disorder molecular- and biological abnormalities is essential for defined by core symptoms affecting perception of the development of hypothesis driven therapy of AML. reality, thinking, behavior and motivation. Based on large scale gene expression and gene methyl- Schizophrenia is challenging in that it forces people ation profile studies (Cancer Cell, 2010) we identified to confront their perception of the world and is AML subtypes with unique genetic and epigenetic associated with significant morbidity and mortality. aberrations and study the biological consequences of Current therapies are partially effective and a these abnormalities. In particular, we discovered a cohesive understanding of pathophysiology is lacking, unique mechanism of transformation of AMLs with even whether one or more disease mechanisms are either an inv(3)(q21q26.2) or a translocation involved. t(3;3)(q21;q26.2) [inv(3)/t(3;3], malignancies asso- Recent progress is illuminating a genetic architecture ciated with a dismal clinical outcome (Cell, 2014). of schizophrenia that involves a substantial contribution Applying functional genomics (4C-seq, ChIP-Seq) and from many common risk variants of small effect, but genome-engineering (TALENS and CRISPR/CAS9), also rare mutations (e.g. copy number variants (CNVs)) we demonstrated reposition of a distal GATA2 enhancer of large effect. This work, in particular the findings from from chromosome 3q21 to the leukemia disease gene CNV studies is making us re-think how we conceptualize EVI1, located at 3q26. This unique translocation of the schizophrenia, and its relationships with other psychiatric GATA2 enhancer causes two simultaneous events, i.e. and brain disorders. The data suggests convergence ectopic activation of EVI1 combined with GATA2 on molecular risk mechanisms implicating synaptic mono-allelic expression. Functional haploinsufficiency plasticity, immune function and chromatin remodeling. arising from inactivating mutations in GATA2 zinc At a molecular level, these mechanisms appear to finger DNA binding domains or in GATA2 promoter overlap across a spectrum of neurodevelopmental sequences has been well established as an underlying disorders. cause of syndromes characterized by predisposition to S16 10th European Cytogenetics Conference 2015 myeloid leukemia. Thus, our data show that structural and KRAS), and cohesin complex (STAG2). Only 6 rearrangements involving single chromosomal reposi- genes (TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and tioning of enhancers can cause deregulation of two RUNX1) are consistently mutated in 10 % or more MDS unrelated distal genes, with cancer as the outcome. patients, whereas a long tail of ∼50 genes are mutated less Importantly, we found that the rearranged enhancer near frequently. Recent studies indicate that the development EVI1 had turned into a BET-inhibitor hypersensitive of MDS reflects the occurrence of somatic mutations in “super-enhancer”.Inv(3)/t(3;3)AMLcellsappeartobe hematopoietic stem cells with aging.13,14 highly sensitive to these BET-inhibitors, which cause References strong downregulation of EVI1 expression accompanied 1. Cazzola M, Della Porta MG, Malcovati L. by a cell cycle block and induction of differentiation and The genetic basis of myelodysplasia and its clinical apoptosis. Our studies emphasize that molecular under- relevance. Blood.2013;122(25):4021–4034. standing of defective transcriptional control may provide 2. Walter MJ, Shen D, Ding L, et al. Clonal architecture leads for hypothesis driven targeting of cancer. of secondary acute myeloid leukemia. NEnglJMed. 2012;366(12):1090–1098. L15 3. Haase D, Germing U, Schanz J, et al. New insights into the prognostic impact of the karyotype in MDS and Myelodysplastic syndromes: from 5q- to somatic correlation with subtypes: evidence from a core dataset mutation of RNA splicing machinery of 2124 patients. Blood. 2007;110(13):4385-4395. 4. Ebert BL, Pretz J, Bosco J, et al. Identification of Mario Cazzola and Luca Malcovati RPS14 as a 5q- syndrome gene by RNA interference Department of Hematology Oncology, Fondazione screen. Nature. 2008;451(7176):335–339. Istituto di Ricovero e Cura a Carattere Scientifico 5. Starczynowski DT, Kuchenbauer F, Argiropoulos (IRCCS) Policlinico San Matteo, and Department of B, et al. Identification of miR-145 and miR-146a as Molecular Medicine, University of Pavia, Pavia-Italy mediators of the 5q- syndrome phenotype. Nat Med. 2010;16(1):49–58. Myelodysplastic syndromes are clonal hematopoietic 6. Barlow JL, Drynan LF, Hewett DR, et al. A p53- stem cell disorders characterized by cytopenia, dependent mechanism underlies macrocytic anemia in a myelodysplasia, ineffective hematopoiesis, and mouse model of human 5q- syndrome. Nat Med. increased risk of progression to acute myeloid leukemia 2010;16(1):59–66. (AML).1 The vast majority of myeloid precursors are 7. Delhommeau F, Dupont S, Della Valle V, et al. clonally derived in MDS.1,2 During the natural course of Mutation in TET2 in myeloid cancers. N Engl J Med. the disease, the acquisition of additional driver muta- 2009;360(22):2289–2301. tions leads to formation of subclones of hematopoietic 8. Bejar R, Stevenson K, Abdel-Wahab O, et al. cells with further impaired differentiation and/or matu- Clinical effect of point mutations in myelodysplastic ration capacity, and eventually to development of AML. syndromes. N Engl J Med. 2011;364(26):2496–2506. Recurrent chromosomal abnormalities are detected in 9. Yoshida K, Sanada M, Shiraishi Y, et al. Frequent about half of patients with MDS,3 and the most common pathway mutations of splicing machinery in aberrations include del(5q), trisomy 8, del(20q), and myelodysplasia. Nature. 2011;478(7367):64–69. monosomy 7.3 While most of these abnormalities are 10. Papaemmanuil E, Cazzola M, Boultwood J, et al. secondary genetic events, the del(5q) of the 5q- syndrome Somatic SF3B1 mutation in myelodysplasia with ring represents a founding event, involving haploinsufficiency sideroblasts. N Engl J Med. 2011;365(15):1384–1395. for RPS14 and miR-145.4–6 11. Papaemmanuil E, Gerstung M, Malcovati L, et al. Our understanding of the molecular basis of MDS Clinical and biological implications of driver mutations in has improved dramatically in the last 5 years.7–12 Driver myelodysplastic syndromes. Blood.2013;122(22):3616– mutant genes include those of RNA splicing (SF3B1, 3627. SRSF2, U2AF1, and ZRSR2), DNA methylation (TET2, 12. Haferlach T, Nagata Y, Grossmann V, et al. DNMT3A,andIDH1/2), chromatin modification Landscape of genetic lesions in 944 patients with (ASXL1 and EZH2), transcription regulation (RUNX1), myelodysplastic syndromes. Leukemia. DNA repair (TP53), signal transduction (CBL, NRAS, 2014;28(2):241–247. 10th European Cytogenetics Conference 2015 S17

13. Genovese G, Kahler AK, Handsaker RE, et al. There is a complex relationship between the evolution Clonal hematopoiesis and blood-cancer risk inferred of segmental duplications (SDs) and rearrangements from blood DNA sequence. NEnglJMed. associated with human disease. The repeat architecture 2014;371(26):2477–2487. of the human genome predisposes certain regions to 14. Jaiswal S, Fontanillas P, Flannick J, et al. Age- non-allelic homologous recombination (NAHR) related clonal hematopoiesis associated with adverse resulting in copy number variants (CNVs). Over the last outcomes. N Engl J Med. 2014;371(26):2488–2498. years, several studies have identified hotspots of recurrent CNVs associated with intellectual disability, L16 autism, epilepsy, and schizophrenia. Most rearrangements are large and flanked by high homologous SDs. Retrotransposable elements, unbalanced transloca- We sought to better understand the mechanisms leading tions and chimerism to genomic instability of previously known disease associated loci by characterizing their SD architecture Joris Vermeesch in humans and non-human primates. We discovered and Hoofd Moleculaire Cytogenetica, UZ Leuven, Campus characterized alternate structural configurations of these Gasthuisberg, Leuven-Belgium regions in humans that often arose as a result of human- specific expansions of SDs in conjunction with inde- The human embryo has been shown to be a cradle pendent evolutionary inversion events. Our findings of chromosomal rearrangements, with multiple cells emphasize the importance of correctly defining alterna- carrying segmental deletions and duplications. To proof tive human genomic configurations in order to assess that those rearrangements can result in chromosomal variable risk of subsequent pathogenic rearrangements. disorders, we reasoned that de novo unbalanced Molecular cytogenetics, genomic approaches, and translocations originating postzygotically could exist out sequencing of long molecules from single haplotypes of compound maternal/paternal derivative chromosomes. remain the only way to correctly resolve these complex Weanalyzed12denovounbalancedtranslocationsand architectures of the human genome. mapped the breakpoints in nine. We demonstrated the existence of compound heterozygotes and estimate L18 that up to 30 % originate postzygotically. In contrast to balanced translocations where large-scale analysis of Chromosome-centric approaches unravel the impact balanced chromosomal translocation breakpoints of polyploidization and interspecific hybridization has shown nonhomologous end joining and microhomology-mediated repair to be the main Jaroslav Dolezel, Jan Vrana, Marie Kubalakova, Petr drivers of interchromosomal structural aberrations, we Capal, Hana Simkova identify nonallelic homologous recombination (NAHR) Institute of Experimental Botany, Centre of the Region between (retro)transposable elements and especially Hana for Biotechnological and Agricultural Research, long interspersed elements (LINEs) as the main Olomouc-Czech Republic mutational mechanism. This finding shows yet another involvement of (retro)transposons in genomic The process of doubling somatic chromosome number, rerangements and exposes a profoundly different or polyploidization, played a major role in plant genome mutational mechanism compared with balanced evolution and speciation. Based on their mode of chromosomal translocations. formation, polyploid species are classified into auto- polyploids, which originate from single species, and L17 allopolyploids, which arise by hybridization between distinct species. Polyploidization is critical for the newly SDs, CNVs and complex regions of the human established hybrids to be fertile and propagate sexually. genome Apart from creating reproductive isolation, increasing the number of chromosome sets results in the increased Francesca Antonacci number of genes, providing substrate for the evolution Department of Biology, University of Bari, Bari-Italy of complex regulatory and metabolic pathways. Recent S18 10th European Cytogenetics Conference 2015 allopolyploids represent suitable models to study highly variable, even among related species. This para- genome changes induced by interspecific hybridization dox is now explained by the well established knowledge and polyploidization, and the impact of these processes that the centromeric function is epigenetically specified. on genome organization, evolution and function. One of This important chromatin domain has so far escaped such species is hexaploid bread wheat, a crop of global comprehensive molecular analysis due to its typical importance. However, until recently, wheat was not association with highly repetitive DNA (satellite considered a suitable model due to huge and complex DNA). Although satellite DNA is a common feature of hexaploid genome comprising three homoeologous mammalian centromeres, we proved that, in Equus chromosome sets. The situation changed with the species (horses, asses and zebras), several centromeres advent of high-throughput next generation sequencing are completely satellite-free [1, 2], thus representing a technologies. After coupling with flow cytometric unique model for studying the organization, function chromosome sorting, which makes it possible to dissect and evolution of mammalian centromeres. nuclear genomes to single chromosomes, a powerful The simple sequence organization of the equid toolbox became available to overcome difficulties satellite-less centromeres is related to the fact that they encountered when analyzing (allo)polyploid genomes were generated recently during evolution through a due to their enormous sequence redundancy. This centromere repositioning process (shifting of the centro- approach permits identification of genic sequences mere function without chromosome rearrangement) [3]. separately on each chromosome and perform comparative We suggest that the DNA component of these evolu- gene analysis of subgenomes and putative parental tionarily young centromeres did not have enough time species. It also facilitates the analysis of transcriptional to acquire the typical complexity of the majority of the activity of homoeologous genes during plant growth mature mammalian centromeres described so far. and development. Although the chromosome-centric The functional organization of the single-copy approaches have been employed most extensively in centromere of horse chromosome 11 was also analysed wheat and other cultivated and wild species of the tribe in different individuals by ChIP-on-chip with an anti- Triticeae, they can be applied in any species from which body against CENP-A, the centromeric histone-H3 mitotic chromosomes can be isolated by flow-sorting. variant [4]. Surprisingly, inter-individual positional This work has been supported by the National Program variation of CENP-A domains was observed; these were of Sustainability (award no. LO 2014) and the Czech scattered in a ~500 kb region, giving rise to “epialleles”. Science Foundation (award no. P501-12-G090). These results proved that the centromeric domains are autonomous relative to the underlying DNA sequence L19 and are characterized by positional instability. We recently investigated the molecular organization Functional organization of satellite-less equid of the donkey centromeres by ChIP-seq and by immune- centromeres FISH on chromatin fibres: 16 satellite-less centromeres were identified and the underlying DNA sequence was SG Nergadze1, F Cerutti1, R Gamba1, FM Piras1,A de novo assembled. The results of a comparative Mazzagatti1, M Corbo1, C Badiale1, E Cappelletti1,J analysis of centromeric and non-centromeric chromatin McCarter2,KSullivan2,ERaimondi1 and Elena will be presented and the possible role of DNA breakage Giulotto1 and methylation in the establishment and function of the 1 Department of Biology and Biotechnology, University centromere will be discussed. of Pavia, Via Ferrata 1, 27100 Pavia, Italy. Taken together, our discoveries demonstrate that, in 2 Centre for Chromosome Biology, School of Natural the genus Equus, centromeres are extraordinarily plastic Sciences, National University of Ireland, Galway, and represent an important driving force in genome Galway, Ireland. evolution. Our results also indicate that the availability of a number of satellite-less centromeres now opens the The centromere is the site of kinetochore assembly way to detailed molecular studies. required for the correct chromosome segregation during [1] Piras et al. (2010) PLoS Genet 6: e1000845. cell division. In centromeric chromatin, despite the evo- [2] Wade et al. (2009) Science 326: 865–867. lutionary conservation of proteins, DNA sequences are [3] Carbone et al. (2006) Genomics 87: 777–782. 10th European Cytogenetics Conference 2015 S19

[4] Purgato et al. (2014) Chromosoma Nov 21 [Epub metaphases including 24-color FISH and array based ahead of print]. comparative genomic hybridization as well as a variety of moleculargenetic techniques such as RT-PCR, quan- L20 titative RT-PCR, fragment analysis, melting curve analysis and sequencing. As high through put sequencing NGS and cytogenetics technologies are available now whole exome and whole genome sequencing can be performed in larger patient Joris A. Veltman cohorts. Thus, data on molecular mutations and their Department of Human Genetics, Radboud University diagnostic and prognostic impact has increased tremen- Nijmegen Medical Centre, Nijmegen-The Netherlands dously in several hematological malignancies. Department of Clinical Genetics, Maastricht University Today the karyotype as assessed by chromosome Medical Centre, Maastricht-The Netherlands banding analysis and molecular mutations play an important role in the diagnosis, classification, prognos- Our genomes are full of variation, including the larger tication and treatment decisions in several hematological structural variations that are the traditional focus in neoplasms. The WHO Classification of tumours of cytogenetics. Genome sequencing technologies are haematopoietic and lymphoid tissues 2008 requires the now allowing us for the first to obtain an integrated karyotype for the diagnosis and classification of several view of all this variation present in a genome. This entities either as the sole feature as in certain AML results in a more complete picture of genome variation subtypes or more frequent in combination with and an integration of the fields of “DNA diagnostics” cytomorphology such as in MDS and MPN. The impact and “cytogenetics” into “genome diagnostics”.In of the karyotype on prognosis in hematological malig- addition, the widespread automation of next generation nancies has best been shown in MDS and AML for sequencing technologies has an impact on the workload which detailed scoring systems have been developed. in a genome diagnostics laboratory, where a switch is More recently also molecular mutations have been required from the “wet lab” to the “dry lab”, the bioin- implemented in prognostic scoring systems in AML formatics interpretation of large amounts of genomic and MDS. variation. In this presentation, I will discuss our switch Treatment decisions are dependent on genetic infor- from exome to genome sequencing in research on mation in a large variety of hematological malignancies. severe intellectual disability. I will demonstrate how this For example the detection of the Philadelphia transloca- has resulted in an enormous improvement in the tion leading to a BCR-ABL1-rearrangement provides the detection of structural variation, also in comparison to basis for a treatment with tyrosine kinase inhibitors. In high resolution genomic microarrays. Also, I will AML and ALL the genetic profile determines whether discuss how a focus on copy number variations may be or not an allogeneic stem cell transplantation is the of enormous value in further understanding the functional treatment of choice. and clinical relevance of genomic variation in the non- From the diagnostic perspective a highly sophisticated coding part of ourgenome, the largely unexplored part characterization of the karyotype/genotype of every of our genome. individual patient has to be aimed at. However, the value of diagnostics is dependent of the therapeutic options L21 that can change patients prognosis. Thus, the more targeted treatments become available the higher the Genetic diagnosis in malignant hemopathies requirements for comprehensive diagnostics gets. The question for tomorrow is whether the “chromo- Claudia Haferlach somal karyotype” will be substituted by a “molecular Munich Leukemia Laboratory, Munich-Germany karyotype”. In AML powerful prognostic models have been developed which are solely based on markers Genetic diagnostics in hematological malignancies detectable by molecular genetic techniques. Further today encompasses several cytogenetic techniques such high through put sequencing techniques challenge the as chromosome banding analysis, fluorescence in situ need for cytogenetics. Whole genome sequencing is hybridization (FISH) on interphase nuclei and on able— as is chromosome banding analysis— to provide S20 10th European Cytogenetics Conference 2015 information on the whole genome and can— as chromo- onto the haploid chromosome set. Interestingly individuals some banding analysis does— detect balanced rear- with Down Syndrome, with constitutional gain of rangements leading to fusion genes as well as unbalanced chromosome 21, have a highly increased risk of rearrangements leading to copy number changes. developing acute leukaemia. Important structural changes Compared to chromosome banding analysis the of chromosome 21 occur in childhood ALL, for example resolution is considerable higher and also molecular the translocation, t(12;21)(p13;q22), which gives rise to mutations are detectable. Thus this techniques has the the ETV6-RUNX1 fusion, accounts for ~25 % of childhood ability to provide the most important information ALL associated with an extremely good prognosis. required in a diagnostic setting. As this technique does More recently we have described an intriguing rear- not provide data on the single cell level clonal evolution rangement of chromosome 21, specifically occurring in can only be calculated and indirectly assumed and not childhood ALL. It was found whilst screening for the separated from independent clones. However, although presence of the ETV6-RUNX1 fusion by FISH. technical feasible in the laboratory, bioinformatics Approximately 2 % childhood ALL patients, in absence necessary to use the sequencing data in a routine setting is of the fusion, showed multiple copies of the RUNX1 still in its infancy. Further, it is still an open question how gene in their interphase cells, which were found to be genetic data should be handled that provides information tandemly duplicated along the long arm of a grossly beyond the primary diagnostic question. chromosome 21 in metaphase. The patients were older The strategies to incorporate the “old” and “new” (median age 9 years) with a high risk of relapse on techniques in the future diagnostic work-flow in hema- standard therapy. Intensive treatment was shown to tological malignancies will be discussed. dramatically improve outcome. The abnormal chromosome 21 was highly heterogeneous between patients as L22 confirmed by SNP arrays, with recurrent amplification of megabase regions of chromosome 21. This abnormality Constitutional and somatic rearrangements of was termed iAMP21 (intrachromosomal amplification of chromosome 21 in acute lymphoblastic leukaemia chromosome 21). We used genomic, cytogenetic and transcriptional analysis, coupled with novel bioinformatic Christine J Harrison approaches, to reconstruct the evolution of iAMP21 Childhood Cancer Cytogenetics, Leukaemia Research ALL. We find that individuals born with the rare Cytogenetics Group, Northern Institute for Cancer constitutional Robertsonian translocation between Research, Newcastle University, Newcastle upon chromosomes 15 and 21, rob(15;21)(q10;q10)c, have Tyne-UK ~2700-fold increased risk of developing iAMP21 ALL compared to the general population. In such cases, Chromosome 21, although the smallest chromosome in amplification was initiated by chromothripsis, simulta- the karyotype, is frequently involved in cancer. It plays a neously involving both sister chromatids of the major role in acute lymphoblastic leukaemia (ALL), Robertsonian chromosome, a novel mechanism for manifesting as a range of chromosomal abnormalities. cancer predisposition. In sporadic iAMP21, breakage- It often occurs as a numerical chromosomal change, fusion-bridge cycles are typically the initiating event, either as the sole gain, giving rise to acquired trisomy often followed by chromothripsis. In both sporadic and 21 or, more frequently, as the gain of two or more copies rob(15;21)c-associated iAMP21, the final stages within a high hyperdiploid karyotype (51–65 chromo- frequently involve large-scale duplications of the abnormal somes with non-random chromosomal gains). Patients chromosome. The end-product is a derivative of chro- with high hyperdiploidy account for approximately mosome 21 or the rob(15;21)c chromosome with gene 30 % of childhood ALL and have an excellent outcome. dosage optimised for leukemic potential, showing In contrast, patients in the rare numerical cytogenetic constrained copy number levels over multiple linked subgroup, near haploidy (25–30 chromosomes)/low genes. Thus, dicentric chromosomes may be an important hypodiploidy (31–39 chromosomes) account for less precipitant of chromothripsis, as rob(15;21)c is than 1 % patients and have a dismal outcome. In this constitutionally dicentric and breakage-fusion-bridge subtype, chromosome 21 is always retained as two copies cycles generate dicentric chromosomes somatically. 10th European Cytogenetics Conference 2015 S21

Furthermore, these findings illustrate that several recognized speciality under the UEMS umbrella. The cancer-specific mutational processes, applied sequen- process of achieving such a clinical speciality was long tially, can co-ordinate to fashion copy number profiles and cumbersome, mainly because approval had to be over large genomic scales, incrementally refining the given by an EU recognition committee, which required fitness benefits of aggregated gene dosage changes. almost unanimous agreement between all 27 EU coun- These changes are major drivers of cancer, engineered tries. To ease the bureaucracy in such certification from a finite, but increasinglywellannotated,repertoire processes, the EU has proposed to establish a profes- of mutational mechanisms, which can potentially generate sional qualifications directive scheme. Hopefully this correlated copy number alterations across hundreds of proposal, where also the ESHG has given substantial linked genes, as exemplified by iAMP21 ALL. input to the process, will be passed within the next years. In order to have a Clinical Laboratory Geneticists (CLG) L23 curriculum ready if such a proposal is passed, an ESHG ad hoc committee has been very active since 2011 under Developmental disease and cancer: Biological and the ESHG umbrella and its work is being continued Clinical Overlaps accordingly. A proposal for a core curriculum (see https://www.eshg.org/clg.0.html) has been finalized in Alfonso Bellacosa 2012, and has been approved by the ESHG Executive Cancer Epigenetics Program, Fox Chase Cancer Center, Committee, the Board and received support of a Philadelphia-USA qualified of the corresponding representatives of European national societies. In 2012/2013 150 Numerous parallelisms exist between development and individuals in 43 countries helped aligning the require- cancer. In this presentation, I will review some of the ments of the CLG as an EU-recognized specialist pro- founding ideas linking development and cancer, and fession with those of their national guidelines of educa- highlight clinical conditions exhibiting features of both tion. Information has been returned by colleagues from developmental derangement and cancer predisposition, 41 countries. Thus, in 2014/15 the first round of appli- including cohesinopathies, rasopathies, phakomatoses, cation for European CLG title was opened to individuals Proteus Syndrome and other overgrowth disorders, holding a national CLG title from the following coun- recessive chromosome breakage syndromes, and domi- tries: Austria, Belgium, Czech Rep, Finland, nant hereditary cancer syndromes. I suggest that these Germany, Hungary, Italy, Lithuania, Macedonia, disorders encompass a continuous spectrum spanning Netherlands, Poland, Portugal, Slovenia, Sweden and clinical genetics and clinical oncology, and derive some Switzerland. >250 applications were received and were general implications that might be useful in the future evaluated. Applicants from other countries could not for the treatment of these diseases. As an additional apply for the European CLG title in 2014/15, but we example, I will discuss the role of active DNA demeth- hope to find ways to open this process also for them in ylation in development and cancer. due course. Members of the European Board of Medical Genetics, Clinical Laboratory Geneticist Professional L24 Branch (CLG) are: Thomas Liehr (Germany— chair), Isabel Carreira (Portugal— co-chair), Dilek Aktas European Standards for Clinical Laboratory (Turkey), Egbert Bakker (The Netherlands), Ana Geneticists Carrio (Spain), Domenico Coviello (Italy), Lina Florentin (Greece), Martina Rincic (Croatia) and Hans Thomas Liehr Scheffer (The Netherlands). Institute of Human Genetics, University Hospital Jena, Jena-Germany L25

There is an increasing need for recognition of also a Changes in ISO 15189: practical implications. laboratory speciality in clinical genetics, in line with last year’s recognition of clinical/medical genetics as an EU Konstantin Miller S22 10th European Cytogenetics Conference 2015

Institute of Human Genetics, Hannover Medical School, L26 Hannover-Germany Mandatory 15189 ISO accreditation for French med- AnewversionoftheEuropeanstandard:Medical ical laboratories: how to best reconcile regulatory and laboratories— Particular requirements for quality normative requirements for cytogenetics? and competence (EN ISO 15189:2007) was published in 2012 and a corrected version followed in Philippe Lochu1, Caroline Pecqueur2 2014. The changes and newly introduced chapters 1LBM Gen-Bio, 8 rue Jacqueline Auriol, 63965 should be taken into consideration by the labora- Clermont-Ferrand Cedex-France, 2Cofrac, 52 rue tories when they apply for accreditation. The Jacques Hillairet, 75012 Paris-France changes concern for example: The French law n°2013-442 of 30 May 2013 has – document control: the identification of changes to completely reformed the French medical biology, documents, affirming its status as a medical speciality. The page number to total number of pages patient is at the centre of the medical practice. – reference to any work referred to other The law has also made the accreditation mandatory, laboratories, according to the standard NF EN ISO 15189 and – programme to introduce new staff, by following a calendar established from 2013 to – periodical review of training and competence of 2020. In 2020, all French medical laboratories, staff, public or private, must be accredited for the totality – feedback on staff suggestions, of their activity. The next administrative deadline – immediate corrective or preventive action in case of will be on 31st of October 2016. All French non-conformities laboratories will have to give proof to the ARS indicated by external reviews, (Regional Health Agencies) that they are – adverse incidents reporting, accredited for 50 % of their activities with at least – recorded reviews of verifications and one examination accredited per field. validations, In addition to the mandatory accreditation of the – automatic selection and reporting of results, medical laboratories, the practice of constitutional and – validation of the laboratory information manage- molecular cytogenetics in France is highly regulated. In ment system. order to practice this discipline i) the biologists must obtain approval from the Agency of Biomedicine, AmajorelementofthenewISOstandardis and ii) the establishment must obtain an adminis- the introduction of risk management as a trative approval issued by the ARS to practice planned process linked to preventive and correc- these examinations. tive actions. In risk management of medical lab- Today, cytogenetic laboratories must prioritize regu- oratories, the impact of work processes and fail- latory requirements and standards which apply to them ures on examination results is evaluated because and that will be assessed. it affects patient safety. This requires the identi- In France, the French Committee for Accreditation fication of risks and their impact by analysing (Cofrac), the national accreditation body designated by non-conformities, errors, incidents or complaints, decree in 2008, is the sole authority able to issue accred- or the application of a failure modes and effect itation. The Healthcare section is in charge of the analysis. A second step is the development of accreditation of medical laboratories. The assessment risk handling strategies that aim at risk reduc- is based on skill assessment conducted by peers. To tion, risk elimination, or acceptance of controlled date, only 7 technical assessors are qualified to assess risk. At the end of this process, monitoring for this discipline. risk control is established. Planned risk manage- To help medical laboratories in the accreditation ment together with anticipatory preventive and process, the Cofrac provides them technical guides reactive corrective actions are the basis of con- including recommendations (SH GTA 01, SH GTA tinuous improvement in the laboratory. 04…) as well as standards (SH REF 02, SH REF 10th European Cytogenetics Conference 2015 S23

04 …)whichencompassregulatoryrequirements In an attempt to improve upon these PPV’s, we in addition to the requirements of the standard NF developed a Laboratory Developed NIPS using EN ISO 15189. massively parallel whole genome sequencing, advanced To date, more than 70 laboratories perform cytoge- automation, GC correction, proprietary bio-statistical netic tests (prenatal diagnosis and genetic activities …) and bioinformatics analysis . We were able to achieve and are mainly located in hospitals (>65 %). a 100 % sensitivity and specificity on a sample set of On April 1st 2015, 14 LBM were accredited for more than 3000 samples and 100 trisomic samples. True performing cytogenetic examinations (4 for somatic Trisomy samples had Z score>10 when fetal fractions cytogenetic, 7 for constitutional cytogenetic and 3 for were >5 % and all unaffected pregnancies demon- both disciplines). strated Z scores<4. We will be contacting each physi- By April 30th 2015, all medical laboratories cian following a positive result and will report the performing genetic examinations must submit the preliminary PPV’s of our test. accreditation request to the Cofrac (Decree N°2015- 205 of 23 February 2015) for at least one constitutional L28 genetic examination and / or one somatic genetic examination. This examination will be either a conventional Prenatal Technologies and Sex ratio or molecular cytogenetic examination or a molecular genetic examination. Kamlesh Madan Department of Clinical Genetics, Leiden University L27 Medical Center, Leiden-The Netherlands

The Positive Predictive Value of Noninvasive It is known that the sex ratio is skewed in favour of Prenatal Screening (NIPS) males in countries in South and East Asia, in particular in China and India. The fact that this is also the case in Charles Strom some countries in Central Asia and East Europe is less Quest Diagnostics Nichols Institute, San Juan well known. In all these countries the skewing of sex Capistrano, CA-USA ratio is unequivocally linked to the availability of prenatal technologies. The preconditions and causes of Recently tremendous progress has been made in sex selection will be discussed. The facts and figures analyzing free floating fetal DNA ffDNA in the from the Census of India and the history of prenatal sex maternal circulation for the presence of aneuploidies. selection in India will be presented. The shortage of Several validation trials has described sensitivities and women of a marriageable age is predicted to peak in specificities >99 %. Since Positive Predictive Value the middle of this century and the problem is expected to (PPV) for any ties is proportional not just to the continue into the next century. The other consequences specificity by also to the prevalence of the disorder, a of shortage of women will be briefly discussed. Wealth specificity of 99 % will lead to a positive predictive and education do not appear to be the solution, on the values of under 50 % for a disorder such as Down contrary. Information on what is being done and what Syndrome in a high risk population with a 1 % could be done will be presented. prevalence. We analyzed data from 161 consecutive prenatal karyotypes where the indication for testing L29 was an high risk NIPS result. The results of this study were positive predictive values of 87 % for The chromosome 22q11.2 rearrangement disorders Trisomy 21, 64 % for Trisomy 18, 48 % for Trisomy 13 and 46 % for sex chromosome Bernice Morrow Aneuploidies. Albert Einstein College of Medicine, Bronx, New When our data is combined with other reports, in York- USA 1,353 samples the PPV’s for Trisomy 21, 18, 13 and Sex chromosome aneuploidies are: 92, 73, 37, and 46 % The chromosome 22q11.2 region is particularly suscep- respectively. tible to meiotic chromosome rearrangements leading to S24 10th European Cytogenetics Conference 2015 birth defect syndromes. DiGeorge syndrome/velo- rearrangements can now be identified so as to cardio-facial syndrome is the most well-known of the have the statistical power to make genotype- 22q11.2 syndromes and is frequently referred to as phenotype correlations. 22q11.2 deletion syndrome (22q11DS). The majority The existence of different chromosome 22q11.2 of affected individuals have developmental delay, rearrangements may explain some of the phenotypic behavioral disorders as well as physical malformations variability connected to this interval, but clearly not such as craniofacial anomalies and congenital heart all, since most patients have the same sized 3 Mb dele- disease. Our lab has been interested in two aspects of tion. As part of an International 22q11.2 Consortium, this syndrome. Firstly, we have been trying to under- we are using Affymetrix 6.0 SNP arrays for genome- stand the reason why the microdeletion occurs in the wide association studies and whole exome sequencing first place, and secondly, why phenotypes are so vari- to uncover genes that may explain heterogeneity in able in affected individuals. disease severity for those with the typical 3 Mb deletion. Over 90 % of 22q11DS patients have a similar sized Once genes are identified, they can be tested for 3 Mb deletion that usually occurs as a de novo event, functional consequences in animal models. which is slightly more frequent in female rather than male meiosis. The deletionresultsfromnon-allelic Oral Abstracts homologous recombination events (NAHR) between low copy repeats termed, LCR22. There are four 1. Clinical Cytogenetics LCR22s in the 3 Mb region (LCR22A, -B, -C, -D) and they are of different sizes. The typical 3 Mb deletion is O1 flanked by LCR22A and LCR22D, which are over 250 kb in size. Each LCR22 is composed of modules Detecting small Copy Number Variations, smaller of pseudogenes that have been duplicated during than 400 kb, improves the diagnostic yield of CMA primate evolution. Because of their recent evolution, in intellectual disability. individual modules are 99 % identical in sequence. This high sequence homology makes them prone to Marie Faoucher1, Eric Fonteneau2, Nicolas Chatron1, NAHR and gene conversion events, making detailed Christel Depienne2, Audrey Labalme1, Elodie Lejeune2, analysis challenging. The current goal is to understand Marianne Till1,CyrilMignot2,CarolineSchluth- whether there are particular hotspots within the LCR22s Bolard1,CarolineNava3,AudreyPutoux1,Gaetan that might be responsible for chromosome rearrange- Lesca1, Fanny Mochel2, Marie-Pierre Alex1, Aurelia ments. If so, it would suggest that individuals with this Jacquette2,MassimilianoRossi1,DelphineHéron2, hotspot might be prone to 22q11.2 rearrangements. To Patrick Edery1,AlexisBrice2,DamienSanlaville1, identify chromosome breakpoints, we are performing Boris Keren2 whole genome sequencing of trio families (proband- Hospices Civils de Lyon Service de Génétique mother-father). This is being followed by analysis of Bron-France 1 GH Pitié-Salpêtrière Département the haplotypes of LCR22s. de Génétique Paris-France 2 GH Pitié-Salpêtrière One important question has been whether there are CRicm UPMC Inserm UMR S975/CNRS UMR varied rearrangements on 22q11.2 mediated by the other 7225 Paris-France3 LCR22s. Relevant to this, syndromic individuals have been identified with proximal, LCR22A-B or LCR22A- Cytogenetic microarray (CMA) allows the elucidation C deletions as well as distal, LCR22B-D or LCR22C-D of 15 to 20 % of remaining unexplained cases of intel- deletions. Finally, patients have been identified with lectual disability. A threshold of 400 kb is reciprocal duplications. Most of these individuals have proposed to consider Copy Number Variants been discovered from clinical microarray testing (CNV). Array use in diagnostic setting could because they had undiagnosed conditions. The fact that detect smaller CNV mostly polymorphic generating 22q11.2 rearrangements are so common, underscores numerous confirmations. Here, we propose to the mutagenic properties of the LCR22s and their evaluate the diagnostic interest of small CNV (inferior importance in causing human disease. It also suggests to 400 kb) in intellectual disability (ID). This that enough individuals with less well-known collaborative study groups an Illumina SNP-array 10th European Cytogenetics Conference 2015 S25 platform and an Agilent CGH-array platform for a total groups, respectively. Proximal deletions result in a of 3330 patients explored for syndromic or isolated variable degree of cognitive impairment, minor facial intellectual disability from 2011 to 2014. dysmorphisms, hypermobility, hypotonia, congenital Over 26,866 CNV detected, 22,683 (84.4 %) were heart defects, hernias, and limb abnormalities. The main smaller than 400 kb. Among the 1440 CNVs considered features seen in intermediate deletions are pre- en as relevant, 850 (40.9 %) were smaller than 400 kb postnatal growth retardation, developmental delay, increasing by 70 % the amount ofnecessaryconfirmation hypotonia, Prader-Willi-like features, facial by another technique. We concluded that 44 of 605 dysmorphisms, limb and cranial abnormalities, congenital (7.3 %) of these CNVs (smaller than 400 kb) were heart defects, and abnormalities of respiratory control. pathogenic. 36 were deletions which involved pathogenic Distal deletions are predominantly characterized by gene in ID and 8 were duplications. It is noteworthy that, intellectual disability, hypotonia, microcephaly, except a MECP2 duplication, all small duplications were corpus callosum agenesis, seizures, and ear, eye, either intragenic or probably truncating an ID gene cardiac, genital and limb anomalies. Other clinical possibly leading to haploinsufficiency. findings may include vertebral and rib anomalies, cleft To conclude, analyzing CNV smaller than 400 kb palate, hydronephrosis, and diaphragmatic hernia. A significantly increases the number of verification but more detailed analysis of the data allowed the enhances the diagnostic yield of 1.3 % in patients with confirmation or identification of candidate genes ID, which is equivalent to 44 patients in our cohort. for specific features, e.g. in congenital heart defects. However, we propose not to consider small non- We also received preliminary information on 6q truncating duplications. While massively parallel duplications (14), 6p deletions (25) and 6p duplications sequencing techniques are becoming routine diagnosis, (8), demonstrating how effective our approach is. This it seems preferable to detect as many pathogenic CNVs prompted us to initiate the development of an interactive as possible using CMA. online database system based on the intelligent collection, combining and presentation of information O2 for chromosome 6, in collaboration with the Chromosome 6 Facebook Group, Unique, ECARUCA The power of social media for karyotype-phenotype and Cartagenia. Eventually, the same approach can be analysis of rare chromosome disorders applied to all chromosome disorders. [email protected] Conny van Ravenswaaij-Arts1, Barbara Frentz1, Trijnie http://www.rug.nl/research/genetics/research/ Dijkhuizen1,PaulineBouman2,MorrisSwertz1, chromosome_6/ Richard Sinke1, Wilhelmina Kerstjens-Frederikse1 University Medical Centre Groningen Genetics O3 Groningen-The Netherlands1 Chromosome 6 Facebook Group Contact parent Utrecht-The Reorganisation of chromosome 9 pericentromeric Netherlands2 segmental duplications in inv(9) carriers and in evolution This presentation is the direct result of a study that was performed on request of a Facebook Group of parents of Marco Wettwer1,MarliesEndlicher1,Michaela children with a chromosome 6 disorder. The successful Neusser1, Ortrud Steinlein1, Stefan Müller1 use of social media in this study demonstrates how eager Ludwig-Maximilians-University Institute of Human parents are to give and receive information about the Genetics, University Hospital Munich-Germany1 condition of their child. In our study we focused on isolated 6q deletions. The evolutionary history of human chromosome 9 is These can be divided into proximal (6q11-q16), inter- complex, and includes structural rearrangements such as mediate (6q15-q25), and distal (6q25-qter). We were inversions and neocentromere formation, accompanied able to collect data through an extensive parent ques- by expansion and reorganisation of large blocks of tionnaire, medical records and detailed breakpoint segmental duplications and of pericentromeric constitu- characterization, of 15, 21 and 27 patients for these three tive heterochromatin. The complexity of these S26 10th European Cytogenetics Conference 2015 evolutionary changes is mirrored by the fact that human Marwijk-Kooy11,PetraMuus12,Gert-Jan chromosome 9 is heteromorphic, with numerous Ossenkoppele13,ArjanvandeLoosdrecht13,Joop structural heterochromatic and euchromatic variants Jansen2 described, including copy number variant segmental Radboud university medical center Human Genetics duplications. Nijmegen-The Netherlands1 Radboud university Here we report on a BAC-FISH study in 11 chromo- medical center Laboratory Medicine Nijmegen-The some 9 pericentric inversion carriers, including patients Netherlands2 VU Univesity Medical Center with clinical manifestations and their healthy relatives, Hematology Amsterdam-The Netherlands3 VU with the aim to more precisely delineate the breakpoints University Medical Center Hematology Amsterdam- and the CNV pattern in these cases. We used a set of The Netherlands4 Erasmus University Medical Center seven BACs from pericentromeric segmental duplica- HOVON Data Center Rotterdam-The Netherlands5 tion and flanking single copy regions, which were University Medical Center Groningen Experimental combined with alphaSat, betaSat and SatIII probes. Hematology Groningen-The Netherlands6 Meander Our results demonstrate at least three distinct inversion Medisch Centrum Internal Medicine Amersfoort-The patterns with breakpoints localized either at the hetero- Netherlands7 Hage Ziekenhuis Internal Medicine The chromatin boundary, or within segmental duplications, Hague-The Netherlands8 Erasmus University Medical accompanied with local duplications or triplications. Center Hematology Rotterdam-The Netherlands9 The patterns observed here could only partly be recon- Medisch Centrum Twente Internal Medicine ciled with previously published inversion patterns, Enschede-The Netherlands10 Isala klinieken Internal suggesting an even more complex inversion scenario Medicine Zwolle-The Netherlands11 Radboud in the human population than previously anticipated. university medical center Hematology Nijmegen-The To better understand the organisation of human chro- Netherlands12 VU Medical Center Hematology mosome 9 pericentromeric CNV in an evolutionary Amsterdam-The Netherlands13 framework, we further included chimpanzee, bonobo, gorilla, orangutan, a gibbon and three Old World In myelodysplastic syndromes (MDS) karyotyping is monkey species in our study. To this end, we confirmed generally considered as the gold standard. However, the recent explosive evolutionary copy number expansion karyotyping is limited by its low resolution (5–10 Mb of chromosome 9 segmental duplications in the last in size). Microarray-based genomic profiling analyses common ancestor of human/African apes, and in allow a genome-wide detection of copy number alter- particular in the human/chimpanzee ancestor. ations (CNAs), down to 100 kb in size, and regions of Further, our results indicate that the evolutionary copy neutral loss of heterozygosity (CNLOH). forces that shaped the human p-arm CNVs may at We have compared karyotyping with microarray- least to some extent be different from those on the based genomic profiling with respect to the detection q-arm side, possibly reflecting their evolutionary yield for genetic abnormalities in bone marrow samples origins in different genomic neighbourhoods. from lower risk MDS patients in a prospective phase II randomized multicenter study (HOVON89). Data 2. Tumour Cytogenetics regarding cytogenetics and microarray were obtained in a fully blinded manner for 91 MDS patients. For O4 microarray-based genomic profiling we used CytoScan HD Array (Affymetrix) platform. Comparison of microarray-based genomic profiling Thirty-two MDS patients had an abnormal karyotype, and karyotyping in myelodysplastic syndromes while 49 patients showed an abnormal array profile. Of (a HOVON89 study) interest, in 16 of these last patients no cytogenetic abnormalities were detected. The abnormalities that were Marian Stevens-Kroef1, Daniel Olde Weghuis1, Najat observed by microarray only were <5 Mb CNAs or ElIdrissi-Zaynoun1,BertvanderReijden2,Eline regions of CNLOH involving regions and genes recur- Cremers3,CAlhan4,TWesters4,HeleenVisser- rently affected in hematological malignancies. Almost all Wisselaar5, D Chitu5, Edo Vellenga6, S Klein7, Pierre CNAs identified by karyotyping (except 2 subclones) Wijermans8, Inge de Greef9, Ron Schaafsma10, M van were also observed by microarray-based genomic 10th European Cytogenetics Conference 2015 S27 profiling. As expected balanced translocations were not allowed for copy number assessment of additional prog- identified by microarray-based genomic profiling. nostic whole chromosomes or segmental regions, not We demonstrate that microarray-based genomic currently tested by FISH. SNP array could not detect profiling allows the identification of almost all copy cytogenetic changes present in minor subclones. The number abnormalities also observed by karyotyping. combined use of the allele track and the copy number In addition, we show that microarray-based genomic track provided accurate identification of aneuploidies, but profiling allows the detection of potential prognostic SNP array analysis had difficulty normalizing copy relevant abnormalities which would not be detected by number for tetraploid tumours. Amplifications of karyotyping. The clinical value of these novel CNAs MYCN in neuroblastoma and CMYC in medulloblastoma and CNLOH will be evaluated within the ongoing were adequately detected, although this was dependent prospective HOVON89 study. upon tumour sampling. SNP array analysis offers advantages and technical challenges as a diagnostic tool O5 for pediatric solid tumours.

Assessment of SNP genomic microarray analysis as O6 an alternative to FISH analysis for the detection of copy number changes in pediatric solid tumours Epigenetic regulation of UNCX, a novel homeobox transcription factor gene activated in acute myeloid Anthony Arnoldo1,JamesStavropoulos2,PaulThorner1, leukemia Cynthia Hawkins1,GinoRSomers1, Mary Shago2 The Hospital for Sick Children Division of Pathology, Giulia Daniele1, Ilaria Iacobucci2, Angelo Lonoce1, Department of Paediatric Laboratory Medicine Toronto- Margherita Perricone2, Mariana Lomiento3, Claudio Canada1 The Hospital for Sick Children Division of Giacinto Atene3, Elena Marasco4, Vilma Mantovani5, Genome Diagnostics, Department of Paediatric Hilmar Quentmeier6,HansGDrexler6,JieDing6, Laboratory Medicine Toronto-Canada2 Orazio Palumbo7,MassimoCarella7,Niroshan Nadarajah8, Rene’ Massimiliano Marsano1, Antonio Medulloblastoma, astrocytoma, and neuroblastoma are Palazzo1,EmanuelaOttaviani2,CarmenBaldazzi2, amongst the most common solid tumours in children. Nicoletta Testoni2,CristinaPapayannidis2,Sergio Recurrent gains or losses of distinct chromosomal Ferrari3, Giovanni Martinelli2, Clelia Tiziana Storlazzi1 regions are prognostic in these three tumours. In medul- University of Bari Department of Biology Bari-Italy1 loblastoma, amplification of CMYC or NMYC is associ- University of Bologna Institute of Hematology “L. e A. ated with a poor prognosis, and loss of chromosome 6 with Seràgnoli” Department of Experimental, Diagnostic and agoodprognosis.A7q34duplicationresultinginBRAF- Specialty Medicine Bologna-Italy2 University of KIAA1549 fusion is associated with good outcome in low Modena and Reggio Emilia Department of Life grade astrocytomas. In neuroblastoma, amplification of Science Modena-Italy3 S. Orsola-Malpighi Hospital NMYC and loss of 1p36 are associated with a poor Center for Applied Biomedical Research (CRBA) prognosis. Currently, we test these loci by FISH analysis, Bologna-Italy4 S. Orsola-Malpighi Hospital University followed by karyotyping if sufficient material remains. of Bologna Center for Applied Biomedical Research We have validated the use of the genomic microarray (CRBA) - Unit of Medical Genetics, Department of platform Affymetrix Cytoscan HD and Chromosome Medical and Surgical Sciences Bologna-Italy5 Leibniz- Analysis Suite software as an alternative to FISH analysis Institute DSMZ-German Collection of Microorganisms for the assessment of copy number changes in these and Cell Cultures Department of Human and Animal tumours. DNA was extracted from frozen samples of 4 Cell Lines Braunschweig-Germany6 IRCCS Medical neuroblastoma, 6 medulloblastoma, and 5 astrocytoma for Genetics Unit San Giovanni Rotondo-Italy7 MLL which FISH (+/- karyotype) information was available. Münchner Leukämielabor GmbH MLL Münchner There was 100 % correlation of SNP array data with Leukämielabor GmbH München-Germany8 FISH/G-banding data in detecting the major clonal unbalanced chromosome abnormalities. SNP array data also Homeobox (HB) genes encode for transcription factors refined G-band results, detected regions of LOH, and showing a crucial role in normal hematopoiesis and in S28 10th European Cytogenetics Conference 2015 leukemogenesis. Interestingly, the deregulation of HB Multiple myeloma (MM) is a plasma cell (PC) malig- genes, often embedded within low-methylated regions nancy characterized by clonal expansion of terminally (≤10 %) called “canyons”, was described in murine differentiated B cells in the bone marrow (BM). It is the hematopoietic stem cells (HSCs) as a consequence of second most common malignant blood disorder in the altered methylation at canyons edges (borders) due to Caucasian population and associated with complex Dnmt3a inactivation. Among them there is Uncx. genetic changes predicting patients’ outcome. UNCX (7p22.3), encoding for a transcription factor Evaluation of genetic lesions is an important tool for involved in somitogenesis and neurogenesis, is specifical- risk assessment and treatment monitoring. ly expressed in eye, brain and kidney. We identified a M5 Due to resolution and low proliferation activity of AML patient with a t(7;10)(p22;p14) translocation as the PC, the detection of chromosomal aberrations by sole cytogenetic anomaly and showing ectopic expression conventional karyotyping has proven to have too strong of UNCX. Since UNCX was never reported in limitations. Whilst molecular cytogenetic approaches association with cancer but only with common myeloid such as SNP-array and iFISH performed on PC enriched cell proliferation and regulation of cell differentiation, we BM samples have shown that genomic alterations are decided to investigate its contribution to leukemogenesis. present in more than 90 % of the MM patients. Recent We observed UNCX ectopic expression in 32.3 % studies demonstrated that disease evolution is associated (20/62) and in 8 % (6/75) of acute myeloid leukemia with an accumulation of specific genome imbalances (AML) patients and cell lines, respectively. Notably, with important prognostic values. Furthermore, one of retroviral-mediated UNCX transfer in CD34+ HSCs the main genetic events in MM is the translocation of the induced a slow-down in their proliferation and differenti- immunoglobulin heavy chain gene complex (IGH) with ation and transduced cells showed a lower growth rate but different chromosomes partners with the gene ahigherpercentageofCD34+stemcellsinliquidculture CCND1 at 11q13 being the most frequently observed. than controls. Additionally, UNCX infected cells Due to the often small quantities of material available displayed a decrease of MAP2K1 proliferation marker after PC enrichment, the assessment of the full MM but increase of KLF4, HOXA10, and CCNA1, associated genome for prognostic markers including balanced trans- with impaired differentiation and pluripotency. Similarly, locations of the IGH gene can be challenging in a routine UNCX-positive patients revealed alteration of gene diagnostic setting. To overcome this, we developed a pathways involved in proliferation, cell cycle control sensitive and reliable analytical protocol allowing whole and hematopoiesis. Moreover, UNCX activation was genome screening with low cell quantities (minimum accompanied by methylation changes at both its canyon 5000 cells) using a combination of SNP-array and a tri- borders, clearly indicating an epigenetic regulation of this colour customized iFISH. This approach interrogates gene. Taken together, our results indicate a novel putative genomic imbalances, CNLOH as well as IGH rearrange- leukemogenic role of UNCX, whose activation may be ments with CCDN1 or other partner and allows rapid ascribed to epigenetic regulators other than DNMT3A, turnaround time of analysis. Workflow, performance and differently from what previously described in murine analytical results will be presented and discussed. HSCs. 3. Prenatal Diagnosis O7 O8 Rapid and comprehensive routine diagnostic approach for prognostic genetic markers in multiple Digital PCR is an efficient alternative to massively myeloma parallel sequencing as a non-invasive, prenatal test for trisomy 21: the results of a pilot study Elodie David1,SandrineBougeon1,ValérieBeyer Stalder1, Laurence Etter1, Kolly Delphine1, Noémie Laïla Allach El Khattabi1,ChristelleRouillac-Le Gesson1,DominiqueMuehlematter1,Jacqueline Sciellour2, Dominique Le Tessier3, Armelle Luscan3, Schoumans1 Raphael Porcher4, Rakia Bhouri3, Juliette Nectoux5, Cancer Genetics Unit, Lausanne University Hospital Valérie Sérazin2, Thibault Quibel6, Vassilis Tsatsaris7, Medical Genetics Lausanne-Switzerland1 François Vialard8, Jean-Michel Dupont1 10th European Cytogenetics Conference 2015 S29

APHP Cochin, INSERM U1016, CNRS UMR8104, O9 Université Paris Descartes Laboratoire de Cytogénétique Paris-France1 CHI Poissy Saint Noninvasive prenatal testing: more caution is needed Germain Fédération de Génétique Poissy-France2 in high risk pregnancies APHP Cochin Laboratoire deCytogénétiqueParis- France3 Hôtel Dieu Centre d’Epidémiologie Clinique Beatrice Oneda1, Katharina Steindl1, Rahim Masood1, Paris-France4 APHP Cochin Laboratoire de Biochimie Irina Reshetnikova1, Pavel Krejci1, Rosa Baldinger1, et Génétique Moléculaire Paris-France5 CHI Poissy Regina Reissmann1, Malgorzata Taralczak1, Adriano Saint Germain Département d’Obstétrique et de Guetg1, Anita Rauch1 Gynécologie Poissy-France6 APHP Cochin, Maternité University of Zürich Institute of Medical Genetics Port-Royal Département d’Obstétrique et de Zurich-Switzerland1 Gynécologie Paris-France7 CHI Poissy Saint Germain, GIG, Faculté des Sciences de la Santé, Université de Purpose: Non-invasive prenatal testing (NIPT) is increas- Versailles Saint-Quentin-en-Yvelines Fédération de ingly being used in prenatal diagnosis. However, published Génétique Poissy-France8 data on confirmatory cytogenetic testing after positive NIPT in order to collect more information on the positive The analysis of circulating cell-free DNA has prompted predictive value for noninvasive testing is still scarce. considerable progress in the field of non-invasive prenatal Methods: Concordance of results among cases testing for fetal aneuploidy. Molecular counting can be with NIPT referred for cytogenetic prenatal and/or achieved with both massivelyparallelsequencingand postnatal studies by karyotyping, fluorescence in digital PCR. However, the large number of PCR reactions situ hybridization and molecular karyotyping was needed to meet statistical requirements has prevented evaluated in 47 consecutive cases. digital PCR from being applied in a clinical setting. Here, Results: True positive and false positive NIPT results we designed an octoplex droplet digital PCR (ddPCR) were obtained in 56.4 % and in 41 % of the cases, approach, which allows increasing the number of available respectively. Inconclusive results followed by cytoge- targets and thus overcomes statistical obstacles. netically pathologic results were encountered in three After technical optimization, we performed a pilot cases. Likely false negative NIPT results were observed study on samples of plasma DNA from 300 pregnant in two cases, one of which ended with the birth of a women for whom a fetal karyotype was available. severely affected girl. The birth of severely affected Molecular counting of circulating cell-free DNA was children occurred in three more cases, in which only performed using a mix of hydrolysis probes targeting NIPT, which resulted normal, was offered, despite chromosome 21 and a reference chromosome. The ultrasound abnormalities. results of our validation experiments showed that Conclusion: The positive predictive value of abnormal ddPCR detected trisomy 21 even when only 5 % of NIPT should not be overestimated and not be confused the DNA was trisomic. In a validation study of plasma with the overall specificity value. In addition, more samples from pregnant women, ddPCR discriminated caution is needed in offering NIPT in high risk clearly between the trisomy 21 group and the euploidy pregnancies with ultrasound anomalies. group. The shape of the receiver operating characteristic curve indicated that our screening test is highly efficient. 4. Animal and Plant Cytogenetics Our results demonstrate that ddPCR is a promising technique for non-invasive prenatal testing of 010 trisomy 21. This approach is technically simple, relatively cheap, easy to implement in a diagnostic Comparative chromosome painting in lacertid lizards: setting and compatible with ethical concerns highly conserved karyotypes but independent origins regarding access to nucleotide sequence informa- of sex chromosomes tion. These advantages make it a potential technique of choice for population-wide screening for Verónica Rojo 1,MalcolmFerguson-Smith2,Jorge trisomy 21 in pregnant women, as well as for Pereira2, Pedro Galán3, Ana González-Tizón1, Andrés other aneuploidies. Martínez-Lage1, Horacio Naveira1 S30 10th European Cytogenetics Conference 2015

Universidade da Coruña Bioloxía Celular e Molecular A Gemma Macchia1, Marco Severgnini2, Ingrid Cifola2, Coruña-Spain1 University of Cambridge Veterinary Monica Zoli3, Stefania Purgato3,OrazioPalumbo4, Medicine Cambridge-England2 Universidade da Coruña Anna Loverro1, Massimo Carella4, Giovanni Perini3, Bioloxía Vexetal, Animal e Ecoloxía A Coruña-Spain3 Florence Pedeutour5,FredrikMertens6,Mariano Rocchi1, Clelia Tiziana Storlazzi1 In this work we applied for the first time high-resolution University of Bari Department of Biology Bari-Italy1 molecular cytogenetic techniques (chromosome painting National Research Council Institute for Biomedical and comparative genomic hybridisation) to carry out com- Technologies Milan-Italy2 University of Bologna parative analyses of sex chromosomes among five lacertid Department of Biology Bologna-Italy3 IRCCS species, namely, Iberolacerta monticola, I. galani, I. bonnali, Casa Sollievo della Sofferenza Hospital Medical Lacerta schreiberi, and Timon lepidus. In addition, flow- Genetics Unit San Giovanni Rotondo-Italy4 Nice sorted chromosome paints from I. monticola were used in University Hospital Laboratory of Solid Tumors genome-wide comparisons with the chromosomal Genetics Nice-France5 University and Regional complements of L. schreiberi and T. lepidus, in order Laboratories Department of Clinical Genetics to detect chromosomal rearrangement and homologies Lund-Sweden6 among the species of the three different genera. Chromosome painting with the W of I. monticola Neocentromeres have been reported as frequently (IMOW) showed that the euchromatic region of the arising on ring/rod-shaped chromosomes, particularly female-specific sex chromosome appears to be con- in sarcomas. It has been reported that the CENP-A served in the other two species of Iberolacerta. In I. centromeric protein is rapidly recruited at double- bonnali, a screening with the remaining flow-sorted strand break (DSB) by the DNA repair machinery. fractions of I. monticola indicates that its biarmed W This finding suggested a potential link between the chromosome most likely has undergone a centric fusion process of DSB repair and neocentromere seeding. We with either chromosome 15 or 16. IMOW and compar- studied four cancer cell lines (three well-differentiated ative genomic hybridisations led to the unequivocal liposarcomas and a lung-sarcomatoid carcinoma) carrying identification of the W chromosome both in L. neocentromeres on ring/rod-shaped chromosomes, to schreiberi and T. lepidus, and indicate a massive accu- investigate this phenomenon. SNP array, FISH, and mulation of female-specific repetitive sequences char- anti-CENP-A ChIP-seq experiments were performed to acteristic of each lineage, leading to the formation of characterize the marker chromosomes and the highly heterochromatic sex chromosomes. neocentromeric domains. Whole genome next- Our preliminary data from chromosome painting generation sequencing was also carried out to finely define using the Z chromosome suggest at least two indepen- the inner structure of the marker chromosomes. Our dent origins of sex chromosomes in these lacertid results revealed that neocentromeres were seeded on a lizards. The study of chromosome synteny with the “patchwork” of short-sized (<100 Kb) amplified remaining IMO chromosome fractions revealed a fragments, some from different chromosomes, high degree of karyotype conservation. The underlining the epigenetic nature of this phenomenon microchromosomes of T. lepidus and L. schreiberi are and suggesting that the massive recruitment of CENPA painted by the IMO fraction containing chromosomes to repair DSB might trigger the neocentromere forma- 11 and 12, thus indicating that a translocation of tion. We are currently evaluating the chromatin status microchromosomes to macrochromosomes generated of the neocentromeric regions by performing targeted this difference in karyotype structure of I. monticola. bisulphite sequencing and H3K4me1-3/H3K4me27 ChIP-seq assays. Furthermore, we are performing 5. Molecular Mechanisms of Chromosome RNA-Seq experiments to identify transcripts likely Rearrangements involved in the neocentromeres seeding and main- O11 tenance. Moreover, two of our liposarcoma cell lines derive from the same primary tumour; this Unraveling the internal structure of neocentromeres will allow us to investigate the evolution of in ring chromosomes harboring genomic amplification marker chromosomes and neocentromere from the in soft tissue tumors original tumour. Finally, several cases of soft tissue 10th European Cytogenetics Conference 2015 S31 tumours carrying neocentromeres will be analysed to molecular analysis allowedascertainingthereal gain a broader view of this phenomenon in primary nature of this CCR. malignant sarcomas. The expected results will unveil For both families, a re-building of the initial rear- the existing link between DSB repair and rangement occurred through meiotic recombination, neocentromere seeding, and add new insight into the giving rise to simpler or more complicated CCR. mechanisms behind the genesis of ring/rod-shaped Combination of next-generation DNA sequencing marker chromosomes. and other standard approaches such as karyotyping and FISH analysis shed light on the complexity of O12 these CCRs. This new approach has improved the quality of genetic counseling. Meiotic recombination and complex chromosome rearrangements: how whole-genome sequencing O13 helps to resolve them. Unbalanced de novo chromosome translocations and Vincent Gatinois1,FranckPellestor1,Geneviève inversions: a two-step origin? Lefort1, Anouck Schneider1, Christine Coubes2, David Genevieve2,PierreSarda2, Wigard Kloosterman3, Maria Clara Bonaglia1, Francesca Novara2, Roberto Jacques Puechberty2 Giorda3,SilvanaBeri4,MarilenaCarmelaDi Hospital A. de Villeneuve— CHRU Chromosomal Giacomo5,EmanuelaFerri5,RobertoCiccone6, Genetics Laboratory Montpellier-France1 Hospital A. Annalisa Vetro7,SaraBertuzzo8,VannaPecile9, de Villeneuve— CHRU Department of Medical Micheala A Aldred10, Cintia Barros Santos-Rebucas11, Genetics Montpellier-France2 University Medical Sabrina Giglio12,PhilipposPatsalis13,Carolina Center Department of Medical Genetics / Genome Sismani14, Diane N Abuelo15, Orsetta Zuffardi6 Diagnostics Utrecht-The Netherlands3 Scientific Institute IRCCS Eugenio Medea Cytogenetics laboratory Bosisio Parini (LC)-Italy1 University of Pavia The presence of at least 3 chromosome breakpoints Molecular Medicine Pavia-Italy2 Scientific Institute defines Complex Chromosome Rearrangements IRCCS Eugenio Medea Molecular Biology Laboratory (CCRs). They can mix translocations, inversions, Bosisio Parini (LC)-Italy3 Scientific Institute IRCCS E. deletions and/or duplications. Complexity of CCRs Medea Molecular Biology Laboratory Bosisio Parini can be very high and the related meiotic distur- (LC)-Italy4 University of Bari Biomedicina dell'Età bances result in important chromosomal imbalances in Evolutiva Bari-Italy5 University of Pavia Departement gametes. So, genetic counseling is tricky for carriers of a of Molecular Medicine Pavia-Italy6 Fondazione IRCCS balanced CCR. Policlinico San Matteo Biotechnology Research We report 2 families with heavy miscarriage histo- Laboratories Pavia-Italy7 Scientific Institute IRCCS E. ries. For them, standard karyotypes revealed CCR. Medea Cytogenetic Laboratory Bosisio Parini Surprisingly, we observed that the degree of complexity (LC)-Italy8 IRCCS Burlo Garofolo Institute for of CCRs changed through generations. Using whole Maternal and Child Health Trieste-Italy9 Case genome sequencing we determined the real number of Western Reserve University School of Medicine breakpoints and specified the position of each of them. Department of Genetics Cleveland-United States10 In the first family, the discovery of a multiple malfor- State University of Rio de Janeiro Department of mation syndrome on ultrasound examination indicated a Genetics Rio de Janeiro-Brazil11 University of prenatal chromosome analysis. The fetal karyotype Florence Medical Genetics Section, Department of showed an unbalanced CCR. Subsequent chromo- Clinical Pathophysiology Florence-Italy12 The some analysis of the mother revealed a much more Cyprus Institute of Neurology and Genetics complex but apparently balanced CCR. For the Translational Genetics Team Nicosia-Cyprus13 The second family, a simple reciprocal translocation Cyprus Institute of Neurology and Genetics was inherited through several generations. For Department of Cytogenetics and Genomics one of the daughters, the rearrangement was more Nicosia-Cyprus14 Brown University Warren Alpert complex involving a third chromosome. Fine School of Medicine Providence-United States15 S32 10th European Cytogenetics Conference 2015

The origin of de novo unbalanced translocations Fengtang Yang1, Sandra Louzada1, Beiyuan Fu1, Ruby and unbalanced rearrangements mimicking a deriv- Banerjee1, Chris Tyler-Smith1 ative from a pericentric inversion (herein called Wellcome Trust Sanger Institute Human Genetics unbalanced inversions), all presenting simultaneous Cambridge-United Kingdom1 deletion and duplication of two terminal chromosomal regions, is still enigmatic although their Molecular Combing is an advanced DNA-stretching frequency, at least for de novo unbalanced trans- technology that allows molecules of 200–600 kb locations, has been estimated to be 22 % at long to be uniformly combed onto a glass surface. amniocentesis and 30 % in subjects with develop- As the stretching is constant along the length of a mental disorders. DNA molecule, when combined with multicolour We have analyzed 35 de novo unbalanced rear- fluorescent in-situ hybridisation (M-FISH), rangements, of which one in mosaic, including Molecular Combing holds the promise for unam- simple translocations (n=25), inv-dup-del translo- biguous characterisation of genomic structure and cations (n=6), and unbalanced inversions (n=4), in variation by direct visualisation. Apart from order to highlight their parental origin and mecha- defining order, orientation, and copy number, this nisms of formation. In most cases, we could char- technique also allows the quantification of length, acterize the rearrangement at the molecular level gap and overlap sizes. However, to visualize large and determine the parental origin of both deleted and complex structural variations and to reduce and duplicated segments. Both imbalances were of potential artefacts associated with broken fibres, maternal origin in 14 subjects, paternal in 8, and much longer molecules (~1000 kb) and references bi-parental in 2. In 6 additional cases, parental marking the two ends of variant regions are often origin was informative for only one of the imbal- required. Here, by modifying the Molecular ances (4 paternal and 2 maternal) while in the Combing protocol, we improved the retention rate remaining 5 no information was available. of longer DNA molecules on combed coverslips. Our findings suggest that partial rescue of an We also introduced dinitrophenol, fluorescein and abnormal chromosome complement consisting in Cy5 haptens, and optimised primary and secondary either (i) a rearranged chromosome, such as a antibody-combinations for use in M-FISH detec- dicentric “mirror” chromosome, or (ii) a supernu- tion, in addition to the widely used biotin and merary chromosome, presumably already present digoxigenin two-colour combination. We have in the zygote, is the most likely mechanism successfully applied thismodifiedfibre-FISH leading to this type of rearrangement. This rescue technique to assembling the pig Y-chromosome may also generate alternative products in different sequence and characterising a series of complex cells of the early embryo, leading to mosaic lines structural and copy-number variation involving of which the most viable one(s) can be detected in large multiallelic gene families or repetitive post-natal tissues. The occurrence of two unbal- sequences identified by array-based assays and anced translocations with the two imbalances next–generation sequencing, including the well- having bi-parental origin supports the proposed studied amylase genes. While confirming the two-step mechanism. In addition, the preferential copy-number changes predicted by alternative maternal origin of both imbalances might indicate methods for the majority of genomic regions being that the final rearrangement results, at least in investigated, we uncovered structural details that some cases, from an original non-disjunction event would be impossible to elucidate using array- although the expected association with increased CGH or short-read sequencing. This approach also maternal age allows pairs of complex haplotypes to be decoded unambiguously. Our results demonstrate that 014 Molecular Combing, when combined with M-FISH, is a definitive approach for the validation Direct visualisation of complex structural and copy- and genotyping of complex structural and copy-number number variation by molecular combing and variation, as well as for assembling regions enriched in multicolour-FISH repeats such as mammalian Y chromosomes. 10th European Cytogenetics Conference 2015 S33

015 ~1,500 ncRNA genes, most of the LRPE-regions that specify the vertebrate body plan, link phenotypes The International Breakpoint Mapping Consortium with disruption/rearrangement of nuclear domains, (IBMC). Systematic mapping of chromosomal and identify human models for their functional breakpoints in the context of phenotypes and nuclear characterization. The project will include participants from genome organization undeveloped, developing and developed countries, and hence facilitate global mobility and technology tranfer. Niels Tommerup1, Malene B. Rasmussen1, Mana M. Mehrjouy1, Iben Bache1, Allan Lind-Thomsen1, Ana 6. Impact of Next Generation Sequencing on Clinical Carolina dos Santos Fonseca2,ChristinaHalgren1, Diagnosis Mads Bak1, Peter Jacky3 University of Copenhagen Department of Cellular and 016 Molecular Medicine Copenhagen-Denmark1 Universidade de São Paulo Departamento de Genética Exome sequencing and SNP arrays as a tool to describe eBiologiaEvolutivaSãoPaulo-Brazil2 Northwest genetic diversity of premalignant and malignant cells: Permanente PC, Emeritus Oregon-United States3 lesson from monoclonal gammopathies

We still lack genotype-phenotype-information for ~80– Aneta Mikulasova1, Brian A. Walker2, Christopher P. 90 % of our protein-coding genes, and for almost all Wardell3, Eileen M. Boyle4, Marketa Wayhelova1, Jan non-coding RNA (ncRNA) genes and regulatory Smetana1, Ludek Pour5, Petr Kuglik1, Roman Hajek6, elements. Even with exome and full genome sequencing, Gareth J. Morgan7 it will take decades and tremendous resources to saturate Faculty of Science, Masaryk University Department of the exome, trancriptome and regulome with mutations/ Experimental Biology Brno-Czech Republic1 Royal variants that can be linked to abnormal and normal Marsden Hospital Centre for Molecular Pathology phenotypes. Furthermore, NGS-based proximity London-United Kingdom2 RIKEN Centre for mapping has revealed a nuclear organization of the Integrative Medical Sciences Laboratory for Genome genome, with ~2–3,000 topologically associating do- Sequencing Analysis Tokyo-Japan3 Institute of Cancer mains (TAD), which can be further organized into loops. Research Division of Molecular Pathology London- To facilitate the functional characterization of TADs, United Kingdom4 University Hospital Brno Department TAD-boundaries and loops, we have initiated a systematic of Internal Medicine, Hematology and Oncology Brno- mapping of constitutional “balanced” chromosomal Czech Republic5 University Hospital Ostrava Department rearrangements (BCR) that must affect this nuclear of Haematooncology Ostrava-Czech Republic6 organization. The first unbiased reexamination of pre- University of Arkansas Medical School Myeloma natally diagnosed de novo BCRs (pBCRdn) show that Institute for Research and Therapy Little Rock, breakpoints affects genomic compartments according to Arkansas-United States7 their size, e.g. protein coding/non-coding genes; regulatory domains associated with long-range position effects Whole exome sequencing (WES) and SNP arrays have (LRPE); unannotated gaps; and TAD and TAD- opened a new landscape to study comprehensive tumour boundaries. We also show that the unbiased morbidity genetic architecture and its evolution during tumour risk of pBCRdn are 2–3 fold higher than previously associated processes at the genome-wide level. estimated (~20 %), due to later onset neurodevelopmental Transformation from monoclonal gammopathy of unde- disorders. This morbidity can in some cases be linked to termined significance (MGUS) to multiple myeloma truncation of known disease genes and LRPE-regions, (MM) can be used as a unique model for cancer devel- but we also highlight the existing gap in genotype- opment studies as an analysis of pure tumour population phenotype knowledge. To address this gap, we have in clearly clinically distinguishable states. In our study, established IBMC, presently involving >150 partners we optimized WES and SNP array protocols from from >50 countries and six continents, with the aim to whole-genome amplified DNA to describe genetic mate-pair map ~10,000 chromosomal breakpoints. This diversity of premalignant and malignant cells at the will target an estimated ~2,700 protein coding genes, genome-wide level. S34 10th European Cytogenetics Conference 2015

Overall, 33 and 69 MGUS patients were included in a otherwise normal sterile male to genital ambiguity and WES and SNP array study, respectively. For WES, variable Turner Syndrome (TS) features. We report on a NEBNext kit and SureSelect Human All Exon V5 male with this rare cause of infertility and discuss the (Agilent) were used, samples were sequenced by inherent consequences regarding assisted reproductive HiSeq2000 (Illumina). Copy number alterations technology and tumor surveillance for gonadoblastoma. (CNAs) were tested by SurePrint G3 CGH+SNP, 4 × Clinical report: A 40 year old man sought the advice 180K (Agilent). Results were compared to 463 and 91 of a urologist for infertility. Anamnesis was unremarkable MM patients analysed by WES and SNP arrays, and precluded external detrimental influences. Clinical respectively. examination showed markedly short stature (150 cm), CNAs and somatic gene mutations (SNVs) were small testicles, normal penis size and normal pubic hair. detected in 68 % (47/69) and 100 % (33/33) of MGUS Semen analysis demonstrated complete azoospermia and patients in comparison to 100 % (91/91, p<10^-4) and low gonadotrophins and testosterone levels. 100 % (463/463) of MM patients, respectively. Lab investigation and results: Standard lymphocyte However, overall number of both CNAs and SNVs per culture from the peripheral blood revealed two cell lines: patient was significantly lower in MGUS (CNAs: a majority of 45,X cells and an abnormal Y chromo- median 2, range 0-15; SNVs: median 89, range 9–315) some in the remaining cells. FISH investigation than in MM (CNAs: median 16, range 2–49, p<10^-18; confirmed a similar distribution with 2 centromeres, 2 SNVs median 123, range 1–897, p<10^-4). We proved SRY, and 2 SHOX signals in the rearranged Y. that complex genetic instability is formed before tumour Multiplex PCR analysis demonstrated microdeletion in clinical manifestation at the gene level followed by the AZFb and AZFc loci. chromosome level. Then, the number of random genetic Discussion: Isodicentric Y chromosomes are the hits increases to form a landscape for significant onco- most commonly reported Y chromosome aberrations, genic hits driving the premalignancy transition to a mainly as mosaics with a 45,X cell line, but their overall clinically manifested tumour disease. Data in detail are prevalence is lower than 0.5 % in chromosomally presented. induced infertility. This Yaberration removes spermato- Support: IGA-MHCZ-NT13492, OPVK-CZ.1.07/ genesis loci resulting most likely in a Sertoli-only 2.3.00/20.0183. syndrome. Timely diagnosis seems important because of a small but significant risk for developing Poster Abstracts gonadoblastoma. In addition, variable features of TS may be observed. Therefore, in similar cases diagnosed 1. Clinical Cytogenetics prenatally (even with apparently normal neonatal appearance) regular follow-up is a responsible task for 1.P1 timely assisting development and education in a condition with unpredictable outcome. 45,X/46,X,idic(Y) in azoospermia: clinical, hormonal and molecular findings in an adult man of very short 1.P2 stature Unusually mild course in trisomy 13 mosaicism with Gabriela Kronberger1,OlafRittinger1,Andreas multiple hemangiomas Jungwirth2 Paracelsus Medical University Clinical Genetics Olaf Rittinger1, Iris Wieser2, Ingrid Bader1, Gabriela Salzburg-Austria1 EMCO Private Clinic Bad Kronberger1, Gabriele Sander3 Dürrnberg-Austria2 PMU Salzburg Department of Pediatrics Salzburg - Austria1 PMU Salzburg Department of Obstetrics Background: In the majority of cases, 45,X/ Salzburg -Austria2 PMU Salzburg Deparment of 46,X,idic(Y) results in normal appearance of the male Pediatrics Salzburg -Austria3 genitalia in the neonatal period; followed later on by infertility. The postnatally developing phenotype is Background. Trisomy 13 mosaicism is rare, occurring in quite variable and unpredictable, ranging from only 5 % of all trisomy 13 cases. Phenotype varies from 10th European Cytogenetics Conference 2015 S35 very severe to milder forms depending on the proportion Kariminejad and Najmabadi Pathology and Genetics of cytogenetically normal and trisomic cells. Several Center Cytogenetics Laboratory Tehran-Iran2 major congenital anomalies are usually associated with University of Social Welfare and Rehabilitation trisomy 13, including capillary hemangiomas. We report Sciences Pediatric Neurorehabilitation Research Center on an 18 month old girl with unexpectedly mild clinical Tehran-Iran3 developmental delay presenting with several hemangiomas of different shapes and sizes, but no other 15q24 microdeletion syndrome is a rare syndrome with malformations. a frequency of about 0.3 % in patients with intellectual Case report. disability and about 0.1–0.2 % in patients with autistic 2nd child of a 44-year old mother; combined test symptom disorder. This syndrome was described for the results in favour of normal fetal development. first time in 2007 by Sharp et al. The reported clinical Dystrophic birth at 28 weeks of gestation, 534 features included growth retardation, intellectual dis- g/33 cm/22 cm (allPatau syndrome, the remarkably mild cogni- Small Deletion of 143 Kb Encompassing Exon 2 of tive delay, postnatal low-normal growth and weight, and the AUTS2: Rise of a New Microdeletion Syndrome? a pattern of different hemangiomas. To our knowledge only one has been reported with similar skin anomalies Silvia Serafim1, Barbara Marques1, Filomena Brito1, without any other organic malformations. Sonia Pedro1, Cristina Ferreira1, Catarina Ventura1, Isabel Gaspar2, Hildeberto Correia1 1.P3 Instituto Nacional de Saude Doutor Ricardo Jorge, I.P. Unidade de Citogenetica, Departamento de Genetica Microdeletion of 15q24 detected by Array-CGH Medica Lisboa-Portugal1 Hospital Egas Moniz, Centro technique, in an Iranian patient with intellectual Hospitalar de Lisboa Ocidental EPE Consulta de disability, autism, seizures, and dysmorphism Genetica Medica Lisboa-Portugal2

Farkhondeh Behjati1, Saghar Ghasemi Firouzabadi1, Chromosome microarray analysis is a powerful diag- Roxana Kariminejad2, Roshanak Vameghi3, Hossein nostic tool and is being used as a first-line approach to Najmabadi1 detect chromosome imbalances associated with University of Social Welfare and Rehabilitation intellectual disability, dysmorphic features and congenital Sciences Genetics Research Center Tehran-Iran1 anomalies. This test enables the identification of new S36 10th European Cytogenetics Conference 2015 copy number variants (CNVs) and their association with mosaic trisomy 22. The patient had the following clin- new microdeletion/microduplication syndromes in ical features: intrauterine growth retardation, mild hypo- patients previously without diagnosis. tonia, cryptorchidism, phimosis, facial asymmetry, Here we report the case of a 17 year-old female with enophthalmus, mild prognathism, bifid uvula, hypoplastic severe intellectual disability, absence of speech, micro- upper limb phalanges, DD including speech delay, and cephaly and congenital abnormalities with a previous ID. Whole genome oligonucleotide microarray CGH normal karyotype performed at a younger age. 105 K (Agilent Technologies) showed a de novo 1 Mb Affymetrix CytoScan HD chromosome microarray interstitial deletion in 22q13.2, confirmed by fluorescence analysis was performed detecting a 143 Kb deletion at in situ hybridization in all cells examined. Moreover, 18 % the 7q11.22 breakpoint, encompassing exon 2 of cells had three chromosome 22 signals in addition to the AUTS2 gene: arr[hg19] 7q11.22(69238957- 22q13.2 deletion. G-banded karyotype was done and 69381975)×1. revealed a trisomy 22 in mosaic. The AUTS2 gene has been recently implicated in Almost all 22q13 deletions published so far have neurodevelopment and is a candidate gene for numerous been terminal deletions with variable sizes (2.7– neurological disorders. Common clinical features 6.9 Mb). Very few cases of smaller, interstitial 22q13.2 described in patients with deletions in AUTS2 gene deletions were reported. In its mosaic form, trisomy 22 include intellectual disability, speech delay and micro- is compatible with life, and there are about 20 reports in cephaly, among others. Thus, the CNV identified in our the literature. It has a variable clinical presentation: patient explains the phenotype observed. growth restriction, dysmorphic features, cardiovascular We compare our patient with other similar reported abnormalities, hemihyperplasia, genitourinary tract cases, adding additional value to the phenotype- anomalies and ID. Neurodevelopmental outcome ranges genotype correlation of deletions in this region. The from normal to severe DD. growing collection of new cases with similar pheno- Our case points out the role of conventional cytoge- types, and the observation of this deletion occurring netic tools in mosaic cases that could be missed by frequently de novo, indicates this CNV as a possible microarray technology. With the advances of CGH, new single gene microdeletion syndrome. new microdeletion and microduplication syndromes are being reported. Data is lacking about the interactions 1.P5 of more than one genetic anomaly detected on a same patient, making the genotype/phenotype correlation Association of structural and numerical anomalies of quite difficult. chromosome 22 in a patient with syndromic intellectual disability. 1.P6

Rania Naoufal1, Marine Legendre2, Dominique Couet1, Mixoploidy combined with aneuploidy in a patient Brigitte Gilbert-Dussardier2, Alain Kitzis1,Frédéric with severe multiple congenital abnormalities and Bilan1, Radu Harbuz1 mental retardation University Hospital Center of Poitiers Genetics Laboratory Poitiers-France1 University Hospital Center Laura J.C.M. van Zutven1,GraziaM.S.Mancini1, of Poitiers Medical Genetics Poitiers-France2 Karen G.C.B. Bindels- de Heus2, Erica L.T. van de Akker2, Lorette. O.M. Hulsman1, Marjan Smit1, H. Array comparative genomic hybridization CGH is now Berna Beverloo1 widely adopted as a first-tier clinical diagnostic test for Erasmus MC Clinical Genetics Rotterdam-The patients with developmental delay/intellectual disability Netherlands1 Erasmus MC— Sophia Pediatrics DD/ID, autism spectrum disorders, and multiple con- Rotterdam-The Netherlands2 genital anomalies. Nevertheless, classic karyotyping still has its impact in diagnosing genetic diseases, We report a 10-year-old female patient born to healthy, particularly mosaic cases. non-consanguineous parents, presenting with metopic We report on a 30 year old patient with syndromic ridge, palatoschisis, dysmorphic features, high-arched intellectual disability, a 22q13.2 microdeletion and eyebrows, multiple congenital contractures, broad 10th European Cytogenetics Conference 2015 S37 thorax with widely separated nipples, thoracic scoliosis, The 22q11.2 Deletion Syndrome (22q11.2 DS) has a short stature, intellectual disability, high pitched voice, prevalence of 1:4000 births and is characterized by nasal speech, hypotonia and failure to thrive. Metabolic multiple anomalies, including several types of congen- investigation shortly after birth was normal. ital heart diseases. The phenotypic spectrum is variable Karyotyping revealed a 45,X karyotype in peripheral and overlaps with other clinical conditions. blood, consistent with Turner syndrome. Consequently, clinical diagnosis is complicated and As the girl’s phenotype could not be explained by several patients with clinical features do not present this Turner karyotype, further genetic analyses were the 22q11.2 deletion. A cohort of 78 patients with performed. ASXL1 mutation analysis for Bohring- clinical suspicion of 22q11.2 DS and congenital heart Opitz trigonocephaly was normal. Subtelomeric disease, without the deletion, previously tested by FISH MLPA and genomic array using DNA from peripheral and/or MLPA, were investigated by whole-genome blood confirmed the 45,X karyotype. Additionally, array Genomic Hybridization (aGH) (CytoScan HD array showed a paternally inherited, small interstitial chip, Affymetrix®). A total of 66 copy number varia- loss on chromosome 4q31.22 (unknown clinical tions (CNVs)≥300 Kb were observed, corresponding to significance). 32 deletions and 34 duplications. Of these, 50 CNVs As still no satisfactory explanation for the girl’s involved genes and are potential loci related with the phenotype was found, a mosaic genetic aberration was phenotype of these individuals. Nine patients showed suspected and genomic array was repeated using DNA CNVs already described in patients with multiple from a skin biopsy. An abnormal profile was observed, congenital anomalies, such as deletions at 4q35.1-q35.2 suggesting the presence of a triploid cell line with loss of (4.5 Mb), 5p15.1-p15.33 (17.4 Mb), 8p23.1 (3.8 Mb), one X chromosome besides the 45,X cell line, which 10q22.3-q23.2 (7.8 Mb), 16p11.2 (611 kb), 17q12 was confirmed using karyotyping on skin fibroblasts. (1.8 Mb) and 22q13.33 (1.1 Mb); and duplications at Retrospectively, the dysmorphic features and congenital 3p26.3 (455 kb and 482 kb) and 3q26.2 (2 Mb). These anomalies resemble earlier descriptions of triploid/ CNVs are potentially pathogenic and dosage effects of diploid mixoploidy syndrome, however in combination genes such as IRX4, BMPR1A, GATA4 and SOX7 can with the Turner phenotype these were difficult to be involved in cardiac development. These results recognize. confirms the wide phenotypic spectrum and the overlap In conclusion, the patient’sphenotypecanbe with different conditions of patients with 22q11.2 DS explained by a tissue specific mosaic abnormal karyo- suspicious. Further studies may contribute for the iden- type with both an abnormal triploid and an abnormal tification of other CNVs, not mapped at 22q11.2 region, diploid cell line being present (mixoploidy). in patients with clinical suspicion of 22q11.2 DS and Mixoploidy in combination with an aneuploidy, as ob- congenital heart diseases. served in this patient, is extremely rare. This observation underscores the importance of re-examination and Financial support: FAPESP, CAPES and CNPq second tissue analysis of patients with multiple congenital anomalies when microarray copy number analysis 1.P8 appears normal. Whole-genome screening in a set of Czech patients 1.P7 with mental retardation and developmental abnormalities using array-CGH technique: our experience Copy number variations (CNVs) in patients with from 2007 to 2014 congenital heart diseases and 22q11.2 DS clinical suspicion Marketa Wayhelova1, Aneta Mikulasova1, Vladimira Vallova1, Katerina Kasikova2, Jan Smetana1, Renata Miriam Coelho Molck1, Milena Simioni1, Társis Paiva Gaillyova2, Petr Kuglik1 Vieira1, Ilária Cristina Sgardioli1, Vera Lúcia Gil-da- Faculty of Science, Masaryk University Department of Silva-Lopes1 Experimental Biology Brno-Czech Republic1 Faculty of Medical Sciences-University of Campinas University Hospital Department of Medical Genetics Department of Medical Genetics Campinas-Brazil1 Brno-Czech Republic2 S38 10th European Cytogenetics Conference 2015

Introduction: Mental retardation (MR) represents a SP-Brazil1 University of Campinas Department of health condition defined as a heterogeneous manifesta- Medical Genetics, Faculty of Medical Sciences tion of CNS dysfunction characterized by significantly Campinas, SP-Brazil2 University of São Paulo subnormal intellectual functions. It affects 1–3 % of Division of Clinical Immunology, Department of Western population and about 0.4 % people suffer from Medicine, Ribeirão Preto Medical School Ribeirão its severe form. MR may be a result of various genetic Preto, SP-Brazil3 events or environmental factors affecting prenatal and postnatal development as well. Despite a rapid progres- Cytogenetic approaches have been applied to under- sion in diagnostic methods and analytical approaches up stand the role of genomic instability in systemic lupus to 50 % MR cases do not have a known etiology. erythematosus (SLE), an autoimmune disease with a Material and methods: In the course of our 7-year strong genetic background. Loss of heterozygosity experience with chromosomal microarray analysis in (LOH) can occur in human cells without evident path- individuals with MR and associated phenotypic abnor- ological consequences. However, LOH has been reported malities we have examined 171 Czech patients at the as a model to explain the susceptibility to some Department of Medical Genetics (University Hospital classes of complex diseases. The purpose of this study Brno) using three different oligonucleotide array-based was to determine the role of LOH in SLE. Blood cell platforms by Agilent Technologies and Oxford Gene lymphocytes from 23 SLE unrelated patients and 59 Technologies. healthy subjects were submitted to the Genome-Wide Results: Overall we found 65 clinically significant Human Cytoscan HD Array (Affymetrix®) in order to CNAs (38 %) and 29 were confirmed as pathogenic screen for LOH regions in genomic DNA. LOH event (17 %). Seven CNAs were evaluated with undetermined was determined by at least 50 contiguous SNPs in a significance (VOUS), seven CNAs were found to be genomic region of 3 Mb. Data analysis was performed likely benign and 22 CNAs remain to be confirmed and in ChAS and Plink software. An average of 4.3 (sd=5.4) evaluated. Totally we detected 43 deletions and 23 LOH events per individual with a medium size of duplications (from 0.67 kb to 31 Mb) and 5 cases of 23.2 Mb (sd=40.9 Mb) were identified in patients, while segmental or whole-chromosome UPD/LOH. controls showed 3.2 LOH events (sd=2.6), spanning a Conclusion: Array-CGH technique represents an effec- region of 14.3 Mb (sd=16.0 Mb). Percentage of LOH in tive tool in the diagnostic algorithm for MR cases in our autosomal chromosomes was also higher in patients laboratory. From this year, patients with negative array- (0.7 %) than controls (0.3 %). We found 35 LOH CGH results will be involved in a pilot study segments that were shared by two or more subjects using NGS technology to analyze mutation status and 37 LOH events that occur once in SLE sample. of MR-associated genes. Based on recent studies, We highlighted a potentially pathogenic LOH region the combination of array-CGH and exome of 16.5 Mb on 9p, present in two patients and absent sequencing technologies leads to the increase of in controls, that covers several genes involved in the diagnostic yield in the cases of idiopathic immune and apoptotic process. This is the first report MR and may help to elucidate their causes. describing LOH events encompassing these regions. Supported by grants CZ.1.07/2.3.00/20.0183 and Our findings show how regions of genomic instability MH CZ–DRO FN Brno, 65269705. could be potential modulators for SLE etiology. Moreover, our results strongly support that SLE patients 1.P9 have more homozygous loci and higher percentage of LOH than healthy individuals. Support: FAPE Copy-neutral loss of heterozygosity pattern in SP(2013/17062-9;2011/23794-7), CNPq(312547/ systemic lupus erythematosus 2009-9),

Fernanda Bueno Barbosa1, Milena Simioni2, Fábio 1.P10 Rossi Torres2, Eduardo Antônio Donadi3, Vera Lúcia Gil-da-Silva-Lopes2, Aguinaldo Luiz Simões1 Prenatal diagnosis and molecular cytogenetic University of São Paulo Department of Genetics, characterization of a de novo duplication of Ribeirão Preto Medical School Ribeirão Preto, 15q24.3-q26.1 10th European Cytogenetics Conference 2015 S39

Isabel Ochando1,MelanieAlonzo2,AnaMaría 1.P11 Serrano2,AntonioUrbano1,MarinaSánchez1, Eduardo Cazorla2, Joaquín Rueda3 Familial supravalvular aortic stenosis due to ELN Hospital Clínica Vistahermosa Unidad de Genética gene disruption by a chromosome 7 paracentric Alicante-Spain1 Hospital Universitario Torrevieja inversion Servicio de Ginecología y Obstetricia Alicante-Spain2 Universidad Miguel Hernández y Hospital Clínica Caroline Schluth-Bolard1,LindaPons2,Audrey Vistahermosa Histología y Anatomía y Unidad de Labalme1,NicolasChatron1,AdrienBuisson1, Genética Sant Joan d’Alacant-Spain3 Clément Narci1,AnneFautrelle1,ChristineBel1, Patrice Bouvagnet3, Damien Sanlaville2 Reported cases of duplications of distal 15q are very Centre de Biologie et de Pathologie Est, Hospices Civils uncommon, in fact, there are around 50 cases described de Lyon Laboratoire de Cytogénétique in the world, an even less de novo duplications like the Constitutionnelle BRON Cedex-France1 Centre de present case report. Most of these mutations are the Recherche en Neurosciences de Lyon TIGER Team, result of unbalance translocations and have an INSERM U1028; CNRS UMR5292 BRON-France2 additional monosomic component. Previous authors Hôpital Louis Pradel, Hospices Civils de Lyon Service have described a distal 15q trisomy syndrome de Cardiologie Pédiatrique BRON-France3 characterized by prenatal and postnatal overgrowth, mental retardation, craniofacial and skeletal Supravalvular aortic stenosis (SVAS) is an autosomal malformations, and genital abnormalities, particu- dominant condition due to haplo-insufficiency of the larly in affected males. Other authors, indicated ELN gene localized in 7q11.23. SVAS has incomplete small-size fetuses. There are only a few reported penetrance and variable expressivity. cases of pure duplication of 15q24-qter region. We report here a two years old boy referred for an The type and severity of reported anomalies isolated SVAS and history of heart murmur in both depend on the length and location of the duplicated branch of the family. region of chromosome 15q. Standard karyotype displayed an apparently balanced We present a prenatal diagnosis of a fetus with paracentric inversion of the long arm of chromosome 7 : single umbilical artery, all the long bones short - 46,XY, inv(7)(q11.2q31). Fluorescent in situ hybridiza- 2.5 SD, but with normal morphology and mineral- tion (FISH) using the Williams Beuren Syndrome ization features. The patient is a 30-year-old (WBS) critical region probe (7q11.2) and RP11- female, 21+1 gestation weeks, low-risk first trimester 644H24 probe (chr7: 73411275-73548266, hg19) screening and normal serology. The fetus karyotype showed that the proximal breakpoint disrupted the showed a 15q duplication visible by conventional ELN locus. Array CGH using a custom oligonucleotide G-banding, not present in the parents’ karyotypes. array densified in the WBS region did not reveal any CGH array showed a de novo duplication imbalance. Massively parallel DNA sequencing is 15q24.3q26.1, of 14 Mb, containing 257 genes underway in order to specify whether the breakpoint (NCBI), 81 of them in the OMIM database. lies within the ELN gene or in its neighbourhood. The fetus reported here had no monosomic compo- The family study showed that the chromosome 7 nent, as reported in the majority of the published cases, inversion was inherited from the father and was also so we can assume that the abnormal phenotype results present in two paternal aunts and in the paternal grand’ from the duplication of the 15q24.3q21.1 segment. Our father. The associated phenotype was highly variable, conclusion is that in case of short long bones (

Anelisa Gollo Dantas1,DiegoMazzotti2,Adriana 1.P13 Bortolai3,MilenyColovati1,RobertaSantos Guilherme1,MarianaMoysés-Oliveira1,VeraAyres Discrepancy between cytogenetic and molecular Meloni1, Chong Ae Kim4, Maria Isabel Melaragno1 results in two cases with isochromosomes Universidade Federal de São Paulo Genetics Division São Paulo-Brazil1 Universidade Federal de São Paulo Naja Becher1, Anne-Bine Skytte1 Psychobiology Department São Paulo-Brazil2 Hospital Aarhus University Hospital Clinical Genetics do Servidor Público Estadual Genetics Division São Aarhus-Denmark1 Paulo-Brazil3 Universidade de São Paulo Instituto da Criança São Paulo-Brazil4 An isochromosome contains two arms with identical DNA sequences and the formation of it must have 22q11.2 deletion syndrome results from a hemizygous happened either at meioses or as a postzygotic event. deletion of chromosome 22 usually flanked by low copy To support the theory of postzygotic formation we repeat (LCR) regions, the 3 and 1.5 Mb deletions being present these two cases: the most frequent. The phenotype may be variable in Case 1: 30-year old gravida 2, para 1 underwent CVS patients with similar deletions, and similar phenotypes at 12+1 weeks of gestation because of enlarged fetal may also be observed in patients with different sized nuchal translucency. Array-CGH was performed on deletions in the 22q11.2 region, even without overlap. DNA from uncultured CVS revealing a 5.6 Mb terminal We conducted cytogenomic investigation by deletion on chromosome 21q (arr[hg19] karyotyping, MLPA and genomic microarray in 29 21q22.2q22.3(42538181-48090317)x1). To estimate patients. Gene expression, analysis of gene interactions recurrence risk the CVS was cultured. Karyotyping and enriched biological processes were evaluated in 14 and Multi-Colour FISH analyses revealed patients and 14 gender and age matched controls. 46,XX,der(21).ish der(21)i(21); one normal chromo- Karyotypes were normal in most patients, with one some 21 and one isochromosome 21 in all metaphases showing a translocation t(6;22), resulting in 22q11.2 analyzed. Array-CGH was then applied to the cultured deletion. MLPA revealed the deletion size: 3 Mb (21 cells and could confirm the 27 Mb duplication and the patients), 1.5 Mb (4 patients) and atypical deletions (4 terminal deletion of 5.6 Mb of chromosome 21q patients). Deletion sizes, size refined by genomic micro- (arr[hg19] 21q11.2q22.2(15390816-4248 array, showed breakpoint variations among patients. 2129)X3,21q22.2q22.3(42518800-48090317)×1). The study of gene expression by microarray identified Case 2: 33-year old man referred for standard reduced expression of 48 transcripts corresponding to chromosomal analyses because of azoospermia. genes of the 22q deleted region, and also of genes Karyotyping and FISH-analysis was performed on flanking the deleted region or genes located in other PFA-stimulated peripheral blood revealing 20 % chromosomes. These data indicate a position effect mosaicism for monosomi X in combination with and the chromatin repositioning in the nucleus, mecha- an iso-dicentric Y-chromosome consisting of two nisms possibly related to gene expression alteration. The copies of Yp (45,X[1]/46,X,der(Y)[9].ish mos X coexpression evaluation by GeneMania software (pter+)(DXYS129x1),i(Y)(pter++)(DXYS15 showed a relationship between the genes of the deleted 3++),idic(Y)(p11.1-q11.1)(DYZ3++),Xcen(DXZ1x1) region and those adjacent to it, between the genes of 1.5 [81]/X(pter+)(DXYS129x1),i(Y)(pter-)(DXYS153-), and 3 Mb deletions regions, and between genes of idic(Y)(p11.1 q11.1)(DYZ3-),Xcen(DXZ1x1)[19]). 10th European Cytogenetics Conference 2015 S41

Subsequently, Array-CGH was performed on DNA collagen. Proper collagen-integrin interactions are extracted from a new peripheral blood sample. Array important in fracture healing, which suggests that confirmed a 10–15 % mosaicism for monosomi X, but ITGA1 plays role in the regulation of mesenchymal could not confirm the presence of an iso-Yp chromo- stem cell and cartilage proliferation. FST encodes a some expecting a duplication of Yp (arr[hg19] monomeric glycoprotein that actively participates in Yp11.31q11.221(2626115-18255512)~0-1,Yq11. the regulation of bone metabolism. Experiments per- 221q11.23(18415668-28689881)x0). formed in mice overexpressing follistatin showed a Conclusion: These two cases illustrate that isochro- decreased quality of skeleton and susceptibility to bone mosomes may be formed postzygotically when fractures. In conclusion, interstitial 5p11q11.2duplication cells try to repair a terminal deletion. We involving ITGA1 and FST could play a causative role in hypothesise that the terminal deletions, but not the iso- familial osteoporosis. chromosomes, were evident in our patients, and that isochromosome-formation was induced by cell culture/ 1.P15 PFA cell-stimulation. A unique case of mosaic male Turners and trisomy 1.P14 13

Familial osteoporosis and interstitial duplication of Omaiyah Al Abdulwahed1, Yousef Al Housawi2 chromosome 5 King Fahad Specialist Hospital P & L Medicine Dammam-Saudi Arabia1 King Fahad Specialist Elisa Tassano1, Natascia Di Iorgi2, Serena Marchese2, Hospital Pediatrics Dammam-Saudi Arabia2 Flavia Napoli2,PatriziaFiorio1,GiorgioGimelli1, Cristina Cuoco1 The incidence of trisomy 13 is 1 in 5000 births and that Istituto Giannina Gaslini Laboratorio di Citogenetica of Turner Syndrome (TS) is 1 in 2500 live born pheno- Genova-Italy1 Istituto Giannina Gaslini UOSD di di typic females. TS is a common autosomal aneuploidy Endocrinologia Clinica e Sperimentale Genova-Italy2 among liveborns. We report a unique case of a male with a Osteoporosis is a systemic bone disease characterized Turner phenotype and trisomy 13. This 25 month by low bone mineral density and structural deterioration old Saudi phenotypically male child was referred of bone tissue leading to an increased risk of fractures. to our outpatient clinic for a genetic workup because of Genetic factors have been recognized to play an impor- dysmorphy. Parents are healthy and are non- tant role in osteoporosis and a number of susceptibility consanguineous. genes have been identified and validated. Here, we A chromosomal microarray profile was found to be describe an 11-year-old girl affected by severe osteopo- consistent with a mosaic trisomy 13. Chromosomal rosis with vertebral fracture and intellectual disability. analysis of 20 metaphases from a stimulated peripheral Her father was affected by a severe form, too. blood culture done in our laboratory showed the Cytogenetic investigations revealed a dicentric chromo- presence of two abnormal cell lines: some 5, rearranged at the centromeric level, present also One had an abnormal derivative of chromosome 13 in her father. The two centromeres were both active, as in the form of a Robertsonian translocation, (13q10), in demonstrated by immunological staining with CENP-C 16 out of 20 metaphases. antibodies. Array-CGH of the proposita showed a large The other cell line had two normal chromosomes 13 proximal duplication at 5p11q11.2 band and a small and a loss of the Y chromosome in 4 metaphases. distal one at 5q11.2, while her father presented a unique No other structural or numerical abnormalities were duplication of 5p11q11.2 bands. The duplicated region detected. This is consistent with the diagnosis of a on chromosome 5 contains ITGA1 (integrin alpha 1), an mosaic trisomy 13 and mosaic TS. osteoporosis susceptibility gene, and FST (follistatin) Also FISH done in our lab to confirm the X/Y status gene. ITGA1 encodes integrin alpha 1 chain that binds showed 69 % of the cells with an XY signal and 31 % to the beta chain to form a receptor involved in cell with only one signal for chromosome X. This result is attachment and neurite outgrowth on laminin and consistent with 45,X/46,XY mosaic TS. S42 10th European Cytogenetics Conference 2015

This is a unique case of mosaic trisomy 13 and not yet described in the literature and it is still unclear Turner syndrome with male phenotype. Although how this aberration contributes to the clinical picture. microarrays can detect small percentage of mosaicisms up to15 %, in this case microarrays failed to detect the 1.P17 mosaic loss of Y chromosome. This abstract was submitted to the 20th International Paternally inherited 7q21.3 microdeletion and Chromosome Conference 2014, University of Kent, maternally inherited 22q11.2 microduplication in a UK.It was accepted as poster and was published as an patient with split-hand/foot malformation, abstract. However, the authors were unable to attend the macrocephaly and developmental delay. conference and the poster was not displayed. Kati Kuuse1,SanderPajusalu1,OlgaŽilina1,Olga 1.P16 Žilina2, Piret Ilisson1, Riin Klade1, Riste Kalamees3, Tiia Reimand1 Case Report: Clinical Expression of an Inherited Tartu University Hospital, United Laboratories Unbalanced Translocation in Chromosome 2 Department of Genetics Tartu-Estonia1 Tartu University Institute of Molecular and Cell Biology Polina Verhovtsova1, Kati Kuuse1, Olga Žilina1, Anu Tartu-Estonia2 Tartu University Hospital Children’s Tiidema1, Airi Visamaa1, Kairit Joost1 Clinic Tartu-Estonia3 Tartu University Hospital, United Laboratories Department of Genetics Tallinn-Estonia1 We report a case of a girl born with a split-hand/foot malformation. At 3 months of age the first signs of Carriers of balanced reciprocal translocations mostly do developmental delay were noticed and an accelerated not have recognizable phenotypic expression. However, head growth had led to macrocephaly. Chromosomal they have an increased risk of producing gametes with microarray analysis (Human CytoSNP-12 BeadChip, unbalanced chromosomal rearrangements, leading to Illumina Inc.) revealed two pathogenic DNA copy miscarriages or children with significant clinical number variations (CNV): a 2.2 Mb deletion in 7q21.3 expression. region associated with split-hand/foot malformation We report a family comprising of healthy type 1 or ectrodactyly–ectodermal dysplasia–cleft parents and their newborn with facial dysmorphism (EEC) syndrome, and a 2.6 Mb duplication in 22q11.2 and feeding difficulties. Conventional GTG- region associated with psychomotor developmental banding (G-bands by trypsin using Giemsa) analysis delay and other clinical features. The patient’s father, of somatic chromosomes identified a balanced who presents merely feet syndactyly, carries the same translocation, 46,XX,t(2;6)(q37.1;q25.2), in the 7q21.3 deletion and the patient’s unaffected mother is a mother and an unbalanced rearrangement, carrier of 22q11.2 duplication. 46,XX,der(2)t(2;6)(q37.1;q25.2)mat, in the child. Both CNVs, 7q21.3 deletion and 22q11.2 dupli- The child has inherited a derivative chromosome 2 with cation, are well-described chromosomal aberrations loss of the segment 2q37.1-qter and gain of 6q25.2-qter. with incomplete penetrance and high clinical vari- The finding was confirmed by FISH (fluorescent in situ ability. The co-occurrence of two familial CNVs hybridization) and chromosomal imbalances were complicates making genotype-phenotype correla- specified by chromosomal microarray analysis tions and providing prognosis for further develop- (Human CytoSNP-12 BeadChip, Illumina Inc.). The ment of the child. amount of deleted material on 2q was ~7.9 Mb and the Diagnostic results of the patient and all affected gain of 6q was ~15.4 Mb. family members are presented and complexity of the 2q37 deletion syndrome is a condition that can affect phenotype will be discussed in the presentation. many parts of the body. This condition is characterized by weak muscle tone (hypotonia) in infancy, mild to 1.P18 severe intellectual disability and developmental delay, behavioral problems, characteristic facial features, and Strategies for dealing with incidental findings in other physical abnormalities. 6q25.2-qter duplication is diagnostic microarray-based copy number analysis 10th European Cytogenetics Conference 2015 S43

Karin Schaeferhoff1, Martin Kehrer1, Angelika Riess1, 1.P19 Ute Grasshoff1, Christopher Schroeder1, Sven Poths1, Vanessa Vosseler1, Andrea Bevot2, Karl Oliver Kagan3, Cytogenomic investigation in patients with Olaf Riess1, Andreas Dufke1 Oculoauriculovertebral Spectrum (OAVS) and University Hospital Tuebingen Institute of Medical candidate loci relevant to phenotype Genetics and Applied Genomics Tuebingen-Germany1 University Hospital Tuebingen Department of Mileny Esbravatti Stephano Colovati1,Silvia Neuropediatrics Tuebingen-Germany2 University Bragagnolo1,RobertaSantosGuilherme1,Anelisa Hospital Tuebingen University Women’sHospital Gollo Dantas1, Maria de Fátima Soares2, Chong Ae Tuebingen-Germany3 Kim3,AnaBeatrizAlvarezPerez1,MariaIsabel Melaragno1 During the past decade, chromosomal microarray-based Universidade Federal de São Paulo Genetics Division copy number analysis has emerged as an important São Paulo-Brazil1 Universidade Federal de São Paulo diagnostic tool for the evaluation of developmental Diagnóstico por Imagem São Paulo-Brazil2 delay, intellectual disability, autism spectrum disorders, Universidade de São Paulo Instituto da Criança São and structural malformations in children. The benefit of Paulo-Brazil3 the high-resolution detection of copy number variants offered by microarray applications comes along with an Oculoauriculovertebral Spectrum (OAVS, OMIM increased risk for the detection of variants of uncertain 164210) is a developmental disorder involving the first clinical significance or unsolicited findings. These and second branchial arches during embyogenesis. This include copy number variants concerning genes not disorder is characterized by a wide spectrum of symp- related to the clinical phenotype of the respective patient toms and physical features that may vary greatly in that could potentially implicate a higher disease risk for range and severity from case to case. The majority of offspring or other family members or present risk factors cases appear to be sporadically and the etiology of the for additional diseases. syndrome is not well understood. Some studies have In our lab, we processed more than 470 detected candidate loci for OAVS by molecular cytoge- Affymetrix Cytoscan HD arrays for diagnostic netics and genetic linkage studies but no gene or locus purposes over the last two years. We mainly analyzed has been specifically attributed to this spectrum. Several patients with developmental delay, intellectual disability, chromosomal abnormalities have been associated with structural malformations and dysmorphic features and OAVS syndrome and the chromosome region most detected pathogenic copy number variations explaining frequently reported is the 22q11.2 region. We studied the disease pattern in 15 % of the samples. In 68 % of the 70 patients with OAVS clinical diagnosis who met the patients, we found no evidence for a genomic minimal criteria (isolated microtia, preauricular tags and imbalance causative for the clinical phenotype. hemifacial microsomia) by karyotyping, genomic array Incidental findings were reported in 2,3 % of the and FISH. All patients showed normal karyotypes, cases (11 patients): Nine patients were found to be except for one: 46,XX,inv(12)(q15q24.1). A derivative carriers of a recessive genetic disorder such as chromosome 4 from a cryptic maternal translocation Cohen syndrome, Joubert syndrome or Usher t(X;4)(p22.31;p15.33) detected by FISH was found in syndrome due to copy number variants in respec- another patient. High resolution oligonucleotide tive genes. An increased risk for cardiovascular genomic arrays (Affymetrix) identified some Copy diseases was discovered in two cases due to copy Number Variations (CNVs): 7 deletions number variants in genes associated with cardiac (4p16.3p15.33, 4q13.3q21.1, 8q13.3, 10q26.2q26.3, disorders. 16p13.3, 22q11.21, Xp22.33) and 6 duplications Here we present selected cases of our incidental (2q32.1, 4p16.1.1, 16p13.11, 17q11.2, and Xp22.33) in findings. We discuss strategies taking pre-analytical different chromosomes. The patient with a ~581 kb counselling, severity and prevalence of the respective 22q11.21 deletion is the seventh patient ever reported with disorder, characteristics of the copy number variant and OAVS and 22q deletion, indicating that 22q11.2 region is the challenges of medical reporting into account, based astrongcandidatelocusforregulationofthebodysym- on our experience. metry and facial development. The other CNVs are S44 10th European Cytogenetics Conference 2015 potentially pathogenic imbalances since they include the whole β-globin gene cluster encompassing genes many genes described in OMIM and genomic regions HBB, HBD, HGB1, HBG2, and HBE1. which may be related to the phenotype in our patients. CONCLUSIONS: Firstly, this case demonstrates that Therefore, CNVs and chromosomal rearrangement CMA could serve as a useful first-tier genetic test for investigation in OAVS may potentially reveal different severe life-threatening neonatal anaemia. Secondly, the pathogenic mechanism in this phenotypically and detected 11p15.4 deletion is clearly pathogenic and has genetically heterogeneous disorder. Financial Support: a size that is just slightly above the resolution limit of FAPESP, Brazil(2013/19897-0). arrays routinely used in clinical diagnostics. Thirdly, we would like to emphasize that some rarer forms of thal- 1.P20 assemia may also be present in Northern-European population and should be included in differential diagnosis Epsilon gamma delta beta thalassemia diagnosed by of severe neonatal anaemia. chromosomal microarray in critically ill neonate 1.P21 Sander Pajusalu1, Pille Tammur1, Imbi Eelmäe2, Helgi Padari2, Olga Zilina3, Tiia Reimand1 Adenovointerstitial6qdeletion:genotype- Tartu University Hospital Department of Genetics, phenotype correlation utilizing array CGH United Laboratories Tartu-Estonia1 Tartu University Hospital Anaesthesiology and Intensive Care Clinic Anila Babameto-Laku1, Dorina Roko1 Tartu-Estonia2 University of Tartu Institute of University Hospital Center Sevice of Medical Genetics Molecular and Cell Biology Tartu-Estonia3 Tirana-Albania1

BACKGROUND: Epsilon gamma delta beta thalasse- Interstitial deletions of the long arm of chromo- mias represent a group of rare autosomal dominant some 6 are relatively rare, with fewer than 100 haemoglobinopathies caused by deletions of the cases reported. Phenotypic variation is in large part β-globin gene cluster. The most prominent clinical due to differences in size and location of the feature is ante- or neonatal anaemia, which might lead segmental aneuploidy. We report a patient, a girl to severe complications. The severe anaemia resolves in 12 months old, with an interstitial deletion of a couple of months after birth. Here we describe a chromosome 6q defined at the molecular level by familial case of (εγδβ)-thalassemia caused by a novel array comparative genomic hybridization (array 11p15.4 microdeletion, which is the first reported case CGH) with CytoChip microarrays oligo 8×60 K. from North-Eastern Europe. In our case the molecular breakpoints differed CASE REPORT: The proband is a girl born with from those indicated by conventional karyotyping, severe non-immune haemolytic anaemia complicated demonstrating the enhanced resolution of array by persistent pulmonary hypertension of the newborn. CGH. The patient had a deletion of 10,1 Mb The baby required blood transfusions, mechanical ven- involving 6q16.1–6q21. The parental karyotypes tilation and extracorporeal membrane oxygenation due were normal. She was the first child of healthy to critical condition. At the age of 2 months the outcome nonconsanguineous parents. Family history was is favourable, although she remains mildly anaemic. unremarkable. She presented developmental delay, The family history was remarkable as her older brother, craniofacial dysmorphology, and functional eye father, paternal grandfather, aunt and cousin have had disorders, suggesting that genes affecting brain anaemia-related health problems of variable severity. and craniofacial development are located in METHODS AND RESULTS: As a first-tier genetic 6q16.2–>q21, as has been previously described in test, chromosomal microarray analysis (CMA) from some cases in the literature. Furthermore, gene(s) DNA sample of the proband was performed using for discordant phenotypic features, such as central Illumina HumanCytoSNP-12 array. CMA revealed a diabetes insipidus, may reside at 6q15. Array CGH 115 kb deletion (arr[hg19] 11p15.4(5,228,708- can help in further refining the deleted region and 5,343,533)×1). The deletion was found also in other thus in the discovery of candidate genes for the five affected family members. The deleted region covers phenotypic characteristics. 10th European Cytogenetics Conference 2015 S45

1.P22 12q13.11, 12q21.2/12q21.33 and 7p21.2–21.3. The second event, pericentric inversion of chromosome 12, Characterization of a de novo complex chromosomal involved two other breakpoints located on 12p13.31- rearrangement with five breakpoints associated with 12p13.2 and 12q21.33/qter. The mapping associated azoospermia with these breakpoints showed several genes of potential interest involved in the reproductive function such as Aurélie Mouka1, Vincent Izard2, Sophie Brisset3, Loïc ARL4, FOXJ2, NEDD1, SYCP3 and NFYB. Array- Drévillon4,JessikaAnasthase4,GérardTachdjian3, CGH analysis did not highlight DNA copy number Lucie Tosca3 variation on estimated breakpoints or elsewhere. Université Paris-Sud, 94276 Le Kremlin-Bicêtre cedex, CCRs predispose to abnormal segregation in meiosis France. AP-HP, Hôpitaux Universitaires Paris-Sud, or gene disruption responsible for reproductive failure Hôpital Antoine Béclère Service d’Histologie, such as azoospermia. This case report underlines the Embryologie et Cytogénétique Clamart-France1 potential of conventional and molecular techniques to AP-HP, Hôpitaux Universitaires Paris-Sud, Hôpital specify mechanism of CCRs formation and to identify Antoine Béclère Service de Gynécologie-Obstétrique breakpoints. et Médecine de la Reproduction Clamart-France2 Université Paris-Sud, 94276 Le Kremlin-Bicêtre cedex, 1.P23 France. AP-HP, Hôpitaux Universitaires Paris-Sud, Hôpital Antoine Béclère Service d’Histologie, Case report: An inherited copy number loss Embryologie et Cytogénétique Clamart-France3 including the ARID1B gene AP-HP, Hôpitaux Universitaires Paris-Sud, Hôpital Antoine Béclère Service d’Histologie, Embryologie et Madalena Avila1, Abhijit Kulkarni2, Sahar Mansour2, Cytogénétique Clamart-France4 Karen Marks2, Rohan Taylor2 St Georges Healthcare NHS Trust South West Thames Complex chromosomal rearrangements (CCRs) are Regional Genetics Service London-United Kingdom1 balanced or unbalanced structural aberrations involving St. George’sHealthcareNHSTrustSouthWest three or more chromosomal breakpoints with exchange Thames Regional Genetics Service London-United of genetic material between two or more chromosomes. Kingdom2 CCRs are rather rare event in the general population and carriers display various phenotypes (normal phenotype, The use of Array CGH (Comparative Genomic infertility, malformations, mental retardation and/or con- Hybridization) as the front line laboratory approach to genital abnormalities). Male carriers are at risk of repro- assess patients with intellectual disability and /or ductive failure as a result of spermatogenesis disruption. congenital anomalies has enabled the identification of Indeed, CCRs can be associated with abnormal segre- new copy number variants (CNVs) and new gation of derivative chromosomes and production of microdeletion / microduplication syndromes. chromosomally abnormal sperm. CNVs frequently encompass a significant number of In this report, we describe a rare and de novo CCR genes, and may provide evidence of association to a associated with azoospermia in a 36 years-old man. The specific phenotype or further delineation of a critical CCR was characterized by karyotype, FISH (fluores- region. However CNVs are often inherited and may cence in situ hybridization) and array-CGH (microarray highlight variable penetrance, or in the case of an unaf- Comparative Genomic Hybridization) Agilent 1 M fected parent may suggest that an alternative mechanism 2.1Kb resolution assays. Successive analyses by FISH might be in place. allowed to estimate the location of breakpoints and thus Coffin-Siris Syndrome (CSS) is characterized by gene mapping of these regions. Results showed that the intellectual disability, severe speech impairment, coarse rearrangement was more complex than initially assumed facial features, microcephaly, developmental delay and with a two-step CCR and five breakpoints were identi- hypoplastic nails on the fifth digits (MIM 135900). The fied. The first event, insertion of a part of an inverted main underlying cause has been suggested to be muta- chromosome 12 into the short arm of a chromosome 7, tions in the ARID1B gene (MIM 614556), a gene involved three breakpoints located on 12p11.1q11/ involved in the remodelling of chromatin. However, S46 10th European Cytogenetics Conference 2015 there are reports of ARID1B mutations in patients retardation and/or inassociationwithautismandepilepsy. referred with intellectual disability with a variable set For this study we analyzed ten patients with normal and/ of clinical features which do not necessarily fit the or abnormal karyotype by Genome Array-CGH and we clinical criteria of CSS. found further cryptic rearrangements in five out of ten Most ARID1B mutation patients have been (50 %). This technique allows us to identify SLC8A3 ascertained through studies of CSS, potentially biasing gene (human gene for member 3 of solute carrier family the documentation of associated clinical features. 8) as a candidate gene for epilepsy (Nucaro et al., 2010). Recently an attempt has been made to further character- It is a sodium-calcium exchanger electively expresses in ize a subset of patients specifically showing involve- brain (Gabellini et al., 2002). The role of ionic channels is ment of ARID1B. well demonstrated in epilepsy. These findings are Here we present a case showing a 956 kb deletion in very important to better genotype/phenotype corre- 6q25.3 encompassing the ARID1B gene, detected by lation and to determine the clinical significance of Array CGH. This patient is affected with microcephaly, these genomic imbalance. In conclusion, this study faltering growth and developmental delay. The CNV is provide evidence that the BAC Genome Array-CGH maternally inherited. We compare this to other patients platform is an important and reliable tool for described in the literature hoping to further delineate the detecting, mapping and characterizing cryptic chromo- role of ARID1B in intellectual disability. some rearrangements.

1.P24 1.P25

Cryptic Chromosome Rearrangements Detected by SOX9 regulatory element duplication and gender BAC Genome Array-CGH. phenotype modularity in a family

Valentina Cabras 1,CaterinaMontaldo2,Rossano Irén Haltrich1, Henriett Pikó2, Andrea Luczay1, Eszter Rossino3, Paola Caria4, Anna Lisa Nucaro5 Kiss1, Veronika Karcagi2, György Fekete1 University Experimental Medical Pathology, Faculty of Semmelweis University 2nd Department of Paediatrics Medicine and Surgery Cagliari-Italy1 University Budapest-Hungary1 National Institute of Environmental Surgical Sciences Cagliari-Italy2 University, Health Dept. of Molecular Genetics and Diagnostics Microcitemico Hospital Pediatric Sciences Cagliari- Budapest-Hungary2 Italy3 University Biomedical Technologies Cagliari- Italy4 CNR, Cittadella Universitaria Genetic and We report two 46,XX SRY-negative male brothers with Biomedical Institute Monserrato, Cagliari-Italy5 incomplete masculinization. Both children were born with bilaterally palpable testes and congenital hypospa- The use of Genome Array-CGH has recently dramati- dias. The older brother’s MRI revealed scrotal right side cally improved the detection of cryptic chromosomal ovotestis, left side atrophic testis and normal size pros- imbalances. This methodology utilizes mapped DNA tate. A testicular biopsy showed decreased number of sequences in a microarray platform for the detection of Sertoli and Leydig cells and germinal cell aplasia. The chromosomal deletions/duplications (Vermeesch et al. histological and MRI examination of the younger 2005; Edelmann and Hirschhorn 2009; Sagoo et al. brother revealed bilateral scrotal gonads with ovarian 2009). We applied the BAC (bacterial artificial chromo- structure and behind the bladder a tubular structure some) Genome Array-CGH platform to study several resembling a closed vagina or Müller duct residue. patients with normal and/or abnormal chromosome kar- The array-CGH analysis identified in the case of yotypes, previously investigated with conventional the older brother a 143 kb duplication 540 kb cytogenetic procedures, and we found further cryptic upstream of SOX9. The younger brother and his rearrangements that had escaped traditional cytogenetic healthy 46,XY father harbored an identical 155 kb analysis, because of low power resolution. Our experi- duplication 605 kb upstream of SOX9. SRY func- ence shows the validity of the BAC platform as a tions as a genetic switch of testicular differentia- reliable method for Genome-wide screening of chromo- tion while all downstream targets promote the somal aberrations in patients with idiopathic mental expression of SOX9. Recent studies have shown 10th European Cytogenetics Conference 2015 S47 that high expression of SOX9 is sufficient to trig- psychomotor retardation and behavioral problems. ger male development in the absence of SRY. The Moreover, our patients do not have an overweight genomic domain regulating SOX9 expression is phenotype, highlighting indeed the role of MYT1L composed of several gonadal regulatory elements in obesity, as none of the deletions encompasses and spans more than 1 Mb upstream of SOX9. MYT1L. The only common disrupted gene is The older brother’s143kbSOX9upstreamdupli- SNTG2, thus redefining the smallest region of cation region overlaps with the minimal critical overlap for autism. SNTG2 is expressed in the size of sex-determining region defined by Benko brain. Its product interacts with neuroligins and Xiao. The hormone and gonadal status of this NLGN3, NLGN4X, and NLGN4Y, which are autism- boy was more appropriate to male phenotype. The related isoforms. Finally, the parental origin of the dele- younger brother’sduplicatedregionwasaway tions seems to be of interest, as all inherited deletions from the mentioned 584–517 kb upstream SOX9 described today appear to be inherited from the paternal critical region. We can hypothesize that the male side. We will discuss the possible imprinting of the 2p25 differentiation has been initiated but probably the region and its role in autism. gonadal SOX9 expression wasn’tsufficientto complete the masculinization. The results of our 1.P27 investigation suggest that gene desert regions with putative regulatory portion of the genome should Xq25 duplication: the crucial role of the STAG2 gene be included in searches and the phenotypic impact in this novel human cohesinopathy of regulatory element alterations might differ substantially Camille Leroy1,Marie-LineJacquemont2,Bérénice Doray3,DenisLamblin4,ValérieCormierDaire5, 1.P26 Anne Philippe5, Sylvie Nusbaum1, Catherine Patrat6, Julie Steffann5,LaurenceColleaux7,Michel 2p25 deletion: involvement of SNTG2 in autism or Vekemans 1,SergeRomana1, Catherine Turleau1, behavioral problems, and parental imprinting. Valérie Malan1 Hôpital Necker-Enfants Malades Service de Morgane Plutino1,HoudaKarmous-Benailly1, Cytogénétique Paris-France1 Centre Hospitalier Fabienne Giuliano1,ChristopheMassol1,Kathy Universitaire de la Réunion Service de Néonatologie Wagner2,VéroniquePaquis-Flucklinger1, Manal Saint-Pierre, La Réunion-France2 Centre Hospitalier Dayem-Quere1 Universitaire de La Réunion, Hôpital Félix Guyon CHU de Nice Service de Génétique Médicale Nice- Service de Génétique Saint-Denis, La Réunion-France3 France1 CHU Lenval Hôpitaux Pédiatriques de Nice Fondation Père Favron CAMSP Saint-Louis, La Nice-France2 Réunion-France4 Hôpital Necker-Enfants Malades Service de Génétique Paris-France5 Groupe Hospitalier Deletions of the terminal region of the short arm Bichat-Claude Bernard Laboratoire de Biologie De la of chromosome 2 have been reported in the liter- Reproduction Paris-France6 Hôpital Necker-Enfants ature in less than 20 patients. These patients share Malades Institut IMAGINE, INSERM UMR_S1163 common clinical features including early-onset Paris-France7 obesity, intellectual disability and behavioral problems or autism. A minimal critical region of The Xq25 duplications syndrome has recently emerged as 1.97 Mb has been estimated, encompassing seven a distinct clinical entity. We report here on six new genes (SH3HYL1, ACP1, TMEM18, SNTG2, patients belonging to two unrelated families and TPO, PXDN, and MYT1L). Many recent studies harbouring an Xq25 microduplication detected by array pointed out MYT1L gene as the main candidate CGH. As in previously reported cases, the phenotype of for ID and obesity. We present three new patients our patients is characterised by delayed milestones, carrying 2p25 deletions: an autistic female child speech disturbance, intellectual disability, abnormal with developmental delay and her depressive behaviours and a characteristic facial dysmorphism. The father, and a third unrelated patient who has common duplicated interval allowed further refinement of S48 10th European Cytogenetics Conference 2015 the shortest region of overlap to 173 kb, including only In vitro fertilization (IVF) is a complex series of proce- one gene, STAG2, which encodes a component of the dures used to treat fertility or genetic problems and assist cohesin complex. We suggest that increased STAG2 gene with the conception of a child, but there are still many copy number and dysregulation of its downstream target couples without achieving successful implantation. genes may be responsible for the specific clinical findings Chromosomal abnormalities, particularly translocations, of this syndrome. Therefore, the Xq25 microduplication are known to be implicated in various forms of repro- could be considered as a novel cohesinopathy, thus ductive failure. The aim of the study was to investigate increasing the group of these disorders. the frequency of chromosomal abnormalities which may impact on pregnancy outcome in the survey of 79 1.P28 couples with in-vitro fertilization implantation failure in Slovakia (2009–2014) using cytogenetic G-banding 18q Deletion Syndrome—Case Report methods. Cytogenetic analyses revealed chromosomal abnormalities in 5/79 (6.3 %) of Slovak couples with Saad Alomar1, Jhumur Pani2 implantation failure, of which 2/79 (2.5 %) were recip- College of Medicine-University of Basra Pathology and rocal translocations and 3/79 (3.8 %) were Robertsonian Forensic Medicine Basra-Iraq1 Oncquest Laboratories ano-malies. Chromosomal findings were detected in Ltd. Cytogenetics New Delhi-India2 5.0 % of women and 1.3 % of men. This study has shown that couples with otherwise unexplained repeated Chromosome 18q deletion syndrome (De Grouchy failure of IVF embryo implantation have a greater than Syndrome) is a cytogenetic disorder that results from a expected chance of carrying a balanced chromosomal deletion of a part of the long arm of chromosome 18. It translocation. Thus it is possible that the presence is classified into 2 types: the first type, a distal deletion, of an unbalanced translocation in some gametes has a deletion up to the terminal part of the long arm, and may predispose to preimplantation failure of in the second group there is an interstitial deletion near embryo development, and increase the risk of the centromere. repeated failure of IVF treatment. Our study has The distal deletion is associated with major clinical demonstrated that balanced parental translocations features that include postnatal growth retardation with may be implicated in the pathogenesis of IVF short stature, mental retardation, microcephaly and implantation failure. Chromoso-mal evaluation midfacial hypoplasia. The incidence is 1:40000 live should be considered as part of the investigation births. Cytogenetically the distal deletion is from of these patients, and genetic counselling and con- 18q21.3 or 18q22.2 to qter. sideration of preimplantation diagnosis should be A 2- year old male child was referred to our genetic an integral part of planning of further treatment clinic because of microcephaly, low set ears, mental strategies. retardation and bilateral undescended testes. The work was supported by project ITMS Peripheral blood karyotyping was done and the result 26110230100 and ITMS 26220120241. was 46,XY,del(18)(q21). Parental karyotyping was normal and no family his- 1.P30 tory of similar clinical features, indicated that the deletion had occurred de novo as a random event during Somatic mosaicism for DNA copy number variations gametogenesis. and copy-neutral loss of heterozygosity in the human genome 1.P29 Olga Zilina1, Marina Koltsina1, Andres Metspalu2, Ants Chromosomal abnormalities in Slovak couples with Kurg1, Neeme Tonisson2, Andres Salumets3 in vitro fertilization implantation failure University of Tartu Institute of Molecular and Cell Biology Tartu-Estonia1 University of Tartu Jarmila Bernasovska1,IvetaBoronova1,Viktoria Estonian Genome Center Tartu-Estonia2 Szabadosova1, Marta Mydlarova Blascakova1 University of Tartu Department of Obstetrics and University of Presov Biology Presov-Slovakia1 Gynaecology Tartu-Estonia3 10th European Cytogenetics Conference 2015 S49

Somatic mosaicism denotes the presence of genetically Chromosomal diseases have a significant impact on distinct populations of somatic cells in one individual the congenital pathology. However, despite the long who has developed from a single fertilised oocyte. history of study and the availability of high- Mosaicism may result from mutations of different scales resolution methods for molecular diagnostics, many that occur during postzygotic development and are issues of their pathogenesis remain unclear. There propagated to only a subset of the adult cells. are several reasons for this situation. First of all, The aim of our study was to investigate somatic there is a significant clinical polymorphism of chro- mosaicism for DNA copy number variations (CNVs) mosomal syndromes. Secondly, the presence of non- and copy-neutral loss of heterozygosity (cn-LOH) specific clinical features is common for patients with events in fully differentiated human tissues. We studied different chromosomal mutations. Additional uncer- panels of tissue samples (11–12 tissues per individual) tainty in the delineation of phenotype-karyotype cor- from four subjects using high-resolution Illumina relations brings contiguous gene syndromes affecting HumanOmniExpress-12 BeadChips. We observed three a wide list of genes with different functions. Finally, CNVs occurring only in a portion of the studied tissues an important limitation is related to the impossibility in one out of four individuals. The length of observed of interpreting clinical effects of chromosomal abnor- somatic CNVs ranged from 123 to 796 kb and they can malities by the analysis of peripheral blood lympho- also be found in general population. In addition to cytes. Induced pluripotent stem cells (iPSCs) with CNVs, we detected five mosaic cn-LOH regions subsequent cell differentiation provide new possibili- >5 Mb in three out of four individuals. Assuming that ties for the study of chromosomal disease pathogen- specific mosaic patterns of CNVand cn-LOH events are esis at the molecular and cell level. Here, we report reflecting their formation during the postzygotic embry- about the production of two iPSCs lines derived onic development of germinal layers and organ systems, from skin fibroblasts of patients with idiopathic we attempted to track the succession of genomic events intellectual disability having a microdeletion or leading to somatic genetic mosaicism. microduplication at 3p26.3, affecting a single gene Our results give further support to the idea that CNTN6 (Kashevarova et al., Mol Cytogenet. 2014). somatic mosaicism for CNVs, but also for cn-LOHs, is The gene product may play a role in the formation a common phenomenon in normal human tissues. Thus, of axon connections in the developing nervous the examination of a single tissue might produce incom- system. The proof of pluripotency was obtained by plete information about CNV and cn-LOH content of a in vitro and in vivo using SCID-mice. The stability whole genome. The frequency and possible phenotypic of the karyotype during reprogramming was also consequences of somatic mosaicism for CNVs and confirmed. Clinical features of our two patients with cn-LOHs remain largely unknown, and obviously 3p26.3 microdeletions and one with microduplications studies that involve larger sample cohorts can answer as well as patients from the DECIPHER database these questions. were summarized. It was shown that the 3p26.3 microdeletions and microduplications are accompa- 1.P31 nied by intellectual disability, growth retardation, delayed psychomotor development, speech delay, Deciphering of pathogenesis of chromosomal low birth weight, microcephaly, epicanthus, wide microdeletion and microduplications syndromes nasal bridge, and abnormalities of the feet. The study using induced pluripotent stem cells was supported by the Russian Scientific Foundation (project 14-15-00772). Igor Lebedev1, Anna Kashevarova1, Nikolay Skryabin1, Lyudmila Nazarenko2, Aleksey Menzorov3, Natalya 1.P32 Matveeva3, Oleg Serov3 Institute of Medical Genetics Laboratory of Two patients with microdeletion and microduplication Cytogenetics Tomsk-Russia1 Institute of Medical involving 1q21.1 Genetics Laboratory of Hereditary Pathology Tomsk- Russia2 Institute of Cytology and Genetics Laboratory Ahmet Cevdet Ceylan1, Gülen Eda Utine2, Mehmet of Developmental Genetics Novosibirsk-Russia3 Alikaşifoğlu1, Koray Boduroğlu2 S50 10th European Cytogenetics Conference 2015

Hacettepe University Department of Medical Genetics Synevo Central Laboratory Cytogenetics Chiajna, Ilfov Ankara-Turkey1 Hacettepe University Department of County-Romania1 Pediatric Genetics Ankara-Turkey2 Introduction: Small supernumerary marker chromo- Molecular cytogenetics helps diagnose patients with somes are structurally abnormal chromosomes which microdeletion/microduplication syndromes. We present cannot be unambiguously identified by conventional two cases with rearrangements involving 1q21.1. cytogenetics methods alone, and are generally equal in A9year-oldfemalepatientwasadmittedfordevel- size or smaller than a chromosome 20 of the same opmental delay. The patient was born to healthy non- metaphase spread. Their incidence has been estimated consanguineous parents from the fifth pregnancy at the to be around 0.14–0.72 per 1000 in the neonatal setting, 40th week of gestation with a birth weight of 2750 g. She and 0.65–1.5 per 1000 in prenatal studies. was able to sit without support at the fifteenth month, Materials and methods/ Results: We present the case walk at three and half years speak at 4 years. Strabismus of a 35-year-old male with infertility. High resolution was diagnosed bilaterally on eye examination and she chromosome analysis revealed a small accessory was operated at 3 years. Head circumferences was bi-satellited acrocentric chromosome found in the 47 cm(<-2sd), weight 17 kg(<3 percentile), length majority of the cells, subsequently identified as - 110 cm(<3 percentile). Physical examination revealed most likely- genetically irrelevant material, part of dysmorphic facial features including bilateral epicanthic chromosome 15, using spectral karyotyping. folds, deep set eyes, wide nasal bridge, bulbous nose, Conclusion: Chromosome painting by SKY is a use- retrognathia. Cranial imaging showed dysmyelinated ful molecular cytogenetics resource, enabling to specify patchy areas of white matter. Routine karyotype analyses the origin of unknown genetic material, such as a marker was 46,XX[72]/47,XX+mar[28], genomic analysis for chromosome: it is a single genome-wide, cost-effective copy number changes using Agilent 60 k chips revealed method with a reasonable resolution, well suited for this 1,3mb deletion at 1q21.1, further analysis showed that situation. Ascertained through infertility in a phenotyp- marker chromosome was derived from chromosome 1. ically normal male, our case is concordant with the The second patient was a 5 year-old girl, born to reports in the literature. healthy second cousin once removed parents at the 29th week of gestation with a birth weight of 940 g. 1.P34 Her twin had died at 24th week of gestation. She wasn’t able to sit without support and walk yet. Refractive error SNP array analysis of 142 Czech patients with autism was diagnosed at eye examination. On physical exami- gives about 10 % diagnostic yield nation head circumference was 46 cm(<-2sd), weight 15 kg(3–10 percentile), length 107 cm(25–50 percen- Eliska Karaskova1, Miroslava Hancarova1, Marketa tile). Developmental delay, dysmorphic facial features Vlckova1, Jana Drabova1, Darina Prchalova1, Marketa including bilateral epicanthic folds, deep set eyes, Havlovicova1, Zdenek Sedlacek1 hypertelorism, triangular face, facial asymmetry were 2nd Faculty of Medicine, Charles University and observed. Cranial imaging showed cerebral hypoplasia, University Hospital in Motol Department of Biology corpus callosum agenesis, brain stem hypoplasia. and Medical Genetics Prague-Czech Republic1 Routine karyotype analyses was 46,XX. Genomic analysis for copy number changes using Agilent 60 k chips Autism spectrum disorders (ASD) affect over 1 % of revealed 2,03mb duplication at 1q21.1. population. They are genetically heterogeneous and have been associated with many copy number and 1.P33 single nucleotide variants (CNVs and SNVs). Hundreds or thousands of genes participate in ASD, Case report: Small Supernumerary Marker most of them still remain unknown. Chromosome 15, cause of male infertility We examined 142 ASD patients from 133 families using Human CytoSNP-12 BeadChips (Illumina). Lucian Oprea1,SandraGrigore1,OanaMantescu1, According to the most significant aberration, the patients Marinela Malageanu1, Viorica Radoi1 were classified into 5 categories (1−repeatedly reported 10th European Cytogenetics Conference 2015 S51 pathogenic aberrations, 2−likely pathogenic aberrations BACKGROUND: The MTHFR C677T polymor- (some literature support), 3−unclear findings, 4 phism may lead to DNA hypomethylation and thus −intergenic aberrations, low-frequency polymorphisms, represent a potential risk factor for chromosome 21 5−frequent polymorphisms). The existence and inheri- nondisjunction. The aim of the present study was tance of category 1 and 2 findings were validated using to assess the association of the MTHFR C677T independent methods. polymorphism with the meiotic stage of chromo- Clearly pathogenic variants found in 10 patients some 21 nondisjunction among mothers of chil- included: large (~10 Mb) deletions in 9p24.3-p23 dren with Down syndrome. and 5q23.3-q31.2, 4.2 Mb duplication in 19q13.42– METHODS: The study included 107 mothers of 43, 160 kb deletion in 9q34.3 (EHMT1), 2.9 Mb children with maternally derived free trisomy 21. The duplication of maternal 15q11.2-15q13.1 (PWS/AS), mean age of the mothers was 32.1±6.5 (range 16–43) 17q12 and 12p13.33 microdeletions, 500 kb duplica- years. Short tandem repeat markers were used to deter- tion of Xq28 (MECP2), and intragenic GRIN2B and mine the meiotic stage of nondisjunction. PCR-RFLP NSD1 deletions. analysis was performed to detect MTHFR C677T poly- Likely pathogenic findings in 6 patients included: morphisms. Statistical analyses were performed and 3 Mb duplication in 2q24.1, 350 kb duplication in reported using the Chi-squared test of independence, 8q11.23 (RB1CC1, proposed new syndrome), combina- odds ratios, and 95 % confidence intervals. Results were tion of three large duplications in one patient, considered statistically significant at p<0.05. intragenic PTPRD deletion, maternal uniparental RESULTS: Meiosis (M) I nondisjunction was isodisomy (UPD) of chr1 and excess of homozy- presented in 86 % (92/107) and MII nondisjunction gosity pointing to consanguinity. These findings in 14 % (15/107) of trisomy 21 cases. The mean are interesting for research but their potential maternal age was not statistically significantly different clinical use needs more study and caution. between maternal MI- and MII- derived cases of Aberrations in category 3 or 4 are difficult to trisomy 21 (31.65±6.65 vs. 34.80±5.22, p=0.084). interpret using the current knowledge. Mothers of children with MI-derived trisomy 21 SNP arrays are an efficient tool for identifying CNVs had a greater frequency of the MTHFR 677CT+ in ASD. CNVs either have an immediate clinical effect TT genotype compared to mothers of children with or can suggest new causal genes. SNP arrays allow MII-derived trisomy (p=0.040). Mothers who are detection of homozygous regions pointing to UPD or carriers of at least one MTHFR 677T allele have distant consanguinity, which can direct further research 3.6 fold higher risk for MI than for MII nondis- towards recessive mutations. junction of chromosome 21. Supported by NT/14200, 00064203, CZ.2.16/3.1.00/ CONCLUSION: These results indicate that the 24022, NF-CZ11-PDP-3-003-2014. MTHFR 677CT+TT genotype could be a risk factor for MI nondisjunction of chromosome 21. However, 1.P35 further analyses on larger sample are required to provide an answer to this question. Maternal MTHFR C677T Polymorphism: A Risk Factor for Meiotic I Nondisjunction of 1.P36 Chromosome 21 AFamilialInterstitial4q35DeletionWithNo Jadranka Vranekovic1,IvanaBabic-Bozovic1, Discernible Clinical Effects Gordana Brumini2,MiljenkoKatunaric3, Bojana Brajenovic-Milic1 Ozden Altiok Clark1, Sezin Yakut Uzuner2, Banu Nur3, School of Medicine, University of Rijeka 1Department Ercan Mihci3, Sibel Berker Karauzum2, Zafer Cetin4 of Biology and Medical Genetics Rijeka-Croatia1 Akdeniz University Medical Genetics Antalya-Turkey1 School of Medicine, University of Rijeka Department Akdeniz University Medical Biology and Genetics of Medical Informatics Rijeka-Croatia2 School of Antalya-Turkey2 Akdeniz University Pediatric Medicine, University of Rijeka Department of Genetics Antalya-Turkey3 Sanko University Medical Pathology Rijeka-Croatia3 Biology Gaziantep-Turkey4 S52 10th European Cytogenetics Conference 2015

Small deletions on the long arm of distal chromosome 4 From all IUFD’s between 2010 and 2014, DNA was do not appear to result in gross congenital isolated and a QF-PCR was performed. In addition, an malformations; the most frequently reported clinical array-CGH was performed if the QF-PCR was normal. findings include mild to moderate mental retardation, In this period, we analysed 333 IUFD products with a learning disabilities and minor dysmorphic features. success rate of 96 %. An abnormal result was found in Here we report a cytogenetically detectable familial 53 samples (16 %). Thirty one out of 53 aberrations interstitial chromosome 4 long arm deletion with no were detected with QF-PCR. Of these 19 were trisomies discernible phenotypic effects in a mother and her two (13, 18, 21), 6 triploidies, 5 monosomy X and 1 XXY. daughters. The karyotypes of the mother and her two Array-CGH showed one or more CNVs in 22 patients daughters were: 46,XX,del(4)(q35.1-q35.2). Based on (7 %). Seven of these were presumed pathogenic or the results of FISH analyses using whole chromosome were de novo. 15 CNV’s were also present in one of specific and subtelomeric probes, the karyotype was the healthy parents and were not considered causal of designated as: 46,XX,del(4)(q35.1-q35.2). ish the IUFD. Thus, if a CNV is detected by array-CGH del(4)(q35-qter)(WCP4+, 36P21+, dJ963K6-). Array- parental follow up and description of the phenotype is CGH analysis showed an interstitial deletion essential for interpretation of this CNV. encompassing 5.75 Mb in the 4q35.1-q35.2 genomic In conclusion, QF-PCR followed by array-CGH is a region (chr4:184.717.878-190.469.337; hg19). This is better approach for genetic analysis of IUFD products the first report of a cytogenetically detectable familial than traditional techniques, because the success rate is interstitial chromosome 4 long arm deletion in which higher (96 % compared to ~50 %). Furthermore, a there are no discernible phenotypic effects. Both our genetic aberration in the IUFD products is detected in findings and a review of the literature suggest that more a higher percentage of samples (23 vs 13 %). detailed molecular analyses are needed in cases with distal chromosome 4 long arm deletions especially those 1.P38 with breakpoints in the 4q35 region to establish a more precise genotype-phenotype correlation. Frequency of uniparental disomy in 836 patients with pathological pehenotype 1.P37 Vanna Pecile 1,LisaCleva1,AntonellaFabretto1, Towards a molecular approach in the genetic Stefania Cappellani1, Luca Bedon2, Elisabetta Lenzini3 analysis of IUFD products. IRCCS Medical Genetics Trieste-Italy1 University of Trieste Life Science Trieste-Italy2 University of Padua Anne-Marie van der Kevie-Kersemaekers1,Karin Department of Child and Mother Health Padua-Italy3 Huijsdens1, Merel van Maarle1, Lia Knegt1 Academic Medical Centre Clinical Genetics Amsterdam- UPD is the inheritance of a chromosome pair derived The Netherlands1 from a single parent. Based on different mechanisms, UPD can present as isodisomy (two copies of one Approximately 15 % of clinically recognized pregnan- parental homolog), heterodisomy (both homologs of cies ends in intrauterine foetal death (IUFD). Fifty per- one parent) or a combination thereof and can involve cent of early IUFD is caused by aneuploidy or triploidy. complete chromosomes or only chromosomal segments. Late IUFD’sshowmorecomplexaberrations. The mode of inheritance can be maternal or paternal. Traditionally, cytogenetic testing in our laboratory UPD is generally thought to occur at a rate of 1:3500 was performed on cultures of chorionic villi, amniotic live births. Besides the detection of submicroscopic fluid or fetal tissue by karyotyping or interphase deletions and duplications, SNP microarrays can be FISH (I-FISH) for chromosomes 13, 18, 21, X and used for detection of uniparental disomy (UPD). While Y. The success rate of karyotyping and I-FISH for SNP-array analysis allows the detection of uniparental IUFD products is low, ~50 % together, compared to isodisomy, uniparental heterodisomy is suspected when molecular approaches (99 %). Moreover karyotyping extended homozygosity regions are found on a single and I-FISH are labour intensive. Therefore a molecu- chromosome, but it is confirmed through the compari- lar approach was introduced in the last 4 years. son of the parent’s genotype. 836 patients with autism, 10th European Cytogenetics Conference 2015 S53

ID, malformation, etc.…were included in this study to UPD and the deletion on maternal chromosome 5q. detect CNVs which are responsible of the pathological Since autistic behaviours is the only consistent finding phenotype. SNP-array analysis was carried out in our patient and two reported patients with a similar with Beadchip (Illumina), the data were analyzed deletion in 5q the comparison can identify the critical with BeadStudio, GenomeStudio and PennCNV. region that is implicated in autism spectrum disorders. We detected isodisomy in 4 of 836 patients, two The isodisomy can be associated with a normal or cases were in homogeneous form (chromosome 5 pathological phenotype. As far as we know one other and 8, two male) and two were in mosaic (chro- patient is described in literature with paternal segmental mosome 15, one male and one female). Two cases uniparental isodisomy and childhood onset schizophre- of segmental UPD (chromosome 14 and 5q) were nia, so in our patient scoliosis and intellectual disability also detected. Furthermore UPD in other five patients may be caused by recessive mutations highlighted by (chromosome 7 three cases, one case for chromosome isodisomy. Exome analysis could highlight mutations in 14 and 15) was suspected, analysis of homozygosity genes on 5q responsible for the phenotype. regions, through the trio analysis, confirmed all five cases. In conclusion frequency of UPD in our study is 1.P40 1,3 %. Our prospective target is to study frequency of segmental or homogeneous UPD with UPDio software Cytogenetic and array CGH findings in patients with in 132 patients and parents, who do not present exten- split hand/foot malformations sive regions of homozygosity. Drahuse Novotná1, Jana Drábová1, Sárka Vejvalková1, 1.P39 Markéta Vlcková1,DanielChudoba1,Markéta Havlovicová1 A patient with segmental isodisomy on 5q11.2-qTER University Hospital Motol Institute of Biology and and deletion 5q11.1-q11.2 Medical Genetics Prague-Czech Republic1

Elisabetta Lenzini1, Vanna Pecile2, Lisa Cleva2, Flavio Split hand/split foot malformation (SHFM), also known Faletra2, Stefania Cappellani2, Luca Bedon3, Daniela as ectrodactyly, is a congenital limb malformation Gambel Benussi2 affecting the central rays of the autopod. It may present University of Padua Department of Child and Mother with syndactyly, median clefts of hands and/or feet and Health Padua-Italy1 IRCCS Medical Genetics Trieste- hypoplasia and/or aplasia of fingers, toes, tarsals and Italy2 University of Trieste Life Science Trieste-Italy3 metatarsals. In severe cases, the hands and feet have a “lobster claw” appearance. Uniparental disomy (UPD) is a genetic condition in The condition is clinically and genetically heteroge- which the offspring receives two chromosomal homo- neous and exhibits usually autosomal dominant inheri- logues from a single parent. If the UPD is present on a tance with variable penetrance and expressivity. It may segment of the homologues, the condition is referred to occur as an isolated defect or as a component of a as a segmental UPD, and if the homologues are identical syndrome involving ectodermic structures. The inci- (copies of a single homologue from one of the parents), dence has been reported to be about 1/18 000 newborn the condition is known as isodisomy. We report on a babies. male patient of 2 years with ID, autism, scoliosis and Six loci SHFM have been mapped: normal karyotype. Analysis conducted with SNP-array SHFM 1 at chromosome 7q21.3-q22.1 (platform Illumina, Human OmnieExpressExome and SHFM 2 at chromosome Xq26 (one reported family) software GenomeStudio) detected a submicroscopic SHFM 3 at chromosome 10q24 (duplication) 5q11.1-q11.2 deletion (49,457,282–53,282,649) of SHFM 4 at chromosome 3q27 (TP63 gene) approximately 3.8 Mb in size. Moreover, an extensive SHFM 5 at chromosome 2q31 (deletion) homozygosity region was detected (53,282,979– SHFM 6 at chromosome 12q13 (WNT10B, autoso- 180,693,127) in chromosome 5q11.2-qter, and UPD mal recessive) was suspected. It was possible to analyze only the SHFM/SHFLD at chromosome 17p13.3 (BHLHA9) mother, for this reason we can speculate a paternal Other loci have been studied. S54 10th European Cytogenetics Conference 2015

Cytogenetic aberrations of 7q chromosome have referred at 1 year 4 months of age with poor growth and been reported, most often deletions or transloca- global developmental delay. The second girl presented tions (balanced, unbalanced, complex), seldom at 13 years of age with delayed secondary sexual char- inversions of 7q. acteristic development, primary amenorrhea. Duplications of SHFM3 are supposed to be the most GTG-banding revealed a mosaic karyotype with a frequent cause of nonsyndromic SHFM in humans. cell line containing a small marker chromosome and a We present two cases: cell line with 45,X. Parents’ karyotypes were normal. 1) A patient who exhibits severe form of Fluorescence in situ hybridization and molecular ectrodactyly, caused by complex aberration of chromo- techniques using STR markers were performed to deter- some 7 consisting of de novo paracentric inversion mine the origin of the marker chromosome and identi- encompassed SHFM1 region and a submicroskopic fied the very small marker as a ring X chromosome. deletion of 7q The presence of a marker chromosome in Turner 2) A patient with an about 0,5 Mb large duplication syndrome generally implicates a sex chromosome of 10q24 (SHFM3) presenting only syndactyly. origin. The severe phenotype of females with tiny Classical cytogenetics as well as array CGH revealed ring X chromosomes can be explained by the inabil- to be useful tools in SHFM diagnosis. ity of these chromosomes to undergo X inactivation Supported by the Project (Ministry of Health, resulting in functional disomy. Also, mosaicism Czech Republic) for conceptual development of resulting in the absence of the ring from some research organization 00064203 (University Hospital tissues, such as the brain, is important for the severity Motol, Prague, Czech Republic). of the phenotype. Acknowledgments: This work was supported by 1.P41 objective 3.3 of Romanian Ministry of Health Program VI and by PN-II-PT-PCCA-2013-4-133 grant Marker chromosome analysis in two patients with Turner syndrome variant resembling Kabuki 1.P42 phenotype Maternal LINE-1 methylation and congenital heart Vasilica Plaiasu1, Diana Aurelia Ochiana1, Gabriela defects in Down syndrome Motei1, Ioana Alina Anca2 IOMC Alfred Rusescu Genetics Bucharest-Romania1 Ivana Babic Bozovic1, Aleksandra Stankovic2, Maja IOMC Alfred Rusescu Pediatrics Bucharest-Romania2 Zivkovic2,JadrankaVranekovic1,VesnaMahulja- Stamenkovic3, Bojana Brajenovic-Milic1 Small marker chromosomes are chromosomal frag- School of Medicine University of Rijeka Department ments whose origins often cannot be determined by of biology and medical genetics Rijeka-Croatia1 conventional cytogenetic methods alone and require VINčA Institute of Nuclear Sciences, University of molecular approaches for definitive characterization. Belgrade Laboratory for Radiobiology and Turner syndrome presents with a characteristic mild Molecular Genetics Belgrade-Serbia2 Rijeka phenotype with some degree of variability. Kabuki syn- University Hospital Department of Obstetrics and drome is a multiple congenital anomaly disorder char- Gynecology Rijeka-Croatia3 acterized by specific facial features, mild to moderate mental retardation, postnatal growth delay, skeletal Background: Down syndrome (DS) is one of the most abnormalities and unusual dermatoglyphic patterns with common chromosomal abnormalities associated with prominent fingertip pads. The association between clin- congenital heart defects (CHD); cardiac defects are ical and cytogenetic findings of Turner syndrome and present in approximately 40–60 % of the cases. The Kabuki phenotype in the same individual is a rare aim of this study was to determine the association occurrence. between maternal global DNA methylation and occur- We present two cases with particular oriental-like rence of CHD in DS. The impact of MTHFR C677T facies, one of them of Tatar ethnicity, originating from polymorphism and dietary intake of folate on global the South-Eastern Region of Romania. First patient was DNA methylation was analyzed. 10th European Cytogenetics Conference 2015 S55

Patients and methods: The study included 94 mothers balanced de novo rearrangements in patients with of children with DS of maternal origin. Among those abnormal phenotype. mothers, 49 % (46/94) have children with DS and CHD The patient is a 1-year -old boy with retarded psy- (DS-CHD+) and 51 % (48/94) of them have children chomotor development and cranium, face and neck with DS without CHD (DS-CHD-). Global DNA meth- asymmetry. A cerebral MRI scan showed a hypo- ylation was analysed in peripheral blood lymphocytes plastic corpus collosum and retrocerebellar cyst. by quantification of LINE-1 methylation using the He is the second child of healthy unrelated MethyLight method. Genotyping of MTHFR C677T parents. Amniocentesis was performed in the 20th polymorphism was performed by PCR-RFLP. gestational weeks because of a nuchal cystic Results: There was no significant difference in values hygromata was visible by fetal ultrasonographic of global DNA methylation between mothers of chil- examination. GTG-banded analysis of 20 metaphase dren with DS-CHD+ and mothers of children with DS- cells from amniocytes revealed an abnormal male CHD- (P=0.951). The combination of MTHFR C677T karyotype with a balanced de novo pericentric inversion genotype/diet significantly influenced global DNA of chromosome 10— inv(10)(p11.2q21). Conventional methylation (R2=4.5 %, P=0.046). The lowest values chromosome analysis was also performed on a periph- of global DNA methylation were determined in mothers eral blood sample from this patient shortly after his with CT+TT genotype and low folate diet. The contri- birth, which confirmed the amniocyte findings. bution of these factors was even higher among mothers Multicolor banding (mBAND) of chromosome 10 was of children with DS-CHD+ (R2=9.9 %, P=0.026), performed to define breakpoints. FISH analysis revealed particularly among those who had children with DS that the distal breakpoint on chromosome 10 was in and septal heart defect (R2=15.4 %, P=0.018). 10q11.2. High-resolution genome-wide array analysis Conclusion: Although global DNA methylation was using Affimetrix CytoscanTM HD array platform not significantly associated with development of CHD showed copy number change in the distal breakpoint. in DS, its influence cannot be completely excluded since The region 10(q11.22q11.23) contained a 5,7 Mb dele- the significant impact of MTHFR genotype and diet on tion. The deletion was considered pathogenic based on global DNA methylation in mothers of DS-CHD+ was the various public web sources, including ISCA and determined. Further analyses on a larger sample are DECIPHER. Thus the patient has apparently balanced needed to provide an answer to this question. de novo pericentric inversion of chromosome 10 accompanied by the clinically relevant CNV. The 5,7-Mb 1.P43 deletion is considered very likely to be causative for the abnormal phenotype. Apparently balanced de novo structural The symbiosis of traditional cytogenetic techniques rerarrangement of chromosome 10 in a boy with molecular cytogenetic techniques, in association with an abnormal phenotype with high-resolution whole-genome CNV analysis, has permitted further investigation of cases of seemingly Nadezda Shilova1,MarinaMinzhenkova1,Ilya balanced chromosomal rearrangements with associated Kanivets2, Yulia Kozlova1, Vladimir Solonitchenko3, phenotypic abnormalities. Zhanna Markova1 Federal State Budgetary Institution laboratory of cyto- 1.P44 genetics Moscow-Russia1 Federal State Budgetary Institution group of molecular cytogenetics Moscow- An unusual combination of an atypical maternally Russia2 Filatov children’s city clinical hospital N13 inherited 0.3 Mb deletion in Williams-Beuren region genetics center Moscow-Russia3 and a de novo 22q11.21 microduplication in an infant with supravalvular aortic stenosis An increasing number of apparently balanced de novo chromosomal rearrangements have been associated with Paola Evangelidou1,GeorgeTanteles2,Angelos phenotypic abnormalities in recent years. The use of Alexandrou3, Ioannis Papaevripidou3, Andria Ketoni3, high-resolution cytogenetic techniques enables detailed Charithea Ioannidou3,NicoleSalameh3,Violetta screening for cryptic imbalance of microscopically Anastasiadou2, Carolina Sismani3 S56 10th European Cytogenetics Conference 2015

The Cyprus Institute of Neurology and Genetics 1.P45 Cytogenetics and Genomics Nicosia-Cyprus1 The Cyprus Institute of Neurology Clinical Genetics Genetic heterogeneity in patients with 22q11.2DS Clinic Nicosia-Cyprus2 The Cyprus Institute of clinical features Neurology Cytogenetics and Genomics Nicosia- Cyprus3 Yulia Kozlova1,NadezdaShilova1,Valentina Antonenko1, Natalia Kotlukova2, Elena Levchenko3, Williams-Beuren syndrome (WBS) is a rare Viktoria Zabnenkova4 multisystemic neurodevelopmental disorder occurring Federal State Budgetary Institution “Research Centre in 1/20,000 live-births. It is caused by a 1.5–1.8 Mb for Medical Genetics” laboratory of cytogenetics deletion on 7q11.23 characterized by supravalvular Moscow-Russia1 Moscow Municipal Clinical Hospital aortic stenosis (SVAS), intellectual disability, №67 Perinatal cardiological Centre Moscow-Russia2 overfriendliness and dysmorphic facial features. Bakoulev Scientific Center for Cardiovascular Surgery The 22q11.21 microduplication syndrome has a of the Russian Academy of Medical Sciences Perinatal variable phenotype which ranges from normal to Cardiological Center Moscow-Russia3 Federal abnormal and has been frequently associated with State Budgetary Institution “Research Centre for congenital heart disease. Medical Genetics” laboratory of DNA diagnosis We present a 1-month old boy referred for a possible Moscow-Russia4 diagnosis of WBS because of SVAS. In addition, he had bilateral pre-auricular pits. The infant A group of 140 probands with clinical features typical of passed away suddenly 10 days after referral possi- 22q11.2DS was analyzed. The most common reason for bly due to severe cardiac complications. Initial FISH analysis referral was the presence of congenital FISH analysis using LPU011 probe (Cytocell) revealed heart defects (CHD), conotruncal and non-conotruncal, normal results. However, subsequent array-CGH facial anomalies, cleft palate, hypocalcemia, immuno- analysis using Cytochip ISCA array (BlueGnome) deficiency and several other abnormalities. All patients revealed two different pathogenic copy number previously underwent standard karyotyping to exclude changes; an atypical deletion of 0.3 Mb within other chromosomal aberrations. Those with normal the WBS region (location: 73,187,643– karyotype underwent FISH analysis with TUPLE1/ 73,501,025) and a duplication of 2.6 Mb within ARSA (Abbott Molecular) probe. 77 samples were the 22q11.21 microduplication syndrome (location: concurrently analyzed by MLPA analysis with 18,894,864–21,505,388). The deletion includes SALSA MLPA P250-B1 DiGeorge (MRC-Holland) CLDN4, WBSCR27, WBSCR28, ELN and the first kit. This analysis was performed in order to indepen- two exons of the LIMK1 gene. MLPA analysis on the dently confirm earlier data, define deletion size and patient and parents showed that the 22q11.21 duplica- attempt to reveal atypical distal deletions/duplications tion was de novo and the 7q11.23 deletion was mater- in 22q11.2 region and deletions in other chromosomes nally inherited. Family studies of the extended family locuses integrated in this specific MLPA kit known to are ongoing. cause similar phenotypes in FISH-negative subgroup. To our knowledge this is the first report of a Three patients within the group were found to have combination of 7q11.23 deletion and 22q11.2 chromosomal abnormalities: 48,XXXX, microduplication. Detailed clinical evaluation of 46,XY,der(4)t(4;8)(q35;q22)mat, mos45,X[42]/ the intellectually normal mother is currently ongo- 47,XXX[8]. FISH analysis revealed 22q11.2 deletion ing which will provide valuable phenotype- in 43 patients, 94 patients were not deleted. Three patients genotype correlation contributing to the limited with normal karyotypes were later diagnosed with Alagille literature of these rare atypical deletions. The con- syndrome (n=1) and Smith-Magenis syndrome (n=2). In tribution of the 22q11.2 to the patient’sphenotype, the subgroup of 77 probands previously tested for 22q11.2 specifically to the severe cardiovascular problem,is microdeletion by FISH MLPA analysis confirmed all 28 difficult to evaluate, however clinical evaluation of FISH-diagnosed cases of 22q11.2 deletion. Most patients the mother might shed some light and define the had common recurrent 3 Mb deletion (22 cases, 79 %), the contribution of the microduplication. remaining patients (21 %) indicated smaller 10th European Cytogenetics Conference 2015 S57

“nested” deletions -and one patient had an elongated region. Further evaluation of the breakpoints is ongoing deletion. In our study MLPA test didn’trevealeither to determine the possible mechanism responsible for the atypical distal deletions or duplications in the remaining derived deletion. The youngest male member of the 49 patients, as well as other deletions in locuses known family has a very mild form of LWD, a phenomenon to cause comparable phenotypes. As clinical spectrum previously observed, that SHOX deficiency is more of 22q11.2DS is known to be rather broad and some pronounced in females than in males and it can also features could only be recognized with child develop- exhibit inter-familial and intra-familial variable expres- ment, some patients of our group were reevaluated and sivity. Extended family studies, detailed clinical evalu- found to have other genetic disorders, which explain ation of the family members are ongoing which will their phenotype. shed more light in the phenotypic effect of this deletion.

1.P46 1.P47

A novel 15.5 kb deletion within the translated Relationship between heterochromatin polymor- sequence of SHOX gene, leading to Leri-Weill phism and FISH studies in sperm in men with idio- Syndrome in a family with emphasizing severity pathic inferitility in females Mª Angeles De Pedro1, Antonio Urbano2,Isabel Angelos Alexandrou1,GeorgeTanteles2,Ioanna Ochando1,JoseJLopez-Galvez3,ManuelLloret3, Alexandrou1,IoannisPapaevripidou1, Carolina Carmen Aviles3, Joaquin Rueda1, Ana Segura3 Sismani1 Hospital Clinica Vistahermosa Unidad Genetica The Cyprus Institute of Neurology and Genetics Alicante-Spain1 Hospital ClinicaVistahermosay Cytogenetics and Genomics Nicosia-Cyprus1 The Universidad Miguel Hernandez Unidad Genetica Sant Cyprus Institute of Neurology and Genetics Clinical Joan D’ALcant-Spain2 Hospital Clinica Vistahermosa Genetics Clinic Nicosia-Cyprus2 Unidad Reproduccion Alicante-Spain3

Leri-Weill dyschondrosteosis (LWD) is a dominantly Heterochromatin polymorphism (HP) is a variant of a inherited genetic disorder characterized by short stature, normal karyotype but which is thought to be more mesomelia and Madelung wrist deformity. The LWD is frequent in infertile men. It has been suggested that there caused by haploinsufficiency of the Short Homeobox is a relationship between HP and infertility, although no contaning gene (SHOX), located in the 2.6 Mb possible mechanism has been described. HP could be pseudoautosomal (PAR1) region. At the heterozygous related to the existence of meiotic alterations that can be level most mutations found are deletions of different detected by Fluorescence in situ hybridization (FISH) size, point mutations within the coding sequence or analysis in decondensed sperm. In the present work, we mutations on regulatory enhancers downstream of the studied FISH results on spermatozoa of infertile men, to SHOX gene. We present the clinical and molecular data evaluate the relation between heterochromatic variants of a three generation family with LWD. The index and male infertility. patient is a 43 year old female with disproportionate Blood karyotype and sperm aneuploidy (disomy and short stature, reduced arm span to height ratio (particu- diploidy) rates for chromosomes 13, 18, 21, X and Y larly mesomelic shortening), a muscular body habitus were evaluated in 251 infertile men without structural and no evidence of a classic Madelung deformity. We chromosomal rearrangements. report a novel 15.5 kb SHOX intragenic deletion detected A total of 84 patients showed abnormalities with by microarray-CGH, followed by MLPA analysis and FISH (33.5 %) and 167 were normal. From the 251 sequencingofthePCRproductencompassingthejunc- patients, 52 patients (20,7 %) showed HP. Of these tions of the deletion. MLPA studies confirmed the patients with HP, 22,6 % showed abnormal FISH results deletion and revealed the same 15.5 kb deletion in her in contrast to 19,7 % of the patients with HP and normal sister, father, daughter and son. Sequencing has deter- sperm FISH. The most frequently observed polymor- mined the exact breakpoints of the intragenic deletion phism was 9qh+(11 %). There were no significant removing exons 3–6containingthehomeodomain differences in abnormal sperm FISH analysis between S58 10th European Cytogenetics Conference 2015 individuals with chromosomal polymorphisms and with deletion or are due to a second pathogenic event. normal karyotype (36.5 % vs 32.7 %; p>0,05). Our case emphasizes the difficulties in finding a Although some studies have suggested that hetero- close correlation between a large deletion and a well chromatin polymorphism is more prevalent in the infer- defined clinical picture. As only five patients with tile population than in the normal population, our data 2p11.2p12 deletions, reported in the literature are has shown that there is no correlation between male characterized by array CGH, further reports will be infertility and the presence of HP. necessary to well define a clinical phenotype related to the 2p11.2p12 microdeletion. 1.P48 1.P49 Interstitial deletion 2p11.2p12. Report of a patient with developmental delay and bilateral choanal Female Sex Chromosome Abnormalities in a atresia Cytogenetics Laboratory: what have we learnt in 30 years? Rosamaria Silipigni1, Silvia Motta1, Marco Baccarin1, Maria Francesca Bedeschi2, Silvana Guerneri1 Cristina Candeias1,NataliaOlivaTeles2,Fernanda Fondazione IRCCS Ca’ Granda Ospedale Maggiore Paula Oliveira1,M.CéuRibeiro1,M.ElisaLopes1, Policlinico Laboratory of Medical Genetics Milano- Silvia Pires1, Manuela Mota Freitas2, Maria da Luz Italy1 Fondazione IRCCS Ca’ Granda Ospedale Fonseca Silva1 Maggiore Policlinico Medical Genetic Milano-Italy2 Centro de Genética Médica Doutor Jacinto Magalhães/ Centro hospitalar do Porto, EPE Genética Porto- De novo interstitial deletions of the short arm of chro- Portugal1 Centro de Genética Médica Doutor Jacinto mosome 2 are rare chromosomal abnormalities. Patients Magalhães/Centro Hospitalar do Porto, EPE/Unidade showing these kind of microdeletions have developmen- Multidisciplinar de Investigação Biomédica Genética tal delay/intellectual disability, minor facial anomalies Porto-Portugal2 including high forehead, frontal bossing, broad nasal bridge, abnormal ears and congenital defects such as Sex chromosome abnormalities are the most frequent skeletal and genital malformations. We describe the sec- cytogenetic anomalies in pre- and postnatal diagnosis. ond child of a healthy and non consanguineous couple The pathologies associated with chromosomes X and Y presenting at birth multiple malformations and minor show a large range of genetic and clinical variability and facial anomalies. Because of the clinical findings, an encompass many cytogenetic anomalies. However the array CGH analysis was performed using Agilent 60 K phenotypes associated with rearrangements of the sex microarray oligonucleotide. The analysis detected a chromosome are generally less severe than those which 9.3 Mb deletion on the short arm of chromosome 2 at are caused by the similar rearrangements in the auto- band p11.2p12 spanning the bases 77,946,599– somes. Infertility is frequently the only clinical manifes- 87,277,610. The five patients previously described dis- tation in adults females with abnormalities of the X. play a minimal common deleted region which explains In 30 years, in the Cytogenetics Unit of CGMJM, the clinical features shared by all of them, while their 27,422 karyotypes were analyzed; of these, 8,730 individual characteristics might be explained by the dif- female cases were selected for this study. Clinical ferent sizes of the deletion. The common deleted region indications for cytogenetic analysis were varied, e.g. involves several genes (CTNNA2, LRRTM1, REEP1), the presence of multiple anomalies, psychomotor highly expressed in the nervous system. The deletion delay, recurrent miscarriages and clinical suspicion found in this case overlaps with most of those reported of Turner Syndrome. Among the selected samples, in literature but our patient displays extra clinical signs 282 showed alterations of the X chromosome such as bilateral choanal atresia and atrial septal defect. It (3,2 %). The abnormal results will be presented in two was impossible to find any direct correlation between the different perspectives: the classification of the karyotype genes involved in the deletion and the choanal atresia by chromosomal abnormality and by the clinical indica- and the heart defect. The question remains open as to tion. Our results will be compared with those described in whether these clinical features are a consequence of the the most recently published papers in medical journals. 10th European Cytogenetics Conference 2015 S59

With this work the authors present a significant num- supraventricular extra sistole, oxyhemoglobin ber of X chromosome anomalies in Portuguese women desaturation during feeding, and laryngeal stridor. and idendity the most frequent abnormality within each Interestingly, one of the HOXA genes, HOXA5, is clinical group. known to be essential to organogenesis and function of respiratory tracts. This case provide new informa- 1.P50 tions about the genotype-phenotype correlation of HOXA genes cluster deletions via the identification HOXA cluster deletions: a clinical challenge and characterization of the smallest deletion reported to date, furthermore opening new discussion and interpre- Silvana Guerneri1,RosamariaSilipigni1,Marco tation cues on the unusual findings detected. Baccarin1, Gloria Sovena1, Donatella Milani2 Fondazione IRCCS Ca’ Granda Ospedale Maggiore 1.P51 Policlinico Laboratory of Medical Genetics Milano- Italy1 Fondazione IRCCS Ca’ Granda Ospedale Illustration of the complementarity between CMA Maggiore Policlinico Pediatric Highly Intensive techniques and conventional cytogenetic techniques: Care Unit, Department of Pathophysiology and a clinical case. Transplantation, Università degli Studi di Milano Milano-Italy2 Gaelle Tachon1,VincentGatinois1,Jacques Puechberty2, Anouck Schneider1, Genevieve Lefort1, HOXA cluster genes play a fundamental role in Franck Pellestor1 embryologic development. Deletion of the entire cluster CHU de Montpellier Laboratoire de Génétique is known to cause a clinically recognizable syndrome. Chromosomique Montpellier-France1 CHU de The clinical manifestations may vary with different sizes Montpellier Département de Génétique Médicale, of deletions of HOXA cluster and flanking regions. Centre de Référence des Anomalies du Développement Smaller deletions of this region usually display mild et Syndromes Malformatifs Montpellier-France2 developmental delay, characteristic facies, and hand- foot-genital syndrome, but larger deletions may cause Background The array comparative hybridization tech- more complex conditions, leading to difficulties of clin- nology (array CGH) has been a technological revolu- ical diagnosis and interpretation. We report a girl born to tion, which had changed our diagnostic approach ahealthyandnon-consanguineousItaliancouple todevelopmental delay pathology. This technology with a deletion involving the entire HOXA cluster enables detection of sub-microscopic chromosomal at 7p15.2-p14.3. To the best of our knowledge, aberrations and has seen its range of applications this is the smallest deletion involving HOXA expanding in the last few years in clinical and genes reported to date. Chromosome analysis of research fields. However,thetechniquepresents the patient revealed an apparently normal karyo- several limitations that hinder the detection of type at the 550 band level. Based on array com- balanced rearrangements and rearrangements involving parative genomic hybridization, a 2.5 Mb intersti- highly repeated satellite DNAs. tial deletion was detected at 7p15.2p14.3 (chr7: Results Herein we report a male patient who presented 26,333,553–28,859,312), involving the entire HOXA psychomotor delay and language disorder associated cluster and a small number of other genes such as with facial features not obviously related to the observed SNX10, SKAP2, EVX1, HIBADH, TAX1BP1, JAZF1, copy number variation. He showed additional satellite and CREB5. The patient in the present study, showed a material on the end of the long arm of the chromosome 4, number of typical manifestations of HOXA13 deletion, clearly visible on the karyotype, although Chromosomal such as speech delay, short stature, left vesicoureteral Microarray Analysis (CMA) revealed only a terminal reflux, small feet with brachydactyly, bilateral mild 435-kb deletion of the 4q35.2. To explain the apparent hypoplasia of distal phalanges of some fingers of discrepancy, a family investigation was carried out and the hands, and some mildly dysmorphic facial fea- indicated that, in fact, the proband carried a derivative tures. However, our patient exhibited a number of chromosome 4 (der4) derived from a reciprocal translo- additional features, such as bradycardia and sporadic cation t(4;21)(q35.2;p11.2) in the mother. S60 10th European Cytogenetics Conference 2015

In parallel, an investigation of a new pregnancy of the consistent with the clinical picture of a Silver- mother revealed a foetus with agenesis of the foetal venous Russell-Syndrome. system and a 47,XY,+der(21)t(4;21)(q35.2;p11.2)mat Examination of the maternal chromosomes confirmed karyotype. these data— the mother is carrier of anearlycrypticrecip- Conclusion With this case, we emphasise that one of rocal translocation t(6;11)(q27;p15.5). Interestingly, a the limitations of the array CGH technology is its pericentric inversion 12 was also diagnosed; the karyo- inability to find an unbalanced rearrangements involving type was therefore 46,XX,t(6;11)(q27;p15.5), satellite material. This study shows that only the combined inv(12)(p11.2~12q13~15). application of array-CGH with conventional and Clinical data and details of the different analyses will molecular cytogenetic approaches could define the be presented. exact nature of chromosomal abnormalities. Moreover, we have disclosed genes which may be involved in the 1.P53 phenotype of mental retardation Establishment of a single-cell RNA/DNA-FISH 1.P52 method for detecting inactivation patterns of structural X chromosome abnormalities Duplication 11p as a result of an unbalanced translocation 46,XY,der(6)t(6;11)(q27;p15.5)mat in a boy Rie Kawamura1, Wakaba Iha1, Yoshimitsu Fukushima1, with Silver-Russell-Syndrome-like symptoms Keiko Wakui1 Shinshu University School of Medicine Department of Sylke Singer1,AngelikaRiess1,MartinKehrer1, Medical Genetics Matsumoto-Japan1 Andreas Tzschach2,MarkusHoopmann3,Rangmar Goelz4, Thomas Eggermann5, Ulrike Mau-Holzmann1 In females, X chromosome inactivation (XCI) is a gene University of Tuebingen Institute of Medical Genetics dosage compensatory mechanism that one of the two and Applied Genomics Tuebingen-Germany1 Xchromosomesisrandomlyinactivatedincells. University of Dresden Institute of Medical Genetics XIST (X-inactive specific transcript, Xq13.2) accumu- Dresden-Germany2 University of Tuebingen lation (called an XIST cloud) on one of the two X Department of Gynaecology Tuebingen-Germany3 chromosomes is the key player of XCI. The purpose University of Tuebingen University Children’sHospital of the present work is to establish a single-cell RNA/ Tuebingen-Germany4 University Hospital RWTH DNA-FISH method for detecting XCI and structural Aachen Institute of Human Genetics Aachen-Germany5 X chromosome abnormalities simultaneously in each individual nucleus. We report on a 16-month old boy who was born in the The subject was a normal phenotype female with an 29th week of gestation with a birth weight of 730 g. interstitial deletion within band Xp21.2 including the Prenatal ultrasound in the 26th week of gestation GK gene (del Xp21 female). We used three kinds of showed intra-uterine growth restriction (IUGR) BAC clones for the simultaneous 3-color RNA/DNA- and hydrocephalus. Chromosome analysis following FISH study onto interphase nuclei of B-lymphoblastoid amniocentesis revealed a normal result at the 400 cell lines; a clone consistent with the XIST gene for band level. After birth, chromosome analysis was detecting XCI, a clone consistent with the UTX gene repeated and a FISH analysis with probes for the (Xp11.3), which is as a reference probe for both normal short and long arm of chromosome 11 was and abnormal Xs, and a clone consistent with the GK performed because of severe thrombocytopenia. gene for distinguishing between normal and abnormal Unexpectedly, no deletion 11q, but an additional Xs of the del Xp21 female. We evaluated inactive X signal of the short arm probe was detected— localized (Xi) by cloud-type XIST RNA signal, and normal on the long arm of a chromosome 6. Array analysis and abnormal Xs by dot-type gene DNA signals on showed a 8 Mb duplication in 11p and a 537 kb the same nucleus. deletion in 6q. Additional molecular genetic analysis We detected cloud-type XIST RNA signal as a revealed a functional duplication of 11p, supposedly marker of Xi, and dot-type DNA signals of GK, of maternal origin. In summary, these findings are UTX, XIST genes on the same nucleus by the 10th European Cytogenetics Conference 2015 S61

RNA/DNA-FISH method, which allowed us to dis- The size and boundaries of structural chromosome tinguish between the normal and the abnormal Xs of anomalies arising de novo are usually difficult to define, the del Xp21 female. In addition, the dot-type UTX based only on classical cytogenetic analysis, due to its and XIST signals were observed at the cloud-type limited resolution. We have compared the clinical fea- XIST RNA signal, and the other UTX, XIST and tures of our proband to other patients carrying a dupli- GK signals were observed at the clear area without cation of proximal part of 16p as described in the liter- cloud-type XIST RNA signal. We confirmed that the ature. Four copies of genes on chromosome 16p11.2- del Xp21 female showed skewed XCI with selective p13.11 seem to result in greater developmental distur- inactivation of the abnormal X. bance than are reported in patients with three doses of the same genes. This is supported by an animal model as 1.P54 well, where research on mice showed that tetrasomy of chromosome 16 had much more deleterious effects on De novo case of a triplication of 16p embryonic development than did trisomy of the same chromosome. Ivana Skrlec1, Silvija Pušeljić2, Luca Lovrečić3, Borut Peterlin3, Jasenka Wagner1 1.P55 Faculty of Medicine, J. J. Strossmayer University of Osijek Medical Genetics Laboratory, Department of Distichiasis in a patient with a deletion downstream Medical Biology and Genetics Osijek-Croatia1 FOXC2 gene University Hospital Osijek Pediatric Clinics Osijek- Croatia2 University Medical Center Ljubljana Clinical Tarik Ait Mouhoub1,EmilieLandais1,Brigitte Institute of Medical Genetics Ljubljana-Slovenia3 Delemer2, Alain Ducasse3, Martine Doco-Fenzy4 CHU Reims Genetic Reims-France1 CHU Reims Chromosome 16p duplication has been observed in Endocrinology Reims-France2 CHU Reims anumberofindividuals,butatriplicationof16p Ophtalmology Reims-France3 CHU Reims— EA3801 has not been reported. Duplication 16p is usually SFR CAP SANTE Reims Reims-France4 observed as a part of complex syndromes among families with structural chromosomal rearrangements. Heterozygous mutation in the coding sequence of Described duplications of the proximal 16p arm varies FOXC2 gene causes a clinical syndrome combining from small, interstitial duplications to whole arm dupli- lymphedema and distichiasis which is a double row of cations. The phenotype of individuals with duplication eyelashes requiring ablation with a risk of keratitis. Less varies from normal to severe, depending on the size of than hundred patients were hitherto described and some the duplication and potential additional chromosomal with renal abnormalities and diabetes mellitus. Several rearrangement. patients suffered from lymphedema with distichiasis Here we present the clinical and cytogenetic findings without FOXC2 gene mutation. One of them carried a in a 6-year-old female referred for genetic evaluation deletion distal to FOXC2 gene with distichiasis, bilateral because of psychomotor delay and dysmorphic vesicoureteral reflux, glomuvenous malformations but features, including low set ears, long philtrum, also microcephaly and intellectual impairment, suggest- gothic palate, wide spaced nipples, clinodactyly ing a regulatory region of FOXC2 gene in this locus and epileptic seizures. Cytogenetic examination of (Butler et al., 2012). More over some studies showed a GTG banded metaphases showed a female karyo- potential regulatory region in 16q24, close to FOXC2 type with additional material on the short arm of gene (Sholto-Douglas-Vernon, 2005— Brice, 2002). chromosome 16. Extended molecular cytogenetic Here, we report a 20 year-old girl patient with analysis (arrayCGH) showed female molecular karyo- distichiasis, myopia, left bifid kidney with vesical type with four copies of the 16p13.11p11.2 region. Size urethral reflux, dysmenorrhea with septate uterus, of the amplification is 11.8±0.1 Mb. The proband’s diabetes mellitus, and mild intellectual delay. She was karyotype can be reported as: 46,XX,add(16)dn. the only case in her family, without parents’ consan- arr[hg19]16p13.11p11.2(16,525,289–28,318,164)x4. guinity. Array CGH study identified a 693 kb deletion Proband’s parents have normal karyotypes. located at 550 kb to FOXC2 gene (Agilent— 180 K) S62 10th European Cytogenetics Conference 2015 encompassing five genes, three pseudogenes and one 46,XX,del(13)(q21.4). Quantitative gene screening using unknown open frame region. Multiplex Ligation dependent Probe Amplification was We describe here the second patient with a 16q24 performed for aneuploidy and we found heterozygous cryptic deletion which could confirm Butler et al. deletion 13q21.4-q34. Both parents were subsequently hypothesis concerning the regulation of FOXC2 gene evaluated by a clinical geneticist and were karyoptyed; by this region. This case allowed us to better map both had normal karyotypes. the potential regulatory region of FOXC2 gene. 1.P57 1.P56 5q distal deletion detected by arrayCGH in a girl A case report: 13q21-qter deletion with digital anom- with learning disability and congenital anomalies alies, duodenal atresia and anal atresia Sónia Custódio1, Sílvia Serafim1, Ana Sousa1, Rosário Yasemin Özen1,HakanGürkan1,ÜlfetVatansever Silveira-Santos1, Ana Berta Sousa1 Özbek2, Emine İkbal Atlı1, Damla Eker1, Betül Acunaş2 Centro Hospitalar Lisboa Norte— Hospital de Santa Trakya University Fac. of Medicine Medical Genetics Maria, Centro Académico de Medicina de Lisboa Edirne-Turkey1 Trakya University Fac. of Medicine Departamento de Pediatria Lisboa-Portugal1 Dept. Pediatrics Edirne-Turkey2 Interstitial deletions of the long arm of chromo- The 13q-syndrome is caused by a deletion of the long some 5 are rare. Clinical features of patients bear- arm of chromosome 13. ing these deletions allowed genotypic classification We report a newborn female infant who was into proximal deletions, encompassing the region delivered at 33 weeks of gestation by Cesarean from q15 to q22, and distal deletions, Section,with a birth-weight of 830 g and severe encompassing the region from q22 to q31. The symmetric intrauterine growth retardation. The clinical presentation of distal deletions has been mother was a 19 year old woman, G2P1A0C1, fairly well established but genotype-phenotype cor- who was followed-up during her pregnancy at a relations have proven hard to define. This lack of 1st level state hospital, initially with normal find- correlation may be due to difficulties in cytogenet- ings then with a suspicion of a cystic lesion in the ically assigning breakpoints in this region due to abdomen of the fetus. The father had previously its banding pattern. had a child from his first marriage who had died Here we describe an eight year old girl with a 40 days after birth because of a major cardiac de novo 12.15 Mb interstitial deletion of anomaly. The mother of our patient was trans- 5q22.3q23.2 region detected by arrayCGH ferred to the university hospital due to preterm (NimbleGen CGX 135 K, PerkinElmer). She pre- rupture of membranes and a risk of congenital sented at birth with cleft palate and feeding diffi- abnormality. The baby required PPV at birth and culties. Posterior clinical examinations revealed a her initial examination at NICU revealed hypertelorism, patent ductus arteriosus, inguinal hernia and hear- flat nose bridge, low-set ears, typical bilateral anterior ing problems, she had speech delay, along with ear tragus, pointed chin, kliteromegali, four digits in specific learning difficulties and deficits in atten- each extremity and bilateral absence of thumbs, and anal tion, motor control and perception (DAMP). atresia. Imaging studies including plain abdominal Craniofacial dysmorphic features, including doli- radiographics, abdominal USG, Doppler echocardiog- chocephaly, prominent ears and downslanting raphy, and transfontanel USG demonstrated duodenal palpebral fissures, were also present. atresia, interatrial septal aneurysm, bilateral mild renal Cases reported in the literature with 5q distal pelvic dilatation and agenesis of corpus callosum. She deletions are usually larger or have a more distal was operated for duodenal atresia, malrotation and location than the present case. This 12 Mb dele- anal atresia but postoperatively developed multiorgan tion with breakpoints well defined by aCGH greatly failure and died on day 5. Karyotyping using her contributes to a more accurate genomic mapping of peripheral blood revealed the chromosomal abnormality, clinical features. 10th European Cytogenetics Conference 2015 S63

1.P58 recombination (NAHR) occurring during meiosis and mediated by LCR. About 90 % of patients have a 3 Mb French collaborative survey of 749 patients with deletion spanning LCR22-A to LCR22-D called the 22q11 deletion «typicallydeletedregion»(TDR).22q11DSis Celine Poirseir1,JustineBesseau-Ayasse2,Anne associated with a wide spectrum of abnormalities, Bazin3, Florence Amblard4, Marie-Christine Deblois5, but little is known about its incidence. Jean-Luc Bresson6, Tiffany Busa7, Chantal Missirian7, Methods and population: Clinical and cytogenetic François Cartault8,MarieCatty9,AgnèsChoiset10, data on the cases diagnosed from 1995 to 2012 were Ghislaine Plessis11,NathalieDouetGuilbert12, collected in 38 French cytogenetic laboratories. 749 Pascalie Letard13,Anne-ClaudeTabet13,Elisabeth cases postnatally diagnosed were collected. Flori14, Mélanie Jimenez15, Bruno Leheup16, Emilie Results: Most cases were diagnosed by FISH Landais17,HakimaLallaoui18,CédricLecaignec19, (98,0 %) and few case (2.0 %) by array-CGH with a James Lespinasse20,DominiqueMartin21,Francis deletion size ranging from 745 to 2904 kb for the latter. Mugneret22,CélinePebrel-Richard23,Marie-France Only 8/15 patients diagnosed by array-CGH had a Portnoi24,AlineReceveur25,CarolineSchluth- deletion involving LCR22-A and LCR22-D corre- Bolard26,JérômeToutain27,CatherineYardin28, sponding to the TDR. Françoise Girard29,PascaleKleinfinger3,François The most frequent reasons for referral in postnatal Vialard30, Martine Doco-Fenzy17 cases were heart defect (48.6 %), facial dysmorphism chu reims Genetique reims-France1 Hôpital Saint (49.7 %) and developmental delay (40.7 %). The signs Germainian Genetique Poissy-France2 Pasteur-Cerba motivating the genetic analysis are different according Genetique Saint-Ouen l-France3 CHU de Grenoble to age at diagnosis and will be described. Some patients Genetique Grenoble-France4 Hôpital Necker with 22q11DS and atypical phenotype are fortuitously Genetique Paris-France5 CHU de Besançon Genetique found with array-CGH. The deletion was inherited in Besancon-France6 CHU de Marseille Genetique 15.4 % of cases mainly of maternal origin. Marseille-France7 CHU Saint-Denis Genetique Saint Conclusion: This is the largest cohort of patients with Denis La Réunion-France8 Biolille Genetique Lille- 22q11DS reported to date in the same population. We France9 Hôpital Saint Vincent de Paul Genetique have contributed to the evaluation of the frequency of Paris-France10 CHU de Caen Genetique Caen-France11 22q11DS diagnosis by cytogenetics laboratories. There CHU de Brest Genetique Brest-France12 CHU Robert are at least 108 affected patients (including fetuses) per Debré Genetique Paris-France13 CHU Strasbourg year in our French population of about 66 Million Genetique Strasbourg-France14 CHU de Tours people. The clinical signs at diagnosis are highly vari- Genetique Tours-France15 CHU de Nancy Genetique able and change with age at diagnosis. Nancy-France16 CHU de Reims Genetique Reims- France17 Cylab Genetique La Rochelle-France18 CHU 1.P59 de Nantes Genetique Nantes-France19 CH de Chambery Genetique Chambery-France20 CH le Mans Genetique Genotype-phenotype correlation in female gonadal Le Mans-France21 CHU de Dijon Genetique Dijon- dysgenesis—experience of Genetics Department of France22 CHU de Clermont-Ferrand Genetique CHU “St. Spiridon”Hospital, Iasi, Romania de Clermont-Ferrand-France23 Hôpital Trousseau Genetique Paris-France24 CHU d’Amiens Genetique Lavinia Caba1, Cornel Bujoran2, Mihaela Gramescu1, Amiens-France25 CHU de Lyon Genetique Lyon- Elena Graur1,RoxanaPopescu1,CarmenNicoleta France26 CHU de Bordeaux Genetique Bordeaux- Gorduza3, Cristina Cristea4, Ligia Gheorghiu5, Letitia France27 CHU de Limoges Genetique Limoges- Leustean4, Maria Christina Ungureanu4, Alina Gatu3, France28 CHU de Strasbourg Genetique Strasbourg- Dumitru Branisteanu4, Voichita Mogos4, Eusebiu Vlad France29 CHU de Poissy Genetique Poissy-France30 Gorduza1 University of Medicine and Pharmacy “Grigore T. The 22q11.2 deletion syndrome (22q11DS) is the most Popa” Medical Genetics Department Iasi-Romania1 recurrent human microdeletion syndrome. The deletion “St. Maria” Hospital Genetics Department Iasi- results more commonly from non allelic homologous Romania2 “St. Spiridon” Hospital Endocrinology S64 10th European Cytogenetics Conference 2015

Department Iasi-Romania3 University of Medicine and Claude Speeg-Schatz3,NadègeCalmels4,Patrick Pharmacy “Grigore T. Popa” Endocrinology Calvas5, Hélène Dollfus2, Jean-Louis Mandel1 Department Iasi-Romania4 Transmed Medical Center Hôpitaux Universitaires de Strasbourg Laboratoire de Expert Endocrinology Iasi-Romania5 Cytogénétique Strasbourg-France1 Hôpitaux Universitaires de Strasbourg Centre de Référence pour Gonadal dysgenesis could be caused by numerical or les Affections Rares en Génétique Ophtalmologique structural abnormalities of the gonosomes; the main (CARGO) Strasbourg-France2 Hôpitaux Universitaires findings are infertility (sterility) and underdeveloped de Strasbourg Service d’Ophtalmologie Strasbourg- sexual characteristics. France3 Hôpitaux Universitaires de Strasbourg Material and methods: Our consists of 7 patients Laboratoire de Diagnostic Génétique Strasbourg- (adolescent or adult) with features suggesting Turner France4 CHU de Toulouse Service de Génétique syndrome (short stature, primary amenorrhea, failure Médicale Toulouse-France5 of sexualisation) clinically diagnosed in the Genetics Department of “St. Spiridon” Hospital, Iasi, Romania. Rieger syndrome (RS) is a rare autosomal dominant These patients were referred from November 2013 to disorder characterized by specific ocular, dental and January 2014 and confirmed by cytogenetic analyses— umbilical anomalies. Mutations in PITX2 and FOXC1 Barr test and chromosomal analysis (using cultures of genes explain about 50 % of RS. Deletions of coding lymphocytes and GTG bands). In one case we used also exons and more rarely chromosomal translocations have FISH. been described. Very recently a deletion involving the Results: In 6 cases, the final diagnosis was Turner upstream non coding region of PITX2 in two RS syndrome (one 45X, one Xp-, two Xq-, one i(Xq), and patients (Volkmann et al. 2011, Reis et al. 2012) has one r(X)). One case of central hypogonadism with ovarian been reported. The conserved non coding elements failure had a 46,XX/47,XX,+mar karyotype. The Barr test upstream of the gene were showed to be involved in was not always concordant with the karyotype. regulation of pitx2 expression in a zebrafish model. The most common clinical finding in our patients We report a girl aged 13 months who presents unilat- with Turner was short stature varying from −2,7 to eral eye anomalies including posterior embryotoxon, −4,8 standard deviations except in one case with Xq irido-corneal adhesions and pupillary anomaly associated deletion where the stature was normal. The stature most with periumbilical skin and anal anteposition. To confirm affected was in patient with X ring chromosome which the suspicion of RS, Sanger sequencing of PITX2 and presents also a development delay and a very rare con- FOXC1 were performed but no mutation was detected. genital anomaly in Turner syndrome— callosal agenesis The Affymetrix SNP array analysis revealed a de novo (described just in five cases associated with Turner 4q25 deletion of 950 kb which contains no gene. The syndrome). deletion began 194 kb upstream of the PITX2 gene In the case of central hypogonadism with ovarian removing cis-regulatory elements of PITX2. failure the cytogenetic finding was a mosaicism with The identification of this additional case highlights normal line and a line with supernumerary marker chro- the interest of using whole-genome chromosomal mosome which was confirmed by FISH to be an X microarray analysis to detect copy number variation in derivative chromosome. gene desert region and the importance of screening the Conclusions: The karyotype with or without FISH is upstream regulatory region of PITX2 involved in RS. an appropriate method to confirm the diagnosis in all cases with gonadal dysgenesis. 1.P61

1.P60 Are there better clinical criteria for requesting molecular analysis for Prader-Willi syndrome diagnosis? Upstream deletion of PITX2 detected by SNP array in a child with Rieger syndrome Catielly Rocha1,LucianaAgostinho1,Leonardo Cordeiro2, Luciano Olivero3, Iane da Silva2, Greice De Sophie Scheidecker1,ValérieKremer1,Françoise Molfetta4, Rosa Martins5, Marcia Ribeiro6, Ney Boa- Girard-Lemaire1,MélanieHild1,ValériePelletier2, Sorte7, Suely dos Santos1, Carmen Lucia A. Paiva2 10th European Cytogenetics Conference 2015 S65

UNIRIO PPGNEURO Rio de Janeiro-Brazil1 UNIRIO Análisis Clínicos Granada-Spain2 Hospital Virgen de PPGBMC Rio de Janeiro-Brazil2 UNIRIO DGBM Rio las Nieves. Servicio de Pediatría Granada-Spain3 de Janeiro-Brazil3 USP FMRP Sao Paulo-Brazil4 UFRJ Hospital Clínico San Cecilio. Instituto Biosanitario. IEDE Rio de Janeiro-Brazil5 UFRJ IPPMG Rio de FIBAO Granada-Spain4 Janeiro-Brazil6 UFBA UMFL Salvador-Brazil7 Autism comprises a clinically heterogeneous group Prader-Willi syndrome (PWS) arises from lack of gene of disorders— collectively referred to as features of expression in paternal chromosomal segment 15q11- impaired social relationship, impaired language and q13 which can result from deletion (65–75 %), maternal comunication, and repetitive behaviors or a narrow uniparental disomy in chromosome 15 (20–30 %) or range of interests. Currently, an etiology can be from a defect in genomic imprinting (~5 %). The diag- identified for between 15 and 20 % of individuals nosis of Prader-Willi syndrome (PWS) is a challenge with autism; in the others the cause remains because several of the signs and symptoms overlap with unknown. those of other genetic syndromes, particularly during Actually, array comparative genomic hibridization early childhood. The aim of this work was to examine (aCGH) has replaced high resolution chrmosome anal- the association between laboratory tests and the clinical ysis as the test of choice for the evaluation of any child criteria established by Holm et al. (1993) and Gunay- with (ASD). For that, we are implement the array CGH Aygun et al. (2001) for diagnosing PWS, and to assess thecnique as first tier test for genetics studies of children the clinical criteria that are important when selecting with ASD. patients for molecular screening of PWS. Forty-five In this study we present the results of array CGH patients suspected of having PWS were evaluated. obtained in 75 patients clinically diagnosed as ASD. Methylation-based PCR (MB-PCR) technique was used The Nimblegen CGX Cytogenetic Microarrays plat- as well as GTG-banding, FISH and microsatellite anal- form, suplied by PerkinElmer, was performed. ysis. Forty-five patients suspected of having PWS were From the total 75 patients, 59 (78,7 %) were evaluated, 13 were MB-PCR positive: 10/13 patients males and 16 (21,3 %) females with a rate nearly met both clinical criteria of Holm et al. (1993) 5to1betweenmalesandfemales,similartothe and Gunay-Aygun et al. (2001), 2/13 met one or rates described previously. Pathologycal results the other set of criteria, and one patient met none were obtained in 8 patients (10,7 %). When the of the criteria. Short stature (χ2=7.89, P=0.004) study was performed by sex, we obtained and feeding problems (χ2=4.70, P=0.03) were the pathologycal results in 5 males (8,5 %) and in 3 only clinical characteristics that differed signifi- females (18,7 %), the higher proportion of cantly between patients with negative and positive pathologycal results in females when compared MB-PCR results. Despite the small sample size of with males in our results, can be due to the small this study, our results indicate that the use of short number of cases and can not be considered stature and feeding problems (poor suck), rather stadisticaly sibnificant. than obesity and mental retardation, as screening Ours results with array CGH for genetic study of criteria for subsequent MB-PCR testing, can ASD are in concordance with the results previously increase the chances of detecting PWS. obtained in other laboratorios and they are improved significantly the genetics diagnosis for ASD patients in 1.P62 our laboratory.

Genetic diagnosis of autism spectrum disorder 1.P63 (ASD) by array CGH. Application of array comparative genomic hybridi- Matías Pérez Sánchez1,AdelardoMoraGujosa2, zation in Hungarian patients with intellectual Susana Roldán3, Amanda Rocío González Ramírez4 disabilities and multiple congenital anomalies Hospital Virgen de las Nieves. Instituto Biosanitario. Servicio Analisis Clínicos Granada-Spain1 Hospital Anikó Ujfalusi1, Katalin Szakszon2, Gabriella P. Szabó2, Virgen de las Nieves. Instituto Biosanitario. Servicio Beáta Bessenyei1, István Balogh1 S66 10th European Cytogenetics Conference 2015

University of Debrecen Department of Laboratory Introduction: Autism spectrum disorders (ASD) are Medicine Debrecen-Hungary1 University of Debrecen neurodevelopmental conditions showing extreme Department of Pediatrics Debrecen-Hungary2 genetic heterogeneity. The CTNNA3 gene (alpha T-catenin) maps to 10q21.3 and codes for a protein Whole genom array comparative genomic hybridization with multiple cellular roles including cell adhesion. (CGH) is recommended as first-tier test for the postnatal The CTNNA3 gene is one of the largest genes in evaluation of individuals with intellectual disability, the human genome and is located in a common developmental delays, autism spectrum disorders and fragile site (FRA10D). Fragiles sites are considered multiple congenital anomalies. Array CGH has hot spots for genomic instability, associated with increased the diagnostic yield of 15–20 % in clinical neuropsychiatric diseases including autism. cytogenetic laboratories. In this study our aim was the Material and Methods: 522 patients were analysed application of array CGH in clinical diagnostics of using the Agilent 4x180K microarrays and cytogenomics patients with intellectual disability, multiple congenital 2.9.2.4 software. The main clinical indications were anomalies and accompanying dysmorphic features. intellectual disability, ASD, epilepsy, and multiple Cytoscan 750 K oligonucleotid array (Affymetrix) was congenital abnormalities. succesfully implemented in a cohort of 18 patients. The Results: Partial deletions of CTNNA3 were found in identified copy number variants were confirmed by 7 of the 522 patients studied and were classified as fluorescence in situ hybridization, multiplex ligation VOUS. The CNVs sizes ranged between 23 Kb and dependent probe amplification and G-banding. In 14 76 Kb with genomic intervals between 68,087,319 and patients with normal karyotypes the array CGH did not 69,235,883 (GRCh37), involving exons 9 to 10, and revealed genomic imbalances.Clinicallysignificant intron 6, 11 and 13 of CTNNA3 gene. Additionally, 2 copy number variants (CNVs)were found in 4/18 out of 7 cases showed other relevant CNVs that were patients with the size ranging from 0.9 to 15 Mb. reported as pathogenic or presumably pathogenic. Two patients had CNVs associated with known Conclusions: Recent studies from Bacchelli et al. syndromes: 3q29 microduplication and 2q32q33 (2014) showed that heterozygous exonic deletions in microdeletion syndrome. Two cases presented rare the CTNNA3 gene are not pathological however, genomic aberrations: unbalanced subtelomeric translo- homozygous or heterozygous compound exonic cation t(8;18)(p23.3;q22.3) causing partial deletion of deletions/mutations affecting α-catenin function may 18q22.3 and duplication of 8p23.3; de novo unbalanced contribute to ASD pathogenesis. Only 3 of 7 patients ins(8;6)(q22;q21q25) with multiple microdeletion on 6q with CTNNA3 deletions presented ASD, all involving and concomitant microdeletion of Xp22.1. All patients deletion of exons 9 and 10. Screening for additional presented multiple congenital abnormalities. Our results mutations in CTNNA3 is warranted to elucidate the further demonstrate the usefulness of chromosomal phenotype in these cases. microarray as first-tier genetic test in the clinical diagnostics of patients with unexplained intellectual dis- 1.P65 abilities, developmental delays and dysmorphic features. Combined Wolf-Hirschhorn and Beckwith- 1.P64 Wiedemann syndrome in a girl with a 4p deletion and a 11p duplication: a recurrent double syndrome CTNNA3 Deletions—Susceptibility to Autism Spectrum Disorders? Karin Nebral1,PetraZeitlhofer2,SörenMai1,Eva Beran-Fleischhacker3, Oskar A. Haas4 Joel Pinto1, Carolina Almeida1, Micaela Guardiano2, Medgen.at GmbH Cytogenetics Vienna-Austria1 Mafalda Sampaio2,SofiaÁgueda2,MiguelLeão3, Medgen.at GmbH Molecular genetics Vienna-Austria2 Sofia Dória1 LK Mistelbach-Gänserndorf Department of Pediatrics Faculty of Medicine Genetics Department Porto- and Adolescent Medicine Mistelbach-Austria3 CCRI, Portugal1 SJoãoHospitalPaediatricsDepartment Children’sCancerResearchInstitute, St. Anna Porto-Portugal2 S João Hospital Genetics Consultation Kinderkrebsforschung Cytogenetics and Molecular Porto-Portugal3 Cytogenetics Vienna-Austria4 10th European Cytogenetics Conference 2015 S67

The co-occurrence of specific chromosome abnormali- two breakpoints located on more than two chromo- ties, which are responsible for recognizable syndromes somes. Most of them are reported to be de novo. In such create phenotypic effects that more or less reflect and cases, CCR involve more breakpoints and more chro- combine the characteristics of each individual one. Such mosomes than in familial cases. CCR can be found in mixed phenotypes may be particularly difficult to clarify phenotypically normal patients presenting, however, in newborns and infants when the particular symptoms recurrent miscarriages or infertility. CCR can also be are not yet that distinctive. We present the case of a 6- found in patients with phenotypic abnormalities due week old girl with various such ambiguous abnormali- to a chromosome imbalance or to genes disruption at ties in whom a CytoScanHD SNP-array (Affymetrix®) the breakpoints. Conventional karyotype generally analysis revealed a 3,98 Mb heterozygous deletion of allows their identification. However, molecular cytoge- telomeric 4p and a 3,4 Mb duplication of telomeric 11p netic methods can reveal subtle rearrangements. We regions. Such abnormalities are typically encountered in report, here, the identification of a de novo CCR Wolf-Hirschhorn (WHS) and, depending on the parental involving chromosome X, 13 and 15 associated with origin of the duplicated segment, in specific forms of a translocation between chromosome 6 and 11, in a Beckwith-Wiedemann (BWS) or Russel-Silver syn- girl presenting a delayed development and a symp- drome (RSS), respectively. To distinguish between the tomatic muscular dystrophy. FISH was performed to later two options we performed a multiplex methylation- characterize this rearrangement, with use of probes specific PCR analysis of the promoter regions of the that hybridize in DMD gene region. We found that H19 and KCNQ1OT1 genes that enabled us to quantify DMD is disrupted by the breakpoint in Xp21.We the copy numbers of the methylated and unmethylated also performed array-CGH that revealed no cryptic alleles. This analysis revealed that the paternal allele imbalance. was duplicated, which is a known but extremely rare Duchenne muscular dystrophy usually affects males. cause of BWS. This result also implies that the girls However, females are also affected in rare instances. unbalanced deletion/duplication abnormality may have The majority of them have been associated with a been inherited from her father who presumably carries a skewed X-inactivation pattern. Of particular importance balanced t(4p;11p). The respective analyses to verify have been those in whom the manifestation of the dis- this notion are currently underway. Our notion is further order has been associated with de novo X;autosome supported by the fact that the one or the other parent of 4 translocations. Although these females carry two X from 5 cases with an analogous unbalanced transloca- chromosomes and therefore have two dystrophin gene tion and a mixed WHS/BWS or WHS/RSS phenotype loci, they manifest DMD because of a skewed X- reported so far was a balanced translocation carrier. The inactivation pattern. Whereas one gene is disrupted by sixth case with such an unbalanced t(4p;11p) reported the translocation and lies on the active X, the other lies herein suggests that such recurrent abnormalities result on the intact, inactive X chromosome. Here, our patient from nonrandom breakpoint events the underlying pre- presented also a developmental delay probably due to disposing genomic structure of which still needs to be the disruption of one or more other genes located in the determined. multiple breakpoints.

1.P66 1.P67

Complex chromosome rearrangement involving Characterization of a rare analphoid supernumer- Xp21 band in a girl with a syndromic Duchenne ary marker chromosome in mosaic muscular dystrophy. Bárbara Marques1, Filomena Brito1, Cristina Alves1, Morgane plutino1, Manal Dayem-Quere1, Annabelle Sónia Pedro1, Cristina Ferreira1,MartaAmorim2, Chaussenot1, Houda Karmous-Benailly1 Hildeberto Correia1 Hôpital Archet 2- CHU NICE Génétique Nice-France1 Instituto Nacional de Saúde Doutor Ricardo Jorge, I.P. Departamento de Genética Humana Lisboa-Portugal1 Complex chromosome rearrangements (CCR) are rare Hospital D. Estefânia, Centro Hospitalar Lisboa structural chromosome aberrations involving more than Central Serviço de Genética Médica Lisboa-Portugal2 S68 10th European Cytogenetics Conference 2015

Analphoid supernumerary marker chromosomes CASE: 22 month old baby was referred to a pediatrician (SMCs) are a rare subclass of SMCs C-band-negative for lack of independent ambulation. At 27 months he and devoid of alpha-satellite DNA. These marker chro- could free run. mosomes cannot be identified unambiguously by con- MATERIAL AND METHODS: Cytogenetic ventional banding techniques alone being necessary to analysis on patient’speripheralbloodwasper- apply molecular cytogenetic methods in favour of a formed, finding an additional material of unknown detailed characterization. origin in the telomeric region of the short arm of In this work we report an analphoid SMC involving chromosome 12. Whole chromosome painting was the terminal long arm of chromosome 7, in 9 years-old made. boy with several dysmorphic features and severe Array CGH technique 60 Kb was performed to development delay. identify the origin of this material and genes Cytogenetic analysis revealed a mosaic karyotype involved. with the presence of an extra SMC, de novo, in 20 % Karyotypes from parents and healthy sister of the of lymphocytes and 73 % of fibroblast cells. patient were analyzed. FISH analysis with alpha-satellite probes for all chro- RESULTS: mosomes, whole chromosome painting probe for chromo- Karyotype: 46,XY,add(12)(p12)[7]/46,XY[7]dn. some 7, and D7S427 and TelVysion 7q probes, allowed WCP: additional material of chromosome 12. establishing the origin of the SMC as an analphoid marker Parents and healthy sister had normal karyotypes. resulting of an invdup rearrangement of 7q36-qter region. CGH array : arr[hg19] 12913.33(230.421- Affimetrix CytoScan HD microarray analysis, 697.269)×1,12p13.33p11.1(835.365-34.756.209)×3 redefined the SMC to arr[hg19] 7q35(143696249- Terminal deletion of 467 Kb in short arm of chromo- 159119707)×2~3, which correspond to a gain of some 12, 12p13,33 region, and interstitial region ampli- 15.42 Mb and encloses 67 OMIM genes, 16 of which fication of 34 Mb in 12p13,33-12p11,1 region. Both are associated to disease. anomalies modify the structure and / or change doses This result, combined with detailed clinical descrip- of reference genes RefSeq, involved in diseases with tion, will provide an important means for better OMIM number. genotype-phenotype correlation and a more suitable DISCUSSION: Interstitial amplification of 34 Mb genetic counselling to the patient and his parents, identified on the short arm of chromosome 12 is asso- despite the additional difficulty resulting from being ciated with Pallister-Killian syndrome (OMIM: a mosaic (expression varies in different tissues). 601803). Analphoid SMCs derived from chromosome 7 are Clinical descriptions of patients with a triplica- very rare, with only three cases reported so far. tion of 12p show the following: newborns have a With this case we hope contribute to a better under- high weight and a high risk of hypoglycemia. standing of this type of chromosome rearrangements Facial anomalies consist of a short neck, flattened which are difficult for genetic counselling. face, anteverted nostrils and everted lower lip. The patients have severe mental retardation involving 1.P68 language and learning and have sleep and behavior disorders. Pallister-Killian syndrome caused by mosaicism of It should be noted that our patient has a duplication of abnormal chromosome 12 but not isochromosome 12 in a mosaic form. 12 p DECIPHER describes two patients with overlapping duplication with our case who have feeding difficulties, Rosa Lobo1, Maria Talavera2, Saturnino Ortiz3, Maria coloboma, prominent nasal septum, micrognathia and Luisa Arranz4 tricuspid regurgitation. Hosp. Rio Hortega Serv, Analisis Clinicos Valladolid- ISCA describes two patients with mental retardation, Spain1 Hosp. Ramon y Cajas Serv. Genetica Madrid- psychomotor retardation and short stature. Spain2 Hsop. Santa Barbara Serv. Pediatria Soria-Spain3 Bibliography: De novo trisomy 12p in twin girls with Hosp. Rio Hortega Serv. Analisis Clinicos Valladolid- different levels of mosaicism, Am J Med Gen A. 2013 Spain4 Jul; 161A(7): 1702–5. 10th European Cytogenetics Conference 2015 S69

1.P69 particularly with the use of intracytoplasmatic sperm injection (ICSI) techniques and the support of appropri- Cytogenetic Approach to Male Infertility Diagnosis: ate genetic counselling. the importance of a karyotype. 1.P70 Maria Elisa Lopes1, Natália Oliva Teles2, Silvia Pires1, Fernanda Paula Oliveira1,MariadoCéuRibeiro1, Discrepancies in results of standard cytogenetics and Cristina Candeias1, Manuela Mota Freitas2, Maria da cytogenetic/molecular evaluation of patients with sex Luz Fonseca e Silva1 chromosome abnormalities Centro de Genetica Medica Jacinto Magalhães/Centro Hospitalar do Porto,E.P.E Unidade de Citogenetica Vyacheslav Chernykh1,LiubovKurilo1,Natalia Porto-Portugal1 Centro de Genetica Medica Jacinto Kuzina2,HanumMagomedova2,YuliaKozlova2, Magalhães/Centro Hospitalar do Porto,E.P.E/UMIB Nadezhda Shilova2,ValentinaAntonenko2,Tatiana Unidade de Citogenetica Porto-Portugal2 Tsvetkova2, Oksana Ryzhkova3,ElenaBliznets3, Alena Chukhrova3, Aleksandr Polyakov3 Introduction: Infertility is a relatively common Federal State Budgetary Institution “Research Centre problem that affects about 15 % of all couples, for Medical Genetics” laboratory of Genetics of with a male-factor identified in approximately half Reproduction Disorders Moscow-Russia1 Federal State of the cases. Although numerous causes may con- Budgetary Institution “Research Centre for Medical tribute to this occurrence, genetic basis including Genetics” Laboratory of Cytogenetics Moscow- chromosomal abnormalitiesand/orgeneticsyn- Russia2 Federal State Budgetary Institution “Research dromes may give rise to gene defects and therefore Centre for Medical Genetics” Laboratory of DNA diag- affect the chances of normal pregnancies. The nosis Moscow-Russia3 examination of male infertility is complex, comprising detailed family history, physical examination, semen The aim of our study was reevaluation of karyotypes in analysis, hormonal screening and genetic analysis of patients with sex chromosome abnormalities and/or somatic cells. Disorders of Sexual Development (DSD). We examined Material and Methods: The study was carried out 453 individuals: 110 Turner syndrome (TS) and 93 between 01.01.2013 and 31.12.2014, in 146 infertile Klinefelter syndrome, 19 46,XX/46,XY karyotype, 21 males, referred to our Cytogenetics Unit for azoosper- XX and 55 XY gonadal dysgenesis, 14 ovotesticular mia, recurrent miscarriages, hypogonadism, infertility, DSD, 56 46,XX-males, and 85 males with Y chromo- oligoasthenospermia (OAT). Chromosomal analysis of some abnormalities and/or mosaicism. peripheral blood lymphocytes was performed according Conventional karyotyping was done by standard to standard protocols. technique. FISH with DXZ1, DYZ3, WCPX, WCPY Results: Among the 146 infertile men studied, 15 and LSI SRYprobes was performed on metaphase chro- revealed constitutional chromosomal abnormalities: 11 mosomes and interphase nuclei of peripheral lympho- cases of Klinefelter Syndrome (47,XXY); three with cytes and buccal smears. Aneuploidy of 13, 18, 21, Х apparently balanced translocations [t(4;6); t(1;11); and Y chromosomes and a chimerism were evaluated by t(20;22)] and one mosaic (45,X/46,XY). QF-PCR for 20 autosomal and 5 gonosomal loci. Y- Conclusions: The present review showed the microdeletions were detected by mPCR for SRY, ZFX/ results and chromosomal abnormalities found in ZFYand 20 Yq markers. X chromosome heterozygosity infertile men, using standard cytogenetic methods, was evaluated by analysis of 13 X-chromosome loci. during a period of 2 years. The anomalies were The karyotypes were refined or redefined in 13.2 % found in 10,2 % infertile males and the most patients. Mostly, it was due to hidden mosaicism and common abnormality was Klinefelter Syndrome, false positive detectionofYchromosomeinthe which is in agreement withpreviouslypublished karyotype. The highest percentage of discrepancies was data. The authors emphasise the importance of detected in XX/XY- and X/XY-mosaics (10.5–22.4 %), cytogenetic studies as a fundamental approach to eval- XX-gonadal dysgenesis (9.5 %) and TS (8.2 %). uate male infertility and improve fertility prospects, Markers were X- or Y-derivates (50 %:50 %) in TS S70 10th European Cytogenetics Conference 2015 patients, and Y-derivate inallmalecarriers.SRY- because of suspicion of an overgrowth syndrome. The negative amplification was found in 9 % XY-gonadal physical examination revealed a flat nasal bridge, long dysgenesis patients, 19 % 46,XX-males were SRY- and pointed chin, long and big hands and feet. However, positive. Cryptic X;Y translocations were found in the clinical features of the case were not consistent with (SRY+)XX-males and 46,X,del(X)(q22) female with an overgrowth syndrome. The unbalanced karyotypes DSD. Yq11.2 microdeletions or cytogenetically detected of the first and second cases resulted from a balanced structural Yabnormalities were found in 40 % individuals t(9;15) translocation in the mothers who were from the with Y chromosome mosaicism, including polysomic same village. The unrelated father of the third case cases, all of them were mosaics. AZF deletions were from a different city was a carrier of the same detected in all idicYp chromosomes, TS females with translocation. The breakpoints of the rearrangements cryptic Y-mosaicism, and two-thirds of Y-derivative were determined by array-CGH and FISH analyses. markers. There is overlapping of certain gonosomal None of these patients had craniosynostosis or over- abnormalities between some DSD forms mainly by growth as previously reported. sex chromosome rearrangements and/or mosaicism. 1.P72 1.P71 Molecular and clinical delineation of the 2p15-16.1 An assessment of three cases with distal 15q microdeletion syndrome and proposal of a new can- duplication didate gene for microcephaly

Muhsin Ozdemir1, Evren Gumus1,AyseNeslihan Jonathan Lévy1, Aurelie Coussement2, Celine Dupont1, Tekin2, Halime Onur Kucuk1, Ebru Erzurumluoglu1, Fabien Guimiot3, Clarisse Baumann4, Geraldine Viot5, Huseyin Aslan1,CoskunYarar2,OguzCilingir1, Sandrine Passemard4, Yline Capri4, Severine Drunat6, Sevilhan Artan1 Camille Leroy1,BrigitteBenzacken1,Jean-Michel Eskişehir Osmangazi University Medical Faculty Dupont2, Anne-Claude Tabet1 Department of Medical Genetics Eskişehir-Turkey1 Robert Debré Cytogenetic Paris-France1 Cochin Eskişehir Osmangazi University Medical Faculty Cytogenetic Paris-France2 Robert Debré Foetopathology Department of Pediatrics Eskişehir-Turkey2 Paris-France3 Robert Debré Clinical Genetic Paris- France4 Cochin Clinical Genetic Paris-France5 Robert Patients with duplications of distal 15q have rarely been Debré Molecular genetic Paris-France6 reported in the literature. A 15q25-qter trisomy syndrome, characterized by unusual features of prenatal Interstitial 2p15-p16.1 microdeletion is a rare syndrome overgrowth, tall stature, macrocephaly and craniosynos- previously reported in 14 patients to our knowledge. It is tosis was proposed by previous authors (Zollino et al. characterized by moderate to severe intellectual disabil- 1999, Am J Med Genet 87:391–394). We report three ity, autism spectrum disorder, short stature, dysmorphic new patients with a duplication of 15q23qter features, structural brain anomalies, congenital organs (46,XY,der(9)(9qter→9p22.1::15q23→15qter)). The defects and microcephaly in some cases. It is considered first case was a fetus with abnormal ultrasonographic acontiguousgenesyndromeinvolvingdeletionof findings of ventriculomegaly and bilateral several genes. Deletions overlapping this region previ- hydronephrosis. Although conventional cytogenetic ously reported are variable in size (from 203 kb to and molecular cytogenetic analyses from amniocytes 6.9 Mb) with non recurrent breakpoints. We report here revealed the abnormality, the parents decided to continue three additional patients with a 2p15p16.1 microdeletion the pregnancy. Postnatal examination of the child identified by SNP-array analysis (Cyto12-SNP, revealed dysmorphic findings as asymmetric face, Illumina) or CGH-array (Agilent 8x60K): one prenatal hypertelorism, prominent philtrum, cleft palate. The case and two postnatal cases. The prenatal and one of second case, a 4-year old girl, was referred because of the postnatal cases shared an overlapping 2p15p16.1 her dysmorphic features including down slanted palpe- deletion of 105 kb (chr2:61671686-61777241, hg19) bral fissures, flat nasal bridge, microretrognathia and including only XPO1 and the distal end of USP34. cleft palate. The third case, a 6 year old girl, was referred Both patients presented features overlapping the clinical 10th European Cytogenetics Conference 2015 S71 spectrum of the previously described 2p15p16 uncultured tissues or unstimulated peripheral blood microdeletion syndrome including mild intellectual dis- cells, the detection of low-level mosaicism, missed by ability in the postnatal case, dysmorphic facial features conventional methods, is more likely and the percentage and brain structural abnormalities. We confirm that one rate of the cell lines included are undistorted. or both genes are probably involved in facial dysmor- From our experience, we present three cases of phic features, cognitive impairment and brain structural mosaicism, two prenatal and one postnatal, that array abnormalities observed in the syndrome. CGH has facilitated to 1) explicate two abnormal The third patient we report on is a 4 years-old male cell lines placental mosaicism, 2) identify the origin with an heterozygous de novo 427 kb deletion at 2p16.1 of a SMC in mosaicism and 3) reveal the existence (chr2:60624940-61051867, hg19) containing BCL11A of a second normal cell in a clinical case with and PAPOLG and a phenotype characterized by speech moderate signs of Williams Syndrome. delay, autistic traits, stereotyped behavior but without BAC array-CGH (Illumina Cytochip Focus microcephaly. Constitutional) was applied and the data were Considering previous reported deletions in the analysed by BlueFuse Multi software. 2p15p16.1 region and our three new cases, we precise Intercellular variation due to chromosomal mosaicism the genotype-phenotype correlation of the microdeletion is frequently hidden and is likely to be involved in the syndrome. Moreover, as the REL gene is the only gene genetic diversity and human pathogenic conditions. Until shared by all patients having a microcephaly, we suggest recently, due to technical difficulties, the significance of that this gene could be considered as a candidate gene chromosomal mosaicism remained under investigation. for microcephaly associated in the syndrome. Today, molecular cytogenetics has the potential to detect low-level mosaicism and thus,futurestudiescanprovide 1.P73 new insight for the uncovering of the role of chromosomal mosaicism in human diseases that will assist in more Genomic imbalances identified by array CGH in informed genetic counseling and patient management. cases with chromosomal mosaicisms 1.P74 Luiza Dimos1, Anca Pavel2, Ruxandra Achim2, Florina Nedelea3, Vasilica Plaiasu4, Rodica Simion1,Danai Microdeletion of 2q24.2 region: clinical and molecu- Stambouli1 lar analysis based on a new case Cytogenomic Medical Laboratory Cytogenetics Bucharest-Romania1 Cytogenomic Medical Marzena Kucharczyk1,MarlenaMłynek1,Agata Laboratory Molecular Bucharest-Romania2 Cieślikowska1, Monika Kugaudo2, Aleksandra Jezela- Cytogenomic Medical Laboratory, Clinical Hospital Stanek1,AnnaGutkowska1,MagdalenaBiałecka1, Filantropia Medical genetics Bucharest-Romania3 Aleksandra Marczak1,DanutaSielska-Rotblum1, Cytogenomic Medical Laboratory,Institute for Mother Małgorzata Krajewska-Walasek1 and Child Medical genetics Bucharest-Romania4 Children’s Memorial Health Institute Department of Medical Genetics Warsaw-Poland1 Children’s At present, array CGH-based techniques are widely Memorial Health Institute; Medical University of used in clinical genetics because they are extremely Warsaw Department of Medical Genetics; Department powerful for the delineation of chromosomal of Child and Adolescent Psychiatry Warsaw-Poland2 breakpoints, identification of new microdeletion/ microduplication syndromes, characterization of extra Microdeletion of 2q24.2 is a rare genomic disorder. To SMC (supernumerary marker chromosome), derivative date, 12 comparable submicroscopic deletions involving chromosomes and uncovering genomic variations in this region have been described. Although this chromo- healthy and disease associated population. somal aberration is associated with a heterogeneous Furthermore, the sensitivity of array CGH has been phenotype, some clinical traits (intellectual disability, documented to detect placental and somatic genomic developmental delay, hypotonia, joint hyperlaxity, intra- mosaicism. Since the application of this method is per- uterine growth restriction, and facial dysmorphy) seem formed on genomic DNA extracted directly from to be recurrent. S72 10th European Cytogenetics Conference 2015

Here, we report on the case of a 4-year-old girl Center Ljubljana Department of Pediatric referred for genetic counseling because of psychomotor Endocrinology, Diabetes and Metabolic Diseases, retardation, speech delay, heart defect (atrial septal University Children’s Hospital Ljubljana-Slovenia3 defect), and dysmorphic features (plagiocephaly, high forehead, short and upslanting palpebral fissures, Technical advances in genetic diagnostics have finally hypertelorism, dysplastic low-set ears, short nose enabled us to analyse the entire genome at once and with flat nasal bridge, long philtrum, high palate, identify disease-associated copy number variants micro-retrognathia). The pregnancy was complicated (CNV). Epilepsy and autism spectrum disorders by maternal infection of the upper airways in the first (ASD) are the two most prevalent subgroups of and third trimester and intrauterine fetal hypotrophy. In neurodevelopmental disorders. By twin and family the neonatal period, the patient presented with feeding studies it was shown that an important part of occurence problems and poor weight gain. At the clinical exami- of both disorders can be explained by genetic factors nation, hypotonia, joint laxity, long fingers and toes as and up to date, numerous genes and some well as abnormal position of the anus were observed. microdeletions/ microduplications have been linked to Ophthalmologic issues included post-inflammatory each of the 2 disorders. Structural variants play a signif- changes on the fundus of the right eye and strabismus icant role in 5–10 % of ASD or epilepsy cases. If there (surgically corrected). The girl showed poor social are additional clinical findings, these percentages are interactions and lack of eye contact. even higher. Using array comparative genomic hybrid- Whole-genome oligonucleotide microarray analysis ization (CGH) we analysed 170 children with epilepsy revealed a 4.93 Mb deletion of 2q24.1q24.3 region (n=80) and ASD (n=90). In our cohort, we found a (chr2:159,684,613–164,611,097; hg19) encompassing patogenic CNV in 5/90 (5.6 %) ASD cases and 12/80 19 OMIM genes; some of them (RBMS1, PSMD14, (15 %) epilepsy cases. Additional 11/90 (12.2 %) ASD TBR1, SLC4A10, DPP4 and KCNH7) could be strong cases and 4/80 (5 %) epilepsy cases had CNV classifed candidate genes for the genotype-phenotype relationship. in the group of variants of unknown significance. The In conclusion, this study contributes additional infor- diagnostic yield is greater in the group of patients where mation for the 2q24.2 microdeletion. Our results also epilepsy co-occur with additional clinical features, such confirm that haploinsufficiency of genes within the as developmental delay, congenital anomalies and deleted region might play a role in the etiology of unexplained epileptic encephalopathy and it has been this genomic disorder. Moreover, the clinical hetero- reported to be as high as 20–30 % (1,2). Indeed, in 10/12 geneity in the patients harbouring this chromosomal of our patients with reported pathogenic CNVs, epilepsy syndrome could be explained by incomplete pene- was accompanied by developmental delay. trance or variable expressivity. Interestingly, some copy number variations are risk This study was supported by the MNiSW Grant No. factors for both epilepsy and ASD. For example 0193/IP1/2013/72. microdeletions in 15q11.2, 15q13.3, and 16p13.11, described in epilepsy patients, also confer a risk for autism 1.P75 (and for other neurodevelopmental disorders, as well). We found all three CNVs in our epilepsy patients and none of Diagnostic utility of arrayCGH in epilepsy and them in ASD group. Our results add to growing evidence autism—Slovenian experience of expected discovery rate and important non-recurrent CNVs in both groups of patients. Luca Lovrecic1, Marija Volk1, Karin Writzl1, Gorazd Rudolf1, Natasa Teran1, Barbara Gnidovec Stražisar2, 1.P76 Sara Bertok3, Borut Peterlin1 University Medical Center Ljubljana Clinical Institute A new chromosomal rearrangement resulting in of Medical Genetics, Division of Obstetrics and Axenfeld-Reiger syndrome Gynaecology Ljubljana-Slovenia1 University Medical Center Ljubljana Department of Child, Adolescent & Laïla El Khattabi1, Ikhlas Ben Ayed1, Christine Ioos2, Developmental Neurology, University Children’s Laurence Perrin3,DominiqueLeTessier1,Aziza Hospital Ljubljana-Slovenia2 University Medical Lebbar1, Jean-Michel Dupont1 10th European Cytogenetics Conference 2015 S73

APHP, Hôpitaux universitaires Paris Centre, France. Introduction: 48, XXYY syndrome, which affects 1 Laboratoire de Cytogénétique, Paris-France1 Hôpitaux in 18,000 to 1 in 50,000 neonates, is a numerical chro- Universitaires Paris Ouest, France. Unité fonctionnelle mosomal abnormality associated with meiotic nondis- de Médecine Physique et Réadaptation de l’enfant, junction during maternal or paternal gametogenesis and Paris-France2 CHU ROBERT DEBRE, Paris, advanced age. This syndrome is characterized by facial France. Unité fonctionnelle de Génétique Clinique, dysmorphism, congenital malformations, mental defi- Paris-France3 ciency and severe behavioral and psychiatric disturbances. The increased stature of affected individuals Axenfeld -Rieger syndrome (ARS) is a developmental and the presence of hypogonadotropic hypogonadism disorder that associates eye abnormalities and multiple led to this condition being considered a variant of congenital malformations. An abnormal migration of Klinefelter syndrome, although both are now recog- neural crest cells results in dysgenesis of the anterior nized as distinct conditions. In this report, we describe segment of the eye. Other manifestations are mild dys- a case of 48,XXYY and the paternal origin of the extra morphic features, dental, cardiac and umbilical anoma- chromosomes. lies. ARS is inherited in an autosomal dominant manner Case report: First child of a young, non- with high penetranceof FOXC1 and PITX2 mutations or consanguineous couple. Maternal depression during deletions. However, over 50 % of cases remain of pregnancy. Post-term cesarean section with perinatal unknown cause. hypoxia. The child showed neurophyschomotor delay, Herein, we report an ARS associated with epi- learning deficiency, convulsions and autism spectrum lepsy in a two year old boy. Clinical examination disorders. Large stature, macrocrania, no eunuchoid found some dysmorphic features and growth delay. habitus, dysphemia. Testicles 10 mL (p50), Tanner stage Ophthalmologic examination revealed a bilateral III pubertal development. Karyotype 48,XXYY, with corectopia, irishypoplasia and a bilateral XYY of paternal origin. Normal hormone levels. No embryotoxon, consistent with ARS eye disorders. fragile X chromosome. Whole genome microarray analysis using Discussion: Aneuploidies of paternal origin are less Nimblegen 135 K array showed a 4.55 Mb de novo common and are usually associated with advanced age. deletion on 4q25q26 bands (nt 112,009,675 to Mental deficiency and behavioral disturbances, the most nt116,558,399 bp – hg18). The deleted region important characteristics seen here, are attributable to encompasses 9 OMIM genes and its distal bound- the 20 % of genes on the X chromosome that escape ary is located 230 Kb upstream of PITX2. In Silico inactivation and/or gene imprinting in maternal/paternal Search we found out the presence of regulatory chromosomes. Unlike Klinefelter syndrome, the sequences of PITX2 in this region. PCR analysis increased stature was not caused by primary confirmed the deletion of these sequences. This is hypogonadism. This case highlights the importance the first case of ARS resulting from a deletion of of investigating the paternal origin of extra chromo- PITX2 gene regulatory sequences. Further studies somes and the mechanism involved in aneuploidy in are carried out to assess the disruption of PITX2 order to establish a prognosis and provide appropriate expression genetic counseling.

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48,XXYY syndrome: case report Cytogenomics Array Database (CAGdb)—a useful tool for cytogeneticists Suely Santos1, Sonia Middleton1, Antonio Francisco Alves da Silva2,FábioMachado2,CintiaSantos- Jack Tarleton1, Pablo Sagaribay1 Rebouças3, Enrique Medina-Acosta2 Mission Health Fullerton Genetics Asheville-United UNIRIO Genetics and Molecular Biology Department States1 Rio de Janeiro-Brazil1 UENF Molecular Identification and Diagnostics Unit Campos-Brazil2 UERJ Genetics The Cytogenomics Array Group (CAG) began as an Department Rio de Janeiro-Brazil3 informal network of colleagues formed to exchange S74 10th European Cytogenetics Conference 2015 information between laboratories regarding constitutional diagnosis was reached in 55 patients: 23 patients diag- microarrays and rare case findings. In order to facilitate nosed as male pseudohermaphrodites, 2 as testicular this exchange, we created a web-accessible database feminization syndrome, 14 as complete gonadal dysgen- (CAGdb) to house each laboratory’s abnormal cases, esis, 8 as female pseudohermaphrodites, 6 as true her- which could then be freely shared in a de-identified maphrodites, 1 as Smith-Lemli-Opitz syndrome and 1 fashion with all participating laboratories. CNV details, Down syndrome, 15 patients were still undiagnosed by interpretation comments, parental and other follow-up the end of the study. data, and clinical features (all fully stripped of patient As conclusion, multidisciplinary approach remains identifiers)are collected and shared for each case in the best for management of such cases. Midwives this site. This database provides a useful laboratory should be well trained. Circumcisions of intersex tool to track experience with in-house microarray patients should be prohibited. Most of the patients cases, network with colleagues, and to share expe- were of low social class thus a non-profit body rience with rare findings. Additionally, this resource should shoulder this responsibility. is meant to facilitate submission to other larger- scale data collections in order to aid in syndrome 2. Tumor Cytogenetics discovery efforts and make information on rare cases more broadly available. 2.P1

1.P79 Improved chromosomal abnormality detection rates in patients with multiple myeloma by karyotyping, Cytogenetic and Molecular Studies of Sudanese fluorescence in situ hybridization and chromosome Patients with Disorders of Sexual Development microarray analysis using plasma cell enrichment

Samia Mahdi1, Imad Fadl-Elmula2 Alvin Soon Tiong Lim1, Tse Hui Lim1, Chuanfei Chen1, Taibah University Medical Laboratories Almadeena Lai Ching Lai1, Evan Rui Long Lin2, Joshua Wei Ming Almonawarah-Saudi Arabia1 Alwatania University Tay3, Xin Hui Ng3, Bryan Yi Wei Teo4, Yunxin Chen4, Pathology Khartoum-Sudan2 Sim Leng Tien1, Sathish Kumar4 Singapore General Hospital Cytogenetics Laboratory, Disorders of sexual differentiation occur in 1–2 of every Pathology Singapore-Singapore1 Nanyang 10,000 births; these present in several forms e.g. male Technological University School of Biological Sciences and female pseudohermaphroditism, gonadal dysgene- Singapore-Singapore2 Temasek Polytechnic School of sis, and true hermaphroditism. The present study aimed Applied Science Singapore-Singapore3 Singapore to explore and characterize cytogenetics, molecular General Hospital Haematology Singapore-Singapore4 alterations, hormonal profile, and clinical aspects of different types of intersex in Sudan. This study com- Aim: To ascertain the efficacy of utilising enriched prised 70 patients: 18 raised as males, 52 as females. plasma cells (PCs) for analysis of bone marrow (BM) Medical history, physical examination, culturing of samples of patients with morphologically-confirmed lymphocytes, chromosomal analysis, PCR, hormonal Multiple Myeloma (MM) profile, ultrasonography, laparoscopic examination Materials and methods: Sixty-three diagnostic BM when required, histopathology, and psychological assess- samples were analysed by FISH, 59 by karyotyping and ment were done. Most patients were above the age of 20 by CMA. The FISH assay employed a 9-probe panel 17 years (40 %) followed by patients younger than 2 years comprising FGFR3/IGH, CCND1/IGH, MAF/IGH, (33 %). The most affected Sudanese tribe was Jaaliyin RB1, TP53, 1q21, and centromeric probes for chromo- tribe (19 %). Half of the patients were referred for genital somes 9, 11 and 15. The CMA assay utilised a 4x180K ambiguity. Other complaints like primary amenorrhea, chip (Agilent SurePrint G3 Cancer CGH+SNP). PCs failure to thrive and short stature were reported. Thirteen were enriched by antibody complexes linked to targeted patients (19 %) were satisfied with their sex of rearing, 5 PCs to the magnetic particles (EasySep™/RoboSep™, patients (6 %) were not satisfied, 16 patients (23 %) could STEMCELL™) and used for FISH (enriched-FISH) not decide; 36 patients (51 %) were children. Final and CMA (enriched-CMA). 10th European Cytogenetics Conference 2015 S75

Results: By karyotyping, only 32 cases patient was taken to the operating room for excision of (54.2 %) were abnormal. With FISH performed the mass and neck dissection. on whole BM (standard-FISH), 53 cases (84.1 %) Histopathological examination revealed a small blue were abnormal. With enriched-FISH, 61 cases round cell neoplasm with multiple foci of perineural (96.8 %) were abnormal. Fourteen cases were analysed invasion involving the sympathetic chain, and metasta- by standard-CMA with 9 being abnormal (64.3 %). sis to two cervical lymph nodes at levels 2 and 3. The However, of the 12 enriched-CMA that were performed, tumor was strongly positive for S100 and focally posi- 100 % were abnormal. tive for synaptophysin and O13 (CD99). The chromo- Discussion: Karyotyping yielded a modest abnormal- some analysis of the resected tumor showed a t(19;22) ity detection rate owing to the indolent nature of PCs in (q13.4;q12.2) confirmed by fluorescence in situ hybrid- culture. The FISH assay is independent of cell division ization analysis using ZNF443 (19p13.13), CRX and consequently a much higher abnormality rate was (19q13.3) and the EWSR1 (22q12.2) locus specific obtained. However, enumeration of signals from whole unique sequence DNA probes. The patient was treated BM invariably includes the scoring of non-PCs. When with chemotherapy followed by autologous stem cell PCs were enriched for FISH, the abnormality rate was transplant. To our knowledge, this is the first extraskeletal further improved. The abnormality rate obtained by ES exhibiting this translocation. standard-CMA on whole BM was only 10 % better than Rare translocations have the potential to aid in karyotyping. This was due to the lower limit of assay the diagnosis and are valuable for correct tumor detection involving mosaicism <20 %. Indeed, when classification and therapeutic purposes. Although CMA was performed on enriched PCs, the abnormality t(11;22)(q24.1;q12.2) anomaly is the hallmark of detection rate reached 100 %. ES, the fusion product of EWS-FLI1 by itself does Conclusion: This study reaffirms the importance of not transform human cells and other mutations utilising enriched PCs for FISH and CMA assays in may appear to be necessary to understand the cell patients with MM. origin of ES. Further studies as well are needed to determine the importance of this chromosome band 2.P2 involvement in ES and other malignancies.

Extraskeletal Ewing sarcoma of the parapharyngeal 2.P3 space with a unique translocation, t(19;22) (q13.4;q12.2) Four genetic lymphoma-specific events (MYC, BCL2, BCL6 and CCND1) identified in a B high Gloria Ramos-Rivera1, Esther Adler1, K H Ramesh1, grade lymphoma case Bradley Schiff2, Mark Suhrland1, Samer Khader1 Montefiore Medical Center Pathology Bronx-United Antoine Ittel1, Catherine Helias1, Marie-Pierre Wissler2, States1 Montefiore Medical Center Otolaryngology, Elise Toussaint3,Marie-PierreChenard2,Laurent Head & Neck Surgery Bronx-United States2 Miguet4, Laurie Monier4, Laurent Mauvieux4, Carine Gervais1 Ewing sarcoma (ES) is a group of small round blue Hôpitaux Universitaires de Strasbourg Laboratoire de cell neoplasms comprising the second most frequent Cytogénétique Hématologique Strasbourg-France1 primary bone malignancy in children and young Hôpitaux Universitaires de Strasbourg Service adults. Around 15 % of ES is estimated to arise in d’anatomo-pathologie Strasbourg-France2 Hôpitaux extraosseous sites. Approximately 90 % of ES demon- Universitaires de Strasbourg Service d’hématologie et strate the translocation t(11;22)(q24.1;q12.2) creating a d’oncologie Strasbourg-France3 Hôpitaux Universitaires fusion gene between EWSR1 and FLI1. Other translo- de Strasbourg Laboratoire d’Hématologie Strasbourg- cations are rare and mainly described as fusions between France4 EWSR1 or EWSR1-related genes. We present a case of a rapidly enlarging mass of the The term « double/triple hit lymphoma » depicts rare left parapharyngeal space, located in the left carotid and aggressive lymphomas displaying either BCL2 and/ space, displacing the carotid bifurcation anteriorly. The or MYC and/or BCL6 gene rearrangements. To our S76 10th European Cytogenetics Conference 2015 knowledge, these rare lymphomas have been described Hematology and Oncology Bratislava-Slovakia2 only twice with four specific genetic events (MYC, Children’s Faculty Hospital Pediatric Oncology and BCL2, BCL6, CCND1). Here we describe the third Hematology Banská Bystrica-Slovakia3 Children’s observation. A 79 years old patient suffering from Faculty Hospital Pediatric Oncology Košice-Slovakia4 paraesthesias was admitted for polyneuritis in a context Faculty of Medicine, Comenius University Institute of of poor general condition. Clinical examination showed Pathological Anatomy Bratislava-Slovakia5 the presence of many scattered nodes and skin infiltration. A skin arm nodule biopsy revealed large prolifer- One of the most common solid tumors in young children ating cells (CD10-, CD20+, CD23-, BCL2+, BCL6+, is neuroblastoma. It is a typical example how important Ki67 80 %) consistent with a DLBCL, NOS. Blood cell role play genetic aberrations in determining the status, film examination showed the presence of pathological disease progression, in discovery of new treatment lymphocytes suggestive of a high grade lymphoma. The options and generally overall patient survival. Non- flow cytometry (CD5 dim, CD10-, CD19+, CD20+, random genetic changes associated with neuroblastoma FMC7+, CD22+, CD23-, CD148+) may be in favour are important prognostic factors and also they are an of pleomorphic mantle cell lymphoma (MCL) variant integral part of disease diagnosis. diagnosis. Blood cytogenetic study found a complex Since 2008 untill 2014, we have analyzed 80 samples hyperdiploid karyotype (47 chromosomes) with a of pediatric patients with age between 1 month to t(3;22)(q27;11) translocation (BCL6/IGL), structural 18 years (median=21 months) with diagnosis of abnormalities of a chromosome 8 resulting in juxtapo- ganglioneuroblastoma and neuroblastoma. Samples sition of 5’MYC and BCL2, a derivative chromosome were collected from three children’scancercenters 18 from a t(14;18)(q32;q21) translocation (IGH/BCL2), within Slovakia. The primary samples were native or and a t(11;14)(q13;q32) complex translocation (IGH/ formalin-fixed paraffin-embedded (FFPE) tumor tissues CCND1). Overexpression of cyclin D1 was detected and also the cultures of bone marrow. We proceed in the white blood cells and in the skin lesion. The according to International Neruroblastoma Risk Group patient was treated with a chemotherapy combining (INRG) Convention and we have focused our analysis rituximab, ifosfamide, cytosine arabinoside and intra- on the main independent prognostic factors— status of thecal methotrexate and died 4 months after the diagno- MYCN (2p24) oncogene, tumor cell ploidy and sis. This third case of quadruple hit lymphoma under- segmental chromosomal aberrations (SCA) as loss of lines the complexity of the classification of such aggres- 1p, 11q and gain of 17q. All samples were analyzed with sive malignancies, particularly DLBCL with expression I-FISH and they were compare with MLPA results. Our of cyclin D1, CCND1 chromosomal rearrangement and primary goal was to monitor the occurrence of above CD5 positivity. Some suggest the existence of a “gray mentioned selected genetic aberrations in correlation zone” in which morphologic, clinical and genetic features with age and available clinical data. are insufficient to segregate these lymphomas between This is the first major study of genetic markers of blastoid MCL and cyclin D1-positive DLBCL. neuroblastoma in Slovakia.

2.P4 2.P5

Molecular cytogenetic analysis of important prog- Bone marrow failure conditions due to chromosome nostic genetic markers among pediatric patients with anomalies neuroblastoma in Slovakia. Emanuela Maserati1, Roberto Valli1, Luciana Vinti2, Martin Cermák1, Katarína Hlinková1, Lívia Rotíková1, Annalisa Frattini3, Marco Fabbri1, Elena De Paoli1, Alena Zákovicová1, Imrich Hikkel1, Zofia Szabóová1, Giuseppe Montalbano1, Franco Locatelli2, Francesco Katarína Balúchová1,LuciaCopáková1,Andrej Pasquali1 Cižmár2,JozefSuvada2,PavelBician3,Viktória Università dell’Insubria Dipartimento di Medicina Halusková3, Ladislav Deák4, Pavel Babál5 Clinica e Sperimentale Varese-Italy1 IRCCS Ospedale National Cancer Institute Medical Genetics Bratislava- Pediatrico Bambino Gesù, Università di Pavia Slovakia1 Children’sUniversityHospitalPediatric Dipartimento di Ematologia e Oncologia Pediatrica 10th European Cytogenetics Conference 2015 S77

Roma, Pavia-Italy2 Università dell’Insubria, CNR G Papanikolaou Hospital Haemotology& HCT Unit Dipartimento di Medicina Clinica e Sperimentale, Thessaloniki-Greece1 Istituto di Ricerca Genetica e Biomedica Varese, Milano-Italy3 Cytogenetic aberrations have important prognostic implications in CLL. Abnormalities of chromo- Two children are reported with bone marrow (BM) some 8q24 are rare and their prognostic signifi- failure but without a precise diagnosis. Patient n. 1 is a cance remains unclear. In a series of 668 patients 3-year-old female with a severe anaemia associated with with CLL successfully karyotypes in our Department several dysmorphic/malformative symptoms. The stan- over the last 12 years, 8 (1.2 %) carried 8q24 aberrations. dard karyotype was normal, but array-CGH showed a These were 7 males and 1 female with a median age of 4 Mb duplication of chromosome 1 short arms of and a 68 years (range 38–80). Binet stage at diagnosis: A-4/B- 2Mbdeletionofchromosome8longarms.The 2/C-1 (unknown, 1). Seven cases had a typical CLL anomaly was constitutional. The lacking region of chro- immunophenotype, while the remaining case was mosome 8 included the locus RUNX1T1 which is considered as atypical. Six and 2 cases carried IGHV- relevant in transcription repression of several genes with unmutated and mutated genes, respectively. In all cases, aroleinmyelopoiesis.TheexpressionofRUNX1T1in conventional cytogenetic analysis was performed prior the BM was down-regulated. We postulate that the chro- to treatment; in 6/8 cases, this analysis was conducted at mosome imbalances are the cause of BM failure and diagnosis, while one case was studied at both diagnosis severe anaemia, besides of all extra-haematological signs. and disease progression. Six cases carried complex Patient n. 2 is a female of less than one year of age karyotypes including 8q aberrations; one case carried with severe pancytopenia, in absence of other symp- t(8;22)(q24.1;q11); the remaining case showed toms. The karyotype showed a constitutional change normal karyotype at diagnosis but developed an abnor- that had been already diagnosed prenatally, and that mal clone at progression showing a t(8;13)(q24;q14). was interpreted as a balanced translocation t(2;8). No Four cases had involvement of chromosome 17p aber- imbalances were disclosed by array-CGH. FISH with rations and two del(11q) as detected by classical cyto- libraries and a number of BAC probes showed that the genetic study and confirmed with FISH. Deletion 13q14 rearrangement was in fact an insertion of two adjacent was detected by FISH in 6/8 cases; 3/6 carried portions of chromosome 2 short arms into the long arms monoallelic deletion, 2/6 carried biallelic deletions and of chromosome 8. The RUNX1T1 gene was intact, but one carried monoallelic and biallelic deletions. included in the portion between the two insertions of Amplification of the c-myc oncogene was found in 3/4 material from chromosome 2. The expression of cases using a MYC break apart rearrangement probe; RUNX1T1 in BM was highly up-regulated, probably the remaining case showed one split signal because of by position effects, and this was interpreted as the cause translocation. After a median follow up 48 months of the pancytopenia. (range 17–154) six of seven patients with available data The two cases reported here, together with other progressed and two of them died. In summary, abnor- similar ones already reported by our group, demonstrate malities 8q24 are rare and associated with complex that a possible and not infrequent cause of BM failure is karyotype and poor prognosis. a chromosome anomaly, both balanced or unbalanced, either constitutional or acquired. 2.P7

2.P6 Prognostic significance of unsuccessful cytogenetics at the time of diagnosis in acute myeloid leukaemia Chronic lymphocytic leukemia with 8q24 aberrations: adistinctdiseasesubgroup? Maria Gaitatzi1,AnastasiaAthanasiadou1,MichalisIskas1, Chrysavgi Lalayanni1,GeorgePapaioannou1,Crysanthi Anastasia Athanasiadou1, George Papaioannou1, Niki Vadikoliou 1,IoannaSakellari1,ZografiaLazarou1,Olga Stavroyanni1, Aliki Tsompanakou1, Maria Gaitatzi1, Asteriou1,RiantSaloum1,AchilleasAnagnostopoulos1 Zografia Lazarou1,OlgaAsteriou1,Chrysavgi G.PapaniKolaou Hospital Haematology & HCT Unit Lalayanni1,RiantSaloum1,AchilleasAnagnostopoulos1 Thessaloniki-Greece1 S78 10th European Cytogenetics Conference 2015

Diagnostic karyotype is one of the most powerful inde- The most frequently observed cytogenetic abnormality pendent prognostic factors in acute myeloid leukemia is monosomy 7 or deletion of chromosome 7 in MDS. (AML). Conventional cytogenetic analysis is unsuc- But very few MDS cases with ring chromosome 7 have cessful in ~10 % of AML patients. The prognostic been reported. In this case report; a 30-year old woman significance of unsuccessful cytogenetics (UC) or unper- with the history of blood transfusion was referred to formed cytogenetics (UPC) on the outcome of AML hospital of Akdeniz University with complaining about patients remains unclear. Recently, two studies showed weakness. Peripheral blood count showed anemia and that UC predicts for poor outcome. Between 1998 and thrombocytopenia. A bone marrow (BM) biopsy was 2013, 393 patients below 74 years of age with AML performed to patient because of unclarified anemia and received intensive treatment. The frequencies of UC thrombocytopenia. The BM biopsy showed decrease in and UPC were 4,6 and 3 %, respectively. No significant granulocyte series and abnormal localization, was differences were noted in the baseline characteristics reported as normocellular BM with dysmegakaryopoiesis. between the patients with UC and complete cytogenetic BM myeloblast percentage was determined 14,3 % by analysis. The early death rates differed between the cyto- flow cytometry immunophenotyping. Chromosomal genetic subgroups (p<0.001) with the highest rates in analysis revealed 46,XX,ring(7)(p22q22)[7]/46,XX,-7,+ patients with UC (11 %) followed by high-risk (HR) mar(7)(p13p22?)[7]/46,XX[23]. FISH analysis results AML, intermediate risk (IR), favorable risk (FR) and indicated that marker chromosome was derived from UPC. However, the complete remission rate was the chromosome 7 with deletion of 7q22 and 7q31 55,55 % in UC and 66 % in UPC and was the same locus. The patient was diagnosed as MDS-RAEB-2 compared with the patients in the successful karyotyping (IPSS:3,highrisk). AML induction type-chemotherapy group (66,6 %, p=ns) and especially with IR AML was initiated (7+3, 100 mg/m2 ARA-C, 12 mg/m2 (68,5 %). The 5-year overall survival(OS) rates were Idarubicin). Minimal residual disease (MRD) was 27,8 % for UC and 33,3 % for UPC, whereas the determined as 5,3 % at BM sample on the 28th corresponding rates for FR, IR and HR AML patients were days after treatment. After therapy, cytogenetic 66,3, 25,9 and 6,2 %, respectively (p<0.001). The proba- analysis revealed 46,XX,del(7)(q22)[5]/46,XX,-7,+ bility 5-year OS was 85 % in UC patients who received mar(7)(p13p22?)[2]/46,XX,ring(7)(p22q22)[1]/ allogeneic hematopoietic cell transplantation(HCT) vs 46,XX[4] and confirmed by FISH. HIDAC treatment 0%inUCpatientswithoutHCT.Higherrelapserates regimen was initiated for re-induction therapy and MRD and shorter disease free survival (DFS) was observed at the was determined as 3,5 % in patient. The patient was than subgroups of UC and HR cytogenetics (p<0.001). In treated with 1 cycle of azacitidine for consolidation summary, the patients in UC subgroup have higher rates therapy. The decision of allogeneic hematopoietic stem of early death and shorter DFS, thus UC should be cell transplantation was received to patient from a HLA considered a high-risk feature. Intensive treatment such full compatible sibling donor and initiated a as allo-SCT could change the poor outcome. myeloablative therapy regimen. Cytogenetic analysis revealed 45,XX,-7[6]/46,XX[9] and monosomy 7 2.P8 was also detected in 31 % of interphase nuclei by FISH. The treatment process of patient is still Monosomy 7 and ring/marker chromosome derived continued at stem cell transplantation unit. To our from chromosome 7 in a patient with MDS-RAEB-2: knowledge,our patient is the fourth MDS patient A case report having ring7 in the literature.

Cigdem Aydin1, Orhan Kemal Yucel2, Ozan Salim2, 2.P9 Levent Undar2,BaharAkkaya3,SibelBerkerKarauzum4 Mehmet Akif Ersoy University Department of Nursing Identification of two novel fusion partners of the Burdur-Turkey1 Akdeniz University Department of MECOM gene in acute myeloid leukemia Hematology Antalya-Turkey2 Akdeniz University Department of Pathology Antalya-Turkey3 Akdeniz Tereza Jancuskova1, Radek Plachy1, Jiri Stika1, Lenka University Department of Medical Biology and Genetics Zejskova1,InkaPraulich2,Karl-AntonKreuzer2, Antalya-Turkey4 Nadezda Kosyakova3, Thomas Liehr3, Sona Pekova1 10th European Cytogenetics Conference 2015 S79 synlab genetics s.r.o. Laboratory synlab genetics 2.P10 s.r.o. Prague-Czech Republic1 University at Cologne Department I of Internal Medicine Cologne-Germany2 Targeted arrayCGH for determining the deleted Jena University Hospital Institute of Human Genetics region on chromosome arm 6q in chronic lympho- Jena-Germany3 cytic leukemia

In acute myeloid leukemia (AML) patients, the Marie Jarosova1,MartinaHruba2,EvaKriegova3, MECOM (MDS and EVI1 complex locus) gene, located Lenka Kruzova1, Alexandra Oltova4, Karla Plevova5, at chromosome band 3q26, can be rearranged with a Milena Holzerova1, Vit Prochazka1, Michael Doubek6, variety of partner chromosomes and partner genes. Here Vera Vozobulova7, Daniel Lysak7, Karel Indrak1, Tomas we report two rare cases of chromosomal translocations Papajik1 involving MECOM – t(3;10)(q26;q21) and Faculty of Medicine and Dentistry, Palacky University t(3;6)(q26;q25) – found in two patients with de novo and University Hospital Olomouc Hemato-oncology AML. Olomouc-Czech Republic1 Medical Faculty and Our aim was to identify the fusion partners on University Hospital Pilzen Intitut of Medical Genetics chromosomes 10q21 and 6q25 and to characterize Pilzen-Czech Republic2 University Hospital Olomouc the precise nucleotide sequence of the chromo- Institute of Immunology Olomouc-Czech Republic3 somal breakpoints, enabling us to design a University Hospital Brno Henatology and oncology clone-specific real-timePCRassayforsensitive Brno-Czech Republic4 University Hospital Brno minimal residual disease (MRD) monitoring in these Hematology and oncology Brno-Czech Republic5 patients. University Hospital Brno Hematology and omcology Karyotypes were investigated by molecular- Brno-Czech Republic6 University Hospital Pilzen cytogenetic techniques (mFISH/mBAND), and involve- Hemato-oncology Pilzen-Czech Republic7 ment of the MECOM gene was confirmed by FISH with the use of a commercially available probe set. Background: Cytogenetic abnormalities have improved The derivative chromosomes were isolated using the current chronic lymphocytic leukemia (CLL) risk fine-needle microdissection followed by whole stratification. 6q deletion is known to occur at a rela- genome amplification (WGA). Amplified chromo- tively low frequency in CLL (3–6 %) and its prognostic somal fragments were sequenced on the GS-Junior significance remains controversial, as is the extent of next-generation sequencing platform. The reads deletion and the candidate genes in the deleted region. obtained were aligned to reference sequences of the Aim: The objectives were to analyze a group of CLL respective chromosomes using in-house developed patients with chromosome 6q deletion using arrayCGH, software. The last mapped reads from both chro- determine the minimally deleted region (MDR) and the mosomes were used as docking sites for primers candidate genes located therein, attempt to determine for long-range PCR to amplify the putative their relative gene expression, and evaluate the clinical breakpoint. The long-range PCR products were characteristics. directly sequenced using Sanger sequencing to Methods: Peripheral blood/bone marrow samples reveal the precise nucleotide sequence of the from a group of 1147 CLL patients in three haematology breakpoint. centers in the Czech Republic (Olomouc, Plzen and Using a combination of cytogenetic and molec- Brno) were analyzed using conventional cytogenetics ular approaches, we mapped both translocations to and FISH. ArrayCGH with chromosome 6 specific the single nucleotide level, revealing a fusion of and oligonucleotide microarrays (Agilent, Santa the MECOM gene (3q26.2) and C10orf107 Clara, USA) and gene expression of FOXO3, NF- (10q21.2) in t(3;10) and LOC101928923 (6q25) kB, TBX21, IL-2 and BCL10 were assessed by in t(3;6). quantititative RT-PCR. The procedure described here enabled us to identify Results: 6q deletion was found in 87 (7.58 %) two previously unreported fusion partners of the patients, arrayCGH was applied in 69 patients. MDR MECOM gene as well as to perform real-time PCR- of 1.4 Mb was determined in the 6q21 region span- based monitoring of MRD. ning eight genes (FOXO3, SOBP, SCML4, SEC63, S80 10th European Cytogenetics Conference 2015

OSTM1, NR2E1, LACE1, and ARMC2). Expression encompassing DLEU gene, in 28 % of patients and profiling of FOXO3 revealed lower number of a-CGH showed in 42.8 % that the deletion comprised FOXO3 transcripts in CLL patients with 6q deletion also the RB1 gene, known to be associated to a worse than in those without 6q deletion (p=0.03) and prognosis. healthy subjects (p<0.0001). Clinical evaluation of The most recurrent anomalies identified only by the deletion showed that 6q deletion was more prevalent a-CGH with a proved or possible prognostic signifi- in males, the patients had unmutated IGVH and more cance included losses of 3p26.3-p25.3, 6q21, 9p21.3, advanced clinical stages (Binet B and C). 10q22.2-q26.3, 14q24.2-q32.33 and 15q14-q15.1. LOH Conclusion: Our study confirmed the low frequency was observed at 5q23.3 and 20q12-q13.2 both in 8 % of of 6q deletion, determined the MDR with low expres- the cases. sion of the FOXO3 gene and higher occurrence of 6q Based on our preliminary results, we would suggest deletion in patients with unfavorable clinical course. to perform a-CGH and conventional cytogenetics that This work was supported by grants: IGA MZ ČR NT can assure the identification of balanced rearrangements 13576 and IGA-LF-2015-001. with a diagnostic significance. Moreover, when a-CGH turn to be normal, FISH should be performed, at least for 2.P11 TP53 deletion associated to the worst prognosis, possibly present in a very low percentage of tumor cells. Comparison between cytogenetics, FISH and a-CGH in CLL evaluation 2.P12

Valentina Achille 1,AnnaVenci1,SaraGandolfi2, The ASXL1 gene alterations in patients with 20q Daniela Bettio1 deletion Humanitas Clinical and Research Center Cytogenetic and Medical Genetic Laboratory, Operative Unit of Jana Brezinova1, Iveta Sarova1, Halka Lhotska2, Sarka Clinical Investigations Rozzano, Milan-Italy1 Ransdorfova1, Silvia Izakova2, Karla Svobodova2, Jana Humanitas Clinical and Research Center Operative Markova3,ZuzanaZemanova2,JaroslavCermak4, Unit of Medical Oncology and Hematology Rozzano, Anna Jonasova5, Kyra Michalova2 Milan-Italy2 Institute of Hematology and Blood Transfusion Cytogenetic Department Prague-Czech Republic1 Chronic lymphocytic leukemia (CLL) is a clinically ULBLD, General University Hospital and 1. Faculty heterogeneous B-cell lymphoid neoplasm, where the of Medicine, Charles University Center of main clinical relevant genetic lesions consists in chro- Oncocytogenetics Prague-Czech Republic2 Institute of mosome gains and losses. Hematology and Blood Transfusion Department of The SurePrint G3 Cancer CGH+SNPs 4x180K plat- Molecular Genetics Prague-Czech Republic3 Institute form (Agilent Technologies) was used to study 25 CLL of Hematology and Blood Transfusion Clinical patients, in addition to conventional cytogenetics and Department Prague-Czech Republic4 General FISH with the FDA-approved panel including probes University Hospital and 1. Faculty of Medicine, specific for ATM and TP53 genes, D13S319(13q14)/ Charles University 1. Medical Department Prague- LSI13q34 loci and chromosome 12 centromere (Abbott). Czech Republic5 Genetic alterations were detected by cytogenetics, FISH and a-CGH in 72, 72 and 92 % of the cases, Deletion 20q is a recurrent abnormality observed in respectively. Conventional cytogenetics detected 2 myeloid disorders. In our previous study we showed a balanced translocations and 1 inversion, not revealed fusion of the ASXL1 and TSHZ2 genes resulting in an by a-CGH. An overall concordance of 92 % was isochromosome of a deleted 20q in a patient with observed between FISH and a-CGH results. In 2 cases myelodysplastic syndrome. The aim of this study was the D13S319 (13q14) deletion detected by FISH was not to determine the frequency of ASXL1 alterations in revealed by a-CGH probably due to the low percentage del(20q) cases. of deleted cells (<10 %). Two cases were normal with the Fluorescence in situ hybridizations (FISH) with locus three methods. FISH detected 13q14 deletion, specific probes for 20q11 and 20q12 regions (Abbott 10th European Cytogenetics Conference 2015 S81

Molecular, Kreatech Diagnostics) confirmed the cyto- The t(7;21)(p22;q22) rearrangement generating a 5′ genetically observed deletions of 20q in a cohort of 25 RUNX1-3′USP42 fusion transcript has been reported in patients with myeloid malignancies. Metaphase FISH two cases of pediatric acute myeloid leukemia AML) mapping with set of 7 bacterial artificial chromosome and further in eight adult cases of myeloid neoplasms. (BAC) probes (BlueGnome) distributed in 20q11.21 We describe the first case of adult AML with a 5′ and 20q13.2 was used for determination of the RUNX1-3′USP42 fusion gene generated by an insertion breakpoints. Array comparative genomic hybridization event instead of chromosomal translocation (CytoChip Cancer 180 K, BlueGnome) was performed Conventional and molecular cytogenetic analyses on DNA samples of bone marrow cells of 14 patients allowed the precise characterization of the chromosomal with suspected ASXL1 gene deletion to find out the rearrangement and breakpoints identification. Gene gene copy number variation. expression analysis was performed by quantitative AweaksignalofRP11-358N2BACprobe real-time PCR experiments, whereas bioinformatic (20q11.21) suggesting the proximal breakpoint of the studies were carried out for revealing structural genomic deletion in the ASXL1 gene was observed in 8 patients characteristics of breakpoint regions. (32 %). In 7 patients (28 %) the signal of this probe was We identified an adult AML case bearing a not present on the derivative chromosome confirming ins(21;7)(q22;p15p22) generating a 5′RUNX1-3′ the ASXL1 gene deletion. In the remaining 10 patients USP42 fusion gene on der(21) chromosome and causing the proximal breakpoint of the deletion was determined USP42 gene over-expression. Bioinformatic analysis of bellow the ASXL1 gene. The distal breakpoint in the the genomic regions involved in TSHZ2 gene was found in one patient only. ins(21;7)/t(7;21) showed the presence of interchro- In our study, ASXL1 gene was altered in 60 % of mosomal segmental duplications (SDs) next to the patients with 20q deletion. Mutations of the ASXL1 USP42 and RUNX1 genes, that may underlie a non- gene are associated with a poor clinical outcome and allelic homologous recombination between chromo- determination of the ASXL1 gene alteration in del(20q) some 7 and 21 in AML. cases may have a prognostic impact. The relations with We report the first case of a 5′RUNX1-3′USP42 clinical data should be studied in a larger cohort of chimeric gene generated by a chromosomal cryptic patients. insertion in an adult AML patient. Our data revealed Supported by MHCR project 00023736, RVO- that there may be a pivotal role for SDs in this very VFN64165, GACR P302/12/G157/1. rare but recurrent chromosomal rearrangement.

2.P13 2.P14

5′RUNX1-3′USP42 chimeric gene in acute myeloid ADAMTS2 gene dysregulation in T/myeloid mixed leukemia can occur through an insertion mechanism phenotype acute leukemia rather than translocation and may be mediated by genomic segmental duplications Giuseppina Tota1,NicolettaCoccaro1,Antonella Zagaria1,LuisaAnelli1, Paola Casieri1,Angelo Antonella Zagaria1, Luisa Anelli1, Nicoletta Coccaro1, Cellamare1,AngelaMinervini1,CrescenzioF Giuseppina Tota1, Paola Casieri1, Angelo Cellamare1, Minervini1, Claudia Brunetti1, Luciana Impera1, Paola Angela Minervini1, Crescenzio F Minervini1, Claudia Carluccio1,CosimoCumbo1,GiorginaSpecchia1, Brunetti1,CosimoCumbo1,GiorginaSpecchia1, Francesco Albano1 Francesco Albano1 University of Bari D.E.T.O. Bari-Italy1 University of Bari D.E.T.O. Bari-Italy1 Mixed phenotype acute leukemias (MPAL) include The runt-related transcription factor 1 (RUNX1) gene is acute leukemias with blasts that express antigens of a transcription factor that acts as a master regulator of more than one lineage, with no clear evidence of mye- hematopoiesis and represents one of the most frequent loid or lymphoid lineage differentiation. T/myeloid targets of chromosomal rearrangements in human (T/My) MPAL not otherwise specified (NOS) is a rare leukemia. leukemia that expresses both T and myeloid antigens, S82 10th European Cytogenetics Conference 2015 accounting for less than 1 % of all leukemias but 89 % of cells, highly relevant to global genome instability, their T/My MPAL. From a molecular point of view, very frequency is usually underestimated due to the limita- limited data are available on T/My MPAL NOS. tions of metaphase cytogenetic methods. In this report we describe a T/My MPAL NOS case The aim of this study was to revise karyotypes of 19 with a complex rearrangement involving chromosomes AML patients with dicentric chromosomes found by 5 and 14, resulting in overexpression of the ADAM conventional G-banding or mFISH using FISH with metallopeptidase with thrombospondin type 1 motif, 2 (multi)centromeric probes (Abbott Molecular, (ADAMTS2) gene due to its juxtaposition to the T cell Kreatech, Metasystems). FISH analyses confirmed 21 receptor delta (TRD) gene segment. out of 27 expected dicentrics and revealed ten others Detailed molecular cytogenetic characterization of which were not cytogenetically detected before. Most the complex rearrangement in the reported T/My frequently involved chromosomes were: 7, 17 (n=5), MPAL case allowed us to observe ADAMTS2 gene 13, 18, 19, 20 and 22 (n=4). In one case, FISH proved overexpression, identifying a molecular marker that only one centromere in suppossed dicentric chromo- may be useful for monitoring minimal residual disease. some, whilst the second centromere was excised and To our knowledge, this is the first evidence of gene observed as a small marker chromosome. dysregulation due to a chromosomal rearrangement in In conclusion, we demonstrate the necessity of T/My MPAL NOS. molecular cytogenetic studies to identify the presence and role of dicentric chromosomes and consequent 2.P15 monosomies in cancer. Neither classical cytogenetics nor mFISH is always sufficient to reveal the presence of Dicentric chromosomes in bone marrow cells of dicentrics, especially in cases when intercentromeric patients with acute myeloid leukemia distance is too small. The evaluation of dicentric chromosomes and monosomies at diagnosis and during the Iveta Sarova1, Jana Brezinova1, Zuzana Zemanova2, course of the disease contributes to the understanding of Sarka Ransdorfova1,SilviaIzakova2,Lenka unclear mechanisms involved in development and pro- Pavlistova2,KarlaSvobodova2,JaroslavCermak3, gression of leukemic transformation and will point out Anna Jonasova4, Magda Siskova4, Kyra Michalova2 to the prognostic significance of dicentric chromosomes Institute of Hematology and Blood Transfusion in AML. Cytogenetic department Prague-Czech Republic1 Supported by MHCR project 00023736, RVO- Institute of Medical Biochemistry and Laboratory VFN64165, GACR-P302/12/G157, PRVOUK-P27/ Diagnostics, General University Hospital and First LF1/1. Faculty of Medicine, Charles University Center of Oncocytogenetics Prague-Czech Republic2 Institute 2.P16 of Hematology and Blood Transfusion Clinical department Prague-Czech Republic3 General Looking for what is not there: An interesting case of University Hospital and 1st Faculty of Medicine, pediatric B-ALL solved by SNP array Charles University 1st Department of Internal Medicine Prague-Czech Republic4 Melanie Beaulieu Bergeron1, Jean McGowan-Jordan1 Children’s Hospital of Eastern Ontario and Department Dicentric chromosomes have been described in many of Pathology & Laboratory Medicine of University of hematological diseases including acute myeloid leuke- Ottawa Ottawa-Canada1 mia (AML). Two mechanisms of formation have been observed: telomere fusion and dicentric translocation. Acute lymphoblastic leukemia (ALL) represents nearly High instability of dicentrics during cell division is 25 % of all pediatric cancers, making it the most usually prevented by secondary chromosomal changes common malignancy. Several cytogenetic abnormalities leading to an inactivation of one of the two centromeres. are well characterized in in pediatric precursor B-cell Missegregation of dicentrics can produce monosomies ALL (pre-B ALL) and have a known prognostic value, which are predictors of poor prognosis in AML. such as high hyperdiploidy (51–65 chromosomes) with Although dicentric chromosomes are known in cancer double trisomy 4 and 10 which is very frequent and of 10th European Cytogenetics Conference 2015 S83 favourable prognosis. On the contrary, the presence of Cytogenetic Nomenclature (ISCN). Chromosome near-haploidy or hypodiploidy in pre-B ALL is rare and analyses in patients with MDS declosed structural and is associated with a very poor prognosis. Unfortunately, numerical abnormalities in 58 (28.3 %) of samples. the presence of a hyperdiploid or near-triploid clone 62.1 % of patients with MDS have only one chromo- resulting from endoreduplication of the near-haploid or somal anomaly, in 15.5 % two chromosomal anomalies hypodiploid clone may mask the latter, causing the pre- was identified, and 22.4 % have the complex karyotype. B ALL to be misclassified as being high hyperdiploid. Trisomy of chromosome 8 was detected in 25.9 %, We present here the case of a 7 year-old male diagnosed deletion of chromosome 5 in 52.2 %, chromosome 7 with pre-B ALL in whom standard chromosome analy- deletion in 25.9 % and chromosome 20 deletion in sis showed the following karyotype: 52,XY,+X,+Y,+ 8.6 %. Cytogenetic analyses revealed in addition to 14,+?14,+21,+21,inc[4]/52,XY,+X,+Y,+14,+21,+21,+ frequently reported chromosomal anomalies novel and ?mar,inc[4]/46,XY[3]. Interphase FISH analysis did not rare chromosomal abnormalities. The results of our detect any of the most common structural rearrange- study proves that cytogenetics is still a valuable tool in ments in pediatric pre-B ALL, nor did it detect the MDS genetic testing, providing important clues for presence of a near-haploid clone. SNP array analysis diagnosis, treatment and clinical managment of on an EDTA blood sample confirmed the observed patients. Detailed detection of cytogenetic changes gains of chromosomes X, Y, 14 and 21 but also demon- may brings insights into molecular mechanisms strated a marked skewing of the alleles on the other underlying myelodysplastic syndromes. chromosomes. These results were consistent with dupli- The work was supported by the project KEGA cation of a single homologue of all chromosomes except 041PU-4/2014. X, Y, 14 and 21 in the abnormal leukemic cells due to endoreduplication of a masked near-haploid clone. We 2.P18 have now changed our procedure so that DNA is extracted from an EDTA blood sample from all new Primary and recurrent diffuse astrocytomas: com- pediatric leukemic patients in order to allow further pared genomic profiles revealed acquisition of aber- analysis by SNP array if required. rations with relevant biological potential.

2.P17 Halka Lhotska1, Zuzana Zemanova1, Hana Cechova2, Sarka Ransdorfova3,LibuseLizcova4,Karla Analysis of cytogenetic characteristics in Slovak Svobodova1, Filip Kramar5, Lenka Pavlistova1, Silvia patients with myelodysplastic syndrome Izakova1, Zdenek Krejcik2, Dagmar Bystricka1, Kyra Michalova1 Iveta Boronova1,JarmilaBernasovska1,Gladys General University Hospital and 1st Faculty of Medicine, Cakanova2, Marta Mydlarova Blascakova1 Charles University Center of Oncocytogenetics, Institute University of Prešov Department of Biology Prešov- of Medical Biochemistry and Laboratory Diagnostics Slovakia1 SEMBID Clinical genetics Prešov-Slovakia2 Prague-Czech Republic1 Institute of Hematology and Blood Transfusion Department of Molecular Genetics Myelodysplastic syndromes (MDS) constitute a hetero- Prague-Czech Republic2 Institute of Hematology and geneous group of clonal disorders characterised by dys- Blood Transfusion Department of Cytogenetics Prague- plasia and innefective haematopoiesis in one or more of Czech Republic3 General University Hospital and First the major myeloid cell lines. MDS is a neoplastic Faculty of Medicine, Charles University Center of disease of bone marrow characterized by non-random Oncocytogenetics, Institute of Medical Biochemistry chromosomal abnormalities. The aim of the study and Laboratory Diagnostics Prague-Czech Republic4 was to analyze the results of cytogenetic examination Central Military Hospital and 1st Faculty of Medicine, of 205 patients with MDS in the Prešov region Charles University Department of Neurosurgery Prague- (1990–2014). Cytogenetic analysis were performed Czech Republic5 from cultured cells of bone marrow without stimulation using GTG banding method and karyotyped Diffuse astrocytomas represent heterogeneous group of according to the International System for Human glial tumours with highly variable biological behaviour. S84 10th European Cytogenetics Conference 2015

Despite radical surgical resection and multidrug chemo- CHU Timone, AP-HM Département de Génétique therapy, recurrent lesions appear after asymptomatic Médicale Marseille-France1 Institut Paoli-Calmettes period in majority of patients and additional intervention Laboratoire d’Oncogénétique Moléculaire Marseille- is necessary. The possibility that tumour may develop France2 CHU Timone, AP-HM Service d’Hématologie novel aberration with relevant biological properties is et d’Oncologie Pédiatrique Marseille-France3 often neglected. In this study we represent two cases of diffuse astrocytomas where additional cytogenetic Glucocorticoid therapy plays a fundamental role in the and epigenetic markers with potential influence on management of lymphoid malignancies, especially in cell proliferation or differentiation were detected at the acute lymphoblastic leukemia. Indeed, glucocorti- relapse. coids induce apoptosis in lymphoid progenitor cells. The biopsies taken from primary and recurrent Several genes play an important role in glucocorticoid- tumours of two patients with astrocytoma were analysed induced response and in particular the NR3C1 gene using I-FISH with VYSIS locus specific and centromeric which codes for human glucocorticoid receptor. probes (Abbott), SNP array (HumanCyto SNP-12 Bead NR3C1 gene deletion is associated to primary glucocor- Chip, Illumina), and MLPA (P370-A1 kit, MRC- ticoid resistance and therefore could represent a poor Holland) to assess copy number variations and the prognosis marker in childhood acute lymphoblastic IDH1 mutations status. Methylation of MGMT and leukemia. MLH3 promoters was investigated by methylation- Here we report an 8-years-old boy with relapsed specific MLPA (ME011 kit). ETV6/RUNX1-positive ALL 3 years after his diag- Both cases were characterised by features specific nosis. At relapse, we also identified by FISH the for diffuse astrocytomas: R132H mutation of IDH1 (12;21)(p13;q22) translocation, and the R banded gene in both, UPD of 17p and/or deletion of karyotype highlighted additional clonal abnormalities CDKN2A gene in one of them. These markers were such as an interstitial heterozygous deletion of the present in primary and recurrent lesions. The addi- long arm of the chromosome 9 and a trisomy 21. tional aberrations, mainly deletions or amplifications The high resolution array-CGH analysis detected of chromosomal segments (such as deletion of RB1 additional copy number variations that were cryptic gene or trisomy 7), acquired UPD and hypermethylation on karyotype : of MGMT and/or MLH3 promoters were detected in the - the NR3C1 heterozygous deletion on the long arm recurrent lesions. of chromosome 5 : del(5)(q31.3q31.3) The IDH1 mutation was the primary event as previ- - the partial trisomy of long arm of chromosome X : ously reported. According to our observation the meth- dup(X)(q26q28) ylation of promoters is the latter event which may along At relapse, as at the diagnosis, assessment of with additional aberrations further disrupt cellular pro- corticosensibility to J7 of prephase of chemotherapy liferation and/or differentiation. Larger cohort of showed corticoresistance. patients with molecularly analysed recurrent lesions is The presence of NR3C1 deletion in leukemic cells necessary for global view of tumour biological behav- clone could explain the persistent corticoresistance iour and may help to define new therapy targets. and suggests that the relapse could be due to persis- Supported by IGA MZ CR NT/13212-4, RVO- tence of an ancestral clone with NR3C1 deletion. VFN64165, PRVOUK-P27/LF1/1. Additional studies are presently being performed in order to identify the presence of ancestral clone on 2.P19 diagnosis. In conclusion, the high resolution array-CGH Highlighting of NR3C1 deletion by array-CGH in analysis may have an impact on the management relapsed childhood ETV6-RUNX1 positive acute of ALL. Indeed, array-CGH allows to highlight the lymphoblastic leukemia presence of copy number abnormalities of genes with a role in resistance to treatment (eg NR3C1 John Boudjarane1, Estelle Balducci1, Emilie Alazard1, in 5q31.3, NR3C2 in 4q31.22, BMF in 15q15.1…) Marina Lafage1, Cornel Popovici2, Gérard Michel3, and might help to guide future personalized Hélène Zattara1 therapies. 10th European Cytogenetics Conference 2015 S85

2.P20 of disease in these patients occur earlier than in patients without aberrations associated with high grade gliomas. Molecular genetic changes in gliomas and their impact This findings are on the border of statistical significance on progression in patients with low grade gliomas (p=0,0519), possibly due to the small number of patients with low grade gliomas in the study group. Irena Urbanovska1,SylvaPitronova2,Jana Dvorackova3, Radim Lipina4, Oldrich Motyka5 2.P21 CGB Laboratory Inc. Laboratory of molecular pathology and genetics Ostrava -Czech Republic1 CGB Acquired uniparental disomy of 17p is associated Laboratory Inc. Laboratory of cytogenetics Ostrava - with mutations of TP53 gene in patients with MDS Czech Republic2 Faculty Hospital Ostrava Institute of and complex karyotypes. pathology Ostrava-Czech Republic3 Faculty Hospital Ostrava Clinic of Neurosurgery Ostrava-Czech Karla Svobodova1,ZuzanaZemanova1,Halka Republic4 VSB- Technical University of Ostrava Lhotska2, Monika Belickova3, Jana Brezinova4, Iveta Nanotechnology Centre Ostrava -Czech Republic5 Sarova4,SilviaIzakova2,LibuseLizcova2,Sarka Ransdorfova4,AdelaBerkova2,MagdaSiskova5, Diffuse gliomas are the most common primary central Anna Jonasova5, Jaroslav Cermak6, Kyra Michalova2 nervous system malignancies. Classification of these General University Hospital and 1st Faculty of Medicine tumors is based on WHO used traditional methods, of Charles University Center of Oncocytogenetics, including cellular morphology, presence of mitoses, Institute of Medical Biochemistry and Laboratory vascular proliferation and necrosis. Using these, tumors Diagnostics Prague 2-Czech Republic1 General are classified in astrocytic, oligodendroglial or University Hospital and 1st Faculty of Medicine of oligoastrocytic categories and specified by a tumor Charles University Center of Oncocytogenetics, grade. Despite the progress in diagnosis and treatment, Institute of Medical Biochemistry and Laboratory gliomas are almost incurable since radical resection is Diagnostic Prague-Czech Republic2 Institute of not possible due to their diffuse nature. It is expected Hematology and Blood Transfusion Department of that the therapy would be based— among others— on the Molecular Genetics Prague-Czech Republic3 knowledge of genetic changes in the individual types of Institute of Hematology and Blood Transfusion tumor (chromosomal abnormalities, activating muta- Department of Cytogenetics Prague-Czech tions of oncogenes and mutations associated with loss Republic4 General University Hospital and 1st of tumorsuppressor gene function). Faculty of Medicine of Charles University 1st In the period of 2007–2012, we examined 127 native Medical Department Prague-Czech Republic5 Institute astrocytic tumor samples and 20 oligodendroglial or of Hematology and Blood Transfusion Clinal oligoastrocytoma tumor samples. In astrocytic tumors, Department Prague-Czech Republic6 FISH analyses of EGFR and MDM2 oncogene statuses, CDKN2A, PTEN, TP53, RB1 tumorsuppressor gene Cytogenetic aberrations are the most important prognos- statuses were performed. In oligodendroglial or tic factors in patients with myelodysplastic syndromes oligoastrocytic tumors, status of 1p/19q codeletion and (MDS). The complex karyotypes are associated with the status of tumorsuppressor genes CDKN2A and PTEN poorest prognosis. However, the pathogenic mecha- were determined by FISH. nisms leading to origin of complex aberrations at diag- In 30 low grade astrocytoma samples, 13 samples nosis of MDS are poorly understood. Segmental were found with chromosomal aberrations associated acquired uniparental disomy (aUPD), occurring as with high grade gliomas (deletion of tumorsuppressor aresultofmitoticrecombination,mightconfer genes CDKN2A, PTEN, monosomy of chromosome increased genomic instability and play a funda- 10, amplification of EGFR gene). In 20 tumors within mental role in tumorigenesis. The aim was to oligodendrogliomas and oligoastrocytomas, 6 tumors evaluate the frequency of aUPD of 17p in bone with histopathological gr. II showed changes associated marrow (BM) cells of newly diagnosed MDS patients with gr. III (deletion of tumorsuppressor genes with complex karyotypes and to assess its correlation CDKN2A, PTEN). It seems that progression or relapse with mutations of TP53 gene (17p13.1). S86 10th European Cytogenetics Conference 2015

Genomic DNA isolated from diagnostic BM samples with nearly 20 identified partner genes. Here we of 68 MDS patients with complex karyotypes (≥3 aber- report a 14-year-old boy with newly diagnosed rations) previously proved by G-banding and/or mFISH AML-M0. He presented with asthenia probably due was analyzed by aCGH/SNP (BlueGnome). Next- to a very low hemoglobin level (4.2 g/dL). Blood generation sequencing on the GS Junior system smears revealed 80 % immature cells and the bone (Roche) was used to detect TP53 mutations. marrow examination showed 95 % blasts positive for The overall incidence of aUPD17p was 13/68 and its CD13, CD34, CD117, CD7 and CD56. Conventional average size was 8–20 Mb always encompassing TP53 cytogenetics identified an abnormal clone with 5q gene. Homozygous mutations of TP53 were detected in deletion. EGR1 (5q31) and CSF1R (5q33-q34) dele- all aUPD17p positive patients (nine missense mutations tions in chromosome 5 were confirmed using FISH in DNA-binding core domain, two frameshift mutations, probes. Systematic interphase FISH using LSI one nonsense mutation, and one substitution in intron RUNX1/RUNX1T1 showed three signals for RUNX1. 4). The most common chromosomal imbalances were FISH studies on metaphases showed the extra RUNX1 loss of 5q (11/13), 3p (8/13), 7q (6/13), monosomy 7 signal on the short arm of chromosome 4. Further char- (2/13), and 12p deletion involving ETV6 gene (8/13). acterization using the subtelomeric 4p probe showed a We confirmed that aUPD17p is a frequent nonran- cryptic t(4;21)(p16;q22) translocation. Our case appears dom finding in MDS with complex karyotypes and is to share similarities with acute myeloid leukemia cases strongly associated with homozygous mutations of with cryptic t(7;21)(p22;q22) recurrent translocation TP53 gene. A heterozygous TP53 mutation and follow- including aberrant CD56 and CD7 lymphoid antigen ing recombination event give rise to homozygosity of expression, loss of 5q material as an additional abnor- mutated gene. This phenomenon may be one of trigger mality, absence of hyperleukocytosis and severe macro- mechanisms of clonal evolution and cause rapid disease cytic anemia. The macrocytic anemia may be related to progression in high risk MDS. 5q deletion and might be associated with the Supported by RVO-VFN64165, GACR P302/12/ haploinsufficiency of the ribosomal genes on the long G157, PRVOUK-P27/LF1/1. arm of chromosome 5, such as RPS14, as described in 5q- myelodysplastic syndromes. To the best of our 2.P22 knowledge, this was the first report of a novel cryptic t(4;21)(p16;q22) translocation involving Identification of a novel cryptic t(4;21)(p16;q22) RUNX1 gene associated with 5q abnormality in a associated with 5q abnormalities in a pediatric childhood AML. Additional studies are being per- case of acute myeloid leukemia formed in order to identify the RUNX1 partner gene implicated in this translocation. Balducci Estelle1, Boudjarane John1, Alazard Emilie1, Loosveld Marie2, Nivaggioni Vanessa2, Marceau Alice3, 2.P23 Oudin Claire4,MichelGérard4,Lafage-Pochitaloff Marina1, Zattara Hélène1 IGH abnormalities detected by I-FISH in selective Laboratoire de cytogénétique onco-hématologique CD138+ plasma cells in multiple myeloma Hôpital de la Timone Marseille-France1 Laboratoire d’hématologie Hôpital de la Timone Marseille- Nadia Voskoboinik1, Svetlana Trestman2, Hilla Kolog1, France2 Laboratoire d’hématologie C.H. Régional Yulia Frug 1,IritAvivi2, Avi Orr-Urtreger1,Rachel Universitaire de Lille Lille-France3 Service Rothman1 d’hématologie et oncologie pédiatrique Hôpital de la TASMC Genetic Institute Tel Aviv-Israel1 TASMC Timone Marseille-France4 Department of Hematology Tel Aviv-Israel2

The runt-related transcription factor 1 (RUNX1) gene, Multiple Myeloma (MM) is a malignant accumu- located in chromosome 21q22, is a transcription lation of monoclonal plasma cells (PC) with low factor that acts as a regulator of hematopoiesis and proliferation activity in the bone marrow (BM). represents one of the most frequent genes involved Conventional cytogenetic analysis of MM has been in chromosomal translocations in human leukemias hampered not only by a low level of bone marrow 10th European Cytogenetics Conference 2015 S87 infiltration with PC and by a low mitotic index of plasma cells in the bone marrow. The genome of MM PC in vitro, but also by the fact that many trans- clonal plasma cells is characterized by genetic changes of locations affecting the IGH locus in 14q32 region prognostic importance. Among the high-risk abnormali- are cytogenetically cryptic in this disease and not visible ties include hypodiploidy with immunoglobulin heavy- in light microscopy. chain (IgH) translocations, including the genes CCND3, MM exhibits a wide variety of numerical and dif- MAF, MAFB and FGFR3/MMSET. Disease progression ferent structural IGH changes involving 14q32 region is accompanied by a number of secondary chromosome translocations with numerous partner chromosomes, aberrations, the most common being MYC rearrange- which might be used for clinical classification. ments, del(13q), del(17p), and the deletion of 1p and/or The main method to detect the chromosomal abnor- amplification of 1q. The aim of our study was to evaluate malities in MM is Interphase Fluorescent In situ and summarize the frequency and types of aberrations on Hybridization (I-FISH) on PC. Enrichment of PC by chromosome 8, the relation to the disease course, and CD138 positive selection prior to I-FISH is one way to finally, to compare the findings with the clinical charac- enhance diagnostic sensitivity. teristics. The analysis was performed on 62 patients with The aim of this study was to characterize the rear- MM at the time of diagnosis and at relapse/progression. rangements of chromosome 14 in group of 109 patients We used FICTION and FISH methods with specific with MM. The specific probe for chromosome 14q32.33 probes for detecting c-MYC, TRAIL-R1, -R2 genes (IGHC/IGHV) was used to detect chromosomal aberra- aberrations. Chromosome 8 aberrations were found in tions. Forty six of the patients (43 %) had at least one 53 % of the patients. At relapse/progression, we detected type of abnormality and the most frequent was IGH c-MYC gene rearrangements in 14 patients, numerical rearrangement (24 %). changes in c-MYC gene in 23 patients and deletion of In this group of patients with 14q32 rearrange- region 8p21.3 in 12 patients. Up to ~73 % of these ment additional probes were further applied to aberrations were detected also at the time of diagnosis. detect t(4;14)(p16.3;q32.3) (LSI FGFR3/IGH), Clinical evaluation of our cohort revealed that the t(11;14)(q13;q32.3) (LSI CCND1/IGH), patients with chromosome 8 aberrations were in t(14;16)(q32.3;q23) (LSI IGH/MAF) or advanced clinical stage, with predominance of t(14;20)(q32.3;q12)(LSI IGH/MAFB). monoclonal IgG. The aberrations of chromosome Interestingly, 21 of the patients (19.2 %) showed only 8weredetectednotonlyatrelapse/progressionbut one signal of IGH, and the effect of this loss on the also at the time of diagnosis, with worsening clinical clinical phenotype is currently being studied. course of the disease. Their frequency and prognostic Our data illustrate that I-FISH together with CD138+ significance demonstrate the importance of their inclu- PC enrichment improve the cytogenetic diagnosis in sion in routine FISH examinations. MM and provide additional tool for MM analysis. This work is supported by the grants: IGA MZ ČR NT 14400 and IGA-LF-2015-001. 2.P24 2.P25 Molecular cytogenetic analysis of selected chromosome 8 regions in patients with multiple myeloma May HER2 amplification be a molecular marker for and its comparison at the time of diagnosis and a subgoup of familial papillary thyroid cancer? following relapse/progression Tinuccia Dettori1,PaolaCaria1,RobertaVanni1, Miroslava Vincova1,MichaelaMikesova1,Lenka Daniela Virginia Frau1, Silvia Cantara2, Furio Pacini2 Kruzova1, Milena Holzerova1, Pavla Mickova1, Tomas University of Cagliari Biomedical sciences Monserrato- Pika1, Vlastimil Scudla1, Marie Jarosova1 Italy1 University of Siena Internal Medicine Siena-Italy2 University Hospital Olomouc Department of Hemato- Oncology Olomouc-Czech Republic1 Introduction and aim. The majority of papillary thyroid carcinoma and follicular thyroid carcinoma are sporadic Multiple myeloma (MM) is a hematologic malignancy (sPTC and sFTC), although familial tumors may characterized by accumulation of terminally differentiated account for approximately 9 % of thyroid carcinoma S88 10th European Cytogenetics Conference 2015 cases. Whether familial papillary thyroid carcinoma Jumping translocations (JT) are a rare cytogenetic (fPTC) has a more severe phenotype than sPTC is phenomenon, the word jumping referring to the translo- still debated, and no specific gene(s) involved in cation of the same segment of the donor chromosome on fPTC has been identified so far. Recently, various to various recipient chromosomes, creating multiple types of novel molecular alterations have been reported related clones in an individual. It has been described as in sPTC, such as ALK and ETV6 genes rearrangements, a constitutional abnormality, in solid neoplasms and and HER2 amplification (HER2amp) in association rarely in various hematological malignancies including with telomere shortening (Pérot et al., 2014; Ricarte- acute myeloid leukemia (AML), acute lymphoblastic Filho et al., 2013; Sugishita et al., 2013). BRAF inver- leukemia, myelofibrosis, chronic myeloid leukemia, sion was already reported in sPTC (Ciampi et al., 2005). lymphomas, and multiple myelomas [McGrattan, On the other hand, our group reported short telomeres 2010; Manola, 2008]. In these patients JT occurred as and chromosome instability in fPTC (Cantara et al., a secondary event leading to progression of disease 2010). These observations prompted us to investigate either as a secondary AML evolving from a previously the frequency of the novel molecular alterations in sPTC existing myeloproliferative neoplasm and and fPTC. myelodysplastic syndrome or a relapse of a disease Results. Interphase FISH retrospective study was previously in remission. JT have commonly been carried out on nuclei isolated from formalin fixed par- described in elderly adults. affin embedded (FFPE) sections. 14 sPTC and 17 fPTC We report a rare case of JT occurring in a 13-year-old (some of which were previously studied by our group girl with RUNX1/RUNX1T1-positive and very high for telomeres length) were examined using home-brew expression WT1+ AML implicating its probable role probe (HER2) and commercially available probes in disease progression. We revealed JTs in bone marrow (BRAF, ETV6 and ALK). We found that HER2amp cells examined at the time of first chemoresistant relapse was higher in fPTC compared to sPTC (p=0.01). No of AML. The findings of conventional cytogenetics rearrangement of the other tested genes was observed. were confirmed by fluorescence in situ hybridization Histologic sections of cases positive for HER2amp were (FISH) with locus specific probes. For precise descrip- then evaluated: HER2amp was mainly present in tion of chromosomal parts involved in JTs, multicolor scattered cells. FISH were performed (MetaSystems). Cytogenetic Conclusions. Our preliminary findings indicate a analysis revealed JTs with chromosomal segments higher HER2amp frequency in fPTC and pinpoint 17q21-17qter translocated to the pericentromeric the need of studying additional cases to verify the regions of chromosomes 13,14,15 and to regions potential of HER2amp in molecularly characterize 1p36 and 2q37. The patient has been performed asubgroupoffPTC.Ifconfirmed,ourfinding allogeneic SCT from haploidentical donor, but child open considering trastuzumab therapy effectiveness died after progression of leukemia. in refractory thyroid carcinoma positive for Her2amp. JTs form a heterogeneous group of rare events Fund: PRIN N.2012Z3F7HE. involving probably random chromosomal segments, they reflect the general chromosomal instability of 2.P26 malignant cells. However, precise analysis of chromosomal breakpoints involved in JTs on a larger series of Jumping translocations in a 13-year-old child patients could contribute to explain their mechanism and with RUNX1/RUNX1T1-positive acute myeloid connection with pathogenesis together with the determi- leukemia nation of clinical significance of these aberrations.

Tatiana Gindina1,NikolayMamaev1,Elena 2.P27 Nikolaeva1, Irina Petrova1, Olga Uspenskaya1, Maria Averyanova1, Sergey Bondarenko1, Boris Afanasyev1 Cytogenetic analysis in myeloid sarcoma Pavlov First Saint-Petersburg State Medical University R.M. Gorbacheva Memorial Institute of Children Arpad Szomor1,ZsuzsannaSzijarto2,Vanessza Oncology, Hematology and Transplantation Saint- Emmert1,RenataCsalodi1,SzabolcsKosztolanyi1, Petersburg-Russia1 Judit Pammer1, Agnes Nagy1, Bela Kajtar3 10th European Cytogenetics Conference 2015 S89

University Pecs First Department of Medicine Tikva-Israel3 Rabin Medical Center, Beilinson Hospital Pecs-Hungary1 University Pecs Department of Hematological Laboratoriesl Petach Tikva-Israel4 Ophthalmology Pecs-Hungary2 University Pecs Institute Sackler School of Medicine, Tel Aviv University of Pathology Pecs-Hungary3 Institute of Hematology, Davidoff Center, Beilinson Hospital, Rabin Medical Center Petach Tikva-Israel5 Myeloid sarcoma (granulocytic sarcoma, chloroma) is Acute promyelocytic leukemia (APL) is a curable an extramedullary mass composed of cells of the mye- form of leukemia characterized by typical morphology, loid lineage. Myeloid sarcoma is reported in 3–8 % of high prevalence of severe bleeding events and distinct patients with acute myeloid leukemia and occurs con- chromosomal aberrations, most commonly t(15;17). comitantly, following, or, rarely, antedating the onset of However, approximately 9 % of APL patients harbor systemic bone marrow leukemia. Potentially it can other translocations involving chromosome 17, such as affect every organ, more often it is found in the t(11;17)(q23;q21), t(5;17)(q35;q12-21), „sanctuary sites”, which are difficult to target with t(11;17)(q13;q21) and der(17). All-trans retinoic acid chemotherapy. Myeloid sarcoma has been described (ATRA) and arsenic trioxide (ATO) have a specific in association with a variety of chromosomal aberra- targeted activity against the PML-RARA fusion protein. tions, complexity of the cytogenetic findings indi- ATRA and ATO have recently been shown to be non- cates poor prognosis. Most common translocation in inferior to chemotherapy and ATRA as induction myeloid sarcoma is t(8;21) and core binding factor therapy for non-high risk APL. The clinical significance involvement is also frequent. During the last 20 years of non t(15:17) APL-related aberrations is controversial, 240 patients with acute myeloid leukemia were treated with conflicting reports regarding sensitivity to modern, in the First Department of Medicine, University Pecs, targeted therapy. Isochromosome 17q (iso(17q)) is a rare Hungary. Nineteen of them (7,9 %) had myeloid cause of APL, usually associated with t(15:17). There is sarcoma. We report on cytogenetic data of 15 patients, no published data regarding the efficacy ATO based where cytogenetic analysis were available. When bone therapy for APL patients with iso(17q). We report a marrow involvement was present, cytogenetic examina- patient with APL with iso(17q) as the sole aberration, tion was performed on bone marrow aspirates. Where without overt t(15;17) translocation but with a cryptic adequate samples were available, conventional PML-RARA transcript, who was treated with ATRA karyotyping, as well as fluorescent in situ hibridization and ATO after failure of chemotherapy and achieved was performed on cells of extramedullary involvement. complete remission. To the best of our knowledge this Most common aberrations were +8; and complex is the first published report of isolated iso(17q) with abnormality (3-3 cases). We also detected translocation cryptic PML-RARA transcript as a cause of APL and t(9;11), t(8;21) and t(8;22). the first reported iso(17q)-related APL successfully treated with an ATO containing regiment. 2.P28 2.P29 Acute promyelocytic leukemia with isochromosome 17q and cryptic PML-RARA successfully treated Potential of aminolevulinic acid/photodynamic with all-trans retinoic acid and arsenic trioxide therapy to induce DNA damage on MCF-7 and HepG2 cell lines Daniel Shepshelovich1, Alina Klein1, Nino Oniashvili2, Josepha Yeshaya2,AdinaAviram3,DorisParnes4, Mona Abo-Zeid1, Amira Gamal-Eldeen2, Mahmoud Esther Rabizadeh3, Pia Raanani5 Abd-Elaziz2, Shady Mostafa2 Genetic Engineering and Biotechnology Division, Sackler School of Medicine, Tel Aviv University National Research Centre Genetics and Cytology Medicine A, Rabin Medical Center, Beilinson Hospital Department Giza-Egypt1 National Research Centre Petach Tikva-Israel1 Raphael Recanati Genetic Institute, Cancer Biology Laboratory Giza-Egypt2 Rabin Medical Center, Beilinson Hospital Cytogenetic Laboratory Petach Tikva-Israel2 Rabin Medical Center, Background: Photodynamic therapy (PDT) is a clinically Beilinson Hospital Hematological Laboratories Petach approved and promising therapeutic procedure involving S90 10th European Cytogenetics Conference 2015 the administration and irradiation of a photosensitizer of acute lymphoblastic leukemia (ALL) with trisomy such as 5-aminolevulinicacid (ALA) to produce singlet 5 as primary chromosomal abnormality. A 32-year-old oxygen. The aim of the present work is to evaluate male was presented in September 2013 with leukocyto- cytotoxic and genotoxic effects induced by ALA/PDT sis, anemia and thrombocytopenia. Smear of peripheral on adenocarcinoma breast cancer (MCF-7) and hepato- blood revealed 81 % blasts and bone marrow aspirate cellular carcinoma (HepG2) cell lines. was hypercellular with 91 % blasts. The case was clas- Methods: ALA (0.5 and 1 mM) was applied to MCF- sified as common ALL according immunophenotype 7 or HepG2 cell lines for 4 h before exposure to laser markers Tdt+,CD19+,CD10+,CD34+. The cytogenetic irradiation (633 nm; 0.25 W) for 4 min and then incu- study showed:47,XY,+5[15] / 48,sl,+13[2]/47,XY,+ bated for 24 h. Cytotoxicity was evaluated with trypan del(5)(q13q31)[2]/46,XY[1] karyotype. The patient blue. The potential to induce DNA damage was recorded attained complete hematological remission after by alkaline comet assay and micronucleus test. one course of induction therapy with CVAD Results: ALA/PDT reduced cell viability in a con- (Cyclophosphamide, Vincristine, Adriamycin and centration dependent manner. ALA/PDT IC50 reached Dexamethazone). Minimal residual disease analysis to 1,74 mM in MCF-7 cells and reached to 5.09 mM in of the bone marrow by flow cytometry showed HepG2 cells. Also, ALA/ PDT induced DNA damage 10 % lymphoblasts. He received consolidation and micronuclei in MCF-7 and HepG2 cell lines and treatment (4 cycle) and MRD was after the first percentages of DNA damage with comet tail and the cycle 1,5 % and after the second cycle 0,1 %. In proportion of tail length were increased highly signifi- March 2014, he received allogeneic hematopoietic cell cantly in comparison to non-treated cells. transplantation from HLA-mishmatced unrelating donor Conclusion: ALA/PDT induced cytotoxic and in CR with positive MRD. Nowadays the patient is alive genotoxic effects on MCF-7 and HepG2 cells. ALA/ in complete remission and maintains complete donor PDT is effective in cancer treatments as it targets DNA chimerism. Despite hematological remission at the end directly leading to induction of DNA damage as evaluated of induction chemotherapy, our patient had evidence of with Comet assay and micronucleus test. Further experi- minimal residual disease and this may have adverse mental studies are needed to investigate mode of cell prognostic significance. A review of this rare abnor- death by ALA/PDT which enables direct DNA damage. mality confirm our findings that trisomy 5 could be associated with a poor prognosis. These data is 2.P30 probably recommend to support the indication of intensive therapies such as allo-SCT in first remission. Trisomy 5 as a sole cytogenetic abnormality in acute lymphoblastic leukemia 2.P31

George Papaioannou1, Maria Gaitatzi1, Ionnis Batsis1, Evaluation of TEL/AML1 fusion and additional Chrysanthi Vadikoliou1,OlgaAsteriou1,Zografia abnormalities involving TEL and/or AML1 genes Lazarou1,AchilleasAnagnostopoulos1,Anastasia using FISH technique in patients with childhood Athanasiadou1 acute lymphoblastic leukemia G.Papanikolaou Hospital Haematology & HCT Unit Thessaloniki-Greece1 Esra Manguoglu1, Çigdem Aydın2, Zafer Cetin3, Funda Tayfun4, Ozden Altiok Clark5, Alphan Küpesiz4, Bahar Numerical chromosomal aberrations are commonly Akkaya6, Sibel Berker Karauzum7 seen in acute lymphoblastic leukemia with specific Akdeniz University, Fakulty of Medicine Department gains of chromosomes (hyperdiploidy), which are asso- of Medical Biology and Genetics Antalya-Turkey1 ciated with an excellent prognosis. Trisomy 5 as sole Mehmet Akif Ersoy University, Bucak School of or primary chromosomal abnormality is very rarely Health Department of Nursing Burdur-Turkey2 detected. A review of the literature shows only 15 Sanko University, Faculty of Medicine Department of patients and 11 of them were children. Some studies Medical Biology Gaziantep-Turkey3 Akdeniz suggest that this rare cytogenetic abnormality may be University, Faculty of Medicine Department of Pediatric associated with a poor outcome. We present a case Hematology Antalya-Turkey4 Akdeniz University, 10th European Cytogenetics Conference 2015 S91

Faculty of Medicine Department of Medical Genetics The aim of this study was to evaluate MDM2 gene Antalya-Turkey5 Akdeniz University, Faculty of copy number alterations in chronic lymphocytic Medicine Department of Pathology Antalya-Turkey6 leukaemia(CLL) patients with fluorescent in situ Akdeniz University, Faculty of Medicine Department of hybridization(FISH) technique by using murine double Medical Biology and Genetics Antalya-Turkey7 minute 2(MDM2) gene-specific probe sets, and also to evaluate the possible relationship between this amplifi- Childhood Acute Lymphoblastic Leukemia (ALL) is cation and the disease course, indication for treatment a malignant disorder of lymphoid progenitor cells and drug resistance. with a peak incidence among 2–5 years of age and Copy number alterations of the MDM2 gene located is the most common type of childhood leukemia. on chromosome 12 were investigated by FISH using The t(12;21)(p13;q22) translocation occurs in 25 % metaphase chromosomes obtained from bone marrow of childhood B-cell precursor ALL. In this study, bone aspiration samples for routine chromosome analysis marrow samples were obtained from 165 patients with in 40 CLL and 20 chronic myeloid leukemia(CML) childhood ALL. We analyzed t(12;21) translocation and patients followed-up at Akdeniz University other related abnormalities by fluorescent in situ hybrid- Department of Haematology. Additionally, routine ization technique using ETV6(TEL)/ RUNX1(AML1) FISH and conventional cytogenetics results of the ES dual color translocation probe and conventional patients were evaluated. cytogenetics analyses were performed. TEL and MDM2 gene amplification was not observed in the AML1 related chromosomal abnormalities were deter- patient cohort (CLL) or in control patients (CML). mined in 42 (25.5 %) of 165 patients with childhood Trisomy 12 FISH signal pattern was observed in 12 ALL. Structural changes were detected in 33 (78.6 %) out of 40 CLL patients (30 %). Furthermore, chromo- and numerical abnormalities in 9 (21.4 %) of a total of somal alterations detected by routine FISH analysis in 42 abnormal karyotypes. Frequencies of FISH abnormal- 40 CLL patients were as follows; 17p13 deletion in 16 ities in pediatric ALL cases were as follows; 8,5 % for cases (40 %), 13q14 deletion in 13 patients (32.5 %), t(12;21)(p13;q22) TEL/AML1 fusion, 6.0 % for AML1 11q22.3 deletion in 6 patients (15 %), and 6q23 deletion amplification, 3.0 % for tetrasomy/trisomy 21, 1.8 % for in 1 case (2.5 %). TEL deletion, 1.21 % for TEL deletion with AML1 Evaluation of MDM2 mRNA expression levels in amplification, 1.21 % for TEL amplification with AML1 CLL patients might be conducted due to absence of amplification, 0.6 % for poliploidy, 0.6 % for AML1 MDM2 gene copy number alterations. Additionally, deletion, 0.6 % for diminished TEL signal. The most evaluation of SNP309 G/G single nucleotide polymor- frequent structural abnormality was t(12;21) translocation phism in advanced stage CLL patients without MDM2 followed by AML1 amplification and TEL deletion while gene amplification might provide important clues for most frequent numerical abnormality was trisomy 21. progression disease. 27 out of the 40 CLL patients (67.5 %) were classi- 2.P32 fied as having early stage disease. We suggest that in the case of progression to advanced stage CLL during the Evaluation of MDM2 gene copy number alterations follow-up period,re-evaluation of MDM2 gene copy in chronic lymphocytic leukaemia patients with fluo- number alterations will be more useful to explain the rescent in situ hybridization relationship between copy number alterations of MDM2 and CLL. Sibel Berker Karauzum1, Sule Darbas1, Cigdem Aydin2, Orhan Kemal Yucel3, Bahar Akkaya4, Ozan Salim3 2.P33 Akdeniz University, Faculty of Medicine Department of Medical Biology and Genetics Antalya-Turkey1 Mehmet The process of malignant transformation in a child Akif Ersoy University, Bucak School of Health Department with acute myeloid leukemia. of Nursing Antalya-Turkey2 Akdeniz University, Faculty of Medicine Department of Hematology Antalya-Turkey3 Monika Lejman1, Dorota Winnicka1, Mariusz Babicz1, Akdeniz University, Faculty of Medicine Department of Borys Styka1 ,IlonaJaszczuk2 ,Katarzyna Pathology Antalya-Turkey4 Wojciechowska2, Marta Holweg1, Jerzy R. Kowalczyk2 S92 10th European Cytogenetics Conference 2015

Department of Pediatric Hematology, Oncology and The treatment was carried out without compli- Transplantology, Cytogenetic Laboratory,Children’s cations. Currently the patient is 4 months after University Hospital Department of Pediatric transplantation in the. Hematology, Oncology and Transplantology, Cytogenetic Laboratory,Children’s University Hospital 2.P34 Lublin-Poland1 Department of Pediatric Hematology, Oncology and Transplantology, Children’s University Application of fluorescence in situ hybridization to Hospital Department of Pediatric Hematology, detect MYCN amplification on paraffin-embedded Oncology and Transplantology, Children’s University tissue sections of neuroblastoma Hospital Lublin-Poland2 Dorota Winnicka1, Monika Lejman2, Mariusz Babicz2, On the basis of genetic changes found in cytogenetic Borys Styka2 ,IlonaJaszczuk3 ,Katarzyna and molecular studies and depending on the response to Wojciechowska3, Marta Holweg2, Jerzy R. Kowalczyk3 treatment, the stratification of patients with acute mye- Department of Pediatric Haematology, Oncology and loid leukemia to therapeutic groups of risk is performed. Transplantology, Cytogenetic Laboratory, Children’s The study reports data of a 5 years old boy with acute University Hospital Department of Pediatric myeloid leukemia (AML) type M5. Cytogenetic exam- Haematology, Oncology and Transplantology, inations of bone marrow with the use of classical Cytogenetic Laboratory, Children’sUniversityHospital method G-banding revealed translocation among three Lublin-Poland1 Department of Pediatric Hematology, chromosomes: 4, 9 and 11. Oncology and Transplantology, Cytogenetic The FISH (fluorescent in situ hybridization) study Laboratory, Children’sUniversityHospitalDepartment was carried out on metaphase slides and interphase of Pediatric Hematology, Oncology and Transplantology, nuclei. The MLL gene rearrangement was detected with Cytogenetic Laboratory,Children’s University Hospital use of the LSI MLL Dual Color, Break Apart Probe Lublin-Poland2 Department of Pediatric Hematology, (Vysis). The existence of the fusion gene (RUNXT1/ Oncology and Transplantology, Children’s University RUNX1) was excluded. Hospital Department of Pediatric Hematology, Further investigations used whole chromosome Oncology and Transplantology, Children’s University painting FISH on 4, 9 and LSI MLL Dual Color, Hospital Lublin-Poland3 Break Apart Probe. To improve genetic diagnosis we applied high resolution comparative genomic hybridiza- Neuroblastoma is a malignant tumor in children aged tions (HR-CGH). 0–15 years. Hybridization method detected deletion of part of p It accounts for 7–10 % of all childhood tumors and arm of chromosome 9-ish cgh dim(9)(p13p21). The study 30–50 % of all infants tumor. Research of the molecular was confirmed by FISH study results with using of LSI biology and cytogenetic are a necessary tool to evaluate p16 probe. No changes were detected in a test for the the advanced stage of the disease and besides the presence of an internal tandem duplication of the FLT3 patient’s age, stage of disease, tumor histology, loca- gene . tion of primary tumor and specific biochemical param- The above studies can determine the direction in eters, are the very important prognostic indicators, which malignant transformation proceeded. The origi- classifying patients to a specific therapeutic group. nal rearrangement had been the translocation (4;11) The most common cytogenetics changes including than appeared the secondary translocation (4;9) and deletions of chromosome 1p, gain of chromosome arm the translocation (9;11) with 9p21 deletion. The fol- 17q, amplification of the oncogene MYCN, ploidy lowing karyotype was received: 46,XY,der(4)t(4;11) changes in tumor cells. (q21;q23),der(9)t(4;9)(q21;p13)der(11)t(9;11) The aim of this study was create a method to (p22;q23). evaluation MYCN amplification on paraffin- embed- Due to complex irregularities of karyotype and renewal ded tissue sections of neuroblastoma patients. Paraffin of blasts, it was decided about hematopoietic cell trans- embedded tissue sections were previously digested plantation after the first remission. Due to lack of related respectively and prepared to apply a molecular probe, donor, it was decided to take cells from unrelated one. using fluorescent in situ hybridization (FISH) technique. 10th European Cytogenetics Conference 2015 S93

The direct fluorochrome- labeled, dual color DNA Translocation of BCL6 gene was observed in 9/49 Probe for the MYCN gene locus (2p24) and α-satellite (18 %) patients with the translocations’ partner from DNA identifying the centromeric region of chromosome chromosomes 2 (IgK gene), 10, 12 and 14 (IgH gene). 2 (Vysis LSI N-MYC (2p24) SpectrumGreen/CEP 2 A total of 14/54 (26 %) patients had copy number SpectrumOrange Probe) was used to estimate MYCN changes at MYC gene, while none of the patients had amplification. MYC rearrangement. Deletion of BLIMP1 was part of Amplification MYCN is detected in 20–30 % neuro- the complex karyotype in 8/46 (17 %) patients. We blastoma patients. It is the strongest marker of poor performed detailed analysis of 24 patients with complex prognosis. It is strongly correlated with advanced stage karyotype and confirmed that chromosomes 1, 3, 6, 9, of disease, rapid tumor progression, resistant to chemo- 10, 14 and 18 were frequently affected by transloca- therapy and poor outcome independent of patient age tions. By using arrayCGH, we examined 38 patients and and stage of advanced disease. found recurrent gains at 1q, 6p, 7, 13q and losses at 6p, The test material were the paraffin embedded tissue 6q and 17p. A comparison of the histological prognostic sections with fresh remove tumors, as well as archival groups with genetic changes shows that GCB-DLBCL sections 32 patients with an established clinical diagno- is associated with rearrangement of chromosome 14, 18, sis of neuroblastoma. and BCL2/IgH positivity. In the group of nonGCB- Positive results of MYCN amplification were DLBCL, the frequently rearranged chromosomes were obtained in 9 patients paraffin-embedded tissue 3, 6, 7, 9, 10 and X. We confirmed that DLBCL is, sections, which represents an 28.125 % of all tested genetically, a heterogeneous disease with number of patients in the analysis. recurrent cytogenetic aberrations that were, in our cohort, detected in 43 (72.8 %) patients. 2.P35 This work was supported by the grant IGA-LF- 2015-001. Molecular cytogenetic analysis of recurrent chromosomal aberrations in diffuse large B-cell lymphoma 2.P36 (DLBCL). FISH and arrayCGH analysis of chromosome Michaela Mikesova1,MilenaHolzerova1,Lenka 13q deletions in patients with chronic lymphocytic Kruzova1,VitProchazka1,TomasPapajik1,Patrik leukemia (CLL) Flodr2, Zuzana Prouzova2, Marie Jarosova1 University Hospital Hemato-oncology clinic Olomouc- Lenka Kruzova1,MilenaHolzerova1,Michaela Czech Republic1 University Hospital Department of Mikesova1, Miroslava Vincova1, Renata Urbanova1, Clinical and Molecular Pathology Olomouc-Czech Tomas Papajik1, Marie Jarosova1 Republic2 University Hospital Hemato-oncology Olomouc- Czech Republic1 DLBCL is the most common non-Hodgkin lymphoma in adults and clinically and genetically it is a heteroge- Cytogenetic abnormalities have confirmed prognostic neous disease. DLBCLs are associated with number of importance in CLL. The most frequent aberration is genetic aberrations. However, the prognostic signifi- deletion of 13q14 detected in ~50 % of patients by cance of some of the recurrent changes, such as BCL2, fluorescence in situ hybridization (FISH). As a single BCL6 or MYC gene rearrangement and deletion of 6p aberration, 13q14 deletion is associated with or 17p13, has been demonstrated. favourable prognosis. However, the prognostic value In our study, we analysed the frequency of BCL6 and of other changes such as 13q14 biallelic deletion, MYC genes rearrangement and BLIMP1 gene deletion monosomy 13 or chromosome 13 translocation remains by fluorescent in situ hybridization (FISH). Multicolor controversial. FISH (mFISH) and array comparative genome hybridi- The aim of our study was to perform arrayCGH zation (arrayCGH) were used to detect recurrent bal- analysis in a group of patients with 13q14 deletion to anced and unbalanced changes in the karyotypes. This determine the extent of the deletion and the common analysis was performed on 59 patients. deleted region (CDR). Additional objectives included S94 10th European Cytogenetics Conference 2015 comparison of the findings in patients with single aber- of Molecular Biology Novy Jicin-Czech Republic3 ration with those with 13q14 deletion and other changes Hospital Novy Jicin Department of Hematology Novy including complex aberrations, and also compare them Jicin-Czech Republic4 with certain clinical parameters. We used FISH with a panel of CLL specific probes Chromosomal abnormalities are important prognostic (Abbott, Illinois, USA; Metasystems, Althusheim, factors in chronic lymphocytic leukemia (CLL). Clonal Germany) and arrayCGH with oligonucleotide micro- evolution (CE), defined as acquisition of new cytoge- array (Agilent, Santa Clara, USA) to perform the netic aberration, may occur in 17–26 % of CLL patients analysis in total of 55 CLL patients with 13q14 and is associated with shorter overall survival. The aim deletion (20 pts with single aberration, 10 patients of our study was to prospectively assess CE frequency with an additional aberration and 25 patients with by FISH and conventional chromosome banding (CBA) complex changes). after stimulation with CpG oligonucleotides and inter- ArrayCGH determined the CDR of 1.67 Mb at leukin-2. The role of other prognostic factors and ther- 13q14.2-13q14.3, containing MIR15a, MIR16-1, apy in the development of poor-prognosis CE (new DLEU1, DLEU2, and DLEU7. We observed that the deletion 17p, new deletion 11q or new complex karyo- large 13q deletion (Type I— including RB1) and the type) was also evaluated. higher percentage of altered nuclei are frequently asso- 140 patients (86 men, 54 women, median age ciated with unmutated IgVH status and complex 64 years) with previously untreated CLL were evaluated changes. by CBA and FISH (11q, 13q, 17p deletions, trisomy 12, Our analysis of 55 CLL patients confirmed signifi- 14q32 rearrangement). Peripheral blood samples of each cant heterogeneity of the extent of the deleted region on patient were provided for baseline and follow-up testing. 13q and the determined CDR. Small number of patients Mutation status of IGVH gene and expression of CD38 in the study has not allowed the determination of clinical and ZAP70 were also analysed. significance of deletions of 13q yet. CE was detected in 15,7 % (22/140) of patients using This work was supported by the grants: IGA MZ ČR FISH, in 28,6 % (40/140) using CBA and in 34,3 % NT 13576 and IGA-LF-2015-001. (48/140) of patients by combining both methods. The median time to CE occurrence was 16 (range 2.P37 6–52) month. Poor-prognosis CE was detected in 15 % (21/140) of patients and was significantly Clonal evolution in chronic lymphocytic leukemia: associated with previous CLL treatment (p=0,013). Combination of FISH and conventional cytogenetics In 10/21 cases CE was shifted into a poor-prognosis after stimulation with CpG oligonucleotides and group according to the hierarchical FISH model, interleukin-2 while in the remaining 11 patients, poor risk CE was presented by complex karyotype without evidence of Lucie Truhlikova1,MartinaStoklasova1,Alena 17p deletion. Burdova1,BronislavaDrevojankova1, Tamara CBA after stimulation with CpG oligonucleotides Sablaturova1,AnetaStrnkova1,YvonaBrychtova2, and interleukin-2 provides more complex information Anna Panovska2,KristinaStepanovska2,Gabriela about cytogenetic abnormalities in CLL than FISH, Vankova 2,KarlaPlevova2,AlexandraOltova2, suggesting that many patients acquire new abnormali- Katerina Horka3,SarkaPospisilova2,JiriMayer2, ties during the course of their disease, especially after Michael Doubek2, Martin Brejcha4 treatment.

AGEL Research and Training Institute Laboratory of 2.P38 Medical Genetics— Department of Cytogenetics Novy Jicin-Czech Republic1 University Hospital, Brno and Cytogenetics in multiple myeloma: Abnormalities of Faculty of Medicine, Masaryk University Department Chromosome 1 of Internal Medicine— Hematology and Oncology Brno-Czech Republic2 AGEL Research and Training Martina Stoklasova1, Lucie Truhlikova1, Bronislava Institute Laboratory of Medical Genetics— Department Drevojankova1,AnetaStrnkova1,AlenaBurdova1, 10th European Cytogenetics Conference 2015 S95

Tamara Sablaturova1,HanaDvorakova1,Cecilia 2.P39 Bodzasova2,JurajDuras2,HanaPlonkova2,Jana Zuchnicka2, Irena Zygulova3, Martin Brejcha4, Marek Cytogenetic and FISH studies of B-cell lymphomas Wrobel4 AGEL Research and Training Institute— Novy Jicin Radka Tomasova1,GabrielaPavlikova1,Ivana Branch, AGEL Laboratories Laboratory of Medical Hojsikova1 Genetics— Department of Cytogenetics Novy Medirex, a.s. Genetics Bratislava-Slovakia1 Jicin-Czech Republic1 University Hospital Ostrava Institute of Clinical Hematology Ostrava-Czech Double-hit B-cell lymphomas (DHL) are rare subtype of Republic2 Silesian Hospital in Opava Department lymphomas usually associated with highly aggressive of Hematology Opava-Czech Republic3 Hospital clinical behavior and poor prognosis. This heterogenous Novy Jicin Department of Hematology Novy Jicin- category is characterized by spectrum of morphological, Czech Republic4 phenotypic, genetic and pathological features overlapping with Burkitt lymphoma (BL), diffuse large B-cell Background: Multiple myeloma (MM) is a B-cell lymphoma (DLBCL), B-lymphoblastic lymphoma/ malignancy of the plasma cells characterized by leukemia (B-LBL) and acute lymphoblastic leukemia/ complex cytogenetic aberrations. Chromosome 1 lymphoma (ALL). DHL are genetically defined by abnormalities are found in almost half of MM obligatory MYC (8q24) rearrangement in combination cases and are associated with aggressive disease. The with another genes, involved in development of lym- most common structural changes that involve chromo- phomas, such as BCL2 (18q21), BCL6 (3q27), CCND1 some 1 are 1p deletion and 1q gain. (11q13) or BCL3 (19q13). The mechanism of proto- Patients and Methods: Between 2009 and 2014, oncogenes activation includes translocation or amplifi- 27 MM patients (6 women, 21 men, median age 68) cation, usually a part of complex karyotypes. Here we showed chromosomal abnormalities by conventional report interesting de novo cases of patients with DHL in chromosome banding (CCB). Bone marrow term of classical and molecular cytogenetics. Details of aspirates were processed using short time cultivation cytogenetic and FISH analysis will be presented. (24–48 h) without stimulation. A minimum of IGH rearrangement and 13q deletion was performed in all 2.P40 cases by cytoplasmic immunoglobin light chain staining (cIg FISH). Chromosome 1 abnormalities were Evaluation of Danusertib effects on cell viability and confirmed by CCB, cIg-FISH or FISH and multicolor cytomorphological parameters in cancer stem cell FISH. lines from glioblastoma multiforme Results: Chromosome 1 aberrations were detected in 15/27 patients. Deletions were identified in 11/15 Chiara Cilibrasi1, Andrèe Guzzi2, Serena Redaelli2, patients: 1p deletions as a result of unbalanced trans- Leda Dalprà3, Angela Bentivegna2 location (8/11), 1p intersticial or terminal deletions University of Milano-Bicocca Department of Surgery (2/11) and 1q deletion (1/11). Gains were caused by and Transaltional Medicine; PhD Program in unbalanced translocation in 10/15 patients: 1q12-qter Neuroscience Monza-Italy1 University of Milano- (5/10), 1q21-qter (3/10), 1q22-qter (1/10) and 1p21- Bicocca Department of Surgery and Translational qter (1/10). Trisomy 1 was presented in 1/15 patient. Medicine Monza-Italy2 University of Milano- Deletion of 1p as well as gain of 1q were were Bicocca; Medical Genetics Laboratory, San Gerardo presented in 7/15 karyotypes. Hospital Department of Surgery and Translational Summary: The 1p was preferentialy involved in Medicine Monza-Italy3 deletions that affect regions 1p11 and 1p13. The 1q gains were mostly in regions 1q12-qter and Glioblastoma multiforme (GBM) is a grade IV astrocy- 1q21-qter. The 1q12–q23 region cover a large number toma and the least successfully treated solid tumor: of possible candidate genes. Our results support the current therapies provide a median survival of 12–15 importance of chromosome1abnormalitiesinthe months after diagnosis, due to the high recurrence rate. patogenesis in MM. Glioma stem cells (GSCs) are believed to be the real S96 10th European Cytogenetics Conference 2015 driving force of tumor initiation, progression and AIM: To investigate the chromosomal abnormalities relapse. Better therapeutic strategies GSC-targeted are present in CML patients at diagnosis and during follow needed. up and to evaluate their long— term response. A number of potential molecular targets for the iden- MATERIALS AND METHODS: 47 newly diag- tification of novel anticancer drugs interferes with the nosed CML patients (median age 56 years (range 26 to cell cycle. Danusertib is a small molecule with strong 89 years) with a male:female ratio 1.14 : 1) treated in our activity against Aurora kinases, a protein kinases family Hospital between 2001 and 2014 were monitored. overexpressed in a variety of human cancers and corre- Conventional cytogenetics and/or Fluorescent in situ lated, also in GBM, with chromosomal instability, tumor Hybridization (FISH) were performed both at diagnosis aggressiveness and poor prognosis. and during follow up. Molecular analyses were used for In this study, we analyzed the effect of Danusertib the monitoring of patients and Molecular Response exposure on cell viability, proliferation and (MR) was defined according to the International Scale. cytomorphological parameters, by means of different RESULTS:. At diagnosis, cytogenetic studies assays (MTT, trypan blue and clonogenic assays, mitotic showed 40/47 (85.11 %) patients with the standard Ph index and ploidy determination, morphological analy- translocation, 4/47 (8.51 %) patients with a variant Ph sis), in four GSC lines from GBM. GBM2 cell line translocation and 2/47 (4.25 %) patients with Additional showed a loss and G166 a gain of Aurora kinases genes, Chromosomal Abnormalities (ACAs) (one with while GliNS2 and G179 cell lines showed no variation. del(7)(p13p15), ider(22)(q10)t(9;22)(q34;q11) and one Results showed that response to Danusertib exposure with +8). In one patient (2.13 %) with normal karyotype, was heterogeneous among GSC lines. Cell viability and FISH revealed the presence of a masked Ph. During proliferation were significantly reduced in GBM2 and follow up, other chromosomal aberrations were detected G179 cell lines, while G166 and GliNS2 were resistant. in 4/47 patients: –Y(n=1) and +8, i(17q) (n=1) in the The analysis of cell and nuclear morphology in GBM2 Ph+clone and –Y(n =1) and +8,+21 (n =1) in and G179 cell lines highlighted the presence of large Ph- patients. Complete and Major MR were obtained multinucleated cells and an increase in the number of in 28 and 12 patients respectively, while no response polymorphic nuclei and micronuclei. At last conven- was observed in 4 patients. However, 3 patients have not tional cytogenetics evidenced a significant increase in yet been tested for response. These results were obtained ploidy in GBM2 and G179. irrespectively of CML phase or treatment used. Expression and mutational analysis of Aurora CONCLUSION: Our findings are consistent kinases and the study of chromosome segregation with other studies, confirming that variant Ph errors are in progress in order to deeply under- translocation, ACAs and masked Ph are rare stand the heterogeneous response to Danusertib events in CML. Conventional cytogenetics, FISH treatment on our GSC. and molecular studies are of great importance at diagnosis and monitoring of CML patients as they 2.P41 provide useful information with prognostic and therapeutic implications. Chromosomal abnormalities in chronic myeloid leukemia patients at diagnosis and during follow up. 2.P42

Elisavet Kouvidi1,IreneMavroudi1,Panagiota An evaluation of array comparative genomic Kanellou2, Helen Papadaki2 hybridisation (aCGH) as a method of improving Medical School of University Hospital of Crete the detection rate of genetic aberrations in Haematology Heraklion, Crete-Greece1 University myelodysplastic syndrome patients. Hospital of Crete Haematology Heraklion, Crete- Greece2 Julie Turbitt1, David Stevenson1, Dominic Culligan2, Paul Batstone1 Chronic Myeloid Leukemia (CML) is a clonal myelo- NHS Grampian Medical Genetics Aberdeen-United proliferative disorder characterized by the presence of Kingdom1 NHS Grampian Haematology Aberdeen- the Philadelphia (Ph) chromosome. United Kingdom2 10th European Cytogenetics Conference 2015 S97

Myelodysplastic syndromes (MDS) consist of a hetero- Italy2 Centre Hospitalier Universitaire Vaudois Unité de geneous group of haematopoietic stem cell disorders cytogénétique du cancer, Service de génétique médicale characterised by dysplastic and ineffective blood cell Lausanne-Switzerland3 University Hospitals Leuven production. Although karyotyping is considered the The Center for Human Genetics Leuven-Belgium4 ‘gold standard’ in the cytogenetic characterisation of MDS, approximately 40–50 % of the patients exhibit a RUNX1 (21q22), encoding a transcription factor crucial normal karyotype. Recent studies have reported the for hematopoiesis, is described as a target of multiple ability of aCGH to detect cryptic copy number alter- translocations in leukemia. More than 55 cytogenetic ations and regions of copy-neutral loss of heterozygosity bands are described as partners, although only 21 of (cnLOH) in MDS. them were identified. DNAwas extracted from stored fixed pellets from 32 We here describe four cases [three AML (cases 1, 2 diagnostic MDS patients with both normal and abnor- and 3) and one CML (4)] showing a t(15;21) transloca- mal karyotypes. Samples were anonymised and proc- tion disrupting RUNX1. FISH analyses revealed that the essed using the Agilent 4x180K Cancer CGH & SNP breakpoints were respectively located between array. 31/32 samples provided an acceptable diagnostic exons 6-7 (case 4) and 7–8 (cases 1 to 3) of the gene. array result. Array CGH detected additional cryptic The mapped breakpoints on chromosome 15 showed copy number alterations and large cnLOH regions, not recurrence in cases 2, 3 and 4 within the basic helix- detected by karyotyping. loop-helix TCF12 transcription factor gene at 15q21.3. Array CGH identified a crytic copy number loss The juxtaposition of TCF12 to RUNX1, with an oppo- (confirmed by FISH) on chromosome 21q, site transcriptional orientation, however, excluded encompassing part of the RUNX1 gene. In contast to the origin of a fusion transcript. Another ALL, the literature suggests that partial deletions of breakpoint was mapped in case 1 within SIN3A RUNX1 are a common finding in MDS. Large terminal (15q24.2) encoding for a transcriptional regulator. regions of cnLOH on chromosome 4q were idenified in The translocation juxtaposed the 5′end of SIN3A 2 MDS patients with an apparently normal karyotype. to the 3′end of RUNX1, with the same transcrip- Recurring cnLOH on chromosome 4q have previously tional orientation. RNA-Seq and RT-PCR experi- been reported in MDS patients and these regions are ments confirmed the occurrence of a SIN3A/ known to include the tumour suppressor gene, TET2. RUNX1 fusion transcript. In the same case, we Array CGH also detected a large cnLOH region on identified another out of frame chimera fusing the chromosome 17p that included TP53. Subsequent 5′ end of RUNX1 to the long non-coding RNA sanger sequencing revealed a TP53 Lys132Gln UBL7-AS1 gene, interrupted at intron 1. Since the two mutation. A balanced translocation present in one genes showed an opposite transcriptional orientation, a patient was not identified by aCGH. submicroscopic inversion event should be envisaged to Array CGH identified all except one of the karyotyp- explain the genesis of such fusion gene. ic abnormalities in our patient cohort, helped to define Thus, we report two novel t(15;21) translocations cytogenetic breakpoints and identified cryptic copy involving RUNX1, one of them as recurrent. number alterations and cnLOH regions not previously Interestingly, in case 1, we identified two chimeric genes detected. involving the 5′ and the 3′ of RUNX1 respectively fused with UBL7-AS1 and SIN3A. Further experiments are 2.P43 presently in progress to elucidate the pathogenetic role of the newly described and, in one case, recurrent rear- Rare recurrent t(15;21) translocations disrupting rangements in myeloid leukemia. RUNX1 in myeloid leukemia. 2.P44 Doron Tolomeo1,AlbertoL’Abbate1,CrocifissaLo Cunsolo2, Paolo Iuzzolino2, Dominique Mühlematter3, Translocation t(6;9)(p22;q34) in a patient with acute Peter Vandenberghe4, Clelia Tiziana Storlazzi1 myeloblastic leukemia University of Bari Department of Biology Bari-Italy1 Ospedale S. Martino UO Anatomia Patologica Belluno- Pelin Tasdemir1, Mine Balasar1, Aynur Acar2 S98 10th European Cytogenetics Conference 2015

Necmettin Erbakan University Medical Genetics 2.P45 Konya-Turkey1 Istanbul Bilim University Molecular Biology Istanbul-Turkey2 Translocation t(12;22)(p13;q11) in a patient with AML M1 INTRODUCTION: The t(6;9)(p23;q34) occurs with an incidence of 1–5%inadultpatientswith Mine Balasar1, Pelin Taşdemir1, Özgür Balasar2, Aynur acute AML with poor prognosis. The t(6;9) is Uğur Bilgin3, Aynur Acar4 reported mostly in young adults. The symptoms Necmettin Erbakan University, Meram School Of are due to the cytopenias often seen in Medicine Department of Medical Genetics Konya- leukemia.t(6;9) results in a chimeric fusion gene Turkey1 Konya Dr. Faruk Sükan Women And between DEK(6p23) and CAN/NUP214(9q34). Children Hospital Department of Medical Genetics Prognosis is usually poor, and complete remission Konya-Turkey2 Necmettin Erbakan University, Meram is achieved with conventional chemotherapy in School Of Medicine Department of Hematology only 50 % of the cases. Konya-Turkey3 İstanbul Bilim University Department MATERIAL AND METHODS: We performed of Molecular Biology and Genetics İstanbul-Turkey4 FISH analysis including PML/RARα,AML1/ ETO,inv16,del5q,MLL, del7q,del20q,p53 probes. Introduction Translocations involving the band 12p13 Karyotype analysis of bone marrow was applied are one of the most commonly observed chromosomal for the patient. Immunophenotypic analysis with abnormalities in human leukemia and myelodysplastic flow cytometry was was made in the diagnose syndrome. stage. ETV6 gene located in 12p13 is often involved in this RESULTS A 35 years old male was diagnosed chromosomal rearrangements. with AML-M1.He came to our attention for a At present 28 translocations have been characterized. marked leukocytosis(28.7×103/μl),anemia(Hb t(12;22)(p13;q11), one of these translocations, has been 7.0 g/dl),thrombocytopenia (102×103/μl) and reported in very few cases so far. Patients reported as hepatosplenomegaly. According to FISH analysis t(12;22)(p13;q11) were diadnosed with AML M0, M2, all the regions were normal. Conventional cytogenetic M4, AML on MDS,CML (Ph-). analyses detected a 46,XY,t(6;9)(p23;q34) karyo- We present here the first case of acute myeloblastic type. The patient was treated with Idarubicin leukemia without maturation (FAB type M1) with HCL(12 mg/m2×3 days) and Cytarabine(100 mg/m2× t(12;22)(p13;q11). 7days).Thenbasedontheabsenceofcompleteresponse Material and Methods Cytogenetic analysis, (CR),the same protocol was repeated. Allogeneic hema- AML-MDS FISH panel (del 5q, PML/RARα, topoietic stem cell transplantation (HSCT) was planned TP53 deletion, AML/ETO, MLL breakapart, del for this patient. 7q,CBFβ /MYH11translocation, del20q), CONCLUSION: Although t(6;9) is mostly associ- TEL(ETV6)/AML1 and BCR/ABL FISH analysis ated with subtypes of AML(M1,M2,M4),considered were performed on bone marrow and peripheral by some researchers to be a separate disease blood samples. entity because of its distinct clinical and morpho- Results The patient diagnosed with AML M1 logic features and poor prognostic implication. was 62 years old. There was no abnormality in the The presence of this karyotype is accepted to be AML-MDS FISH panel. The result of cytogenetic an indicator for allogeneic hematopoietic stem analysis performed on bone marrow sample was cell transplantation (HSCT). Molecular follow-up 46, XY, t(12;22)(p13;q11). BCR/ABL and of minimal residual disease is helpful in manag- TEL(ETV6)/AML1 FISH analysis were performed ing the disease. WHO classification of hemato- to define breaking points on derivative chromo- poietic tumors emphasizes the importance of somes 22 and 12. BCR signals were normal on prognostic impacts of cytogenetic abnormalities. bone marrow cells but the rearrangement of ETV6 AML with t(6;9)(p23;q34) is a rare status but (12p13) region was observed in 15 % of peripheral associated with distinctive morphologic and clinical blood cells examined. Molecular analyzes were features. planned. 10th European Cytogenetics Conference 2015 S99

Conclusion We identified t(12;22)(p13;q11) involv- transplantation and remained in remission for 17 months. ing ETV6 gene in a AML M1 patient with cytogenetic In November 2013 the patient had a relapse of the AML analysis and FISH method. In the literatue, with the following karyotype: 45,XY,del(6)(q13),-7[9]/ t(12;22)(p13;q11) in hematologic malignancies is asso- 46,XY[17]. FISH analysis revealed 33 % cells with ciated with MN1-ETV6 fusion gene. MN1 is a tran- monosomy 7, but no presence of BCR/ABL1. The scription co-activator of the retinoic acid receptor patient received two cycles of chemotherapy and in (RAR). RAR is a key regulator of proliferation and February 2014 an allogeneic stem cell transplant differentiation of blood cell lineages. The MN1-ETV6 from a related donor.In Feb.2015 the patient is still fusion protein is a dominant-negative suppressor of in complete remission. MN1. Thus, MN1-ETV6 fusion gene contributes to Transformation of a Ph-negative clone with mono- the development of leukemia. somy 7 and no clinical signs of MDS into overt To our knowledge, AML M1 with t(12;22)(p13;q11) AML is a rare but recurrent phenomenon. So, patients is the first case in the literature. showing monosomy 7 during imatinib treatment should be closely monitored both cytogenetically 2.P46 and clinically for signs of MDS or AML.

Cytogenetic studies on a chronic myeloid leukemia 2.P47 case developing into acute myeloid leukemia after treatment with imatinib. Loss of the Y chromosome in myelodysplastic syndromes W. Kroes1, E. den Ouden1, M. Baasten1, L. Saris1, L. Kerkhoffs2, P. von dem Borne3 Dolors Costa1, Meritxell Nomdedeu2, Xavier Calvo2, LUMC Clinical Genetics Leiden-The Netherlands1 Concha Munoz1, Ana Carrio1, Amparo Arias1, Candida HAGA Hospital Hematology the Hague-The Gomez1, Elias Campo1, Benet Nomdedeu2 Netherlands2 LUMC Hematology Leiden-The Hospital Clinic Unitat D’Hematologia Barcelona- Netherlands3 Spain1 Hospital Clinic Departament D’Hematologia Barcelona-Spain2 A 34-year-old male was diagnosed with CML in April 2006. All metaphases in the bone marrow showed Introduction: The clinical association between loss of t(9;22)(q34;q11.2). After one year of daily treatment the Y chromosome and MDS has been debated, because with 400 mg imatinib no Philadelphia-chromosome both phenomena are related to aging. The objective of positive metaphases were found in the bone marrow. this study was to asses if the the mean age is related to FISH showed 2.5 % of the 200 interphase nuclei posi- the loss of the Y chromosome. tive for BCR/ABL1. Surprisingly, a monosomy 7 was Material and Methods. From 1986 to July of 2014, detected in 15 out of 20 metaphases, whereas at this time 875 patients were diagnosed of Myelodysplastic no clinical signs of MDS or AML were present. Three Syndrome (MDS) in the Hospital Clinic of Barcelona months later, the bone marrow showed 3 clones: and 275 (55 %) were male. ANOVA was used for age 46,XY,t(9;22)(q34;q11.2)[2]/45,XY,-7[13]46,XY[5]. comparison between groups. There were still no morphological signs of MDS or Results. Twenty-six (6 %) patients showed a loss AML. After three years, monosomy 7 was detected in of the Y chromosome, in most of them as a sole all metaphases and by FISH in 90 % of 200 interphase abnormality. According to the WHO classification nuclei. Again FISH for BCR/ABL1 was negative. Bone about half of these 28 patients were affected of marrow cytology showed AML. The patient received refractory cytopenia with multilineage dysplasia induction therapy with cytarabine and daunorubicine (RCMD) (n=16). The mean age in patients with loss of and one cycle of amsacrine and cytarabine. Then cyto- the Y chromosome was 77 years old (95 %CI: 74 to 80), genetic studies of the bone marrow revealed 3 clones: significantly higher than that observed in patients with 45,XY,-7[3]/47,XY,+Y[5],46,XY[22]. FISH was posi- either a normal karyotype (71 y.o.; 95 %CI: 70 to 73) or tive again for BCR/ABL in 2,5 % of the 200 interphase with an abnormal karyotype without loss of the Y chro- nuclei. In June 2012 the patient underwent a double cord mosome (61 y.o.; 95 %CI: 65 to 70). S100 10th European Cytogenetics Conference 2015

Conclusion. Based on our results, the loss of the Y Vysis: LSI EGR1/D5S23, D5S721 Dual Color Probe, chromosome is observed more frequently in a group of D7S486/CEP7 FISH Probe Kit, CEP 8 (D8Z2) elder MDS patients. SpectrumGreen Probe, TP53 /CEP 17 FISH Probe Kit and LSI D20S108, 20q12, Spectrum Orange. 20q dele- 2.P48 tion was found in 13 % of cells. With all the data collected, the final diagnosis was FISH as a useful tool for diagnosing a hematologic Refractory anemia with 20q deletion (IPSS-R: very disorder low, 1)+Monoclonal gammopathy with undetermined significance IgA Lambda. María Talavera1, Dolores Rey1, Miguel Piris2, Ana In this case the molecular cytogenetics has not only Vallés2, Monica García-Cosío3, Ernesto Roldán4, Rosa served like prognostic indicator, it was the real key tool Lobo5, Jesús Villarrubia2 for diagnosis. University Hospital Ramon y Cajal Genetics Madrid- Spain1 University Hospital Ramon y Cajal Hematology 2.P49 Madrid-Spain2 University Hospital Ramon y Cajal Pathology Madrid-Spain3 University Hospital Ramon y Genetic alterations distinguishing molecular sub- Cajal Inmunology Madrid-Spain4 University Hospital types of papillary thyroid tumors may influence the Rio Hortega Clinical Analysis Madrid-Spain5 behaviour of corresponding stem-like cancer cells

In myelodysplastics syndromes (MSD) Fluorescence In Paola Caria1, Rita Pillai2, Tinuccia Dettori1, Daniela Situ Hybridization (FISH) is a powerful tool for patient Virginia Frau1, Valeria Sogos1, Roberta Vanni1 prognosis. We describe a case in which this technique University of Cagliari Dept of Biomedical Sciences was used to something more. Cagliari-Italy1 University of Cagliari Research and 85-year-old patient referred to our service for Territory Division Cagliari-Italy2 Monoclonal gammopathy with undetermined significance (MGUS) to reject progression. Introduction and aim. RET/PTC1 activation and Marrow cytology showed a moderately reactive with BRAFV600E mutation distinguish two molecular sub- mild dysplastic changes. types of papillary thyroid tumors and confer distinct Flow cytometry detected slight alterations in ery- biological behaviour to the associated carcinoma. throid size accompanied of 0.6 % of proerythroblasts, These genetic alterations are maintained in the TPC-1 relative to the total cellularity. Myeloid cell population and B-CPAP PTC-derived cell lines, respectively. From moderately hypogranular without antigenic alterations. these cell lines (CLs), we have recently isolated 0,7 % clonal plasmatic cells. Final immunodiagnosis: thyrospheres (ThySp), with stem-like tumor cell Monoclonal gammopathy MGUS type in the context of properties, still bearing the genetic alterations of discrete cellular dysplasia possibly associated with the parental adherent cells (AdhC). To explore the MDS. possible influence of the specific alterations on The pathologist reported a hypercellular bone marrow ThySp behaviour, we compared some functional (60 %) with mild reticulin fibrosis (grade 1/3) and and molecular aspects of ThySp isolated from the osteoesclerosis. Hyperplasia was found in all three series. two CLs. Specifically, we compared self-renewal Granulocytic series with proper maturation; red series of (SR) and sphere-forming efficiency (SFE), expression of dispersed location and megakaryocytic series constituted stemness (OCT4/Nanog/ABCG2/ALDH1-A1) and by large forms that are not added. Blast percentage did epithelial/ thyroid differentiation markers (CK19/TTF1/ not exceed 3 %. The pathologist’s definitive diagnosis: PAX8) in both AdhC and ThySp. clinical and laboratory correlation is necessary for diag- Results FISH and real time-PCR confirmed RET/ nosis of myelodysplastic syndrome. Plasmacytosis inter- PTC1 and BRAFV600E in TPC-1 and B-CPAP stitial (15 %) CD138 +, CD56 -, Lambda+. Compatible ThySp, respectively. B-CPAP ThySp maintained SR diagnosis with MGUS. and SFE for a significantly higher number of genera- Conventional cytogenetics showed a normal karyo- tions compared to TPC-1 ThySp. The main difference in type. FISH analyses were performed with probes from the molecular behaviour between the CLs was the lack 10th European Cytogenetics Conference 2015 S101 of expression of early and late differentiation markers in with diagnosed carcinoma of the endometrium. The TPC-1 AdhC and ThySp. Indeed, CK19 marker was studies were supported by the use of a custom-made present in B-CPAP AdhC but not in ThySp, which 4x180K microarray of BlueGnome. expressed TTF1 at lower level than AdhC. ALDH1- Results: The patients with no endometrial hyperpla- A1 was negative in both TPC-1 ThySp and AdhC, sia presented with copy number variations (CNV), higher expression was seen in B-CPAP ThySp than which are normally found with varying frequency AdhC. in the genome of the healthy female population. A Conclusions. Our molecular and functional findings significant genome imbalance was identified in the indicate that B-CPAP express a wider panel of stemness twenty-six (26) (100 %) patients with diagnosed markers and that B-CPAP ThySp rather than TPC-1 hyperplasia and in eleven (11) (84.6 %) with diag- ThySp contain cells behaving as stem-like tumor nosed endometrial cancer. Other, not yet reported cells. On the strength of our results, we cannot ruled changes were also observed in characteristic regions out the hypothesis that BRAFV600E and RET/PTC1 of the genome: 14q32.33 and 15q11.2. may influence the functional and molecular features Conclusions: A study of chromosomal instability, of B-CPAP and TPC-1 ThySp. Fund: PRIN N° which determines the type and extent of chromosomal 2012Z3F7HE and RAS. changes, is sensitive enough to uncover critical regions and, consequently, isolate genes or groups of genes 2.P50 supporting the formation and development of endometrial malignancy. The degree of genome imbalance Chromosomal aberrations, mutations and polymor- can thus be considered as a risk factor for endome- phisms as prognostic factors in endometrial cancer trial cancer. development 2.P51 Bogdan Kaluzewski1,MichalBednarek1,Tadeusz Kaluzewski1, Maria Constantinou2, Agata Iwaniuk1, Distrubution of chromosomal abnormalities in adult Slawomir Jedrzejczyk3,KatarzynaLau3,Borowiec acute myeloid leukemia patients: A single center Maciej2 study

GENOS Non-Public Healthcare Institution, member of AyşeÇırakoglu1, Şükriye Yılmaz1, R.Dilhan Kuru1, the Polish Technology Platform for Innovative Serdar Şahin2,Fırat Fevzi Yılmaz2,YeldaTarkan- Medicine, of the National Centre for Research and Argüden1,Tuğrul Elverdi2, Seniz Ongoren2,Ayhan Dev Medical Genetics Zapolice-Poland1 University of Deviren1, Zafer Başlar2, Seniha Hacıhanefioğlu1 Lodz Genetic Outpatient Clinic; Chair of Clinical and Cerrahpasa Faculty of Medicine, Istanbul University Laboratory Genetics, Department of Clinical Genetics Medical Biology Istanbul-Turkey1 Cerrahpasa Faculty Lodz-Poland2 Henryk Jordan’s Lodz IVth City Hospital of Medicine, Istanbul University Hematology Istanbul- Department of Gyneacology Lodz-Poland3 Turkey2 Objectives: The primary end-point of the research project, undertaken in a group of patients with endome- Acute myeloid leukemia (AML) is a heterogenous trial hyperplasia, was an identification of the earliest and group. Cytogenetic abnormalities are found in more specific genetic changes which could be associated with than 50 % of adult AML patients. Cytogenetics has an increased risk of transformation into malignancy. The significant role in classification of AML according the secondary end-point of the reported research was a World Health Organization (WHO) classification. characteristics of genetic processes, associated with In this study, we present our cytogenetic data of 303 advanced malignant formations. adult AML patients retrospectively. The study included Material and methods: The studies involved forty- 16 years and older patients (range 16–89, median 45) four (44) female patients, including five (5) with no who were reffered to our laboratory between 1996 and histopathologically confirmed hyperplastic features, 2014. Cytogenetic analysis was carried out succesfully twenty-six (26) with histopathologically confirmed in 247 patients (81.5 %). We detected clonal chromo- endometrial hyperplasia and thirteen (13) patients somal abnormalities in 55 % of analyzed patients. S102 10th European Cytogenetics Conference 2015

Reccurent abnormalities for AML; t(8;21) and t(15;17) patients, no complete monoclonal immunoglobulin were found in 4 and 3.2 % of the patients respectively. can be detected in the serum; these patients produce −5/del(5q) were found in 5.26 % of the patients while - large amounts of light chains. These patients are 7/del(7q) were 7.28 %. 11q23 rearrangements were commonly referred to as having light-chain MM. In present in 2.4 % and +8 were detected in 4.45 % most of these patients, large amounts of monoclonal of the patients. Abnormalities of 12p, −13/del(13q) light chains are detected in the urine and to a lesser and del(9q) were observed in 2 % of the patients extent in the serum. These light-chain MM (LC-MM) each. t(4;19)(q21;p or q13), which was not reported patients have in two thirds of the cases a κ-LC type, previously in AML with these breakpoints, was observed whereas the remaining one third of the patients produces in two patients in our study. λ-LC. Although improved predictive risk stratification Most of the abnormalities reported above were part models which have included international staging sys- of a complex karyotype. In 12.5 % of the patients found tems as well as FISH-detected chromosomal changes complex karyotype. Recent studies identified used for guiding treatment algorithms in MM, the monosomal karyotype (MK) which is defined by pres- prevalence of genetic prognostic factors for these ence of a clone with at least two separate autosomal LC-MM cases is unknown. Because of the prognostic monosomies or one monosomy beside one or more significance of 13q deletions in multiple myeloma, structural abnormalities. MK were shown in 36 we sought to define the frequency and significance (14.5 %) of our patients. The majority of these patients of this abnormality in LC-MM patients tested in the also had a complex karyotype. MK was reported ~10 % routine clinical laboratory. We reviewed laboratory in all AML and up to 20 % in older patients by previous characteristics of patients with LC-MM and with studies. monoclonal gammopathy who had FISH analysis for In accordance to literature, we concluded that 13q deletion performed as part of their routine clinical frequency of balanced abnormalities were more com- testing using the standard screening multiple myeloma mon in younger group (16–34 years) and frequencies panel employed at our laboratory. Of the bone marrow of unbalanced abnormalities, complex karyotypes and specimens from monoclonal gammopathy patients, monosomal 10 % had 13q deletions. The corresponding frequencies were significantly different between MM and LC-MM 2.P52 groups: 6 and 20 %, respectively.

13q deletion and survival of patients with light chain 2.P53 multiple myeloma Follow-up of del(5)q MDS patients with or without Ajlan Tukun1, Gizem Gıran2,Çağatay Kundak3, Gülçin other chromosomal abnormalities Sungar2, Yahya Laleli3 Ankara University Faculty of Medicine Medical Özlem Türedi1,Pınar Ataca2, Timur Tuncalı1, Erden Genetics Ankara-Turkey1 Duzen Laboratories Group Atilla2, Arzu Vicdan1, Ceren Damla Durmaz1, Klara Medical Genetics Ankara-Turkey2 Duzen Laboratories Dalva2,NüketYürürKutlay1,HalilGürhan Group Clinical Biochemistry Ankara-Turkey3 Karabulut1, Hatice Ilgın Ruhi1, Önder Arslan2, Muhit Özcan2, Osman İlhan2, Günhan Gürman2, Ajlan Tükün1 The recent advances in investigative techniques that Ankara University School of Medicine Medical have helped refine the diagnostic work up in multiple Genetics Ankara-Turkey1 Ankara University School of myeloma (MM) includes use of serum free light Medicine Hematology Ankara-Turkey2 chains, and use of cytogenetic and FISH to determine prognosis. 14q rearrangements, del13q, ampli- Myelodysplastic syndrome is the most common fication of 1q21 and del17p are especially important hematologic malignancy characterized by ineffec- prognostic markers which are used for risk stratifi- tive hematopoiesis leading to persistent peripheral cation for MM patients. Although most MM cases blood cytopenia and predisposition to acute myeloid are characterized by the detection of a monoclonal leukemia whereas interstitial deletion of chromosome immunoglobulin in the serum, in about 15 % of the 5q is the most common cytogenetic abnormality in this 10th European Cytogenetics Conference 2015 S103 syndrome. Lenalidomide, is a second generation, immu- (CpG-IL2) has been proposed for stimulation of B- nomodulatory drug that was approved for treatment of lymphocytes division in growth media. We assessed transfusion dependent anemia in patients with lower risk how the introduction of CpG-IL2 influences the routine MDS with interstitial deletion of the long arm of cytogenetic analysis in CLL. chromosome 5. The experiences with lenalidomide in 24 samples (peripheral blood - 7, bone marrow patients with lower risk MDS with deletion 5q aspirate - 17) were cultivated in parallel with or showed cytogenetic responses, independent of the without addition of CpG-IL2. Mitotic indexes were presence or absence of additional cytogenetic abnor- determined in each sample to evaluate proliferation malities. Therefore 5q deletion is drawing increasing rates. To evaluate the influence of CpG-IL2 stimulation attention. on cytogenetics results we analyzed two groups of dif- We analyzed clinical and laboratory characteristics ferent samples (55 each) which were cultivated with or and follow up information of 50 MDS patients with 5q without addition of CpG-IL2. FISH analysis for recur- deletion. All these patients were scored according to rent aberrations (del(11q), +12, del(13q), del(17p), IGH the revised International Prognostic Scoring System rearrangement) was done in all samples. In 36 samples (R-IPSS). In our study, 9 patients (18 %) had iso- of each group G-banding analysis was additionally lated del5q and the remaining 41 patients (82 %) performed. had additional chromosomal abnormalities. In our When we evaluate proliferation rates of same study the most frequent chromosomal abnormality samples cultivated with or without addition of concomitant with del(5)(q) was 7q deletion. During CpG-IL2, we found that mitotic indexes were sig- the follow-up transformation of myelodysplastic syn- nificantly increased in blood samples only drome into acute myeloid leukemia rates in the (p<0,05). The importance of CpG-IL2 stimulation patients with del(5)(q) alone and del(5)(q) with other was revealed when the results of banding analysis chromosomal abnormalities were 66.7 and 65.9 % were evaluated. Unsuccessful banding analysis has respectively. Twenty nine of the patients were under the been reduced from 22 % to less than 3 %. Since treatment and three of them received lenalidomide ther- the effect of CpG-IL2 on malignant B-lymphocytes apy. In the group of del(5)(q) alone 22 % of the patients is selective the proportion of false negative results were in remission with a 12 months follow up. At the was considerably reduced. Proportion of normal same period, the remission rate was 7 % the in patients karyotypes in patients with recurrent abnormalities with del5q and other chromosomal abnormalities. The detected by FISH has decreased from 20 to 3 %. median survival was 20 months in patient group with Additionally, proportion of CLL patients with chromo- del(5)(q) alone, and 12 months in the group with addi- somal aberrations that were revealed only by banding tional abnormalities accompanying del(5)(q). analysis has increased from 14 to 33 %. Here, we present genetic abnormalities and clinical Our study confirmed that cultivation of CLL samples implications of 50 MDS patients with del(5)(q). with CpG-IL2 stimulation is mandatory when banding analysis is performed. It enables fulfillment of the latest 2.P54 ECA quality standards for bone marrow aspirates as well as for peripheral blood. The influence of CpG-oligodeoxynucleotide/IL2 stimulation on quality of routine cytogenetic analysis 2.P55 in CLL Study of fluorescence in situ hybridization in malig- Helena Podgornik1,JasminaFarič1,AnaDoplihar nant melanoma Kebe1 University Medical Centre Ljubljana Department of Beyhan Durak Aras1, Sevgi Işık1, Tolga Töre1, Funda Haematology Ljubljana-Slovenia1 Canaz2,HülyaYılmaz3,MuhsinÖzdemir1,Oğuz Çilingir1, Hüseyin Aslan1, Sevilhan Artan1 Chromosome banding analysis in CLL is hampered by a Eskişehir Osmangazi University Medical Faculty low in vitro proliferation rate of B-CLL lymphocytes. Medical Genetics Eskişehir-Turkey1 Eskişehir Combination of CpG-oligodeoxynucleotides and IL-2 Osmangazi University Medical Faculty Pathology S104 10th European Cytogenetics Conference 2015

Eskişehir-Turkey2 Eskişehir Osmangazi University and gain of 5p and type 2 includes extra i(5p) and two Medical Faculty Biostatistics Eskişehir-Turkey3 normal homolog chromosomes 5. To date, i(5)(p10) has been reported in 24 cases. Of them, 11 were type 1 and Morbidity and mortality rates of malignant melanomas 13 were type 2. Here we reported 2 cases with i(5p). The are reported to be increasing every year. Since there are a first case who had been treated for bladder cancer before subset of cases where histopathologic discrimination developed MDS, diagnosed as RAEB-II. Cytogenetic between malignant melanoma and melanocytic nevi and FISH analyses of the bone marrow revealed an may be exceedingly difficult, genetic and genomic anal- aberrant karyotype, showing type 1 i(5p) and a deletion ysis have yielded new opportunities for improvements in 12p. Following two cycles of successful treatment in diagnostic accuracy for the distinction of nevus from with 5-azacytidine, the case died at home and therefore melanoma and better selection of patients affected by the death reason could not be determined. In the second melanoma for targeted treatment. In this study, we case, the diagnosis of AML was established. aimed to determine the ability of FISH analysis targeting Cytogenetic and FISH analyses showed both type 2 CCND1, MYB, RREB1, CEP6 regions in the samples i(5p) and trisomy 8. The case died due to severe pneu- from 18 malignant melanoma and 24 melanocytic nevi. monia. The presence of type 2 i(5p) accompanied by The results showed copy number alterations in 15 and 4 trisomy 8 could have been reported in only 9 cases. It is cases with malignant melanoma and melanocytic nevi, controversial whether i(5p) is the seconder abnormality. respectively. In contrast to the literature, the CCND1 In the second case, the clones with either pure i(5p) or gene amplification was significant (p<0,001). The other pure trisomy 8 were seen. Although poor prognosis of aim of the study was to determine the relation between the patients with i(5p) clones have been reported, the copy number alterations and prognosis of the dis- reporting and collecting data of rare chromosomal eases. The FISH analysis specific to EGFR, P16, P53 abnormalities will add information to disease pro- and MDM2 genes were performed. The EGFR gene gression and prognosis. amplification showed significant difference between malignant melanoma and nevi cases (p<0,001). No 2.P57 relation between genomic copy alterations and prognostic values (ulcer, lenf infiltration etc.) was seen. We Frequency of genetic changes in pediatric leukemias concluded that CCND1, MYB, RREB1 and EGFR diagnosed with the help of FISH at Paraná state alterations have a potential to discriminate between hospital malignant melanoma and melanocytic nevi in challenging cases. Bruno Osternack1, Mara Pianovski2, Roberto Rosati3 Faculdades Pequeno Príncipe molecular biology 2.P56 Curitiba-Brazil1 Universidade Federal do Paraná onco- hematology Curitiba-Brazil2 Instituto de Pesquisa Pelé Isochromosome 5p—ararechromosomeabnormality Pequeno PríncipeInstituto de Pesquisa Pelé Pequeno in myeloid disorders Príncipe molecular biology Curitiba-Brazil3

Beyhan Durak Aras1, Sevgi Işık1, Neslihan Andıç2, FISH is a valuable tool for the identification of genetic Oğuz Çilingir1, Hüseyin Aslan1, Muhsin Özdemir1, changes, because leukemic blasts often give poor qual- Sevilhan Artan1, Meltem Olga Akay2 ity metaphases or none at all. In this sense, FISH is Eskişehir Osmangazi University Medical Faculty recommended by WHO as a method that can be used Medical Genetics Eskişehir-Turkey1 Eskişehir on interphase nuclei, and replace techniques that have Osmangazi University Medical Faculty Heamatology false negative results, technical problems and to confirm Eskişehir-Turkey2 other techniques. This research aimed to evaluate the incidence of genetic alterations presented in pediatric Although loss of chromosome 5q is a common abnor- patients diagnosed with acute leukemia, and associate mality in patients with AML and MDS, isochromosome these changes with age, gender and blood test results, of chromosome 5p is a rare abnormality. Two types of classical cytogenetics and immunophenotyping. Thus, a i(5p) have been reported: type 1 is formed by loss of 5q quantitative cross sectional study was performed with 10th European Cytogenetics Conference 2015 S105 retrospective data collected from medical records from 84 year-old male with acute myeloid leukemia with beginning FISH analysis, from January 2013 to multilineage dysplasia). December 2014. Seventy four samples were analyzed, In order to assess the del20q CDR, we per- 58 (79 %) of which from the Hospital Infantil Pequeno formed FISH using the 20q12 probe (Vysis, Príncipe and 16 (21 %) from the Hospital de Clínicas da Abbott, Rungis, France) and a library of Bacterial Universidade Federal do Paraná; 36 (48 %) samples Artificial Chromosome (BAC) clones located in were obtained from male patients and 38 (52 %) from bands 20q11 and 20q12. female patients. The analysis by FISH showed 20 sam- The CDR encompassed 5.6 Megabases (Mb), ples with alterations, being 2 BCR/ABL1 positive, 6 between bands q11.1 and q11.23, and 8.6 Mb, MLL positive and 5 ETV6/RUNX1 positive samples. between bands q11.21 and q12, for patients 1 and 2, The incidence of MLL gene rearrangement, BCR/ABL1 respectively. No recurrent breakpoint was found. and ETV6/RUNX1 gene fusions was 30, 10 and 25 %, D20S108 locus was retained and 20q deletions were respectively. Data analysis indicated that the fusion large, interstitial and proximal in 2 cases. gene BCR/ABL1 had similar frequencies in all age In our patients, the 20q deleted regions included groups up to age 15, the fusion genes ETV6/RUNX1 several genes, notably HCK, ASXL1 and DNMT3B. and MLL had the highest until the fifth year of life Haploinsufficiency of these genes could play a role in and decreasing with age; but ETV6/RUNX1 was the myeloid disorder. most frequente finding in this study. Two BCR/ABL1 20q deletion is a recurrent cytogenetic abnormality positive cases with a confirmation by cytogenetics. with a favorable outcome according to the revised Six MLL positive but 2 cases confirmed by cytoge- International Prognostic Scoring System. Although netics. 5 ETV6/RUNX1 positive cases had no trans- very rare, atypical proximal del20q is a recurrent location by cytogenetics. The results of PML/RARα abnormality. It could be underestimated without use were negative by FISH. of FISH probe focusing on D20S108. Whether the leukemia process and prognosis of this anomaly is 2.P58 identical to the classic 20q deletion in myeloid pathologies remain to be established. Atypical proximal deletions of the long arm of chromosome 20 retaining D20S108 locus in myeloid 2.P59 malignancies. Associations between HER2/neu,TOP2A, chromo- Nathalie Douet-Guilbert1, Nadia Guéganic1, Delphine some 17 copy numbers, and CDH1 and GSTP1 gene Bernard1,AudreyBasinko2,Marie-JoséeLeBris2, promotor hypermethylations of patients with breast Corinne Tous2, Frédéric Morel1, Marc De Braekeleer1 cancer Faculty of Medicine Laboratory of Cytogenetics Brest- France1 CHRU Brest Laboratory of Cytogenetics Brest- Onur Eroglu1, Ayse Feyda Nursal2, Mine Erci Baysak3, France2 Oguz Cilingir3,BeyhanDurakAras3,Muhsin Ozdemir3, Sevilhan Artan3 Deletion of the long arm of chromosome 20 (del20q) is Bilecik Seyh Edebali University Molecular Biology and a common finding in myeloid disorders. The 20q dele- Genetics Bilecik-Turkey1 Giresın University Medical tion breakpoints are heterogeneous and the commonly Faculty of Medical Genetics Giresun-Turkey2 deleted region (CDR) is different in various myeloid Eskisehir Osmangazi University Medical Faculty of pathologies but overlaps within 20q12 and D20S108 Medical Genetics Eskisehir-Turkey3 locus. Our objective was to focus on and characterize the deletion size of atypical proximal 20q deletion in 2 Introduction: Breast cancer is an important public health myeloid patients using fluorescent in situ hybridization problem worldwide. The HER2 /neu protooncogene is (FISH). amplified and overexpressed in approximately 25–30 % In the past 10 years, we identified two patients with of invasive breast carcinomas. DNA topoisomerase 2- myeloid malignancies and atypical 20q deletion (a 74- alpha enzyme controls and alters the topologic states of year old male with myelodysplastic syndrome and a DNA during transcription. GSTP1 plays an important S106 10th European Cytogenetics Conference 2015 role in protecting cells from cytotoxic and carcinogenic analysis. It has been shown to improve detection rate agents and it is expressed in normal tissues at variable of cryptic aberrations in apparently normal samples levels in different cell types. CDH1 plays a critical role and the accuracy in resolving complex chromosomal for establishment and maintenance of polarity and dif- rearrangements. It is also an invaluable diagnostic ferentiation of epitheliumduringthedevelopment tool for establishing the origin of small supernumerary period. Also, it plays an important role in signal trans- marker chromosomes (sSMCs). SMCs characterization duction, differentiation, gene expression, cell motility is of utmost importance for genetic counselling in pre- and inflammations. natal diagnosis but also for prognostication in hemato- Method: In this study we analysed retrospective logical malignancies. HER2/neu, TOP2A gene and Chromosome17 copy Short term cultivated bone marrow cells from number alterations by fluorescence in situ hybridization four acute leukemia patients were analyzed by (FISH) in primary tumor core biopsies from 100 conventional banding and FISH applying different high-risk primary breast cancer patients (tumors DNA probes. G-banding revealed one or two ≥2cmand/orlenfaticmetastaseand/ordistantmetastases marker chromosomes as an isolated anomaly in and/or under 40 years) . The methylation levels of all patients. FISH did not resolve the origin of the CDH1 and GSTP1 gene promoters were markers. Therefore, we decided to apply SKY assessed Methylation Sensitive High Resolution (Applied Spectral Imaging, Carlsbad, CA) followed by Melting Analysis (MS-HRM). FISH to confirm SKY findings. Several methaphases of Results: In our study, HER2/neu amplifications sufficient quality were analyzed in each patient. were identified in 22 % and TOP2A amplifications In one patient with two distinct markers SKY in 21 % and deletions in 4,8 % of patients. clearly showed both markers to belong to chromo- HER2/neu and TOP2A amplifications are found some 8. In other three patients SMCs origin was to be associated with IDC tumor type and high not unequivocally resolved, due to insufficient grade also HER2/neu amplifications is associated chromosomal material (<6–10 Mb in size) or het- with PR(-), TOP2A amplifications is associated erogeneity of markers. with ER(+). TOP2A deletions is associated with Although SKY is an elegant technique which enables ER(-) and PR(-). Polysomy17 was present in 22 % simultaneous analysis of all chromosomes, it has limi- and monosomy 11 % of patients CDH1 and tations in routine analysisofmalignantdiseases. GSTP1 methylation frequencies were 83,7 and 74,1 % Metaphase spreads of sufficient quality and quantity respectively. are needed to capture different possible clones. Albeit Conclusions:. Our study is important as being these conditions are fulfilled the technique can help to the first study that analyzes association between resolve only larger SMCs. In some patients however, HER2/neu, TOP2A gene copy numbers and CDH1, SKY can contribute to a more accurate evaluation GSTP1 gene promotor methylation status in Turkish of prognostic significance of chromosomal aberra- population. tion observed consequently influencing also therapeutic decisions. 2.P60 2.P61 SKYanalysis of small marker chromosomes in acute leukemia patients A case of TdT+ “triple-hit”lymphoma

Ana Doplihar Kebe1,NušaKosednar1,Katarina Tatiana Obukhova1,LubovGrebenyuk1,Galina Reberšek1, Helena Podgornik1 Alimova1, Lubov Shishigina1, Alla Kovrigina2, Olga University Medical Centre Ljubljana Department of Gavrilina3, Sergey Kravchenko3 Haematology Ljubljana-Slovenia1 National Center for Hematology Cytogenetic Laboratory Moscow-Russia1 National Center for In combination with the conventional banding and Hematology Department of Pathology Moscow- FISH, Spectral karyotyping (SKY) has become an Russia2 National Center for Hematology Department important technique that improves the chromosome of Hematology and Intensive Care Moscow-Russia3 10th European Cytogenetics Conference 2015 S107 t(c-MYC/8q24) are the hallmark of Burkitt lymphoma 2.P62 (BL) but occur in subset of other mature-B-cell lymphomas as secondary aberrations. “Double-hit” or “triple- Optimal strategy for obtaining chromosome and hit” lymphomas (DHL and THL) are rare subtype of fluorescence in situ hybridization results for plasma lymphomas characterized by concurrent chromosomal cell dyscrasias by using both positive and negative rearrangement of c-MYC/8q24 with BCL2/18q21 and/ fractions of CD138 enriched plasma cells or BCL6/3q27, complex karyotype, mature B-cell immunophenotype, Ki67~100 %, aggressive clinical Veronica Ortega1, Gihan Mohamed1, William Ehman, behavior and poor prognosis. Jr1,MeiqingZhu1,ChristinaMendiola1, Gopalrao We report on a case of THL with immunophenotype Velagaleti1 of B-lymphoblastic leukemia/lymphoma University of Texas Health Science Center Dept. (CD10+,CD38+,CD45+,PAX5+,CD79a+,CD99+ Pathology San Antonio, TX-United States1 ,BCL-2+,BCL-6(-/+),TdT(-/+),CD34-,Ki-67-80-90 %) in 39-year-old male patient. Lymph node biopsy cells Plasma cell dyscrasias (PCD) comprise a genetically were studied at diagnosis. diverse group of diseases such as monoclonal Conventional cytogenetic analysis revealed complex gammopathy of undetermined significance, karyotype with typical for follicular lymphoma (FL) plasmacytoma, smoldering myeloma, indolent myeloma, t(14;18)(q32;q21) and presumably c-MYC and BcL6 and plasma cell myeloma. FISH is superior to chromo- translocations: 48,XY,dup(1)(pter→q31::q21→ some analysis (CA) in detecting important qter),t(3;?22)(q27;?q11),del(6)(q21),+7,+ prognostic genetic abnormalities in PCD, however, 8,t(8;?22)(q24;?q11),t(14;18)(q32;q21),der(14) its sensitivity is hampered due to paucity of plasma t(14;18)(q32;q21). cells (PC) in whole bone marrow (WBM) and We performed molecular cytogenetic analysis that false-negative results when frequency of abnormal included interphase and metaphase FISH, M-FISH and cells is below cut-off values. Studies have shown M-BAND with probes: LSI c-MYC BAP, LSI c-MYC/ that abnormality detection rate in EPC is greater IGH/cep8 DFTP, LSI Bcl2 B.P. LSI Bcl2/IGH DFTP, than unselected plasma cells (UPC), but purifica- LSI Bcl6 BAP (ABBOTT), XL IGL BAP, XL IGK BAP, tion techniques are limiting to FISH when sample 24XCyte Human Multicolor ProbeKit and Human volumes are inadequate. The inability to perform mBAND ProbeKit (MetaSystems). Molecular cytoge- CA compromises patient care since CA is equally netic study confirmed the presence of “triple-hit”— important for detecting non-PC related abnormalities, t(BcL2/18q21) and c-MYC/8q24 and BCL6/3q27 such as secondary myelodysplastic syndrome. To translocations to both alleles of IgL/22q11. Furthermore, optimize limited sample volumes, we designed a study in our case it was shown that: the original clone contained where an immnuno-magnetic CD138 enriched positive atypicalt(14;18)(q32;q21)involvingonealleleof selection was used for FISH while the myeloid chromosome 14, but there was a loss of normal elements in the negative selection were used for chromosome 14 and doubling of its derivative in CA. FISH studies were performed on EPC, and tumor progression at the preclinical level; Bcl6 parallel karyotyping by WBM and the negative rearrangement is represented by duplication of selection. Purity of EPC was confirmed by flow- 3q27 region and translocation BcL6/3q27 to IgL/ cytometry (47.3 to 96.9 %). FISH results showed 22q11 locus with deletion of its centromeric region on that the abnormality rate of EPC (79 %) doubled chromosome 22; c-MYC locus break point is located compared to UPC (34 %), and CA displayed more telomeric than in typical Burkitt lymphoma’s 100 % (154/154) success rate using the discarded translocations; duplication of 1q is inverted. Detailed myeloid elements. PCD abnormalities were cytogenetic and molecular cytogenetic study is neces- confined to WBM while non-PCD abnormalities sary for differential diagnosis of DHL and THL with were found in both WBM and supernatant confirming BL, FL, diffuse large B-cell lymphoma as well as due to effective removal of PC by isolation and efficient utility the possible aberrant “immature” immunophenotype of the discarded myeloid elements for CA. Our results with lymphoblastic lymphoma, to search for adequate demonstrate the feasibility of using the negative fraction treatment strategies for these patients. for CA. S108 10th European Cytogenetics Conference 2015

3. Prenatal Diagnosis allowing us the detection of CNAs across the whole genome in single experiment. The utilization of a novel 3.P1 generation of microarrays in PGD is suitable to detect structural CNAs with a resolution vastly overcoming High resolution array-CGH screening of 5-day previous generation of BAC arrays. This study was embryos: focus on interstitial structural CNAs supported by OPVK-CZ.1.07/2.3.00/20.0183. with 8x60k Agilent platform 3.P2 Jan Smetana1, Aneta Mikulasova1, Vladimira Vallova2, Nemcova Darja3, Marek Koudelka3, Petr Kuglik1 Prenatal diagnosis and molecular characterization Masaryk University Brno, Faculty of Science Institute of a maternally inherited derivative chromosome 9 of Experimental Biology, Laboratory of Molecular resulting in a partial deletion 9p(9p22-pter) and par- Cytogenetics Brno-Czech Republic1 University tial duplication 18q23. Hospital Brno Department of Medical Genetics Brno- Czech Republic2 Reprofit International Reprofit Ariadna Escalona1, Mònica Sureda1, Joana Fortuño1, International Brno-Czech Republic3 Jordi Duran2,SusanaCaballero1,LeonorMarín1, Fernando Ayensa3, Sandra Andreu1 The utilization of genome-wide screening techniques in mdb Laboratori d’anàlisis clíniques, S.A. Departament PGD brings novel information about the incidence of de Citogenètica i Biologia Molecular Barcelona-Spain1 copy number aberrations (CNAs). However, most of mdb Laboratori d’anàlisis clíniques, S.A. Departament the screening platforms currently used are focused Direcció Mèdica Barcelona-Spain2 Mútua Nuestra on the detection of aneuploidies, but not structural Senyora del Carmen Departament Tocoginecologia CNAs. In this study, we validated and used a high Granollers-Spain3 resolution microarray platform (Agilent 8x60k) to evaluate the incidence of structural CNAs in a cohort Simultaneous occurrence of partial 9p deletion and of 5-day embryos from patients and healthy donors partial 18q duplication is very rare, with only a few (DEMs). cases published in the literature. This chromosomal In total, 383 (56 DEMs, 327 patient’s) 5-day embryos imbalance has been associated with different patterns were analyzed from 86 patients and 18 DEMs. Array- of dysmorphisms as well as anorectal malformations. CGH screening was successfully performed in 96.1 % Here, we report prenatal diagnosis and rapid charac- (368/383). CNAs were detected in both patient and terization by subtelomere FISH of an additional case. DEM samples in 35.3 % (128/368). A 39 year-old pregnant woman carrier of a balanced Structural CNAs were observed in 2 DEM cases translocation between chromosomes 9 and 18 was (4.3 %; 2/47) and 8.4 % of patient samples (12.5 %; referred to our laboratory for prenatal diagnosis 40/321). Overall, we identified 42 different structural because of an abnormal maternal serum screening CNAs (23 regions of gain, 19 regions of loss of genetic result (1/141) and intrauterine growth retardation material). Median of size was 52.7Mbp. Most frequently (IUGR) at 15th weeks of gestation. Conventional affected were chromosomes 5 and 8 (both 11.9 %; 5/42, cytogenetic analysis of cultured amniocytes revealed a respectively). maternally inherited derivative of chromosome 9. Aneuploidies were found in 12.8 % of DEMs (6/47) Further FISH analysis with subtelomeric 9p and 18q and in 31.8 % of all patient’ssamples(102/321). probes demonstrated an unbalanced karyotype Overall, we detected 190 cases of CNAs affecting whole consisting on a partial 9p(9p22->pter) deletion and a chromosome (89 cases of trisomy, 101 cases of mono- partial 18q23 duplication, reformulating the conven- somy). The most common gains of a whole chromo- tional karyotype as 46,XX,der(9)t(9;18)(p22;q23) some were trisomies 15 and 20 (5.3 %; 10/190, respec- mat.ish der(9)t(9;18)(305J7-T7-,VIJyRM2050+). We tively), while frequent loss was observed as monosomy were informed that our patient already had a previ- 16 and 22 (both 5.8 %, 11/189). ously born child with this chromosomal rearrange- Taking together, array-CGH with high resolution ment who is severely affected with clinical features that microarrays is a robust and cost effective technique could be associated with both, the 9p deletion syndrome 10th European Cytogenetics Conference 2015 S109 and the Edwards syndrome. In this work, we review tetraploidy. Among the few reported cases of the literature of prenatal diagnosis of partial 9p 92,XXXY tetraploidy, those associated to partial moles deletion and partial 18q duplication and we discuss show a PPPM genotype (3 paternal and 1 maternal the unexpected high recurrence risk in this family, alleles), and the only case with PPMM genotype was as well as candidate genes that are possibly involved in found in a spontaneously aborted fetus, similar to our the clinical features detected in our patient’sborn case. We are not aware of other cases with combination child. of a digynic triploidy and a tetraploidy with PPMM contribution. Our case adds evidence of the influence 3.P3 of the balance between paternal and maternal genomic doses on the phenotype. 69,XXYand 92,XXXY in two consecutive pregnancies 3.P4 Anna Soler1, Cèlia Badenas1, Madrigal Irene1, Muñoz Miriam2, Borobio Virginia2, Margarit Ester1, Vallecillos Comparison of two academic software (RAPIDR M. Angeles3, Sánchez Aurora1 and WISECONDOR) for aneuploidies detection Hospital Clínic Bioquímica i Genètica Molecular, using semiconductor sequencing data in a IDIBAPS Barcelona-Spain1 Hospital Clínic Medicina NIPT process. Fetal Barcelona-Spain2 Hospital Clínic Bioquímica i Genètica Molecular Barcelona-Spain3 Nicolas Chatron1, Laila El Khattabi2, Sacha Schutz3, Juliana Pipoli da Fonseca4,StéphanieBrun5,Paul Triploidy is one of the most frequent chromosome Gueguen3, Juliette Nectoux6, Arthur Sorlin7, Manal aberrations in first trimester spontaneous abortions, Quere8,SylvieBannwarth8,JohnBoudjarane9, but a few triploid conceptions survive into the second Amandine Campan-Fournier10,CélineBonnet7, trimester. Tetraploidy is also a common finding in Franck Letourneur4,ErwanGuichoux11,Arnaud early abortions, although its prevalence is much lower Lagarde9,CarolineSchluth-Bolard1,Philippe than triploidy. Among tetraploidies, 92,XXXY karyo- Jonveaux7,VéroniquePaquis-Flucklinger8,Michel types are extremely rare. To our knowledge only six Goosens6, Catherine Badens9, Michel Vidaud6, Benoit cases have been reported, five of them related to Arveiler12, Damien Sanlaville1, Jean-Michel Dupont2, hydatidiform moles. Their origin can not obviously Caroline Rooryck12, Claude Ferec3, Claire Bardel10 be due to an endoreduplication of the normal diploid Hospices Civils de Lyon, UCBL1 Service de Génétique set of chromosomes. The patient was referred for Lyon-France1 APHP-HUPC, Université Paris Descartes prenatal diagnosis due to the sonographic finding of Service de Cytogénétique, INSERM U1016 Paris- a polymalformed male fetus. An amniocentesis was France2 CHRU de Brest Laboratoire de génétique performed before TOP. The autopsy confirmed the moléculaire, INSERM U1078 Brest-France3 APHP- sonographic findings, and the pathological study of HUPC, Université Paris Descartes Plateforme the placenta did not show morphological alterations. génomique – Inserm U1016 Paris-France4 CHU In her next pregnancy, sonographic examination Bordeaux Maternité Centre Aliénor d’Aquitaine disclosed a missed abortion at 6 weeks, with a visible Bordeaux-France5 HUPC Hôpital Cochin Service de embryo. A chorionic villi sample was obtained for Biochimie et Génétique Moléculaire Paris-France6 cytogenetic analysis before evacuation. Macroscopic CHRU Nancy - Université de Lorraine Service de examination of the villi sample did not reveal molar génétique, INSERM U954 Nancy-France7 CHU de vesicular appearance. Nice Service de Génétique Médicale, Hôpital de QF-PCR and cytogenetic analyses were performed l’Archet II Nice-France8 CHU Timone Département de on amniotic fluid (first pregnancy) and chorionic villi Génétique Médicale Marseille-France9 Hospices sample (second pregnancy). A 69,XXY and 92,XXXY Civils de Lyon, UCBL1 Service de biostatistique, karyotypes were respectively found. QF-PCR results CNRS UMR 5558 Lyon-France10 INRA Cestas disclosed two maternal and one single paternal alleles Plateforme Génome Transcriptome de Bordeaux in the first pregnancy (digynic triploidy), and double Bordeaux-France11 CHU Bordeaux Service de maternal and double paternal contribution to the Génétique médicale Bordeaux-France12 S110 10th European Cytogenetics Conference 2015

Based on a statistical analysis of low coverage genome Claire Bardel11, Arnaud Lagarde9, Amandine Campan sequencing data, non-invasive prenatal testing of aneu- Fournier11, Franck Letourneur1, Philippe Jonveaux7, ploidies is being provided in a growing numbers of Véronique Paquis-Fluckinger8, Michel Vidaud6, countries. It has proved a major improvement versus Benoît Arveiler12,MichelGoossens6,Caroline classic screening strategies but still requires invasive Rooryck12, Damien Sanlaville4, Jean-Michel Dupont2, procedures when positive. Using 500 samples included Claude Ferec5 in our French multicenter study, we aim to validate two different published bioinformatics tools for aneuploidy Inserm U1016 Plateforme Génomique Paris-France1 calling. All patients included had an indicated invasive APHP-HUPC, INSERM U1016, Université Paris sampling to achieve fetal karyotype in parallel. Descartes Service de Cytogénétique Paris-France2 WISECONDOR (WIthin SamplE COpy Number CHU Bordeaux Maternité Centre Aliénor d’Aquitaine aberration DetectOR) and RAPIDR (Reliable Accurate Bordeaux-France3 HCL, UCBL1 Service de Génétique Prenatal non-Invasive Diagnosis R package) both Lyon-France4 CHRU de Brest Laboratoire de Génétique require a reference set of euploid samples. RAPI Moléculaire, INSERM U1078 Brest-France5 HUPC DR establishes a baseline for confrontation with Hôpital Cochin Service de Biochimie et Génétique unknown samples. WISECONDOR uses a “within Moléculaire Paris-France6 CHRU Nancy-INSERM sample” normalization algorithm made from the U954-Université de Lorraine Service de Génétique reference set and preventing from inter-individual Nancy-France7 Hôpital de l’Archet II, CHU de Nice variation. They both use a bin segmentation approach to Service de Génétique Médicale Nice-France8 CHU de take into account the GC-content bias. Different QC Marseille Département de Génétique Moléculaire metrics are used. Fetal fraction, a well-known cause Marseille-France9 INRA Cestas Plateforme Génome for false negative, can be estimated for male preg- Transcriptome Bordeaux-France10 HCL, CNRS UMR nancies via RAPIDR. WISECONDOR uses an inter- 5558, Université Lyon 1 Service de biostatistique chromosomal concordance test to reduce technical Lyon-France11 CHU Bordeaux Service de Génétique noise. A training set of 50 samples has shown Médicale Bordeaux-France12 comparable performances regarding False Negative and False Positive rates for main aneuploidies (T21, Combined first-trimester screening has improved prena- T18, T13). The study will focus on the practicality tal screening for trisomy 21. However the number of for use in routine diagnosis (time of calculation, unnecessary invasive diagnostic procedures still necessary resources, setting up), reliability of QC remains high. Non-Invasive Prenatal Testing (NIPT) metrics and potential discrepancies. using massively parallel sequencing of cell-free fetal Both applications were initially developed using data DNA from maternal plasma, which is now part of the from Illumina dye sequencing technologies. In this prenatal landscape, should drastically diminish the risk study we will demonstrate that semiconductors associated with invasive techniques. Several publica- sequencing data fit to these two turnkey methods. tions have established NIPT effectiveness using mainly the Illumina sequencing technology. 3.P5 AFrenchconsortiumofsevenacademichospitalscol- laborates to validate a commonprotocolandtoevaluate Non-Invasive Prenatal Testing for the most common the efficiency and reliability of NIPT of the most common aneuploidies (trisomies 21, 18, and 13) using a chromosomal aneuploidies using a semiconductor- semiconductor-sequencing platform: a French mul- sequencing platform. Indeed many French laboratories ticenter pilot study are already equipped with this technology. A total of 500 pregnant women (between 12.3 and Juliana Pipoli da Fonseca1, Laïla El Khattabi2, 35 weeks of gestation) who presented a high risk of Stéphanie Brun3, Nicolas Chatron4, Paul Gueguen5, aneuploidy and underwent fetal karyotyping were Juliette Nectoux6, Florent Dumont1, Sacha Schutz5, included in a prospective study. 15 % of these Arthur Sorlin7,ManalQuere8,JohnBoudjarane9, patients presented a fetus with one of the most Erwan Guichoux10,SylvieBannwarth8,Céline common aneuploidies: trisomies 21, 18 and 13. The Bonnet7, Catherine Badens9, Caroline Schluth Bolard4, NIPT results matched the fetal karyotyping results in 10th European Cytogenetics Conference 2015 S111 all of the cases: all trisomies were detected. The analysis (2.7 %) and one 46,XY/47,XY,+20. In the same period of the whole genome sequencing data (including nota- we found 33 other cases with T21 by PD. PD was bly librairies quantification, total raw reads per sample, performed because of echographic anomalies (18 cases), estimate of plasma fetal DNA fraction) enabled us to advanced maternal age [AMA] (6 cases), nuchal trans- establish the quality criteria required for its use in rou- lucency [NT] (5 cases), AMA+NT (3 cases), and a tine diagnosis. previous child with T21 (1 case). Our results— 1.54 % NIPT using a semiconductor-sequencing platform is correlation between BPS and PD— could be explained a rapid and cost-effective alternative to invasive proce- by errors in storage or manipulation of blood samples, dures, and represents an attractive approach for large wrong interpretation of results or gestational age. All scale population NIPT. these aspects must be improved in the future for amelioration of the detection of T21 in pregnancies without 3.P6 high risk.

Correlation between biochemical prenatal screening 3.P7 for trisomy 21 and the results of prenatal chromosomal analysis – retrospective study on the 11 years Aneuploidy screening using circulating fetal cells in (2004–2014) in the „ Cuza Vodă”Maternity Iasi, maternal blood by dual-probe FISH protocol Romania Giuseppe Calabrese1,DonatellaFantasia1,Melissa Eusebiu Vlad Gorduza1, Violeta Martiniuc2, Mircea Alfonsi1, Elisena Morizio1, Claudio Celentano2, Paolo Onofriescu3,ElenaMihălceanu3,GheorgheIliev4, Guanciali Franchi1, Giandomenico Palka1, Peter Benn3, Dragoş Nemescu3,MihaelaGrigore3,Mihaela Gianmaria Sitar4 Grămescu1 University of Chieti Genetica Medica Chieti-Italy1 ASL University of Medicine and Pharmacy Medical Genetics Chieti Hospital Clinica Ostetrica e Ginecologica Chieti- Iasi-Romania1 Obstetric and Gynecology Hospital Italy2 University of Connecticut Health Center Prenatal Diagnosis Iasi-Romania2 University of Farmington Farmington, CT-United States3 Università Medicine and Pharmacy Obstetric and Gynecology Pavia Policlinico S. Matteo Pavia-Italy4 Iasi-Romania3 Obstetric and Gynecology Hospital Obstetric and Gynecology Iasi-Romania4 Background: A long sought goal of medical genetics has been the replacement of invasive procedures for We made a retrospective study concerning biochemical detection of chromosomal aneuploidies by isolating prenatal screenings (BPS) for trisomy 21 (T21). The and analyzing fetal cells or free fetal DNA from study is based on the data from Genetic Department of maternal blood thereby avoiding any risk to the “Cuza Vodă” Maternity Iaşi, Romania and covers the fetus. However, a rapid, simple, consistent and low period 2004–2014. BPS was made by triple test (TT) cost procedure suitable for routine clinical practice double test (DT) and association of these analyses (TT+ has not yet been achieved. The purpose of this study DT). Prenatal diagnosis (PD) was made by by FISH (all was to assess the feasibility of predicting fetal aneu- period) and/or karyotyping (in the last three years). ploidy by applying a dual-probe FISH protocol to During this period 1974 prenatal analyses were made. fetal cells isolated from maternal blood. 1034 (52.38 %) of them were performed because of an Methods: 102 pregnant women received prospective abnormal BPS for T21: 678 TT, 319 DT, 37 cases TT+ testing for fetal aneuploidy through FISH analysis of DT. In cases with abnormal BPS we found a chromo- fetal cells isolated from maternal blood. Results were somal anomaly in 29 cases (2.8 %), including 16 T21 compared with the karyotype determined by invasive (1.54 %). For abnormal TT we identified 16 chromo- procedures, or at birth. somal anomalies: 7T21 (1.05 %), 5 trisomies 18, 3 cases Results: Five out of 102 samples showed a fetal of XXY and one balanced translocation t(11;22). For aneuploidy which was confirmed by invasive pre- abnormal DT we found 11 chromosomal anomalies: 8 natal diagnosis procedures. In pregnancies with an T21 (2.19 %) 2 monosomies X, and one triploidy aneuploid fetus the frequency of trisomic cells (XXX). For abnormal TT+DT we identified 1 T21 (range, 0.38–0.90 %) was at least double that seen S112 10th European Cytogenetics Conference 2015 in samples from normal pregnancies (<0.18 %). Chromosome 7p duplication syndrome is a rare chro- One false positive and one false negative results mosomal disorder associated with typical craniofacial were obtained. anomalies: asymmetric skull, dolichocephaly, large Conclusions: Non invasive prenatal genetic screening fontanels, high forehead, micrognathia, low set malfor fetal aneuploidy is feasible using fetal cells isolated formed ears, hypertelorism. Features vary depending from maternal blood and this could substantially reduce on the size of the duplication and can include car- the need for invasive procedures. diac malformations, hypermobile joints and, in most cases, intellectual disability. 3.P8 The parents decided to terminate the pregnancy. Clinical features of the fetus and the origin of Fetus with two de novo chromosome aberrations: the extra X chromosome and the duplicated chro- XXX and duplication 7p mosome 7 will be discussed based on clinical examination of the aborted fetus and microsatellite Andreja Zagorac1, Ana Blatnik1, Faris Mujezinović2, analysis of genomic DNA extracted from fetal Boris Zagradišnik1, Alenka Erjavec Škerget1, Špela tissue as well as from blood of both parents. Stangler Herodež1, Nadja Kokalj Vokač1 University Medical Centre Maribor Laboratory of 3.P9 Medical Genetics Maribor-Slovenia1 University Medical Centre Maribor Department of Perinatology Unusually large familial euchromatic variant Maribor-Slovenia2 16p11.2 discovered at amniocentesis: a case report

Double chromosome aberrations involving both, a Triin Mölter-Väär1, Tatjana Zõrjanova1, Kati Kuuse1, de novo structural and a numerical aberration are Anu Tiidema1, Elvira Kurvinen1, Polina Verhovtsova1, rare. Airi Visamaa1, Kairit Joost1 We report on a case in which the de-novo duplication United Laboratories, Tartu University Hospital of an autosome and a sex chromosome aneuploidy were Department of Genetics Tallinn-Estonia1 detected. The patient was referred to our institution for fetal Aim. To report an unusually large familial euchromatic karyotyping due to a positive ultrasound examination at variant of 16p11.2 discovered in cytogenetic studies on 20th week of gestation: two soft markers for aneuploidy— amniotic fluid. asingleumbilicalarteryandanechogenicintracardiac Case. Amniocentesis in 42 year old woman was focus were noticed. QF-PCR for the rapid prenatal performed due to mother’s concern and complicated diagnosis of fetal aneuploidies after amniocentesis family and obstetric history. Cytogenetic studies by detected a sex chromosome aneuploidy: triple X. G-banding revealed an unusually large variant 16p+ The karyotype revealed an extra X chromosome and chromosome (p arm twice as long as normal 16p) in in addition a derivative chromosome 7: 47,XXX,der(7), the fetus. The finding was specified using FISH with with additional genetic material on 7p. chromosome 16 specific probes (Cytocell) and the ge- FISH using a subtelomeric 7ptel DNA probe showed nomic content of the duplicated region was clarified a normal FISH pattern. To define the rearranged 7p using chromosomal SNP microarray analysis (Illumina region Array-CGH was performed indicating three Inc.). Chromosomal microarray analysis could copies of 7p21.3p22.3, 7.65 Mb in range and con- identify merely ~1,4 Mb triplication of harmless firmed an extra X chromosome. euchromatic region suggesting the presence of After aCGH, the karyotype was re-written: heterochromatic material as well. Cytogenetic studies 47,XXX,der(7).arr (X)x3,7p22.3p21.3(1,370,653- of parental peripheral blood revealed the phenotypi- 9,023,662)x3 cally normal father to be a carrier of var(16p+). The clinical features of triple X syndrome are subtle Conclusion. It is important to differentiate and exclude and can be variable: tall stature, normal sexual develop- harmless euchromatic variants of proximal 16p11.2 from ment and puberty. Mental retardation is rare but learning microscopically similar pathogenic 16p11.2-p12.2 dupli- disabilities are quite common. cations. In case of documented familial 16p+variation, 10th European Cytogenetics Conference 2015 S113 that is ascertained with chromosomal microarray scan at 22 weeks was normal and the couple was analysis, the predicted outcome of the pregnancy counselled about the possible outcomes; they decided to can be reported as normal. continue the pregnancy. Results from postnatal testing are awaited. 3.P10 We will review the different tests (NIPT, QFPCR and chromosome analysis) comparing their Limitations of NIPT: a case of confined placental methodologies and potential impact on prenatal mosaicism diagnosis. We highlight the importance of understanding the positive predictive values of a screen- Abhijit Kulkarni1, Madalena Avila1, Celia Donaghoue2, ing test such as NIPT. Celia Brown2, Ramesh Ganapathy3, Rohan Taylor1, Kathy Mann2 3.P11 SW Thames Regional Genetics Laboratory, St George’s University Hospitals, NHS Foundation Trust Genetics Chromothripsis detected by arrayCGH in a prenatal London-United Kingdom1 Guy’sandStThomas’ case Hospital NHS Foundation Trust Viapath Analytics London-United Kingdom2 Epsom and St. Helier Elisabet Lloveras1,AnnaCanellas1,Vincenzo University Hospitals, NHS Trust Fetal Medicine Unit Cirigliano1,CarmenMartin2,MonicaMenendez3, London-United Kingdom3 Laura Barranco1, Elena Ordoñez1, Begoña Mendez1, Daniel Fernández1, Meritxell Pique1, Alberto Plaja4 Non-invasive prenatal testing (NIPT) is becoming LABCO-Iberia genètica i genètica molecular increasingly popular with companies emphasizing Barcelona-Spain1 LABCO-Iberia genética y high sensitivity and specificity as marketing tools. genética molecular Madrid-Spain2 Hospital de These statistics may be misunderstood; especially Fuenlabrada Ginecología Madrid-Spain3 LABCO- in the case of high risk results where a positive Iberia. Hospital Vall d’Hebron genètica i genètica predictive value would be a more useful measure. molecular. Area de Medicina molecular y genética There may be discordance between the result of Barcelona-Spain4 the NIPT and the fetal karyotype due to feto- placental mosaicism. Mosaicism is detected in The term chromothripsis accounts for an extraordinary about 1 % of chorionic villus samples taken at one-step catastrophic genomic event that generates mul- 10 to 11 weeks. tiple complex chromosome rearrangements within one, We present a case of a discrepant NIPT result due to or a handful, of genomic regions. To date, the causes and confined placental mosaicism. A chorionic villus the mechanisms of formation of these events remains sampling was performed in a 45 year old female unclear. following a high risk NIPT result for trisomy 18. We present the first prenatal case of chromothripsis The QFPCR aneuploidy test indicated mosaicism for on chromosome 18 identified by array-CGH. The trisomy 18; results were consistent with a trisomy 18 parental origin of the restructured chromosome conception followed by trisomy rescue generating a was investigated by QF-PCR and further charac- disomic cell line, as the abnormal cell line was terization of the rearranged chromosome was present in about 80 % of the cells in the sample. achieved by FISH and conventional cytogenetic Karyotype analysis of multiple chorionic villus cul- analyses. tures confirmed the mosaicism detected by QFPC A38-years-oldwomanwasreferredforprenatal R; 24 out of 31 cells studied showed trisomy 18. cytogenetic diagnosis at 12 weeks of gestational age An amniocentesis was performed to determine the because of increased nuchal translucency (4 mm) and status of the fetus. QFPCR analysis of the heavily high risk first trimester screening (1:3 for trisomy 21 and blood stained amniotic fluid showed two geno- 1:422 for trisomy 18). Chorionic villus sample was types consistent with MCC, however chromosome obtained and QF-PCR resulted in a female fetus with analysis of 50 cells from two independent cultures normal chromosome 13, 18 and 21copy number. Cell yielded a normal male karyotype. An ulta sound culture failed and no cytogenetic result was available. S114 10th European Cytogenetics Conference 2015

Nuchal translucency of 7 mm and others ultrasound Array-CGH analysis on DNA from cultured markers were detected at 16 weeks of gestation. Amniotic amniocytes characterized two mosaic alterations of fluid sample was collected and QF-PCR, conventional chromosome 18 (one case with triplication of 14 Mb at cytogenetic, FISH and array-CGH results detected a 18(p11.32p11.21) and the other case with an amplifica- highly rearranged dicentric chromosome 18 with loss of tion involving whole 18p and part of 18q of about the entire short arm and multiple gains and losses of the 26 Mb and a 17 Mb amplification at 6(p12.1q13). long arm, with at least 11 breakpoints. The majority of sSMC are derived from the acrocen- The present case highlights the usefulness of tric chromosomes. The sSMC derived from other auto- performing array-CGH in prenatal samples. somes, as the ones in the present study, generally have poor genotype-phenotype correlations, with phenotypes 3.P12 ranging from normal to severely abnormal, being their euchromatic content the most accurate predictor of Molecular characterization of prenatally detected phenotype. small supernumerary marker chromosomes: Array-CGH analysis is an effective tool to characterize improving the genotype-phenotype correlations sSMC. Using this approach the origin and euchromatic content of sSMC can be identified. The measurement Isabel Marques Carreira1, Marta Coelho Pinto1, Ana of the sSMC size, genetic material and associated Jardim1, Susana Isabel Ferreira1, Lúcia Simões1, Nuno genes allows the improvement of prenatal genetic Lavoura1, Alexandra Mascarenhas1, Eulália Galhano2, counseling with a more accurate delineation of the Helena Gonçalves3, Fabiana Ramos4, Joana Barbosa genotype-phenotype correlation. Melo1 Faculdade de Medicina da Universidade de Coimbra 3.P13 Laboratório de Citogenética e Genómica Coimbra- Portugal1 Centro Hospitalar e Universitário de Dealing with de novo apparently balanced rear- Coimbra EPE Maternidade Bissaya Barreto Coimbra- rangements in the era of Array-CGH Portugal2 Centro Hospitalar e Universitário de Coimbra EPE Maternidade Daniel de Matos Coimbra-Portugal3 Ana Jardim1,JoanaBarbosaMelo1,Alexandra Hospital Pediátrico de Coimbra, Centro Hospitalar e Mascarenhas1, Luís Miguel Pires1, Nuno Lavoura1, Universitário de Coimbra EPE Serviço de Genética Fabiana Ramos2, Eulália Galhano3,IsabelMarques Médica Coimbra-Portugal4 Carreira1 Faculdade de Medicina da Universidade de Coimbra Small supernumerary marker chromosomes (sSMC) are Laboratório de Citogenética e Genómica Coimbra- defined as structurally abnormal chromosomes that can- Portugal1 Hospital Pediátrico Coimbra, Centro not be characterized by conventional banding cytoge- Hospitalar e Universitário de Coimbra EPE Serviço de netics and are generally equal in size, or smaller, than the Genética Médica Coimbra-Portugal2 Centro Hospitalar chromosome 20 of the same metaphase spread. The e Universitário de Coimbra EPE Maternidade Bissaya identification of a sSMC at prenatal diagnosis is a great Barreto Coimbra-Portugal3 challenge for genetic counseling because of missing or little clinical and prognostic information. Array-CGH De novo apparently balanced chromosome rearrange- (aCGH) has been shown to be a valuable tool in genetic ments (dnABCRs) are detected in approximately 1:8001 counseling for prenatally detected sSMC allowing the to 1:10002 prenatal tests. Whenever a dnABCR is iden- identification of sSMC, its euchromatic content and the tified, the outcome for the fetus is difficult to predict associated genes involved, contributing for a better because there is no evidence for a normal phenotype in a genotype-phenotype correlation. carrier parent and the rearrangement may not be truly Three cases of mosaic de novo sSMC, detected by balanced. Phenotypic abnormalities observed in the conventional cytogenetics, were analysed, during the patients with dnABCRs can be due to 1) submicro- last year, with Agilent whole genome array; two of them scopic deletions or duplications near the breakpoints, using the 180 K oligonucleotide array-CGH and the 2) submicroscopic deletions or duplications unrelated other with the 60 K oligonucleotide array-CGH. to the ABCR, 3) gene disruption at the breakpoints, 10th European Cytogenetics Conference 2015 S115

4) a position effect, 5) uniparental disomy, or 6) Chorionic villus sampling performed at 11 weeks another unidentified genetic or environmental factor. showed a normal female karyotype 46,XX. Persistence We report on five recent prenatal cases carrying a of cystic hygroma in two other ultrasound examinations dnABCRs detected with conventional karyotyping. leads the medical team to sample amniotic fluid at Three of the cases had reciprocal translocations, one 18 weeks. Conventional karyotyping confirmed an had a pericentric inversion and another presented both apparently normal 46,XX karyotype. An oligonu- an inversion and a translocation. Array-CGH was per- cleotide based-array comparative genomic hybridi- formed to search for genomic imbalances that were zation Agilent 105 K was performed in order to revealed in two of five the cases. Only one of the detect any imbalance. Array-CGH showed a de imbalances identified was near one of the breakpoints novo 2.5 Mb deletion in 5q14.2q14.3, confirmed and all the imbalances detected were de novo. by fluorescent in situ hybridation (FISH). This Interpretation of dnABCRs in a prenatal setting and deletion included the VCAN gene. the ensuing genetic counseling is challenging due to the Heterozygous mutation of the VCAN gene is limited resolution of routine cytogenetics. The cases associated with vitreoretinopathy (Wagner syndrome presented provide further evidence that in case of pre- OMIM#143200 and erosive vitreoretinopathy). The natally detected dnABCRs, array-CGH is useful not penetrance of Wagner syndrome is complete and only in identifying the genomic imbalances at the associated with variable phenotype including retinal breakpoints, but also in detecting unexpectedly complex detachment, myopia, presenile cataract and progres- rearrangements in other chromosomes. sive chorioretinal atrophy. Wagner syndrome was Seeing as the increased risk for phenotypic anomalies associated with heterozygous mutation of the has been mainly attributed to the gain or loss of genetic VCAN gene but no deletion was reported. material, the diagnosis of these imbalances is paramount After genetic and ophthalmologic counseling, the and, therefore, in prenatal diagnosis, array-CGH should patients decided to continue with the pregnancy. At always be proposed to fetuses with dnABCRs, even if birth, the child will be referred to an ophthalmologist not associated to malformations, so that an accurate risk for medical supervision. assessment can be provided. 3.P15 3.P14 Prenatal diagnosis of Simpson-Golabi-Behmel Prenatal diagnosis of unexpected de novo 5q14 dele- Syndrome Type 1 by array CGH in a second trimes- tion encompassing VCAN gene identified by array- ter twin pregnancy CGH in a fetus with cystic hygroma Denise Morotti1,SilviaMotta1,MarcoBaccarin1, Naud Marie-Emmanuelle1,MetayCorinne2,Tosca Rosamaria Silipigni1,SimonaBoito2,Federica Lucie1, Colmant Claire3, Senat Marie-Victoire3, Folliot Natacci3, Silvana Guerneri1 Patrick2,PineauDominique2,TachidjianGérard1, Foundation IRCCS Ca’ Granda Ospedale Maggiore Brisset Sophie1 Policlinico Lab of Medical Genetics Milan-Italy1 AP-HP Hôpital Antoine Béclère, Université Paris Sud Foundation IRCCS Ca’ Granda Ospedale Maggiore Histologie-Embryologie-Cytogénétique Clamart- Policlinico Division of Prenatal Diagnosis Milan- France1 AP-HP Hôpital Antoine Béclère Histologie- Italy2 Foundation IRCCS Ca’ Granda Ospedale Embryologie-Cytogénétique Clamart-France2 AP-HP Maggiore Policlinico Medical Genetic Unit Milan- Hôpital Bicêtre Gynécologie Obstétrique Le Kremlin Italy3 Bicêtre-France3 Simpson-Golabi-Behmel type 1 (SGBS1) is a rare X- A 31 year old woman was referred to our Prenatal linked syndrome characterized by multiple congenital Diagnosis Center at 11 weeks gestation for fetal cystic abnormalities, pre-postnatal overgrowth and a wide hygroma measuring 6.6 mm. This pregnancy occured spectrum of clinical manifestations. The prevalence after 2 early miscarriages and the couple was non of the condition is unknown, since milder cases consanguineous. probably are underdiagnosed. S116 10th European Cytogenetics Conference 2015

SGB1 is caused by a deletion or mutation in the Materials and Methods: Array-CGH was performed GPC3 gene which maps to Xq26.2. in 100 prenatal samples. The indications for the analysis Here we report a case of a twin bichorionic were diverse, ranging from ultrasound anomalies/major pregnancy of a 30 years old woman who abnormalities, medical interruptions due to major anom- underwent CVS chromosomal analysis (13.3 week) alies, carrier progenitors of genomic imbalances and due to an increased nucal traslucency(NT) in one even to clarify conventional cytogenetic findings. fetus. Karyotype analysis revealed a 46,XY result Array-CGH was performed using Agilent oligonucleo- in both fetuses. A further US control at 17 week tide 180 K in DNA obtained in the majority of the detected in the first twin heart defects, hyperechogenic samples from amniotic fluid and chorionic villus, but kidneys, ventricularmegalia and hypospadia. For these also from fetal blood and skin biopsy of the death fetus. reasons a CGH array (Agilent, SurePrint G3 Human Each sample was hybridized against a sex-matched CGH Microarray Kit,60 k) was performed revealing a commercial control and the analysis was performed to 152.8 kb deletion, involving exons 4-5-6-7 of GPC3 detect imbalances above 400Kb in size, except in the gene at Xq26.2 band in the malformed fetus, the cases where a specific familiar imbalance was being second fetus was found to be normal while the mother, evaluated. showing a mild phenotype, resulted to be a carrier of the Results: Of the samples analyzed due to cytogenetic same deletion. After genetic counselling, the couple findings, we were able to: characterize marker and opted for a selective abortion. We report a detailed derivative chromosomes, exclude genomic imbal- description of the relation between the size of the dele- ances in translocations and inversions, detect geno- tion, the ultrasound findings as well as the postmortem mic imbalances in apparently balanced translocations examination. and detect mosaic trisomy for chromosomes 13 and Our case confirmed that SGBS1 could have a recog- 21 in a 13;21 Robertsonian translocation. Of the nizable prenatal phenotype and could be diagnosed also samples tested due to carrier progenitors of genomic in the second trimester giving the couple the basis of an imbalances, we defined the origin of the imbalance appropriate genetic counselling. A better knowledge of in most of the cases. In the remaining samples, SGBS1 phenotype could address more efficiently the imbalances were observed: maternal, paternal, de molecular testing. novo and 1 imbalance resulting from a maternal balanced translocation. 3.P16 Conclusions: Array-CGH is valuable not only to establish a diagnosis in samples with ultrasound anom- Array-CGH in prenatal diagnosis—reflexions over alies but also to characterize cytogenetic findings, show- the first 100 cases ing to be a good tool for genetic counseling. The use of CGH array in high-risk pregnancies in conjunction with Alexandra Mascarenhas1, Susana Isabel Ferreira1, Luis the karyotype analysis seems to be best strategy in Miguel Pires1, Cláudia Pais1, Patrícia Paiva1, Miguel prenatal diagnosis. Branco2, Ana Isabel Rei2, Joana Barbosa Melo1, Isabel Marques Carreira1 3.P17 Faculdade de Medicina da Universidade de Coimbra Laboratório de Citogenética e Genómica Coimbra- Routine prenatal diagnosis in a tertiary public hos- Portugal1 Centro Hospitalar e Universitário de pital: first 3 years of experience using CGH- Array Coimbra EPE Maternidade Bissaya Barreto Coimbra- Portugal2 Marina Giralt1, Carmen Mediano1, Cristina Hernando2, Carlota Rodó3,MªÁngelesSánchez-Durán4,Mar Introduction: Conventional cytogenetics has been the Borregán1, Eduardo Tizzano1 main approach for the detection of chromosomal abnor- Hospital Vall Hebrón Genètica Barcelona-Spain1 malities in prenatal diagnosis, however is being supple- qGenomics Laboratory Genètica Barcelona-Spain2 mented and in some cases, especially in cases with Hospital Vall Hebrón Unitat Medicina Fetal Barcelona- ultrasound anomalies, replaced by chromosomal micro- Spain3 Hospital Vall Hebrón Unitat Diagnòstic Prenatal array analysis. Barcelona-Spain4 10th European Cytogenetics Conference 2015 S117

CGH-Array has had a major impact on genetic diagnosis Praenatal-Medizin Muenchen Human Genetics Munich- over the last years. As a public tertiary hospital our Germany1 Service routinely offers QF-PCR and karyotype to all prenatal invasive tests. The use of CGH-array technology Complete fetal trisomy 16 results in early miscarriage. has been progressively introduced from 2012 onward. Only fetal mosaic trisomy 16 is compatible with surviv- Data collected between January 2012 and December ing beyond the first trimester of pregnancy. Trisomy 16 2014 account for 324 CGH-array tests out of 1675 inva- has recently been included in noninvasive prenatal tests. sive procedures (19.42 %). Due to trisomy rescue events maternal uniparental Referral indications (CGH-array group) included fetal disomy (UPD) is relatively common und must be inves- structural abnormalities detected on ultrasound (83.6 %), tigated. There is a high risk of abnormal outcome even genetic family history (14.4 %) and others (2 %) in cases with confined placental mosaicism without 60 K custom oligonucleotide-based CGH array UPD 16. Intrauterine growth retardation, fetal death, (qChip Pre v1.1 Complete), that allows an average preeclampsia, preterm delivery, neonatal death, devel- resolution of 100–125 Kb in regions causing genomic opmental delay, congenital heart defects and additional disorders, was used over fresh/cultured AF(n=287) or minor anomalies can be observed. Abnormal serum CVS(n=37) samples. Results were obtained at an aver- parameters in first trimester screening with a very low age time of 7 days. level of PAPP A are correlated with trisomy 16. Pathologic results including QF-PCR+karyotype We present detailed ultrasound, cytogenetic and UPD and CGH-array were found in 48 cases in 2012 test results together with outcome data of 17 pregnancies (8.05 %) and 7 of them were exclusively diagnosed by with trisomy 16 seen in a single center over the last CGH-array (increasing rate 1.17 %). 20 years. There were only 6 live births, all with severe 52 positive cases in 2013 (9.98 %) showed only 4 of intrauterine growth retardation and other pregnancy diagnosed by CGH-array (increasing rate of 0.77 %). complications. Intrauterine death happened in three Finally, from 60 positive cases in 2014 (10.58 %) only 4 cases. In the remaining 8 cases pregnancies were termi- were diagnosed by CGH-array (increasing rate of nated. Detailed pathology reports will be presented and 0.71 %). results will be compared with reported cases. Global results give us a 0.9 % incremental yield of Pregnancies with prenatally diagnosed trisomy 16 are pathological detection by CGH-array within the context at high risk of pregnancy complications especially of all prenatal diagnosis procedures and a 4.6 % consid- severe growth retardation and adequately monitoring ering the 324 CGH-arrays. Among pathological results is difficult. Extensive counselling has to be provided with CGH-array there was a significant incidence of to help the patients understand the many implications cases with cardiac and neural tube defects (46.7 %). It this diagnosis has for the ongoing pregnancy. was also very useful for familial reciprocal translocations carriers given faster results in comparison with 3.P19 karyotype. We conclude that although CGH-array represents a Deletion 19p13.3 and duplication 19q13.3qter in valuable diagnostic tool, indications for testing in the Prenatal Diagnosis—a case report context of prenatal diagnosis should be carefully evaluated with reliable and debated protocols. Manuela Mota Freitas1,SilviaPires2,Cristina Candeias2,NataliaOlivaTeles1,MariadoCéu 3.P18 Rodrigues3, Maria da Luz Fonseca e Silva2 Centro de Genética Médica Doutor Jacinto Prenatal diagnosis of trisomy 16: Difficult counsel- Magalhães/Centro Hospitalar do Porto, EPE/Unidade ling and poor pregnancy outcome Multidisciplinar de Investigação Biomédica Genética Porto-Portugal1 Centro de Genética Médica Doutor Cornelia Daumer-Haas1,DanielaLiebrecht1,Vera Jacinto Magalhães/Centro Hospitalar do Porto, EPE Gloning1,MatthiasLindner1,MoneefShoukier1, Genética Porto-Portugal2 Centro Materno Infantil do Christof Bagowski1,SabineMinderer1,Thomas Norte, Centro Hospitalar do Porto, EPE Consulta de Schramm1, Karl- Philipp Gloning1 Diagnóstico pré-natal Porto-Portugal3 S118 10th European Cytogenetics Conference 2015

Introduction: The presence of, concomitantly, duplica- Current single-cell PCR or FISH based PGD-assays tion and deletion of different regions in the same chro- require family-specific designs and labor-intensive mosome is considered a complex chromosomal rear- workup. Array comparativegenomichybridization rangement. Usually this type of anomaly results from a (aCGH)-based methods enable genome-wide aneu- meiotic crossing-over of a parental chromosomal inver- ploidy detection of large fragments, but do not allow sion and is defined as a recombinant chromosome. diagnosing single gene disorders or submicroscopic When it is “de novo” the abnormal chromosome is imbalances. considered a derivative. The authors present a rare case Here, we present the transition in the clinic from of a derivative chromosome 19 observed in prenatal traditional cytogenetic and molecular cytogenetic diagnosis. methods to a novel generic SNP-array-based method Material and methods: A pregnant woman was that allows us to detect in single blastomeres not only referred for karyotyping due to positive biochemi- the inheritance of monogenic disorders (recessive and cal screening, neural tube defects and fetal dominant) genome wide, but also chromosomal rear- malformations. Chromosome analysis with high rangements and aneuploidies, including their parental resolution GTL-banding was performed on meta- origin as well as the meiotic or mitotic nature of chro- phases obtained from cultured amniocytes. FISH mosomal trisomies. The developed analysis method studies included subtelomeric and whole chromo- called “haplarithmisis” is embedded in a broad compu- some painting probes, both specific for chromo- tational pipeline for single-cell haplotyping and imputa- some 19. MLPA techniques were performed using kits tion of linked disease variants (siCHILD). P036 and P070 (MRC-Holland) for the subtelomeric We will discuss results from clinical cases for differ- regions. ent indications including Mendelian disorders, chromo- Results: The fetal karyotype revealed a structurally somal imbalances and complex rearrangements. abnormal chromosome 19 with additional material This novel generic method not only facilitates the in the terminal band of the short arm. FISH using genetic selection of unaffected embryos, but also subtelomeric probes (Vysis) and whole chromo- broadens the range of classic PGD, enabling us to help some painting (Cytocell), both for chromosome couples that could not be assisted so far. 19, showed a 19pter deletion and a 19q13.3qter The ability to characterize the entire genome of duplication, confirmed by MLPA. Since parental asinglehumancellforSNPsandcopynumber karyotypes were normal, this anomaly was considered variants in a single assay is a great improvement “de novo”. in PGD practice. Discussion: We compare the clinical features in this fetus with the previously reported cases with either 3.P21 terminal deletion and/or a duplication of distal 19q. The authors enhance the importance of the combination Placental mesenchymal dysplasia: important differ- of high resolution banding with appropriate molecular ential diagnosis for partial moles and frequently cytogenetic techniques in the characterization of com- associated with Beckwith-Wiedemann syndrome plex chromosomal rearrangements. Ulrike A. Mau-Holzmann1,ThomasEggermann2, 3.P20 Susanne Mackensen-Haen3, Rangmar Goelz4, Andreas Tzschach5, Karl Oliver Kagan6, Sylke Singer1 Preimplantation Genetic Diagnosis: one assay that University of Tuebingen Medical Genetics & Applied fits all. Genomics Tuebingen-Germany1 RWTH Aachen, University Institut for Human Genetics Aachen- Cindy Melotte1, Eftychia Dimitriadou1, Masoud Zamani Germany2 University of Tuebingen Institute for Esteki1, Sophie Debrock2, Thomas D’Hooghe2, Thomy Pathology Tuebingen-Germany3 University of de Ravel1, Thierry Voet1, Joris Vermeesch1 Tuebingen Neonatology Tuebingen-Germany4 Center for Human Genetics UZ Leuven Leuven- University of Dresden Institute for Human Genetics Belgium1 Leuven University Fertility Center UZ Leuven Dresden-Germany5 University of Tuebingen Department Leuven-Belgium2 for Gynecology Tuebingen-Germany6 10th European Cytogenetics Conference 2015 S119

Placental mesenchymal dysplasia (PMD) is a rare date, more than 40 cases with 3p25-p26 deletions have placental anomaly. Prenatal ultrasound indicates been described and only two, a mother and daughter, placentomegaly and cystic structures resembling a were reported without phenotypic effect. partial mole, but with apparently normal fetal mor- We presented a family with a 3p deletion in the phology. PMD is associated with intra-uterine growth mother and two children. restriction (IUGR), intra-uterine mortality, prematurity The 3p deletion was detected in a prenatal diagnosis and in about 20 % of the cases with Beckwith- of the third child. Cytogenetic analysis showed a female Wiedemann syndrome (BWS), respectively. karyotype with a subtelomeric deletion on the short arm The underlying cause is unusual and interesting as of chromosome 3, confirmed by FISH technique PMD is a special type of mosaicism. One part of the (subtelomeric probes for chromosome 3 - Vysis). placenta is normal with a biparental diploid genotype Cytogenetic and FISH studies were carried out on the (preponderance of females). The dysplastic part is also couple and another child. The same subtelomeric dele- diploid, but of paternal uniparental origin. tion was found in the mother and her son. Array analy- We present a case of a prematurely born female with ses revealed a loss of 7.4 Mb in 3p26.3p26.1. typical signs of BWS. PMD was suspected prenatally The son is now followed in a genetic counseling (confirmed pathologically). Molecular studies con- consultation and a mild mental retardation was firmed the diagnosis of BWS (UPD11pat) and observed. The daughter and the mother show a showed a genome wide paternal uniparental disomy normal development. in the majority of cells, with a low degree of bipa- The presence of the same chromosome deletion in rental cells in blood. normal as well as abnormal phenotypes has been inves- PMD should be considered in cases with partial tigated. Several models could explain the different phe- mole— especially if there are no major anomalies indi- notypes. These include regions of low gene density, cating triploidy. absence of dosage sensitivity, and functional redundancy Gynaecologists, cytogeneticists and pathologists of genes that have additional or related copies outside of should be aware of this rare differential diagnosis and the deleted region. initiate respective studies. The authors present a literature review of subtelomeric deletion of chromosome 3 and its implications. 3.P22 3.P23 Subtelomeric deletion of chromosome 3: what is it’s significance? Prenatal cytogenetic diagnosis by CGH array: our 2014 experience Marta Souto1, Pedro Botelho1, Regina Severo2, Marcia Martins3, Osvaldo Moutinho2, Rosário Pinto Leite1 Bruno Pichon1,ChristineHans1,NicolasLufin1, Centro Hospitalar de Trás-os-Montes e Alto Douro Catheline Vilain2, Anne De Leener2 Laboratorio de Citogenética Vila Real-Portugal1 ULB-Erasme laboratory of cytogenetics Brussels- Centro Hospitalar de Trás-os-Montes e Alto Douro Belgium1 ULB-Erasme Human genetics centre Brussels- Serviço de Ginecologia e Obstetricia Vila Real- Belgium2 Portugal2 Centro Hospitalar de Trás-os-Montes e Alto Douro Serviço de Genética Vila Real-Portugal3 Since 2014, karyotyping has been replaced by the high resolution CGH array method for cytogenetic analysis Subtelomeric deletions are responsible for birth defects of amniotic fluid. CGH array was performed on the 60 K and mental retardation and several syndromes have ISCA bluegnome slides. The resolution of the analysis already been described. The 3p deletion syndrome is was 400 kb in the backbone of the genome and higher in one of these disorders with a variable spectrum of the ISCA loci. defects depending upon the overall size of the deleted 544 cytogenetic analyses were performed on amni- segment. Characteristic features of distal 3p- syndrome otic fluid samples in our laboratory in 2014. Rapid FISH include low birth weight, microcephaly, trigonocephaly, aneuploidy screening test detected 29 viable autosomal hypotonia, psychomotor and growth retardation. To aneuploidies and 5 gonosomal aneuploidies. Cases with S120 10th European Cytogenetics Conference 2015 autosomal aneuploidies were confirmed by karyotype derived causes of error. Importantly, it differentiates and cases with gonosome aneuploidies were further high z-scores due to fetal trisomies from those due to analyzed by CGH array that didn’t detect any other local maternal CNVs causing false positives. genetic abnormality. Following routine clinical analysis of over 5000 pro- Among the 510 remaining cases, 414 cases (81.2 %) spective pregnancies, several other genomic imbalances were not reported: 404 cases were normal (only benign were found in addition to detection of the common CNVs), and 10 cases were consisted as susceptibility autosomal aneuploidies. These findings include (i) other CNVs not to be communicated in accordance with the (segmental) aneuploidies (0,3 % of cases), 4 of which Belgian guidelines and 2 cases (0.4 %) were not could be confirmed to be fetal (1 mosaic trisomy 16 and contributory. 1 mosaic trisomy 15 which contained a uniparental For the remaining 94 cases (18.4 %) the pathogenicity disomy cell line, 2 partial duplications 18 and 1 terminal of the CNVs was evaluated by at least 2 geneticists. 74 5p deletion) and (ii) maternal imbalances. Importantly, cases (14.5 %) were not reported because of lack of three aberrant genome representation profiles were evidence for pathogenicity. 20 cases (3.9 %) were consid- observed that could not be attributed to the mater- ered abnormal or possibly pathogenic and were reported. nal nor the fetal genomic constitution. Whole body Among the reported cases: 6 cases corresponded to defi- diffusion-weighted magnetic resonance imaging nitely pathologic CNVs (deletions of/in genes of known and subsequent pathologic and genetic investiga- autosomal dominant or X linked diseases and large deletion uncovered the presence of respectively an tions), 2 cases corresponded to susceptibility CNVs to be ovarian carcinoma, a follicular lymphoma and a communicated in accordance with the Belgian guidelines, nodular sclerosis classical Hodgkin lymphoma. 9casescorrespondedtounknownCNVswithpossible The copy number variations in those tumors were pathologic effects (duplications of part of genes of known concordant with the NIPT plasma profile. NIPT autosomal dominant or X linked diseases, high OMIM thus enables the accurate presymptomatic detection genes content) and 3 cases corresponded to carriers of of maternal tumors. These incidental findings are CNVs with possible pathologic effects (duplication of an unsuspected added benefit of NIPT. part of genes of known X linked diseases in females). In some cases, pathogenicity was re-evaluated after 3.P25 familial investigation. Prenatal finding of pericentric inversion of chromo- 3.P24 some 3

Incidental findings of genome wide non-invasive Pavel Tesner1,JanaDrabova1,MiroslavStolfa1, fetal aneuploidy detection: presymptomatic identi- Drahuse Novotna1, Eduard Kocarek1 fication of maternal cancers Charles University in Prague— 2nd Faculty of Medicine and University Hospital in Motol Dept. of Biology and Kris Van Den Bogaert1, Nathalie Brison1, Paul Brady1, Medical Genetics Prague-Czech Republic1 Luc Dehaspe1, Iwona Wlodarska1, Thomy de Ravel1, Hilde Peeters1, Hilde Van Esch1, Koen Devriendt1, Eric Every prenatal chromosomal finding of uncertain path- Legius1, Joris Vermeesch1 ogenicity is a very difficult situation in genetic counsel- University Hospitals Leuven Department of Human ling. Even postnatally, it is often impossible to clearly Genetics Leuven-Belgium1 correlate the cytogenetic finding and eventual phenotypic abnormality. Non-invasive prenatal testing (NIPT) for fetal aneuploidy We present a case of Czech patient with prenatal detection is increasingly being offered in the clinical finding of de novo pericentric inversion 46,XX,inv(3) setting following studies demonstrating high sensitivities (p11.2q21) in the amniotic cells. Amniocentesis was and specificities for trisomies 21, 18 and 13 detection. performed due to the advanced maternal age— there However, a baseline false positive and false negative rate were no other risk indicators (apart from lower free ß- remains. We introduced an analysis pipeline which hCG level which was analogous to previous pregnancy addresses some of the technical and biologically- resulting in a cytogenetically normal healthy daughter). 10th European Cytogenetics Conference 2015 S121

There is only one similar case presented by Spedicato venosus) identified a high risk pregnancy and an inva- FS et al.: Pericentric inversion inv(3)(p11q21). [J Med sive test was proposed and accepted. Rapid test (FISH) Genet. 1984 Oct;21(5):396.] The inversion was detected of amniotic fluid showed two signals for chromosomes in a newborn with a prominent forehead and receding 7(7q11.23),13(13q14),18(18p11.1q11.1),21 chin, a left preauricular tag, a long philtrum, and pen- (21q22.13q22.2), 22 (22q11.2) and cell culture and dulous cheeks. Its familial occurrence in other relatives subsequent cytogenetic analysis, established a normal with normal phenotype (brother, father, paternal aunt female karyotype (approx. 400 bphs). and her daughter) emerged later with examination of At 18w, after information of the echocardiographic the family. findings (dysplastic pulmonary valve, pulmonary steno- In addition to the case from 1984, we got also the sis, moderate pulmonary insufficiency and pulmonary result of array comparative genomic hybridization trunk dilation, cervical lymphangiomas, persistence of which was normal, without any deletion in breakpoints nuchal translucency, and cerebral ventriculomegaly) or anywhere else. The pregnancy proceeds without any and their clinical significance, the couple decided to complication and apparently healthy daughter was born abort. in term in the middle of February. The prenatal cytoge- An aCGH (Agilent Technologies 8x60K) performed netic finding was verified at the moment and it will be on the fetal tissue and analyzed with CytoGenomics 2.9 extended by further molecular cytogenetic analysis of software revealed the presence of a 19,3 Mb deletion in the peripheral blood. The child will be clinically exam- 5p15.33-5p14.3 and a 14,5 Mb duplication in 11p15.5- ined thoroughly again before the conference (at her age 11p15.2. This result was confirmed by FISH on meta- of 4 months). All of these results will be presented on phase chromosomes, using MD Cri-du-Chat CTNND the poster. 5p15 (red)/5q31 (green) and ST 11pter (blue) probes, Our work is supported by the project (Ministry of and the formule was stablished as: 46,XX.ish Health, Czech Republic) for conceptual development of der(5)t(5;11)(p14.3;p15.2)pat(CTNND-,D11S1363+). research organization 00064203 (University Hospital The cardiopathy, lymphangiomas, and persistence of Motol, Prague, Czech Republic), Grant Agency of nuchal translucency may suggest a Noonan syndrome. Charles University (no. 264811), and Technology However, the exclusion of chromosome aberrations Agency of the Czech Republic (no. TA01010931). continues to be necessary in order to provide suitable genetic counseling. 3.P26 3.P27 Unbalanced translocation in a fetus with normal nuchal translucency in first trimester screening and Conflicting results QF-PCR and karyotyping due to normal prenatal karyotype structural aberration of the Y-chromosome

Estibaliz Santamaría1,BeatrizBarreña1,Alvaro K.B.M. Hansson1,N.S.denHollander1,M.vanHuizen2, Gorostiaga2,FernandoMozodeRosales2,Majo J. Lind2,L.Groenewegen1,M.Hoogenboom1,J. Fernandez-Mellado2, Laura Román1, Marian Fiaño1, Zonneveld1,M.J.V.Hoffer1 M. Asun Tapia1,EstherSarasola1, María García- LUMC Clinical Genetics Leiden-The Netherlands1 Barcina1 LUMC Gynaecology and Obstetrics Leiden-The H. Universitario Basurto (Osakidetza) Genetics Bilbao- Netherlands2 Spain1 H. Universitario Basurto (Osakidetza) Obstetrics Bilbao-Spain2 Quantitative fluorescent polymerase chain reaction (QF- PCR) is an accurate and efficient technique for rapid We describe the case of a pregnant woman with normal prenatal diagnosis of trisomy 13, 18 and 21 and aneu- nuchal translucency (NT) (<3 mm) in the first trimester ploidies of the sex chromosomes. Discordant results and a triple screening risk of 1/547. The secondary between QF-PCR and karyotyping have occasionally markers at 14 weeks of gestation (NT: 3,1 mm (>p99), been reported, mostly due to mosaicism. hypoplastic nasal bone, significant tricuspid regurgita- A 20-year old female underwent chorionic villus tion and the “a” wave absent/reversed in the ductus sampling at 13 weeks’ gestation because of an increased S122 10th European Cytogenetics Conference 2015 risk of a chromosomal aberration after first trimester had a normal karyotype, as did the parents. Further screening. Ultrasound investigation demonstrated no testing was done using MLPA and FISH techniques, in abnormalities. The results from the QF-PCR analysis order to clarify the abnormal chromosome 18. MLPA were interpreted as a possible mosaic 45,X/46,XY studies revealed a deletion of the subtelomeric because of the low contribution of the Y chromo- region of the long arm of chromosome 18 and a some. Conventional karyotyping of the LTC demon- duplication of the subtelomeric region of the long strated a non-mosaic 45,X. Since no abnormal findings arm of chromosome 17; FISH analysis confirmed were detected on ultrasound, a confined placental that the duplicated (17q) sequence was translocated mosaicism was suggested as a possible explanation into the long arm of the abnormal chromosome 18. for the discordant findings. A subsequent amniocen- In conclusion, this fetus had a der(18), due to an tesis revealed a normal male genotype with QF-PCR. initial translocation t(17;18)(q24;q21.3), resulting Karyotyping, on the contrary, demonstrated a mosaic in partial deletion of the long arm of chromosome pattern with 45,X and a structural rearranged Y chro- 18 (18q21.3→qter) and a partial duplication of the mosome, probably an i(Yp). Additional FISH con- long arm of chromosome 17 (17q24→qter). firmed the presence of an isochromosome of the A review of the literature indicated a recent and short arm of the Y chromosome. The case presented similar case, where the phenotype associated to the here demonstrates that caution should be taken when derivative chromosome 18 included mental retardation, conflicting results are observed in CVS— even if a several dysmorphic features and congenital anomalies. normal profile is obtained with QF-PCR in a follow-up Parents were offered adequate genetic counselling and amniocentesis. This case further illustrate that mosai- decided not to terminate the pregnancy. cism for an abnormal cell line can result in a normal QF-PCR profile. 3.P29

3.P28 Prenatal diagnosis of 16p duplication presenting with ultrasound abnormalities Derivative chromosome 18 in prenatal diagnosis: a case report Oguz Cilingir1, Ebru Erzurumluoglu, Halime Kucuk1, Melih Velipasaoglu2,HuseyinAslan1,Muhsin Fernanda Paula Oliveira1, Cristina Candeias1, Manuela Ozdemir1,AycaDemirel1,MerveSakaGuvenc1, Mota Freitas2,NatáliaOlivaTeles2,MariadoCéu Sevilhan Artan1 Rodrigues3, Céu R. Mota4, Maria da Luz Fonseca e Eskisehir Osmangazi University Faculty of Medicine Silva1 Department of Medical Genetics Eskisehir-Turkey1 Centro de Genetica Medica Jacinto Magalhães/ Centro Eskisehir Osmangazi University Faculty of Medicine Hospitalar do Porto, E.P.E. Unidade de Citogenetica Obsterics and Gynecology Eskisehir-Turkey2 Porto-Portugal1 Centro de Genetica Medica Jacinto Magalhães/ Centro Hospitalar do Porto, E.P.E./UMIB Duplication of the short arm of chromosome 16 is a Unidade de Citogenetica Porto-Portugal2 Centro rarely seen genomic instability with well described Materno Infantil do Norte/Centro Hospitalar do Porto, symptoms including mental and growth retardation, E.P.E. Consulta de Diagnostico Pre-Natal Porto- craniofacial, cardiac, urogenital abnormalities, finger Portugal3 Centro Materno Infantil do Norte/Centro anomalies and pulmonary vascular diseases. Most of Hospitalar do Porto, E.P.E. Consulta de Genetica the cases are derived from a chromosomal translocation, Porto-Portugal4 usually due to maternal rseciprocal translocation. A 19-year-old woman, gravida 2, abortus 1, was We report a twin pregnancy obtained by in vitro fertil- referred to our clinic at 13 weeks of gestation because ization (IVF) techniques referred to our center for pre- of abnormal USG findings including cystic hygroma, natal cytogenetic studies. Fetus 1 presented with a pos- significantly increased nuchal translucency (NT) itive biochemical screening. The conventional cytoge- (10 mm) and short limbs. Chorionic villi sampling netic analysis with GTL banding revealed a female fetus (CVS) was performed after genetic counseling. The with a structurally abnormal chromosome 18. Fetus 2 results of the QF-PCR rapid aneuploidy screening were 10th European Cytogenetics Conference 2015 S123 normal. Array CGH (Agilent, 60 K) analysis demon- gene, with or without YWHAE or CRK genes. The strated a 26 Mb duplication of 16p13.3- p12.1 current case, according to Bruno’s classification, belongs (1100695-27109354) and the findings were confirmed to the Class I 17p13.3 microduplications. by follow-up FISH analysis. Subsequent parental After genetic counselling the parents decided to ter- karyotyping revealed 46,XY and 46,XX,t(6;16) minate the pregnancy. Even though the most typical (q27q12.1) for father and mother, respectively. The characteristics for this syndrome such as developmental 6qter deletion could not be determined by array-CGH and psychomotor delay appear after birth, some of the but demonstrated by subtelomeric FISH analysis. The phenotypic features of the syndrome could be identified fetal karyotype was 46,XY,der(6)t(6;16)(q27q12.1)mat in the fetus. These were similar to some previously The pregnancy was terminated at 17 weeks. The pheno- described patients, including broad nasal bridge, typic assessment could not be performed due to exten- hypertelorism, micrognathia, fleshy earlobes, thin upper sive edema in abortion material. lip, cardiopathy. We could not confirm the hand/foot To our knowledge, coexistence of 16p13.3p12.2 anomalies (CRK is known to interact with signal path- duplication and subtelomeric 6q deletion has not been ways involved in limb development) nor cleft lip and described previously in the literature. Besides, the case palate (YWHAE has been implicated in the etiology of also emphasizes the importance of a detailed 11– orofacial clefts) often seen in Class I duplications. 14 week ultrasound assessment in diagnosing fetal chro- We report a fetus with a de novo 417 kb mosomal aberrations in combination with the modern microduplication 17p13.3. We made a genotype- aspects of array CGH, thus providing more precise and phenotype correlation between our case and patients rapid prenatal diagnosis. described in literature. The present case illustrates the importance of micro-array analysis in prenatal genetic 3.P30 diagnosis. Additional reports are needed for the complete delineation of the clinical spectrum of 17p13.3 Afetuswitha417kbdenovo17p13microduplication. microduplications and the genotype-phenotype correlation concerning Class I and II patients. A. Van Den Bogaert1, M. Leyder2, C. Staessen1, H. De Wilde2, E. Paeps1, I. Villacorta1, A. Monsaert1, S. De 3.P31 Winter1, G. Melotte1, E. Vandermot1, M. Breugelmans2, E. Denayer1, M. De Rademaeker1, K. Keymolen1, K. Cell-free fetal DNA extraction: evaluation of two Van Berkel1 methods based on silica-columns and magnetic UZbrussel Centre for Medical Genetics, Reproduction beads. and Genetics, Reproduction Genetics and Regenerative Medicine Brussels-Belgium1 UZbrussel Department of Paula Santibáñez1, María López1, A. Belén Rodríguez- Prenatal Medicine Brussels-Belgium2 Martínez2, Esther Sarasola2, Cristina Cervera-Acedo1, María J. García-Barcina2, Elena Domínguez-Garrido1 A 28 year old woman underwent amniocentesis because Rioja Salud Foundation-CIBIR Molecular Diagnostic fetal ultrasound revealed single umbilical artery at Unit Logroño-Spain1 Basurto University Hospital 12 weeks. At 16 weeks a cardiopathy including aortic Osakidetza Genetic Unit Bilbao-Spain2 valve stenosis, cardiomegaly and fibroelastosis and hyperechogenic bowel was diagnosed. The pregnancy Introduction: The presence of cell-free fetal DNA was obtained by intracytoplasmatic sperm injection after (cffDNA) in the maternal circulation offers a non- in vitro maturation of oocytes. invasive source of fetal genetic material for prenatal Using 44 K Agilent oligo array, we detected a 417 kb diagnosis. This includes fetal sex determination by spe- microduplication 17p13.3. This duplication concerns a cific Y chromosome sequence detection, such as SRYor de novo phenomenon and encompasses 6 genes, namely DYS14. This determination is particularly important YWHAE, ABR, CRK, TUSC5, MYO1C and BHLHA9 when a fetus may be affected by an X-linked disorder, . Bruno et al. (2010) proposed two classes of 17p13.3 such as haemophilia. Due to the low percentage of fetal microduplications. Class I involves YWHAE gene but DNA present in maternal plasma (<10 %) during early not PAFAH1B1, while class II involves PAFAH1B1 gestation, efficient extraction processes are required for S124 10th European Cytogenetics Conference 2015 successful downstream detection applications in non- We report here a case of discordant results between invasive prenatal testing (NIPT). NIPT and fetal karyotyping due to confined placental Objective: Evaluate, using qPCR, the cffDNA mosaicism of sex chromosomes. In addition, in this extraction efficiency of 2 different methods: QIAamp® case, an unexpected finding in the fetal karyotype DNA MIDI kit (method A) and Chemagic circulating required a second amniocentesis to reach the final NA kit (method B). conclusion about the prenatal result. Material & Methods: Circulating DNAwas extracted from 800 μL-plasma samples from 70 pregnant women 3.P33 carrying a male fetus. Protocols used followed manu- facturer’s instructions for method A and a modified False positive and false negative results of cell free protocol for method B. qPCR targeting GAPDH and DNA testing SRY genes was carried out in order to determine the total cell-free DNA (cfDNA) and the fetal fraction Seher Basaran1,AtilYuksel2,RecepHas2,Melike extracted, respectively. Ct values and standard devia- Kirgiz3, Tahir Dehgan3, Birsen Karaman1 tion were evaluated. Istanbul Medical Faculty Medical Genetics Istanbul- Results: In terms of cfDNA, GAPDH assays showed Turkey1 Istanbul Medical Faculty Gynecology and Ct and SD values of 33.3 (0.76) by method A and 29.3 Obstetrics Istanbul-Turkey2 Premed Genetic Diagnosis (0.22) by method B. SRY assays yields a Ct and SD Center Medical Genetics Istanbul-Turkey3 values of 34.3 (1.33) for method A and 32.5 (0.30) and method B. No SRY amplification was obtained for 4 The most exciting development in prenatal practice is samples processed by method A. the introduction of cell free DNA testing (cf-DNA) for Conclusion: Modified-Chemagic circulating NA the screening of common aneuploidies, which offers protocol is the best approach for cffDNA from highest sensibility and specificity. Since cf-DNA maternal plasma. This modified protocol results originates from the cytotrophoblasts of the placenta, robust and accurate for both cffDNA and cfDNA it reflects the genotype of the cytotrophoblasts, extraction. which might differ from the fetus itself. Therefore, it’s mandatory to confirm the results by invasive 3.P32 approach. The percentages of false positive and negative results of cf-DNA are still not clear. Prenatal screening for aneuploidy in maternal Here we present our confirmation results of 33 inves- blood: an unexpected finding tigations with nine false positive and one false negative case. Concepcion Villalón Villarroel1, Eva García-Galloway1, 18/19 cases with a risk for trisomy 21 obtained M José Cabrejas Nuñez1, Irene Pelayo Delgado1, José by cf-DNA test were confirmed, one was normal M. García-Sagredo1, Miguel Angel Moreno Pelayo1 in amniocytes (1/19 false positive for trisomy 21). University Hospital Ramón y Cajal Genetics Department Another case, with pathological USG and normal Madrid-Spain1 cf-DNA, had mosaic trisomy 21 (70 %) by karyotyping (one false negative for trisomy 21). Noninvasive prenatal test (NIPT) for the most fre- 2/3 cases with a risk for trisomy 18 were con- quent foetal chromosomal abnormalities in mater- firmed, but one was not (1/3 false positive for nal blood is becoming the most demanded labora- trisomy 18). tory tool for pregnant women, replacing the clas- Two cases with a risk for trisomy 13 could not be sical prenatal invasive test. The cell-free fetal confirmed (2/2 false positive for trisomy 13). DNA in the pregnant woman’splasmaoriginates 2/5 cases with a risk for monosomy X were con- from the placenta. An abnormal NIPT result could firmed. The other three cases were normal in amniocytes therefore be due to confined placental mosaicism. by karyotyping and interphase-FISH (3/5 false positive This is one of the limitations of the test. All for monosomy X). abnormal NIPT results must be confirmed by a 1/3 case with Klinefelter syndrome was confirmed in conventional prenatal invasive test. amniocytes (2/3 false positive for 47,XXY). 10th European Cytogenetics Conference 2015 S125

Discordant findings between cf-DNA and karyotyping ChIP assay using H4 hyperacetylated antibody for can be explained by confined placental mosaicism etc. the 5′coding region of Prm1 and Prm2 genes, suggests a We believe that these cases and their clinical findings crosstalk between histone acetylation and DNA meth- should be reported to understand the biological mech- ylation. The regulation of histone deacetylase activity anism of discrepancy and also to determine the per- appears to play a major role in histone replacement centages of false positive and negative results, which during spermiogenesis and testes specific genes expres- are important in genetic counselling regarding cf-DNA sion in round spermatids. testing. 4.P2 4. Animal and Plant Cytogenetics Microdissection of chicken lampbrush chromosome 4.P1 regions for FISH-probes generation and high- throughput sequencing Distinct DNA methylation and chromatin dynamics of spermatogenic cell-specific genes in mouse Anna Zlotina1,NadezdaKosyakova2, Tatiana Kulikova1, Thomas Liehr2, Alla Krasikova1 Liliana Burlibasa1, Horatiu Paul Domnariu2, Carmen Saint-Petersburg State University Department of Domnariu3, Petru Armean4, Ioana Hasegan1 Cytology and Histology Saint-Petersburg -Russia1 University of Bucharest Genetics Department Jena University Hospital Institute of Human Genetics Bucharest-Romania1 Ulm University Faculty of Jena-Germany2 Medicine Ulm-Germany2 University of Sibiu, Lucian Blaga Faculty of Medicine Sibiu-Romania3 UMF Carol Microdissection represents a reliable approach for Davila Urology Department Bucharest-Romania4 investigation of chromosomes and their regions. Microdissection technique allows to get broad panels To understand the role of chromatin structure in of locus-specific and whole-chromosome painting the expression of protamine 1, protamine 2 genes FISH-probes that are widely used for chromosome during mouse spermatogenesis, we have examined identification, mapping of evolutionary and clinical the epigenetic profile of these specific genes cor- breakpoints, revealing the orthologous regions in related with global epigenetic changes during karyotypes of related species, and for investigation sperm differentiation. of spatial genome organization. Spermatogenic cells were obtained from 9- to 11- At the same time, keeping in mind relatively small week-old males by centrifugation on a BSA gradient. physical size of mitotic chromosomes of higher verte- The dynamics of H4 hypercetylation and DNA meth- brates, the standard microdissection procedure proves to ylation during murine spermatogenesis have been be inappropriate for analysis of tiny chromosomal investigated. regions and functional domains. In this regard, it is The transmission electron microscopy analysis, quite reasonable to use extended meiotic lampbrush immunohistochemistry technique and specific molec- chromosomes (LBCs) as initial material for micro- ular investigations were employed for deciphering dissection. LBCs are highly decondensed because of some peculiar aspects of chromatin organization their transcriptional activity that makes the microdis- and evolution in mouse spermatogenesis. section procedure much simpler. In present work we Spermatogonia, preleptotene spermatocytes and developed an approach for LBCs mechanical micro- spermatids contained global hyperacetylated H4, dissection, amplification of isolated material followed whereas no acetylated H4 was noticed in leptotene by highly specific FISH-probes preparation and high- or pachytene spermatocytes. throughput sequencing procedure. In particular, the Using the bisulfite mutagenesis technique, we found chicken (Gallus g. domesticus) chromosome regions that in the 5′ coding region, both Prm1 and 2 were fully as small as a single chromomere (~2 Mb in size) methylated in pachytene spermatocytes and spermato- were successfully dissected and applied for FISH zoa and are unmethylated when they are expressed in and next-generation sequencing (NGS), with only round spermatids. one chromosomal copy being used as input material. S126 10th European Cytogenetics Conference 2015

LBCs are enriched with unusual marker structures also for the first time demonstrated that the migrating including so-called «complex loops», whose organi- chromatin contains normal structures of the synaptonemal zation, precise genomic location, molecular composi- complex and lacks any signs of apoptosis. As has been tion and functions are still obscure. Here, we dis- shown, the chromatin migrating between cells in sected the material of the marker structures from cytomixis is neither selectively heterochromatized nor chicken lampbrush macrochromosomes 2 and 3, pre- degraded both before its migration to another cell and pared specific FISH-probes and deciphered the sam- after it enters a recipient cell as micronuclei. We have also ples using NGS. Such a complex comprehensive demonstrated that the cytomictic chromatin retains approach allows to assign unambiguously the posi- anormaltranscriptionactivitylevelaswellasthe tion of individual chromomeres and cytological normal processes of condensation, conjugation, and markers of lampbrush chromosomes to genomic recombination corresponding to the current meiotic coordinates. stage. The results admit the possibility that cytomixis The work was partially performed using experimen- may be the reason underlying a change in the karyotype tal equipment of the Research Resource Centers of produced pollen. ‘Chromas’ and ‘Molecular and cell technologies’ of The work was supported by the Russian Foundation Saint-Petersburg State University. for Basic Research (grant no. 14-04-31567 mol_a).

4.P3 4.P4

Histone modifications during cytomixis in tobacco Characterization of four species of Bovidae using satellite I DNA probes Sergey Mursalimov1,DmitriDemidov2,Natalya Permyakova1, Elena Deineko1, Andreas Houben2 Mariella Nieddu1,RobertoMezzanotte2,Renato Institute of Cytology and Genetics, Siberian Robledo2 Branch, Russian Academy of Sciences Molecular Univerity of Cagliari Biomedical Sciences Monserrato— genetics, cell biology and bioinformatics Cagliari-Italy1 Univerity of Cagliari Biomedical Science Novosibirsk-Russia1 Leibniz Institute of Plant Monserrato— Cagliari-Italy2 Genetics and Crop Plant Research Breeding Research Gatersleben-Germany2 Taxonomy of the Bovidae family is difficult and the evolutionary relationship among most bovid sub- Cytomixis is migration of the nuclei between plant families remains uncertain. We used a molecular cells through intercellularchannelsofaspecial cytogenetic approach which allows the investiga- type (cytomictic channels), differing from the plas- tion of peculiar DNA fractions, belonging to the modesmata in their structure and size. This unique category of highly repeated DNA sequences, often phenomenon was discovered over a century ago located at or near the centromere. Such DNAs are and to date cytomixis has been observed in hun- known to be reliable evolutionary markers, as dreds of various higher plant species, mostly in changes in satellite DNA composition may coin- microsporogenesis. cide with speciation and are useful as phylogenetic It is so far unknown what drives cytomixis and what markers. In this studies we isolated two clones, is the functional state of the chromatin migrating Bt1 from Bos taurus and Om1 from Ovis orientalis between cells. Using immunostaining, we have ana- musimon, and used them as probes for hybridiza- lyzed the distribution of main histone modification tion on both genomic DNA and chromosomes in types (methylation, acetylation, and phosphorylation) members of Bovini and Caprini tribes, respectively Bos that reflect the functional state of chromatin in the taurus, Bubalus bubalis, Ovis orientalis musimon and tobacco microsporocytes involved in cytomixis. We Capra hircus. The two sequences were analyzed and have pioneered in demonstrating that the chromatin compared to those present in the database, and turned in the cytomictic cells does not differ from the out to be almost identical to DNA families 1.714 and chromatin in intact microsporocytes according to all 1.715, which belong to satellite I DNA. Om1 is homol- 14 analyzed histone modification types. We have ogous to family 1.714, while Bt1 is homologous to 10th European Cytogenetics Conference 2015 S127 family 1.715. Probes showed completely coherent GTG-banding. Conventional and G-banded karyotypes results in Southern blots as well as in FISH: Bt1 were arranged respectively. sequence hybridized to Bos taurus and Bubalus All cells that were observed showed an inter- bubalis chromosomes and genomic DNAs, while mediate karyotype between sheep and goat with 57 no signal was detected in Capra hircus and Ovis chromosomes in total. The G-banding karyotype orientalis musimon. Om1 sequence hybridized to revealed the presence of 3 metacentric and 54 Capra hircus and Ovis orientalis musimon chromo- acrocentric chromosomes. The autosomes involved somes and genomic DNAs, while no hybridization in the hybrid combination were CHI1,3, CHI2,8 was observed in Bos taurus and Bubalus bubalis. and CHI5,11 corresponding to the metacentric Moreover, no signal has been detected on sex chromosomes OAR1, OAR2 and OAR3. The sex chromosomes and, likewise, a lack of positive FISH chromosomes were correctly arranged and no fur- signal was observed in metacentric/submetacentric ther morphological differences were evidenced by autosomes. The results obtained clearly show that classical cytogenetic investigation. Further molecu- Bovini and Caprini belong to two very distant sub- lar cytogenetic and genetic analysis are in progress families of Bovidae, still retaining some degree of to clarify the recombination events occurred in this homology at the level rare interspecies hybrid.

4.P5 4.P6

Cytogenetic characterization of an hybrid goat-sheep Non-coding RNA derived from a conservative by GTG-banding subtelomeric tandem repeat in embryos and adult tissues of Galliformes species revealed by fluorescent Alfredo Pauciullo1, C Knorr2, Angela Perucatti3,A in situ hybridization approach Stölzl2,DPieper2,AlessandraIannuzzi3,Leopoldo Iannuzzi3, Georg Erhardt1 Irina Trofimova1, Darya Popova1, Alla Krasikova1 Justus-Liebig-University Institute for Animal Breeding Saint-Petersburg State University Cytology and and Genetics Giessen-Germany1 Georg-August- Histology Saint-Petersburg-Russia1 University Department for Animal Science Göttingen- Germany2 National Research Council ISPAAM Transcription of tandemly organized DNA at ter- Laboratory of Animal Cytogenetics and Gene Mapping minal chromosomal regions is poorly investigated. Naples-Italy3 Previously we demonstrated transcription of the conservative subtelomeric tandem PO41 (“pattern In mammals interspecies hybridization occurs rarely of 41 bp”)repeatinchickenmalignantcells.PO41 under natural conditions. Sheep (Ovis aries, 2n=54) RNAs distribution and secondary structure in interphase and goats (Capra hircus, 2n=60) are genetically and through the mitosis were characterized. In this different and do not readily interbreed, although study transcription of PO41 repeat in chicken (Gallus information of such interspecies matings exists. gallus domesticus) and Japanese quail (Coturnix Cytogenetic incompatibility is one of the causes coturnix japonica) somatic tissues and chicken embryos of embryo death due to incorrect chromosome was examined in detail. paring during the zygote formation and/or aneuploidy Using RNA-FISH we demonstrated that both strands occurrence during the zygote division. In this study we of PO41 repeat are transcribed in identical pattern in report a preliminary cytogenetic characterization of an somatic cells of chicken and quail tissues (brain, healthy female hybrid goat-sheep born in a flock under muscles, oviduct, intestine). Chicken embryos were natural conditions. staged according to Hamburger and Hamilton (1951) Peripheral blood sample cultures were performed to and collected from 4-6 to 20 stages. Sense and get normal lymphocyte cell cultures. After the fixation, a antisense strands of PO41 repeat were found to be part of the obtained metaphases was stained with a 5 % transcribed at each stage. Transcripts were distributed Giemsa solution, whereas a part was treated with 0.05 % in all nuclei of developing embryos in pattern being of trypsin solution and Giemsa staining to obtain the identical to somatic and malignant cells: one-two or S128 10th European Cytogenetics Conference 2015 dispersed foci were detected in euchromatin or close fragments or whole chromosomes. We reported different to chromocenters. In embryo dividing cells PO41 ploidy levels in the endosperm nuclei. The expected 3n RNAs distribute between condensed chromosomes endosperm was observed in diploid and tetraploid oats till anaphase, when they concentrate at the cleavage and sporadically in hexaploids. The formation of 2n plane. At telophase, transcripts envelop terminal endosperm was documented in all studied oats. This regions of chromosomes, the results being similar indicates that endosperm may be formed without the to those obtained in malignant cells. RNase A treat- participation of sperm cell. Another possibility is the ment before or RNase H after whole-mount FISH degeneration of one of the central cell nuclei during (WFISH) and DNA-WFISH were performed as control the early stages of embryo sac development. experiments. Moreover, a number of intermediate ploidy levels, Taken together extremely uniform character of typical for aneuploidy, were found. We documented PO41 repeat transcription and transcripts distribution the loss of chromosomes of different genomes so it at different stages of development and cell cycle, seems that the elimination of chromosomes in endo- among species and cell types shown here, we con- sperm is random. clude that PO41 ncRNAs have universal mediator or regulatory functions. 4.P8 The work was partially performed using equipment of the Research Resource Centers ‘Chromas’ and The monitoring of the environmental pollution in ‘Molecular and cell technologies’ of Saint-Petersburg Romanian bovine farms by cytogenetic investigations State University. Ioana Nicolae1,AdrianBota2,LucianHarceaga3, 4.P7 Daniela Vidmichi1, Ionut Hoportean1 R&D Institute for Bovine Cytogenetics Balotesti- The A, B, C and D genomes in endosperm of oat Romania1 R&D Station for Buffalo Reproduction amphiploids. Sercaia-Romania2 Semtest-BVN Reproduction Tg.Mures-Romania3 Paulina Tomaszewska1, Romuald Kosina2 University of Wroclaw Institute of Experimental The monitoring of the environmental pollution by using Biology Wroclaw-Poland1 University of Wroclaw the cytogenetic investigation in Romanian cattle and Institute of Environmental Biology Wroclaw-Poland2 buffalo farms has been developed in the last 4 years. The main goal of our study was to assess the pollutants In situ hybridization of total genomic DNA was used effects on the genetic material integrity of farm animals to analyse the free nuclear endosperm of six oat like cattle and buffalo. The cytogenetic investigation of amphiploids derived from crosses of 2x, 4x, and 6x 350 heads (287 cattle and 63 buffaloes) reared in differ- species in various combinations. The parental species ent farms from all over the country carried out. We were included in the study. Total genomic DNA identified chromosomal instability for 42 animals probes derived from A. nuda L. and A. eriantha (31 cattle and 11 buffaloes), represented by a large Dur. were used to distinguish the A and C genomes, number of mono-and bi-chromatidic breakages on respectively. The study was focused on frequency of autosomes and heterosomes, loss of chromosome endosperm cytogenetic anomalies, the number and fragments and gaps. All females with chromosomal arrangement of chromosomes and relationships between instability presented reproductive disorders. SCEs-test, the A, B, C and D genomes. The results showed which is a specific test for identifying the effects of toxic frequent cytogenetic disorders in endosperm of agents on the genetic material integrity, has been amphiploids as well as their parental species, including applied. For animals with many chromosomal break- anaphase and telophase bridges, multipolar nuclei divi- ages the number of sister chromatid exchanges sions, merging of nuclei, ring and frying-pan chromo- (SCEs) was very high for cattle (8–16 SCEs/cell) somes, laggards, hiperploidy and numerous transloca- and buffaloes (10–17 SCEs/cell) compared to the tions. The increased frequency of micronuclei was dem- normal animals. The identification and quantification onstrated in amphiploids, suggesting elimination of of the total aflatoxin and heavy metals present in the 10th European Cytogenetics Conference 2015 S129 environment and, especially, in animal feed revealed zonkey has received a haploid set of chromosomes high level then maximum permitted by European from each parent. and Romanian law in force. Acknowledgments: This study has been partially Acknowledgements: This study was supported by supported by the CISIA-VARIGEAV project, National the Sectorial Project 7.3.1./2011 Research Council (CNR) of Italy.

4.P9 4.P10

M-FISH in a rare hybrid foaled by a donkey and a Molecular karyotyping the avian genome in a single zebra sire fluorescence in situ hybridisation experiment

Alessandra Iannuzzi1,VivianaGenualdo1,Angela Gothami Fonseka1,RebeccaOConnor1,Richard Perucatti1,AlfredoPauciullo2,JorgePereira3, Frodsham2, Martin Lawrie2, Darren Griffin1 Leopoldo Iannuzzi1, Malcom Ferguson-Smith3 University of Kent School of Biosciences Canterbury- CNR-ISPAAM Agrifood Naples-Italy1 University United Kingdom1 Cytocell Ltd Research and of Torino Department of Agricultural, Forest and Development Cambridge-United Kingdom2 Food Science (DISAFA) Grugliasco (TO)-Italy2 Cambridge University Department of Veterinary The avian karyotype is extremely complex, consisting Medicine Cambridge-United Kingdom3 mostly of large numbers of microchromosomes that confound characterisation by classical means. M-FISH (Multicolor fluorescence in situ hybridization) Particularly, in most birds, chromosomes from is one of the newest and powerful molecular cyto- number 7 onwards are extremely difficult to dis- genetic techniques. It can be used as a source of tinguish unless molecular cytogenetics is used. To information in many ways and for a precise descrip- circumvent this problem, we developed fluores- tion of chromosomal rearrangements in particular. In cence in situ hybridisation based chromosome the present study, for the first time in a hybrid, we macro-array, which we have called the “Chicken report a new way to study chromosome rearrange- Multiprobe System”,inordertoidentifyallofthe ments on a male zonkey foaled by a donkey (Equus chromosomes in the Chicken karyotype. This device has asinus, 2n=62, XX) and a zebra sire (Equus a number of utilities to address a series of scientific burchelli, 2n=44, XY) using M-FISH. We have questions. The design of the device consists of established that the somatic cells of the hybrid pos- 24 squares, with either 3 colour macro chromo- sess 53 chromosomes, making very difficult pairing some paints (generated by flow cytometry) or, for of homologous chromosomes. For this reason, we the microchromosomes, subtelomeric BAC probes have applied the multi-colour Zoo-FISH using a total in red and green fluorophores, or (the third colour of 29 whole chromosome painting probes (wcps) for macrochromosomal paints a blue fluorescent dye). obtained through fow-sorted zebra and horse chro- The probes are reversibly dried on to the device, then mosomes and, later DOP-PCR amplified and rehydrated in hybridisation buffer before being exposed labelled. In this case, five different pools of six to a corresponding glass slide with 24 equivalent wcps each were prepared, making possible five squares, each spotted with the fixed cell sample sequential FISH performed on the same slide for containing metaphase spreads. the identification of almost all chromosomes. The Applications of this multi-hybridisation system acquired images were then superimposed enabling include detection of translocations in individual individual chromosomes to be classified based on birds; characterisation of DT40 cell lines; and the fluor composition, in accordance with the com- comparative genomics on the metaphases of other binatorial labeling scheme of the M-FISH probe species. In recent experiments we have established pool used. Finally, we have analyzed in detail that the system is effective on the chromosomes of these digitally acquired and processed images to other Galliform species. The devices can be make the identification of all chromosomes for the customised to include bespoke probes and we have first time in this rare hybrid. We show that the used it to map the chromosomal locations of viral S130 10th European Cytogenetics Conference 2015 integration sites by co-hybridising the viral sequences 4.P12 on the Chicken Multiprobe System. Intraspecific variation revealed by chromosomal 4.P11 mapping of microsatellite in stingless bee Trigona spinipes Chromosomal mapping using microsatellite probes in two stingless bees species of region Amazon Ríudo de Paiva Ferreira1, Camila Moura Novaes1, Natália Martins Travenzoli1, Denilce Meneses Lopes1 Camila Moura Novaes1, Natália Martins Travenzoli1, Universidade Federal de Viçosa Departamento de Ríudo Paiva Ferreira1, Denilce Meneses Lopes1 Biologia Geral Viçosa-Brazil1 Universidade Federal de Viçosa Departamento de Biologia Geral Viçosa-Brazil1 The stingless bee Trigona spinipes is widely distributed in South America. Populations of this specie have been The Amazon has a great diversity with a high number of reduced by agricultural landscapes because this species species, including bees of the tribe Meliponini. These damages flowers of some crops. The aim of study was to bees are known as indigenous stingless bees and are characterize T. spinipes by classical and molecular important pollinators of crops and natural vegetation. (chromosomal mapping of 18S gene and of repetitive The construction of hydropower plants, deforestation DNA sequence ((GA)15, (CGG)10 and (CAA) 10) and expansion of agriculture represent threats to local cytogenetic. Metaphase chromosomes were obtained diversity. So knowledge about their biology and genet- from cerebral ganglia of T. spnipes larvae from three ics are important to assist in management plans and cities of Southeastern Brazil. The chromosome clas- conservation. This work aimed to increase the cytoge- sification was based on distribution of heterochro- netic knowledge of Amazonian species, through the matin according to Minimum Interaction Theory physical mapping of repetitive DNA sequences in proposed by Imai (1988). Chromosome number of Nannotrigona punctata and Scaptotrigona bipunctata T. spinipes was 2n=34, one euchromatic acrocen- bees. The metaphase chromosomes were obtained from tric pair (Ae) and 16 pseudoacrocentric pairs (Am). the cerebral ganglia in larvae defecation stage according Signal of 18 s probe was distributed in three pairs to a specific protocol. For chromosomal mapping were in terminal region of heterochromatic arm and one used probes 18S rDNA and microsatellite GA(15) for chromosomal pair in interstitial region just below fluorescence in situ hybridization (FISH). telomere in all samples. Only the probe (GA)15 Approximately 10 metaphases were analyzed per presented signal. This probe marked euchromatic individual to determine the diploid number of chro- regions, being distributed (i) the long arm of chromosomes and karyotype structure. The chromosome mosome Ae and (ii) in each eucromatic arm in number of 2n=34 in both species. The microsatel- chromosomes Am and (iii) in some heterochromatic lite GA showed signs in all chromosomes, marking arm in Am pairs. The colonies revealed similar regions euchromatic of chromosome. The same pat- pattern in chromosome number, morphology, the tern was found for the two species, also similar to distribution of heterochromatin and regions of the 18S that seen in other species of stingless bees, which rDNA. Only the location of microsatellite probes seems to be a feature retained for this group. The revealed differences among T. spinipes colonies, an 18S rDNA scored only one pair of chromosomes in important highlight. This is first chromosomal the terminal portion of the short arm of mapping of microsatellite for genus Trigona. The Nannotrigona punctata. On the other hand, showed colonies are located along the Atlantic Forest, two labeled Scaptotrigona bipunctata pairs. Despite however environmental changes, altitude and the great diversity of species found in Brazil, only temperature, over this biome may have originated asmallnumberwasanalyzedusingtechniquesof variations detected. Although we do not have sufficient molecular cytogenetics. Thus, this study increase information to state that observed variations reflect local the known about bees, providing important infor- adaptations. Ours data demonstrate that a new tool, mation on the cytogenetic characteristics of species chromosomal mapping of microsatellites, may be used in the Amazon region. for cytogenetic and population studies. 10th European Cytogenetics Conference 2015 S131

4.P13 4.P14

Molecular cytogenetic demonstrate variation in the Sequence characterization and genomic distribution repetitive DNA of stingless bees of the genus of Panax ginseng tandem repeats (PgTR) reveal Melipona utility in chromosome identification and possible role in heterochromatin maintenance Natália Martins Travenzoli1, Ríudo Paiva Ferreira1, Camila Moura Novaes1, Denilce Meneses Lopes1 Nomar Espinosa Waminal1, Hong-Il Choi1, Junki Lee1, Universidade Federal de Viçosa Biologia Geral Tae-Jin Yang1, Hyun Hee Kim2 Viçosa-Brazil1 Seoul National University Department of Plant Sciences Seoul-Korea1 Sahmyook University Life Science The bees Melipona genus are social insects belonging to Department Seoul-Korea2 the tribe Meliponini. Distributed in the Neotropical region, they have economic importance and ecology Repetitive elements make up a large portion of plant because they are pollinators of native plants and grown genomes and play major roles in molecular regulatory in their areas of occurrence. Cytogenetic studies indicate functions. In particular, tandem repeats have been asso- diploid number 2n=18 chromosomes conserved for the ciated with heterochromatin maintenance and gene species of the genus Melipona, except M. seminigra expression. Here, we characterized the sequence and (2n=20). However the pattern of distribution and quan- genomic distribution of a 167-bp Panax ginseng tandem tity of heterochromatin allowed a division the genus into repeat (PgTR), consisting of 9.6 repeat units (1,603 bp), two groups: the first group characterized by species with from bacterial artificial chromosome (BAC) low heterochromatic content while the second would PgH005J07. This locus is flanked by nested fragments present species with high heterochromatin content. The of DNA transposons in a Ty3/Gypsy LTR objective of this study was to evaluate the distribution of retrotransposons, implying a retrotransposon derivative microsatellite loci GA15 using the fluorescent in situ of this repeat. Nine PgTR variants were identified and hybridization in species with high (M. fuliginosa and categorized into two subclasses; the most diverged M. flavolineata) and low heterochromatic content variant was PgTR1, which comprised its own subclass (M. bicolor and M. puncticollis) and compare the results and had at most 86 % sequence identity to the other with the heterochromatic pattern in order to contribute to variants. All repeat unit variants possessed high AT evolutionary studies of the group. The distribution of content between nt 100 and nt 125, with curvature microsatellites GA15 confirms the division of propensity peaks corresponding to this region, implying Meliponidae: the species with high heterochromatin that PgTRs play a role in heterochromatin folding. content, M. fuliginosa and M. flavolineata, marked the Whole-genome sequence (WGS) read mapping subtelomeric region in almost all chromosome arms, revealed that the PgTR units varied in genomic abun- coinciding with euchromatin; already the species with dance. Additionally, all units possessed cis-acting regu- low content M. punctiollis and M. bicolor, plus some latory elements associated with biotic and abiotic stress subtelomeric markings, have submitted a pair of response. WGS read mapping covered only <1 % of the chromosomes with stronger and bigger bands in genomic PgTR while FISH analysis covered about 8 % the pericentromeric region and absence of mark- and revealed a genomic distribution unique to ings on some chromosomes. This indicates that the individual chromosomes. Thus, PgTRs are reliable euchromatin appears in both groups have a pattern cytogenetic markers and can be used to discrimi- of distribution of different microsatellites, probably nate between chromosomes. As such, PgTRs reflecting different evolutionary histories. Thus, the will contribute to comparative cytogenetics among distribution of microsatellite patterns were impor- Panax spp., increasing our understanding of tant cytogenetic markers that allowed genetic dif- genome evolutionary dynamics. Furthermore, our ferentiation among species of high and low DNA con- results should provide a foundation for future tent. It is expected that this tool can contribute to a better research on the role of tandem repeats in gene understanding of the evolution pattern between species expression, as well as in the shaping and maintenance of the genus Melipona. of the ginseng genome. S132 10th European Cytogenetics Conference 2015

4.P15 Universidade Estadual Paulista-UNESP Departamento de Biologia, Instituto de Biociencias São Paulo-Brazil1 Effects of chromosomal translocations on DNA University of Cambridge Department of Veterinary methylation and consequences on spermatogenesis Medicine Cambridge-United Kingdom2 Cytocell Ltd in the germline of male pigs Cambridge-United Kingdom3 Universite Paris-Sud Laboratoire Evolution, Genomes, Comportment, Valentine Marquet1, Alain Pinton1, Harmonie Barasc1, Ecologie CNRS Paris-France4 Hervé Acloque1 INRA-ENVT UMR GenePhySE Castanet-Tolosan- The african western clawed frog (Xenopus tropicalis, France, Metropolitan1 XTR), also known as the tropical clawed frog, is an important model organism for genetic and developmen- Infertility is a major concern for both human and live- tal studies. X. tropicalis, has a small genome, about 1.7 stock. Epigenetic analyses have highlighted abnormali- Gbp, with 10 chromosome pairs (2n=20). We have ties in the methylation of several genes in the germline generated chromosome specific painting probes for 8 of infertile subjects, providing new informations on of these chromosome pairs from flow-sorted chromo- genetic causes of infertility. Furthermore, it has been somes, amplified and labelled by DOP-PCR, and recently hypothetized that one of the mechanisms assigned by hybridization to XTR chromosomes 1, 2, through which balanced chromosomal translocations 3, 5, 7, 8, 9 and 10. A comparative genome study is affect fertility is the induction in germ cells of epigenetic underway using cross-species painting with modifications on genes critical for meiosis. Our team chromosome-specific XTR probes on X. laevis (XLA), previously found an abnormal methylation in an whose allotetraploid genome (3.1 Gbp, 2n=36) is sep- imprinted locus in three out of eight infertile boars, arated from XTR by 50–65 million years, as part of a including the two boars carrying a chromosomal trans- project on karyotype evolution in amphibians. Cross- location. We thus aim to explore the impact of chromo- species painting using microdissected XTR probes on somal translocations on DNA methylation during male XLA has been reported previously, but this seems to be meiosis, in a context of altered spermatogenesis. We will the first study using amphibian whole chromosome- study genome-wide DNA methylation in the germline specific DNA from flow-sorted chromosomes. of these three boars and local methylation around the breakpoints in the translocation carriers, compared to a 4.P17 fertile non-translocated control. For these purposes, we will confirm high-throughput sequencing data using Genomic in situ hybridization identifies genomic pyrosequencing on bisulfite converted DNA extracted relationships of Sorghum bicolor and closely related from sperm and from meiocytes and spermatogonies species (Poaceae) sorted by FACS. We will then study their transcriptome and focus on the expression of genes with methylation Qing Liu1, Huan Liu1 alterations and genes located at the translocations South China Botanical Garden, Chinese Academy of breakpoints. This work should provide insights on a Sciences Botany Guangzhou-China1 newly discovered epigenetic mechanism through which chromosomal translocations affect spermatogen- Cultivated sorghum [Sorghum bicolor (L.) Moench] esis and fertility. ranks fifth in both production and planted area of cereal crops worldwide, only behind wheat, rice, maize, and 4.P16 barley, but the degree of relatedness of S. bicolor with closely related species in the genus is unverified. Preparation of chromosome-specific paints by flow- Multicolor genomic in situ hybridization (McGISH) sorting of Xenopus tropicalis was used to study the genome organization and evolution of S. bicolor. Total genomic DNA from closely- Thiago Gazoni1, Jorge Pereira2,JorgePereira3, related species (two probe pairs from S. arundinaceum Nicolas Pollet4,PatriciaParise-Maltempi1,Malcom and S. × almum, and S. arundinaceum and S. Ferguson-Smith2 propinquum) were labeled and hybridized in the 10th European Cytogenetics Conference 2015 S133 presence of blocking DNA onto metaphase spreads of hybridization and polymerase chain reaction-based tech- S. bicolor. The probe pair of S. arundinaceum and niques, we inferred the fine structure of MYC- containing S. × almum painted sharp signals and peak appeared dmin/hsr amplicons harboring sequences from several in the centromere region of 16 chromosomes from different chromosomes in seven tumor cell lines, and S. bicolor, whereas the probe pair of S. arundinaceum characterized an unprecedented number of hsr insertion and S. propinquum delineated uniform signals without sites. Local chromosome shattering involving a single- peak located on 16 chromosomes of S. bicolor. The step catastrophic event (chromothripsis) was recently McGISH evidence support that 1) the diploid proposed to explain clustered chromosomal rearrange- S. arundinaceum and S. propinquum are the primary ments and genomic amplifications in cancer. Our bioin- ancestor of S. bicolor, 2) probe of S. × almum formatics analyses based on thelistedcriteriatodefine (progenitor) produced strong hybridization signals chromothripsis led us to exclude it as the driving force on 16 chromosomes of S. bicolor (ancestor), and underlying amplicon genesis in our samples. Instead, the 3) recurrent hybridization between diploid ances- finding of coexisting heterogeneous amplicons, differing tors formed the diploidization S. bicolor. in their complexity and chromosome content, in cell lines derived from the same tumor indicated the occurrence of 5. Molecular Mechanisms of Chromosome amultistepevolutionaryprocessinthegenesisof Rearrangements dmin/hsr. Our integrated approach allowed us to gather a complet view of the complex chromosome 5.P1 rearrangements occurring within MYC amplicons, suggesting that more than one model may be invoked to Genomic organization and evolution of double explain the origin of dmin/hsr in cancer. Finally, we minutes/homogeneously staining regions with MYC identified PVT1 as a target of fusion events, confirming amplification in human cancer its role as breakpoint hotspot in MYC amplification.

Alberto L’Abbate1,GemmaMacchia1,Pietro 5.P2 D’Addabbo1,AngeloLonoce1,DoronTolomeo1, Domenico Trombetta2,KlaasKok3,Christoph Molecular mechanisms related to abnormal phenotype Bartenhagen4,ChristopherW.Whelan5,Orazio of balanced chromosome rearrangements carriers Palumbo6, Marco Severgnini7, Ingrid Cifola7, Martin Dugas4,MassimoCarella6,GianlucaDeBellis7, Milena Simioni1,FrançoisArtiguenave2,Nilma Mariano Rocchi1,LuciaCarbone5, Clelia Tiziana Viguetti-Campos1, Vera L. Gil-da-Silva-Lopes1 Storlazzi1 University of Campinas Medical Genetics Campinas- University of Bari Department of Biology Bari-Italy1 Brazil1 Commissariat A L’ Energie Atomique Genetics IRCCS Casa Sollievo della Sofferenza Hospital Paris-France2 Laboratory of Oncology San Giovanni Rotondo-Italy2 University of Groningen Department of Genetics Balanced chromosomal rearrangements (BCR) occur in Groningen-The Netherlands3 University of Munster approximately 1:2000 newborns and are associated with Institute of Medical Informatics Munster-Germany4 abnormal phenotypes in around 6 % of cases. Abnormal Beaverton National Primate Research Center Oregon- phenotypes can be caused by disruption of genes at the United States5 IRCCS Casa Sollievo della Sofferenza breakpoints, small duplications or deletions, positional Hospital Medical Genetics Unit San Giovanni Rotondo- effects. Conventional cytogenetic techniques have lim- Italy6 National Research Council Institute for ited resolution and enables complete clinical investiga- Biomedical Technologies Milan-Italy7 tion. Molecular techniques applied to BCR carriers can contribute to characterization of this type of chromo- The mechanism for generating double minutes chromosome rearrangement and, also to phenotype-genotype somes (dmin) and homogeneously staining regions (hsr) correlation. From last 10 years (from 2004 to 2014), 35 in cancer is still poorly understood. Through an integrated individuals with abnormal phenotype and BCR were approach combining next generation sequencing, single registered in our service and 15 were selected to inves- nucleotide polymorphism array, fluorescent in situ tigation by molecular techniques. Chromosomes S134 10th European Cytogenetics Conference 2015 rearrangements involved 11 reciprocal translocation, 3 formation and p53 inactivation might cause inversions and one balanced insertion. First, array geno- chromothripsis. The remarkable point is that all mic hybridization (AGH) was performed and genomic these plausible mechanisms have been identified imbalances were detected in 20 % of cases, one at in the field of human reproduction as causal fac- rearrangement breakpoint while other two in unrelated tors for reproductive failures and chromosomal chromosomes. Alterations were further confirmed by abnormalities genesis. Specific features of gameto- fluorescence in situ hybridization and were associated genesis and early embryonic development such as to the phenotype of carriers. In patients that AGH did the weakness of cell cycle and mitosis checkpoints not show any genomic imbalance, second step was and the rapid kinetics of division in germ cells and investigate chromosome breakpoints at base pair level early cleavage embryos, may contribute to the by next generation sequence. Chromosomes involved in emergency of chromothripsis. All the data support rearrangement were directly microdissected from meta- the assumption that this unanticipated catastrophic phase slides, amplified by a commercial kit, and suffi- phenomenon may arise more frequently than pre- cient material for sequencing was obtained. Genomic viously thought in both gametogenesis and human libraries were prepared following the Illumina Nextera embryogenesis, but also confirmed its compatibility XT protocol and paired-end sequence was carried out in with viability and transmission to subsequent Illumina HiSEQ 2000. Currently, bioinformatics tools generations. of alignment are analyzing sequence reads and will be The complexity of chromothripsis and the diversity presented in detail. The strategy applied in this study of its causative mechanisms raises questions about the seems to be efficient to elucidate the genomic mecha- origin of this phenomenon, its ties to chromosome nisms responsible for abnormal phenotype of BCR car- instability and its implication in human reproduction riers. This is the first study in our laboratory using this and congenital disorders formation. Further in-depth approach, which probably will improve the manage- analyses on large samples of patients and cells (gametes ment of patients carrying BCR. and blastomeres) are needed to appreciate the contribu- Supported by Fapesp (2011/23794-7 and tion of this cataclysmic process to altered human devel- 2012/10071-0) and CNPq (304455/2012-1). opment and the generation of congenital diseases. This constitutes a great challenge and implies further efforts 5.P3 to develop and adapt new techniques, especially next- generation sequencing, to the precise identification of Chromothripsis: a plausible mechanism for massive genomic rearrangements. reproductive failures and complexe chromosomal rearrangement genesis. 5.P4

Franck Pellestor1,VincentGatinois1,Jacques Use of 5-ethynyl-2′-deoxyuridine (EdU) incorpo- Puechberty1, David Geneviève1, Geneviève Lefort1 ration for identification of the late replicating CHU Montpellier Chromosomal Genetics X-chromosome in balanced and unbalanced Montpellier-France1 alterations

Over the last 4 years, the discovery of an unexpected Luiza Sisdelli1,MarianaMoyses-Oliveira1,Isabel type of massive chromosomal rearrangements, called Melaragno1, Gianna Carvalheira1 chromothripsis, came revolutionized our view of the UNIFESP Morphology and Genetics Sao Paulo-Brazil1 genesis of complex chromosomal rearrangement in cancers and congenital disorders. X-chromosome inactivation usually occurs randomly in Chromothripsis is characterized by the shattering of normal female cells, being either maternal or paternal one (or a few) chromosome segment(s) followed by a X-chromosome methylated. However, in women with chaotic reassembly of the fragments generated through a X- chromosome alterations, the inactivation is not ran- single initial catastrophic event. dom: in X-autosome balanced translocations, normal Various mechanisms, involving abortive apopto- X is preferentially inactivated, while in unbalanced X sis, telomere erosion, mitotic errors, micronuclei chromosome alterations, the aberrant X is usually 10th European Cytogenetics Conference 2015 S135 inactivated. X inactivation pattern has been identi- Copy number variants (CNVs) of the Williams–Beuren fied by different approaches, such as: cytogeneti- syndrome (WBS) 7q11.23 region are responsible for cally, using 5-bromo-2′deoxyuridine (BrdU) or neurodevelopmental disorders with multi-system antibodies to the acetylated histones; and molecu- involvement and variable expressivity. Here we describe larly, using HUMARA assay or methylation three patients with structural reorganizations of the analyses. Here we present X-chromosome late 7q11.23 region sharing a common breakpoint between replication data from seven patients: four with LIMK1 and EIF4H/WBSCR1 genes indicating the X-autosome balanced translocations; two with Xp existence of a previously unrecognized recurrent deletion and one with Xq isochromosome. breackpoint responsible for rearrangements in the Lymphocyte cultures were exposed to 5-ethynyl- WBS region. One patient had WBS phenotype and 2′-deoxyuridine (EdU) for 2 h before preparation, although testing with a commercially available FISH using Alexa Fluor® 488 and DAPI staining, assay was negative for deletion, aCGH showed an atyp- according to modifications of Click-iT® EdU ical deletion in the WBS region with atypical Assay kit (Invitrogen). Because EdU is a labeled breakpoints. Our findings point to the existence of a nucleoside analog of thymidine, its incorporation previously unrecognized recurrent breakpoint responsi- during active DNA synthesis can reveal late repli- ble for rearrangements in the WBS region. Most com- cation regions and the inactive X-chromosome. In mercial FISH assays include probes flanking this novel order to better distinguish the derivative X chro- breakpoint and, as consequence, fail to detect reorgani- mosomes, fluorescent in situ hybridization, with zations with this breakpoint. Further testing with aCGH centromere probes and BAC probes (red labeled) array should be performed in patients with WBS and for different parts of the X chromosome, was used negative FISH results. simultaneously with EdU procedure. As expected, we found that the normal X-chromosome was 5.P6 preferentially inactivated in women with X-autosome balanced translocations; while prefer- Chromosomal integrity associated with different ential inactivation occurred in the aberrant approaches for in vitro stem cells derivation X-chromosome in unbalanced alterations. The inactivated X presented a strong green fluores- Maria Sobol1, Doroteya Raykova1, Lucia Cavelier1, cence. This EdU assay permitted an accurate and Ayda Khalfallah1, Jens Schuster1, Niklas Dahl1 efficient cytogenetic detection of the late replication Uppsala University Dep. Immunology, Genetics and regions, being faster and more sensitive in the evalua- Pathology Uppsala-Sweden1 tion of X inactivation than BrdU incorporation methods. Stem cells have brought great promises for disease 5.P5 modelling and cell-based therapies where special place have been taken by induced pluripotent stem cells AnovelrecurrentbreakpointintheWilliams– (iPSCs). This technique allows to derive individualised Beuren region as a cause of FISH pitfalls in the stem cell lines in vitro by reprograming differentiated diagnosis of Williams syndrome cells e.g. fibroblasts to pluripotent cells. One concern related to the use of reprogrammed somatic cells is the Alberto Plaja1, Elisabet Lloveras1, Luis Izquierdo2, loss of genomic integrity and chromosome stability, that Elena Ordoñez3,LauraBarranco1,AnnaCanellas1, is a hallmark for cancer and many other human disorders. Vincenzo Cirigliano3, Anna Cueto-González4, Miguel We investigated 16 human iPSC lines reprogrammed del Campo4, Neus Castells4, Teresa Vendrell4, M. Mar by non-integrative Sendai virus (SeV) and another 16 Borregán4, Eduardo Tizzano4 iPSC lines generated by integrative lentivirus for genetic LABCO Departamento de Citogenética y Arrays changes. At early passages the cytogenetic rearrange- Barcelona-Spain1 Hospital La arzuela Unidad Genética ments were detected in 44 % (7/16) and 6 % (1/16) of Madrid-Spain2 LABCO Departamento Biología iPSC lines generated by lentiviral and SeV integration Molecular Barcelona-Spain3 Hospital Vall d’Hebron correspondingly. We found that these numerical and/or Àrea de Genètica Clínica y Molecular Barcelona-Spain4 structural rearrangements were associated with S136 10th European Cytogenetics Conference 2015 chromosomes 5 and 12 known to be as the most fre- situ hybridization (FISH) with a centromeric probe, quently involved chromosomes. We present herein the and by the analysis of structural chromosomal aber- karyotypic aberrations in the iPSC lines including a rations (sCAs) and expression of fragile sites (FS). duplication on chromosome 5q13-q33 that restricts a The in vivo study was carried out in 55 hypertensive candidate region for growth advantage. patients while the in vitro study was performed in We conclude that the use of integrative lentivirus 10 control individuals by adding the drugs to the confers a higher risk for cytogenetic abnormalities at culture medium at a final concentration similar to the early passages when compared to SeV based levels found in plasma in patients. In relation to the reprogramming. Thus, SeV based generation of iPSC CBMN assay, our results showed a significant maintains genomic integrity better than integrative len- increase in the frequencies of MN and binucleated tivirus. Therefore our results suggest that SeV has better cells with MN (BNMN) in vivo and especially perspectives to be used for stem iPSCs derivation espe- in vitro. FISH analysis revealed no significant differ- cially for purposes related to cell-based therapies. ences between patients and control individuals in the Additionally our findings expand the knowledge on frequency of centromeric signals. Regarding the acquired cytogenetic aberrations in iPSC after incidence of sCA, the data revealed again a signifi- reprogramming and during culture. cantly higher rate of sCA in vivo, and mostly in vitro. Chomosomal location analysis pointed at 5.P7 chromosomes 2 and 17 as preferential targets for the clastogenic effect of ARBs drugs. Several FS Evaluation of genotoxic effects of antihypertensive emerged as specific; among them, the locus 17q21, drugs: Angiotensin II receptor blockers recently proposed as a new FS, was only expressed in ARBs-treated patients, supporting the findings Iratxe Huerta1, Maitane Barasoain1, Mercedes Télez1, for a linkage between hypertension and a telomeric Eduardo Ortiz-Lastra2, Javier González2, Mikel Longa3, locus on chromosome 17. Javier Muga3, Gorka Barrenetxea2, Begoña Criado4, These results provide evidence for an association Isabel Arrieta1 among the antihypertensive therapy, arterial hyperten- Faculty of Science and Technology, University of the sion and DNA damage in human lymphocytes. Basque Country Genetics, Physical Anthropology and Animal Physiology Bilbao-Spain1 Faculty of Medicine, 5.P8 University of the Basque Country Medical-Surgical Specialities Bilbao-Spain2 Virgen de Begoña Clinical Derivative from de novo unbalanced translocations: Analysis Laboratory (Medikosta) Molecular Genetics overall frequency, parental origin and mechanism of Erandio-Spain3 Cooperativa de Ensino Superior formation Politecnico e Universitario (CESPU) Biomedical Sciences Porto-Portugal4 Francesca Malvestiti1,CristinaAgrati1,Barbara Malvestiti1, Francesca Dulcetti1, Rosaria Liuti1, Anna Arterial Hypertension is commonly treated with antihy- Ruggeri1, Sara Chinetti1, Federico Maggi1, Giuseppe pertensive drugs combined with appropriate changes in Simoni1, Francesca Romana Grati1 life-style. The large majority of hypertensive patients TOMA Advanced Biomedical Assays S.p.A. Research need long-term therapy and documentation of safety and Development, Cytogenetics and Molecular Biology and efficiency is required, including sensitive indices Busto Arsizio, Varese-Italy1 of genotoxic damage. In this study, the potential genotoxic effect of five Derivative chromosome from de novo unbalanced Angiotensin II receptor blockers (ARBs: translocation (UT) is defined as a chromosome compris- Candesartan, Telmisartan, Eprosartan, Valsartan, ing terminal duplication and deletion of a terminal seg- Olmesartan) was analyzed in vivo and in vitro in ment of another chromosome. Few studies investigated human peripheral blood lymphocytes (PBLs) by the breakpoint sequences of de novo UTs and the means of the cytokinesis-block micronucleous parental origin of the unbalanced segments. Previous (CBMN)assay in combination with fluorescence in studies hypothesized a mechanism of formation 10th European Cytogenetics Conference 2015 S137 different from a parental germline or low-level somatic role, as well. Recently, array-CGH was found to be a mosaicism for balanced translocations. We present six very useful tool in the identification of candidate genes new cases, five of them detected during prenatal diag- in different conditions. Therefore, we performed array- nosis, with de novo UTs with the aim to: i) calculate CGH analysis in a total of 55 POF (30,4±6,36 years) their frequency in prenatal samples; ii) determine the patients with normal chromosome constitutions and parental origin of the unbalanced segments and iii) Fragile X specific CGG repeats. Totally, 16 different discuss the mechanism of formation. copy number variations (62.5 % deletions) were Karyotyping were performed following the standard detected in 42 % of the patients. The deletion of the methods and the European Guidelines. After detecting ASMTL, P2RY8,SHOX (chrXp22.33) and MSX1 an UT, parental metaphases were analyzed by (chr4p16.2) genes whose involvement in POF have karyotyping and/or FISH using specific probes to define been reported previously were also mostly seen CNVs the origin and the recurrence risk. The de novo deriva- in the study. Besides, it was striking to note that the tive chromosomes were characterized by chromosomal chromosome 15q26.3 deletion was detected in 5 cases. microarrays and the origin of the unbalances were The gene PCSK6, located in this locus, is a member of investigated by segregation microsatellite analysis. proprotein convertase (PC) family that is expressed in The incidence of UTs in prenatal samples is 0.053 % numerous ovarian cell types including granulosa cells (95 %CI: 0.032–0.085 %); in agreement with previous and oocytes. Previous studies have reported its role in studies ~30 % of them are de novo and have an inci- maintaining normal cellularandtissuehomeostasis dence of 0.016 % (1:6094) (95 %CI: 0.007–0.038). As in the ovary. We suggested that PCSK6 activity should expected, in patients with developmental disabilities the be examined in human ovarian pathology to ascertain reported frequency is 0.23 %, higher than in general further the clinical role that altered PC activity may prenatal population (p<0.01). Four out of six reported contribute to human ovarian function. cases showed a cryptic aberration, not detectable by conventional karyotyping due to the small size 5.P10 of the deleted or duplicated segment. We’ll discuss the possible mechanisms ofderivativeformation Genetic and epigenetic biomarkers of folate (meiotic or mitotic) in relation to the parental origin metabolism and trisomy 21 and a literature review. Lucia Migliore1, Fabio Coppede’1 5.P9 University of Pisa Department of Translational Research and New Technologies in Medicine and Copy Number Variants In Premature Ovarian Surgery Pisa-Italy1 Failure At the end of the last Century it was hypothesized that Halime Kucuk1, Sevilhan Artan1, Yunus Aydin2, Ebru impaiments in folate metabolism, resulting from the Erzurumluoglu1, Huseyin Aslan1, Hikmet Hassa2 presence of polymorphisms in metabolic genes, could Eskisehir Osmangazi University Medical Faculty impair DNA methylation at centromeric regions, thus Faculty of Medicine, Department of Medical Genetics fovouring chromosome 21 malsegregation and the birth Eskisehir-Turkey1 Eskisehir Osmangazi University of child with Down Syndrome (DS). We subsequently Medical Faculty Faculty of Medicine, Department of screened peripheral blood lymphocytes of women who Obstetrics and Gynecology Eskisehir-Turkey2 had a child with DS (MDS) in young age (<35 years) observing an increased tendency to chromosome Premature ovarian failure (POF) is a complex heteroge- malsegregation also in somatic cells (evaluated by neous clinical disease that may be affected by the mul- means of the micronucleus assay coupled with FISH tiple genes that control follicle formation and is charac- technique). In order to shed light on the contribution of terized by an early loss of the ovarian function. The folate metabolism to the observed increased frequency etiology of POF generates 20 % of familial genetic of micronucleated lymphocytes in MDS we applied causes and 5 % of chromosome X abnormalities. artificial neural networks (ANNs) that revealed impor- Autosomal chromosome abnormalities may also play a tant connections between the cytogenetic damage and S138 10th European Cytogenetics Conference 2015 common polymorphisms in genes of the folate metabol- in situ hybridization (FISH), to determine the TERT and ic pathway, namely MTHFR 677C>T, MTRR 66A>G, TERC copy number genes, is applied to cultured RFC1 80G>A, MTR 2756A>G, TYMS 28 bp repeats amniocytes retrieved from nine samples with various and 1494 6 bp deletion polymorphisms. Subsequent chromosome abnormalities and from a control group case–control studies and/or literature meta-analyses by of 20 samples with a normal karyotype. The rearranged us confirmed a contribution of both RFC1 80G>A and amniocytes showed increased TERC gene copy number MTRR 66A>G polymorphisms to the maternal risk of and enhanced gathering of the signal compared to the birth of a child with DS. Moreover, we recently normal amniocytes. Gene expression analysis observed association of promoter polymorphisms in performed on all cultures revealed that hTERT is not the DNMT3B gene, coding for a DNA methyltransfer- expressed in 1/3 of the samples while the gene hTERC ase protein, with the risk of birth of a child with DS, as is always expressed. The expression of hTERC is higher well as increased MTHFR promoter methylation in than that of hTERT. However there was a greater blood DNA of MDS with respect to control mothers. expression of the amniotic samples with normal karyo- Overall, our data suggest that both genetic and epige- type, than those with netic biomarkers of the folate metabolic pathway could represent a maternal risk factor for trisomy 21. 5.P12

5.P11 Unusual chromosomal rearrangement resulted in interstitial monosomy 9p: case report Copy number variation and expression of hTERT and hTERC genes in amniocyte cultured samples Ceren Damla Durmaz1,KanayYararbaş2,Özlem with normal and abnormal karyotype Türedi1, İsmigül Akın2,CansuGürbüz1,Günay Karataş2, Ajlan Tükün1 Giulia Savio1, Giovanna Piovani1 Ankara University School of Medicine Medical University Department of Molecular and Translational Genetics Ankara-Turkey1 Düzen Laboratories Group Medicine Brescia-Italy1 Medical Genetics İstanbul-Turkey2

Telomeres are located at the ends of chromosomes and Chromosomal rearrangement is a chromosome abnor- protect them from fusion and degradation. Telomerase is mality in the structure of the chromosome that encom- aribonucleoproteinenzymecomplexthatadds passes several different classes of events, like transloca- telomeric repeats, TTAGGG, to the ends of chromo- tions, inversions, deletions and duplications. Structural somes. The telomerase components are the telomerase chromosomal abnormalities occur in approximately reverse transcriptase (TERT) catalytic subunit, located 0.5 % of newborns. On the other hand, complex chro- at chromosome band 5p15.33, and the telomerase RNA mosomal rearrangements are much more rare structural (TERC) template subunit, at 3q26.3. Functional telo- anomalies. At least three break points, including genetic meres are essential for normal maintenance of chromo- material exchange between two or more chromosomes, somes during mitotic and meiotic division. Clonal evo- are required for complex chromosomal rearrangements lution of chromosome rearranged cells with increased (CCRs). Genomic gains and losses frequently TERT or TERC copy number has been observed, sug- accompany complex chromosomal rearrangements. gesting a growth advantage in cells with that increased Whether the amount of genetic material changes or gene dosage. The aim of our work is to study if there is not, clinical results may occur due to break points. any relationship between the hTERT and hTERC copy Overall, CCRs are often associated with mental number variation in cultured amniocyte samples with retardation, congenital abnormalities and recurrent normal and abnormal karyotype, to investigate whether abortions. Here, we report a case with interstitial there is correlation between a change in the number monosomy 9p in a complex de novo rearrangement. copies of the two genes together and the second step The patient presented with craniofacial dysmorphism, was check and compare TERT and TERC mRNA delayed psychomotor development and various congen- expressions, detected by relative quantifications with ital malformations. He had also bilateral nephrolithiasis RT-PCR. Basic research study, interphase fluorescence and cryptorchidism. 10th European Cytogenetics Conference 2015 S139

We combined cytogenetic studies and FISH analyses are well documented, very few patients with mosaicism to delineate the deletion. The result of our cytogenetic have been reported so far. studies was 46,XY,der(9)(p22→pter). In order to con- We describe a new case of mosaic for at least two cell firm deletion we also performed FISH analysis and lines carrying de novo different terminal 22q13 deletions these analyses showed that 9p subtelemeric region has detected by CGH-array (Agilent 180 K(Hg19)). All the inserted into 13th chromosome. Molecular cells have a 2, 7 Mb terminal deletion (from 48 388 703 karyotyping was performed to describe the exact to 51,178,264 bp), while only 40 % as confirmed by genomic points of rearrangement. We confirmed FISH with clone CTA-268H5 have a larger deletion that the arrangement occurred in this case is an (6,3 Mb) (from 44 864 865 to 48 31 5 347 bp). insertion/deletion complex abnormality which has This mosaicism may be due to distinct stabilizing not been reported earlier. events, occurring in different cells of the early embryo, Insertional translocations (ITs) are rare chromosomal of the same unstable terminal deletion. Previous studies rearrangements and at least three breaks are required for in subjects with 22q13 deletion unraveled variety of formation of ITs. The exact cytogenetic mechanisms mechanisms for chromosome repair of terminal dele- underlying the origin of ITs and CCRs are unclear but tions, including de novo telomere synthesis and telo- several mechanisms have been proposed to explain the mere capture. In this case, we hypothesize that a termi- reasons of them, such as non-allelic homologous recom- nal deletion happened in meiosis, which was bination, non-homologous end joining, fork stalling and imperfectly repaired by telomere capture or synthesis, template switching micro homology mediated break- leading to an unstable chromosome which subsequently induced replication and chromothripsis. Elucidation of broke again in a subset of blastomeres. these mechanisms will facilitate our understanding of While these cryptic mosaics may lead to a more genomic rearrangement. complex than expected correlation between deletion size and severity of phenotype, SHANK3 is up to now 5.P13 the only candidate gene responsible for the neurological features of the PMS phenotype which in this case was A mosaic of two cell lines carrying different terminal not confounding. 22q13 deletions: a case report 6. Impact of Next Generation Sequencing on Clinical Ikhlas BenAyed1, Laila El Khattabi1, Geraldine Viot2, Diagnosis Edwige Dakouri3,DominiqueLeTessier1,Aziza Lebbar1, Jean-Michel Dupont1 6.P1 APHP, Hôpitaux universitaires Paris Centre, France Laboratoire de Cytogénétique Paris-France1 APHP, Impact of Array Painting and Whole Genome Hôpitaux universitaires Paris Centre, France Unité de Sequencing on Cytogenetic Diagnosis: from Matérnité Paris-France2 centre Hospitalier Intercommunal Complex Chromosome Rearrangements to de Villeneuve St Georges service de pédiatrie et de chromothripsis néonatologie Villeneuve St Georges-France3 Anouck Schneider1, Patricia Blanchet2, Pauline Bouret1, Phelan-McDermid Syndrome (PMS) is a Mélanie Di Nicola1,DavidGenevieve2,Franck neurodevelopmental disordercaused by the deletion or Pellestor1,BeeLingNg3,WigardKloosterman4, disruption of a 22q13 region. The clinical features Jacques Puechberty2 ofPMS are highly variable and include develop- Laboratoire de Génétique Chromosomique Hôpital mental delay, expressivespeech/language delay, neona- Arnaud De Villeneuve Montpellier-France1 tal hypotonia, autistic traits and mild dysmorphic Département de Génétique Médicale Hôpital Arnaud features. De Villeneuve Montpellier-France2 Cytometry Core 22q13 monosomy can result from simple terminal Facility The Wellcome Trust Sanger Institute deletions (70 %), ring chromosomes (14 %), unbalanced Cambridge-United Kingdom3 Department of Medical translocations (7 %) or interstitial deletions (17–74 kb) Genetics, Center for Molecular Medicine University in 9 % of the patients. Although 22q terminal deletions Medical Center Utrecht AB Utrecht-The Netherlands4 S140 10th European Cytogenetics Conference 2015

Complex chromosomal rearrangements (CCR) are Complex chromosomal rearrangements (CCRs) are defined as balanced or unbalanced structural abnormal- structural abnormalities involving at least three ities of chromosomes with 3 breakpoints on 2 different breakpoints with the exchange of chromosomal seg- chromosomes. CCR are rarely reported in constitutional ments. The great majority of CCRs are associations of cytogenetics and mainly include associations of translocations. translocations. We report the case of a female child in inten- We report the case of a patient with early intellectual sive care for intrauterine growth retardation, neo- deficiency associated with absence of language, hypo- natal respiratory distress, sucking and swallowing tonia and postural delay despite a good understanding difficulties, global hypertonia and malformation and a good motor autonomy. Karyotype revealed a de syndrome (patent malformative ductus arteriosus, novo complex translocation to chromosomes 3: cortical giration and white matter abnormalities, 46,XY,t(3;15;5)(p11;q2?5;q23)dn. ocular abnormalities, bilateral renal cortical microcysts, To better characterize this RCC, we implemented abnormalities of hands and feet). The child died at various techniques such as FISH, Array Painting and 3 months of life in a context of seizures, respiratory Whole Genome Sequencing. These different distress and heart failure. approaches led to the identification of a much complex The initial karyotype suggested a de novo unbal- and massive genomic rearrangement which may be anced CCR with what resembled a 5p isochromosome, classified as a chromothripsis. This constitutes a new a chromosome 1 derivative from a t(1p36;5q13) and a class of complex chromosomal rearrangements based 1p36.3 terminal deletion. on the shattering of one or a few chromosomal Chromosomal microarray (CMA) studies revealed segment(s) followed by a chaotic reassembly of the additional de novo imbalances with a 547-kb deletion chromosomal segments. in 2p11.2, a 2509-kb duplication in 5q11.1q11.2 and The study of this patient confirms the complex- 250-kb and 1190-kb deletions in 5q31.1. The 2p11.2 ity of the genesis mechanisms of CCR and then deletion includes the REEP1 gene responsible for auto- subsequent diagnosis. The implementation of new somal dominant distal hereditary motor neuropathy type techniques has upset our knowledge with the dis- VB (HMNVB, omim: #614751). covery of the unanticipated phenomena coined Whole genome sequencing (WGS) clarified the chromothripsis. We emphasize the difficulties of breakpoints of the chromosomal rearrangements interpretation of these new techniques, particularly and revealed new ones, further complicating the Whole Genome Sequencing and therefore the cytogenetics of the CCR. The identification of these importance of the role of FISH at present in numerous breakpoints on two chromosomes sug- everyday life. gests chromothripsis or chromoanasynthesis, two unanticipated phenomenon recently evidenced in constitu- 6.P2 tional chromosomal disorders. The use of recent technologies in cytogenetics Characterization of complex chromosomal rear- (CMA, WGS) has brought a new dynamic to this disci- rangements through new cytogenetic technologies: pline by both increasing the number of etiological diag- from discovery to discovery. noses made and also by apprehending the mechanisms leading to chromosomal aberrations. Ultimately a lot of Jacques Puechberty1,ChristineCoubes1,Anouck CCRs are more complex than initially suggested by Schneider2, Wigard Kloosterman3, Geneviève Lefort2, conventional cytogenetic analysis. Magali Tournaire2, Manon Girard2, Mélanie Di Nicola2, Emmanuelle Haquet1, Sylvie Tavaiaux2,David 6.P3 Genevieve1, Franck Pellestor2 Centre Hospitalier Régional et Universitaire Génétique Genetic non invasive prenatal testing: a clinical and Médicale Montpellier-France1 Centre Hospitalier technical experience of 3.000 cases with follow-up. Régional et Universitaire Génétique Chromosomique Montpellier-France2 University Medical Center Yaima Torres1, Javier Suela1, Juan C Cigudosa1 Biomedical Genetics Utrecht-The Netherlands3 NIMGenetics Genomics Madrid-Spain1 10th European Cytogenetics Conference 2015 S141

Non Invasive Prenatal Testing (NIPT) is a fetal cell-free Children’s Clinical University Hospital Department of DNA genomic based screening for an accurate detection Laboratory Medicine Riga-Latvia1 Children’s Clinical of chromosomal abnormalities, including chromosome University Hospital Pediatric Clinic Riga-Latvia2 21, 18 and 13 trisomies, X and Y aneuploidies. Because Children’sClinicalUniversityHospitalMedical NIPT is very accurate (>99 % accuracy in 21, 18 and 13 Genetics and Prenatal Diagnosis Clinic Riga-Latvia3 trisomies) and is not life threatening for the fetus, has been recently introduced in prenatal care as a Introduction. Chromosome instability is a hallmark powerful tool in cases where invasive techniques of some rare inherited monogenic disorders, which do not seem to be necessary (i.e. advanced maternal age are characterized by failuretothrive,developmen- with normal ultrasound). Here we show a NIPT tal delay, congenital anomalies, immunodeficiency, protocol, called TrisoNIM®, which has been partially and predisposition to malignancy. performed in our laboratory, based in massive parallel The aim of the study is to reveal the chromo- sequencing. some aberrations, clinical features and disturbances Atotalof3000sampleswereincluded.DNA of the immune system of Nijmegen breakage from maternal plasma was extracted and sequenced syndrome (NBS), ataxia— telangiectasia syndrome using Ion Proton® protocols, optimized for the (A-T) and Fanconi anemia (FA) patients from detection of trisomies 21, 18, 13. In collaboration Latvia diagnosed in a single tertiary referral with BGI Europe, sequenced data were uploaded hospital (2006–2014). to the BGI cloud and analyzed using the NIFTY Methods. NBS was diagnosed in two girls and algorithm. Clinical information at birth of all cases two boys (average age 6.83 years, range 0.75– was available for follow-up. 17.75 years) from three families by standard cyto- High risk results for aneuploidies were detected in 36 genetic analysis. A-T was detected in two girls cases out of 3000 (1.2 %) including: 25 trisomy 21 (7.33 and 9.50 years). Four girls and ten boys (0.83 %), 7 trisomy 18 (0.23 %) and 8 sexual aneu- (average age 10 years, range 4.25–15.9 years) with ploidies (0.27 %). Confirmation results was performed various hematological disorders (bone marrow fail- in all instances by amniocentesis with prenatal ure, anemia, thrombocytopenia, leukopenia and/or array-CGH. 24/25 trisomy 21, 5/7 trisomy 18 and 5/8 malignancy) were evaluated by chromosome break- sexual aneuploidies were confirmed. False positive in age test using diepoxybutane (DEB). trisomy 21 was explained due to a vanishing twin effect; Results. Characteristic chromosomal rearrange- false positives in T18 and sexual aneuploidies were ments involving breakpoints at 7p13, 7q35, suggested to be caused by placental mosaicism 14q11.2 and 14q32 in standard blood cultures were (0.16 % of general population). No false negative observed in 13.6–30.0 % and 6.3 % of analysed were found. cells in patients with NBS and A-T, respectively. TrisoNIM is a highly accurate technique for testing NBS and A-T patients had typical clinical features. prenatal chromosomal aneuploidies. However, the pres- There were different changes in immunoglobulin ence of false positives due to placental biological pro- level (mostly IgA, IgG) and cell immunity in cesses such as placental mosaicism highlights the need NBS and A-T patients. FA was diagnosed in two for an invasive confirmation in every high girls and two boys (average age 9.17 years, range 8.17–10.17 years). Spontaneous chromosome aber- 7. Other rations were observed in 27.00±9.31 % (16–38 %) of analysed cells from FA patients compared to 7.P1 2.00±2.71 % (0–8%)innormalcontrolpopulation. DEB-induced chromosome aberrations were seen in Clinical, immunological and cytogenetic findings in 85.00±6.83 % (76–92 %) of analysed cells from children with chromosome instability syndromes FA patients compared to 7.31±3.35 % (4–12 %) in from Latvia. normal control population. Conclusions. Detection of underlying genetic defect Aigars Dzalbs1, Tatjana Prokofjeva2, Ieva Grinfelde3, provides the appropriate family genetic counseling and Liene Kornejeva3, Ieva Micule3, Zita Krumina3 subsequent management. S142 10th European Cytogenetics Conference 2015

7.P2 Andrei Tikhonov1, Olga Efimova2, Anna Pendina2, Irina Fedorova3,MikhailKrapivin1,OlgaChiryaeva2, Characterization by molecular cytogenetics, Tatyana Kuznetzova2, Vladislav Baranov2 array-CGH and whole transcriptome analysis St. Petersburg State University Genetics and of different batches of HeLa cells: genomic Biotechnology St.Petersburg-Russia1 D.O. Ott variability and divergent gene expression Research Institute of Obstetrics, Gynecology and Reproductology Laboratory for Prenatal Diagnosis of Roberto Valli1, Annalisa Frattini2, Marco Fabbri1, Elena Human Inborn and Inherited Diseases De Paoli1, Giuseppe Montalbano1, Laura Gribaldo3, St.Petersburg-Russia2 D.O. Ott Research Institute of Francesco Pasquali1, Emanuela Maserati1 Obstetrics, Gynecology and Reproductology Università dell’Insubria Dipartimento di Medicina Department of Assisted Reproductive Technologies Clinica e Sperimentale Varese-Italy1 Università St.Petersburg-Russia3 dell’Insubria, CNR Dipartimento di Medicina Clinica e Sperimentale, Istituto di Ricerca We have studied chromosome hydroxymethylation and Genetica e Biomedica Varese, Milano-Italy2 methylation patterns in metaphase chromosomes from European Commission Institute for Health and IVF-produced human triploid zygotes and blastomeres Consumer Protection Ispra-Italy3 of cleavage-stage embryos. To obtain metaphase chromosomes, zygotes and embryos were treated with 0.1 % The HeLa cell line is one of the best-characterized and colchicines, 0.9 % sodium citrate and fixed with used cell line in biomedical research. We compared the freshly prepared 3:1 methanol:acetic acid. Using genomic profile of different HeLa batches by chromo- indirect immunofluorescence, we have analyzed the some analysis, FISH and array-CGH with the aim to localization of 5-hydroxymethylcytosine (5hmC) and verify their genomic variation. The batches under study its co-distribution with 5-methylcytosine (5mC) on the were four, and they have been obtained from four QFH-banded metaphase chromosomes. different Italian laboratories: two of them derived from We observed the sequential changes in 5hmC and the same batch but then they were cultured in two 5mC patterns in the genome of human preimplantation different laboratories for about 8 years, as well as the embryos. In zygotes, 5hmC accumulates in both paren- two other batches, that derived from the same source, tal chromosome sets, but hydroxymethylation is more but were then cultured in two different laboratories for intensive in the poorly methylated paternal set. In the about 12 years. maternal set, chromosomes are highly methylated, but The results demonstrate that the four different contain little 5hmC. Hydroxymethylation is highly batches analyzed exhibit a time-dependent divergence region specific in both parental chromosome sets: in chromosome anomalies and rearrangements, with hydroxymethylated loci correspond to R-bands, but substantial differences in DNA content. Since genomic not G-bands, and have well-defined borders, which gains or losses or whole-chromosome aneuploidy can coincide with the R/G-band boundaries. The centro- imply drastic consequences in gene expression, we meric regions and heterochromatin at 1q12, 9q12, performed whole transcriptome analysis in order to 16q11.2, and Yq12 contain little 5mC and no 5hmC. verify the transcriptional effect of the different We hypothesize that 5hmC may mark structural/ aneuploidies and rearrangements of our clones. functional genome ‘units’ corresponding to chromo- We concluded that the genomic difference is paralleled some bands in the newly formed zygotic genome. by a severe alteration of the gene expression pro- Also, we suggest that the hydroxymethylation of file. Our results highlight that the use of HeLa R-bands in zygotes can be treated as a new characteristic cells might lead to faulty conclusions and to distinguishing them from G-bands. At cleavages, unreproducible results. chromosomes with asymmetrical hydroxymethylation of sister chromatids appear. They decrease in num- 7.P3 ber during cleavages, whereas totally non- hydroxymethylated chromosomes become numerous. Chromosome hydroxymethylation patterns in Our findings suggest that, in the zygotic genome, human zygotes and preimplantation embryos 5hmC is distributed selectively and its pattern is 10th European Cytogenetics Conference 2015 S143 determined by both parental origin of chromo- 7.P5 somes and type of chromosome bands— R, G, or C. At cleavages, chromosome hydroxymethylation pattern is Clonal chromosomal alterations during the mesen- dynamically changed due to passive and non-selective chymal stem cell cultivation- case report overall loss of 5hmC, which coincides with that of 5mC. Supported by RFBR&scholarship from RF Tamara Borgonovo1,MariaMarleneSolarewicz1, President. Isadora May Vaz1, Carmen Lucia Kunioshi Rebelatto1, Alexandra Cristina Senegaglia1, Debora Regina Daga1, 7.P4 Enilze Ribeiro2, Iglenir João Cavalli2, Paulo Roberto Slud Brofman1 The DNA Integrity Number: A novel approach for PUCPR CTC - Centro de Tecnologia Celular objective integrity classification of genomic DNA Curitiba-Brazil1 UFPR Departamento de Genetica samples Curitiba-Brazil2

Catherine Tissot1, Eva Schmidt1, Isabel Pechtl1, Marcus The use of human mesenchymal stem cells (MSC) has Gassmann1 been shown to be a promising strategy for cell therapy in Agilent Technologies R and D and Marketing injured tissues recovery. However, it has been described Waldbronn-Germany1 that MSCs can acquire genetic changes when cultivated in vitro and they would be more susceptible to undergo Genomic DNA (gDNA) is used as starting material in neoplastic transformation. Thus, the application of the experimental workflow of many applications in cytogenetic techniques is needed to evaluate the integ- molecular biology. The integrity of the DNA critically rity of MSC genetic material used in cell therapy. As affects the success of many downstream experiments quality control of the cells, at the Center for Cell like array CGH or sequencing. Initial electrophoretic Technology (CTC-PUCPR), we performed regularly analysis of the sample is highly recommended as the cytogenetic analyses (G-banding karyotyping). We respective downstream applications can be expensive report on a 67-year-old patient, male, selected to join and time consuming. The Agilent Genomic DNA the project: Autologous transplantation of mesenchymal ScreenTape Assay has been primarily developed for cells for treatment of severe and refractory isquemic the electrophoretic analysis of genomic DNA samples. cardiomyopathy. To evaluated chromosomal alterations, A ScreenTape is a pre-packaged microfluidic device one bone marrow and five MSC in different passages designed for performing electrophoretic applications in (P) were used. The bone marrow sample showed a a microscale format. It is used in combination with the normal karyotype: 46,XY[20], however, the MSC at Agilent 2200 TapeStation instrument. Degradation of P2 showed a hyperdiploid clone, with composite karyo- gDNA is typically a gradual process in which high- type: 43~52,XY,+X,+4,+5,+8[cp4] and non-clonal molecular weight DNA is fragmented into smaller spe- trissomies: +2, +10, +12, +20. Following pre- cies. It can occur either enzymatically, chemically or established criteria in CTC-PUCPR, this sample was mechanically. Judging the integrity of DNA by visual not approved for infusion. After that, the cells showed evaluation of the electropherogram trace is subjective normal karyotype at P4 and P5. A mononuclear cells and can be error-prone. In order to standardize this a sample of the same patient, which had been frozen for novel algorithm was developed to score gDNA samples backup, was cultivated, and at P3 showed a clonal on the 2200 TapeStation. The DNA integrity number translocation t(9;18)(p24;q11)[8]. At P5 the karyotype (DIN) is calculated from several features obtained from was normal. At this moment, from all cytogenetic anal- the electrophoretic trace and ranges from 1 to 10. ysis performed in CTC-PUCPR (152 samples), this was Here we show data demonstrating the reproducibil- the only case to show a clonal cytogenetic abnormality, ity, scalability and linearity of the DIN. The DIN and it was discarded. There is controversy about the use is independent from instrument, reagent and sam- of cells with cytogenetic abnormality for therapy, ple concentration variability and can be used as because of their tumorigenic doubtful capacity. Despite objective measure for determining the integrity of this, we decided, like Muntion et al. (2012), not to use gDNA. these cells for regenerative medicine purposes. S144 10th European Cytogenetics Conference 2015

7.P6 the Federal University of Parana. For this test, we calculated the rate of breaks per cell analysed as described by Cytogenetic analysis in Fanconi anemia Auerbach (1989). Bone marrow samples were studied by G-banding karyotyping and metaphases were analyzed Ana Teresa Schmid-Braz1, Miriam Lacerda Barbosa1, and interpreted according to ISCN— International Michele Moura Henrique1, Lismeri Wuicik-Merfort1, System for Human Cytogenetic Nomenclature (2013). Tamara Borgonovo1,ValderezRavaglioJamur1, Atotalof1839patientswerereferredfortheDEBtest. Carmem Bonfim1 Of these, 451 (25 %) were positive and 1388 (75 %) were UFPR Hospital De Clinicas Curitiba-Brazil1 negative. The bone marrow karyotypes was obtained in 471 samples of patients who tested positive for the DEB Fanconi Anemia (FA) is a rare autosomal recessive or test (some patients have more than one sample). In 129 X-linked disease characterized by chromosomal instabil- (27 %) we detected clonal chromosomal aberrations and ity, congenital abnormalities, progressive bone marrow most of them were observed on chromosomes: 1, 5, 7, 17, failure and predisposition to developing myelodysplastic 21, 22, 8, 9, 11, 15 and X. Our frequency of positive DEB syndrome, leukemias or solid tumors. The diepoxybutane tests (25 %) was similar to the one reported in the (DEB) test is the recommended method to confirm the literature. Cytogenetic analysis of bone marrow cells in diagnosis of FA. The DEB test was performed in patients FA patients is important to understand the progression to with bone marrow failure syndromes and/or the possibil- malignant hematological diseases although the role ity of having FA (clinical examination/family history). of these chromosomal aberrations in tumoral pro- From January 1996 to August 2014 we analyzed periph- gression remains controversial. Our data indicates eral blood (DEB test) and bone marrow samples that aberrations in chromosomes 1,5 and 7 are (G-banding karyotyping) from patients that were referred among the most frequently described in agreement with to the Cytogenetic Laboratory at the Clinical Hospital of literature. 10th European Cytogenetics Conference 2015 S145

AUTHOR INDEX Anasthase, Jessika 1.P22 Babicz, Mariusz 2.P33, 2.P34 Anca, Ioana Alina 1.P41 Baccarin, Marco 1.P48, 1.P50, Abd-Elaziz, Andıç, Neslihan 2.P56 3.P15 Mahmoud 2.P29 Andreu, Sandra 3.P2 Bache, Iben O15 Abo-Zeid, Mona 2.P29 Anelli, Luisa 2.P13, 2.P14 Badenas, Cèlia 3.P3 Abuelo, Diane N O13 Antonacci, Baden, Catherine 3.P4, 3.P5 Acar, Aynur 2.P44, 2.P45 Francesca L17 Bader, Ingrid 1.P2 Achille, Valentina 2.P11 Antonenko, Valentina 1.P45, 1.P70 Badiale, C L19 Achim, Ruxandra 1.P73 Arias, Amparo 2.P47 Bagowski, Christof 3.P18 Acloque, Hervé 4.P15 Armean, Petru 4.P1 Bak, Mads O15 Acunaş, Betül 1.P56 Arnoldo, Anthony O5 Balasar, Mine 2.P44, 2.P45 Adler, Esther 2.P2 Arranz, Maria Luisa 1.P68 Balasar, Özgür 2.P45 Ae Kim, Chong 1.P19 Arrieta, Isabel 5.P7 Baldazzi, Carmen O6 Afanasyev, Boris 2.P26 Arslan, Önder 2.P53 Baldinger, Rosa O9 Agostinho, Luciana 1.P61 Artan, Sevilhan 1.P71, 2.P55, Balducci, Estelle 2.P19 Agrati, Cristina 5.P8 2.P56, 2.P59, Balogh, István 1.P63 Águeda, Sofia 1.P64 3.P29, 5.P9 Balúchová, Katarína 2.P4 Ait Mouhoub, Tarik 1.P55 Artiguenave, François 5.P2 Banerjee, Ruby O14 Akay, Meltem Olga 2.P56 Arveiler, Benoit 3.P4, 3.P5 Bannwarth, Sylvie 3.P4, 3.P5 Akın, İsmigül 5.P12 Aslan, Huseyin 1.P71, 2.P55, Baranov, Vladislav 7.P3 Akkaya, Bahar 2.P8, 2.P31, 2.P56, 3.P29, Barasc, Harmonie 4.P15 2.P32 5.P9 Barasoain, Maitane 5.P7 Al Abdulwahed, Asteriou, Olga 2.P6, 2.P7, Barbosa Melo, Joana 3.P12, 3.P13 Omaiyah 1.P15 2.P30 Bardel, Claire 3.P4, 3.P5 Al Housawi, Yousef 1.P15 Ataca, Pınar 2.P53 Barranco, Laura 3.P11, 5.P5 Alazard, Emilie 2.P19 Atene, Claudio Barreña, Beatriz 3.P26 Albano, Francesco 2.P13, 2.P14 Giacinto O6 Barrenetxea, Gorka 5.P7 Aldred, Micheala A O13 Athanasiadou, Barros Alex, Marie-Pierre O1 Anastasia 2.P6, 2.P7, Santos-Rebucas, Alexandrou, Angelos 1.P44, 1.P46 2.P30 Cintia O13 Alexandrou, Ioanna 1.P46 Atilla, Erden 2.P53 Bartenhagen, Alfonsi, Melissa 3.P7 Atlı, Emine İkbal 1.P56 Christoph 5.P1 Alhan, C O4 Aurora, Sánchez 3.P3 Basinko, Audrey 2.P58 Alice, Marceau 2.P22 Averyanova, Maria 2.P26 Başaran, Seher 3.P33 Alikaşifoğlu, Mehmet 1.P32 Avila, Madalena 1.P23, 3.P10 Başlar, Zafer 2.P51 Alimova, Galina 2.P61 Aviles, Carmen 1.P47 Batsis, Ionnis 2.P30 Almeida, Carolina 1.P64 Aviram, Adina 2.P28 Batstone, Paul 2.P42 Alomar, Saad 1.P28 Avivi, Irit 2.P23 Baumann, Clarisse 1.P72 Alonzo, Melanie 1.P10 Aydın, Çigdem 2.P8, 2.P31, Bazin, Anne 1.P58 Altiok Clark, Ozden 1.P36, 2.P31 2.P32 Beaulieu Bergeron, Alvarez Perez, Aydin, Yunus 5.P9 Melanie 2.P16 Ana Beatriz 1.P19 Ayensa, Fernando 3.P2 Becher, Naja 1.P13 Alves, Cristina 1.P67 Bedeschi, Maria Amblard, Florence 1.P58 Baasten, M. 2.P46 Francesca 1.P48 Amorim, Marta 1.P67 Babál, Pavel 2.P4 Bednarek, Michal 2.P50 Anagnostopoulos, Babameto-Laku, Bedon, Luca 1.P38, 1.P39 Achilleas 2.P6, 2.P7, Anila 1.P21 Behjati, Farkhondeh 1.P3 2.P30 Babic Bozovic, Bel, Christine 1.P11 Anastasiadou, Violetta 1.P44 Ivana 1.P35, 1.P42 Belickova, Monika 2.P21 S146 10th European Cytogenetics Conference 2015

Bellacosa, Alfonso L23 Boudjarane, John 2.P19, 3.P4, Calvas, Patrick 1.P60 Ben Ayed, Ikhlas 1.P76, 5.P13 3.P5 Calvo, Xavier 2.P47 Benn, Peter 3.P7 Bougeon, Sandrine O7 Campan Fournier, Bentivegna, Angela 2.P40 Bouman, Pauline O2 Amandine 3.P4, 3.P5 Benzacken, Brigitte 1.P72 Bouret, Pauline 6.P1 Campo, Elias 2.P47 Beran-Fleischhacker, Bouvagnet, Patrice 1.P11 Canaz, Funda 2.P55 Eva 1.P65 Boyle, Eileen M. O16 Candeias, Cristina 1.P49, Beri, Silvana O13 Brady, Paul 3.P24 1.P69, Berker Karauzum, Bragagnolo, Silvia 1.P19 3.P19, Sibel 1.P36, 2.P8, Brajenovic-Milic, 3.P28 2.P31, Bojana 1.P35, 1.P42 Canellas, Anna 3.P11, 5.P5 2.P32 Branco, Miguel 3.P16 Cantara, Silvia 2.P25 Berkova, Adela 2.P21 Branisteanu, Dumitru 1.P59 Capal, Petr L18 Bernard, Delphine 2.P58 Brejcha, Martin 2.P37, Cappellani, Stefania 1.P38, Bernasovska, Jarmila 1.P29, 2.P17 2.P38 1.P39 Bertok, Sara 1.P75 Bresson, Jean-Luc 1.P58 Cappelletti, E L19 Bertuzzo, Sara O13 Breugelmans, M. 3.P30 Capri, Yline 1.P72 Besseau-Ayasse, Brezinova, Jan. 2.P12, Carbone, Lucia 5.P1 Justine 1.P58 2.P15, Carella, Massimo O6, O11, Bessenyei, Beáta 1.P63 2.P21 5.P1 Bettio, Daniela 2.P11 Brice, Alexis O1 Caria, Paola 1.P24, Beverloo, H. Berna 1.P6 Brison, Nathalie 3.P24 2.P25, Bevot, Andrea 1.P18 Brisset, Sophie 1.P22 2.P49 Beyer Stalder, Valérie O7 Brito, Filomena 1.P4, 1.P67 Carluccio, Paola 2.P14 Bhouri, Rakia O8 Brown, Celia 3.P10 Carreira, Isabel Bialecka, Magdalena 1.P74 Brumini, Gordana 1.P35 Marques 3.P16 Bician, Pavel 2.P4 Brun, Stéphanie 3.P4, 3.P5 Carrio, Ana 2.P47 Bilan, Frédéric 1.P5 Brunetti, Claudia 2.P13, Cartault, François 1.P58 Bilgin, Aynur Uğur 2.P45 2.P14 Carvalheira, Gianna 5.P4 Bindels-de Heus, Brychtova, Yvona 2.P37 Casieri, Paola 2.P13, Karen G.C.B. 1.P6 Bueno Barbosa, 2.P14 Blanchet, Patricia 6.P1 Fernanda 1.P9 Castells, Neus 5.P5 Blatnik, Ana 3.P8 Buisson, Adrien 1.P11 Catty, Marie 1.P58 Bliznets, Elena 1.P70 Bujoran, Cornel 1.P59 Cavalli, Iglenir João 7.P5 Boa-Sorte, Ney 1.P61 Burdova, Alena 2.P37, Cavelier, Lucia 5.P6 Boduroğlu, Koray 1.P32 2.P38 Cazorla, Eduardo 1.P10 Bodzasova, Cecilia 2.P38 Burlibasa, Liliana 4.P1 Cazzola, Mario L15 Boito, Simona 3.P15 Busa, Tiffany 1.P58 Cechova, Hana 2.P18 Bonaglia, Maria Clara O13 Bystricka, Dagmar 2.P18 Celentano, Claudio 3.P7 Bondarenko, Sergey 2.P26 Cellamare, Angelo 2.P13, Bonfim, Carmem 7.P6 Caba, Lavinia 1.P59 2.P14 Bonnet, Céline 3.P4, 3.P5 Caballero, Susana 3.P2 Cermak, Jaroslav 2.P12, Borgonovo, Tamara 7.P5, 7.P6 Cabras, Valentina 1.P24 2.P15, Boronova, Iveta 1.P29, 2.P17 Cabrejas Nuñez, 2.P21 Borregán, Mar 3.P17, 5.P5 M José 3.P32 Cermák, Martin 2.P4 Bortolai, Adriana 1.P12 Cakanova, Gladys 2.P17 Cerutti, F L19 Bota, Adrian 4.P8 Calabrese, Giuseppe 3.P7 Cervera-Acedo, Botelho, Pedro 3.P22 Calmels, Nadège 1.P60 Cristina 3.P31 10th European Cytogenetics Conference 2015 S147

Cetin, Zafer 1.P36, Costa, Dolors 2.P47 Deák, Ladislav 2.P4 2.P31 Coubes, Christine O12, 6.P2 Deblois, Ceylan, Ahmet Cevdet 1.P32 Couet, Dominique 1.P5 Marie-Christine 1.P58 Chatron, Nicolas O1, 1.P11, Coussement, Aurelie 1.P72 Debrock, Sophie 3.P20 3.P4, 3.P5 Cremers, Eline O4 Dehaspe, Luc 3.P24 Chaussenot, Annabelle 1.P66 Criado, Begoña 5.P7 Dehgan, Tahir 3.P33 Chen, Chuanfei 2.P1 Cristea, Cristina 1.P59 Deineko, Elena 4.P3 Chen, Yunxin 2.P1 Cristina Sgardioli, del Campo, Miguel 5.P5 Chenard, Marie-Pierre 2.P3 Ilária 1.P7 Delemer, Brigitte 1.P55 Chernykh, Vyacheslav 1.P70 Csalodi, Renata 2.P27 Delphine, Kolly O7 Chinetti, Sara 5.P8 Cueto-González, Anna 5.P5 Delwel, Ruud L14 Chiryaeva, Olga 7.P3 Culligan, Dominic 2.P42 Demidov, Dmitri 4.P3 Chitu, D O4 Cumbo, Cosimo 2.P13, 2.P14 Demirel, Ayca 3.P29 Choi, Hong-Il 4.P14 Cuoco, Cristina 1.P14 den Hollander, N.S. 3.P27 Choiset, Agnès 1.P58 Custódio, Sónia 1.P57 den Ouden, E. 2.P46 Chudoba, Daniel 1.P40 Çırakoglu, Ayşe 2.P51 Denayer, E. 3.P30 Chukhrova, Alena 1.P70 Çilingir, Oğuz 2.P55, 2.P56 Depienne, Christel O1 Ciccone, Roberto O13 Dettori, Tinuccia 2.P25, 2.P49 Cieslikowska, Agata 1.P74 da Silva, Iane 1.P61 Deviren, Ayhan 2.P51 Cifola, Ingrid O11, 5.P1 D’Addabbo, Pietro 5.P1 Devriendt, Koen 3.P24 Cigudosa, Juan C 6.P3 Daga, Debora Regina 7.P5 D’Hooghe, Thomas 3.P20 Cilibrasi, Chiara 2.P40 Dahl, Niklas 5.P6 Di Giacomo, Cilingir, Oguz 1.P71, Dakouri, Edwige 5.P13 Marilena Carmela O13 2.P59, Dalprà, Leda 2.P40 Di Iorgi, Natascia 1.P14 3.P29 Dalva, Klara 2.P53 Di Nicola, Mélanie 6.P1, 6.P2 Cirigliano, Vincenzo 3.P11, Daniele, Giulia O6 Dijkhuizen, Trijnie O2 5.P5 Dantas, Anelisa Gollo 1.P12 Dimitriadou, Eftychia 3.P20 Cizmár, Andrej 2.P4 Darbas, Sule 2.P32 Dimos, Luiza 1.P73 Claire, Oudin 2.P22 Darja, Nemcova 3.P1 Ding, Jie O6 Claire, Colmant 3.P14 Daumer-Haas, Doco-Fenzy, Martine 1.P55, 1.P58 Cleva, Lisa 1.P38, Cornelia 3.P18 Dolezel, Jaroslav L18 1.P39 David, Elodie O7 Dollfus, Hélène 1.P60 Coccaro, Nicoletta 2.P13, Dayem-Quere, Manal 1.P26, 1.P66 Domínguez-Garrido, 2.P14 De Bellis, Gianluca 5.P1 Elena 3.P31 Coelho Molck, Miriam 1.P7 De Braekeleer, Marc 2.P58 Dominique, Pineau 3.P14 Coelho Pinto, Marta 3.P12 de Fréminville, Domnariu, Carmen 4.P1 Colleaux, Laurence 1.P27 Bénédicte L3 Domnariu, Colovati, Mileny 1.P12 de Greef, Inge O4 Horatiu Paul 4.P1 Constantinou, Maria 2.P50 De Leener, Anne 3.P23 Donadi, Eduardo Copáková, Lucia 2.P4 De Molfetta, Greice 1.P61 Antônio 1.P9 Coppede’, Fabio 5.P10 de Paiva Ferreira, Donaghoue, Celia 3.P10 Corbo, M L19 Ríudo 4.P12 Doplihar Kebe, Ana 2.P54, Cordeiro, Leonardo 1.P61 De Paoli, Elena 2.P5, 7.P2 2.P60 Corinne, Metay 3.P14 De Pedro, Mª Angeles 1.P47 Doray, Bérénice 1.P27 Cormier Daire, Valérie 1.P27 De Rademaeker, M. 3.P30 Dória, Sofia 1.P64 Correia, Hildeberto 1.P4, de Ravel, Thomy 3.P20, 3.P24 dos Santos, Suely 1.P61 1.P67 De Wilde, H. 3.P30 dos Santos Fonseca, Corvin, Aiden L13 De Winter, S. 3.P30 Ana Carolina O15 S148 10th European Cytogenetics Conference 2015

Doubek, Michael 2.P10, Emmert, Vanessza 2.P27 Foijer, Floris L11 2.P37 Endlicher, Marlies O3 Fonseca e Silva, Douet Guilbert, Erci Baysak, Mine 2.P59 Maria da Luz 1.P49, Nathalie 1.P58, Erhardt, Georg 4.P5 1.P69, 2.P58 Erjavec Skerget, 3.P19, Drabova, Jan. 1.P34, 1.P40, Alenka 3.P8 3.P28 3.P25 Eroglu, Onur 2.P59 Fonseka, Gothami 4.P10 Drévillon, Loïc 1.P22 Erzurumluoglu, Fonteneau, Eric O1 Drevojankova, Ebru 1.P71, Fortuño, Joana 3.P2 Bronislava 2.P37, 2.P38 3.P29, Frattini, Annalisa 2.P5, 7.P2 Drexler, Hans G O6 5.P9 Frau, Daniela Drunat, Severine 1.P72 Esbravatti Stephano Virginia 2.P25, Ducasse, Alain 1.P55 Colovati, Mileny 1.P19 2.P49 Ducrocq, Marie L3 Escalona, Ariadna 3.P2 Frentz, Barbara O2 Dufke, Andreas 1.P18 Estelle, Balducci 2.P22 Frodsham, Richard 4.P10 Dugas, Martin 5.P1 Ester, Margarit 3.P3 Froguel, Philippe L8 Dulcetti, Francesca 5.P8 Etter, Laurence O7 Frug, Yulia 2.P23 Dumont, Florent 3.P5 Evangelidou, Paola 1.P44 Fu, Beiyuan O14 Dupont, Celine 1.P72 Fukushima, Dupont, Fabbri, Marco 2.P5, 7.P2 Yoshimitsu 1.P53 Jean-Michel O8, 1.P72, Fabretto, Antonella 1.P38 1.P76, 3.P4, Fadl-Elmula, Imad 1.P79 Gaillyova, Renata 1.P8 3.P5, 5.P13 Faletra, Flavio 1.P39 Gaitatzi, Maria 2.P6, 2.P7, Durak Aras, Fantasia, Donatella 3.P7 2.P30 Beyhan 2.P55, Faoucher, Marie O1 Galán, Pedro O10 2.P56, Faric, Jasmina 2.P54 Galhano, Eulália 3.P12, 3.P13 2.P59 Fautrelle, Anne 1.P11 Gamal-Eldeen, Amira 2.P29 Duran, Jordi 3.P2 Fedorova, Irina 7.P3 Gamba, R L19 Duras, Juraj 2.P38 Fekete, György 1.P25 Gambel Benussi, Durmaz, Ceren Damla 2.P53, 5.P12 Ferec, Claude 3.P4, 3.P5 Daniela 1.P39 Dvorackova, Jan. 2.P20 Ferguson-Smith, Ganapathy, Ramesh 3.P10 Dvorakova, Hana 2.P38 Malcom O10, Gandolfi, Sara 2.P11 Dzalbs, Aigars 7.P1 4.P9, García-Barcina, María 3.P26, 3.P31 4.P16 García-Cosío, Monica 2.P48 Edery, Patrick O1 Fernández, Daniel 3.P11 García-Galloway, Eva 3.P32 Eelmäe, Imbi 1.P20 Fernandez-Mellado, García-Sagredo, Efimova, Olga 7.P3 Majo 3.P26 José M. 3.P32 Eggermann, Thomas 1.P52, 3.P21 Ferrari, Sergio O6 Gaspar, Isabel 1.P4 Ehman, William 2.P62 Ferreira, Cristina 1.P4, Gassmann, Marcus 7.P4 Eker, Damla 1.P56 1.P67 Gatinois, Vincent O12, El Khattabi, Laïla O8, 1.P76, Ferreira, Susana Isabel 3.P12, 1.P51, 3.P4, 3.P5, 3.P16 5.P3 5.P13 Ferri, Emanuela O13 Gatu, Alina 1.P59 ElIdrissi-Zaynoun, Fiaño, Marian 3.P26 Gavrilina, Olga 2.P61 Najat O4 Fiorio, Patrizia 1.P14 Gazoni, Thiago 4.P16 Elverdi, Tuğrul 2.P51 Flodr, Patrik 2.P35 Genevieve, David O12, 5.P3, Emilie, Alazard 2.P22 Flori, Elisabeth 1.P58 6.P1, 6.P2 10th European Cytogenetics Conference 2015 S149

Genualdo, Viviana 4.P9 Gribaldo, Laura 7.P2 Hélène, Zattara 2.P22 Gérard, Michel 2.P22 Griffin, Darren 4.P10 Helias, Catherine 2.P3 Gérard, Tachidjian 3.P14 Grigore, Sandra 1.P33 Hernando, Cristina 3.P17 Gervais, Carine 2.P3 Grigore, Mihaela 3.P6 Héron, Delphine O1 Gesson, Noémie O7 Grinfelde, Ieva 7.P1 Hestand, MS L9 Ghasemi Firouzabadi, Groenewegen, L. 3.P27 Hikkel, Imrich 2.P4 Saghar 1.P3 Guanciali Franchi, Hild, Mélanie 1.P60 Gheorghiu, Ligia 1.P59 Paolo 3.P7 Hlinková, Katarína 2.P4 Gıran, Gizem 2.P52 Guardiano, Micaela 1.P64 Hoffer, M.J.V. 3.P27 Giglio, Sabrina O13 Guéganic, Nadia 2.P58 Hojsikova, Ivana 2.P39 Gilbert-Dussardier, Gueguen, Paul 3.P4, 3.P5 Holweg, Marta 2.P33, Brigitte 1.P5 Guerneri, Silvana 1.P48, 2.P34 Gil-da-Silva-Lopes, 1.P50, Holzerova, Milena 2.P10, Vera Lúcia 1.P7, 1.P9, 3.P15 2.P24, 5.P2 Guetg, Adriano O9 2.P35, Gimelli, Giorgio 1.P14 Guichoux, Erwan 3.P4, 3.P5 2.P36 Gindina, Tatiana 2.P26 Guilherme, Roberta Hoogenboom, M. 3.P27 Giorda, Roberto O13 Santos 1.P12 Hoopmann, Markus 1.P52 Giralt, Marina 3.P17 Guimiot, Fabien 1.P72 Hoportean, Ionut 4.P8 Girard, Françoise 1.P58, 1.P60 Gumus, Evren 1.P71 Horka, Katerina 2.P37 Girard, Manon 6.P2 Gutkowska, Anna 1.P74 Houben, Andreas 4.P3 Giuliano, Fabienne 1.P26 Guzzi, Andrèe 2.P40 Hruba, Martina 2.P10 Giulotto, Elena L19 Gürbüz, Cansu 5.P12 Huerta, Iratxe 5.P7 Gloning, Karl- Philipp 3.P18 Gürkan, Hakan 1.P56 Huijsdens, Karin 1.P37 Gloning, Vera 3.P18 Gürman, Günhan 2.P53 Hulsman, Lorette. O.M. 1.P6 Gnidovec Strazišar, Iacobucci, Ilaria O6 Barbara 1.P75 Haas, Oskar A. 1.P65 Iannuzzi, Alessandra 4.P5, 4.P9 Goelz, Rangmar 1.P52, 3.P21 Hacıhanefioğlu, Seniha 2.P51 Iannuzzi, Leopoldo 4.P5, 4.P9 Gollo Dantas, Anelisa 1.P19 Haferlach, Claudia L21 Iha, Wakaba 1.P53 Gomez, Candida 2.P47 Hajek, Roman O16 Iliev, Gheorghe 3.P6 Gonçalves, Helena 3.P12 Halgren, Christina O15 Ilisson, Piret 1.P17 González, Javier 5.P7 Haltrich, Irén 1.P25 Impera, Luciana 2.P14 González Ramírez, Halusková, Viktória 2.P4 Indrak, Karel 2.P10 Amanda Rocío 1.P62 Hancarova, Miroslava 1.P34 Ioannidou, Charithea 1.P44 González-Tizón, Ana O10 Hans, Christine 3.P23 Ioos, Christine 1.P76 Goosens, Michel 3.P4, 3.P5 Hansson, K.B.M. 3.P27 Irene, Madrigal 3.P3 Gorduza, Carmen Haquet, Emmanuelle 6.P2 Iskas, Michalis 2.P7 Nicoleta 1.P59 Harbuz, Radu 1.P5 Isrie, M L9 Gorduza, Eusebiu Harceaga, Lucian 4.P8 Işık, Sevgi 2.P55, Vlad 1.P59, 3.P6 Harrison, Christine J. L22 2.P56 Gorostiaga, Alvaro 3.P26 Has, Recep 3.P33 Ittel, Antoine 2.P3 Gramescu, Mihaela 1.P59, 3.P6 Hasegan, Ioana 4.P1 Iuzzolino, Paolo 2.P43 Grasshoff, Ute 1.P18 Hassa, Hikmet 5.P9 Iwaniuk, Agata 2.P50 Grati, Francesca Hassold, Terry L1 Izakova, Silvia 2.P12, Romana 5.P8 Havlovicova, Marketa 1.P34, 1.P40 2.P15, Graur, Elena 1.P59 Hawkins, Cynthia O5 2.P18, Grebenyuk, Lubov 2.P61 Heinimann, Karl L7 2.P21 S150 10th European Cytogenetics Conference 2015

Izard, Vincent 1.P22 Kawamura, Rie 1.P53 Krapivin, Mikhail 7.P3 Izquierdo, Luis 5.P5 Kehrer, Martin 1.P18, Krasikova, Alla 4.P2, 4.P6 İlhan, Osman 2.P53 1.P52 Kravchenko, Sergey 2.P61 Jacky, Peter O15 Kemal Yucel, Orhan 2.P32 Krejci, Pavel O9 Jacquemont, Marie-Line 1.P27 Keren, Boris O1 Krejcik, Zdenek 2.P18 Jacquemont, Sebastien L12 Kerkhoffs, L. 2.P46 Kremer, Valérie 1.P60 Jacquette, Aurelia O1 Kerstjens-Frederikse, Kreuzer, Karl-Anton 2.P9 Jancuskova, Tereza 2.P9 Wilhelmina O2 Kriegova, Eva 2.P10 Jansen, Joop O4 Ketoni, Andria 1.P44 Kroes, W. 2.P46 Jardim, Ana 3.P12, Keymolen, K. 3.P30 Kronberger, Gabriela 1.P1, 1.P2 3.P13 Khader, Samer 2.P2 Krumina, Zita 7.P1 Jarosova, Marie 2.P10, Khalfallah, Ayda 5.P6 Kruzova, 2.P24, Kim, Chong Ae 1.P12 Lenka 2.P10, 2.P35, Kim, Hyun Hee 4.P14 2.P24, 2.P36 Kirgiz, Melike 3.P33 2.P35, Jaszczuk, Ilona 2.P33, Kiss, Eszter 1.P25 2.P36 2.P34 Kitzis, Alain 1.P5 Kubalakova, Marie L18 Jedrzejczyk, Slawomir 2.P50 Klade, Riin 1.P17 Kucharczyk, Marzena 1.P74 Jezela-Stanek, Klein, Alina 2.P28 Kucuk, Halime 3.P29, Aleksandra 1.P74 Klein, S O4 5.P9 Jimenez, Mélanie 1.P58 Kleinfinger, Pascale 1.P58 Kugaudo, Monika 1.P74 John, Boudjarane 2.P22 Kloosterman, Kuglik, Petr O16, 1.P8, Jonasova, Anna 2.P12, Wigard O12, 6.P1, 3.P1 2.P15, 6.P2 Kulikova, Tatiana 4.P2 2.P21 Knegt, Lia 1.P37 Kulkarni, Abhijit 1.P23, Jonveaux, Philippe 3.P4, 3.P5 Knorr, C 4.P5 3.P10 Joost, Kairit 1.P16, Kocarek, Eduard 3.P25 Kumar, Sathish 2.P1 3.P9 Kok, Klaas 5.P1 Kundak, Çağatay 2.P52 Jungwirth, Andreas 1.P1 Kokalj Vokac, Nadja 3.P8 Kunioshi Rebelatto, Kagan, Karl Oliver 1.P18, Kolog, Hilla 2.P23 Carmen Lucia 7.P5 3.P21 Koltsina, Marina 1.P30 Kurg, Ants 1.P30 Kajtar, Bela 2.P27 Kornejeva, Liene 7.P1 Kurilo, Liubov 1.P70 Kalamees, Riste 1.P17 Kosednar, Nuša 2.P60 Kuru, R.Dilhan 2.P51 Kaluzewski, Bogdan 2.P50 Kosina, Romuald 4.P7 Kurvinen, Elvira 3.P9 Kaluzewski, Tadeusz 2.P50 Kosyakova, Nadezda 2.P9, 4.P2 Kutlay, Nüket Yürür 2.P53 Kanellou, Panagiota 2.P41 Kosztolanyi, Szabolcs 2.P27 Kuuse, Kati 1.P16, Kanivets, Ilya 1.P43 Kotlukova, Natalia 1.P45 1.P17, Karabulut, Halil Gürhan 2.P53 Koudelka, Marek 3.P1 3.P9 Karaman, Birsen 3.P33 Kouvidi, Elisavet 2.P41 Kuzina, Natalia 1.P70 Karaskova, Eliska 1.P34 Kovac, Michal B. L7 Kuznetzova, Tatyana 7.P3 Karataş, Günay 5.P12 Kovrigina, Alla 2.P61 Küpesiz, Alphan 2.P31 Karcagi, Veronika 1.P25 Kowalczyk, Jerzy R. 2.P33, Labalme, Audrey O1, 1.P11 Kariminejad, Roxana 1.P3 2.P34 L’Abbate, Alberto 2.P43, Karmous-Benailly, Kozlova, Yulia 1.P43, 5.P1 Houda 1.P26, 1.P45, Lacerda Barbosa, 1.P66 1.P70 Miriam 7.P6 Kashevarova, Anna 1.P31 Krajewska-Walasek, Lafage, Marina 2.P19 Kasikova, Katerina 1.P8 Malgorzata 1.P74 Lagarde, Arnaud 3.P4, 3.P5 Katunaric, Miljenko 1.P35 Kramar, Filip 2.P18 Lai, Lai Ching 2.P1 10th European Cytogenetics Conference 2015 S151

Lalayanni, Chrysavgi 2.P6, 2.P7 Liehr Thomas L24, Magomedova, Hanum 1.P70 Laleli, Yahya 2.P52 2.P9, Mahdi, Samia 1.P79 Lallaoui, Hakima 1.P58 4.P2 Mahulja-Stamenkovic, Lamblin, Denis 1.P27 Lim, Alvin Soon Tiong 2.P1 Vesna 1.P42 Landais, Emilie 1.P55, Lim, Tse Hui 2.P1 Mai, Sören 1.P65 1.P58 Lin, Evan Rui Long 2.P1 Malageanu, Marinela 1.P33 Larmuseau, MHD L9 Lind, J. 3.P27 Malan, Valérie 1.P27 Lau, Katarzyna 2.P50 Lindner, Matthias 3.P18 Malcovatti, Luca L15 Lavoura, Nuno 3.P12, Lind-Thomsen, Allan O15 Malvestiti, Francesca 5.P8 3.P13 Ling, Ng Bee 6.P1 Malvestiti, Barbara 5.P8 Lawrie, Martin 4.P10 Lipina, Radim 2.P20 Mamaev, Nikolay 2.P26 Lazarou, Zografia 2.P6, Liu, Qing 4.P17 Mancini, Grazia M.S. 1.P6 2.P7, Liu, Huan 4.P17 Mandel Jean-Louis 1.P60 2.P30 Liuti, Rosaria 5.P8 Manguoglu, Esra 2.P31 Le Bris, Marie-Josée 2.P58 Lizcova, Libuse 2.P18, Mann, Kathy 3.P10 Le Tessier, 2.P21 Mansour, Sahar 1.P23 Dominique O8, Lloret, Manuel 1.P47 Mantescu, Oana 1.P33 1.P76, Lloveras, Elisabet 3.P11, Mantovani, Vilma O6 5.P13 5.P5 Marasco, Elena O6 Leão, Miguel 1.P64 Lo Cunsolo, Crocifissa 2.P43 Marchese, Serena 1.P14 Lebbar, Aziza 1.P76, Lobo, Rosa 1.P68, Marczak, Aleksandra 1.P74 5.P13 2.P48 Marie, Loosveld 2.P22 Lebedev, Igor 1.P31 Locatelli, Franco 2.P5 Marie-Emmanuelle, Lecaignec, Cédric 1.P58 Lochu, Philippe L26 Naud 3.P14 Lee, Junki 4.P14 Lomiento, Mariana O6 Marie-Victoire, Senat 3.P14 Lefort, Genevieve O12, 1.P51, Longa, Mikel 5.P7 Marín, Leonor 3.P2 5.P3, 6.P2 Lonoce, Angelo O6, 5.P1 Marina, Legendre, Marine 1.P5 Lopes, M. Elisa 1.P49, Lafage-Pochitaloff 2.P22 Legius, Eric 3.P24 1.P69 Markova, Zhanna 1.P43 Leheup, Bruno 1.P58 López, María 3.P31 Markova, Jan. 2.P12 Lejeune, Elodie O1 Lopez-Galvez, Jose J 1.P47 Marks, Karen 1.P23 Lejman, Monika 2.P33, Louzada, Sandra O14 Marques, Barbara 1.P4, 2.P34 Loverro, Anna O11 1.P67 Lenzini, Elisabetta 1.P38, Lovrecic, Luca 1.P54, Marques Carreira, Isabel 3.P12, 1.P39 1.P75 3.P13 Leroy, Camille 1.P27, Lucie, Tosca 3.P14 Marquet, Valentine 4.P15 1.P72 Luczay, Andrea 1.P25 Marsano, Rene’ Lesca, Gaetan O1 Lufin, Nicolas 3.P23 Massimiliano O6 Lespinasse, James 1.P58 Luscan, Armelle O8 Martin, Dominique 1.P58 Letard, Pascalie 1.P58 Lysak, Daniel 2.P10 Martin, Carmen 3.P11 Letourneur, Franck 3.P4, 3.P5 M.Angeles, Vallecillos 3.P3 Martinelli, Giovanni O6 Leustean, Letitia 1.P59 Macchia, Gemma O11, Martínez-Lage, Andrés O10 Levchenko, Elena 1.P45 5.P1 Martiniuc, Violeta 3.P6 Lévy, Jonathan 1.P72 Machado, Fábio 1.P77 Martins, Rosa 1.P61 Leyder, M. 3.P30 Maciej, Borowiec 2.P50 Martins, Marcia 3.P22 Lhotska, Halka 2.P12, Mackensen-Haen, Martins Travenzoli, 2.P18, Susanne 3.P21 Natália 4.P11, 2.P21 Madan Kamlesh, L28 4.P12, Liebrecht, Daniela 3.P18 Maggi Federico, 5.P8 4.P13 S152 10th European Cytogenetics Conference 2015

Mascarenhas, Migliore, Lucia 5.P10 Moutinho, Osvaldo 3.P22 Alexandra 3.P12, Mignot, Cyril O1 Moyses-Oliveira, 3.P13, Miguet, Laurent 2.P3 Mariana 1.P12, 5.P4 3.P16 Mihalceanu, Elena 3.P6 Mozo de Rosales, Maserati, Emanuela 2.P5, Mihci, Ercan 1.P36 Fernando 3.P26 7.P2 Mikesova, Michaela 2.P24, 2.P35, Mölter-Väär, Triin 3.P9 Masood, Rahim O9 2.P36 Muehlematter, Massol, Christophe 1.P26 Mikulasova, Aneta O16, 1.P8, Dominique O7 Matveeva, Natalya 1.P31 3.P1 Muga, Javier 5.P7 Mau-Holzmann, Milani, Donatella 1.P50 Mugneret, Francis 1.P58 Ulrike 1.P52, Miller, Konstantin L25 Mujezinovic, Faris 3.P8 3.P21 Minderer, Sabine 3.P18 Munoz, Concha 2.P47 Mauvieux, Laurent 2.P3 Minervini, Angela 2.P13, 2.P14 Mursalimov, Sergey 4.P3 Mavroudi, Irene 2.P41 Minervini, Muus, Petra O4 May Vaz, Isadora 7.P5 Crescenzio F. 2.P13, 2.P14 Mühlematter, Mayer, Jiri 2.P37 Minzhenkova, Marina 1.P43 Dominique 2.P43 Mazzagatti, M L19 Miriam, Muñoz 3.P3 Müller, Stefan O3 Mazzotti, Diego 1.P12 Missirian, Chantal 1.P58 Mydlarova McCarter, J L19 Mlynek, Marlena 1.P74 Blascakova, Marta 1.P29, 2.P17 McGowan-Jordan, Mochel, Fanny O1 Jean 2.P16 Mogos, Voichita 1.P59 Nadarajah, Niroshan O6 Mediano, Carmen 3.P17 Mohamed, Gihan 2.P62 Nagy, Agnes 2.P27 Medina-Acosta, Monier, Laurie 2.P3 Najmabadi, Hossein 1.P3 Enrique 1.P77 Monsaert, A. 3.P30 Naoufal, Rania 1.P5 Mehrjouy, Mana M. O15 Montalbano, Giuseppe 2.P5,7.P2 Napoli, Flavia 1.P14 Melaragno, Maria Montaldo, Caterina 1.P24 Narci, Clément 1.P11 Isabel 1.P12, 1.P19, Mora Gujosa, Natacci, Federica 3.P15 5.P4 Adelardo 1.P62 Nava, Caroline O1 Melo, Joana Barbosa 3.P16 Morel, Frédéric 2.P58 Naveira, Horacio O10 Meloni, Vera Ayres 1.P12 Moreno Pelayo, Nazarenko, Melotte, Cindy 3.P20 Miguel Angel 3.P32 Lyudmila 1.P31 Melotte, G. 3.P30 Morgan, Gareth J. O16 Nebral, Karin 1.P65 Mendez, Begoña 3.P11 Morizio, Elisena 3.P7 Nectoux, Juliette O8, 3.P4, Mendiola, Christina 2.P62 Morotti, Denise 3.P15 3.P5 Menendez, Monica 3.P11 Morrow, Bernice L29 Nedelea, Florina 1.P73 Meneses Lopes, Mostafa, Shady 2.P29 Nemescu, Dragoş 3.P6 Denilce 4.P11, 4.P12, Mota Freitas, Nergadze, SG L19 4.P13 Manuela 1.P49, 1.P69, Neusser, Michaela O3 Mensah, Martin A. L9 3.P19, 3.P28 Ng, Xin Hui 2.P1 Menzorov, Aleksey 1.P31 Motei, Gabriela 1.P41 Nicolae, Ioana 4.P8 Mertens, Fredrik O11 Motta, Silvia 1.P48, 3.P15 Nieddu, Mariella 4.P4 Metspalu, Andres 1.P30 Motyka, Oldrich 2.P20 Nikolaeva, Elena 2.P26 Mezzanotte, Roberto 4.P4 Mouka, Aurélie 1.P22 Nomdedeu, Benet 2.P47 Michalova, Kyra 2.P12, 2.P15, Moura, Henrique Nomdedeu, 2.P18, 2.P21 Michele 7.P6 Meritxell 2.P47 Michel, Gérard 2.P19 Moura Novaes, Novara, Francesca O13 Mickova, Pavla 2.P24 Camila 4.P11, Novotna, Drahuse 1.P40, 3.P25 Micule, Ieva 7.P1 4.P12, Nucaro, Anna Lisa 1.P24 Middleton, Sonia 1.P77 4.P13 Nur, Banu 1.P36 10th European Cytogenetics Conference 2015 S153

Nursal, Ayse Feyda 2.P59 Pajusalu, Sander 1.P17, Pelletier, Valérie 1.P60 Nusbaum, Sylvie 1.P27 1.P20 Pembrey, Marcus L5 Palazzo, Antonio O6 Pendina, Anna 7.P3 O Connor, Rebecca 4.P10 Palka, Giandomenico 3.P7 Pereira, Jorge O10, 4.P9, Obukhova, Tatiana 2.P61 Palumbo, Orazio O6, O11, 4.P16 Ochando, Isabel 1.P10, 1.P47 5.P1 Pérez Sánchez, Ochiana, Diana Pammer, Judit 2.P27 Matías 1.P62 Aurelia 1.P41 Pani, Jhumur 1.P28 Perini, Giovanni O11 Olde Weghuis, Panovska, Anna 2.P37 Permyakova, Daniel O4 Papadaki, Helen 2.P41 Natalya 4.P3 Oliva Teles, Papaevripidou, Perricone, Natalia 1.P49, 1.P69, Ioannis 1.P44, Margherita O6 3.P19, 3.P28 1.P46 Perrin, Laurence 1.P76 Oliveira Fernanda, Papaioannou, Perucatti, Angela 4.P5, 4.P9 Paula 1.P49, 1.P69, George 2.P6, 2.P7, Peterlin, Borut 1.P54, 3.P28 2.P30 1.P75 Olivero, Luciano 1.P61 Papajik, Tomas 2.P10, Petrova, Irina 2.P26 Oltova, Alexandra 2.P10, 2.P37 2.P35, Philippe, Anne 1.P27 Oneda, Beatrice O9 2.P36 Pianovski, Mara 2.P57 Ongoren, Seniz 2.P51 Papayannidis, Cristina O6 Pichon, Bruno 3.P23 Oniashvili, Nino 2.P28 Paquis-Flucklinger, Pieper, D 4.P5 Onofriescu, Mircea 3.P6 Véronique 1.P26, 3.P4, Pika, Tomas 2.P24 Onur Kucuk, Halime 1.P71 3.P5 Pikó, Henriett 1.P25 Oprea, Lucian 1.P33 Parise-Maltempi, Pillai, Rita 2.P49 Ordoñez, Elena 3.P11, 5.P5 Patricia 4.P16 Pinto, Joel 1.P64 Orr-Urtreger, Avi 2.P23 Parnes, Doris 2.P28 Pinto Leite, Rosário 3.P22 Ortega, Veronica 2.P62 Pasquali, Francesco 2.P5, 7.P2 Pinton, Alain 4.P15 Ortiz, Saturnino 1.P68 Passemard, Sandrine 1.P72 Piovani, Giovanna 5.P11 Ortiz-Lastra, Eduardo 5.P7 Patrat, Catherine 1.P27 Pipoli da Fonseca, Ossenkoppele, Patrick, Folliot 3.P14 Juliana 3.P4, 3.P5 Gert-Jan O4 Patsalis, Philippos O13 Pique, Meritxell 3.P11 Osternack, Bruno 2.P57 Pauciullo, Alfredo 4.P5, 4.P9 Piras, FM L19 Ottaviani, Emanuela O6 Pavel, Anca 1.P73 Pires, Silvia 1.P49, 1.P69, Ozdemir, Muhsin 1.P71, 2.P55, Pavlikova, Gabriela 2.P39 3.P19 2.P56, 2.P59, Pavlistova, Lenka 2.P15, Pires, Luís 3.P29 2.P18 Miguel 2.P48, 3.P13, Özcan, Muhit 2.P53 Pebrel-Richard, Céline 1.P58 3.P16 Özen, Yasemin 1.P56 Pechtl, Isabel 7.P4 Pitronova, Sylva 2.P20 Pecile, Vanna O13, 1.P38, Plachy, Radek 2.P9 P. Szabó, Gabriella 1.P63 1.P39 Plaiasu, Vasilica 1.P41, 1.P73 Pacini, Furio 2.P25 Pecqueur, Caroline L26 Plaja, Alberto 3.P11, 5.P5 Padari, Helgi 1.P20 Pedeutour, Florence O11 Plessis, Ghislaine 1.P58 Paeps, E. 3.P30 Pedro, Sonia 1.P4, 1.P67 Plevova, Karla 2.P10, 2.P37 Pais, Cláudia 3.P16 Peeters, Hilde 3.P24 Plonkova, Hana 2.P38 Paiva, Carmen Pekova, Sona 2.P9 Plutino, Morgane 1.P26, 1.P66 Lucia A. 1.P61 Pelayo Delgado, Irene 3.P32 Podgornik, Helena 2.P54, 2.P60 Paiva, Patrícia 3.P16 Pellestor, Franck O12, 1.P51, Poirseir, Celine 1.P58 Paiva Ferreira, Ríudo 4.P11, 4.P13 5.P3, 6.P1, Pollet, Nicolas 4.P16 Paiva Vieira, Társis 1.P7 6.P2 Polyakov, Aleksandr 1.P70 S154 10th European Cytogenetics Conference 2015

Pons, Linda 1.P11 Ribeiro, Maria Sánchez-Durán, Popescu, Roxana 1.P59 do Céu 1.P49, 1.P69 Mª Ángeles 3.P17 Popova, Darya 4.P6 Ribeiro, Marcia 1.P61 Sander, Gabriele 1.P2 Popovici, Cornel 2.P19 Ribeiro, Enilze 7.P5 Sanlaville, Damien L2, O1, Porcher, Raphael O8 Riess, Angelika 1.P18, 1.P11, Portnoi, Marie-France 1.P58 1.P52 3.P4, Pospisilova, Sarka 2.P37 Riess, Olaf 1.P18 3.P5 Poths, Sven 1.P18 Rittinger, Olaf 1.P1, 1.P2 Santamaría, Estibaliz 3.P26 Pour, Ludek O16 Robledo, Renato 4.P4 Santibáñez, Paula 3.P31 Praulich, Inka 2.P9 Rocchi, Mariano O11, 5.P1 Santos, Suely 1.P77 Prchalova, Darina 1.P34 Rocha, Catielly 1.P61 Santos Guilherme, Prochazka, Vit 2.P10, 2.P35 Rodó, Carlota 3.P17 Roberta 1.P19 Prokofjeva, Tatjana 7.P1 Rodrigues, Santos-Rebouças, Prouzova, Zuzana 2.P35 Maria do Céu 3.P19, 3.P28 Cintia 1.P77 Puechberty, Jacques O12, 1.P51, Rodriguez Perales, Sarasola, Esther 3.P26, 3.P31 5.P3, 6.P1, Sandra L6 Sarda, Pierre O12 6.P2 Rodríguez-Martínez, Saris, L. 2.P46 Purgato, Stefania O11 A. Belén 3.P31 Sarova, Iveta 2.P12, 2.P15, Pušeljic, Silvija 1.P54 Rojo, Verónica O10 2.P21 Putoux, Audrey O1 Roko, Dorina 1.P21 Savio, Giulia 5.P11 Roldán, Susana 1.P62 Schaafsma, Ron O4 Quentmeier, Hilmar O6 Roldán, Ernesto 2.P48 Schaeferhoff, Karin 1.P18 Quere, Manal 3.P4, 3.P5 Román, Laura 3.P26 Scheidecker, SophieS 1.P60 Quibel, Thibault O8 Romana, Serge 1.P27 Schiff, Bradley 2.P2 Rooryck, Caroline 3.P4, 3.P5 Schluth-Bolard, R. Mota, Céu 3.P28 Rosati, Roberto 2.P57 Caroline O1, 1.P11, Raanani, Pia 2.P28 Rossi, Massimiliano O1 1.P58, 3.P4, Rabizadeh, Esther 2.P28 Rossi Torres, Fábio 1.P9 3.P5 Radoi, Viorica 1.P33 Rossino, Rossano 1.P24 Schmid-Braz, Raimondi, E L19 Rothman, Rachel 2.P23 Ana Teresa 7.P6 Ramesh, K H 2.P2 Rotíková, Lívia 2.P4 Schmidt, Eva 7.P4 Ramos, Fabiana 3.P12, 3.P13 Rouillac-Le Sciellour, Schneider, Anouck O12, 1.P51, Ramos-Rivera, Gloria 2.P2 Christelle O8 6.P1, 6.P2 Ransdorfova, Sarka 2.P12, 2.P15, Rudolf, Gorazd 1.P75 Schoumans, 2.P18, 2.P21 Rueda, Joaquín 1.P10, 1.P47 Jacqueline O7 Rasmussen, Ruggeri, Anna 5.P8 Schramm, Thomas 3.P18 Malene B. O15 Ruhi, Hatice Ilgın 2.P53 Schroeder, Rauch, Anita O9 Ryzhkova, Oksana 1.P70 Christopher 1.P18 Ravaglio, Jamur Schuster, Jens 5.P6 Valderez 7.P6 Sablaturova, Tamara 2.P37, 2.P38 Schutz, Sacha 3.P4, 3.P5 Raykova, Doroteya 5.P6 Sagaribay, Pablo 1.P78 Schwarzacher, Trude L4 Reberšek, Katarina 2.P60 Saka Guvenc, Merve 3.P29 Scudla, Vlastimil 2.P24 Receveur, Aline 1.P58 Sakellari, Ioanna 2.P7 Sedlacek, Zdenek 1.P34 Redaelli, Serena 2.P40 Salameh, Nicole 1.P44 Segura, Ana 1.P47 Rei, Ana Isabel 3.P16 Salim, Ozan 2.P8, 2.P32 Senegaglia, Reimand, Tiia 1.P17, 1.P20 Saloum, Riant 2.P6, 2.P7 Alexandra Cristina 7.P5 Reissmann, Regina O9 Salumets, Andres 1.P30 Serafim, Silvia 1.P4, 1.P57 Reshetnikova, Irina O9 Sampaio, Mafalda 1.P64 Sérazin, Valérie O8 Rey, Dolores 2.P48 Sánchez, Marina 1.P10 Serov, Oleg 1.P31 10th European Cytogenetics Conference 2015 S155

Serrano, Ana María 1.P10 Soler, Anna 3.P3 Szabadosova, Severgnini, Marco O11, 5.P1 Solonitchenko, Viktoria 1.P29 Severo, Regina 3.P22 Vladimir 1.P43 Szabóová, Zofia 2.P4 Shago, Mary O5 Somers, Gino R O5 Szakszon, Katalin 1.P63 Shepshelovich, Sophie, Brisset 3.P14 Szijarto, Zsuzsanna 2.P27 Daniel 2.P28 Sorlin, Arthur 3.P4, 3.P5 Szomor, Arpad 2.P27 Shilova, Nadezda 1.P43, 1.P45, Sousa, Ana 1.P57 Şahin, Serdar 2.P51 1.P70 Souto, Marta 3.P22 Tabet, Anne-Claude 1.P58, 1.P72 Shishigina, Lubov 2.P61 Sovena, Gloria 1.P50 Tachdjian, Gérard 1.P22 Shoukier, Moneef 3.P18 Specchia, Giorgina 2.P13, Tachon, Gaelle 1.P51 Sielska-Rotblum, 2.P14 Talavera, Maria 1.P68, 2.P48 Danuta 1.P74 Speeg-Schatz, Claude 1.P60 Tammur, Pille 1.P20 Silipigni, Rosamaria 1.P48, 1.P50, Staessen, C. 3.P30 Tanteles, George 1.P44, 1.P46 3.P15 Stambouli, Danai 1.P73 Tapia, M. Asun 3.P26 Silva, Antonio Stangler Herodez, Taralczak, Francisco Alves da 1.P77 Spela 3.P8 Malgorzata O9 Silveira-Santos, Stankovic, Aleksandra 1.P42 Tarkan-Argüden, Rosário 1.P57 Stavropoulos, James O5 Yelda 2.P51 Simion, Rodica 1.P73 Stavroyanni, Niki 2.P6 Tarleton, Jack 1.P78 Simioni, Milena 1.P7, 1.P9, Steffann, Julie 1.P27 Tasdemir, Pelin 2.P44, 2.P45 5.P2 Steindl, Katharina O9 Tassano, Elisa 1.P14 Simkova, Hana L18 Steinlein, Ortrud O3 Tavaiaux, Sylvie 6.P2 Simões, Aguinaldo Stepanovska, Kristina 2.P37 Tay, Joshua Wei Luiz 1.P9 Stevens-Kroef, Marian O4 Ming 2.P1 Simões, Lúcia 3.P12 Stevenson, David 2.P42 Tayfun, Funda 2.P31 Simoni, Giuseppe 5.P8 Stika, Jiri 2.P9 Taylor, Rohan 1.P23, 3.P10 Singer, Sylke 1.P52, Stoklasova, Martina 2.P37, Tekin, Ayse Neslihan 1.P71 3.P21 2.P38 Télez, Mercedes 5.P7 Sinke, Richard O2 Stolfa, Miroslav 3.P25 Teo, Bryan Yi Wei 2.P1 Sisdelli, Luiza 5.P4 Storlazzi, Clelia Teran, Natasa 1.P75 Siskova, Magda 2.P15, 2.P21 Tiziana O6, O11, Tesner, Pavel 3.P25 Sismani, Carolina O13, 2.P43, 5.P1 Testoni, Nicoletta O6 1.P44, Stölzl, A 4.P5 Thorner, Paul O5 1.P46 Strnkova, Aneta 2.P37, Tien, Sim Leng 2.P1 Sitar, Gianmaria 3.P7 2.P38 Tiidema, Anu 1.P16, 3.P9 Škrlec, Ivana 1.P54 Strom, Charles L27 Tikhonov, Andrei 7.P3 Skryabin, Nikolay 1.P31 Styka, Borys 2.P33, Till, Marianne O1 Skytte, Anne-Bine 1.P13 2.P34 Tissot, Catherine 7.P4 Slud Brofman, Paulo Suela, Javier 6.P3 Tizzano, Eduardo 3.P17, 5.P5 Roberto 7.P5 Suhrland, Mark 2.P2 Tolomeo, Doron 2.P43, 5.P1 Smetana, Jan O16, 1.P8, Sullivan, K L19 Tomasova, Radka 2.P39 3.P1 Sungar, Gülçin 2.P52 Tomaszewska, Smit, Marjan 1.P6 Sureda, Mònica 3.P2 Paulina 4.P7 Soares, Maria de Suvada, Jozef 2.P4 Tommerup, Niels O15 Fátima 1.P19 Svobodova, Karla 2.P12, Tonisson, Neeme 1.P30 Sobol, Maria 5.P6 2.P15, Torres, Yaima 6.P3 Sogos, Valeria 2.P49 2.P18, Tosca, Lucie 1.P22 Solarewicz, Maria 2.P21 Tota, Giuseppina 2.P13, 2.P14 Marlene 7.P5 Swertz, Morris O2 Touraine, Renaud L3 S156 10th European Cytogenetics Conference 2015

Tournaire, Magali 6.P2 van Huizen, M. 3.P27 Vlckova, Marketa 1.P34, 1.P40 Tous, Corinne 2.P58 van Maarle, Merel 1.P37 Voet, Thierry 3.P20 Toussaint, Elise 2.P3 van Marwijk-Kooy, M O4 Volk, Marija 1.P75 Toutain, Jérôme 1.P58 van Ravenswaaij-Arts, von dem Borne, P. 2.P46 Töre, Tolga 2.P55 Conny O2 Voskoboinik, Nadia 2.P23 Trestman, Svetlana 2.P23 van Zutven, Laura Vosseler, Vanessa 1.P18 Trofimova, Irina 4.P6 J.C.M. 1.P6 Vozobulova, Vera 2.P10 Trombetta, Vandenberghe, Peter 2.P43 Vrana, Jana L18 Domenico 5.P1 Vanderheyden, N L9 Vranekovic, Truhlikova, Lucie 2.P37, 2.P38 Vandermot, E. 3.P30 Jadranka 1.P35, 1.P42 Tsatsaris, Vassilis O8 Vanessa, Nivaggioni 2.P22 Tsompanakou, Aliki 2.P6 Vankova, Gabriela 2.P37 Wagner, Kathy 1.P26 Tsvetkova, Tatiana 1.P70 Vanni, Roberta 2.P25, Wagner, Jasenka 1.P54 Tukun, Ajlan 2.P52, 2.P53, 2.P49 Wakui, Keiko 1.P53 5.P12 Vatansever Özbek, Walker, Brian A. O16 Tuncalı, Timur 2.P53 Ülfet 1.P56 Waminal, Nomar Turbitt, Julie 2.P42 Vejvalková, Sárka 1.P40 Espinosa 4.P14 Turleau, Catherine 1.P27 Vekemans, Michel 1.P27 Wardell, Türedi, Özlem 2.P53, 5.P12 Velagaleti, Gopalrao 2.P62 Christopher P. O16 Tyler-Smith, Chris O14 Velipasaoglu, Melih 3.P29 Wayhelova, Marketa O16, 1.P8 Tzschach, Andreas 1.P52, 3.P21 Vellenga, Edo O4 Westers, T O4 Ujfalusi, Anikó 1.P63 Veltman, Joris A. L20 Wettwer, Marco O3 Undar, Levent 2.P8 Venci, Anna 2.P11 Whelan, Ungureanu, Maria Vendrell, Teresa 5.P5 Christopher W. 5.P1 Christina 1.P59 Ventura, Catarina 1.P4 Wieser, Iris 1.P2 Urbano, Antonio 1.P10, 1.P47 Verhovtsova, Polina 1.P16, 3.P9 Wijermans, Pierre O4 Urbanova, Renata 2.P36 Vermeesch, Joris L16, Winnicka, Dorota 2.P33, 2.P34 Urbanovska, Irena 2.P20 3.P20, Wissler, Marie-Pierre 2.P3 Uspenskaya, Olga 2.P26 3.P24 Wlodarska, Iwona 3.P24 Utine Gülen, Eda 1.P32 Vetro, Annalisa O13 Wojciechowska, Vadikoliou, Vialard, François O8, 1.P58 Katarzyna 2.P33, Chrysanthi 2.P7, 2.P30 Vicdan, Arzu 2.P53 2.P34 Vallés, Ana 2.P48 Vidaud, Michel 3.P4, 3.P5 Writzl, Karin 1.P75 Valli, Roberto 2.P5, 7.P2 Vidmichi, Daniela 4.P8 Wrobel, Marek 2.P38 Vallova, Vladimira 1.P8, 3.P1 Viguetti-Campos, Wuicik-Merfort, Vameghi, Roshanak 1.P3 Nilma 5.P2 Lismeri 7.P6 Van Berkel, K. 3.P30 Vilain, Catheline 3.P23 van de Akker, Villacorta, I. 3.P30 Yakut Uzuner, Sezin 1.P36 Erica L.T. 1.P6 Villalón Villarroel, Yang, Tae-Jin 4.P14 van de Loosdrecht, Concepcion 3.P32 Yang, Fengtang O14 Arjan O4 Villarrubia, Jesús 2.P48 Yarar, Coskun 1.P71 Van Den Bogaert, Vincova, Miroslava 2.P24, Yararbaş, Kanay 5.P12 Kris 3.P24 2.P36 Yardin, Catherine 1.P58 Van Den Bogaert, A. 3.P30 Vinti, Luciana 2.P5 Yeshaya, Josepha 2.P28 van der Kevie- Viot, Geraldine 1.P72, 5.P13 Yılmaz, Fırat Fevzi 2.P51 Kersemaekers, Virginia, Borobio 3.P3 Yılmaz, Şükriye 2.P51 Anne-Marie 1.P37 Visamaa, Airi 1.P16, 3.P9 Yılmaz, Hülya 2.P55 van der Reijden, Bert O4 Visser-Wisselaar, Yucel, Orhan Kemal 2.P8 Van Esch, Hilde 3.P24 Heleen O4 Yuksel, Atil 3.P33 10th European Cytogenetics Conference 2015 S157

Zabnenkova, Zattara, Hélène 2.P19 Zivkovic, Maja 1.P4 Viktoria 1.P45 Zeitlhofer, Petra 1.P65 Zlotina, Anna 4.P2 Zagaria, Antonella 2.P13, 2.P14 Zejskova, Lenka 2.P9 Zoli, Monica O11 Zagorac, Andreja 3.P8 Zemanova, Zuzana 2.P12, 2.P15, Zonneveld, J. 3.P27 Zagradisnik, Boris 3.P8 2.P18, 2.P21 Zõrjanova, Tatjana 3.P9 Zákovicová, Alena 2.P4 Zhu, Meiqing 2.P62 Zuchnicka, Jan. 2.P38 Zamani Esteki, Zilina, Olga 1.P16, 1.P17, Zuffardi, Orsetta O13 Masoud 3.P20 1.P20, 1.P30 Zygulova, Irena. 2.P38