Original article J Med Genet: first published as 10.1136/jmg.2007.054007 on 15 November 2007. Downloaded from Duplications in addition to terminal deletions are present in a proportion of ring chromosomes: clues to the mechanisms of formation E Rossi,1* M Riegel,2* J Messa,1 S Gimelli,1 P Maraschio,1 R Ciccone,1 M Stroppi,3 P Riva,3 C S Perrotta,4 T Mattina,4 L Memo,5 A Baumer,2 V Kucinskas,6 C Castellan,7 A Schinzel,2 O Zuffardi1,8 1 Biologia Generale e Genetica ABSTRACT ‘‘ring syndrome’’1 that in cases with intact ring Medica, Universita`di Pavia, 2 Background and methods: Ring chromosomes are chromosomes is characterised, independently of Pavia, Italy; Institut fuer often associated with abnormal phenotypes because of Medizinische Genetik der the chromosome involved, by severe growth fail- Universitaet Zuerich, loss of genomic material at one or both ends. In some ure, minor dysmorphic features, and mild to Schwerzenbach, Switzerland; cases no deletion has been detected and the abnormal moderate mental retardation, without major mal- 3 Dipartimento di Biologia e phenotype has been attributed to mitotic ring instability. formations. In a review of 207 cases, Kosztola´nyi2 Genetica, Universita`di Milano, We investigated 33 different ring chromosomes in Milano, Italy; 4 Divisione di estimated that one fifth of subjects with auto- Genetica Medica, Universita`di patients with phenotypic abnormalities by array based somal rings are affected by the ‘‘ring syndrome’’ Catania, Catania, Italy; 5 UO comparative genomic hybridisation (CGH) and fluores- phenotype. Indeed, more recent papers have Patologia Neonatale, Ospedale cence in situ hybridisation (FISH). demonstrated that intact ring chromosomes may Ca`Foncello, Treviso, Italy; Results: In seven cases we found not only the expected 6 Human Genetics Center, cause areas of hypopigmentation along the lines of Santariskiu Hospital, University terminal deletion but also a contiguous duplication. FISH Blaschko as the only sign of ring-induced mosai- of Vilnius, Vilnius, Lithuania; analysis in some of these cases demonstrated that the cism,3 or specific features such as a characteristic 7 Genetische Beratungsstelle, duplication was inverted. Thus these ring chromosomes 8 type of epilepsy and electroencephalographic pat- Bozen, Italy; Fondazione IRCCS derived through a classical inv dup del rearrangement tern as reported for several ring (20) chromosomes,4 Policlinico San Matteo, Pavia, consisting of a deletion and an inverted duplication. Italy thus weakening the hypothesis of the ‘‘ring Discussion: Inv dup del rearrangements have been syndrome’’. Moreover, fluorescent in situ hybridi- Correspondence to: reported for several chromosomes, but hardly ever in ring sation (FISH) analysis at first, and more recently Dr O Zuffardi, Genetica Medica, chromosomes. Our findings highlight a new mechanism Universita’ di Pavia, Pavia, Italy; whole genome array screenings, have demon- [email protected] for the formation of some ring chromosomes and show strated that in most of the cases a cryptic deletion that inv dup del rearrangements may be stabilised not is at the basis of the phenotypic abnormalities in *The first two authors contrib- only through telomere healing and telomere capture but apparently intact rings.5–7 Recently the case of an uted equally to the work also through circularisation. This type of mechanism must r(14) has been reported with the combination of an http://jmg.bmj.com/ Received 22 August 2007 be kept in mind when evaluating possible genotype– inverted duplication with a terminal deletion Revised 30 October 2007 phenotype correlations in ring chromosomes since in characterised using high resolution molecular Accepted 31 October 2007 these cases: (1) the deletion may be larger or smaller karyotyping and FISH.8 The patient presented Published Online First than first estimated based on the size of the ring, with a 15 November 2007 overlapping clinical features described in terminal different impact on the phenotype; and (2) the associated deletion, duplication and ring chromosome 14 duplication will in general cause further phenotypic cases. By examining 33 probands with ring anomalies and might confuse the genotype–phenotype chromosomes through array based comparative on September 25, 2021 by guest. Protected copyright. correlation. Moreover, these findings explain some genomic hybridisation (CGH) we detected the phenotypic peculiarities which previously were attributed same situation in seven of them, suggesting both to a wide phenotypic variation or hidden mosaicism a new mechanism of ring formation and a warning related to the instability of the ring. for clinical geneticists to consider this possibility while performing genotype–phenotype correla- tions. Ring chromosomes are usually associated with abnormal phenotypes due to the loss of material at MATERIALS AND METHODS both or at least one chromosome end. Thus, in principle, the abnormal phenotypes are essentially Patients due to haploinsufficiency of those dosage sensitive The initial chromosome examination which genes contained in the deleted segment(s). The detected a ring chromosome was performed in 17 finding of ring chromosomes without apparent loss cases in different cytogenetic laboratories in Italy of genetic material in subjects with abnormal and in 16 cases in Zurich, Switzerland. In one case phenotypes led investigators to hypothesise that (case 33, from Zurich), the ring chromosome was the ring formation with the related difficulties in transmitted from a mildly affected mother with the sister chromatid separation at cell division low mosaicism to a non-mosaic daughter, while in induced the generation of secondary aneuploid all the other cases the ring formation had occurred This paper is freely available 9 online under the BMJ Journals cells. Some aneuploidies, being lethal at the cellular de novo. Three cases, 8 (case 14 in Ballarati et al ), 9 9 10 unlocked scheme, see http:// level, would in turn give rise to increased cell death (case 10 in Ballarati et al and Guala et al ) and 25 jmg.bmj.com/info/unlocked.dtl rate. Altogether this situation should lead to the (Baumer et al11) had already been published. J Med Genet 2008;45:147–154. doi:10.1136/jmg.2007.054007 147 Original article J Med Genet: first published as 10.1136/jmg.2007.054007 on 15 November 2007. Downloaded from Array CGH and 13) in which, thanks to the availability of lymphoblastoid In 28 cases array based CGH was performed using the Agilent cell lines, we demonstrated by dual colour FISH that the Human Genome CGH Microarray Kit 44B (Agilent duplication was inverted. In case 7 the BACs used were RP11- Technologies, Santa Clara, California, USA), and in six cases 164b1 (biotin labelled) and RP11-122a8 (digoxigenin labelled) the 244A Kit. In one case (case 21) we used both platforms. (fig 2A); in case 13 the inversion was identified by BAC RP11- These platforms are high resolution oligonucleotide based 1072p10 (biotin labelled) and RP11-522b15 (digoxigenin microarrays that allow genome-wide survey and molecular labelled) (fig 2B). The orientation of the duplication could not profiling of genomic aberrations with a resolution of about be investigated in the five cases with a ‘‘dup del’’ rearrangement 100 kb and 20 kb, respectively. Labelling and hybridisation were (cases 5, 10, 21, 26, 33). The chromosomes involved were: performed following the protocols provided by Agilent. Briefly, chromosome 13 (cases 5, 7 and 10), chromosome 15 (case 13), 500 ng of purified DNA of a patient and of a control (Promega chromosome 18 (cases 21 and 26) and chromosome 22 (case 33). Corporation, Madison, Wisconsin, USA) were double-digested Data on array-CGH results and parental origin of the 33 cases with RsaI and AluI for 2 h at 37uC. After 20 min at 65uC, DNA are given in table 1. of each digested sample was labelled, by the Agilent random The clinical features of the seven patients with inv dup del or primers labelling kit, for 2 h using Cy5-dUTP for the patient dup del ring chromosomes and the array CGH findings with the DNA and Cy3-dUTP for the control DNA. Labelled products resulting genomic imbalance are listed in table 2. were column purified and prepared according to the Agilent In the two inv dup del cases (cases 7 and 13) microsatellite protocol. After probe denaturation and pre-annealing with analysis in patients and parents with markers mapping to the 50 mg of Cot-1 DNA (Invitrogen, Carlsbad, California, USA), deleted regions (D13S173, D13S1315: case 7; D15S642, hybridisation was performed at 65uC with rotation for 40 h. D15S107: case 13) indicated a paternal origin of the rearrange- After two washing steps the arrays were analysed with the ment in both cases (fig 3). Moreover, microsatellite markers of Agilent scanner and the Feature Extraction software (v8.0; the duplicated region showed that the rearrangement was v9.1.3). Graphical overview was obtained using the CGH intrachromosomal since only two alleles, the paternal one analytics software (v3.1; v3.4). All the breakpoint sequences showing a double peak area, had been detected for all the were analysed with self chain and segmental duplication repeats informative markers (D13S1283, D13S790, D13S1323; 15DUP3, tools from UCSC (http://genome.ucsc.edu/). 15DUP4, 15DUP11, 15DUP13, 15DUP15) (fig 3A,B). In case 33, with a dup del(22q), the rearrangement was FISH probably maternal in origin. We did not perform microsatellite To confirm array CGH results and demonstrate the inversion of analysis since parental DNA was not available. However, the the duplication in patients 7 and 13, FISH experiments were mother who had some phenotypic abnormalities showed a low performed. All bacterial artificial chromosome (BAC) probes mosaicism (1 out of 50) for the same ring in lymphocytes. used were selected according to the UCSC Human Genome In case 21 with an inv dup del(18p), the array CGH results Browser and were obtained from the human library RPCI-11.