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The North East Thames Regional Genetics Service is based at Great Ormond Street Hospital. Cytogenetics Service Comprised of the Molecular and Cytogenetics laboratories along with Clinical Genetics we Level 5, York House, serve a population of approximately 5 million across North East London and Essex. 37 Queen Square London. WC1N 3BH. The service has a staff of approximately 80 including consultant clinical geneticists, state T +44 (0) 20 7829 8870 registered clinical scientists, genetic technologists and administrative support staff. The staff F +44 (0) 20 7813 8578 work closely with clinical colleagues and other healthcare scientists in pathology as well as with research staff at the Institute for Child Health. Jonathan Waters PhD FRCPath The laboratories provide an extensive range of diagnostic testing services and have Head of Service full CPA (UK) accreditation. The laboratory was inspected for UKAS accreditation Deborah Morrogh, under standard ISO15189 in May 2013, and is still waiting for a formal accreditation DipRCPath status response from UKAS. The service is a member of the South East of England Deputy Head Genetics Network (SEEGEN) and the United Kingdom Genetics Testing Network (UKGTN). Molecular The laboratory service processes over 26,500 samples per year and issues Genetics Service approximately 18,500 analytical reports. The service repertoire is regularly updated, but includes a regional service for postnatal and prenatal microarray, karyotyping Level 6, York House and a number of single gene disorders. Patients with developmental delay and/or 37 Queen Square dysmorphism, or patients with an anomalous ultrasound scan during pregnancy are London WC1N 3BH offered a 750K microarray analysis. Karyotyping is performed for a smaller number T +44 (0) 20 7762 6888 of patients, whilst samples from pregnancy losses are analysed using molecular F +44 (0) 20 7813 8578 methods. www.labs.gosh.nhs.uk The Unit receives nationally commissioned funding for a number of specialised Lucy Jenkins FRCPath services including Bardet-Biedl syndrome, craniosynostoses, lysosomal storage Head of Service disorders and severe combined immunodeficiencies. Sam Loughlin DipRCPath The laboratory also provides both a national and international service for non- Deputy Head invasive prenatal diagnosis, deafness, familial hypercholesterolaemia, surfactant deficiency, craniofacial, metabolic and primary immune deficiency disorders. Clinical Genetics Referrals are received for diagnostic, predictive, carrier and prenatal testing. Great Ormond Street Hospital, We undertake externally funded research projects and accept private referrals. Level 4, York House, Please contact the laboratory for further information. 37 Queen Square, London. WC1N 3BH. Research and development is a key objective of the Unit, a number of staff having joint T +44 (0) 20 7762 6856 academic appointments with University College London. The service has a strong F +44 (0) 20 7813 8141 commitment to education and training and public and patient engagement and participates in clinician, scientist and technologist training programmes. A number of staff are also actively Dr Jane Hurst involved at a regional and national level in policy development, training and examination. Lead Clinician
The laboratory provides a DNA banking service and can forward samples to other centres for Other contacts approved requests provided funding is available. A complete list of in-house services and corresponding information sheets can be found in this service pack. Further details regarding Nick Lench PhD FRCPath tests which may be available from other laboratories can be found on pages 3 and 4. Director Jonathan Northfield Laboratory Opening Hours Service Manager Mike Tinsley The laboratory is staffed Monday - Friday, 8.00am – 6.00pm excluding bank IT Manager and Data Analyst holidays.
Sample reception is open from 8.30am to 5.30pm. Specimens arriving outside these hours are refrigerated / frozen prior to processing. There is no out-of-hours service. Please send samples to the address shown in the right hand panel.
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Sample Requirements Cytogenetics Service Level 5, York House, For sample requirements, please refer to our request card which can be found at: 37 Queen Square London. WC1N 3BH. http://www.labs.gosh.nhs.uk/laboratory-services/genetics/sending-a-sample T +44 (0) 20 7829 8870 It is the responsibility of the patient’s clinician to ensure that all requests meet F +44 (0) 20 7813 8578 testing criteria, that samples are correctly labelled and request forms are completed to a minimum standard. Jonathan Waters PhD FRCPath In submitting samples the clinician confirms that consent for testing and possible Head of Service storage has been obtained. Deborah Morrogh, Samples DipRCPath Deputy Head 5 ml venous blood in plastic EDTA bottles (>1 ml from neonates) For DNA samples, it is requested that the referral laboratory provides sufficient Molecular DNA for the analysis being requested. Genetics Service
Samples must be labelled with: Level 6, York House 37 Queen Square • Patient’s full name (surname/family name and first/given name) London WC1N 3BH • Date of birth and unique hospital/NHS number T +44 (0) 20 7762 6888 • It is also desirable to have the date and time sample was taken and/or location F +44 (0) 20 7813 8578 www.labs.gosh.nhs.uk Prenatal Samples Lucy Jenkins FRCPath Head of Service Please contact the laboratory in advance of arranging a prenatal sample. Sam Loughlin DipRCPath Cytogenetic analysis of prenatal samples is routinely available. The type of test Deputy Head offered is dependent on criteria such as the patient having a serum screen risk only, or having abnormalities detected on ultrasound scan or individual service level Clinical Genetics agreements. Great Ormond Street Hospital, Prenatal diagnosis for molecular genetic disorders can only be offered by prior Level 4, York House, arrangement where diagnosis has been confirmed by molecular means and 37 Queen Square, parental samples are fully informative. It is standard practice for the laboratory to London. WC1N 3BH. exclude maternal cell contamination of all fetal samples; a maternal blood sample is T +44 (0) 20 7762 6856 required for this analysis. F +44 (0) 20 7813 8141 CVS / Amniocentesis: Tissue type and date of biopsy should be clearly Dr Jane Hurst documented on the referral information. In the case of twins, special attention must Lead Clinician be given to the identity of each sample. Other contacts Cell Free Fetal DNA Analysis: 20ml of maternal blood in EDTA is required. Sample date & gestation as confirmed by ultrasound scan must be provided along with a Nick Lench valid clinical indication for early gender determination or single gene testing. This PhD FRCPath Director test does not apply to twin pregnancies. Jonathan Northfield Please see pages 34 - 37 for further details. Service Manager Minimum sample labelling criteria: Mike Tinsley IT Manager and Data Analyst • Patient’s full name and date of birth • Unique hospital/NHS number
Request form The Regional Genetics Laboratory has its own referral card; an electronic version is available on our website.
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Alternative referral cards / letters are acceptable; it is preferable that any referral Cytogenetics Service card is fully completed. However, referral documents must provide the minimum Level 5, York House, criteria of: 37 Queen Square • Patient’s full name and date of birth London. WC1N 3BH. • NHS number (essential) and hospital number T +44 (0) 20 7829 8870 F +44 (0) 20 7813 8578 • Full name and address of referring clinician/consultant • Patient’s postcode Jonathan Waters PhD FRCPath • Patient’s GP name and address Head of Service • Clearly mark if referral is for a non-NHS patient Deborah Morrogh, • Analysis can only be carried out if a specific disease / gene test(s) is requested DipRCPath Deputy Head • For family / targeted mutation tests, a mutation report or GOS genetics family ID is required along with the relationship of your patient to family members Molecular previously tested. Genetics Service Sending samples Level 6, York House Samples sent by Royal Mail or courier must comply with PI 650 for category B 37 Queen Square substances. London WC1N 3BH +44 (0) 20 7762 6888 • This is a triple layer system which comprises a primary leak-proof receptacle T within a secondary leak-proof receptacle contained in a rigid outer package. F +44 (0) 20 7813 8578 The packaging should be strong enough to withstand a 95 kPA pressure www.labs.gosh.nhs.uk differential and a drop of 1.2 m. Lucy Jenkins FRCPath • There should be sufficient absorbent material between the primary and Head of Service secondary packaging to absorb any spillage. The primary container and Sam Loughlin DipRCPath absorbent material must be placed into a single bag with the request form Deputy Head in the pouch. • The package should be clearly labelled ‘diagnostic specimen UN3373’. Clinical Genetics Great Ormond Street Hospital, Pricing Level 4, York House, 37 Queen Square, London. WC1N 3BH. NHS Provider to Provider and Private / Overseas test prices T +44 (0) 20 7762 6856 Please contact the laboratory for an up-to-date price list. Prices are also available F +44 (0) 20 7813 8141 on our UKGTN listing. Dr Jane Hurst http://ukgtn.nhs.uk/find-a-test/search-by-laboratory/laboratory/london-north-east-rgc-gosh-43/ Lead Clinician
Tests carried out by other laboratories Other contacts Nick Lench PhD FRCPath UK Genetic Testing Network www.ukgtn.nhs.uk Director The United Kingdom Genetic Testing Network (UKGTN) is a collaborative group of Jonathan Northfield UK laboratories and their clinicians, commissioners and patient representatives. Service Manager The network, which is overseen by the Department of Health, aims to ensure that Mike Tinsley the UKGTN services provided by the member laboratories are of high quality, that IT Manager and Data Analyst new services are evaluated for effectiveness and that the NHS commissioning mechanisms are appropriately informed in order to promote equity of access. Subject to meeting recognised referral criteria and available funding for specific tests, DNA may be extracted and forwarded to the relevant UKGTN laboratory for tests not available in-house. The UKGTN website lists currently evaluated tests (including a search function and alphabetical list of tests), tests currently undergoing evaluation and tests likely to be submitted in the near future.
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The current accreditation status of UK laboratories registered with CPA (UK) Ltd. Cytogenetics Service can be checked at http://www.cpa-uk.co.uk/ Level 5, York House, 37 Queen Square Testing carried out by laboratories outside the UKGTN London. WC1N 3BH. Molecular genetic screening for some disorders may not currently be available from T +44 (0) 20 7829 8870 UKGTN laboratories. These tests may be available at other diagnostic laboratories F +44 (0) 20 7813 8578 within and outside the UK and in some cases samples can be forwarded provided Jonathan Waters funding is available. Please contact the laboratory for further information. PhD FRCPath Head of Service Deborah Morrogh, DipRCPath Deputy Head
Molecular Genetics Service
Level 6, York House 37 Queen Square London WC1N 3BH T +44 (0) 20 7762 6888 F +44 (0) 20 7813 8578 www.labs.gosh.nhs.uk Lucy Jenkins FRCPath Head of Service Sam Loughlin DipRCPath Deputy Head
Clinical Genetics Great Ormond Street Hospital, Level 4, York House, 37 Queen Square, London. WC1N 3BH. T +44 (0) 20 7762 6856 F +44 (0) 20 7813 8141 Dr Jane Hurst Lead Clinician
Other contacts Nick Lench PhD FRCPath Director Jonathan Northfield Service Manager Mike Tinsley IT Manager and Data Analyst
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Core disorders
Disorder Gene Locus OMIM Disease OMIM Gene Page
Angelman syndrome 15q11-q13 #105830 *606945 12
Bardet-Biedl syndrome Various – see NGS panel section #209900 13
Beckwith Wiedemann syndrome 11p15.5 #130650 14
Familial breast/ovarian cancer BRCA1 17q21 #604370 +113705 15 BRCA2 13q13.1 #612555 *600185
Congenital Tufting Enteropathy EPCAM 2p21 #613217 *185535 16
Cystic Fibrosis CFTR 7q31.2 #219700 *602421 17
Cystic Fibrosis Non-Invasive Prenatal CFTR 7q31.2 37 Diagnosis NGS Panel
Early Infantile Epileptic Encephalopathy Various – see NGS panel section 18 (EIEE)
Fragile X syndrome FMR1 Xq27.3 +309550 19
GNAS disorders GNAS1 20q13.2 +139320 20
IL-10 related inflammatory bowel IL10 1q32.1 #266600 *124092 21 disease IL10RA 11q23.3 #613148 *146933 IL10RB 21q22.11 #123889 *612567
Kabuki syndrome MLL2 12q13.12 #147920 22
Nicolaides-Baraitser Syndrome SMARCA2 9p24 #601358 *600014 23
Pulmonary Surfactant SFTPB 2p12-p11.2 #265120 (SMDP1) *178640 24 Metabolism Dysfunction 1, 2 & 3 SFTPC 8p21 #610913 (SMDP2) *178620 24 ABCA3 16p13.3 #610921 (SMDP3) *601615 24
Popliteal Pterygium syndrome IRF6 1q32-q41 #119500 *607199 25
Prader-Willi syndrome 15q11-q13 #176270 26
Primary congenital glaucoma CYP1B1 2p22.2 #231300 *601771 27
Rhabdoid tumour SMARCB1 22q11.23 #609322 *601607 28 predisposition syndrome
Rett syndrome MECP2 Xq28 #312750 *300005 29
Silver Russell syndrome 11p15.5 #180860 30 7p11.2
Van der Woude syndrome IRF6 1q32-q41 #119300 *607199 25
Very Early Onset Inflammatory Bowel Various – see NGS panel section 31 Disease (VEO-IBD)
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Core disorders
Disorder Page
X-inactivation 32
Zygosity testing 33
Cell-free fetal DNA testing
Disorder Page
Cell-free fetal DNA sex determination See information sheet for full test details 34
FGFR3-related skeletal dysplasia Non- See information sheet for full test details 35 Invasive Prenatal Diagnosis NGS Panel
Apert syndrome Non-Invasive Prenatal See information sheet for full test details 36 Diagnosis NGS Panel
Cystic Fibrosis Non-Invasive Prenatal See information sheet for full test details 37 Diagnosis NGS Panel
Cardiovascular Disorders
Disorder Gene Locus OMIM Disease OMIM Gene Page
Familial Hypercholesterolaemia LDLR 19p13.2 #143890 *606945 38 APOB 2p24 #144010 +107730 38 PCSK9 1p32.3 #603776 *607786 38 LDLRAP1 1p36.11 #603813 *605747 38
Hypertrophic Cardiomyopathy MYBPC3 11p11.2 #192600 *600958 39 MYH7 14q12 *160760 39 TNNT2 1q32 *191045 39 TNNI3 19q13.4 +191045 39
Loeys Dietz syndrome TGFBR1 9q22.33 #609192 *190181 40 #608967 TGFBR2 3p22 #610168 +190182 40 #610380
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Skeletal Dysplasias
Disorder Gene Locus OMIM Disease OMIM Gene Page
Achondroplasia FGFR3 4p16.3 #100800 *134934 41
Hypochondroplasia FGFR3 4p16.3 #146000 *134934 41
Thanatophoric Dysplasia FGFR3 4p16.3 #187600 *134934 42
Non-Invasive Prenatal Diagnosis NGS FGFR3 4p16.3 *134934 35 Panel FGFR3-related skeletal dysplasia
Craniosynostosis
Disorder Gene Locus OMIM Disease OMIM Gene Page
Apert syndrome FGFR2 10q26 #101200 *176943 43
Apert syndrome Non-Invasive Prenatal FGFR2 36 Diagnosis NGS Panel
CATSHL FGFR3 4p16.3 #610474 *134934 43
Craniosynostosis type 3 TCF12 15q21 #615314 *600480 43 (TCF12-related craniosynostosis)
Craniosynostosis type 4 ERF 19q13 #600775 *611888 43 (ERF-related craniosynostosis)
Crouzon syndrome FGFR2 10q26 #123500 *176943 43 FGFR3 4p16.3 *134934
Crouzon with acanthosis nigricans FGFR3 4p16.3 #612247 *134934 43
Muenke syndrome FGFR3 4p16.3 #602849 *134934 43
Pfeiffer syndrome FGFR1 8p11 #101600 *136350 43 FGFR2 10q26 *176943
Saethre-Chotzen syndrome TWIST 7p21 #101400 *601622 43
Deafness
Disorder Gene Locus OMIM Disease OMIM Gene Page
Aminoglycoside induced deafness MTRNR1 Nt648-1601 #580000 *561000 44
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Branchio-oto-renal syndrome EYA1 8q13.3 #113650 *601653 45 SIX1 45 SIX5 45
Connexin 26 (DFNB1) GJB2 13q11-q12 #220290 *121011 46
EAST syndrome KCNJ10 1q23.2 #612780 *602208 47
Non-Syndromic Hearing Loss Various – see NGS panel section 48
Pendred syndrome SLC26A4 7q31 #274600 *605646 49
Usher Syndrome Various – see NGS panel section 48
Waardenburg syndrome PAX3 2q35 #193500 *606597 50 Types 1 - 4 #148820 50
X-Linked Deafness (DFN3) POU3F4 Xq21.1 51
Metabolic Disorders
Disorder Gene Locus OMIM Disease OMIM Gene Page
Carbamoylphosphate CPS1 2q35 #237200 *608307 52 synthetase 1 deficiency
Fabry disease GLA Xq22 +301500 53
Gaucher disease GBA 1q21 #230800 (Type1) *606463 54 #230900 (Type2) 54 #231000 (Type 3) 54
Glycogen Storage disease type 1a G6PC 17q21 +232300 55
Glycogen storage disease type 2 (Pompe GAA 17q25.2-q25.3 #232300 *606800 56 disease)
Krabbe disease GALC GALC 14q31 #245200 *606890 57
Long-chain, deficiency of HADHA 2q23 #609016 *600890 58 Acyl-CoA dehydrogenase
Medium-chain, deficiency ACADM 1p31 #201450 *607008 59 of Acyl-CoA dehydrogenase
Metachromatic Leukodystrophy (incl. ARSA 22q13.31-qter #250100 *607574 60 pseudodeficiency of arylsulphatase A)
Mucopolysaccharidosis IDUA 4p16.3 #607014 (Hurler) *252800 61 type 1 (Hurler / Scheie) #607015 (H/S) 61 #607016 (Scheie) 61
Mucopolysaccharidosis IDS Xq28 +309900 62 type 2 (Hunter)
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Metabolic Disorders
Disorder Gene Locus OMIM Disease OMIM Gene Page
Mucopolysaccharidosis type 3 (Sanfilippo) SGSH 17q25.3 #252900 (MPS3A) *605270 63 NAGLU #252920 (MPS3B) *609701 63
Neuronal Ceroid Lipofuscinosis type 1(incl. PPT1 1p32 #256730 *600722 64 infantile Batten disease)
Neuronal Ceroid Lipofuscinosis type 2 TPP1 11p15.5 #204500 *607998 65 (late-infantile Batten)
Neuronal Ceroid Lipofuscinosis type 3 CLN3 16p12.1 #204200 *607042 66 (juvenile Batten)
Lipofuscinosis type 5 CLN5 13q21.2-q32 #256731 *608102 67 (variant late-infantile Batten)
Neuronal Ceroid Lipofuscinosis type 6 CLN6 15q21-q23 #601780 *606725 67 (variant late-infantile Batten)
Neuronal Ceroid Lipofuscinosis type 7 CLN7 4q28.2 #610951 *611124 67 (variant late-infantile Batten)
Neuronal Ceroid Lipofuscinosis type 8 CLN8 8pter-p22 #600143 *607837 67 (variant late-infantile Batten)
Ornithine transcarbamylase deficiency OTC Xp21.1 #311250 *300461 68
Osteopetrosis, autosomal recessive TCIRG1 11q13.4-q13.5 #259700 *604592 69
Pyridoxine Dependent Epilepsy ALDH7A1 5q23.2 #266100 *107232 70
Schindler disease NAGA 22q13.2 #609241 *104170 71 (Kanzaki disease) #609242
Renal Disorders
Disorder Gene Locus OMIM Disease OMIM Gene Page
CFHR5 Nephropathy CHFR5 1q31.3 *608593 72
Cystinosis CTNS 17p13 #219800 *606272 73 (Adult) #219750 73 (Juvenile) #219900 73
Juvenile Nephronophthisis NPHP1 2q13 #256100 *607100 74
Steroid-resistant NPHS2 1q25-q31 #600995 *604766 75 nephrotic syndrome
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Immunodeficiencies
Disorder Gene Locus OMIM Disease OMIM Gene Page
Autoimmune lymphoproliferative syndrome TNFRSF6 10q24.1 #601859 *134636 76 (ALPS)
Cartilage Hair Hypoplasia RMRP 9p21-p12 #250250 *157660 77
Familial hemophagocytic PRF1 10q22 #603553 *170280 78 lymphohistiocystosis
Interleukin 7 receptor alpha severe IL7Ra 5p13 #600802 *146661 79 combined immunodeficiency
JAK3-deficient severe combined JAK3 19p13.1 #600802 *600173 80 immunodeficiency
Lymphoproliferative syndrome 1 ITK 5q33.3 #613011 *186973 81 (ITK deficiency)
Netherton Syndrome SPINK5 5q32 #256500 *605010 82
Primary immunodeficiency Various – see NGS panel section 83
Radiation-sensitive SCID Artemis 10p #602450 *605988 84 (DCLREC1C)
RAG-deficient severe combined RAG1 11p13 #601457 *179615 85 immunodeficiency RAG2 11p13 *179616 85
Wiskott-Aldrich syndrome WAS Xp11.23- #301000 *300392 86 p11.22
X-linked agammaglobulinaemia BTK Xq21.3-q22 #307200 +300300 87
X-linked Hyper IgM syndrome (HIGM) CD40LG Xq26 #308230 *300386 88
X-linked Lymphoproliferative syndrome SAP Xq25 #308240 *300490 89 XIAP Xq25 #300635 *300079 89
X-linked Severe combined IL2RG Xq13 #300400 *308380 90 immunodeficiency
Next generation sequencing panels
Disorder Number of Page genes tested
Bardet-Biedl syndrome 13 See information sheet for full details 13
Early Infantile Epileptic Encephalopathy 46 See information sheet for full details 18
Familial breast/ovarian cancer 2 See information sheet for full details 15
Familial Hypercholesterolaemia 4 See information sheet for full details 38
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Very early onset inflammatory bowel 40 See information sheet for full details 31 disease
Inherited hearing loss panel 95 See information sheet for full details 48
Primary immunodeficiency 72 See information sheet for full details 83
Cytogenetics whole genome or targeted copy number testing and positional information
Primary postnatal tests Major referral category
Microarray (750 K SNP) Dysmorphism / developmental delay
Karyotyping Infertility
Mosaicism screening As requested & for selected query sex chromosome abnormalities/ Turner syndrome referrals
FISH Rapid aneuploidy testing and chromosomal sex assignment
QF-PCR and MLPA † Products of conception (3rd or subsequent miscarriage in 1st trimester; any thereafter)
Primary prenatal tests Major referral category
QF-PCR only Increased risk of Down Syndrome
QF-PCR and microarray (750K SNP) Abnormalities on scan or raised NT (>3 mm at 12-14 weeks)
† Note that this service is offered in place of routine parental karyotyping of couples with a history of recurrent (3 or more) miscarriages in line with RCOG Green Top Guidelines (2010).
OMIM notes An asterisk (*) before an entry number indicates a gene of known sequence. A number symbol (#) before an entry number indicates that it is a descriptive entry, usually of a phenotype, and does not represent a unique locus. Discussion of any gene(s) related to the phenotype resides in another entry (entries) as described in the first paragraph. A plus sign (+) before an entry number indicates that the entry contains the description of a gene of known sequence and a phenotype. A percent sign (%) before an entry number indicates that the entry describes a confirmed Mendelian phenotype or phenotypic locus for which the underlying molecular basis is not known.
Angelman Syndrome
Contact details Introduction Molecular Genetics Service Angelman syndrome (MIM 105830) occurs in 1/15000 - 1/20000 individuals. It is characterized Level 6, York House by severe motor and intellectual retardation, seizures associated with characteristic EEG traces, 37 Queen Square microcephaly, ataxia, frequent jerky limb movements and flapping of the arms and hands, hypotonia, hyperactivity, hypopigmentation (39%), absence of speech, characteristic face shape, London, WC1N 3BH and episodes of paroxysmal laughter. The AS phenotype results from the lack of a maternal T +44 (0) 20 7762 6888 contribution at chromosome 15q11-q13. This can be caused by deletion (~75%), paternal F +44 (0) 20 7813 8578 uniparental disomy (UPD) (~2%) or mutations in the imprinting centre (IC) (~5%) that cause abnormal methylation at exon alpha of the SNRPN gene at 15q11-13. These mutations are all detected by disrupted methylation. About 20% of AS patients have a normal methylation pattern Samples required and are believed to have a mutation in a putative Angelman gene (UBE3A). Deletions and UPD • 5ml venous blood in plastic EDTA are usually de novo events, associated with low recurrence risks, although it is important to bottles (>1ml from neonates) determine whether either parent of an affected child has a predisposing chromosome translocation. There is a recurrence risk of up to 50% in families with confirmed AS who do not • Prenatal testing must be arranged show maternal deletion or UPD. in advance, through a Clinical Genetics department if possible. Referrals • Amniotic fluid or CV samples should be sent to Cytogenetics for • Confirmation of clinically suspected AS in children/adults. dissecting and culturing, with • Investigation of the molecular defect in genetically confirmed AS cases (parental instructions to forward the sample samples required). to the Regional Molecular Genetics • laboratory for analysis Carrier testing in adult relatives of confirmed (genetic) AS patients who are suspected of having an IC mutation (samples from appropriate family members • A completed DNA request card are required). should accompany all samples Prenatal testing Patient details Prenatal diagnosis is available to couples where AS has been confirmed in the family To facilitate accurate testing and and to couples at risk of having a child affected with AS due a balanced chromosomal reporting please provide patient demographic details (full name, date of rearrangement involving chromosome 15 in one of the parents. Please contact the birth, address and ethnic origin), details laboratory to discuss, prior to sending prenatal samples. of any relevant family history and full contact details for the referring clinician Service offered Confirmation of AS by methylation analysis and microsatellite analysis to determine the underlying cause in confirmed cases and carrier testing for adults (requires samples from appropriate family members). UBE3A mutation analysis is not offered in this laboratory. Technical For diagnostic referrals the initial test is to determine the methylation status of exon alpha of the SNRPN gene. Methylation analysis is performed by methylation specific PCR following bisulphite modification of genomic DNA. Normal individuals yield a 313bp maternally derived fragment and a 221bp paternally derived fragment. Patients with AS show a single 221bp paternal fragment only. In AS patients with abnormal methylation, further analysis is recommended to characterise the nature of the mutation. This involves the use of chromosome 15 microsatellite markers from within and flanking the commonly deleted region. Cytogenetic analysis is also helpful in identifying deletions and predisposing parental translocations. NB. Similar analysis is undertaken for Prader-Willi syndrome Target reporting time Routine analysis - the initial methylation test takes up to 4 weeks. Microsatellite marker analysis takes 8 weeks from receipt of parental samples. Please contact the laboratory for urgent cases.
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Bardet-Biedl syndrome
Contact details Introduction Molecular Genetics Service Level 6, York House Bardet-Biedl syndrome (BBS, OMIM #209900) is an autosomal recessive disorder 37 Queen Square comprised of postaxial polydactyly, retinitis pigmentosa, genito-urinary and renal London, WC1N 3BH abnormalities, obesity and learning difficulties. Patients may also have diabetes, Hirschprung disease, hypertension and neurological deficits. One third of patients will T +44 (0) 20 7762 6888 develop renal failure and about 10% will progress to end-stage renal failure requiring F +44 (0) 20 7813 8578 dialysis and/or transplantation. Diagnosis of the condition is often delayed until early to mid-teens only when visual impairment emerges. Samples required The condition is rare with an estimated incidence of 1 in 125,000 in the UK although a • 5ml venous blood in plastic EDTA higher incidence is seen in communities in which consanguineous marriage is bottles (>1ml from neonates) common. BBS is a heterogeneous condition with at least 18 different genes • Prenatal testing must be arranged associated with the syndrome. in advance, through a Clinical Genetics department if possible. • Amniotic fluid or CV samples should be sent to Cytogenetics for Referrals dissecting and culturing, with instructions to forward the sample NCG funded analysis is available to patients resident in England and Scotland. For to the Regional Molecular Genetics non-NCG referrals, please contact the laboratory. laboratory for analysis • A completed DNA request card should accompany all samples Prenatal testing Patient details Prenatal testing may be available for families following molecular confirmation of To facilitate accurate testing and diagnosis in the proband - please contact the laboratory to discuss. reporting please provide patient demographic details (full name, date of birth, address and ethnic origin), details of any relevant family history and full Service offered contact details for the referring clinician Next generation sequencing of a panel of 13 BBS genes (BBS1, BBS2, ARL6, BBS4, BBS5, MKKS, BBS7, TTC8, BBS9, BBS10, BBS12, MKS1 and ALMS1). Mutations in these genes account for ~75% of BBS cases.
Technical Mutation screening is carried out by next generation sequencing with library preparation using a TruSeq custom amplicon kit followed by sequencing on the Illumina MiSeq. Data is analysed using an in-house pipeline with all mutations confirmed by Sanger sequencing.
Target reporting time Four months for a full mutation screen in an index case. Two weeks for familial mutation testing. Please contact the laboratory for urgent cases.
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Beckwith-Wiedemann Syndrome
Contact details Introduction Molecular Genetics Service Level 6, York House Beckwith-Wiedemann Syndrome (MIM 130650) occurs in approximately 1/13,700 37 Queen Square individuals. Beckwith-Wiedemann Syndrome is a growth disorder characterised by London, WC1N 3BH macrosomia, macroglossia, visceromegaly, embryonal tumours, hemihyperplasia, omphalocele, neonatal hypoglycemia, ear creases/pits, adrenocortical cytomegaly T +44 (0) 20 7762 6888 and renal abnormalities. F +44 (0) 20 7813 8578 BWS is a complex, multigenic disorder caused by alterations in growth regulatory Samples required genes on chromosome 11p15. Approximately 85% of BWS cases are sporadic and are mainly caused by epigenetic alterations in one of the two imprinting control • 5ml venous blood in plastic EDTA regions on chromosome 11p15 (ICR1 and ICR2). Decreased methylation at KvDMR bottles (>1ml from neonates) (ICR2) accounts for the majority of BWS cases (~50%). Approximately 2-7% of BWS • Prenatal testing must be arranged patients have increased methylation at the H19DMR (ICR1) locus. Paternal in advance, through a Clinical uniparental disomy of 11p15.5 results in increased methylation at H19DMR and Genetics department if possible. decreased methylation at KvDMR and accounts for 10-20% of patients. Mutations in • Amniotic fluid or CV samples the CDKN1C gene are a further cause of BWS and are inherited in an autosomal should be sent to Cytogenetics for dominant manner; these account for 5-10% of sporadic and 40% of familial BWS dissecting and culturing, with cases. Parent of origin specific chromosome rearrangements involving 11p15 are also instructions to forward the sample to the Regional Molecular Genetics associated with the BWS phenotype. Translocations and inversions show maternal laboratory for analysis inheritance whereas duplications typically show paternal inheritance. Chromosomal rearrangements account for less than 1% of cases. • A completed DNA request card should accompany all samples Referrals
Confirmation of clinically suspected BWS in children/adults Patient details To facilitate accurate testing and Prenatal testing reporting please provide patient demographic details (full name, date of Prenatal testing to confirm a diagnosis of BWS suspected on antenatal ultrasound birth, address and ethnic origin), details scan. Please contact the laboratory to discuss, prior to sending prenatal samples. of any relevant family history and full contact details for the referring clinician Service offered Confirmation of BWS by dosage and methylation analysis of chromosome 11p15. CDKN1C mutation analysis is not offered in this laboratory. Technical For diagnostic referrals, testing involves dosage and methylation analysis of the chromosome 11p15 region using methylation specific MLPA (MS-MLPA) analysis. Unaffected individuals will show normal dosage and methylation at both imprinting control regions. Patients with BWS will show either increased methylation at ICR1 or reduced methylation at ICR2. Patients with BWS caused by paternal UPD11p15.5 will show increased methylation at ICR1 and reduced methylation at ICR2. Cytogenetic analysis can be helpful for the identification of chromosomal rearrangements. Target reporting time Routine analysis – dosage and methylation analysis by MS-MLPA takes up to 4 weeks. Please contact the laboratory if urgent testing is required.
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Familial breast/ovarian cancer
Contact details Introduction Molecular Genetics Service Level 6, York House Hereditary breast and ovarian cancer due to mutations in BRCA1 and BRCA2 genes 37 Queen Square is the most common cause of hereditary forms of both breast and ovarian cancer. The London, WC1N 3BH prevalence of BRCA1/2 mutations is ~1/400 to 1/800; however this varies depending on ethnicity. Notably, in Ashkenazi Jewish populations there are three well-described T +44 (0) 20 7762 6888 founder mutations and their combined frequency in this population is 1/40. F +44 (0) 20 7813 8578 Mutations in the BRCA1 and BRCA2 genes are also associated with other forms of cancer including fallopian tube carcinoma and primary papillary serous carcinoma of the peritoneum. The risk of prostate cancer in male BRCA1 carriers is increased with Samples required a relative risk of ~1.8; however the age at diagnosis is comparable to that of sporadic prostate cancer. Prostate cancer is more strongly associated with BRCA2 mutations, • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) with a relative risk of 4.6 in male carriers as well as evidence of earlier age of onset. • Prenatal testing must be arranged Referrals in advance, through a Clinical Genetics department if possible. Referrals are accepted only via a Consultant Clinical Geneticist. • Amniotic fluid or CV samples should be sent to Cytogenetics for Service Offered dissecting and culturing, with • Complete screening of the coding exons including flanking intronic regions of instructions to forward the sample the BRCA1 and BRCA2 genes is offered to individuals who meet the criteria to the Regional Molecular Genetics defined by the NICE and Pan Thames guidelines. laboratory for analysis • A completed DNA request card • Common mutation screening is offered to individuals of populations where should accompany all samples there is a high frequency of BRCA1/2 mutations: BRCA1 c.68_69delAG Patient details Ashkenazi Jewish panel BRCA1 c.5266dupC To facilitate accurate testing and BRCA2 c.5946delT reporting please provide patient BRCA1 c.181T>G demographic details (full name, date of birth, address and ethnic origin), details Polish panel BRCA1 c.4035delA of any relevant family history and full BRCA1 c.5266dupC contact details for the referring clinician • Predictive testing is offered to individuals who have a known family mutation in either the BRCA1 or BRCA2 gene. A family mutation control is required for analysis. Technical Complete screening of the BRCA1/2 genes is carried out by Next Generation Sequencing using the Multiplicom BRCA MASTR Dx kit and the Illumina MiSeq platform. Mutations are confirmed by Sanger sequencing. MLPA analysis is used to detect large gene deletions and duplications Common mutation screens are carried out by Sanger sequencing of the relevant exons. Predictive testing is carried out by Sanger sequencing or MLPA as appropriate Target reporting time 8 weeks for a BRCA1/2 complete screen 4 weeks for a common mutation screen 2 weeks for a predictive test Please contact the laboratory for urgent cases
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Congenital Tufting Enteropathy
Contact details Introduction Molecular Genetics Service Level 6, York House Congenital Tufting Enteropathy (CTE; OMIM #613217) is a rare, neonatal-onset 37 Queen Square autosomal recessive condition characterised by chronic intractable diarrhoea leading London, WC1N 3BH to severe malabsorption and significant morbidity and mortality. The severity of intestinal malabsorption in most cases results in complete dependence on long-term T +44 (0) 20 7762 6888 parenteral nutrition. F +44 (0) 20 7813 8578 Histologically, affected individuals display focal epithelial tufts in the duodenum and Samples required jejunum composed of closely packed enterocytes. • 5ml venous blood in plastic EDTA The prevalence of CTE is estimated between 1/50’000 to 1/100’000 in Western bottles (>1ml from neonates) Europe but is higher in consanguineous populations and patients of Arabic ethnic • Prenatal testing must be arranged origin. in advance, through a Clinical Congenital Tufting Enteropathy is caused by mutations in the EPCAM gene at 2p21, Genetics department if possible. with a founder EPCAM mutation reported in several families of Arabic ethnic origin. • Amniotic fluid or CV samples should be sent to Cytogenetics for The EPCAM gene (NM_002354.2, ENST00000263735) consists of 9 coding exons dissecting and culturing, with and encodes an epithelial cell adhesion molecule. instructions to forward the sample to the Regional Molecular Genetics laboratory for analysis Referrals • A completed DNA request card should accompany all samples Referrals are accepted from Consultant Clinical Geneticists and Consultant Paediatric Gastroenterologists in the following: Patient details • Patients with clinically suspected Congenital Tufting Enteropathy To facilitate accurate testing and reporting please provide patient • Carrier testing in family members for known familial mutations demographic details (full name, date of birth, address and ethnic origin), details For clinical enquiries, please contact Dr Neil Shah, Gastroenterology, GOSH of any relevant family history and full contact details for the referring clinician Tel: +44 (0) 20 7405 9200 ext 5949, email: [email protected]
Prenatal testing Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken - please contact the laboratory to discuss. Service offered Analysis for point mutations and small insertions / deletions is by direct Sanger sequencing of the coding region in 9 amplicons. Technical Mutation screening is carried out by direct sequencing analysis. Target reporting time The target reporting time is 8 weeks for an EPCAM mutation screen and 2 weeks for carrier testing. Please contact the laboratory for urgent cases.
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Cystic Fibrosis
Contact details Introduction Molecular Genetics Service Level 6, York House Cystic fibrosis (MIM 219700) is an autosomal recessive condition caused by 37 Queen Square mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. To date over London, WC1N 3BH 1000 mutations with varying frequency have been identified in this gene. The ethnic origin of the patient influences the incidence of CF in the population and the mutations T +44 (0) 20 7762 6888 most commonly identified. F +44 (0) 20 7813 8578 Referrals • Confirmation of diagnosis in individuals clinically suspected of having CF. A sweat Samples required test should be undertaken prior to molecular genetic analysis wherever possible. • 5ml venous blood in plastic EDTA • Testing in individuals who may have a mild variant form of CF, e.g. congenital bottles (>1ml from neonates) bilateral absence of the vas deferens (CBAVD), bronchiectasis and pancreatitis. • Prenatal testing must be arranged • Carrier testing in pregnant couples with fetal echogenic bowel in advance, through a Clinical • Carrier testing in individuals at increased risk (above the population risk) of having an Genetics department if possible. affected pregnancy, for example a family history of CF, a partner shown to be a • Amniotic fluid or CV samples carrier or first cousin partnerships. Accurate carrier testing in CF families ideally should be sent to Cytogenetics for requires either a sample from an affected family member or information regarding the dissecting and culturing, with mutations carried in the family. Without this information the extent to which we can instructions to forward the sample reduce an individual’s carrier risk is less than if information on family mutations is to the Regional Molecular Genetics available. laboratory for analysis • In accordance with UK genetic testing guidelines carrier testing is only exceptionally • A completed DNA request card undertaken in minors. should accompany all samples Prenatal testing Patient details Prenatal testing is available for couples in whom specific mutations have been To facilitate accurate testing and identified - please contact the laboratory to discuss. reporting please provide patient demographic details (full name, date of Service offered birth, address and ethnic origin), details of any relevant family history and full 32 mutation screen and the partially penetrant intron 8 polyT mutation in cases contact details for the referring clinician referred for CFTR-related disease such as confirmed CBAVD, bronchiectasis and pancreatitis as well as CF referrals where the p.Arg117His mutation has been detected. Linked marker analysis is available in families where we are unable to identify a mutation in a clinically affected individual; this relies on the clinical diagnosis and sample availability from the affected individual and appropriate family members. Technical The mutation detection system in use in this laboratory is a kit based oligonucleotide ligation assay (OLA). As only 32 of the most commonly identified mutations are covered by this analysis failure to identify a mutation cannot exclude affected/carrier status, a residual risk to the individual is therefore calculated and reported wherever possible. In the North European population this system detects approximately 90% of cystic fibrosis mutations. Information regarding the ethnic origin of the patient is important for calculation of residual risk as the mutation spectrum, and hence the detection rate of the assay used, varies in different populations. Target reporting time 2 weeks for routine analysis of the 32 mutations. Please contact the laboratory if urgent or prenatal testing is required.
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Early Infantile Epileptic Encephalopathy (EIEE)
Contact details Introduction Molecular Genetics Service The early infantile epileptic encephalopathies (EIEE) are a group of disorders Level 6, York House characterized by early onset seizures and developmental delay. Many are associated 37 Queen Square with intractable seizures, severe developmental delay and require lifelong care. Early London, WC1N 3BH mortality is common amongst severely affected individuals due to seizures and/or T +44 (0) 20 7762 6888 respiratory tract infections. F +44 (0) 20 7813 8578 An increasing number of individual genetic disorders are now recognised to cause EIEE. In only a small subset of the disorders is the clinical phenotype sufficiently Samples required recognisable or distinctive to allow targeted testing of specific genes. • 5ml venous blood in plastic EDTA The annual incidence of EIEE has recently been estimated as approximately bottles (>1ml from neonates) 27/100,000. • A completed DNA request card should accompany all samples Referrals Patient details • Patients with EIEE and insufficient clinical features to target gene testing to a To facilitate accurate testing and single gene. A completed proforma must accompany all referrals reporting please provide patient (https://www.labs.gosh.nhs.uk/media/529300/severe_delay_- demographic details (full name, date of birth, address and ethnic origin), details _seizure_panel_pre-test_proforma_2014.doc). of any relevant family history and full • Mutation testing can be offered to the relatives of EIEE patients once a disease contact details for the referring clinician causing mutation has been identified.
Service offered Next generation sequencing of 45 genes (see list below) with mutation confirmation by Sanger sequencing. List of genes included in panel ADSL; ALG13; ARHGEF9; ARX; ATP1A3; ATRX; CDKL5; CHD2; CHRNA2; CHRNA4; CHRNB2; CNTNAP2; EHMT1; FOXG1; GABRB3; GRIN2A; GRIN2B; KCNQ2; KCNT1; KIAA1279; LGI1; MAGI2; MBD5; MECP2; MEF2C; NRXN1; PCDH19; PLCB1; PNKP; POLG; PRRT2; SCN1A; SCN2A; SCN8A; SIP1 [ZEB2]; SLC16A2; SLC25A22; SLC2A1 [GLUT1]; SLC9A6; SPTAN1; STXBP1; SYNGAP1; TBC1D24; TCF4; UBE2A; UBE3A
Technical Mutation screening is carried out by next generation sequencing with library preparation using a Sure Select XT custom kit followed by sequencing on the Illumina MiSeq. Data is analysed using an in-house pipeline with all mutations confirmed by Sanger sequencing.
Target reporting time 4 months for a full mutation screen in an index case (next generation sequencing). 2 weeks for familial mutation testing. Please contact the laboratory for urgent cases.
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Fragile X Syndrome
Contact details Introduction Molecular Genetics Service Fragile X syndrome (MIM 309550) is an X-linked mental retardation syndrome Level 6, York House associated with dysmorphic features (large everted ears, coarse facies, elongated 37 Queen Square face, macro-orchidism) in a proportion of cases. Around 1 in 5000 of the population is London, WC1N 3BH affected with fragile X, they are predominantly male but females can also be affected. T +44 (0) 20 7762 6888 The majority of fragile X cases are caused by expansion of the (CGG)n repeat in the F +44 (0) 20 7813 8578 promoter region of the FMR1 gene on chromosome Xq27.3 (FRAX A cases). Expansion of the (CGG)n repeat sequence to >200 repeats accompanied by
methylation of the adjacent CpG island extinguishes the FMR1 gene expression (full mutation expansion). Premutation alleles with 59-200 (CGG)n repeats are unstable Samples required at meiosis and can lead to full expansion mutations in subsequent generations. • 5ml venous blood in plastic EDTA Intermediate alleles (46-58 repeats) are not believed to be associated with fragile X bottles (>1ml from neonates) syndrome, but may display size instability in future generations. FMR1 point mutations • Prenatal testing must be arranged and deletions are rare causes of the syndrome. Premutation allele carriers can in advance, through a Clinical display additional phenotypes such as premature ovarian failure (POF) and a Genetics department if possible. neurodegenerative disorder of older adults, fragile X associated tremor/ataxia • Amniotic fluid or CV samples syndrome (FXTAS). should be sent to Cytogenetics for dissecting and culturing, with Referrals instructions to forward the sample to the Regional Molecular Genetics Children/adults (male or female) in whom a diagnosis of fragile X syndrome is laboratory for analysis suspected. Adults with a suspected clinical diagnosis of POF and FXTAS. Carrier testing for adults with a confirmed or suspected family history of fragile X syndrome. • A completed DNA request card should accompany all samples Prenatal samples (see below). Prenatal testing Patient details To facilitate accurate testing and Prenatal testing is available for confirmed fragile X carriers - analysis can be carried reporting please provide patient out on prenatal samples by direct analysis of the FMR1 (CGG)n repeat and/or by demographic details (full name, date of linked marker analysis if samples from the relevant family members are available. birth, address and ethnic origin), details of any relevant family history and full Service offered contact details for the referring clinician Direct analysis of the FMR1 (CGG)n repeat to identify intermediate alleles, premutations and full mutations. Linked marker analysis is available in families where we are unable to identify a mutation in a clinically affected individual; this relies on the clinical diagnosis being correct and sample availability from the affected individual and appropriate family members. Technical DNA is analysed by PCR of the (CGG)n repeat within the 5’ untranslated region of the FMR1 gene. The AmplideX™ FMR1 PCR kit is used to detect large premutations and full mutations which cannot be detected using the routine PCR assay. Neither of these assays is able to detect point mutations or deletions within the FMR1 gene, and they are also unable to exclude mosaicism. Please note that the PCR/ AmplideX™ assay are not methylation sensitive and provide no information on the methylation status of the FMR1 promoter. Target reporting time Routine analysis- 2 weeks for the initial PCR-based mutation screen, and 4 weeks if AmplideX™ is required. For urgent samples please contact the laboratory.
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GNAS1 gene mutation disorders (AHO/PHP1a/PPHP/ McCune Albright’s disease)
Contact details Introduction Molecular Genetics Service Albright’s hereditary osteodystrophy (AHO) is an autosomal dominant disorder Level 6, York House characterised by short stature, obesity, brachydactyly, subcutaneous ossifications and 37 Queen Square mental defects. There is a 2:1 ratio of affected females to males. AHO can present in London, WC1N 3BH one of two ways: with the somatic features of AHO alone T +44 (0) 20 7762 6888 (pseudopseudohypoparathyroidism, PPHP); or with AHO plus resistance to multiple F +44 (0) 20 7813 8578 hormones which increase cAMP in their target organs (pseudohypoparathyroidism type 1a, PHP 1a). Both PHP 1a and PPHP are caused by inactivating mutations in the GNAS1 gene. PHP1a is usually caused by mutations in maternal GNAS1, PPHP Samples required in paternal allele. • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) McCune Albright syndrome (MAS) is characterised by precocious puberty, café au lait • Prenatal testing must be arranged spots and polyostic fibrous dysplasia of bone where the normal interior of bone is in advance, through a Clinical replaced by fibro-osseous connective tissue. McCune Albright syndrome is caused Genetics department if possible. by somatic activating mutations in exons 8 and 9 of GNAS1. All MAS patients are • Amniotic fluid or CV samples mosaics. should be sent to Cytogenetics for GNAS1 encodes the α subunit of the G protein Gs. The G proteins are a family of dissecting and culturing, with instructions to forward the sample guanine nucleotide binding proteins involved in transmembrane signalling. They form to the Regional Molecular Genetics heterotrimers of α, β and γ. laboratory for analysis GNAS1 (located on 20q13.3) has 13 exons, 6 polyadenylation sites 3’ and 4 isoforms • A completed DNA request card (due to differential splicing of exons 3 and 4). There are two alternatively spliced should accompany all samples transcripts using exons upstream of GNAS1 (termed XLαs and NESP55) spliced to GNAS1 ex2-13 (+/- exon 3) expressed in most fetal tissue. Although the gene is Patient details biallelically expressed in most fetal tissue, XLαs is only expressed from the paternal To facilitate accurate testing and chromosome and NESP55 only expressed from the maternal chromosome. reporting please provide patient demographic details (full name, date of Referrals birth, address and ethnic origin), details of any relevant family history and full • Patients with clinical symptoms as above. contact details for the referring clinician • Carrier testing for family members for the familial mutation Prenatal testing Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken- please contact the laboratory to discuss. Service offered AHO: Sequencing analysis of all 13 exons. Approximately 80% of inactivating mutations will be detected by this method. MAS: Sequencing of exon 8 using COLD PCR amplification and exon 9 by standard sequencing analysis. The exon 8 COLD PCR amplifies the c.601C>T and c.602G>A variant alleles at greater efficiency than the wild type, thereby increasing sensitivity for low level mosaicism. DNA from an affected tissue such as bone has given more successful results than DNA extracted from lymphocytes. Target reporting time The target reporting time is 8 weeks for a GNAS1 mutation screen, 4 weeks for MAS mutation screening and 2 weeks for carrier testing. Please contact the laboratory for urgent cases.
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IL10-related Infantile Inflammatory Bowel Disease
Contact details Introduction Molecular Genetics Service Inflammatory bowel disease (OMIM # 266600) is a heterogeneous group of disorders Level 6, York House commonly presenting in adolescence or adulthood, but may present in infancy and be 37 Queen Square inherited as an autosomal recessive condition. London, WC1N 3BH Interleukin-10 (IL10) is an anti-inflammatory cytokine secreted by several cell types T +44 (0) 20 7762 6888 that is essential for the regulation of immune homeostasis in the gastro-intestinal tract. F +44 (0) 20 7813 8578 IL10 activates downstream signalling pathways by binding to the IL10 receptor, comprised of 2 α subunits (encoded by IL10RA) and 2 β subunits (encoded by Samples required IL10RB). • 5ml venous blood in plastic EDTA Loss-of-function mutations in IL10 at 1q32.1 (NM_000572.2), IL10RA at 11q23.3 bottles (>1ml from neonates) (NM_001558.3) and IL10RB (NM_000628.3) are associated with hyper-inflammatory • Prenatal testing must be arranged immune responses in the intestine resulting in severe, infantile-onset inflammatory in advance, through a Clinical bowel disease. Genetics department if possible. • Amniotic fluid or CV samples should be sent to Cytogenetics for dissecting and culturing, with Referrals instructions to forward the sample Referrals are accepted from Consultant Clinical Geneticists and Consultant Paediatric to the Regional Molecular Genetics laboratory for analysis Gastroenterologists in the following: • A completed DNA request card • Patients with clinically suspected IL10-related inflammatory bowel disease should accompany all samples • Carrier testing in family members for known familial mutations Patient details For clinical enquiries, please contact Dr Neil Shah, Gastroenterology, GOSH To facilitate accurate testing and Tel: +44 (0) 20 7405 9200 ext 5949, email: [email protected] reporting please provide patient demographic details (full name, date of birth, address and ethnic origin), details Prenatal testing of any relevant family history and full contact details for the referring clinician Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken - please contact the laboratory to discuss. Service offered Analysis for point mutations and small insertions / deletions is by direct Sanger sequencing analysis of the coding regions of IL10, IL10RA and IL10RB in 21 amplicons. Technical Mutation screening is carried out by direct sequencing analysis. Target reporting time The target reporting time is 8 weeks for an IL10 / IL10RA / IL10RB mutation screen and 2 weeks for carrier testing. Please contact the laboratory for urgent cases.
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Kabuki Syndrome-1 (KABUK1)
Contact details Introduction Molecular Genetics Service Kabuki syndrome-1 (KABUK1; MIM# 147920) is a rare congenital mental retardation Level 6, York House syndrome with distinct dysmorphic features and growth retardation. Kabuki 37 Queen Square Syndrome-1 is an autosomal dominant condition that occurs in 1 in approximately London, WC1N 3BH 32,000 live births. Most cases appear to be de novo, but familial cases are reported. T +44 (0) 20 7762 6888 Kabuki syndrome-1 is caused by mutations in the MLL2 gene, encoding an H3K4 F +44 (0) 20 7813 8578 histone methyl transferase which acts as an epigenetic transcriptional activator during growth and development. The majority of the reported mutations are truncating mutations (nonsense or frame shift mutations) but missense and splicing mutations Samples required have also been identified in Kabuki Syndrome patients. • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) • Prenatal testing must be arranged Referrals in advance, through a Clinical Genetics department if possible. Asymptomatic (carrier) testing can be offered to relatives of affected patients once a • Amniotic fluid or CV samples disease causing mutation has been identified. (Referrals for full screening are sent to should be sent to Cytogenetics for the Manchester Regional Genetics Laboratory). dissecting and culturing, with instructions to forward the sample to the Regional Molecular Genetics Prenatal testing laboratory for analysis • A completed DNA request card Prenatal testing is available for families in whom specific mutations have been should accompany all samples identified - please contact the laboratory to discuss.
Patient details Service offered To facilitate accurate testing and reporting please provide patient Mutation specific testing for previously identified family mutations is available for demographic details (full name, date of family members. birth, address and ethnic origin), details of any relevant family history and full contact details for the referring clinician Technical Mutation screening is carried out by direct fluorescent sequencing.
Target reporting time Mutation-specific test: 2 weeks. Please contact the laboratory for urgent cases.
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Nicolaides-Baraitser Syndrome
Contact details Introduction Molecular Genetics Service Nicolaides-Baraitser Syndrome (NBS; MIM # 601358) is an autosomal dominant Level 6, York House condition manifesting primarily as severe developmental delay with short stature, 37 Queen Square microcephaly, seizures, and a characteristic facial dysmorphism. Notable features London, WC1N 3BH include sparse hair on the scalp, and progressive skin wrinkling; additionally, the T +44 (0) 20 7762 6888 hands tend to show brachydactyly, inter-phalangeal joint swellings and broad distal F +44 (0) 20 7813 8578 phalanges. It is generally caused by de novo heterozygous mutations within the SMARCA2 gene, located on chromosome 9p24.3. This encodes the SMARCA2 protein which forms part of the SWI/SNF chromatin remodeling complex. The main Samples required function of this complex is activation of transcription by altering nucleosome structure, • 5ml venous blood in plastic EDTA making the DNA more accessible to transcription factors and transcription apparatus. bottles (>1ml from neonates) The majority of mutations reported so far are heterozygous missense mutations within • Prenatal testing must be arranged the ATPase/helicase domain of SMARCA2. in advance, through a Clinical Genetics department if possible. Referrals • Amniotic fluid or CV samples should be sent to Cytogenetics for Prospective patients should have a probable diagnosis of Nicolaides-Baraitser dissecting and culturing, with syndrome based on the presence of: instructions to forward the sample • Developmental delay to the Regional Molecular Genetics laboratory for analysis • AND characteristic features suggestive of Nicolaides-Baraitser Syndrome, e.g. • A completed DNA request card o Sparse hair on the scalp should accompany all samples o Short stature Patient details o Prominent interphalangeal joints To facilitate accurate testing and Epilepsy reporting please provide patient o demographic details (full name, date of Prenatal testing birth, address and ethnic origin), details of any relevant family history and full Prenatal testing may be available for families in whom specific mutations have been contact details for the referring clinician identified - Please contact the laboratory to discuss. Service offered Mutation screening is carried out in two stages. 1) Analysis of the commonly mutated exons 15-21 and 23-25 of SMARCA2. 2) In patients normal for level 1, a level 2 analysis can be carried out to screen the remaining coding exons. Mutation specific testing for previously identified family mutations is available in family members. Technical Mutation screening is carried out by bidirectional fluorescent Sanger sequencing. Target reporting time Mutation screening: 8 weeks. Mutation-specific test: 2 weeks. For urgent samples please contact the laboratory.
Version 6
Pulmonary Surfactant Metabolism Dysfunction 1, 2 and 3 (Genes: SFTPB, SFTPC and ABCA3)
Contact details Introduction Molecular Genetics Service Pulmonary Surfactant Metabolism Dysfunction comprises a genetically Level 6, York House heterogeneous group of disorders that result in severe respiratory insufficiency or 37 Queen Square failure in full-term neonates or infants. These disorders are associated with various London, WC1N 3BH pathologic entities, including pulmonary alveolar proteinosis (PAP), desquamative T +44 (0) 20 7762 6888 interstitial pneumonitis (DIP), or cellular nonspecific interstitial pneumonitis (NSIP). F +44 (0) 20 7813 8578 Type 1 (MIM 265120) is caused by recessive mutations in the pulmonary associated surfactant protein B (SFTPB) gene (MIM 178640) at 2p12-11.2, which encodes the Samples required pulmonary-associated surfactant protein B (SPB) a crucial component of pulmonary • 5ml venous blood in plastic EDTA surfactant, the mixture of lipids and specific proteins, which reduces surface tension at bottles (>1ml from neonates) the alveolar air-liquid interface. • Prenatal testing must be arranged Type 2 (MIM 610913) is caused by dominant mutations in the pulmonary associated in advance, through a Clinical surfactant protein C (SFTPC) gene (MIM 178620) at 8p21, which encodes pulmonary- Genetics department if possible. associated surfactant protein C (SPC) a crucial component of pulmonary surfactant • Amniotic fluid or CV samples which also reduces surface tension at the alveolar air-liquid interface. should be sent to Cytogenetics for dissecting and culturing, with instructions to forward the sample Type 3 (MIM 610921) is caused by recessive mutations in the ATP binding cassette to the Regional Molecular Genetics (ABC), subfamily A, member 3 (ABCA3) (MIM 601615) gene at 16p13.3, which is a laboratory for analysis member of the ABC transporter family. ABCA3 is expressed predominantly at the • A completed DNA request card limiting membrane of the lamellar bodies in lung alveolar type II cells and is thought to should accompany all samples be involved in surfactant secretion.
Patient details Referrals To facilitate accurate testing and reporting please provide patient Full term neonates with severe respiratory distress of unknown aetiology. demographic details (full name, date of Occasionally older children with respiratory distress of unknown cause. Carrier birth, address and ethnic origin), details testing for parents, and other adult relatives. A completed clinical information sheet is of any relevant family history and full required prior to analysis. contact details for the referring clinician Prenatal testing Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken; please contact the laboratory to discuss. Service offered Direct sequence analysis of the coding regions of the SFTPB gene (exons 1 to 10), SFTPC gene (exons 1 to 5) and of the ABCA3 gene (exons 4-33). Linked marker analysis is also available as appropriate for SFTPB. Technical SFTPB: Direct sequencing of coding exons 1 to 10. Six linked markers are available. SFTPC: Direct sequencing of coding exons 1 to 5. ABCA3: Direct sequencing of coding exons 4 to 33 and one known intronic mutation. Target reporting time 8 weeks for a whole gene screen. 2 weeks for carrier testing. Please contact the laboratory for urgent cases.
Version 6
Interferon Regulatory Factor 6 (IRF6) gene disorders (Popliteal Pterygium syndrome and Van der Woude syndrome)
Contact details Introduction Molecular Genetics Service Level 6, York House Van der Woude syndrome (VWS; MIM) and Popliteal Pterygium syndrome (PPS) are 37 Queen Square allelic autosomal dominant disorders caused by mutations in the interferon regulatory London, WC1N 3BH factor 6 gene (IRF6; MIM *607199). VWS is the most common form of cleft lip and/or palate accounting for 1-2% of cases. Lip pits and/or sinuses are cardinal features of T +44 (0) 20 7762 6888 the syndrome present in 70-80% of patients. PPS combines the symptoms of VWS F +44 (0) 20 7813 8578 with popliteal webs, unusual nails, syndactyly, ankyloblepharon and genital abnormalities. Samples required It has been proposed that orofacial development is affected in VWS as a result of • 5ml venous blood in plastic EDTA haploinsufficiency with protein truncating mutations commonly identified throughout bottles (>1ml from neonates) the IRF6 gene. The features of PPS are thought to result from dominant negative • Prenatal testing must be arranged mutations (generally missense) in the DNA binding domain of the protein. in advance, through a Clinical Confirmation of diagnosis enables prenatal testing for PPS and clarification of Genetics department if possible. recurrence risk for VWS (50% as opposed to 3-5% for isolated cleft/lip palate • Amniotic fluid or CV samples families). should be sent to Cytogenetics for dissecting and culturing, with The IRF6 gene (1q32-q41) has 9 exons (exons 1 and 2 are non-coding). c.250C>T instructions to forward the sample (p.Arg84Cys) and c.251G>A (p.Arg84His) are recurrent mutations identified in PPS to the Regional Molecular Genetics patients. A variety of point mutations and small deletions have been identified in VWS laboratory for analysis located throughout the IRF6 gene. • A completed DNA request card should accompany all samples Referrals Patient details • Referrals are only accepted via a Clinical Geneticist or cleft surgeon. To facilitate accurate testing and reporting please provide patient • Testing of other family members will be possible upon identification of a demographic details (full name, date of causative mutation in the index case. birth, address and ethnic origin), details of any relevant family history and full contact details for the referring clinician Prenatal testing for Popliteal Pterygium syndrome Prenatal testing is available for families in whom mutations causing PPS have been identified or in whom appropriate family studies have been undertaken. Please contact the laboratory to discuss.
Service offered • Direct sequencing of all coding exons (3-9) and intron-exon boundaries. • Testing for known mutations in relatives of patients with confirmed PPS / VWS mutations by sequencing.
Contact details for Consultant Cleft Geneticist at Great Ormond Street Dr M Lees, Clinical Genetics, Great Ormond Street Hospital, Level 4 York House, 37 Queen Square, London WC1N 3BH
Target reporting time 8 weeks for routine mutation screen in an index case. 2 weeks for carrier testing. Please contact the laboratory for urgent cases.
Version 6
Prader-Willi Syndrome
Contact details Introduction Molecular Genetics Service Prader-Willi syndrome (PWS) (MIM 176270), occurring in 1/15000 - 1/20000 Level 6, York House individuals, is characterised by diminished fetal activity, obesity, muscular hypotonia, 37 Queen Square developmental delay, short stature, hypogonadotropic hypogonadism, and small London, WC1N 3BH hands and feet. The PWS phenotype results from the lack of a paternal contribution at 15q11-q13. This can be caused by a deletion (~70%), maternal uniparental disomy T +44 (0) 20 7762 6888 (UPD) (25-30%) and rarely due to mutations in the imprinting centre (IC) that cause F +44 (0) 20 7813 8578 abnormal methylation at exon alpha of the SNRPN locus. These mutations are all detected by disrupted methylation. Deletions and UPD are usually de novo events, Samples required associated with low recurrence risks, although it is important to determine whether • 5ml venous blood in plastic EDTA either parent of an affected child has a predisposing chromosome translocation. bottles (>1ml from neonates) There is a recurrence risk of up to 50% in families with confirmed PWS who do not • Prenatal testing must be arranged have a deletion or UPD and are therefore likely to have an IC mutation. in advance, through a Clinical Referrals Genetics department if possible. Confirmation of clinically suspected PWS in children/adults. • Amniotic fluid or CV samples should be sent to Cytogenetics for Investigation of the molecular defect in confirmed PWS cases, distinguishing between dissecting and culturing, with UPD, deletion and IC mutations (parental samples required). instructions to forward the sample Carrier testing in adult relatives of confirmed PWS patients who are suspected of to the Regional Molecular Genetics having an IC mutation (samples from appropriate family members are required). laboratory for analysis Prenatal testing • A completed DNA request card should accompany all samples Prenatal diagnosis is available to couples where PWS has been confirmed in the family and to couples at risk of having a child affected with PWS due a balanced Patient details chromosomal rearrangement involving chromosome 15 in one of the parents. Please To facilitate accurate testing and contact the laboratory to discuss each case prior to sending prenatal samples to the reporting please provide patient laboratory. demographic details (full name, date of birth, address and ethnic origin), details Service offered of any relevant family history and full contact details for the referring clinician Confirmation of a PWS diagnosis by methylation analysis and microsatellite analysis to determinate the molecular defect in confirmed cases (requires samples from appropriate family members). Technical For diagnostic referrals the initial test is to determine the methylation status of exon alpha of the SNRPN gene. Methylation analysis is undertaken by methylation specific PCR following bisulphite modification of genomic DNA. Normal individuals yield a 313bp maternally derived fragment and a 221bp paternally derived fragment. Patients with Prader-Willi syndrome show a single 313bp maternal fragment only. In PWS patients with abnormal methylation, further analysis is recommended to characterise the nature of the mutation, this affects the recurrence risk for the parents (parental samples required), and involves the use of chromosome 15 microsatellite markers from within the commonly deleted region and markers flanking this region. Cytogenetic analysis is also helpful in identifying deletions and predisposing parental translocations. NB. A similar testing process is undertaken for Angelman syndrome. Target reporting time Routine analysis - the initial methylation test takes up to 4 weeks. Microsatellite marker analysis takes 8 weeks from receipt of parental samples. Please contact the laboratory for urgent cases.
Version 6
Primary Congenital Glaucoma (PCG)
Contact details Introduction Molecular Genetics Service Primary congenital glaucoma (PCG; MIM: #231300) is a congenital or infantile onset condition, Level 6, York House characterised by raised intraocular pressure. Signs of this may include enlargement of the globe 37 Queen Square (buphthalmos), cloudy/hazy corneas, lacrimation and optic disc changes. The condition causes irreversible optic nerve damage and blindness if untreated. London, WC1N 3BH T +44 (0) 20 7762 6888 This autosomal recessive condition has an incidence of 1/10,000 and is caused by mutations in the CYP1B1 gene (MIM:*601771) located at 2p22.2. CYP1B1 consists of 3 exons, 2 of which are F +44 (0) 20 7813 8578 coding (NM_000104.3). Patients with primary congenital glaucoma (PCG) are defined by elevated intraocular pressure Samples required (IOP) >21 mm Hg and/or signs consistent with elevated IOP, including • • 5ml venous blood in plastic EDTA disc cupping >0.3 or disc asymmetry ≥0.2 bottles (>1ml from neonates) • progressive disc cupping • buphthalmos (prominent, enlarged eye) • Prenatal testing must be arranged • enlarged corneal diameter (>11 mm in newborn, >12 mm in a child <1 year, or >13 mm in a in advance, through a Clinical child >1 year) Genetics department if possible. • corneal edema • Amniotic fluid or CV samples • Descemet's membrane splits (Haab's striae) should be sent to Cytogenetics for • visual field defects dissecting and culturing, with • progressive myopia in a child < 2 years of age. instructions to forward the sample to the Regional Molecular Genetics Sensitivity: 50% of patients with the symptoms described above will be expected to have a laboratory for analysis mutation in CYP1B1. No other genes have yet been described. • A completed DNA request card Later-onset disease (e.g. in middle childhood/adolescence) caused by CYP1B1 mutations is should accompany all samples rarely reported. Referrals Patient details To facilitate accurate testing and Referrals are accepted from Consultant Clinical Geneticists and Consultant Paediatric reporting please provide patient Ophthalmologists in the following: demographic details (full name, date of • Patients with the symptoms detailed above birth, address and ethnic origin), details • Carrier testing in family members for familial mutations of any relevant family history and full contact details for the referring clinician Prenatal testing Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken - please contact the laboratory to discuss. Service offered Analysis for point mutations and small insertions / deletions is by direct sequencing of the coding region in 6 amplicons. A wide variety of mutations have been reported, occurring throughout the coding region but no common mutations have been described. A single case has been identified with a whole gene deletion; analysis for large deletions and duplications however is not currently available. Technical Direct Sanger sequencing analysis of exons 2 and 3 of the coding region. Target reporting time The target reporting time is 8 weeks for a CYP1B1 mutation screen and 2 weeks for carrier testing. For urgent samples please contact the laboratory.
Version 6
SMARCB1 – Rhabdoid Tumour
Contact details Introduction Molecular Genetics Service Rhabdoid tumour predisposition syndrome (MIM# 609322) is a rare syndrome Level 6, York House occurring in infancy and early childhood. The incidence is not currently known but is 37 Queen Square thought to occur in ~ 0.1-05 per million children per year. Rhabdoid tumours are highly London, WC1N 3BH malignant neoplasms that develop in the brain and spinal cord (atypical teratoid T +44 (0) 20 7762 6888 rhabdoid tumour), kidney and/or soft tissue (malignant rhabdoid tumour or extra-renal F +44 (0) 20 7813 8578 rhabdoid tumour). Patients may present with apparently sporadic tumours in one site or with multiple tumours arising in the brain, kidney and/or soft tissues. Approximately 1/3 of rhabdoid tumours are due to germline mutations in the SMARCB1 gene. The Samples required SMARCB1 gene encodes the INI-1/hSNF5 protein which is a member of the • 5ml venous blood in plastic EDTA SW1/SNF chromatin re-modelling complex and functions in controlling gene bottles (>1ml from neonates) transcription. • Prenatal testing must be arranged in advance, through a Clinical Rhabdoid tumours are highly resistant to conventional chemotherapies and Genetics department if possible. radiotherapy; patients frequently succumb rapidly to disease. • Amniotic fluid or CV samples Loss of protein expression of SMARCB1 gene can be detected by should be sent to Cytogenetics for immunohistochemistry, however a small number of tumours show expression. dissecting and culturing, with instructions to forward the sample to the Regional Molecular Genetics laboratory for analysis Referrals • A completed DNA request card should accompany all samples Referrals are accepted only via a Consultant Clinical Geneticist or Consultant Neurologist. Patient details To facilitate accurate testing and Prenatal testing reporting please provide patient demographic details (full name, date of Prenatal testing is available for families in whom specific mutations have been birth, address and ethnic origin), details identified on referral from a Clinical Genetics unit only. of any relevant family history and full contact details for the referring clinician Service offered Mutation screening by direct sequencing is offered for the 9 exons of the SMARCB1 gene. MLPA analysis is available to look for large deletions / duplications.
Target reporting time 8 weeks for mutation screen in index case. 2 weeks for targeted testing using mutation specific tests. For urgent samples please contact the laboratory.
Version 6
Rett Syndrome
Contact details Introduction Molecular Genetics Service Rett syndrome (MIM 312750) is an X-linked dominant neurodevelopmental disorder that occurs Level 6, York House mainly in females. It is characterized by arrested development between 6 and 18 months of age, regression of acquired skills, loss of speech, stereotypical movements (classically of the hands), 37 Queen Square microcephaly, seizures, and mental retardation. London, WC1N 3BH >90% of cases with classical RTT are caused by mutations in the methyl-CpG-binding-protein 2 T +44 (0) 20 7762 6888 (MECP2) gene on Xq28 (MIM 300005). MECP2 mutations have also been reported in individuals with Angelman-like syndrome, non-specific mental retardation and neonatal severe F +44 (0) 20 7813 8578 encephalopathy. A severe atypical form of Rett syndrome (EIEE2 – MIM300672) is caused by mutations in the cyclin dependent-like kinase 5 (CDKL5) gene on Xp22 (OMIM 300203) and has Samples required been reported in both males and females. • 5ml venous blood in plastic EDTA Referrals bottles (>1ml from neonates) • Confirmation of diagnosis in individuals clinically suspected of having a MECP2-related • Prenatal testing must be arranged disorder. in advance, through a Clinical • Confirmation of diagnosis in individuals clinically suspected of having a CDKL5-related Genetics department if possible. disorder. • Amniotic fluid or CV samples • Maternal carrier testing once a mutation has been identified in the proband (a mother who should be sent to Cytogenetics for has a MECP2 or CDKL5 mutation may have favourably skewed X-chromosome inactivation that dissecting and culturing, with results in her being unaffected or mildly affected). instructions to forward the sample to the Regional Molecular Genetics Prenatal testing laboratory for analysis Prenatal testing is available for couples 1) when the mother has been found to be a carrier 2) to • A completed DNA request card rule out recurrence due to germ-line mosaicism in either parent when a mutation has been should accompany all samples identified in their affected child - please contact the laboratory to discuss. Service offered Patient details To facilitate accurate testing and MECP2 reporting please provide patient demographic details (full name, date of MLPA analysis of the MECP2 gene to identify large deletions and duplications Mutation screening covering the 5’UTR and all four exons and intron-exon boundaries (including birth, address and ethnic origin), details MECP_e2 exon 2). of any relevant family history and full contact details for the referring clinician CDKL5 MLPA analysis of the CDKL5 gene to identify large deletions and duplications. If required, samples are exported for sequence analysis. Technical Dosage analysis is by Multiplex Ligation-dependent Probe Amplification (MLPA). MECP2 uses kit P015-F1 from MRC-Holland where 13 MECP2 probes cover all 4 exons. CDKL5 uses kit P189-C1 from MRC-Holland where 23 probes cover all exons of the CDKL5 major transcript NM_003159.2. Mutation screening is carried out by bi-directional sequence analysis. Nomenclature Nomenclature for the MECP2 gene is based on reference sequence NM_001110792.1 - this encodes MECP2-e1, is the longest isoform of the MECP2 protein and encompasses exon 1 but not exon 2 of the MECP2 gene. It has been shown that this is the predominant isoform expressed in the brain and central nervous system. NM_004992 encodes MECP2_e2 which encompasses exons 2, 3 and 4 and was the originally identified transcript. It was later shown that expression of this transcript is mainly confined to fibroblast and lymphoblast cells. Target reporting time 8 weeks for routine sequence and dosage analysis of MECP2. 4 weeks for routine dosage analysis of CDKL5. 2 weeks for known familial mutations. Please contact the laboratory if urgent or prenatal testing is required.
Version 6
Silver Russell Syndrome
Contact details Introduction Molecular Genetics Service Silver Russell Syndrome (SRS) (MIM 180860), occurring in ~1/100’000 individuals, is Level 6, York House characterised by severe intrauterine and postnatal growth retardation. The growth 37 Queen Square failure in SRS is frequently associated with failure to thrive and very low body mass London, WC1N 3BH index. Craniofacial symptoms include a characteristic small triangular face with a T +44 (0) 20 7762 6888 prominent forehead and micrognathia, down turned corners of the mouth and ear F +44 (0) 20 7813 8578 abnormalities. In more than 50% of patients, limb and body asymmetry is present. SRS is a genetically heterogeneous disorder. Up to 50% of cases are due to Samples required abnormal methylation of chromosome 11p15, particularly decreased methylation at • 5ml venous blood in plastic EDTA the H19DMR imprinting control centre (ICR1). Approximately 5 -10% of individuals bottles (>1ml from neonates) with SRS have maternal uniparental disomy of chromosome 7. In 1-2% of SRS • Prenatal testing must be arranged patients microscopic chromosome aberrations have been described. Maternal UPD7 in advance, through a Clinical and methylation abnormalities are usually de novo events associated with low Genetics department if possible. recurrence risk however, familial cases have been reported. • Amniotic fluid or CV samples Referrals should be sent to Cytogenetics for dissecting and culturing, with Confirmation of clinically suspected SRS in children/adults. instructions to forward the sample to the Regional Molecular Genetics Investigation for maternal UPD7 (parental samples required). laboratory for analysis • A completed DNA request card should accompany all samples Prenatal testing Due to the low recurrence risk associated with Silver Russell Syndrome prenatal Patient details diagnosis is not routinely offered. To facilitate accurate testing and reporting please provide patient Service offered demographic details (full name, date of birth, address and ethnic origin), details Confirmation of SRS by dosage and methylation analysis of chromosome 11p15. If of any relevant family history and full normal, microsatellite analysis of chromosome 7 is carried out to detect UPD7; contact details for the referring clinician samples from both parents are required for microsatellite analysis. Technical For diagnostic referrals the initial test involves methylation and dosage analysis of the chromosome 11p15 region using methylation specific MLPA (MS-MLPA) analysis. Unaffected individuals will show normal dosage and methylation at both imprinting control regions. Patients with Silver Russell syndrome will show reduced methylation at the H19DMR imprinting control region (ICR1). In patients with a normal methylation pattern in the chromosome 11p15 region, further analysis using microsatellite markers for chromosome 7 is recommended to exclude maternal UPD7 as a cause of SRS (parental samples required). Cytogenetic analysis can be helpful for the identification of predisposing parental translocations. Target reporting time Routine analysis - the initial methylation and dosage (MS-MLPA) test takes up to 4 weeks. Microsatellite marker analysis for UPD7 takes 8 weeks from receipt of parental samples. Please contact the laboratory if urgent testing is required.
Version 6
Very Early Onset Inflammatory Bowel Disease
Contact details Introduction Molecular Genetics Service Very early onset inflammatory bowel disease (VEO-IBD) affects the gut but also other Level 6, York House tissues and as a consequence of persistent inflammation and treatment side effects 37 Queen Square the function of organs such as the liver may be impeded. Children are often not able to tolerate food and frequently rely on parenteral nutrition. Data is sparse but suggests London, WC1N 3BH that patients presenting in the first two years of life generally have a very poor T +44 (0) 20 7762 6888 prognosis with high mortality and morbidity (1). F +44 (0) 20 7813 8578 VEO-IBD (children with disease-onset before 6 years of age) has an estimated incidence of 4.37/100,000 and a prevalence of 14/100,000. Recent discoveries and Samples required published reviews (2) suggest that many monogenic diseases can present with an IBD-like phenotype (monogenic IBD). • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) Ref: 1. Cannioto, Z., et al., Eur J Pediatr, 2009. 168(2): 149-55 2. Uhlig HH. Gut 2013;62:1795–1805 • Prenatal testing must be arranged in advance, through a Clinical Referrals Genetics department if possible. • Amniotic fluid or CV samples Referrals are accepted from Consultant Clinical Geneticists and Consultant Paediatric should be sent to Cytogenetics for Gastroenterologists with presentation: dissecting and culturing, with • Patients aged under 6 years of age at onset with bloody diarrhoea & severe failure instructions to forward the sample to thrive to the Regional Molecular Genetics • Severe intestinal inflammation (macro- and microscopic) on upper and/or lower laboratory for analysis endoscopy • A completed DNA request card • Histology consistent with chronic inflammatory intestinal pathology should accompany all samples • All referrals must be accompanied by a completed proforma Patient details (www.labs.gosh.nhs.uk/media/528431/VEO-IBD Panel Proforma.doc) For clinical enquiries, please contact Dr Neil Shah, Gastroenterology, GOSH To facilitate accurate testing and Tel: +44 (0) 20 7405 9200 ext 5949, email: [email protected] reporting please provide patient demographic details (full name, date of Prenatal testing birth, address and ethnic origin), details of any relevant family history and full Prenatal testing is available for families in whom specific mutations have been contact details for the referring clinician identified or in whom appropriate family studies have been undertaken - please contact the laboratory to discuss. Service offered Analysis of coding regions and intron/exon boundaries of 40 genes (see list below); variant confirmation and familial tests by Sanger sequencing. ADA, ADAM17, AICDA, BTK, CD3γ, CD40LG, CYBA, CYBB, DCLRE1C, DOCK8, EPCAM, FOXP3, GUCY2C, HPS1, HPS4, HPS6, ICOS, IKBKG, IL2RG, IL10, IL10RA, IL10RB, ITGB2, LIG4, LRBA, NCF1, NCF2, NCF4, PIK3R1, PLCG2, RAG2, RET, SH2D1A, SKIV2L, SLC37A4, STXBP2, TTC37, WAS, XIAP, ZAP70 Technical Mutation screening is carried out by next generation sequencing with library preparation using a Sure Select XT custom kit followed by sequencing on the Illumina MiSeq. Data is analysed using an in-house pipeline with all mutations confirmed by Sanger sequencing. Target reporting time 4 months for a full mutation screen in an index case (next generation sequencing). 2 weeks for familial mutation testing. Please contact the laboratory for urgent cases.
Version 6
X-Inactivation
Contact details Introduction Molecular Genetics Service The X-inactivation status of females may be determined using X-linked methylation Level 6, York House sensitive polymorphic markers. This information may be useful to explain the 37 Queen Square manifestation of X-linked recessive conditions in females or to indicate carrier status London, WC1N 3BH for certain X-linked disorders. T +44 (0) 20 7762 6888 In females, random X-inactivation/lyonisation occurs where one of the two X F +44 (0) 20 7813 8578 chromosomes is randomly inactivated in every somatic cell. Hence the expression levels of most genes on the X chromosome are similar in males and females. Samples required However, 5-20% of the normal female population appear to have non-random or • 5ml venous blood in plastic EDTA skewed X-inactivation. Non-random X-inactivation is also thought to increase with bottles (>1ml from neonates) age. In certain conditions, if a female has a mutation in a given gene on one X • Prenatal testing must be arranged chromosome then non-random X-inactivation can occur, but this can be tissue in advance, through a Clinical dependent and therefore care must be taken to ensure the most appropriate tissue is Genetics department if possible. analysed. • Amniotic fluid or CV samples should be sent to Cytogenetics for dissecting and culturing, with The technique can be applied to any appropriate condition, however in this instructions to forward the sample laboratory X-inactivation studies are most commonly used to indicate carrier status to the Regional Molecular Genetics for the immunodeficiency conditions Wiskott Aldrich syndrome (WAS), X-linked laboratory for analysis severe combined immunodeficiency (XSCID) and X-linked agammaglobulinaemia • A completed DNA request card (XLA). In these conditions carrier females have unilateral X-inactivation patterns in should accompany all samples their whole blood, T cells only, or B cells only, respectively (separated cells will be required for this analysis, please see information below). Patient details Referrals To facilitate accurate testing and reporting please provide patient • To indicate carrier status of females with a suspected family history of the demographic details (full name, date of immunodeficiency disorders, WAS, XSCID and XLA, where no sample is available birth, address and ethnic origin), details from the affected male or where no mutation has been identified. of any relevant family history and full • For studies in other X-linked recessive conditions, please contact the laboratory to contact details for the referring clinician discuss. Service offered X-inactivation status at the androgen receptor (AR) gene, Xq11-q12 (MIM 313700).
Technical A methylation sensitive restriction enzyme is used to detect differential methylation patterns between the inactive and active X chromosomes. The methylation sensitive sites are in close proximity to the polymorphic site allowing the two X chromosomes to be distinguished. The androgen receptor is very informative and has a heterozygosity of 90%.
Target reporting time Routine analysis (immunodeficiency disorders) - 4 weeks. For urgent samples please contact the laboratory. For studies in other diseases please contact the laboratory to discuss the utility of the analysis, the samples required and the expected reporting time.
Version 6
Zygosity testing
Contact details Introduction Molecular Genetics Service Level 6, York House Molecular analysis of polymorphic markers can be used for ‘DNA fingerprinting’ to 37 Queen Square determine the zygosity of twins. Monozygotic (identical) twins will inherit the same London, WC1N 3BH alleles from their parents for all of the markers tested, whereas dizygotic twins are likely to inherit different alleles (but they may inherit the same alleles by chance). The T +44 (0) 20 7762 6888 likelihood of monozygosity can be determined by testing both parents and twins and F +44 (0) 20 7813 8578 calculating the likelihood of the same alleles being inherited by chance. If parents are not available then samples from the twins alone may be used and allele frequencies Samples required used to calculate the likelihood that the same alleles have been inherited by chance, • 5ml venous blood in plastic EDTA however there is limited data available for allele frequencies in different ethnic groups. bottles (>1ml from neonates) • Prenatal testing must be arranged in advance, through a Clinical Genetics department if possible. • Amniotic fluid or CV samples Referrals should be sent to Cytogenetics for dissecting and culturing, with Zygosity studies are often requested when one twin has developed a clinical instructions to forward the sample phenotype which is thought to be genetic in origin. These studies may help to to the Regional Molecular Genetics establish the recurrence risk. laboratory for analysis • A completed DNA request card should accompany all samples Service offered Patient details 9 highly polymorphic markers plus the amelogenin locus (a segment of the X-Y To facilitate accurate testing and pseudoautosomal region) are analysed. reporting please provide patient demographic details (full name, date of birth, address and ethnic origin), details of any relevant family history and full Technical contact details for the referring clinician Zygosity analysis makes use of an AmpflSTR Profiler Plus PCR amplification kit (manufactured by Applied Biosystems). This contains primers to amplify by PCR 9 polymorphic markers on 9 different chromosomes, plus the amelogenin locus. Different alleles are detected by size differentiation and analysed on a genetic analyser. Allele frequencies for the US Caucasian and Afro-American population have been determined and can be used to calculate the likelihood of monozygosity to greater than 99% probability in these ethnic groups, when parents are not available.
Target reporting time Routine analysis 4 weeks. Please contact the laboratory for urgent cases.
Version 6
Cell-Free Fetal DNA Sex Determination
Contact details Introduction Regional Genetics Service Free fetal DNA may be detected in maternal plasma from early in gestation and used Level 5/6, York House for determination of fetal gender. The sex of the fetus is determined by the presence 37 Queen Square of Y-specific sequence for a male fetus and the absence of Y specific material in the London, WC1N 3BH cell free DNA extract in the case of a female fetus. T +44 (0) 20 7762 6888 The analytical sensitivity and specificity of the Real Time PCR assay was measured in F +44 (0) 20 7813 8578 189 pregnancies (394 tests) over a period of 2 years from April 2007 to March 2009. When audited against pregnancy outcome there were 145 cases with a known Samples required outcome and in these cases the test demonstrated 100% (95% CI 97.5-99.9) • Pregnant Women concordance with no false positives or false negatives. 2x 10mls venous blood in This is achieved by testing two separate maternal samples for the presence of SRY plastic EDTA bottles or glass Streck tubes* and by stipulating that the fetus is at least 7 weeks gestation at the time of sampling. Referrals • Testing must be arranged in advance, through your Local All referrals should be made via a Clinical Genetics Department or Fetal Medicine Clinical Genetics Department or Unit, please contact the laboratory in advance of sending a sample. Fetal Medicine Unit Samples are accepted from patients from 7 weeks gestation (confirmed by scan) at • A completed DNA request card which time there should be a sufficient concentration of free fetal DNA in the should accompany all samples circulation. with an appropriate telephone number and a secure fax *From 7 to 9 weeks gestation, 2 x 10ml samples are required, taken one week apart. number. At 9+ weeks gestation 2 x 10ml samples may be taken at the same time. Samples should be sent to arrive in the laboratory within 24 hours of sampling (24-48 hours for Patient details Streck tubes) if possible. Advance notice is required so samples can be processed To facilitate accurate testing and rapidly upon receipt. Information on the outcomes of the pregnancy will be requested reporting please provide patient as part of a national ethically approved audit. Information sheets for parents and the demographic details (full name, date of audit are available on our laboratory website. birth, address and ethnic origin), details of any relevant family history and full Service offered contact details for the referring clinician We offer this service to pregnancies at risk of X-linked disorders or congenital adrenal hyperplasia. It is not available for non-medical indications. This test may not be applicable in multiple pregnancies including those with a possible vanishing twin. A male fetus is detected by the presence of SRY-specific sequence. The assay cannot distinguish between a lack of SRY indicative of a female fetus and a failure to extract sufficient free fetal DNA for analysis. A second sample ideally at later date but dependent on the gestation age is therefore required to repeat the analysis. Consistent absence of SRY in the presence of the control marker is taken as evidence that the fetus is female. Technical Maternal EDTA blood is separated as rapidly as possible after collection. Cell free DNA is then extracted from the plasma. Molecular analysis is performed using real time PCR and Taqman assays for the SRY marker and a CCR5 control marker. Results of the duplicate analysis will be released following analysis of the second sample. Target reporting time The results of the Y-specific probe analysis should be available within 3 days of the laboratory receiving the second sample.
Version 6
NIPD for FGFR3-related skeletal dysplasias
Contact details Introduction Regional Genetics Service Achondroplasia (ACH) (MIM 100800) is an autosomal dominant skeletal disorder due Level 5/6, York House to mutations in the FGFR3 gene on chromosome 4p16.3. Around 80-90% of cases 37 Queen Square are sporadic. Thanatophoric Dysplasia (TD), a sporadic neonatal lethal skeletal London, WC1N 3BH dysplasia, is divided into two subsets based upon radiological findings. TD type I (MIM T +44 (0) 20 7762 6888 187600) is associated with curved femora and variable but milder craniosynostosis F +44 (0) 20 7813 8578 and TD type II (MIM 187601) with straight femora and often cloverleaf skull. Mutations in the FGFR3 gene have been identified in almost 100% of confirmed cases of TD. A single mutation, p.Lys650Glu, accounts for all TD type II patients reported to date. Samples required Several recurrent mutations have been identified in TD type I. Non-invasive prenatal • Pregnant Women genetic diagnosis (NIPD) by next generation sequencing (NGS) is possible using cell 2x 10mls venous blood in plastic free fetal DNA (cffDNA) in pregnancies at risk of ACH or TD. EDTA bottles or glass Streck tubes, this should ideally reach the Referrals laboratory within 24-48 hours of sampling All referrals should be made via a Clinical Genetics Department or Fetal Medicine Unit and will be accepted in either of the categories given below. If you wish to refer a case • The minimum gestation (by scan) which does not fulfil these criteria please contact Professor Lyn Chitty is 9wks for accepting a sample. If ([email protected]) (Clinical) or Fiona McKay ([email protected]) earlier than 18wks then 2 blood samples a week apart may be (Laboratory) required 1. At risk pregnancy
• Testing must be arranged in • Paternal Achondroplasia OR advance, through your Local • a previous pregnancy has been confirmed to have ACH or TD, thus there is a very Clinical Genetics Department or small risk of recurrence due to germline mosaicism Fetal Medicine Unit 2. Abnormal ultrasound findings • A completed DNA request card and ultrasound report should Achondroplasia accompany all samples with an • appropriate telephone number Femoral length on or above the 3rd percentile (i.e. within normal range) at routine and a secure fax number. 18-20 week scan AND femur length and all long bones below 3rd percentile after 25 weeks gestation AND head circumference and abdominal circumference within • Pregnancy outcome or above the normal range for gestation at diagnosis. Fetal and maternal dopplers Details of pregnancy outcome will should be normal. be required for confirmation of laboratory results as part of the Thanatophoric dysplasia ongoing validation of new tests • The following features must be present: All long bones below the 3rd percentile AND small chest with short ribs • Additional features include polyhydramnios, bowed femora, relative macrocephaly, Patient details cloverleaf skull, short fingers To facilitate accurate testing and reporting please provide patient Service offered demographic details (full name, date of birth, address and ethnic origin), details NGS for 29 FGFR3 mutations associated with skeletal dysplasia. of any relevant family history and full contact details for the referring clinician Technical Maternal EDTA blood spun as soon as possible after collection, cffDNA extracted from plasma. Molecular analysis by PCR to amplify 5 amplicons of FGFR3, followed by NGS (Illumina MiSeq). Amplification of fetal DNA is confirmed using HLA markers, or SRY-specific sequences. Target reporting time
Results are normally available within 5 days of sample receipt
Version 6
NIPD for Apert syndrome
Contact details Introduction Regional Genetics Service Apert syndrome (MIM 101200) is a congenital disorder characterized primarily by Level 5/6, York House craniosynostosis, midface hypoplasia, and syndactyly of the hands and feet with a 37 Queen Square tendency to fusion of bony structures. Most cases are sporadic, but autosomal London, WC1N 3BH dominant inheritance has been reported. Apert syndrome can be very severe and is T +44 (0) 20 7762 6888 easily distinguishable from other craniosynostosis syndromes. Two mutations in F +44 (0) 20 7813 8578 FGFR2 exon 8, c.755C>G (p.Ser252Trp) and c.758C>G (p.Pro253Arg), account for over 98% of reported cases. Samples required Non-invasive prenatal genetic diagnosis (NIPD) is now possible using cell free fetal • Pregnant Women DNA (cffDNA) in pregnancies at risk of Apert syndrome. 2x 10mls venous blood in plastic EDTA bottles or glass Streck Referrals tubes, this should ideally reach the laboratory within 24-48 All referrals should be made via a Clinical Genetics Department or Fetal Medicine Unit hours of sampling and will be accepted in either of the categories given below. If you wish to refer a case which does not fulfil these criteria please contact Professor Lyn Chitty • The minimum gestation (by scan) ([email protected]) (Clinical) or Fiona McKay ([email protected]) is 9wks for accepting a sample. If (Laboratory). earlier than 18wks then 2 blood samples a week apart may be 1. At risk pregnancy required • Paternal Apert syndrome OR • Testing must be arranged in • a previous pregnancy has been confirmed to have Apert syndrome, thus there advance, through your Local Clinical Genetics Department or is a very small risk of recurrence due to germline mosaicism Fetal Medicine Unit 2. Abnormal ultrasound findings
• A completed DNA request card • Acrocephaly AND and ultrasound report should accompany all samples with an • Symmetrical syndactyly appropriate telephone number and a secure fax number. Service offered
• Pregnancy outcome Targeted next generation sequencing (NGS) for p.Ser252Trp (c.755C>G) and Details of pregnancy outcome will p.Pro253Arg (c.758C>G) mutations in FGFR2. be required for confirmation of laboratory results as part of the Technical ongoing validation of new tests Maternal EDTA blood is spun as soon as possible after collection, cffDNA is extracted from plasma. Molecular analysis is performed by PCR, followed by NGS (Illumina Patient details MiSeq). Amplification of fetal DNA will be confirmed using HLA markers, or SRY- specific sequences. To facilitate accurate testing and reporting please provide patient Target reporting time demographic details (full name, date of birth, address), details of any relevant Results are normally available within 5 days of sample receipt. family history and full contact details for the referring clinician
Version 6
NIPD for Cystic Fibrosis
Contact details Introduction Regional Genetics Service Cystic fibrosis (MIM 219700) is an autosomal recessive condition caused by Level 5/6, York House mutations in the cystic fibrosis conductance regulator (CFTR) gene. CF affects 37 Queen Square epithelia of the respiratory tract, exocrine pancreas, intestine, male genital tract, London, WC1N 3BH hepatobiliary system, and exocrine sweat glands, resulting in complex multisystem T +44 (0) 20 7762 6888 disease. Pulmonary disease is the major cause of morbidity and mortality in CF. F +44 (0) 20 7813 8578 Paternal mutation exclusion by non-invasive prenatal genetic diagnosis (NIPD) is now possible using cell free fetal DNA (cffDNA) in pregnancies at risk of cystic fibrosis for carrier couples where the parental mutations differ and the paternal mutation is one of Samples required 10 mutations covered by the panel. • Pregnant Women 2x 10mls venous blood in plastic Referrals EDTA bottles or glass Streck tubes, this should ideally reach the All referrals should be made via a Clinical Genetics Department or Fetal Medicine laboratory within 24-48 Unit. Both parental mutations must have been confirmed by molecular genetic testing. hours of sampling This test is only applicable to couples :
• The minimum gestation (by scan) 1) who are known carriers of different CF mutations AND is 9wks for accepting a sample. If earlier than 18wks then 2 blood 2) the paternal mutation is one of the 10 mutations listed below samples a week apart may be • c.1521_1523delCTT p.(Phe508del) required • c.489+1G>T • Testing must be arranged in • c.1624G>T p.(Gly542*) advance, through your Local Clinical Genetics Department or • c.1652G>A p.(Gly551Asp) Fetal Medicine Unit • c.3846G>A p.(Trp1282*)
• • A completed DNA request card c.1657C>T p.(Arg553*) and ultrasound report should • c.1519_1521delATC p.(Ile507del) accompany all samples with an • appropriate telephone number c.1679G>C p.(Arg560Thr) and a secure fax number. • c.1646G>A p.(Ser549Asn)
• • Pregnancy outcome c.1647T>G p.(Ser549Arg) Details of pregnancy outcome will The absence of a paternal mutation indicates that the fetus is at most only a carrier for be required for confirmation of CF and is therefore predicted to be unaffected. This test is not applicable to couples laboratory results as part of the ongoing validation of new tests who carry the same CF mutation, or where the CF mutations have not been characterised. If you wish to refer a case which does not fulfil these criteria please contact Professor Lyn Chitty ([email protected]) (Clinical) or Fiona McKay Patient details ([email protected]) (Laboratory). To facilitate accurate testing and Service offered reporting please provide patient demographic details (full name, date of Targeted next generation sequencing (NGS) for 10 mutations in CFTR. birth, address), details of any relevant family history and full contact details for Technical the referring clinician Maternal EDTA blood is spun as soon as possible after collection, cffDNA is extracted from plasma. Molecular analysis is performed by PCR, followed by NGS (Illumina MiSeq). Amplification of fetal DNA will be confirmed using HLA markers, or SRY- specific sequences. Target reporting time Results are normally available within 5 days of sample receipt.
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Familial Hypercholesterolaemia
Contact details Introduction Molecular Genetics Service Familial hypercholesterolaemia (FH) (MIM 143890) is a relatively frequent autosomal Level 6, York House dominant condition, characterised clinically by elevations in low-density lipoprotein 37 Queen Square cholesterol (LDL-C), tendon xanthomata (TX) and premature coronary heart disease London, WC1N 3BH (CHD). Heterozygous FH has an incidence of around 1/500 individuals in the UK, and T +44 (0) 20 7762 6888 severe homozygous FH affects 1/1000 000 individuals. FH is genetically F +44 (0) 20 7813 8578 heterogeneous; however the primary genetic defect in FH is a mutation in the gene encoding the LDL-receptor (LDLR). LDLR has 18 exons and family specific mutations are found throughout the gene, although some recurrent mutations are reported. Samples required Large deletions or duplications encompassing one or more exons accounts for 5% of • 5ml venous blood in plastic EDTA mutations in LDLR. A clinically indistinguishable disorder, familial defective bottles (>1ml from neonates) apolipoprotein B100 (FDB), is due to a mutation in the gene encoding apolipoprotein • A completed DNA request card B (APOB), which is one of the ligands of the LDL-receptor. The majority of FDB should accompany all samples cases (2-5% of hypercholesterolaemic individuals) have a single mutation, p.Arg3527Gln. Mutations causing FH have also been identified in the PCSK9 and Patient details LDLRAP1 genes that account for a small proportion of cases. To facilitate accurate testing and Referrals reporting please provide patient demographic details (full name, date of Referral criteria for testing are as determined by the Simon Broome Steering birth, address and ethnic origin), details Committee: of any relevant family history and full contact details for the referring clinician a) Total cholesterol >7.5mmol/l or LDL-C >4.9mmol/l if >16yrs. If <16yrs total cholesterol >6.7mmol/l or LDL-C >4.0mmol/l b) TX in patient or in first or second degree relative c) Family history of myocardial infarction (MI) <60yrs in first degree relative or family history of MI <50yrs in second degree relative d) Family history of total cholesterol >7.5mmol/l in first or second degree relative Patients are separated into two groups, ‘definite FH’ and ‘possible FH’. For a diagnosis of ‘definite FH’ both a) & b) must be present, but for ‘possible’ FH both a) & c) or a) & d) must be observed. Both groups are appropriate for genetic testing. Mutation testing can be offered to the relatives of FH patients once a disease causing mutation has been identified. Service offered Next generation sequencing of the LDLR, APOB, PCSK9 and LDLRAP1 genes with mutation confirmation by Sanger sequencing. MLPA to detect large deletions and duplications in the LDLR gene. Mutation specific testing for previously identified mutations is available to other family members. Technical Mutation screening is carried out by next generation sequencing with library preparation using a TruSeq custom amplicon kit followed by sequencing on the Illumina MiSeq. Data is analysed using an in-house pipeline with all mutations confirmed by Sanger sequencing. Dosage analysis is by Multiplex Ligation- dependent Probe Amplification (MLPA) analysis. Target reporting time 8 weeks for a full mutation screen in an index case (next generation sequencing and MLPA). 2 weeks for familial mutation testing.
Version 6
Hypertrophic cardiomyopathy (MYBPC3, MYH7, TNNT2, TNNI3)
Contact details Introduction Molecular Genetics Service Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterised Level 6, York House by unexplained hypertrophy of the left ventricle and is one of the leading causes of 37 Queen Square premature sudden death in young adults (especially between 10 and 30 years). The London, WC1N 3BH disease is mainly caused by mutations in genes encoding protein components of the T +44 (0) 20 7762 6888 cardiac sarcomere. There is a wide heterogeneity with at least fourteen genes F +44 (0) 20 7813 8578 responsible for HCM. There is also a variation in expressivity and penetrance.
Samples required Referrals • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) Predictive mutation analysis is available for family members in whom the causative • Prenatal testing must be arranged mutation has been confirmed in a CPA accredited laboratory. in advance, through a Clinical Genetics department if possible. Confirmatory mutation analysis can be carried out for patients in whom a mutation has been detected in a research laboratory. • Amniotic fluid or CV samples should be sent to Cytogenetics for dissecting and culturing, with Prenatal testing instructions to forward the sample to the Regional Molecular Genetics Prenatal testing is available for families in whom specific mutations have been laboratory for analysis identified or in whom appropriate family studies have been undertaken; please contact • A completed DNA request card the laboratory to discuss. should accompany all samples
Patient details Service offered Predictive and confirmatory testing can be offered to families where a mutation has To facilitate accurate testing and been identified in the MYBPC3, MYH7, TNNT2 or TNNI3 genes. reporting please provide patient demographic details (full name, date of birth, address and ethnic origin), details Technical of any relevant family history and full contact details for the referring clinician Testing is carried out by direct sequencing analysis.
Target reporting time The turn around time for familial mutations (including predictive tests) is 2 weeks. Please contact the laboratory for urgent cases.
Version 6
Loeys Dietz Syndrome
Contact details Introduction Molecular Genetics Service Loeys-Dietz syndrome (LDS) is an aortic aneurysm syndrome characterised by widely Level 6, York House spaced eyes (hypertelorism), bifid uvula and/or cleft palate and generalised arterial 37 Queen Square tortuosity with ascending aortic aneurysm and dissection. Affected patients have a London, WC1N 3BH high risk of aortic dissection or rupture at an early age and at aortic diameters that T +44 (0) 20 7762 6888 ordinarily would not be predictive of these events. LDS shows autosomal dominant F +44 (0) 20 7813 8578 inheritance and variable clinical expression. Approximately 25% of individuals diagnosed with LDS have an affected parent, with the remaining 75% due to de novo mutations. LDS is caused by mutations in TGFBR1 and TGFBR2. Both proteins are Samples required transmembrane serine/threonine receptor kinases. TGFBR1 contains 9 exons and • 5ml venous blood in plastic EDTA spans approximately 53 kb. TGFBR2 contains 8 exons in the longest transcript, bottles (>1ml from neonates) including the alternatively spliced exon 1A, and spans approximately 91 kb. • Prenatal testing must be arranged Mutations throughout the coding region of both genes have been reported including in advance, through a Clinical frame shift, nonsense, missense and splice site mutations as well as small insertions Genetics department if possible. or deletions. Most mutations are missense mutations in or immediately flanking the • Amniotic fluid or CV samples serine-threonine kinase domains of either receptor. Analysis is by sequencing of all should be sent to Cytogenetics for coding exons of both genes and will detect >95% of mutations in individuals with dissecting and culturing, with typical findings of LDS. instructions to forward the sample to the Regional Molecular Genetics laboratory for analysis • A completed DNA request card Referrals should accompany all samples • New referrals should fulfill the UKGTN testing criteria. Patient details • Mutation testing can be offered to the relatives of LDS patients once a disease To facilitate accurate testing and causing mutation has been identified. reporting please provide patient demographic details (full name, date of birth, address and ethnic origin), details Prenatal testing of any relevant family history and full contact details for the referring clinician Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken; please contact the laboratory to discuss.
Service offered Mutation screening of all the coding exons of the TGFBR1 and TGFBR2 genes in affected patients. Mutation specific testing for previously identified family mutations is available to other family members.
Technical Screening is carried out by direct sequencing analysis.
Target reporting time The target reporting time for both genes is 8 weeks. The target turnaround time for familial mutations (incl. predictive tests) is 2 weeks. Please contact the laboratory for urgent cases.
Version 6
Achondroplasia (ACH) and Hypochondroplasia (HCH)
Contact details Introduction Molecular Genetics Service Achondroplasia (MIM 100800) and hypochondroplasia (MIM 146000) are autosomal Level 6, York House dominant skeletal disorders with mutations in the FGFR3 gene on chromosome 37 Queen Square 4p16.3. London, WC1N 3BH Achondroplasia (ACH) has a birth incidence of between 1/15,000 and 1/77,000. T +44 (0) 20 7762 6888 Around 80-90% of cases are sporadic and there is an association with increased F +44 (0) 20 7813 8578 paternal age at the time of conception, suggesting that new mutations are generally of paternal origin. There are rare familial forms, as well as reported cases of germline Samples required and somatic mosaicism. • 5ml venous blood in plastic EDTA Hypochondroplasia (HCH) is genetically distinct from ACH and is clinically less bottles (>1ml from neonates) severe, with no associated craniofacial abnormalities. Because of its mild nature, • Prenatal testing must be arranged HCH can be difficult to diagnose and may be genetically heterogeneous. in advance, through a Clinical Approximately 70% of HCH patients have one of two mutations in the FGFR3 gene. Genetics department if possible. Of the remaining 30%, some families are reported that do not link to chromosome • Amniotic fluid or CV samples 4p16.3 should be sent to Cytogenetics for dissecting and culturing, with Referrals instructions to forward the sample to the Regional Molecular Genetics We offer testing for confirmation of diagnosis in affected individuals and family laboratory for analysis members. • A completed DNA request card should accompany all samples Prenatal testing 1) Prenatal testing is available to families in whom specific mutations have been Patient details identified - please contact the laboratory to discuss. To facilitate accurate testing and reporting please provide patient 2) Prenatal testing to confirm a diagnosis of ACH suspected on antenatal ultrasound demographic details (full name, date of scan birth, address and ethnic origin), details of any relevant family history and full Service offered contact details for the referring clinician Achondroplasia: Testing for the common p.Gly380Arg (c.1138G>A and c.1138G>C) mutations in exon 8 of FGFR3. Together these account for around 99% of mutations.
Hypochondroplasia: Mutation screening of exons 6, 8, 11 and 13 of FGFR3 will detect approximately 75% of reported mutations. Includes testing for the common p.Asn540Lys (c.1620C>A and c.1620C>G) mutations in exon 11 (which account for around 70% of mutations) and the common achondroplasia p.Gly380Arg (c.1138G>A and c.1138G>C) mutations.
Technical Direct sequence analysis of exons 8 (ACH) and 6, 8, 11 and 13 (HCH) detects the common mutations. This will also detect other mutations that may be present in these exons. Target reporting time 2 weeks for routine ACH analysis and 4 weeks for routine HCH analysis. For urgent samples please contact the laboratory.
Version 6
Thanatophoric dysplasia (TD) type I and type II
Contact details Introduction Molecular Genetics Service Thanatophoric Dysplasia, a sporadic neonatal lethal skeletal dysplasia, is divided into Level 6, York House two subsets based upon radiological findings. TD type I (MIM 187600) is associated 37 Queen Square with curved femora and variable but milder craniosynostosis and TD type II (MIM London, WC1N 3BH 187601) with straight femora and often cloverleaf skull. Mutations in the FGFR3 gene T +44 (0) 20 7762 6888 on chromosome 4 have been identified in almost 100% of confirmed cases of TD. A F +44 (0) 20 7813 8578 single mutation, p.Lys650Glu, has been identified in all of the TD type II patients reported to date. Several recurrent mutations have been identified in TD type I involving the gain of a cysteine residue, as well as rare mutations. Samples required • 5ml venous blood in plastic EDTA Referrals bottles (>1ml from neonates) We offer testing for confirmation of diagnosis in affected individuals and if requested, • Prenatal testing must be arranged family members. in advance, through a Clinical
Genetics department if possible. • Amniotic fluid or CV samples Prenatal testing should be sent to Cytogenetics for dissecting and culturing, with 1) Prenatal testing is available to families in whom specific mutations have been instructions to forward the sample identified - please contact the laboratory to discuss. to the Regional Molecular Genetics laboratory for analysis 2) Prenatal testing to confirm a TD diagnosis suspected on antenatal ultrasound scan • A completed DNA request card Service offered should accompany all samples TD type I - Several recurrent mutations have been identified involving the gain of a Patient details cysteine residue, including p.Arg248Cys (55% of cases), p.Tyr373Cys (24%), and To facilitate accurate testing and p.Ser249Cys (6%). Rarer mutations including p.Lys650Met, p.Gly370Cys, and reporting please provide patient p.Ser371Cys have also been reported. Mutation of the stop codon accounts for 10% demographic details (full name, date of of TD type I patients. birth, address and ethnic origin), details of any relevant family history and full TD type II - A single mutation, p.Lys650Glu, has been identified in all of the TD type II contact details for the referring clinician patients reported to date. Reference: Passos-Bueno et al; Human Mutation 14: 115-125, 1999 Technical The common mutations listed above are detected by direct sequencing analysis of exons 6, 8, 13 and 17. This may also detect other mutations present in these exons. The common achondroplasia p.Gly380Arg (c.1138G>A and c.1138G>C) mutations will also be detected by this analysis. Target reporting time 4 weeks for routine analysis. For urgent samples please contact the laboratory.
Version 6
Craniosynostosis
Contact details Introduction Molecular Genetics Service Craniosynostosis is the premature fusion of one or more of the cranial sutures, resulting in Level 6, York House abnormal skull growth, and affects approximately 1 in 2500 individuals. Craniosynostosis 37 Queen Square represents a heterogeneous group of disorders arising from both genetic and environmental factors. The craniosynostosis syndromes are usually sporadic, autosomal dominant disorders London, WC1N 3BH that have significant clinical overlap. T +44 (0) 20 7762 6888 F +44 (0) 20 7813 8578 Referrals We offer testing for confirmation of diagnosis in affected individuals and family members. Supra Samples required regional funding covers referrals for the following: • 5ml venous blood in plastic EDTA Muenke Syndrome / Non-syndromic Craniosynostosis MIM 602849 bottles (>1ml from neonates) Saethre-Chotzen Syndrome MIM 101400 • Prenatal testing must be arranged in advance, through a Clinical Pfeiffer Syndrome MIM 101600 Genetics department if possible. Crouzon Syndrome MIM 123500 • Amniotic fluid or CV samples should be sent to Cytogenetics for Apert Syndrome MIM 101200 dissecting and culturing, with instructions to forward the sample Prenatal testing to the Regional Molecular Genetics laboratory for analysis Prenatal testing is available for families in whom specific mutations have been identified - please contact the laboratory to discuss. • A completed DNA request card should accompany all samples Service offered
Patient details Muenke Syndrome / Non syndromic craniosynostosis: p.Pro250Arg in FGFR3 is the only testing offered To facilitate accurate testing and reporting please provide patient Pfeiffer Syndrome: p.Pro250Arg in FGFR3, p.Pro252Arg in FGFR1 and mutation screening demographic details (full name, date of initially across exons 8 and 10 of FGFR2 and then exons 3, 5, 11, 14-17 birth, address and ethnic origin), details of any relevant family history and full Crouzon Syndrome: p.Pro250Arg in FGFR3 then mutation screening, initially across exons 8 and contact details for the referring clinician 10 of FGFR2 and then exons 3, 5, 11, 14-17 Crouzon Syndrome with acanthosis nigricans: p.Pro250Arg and p.Ala391Glu in FGFR3 then mutation screening, initially across exons 8 and 10 of FGFR2 and then exons 3, 5, 11, 14-17 Saethre-Chotzen Syndrome: p.Pro250Arg in FGFR3 then mutation screening across exon 1 of TWIST and MLPA analysis for TWIST gene deletions Apert Syndrome: targeted analysis for common mutations: p.Ser252Trp (c.755C>G) and p.Pro253Arg (c.758C>G) in exon 8 of FGFR2 Coronal Synostosis: p.Pro250Arg in FGFR3, exons 8 and 10 of FGFR2 then mutation screening across exon 1 of TWIST and MLPA analysis for TWIST gene deletions followed by screening of the TCF12 gene Sagittal Synostosis: p.Pro250Arg in FGFR3, exons 8 and 10 of FGFR2 followed by screening of the ERF gene Technical Direct sequence analysis of the appropriate exons is carried out to detect the common mutations and to screen for unknown mutations in FGFR1, FGFR2, FGFR3 and TWIST, ERF and TCF12. TWIST gene deletions are detected by MLPA dosage analysis. Target reporting time Routine analysis: 2 - 4 weeks for targeted mutation testing and 8 weeks for mutation screen and MLPA analysis. For urgent samples please contact the laboratory.
Version 6
Mitochondrial testing m.1555A>G
Contact details Introduction Molecular Genetics Service Some mitochondrial point mutations have been associated with deafness, the most Level 6, York House commonly reported being m.1555A>G. 37 Queen Square London, WC1N 3BH The homoplasmic mutation m.1555A>G in the mitochondrial MT-RNR1 (12S rRNA) gene has been associated with aminoglycoside-induced and nonsyndromic T +44 (0) 20 7762 6888 sensorineural deafness (Estivill X et al, Am J Hum Genet 62(1): 27-35, 1998; Prezant F +44 (0) 20 7813 8578 TR et al., Nat Genet 4 (3): 289-294, 1993).
Samples required The mutation has been detected in families with maternally transmitted deafness and seems to have an age dependent penetrance for deafness, which is enhanced by • 5ml venous blood in plastic EDTA treatment with aminoglycosides. bottles (>1ml from neonates) • Prenatal testing must be arranged in advance, through a Clinical Referrals Genetics department if possible. • • Amniotic fluid or CV samples Patients with hearing loss for m.1555A>G mutation analysis. should be sent to Cytogenetics for • Patients who may require aminoglycosides. dissecting and culturing, with instructions to forward the sample • Maternal relatives of patients with the m.1555A>G mutation. to the Regional Molecular Genetics laboratory for analysis • A completed DNA request card Service offered should accompany all samples Mutation analysis for the m.1555A>G mutation. Patient details To facilitate accurate testing and Technical reporting please provide patient demographic details (full name, date of Restriction enzyme assay is performed to detect the m.1555A>G mutation. All birth, address and ethnic origin), details mutation positive results are confirmed by sequence analysis. of any relevant family history and full contact details for the referring clinician Target reporting time Two weeks for routine testing of m.1555A>G mutation in index case. Two weeks for maternal relatives of patients with the m.1555A>G mutation. Please contact the laboratory for urgent cases.
Version 6
Branchio-oto-renal syndrome (BOR)
Contact details Introduction Molecular Genetics Service Branchio-oto-renal syndrome (BOR) (MIM 113650) is an autosomal dominant Level 6, York House condition that manifests with the following phenotypes: 37 Queen Square London, WC1N 3BH Hearing loss T +44 (0) 20 7762 6888 Preauricular pits (“ear pits”) F +44 (0) 20 7813 8578 Pinnae abnormalities Samples required Branchial fistulae (lateral fistula of the neck) • 5ml venous blood in plastic EDTA Renal anomalies bottles (>1ml from neonates) • Prenatal testing must be arranged BOR has an incidence of approximately 1/40,000, accounts for about 2% of in advance, through a Clinical profoundly deaf children, and can be caused by mutations in the EYA1 gene or, more Genetics department if possible. rarely, in SIX1 or SIX5 genes. EYA1 has 16 exons with most mutations identified in • Amniotic fluid or CV samples exons 8-16. SIX1 and SIX5 have 2 and 3 coding exons respectively. should be sent to Cytogenetics for dissecting and culturing, with Referrals instructions to forward the sample to the Regional Molecular Genetics Prospective patients should have at least three of the four following major features: laboratory for analysis hearing loss, branchial defects, ear pits and renal anomalies. • A completed DNA request card Asymptomatic (carrier) testing can be offered to relatives of affected patients once a should accompany all samples disease causing mutation has been identified.
Patient details Prenatal testing To facilitate accurate testing and Prenatal testing is available for families in whom specific mutations have been reporting please provide patient identified - please contact the laboratory to discuss. demographic details (full name, date of birth, address and ethnic origin), details Service offered of any relevant family history and full contact details for the referring clinician Testing involves MLPA analysis of the EYA1 gene to identify larger deletions and duplications and mutation screening of the 16 exons (including alternative exon 1) of the EYA1 gene. Patients testing negative for EYA1 mutations can be tested for mutations in the SIX1 and SIX5 genes by sequencing analysis. Technical Dosage analysis is by Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. Mutation screening is carried out by direct sequencing. Target reporting time EYA1 MLPA analysis and mutation screening of exons 1-16 takes 8 weeks. SIX1 and SIX5 analysis takes 8 weeks. Mutation-specific tests take 2 weeks. Please contact the laboratory for urgent cases.
Version 6
Connexin 26
Contact details Introduction Molecular Genetics Service Pre-lingual non-syndromic sensorineural hearing loss (NSSNHL) is predominantly due Level 6, York House to recessive mutations. DFNB1 was the first locus described for autosomal recessive 37 Queen Square NSSNHL and accounts for a high proportion of cases. London, WC1N 3BH The GJB2 gene (located at 13q11-q12) encodes the gap junction protein, beta 2 - T +44 (0) 20 7762 6888 also known as connexin 26. GJB2 mutations may account for 10-30% of sporadic F +44 (0) 20 7813 8578 non-syndromic deafness. The c.35delG mutation is the most common GJB2 mutation described so far and is found in the majority of families linked to DFNB1. Other Samples required common mutations have been detected in specific ethnic groups. • 5ml venous blood in plastic EDTA A small proportion of individuals with DFNB1 have one identifiable GJB2 mutation and bottles (>1ml from neonates) one of two large deletions (del(GJB6-D13S1830) – 309kb, del(GJB6-D13S1854) – • Prenatal testing must be arranged 232kb) that include a part of GJB6 (encoding connexin 30) inherited on the opposite in advance, through a Clinical chromosome (del Castillo et al., J Med Genet (2005) 42:588-594). Genetics department. • Amniotic fluid or CV samples Specific heterozygous GJB2 mutations have also been described in patients with should be sent to Cytogenetics for idiopathic autosomal dominant hearing loss and rare cases of hearing loss associated dissecting and culturing, with with skin phenotypes: (Keratoderma ichthyosis and deafness syndrome (KID), instructions to forward the sample Vohwinkel syndrome, and palmoplantar keratoderma (PPK) and deafness. to the Regional Molecular Genetics laboratory for analysis Referrals • A completed DNA request card • should accompany all samples Patients with hearing loss for mutation screening of connexin 26 • Patients with hearing loss and a relevant skin phenotype for mutation screening Patient details of connexin 26. To facilitate accurate testing and • reporting please provide patient Adult relatives of patients with connexin 26 mutations for carrier status. demographic details (full name, date of birth, address and ethnic origin), details of any relevant family history and full Service offered contact details for the referring clinician Mutation screening of connexin 26 coding exon 2. Analysis for the 309kb deletion (GJB6-D13S1830) and 232bk deletion (GJB6-D13S1854), connexin 26 intron 1 splice donor site mutation (c.-23+1G>A) and splice acceptor site mutation (c.1-24A>C). Detection of known mutations in relatives of patients with confirmed connexin 26 mutations. Technical Direct sequencing analysis of connexin 26 exon 2 which covers the 681bp coding region and the c.-24A>C splice acceptor site mutation. Size separation assay for the 309kb and 232kb deletions and restriction digest assay for the c.-23+1G>A splice donor site mutation. Target reporting time Routine mutation screen in index case takes 8 weeks. Routine testing of specific mutations takes 2 weeks. Please contact the laboratory for urgent cases.
Version 6
EAST Syndrome
Contact details Introduction Molecular Genetics Service EAST syndrome (OMIM #612780) is an autosomal recessive condition that manifests Level 6, York House with the following phenotypes: 37 Queen Square • epilepsy London, WC1N 3BH T +44 (0) 20 7762 6888 • ataxia F +44 (0) 20 7813 8578 • sensorineural hearing loss Samples required • tubulopathy (electrolyte imbalance) • 5ml venous blood in plastic EDTA EAST syndrome is a rare disorder caused by mutations in the KCNJ10 gene (OMIM bottles (>1ml from neonates) *602208). KCNJ10 has 1 large coding exon. • Prenatal testing must be arranged in advance, through a Clinical Genetics department if possible. Referrals • Amniotic fluid or CV samples • Prospective patients should have a clear clinical phenotype, preferably referred by should be sent to Cytogenetics for clinical genetics or specialist renal departments dissecting and culturing, with instructions to forward the sample to the Regional Molecular Genetics • Asymptomatic (carrier) testing can be offered to relatives of affected patients once laboratory for analysis a disease causing mutation has been identified. • A completed DNA request card should accompany all samples Prenatal testing
Patient details Prenatal testing is available for families in whom specific mutations have been identified - please contact the laboratory to discuss. To facilitate accurate testing and reporting please provide patient Service offered demographic details (full name, date of birth, address and ethnic origin), details • Mutation screening of the single large coding of the KCNJ10 gene in three of any relevant family history and full overlapping fragments contact details for the referring clinician • Detection of known mutations: In relatives of patients with confirmed KCNJ10 mutations. Technical Mutation screening is carried out by direct fluorescent sequencing. Target reporting time 8 weeks for mutation screening of an index case. 2 weeks for routine testing of specific mutations (carrier testing). For urgent samples please contact the laboratory
Version 6
Inherited hearing loss panel
Contact details Introduction Molecular Genetics Service Inherited or familial hearing loss can present in isolated (non-syndromic) form or as syndromic Level 6, York House hearing loss, in combination with other specific phenotypic features. 37 Queen Square The genes included in this test contribute to non-syndromic hearing loss as well as those London, WC1N 3BH associated with the following syndromic forms: Usher Syndrome, Waardenburg Syndrome, T +44 (0) 20 7762 6888 Pendred Syndrome, Perrault Syndrome, Chudley-McCullough Syndrome, Wolfram Syndrome and Branchio-oto-renal Syndrome. F +44 (0) 20 7813 8578 The presentation for patients with these conditions is variable and in some cases may appear Samples required to be isolated hearing loss. Prognosis is therefore also variable. • 5ml venous blood in plastic EDTA Inheritance of familial hearing loss is gene dependent, and may show autosomal dominant bottles (>1ml from neonates) (inherited or de novo), autosomal recessive or X-linked inheritance. • Prenatal testing must be arranged The incidence of pre-lingual severe hearing loss at birth or during early childhood is in advance, through a Clinical approximately one per 1000 with a further 1/1000 children becoming deaf before adulthood. Genetics department if possible. • Amniotic fluid or CV samples Referrals should be sent to Cytogenetics for • Patients presenting with moderate, severe or profound hearing loss in isolation or in dissecting and culturing, with combination with syndromic features consistent with one of the syndromes listed above instructions to forward the sample to the Regional Molecular Genetics • Referrals must originate from either clinical genetics or audiology departments laboratory for analysis • • A completed DNA request card All referrals must be accompanied by a completed proforma should accompany all samples (https://www.labs.gosh.nhs.uk/media/532491/syndromic_non-syndromic_hl_panel_pre- test_proforma_2014v1.1.doc) Patient details Prenatal testing To facilitate accurate testing and Please contact the laboratory regarding prenatal testing if it is considered appropriate. reporting please provide patient demographic details (full name, date of Service offered birth, address and ethnic origin), details of any relevant family history and full Analysis of coding regions and intron/exon boundaries of 95 genes (see list below); variant contact details for the referring clinician confirmation and familial tests by Sanger sequencing. Please note that genes implicated in Usher Syndrome (indicated with an asterisk below) may be requested separately, or in addition to the other genes on this panel. They are not analysed without specific indication. ACTG1, ATP2B2, BDP1, CABP2, CCDC50, CEACAM16, CLDN14, COCH, COL11A2, COL4A6, CRYM, DFNA5, DFNB31, DFNB59, DIABLO, DIAPH1, DIAPH3, ESPN, ESRRB, EYA4, GIPC3, GJB2, GJB3, GJB6, GRHL2, GRXCR1, HGF, ILDR1, KARS, KCNQ4, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MIR96, MSRB3, MYH14, MYH9, MYO15A, MYO1A, MYO3A, MYO6, OTOA, OTOF, OTOG, PNPT1, POU3F4, POU4F3, PRPS1, PTPRQ, P2RX2, RDX, RPGR, SERPINB6, SLC17A8, SLC26A5, SLC4A11, SMPX, STRC, TECTA, TJP2, TMC1, TMIE, TMPRSS3, TPRN, TRIOBP, EYA1, SIX1, SIX5, KCNJ10, SLC26A4, GPSM2, LARS2, HSD17B4, HARS2, CLPP, PAX3, SOX10, MITF, SNAI2, KIT, EDNRB, EDN3, WFS1, CDH23*, CIB2*, CLRN1*, GPR98*, HARS*, MYO7A*, PCDH15*, PDZD7*, USH1C*, USH1G*, USH2A* Technical Mutation screening is carried out by next generation sequencing with library preparation using a SureSelect XT custom kit followed by sequencing on the Illumina MiSeq. Data is analysed using an in-house pipeline with all mutations confirmed by Sanger sequencing. Target reporting time 4 months for a full mutation screen in an index case (next generation sequencing). 2 weeks for familial mutation testing. Please contact the laboratory for urgent cases.
Version 6
Pendred Syndrome
Contact details Introduction Molecular Genetics Service Level 6, York House Pendred syndrome is an autosomal recessive form of hearing loss due to mutations in 37 Queen Square the SLC26A4 gene on chromosome 7q31 that presents with other features including London, WC1N 3BH goitre, enlarged vestibular aqueducts (EVA) and Mondini malformation. The estimated frequency of Pendred syndrome related hearing loss is 7%. T +44 (0) 20 7762 6888 F +44 (0) 20 7813 8578 Mutations in SLC26A4 disrupt ion exchange activity of the polypeptide pendrin. Pendrin is expressed in non-sensory epithelia of the inner ear and in thyroid Samples required folliculocytes. • 5ml venous blood in plastic EDTA SLC26A4 has 21 exons; mutations have been reported across the gene including a bottles (>1ml from neonates) small number that appear to be recurrent. p.Leu236Pro, p.Gly209Val, c.1001+1G>A, • Prenatal testing must be arranged p.Glu384Gly, p.Thr410Met and p.Thr416Pro have been reported amongst Western in advance, through a Clinical patients (Coyle et al , Hum Mol Genet 1998, 7:7 1105-1112). c.919-2A>G, Genetics department if possible. p.His723Arg, p.Ser90Leu and p.Leu676Gln have been reported to be recurrent in • Amniotic fluid or CV samples particular Asian populations (Park et al, J Med Genet, 2003; 40:242-248). should be sent to Cytogenetics for dissecting and culturing, with instructions to forward the sample to the Regional Molecular Genetics Referrals laboratory for analysis • A completed DNA request card • Patients with a clinical diagnosis / a strong likelihood of PDS should accompany all samples • Adult relatives of patients with SLC26A4 mutations for carrier status
Patient details To facilitate accurate testing and Prenatal testing reporting please provide patient demographic details (full name, date of Prenatal testing may be available for families in whom specific mutations have been birth, address and ethnic origin), details identified or in whom appropriate family studies have been undertaken- please of any relevant family history and full contact the laboratory to discuss. contact details for the referring clinician Service offered • Mutation Analysis: Direct sequencing of the 21 exons (including the non-coding exon 1). • Detection of known mutations: In relatives of patients with confirmed SLC26A4 mutations.
Technical Mutation screening is carried out by direct fluorescent sequencing.
Target reporting time 8 weeks for mutation screening of an index case. 2 weeks for routine testing of specific mutations (carrier testing). For urgent samples please contact the laboratory
Version 6
Waardenburg Syndrome types 1-4 (PAX3, MITF, SOX10 genes)
Contact details Introduction Molecular Genetics Service Waardenburg syndrome (WS) is an auditory-pigmentary disorder consisting of four Level 6, York House clinical subtypes with an annual incidence of 1/270 000 births. WS comprises 37 Queen Square approximately 3% of congenitally deaf children. WS1 (MIM #193500) and WS3 (MIM London, WC1N 3BH #148820) are defined by deafness, depigmentation features and dysmorphology. T +44 (0) 20 7762 6888 WS3 individuals also have musculoskeletal abnormalities of the upper limbs. F +44 (0) 20 7813 8578 The Paired Box Gene 3 (PAX3) on chromosome 2q35 is the only gene known to be associated with WS1 and WS3 with point mutations identified in more than 90% of Samples required affected individuals. No common mutations are known. Partial and total gene • 5ml venous blood in plastic EDTA deletions have also been described and may represent 10% of cases without bottles (>1ml from neonates) identified point mutations. • Prenatal testing must be arranged WS1 is autosomal dominant but may arise de novo and demonstrates variable in advance, through a Clinical expressivity. In WS3 homozygous and compound heterozygous (severe phenotype) Genetics department if possible. or heterozygous (moderate phenotype) mutations are seen. • Amniotic fluid or CV samples should be sent to Cytogenetics for Waardenburg syndrome Type 2 (WS2) and Type 4 (WS4) are genetically dissecting and culturing, with heterogeneous disorders. Partial and full gene deletions of MITF have been reported instructions to forward the sample in WS2 patients and in patients with Tietz syndrome, a severe WS2 phenotype to the Regional Molecular Genetics characterised by uniform hypopigmentation. SOX10 deletions have been reported in laboratory for analysis patients with WS2 and WS4. • A completed DNA request card should accompany all samples Referrals Patients with suspected WS1/WS3 for mutation screening and MLPA analysis of Patient details PAX3 To facilitate accurate testing and reporting please provide patient Adult relatives of patients with PAX3 mutations for carrier status. demographic details (full name, date of birth, address and ethnic origin), details Patients with suspected WS2, WS4 or Tietz syndrome can also be screened for of any relevant family history and full deletions in the MITF and SOX10 genes by MLPA. contact details for the referring clinician Service offered Mutation screening of PAX3 gene. Dosage analysis by MLPA (PAX3, MITF, SOX10). Detection of known mutations in relatives of patients with PAX3 mutations. Sequence analysis of the MITF and SOX10 genes is not available. Technical Direct Sanger sequencing analysis of PAX3 covering the 5’UTR and all ten exons present in the longest transcript. This analysis also covers the alternatively spliced 3’ end of the major isoform. Large scale deletions detected in PAX3, MITF and SOX10 by MLPA.
Target reporting time Routine mutation screen in index case takes 8 weeks. Routine testing of specific mutations takes 2 weeks. For urgent samples please contact the laboratory.
Version 6
X-linked deafness (POU3F4)
Contact details Introduction Molecular Genetics Service Hearing loss affects 1 to 3 in 1000 newborns with 50% of these cases because of Level 6, York House genetic causes. The majority of these are nonsyndromic (70%) and 1-2% are X- 37 Queen Square linked. The POU3F4 gene, DFN3 locus, (located at Xq21.1) encodes a transcription London, WC1N 3BH factor of the POU-domain family, comprised of two domains: POU-specific domain T +44 (0) 20 7762 6888 and a homeodomain. X-linked hearing loss can also exhibit as part of a syndrome; F +44 (0) 20 7813 8578 e.g. Norrie disease. Clinically DFN3 is characterised by congenital sensorineural or mixed hearing loss with an abnormally wide opening in the bone separating the basal turn of the cochlea and the inner auditory meatus, a fixation of the stapes with a Samples required perilymphatic gusher occurring during stapes surgery. The condition produces • 5ml venous blood in plastic EDTA characteristic radiological findings. There are no common mutations but micro and bottles (>1ml from neonates) large deletions 5’ of the POU3F4 gene account for ~50% of all mutations. • Prenatal testing must be arranged in advance, through a Clinical Referrals Genetics department if possible. • Patients with hearing loss for mutation screening of POU3F4. • Amniotic fluid or CV samples should be sent to Cytogenetics for • Adult relatives (females) of patients with POU3F4 mutations for carrier status. dissecting and culturing, with instructions to forward the sample Service offered to the Regional Molecular Genetics laboratory for analysis Mutation screening of the single POU3F4 exon by sequencing analysis in 4 • A completed DNA request card overlapping fragments. Normal or ‘failed’ PCR analyses will be screened for deletions should accompany all samples and insertions by MLPA. Detection of known mutations in relatives of patients with confirmed POU3F4 Patient details mutations. To facilitate accurate testing and reporting please provide patient Technical demographic details (full name, date of birth, address and ethnic origin), details Direct sequencing analysis of POU3F4 exon 1 in 4 fragments which covers the of any relevant family history and full 1087bp coding region (361 amino acids) and splice acceptor and donor sites. contact details for the referring clinician The MLPA kit covers the exon plus three regions of putative enhancer sites 5’ to the exon. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for routine testing of specific mutations. For urgent samples please contact the laboratory.
Version 6
Carbamoylphosphate synthetase 1 (CPS1) deficiency
Contact details Introduction Molecular Genetics Service Carbamoylphosphate synthetase 1 deficiency (MIM 237300) is a rare autosomal Level 6, York House recessive metabolic disorder. CPS1 deficiency affects the first enzymatic step in the 37 Queen Square urea cycle and results in hyperammonemia that can lead to lethargy, vomiting, coma London, WC1N 3BH and premature death. The clinical presentation is varied from neonatal onset, where T +44 (0) 20 7762 6888 patients have severe hyperammonemia which is fatal in the first few days of life, to a F +44 (0) 20 7813 8578 case reported where a woman in her third decade of life collapsed and died after a normal pregnancy and delivery. The onset of CPS1 deficiency may also be exacerbated by infection, metabolic stress or excessive protein intake. Samples required • 5ml venous blood in plastic EDTA The CPS1 gene (2q35) consists of 38 exons. bottles (>1ml from neonates) • Prenatal testing must be arranged Referrals in advance, through a Clinical Genetics department if possible. Prior to genetic analysis, clinically affected patients should wherever possible be • Amniotic fluid or CV samples confirmed as having CPS1 deficiency by enzyme analysis on a liver biopsy. Linkage should be sent to Cytogenetics for analysis may be requested in the affected proband of a family, please supply details dissecting and culturing, with of biochemical testing undertaken, clinical details and any relevant pedigree. instructions to forward the sample to the Regional Molecular Genetics Linkage analysis can be offered to the siblings for diagnostic testing and to adult laboratory for analysis relatives for carrier testing of CPS1 patients once an informative haplotype has been • A completed DNA request card identified. should accompany all samples
Patient details Prenatal testing To facilitate accurate testing and Prenatal testing is available for confirmed CPS1 families in whom linkage analysis has reporting please provide patient been shown to be informative - please contact the laboratory to discuss. demographic details (full name, date of birth, address and ethnic origin), details of any relevant family history and full contact details for the referring clinician Service offered Linkage analysis of the CPS1 gene region is undertaken in the affected patient and their parents.
Technical There are eight microsatellite markers available spanning the CPS1 region which may be useful for family studies, please contact the laboratory to discuss.
Target reporting time 8 weeks for linkage analysis in the index case and parents. Please contact the laboratory for urgent cases.
Version 6
Fabry disease (α-galactosidase A deficiency)
Contact details Introduction Molecular Genetics Service Fabry disease (MIM 301500) is an X-linked lysosomal storage disorder affecting Level 6, York House ~1/40,000 males. It is due to a deficiency of the lysosomal hydrolase, α-galactosidase 37 Queen Square A. Males with classical Fabry disease have no residual enzyme activity, whereas London, WC1N 3BH atypical patients, usually with symptoms confined to the heart (cardiac variant), have T +44 (0) 20 7762 6888 varying degrees of residual activity. These enzyme activity levels allow the clinical F +44 (0) 20 7813 8578 diagnosis to be confirmed. The symptoms of Fabry disease begin during childhood or teenage years and include angiokeratoma, acroparesthesia and ocular features. Cerebrovascular, cardiovascular and renal malfunction may develop later. Clinical Samples required manifestation in carrier females can range from being asymptomatic to being as • 5ml venous blood in plastic EDTA severely affected as affected males. Enzyme replacement therapy for Fabry disease bottles (>1ml from neonates) is now well established and in wide use. • Prenatal testing must be arranged in advance, through a Clinical The gene encoding α-galactosidase A (GLA) (Xq22.1) consists of 7 exons and family Genetics department if possible. specific mutations are found throughout the gene, although some recurrent mutations • Amniotic fluid or CV samples are reported and one mutation, p.(Asn215Ser), is commonly found in patients with the should be sent to Cytogenetics for cardiac variant. dissecting and culturing, with instructions to forward the sample Referrals to the Regional Molecular Genetics laboratory for analysis Clinically affected male patients should have their diagnosis confirmed by biochemical analysis; this should be arranged either locally or with the Enzyme Unit, Chemical • A completed DNA request card Pathology, Great Ormond Street Hospital (tel: 0207 4059200 ext 1785/6751). should accompany all samples Biochemically confirmed patients can be referred for mutation analysis. Patient details Clinically affected female patients can be referred directly for mutation analysis (due to To facilitate accurate testing and unreliability of heterozygote detection by biochemical testing). reporting please provide patient Mutation testing can be offered to relatives of affected patients once a disease demographic details (full name, date of birth, address and ethnic origin), details causing mutation has been identified. of any relevant family history and full contact details for the referring clinician Prenatal testing Prenatal testing is available, if required, for families where specific mutations have been identified - please contact the laboratory to discuss. Service offered Mutation screening of all 7 exons and intron-exon boundaries of the GLA gene is undertaken by Sanger sequence analysis in affected patients followed by MLPA dosage analysis to detect large deletions/duplications of the GLA gene. Mutation specific testing for previously identified mutations is also available in family members. Target reporting time 8 weeks for routine mutation screen (including MLPA) in index case. 2 weeks for family mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
Gaucher disease (β-glucocerebrosidase deficiency)
Contact details Introduction Molecular Genetics Service Gaucher disease (MIM 230800) is an autosomal recessive condition caused by a Level 6, York House deficiency of the lysosomal enzyme β-glucocerebrosidase (GBA) and the resultant 37 Queen Square accumulation of its undegraded substrate, glucosylceramide, in the lysosomes. London, WC1N 3BH Biochemical enzyme analysis confirms a clinical diagnosis in affected individuals. The T +44 (0) 20 7762 6888 disease can be broadly divided into three clinical forms on the basis of the absence F +44 (0) 20 7813 8578 (type 1) or presence (types 2 and 3) of primary CNS involvement although there is actually likely to be a clinical continuum. Type 2 is considered to be the most severe form and type 1, the least severe. All forms are characterised by hepatosplenomegaly Samples required and anaemia with bone involvement common in types 1 and 3. Treatment involves • 5ml venous blood in plastic EDTA bone marrow transplantation or enzyme replacement therapy. Type 1 is the most bottles (>1ml from neonates) prevalent form and is particularly common in the Ashkenazi Jewish population with an • Prenatal testing must be arranged incidence of ~1/855 individuals. Type 1 shows a broad spectrum of severity ranging in advance, through a Clinical from severely affected individuals to asymptomatic, presenting in childhood or Genetics department if possible. adulthood. Types 2 and 3 are rarer. • Amniotic fluid or CV samples should be sent to Cytogenetics for All three subtypes are caused by mutations in the GBA gene; the phenotypic dissecting and culturing, with heterogeneity correlates to some extent with the different nature of the mutations instructions to forward the sample identified. The GBA gene (1q21) comprises 12 exons. Although many novel mutations to the Regional Molecular Genetics are known, there are ‘common’ mutations within the gene, particularly in the laboratory for analysis Ashkenazi Jewish population. • A completed DNA request card should accompany all samples Referrals Clinically affected patients should have their diagnosis confirmed by biochemical Patient details analysis; this should be arranged locally, or with the Enzyme Unit, Chemical To facilitate accurate testing and Pathology, Great Ormond Street Hospital (tel: 0207 4059200 ext 1785/6751). reporting please provide patient Biochemically confirmed patients can be referred for mutation analysis. demographic details (full name, date of birth, address and ethnic origin), details Carrier testing can be offered to adult relatives of affected patients once a disease of any relevant family history and full causing mutation has been identified. contact details for the referring clinician Prenatal testing Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken - please contact the laboratory to discuss. Service offered Testing is offered for the following recurrent GBA mutations which account for ~86% mutations in the Ashkenazi population and 70% of mutations in the non-Jewish UK population. Analysis is carried out by PCR & restriction enzyme digest, ARMS PCR and nested PCR analysis: p.(Asn409Ser), p.(Leu483Pro), p.(Arg502Cys), p.(Asp448His), c.84dupG, c.(1263_1319)del55 and c.115+1G>A. Target reporting time 4 weeks for routine mutation screen. 2 weeks for carrier testing using mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
Glycogen storage disease type 1a (GSD1a)
Contact details Introduction Molecular Genetics Service Glycogen storage disease type 1a (GSD1a, MIM 232200), also known as Von Gierke Level 6, York House disease, is an autosomal recessive inborn error of glycogen metabolism, occurring in 37 Queen Square ~1/100,000 live births worldwide. The condition usually manifests during the first year London, WC1N 3BH of life with severe hypoglycemia, growth retardation, hepatomegaly, bleeding T +44 (0) 20 7762 6888 diathesis, lactic acidemia, hyperlipidemia and hyperuricemia. Long-term complications F +44 (0) 20 7813 8578 include gout, hepatic adenomas, osteoporosis and renal disease. GSD1a is caused by a deficiency of the enzyme glucose-6-phosphatase (G6Pase), Samples required which has an important role in glycogen metabolism and blood glucose homeostasis. • 5ml venous blood in plastic EDTA G6Pase is normally expressed in the liver, kidney and intestinal mucosa and absence bottles (>1ml from neonates) of G6Pase activity is associated with the excessive accumulation of glycogen in these • Prenatal testing must be arranged organs. A clinical diagnosis of GSD1a can be confirmed by enzyme analysis on a liver in advance, through a Clinical biopsy. The G6PC gene consists of 5 exons and family specific mutations are found Genetics department if possible. throughout the gene, however, ethnic specific mutations are recognised and • Amniotic fluid or CV samples information regarding ethnic origin is a useful indicator. In the North European should be sent to Cytogenetics for Caucasian population two mutations, namely p.(Gln347*) and p.(Arg83Cys) account dissecting and culturing, with for approximately 62% of all mutations. instructions to forward the sample to the Regional Molecular Genetics Referrals laboratory for analysis Clinically affected patients can have their diagnosis confirmed by biochemical • A completed DNA request card demonstration of a deficiency of G6Pase activity on liver biopsy. This should be should accompany all samples arranged locally, or with the Enzyme Unit, Chemical Pathology, Great Ormond Street Hospital (tel: 0207 4059200 ext 1785/6751). Patient details To facilitate accurate testing and Affected patients can be referred for mutation analysis. If the necessary patient reporting please provide patient samples are unavailable genetic testing can be undertaken in the parents of the demographic details (full name, date of affected child. Information regarding ethnic origin is useful. birth, address and ethnic origin), details of any relevant family history and full Carrier testing can be offered to the adult relatives of affected patients once a disease contact details for the referring clinician causing mutation has been identified. Prenatal testing Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken - please contact the laboratory to discuss. Service offered Sanger sequencing of the 5 exons and intron-exon boundaries is carried out. Mutation specific testing for previously identified family mutations is also available in family members by Sanger sequencing. Target reporting time 8 weeks for mutation screen in index case. 2 weeks for carrier testing using mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
Glycogen storage disease type 2 (GSD2) / Pompe disease
Contact details Introduction Molecular Genetics Service Glycogen storage disease type 2 (GSD2, MIM 232300) is an autosomal recessive Level 6, York House inborn error of glycogen metabolism caused by deficiency of acid α-glucosidase 37 Queen Square (GAA), which is required for the degradation of lysosomal glycogen. More commonly London, WC1N 3BH used names for this disorder include Pompe disease, acid maltase deficiency and T +44 (0) 20 7762 6888 glycogenosis type 2. GSD2 is characterised by lysosomal accumulation of glycogen in F +44 (0) 20 7813 8578 many body tissues as opposed to the exclusive cytoplasmic accumulation of glycogen that occurs in most other glycogen storage disorders. Samples required Clinical presentation varies from a rapidly fatal infantile disease to a slowly • 5ml venous blood in plastic EDTA progressive late-onset myopathy frequently associated with respiratory insufficiency. bottles (>1ml from neonates) Generally there is a correlation between the severity of the disorder and the amount of • Prenatal testing must be arranged residual GAA activity. Incidence varies by ethnicity; in the Caucasian population the in advance, through a Clinical frequency of infantile disease is between 1:100,000 and 1:200,000 and late-onset Genetics department if possible. disease possibly as high as 1:60,000. Enzyme replacement therapy is available which • Amniotic fluid or CV samples may slow or reverse symptoms of the disease. should be sent to Cytogenetics for The GAA gene consists of 20 exons (exon 1 is non-coding) and family specific dissecting and culturing, with instructions to forward the sample mutations are found throughout the gene. A mild splicing mutation in intron 1 (c.-32- to the Regional Molecular Genetics 13T>G) in combination with a more severe mutation is commonly associated with the laboratory for analysis late-onset phenotype in Caucasians. • A completed DNA request card Referrals should accompany all samples Clinically affected patients should have their diagnosis confirmed by enzyme analysis; Patient details this should be arranged locally, or with the Enzyme Unit, Chemical Pathology, Great To facilitate accurate testing and Ormond Street Hospital (tel: 0207 4059200 ext 1785/6751). Biochemically confirmed reporting please provide patient patients can be referred for mutation analysis. If the necessary patient samples are demographic details (full name, date of unavailable genetic testing can be undertaken in the parents of the affected child. birth, address and ethnic origin), details Information regarding ethnic origin is useful. of any relevant family history and full contact details for the referring clinician Carrier testing can be offered to adult relatives of affected patients once a disease causing mutation has been identified. Prenatal testing Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken - please contact the laboratory to discuss. Service offered Mutation screening of exons 2-20 by Sanger sequencing analysis. Mutation specific testing for previously identified family mutations is also available by Sanger sequencing. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for carrier testing using mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
Krabbe disease (Globoid Cell Leukodystrophy)
Contact details Introduction Molecular Genetics Service Krabbe disease (MIM #245200) is an autosomal recessive inborn error of metabolism Level 6, York House caused by deficiency of the enzyme galactosylceramidase (galactocerebrosidase). 37 Queen Square Galactosylceramidase (EC 3.2.1.46) is a lysosomal enzyme involved in the catabolism of galactosylceramide, a major lipid in myelin, kidney, and epithelial cells London, WC1N 3BH of the small intestine and colon. Enzyme deficiency results in the build-up of T +44 (0) 20 7762 6888 undigested fats affecting growth of the nerve’s protective myelin sheath and causes F +44 (0) 20 7813 8578 severe degeneration of mental and motor skills. The disease may be diagnosed by its characteristic grouping of certain cells (multinucleated globoid cells), nerve Samples required demyelination and degeneration, and destruction of brain cells. Special stains for myelin (e.g.; luxol fast blue) may be used to aid diagnosis. Definitive testing is by • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) direct enzyme analysis. Infants with Krabbe disease are normal at birth. Symptoms begin between the ages of • Prenatal testing must be arranged 3 and 6 months with irritability, inexplicable crying, fevers, limb stiffness, seizures, in advance, through a Clinical Genetics department if possible. feeding difficulties, vomiting, and slowing of mental and motor development. In infants, the disease is generally fatal before age 2. There are also juvenile- and adult- • Amniotic fluid or CV samples onset cases of Krabbe disease, which have similar symptoms but slower progression should be sent to Cytogenetics for dissecting and culturing, with and significantly longer lifespan. Although there is no cure for Krabbe disease, bone instructions to forward the sample marrow transplantation has been shown to benefit mild cases early in the course of to the Regional Molecular Genetics the disease. The incidence of Krabbe disease is around 1 in 100,000–200,000 births. laboratory for analysis The GALC gene is situated at 14q31 and consists of 17 exons. A recurrent 30kb • A completed DNA request card deletion has been described which extends from intron 10 to intron 17 of the GALC should accompany all samples gene and in the homozygous state is associated with infantile onset disease. The allele frequency of this deletion in Krabbe patients is reported to be approximately Patient details 50% in Dutch patients and 35% in non-Dutch European patients (Kleijer, WJ et al. (1997) J Inher Metab Dis 20:587-594). To facilitate accurate testing and reporting please provide patient Referrals demographic details (full name, date of birth, address and ethnic origin), details • Clinically affected patients should have their diagnosis confirmed by enzyme of any relevant family history and full analysis; this should be arranged locally or with the Enzyme Unit, Chemical contact details for the referring clinician Pathology, Great Ormond Street Hospital (tel: 0207 4059200 ext 1785/6751). Biochemically confirmed patients can be referred for mutation analysis. If the necessary patient samples are unavailable, genetic testing can be undertaken in the parents of the affected child. Information regarding ethnic origin is useful. • Carrier testing can be offered to adult relatives of affected patients once a disease causing mutation has been identified. Prenatal testing Prenatal testing is available for families in whom the 30kb deletion has been identified or in whom appropriate family studies have been undertaken - please contact the laboratory to discuss. Service offered Testing for the common 30kb deletion by three-primer PCR analysis. Other disease causing mutations are heterogeneous and testing is not currently offered as part of this diagnostic service. Target reporting time 2 weeks for routine deletion mutation test in the index case and family member carrier testing. Please contact the laboratory for urgent cases.
Version 6
Long chain acyl-CoA dehydrogenase (LCHAD) deficiency
Contact details Introduction Molecular Genetics Service Long chain acyl-CoA dehydrogenase (LCHAD) deficiency is an autosomal recessive Level 6, York House disorder of fatty acid metabolism (MIM 201460), caused by a deficiency of the long- 37 Queen Square chain hydroxyacyl-CoA dehydrogenase (HADHA) enzyme. Tandem mass London, WC1N 3BH spectrometry of organic acids in urine, and carnitines in blood spots, allows the T +44 (0) 20 7762 6888 diagnosis to be unequivocally determined. F +44 (0) 20 7813 8578 LCHAD deficiency is clinically heterogeneous but is often characterised by cardiomyopathy, skeletal myopathy, hypoglycemia, pigmentary retinopathy or sudden Samples required infant death. An additional clinical complication can occur in the pregnant mothers of • 5ml venous blood in plastic EDTA affected fetuses; they may experience maternal acute fatty liver of pregnancy (AFLP) bottles (>1ml from neonates) syndrome or hypertension/haemolysis, elevated liver enzymes and low platelets • Prenatal testing must be arranged (HELLP) syndrome. in advance, through a Clinical The gene encoding the HADHA enzyme is located at 2p23. The mutation, Genetics department if possible. c.1528G>C, causes the replacement of the amino acid glutamic acid with glutamine at • Amniotic fluid or CV samples codon 510 p.(Glu510Gln), this results in loss of LCHAD activity and accounts for should be sent to Cytogenetics for approximately 87% of mutant alleles. dissecting and culturing, with instructions to forward the sample Referrals to the Regional Molecular Genetics laboratory for analysis • Clinically affected patients should have their diagnosis confirmed by • A completed DNA request card biochemical analysis; this should be arranged either locally or with Chemical should accompany all samples Pathology, Great Ormond Street Hospital (tel: 0207 4059200 ext 5009). Affected patients can then be referred for mutation testing. If the necessary patient samples Patient details are unavailable, genetic testing can be undertaken in the parents of an affected child. To facilitate accurate testing and • Pregnant patients who have AFLP or HELLP can be referred for carrier reporting please provide patient testing, along with their partners. demographic details (full name, date of birth, address and ethnic origin), details • Carrier testing can be offered to the adult relatives of affected patients once a of any relevant family history and full disease causing mutation has been identified and partner testing is offered to contact details for the referring clinician confirmed carriers. Prenatal testing Prenatal testing, by genetic analysis, is available to couples that have both previously been shown to be carriers of the common mutation. Prenatal diagnosis is also offered by biochemistry regardless of mutation. Please contact the laboratory to discuss. Service offered Testing for the presence of the common c.1528G>C p.(Glu510Gln) mutation in the HADHA gene by Sanger sequencing. Screening of the remainder of the gene is not yet available. Target reporting time Routine analysis - 2 weeks. Please contact the laboratory for urgent cases.
Version 6
Medium chain acyl-CoA dehydrogenase (MCAD) deficiency
Contact details Introduction Molecular Genetics Service Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is an autosomal Level 6, York House recessive disorder of fatty acid metabolism (MIM 201450), caused by a deficiency of 37 Queen Square the MCAD enzyme. Tandem mass spectrometry of organic acids in urine, and London, WC1N 3BH carnitines in blood spots, allows the diagnosis to be unequivocally determined. T +44 (0) 20 7762 6888 MCAD deficiency has an incidence in the UK of approximately 1/10,000 live births. It F +44 (0) 20 7813 8578 is clinically heterogeneous but often presents as an episodic disease resembling Reye syndrome, with vomiting, lethargy and coma after metabolic stress, prolonged Samples required fasting or infection. Patients may also have cardiomyopathy and/or skeletal myopathy, • 5ml venous blood in plastic EDTA and some patients present as sudden infant death cases. Between episodes, patients bottles (>1ml from neonates) can appear normal and biochemical abnormalities can be absent. The gene encoding • Prenatal testing must be arranged the MCAD enzyme (ACADM) is located at 1p31 and the mutation c.985A>G causes in advance, through a Clinical the replacement of the amino acid lysine with glutamic acid at codon 329, Genetics department if possible. p.(Lys329Glu). This causes loss of MCAD activity and accounts for approximately • Amniotic fluid or CV samples 90% of mutant alleles in clinically affected North European patients, and 71% of should be sent to Cytogenetics for alleles in newborn screening. dissecting and culturing, with instructions to forward the sample Referrals to the Regional Molecular Genetics • laboratory for analysis Clinically affected patients should have their diagnosis confirmed by biochemical analysis; this should be arranged either locally or with Chemical Pathology, • A completed DNA request card Great Ormond Street Hospital (tel: 0207 4059200 ext 5009). Affected patients should accompany all samples can then be referred for mutation testing. If the necessary patient samples are unavailable, genetic testing can be undertaken in the parents of an affected Patient details child. To facilitate accurate testing and reporting please provide patient • Carrier testing may be offered to the adult relatives of affected patients once a demographic details (full name, date of disease causing mutation has been identified and partner testing for the birth, address and ethnic origin), details common c.985A>G mutation can be offered if appropriate. of any relevant family history and full contact details for the referring clinician Prenatal testing Prenatal testing, by genetic analysis, is available to couples that have both been shown to be carriers of disease-causing mutations. Prenatal diagnosis is also offered by biochemistry regardless of mutation. Please contact the laboratory to discuss. Service offered Level 1 mutation analysis - testing for the common c.985A>G mutation by Sanger sequence analysis. Level 2 mutation analysis - Sanger sequencing of the remaining exons of the ACADM gene. Target reporting time 2 weeks for routine level 1 screen in index case, 8 weeks for level 2 screen, 2 weeks for carrier testing using mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
Metachromatic Leukodystrophy & Pseudodeficiency of arylsulphatase A
Contact details Introduction Molecular Genetics Service Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disorder caused by deficiency of the enzyme arylsulphatase A which catalyses the first step in the Level 6, York House degradation of the sphingolipid 3-O-sulphagalactosyl ceramide (sulphatide). Accumulation of 37 Queen Square sulphatide in the brain leads to progressive demyelination of the central and peripheral nervous London, WC1N 3BH systems causing a variety of neurological symptoms including gait disturbances, ataxias, optical atrophy, dementia, seizures and spastic tetraparesis. Disease severity can range from mild to T +44 (0) 20 7762 6888 severe and can be broadly grouped into 3 subtypes (late-infantile, juvenile and adult). The F +44 (0) 20 7813 8578 majority of patients with arylsulphatase A deficiency and signs of MLD will have mutations in the ARSA gene however there is a much less common form of MLD caused by deficiency of saposin Samples required B, a non-enzymatic sphingolipid activator protein. Arylsulphatase A is also defective in multiple sulphatase deficiency due to mutations in SUMF1. The ARSA gene (22q13.31-qter) comprises 8 • 5ml venous blood in plastic EDTA exons. Although many novel mutations are known, there are ‘common’ mutations within the bottles (>1ml from neonates) gene, particularly the c.459+1G>A and c.1277C>T, p.(Pro426Leu) mutations, which account for • Prenatal testing must be arranged around 50% of disease alleles in the Northern European population. in advance, through a Clinical Genetics department if possible. Pseudodeficiency of arylsulphatase A (PDASA) • Amniotic fluid or CV samples Pseudodeficiency of arysulphatase A is a condition of reduced arylsulphatase A activity (<15% should be sent to Cytogenetics for normal) without clinical consequence, which can complicate the biochemical diagnosis of MLD. dissecting and culturing, with PDASA is caused by two sequence variants in the ARSA gene, namely PD2 (Poly A / c.*96A>G) instructions to forward the sample and PD1 (NGly / p.(Asn350Ser). PD2 is almost invariably seen on a background with PD1 but to the Regional Molecular Genetics PD1 can occur independently of PD2 and its effect on causing PDASA is controversial. laboratory for analysis Referrals • A completed DNA request card should accompany all samples PDASA testing is used to assist the interpretation of arylsulphatase A activity results. Referrals are generally via the Enzyme Unit, Great Ormond Street Hospital however Patient details referrals may be accepted from other centers who carry out biochemical testing for To facilitate accurate testing and arylsulphatase A. Biochemical confirmation of arylsulphatase A deficiency can only be reporting please provide patient confirmed after PDASA testing. In families with PDASA, prenatal testing by enzyme demographic details (full name, date of analysis can be complicated and in many cases impossible. For these families genetic birth, address and ethnic origin), details testing is particularly useful but this can also mean that in some cases testing for MLD of any relevant family history and full may have to be performed without biochemical confirmation. In these cases a very contact details for the referring clinician strong clinical picture of MLD must be present. Prenatal testing Prenatal testing is available for families in whom mutations have been identified or in whom appropriate family studies have been undertaken. Prenatal testing for PDASA may also be requested by the Enzyme Unit, Great Ormond Street Hospital. Service offered • PDASA: Testing for the presence of PD1 and PD2 by Sanger sequencing. • MLD Level 1 analysis: testing for the common mutations c.459+1G>A and c.1277C>T, p.(Pro426Leu) by Sanger sequencing. • MLD Level 2 analysis: Sanger sequencing of all 8 coding exons and intron-exon boundaries. Target reporting time 4 weeks for PDASA testing and MLD routine level 1 screen in index case, 8 weeks for level 2 screen. 2 weeks for routine testing of specific mutations. Please contact the laboratory for urgent cases.
Version 6
Mucopolysaccharidosis 1 (MPS1) (Hurler / Scheie syndrome)
Contact details Introduction Molecular Genetics Service MPS1 (MIM 252800) is an autosomal recessive lysosomal storage disorder, also Level 6, York House known as Hurler syndrome (severe) or Scheie syndrome (milder variant). The 37 Queen Square condition is caused by a deficiency of the enzyme alpha-L-iduronidase (IDUA), which is required for lysosomal degradation of the glycosaminoglycans, heparin sulphate London, WC1N 3BH and dermatan sulphate. Affected individuals have a characteristic pattern of urine T +44 (0) 20 7762 6888 metabolites and a deficiency in the IDUA enzyme activity; biochemical enzyme F +44 (0) 20 7813 8578 analysis utilises these features to confirm a clinical diagnosis. Hurler patients are usually diagnosed by the age of 2 years and characteristically Samples required have short stature, coarse facial features, developmental delay, heart defects and hepatosplenomegaly. Scheie patients can present at a later age, and have a milder • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) course of symptoms, including joint stiffness, corneal clouding and aortic valve disease. Other patients have an intermediate phenotype. The phenotypic • Prenatal testing must be arranged heterogeneity correlates to some extent with the different nature of the mutations in advance, through a Clinical Genetics department if possible. identified in the IDUA gene, although many novel mutations are known, there are ‘common’ mutations within the gene. • Amniotic fluid or CV samples The IDUA gene (4p16.3) has 14 exons and mutations have been found throughout should be sent to Cytogenetics for dissecting and culturing, with the gene. The recurrent mutations p.(Gln70*), p.(Ala327Pro) and p.(Trp402*) account instructions to forward the sample for approx. 70% of disease alleles in the Northern European population. The to the Regional Molecular Genetics p.(Trp402*) and p.(Gln70*) are the most common mutations seen in Hurler patients. laboratory for analysis p.(Arg89Gln) and c.590-7G>A are generally associated with Scheie syndrome. • A completed DNA request card should accompany all samples Referrals • Clinically affected patients should have their diagnosis confirmed by biochemical Patient details analysis; this should be arranged either locally or with the Enzyme Unit, Great To facilitate accurate testing and Ormond Street Hospital (tel: 0207 4059200 ext 1785/6751). Biochemically reporting please provide patient confirmed patients can be referred for mutation analysis. If the necessary patient demographic details (full name, date of samples are unavailable genetic testing can be undertaken in the parents of a birth, address and ethnic origin), details child. of any relevant family history and full • Carrier testing can be offered to the adult relatives of affected patients once a contact details for the referring clinician disease causing mutation has been identified. Prenatal testing Prenatal testing is available for families in whom mutations have been identified or in whom appropriate family studies have been undertaken. This service is also offered by biochemical analysis. Please contact the laboratory to discuss. Service offered • Level 1 mutation analysis: detection of the ‘common’ mutations p.(Gln70*), p.(Ala327Pro) and p.(Trp402*) by Sanger sequence analysis. • Level 2 mutation analysis: Sanger sequencing of all 14 coding exons and intron- exon boundaries. • Detection of known mutations in relatives of patients with confirmed MPS1 mutations by Sanger sequencing. Target reporting time 4 weeks for routine level 1 screen in index case, 8 weeks for level 2 screen. 2 weeks for carrier testing using mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
Mucopolysaccharidosis type 2 (MPS2) (Hunter syndrome)
Contact details Introduction Molecular Genetics Service Hunter syndrome (MIM 309900) is an X-linked recessive lysosomal storage disorder. Level 6, York House The condition is caused by a deficiency of the enzyme iduronate-2-sulphatase (IDS), 37 Queen Square which is required for the lysosomal degradation of the glycosaminoglycans, heparan London, WC1N 3BH sulphate and dermatan sulphate. Affected males have a characteristic pattern of T +44 (0) 20 7762 6888 urine metabolites and a deficiency of the IDS enzyme; biochemical enzyme analysis F +44 (0) 20 7813 8578 utilises these features to confirm a clinical diagnosis. Hunter syndrome is clinically heterogeneous, but the predominant clinical features Samples required include coarse facial features, stiff joints, hepatosplenomegaly, cardiovascular and • 5ml venous blood in plastic EDTA respiratory disorders, developmental delay and mental retardation. The IDS gene bottles (>1ml from neonates) consists of 9 exons and family specific mutations are found throughout the gene. • Prenatal testing must be arranged Homologous recombination between the IDS gene and the unexpressed IDS in advance, through a Clinical pseudogene, located 20kb telomeric of IDS, leads to inversions and deletions, a Genetics department if possible. common inversion accounts for ~10% of Hunter cases. • Amniotic fluid or CV samples Referrals should be sent to Cytogenetics for dissecting and culturing, with • Clinically affected patients should have their diagnosis confirmed by biochemical instructions to forward the sample analysis; this should be arranged either locally or with the Enzyme Unit, Great to the Regional Molecular Genetics laboratory for analysis Ormond Street Hospital (tel: 0207 4059200 ext 1785/6751). Such patients may then be referred for mutation analysis. If the necessary patient samples are • A completed DNA request card unavailable genetic testing can be undertaken in the mother of the affected child. should accompany all samples • Carrier testing can be offered to female relatives of affected patients once a Patient details disease causing mutation has been identified. To facilitate accurate testing and Prenatal testing reporting please provide patient demographic details (full name, date of Prenatal testing is available for families in whom specific mutations have been birth, address and ethnic origin), details identified. This service is also offered by biochemistry. Please contact the laboratory of any relevant family history and full contact details for the referring clinician to discuss. Service offered All confirmed Hunter patients (or their mothers if no sample is available from the affected male) are first tested for the presence of the common inversion, which has been shown to occur in ~10% of Hunter patients. Mutation screening is then undertaken in the remainder of the gene including MLPA analysis to detect large deletions and duplications. Mutation specific testing for previously identified mutations is also available in family members. Target reporting time 2 weeks for inversion test. 8 weeks for routine mutation screen and MLPA in index case. 2 weeks for carrier testing using mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
Mucopolysaccharidosis type 3 (MPS3) (Sanfilippo syndrome)
Contact details Introduction Molecular Genetics Service Mucopolysaccharidosis type 3 (MPS3 / Sanfilippo syndrome MIM #252900) is an Level 6, York House autosomal recessive lysosomal storage disorder caused by impaired degradation of 37 Queen Square heparan sulphate (found in the urine of affected patients). The syndrome is London, WC1N 3BH characterised by severe central nervous system degeneration, but only mild somatic T +44 (0) 20 7762 6888 disease (moderately severe claw hand and visceromegaly, little or no corneal F +44 (0) 20 7813 8578 clouding or skeletal change). Onset of clinical features usually occurs between 2 and 6 years; severe neurologic degeneration occurs in most patients between 6 and 10 years of age leading to a vegetative state, and death occurs typically during the Samples required second or third decade of life. Type A is reported to be the most severe of the 4 • 5ml venous blood in plastic EDTA subtypes of Sanfilippo syndrome with earlier onset and rapid progression of bottles (>1ml from neonates) symptoms and shorter survival (typically during the teens). • Prenatal testing must be arranged in advance, through a Clinical Affected patients have a characteristic pattern of urine metabolites and a deficiency in Genetics department if possible. one of the enzymes involved in heparan sulphate degradation; biochemical enzyme • Amniotic fluid or CV samples analysis utilises these features to confirm a clinical diagnosis. should be sent to Cytogenetics for Testing is currently available for types A and B which are due to deficiencies of the dissecting and culturing, with enzymes N-sulfoglucosamine sulfohydrolase (SGSH) and alpha-N- instructions to forward the sample to the Regional Molecular Genetics acetylglucosaminidase (NAGLU), respectively. There have been several recurrent laboratory for analysis mutations identified in both the SGSH and NAGLU genes although these are generally population specific. • A completed DNA request card should accompany all samples Referrals
Patient details • Clinically affected patients should have their diagnosis confirmed by biochemical To facilitate accurate testing and analysis (including disease subtype e.g. A); this should be arranged either locally reporting please provide patient or with the Enzyme Unit, Great Ormond Street Hospital (tel: 0207 4059200 ext demographic details (full name, date of 1785/6751). Such patients may then be referred for mutation analysis. If the birth, address and ethnic origin), details necessary patient samples are unavailable genetic testing can be undertaken in of any relevant family history and full the parents of the affected child. contact details for the referring clinician • Carrier testing can be offered to the adult relatives of affected patients once a disease causing mutation has been identified. Prenatal testing Prenatal testing is available for families in whom mutations have been identified or in whom appropriate family studies have been undertaken. This service is also offered by biochemical analysis. Please contact the laboratory to discuss. Service offered
• Mutation screening: Sanger sequencing of all coding exons and intron-exon boundaries. • Detection of known mutations in relatives of patients with confirmed MPS3A or MPS3B mutations by Sanger sequencing. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for carrier testing using mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
Neuronal Ceroid-Lipofuscinosis type 1 (NCL1) Infantile neuronal ceroid-lipofuscinosis (INCL)
Contact details Introduction Molecular Genetics Service Neuronal ceroid-lipofuscinosis type 1 (NCL1 / Batten disease) (MIM #256730) is a Level 6, York House rare autosomal recessive neurodegenerative disorder caused by mutations in the 37 Queen Square PPT1 gene, which encodes the enzyme palmitoyl–protein thioesterase-1 (PPT1; MIM 600722). NCL1 is one of at least eight genetically distinct diseases associated with London, WC1N 3BH the NCL disease spectrum. Onset is typically infantile (INCL) however juvenile and T +44 (0) 20 7762 6888 adult onset cases have also been described. The differential diagnosis of NCL1 from F +44 (0) 20 7813 8578 other NCL types is based on age of onset, clinical phenotype, ultra structural characterisation of the storage material and PPT1 enzyme activity. NCL1 is Samples required characterised by the accumulation of auto fluorescent lipopigment in granular osmiophilic deposits (GROD) in neurons and other cell types using electron • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) microscopy and loss of palmitoyl protein thioesterase-1 (PPT1) enzyme activity in leucocytes and fibroblasts. Typical clinical features of INCL are retarded head growth • Prenatal testing must be arranged from about 5 months, hyper excitability (including sleep problems), muscular in advance, through a Clinical Genetics department if possible. hypotonia and reduced development of fine motor skills between 10-18 months of age. INCL usually progresses with visual loss (by 18 months - 2 years), loss of motor • Amniotic fluid or CV samples skills, and premature death between 8-13 years. should be sent to Cytogenetics for dissecting and culturing, with The PPT1 gene (1p32) consists of 9 exons and mutations have been found instructions to forward the sample throughout the gene. The four most common PPT1 mutations are p.(Arg122Trp) to the Regional Molecular Genetics (Finnish-specific), p.(Arg151*), p.(Thr75Pro) and p.(Leu10*). The p.(Arg151*) and laboratory for analysis p.(Leu10*) mutations may account for up to 75% of mutations in certain populations. • A completed DNA request card should accompany all samples Referrals • Clinically affected patients should, wherever possible, have their diagnosis Patient details confirmed by analysis of PPT1 enzyme activity in leucocytes and fibroblasts. This To facilitate accurate testing and should be arranged locally or with the Enzyme Unit at Great Ormond Street reporting please provide patient Hospital; (tel: 0207 4059200 ext 1785/6751). Such patients may then be referred demographic details (full name, date of for mutation analysis. If the necessary patient samples are unavailable genetic birth, address and ethnic origin), details testing can be undertaken in the parents of a child. of any relevant family history and full contact details for the referring clinician • Carrier testing can be offered to the adult relatives of NCL1 patients once a disease causing mutation has been identified. Prenatal testing Prenatal testing is available for families in whom the diagnosis of NCL1 has been confirmed by the identification of a mutation or in whom appropriate family studies have been undertaken - please contact the laboratory to discuss. Service offered
• Level 1 mutation analysis: detection of recurrent mutations p.(Arg151*) and p.(Leu10*) by Sanger sequencing analysis. • Level 2 mutation analysis: Sanger sequencing of all 9 coding exons and intron- exon boundaries. • Detection of known mutations in relatives of patients with confirmed NCL1 mutations by Sanger sequencing. Target reporting time 4 weeks for routine level 1 screen in index case, 8 weeks for level 2 screen. 2 weeks for carrier testing using mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
Neuronal Ceroid-Lipofuscinosis type 2 (NCL2) Late infantile neuronal ceroid-lipofuscinosis (LINCL)
Contact details Introduction Molecular Genetics Service Neuronal ceroid-lipofuscinosis type 2 (NCL2 / Batten disease) (MIM #204500) is a Level 6, York House rare autosomal recessive neurodegenerative disorder caused by mutations in the 37 Queen Square TPP1 gene which encodes the lysosomal enzyme tripeptidyl peptidase 1. NCL2 is one of at least eight genetically distinct diseases associated with the NCL disease London, WC1N 3BH spectrum. NCL2 is generally referred to as late-infantile NCL (LINCL) due to typical T +44 (0) 20 7762 6888 onset of symptoms between the ages of 2 and 4 years. Variant forms of LINCL F +44 (0) 20 7813 8578 (vLINCL) have been reported to be caused by mutations in the CLN1, CLN5, CLN6, CLN7 and CLN8 genes. Clinical features of LINCL are normal development until the Samples required onset of seizures, ataxia and myoclonus between 2 and 4 yrs. LINCL usually progresses with visual loss (by 5-6 yrs), chair bound by 4-6 yrs with poor prognosis. • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) The differential diagnosis of NCL2 from the other NCL types is based on age of onset, clinical phenotype, ultra structural characterisation of the storage material and TPP1 • Prenatal testing must be arranged enzyme levels. A clinical diagnosis of NCL2 is confirmed biochemically by loss of in advance, through a Clinical Genetics department if possible. tripeptidylpeptidase I (TPP1) enzyme activity in leucocytes and fibroblasts or accumulation of auto fluorescent lipopigment with a curvilinear profile in neurons and • Amniotic fluid or CV samples other cell types. should be sent to Cytogenetics for dissecting and culturing, with TPP1 (11p15) consists of 13 exons. The two most common mutations are c.509- instructions to forward the sample 1G>C (~33% of LINCL chromosomes) and p.(Arg208*) (~26% of LINCL to the Regional Molecular Genetics chromosomes). Other disease causing mutations are family specific and found laboratory for analysis throughout the gene. • A completed DNA request card should accompany all samples Referrals • Clinically affected patients should, wherever possible, have their diagnosis Patient details confirmed by analysis of TPP1 enzyme activity in leucocytes and fibroblasts. This To facilitate accurate testing and should be arranged locally or with the Enzyme Unit, Great Ormond Street reporting please provide patient Hospital; (tel: 0207 4059200 ext 1785/6751). Biochemically confirmed patients demographic details (full name, date of can be referred for mutation analysis. If the necessary patient samples are birth, address and ethnic origin), details unavailable genetic testing can be undertaken in the parents of an affected child. of any relevant family history and full contact details for the referring clinician • Carrier testing can be offered to the adult relatives of NCL2 patients once a disease causing mutation has been identified. Prenatal testing Prenatal testing is available for families in whom the diagnosis of NCL2 has been confirmed by the identification of a mutation or in whom appropriate family studies have been undertaken - please contact the laboratory to discuss. Service offered
• Level 1 mutation analysis: detection of the recurrent mutations c.509-1G>C and p.(Arg208*) by Sanger sequencing analysis. • Level 2 mutation analysis: Sanger sequencing of all 13 coding exons and intron- exon boundaries. • Detection of known mutations in relatives of patients with confirmed TPP1 mutations by direct sequencing. Target reporting time 2 weeks for routine level 1 screen in index case, 8 weeks for level 2 screen. 2 weeks for carrier testing using mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
Neuronal Ceroid-Lipofuscinosis type 3 (NCL3) Juvenile neuronal ceroid-lipofuscinosis (JNCL)
Contact details Introduction Molecular Genetics Service Neuronal ceroid-lipofuscinosis type 3 (NCL3 / Batten disease) (MIM #204200) is a Level 6, York House rare autosomal recessive neurodegenerative disorder caused by mutations in the 37 Queen Square CLN3 gene. NCL3 is one of at least eight genetically distinct diseases associated with London, WC1N 3BH the NCL disease spectrum. NCL3 is generally referred to as juvenile NCL (JNCL) due T +44 (0) 20 7762 6888 typical onset of symptoms between the ages of 4 and 7 years. A rare variant form of F +44 (0) 20 7813 8578 JNCL (vJNCL) has been associated with mutations in the CLN1 gene (usually associated with the infantile form of the disease). NCL3 is typically characterised by normal development until the onset of visual failure due to retinal degeneration Samples required between 4 and 7 yrs. Progression of visual loss is usually rapid. Other clinical features • 5ml venous blood in plastic EDTA include seizures and psychomotor deterioration; prognosis is poor. The differential bottles (>1ml from neonates) diagnosis of NCL3 from the other NCL types is based on age of onset, clinical • Prenatal testing must be arranged phenotype and ultra-structural characterisation of the storage material. NCL3 is in advance, through a Clinical characterised by the accumulation of auto fluorescent lipopigment with a fingerprint Genetics department if possible. profile in neurons and other cell types and the presence of vacuolated lymphocytes in • Amniotic fluid or CV samples blood smears. should be sent to Cytogenetics for dissecting and culturing, with The CLN3 gene (16p12) consists of 15 exons spanning 15kb of genomic DNA. A instructions to forward the sample 1.02kb deletion (introns 6-8) is reported to account for ~69% of JNCL alleles (~85% in to the Regional Molecular Genetics Finnish population). Other disease causing mutations are family specific and found laboratory for analysis throughout the gene. • A completed DNA request card should accompany all samples Referrals • Clinical and histopathological review of the affected patient is recommended to Patient details indicate a diagnosis of NCL3. Please supply details of histopathological testing To facilitate accurate testing and undertaken, clinical details and any relevant pedigree. If the necessary patient reporting please provide patient samples are unavailable genetic testing can be undertaken in the parents of an demographic details (full name, date of affected child. birth, address and ethnic origin), details of any relevant family history and full • Carrier testing can be offered to the adult relatives of NCL3 patients once a contact details for the referring clinician disease causing mutation has been identified. Prenatal testing Prenatal testing is available for families in which the diagnosis of NCL3 has been confirmed by mutation analysis. Please contact the laboratory to discuss. Service offered Level 1: Testing for the common 1.02kb deletion. Level 2: Screening of the CLN3 gene by Sanger sequence analysis. Target reporting time 2 weeks for routine level 1 screen in index case, 8 weeks for level 2 screen. 2 weeks for carrier testing using mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
Variant Neuronal Ceroid-Lipofuscinosis (CLN5, 6, 7 & 8) Adult Neuronal Ceroid-Lipofuscinosis (CLN6)
Contact details Introduction Molecular Genetics Service Variant neuronal ceroid-lipofuscinosis (also generally referred to as variant late- Level 6, York House infantile Batten disease) is a rare autosomal recessive neurodegenerative disorder 37 Queen Square which can be caused by mutations in one of several genes including CLN5, CLN6, London, WC1N 3BH MFSD8 (CLN7) and CLN8. The CLN6 gene is also associated with an adult form of T +44 (0) 20 7762 6888 NCL known as Kufs disease. The neuronal ceroid-lipofuscinoses are a group of at F +44 (0) 20 7813 8578 least eight genetically distinct diseases associated with a similar phenotype but variable age of onset. Samples required Variant late-infantile NCL (vLINCL) is so called due to the similarity of clinical • 5ml venous blood in plastic EDTA presentation and age of onset to the classic late-infantile form of NCL. The differential bottles (>1ml from neonates) diagnosis of variant NCL from other NCL types is based on age of onset, clinical • Prenatal testing must be arranged phenotype and ultra-structural characterisation of the storage material. Characteristic in advance, through a Clinical accumulation of auto fluorescent lipopigment with mixed fingerprint/curvilinear/ Genetics department if possible. rectilinear profiles is seen in neurons and other cell types and there is an absence of • Amniotic fluid or CV samples vacuolated lymphocytes on a blood smear (differentiating this type of NCL from should be sent to Cytogenetics for NCL3). Adult NCL (ANCL) has a similar presentation to the other NCLs but without dissecting and culturing, with visual loss and with a later onset. instructions to forward the sample to the Regional Molecular Genetics The CLN5 gene (13q21.1-q32) consists of 4 exons, CLN6 (15q21-q23) consists of 7 laboratory for analysis exons, MFSD8 (CLN7) (4q28.2) consists of 13 exons, and CLN8 (8pter-p22) consists of 3 exons. Mutations are generally family specific and found throughout each gene. • A completed DNA request card should accompany all samples Referrals
Patient details • Clinical and histopathological review of the affected patient is recommended to To facilitate accurate testing and indicate a diagnosis of variant NCL. Please supply details of histopathological reporting please provide patient testing undertaken, clinical details and any relevant pedigree. If the necessary demographic details (full name, date of patient samples are unavailable genetic testing can be undertaken in the parents of birth, address and ethnic origin), details an affected child. of any relevant family history and full contact details for the referring clinician • Carrier testing can be offered to the adult relatives of variant NCL patients once a disease causing mutation has been identified. Prenatal testing Prenatal testing is available for families in whom the diagnosis of variant NCL has been confirmed by the identification of a mutation or in whom appropriate family studies have been undertaken - please contact the laboratory to discuss. Service offered Sanger sequencing analysis of all coding exons and intron-exon boundaries of the CLN5, CLN6, MFSD8 (CLN7) and CLN8 genes. Genes will be sequenced at the same time; however, a particular gene can be tested on request. Adult-onset cases will be screened for mutations in the CLN6 gene. Target reporting time 8 weeks for routine mutation screening. 2 weeks for routine testing of specific mutations. Please contact the laboratory for urgent cases.
Version 6
Ornithine transcarbamylase (OTC) deficiency
Contact details Introduction Molecular Genetics Service Ornithine transcarbamylase (OTC) deficiency (MIM 311250) is a rare X-linked Level 6, York House recessive disorder. Females also frequently manifest the condition, presumably due to 37 Queen Square non-random X chromosome inactivation in their liver cells. Deficiency of OTC causes London, WC1N 3BH a defect in the urea cycle and results in hyperammonemia, leading to lethargy, T +44 (0) 20 7762 6888 vomiting, coma and premature death. The clinical presentation is variable. In males, F +44 (0) 20 7813 8578 there are generally accepted to be two forms of OTC deficiency - a neonatal form, where patients have severe hyperammonemia which is fatal in the first few days of life and a late onset form which occurs at any point after this initial neonatal period and Samples required can be exacerbated by infection, metabolic stress or excessive protein intake. Female • 5ml venous blood in plastic EDTA carriers can also experience this full range of clinical symptoms, varying from bottles (>1ml from neonates) apparently unaffected to neonatal death. • Prenatal testing must be arranged in advance, through a Clinical The OTC gene (Xp21.1) consists of 10 exons and family specific mutations are found Genetics department if possible. throughout the gene, although some recurrent mutations at CpG sites in exons 1, 3, 5 • Amniotic fluid or CV samples and 9 are reported and some late onset specific mutations are known. A whole gene should be sent to Cytogenetics for deletion accounts for approximately 10% of OTC cases. dissecting and culturing, with instructions to forward the sample Referrals to the Regional Molecular Genetics • laboratory for analysis Prior to genetic analysis, clinically affected patients should, wherever possible, be confirmed as having OTC deficiency by enzyme analysis on a liver biopsy or by • A completed DNA request card finding elevated orotic acid levels by biochemical analysis. Allopurinol or protein should accompany all samples load tests can be used to indicate female carrier status, but these are not always conclusive. Mutation analysis can be requested in the affected proband of a Patient details family, please supply details of biochemical testing undertaken, clinical details and To facilitate accurate testing and any relevant pedigree. reporting please provide patient demographic details (full name, date of • If there is no sample available from an affected individual testing can be birth, address and ethnic origin), details undertaken in the mother of an affected child (however, it should be noted that, of any relevant family history and full unless there are additional affected family members, they are not necessarily contact details for the referring clinician mutation carriers). • Carrier testing can be offered to the female relatives of OTC patients once a disease causing mutation has been identified. Prenatal testing Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken - please contact the laboratory to discuss. Service offered Entire gene deletions account for ~10% of neonatal OTC cases and are tested for by MLPA analysis in both males and females. This is then followed by mutation screening of the gene by Sanger sequence analysis of the 10 exons and intron-exon boundaries. This testing strategy detects approximately 84% of mutations in patients with enzymatically confirmed OTC deficiency. Mutation specific testing for previously identified mutations is also available in family members by Sanger sequencing or MLPA. Target reporting time 8 weeks for mutation screen including MLPA in index case. 2 weeks for carrier testing using mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
Osteopetrosis
Contact details Introduction Molecular Genetics Service Autosomal recessive malignant osteopetrosis (MIM 259700) is a rare congenital Level 6, York House disorder of bone resorption affecting 1/200,000 individuals. The condition is caused by 37 Queen Square failure of osteoclasts to resorb immature bone. This results in abnormal bone marrow London, WC1N 3BH cavity formation and bone marrow failure. T +44 (0) 20 7762 6888 Clinical features of osteopetrosis include fractures (especially of the long bones), F +44 (0) 20 7813 8578 visual impairment, nerve compression resulting in headaches, blindness and deafness, haematological difficulties, unusual dentition, frequent infections, failure to Samples required thrive, and growth retardation. It is diagnosed immediately/shortly after birth and death • 5ml venous blood in plastic EDTA can occur by 2 years due to severe anaemia, bleeding and /or infection. bottles (>1ml from neonates) Osteopetrosis is generally diagnosed through skeletal X-rays. Bones appear • Prenatal testing must be arranged unusually dense on X-rays with a chalky white appearance. Bone density tests and in advance, through a Clinical bone biopsies can also confirm a diagnosis. At present, bone marrow transplantation Genetics department if possible. is the only treatment that has been proven to significantly alter the course of the • Amniotic fluid or CV samples disease. should be sent to Cytogenetics for dissecting and culturing, with The TCIRG1 gene located at 11q13, consists of 20 exons and encodes an a3 subunit instructions to forward the sample of the vacuolar pump, which mediates acidification of bone/osteoclast interface. to the Regional Molecular Genetics Mutations of this gene have been found in ~50% of autosomal recessive laboratory for analysis osteopetrosis patients. • A completed DNA request card should accompany all samples Referrals • Clinically affected patients should if possible have their diagnosis confirmed by X- Patient details ray analysis, bone density tests and bone biopsies. To facilitate accurate testing and reporting please provide patient • Carrier testing can be offered to adult relatives of affected patients once a disease demographic details (full name, date of causing mutation has been identified. birth, address and ethnic origin), details of any relevant family history and full Prenatal testing contact details for the referring clinician Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken- please contact the laboratory to discuss. Service offered Mutation screening by Sanger sequencing is offered for exons 2 to 20 of the TCIRG1 gene in affected individuals. If no sample is available from the affected individual testing can be undertaken in their mother and father. Mutation specific testing for previously identified mutations is also available in family members by Sanger sequencing. Target reporting time 8 weeks for mutation screen in index case. 2 weeks for carrier testing using mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
Pyridoxine-Dependent Epilepsy (Antiquitin – ALDH7A1)
Contact details Introduction Molecular Genetics Service Pyridoxine-dependent epilepsy (PDE MIM 266100) is an autosomal recessive disease Level 6, York House caused by mutations in the alpha-aminoadipic semialdehyde dehydrogenase 37 Queen Square (ALDH7A1 – antiquitin) gene located on chromosome 5q31. Alpha-aminoadipic semialdehyde (AASA) dehydrogenase is involved in the cerebral lysine degradation London, WC1N 3BH pathway. Deficiency of the enzyme leads to the accumulation of AASA and T +44 (0) 20 7762 6888 piperideine-6-carboxylate (P6C), elevated levels of which can be detected in urine, F +44 (0) 20 7813 8578 plasma, and CSF of affected patients. Patients usually present with neonatal seizures that are unresponsive to conventional anti-convulsant therapy, but which can be Samples required controlled by treatment with pyridoxine (vitamin B6). Less commonly, later-onset cases present during the second and third years of life. Patients also display varying • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) degrees of developmental delay, which may, in part, be independent of the timing of pyridoxine therapy. In affected patients, P6C inactivates pyridoxalphosphate (PLP), • Prenatal testing must be arranged the active vitamer of pyridoxine, leading to severe PLP deficiency. As PLP is a in advance, through a Clinical Genetics department if possible. cofactor of various enzymes in the CNS, seizures in PDE are most probably due to perturbation in the metabolism of cerebral amino acids and neurotransmitters. • Amniotic fluid or CV samples Pyridoxine is a precursor of PLP and it can therefore reduce the effect of PLP should be sent to Cytogenetics for dissecting and culturing, with deficiency in PDE patients instructions to forward the sample The ALDH7A1 gene consists of 18 exons and mutations have been found throughout to the Regional Molecular Genetics the gene. Two mutations, c.834G>A, p.(=) and c.1279G>C, p.(Glu427Gln), account laboratory for analysis for approximately 5% and 33%, respectively, of mutant alleles in the Caucasian • A completed DNA request card population. should accompany all samples Referrals • Clinically affected patients, if possible, should have their diagnosis confirmed by Patient details urinary analysis of alpha-aminoadipic semialdehyde (AASA); this should be To facilitate accurate testing and arranged either locally or with Dr Philippa Mills, Biochemistry Department, UCL reporting please provide patient Institute of Child Health (tel: 0207 242 9789 (ext 2108). Biochemically confirmed demographic details (full name, date of birth, address and ethnic origin), details patients can be referred for mutation analysis. If the necessary patient samples of any relevant family history and full are unavailable genetic testing can be undertaken in the parents of the affected contact details for the referring clinician child. Information regarding ethnic origin is useful. • Carrier testing can be offered to adult relatives of affected patients once a disease causing mutation has been identified. Prenatal testing Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken - please contact the laboratory to discuss. Service offered • Level 1 mutation analysis: detection of recurrent mutations c.834G>A and c.1279G>C by Sanger sequencing analysis. • Level 2 mutation analysis: Sanger sequencing of all remaining coding exons and intron-exon boundaries. • Mutation specific testing for previously identified family mutations is also available by Sanger sequencing. Target reporting time 4 weeks for routine level 1 screen in index case, 8 weeks for level 2 screen. 2 weeks for carrier testing using mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
Schindler Disease
Contact details Introduction Molecular Genetics Service Schindler disease (MIM 609241) is a rare autosomal recessive lysosomal storage Level 6, York House disease, which is caused by a deficiency of the enzyme, alpha-N- 37 Queen Square acetylgalactosaminidase (NAGA). NAGA is a lysosomal glycohydrolase that cleaves London, WC1N 3BH alpha-N-acetylgalactosaminidase moieties from glycoconjugates inside lysosomes. T +44 (0) 20 7762 6888 Schindler disease is clinically heterogeneous with 3 main phenotypes; type 1 is an F +44 (0) 20 7813 8578 infantile-onset neuroaxonal dystrophy; type 2, also known as Kanzaki disease (MIM 609242), is an adult onset disorder characterised by angiokeratoma corporis diffusum and mild intellectual impairment; and type 3 is an intermediate disorder with mild to Samples required moderate neurological manifestations. • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) Affected patients have an abnormal urinary oligosaccharide and glycopeptide profile • Prenatal testing must be arranged and the diagnosis is confirmed by a deficiency of the NAGA enzyme in plasma, in advance, through a Clinical leucocytes, or fibroblasts. Genetics department if possible. The NAGA gene is located on chromosome 22q13.2 and consists of 9 exons, and • Amniotic fluid or CV samples family specific mutations are found throughout the gene. To date, 14 patients from ten should be sent to Cytogenetics for families are known and ethnic specific mutations are recognised, information dissecting and culturing, with regarding ethnic origin is therefore a useful indicator. instructions to forward the sample to the Regional Molecular Genetics Referrals laboratory for analysis • Clinically affected patients should have their diagnosis confirmed by biochemical • A completed DNA request card analysis; this should be arranged either locally or with the Enzyme Unit, Great should accompany all samples Ormond Street Hospital; (tel: 0207 4059200 ext 1785/6751). Such patients may then be referred for mutation analysis. If the necessary patient samples are Patient details unavailable genetic testing can be undertaken in the parents of the affected child. To facilitate accurate testing and • Carrier testing can be offered to the adult relatives of affected patients once a reporting please provide patient disease causing mutation has been identified. demographic details (full name, date of birth, address and ethnic origin), details Prenatal testing of any relevant family history and full contact details for the referring clinician Prenatal testing is available for families in whom mutations have been identified or in whom appropriate family studies have been undertaken – please contact the laboratory to discuss. Service offered Mutation screening of exons 1 to 9 by Sanger sequencing analysis. Mutation specific testing for previously identified family mutations is also available by Sanger sequencing. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for carrier testing using mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
CFHR5 Nephropathy
Contact details Introduction Molecular Genetics Service Affected individuals display persistent microscopic haematuria with episodes of Level 6, York House macroscopic haematuria associated with intercurrent infections (commonly of the 37 Queen Square respiratory tract). Renal biopsy demonstrates C3 glomerulonephritis (C3GN, a type of London, WC1N 3BH mesangiocapillary glomerulonephritis (MCGN)). 80% men and 20% affected women th T +44 (0) 20 7762 6888 develop renal failure by the 8 decade of life. The incidence of CFHR5 nephropathy in F +44 (0) 20 7813 8578 the Cypriot population is estimated at 1/1000 to 1/8000; prevalence in the UK Caucasian population is low (<1:100,000) since C3GN is a very rare diagnosis. This autosomal dominant condition is caused by a mutation in the Complement Factor H- Samples required Related gene 5 (CFHR5; MIM: *608593). The gene is homologous to Complement • 5ml venous blood in plastic EDTA Factor H and Complement Factor-H Related genes 1-4 which lie at neighbouring loci. bottles (>1ml from neonates) CFHR5 consists of 10 exons which code for 9 homologous short consensus repeat • A completed DNA request card domains, each of which has two internal disulphide bridges. The protein product of should accompany all samples CFHR5 has the ability to co-localise with (and regulate activation of) complement C3 in the kidney. Patient details The most common CFHR5 gene mutation is a duplication of exons 2 and 3 (c.59- To facilitate accurate testing and 1808_430+3242dup) described in the Cypriot population. Only one other mutation has reporting please provide patient been published, a frameshift in exon 4 identified in one non-Cypriot C3GN patient. demographic details (full name, date of birth, address and ethnic origin), details Referrals of any relevant family history and full contact details for the referring clinician Referrals are accepted from Consultant Clinical Geneticists and Consultant Nephrologists in the following patients: Cypriot origin with unexplained haematuria or renal disease. Patients of any ethnicity may be referred if C3GN or MCGN is present. At risk family members where the familial mutation is known. Service offered Detection of exon 2-3 duplication: A single PCR reaction incorporates primers that amplify both a 298bp fragment of the wild type sequence and a 222bp fragment unique to the duplication. Detection of other mutations: PCR amplification of all 10 exons of CFHR5 followed by Sanger sequence analysis. Target reporting time 2 weeks for duplication analysis. 8 weeks for full sequencing analysis of CFHR5. Please contact the laboratory for urgent cases.
Version 6
Cystinosis
Contact details Introduction Molecular Genetics Service Cystinosis (MIM 219800, 219900 and 219750) is a rare autosomal recessive disorder Level 6, York House affecting 1/175,000 individuals. The condition is caused by the failure to transport the 37 Queen Square amino acid cysteine out of the lysosomes. Cystine (a dimer of two cysteine London, WC1N 3BH molecules) accumulates forming crystals which causes cell and tissue destruction in T +44 (0) 20 7762 6888 all systems of the body. Excess cystine can be detected by cystine binding protein F +44 (0) 20 7813 8578 assays which can be used to confirm a clinical diagnosis. Three forms of cystinosis have been defined by age of onset and severity of symptoms. The most common form is infantile nephropathic cystinosis (95% of cases) that has an age of onset of 6- Samples required 12 months. Features include renal proximal tubular dysfunction (renal Fanconi • 5ml venous blood in plastic EDTA syndrome), without treatment affected children suffer worsening growth retardation bottles (>1ml from neonates) and develop end stage renal failure by ~10 years. The juvenile form of cystinosis • Prenatal testing must be arranged occurs in around 4-5% of affected individuals. Age of onset is between 12-15 years in advance, through a Clinical and individuals usually present with proteinuria and glomerular renal impairment, but Genetics department if possible. do not suffer from such profound tubular dysfunction or growth retardation. The • Amniotic fluid or CV samples benign form of cystinosis occurs in adulthood; individuals do not suffer from any renal should be sent to Cytogenetics for disease and grow normally. They require no treatment and have a normal life dissecting and culturing, with expectancy and quality, except perhaps for photophobia due to cystine crystals in the instructions to forward the sample cornea. The CTNS gene consists of 12 exons. The most common mutation is a 57kb to the Regional Molecular Genetics deletion which accounts for approximately 60% of alleles in North Europeans, and laboratory for analysis makes up around one third of all mutations found in individuals with cystinosis. The • A completed DNA request card rest of the mutations reported are spread throughout the coding area of the gene and should accompany all samples include insertions, small deletions, nonsense, splicing and missense mutations. No mutation hotspots have been identified. Patient details To facilitate accurate testing and Referrals reporting please provide patient • demographic details (full name, date of Clinically affected patients should have their diagnosis confirmed by biochemical birth, address and ethnic origin), details analysis; this should be arranged either locally or with the Enzyme Unit, Great of any relevant family history and full Ormond Street Hospital; (tel: 0207 4059200 ext 1785/6751). Biochemically contact details for the referring clinician confirmed patients can be referred for mutation analysis. • Carrier testing can be offered to adult relatives of affected patients once a disease causing mutation has been identified. Prenatal testing Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken - please contact the laboratory to discuss. Service offered • Level 1 mutation analysis: testing for the common 57kb deletion by PCR analysis. • Level 2 mutation analysis: Sanger sequencing of exons 3 to 12 of the CTNS gene (exons 1 & 2 are non-coding). • Detection of known mutations in relatives of patients with confirmed CTNS mutations by Sanger sequencing or deletion PCR analysis. Target reporting time 2 weeks for routine level 1 screen in index case, 8 weeks for level 2 screen. 2 weeks for routine testing of specific mutations. Please contact the laboratory for urgent cases.
Version 6
Juvenile nephronophthisis (NPH1)
Contact details Introduction Molecular Genetics Service Familial juvenile nephronophthisis (NPH1, MIM 256100) is an autosomal recessive Level 6, York House condition accounting for 2-10% of childhood chronic renal failure. It is caused by 37 Queen Square mutations in the NPHP1 gene on chromosome 2. Approximately 80% of familial, and London, WC1N 3BH 65% of sporadic nephronophthisis patients with purely renal symptoms have been T +44 (0) 20 7762 6888 shown to be homozygous for a 250kb deletion of chromosome 2q13, including almost F +44 (0) 20 7813 8578 the entire NPHP1 gene. A subset of individuals affected with a mild form of Joubert syndrome have also been Samples required reported to have this homozygous deletion (Parisi MA et al. Am J Hum Genet. 2004 • 5ml venous blood in plastic EDTA Jul;75(1):82-91). bottles (>1ml from neonates) • Prenatal testing must be arranged in advance, through a Clinical Referrals Genetics department if possible. • Amniotic fluid or CV samples We offer testing for confirmation of diagnosis in affected probands. should be sent to Cytogenetics for Carrier testing is NOT available. dissecting and culturing, with instructions to forward the sample to the Regional Molecular Genetics laboratory for analysis Prenatal testing • A completed DNA request card Prenatal testing may be available for families following analysis of the affected should accompany all samples proband - please contact the laboratory to discuss.
Patient details To facilitate accurate testing and Service offered reporting please provide patient demographic details (full name, date of Detection of the homozygous 250kb deletion in affected probands only. birth, address and ethnic origin), details of any relevant family history and full contact details for the referring clinician Target reporting time 2 weeks for routine analysis of the NPHP1 deletion. Please contact the laboratory for urgent cases.
Version 6
Steroid-Resistant Nephrotic Syndrome (NPHS2)
Contact details Introduction Molecular Genetics Service Nephrotic syndrome is a condition that is caused by any of a group of diseases that Level 6, York House damage the kidneys' filtering system, the glomeruli. The structure of the glomeruli 37 Queen Square prevents most protein from getting filtered through into the urine. Nephrotic syndrome London, WC1N 3BH is characterised by proteinuria (abnormally high loss of protein in the urine), T +44 (0) 20 7762 6888 albuminemia and hypercholesterolemia. Ultimately, there is rapid progression to end- F +44 (0) 20 7813 8578 stage renal disease where the kidneys are irreversibly damaged, resulting in death if untreated. Samples required Treatment is based on relieving symptoms, preventing complications and delaying • 5ml venous blood in plastic EDTA progressive kidney damage. Patients with nephrotic syndrome are typically treated bottles (>1ml from neonates) with steroids, of which about 80% have a good response; the rest are considered to • Prenatal testing must be arranged be steroid-resistant and may require renal transplant. One of the main features in in advance, through a Clinical steroid-resistant nephrotic syndrome (SRNS; MIM 600995) is focal segmental Genetics department if possible. glomerulosclerosis (FSGS). • Amniotic fluid or CV samples Mutations in the podocin gene, NPHS2, are associated with autosomal recessive should be sent to Cytogenetics for steroid-resistant nephrotic syndrome (SRNS), including focal segmental dissecting and culturing, with glomerulosclerosis (FSGS). Around half of familial forms and 10-30% of sporadic instructions to forward the sample to the Regional Molecular Genetics forms of SRNS are found to have NPHS2 mutations in both alleles. The gene is laboratory for analysis located on chromosome 1q25-31 and consists of 8 exons. • A completed DNA request card Referrals should accompany all samples Affected patients should fulfill the following criteria: Patient details Presence of nephrotic syndrome (serum albumin < 25g/l and urine albumin > 4 To facilitate accurate testing and mg/m2/h or urine albumin/creatinine ratio > 100 mg/mmol ), that is either: reporting please provide patient demographic details (full name, date of • resistant to treatment with steroids, or birth, address and ethnic origin), details of any relevant family history and full • present in the first 3 months of life, or contact details for the referring clinician • have a histological picture of FSGS on biopsy. Please also send a completed clinical information sheet (available on our laboratory website). Carrier testing can be offered to adult relatives of affected patients once a disease causing mutation has been identified. Prenatal testing Prenatal testing may be available for families following analysis of the affected proband - please contact the laboratory to discuss. Service offered Mutation screening of the 8 exons and exon/intron boundaries of the NPHS2 gene by Sanger sequencing. Target reporting time 8 weeks for routine screen in index case. 2 weeks for carrier testing using mutation specific tests. Please contact the laboratory for urgent cases.
Version 6
Autoimmune lymphoproliferative syndrome (ALPS)
Contact details Introduction Molecular Genetics Service ALPS is a rare immunodeficiency disorder associated with inherited mutations in the Level 6, York House FAS gene encoding Fas (also known as Apo-I or CD95) receptor protein (ALPS type 37 Queen Square IA); others have mutations in the FASLG gene encoding Fas ligand (ALPS type IB), or London, WC1N 3BH in the CASP10 and CASP8 genes encoding caspase 10 and 8 protease (ALPS type T +44 (0) 20 7762 6888 IIa and IIb, respectively). F +44 (0) 20 7813 8578 Most patients have ALPS type IA (MIM 134637) due mainly to dominant-negative highly penetrant mutations in the Fas death domain encoded by exon 9 of the FAS Samples required gene. ALPS is characterised by splenomegaly, defective lymphocyte apoptosis, • 5ml venous blood in plastic EDTA lymphadenopathy, hypergammaglobulinaemia (IgG and IgA), autoimmunity and bottles (>1ml from neonates) accumulation of a polyclonal population of T cells called double-negative CD4-CD8- T • Prenatal testing must be arranged cells. Affected individuals can be diagnosed on the basis of the presence of these in advance, through a Clinical double-negative α/β T cells. Genetics department if possible. Referrals • Amniotic fluid or CV samples • should be sent to Cytogenetics for Affected patients should be referred to the Molecular Immunology laboratory at dissecting and culturing, with GOSH for T cell analysis. This requires prior arrangement and completion of instructions to forward the sample specific request forms (contact Dr Kimberly Gilmour - see details below). We to the Regional Molecular Genetics liaise closely with this department and will undertake mutation screening in laboratory for analysis appropriate patients. • A completed DNA request card • Carrier testing can be offered to relatives of ALPS patients once a disease should accompany all samples causing mutation has been identified. Prenatal testing Patient details To facilitate accurate testing and Prenatal testing is available for families in which a mutation has been identified - reporting please provide patient please contact the laboratory to discuss. demographic details (full name, date of birth, address and ethnic origin), details Service offered of any relevant family history and full contact details for the referring clinician Mutation screening of the 9 exons of the FAS gene in affected individuals found to have double-negative α/β T cells and based on their clinical details. Cases found to have normal numbers of double-negative α/β T cells may have further investigations such as functional apoptosis assays (contact Prof. Adrian Thrasher - see details below). If DNA from an affected individual is unavailable the parents can be screened for mutations where appropriate. Mutation-specific tests for family members are also available. Technical Mutation screening is undertaken by sequence analysis of exons 1 to 9 of the FAS gene. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for carrier testing for known family mutations. For urgent samples please contact the laboratory. To arrange double-negative α/β T cell analysis, or functional apoptosis studies, please contact: Dr. Kimberly Gilmour, Molecular Immunology, Great Ormond Street Hospital tel.: +44 (0) 20 7829 8835 email: [email protected]
Version 6
Cartilage hair hypoplasia (CHH)
Contact details Introduction Molecular Genetics Service Mutations in the untranslated RMRP gene on chromosome 9p13-p12 (encoding the Level 6, York House RNA component of RNase MRP endoribonuclease) lead to a wide spectrum of 37 Queen Square autosomal recessive skeletal dysplasias, ranging from the milder phenotypes London, WC1N 3BH metaphyseal dysplasia without hypotrichosis (MDWH) and cartilage hair hypoplasia T +44 (0) 20 7762 6888 (CHH) to the severe anauxetic dysplasia (AD). This clinical spectrum includes F +44 (0) 20 7813 8578 different degrees of short stature, hair hypoplasia, defective erythrogenesis, and immunodeficiency. Samples required Mutations in RMRP are found in both the transcribed region and the promoter region • 5ml venous blood in plastic EDTA (from the TATA box to the transcription initiation site). A founder mutation, 70A>G, is bottles (>1ml from neonates) present in 92% of Finnish and 48% of non-Finnish patients with CHH (Thiel et al • Prenatal testing must be arranged (2007) Am. J. Hum. Genet 81: 519-529). in advance, through a Clinical Nomenclature: Please note, although HGVS recommendations for the description of Genetics department if possible. DNA sequence variants (den Dunnen JT and Antonarakis SE (2000). Hum.Mutat. • Amniotic fluid or CV samples 15: 7-12) state that numbering should start with 1 at the first nucleotide of the should be sent to Cytogenetics for database reference file (genomic Reference Sequence M29916.1), it is common dissecting and culturing, with practice in the literature for RMRP numbering to start with 1 at the transcription start instructions to forward the sample to the Regional Molecular Genetics site. laboratory for analysis Referrals • A completed DNA request card • Patients with suspected RMRP-related disorder for mutation screening of RMRP. should accompany all samples • Adult relatives of patients with RMRP mutations for carrier status. • Testing is available for minor siblings to establish carrier status prior to bone Patient details marrow/stem cell donation. To facilitate accurate testing and reporting please provide patient Prenatal testing demographic details (full name, date of birth, address and ethnic origin), details Prenatal testing is available for families in whom specific mutations have been of any relevant family history and full identified or in whom appropriate family studies have been undertaken- please contact details for the referring clinician contact the laboratory to discuss.
Service offered Mutation screening of the RMRP gene. Detection of known mutations in relatives of patients with confirmed RMRP mutations. Technical Direct sequencing analysis of the RMRP gene - promoter region and the transcribed region. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for routine testing of specific mutations. For urgent samples please contact the laboratory.
Version 6
Familial hemophagocytic lymphohistiocytosis (FHL)
Contact details Introduction Molecular Genetics Service Familial hemophagocytic lymphohistiocystosis (FHL) due to perforin gene defects Level 6, York House (MIM 603553) is a rare autosomal recessive immunodeficiency characterised by 37 Queen Square defective or absent T and natural killer (NK) cell cytotoxicity. Affected individuals can London, WC1N 3BH be diagnosed on the basis of very low or absent perforin protein. The perforin gene, T +44 (0) 20 7762 6888 PRF1 has 3 exons of which exons 2 and 3 are coding. Mutations are found F +44 (0) 20 7813 8578 throughout the gene with some evidence of founder mutations. Only 20-40% of FHL cases are due to defects in the perforin gene. FHL disease-causing mutations have been identified in several other genes: UNC13D (FHL3), STX11 (FHL4), and STXBP2 Samples required (FHL5). • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) Referrals • Prenatal testing must be arranged • Affected patients should be referred to the Molecular Immunology laboratory at in advance, through a Clinical GOSH for perforin protein analysis. This requires prior arrangement and Genetics department if possible. completion of specific request forms (see contact information below). We work • Amniotic fluid or CV samples closely with this department and will undertake mutation screening in should be sent to Cytogenetics for appropriate patients. dissecting and culturing, with • Carrier testing can be offered to the relatives of FHL/PRF1 patients once a instructions to forward the sample disease causing mutation has been identified. However, due to the rarity of the to the Regional Molecular Genetics disorder partner screening is not offered unless there is consanguinity or a laboratory for analysis family history of FHL in the partner. • A completed DNA request card should accompany all samples Prenatal testing
Patient details Prenatal testing is available for families in which a mutation has been identified - please contact the laboratory to discuss. To facilitate accurate testing and reporting please provide patient Service offered demographic details (full name, date of birth, address and ethnic origin), details Mutation screening of the PRF1 gene in affected individuals found to have absent or of any relevant family history and full abnormal perforin expression. For cases where there is strong clinical indication of contact details for the referring clinician FHL but where evaluation of perforin protein is either not possible or where expression is normal, mutation testing may still be undertaken but will be considered on a case by case basis. If DNA from the affected individual is unavailable and there is a strong clinical indication of FHL, then screening can be undertaken in the parents. Mutation-specific tests for family mutations are also available. Technical Mutation screening is undertaken by sequence analysis of the PRF1 gene. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for carrier testing using mutation-specific tests. For urgent samples please contact the laboratory.
To arrange perforin expression studies please contact Dr Kimberly Gilmour in Molecular Immunology, GOSH - Tel: +44 (0) 20 7829 8835, Email: [email protected]
Version 6
IL7R-alpha severe combined immunodeficiency (IL7RαSCID)
Contact details Introduction Molecular Genetics Service Interleukin 7 receptor alpha severe combined immunodeficiency (IL7Rα-SCID, MIM Level 6, York House 608971) is a rare autosomal recessive immunodeficiency characterised by a lack of 37 Queen Square circulating T cells, but normal B and natural killer cells (T-B+NK+). Affected individuals London, WC1N 3BH can be diagnosed on the basis of an abnormality or deficiency of the IL7Rα protein. T +44 (0) 20 7762 6888 The IL7Rα gene has 8 exons and family specific mutations have been found F +44 (0) 20 7813 8578 throughout the gene. Referrals Samples required • Affected patients should be referred to the Molecular Immunology laboratory at • 5ml venous blood in plastic EDTA GOSH for IL7Rα protein analysis. This requires prior arrangement and bottles (>1ml from neonates) completion of specific request forms (see contact information below). We work • Prenatal testing must be arranged closely with this department and will undertake mutation screening in in advance, through a Clinical appropriate patients. Genetics department if possible. • Carrier testing can be offered to the relatives of IL7Rα-SCID patients once a • Amniotic fluid or CV samples disease causing mutation has been identified. However, due to the rarity of the should be sent to Cytogenetics for disorder, partner screening is not offered unless there is consanguinity or a dissecting and culturing, with family history of IL7Rα-SCID in the partner. instructions to forward the sample to the Regional Molecular Genetics Prenatal testing laboratory for analysis • A completed DNA request card Prenatal testing is available for families in whom specific mutations have been should accompany all samples identified or in whom appropriate family studies have been undertaken - please contact the laboratory to discuss. Patient details Service offered To facilitate accurate testing and reporting please provide patient Mutation screening of the IL7Rα gene in affected individuals found to have absent or demographic details (full name, date of abnormal IL7Rα expression. For cases where there is a strong clinical indication of birth, address and ethnic origin), details IL7Rα-SCID but where evaluation of IL7Rα protein is either not possible or where of any relevant family history and full expression is normal, mutation testing may still be undertaken but will be considered contact details for the referring clinician on a case-by-case basis. If DNA from an affected individual is unavailable then screening can be undertaken in the parents. Mutation-specific tests for family members are also available. Technical Mutation screening is undertaken by sequence analysis of the IL7Rα gene. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for carrier testing using mutation-specific tests. For urgent samples please contact the laboratory. To arrange IL7Rα expression studies please contact Dr Kimberly Gilmour in Molecular Immunology, GOSH – Tel: +44 (0) 20 7829 8835, Email: [email protected]
Version 6
JAK3-deficient severe combined immunodeficiency (JAK3-SCID)
Contact details Introduction Molecular Genetics Service JAK3-SCID (MIM 600802) is an autosomal recessive immunodeficiency characterised Level 6, York House by a lack of circulating T cells. Affected individuals can be diagnosed on the basis of 37 Queen Square an abnormality or deficiency of the Janus 3 kinase protein (JAK3). The JAK3 gene London, WC1N 3BH has 24 exons (23 coding) and family specific mutations are found throughout the T +44 (0) 20 7762 6888 gene. F +44 (0) 20 7813 8578 Referrals Samples required • Affected patients should be referred to the Molecular Immunology laboratory at • 5ml venous blood in plastic EDTA GOSH for JAK3 protein analysis. This requires prior arrangement and bottles (>1ml from neonates) completion of specific request forms (see contact information below). We work closely with this department and will undertake mutation screening in • Prenatal testing must be arranged in advance, through a Clinical appropriate patients. Genetics department if possible. • Carrier testing can be offered to the relatives of JAK3 patients once a disease • Amniotic fluid or CV samples causing mutation has been identified, however due to the rarity of the disorder should be sent to Cytogenetics for partner screening is not offered unless there is consanguinity or a family dissecting and culturing, with history of JAK3-SCID in the partner. instructions to forward the sample
to the Regional Molecular Genetics laboratory for analysis Prenatal testing • A completed DNA request card should accompany all samples Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken - please Patient details contact the laboratory to discuss. To facilitate accurate testing and Service offered reporting please provide patient demographic details (full name, date of Mutation screening of the JAK3 gene in affected individuals found to have absent or birth, address and ethnic origin), details abnormal JAK3 expression. Cases found to have JAK3 expression may be screened of any relevant family history and full if there is a strong clinical indication for a diagnosis of JAK3-SCID. If DNA from the contact details for the referring clinician affected individual is unavailable screening can be undertaken in the parents. Mutation-specific tests for family members are available. Technical Mutation screening is undertaken by sequence analysis of the JAK3 gene. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for carrier testing using mutation-specific tests. For urgent samples please contact the laboratory. To arrange JAK3 expression studies please contact Dr Kimberly Gilmour in Molecular Immunology, GOSH Tel: +44 (0) 20 7829 8835 Email: [email protected]
Version 6
Lymphoproliferative syndrome 1 (ITK deficiency)
Contact details Introduction Molecular Genetics Service EBV-associated autosomal lymphoproliferative syndrome (ITK deficiency) presents Level 6, York House with a similar but not identical phenotype to X-linked lymphoproliferative disease 37 Queen Square (XLP) characterised by lymphoproliferation and severe immune dysregulation London, WC1N 3BH following Epstein-Barr virus (EBV infection) and is inherited in an autosomal recessive T +44 (0) 20 7762 6888 manner. As in XLP, not all 3 classic clinical manifestations (fulminant infectious F +44 (0) 20 7813 8578 mononucleosis, lymphoproliferative disorder and dysgammaglobulinaemia) may develop and patients can develop different immune mediated syndromes including fatal haemophagocytosis, hypogammaglobulinaemia and autoimmune phenomena Samples required presenting as renal disease. While in XLP the predominant lymphoproliferation is • 5ml venous blood in plastic EDTA Burkitt’s lymphoma, 4 out of 5 published patients with ITK deficiency had Hodgkin’s bottles (>1ml from neonates) disease. Infection with EBV is a universal component of the phenotype. • Prenatal testing must be arranged in advance, through a Clinical Referrals Genetics department if possible. • Affected patients should be referred to the Molecular Immunology laboratory at • Amniotic fluid or CV samples GOSH for ITK protein analysis. This requires prior arrangement and should be sent to Cytogenetics for completion of specific request forms (see contact information below). We work dissecting and culturing, with closely with this department and will undertake mutation screening in instructions to forward the sample appropriate patients. to the Regional Molecular Genetics • Carrier testing can be offered to the female relatives of ITK patients once a laboratory for analysis disease causing mutation has been identified. • A completed DNA request card should accompany all samples Prenatal testing
Patient details Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken. Please To facilitate accurate testing and contact the laboratory to discuss. reporting please provide patient demographic details (full name, date of Service offered birth, address and ethnic origin), details of any relevant family history and full Mutation screening of the ITK gene in affected individuals found to have absent or contact details for the referring clinician abnormal ITK expression. Cases found to have ITK expression will be screened due to lack of sensitivity data for the protein test. Technical Mutation screening is undertaken by sequence analysis of the 17 exons and exon/intron boundaries. MLPA analysis for deletion/duplication testing. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for carrier testing using mutation-specific tests. For urgent samples please contact the laboratory.
To arrange ITK expression studies please contact Dr Kimberly Gilmour, Molecular Immunology, GOSH Tel: +44 (0) 20 7829 8835 Email: [email protected]
Version 6
Netherton syndrome (NS)
Contact details Introduction Molecular Genetics Service Netherton syndrome (Comèl-Netherton syndrome) (NS) (MIM 256500) is an Level 6, York House autosomal recessive multisystemic disorder characterised by localised or generalised 37 Queen Square congenital ichthyosis, hair shaft abnormalities (trichorrhexis invaginata), atopic London, WC1N 3BH diathesis, immune deficiency and markedly elevated IgE levels. The condition T +44 (0) 20 7762 6888 predominantly affects females. Some infants with Netherton syndrome develop F +44 (0) 20 7813 8578 progressive hypernatremic dehydration, failure to thrive, and enteropathy. These complications can be fatal. NS is caused by mutations in the SPINK5 gene on chromosome 5q32, encoding the serine protease inhibitor LEKTI. Samples required • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) Referrals • Prenatal testing must be arranged • Confirmation of diagnosis in individuals clinically suspected of having in advance, through a Clinical Netherton syndrome. Genetics department if possible. • Carrier testing can be offered to relatives of affected patients once the disease • Amniotic fluid or CV samples causing mutations in the family have been identified. should be sent to Cytogenetics for dissecting and culturing, with instructions to forward the sample Prenatal testing to the Regional Molecular Genetics laboratory for analysis Prenatal testing is available for couples in whom specific mutations have been • A completed DNA request card identified - please contact the laboratory to discuss. should accompany all samples Service offered Patient details To facilitate accurate testing and 1) Bi-directional sequence analysis of all 34 coding exons and intron-exon boundaries reporting please provide patient in SPINK5. demographic details (full name, date of birth, address and ethnic origin), details 2) The c.2468dup, p.LysGlufs*4 mutation can be detected by fluorescent fragment of any relevant family history and full analysis of the (A)10 homopolymer tract in exon 26 of the SPINK5 gene. Two contact details for the referring clinician alternative forward primers are used to allow accurate sizing of the repeat region.
Target reporting time 8 weeks for routine sequence analysis of the SPINK5 gene and fragment analysis for the c.2468dup, p.LysGlufs*4 mutation. 2 week turnaround time for testing of a known familial mutation. Please contact the laboratory if urgent or prenatal testing is required.
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Primary immunodeficiency (PID)
Contact details Introduction Molecular Genetics Service The primary immunodeficiencies (PID) are a heterogeneous group of >150 disorders Level 6, York House that result from defects in immune system development and/or function. PIDs are 37 Queen Square broadly classified as disorders of adaptive immunity (i.e., T-cell, B-cell or combined immunodeficiencies) or of innate immunity (e.g., phagocyte and complement London, WC1N 3BH disorders). T +44 (0) 20 7762 6888 F +44 (0) 20 7813 8578 The clinical presentation of PIDs is highly variable; however, most disorders involve increased susceptibility to infection. The type and pattern of infection depends on which Samples required part(s) of the host defences are missing or defective since some defences are more important against some pathogens than others. Defects in phagocyte function or • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) humoral immunity (B cell deficiency) result in infections with common and unusual bacteria. Defects in T cell immunity usually present with recurrent viral, fungal, or • Prenatal testing must be arranged protozoal infections. Very serious inherited immunodeficiencies become apparent in advance, through a Clinical Genetics department if possible. shortly after birth or in the first year of life, and can lead to death if untreated. Others (usually the milder forms) may not present until people reach their twenties and thirties. • Amniotic fluid or CV samples should be sent to Cytogenetics for dissecting and culturing, with The minimum incidence of PID peaked in 2000–08 at 12·5 per 100,000 live births. instructions to forward the sample to the Regional Molecular Genetics laboratory for analysis Referrals • • A completed DNA request card Patients with primary immunodeficiency. A completed proforma must should accompany all samples accompany all referrals (http://www.labs.gosh.nhs.uk/media/528387/PID panel pre-test proforma_2014.doc) • Patient details Mutation testing can be offered to the relatives of PID patients once a disease causing mutation has been identified. To facilitate accurate testing and reporting please provide patient Service offered demographic details (full name, date of birth, address and ethnic origin), details Next generation sequencing of 72 genes (see list below) with mutation confirmation by of any relevant family history and full Sanger sequencing. contact details for the referring clinician List of genes included in panel ADA, AICDA, AIRE, BTK, CASP8, CASP10, CD27, CD247, CD3D, CD3E, CD3G, CD40, CD40LG, CIITA, CORO1A, CYBA, CYBB, DCLRE1C, DOCK8, FAS, FASLG, FOXP3, HPS1, HPS4, HPS6, ICOS, IKBKG, IL2RG, IL7R, IL10, IL10RA, IL10RB, ITK, JAK3, LIG4, LRBA, LYST, MAGT1, MYO5B, NCF1, NCF2, NCF4, NHEJ1, NRAS, ORAI1, PNP, PRF1, PRKDC, PTPRC, RAB27A, RAG1, RAG2, RFX5, RFXANK, RFXAP, RMRP, SH2D1A, STAT1, STAT3, STAT5A, STAT5B, STX11, STXBP2, TAP1, TAP2, TAPBP, TNFRSF13B, UNC13D, UNG, WAS, XIAP, ZAP70 Technical Mutation screening is carried out by next generation sequencing with library preparation using a Sure Select XT custom kit followed by sequencing on the Illumina MiSeq. Data is analysed using an in-house pipeline with all mutations confirmed by Sanger sequencing. Target reporting time 4 months for a full mutation screen in an index case (next generation sequencing). 2 weeks for familial mutation testing. Please contact the laboratory for urgent cases.
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Radiation-sensitive severe combined immunodeficiency (RS-SCID)
Contact details Introduction Molecular Genetics Service Severe combined immunodeficiency (SCID) is a group of genetically and Level 6, York House phenotypically heterogeneous disorders that can be immunologically classified by the 37 Queen Square absence or presence of T, B, and natural killer (NK) cells. The most severe form of SCID has the T-B-NK+ phenotype, accounting for ~20% of all cases in which patients London, WC1N 3BH present with a virtual absence of both circulating T and B cells, while maintaining a T +44 (0) 20 7762 6888 normal level and function of NK cells. This form of SCID is caused by autosomal F +44 (0) 20 7813 8578 recessive mutations in at least three primary genes necessary for V(D)J recombination, RAG1, RAG2, and DCLRE1C (ARTEMIS). DCLRE1C mutations Samples required cause a T and B cell deficient form of SCID that is clinically indistinguishable from a RAG1/RAG2 disorder. Infants present with severe recurrent viral, bacterial or fungal • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) infections and failure-to-thrive. Defects in DCLRE1C can be distinguished from RAG defects because the former has the additional feature of increased sensitivity to • Prenatal testing must be arranged ionising radiation in bone marrow and fibroblast cells. in advance, through a Clinical Genetics department if possible. The DNA crosslink repair 1C gene (DCLRE1C; MIM 605988) encodes ARTEMIS which is an essential factor of V(D)J recombination during lymphocyte development • Amniotic fluid or CV samples and in the repair of DNA double-strand breaks (DSB) by the non-homologous end should be sent to Cytogenetics for dissecting and culturing, with joining (NHEJ) pathway. Patients with mutations in the DCLRE1C gene, suffer from instructions to forward the sample radiosensitive SCID (RS-SCID; MIM 602450) or radiosensitive Omenn syndrome to the Regional Molecular Genetics (MIM 603554). laboratory for analysis The DCLRE1C gene (10p13) has 14 exons and mutations have been found • A completed DNA request card throughout the gene. The most frequent mutations reported are gross deletions (59%) should accompany all samples due to homologous recombination of the wild-type DCLRE1C gene with a pseudo- DCLRE1C gene located 61.2 kb 5’ to the DCLRE1C start codon. Patient details Referrals To facilitate accurate testing and • Patients with suspected RS-SCID. reporting please provide patient • demographic details (full name, date of Carrier testing can be offered to the relatives of RS-SCID or Omenn syndrome birth, address and ethnic origin), details patients once a disease-causing mutation has been identified. of any relevant family history and full • Radiation sensitivity testing can be performed by MRC Sussex (Professor contact details for the referring clinician Penny Jeggo). Please contact the laboratory to discuss. Prenatal testing Prenatal testing is available for families in whom specific mutations have been identified or in whom appropriate family studies have been undertaken. Please contact the laboratory to discuss. Service offered • Sequencing analysis is performed of the coding regions and splice sites of the DCLRE1C gene. • MLPA analysis for deletion/duplication testing Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for carrier testing using mutation-specific tests. For urgent samples please contact the laboratory.
Contact details for Molecular Immunology / Enzyme unit: Dr Kimberly Gilmour, Molecular Immunology, GOSH - Tel: +44 (0) 20 7829 8835, Email: [email protected] Enzyme Unit, Chemical Pathology, Great Ormond Street Hospital, London WC1N 3JH Tel: +44 (0) 207405 9200 (x2509)
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RAG-deficient severe combined immunodeficiency (RAG-SCID)
Contact details Introduction Molecular Genetics Service RAG-deficient severe combined immunodeficiency (RAG-SCID, MIM 601457) is an Level 6, York House autosomal recessive immunodeficiency characterised by a lack of circulating T and B 37 Queen Square cells. Affected individuals can be diagnosed on the basis of an abnormality or London, WC1N 3BH deficiency of the V(D)J recombinase subunits, RAG1 and RAG2. The RAG1 and T +44 (0) 20 7762 6888 RAG2 genes have 2 exons of which exon 2 is coding. Family specific mutations have F +44 (0) 20 7813 8578 been found throughout the coding region of both genes. Omenn syndrome (MIM 603554) is a leaky TlowB-SCID characterised by reticuloendotheliosis and eosinophilia. It is caused by mutations in RAG1 and RAG2 that result in a partially Samples required functional recombinase. • 5ml venous blood in plastic EDTA bottles (>1ml from neonates) Referrals • Prenatal testing must be arranged • Patients with suspected RAG-SCID. in advance, through a Clinical • Carrier testing can be offered to the relatives of RAG-SCID or Omenn Genetics department if possible. syndrome patients once a disease-causing mutation has been identified. • Amniotic fluid or CV samples However, due to the rarity of the disorder, partner screening is not offered should be sent to Cytogenetics for unless there is consanguinity or a family history of RAG-SCID or Omenn dissecting and culturing, with syndrome in the partner. instructions to forward the sample • RAG1 and RAG2 protein analysis can be performed by the Molecular to the Regional Molecular Genetics laboratory for analysis Immunology laboratory at GOSH but requires a bone marrow sample from affected patients. This requires prior arrangement and completion of specific • A completed DNA request card request forms (see contact information below). should accompany all samples Prenatal testing Patient details Prenatal testing is available for families in whom specific mutations have been To facilitate accurate testing and identified or in whom appropriate family studies have been undertaken. Please reporting please provide patient demographic details (full name, date of contact the laboratory to discuss. birth, address and ethnic origin), details of any relevant family history and full Service offered contact details for the referring clinician Mutation screening of the RAG1 and RAG2 genes in affected individuals. Due to the requirement of a bone marrow sample for protein analysis, mutation screening will be carried out in the absence of protein testing if clinically indicated. If DNA from the affected individual is unavailable then screening can be undertaken in the parents. Technical Mutation screening is undertaken by sequence analysis of the RAG1 and RAG2 genes. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for carrier testing using mutation-specific tests. For urgent samples please contact the laboratory. To arrange RAG expression studies please contact Dr Kimberly Gilmour, Molecular Immunology, GOSH Tel: +44 (0) 20 7829 8835, Email: [email protected]
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Wiskott-Aldrich syndrome (WAS)
Contact details Introduction Molecular Genetics Service WAS (MIM 301000) is an X-linked immunodeficiency characterised by Level 6, York House thrombocytopenia and abnormal B- and T-cell functions. In carrier women this 37 Queen Square manifests as a skewed X-inactivation pattern in their whole blood. Affected individuals London, WC1N 3BH can be diagnosed on the basis of an abnormality or deficiency of the Wiskott-Aldrich T +44 (0) 20 7762 6888 syndrome protein (WASP). The WAS gene (encoding WASP) has 12 exons and F +44 (0) 20 7813 8578 family specific mutations are found throughout the gene. Referrals Samples required • Affected patients should be referred to the Molecular Immunology laboratory at • 5ml venous blood in plastic EDTA GOSH for WAS protein analysis. This requires prior arrangement and bottles (>1ml from neonates) completion of specific request forms (see contact information below). We work • Prenatal testing must be arranged closely with this department and will undertake mutation screening in in advance, through a Clinical appropriate patients. Genetics department if possible. • Carrier testing can be offered to the female relatives of WAS patients once a • Amniotic fluid or CV samples disease causing mutation has been identified. should be sent to Cytogenetics for dissecting and culturing, with Prenatal testing instructions to forward the sample to the Regional Molecular Genetics Prenatal testing is available for families in whom specific mutations have been laboratory for analysis identified or in whom appropriate family studies have been undertaken. Please • A completed DNA request card contact the laboratory to discuss. should accompany all samples Service offered Patient details Mutation screening of the WAS gene in affected individuals found to have absent or To facilitate accurate testing and abnormal WASP expression. Cases found to have WASP expression may be reporting please provide patient screened if there is a strong clinical indication for a diagnosis of WAS. If DNA from an demographic details (full name, date of affected male is unavailable the mother can be tested for her X-inactivation status and birth, address and ethnic origin), details subsequent mutation screening carried out where appropriate. of any relevant family history and full contact details for the referring clinician Technical Mutation screening is undertaken by sequencing analysis. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for carrier testing using mutation-specific tests. For urgent samples please contact the laboratory. To arrange WASP expression studies please contact Dr Kimberly Gilmour, Molecular Immunology, GOSH Tel: +44 (0) 20 7829 8835, Email: [email protected]
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X-linked agammaglobulinaemia (XLA)
Contact details Introduction Molecular Genetics Service XLA (MIM 300300) is an X-linked immunodeficiency characterised by a lack of Level 6, York House circulating B cells. In carrier women this manifests as a skewed X-inactivation pattern 37 Queen Square in their B cells. Affected individuals can be diagnosed on the basis of an abnormality London, WC1N 3BH or deficiency of the Bruton’s tyrosine kinase protein (BTK). The BTK gene has 19 T +44 (0) 20 7762 6888 exons and family specific mutations are found throughout the gene. F +44 (0) 20 7813 8578 Referrals • Affected patients should be referred to the Molecular Immunology laboratory at Samples required GOSH for BTK protein analysis. This requires prior arrangement and • 5ml venous blood in plastic EDTA completion of specific request forms (see contact number below). We work bottles (>1ml from neonates) closely with this department and will undertake mutation screening in • Prenatal testing must be arranged appropriate patients. in advance, through a Clinical • Carrier testing can be offered to the female relatives of XLA patients once a Genetics department if possible. disease causing mutation has been identified. • Amniotic fluid or CV samples should be sent to Cytogenetics for Prenatal testing dissecting and culturing, with instructions to forward the sample Prenatal testing is available for families in whom specific mutations have been to the Regional Molecular Genetics identified or in whom appropriate family studies have been undertaken. Please laboratory for analysis contact the laboratory to discuss. • A completed DNA request card Service offered should accompany all samples Mutation screening of the BTK gene in affected individuals found to have absent or Patient details abnormal BTK expression. Cases found to have BTK expression may be screened if To facilitate accurate testing and there is a strong clinical indication for a diagnosis of XLA. If DNA from an affected reporting please provide patient male is unavailable the mother can be tested for her X-inactivation status and demographic details (full name, date of subsequent mutation screening carried out where appropriate. birth, address and ethnic origin), details of any relevant family history and full Technical contact details for the referring clinician Mutation screening is undertaken by sequence analysis of the 19 exons and exon/intron boundaries of the BTK gene. MLPA analysis for deletion/duplication testing. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for carrier testing using mutation-specific tests. For urgent samples please contact the laboratory. To arrange BTK expression studies please contact Dr Kimberly Gilmour, Molecular Immunology, GOSH Tel: +44 (0) 20 7829 8835, Email: [email protected]
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X-linked hyper IgM syndrome (X-HIGM)
Contact details Introduction Molecular Genetics Service Hyper IgM syndrome is a primary immunodeficiency characterised by an inability to Level 6, York House produce immunoglobulin isotypes other than IgM and IgD resulting in susceptibility to 37 Queen Square bacterial and opportunistic infections. The disease is genetically heterogeneous with London, WC1N 3BH both X-linked recessive and autosomal recessive forms. X-linked HIGM (MIM 308230) T +44 (0) 20 7762 6888 is the most common form and affected individuals can be diagnosed on the basis of F +44 (0) 20 7813 8578 an abnormality or deficiency of the CD40 ligand protein, CD154. The CD40LG gene (MIM 300386) has 5 exons and family specific mutations are found throughout the gene. Samples required • 5ml venous blood in plastic EDTA Referrals bottles (>1ml from neonates) • Affected patients should be referred to the Molecular Immunology laboratory at • Prenatal testing must be arranged GOSH for CD154 protein analysis. This requires prior arrangement and in advance, through a Clinical completion of specific request forms (see contact information below). We work Genetics department if possible. closely with this department and will undertake mutation screening in appropriate • Amniotic fluid or CV samples patients. should be sent to Cytogenetics for • Carrier testing can be offered to the female relatives of X-linked HIGM patients dissecting and culturing, with once a disease causing mutation has been identified. instructions to forward the sample to the Regional Molecular Genetics Prenatal testing laboratory for analysis • A completed DNA request card Prenatal testing is available for families in whom specific mutations have been should accompany all samples identified or in whom appropriate family studies have been undertaken. Please contact the laboratory to discuss. Patient details Service offered To facilitate accurate testing and reporting please provide patient Mutation screening of the CD40LG gene in affected individuals found to have absent demographic details (full name, date of or abnormal CD154 expression. Cases found to have CD154 expression may be birth, address and ethnic origin), details screened if there is a strong clinical indication for a diagnosis of HIGM. If DNA from an of any relevant family history and full affected male is unavailable screening can be undertaken in the mother. contact details for the referring clinician Technical Mutation screening is undertaken by sequence analysis of the 5 exons and exon/intron boundaries of the CD40LG gene. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for carrier testing using mutation-specific tests. For urgent samples please contact the laboratory.
To arrange CD154 expression studies please contact Dr Kimberly Gilmour, Molecular Immunology, GOSH Tel: +44 (0) 20 7829 8835, Email: [email protected]
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X-linked lymphoproliferative disease (XLP)
Contact details Introduction Molecular Genetics Service XLP 1 (MIM 308240) and XLP 2 (MIM 300635) are X-linked immunodeficiencies Level 6, York House characterised by extreme sensitivity to the Epstein Barr virus (EBV). Affected 37 Queen Square individuals can be diagnosed on the basis of an abnormality or deficiency of the London, WC1N 3BH SLAM associated protein (SAP) or XIAP protein. The SH2D1A gene (encoding SAP) T +44 (0) 20 7762 6888 has 4 exons and family specific mutations are found throughout the gene. The XIAP F +44 (0) 20 7813 8578 gene has 7 exons (6 coding). Referrals Samples required • Affected patients should be referred to the Molecular Immunology department • 5ml venous blood in plastic EDTA at GOSH for SAP/XIAP protein analysis. This requires prior arrangement and bottles (>1ml from neonates) completion of specific request forms (see contact information below). We work • Prenatal testing must be arranged closely with this department and are able to undertake mutation screening in in advance, through a Clinical appropriate patients. Genetics department if possible. • Carrier testing can be offered to the female relatives of XLP patients once a • Amniotic fluid or CV samples disease causing mutation has been identified. should be sent to Cytogenetics for dissecting and culturing, with Prenatal testing instructions to forward the sample to the Regional Molecular Genetics Prenatal testing is available for families in whom specific mutations have been laboratory for analysis identified or in whom appropriate family studies have been undertaken. Please • A completed DNA request card contact the laboratory to discuss. should accompany all samples Service offered Patient details Mutation screening of the SH2D1A or XIAP genes in affected individuals found to To facilitate accurate testing and have absent or abnormal SAP or XIAP expression. Cases found to have SAP or XIAP reporting please provide patient expression may be screened if there is a strong clinical indication for a diagnosis of demographic details (full name, date of XLP. If DNA from an affected male is unavailable screening can be undertaken in the birth, address and ethnic origin), details mother. Mutation-specific tests for family members are also available. of any relevant family history and full contact details for the referring clinician Technical Mutation screening is undertaken by sequence analysis of the 4 exons and exon/intron boundaries for the SH2D1A gene. This detects approximately 43% of mutations in patients shown to have abnormal or deficient SAP. This suggests that there is an as yet unidentified molecular defect in some of these patients, which may or may not be in the SH2D1A gene. Mutation screening of the 7 exons of the XIAP gene is undertaken by sequence analysis. MLPA analysis for deletion/duplication analysis. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for carrier testing using mutation-specific tests. For urgent samples please contact the laboratory. To arrange SAP/XIAP expression studies please contact Dr Kimberly Gilmour, Molecular Immunology, GOSH Tel: +44 (0) 20 7829 8835, Email: [email protected]
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X-linked severe combined immunodeficiency (XSCID)
Contact details Introduction Molecular Genetics Service XSCID (MIM 300400) is an X-linked immunodeficiency characterised by a lack of Level 6, York House circulating T cells. In carrier women this manifests as a skewed X-inactivation pattern 37 Queen Square in their T cells. Affected individuals can be diagnosed on the basis of an abnormality London, WC1N 3BH or deficiency of the common gamma chain protein (γc). The IL2RG gene (encoding T +44 (0) 20 7762 6888 γc) has 8 exons and family specific mutations are found throughout the gene. F +44 (0) 20 7813 8578 Referrals • Affected patients should be referred to the Molecular Immunology laboratory at Samples required GOSH for γc protein analysis. This requires prior arrangement and completion • 5ml venous blood in plastic EDTA of specific request forms (see contact information below). We work closely with bottles (>1ml from neonates) this department and will undertake mutation screening in appropriate patients. • Prenatal testing must be arranged • Carrier testing can be offered to the female relatives of XSCID patients once a in advance, through a Clinical disease causing mutation has been identified. Genetics department if possible. • Amniotic fluid or CV samples Prenatal testing should be sent to Cytogenetics for dissecting and culturing, with Prenatal testing is available for families in whom specific mutations have been instructions to forward the sample identified or in whom appropriate family studies have been undertaken. Please to the Regional Molecular Genetics contact the laboratory to discuss. laboratory for analysis • A completed DNA request card Service offered should accompany all samples Mutation screening of the IL2RG gene in affected individuals found to have absent or abnormal γc expression. Cases found to have γc expression may be screened if there Patient details is a strong clinical indication for a diagnosis of XSCID. If DNA from an affected male is To facilitate accurate testing and unavailable the mother can be tested for her X-inactivation status and subsequent reporting please provide patient mutation screening where appropriate. demographic details (full name, date of birth, address and ethnic origin), details Technical of any relevant family history and full contact details for the referring clinician Mutation screening is undertaken by sequence analysis of the 8 exons and exon/intron boundaries. This detects approximately 90% of mutations in patients shown to have abnormal or deficient γc. Target reporting time 8 weeks for routine mutation screen in index case. 2 weeks for carrier testing using mutation-specific tests. For urgent samples please contact the laboratory. To arrange γc expression studies please contact Dr Kimberly Gilmour, Molecular Immunology, GOSH Tel: +44 (0) 20 7829 8835, Email: [email protected]
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