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Development of a UK diagnostic service for Meckel-Gruber syndrome Helen Lindsay, Kimberley Flintoff, David Cockburn, Ruth Charlton and Colin Johnson Yorkshire Regional DNA Laboratory Overview • Meckel-Gruber syndrome (MKS) – Clinical features – Genetic aspects • MKS mutation spectrum • Screening test strategy • Achievements to date Meckel-Gruber syndrome (MKS) Clinical features • Lethal developmental disorder • UK incidence approximately 1 in 30,000 • ‘Classic triad’ of features (Salonen, 1984): – occipital encephalocele (or other CNS abnormality) – bilateral large multicystic kidneys – fibrotic changes in the liver • other features: bilateral postaxial polydactyly, microphthalmia, cleft lip and palate, heart defects, genetic abnormalities, bowing of long bones, situs inversus, low set ears etc. Diagnosis • Via ultrasound at 11-14 weeks • Often an autopsy is necessary Genetic aspects • • • • Associated with the dysfunction of primary cilia Autosomal recessive Genetic and clinical heterogeneity Oligogenic inheritance Map Function Reference MKS1 17q22 Kyttälä et al. (2006) MKS2 11q13 novel: cytoplasmic protein, contains B9 domain, localises to basal bodies - MKS3/TMEM67 8q22.1 Smith et al. (2006) MKS4/CEP290/ NPHP6 12q21 novel: Frizzled-like receptor, localises to primary cilia & basal bodies centrosomal protein; causative of other ‘ciliopathies’ MKS5/RPGRIP1L 16q12.2 Roume et al. (1998) Baala et al. (2007) novel: colocalises and interacts Delous et al. (2007) with other ciliary proteins Genetic aspects • • • • Associated with the dysfunction of primary cilia Autosomal recessive Genetic and clinical heterogeneity Oligogenic inheritance Map Function Reference MKS1 17q22 Kyttälä et al. (2006) MKS2 11q13 novel: cytoplasmic protein, contains B9 domain, localises to basal bodies - MKS3/TMEM67 8q22.1 Smith et al. (2006) MKS4/CEP290/ NPHP6 12q21 novel: Frizzled-like receptor, localises to primary cilia & basal bodies centrosomal protein; causative of other ‘ciliopathies’ MKS5/RPGRIP1L 16q12.2 Roume et al. (1998) Baala et al. (2007) novel: colocalises and interacts Delous et al. (2007) with other ciliary proteins MKS1 and MKS3 mutations MKS3/Meckelin MKS1 B9 domain Khaddour et al. (2007) Human Mutation 28(5); 523-4 The need for a diagnostic service for MKS • Prior to this project, no CPA accredited laboratory offered MKS testing • Mutation scanning performed on a research basis by Dr Colin Johnson at the Leeds Institute of Molecular Medicine – approximately 50 requests, nationally and internationally, for screening annually • In the local population the incidence of MKS may be as high as 1 in 3000 • A diagnostic service would allow – – – – accurate diagnosis confirmation of research results carrier testing in at-risk individuals prenatal testing Proposed test strategy Referrals to DNA Lab Pakistani origin Other origin Screen for common MKS3 splice-site mutations Autozygosity or linkage analysis at MKS1 & MKS3 Targeted mutation screen by sequencing • Clinical sensitivity of testing for mutations in MKS1 and MKS3 in the general population is approximately 15% • In the local Pakistani population sensitivity for the MKS3 c.1575+1G>A mutation alone is estimated at 40% The story so far... • Bidirectional sequencing optimised for: – entire coding region of MKS1 (18 exons) – exons 1-18 of MKS3 • Microsatellite analysis for MKS1 and MKS3 loci optimised: – MKS1 17q22 – MKS3 8q22 D17S1853 and D17S1290 D8S1818 and D8S1699 • Reports issued: – 35 confirmations of research findings • 30 locally • 5 nationally – it is anticipated that these results will lead to cascade carrier testing and prenatal diagnosis requests • Gene dossier to be submitted to UKGTN April 2008 Mutations reported 482 483 484 485 486 487 488 Thr Thr Gly Thr Val Thr Phe MKS1 • c.1451_1453dupGGCA (p.Thr485fs) – Pakistani – 4bp duplication in exon 16 Thr Thr Gly Arg His Cys His MKS3 • c.1674+1G>A – Pakistani – mutation abolishes exon 16 splice donor site Upper panel: wild-type Lower panel: homozygous mutant Exon16 | Intron 16 Mutations reported Exon 15 | Intron 15 MKS3 • c.1575+1G>A – Pakistani – mutation abolishes exon 15 splice donor site – Estimated allele frequency in the local Pakistani population is 0.016; carrier frequency approximately 1/32 – Variable phenotype e.g. CNS, polydactyly. Inter- and intra-familial variation • c.870-2A>G – Pakistani – mutation abolishes exon 9 splice acceptor site Upper panel: wild-type Lower panel: homozygous mutant Intron 8 | Exon 9 MKS testing costs Two common Pakistani mutations £200 Microsatellite analysis £100 per person Sequencing (per gene) up to £1000 Known mutation £150 Please contact the laboratory for further information on testing for Meckel-Gruber syndrome. [email protected] Acknowledgements Leeds Institute of Molecular Medicine • Colin Johnson Yorkshire Regional DNA Laboratory • Kim Flintoff, David Cockburn, Ruth Charlton Yorkshire Clinical Genetics Service • Chris Bennett