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

• Meckel-Gruber syndrome (MKS)– Clinical features– Genetic aspects

• MKS mutation spectrum

• Screening test strategy

• Achievements to date

Overview

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 novel: cytoplasmic protein, contains B9 domain, localises to basal bodies

Kyttälä et al. (2006)

MKS2 11q13 - Roume et al. (1998)

MKS3/TMEM67 8q22.1 novel: Frizzled-like receptor, localises to primary cilia & basal bodies

Smith et al. (2006)

MKS4/CEP290/ NPHP6

12q21 centrosomal protein; causative of other ‘ciliopathies’

Baala et al. (2007)

MKS5/RPGRIP1L 16q12.2 novel: colocalises and interacts with other ciliary proteins

Delous et al. (2007)

Genetic aspects

• Associated with the dysfunction of primary cilia• Autosomal recessive• Genetic and clinical heterogeneity• Oligogenic inheritance

Map Function Reference

MKS1 17q22 novel: cytoplasmic protein, contains B9 domain, localises to basal bodies

Kyttälä et al. (2006)

MKS2 11q13 - Roume et al. (1998)

MKS3/TMEM67 8q22.1 novel: Frizzled-like receptor, localises to primary cilia & basal bodies

Smith et al. (2006)

MKS4/CEP290/ NPHP6

12q21 centrosomal protein; causative of other ‘ciliopathies’

Baala et al. (2007)

MKS5/RPGRIP1L 16q12.2 novel: colocalises and interacts with other ciliary proteins

Delous et al. (2007)

Khaddour et al. (2007) Human Mutation 28(5); 523-4

MKS1 and MKS3 mutations

MKS1 MKS3/Meckelin

B9 domain

• 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

The need for a diagnostic service for MKS

Proposed test strategy

• 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%

Referrals to DNA Lab

Pakistani origin Other origin

Autozygosity or linkage analysis at MKS1 & MKS3

Targeted mutation screen by sequencing

Screen for common MKS3 splice-site mutations

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 D17S1853 and D17S1290– MKS3 8q22 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

MKS1

• c.1451_1453dupGGCA (p.Thr485fs)– Pakistani– 4bp duplication in exon 16

MKS3

• c.1674+1G>A– Pakistani– mutation abolishes exon 16 splice donor site

Mutations reported

Exon16 | Intron 16

Thr Thr Gly Thr Val Thr Phe

Thr Thr Gly Arg His Cys His

482 483 484 485 486 487 488

Upper panel: wild-typeLower panel: homozygous mutant

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

Exon 15 | Intron 15Mutations reported

Intron 8 | Exon 9

Upper panel: wild-typeLower panel: homozygous mutant

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.

helen.lindsay@leedsth.nhs.uk

Acknowledgements

Leeds Institute of Molecular Medicine• Colin Johnson

Yorkshire Regional DNA Laboratory• Kim Flintoff, David Cockburn, Ruth Charlton

Yorkshire Clinical Genetics Service• Chris Bennett