development of a new method to prioritise gene analysis in familial hypertrophic cardiomyopathy...

Development of a New Method to Prioritise Gene Analysis in

Familial Hypertrophic Cardiomyopathy

Jayne Duncan

West of Scotland Regional Genetics Service, Glasgow

Familial Hypertrophic Cardiomyopathy (FHC)

• Autosomal dominant disorder showing variable penetrance and age of onset.

• Affects approximately 1/500 adults and is the most common cause of sudden death in young healthy individuals.

• So far mutations in over 20 genes have been associated with FHC

Primary Clinical Features of FHC

• Left ventricular hypertrophy, “a thickening of the tissue due to increased size of the constituent cells”.

• Myocyte/myofibrillar disarray caused by the abnormal shapes, intracellular connections and arrangement of the hypertrophic myocytes and fibrosis.

1 http://www.maxshouse.com2 Arad et al 2002 Hum Mol Genet. 11. (20) 2499-2506

Genotype Phenotype Correlation

The Heterogeneous Nature of FHC

• HCM is caused by dominant mutations in the sarcomeric genes.

• de novo mutations occur rarely and account for approximately 10% of cases.

• Mutations in the sarcomeric genes account for ~55% of cases of HCM.

• Syndromes such as the Glycogen storage disorders and Friedreich ataxia can mimic HCM.

Glasgow Linkage Exclusion Analysis Method (GLEAM)

• Glasgow Linkage Exclusion Analysis Method (GLEAM)

• Novel method to prioritise gene analysis in heterogeneous disorders

• A gene is excluded from analysis when affected relatives are oppositely homozygous for SNPs in and around the gene of interest

GLEAM

• A and B represent alleles at a susceptibility locus for a dominantly inherited disorder affecting individuals II:2, II:3, III:1 and III4.

• Since III:1 has no allele in common with II:3 or III:4 it effectively rules out this locus as being responsible for the disease in this family.

I:1 I:2

II:2II:1 II:3 II:4

III:1 III:2 III:3 III:4

BB AB AA AB

BB AB AB AA

Genes analysed in the FHC Project

Gene Name Chromosome No Exons No SNPs

TTN 2 363 212

MYH7 14 38 76

MYH6 14 37 77

MYBPC3 11 35 147

RAF1 3 16 163

PRKAG2 7 16 146

TPM1 15 16 132

TNNT2 1 15 116

MYLK2 20 12 94

TNNI3 19 8 92

MYL3 3 7 89

MYL2 12 7 94

CAV3 3 2 98

SNP Analysis Platform

• 96 fibre optic bundles on each plate• Each fibre contains a bead that corresponds to

each SNP• Image taken from www.Illumina.com

Sentrix Array Matrix

Results- Raw Data

Raw data for one patient sample

Results- Raw Data

Clustered patient SNP data for a single SNP locus

AA BBAB

Results- Genotype Comparisons

Results

Relationship Number of pairs Average numberof genes excluded

Sibs 49 3

Aunt/NieceNephew

22 5

First Cousins 10 7

First Cousins once removed

5 8

Second Cousins 9 7

Grandparent/Grandchild

1 2

ResultsGene Number of times

gene excluded Percentagenumber of timesgene excluded

TTN 34/96 35%

MYH6 and MYH7 33/96 34%

MYBPC3 28/96 29%

RAF1 23/96 24%

PRKAG2 47/96 49%

TPM1 29/96 30%

TNNT2 39/96 41%

MYLK2 40/96 42%

TNNI3 34/96 35%

MYL3 33/96 34%

MYL2 26/96 27%

CAV3 40/96 42%

Interesting Case

I:1 I:2

II:1 II:3II:2

III:2III:1

IV:1 IV:2 IV:3

II:4

III:4III:3 III:5 III:7III:6

IV:4 IV:5 IV:6

H15.1 H15.4 H15.7 H15.14 H15.15 H15.16 H15.12

TNNI3/ TNNI3/ MYBPC3 TNNI3 TNNI3 TNNI3 TNNI3 MYBPC3 MYBPC3

Interesting Case

• Familial mutation in TNNI3 was not excluded in all affected family members.

• Comparisons between H15.1, H15.4 and H15.7 did not exclude MYBPC3.

• MYBPC3 was excluded when H15.1, H15.4 and H15.7 were compared against other family members who did not have this mutation.

• Testing for the TNNI3 mutation in H15.7 would have been negative and suggested a second mutation prompting further analysis.

Conclusions

• For all the pedigrees with one known mutation, this gene was not excluded in any of the analyses performed.

• More genes tend to be excluded when more distantly related individuals such as first cousins or aunt/niece, nephew pairs are considered, rather than more closely related sibs

• GLEAM can be used to determine the order in which genes are sequenced in heterogeneous disorders

Acknowledgements

• Scottish Health Innovations Ltd

• Dr Wai Lee & Dr Stewart Lang, British Heart Foundation, Glasgow Cardiovascular Research Centre, University of Glasgow.

Dr Petros Syrris, Department of Medicine, University College London