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