Genotype-phenotype correlations in hypertrophic cardiomyopathy can be confounded by double mutations in a single disease gene

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease arising from mutations in at least 9 sarcomeric protein genes. Genotype:phenotype correlations may be of prognostic use and certain mutations have been associated with a low risk of sudden death; however, in a minority of families the same mutations are associated with adverse prognosis. Mutations in FHC genes can efficiently be found using a combination of linkage analysis and temperature modulated heteroduplex analysis (TMHA). We have undertaken mutation detection in families with FHC to investigate genotype-phenotype correlations. Linkage in "Family A"' was consistent with a β-myosin heavy chain causative mutation. Screening of this gene using TMHA revealed variants in exons 3, 16 and 20. The variant in exon 3 is a common polymorphism and does not segregate with the disease. Haplotype analysis showed that the other two variants segregated with disease, with both on the same parental chromosome. Sequencing revealed the previously described V606M mutation in exon 16 and an A728V mutation in exon 20; we screened 200 normal chromosomes by typing of an Mscl site introduced by the A728V mutation and excluded the possibility that it was a common polymorphism. The alanine residue has been conserved in diverse myosin isoforms from drosophila to man and lies close to the essential light chain interface. These data suggest that both the V606M and A728V are pathogenic. The V606M has been considered a "benign" mutation: however 2 of 8 affected individuals in "Family A" died suddenly at a young age. This "malignant" phenotype could be caused by the A728V acting alone or in combination with the V606M. We conclude that genotype-phenotype correlations in FMC can be confounded by the presence of more than one mutation in the same copy of a disease gene. We propose that, contrary to current practice, a complete screen for variants is required even when a mutation has been identified before a genotype-phenolype correlation is proposed.