Genotype-phenotype correlations and genetic family screening in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. Clinical presentation is heterogeneous, outcome ranging from benign asymptomatic forms to more malignant expressions resulting in sudden or heart failure death. To date, more than 450 mutations have been reported in genes encoding sarcomeric proteins, proteins of the Z-disc, intercalated discs and in genes involved in cardiac metabolism. DNA testing is helpful for confirming diagnosis in ambiguous situations, can give some prognostic information and represents the gold standard for preclinical diagnosis in family members. However, mutation screening in HCM allows positive results in a small percentage of probands and is expensive and time-consuming. Aim of this study was to identify pathogenic mutations in the most common HCM related genes and to correlate molecular defect with clinical-morphological phenotypic pattern in a large cohort of HCM probands from a single centre. Since the mutation in the index case has been identified, a cascade screening has been performed in the first degree family members searching for carriers. Genetic screening for the 4 most commonly HCM-related sarcomeric genes (i.e. MYBPC3, MYH7, TNNT2, TNNI3) was performed by denaturing high performance liquid chromatography (DHPLC) and direct sequencing in 83 HCM index-cases from large and/or “malignant” families. A subgroup of 30 probands underwent more extensive mutation screening by 12 genes (i.e. MYH7, MYBPC3, MYL2, MYL3, TNNT2, TNNI3, TNNC1, TPM1, ACTC, CSRP3, PLN and PRKAG2) array-based DNA resequencing assay. If the mutations found were novel, these were searched in a healthy control population to rule out the possibility that they represent single nucleotide polymorphisms (SNPs). Including both screening methods, our population consisted of 99 index cases (age at diagnosis 31±17 years, age at last control 45±17 years, 70% males, 65% familial cases, 38% with obstruction). Twenty-seven pathogenic HCM-causing mutations were found in 30 probands (30%). Percentage of mutation positive patients was not different in probands with HCM family history (21/64 probands, 33%) versus sporadic cases (9/35 probands, 26%; p=0.46), and irrespective of the screening method used (25/83 probands, 30%, by DHPLC and direct sequencing vs 8/30 probands, 27%, by DNA resequencing array; p=0.72). In 14 index-cases screened both by DHPLC and DNA resequencing array, there was agreement between the two different screening methods (3 mutations in 3 patients found by both methods, and just an intronic mutation “missed” by DNA resequencing array). Genes more frequently involved were MYH7 coding for beta-myosin heavy chain (11 mutations in 11 patients) and MYBPC3 for cardiac myosin-binding protein C (8 mutations in 12 patients). In a minority of probands HCM-causing genes were TNNI3, TNNT2 and MYL3, respectively in 4, 3 and one patients. Two patients had double mutation in compound heterozygous. Wide heterogeneity in clinical presentation and evolution was present in spite of genotype characterization, but when multiple mutations were detected, they were associated with particularly severe phenotype. Fifty-one members from 16 different families were screened for the mutation(s) found in their family proband and 23 (45%) resulted carriers. Eight carriers had phenotypic expression fulfilling diagnostic criteria for HCM (i.e. maximal left ventricular wall thickness, MLVWT ≥ 13 mm), whereas 10 had only minor signs suggestive of HCM (such as ECG abnormalities, MLVWT=12-13 mm, abnormal left ventricular filling pattern at echo-Doppler), and 5 were healthy carriers. Tissue Doppler Imaging seemed to be useful for preclinical diagnosis, but a multiparametric evaluation is needed to identify mutation carriers before phenotypic expression of HCM. Nowadays, mutation screening is becoming part of diagnostic and clinical management of HCM patients and family members. The spectrum of HCM-associated genes has moved outside the myofilaments of the sarcomere to encompass additional subgroups of proteins involved in the pathogenesis of HCM. Mutation carriers without HCM phenotype represent a new subgroup of patients at risk for developing disease, whose clinical and prognostic profile remains unresolved, but of particular interest as possible target for preventive therapeutic strategies that can change the natural history of this disease.