Sarcomeric hypertrophic cardiomyopathy (HCM) is the most prevalent genetic cardiovascular disease and one of the leading causes of sudden cardiac death, particularly in young people. The disease is defined by the presence of increased ventricular wall thickness or mass in the absence of loading conditions sufficient to cause the observed abnormality.1 The usual clinical marker of the disease is unexplained ventricular hypertrophy, usually identified on echocardiographic examination. Sarcomeric HCM is associated with mutations in genes encoding sarcomere or sarcomere-related proteins. Several different mutations associated with the disease have been identified in at least 12 genes, the myosin-binding protein C (MYBPC3) and beta-myosin heavy chain (MYH7) genes being the most common. Mutations in these two genes and also in the cardiac troponin T (TNNT2) and cardiac troponin I (TNNI3) genes are associated with the disease in up to 62% of unrelated sarcomeric HCM patients in various cohorts around the world.2–9

The disease has an autosomal dominant pattern of transmission, and its penetrance is variable and age-related. As hypertrophy may develop later in life, the commonly accepted prevalence of 0.2%10 may be an underestimation. The clinical course of HCM is highly unpredictable and there is a risk of sudden death, particularly in individuals considered to have a high clinical risk profile. The risk for those only genetically affected but with no phenotype (carriers) is not well defined.

Although it is believed that specific gene mutations with distinct functional characteristics may contribute to different phenotypes and prognosis, for most causal mutations prognostication will be difficult, given their private character in affected families. On the other hand, if it is assumed that the true prognostic value of HCM genetic testing simply relies on a positive test (identification of a pathogenic sarcomeric mutation)9 and not on the type or location of a particular mutation, then periodic evaluation for risk stratification is recommended to all genetically affected individuals.

Molecular diagnosis distinguishes between sarcomeric HCM and other hypertrophic heart disorders with distinct clinical courses and prognosis. The correct diagnosis of HCM should therefore be the first step in cases of unexplained left ventricular hypertrophy (LVH). If the genetic test is positive – a causal sarcomeric mutation is found in about 60% of affected individuals2 – a diagnosis is made and clinical workup is performed in order to help decision-making, particularly to prevent sudden death. Genetic screening of the family follows (in a cascade approach) to identify other at-risk family members. Absence of the causal mutation in relatives will reassure them, since in this case there is at present considered to be no risk of developing the disease.

In 30–40% of index patients, no mutation-associated disease is identified.11 In these cases, although the prognosis appears to be better,9 all first-degree relatives should be considered at risk and serial clinical screening is needed.

HCM is a global disease and genotype profiles regarding HCM-associated mutations have been described for several HCM populations in different countries worldwide.4–9,12–15 However, data on the Portuguese population are scarce16,17 and the prevalence of the most commonly involved genes is unknown. The main objective of this paper is to report the mutation profile in a Portuguese cohort of unrelated consecutive patients with sarcomeric HCM, followed at a large referral center.

The present study reports the frequency and profile of single and multiple mutations in a Portuguese cohort of unrelated HCM index patients, most with an established diagnosis for many years (mean follow-up 12.7 years). The entire coding regions of five sarcomeric genes were screened for alterations in order to identify all described and as yet unreported mutations. Our results demonstrated an overall mutation detection rate of 53%. This is in agreement with previously reported data in other population studies.4–6,8,9,12,15 All mutations in our cohort were identified in the four genes most commonly associated with HCM (MYBPC3, MYH7, TNNT2 and TNNI3). Familial HCM showed the highest rate of mutations, comprising 71% of the population with a positive genotype.

In a recent study that included only HCM index patients genotyped for these four genes,15 a higher rate of mutations in familial disease (64%) was found in a French population compared to sporadic (29%) or inconclusive disease (46%). These data corroborate previously published studies in the French population showing a 70% frequency of sarcomeric mutations in familial HCM.5 In our cohort, only 7% (three out of 16 patients) of sporadic HCM and 22% of patients (9 out of 20) with inconclusive disease had an identified mutation.

Differences concerning sporadic or inconclusive disease may be explained by differences in the populations studied, as is always the case with HCM. The number of relatives available for studying cosegregation with the disease in each family is highly variable in different published series, as is the information on a diagnosis of HCM in relatives. Additionally, a different number of genes are screened in each population studied. Studies that include predominantly familial HCM have a high rate of genotype-positive patients,12 as is the case with our study. Sporadic HCM was found in only 21% of our cohort; it is less frequent than familial HCM and is diagnosed later.

Our findings show that many of the inconclusive HCM cases are in fact familial. The study of as many relatives as possible in each family should be the goal in HCM genetic population studies.

In 36 patients (46%) – including 29% of those with familial HCM – no mutations were identified in the five genes screened.

We are aware that screening only five sarcomeric genes may have missed some genotype cases in other genes, although most myofilament mutations associated with HCM are usually identified in the four genes mutated in our population. No mutations were found in MYL2 in our population, and the reported mean frequency of mutations in TPM1 (encoding α-tropomyosin), MYL3 (myosin essential light chain) and ACTC (encoding α-actin) is very low, 1.21%, 0.11% and 0.49%, respectively.12 It is commonly thought that since HCM is a genetically heterogeneous disease, patients with HCM and no identified mutations in the known myofilament coding genes may in fact have mutations in unexplored regions of these genes. Alternatively mutations may exist in novel myofilament encoding genes (like titin, muscle LIM protein, telethonin, myozenin 2 or vinculin) or in other HCM-susceptibility genes that were not screened in our cohort.

Some differences were observed in our population between patients with and without myofilament identified mutations. Patients with a positive genotype were younger at the time of diagnosis and showed more severe septal hypertrophy (at diagnosis and after the same follow-up time). The implications of these findings need further study.

Mutations in MYBPC3 were the most prevalent cause of HCM in our cohort, with 35% of probands affected. The frequency of mutations in MYBPC3 has been reported as ranging from 14% to 26% in HCM.12,15 A recent study of an Italian cohort including 203 unrelated HCM patients9 found that 34% had mutations in MYBPC3, a similar frequency to ours. Although defining the pathogenicity of mutations in MYBPC3 (especially for missense mutations) is particularly challenging, the criteria applied in our study for the novel variants identified support the idea that most of the missense variants identified are indeed pathogenic and their effects are not overestimated.

The prevalence of patients with mutations in the MYH7 gene was 11.7%. Findings in other studies ranged from 2.8% to 25%.12 The number of patients with mutations in MYH7 in our study is unlikely to be underestimated as the entire coding sequence of the gene was analyzed. Mutations in this gene and in MYBPC3 were found in 87% of genotype-positive patients, a frequency close to that reported in other studies.5,9,15

The TNNT2 gene was affected in 6.5% of HCM probands (12% of genotype-positive patients). Mutations in this gene are the most commonly involved in thin filament-HCM, with reported values for affected probands ranging from 0% to 6.67%.22,35 Mutations in the TNNI3 gene were also found in 2.6% of HCM patients (4.9% of those with identified mutations), in agreement with previously reported rates (ranging from 0% to 6.5%).12,15

Multiple mutations were detected in 7% of index patients with a positive genotype. This finding is in line with previously published studies4,5,7,9 and indicates that once a single sarcomeric mutation is identified in a patient a continued search for other mutations is necessary, at least in MYBPC3 and MYH7, the two genes most commonly associated with sarcomeric HCM.

Comparison of clinical data of patients with single mutations in different genes showed no significant differences except for a higher degree of hypertrophy after follow-up in patients with MYBPC3 gene mutations. The possible interpretations of this finding are beyond the scope of this study, since the rate of different private mutations in this gene was very high and the specific functional effect of each mutation on disease expression is unknown. Furthermore, it is not the purpose of this study to establish associations between specific genotypes and phenotypes in HCM or to put forward considerations about individual prognosis.

A genetic diagnosis establishing a causal relationship with HCM is important for several reasons. First, it identifies affected individuals (excluding other less prevalent genetic causes of cardiac hypertrophy with different outcomes). Second, it enables identification of family members with a positive genotype at risk of developing HCM who require rigorous and serial follow-up for risk stratification. Third, knowledge of the genetic cause of HCM not only may in the near future help direct treatment strategies in already clinically affected patients36 but also, within families with different mutations, the identification of carriers (in a preclinical state) may enable preventive interventions to limit disease development, a possibility that is already under study (http://clinicaltrials.gov/ct2/show/record/NCT00319982). Additionally genetic study should be performed to reassure genotype-negative family members.

The molecular screening of five sarcomeric genes associated with HCM in a Portuguese population enabled a genetic diagnosis in 53% of a consecutive cohort of unrelated patients. The highest yield was observed in familial HCM (71%). The most commonly mutated gene was MYBPC3, but since mutations in this gene and in MYH7 account for most genotype-positive patients and more than one mutation was found in 4% of HCM patients, both genes should be systematically screened as a first genetic approach by testing the entire coding regions of these genes. Private mutations were the rule and novel mutations were a frequent finding, illustrating the difficulty of making genotype-phenotype correlations in HCM on an individual basis for each mutation. Independently of the gene affected, genotype-positive patients were younger at the time of diagnosis and had more severe hypertrophy than patients with a negative genotype, although the significance of these findings and their impact on prognosis needs further evaluation. The identification of a positive genotype in probands led to confirmation of the disease in a substantial number of relatives, including 25 carriers (genetically positive, preclinical state) with implications for follow-up, risk stratification and genetic counseling.

The authors have no conflicts of interest to declare.