Multiple mutations in desmosomal proteins encoding genes in arrhythmogenic right ventricular cardiomyopathy/dysplasia

In this article a cohort of 42 consecutive index cases with a typical form of ARVC/D was investigated at the Department of Cardiothoracic and Vascular Sciences of the University of Padua, Italy.
The study protocol included: physical examination, family history, 12-lead electrocardiogram (ECG), signal-averaged ECG, 24-hour Holter ECG and 2-dimensional echocardiogram. Diagnosis was made according to the past Task Force criteria. Moreover, family members of these 42 subjects were also analyzed using the same clinical protocol. The index case patients were screened for mutations in PKP2, DSP, DSG2, DSC2, JUP, and TGF3 genes by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing.
A control group of 250 healthy and unrelated subjects (500 alleles) from the Italian population was used to exclude that the detected mutations were DNA polymorphisms. Among these 42 unrelated ARVC/D index cases, 7 (16.6%) carried a PKP2 mutation, 5 (11.9%) a DSP mutation, 4 (9.5%) a DSG2 mutation, 2 (4.8%) a DSC2 mutation, and 2 (4.8%) a TGF3 mutation. No one carried JUP mutations. A potential disease causing mutation was found in 38% of the cases.

Overall, 3 index cases (7.1%) carried multiple mutations in the same gene or in different genes. Family screening identified an additional 7 multiple-mutation carriers.  Among the 7 double heterozygotes for mutations in different genes, 2 were clinically unaffected, 2 were affected, and 3 showed some clinical signs of ARVC/D even if they did not fulfill the diagnostic criteria.

Two compound heterozygotes for mutations in the same gene and 1 subject carrying 3 different mutations showed a severe form of the disease with heart failure onset at a young age. Moreover, multiple-mutation carriers showed a higher prevalence of left ventricular involvement (P=.025) than single-mutation carriers.

The authors conclude that the occurrence of compound and double heterozygotes in ARVC/D index cases is particularly relevant to mutation screening strategy and to genetic counseling. Even if multiple-mutation carriers show a wide variability in clinical expression, the extent of the disease is higher compared to that in single-mutation carriers.

Commentary

Critical review of the article suggests us to focus our attention on three important aspects:

1. Multiple mutations in the same gene or in different genes in ARVC/D.

The presence of multiple mutations has been found to occur in either the same gene (compound heterozygotes) or in different genes (double heterozygotes) in different inherited cardiomyopathies. Thus in HCM multiple mutation carriers account approximately up to 5%. It is known that individuals who carry 2 disease-causing familial HCM mutations have a more severe disease than patients with single mutations, including earlier onset, more severe left ventricular hypertrophy and heart failure and a higher rate of sudden death events (resuscitated cardiac arrest or sudden death). Recently an article by Girolami et al described a more complex genotype (triple sarcomeric mutations) in a cohort of patients with HCM, showing an increased risk of end-stage progression and ventricular arrhythmias in these patients, supporting an association between multiple sarcomere defects and adverse outcome.
Identification of complex genotypes when studying inherited cardiomyopathies is not uncommon, and probably it’s becoming evident with the widespread use of genetic studies. It sounds plausible that the more complex genotype, the more complex phenotype would be expected, but it cannot be considered as a general rule. A highly malignant mutation may have a more severe phenotype than the presence of a combination of less severe variants.

2. Genetic study methodology in ARVC/D.

On the basis of the reported findings, genetic screening of desmosomal ARVC/D genes should not be stopped after the identification of one mutation in one gene and should be continued at least on the main desmosomal ARVC/D genes (PKP2, DSP, DCS-2 and DSG-2). Probably this genotyping strategy should include other genes as TMEM 43 that have been related with the disease.

 3. New ARVC/D diagnostic criteria

With the proposed modification of the Task Force, genetic studies have come to represent a major criterion in the diagnosis of the disease. Identification of a pathogenic mutation categorized as associated or probably associated with ARVC/D in the patient under evaluation is considered to be a major criterion on ARVC/D diagnosis. The new Task Force consider a pathogenic mutation a DNA alteration associated with ARVC/D that alters or is expected to alter the encoded protein, is unobserved or rare in a large non-ARVC/D control population, and either alters or is predicted to alter the structure or function of the protein or has demonstrated linkage to the disease phenotype in a conclusive pedigree.

But this is not as simple as it sounds. It is often difficult to establish whether a mutation is pathogenic or not. There are several limitations:  

• functional studies are usually not conclusive,
•  the number of ethnically matched controls is usually small,
•  there are multiple problems to establish the cosegregation of the genetic variant in the family, including incomplete penetrance and the presence of double mutations in the same family

It’s therefore essential to obtain information about the clinical and genetic data of all the genetic variants. It’s very important to publish articles like this of Bauce et al, which provide clinical, genetical and complete family studies of ARVC/D.  

The presence of multiple mutations has been found to occur in either the same gene (compound heterozygotes) or in different genes (double heterozygotes) in different inherited cardiomyopathies.

Thus in HCM multiple mutation carriers account approximately up to 5%. It is known that individuals who carry 2 disease-causing familial HCM mutations have a more severe disease than patients with single mutations, including earlier onset, more severe left ventricular hypertrophy and heart failure and a higher rate of sudden death events (resuscitated cardiac arrest or sudden death).

Recently an article by Girolami et al described a more complex genotype (triple sarcomeric mutations) in a cohort of patients with HCM, showing an increased risk of end-stage progression and ventricular arrhythmias in these patients, supporting an association between multiple sarcomere defects and adverse outcome. Identification of complex genotypes when studying inherited cardiomyopathies is not uncommon, and probably it’s becoming evident with the widespread use of genetic studies. It sounds plausible that the more complex genotype, the more complex phenotype would be expected, but it cannot be considered as a general rule. A highly malignant mutation may have a more severe phenotype than the presence of a combination of less severe variants.

On the basis of the reported findings, genetic screening of desmosomal ARVC/D genes should not be stopped after the identification of one mutation in one gene and should be continued at least on the main desmosomal ARVC/D genes (PKP2, DSP, DCS-2 and DSG-2).  Probably this genotyping strategy should include other genes as TMEM 43 that have been related with the disease.
With the proposed modification of the Task Force, genetic studies have come to represent a major criterion in the diagnosis of the disease. Identification of a pathogenic mutation categorized as associated or probably associated with ARVC/D in the patient under evaluation is considered to be a major criterion on ARVC/D diagnosis. The new Task Force consider a pathogenic mutation a DNA alteration associated with ARVC/D that alters or is expected to alter the encoded protein, is unobserved or rare in a large non-ARVC/D control population, and either alters or is predicted to alter the structure or function of the protein or has demonstrated linkage to the disease phenotype in a conclusive pedigree.

But this is not as simple as it sounds. It is often difficult to establish whether a mutation is pathogenic or not. There are several limitations: It’s therefore essential to obtain information about the clinical and genetic data of all the genetic variants. It’s very important to publish articles like this of Bauce et al, which provide clinical, genetical and complete family studies of ARVC/D.  

Conclusion:

We know that in medicine no single test is able to provide a definite diagnosis in any disease. In inherited cardiomyopathies a correct diagnosis requires the integration of clinical, morphological and familial studies, together with the genetic tests.