The core approach to human genetic studies remains the same: the careful and comprehensive phenotyping of subjects and their family members, and then correlating those phenotypes with genetic information. The challenge of this approach is to assure oneself that the genetic variation identified is causative of the phenotype of interest.
The term ‘mutation’ is most commonly applied in Mendelian disease to one or a short string of variants in coding DNA (). The most common are missense mutations, but less common types include nonsense, splice site, and short insertion or deletion mutations (). Synonymous variants do not change the amino acid of that codon, while nonsynonymous variants do change the amino acid of that codon.
Considerations for Molecular Genetic Testing
Classifying a variant as a disease-causing mutation
Ascertaining whether any one specific variant is causing the phenotype of interest requires weighing several types of evidence, and achieving a high level of certainty for any one variant is challenging, especially if that variant is novel (1
) (). In most cases, the sum of all of the evidence is required to decide if the identified variants are relevant (). As noted below, with next generation sequencing (NGS) approaches, the unique genetic variants identified in an individual affected with a specific phenotype can be many – hundreds to thousands – creating new challenges.
The term ‘Mendelian disease’ has been applied to heritable genetic disease, usually familial, with identifiable inheritance patterns (dominant or recessive, and autosomal, X-linked or mitochondrial) (1
). Many Mendelian diseases are uncommon to rare, with population frequencies well below 1%. For Mendelian disease demonstrating autosomal dominant inheritance (which is the case in most FDC families) (1
), the most powerful evidence that a putative mutation is indeed disease-causing is segregation of the variant of interest with the disease phenotype in at least one large, multigenerational family with multiple affected individuals who carry the variant and multiple unaffected individuals who do not carry the variant (). Multiple large families available to assess segregation increases the strength of evidence. While this concept is superficially simple, certain features of adult-onset Mendelian disease commonly observed with FDC complicates this approach.
One feature is incomplete penetrance, which refers to individuals who carry a mutation but do not manifest any evidence of the disease phenotype. Thus, in gene discovery studies, the absence of a DCM phenotype in someone carrying a putative disease-causing variant can never be considered absolute evidence that the variant is not relevant: the individual in question may simply be manifesting incomplete penetrance. A key corollary for clinicians caring for at-risk family members is that a negative clinical cardiovascular evaluation at any age does not rule out the possibility that the family member may develop later disease. This provides the rationale for the periodic rescreening of at-risk family members who have normal evaluations.
A related concept, ‘age-dependent’ or ‘age-related’ penetrance, is also observed with FDC, where a disease-causing mutation usually manifests a disease phenotype only in the adult years, most commonly in the 4th to 6th decades or later.
Another feature that complicates FDC assessment is variable expressivity, which means that only some aspects of the DCM phenotype are present. For example, only mild left ventricular enlargement (LVE) without systolic dysfunction, or the onset of arrhythmia or conduction system disease with only borderline DCM may be observed. Also, age of onset can vary significantly, with variable severity of disease progression. Thus, within a large FDC family a wide range of clinical findings may be present without fully developed DCM. Reliance on endophenotypes (partial or sub-phenotypes) as an indication of genetic DCM/FDC also has been problematic, in part because subtle clinical changes may result from other more common causes of CV disease, making it difficult to decipher genetic from non-genetic cause.
While usually nonsyndromic, DCM can be included in syndromic disease involving various organ systems, but most commonly skeletal muscle disease (muscular dystrophy) (12
Other criteria to assign causality (), in addition to segregation of the variant with the phenotype, include its relative rarity in control DNAs (commonly <<1%). The rationale for this is that if it were common in the population, it would be unlikely to cause a rare genetic disease. Nevertheless, how rare is rare (<0.01, <0.005, < 0.001, <0.0001)? Some analyses have suggested that the majority of rare alleles (0.001 – 0.003) may be injurious (91
). The caveat with control DNAs is that they should be representative of the race and/or ethnicity of the DCM family, as variants observed to be common (>1%) in one population can be rare in a different population.
Conservation of the amino acid or nucleotide (i.e., lack of variation in the protein structure or specific nucleotide sequence (92
) of lower species) is also used to assess variants, with the rationale that an amino acid or a nucleotide position with greater variation in lower species may have increased tolerance to variants at that position and are therefore less likely to be disease-causing. Other features are also relevant ().
Much of this, vital for discovery efforts, is also relevant for FDC clinical genetics. These fundamental principles of human genetics investigations have not changed, but with NGS the quantity of data to which they are applied has changed dramatically.
Text limitations do not permit a reiteration of the components and importance of skilled genetic counseling, especially for difficult, confusing or syndromic cases, supported by geneticist consultations as needed (1
). Unlike most cardiologists, genetic counselors are trained to deal with the family as a unit of inquiry rather than the individual patient, an essential quality for genetic medicine. Genetic counselors are also trained to emphasize disease prevention in contrast to the focus on disease treatment taken by most cardiovascular specialists. Both of these qualities are particularly relevant for facilitating genetic risk assessment. The availability of genetic counselors with cardiovascular training or experience can provide the support needed to initiate the practice of cardiovascular genetic medicine. We refer the reader to several citations that deal with these important points (1