A glimpse into multi-gene rare variant genetics: triple mutations in hypertrophic cardiomyopathy

Four remarkable hypertrophic cardiomyopathy (HCM) pedigrees, each containing one individual harboring 3 HCM mutations, have been published by Girolami and colleagues in this issue of the Journal (1). With a backdrop of the cardiovascular phenotypes and genotypes of their family members, these 4 individuals give us a glimpse into the effects of multi-gene rare variant genetics. The HCM genetic variants identified fit into a rare variant paradigm (Table 1), and each met usual criteria to be considered a disease-causing mutation: the variant was found in a known HCM gene, it was rare in the population, it changed a key amino acid, and in some cases it had been reported previously in association with HCM. Variants in dilated cardiomyopathy (DCM) and long QT syndrome (LQTS) also may be classified using a similar set of criteria (Table 1).

Table 1. Characteristics of Rare versus Common Variant Genetic Disease Paradigms^*.

	Rare variant paradigm			Common variant common disease paradigm
	Archetypal Mendelian rare variant	HCM, LQTS	DCM
Characteristic
Familial concentration	Always, unless lethal prior to reproduction	Usually	Commonly, 20-50% of probands	Usually <1-2%
Penetrance	Very high, > 95%	Mostly high	Variable	Low
Clinical heterogeneity	Low	Low to high	Low to high	High
Locus heterogeneity	Occasional	Minimal (3-5 principal genes)	Moderate (10-30 principal genes)	Many
Minor allele frequency	<0.1%	Usually < 0.2%	Usually <0.2%	>5%
Variant's strength of biological effect	Very strong	Strong	Strong to less strong	Weak
The variant's contribution to understanding disease etiology	Definitive	Usually definitive	Definitive to questionable	Questionable
Relationship of variant to other putative disease- associated rare variants	Effects of other variants not necessary to explain phenotype	Needs to be determined experimentally	Needs to be determined experimentally	Usually in linkage disequilibrium with a known functional variant (common or rare)
Genetic disease mechanism	One rare variant	Usually one rare variant; multiple mutations in 2-8%	Usually one rare variant; frequency of multiple mutations and contribution to phenotype unknown	Increased susceptibility of disease risk, presentation or progression
Primary approach, genome-wide association study	No	No	No	Yes
Primary approach, linkage study	Yes	Yes, in large families	Yes, in large families	No
Primary approach, resequencing study	Possibly	Yes	Yes	No

^*Adapted from (2).

In patients with HCM, a putative disease-causing mutation can be identified in 50-60% of patients, and of these, 80-85% of mutations, usually missense, are found in either MYH7, encoding beta myosin heavy chain, or MYBPC3, encoding myosin binding protein C (2). Mutations in TNNT2 or TNNI3, encoding cardiac troponin T or I, constitute another 10% of HCM mutations (2). An individual harboring 2 mutations in HCM is not new, and has been reported in 2.5% (3), 3.5% (4), or 5% (5) of HCM cases in association with earlier onset disease.

What added insights can be gained from these unusual triple mutation HCM pedigrees? They strengthen the concept that multiple rare variants acting in concert actually do modulate the HCM phenotype. They also show us that the type of mutation (missense or nonsense), its relationship to other mutations in the same gene (cis or trans) and the combinations of different mutations (in the same or different genes relevant for HCM) result in different phenotypes. In Cases 1-3, the individuals who carried 3 mutations had an earlier onset (ages 18, 29, and 24 years) and more aggressive disease (heart failure, cardiac arrest, heart transplant, etc), compared to their family members who carried 1 or 2 mutations, or when compared to the remainder of the 484 probands. But 3 mutations did not always cause more severe disease, as the individual in the fourth family with 3 mutations had an HCM phenotype with a 19 mm septum but was asymptomatic and otherwise normal. She was a 48 year old female who underwent screening because her brother, who carried two mutations, was diagnosed with HCM after presenting with minor symptoms. Her 3 daughters, all in their 30's, who carried combinations of 1 or 2 of their mother's mutations, had normal cardiovascular screening. Notably, each of these mutations, one in MYH7 and the other two in MYBPC3, had been reported previously as disease-causing when observed as solo mutations.

More generally, aside from multiple mutations in genes known to cause HCM, the variability of phenotypes between closely related family members carrying the same rare variant is commonly observed in genetic cardiomyopathy, whether HCM or DCM (2). This phenotypic variability has been attributed to environmental influences (e.g., blood pressure, diet, activity, etc), and the individual's specific genetic background, whether from common or rare variants. Efforts have been made to identify this genetic background in animal models and in humans (6). While common variant genetics has received a great deal of attention, principally to find risk factors for disease (Table 1) (7), the magnitude of risk identified has been limited (8).

We all carry many variants that could cause or influence genetic disease, but experimental issues have limited our abilities to identify them. The sequencing of Craig Venter's DNA in 2007 using conventional capillary-based sequencing methods at great expense showed that he carried an estimated 1.3 million novel variants, including 11,718 heterozygous and 9434 homozygous coding variants; these were dispersed over 4,107 genes that had 6,114 nonsynonymous single nucleotide variants, including missense, nonsense and insertion/deletion (indel) mutations (9). A more recent and much less cost intensive study, using exome capture followed by massively parallel next generation sequencing, gave similar results (10), where the exome is defined as the 1% of the genome that encodes known proteins. From 12 individuals of varied ethnicities, an average of 9449 synonymous, 7777 nonsynonymous, 46 nonsense, 17 splice site variants and 52 frameshift indels were identified per individual (10).

So what does the future hold? Exome sequencing, a major technological advance, will help to unravel the complexity of rare variant genetics. It is more tractable than current methods, efficiently enriching exomic DNA, which is then applied to next generation sequencing platforms that are more cost effective than conventional methods. Using this approach, variants can be identified in genes beyond the known HCM genes that are relevant to HCM risk, causation, presentation or progression. Such new information will have an enormous impact on the field. This is also likely to be the case for DCM genetics based on inferences from preliminary studies (11, 12), as rare variants in > 30 genes have been implicated to cause DCM, yet they explain no more than one-third of its genetic basis (2).

These are exciting times for cardiovascular rare variant genetics - stay tuned!