Sarcomeric Gene Mutations in Humans with HCM

Hypertrophic cardiomyopathy (HCM) is a primary myocardial disease characterized by myocardial wall thickening (almost always confined to the left ventricle). In humans, HCM is a genetic disease and is usually inherited in an autosomal dominant pattern. The first mutation (in the β-myosin heavy chain gene [β-MHC]) responsible for HCM in humans was identified in 1989, and since then, over 600 mutations in 10 genes that encode for sarcomeric proteins have been identified in human families with HCM.¹ They include the β-myosin heavy chain, α-tropomyosin, cardiac troponins I, C, and T, myosin binding protein C, essential and regulatory light chains, titin, and actin genes. The genes with the most mutations described and the ones that most commonly produce disease are the β-MHC and cardiac myosin binding protein C (MYBPC3) genes, which account for roughly 60% of the mutations identified, and for 60% of HCM cases. It is now known that sarcomeric gene mutations actually cause HCM since several mutations that have been identified in human families with HCM have been placed in transgenic mice and the disease reproduced (at least partially), thus fulfilling Koch's postulates.²

Mutations in the MYBPC3 gene in humans with HCM were first identified in 1995 and it was the fourth causal gene to be identified.³ To date, approximately 200 mutations in MYBPC3 that cause HCM have been identified in humans. Compared with mutations in β-MHC, a larger number of the MYBPC3 mutations are associated with a more benign clinical course (later average age at onset of symptoms and a lower incidence of sudden death). Nonetheless, MYBPC3 mutations are a significant cause of morbidity and mortality for millions of people worldwide. For instance, a single MYBPC3 founder mutation found in people in South Asia (India, Pakistan, Sri Lanka, Indonesia and Malaysia; close to 5% of this population and upwards of 40 million people) makes them up to 7 times more at risk for development of cardiac dysfunction and heart failure than normal individuals. Typically, individuals affected with this mutation are free of HCM through the third decade of life but 90% of older individuals eventually develop cardiac complications.

The majority of MYBPC3 mutations are splice site donor/acceptor or other insertion/deletion mutations that are predicted to lead to reading frame shifts, premature stop codons, and truncated proteins. However, approximately 40% of the mutations in MYBPC3 are due to point mutations (single base pair changes resulting in single amino acid substitutions). When compared to truncation/frame shift/splice variants, point mutations more commonly result in a more benign clinical course although there are those that produce severe disease.

The functional pathophysiology of missense mutations in MYBPC3 is not yet fully known, but potentially they can alter cMyBP-C structure, disrupt cardiac myosin binding C (cMyBP-C) protein function or alter interactions with other proteins.³ Missense mutations could result in so-called haploinsufficiency where one allele is rendered nonfunctional, resulting in only half the protein product being produced, and so in sarcomere dysfunction, as can occur with truncation mutations. Alternatively, a protein that is produced by a mutated gene can be so altered that it is lysed within the cell before it ever is incorporated into a sarcomere. A missense mutation can also just result in a dysfunctional protein that is incorporated into a sarcomere. For example, the N775K mutation in humans allows the C5 domain of the protein to unfold rendering it dysfunctional. In other instances a mutation alters the ability of the protein (cMyBP-C) to interact with other proteins. For example, domains C7 to C10 anchor cMyBP-C to myosin and titin and disruptions of this interaction could impair the function of cMyBP-C. Lastly, missense mutations can result in a dominant-negative effect where an altered protein produced by a mutated gene has a detrimental effect on the normal protein.

The manner by which HCM is produced by sarcomeric mutations is relatively unknown and still controversial. One theory is that the abnormal protein produced by a mutated gene results in dysfunctional sarcomeres through any of the means discussed in the previous paragraph. Those dysfunctional sarcomeres contract poorly and this either forces functional sarcomeres to bear a larger work load (if, for example half of the sarcomeres are rendered dysfunctional) or all of the sarcomeres together can do less work (generate less force; if, for example, all of the sarcomeres are working at 50% capacity). The myocardium then has to compensate by replacing the dysfunctional sarcomeres with additional functional ones or has to add in more dysfunctional sarcomeres to help carry the load. The net result is that the myocardium adds in new sarcomeres within myocytes resulting in the cells growing wider. Because myocytes make up a large part of the heart muscle, the thicker myocytes result in the wall of the left ventricle increasing in thickness.

Familial Feline HCM

The first "family" of cats with an inherited form of HCM was identified in a research colony of Maine coon cats in 1992 and first reported in 1999.⁴ The disease appeared to be inherited as a simple autosomal dominant trait in this breed with 100% penetrance (all cats affected with HCM). However, this colony was particularly inbred and, so, genetic modifying factors were probably uniform and a number of cats were probably homozygous for the subsequently identified mutation. In Maine coon cats in the real world, it is now known that the disease is not 100% penetrant. Two lines of evidence support this. First, it is known that when an affected cat is identified (proband) that it is possible that neither parent may have echocardiographic evidence of the disease. Secondly, Maine Coon cats screened for the identified mutation and for HCM often have the mutation but no HCM, especially when the screening clinic includes only young cats.

The disease has been reproduced in Maine coon cats in the original research colony by mating affected to unaffected and affected to affected cats as well as by breeding affected Maine coon male cats to domestic shorthair female cats. The fact that HCM can be produced in offspring from mating a Maine Coon cat with HCM to a normal domestic shorthair cat proves that the disease is inherited as an autosomal dominant trait. The course of the disease can be accelerated by mating affected to affected cats, probably because cats that are homozygous for the mutation are produced. Penetrance is age related and the disease is progressive over months to years. In many affected Maine coon cats in the research colony, HCM is not apparent during the first year of life but becomes apparent by 2 years of age in males. Females tend to get the disease later, with many manifesting the disease by 3–4 years of age but some not showing evidence of HCM until 6 or 7 years of age and, others not until 10 and 13 years of age (most recently identified in two cats with the identified mutation in the research colony). When both parents have HCM, an affected Maine coon kitten may have echocardiographic evidence of the disease as early as 6 months of age and have severe disease by one year of age, whether male or female. Again, these are assumed to be cats that are homozygous for the subsequently identified mutation.

In 2005, Dr. Kate Meurs identified the first gene mutation responsible for HCM in Maine Coon cats from our research colony.⁵ This mutation is in exon 3 of MYBPC3. The exact location is codon 31 of the gene where a single point base pair (missense) mutation changes alanine to proline in the encoded protein (A31P). This region of the gene is highly conserved across species and the resultant amino acid change results in a change in the computed protein structure. Several studies have now shown that this mutation is only found in Maine Coon cats (not in other purebred cats). Prevalence of the mutation across continents is generally in the 30–40% range (highly prevalent).

In 2007, a mutation in the same gene (MYBPC3) was identified in Ragdoll cats with HCM (R820W).⁶ This mutation is at a completely different location on the gene, but it again occurs in a highly conserved region. Ragdoll cats have long been known to have a particularly malignant form of the disease where they often die before reaching one year of age.

Several studies have now been done to look at the penetrance of the A31P mutation in Maine Coon cats (to determine what percent of cats with the mutation have HCM). To date, these have all been done as part of echocardiographic clinics to screen for HCM. Unfortunately, this type of study is inherently biased since the cats being screened are chosen by the breeder, not the investigator. This has resulted in populations of younger and female-predominant cats.

The first study looked at a group of Maine Coon cats during a screening clinic and correlated the genetic findings with echocardiographic findings.⁷ The investigators found that many Maine Coon cats with the A31P mutation did not have echocardiographic evidence of HCM, and a few cats without the A31P mutation had HCM. The veiled conclusion by the authors was that the A31P mutation might not be causal and that genetic testing for the mutation in this breed may not be warranted. However, what was shown instead is that the penetrance of this mutation is relatively low, at least in young cats, which is not surprising since most MYBPC3 mutations in humans have a low and an age-related penetrance. They also showed that there appears to be at least one more cause of HCM in this breed. This has been apparent in the colony at UC Davis for a number of years.

Another study made observations on the echocardiographic appearance of hearts from Maine coon cats with the A31P MYBPC3 mutation.⁸ In this study, echocardiography was performed on 96 Maine coon cats presented for screening for HCM. Both two-dimensional and tissue Doppler imaging echocardiography were performed. Cats had to have an LV wall thickness greater than 6 mm to make the diagnosis of HCM. Of the 96 cats, 44 of the cats had the A31P mutation (38 were heterozygous and 6 were homozygous). Unfortunately, 45 of the 96 cats were less than 2 years of age and so too young to have evidence of HCM if they were heterozygous. These young cats probably should have been excluded from analysis if they were heterozygous. Of the 38 heterozygous cats, four had clear evidence of at least moderate HCM. However, only 10 of the 34 heterozygous cats that did not have HCM were over 4 years of age. Of the 44 cats that had the mutation, 13 were male and 31 were female. This produced an additional bias, since female Maine coon cats get HCM at a later age and get less severe disease. Still, this study clearly suggests that the A31P mutation is not nearly 100% penetrant in Maine coon cats when a cat is heterozygous for the mutation (11% in this study). As expected, homozygous cats appear to be a different story. Of the 6 cats that were homozygous for the A31P MYBPC3 mutation in this same study, 4 had clear echocardiographic evidence of HCM and the other two had abnormal diastolic function as assessed by TDI, an abnormality previously shown to be present in cats that go on to develop HCM. Consequently, it appears that all 6 of these cats had HCM, which once again proves the mutation to be causal. Two cats in this study without the known mutation also had HCM. This once again documents that there is at least one more cause of HCM in Maine coon cats. Lastly, some of the cats heterozygous for the A31P mutation had evidence of regional diastolic dysfunction, showing that their myocardium was not normal even when they had no evidence of hypertrophy.

The most recent study into this subject looked at 332 cats.⁹ They similarly showed that penetrance for Maine Coon cats heterozygous for the mutation is low (6%), that most but not all cats homozygous for the mutation get HCM (penetrance is high) and that most of the Maine Coon cats that get severe HCM prior to 6 years of age do so because they are homozygous for the mutation. The prevalence of HCM was 6%. Eighteen cats were homozygous and 89 cats were heterozygous for the mutation. The odds ratio for having HCM for homozygous cats was 21.6 (95% confidence interval 7–66). Overall, 50% of the cats that were homozygous for the mutation had HCM. Only two cats over four years were homozygous and both had HCM.

The previously referenced studies have only made a preliminary attempt to look at penetrance of the A31P mutation in Maine coon cats that are heterozygous for the A31P mutation. What really needs to be done is a longitudinal study looking at the same mutated cats until they are at least 10 years of age to tell what the true penetrance is for heterozygous cats rather than looking at a group of cats once at one point in time.

We are currently examining the cellular effects of this mutation and have preliminary evidence to show clear abnormalities produced by this mutation (e.g., myocardium from cats homozygous for the A31P mutation has no myosin binding protein C). Our preliminary findings clearly show that the A31P mutation causes intracellular derangement. It has also been shown that the mutated protein in cats with the A31P mutation is incorporated into the sarcomere, even in cats homozygous for the mutation.³ This means the mutation does not result in haploinsufficiency.

There are currently several labs that test for the A31P MYBPC3 mutation in Maine Coon cats (one in the USA and at least two in Europe). This service has been set up so that breeders can try to rid their breed of this mutation and the HCM caused by this mutation. Many breeders have been reluctant to do testing and even more reluctant not to breed mutated cats. To be fair, the method of dealing with this problem is controversial (discussed below). In Dr. Meurs' lab, approximately 35% of the DNA samples submitted have had the mutation.¹⁰

Concern has been expressed about what would happen if all of these cats were removed from the breeding pool. The concern has to do with decreasing the size of the gene pool and so producing more recessive traits in this breed. In other words, if 35% of the cats could no longer be bred, the remaining cats would have to make up the breeding pool and by shrinking that pool, the breed may be worse off because of increased inbreeding. The author's counter to that argument is as follows. Breeders commonly only use around 10% of cats in a purebred population for breeding. That means they already exclude 90% of cats from being bred. So if we recommend that they don't breed any cat with a mutation, it means we're recommending that they not use 35% of 10% or 3.5% of the entire population. Yes, that does mean that 35% of the cats they would normally breed (the good-looking 10%) would be no longer eligible for breeding. But it also means that they could still use the 65% of the cats they normally don't breed (the less than good-looking 90%) to breed. What would they sacrifice? Maybe their breed wouldn't be quite as visually attractive as before - but it also might be healthier without the mutation. Of course, getting breeders to breed cats they don't think look quite as good as some others is probably an impossible task. So one recommendation has been made that if a cat is heterozygous for the A31P mutation and it is very good looking, it can be bred once. Its kittens then should be tested for the mutation and only those without should be bred. The question then comes, what do you do with the mutant kittens that have been produced in this process? And this recommendation was first made 3 years ago, meaning breeders have had plenty of time to do this and should no longer be relying on this method.

Lastly, it should be mentioned that the Ragdoll mutation (R820W) has also now been identified in humans. In humans, it also produces HCM but can also produce left ventricular noncompaction in some individuals. Another mutational variant affecting the same codon (R820Q) also produces HCM and one that progresses to the so-called burnout phase (i.e., DCM) more frequently. The A31P mutation has not been identified in humans.

References

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4.  Kittleson MD, Meurs KM, Munro MJ, Kittleson JA, Liu SK, Pion PD, et al. Familial hypertrophic cardiomyopathy in Maine Coon cats: an animal model of human disease. Circulation. 1999;99:3172–3180.

5.  Meurs KM, Sanchez X, David RM, Bowles NE, Towbin JA, Reiser PJ, et al. A cardiac myosin binding protein C mutation in the Maine Coon cat with familial hypertrophic cardiomyopathy. Hum Mol Genet. 2005;14:3587–3593.

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7.  Wess G, Schinner C, Weber K, Hartmann K. Genetic Association of the A31P and A74T polymorphism in the MYBPC3 gene and hypertrophic cardiomyopathy in Maine Coon Cats. J Vet Intern Med. 2008;22:717.

8.  Carlos Sampedrano C, Chetboul V, Mary J, Tissier R, Abitbol M, Serres F, et al. Prospective echocardiographic and tissue Doppler imaging screening of a population of Maine Coon Cats Tested for the A31P mutation in the myosin-binding protein C gene: a specific analysis of the heterozygous status. J Vet Intern Med. 2009;23:91–99.

9.  Godiksen MT, Granstrom S, Koch J, Christiansen M. Hypertrophic cardiomyopathy in young Maine Coon cats caused by the p.A31P cMyBP-C mutation - the clinical significance of having the mutation. Acta Vet Scand. 2011;53:7.

10. Fries R, Heaney AM, Meurs KM. Prevalence of the myosin-binding protein C mutation in Maine Coon cats. J Vet Intern Med. 2008;22:893–896.