ClinVar Genomic variation as it relates to human health

The p.Glu143Lys variant (rs104893750) has been previously identified in two families of Latin American ethnicity (Caleshu 2011 and Olson 2002), and in a large cohort of patients referred for testing due to cardiomyopathy (Walsh 2017). Pathogenic variants in MYHL3 are typically associated with dominantly inherited hypertrophic cardiomyopathy (HCM8; MIM: 608751). However, in the two families described in Caleshu et al (2011) and Olson et al (2002), affected individuals were homozygous for the p.Glu143Lys, whereas a total of five heterozygous carriers between both families (ages 7-70) were clinically unaffected. Consanguinity was confirmed in the family described in Olsen et al (2002), and the authors suggested the p.Glu143Lys variant acts in a recessive manner, likely through a mechanism distinct from other dominantly inherited pathogenic MYL3 variants. At least one functional study of several pathogenic variants in MYL3, including p.Glu143Lys, revealed similar defects compared to wild-type in the binding of variant MYL3 protein to myosin heavy chain. However, these functional observations are difficult to interpretation, as the exact molecular mechanisms underlying the physiological defects associated with MYL3 remain unknown. Furthermore, to our knowledge, no other recessive-acting pathogenic alleles have been identified in MYL3, and all clinical laboratories submitting to ClinVar also classify the p.Glu143Lys variant as being of uncertain clinical significance (Variation ID: 14063). The glutamic acid at codon 143 is highly conserved considering 14 species up to C. elegans (Alamut software v2.9), and computational analyses suggest this variant has a significant effect on MYL3 protein structure/function (SIFT: damaging, PolyPhen2: possibly damaging, and Mutation Taster: disease causing). In summary, based on the available information, the clinical significance of the p.Glu143Lys variant cannot be determined with certainty.

The MYL3 c.427G>A variant is predicted to result in the amino acid substitution p.Glu143Lys. This variant has been reported in many individuals affected with hypertrophic cardiomyopathy (HCM) (Walsh R et al 2016. PubMed ID: 27532257; Gómez J et al 2017. PubMed ID: 28356264; McNamara JW et al 2017. PubMed ID: 28658286; Mademont-Soler I et al 2017. PubMed ID: 28771489; Yadav S et al 2019. PubMed ID: 30706179; Miller RJH et al 2019. PubMed ID: 31199839; Yoneda ZT et al 2021. PubMed ID: 34495297). In at least two families, this variant was reported as homozygotes in the affected individuals with restrictive cardiomyopathy whereas heterozygous carriers were clinically unaffected (Olson et al 2002. PubMed ID: 12021217; Caleshu C et al 2011. PubMed ID: 21823217). Functional studies suggested this variant reduces the binding affinity for the cardiac myosin heavy chain, and transgenic mice overexpressing the p.Glu413Lys variant exhibit clinical features of restrictive cardiomyopathy (Lossie J et al 2011. PubMed ID: 22131351; Sahni N et al 2015. PubMed ID: 25910212; Yuan CC et al 2017. PubMed ID: 28371863). This variant is reported in 0.011% of alleles in individuals of Latino descent in gnomAD (http://gnomad.broadinstitute.org/variant/3-46901019-C-T). This variant is interpreted as likely pathogenic.

The p.Glu143Lys variant in MYL3 has been reported in the heterozygous state in >35 individuals with hypertrophic cardiomyopathy (HCM), many of whom are of reported Latino ancestry, suggesting it may be a founder mutation in that population (Gomez 2014 PMID: 25342278, McNamara 2017 PMID: 28658286, LMM data, GeneDx pers. comm., Invitae pers. comm., Ambry pers. comm.). A few of these individuals also carried a likely pathogenic variant in another HCM-associated gene. This variant has also been identified in the homozygous state in at least 5 individuals with early onset HCM or restrictive cardiomyopathy (RCM) and in 3 affected siblings who also had early onset disease (Olson 2002 PMID: 12021217, Caleshu 2011 PMID: 21823217, LMM data, Ambry pers. comm., Invitae pers comm.). Relatives who were heterozygous carriers of this variant were clinically unaffected (Olson, 2002 PMID: 12021217, Caleshu 2011 PMID: 21823217) suggesting reduced penetrance. Additionally, it has been reported in ClinVar (Variant ID: 14063) and has been identified in 0.01% (4/35436) of Latino chromosomes by gnomAD (http://gnomad.broadinstitute.org). In vitro and in vivo functional studies, including transgenic mice expressing the p.Glu413Lys human variant that had clinical features of RCM, support an impact on protein function (Lossie 2012 PMID: 22131351, Sahni 2015 PMID: 25910212, Yuan 2017 PMID: 28371863) and computational prediction tools and conservation analyses are consistent with pathogenicity. In summary, although additional studies are required to fully establish its clinical significance, this variant meets criteria to be classified as likely pathogenic for autosomal dominant HCM with reduced penetrance and is associated a more severe presentation when a pathogenic variant is also found on the second copy of the gene. ACMG/AMP Criteria applied: PS4, PS3_Moderate, PP3.

Variant summary: MYL3 c.427G>A (p.Glu143Lys) results in a conservative amino acid change located in the EF-hand domain (IPR002048) of the encoded protein sequence. Four of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 1.2e-05 in 251486 control chromosomes (gnomAD). c.427G>A has been reported in the literature in multiple individuals affected with Hypertrophic Cardiomyopathy (HCM) (e.g. Gomez_2017, Ho_2018, Mademont-Soler_2017, McNamara_2017, Miller_2019, Olson_2002, Walsh_2017). At least two studies report individuals affected with childhood-onset HCM or early-onset restrictive cardiomyopathy that were homozygous for the variant, while multiple heterozygote family members were reported as unaffected with no evidence of cardiomyopathy (Caleshu_2011, Olson_2002). Olson et al (2002) postulated that the variant causes recessive cardiomyopathy and acts through a different molecular mechanism of disease than other dominantly-inherited MYL3 pathogenic variants. Additional studies reported the variant in HCM individuals co-occurring with variants from other cardiomyopathy genes (e.g. MYBPC3 p.I539V, TTN p.T6325A and p.R24188T, MYBPC3 p.D770N) (Gomez_2017, Mademont-Soler_2017, McNamara_2017). At least one of these is a known pathogenic variant (MYBPC3 p.D770N) and through their studies on the super-relaxed state (SRX) of myosin, the authors suggested that the combination of the two variants may cause a compound heterozygous effect (McNamara_2017). These data indicate that the variant is very likely to be associated with disease. Experimental evidence demonstrated the variant affects protein function (e.g. Lossie_2012, Sahni_2015). Eight ClinVar submitters (evaluation after 2014) cite the variant as pathogenic/likely pathogenic (n=7) and as uncertain significance (n=1). Based on the evidence outlined above, the variant was classified as pathogenic.

MYL3 NM_000258.2 exon 4 p.Glu143Lys (c.427G>A): This variant has been reported in the literature in the heterozygous state in at least 5 individuals with HCM, in the homozygous state in two siblings with early-onset HCM, and in the homozygous state in one individual with RCM (Olson 2002 PMID:12021217, Caleshu 2011 PMID:21823217, Gomez 2014 PMID:25342278, McNamara 2017 PMID:28658286, Walsh 2017 PMID:27532257). Of note, at least one of these individuals was also identified to carry an additional clinically significant cardiogenetic variant (McNamara 2017 PMID:28658286). This variant is present in 0.008% (3/34420) of Latino alleles in the Genome Aggregation Database (http://gnomad.broadinstitute.org/variant/3-46901019-C-T) and is present in ClinVar, with multiple labs classifying this variant as pathogenic or likely pathogenic (Variation ID:14063). Evolutionary conservation and computational predictive tools suggest that this variant may impact the protein. An in vitro functional study on rat cardiomyocytes demonstrated significantly reduced binding affinity of this protein to myosin heavy chains when compared to the wildtype, supporting that this variant impacts the protein (Lossie 2012 PMID:22131351). However, this study may not accurately represent in vivo biological function. In summary, data on this variant is highly suspicious for disease, but requires further evidence for pathogenicity. Therefore, this variant is classified as likely pathogenic.

This sequence change replaces glutamic acid, which is acidic and polar, with lysine, which is basic and polar, at codon 143 of the MYL3 protein (p.Glu143Lys). This variant is present in population databases (rs104893750, gnomAD 0.009%). This missense change has been observed in individuals with autosomal dominant hypertrophic cardiomyopathy and/or autosomal recessive restrictive cardiomyopathy (PMID: 12021217, 21823217, 27532257; Invitae). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 14063). An algorithm developed to predict the effect of missense changes on protein structure and function (PolyPhen-2) suggests that this variant is likely to be disruptive. Experimental studies have shown that this missense change affects MYL3 function (PMID: 22131351). For these reasons, this variant has been classified as Pathogenic.

This missense variant replaces glutamic acid with lysine at codon 143 in the EF-hand domain of the MYL3 protein. Computational prediction tool suggests that this variant may have a deleterious impact on protein structure and function (internally defined REVEL score threshold >= 0.7, PMID: 27666373). An experimental study has shown that this variant may reduce affinity for the cardiac myosin heavy chain in vitro (PMID: 22131351). In a transgenic mouse model, this variant caused a phenotype consistent with severe restrictive cardiomyopathy, including increased ventricular stiffness, sarcomere abnormalities, and interstitial cardiac fibrosis (PMID: 28371863). This variant has been identified in heterozygosity in more than 30 individuals affected with hypertrophic cardiomyopathy (PMID: 27532257, 30706179, 30847666, 33495597, 35026164; communication with external laboratories: ClinVar variation ID: 14063). Some of these individuals were reported to carry other variants associated with cardiomyopathy. This variant has also been reported in homozygosity in two unrelated individuals affected with restrictive cardiomyopathy (PMID: 12021217, 21823217). Five heterozygous individuals from these two families were unaffected suggesting reduced penetrance. This variant has been identified in 4/282890 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Likely Pathogenic.

This missense variant replaces glutamic acid with lysine at codon 143 in the EF-hand domain of the MYL3 protein. Computational prediction tool suggests that this variant may have a deleterious impact on protein structure and function (internally defined REVEL score threshold >=0.7, PMID: 27666373). An experimental study has shown that this variant may reduce affinity for the cardiac myosin heavy chain in vitro (PMID: 22131351). In a transgenic mouse model, this variant caused a phenotype consistent with severe restrictive cardiomyopathy, including increased ventricular stiffness, sarcomere abnormalities, and interstitial cardiac fibrosis (PMID: 28371863). This variant has been identified in heterozygosity in more than 30 individuals affected with hypertrophic cardiomyopathy (PMID: 27532257, 30706179, 30847666, 33495597, 35026164; communication with external laboratories: ClinVar variation ID: 14063). Some of these individuals were reported to carry other variants associated with cardiomyopathy. This variant has also been reported in homozygosity in two unrelated individuals affected with restrictive cardiomyopathy (PMID: 12021217, 21823217). Five heterozygous individuals from these two families were unaffected suggesting reduced penetrance. This variant has been identified in 4/282890 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Likely Pathogenic.

The p.E143K variant (also known as c.427G>A), located in coding exon 4 of the MYL3 gene, results from a G to A substitution at nucleotide position 427. The glutamic acid at codon 143 is replaced by lysine, an amino acid with similar properties. This alteration has been reported in the homozygous state in multiple unrelated individuals with early onset cardiomyopathy with restrictive physiology and/or hypertrophic cardiomyopathy (HCM) and has segregated with disease in affected homozygous siblings, while tested heterozygous family members were unaffected (Olson TM et al. Circulation. 2002;105(20):2337-40; Caleshu C et al. Am J Med Genet. A 2011;155A(9):2229-35; Ambry internal data; external communication). This alteration has also been detected in the heterozygous state in numerous individuals with HCM or who were referred for HCM genetic testing (McNamara JW et al. PLoS ONE. 2017;12(6):e0180064; Walsh R et al. Genet. Med. 2017;19:192-203; Ambry internal data; external communication). The vast majority of reported cases are of Hispanic ethnicity, and based on data from gnomAD, this alteration is present at a low frequency of 0.01% (4/35436) in the Latino sub-population. Functional studies suggest this alteration impacts a variety of mechanistic properties of myosin, and transgenic mice overexpressing E143K exhibit a phenotype consistent with restrictive cardiomyopathy; however, the clinical relevance of these results is unclear (Lossie J et al. Cardiovasc Res. 2012;93(3):390-6; Yuan CC et al. Cardiovasc Res. 2017;113(10):1124-1136; Wang Y et al. Open Biol. 2018;8(4):170240). This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Based on the majority of available evidence to date, this variant is likely to be pathogenic; however, in the heterozygous state, this variant may have reduced penetrance.

Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 21823217, 22957257, 28420666, 30297972, 25342278, 23594557, 27574918, 26443374, 21415409, 28658286, 25910212, 27532257, 28356264, 28771489, 31199839, 30706179, 28371863, 29669825, 31447099, 37511838, 22131351, 12021217, 34014247, 35288424, 34495297, 37652022, 30847666, 35026164, 33495597)

PP3, PM2, PM3, PS3_supporting, PS4_moderate

Note this variant was found in clinical genetic testing performed by one or more labs who may also submit to ClinVar. Thus any internal case data may overlap with the internal case data of other labs. The interpretation reviewed below is that of the Stanford Center for Inherited Cardiovascular Disease. p.Glu143Lys (c.427G>A) We last reviewed this variant in 2013. In August 2015 we updated the review with ClinVar and ExAC. We did not look for additional case data given that the available data is conflicting. Per ClinVar, GeneDx and LMM both classify it as a variant of uncertain significance. The variant has been seen in at least 8 maybe 9 cases of cardiomyopathy. At least 5 of these individuals are Hispanic. At least three have another pathogenic variant. Olsen and colleagues report this variant in a family in which three siblings presented with restrictive cardiomyopathy in the second decade of life with an apparent autosomal recessive mode of inheritance. There was known consanguinity (parents were second cousins). The phenotype includes mid-cavitary left ventricular hypertrophy with mild dynamic obstruction in systole. Systolic function was normal, but restrictive physiology was diagnosed on the basis of Doppler measurements, severe biatrial enlargement, and mild pulmonary hypertension. Two siblings were found to be homozygous for this variant (no sample was available for the third). A fourth sibling, parents (age 40) and paternal grandfather (age 70) were all found to be heterozygous and clinically unaffected. The ancestry of this patient was not reported. (Olsen T et al., 2002). This variant was also described in the homozygous state in a patient from El Salvador with restrictive cardiomyopathy and no signs of left ventricular hypertrophy. This individual also carried the p.Gly57Glu in MYL2 gene. The patient’s mother was the only family member available for evaluation. On genetic testing she was found to be a double heterozygote for the p.Glu143Lys mutation in MYL3 and the p.Gly57Glu mutation in MYL2. She had a normal transthoracic echocardiogram, electrocardiogram, and physical exam at 45 years of age (Caleshu et al., 2011) This variant is in coding exon 4 of 7 exons. MYL3 is a calcium binding protein and acts as a stabilizer of the myosin head. In a functional study of rat cardiomyopcytes, recombinant protein produced with this alteration was shown to significantly lower the binding affinity to the myosin heavy chain (Lossie J et al., 2012). In silico analysis with PolyPhen-2 predicts the variant to be possibly damaging. The Glutamic acid at codon 143 is conserved across species, as are neighboring amino acids. The variant was entered into dbSNP(build 36) as rs104893750 based on the report by Olson et al. but has not been found independently by any other SNP discovery efforts. SNP rs104893750 is absent from HapMap data release 28. There is no nonsynonymous variation at codon 143 listed in the Exome Aggregation Consortium dataset (http://exac.broadinstitute.org/), which currently includes variant calls on ~64,000 individuals of European, African, Latino and Asian descent (as of August 27th, 2015). Of note given the patient's ancestry and the case data, this includes 5789 Latino individuals. I checked coverage at that site and the mean and median coverage were over 90 (3-46901019-C-T).