ClinVar Genomic variation as it relates to human health

This c.170C>G (p.Ala57Gly) variant has previously been detected in several patients and families with hypertrophic cardiomyopathy [PMID 11174330, 20641121]. The penetrance of the disorder was estimated between 63 and 78% in carriers over 18 years of age [PMID 11174330, 20641121]. In vitro assays showed that the mutant protein has reduced binding affinity to myosin [PMID 22131351]. Transgenic mice expressing the mutant allele showed hypertrophic cardiomyopathy, consistent with the human phenotype [PMID 23748425]. This variant has been reported in 11 heterozygous individuals from the ExAC database (http://exac.broadinstitute.org/variant/3-46902303-G-C). This variant is conserved in mammals. Computer based prediction algorithms (SIFT and Polyphen-2) yield discordant results regarding the pathogenicity of this change. Nevertheless, based on reported patients and functional data, this variant is classified as pathogenic. Pathogenic variants in the MYL3 gene are considered medically actionable [ACMG59, PMID 27854360].

Variant summary: MYL3 c.170C>G (p.Ala57Gly) results in a non-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 (ACMG PP3). The variant allele was found at a frequency of 7.1e-05 in 252370 control chromosomes (gnomAD). The observed variant frequency is approximately 2.9- fold the estimated maximal expected allele frequency for a pathogenic variant in MYL3 causing Hypertrophic Cardiomyopathy phenotype (2.5e-05), suggesting that the variant is benign. This data should be interpreted with caution in regard to cardiac phenotypes, however, as gnomAD control data includes several well-phenotyped cardiac cohorts (e.g. Jackson Heart Study, Myocardial Infarction Genetics Consortium, etc.) and no phenotypic information about the individuals who had this variant are provided in this database (ACMG BS1, not engaged). c.170C>G has been reported in the literature in multiple individuals affected with Hypertrophic Cardiomyopathy. Most notably, it was found in two unrelated Korean families with HCM in which affected individuals presented with a classic asymmetric septal hypertrophy (Lee_2001, Choi_2010). The variant was shown to cosegregate with disease in both of these families, with one family having five affected family members carrying the variant over two generations, although the variant appeared to demonstrate incomplete penetrance as one unaffected family member carried the variant (age 48 at the time of Choi_2010 publication). The variant has also been reported in other HCM patients, although with limited evidence (such as cosegregation data) for causality (examples- Lee_2001, Murakami_2001, Weissler-Snir_2017, Ho_2018, Robyns_2020, Chung_2020, Kim_2020). Overall, these data indicate that the variant may be associated with disease (ACMG PP1 moderate). However, the variant has also been reported in at least one additional unaffected individual (Natarajan_2016). Several publications report experimental evidence evaluating an impact on protein function. Although several studies report statistically significant differences in structure (e.g., fibrosis and myofilament disarray in a transgenic mouse, Muthu_2011) and function (e.g., increased Ca2+ sensitivity and decreased maximal tension, Kazmierczak_2013), the differences between the variant and controls in most of the data are relatively small and the biological significance is unknown (ACMG PS3, not engaged). Seven other clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. Multiple laboratories reported the variant with conflicting assessments (uncertain significance, n=4; pathogenic, n=3). This variant was re-classified following a discrepancy resolution discussion initiated by Dr. Birgit Funke and Megan Crawley to all ClinVar submitter labs in June-2018. As of Dec-2018, this variant was slated for finalization as a consensus VUS by the ClinGen expert review panel (personal correspondence, Dr. Funke, Melissa A. Kelly). Based on the evidence outlined above, the variant was re-evaluated to retain its previous classification as VUS-possibly pathogenic.

The variant was co-segregated with Cardiomyopathy, hypertrophic, 8 in multiple affected family members (PMID: 11174330, PP1_P). The variant has been observed in at least two similarly affected unrelated individuals (PMID: 11174330, 20641121, 29121657, 27532257, PS4_M). Functional studies provide supporting evidence of the variant having a damaging effect on the gene or gene product(PMID: 22131351, 23748425) (PS3_P). In silico tool predictions suggest damaging effect of the variant on gene or gene product (REVEL: 0.78, 3CNET: 0.96, PP3_P). A missense variant is a common mechanism associated with Cardiomyopathy (PP2_P). It is observed at an extremely low frequency in the gnomAD v2.1.1 dataset (total allele frequency: 0.000072, PM2_M). Therefore, this variant is classified as likely pathogenic according to the recommendation of ACMG/AMP guideline.

The heterozygous c.170C>G (p.Ala57Gly) missense variant in the MYL3 gene has been reported as heterozygous in multiple individuals affected with hypertrophic cardiomyopathy [HCM; PMID: 27532257, 29121657, 27831900, 32492895]. This missense variant was reported in two unrelated Korean families with HCM and co-segregated the disease in both families [PMID: 11174330, 20641121]. However, one unaffected family member (48 years old at the time ofclinical evaluation) with heterozygous variant revealed no HCM phenotype [PMID: 20641121]. This variant has been reported in the ClinVar database [Variation ID:31780] with conflicting interpretations [Uncertain significance = 5 and pathogenic =3]. Functional studies suggest a reduced binding affinity of mutated MYL3(p.Ala57Gly) to the cardiac myosin heavy chain [PMID: 22131351] and the disruption of myofilament function leading to hypertrophy in a transgenic mice expressing the mutated MYL3 (p.Ala57Gly) [PMID: 23748425]. However, these types of functional studies may not accurately determine the true biological effect(s) and are not validated in clinical diagnostic laboratory setting. The variant has 0.00002630 allele frequency in the gnomAD (v3) database (4 out of 152078 heterozygous alleles,no homozygotes) and 0.00007158 allele frequency in the gnomAD(v2) database (18 out of 251470 heterozygotes, 0.027% allele frequency in East Asiansub-population), which is higher than the maximum expected allele frequency for a pathogenic variant in the MYL3-related dominant HCM. The variant affects a conserved residue [Ala57] located in the EF-hand domain of MYL3 gene. The variant is predicted deleterious by multiple In silico prediction tools (CADD score = 26.6,REVEL score = 0.780). Based on the available evidence, the heterozygous c.170C>G (p.Ala57Gly) missense variant identified in the MYL3 gene is reported as a Variant of Uncertain Significance.

The p.A57G variant (also known as c.170C>G), located in coding exon 3 of the MYL3 gene, results from a C to G substitution at nucleotide position 170. The alanine at codon 57 is replaced by glycine, an amino acid with similar properties. This variant has been detected in multiple unrelated patients with hypertrophic cardiomyopathy and was reported to co-segregate with disease in two unrelated families (Lee W et al., Am. Heart J. 2001 Feb; 141(2):184-9; Choi JO et al., Clin Cardiol 2010 Jul; 33(7):430-8; Murakami C et al., Kitasato Med J 2014; 44:47-55; Weissler-Snir A et al. Circ Cardiovasc Imaging, 2017 Feb;10; Ambry internal data; LMM pers. comm.). However, this alteration has also been identified in unaffected individuals and as a secondary finding in individuals who underwent whole exome sequencing for non-cardiovascular indications (Jang MA et al. Genet. Med., 2015 Dec;17:1007-11; Maxwell KN et al. Am. J. Hum. Genet., 2016 May;98:801-817; Natarajan P et al. Sci Transl Med, 2016 11;8:364ra151; Ambry internal data). Furthermore, based on data from gnomAD, the frequency for this variant is above the maximum credible frequency for a disease-causing variant in this gene based on internally established thresholds (Karczewski et al. Nature. 2020 May;581(7809):434-443; Whiffin et al. Genet Med. 2017 10;19:1151-1158). Multiple functional studies suggest that this alteration may impact MYL3 structure and function, but the observed differences are relatively minor and the clinical relevance is uncertain (Muthu P et al., FASEB J. 2011 Dec; 25(12):4394-405; Lossie J et al., Cardiovasc. Res. 2012 Mar; 93(3):390-6; Kazmierczak K et al., Am. J. Physiol. Heart Circ. Physiol. 2013 Aug; 305(4):H575-89; Ma N et al. Circulation, 2018 Dec;138:2666-2681; Wang Y et al. Open Biol, 2018 04;8). This amino acid position is well conserved in available vertebrate species. In addition, the in silico prediction for this alteration is inconclusive. Since supporting evidence is conflicting at this time, the clinical significance of this alteration remains unclear.

In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; Published functional studies are conflicting; one mouse model showed cardiac fibrosis and hypertrophy, however the same model has been previously reported to lack a hypertrophic phenotype (PMID: 23748425, 21885653); This variant is associated with the following publications: (PMID: 11174330, 26443374, 27831900, 34217267, 33288880, 34293104, 34014247, 33726816, 33087929, 33407484, 17142342, 22131351, 22957257, 21415409, 20641121, 27153395, 26385864, 25856671, 27532257, 28518168, 21885653, 29914921, 32034976, 31513939, 31447099, 32492895, 32380161, 32686758, 35626289, 33803477, 28193612, 29121657, 30706179, 33935716, 33495596, 37728764, 30105547, 28241245, 37629714, 31905684, 31131433, 37583586, 37929589, 38175910, 37652022, 29669825, 26668058, 23748425)

ACMG score pathogenic

This missense variant replaces alanine with glycine at codon 57 of the MYL3 protein. Computational prediction 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 lowers the binding capacity of the MYL3 protein to the myosin lever-arm in vitro (PMID: 22131351). In addition, transgenic mice expressing this variant showed decreased maximal force generation, high levels of heart fibrosis, and hypertrophy compared to wild-type (PMID: 23748425, 32034976). This variant has been reported in a three-generation Korean family affected with hypertrophic cardiomyopathy (PMID: 11174330, 20641121). Among 12 carriers in this family, 5 individuals were affected with late-onset hypertrophic cardiomyopathy, 6 individuals were affected with late-onset atrial fibrillation, heart failure and sudden cardiac death, and one adult individual had normal ECG and echocardiographic findings. This variant has also been reported to show 50% penetrance in a small family affected with hypertrophic cardiomyopathy (PMID: 29121657). This variant has been reported in another five unrelated individuals affected with hypertrophic cardiomyopathy (PMID: 30105547, 32492895, 33495596, 35626289, Irie et al. 2011, doi:10.1016/j.fsigss.2011.08.072). However, this variant has also been identified in 18/251470 chromosomes in the general population by the Genome Aggregation Database (gnomAD). In addition, in multiple case-control studies recently conducted, this variant has not shown a significant association with hypertrophic cardiomyopathy (communication with an external laboratory; ClinVar SCV000199362.6). Although there is a suspicion that this variant may be associated with disease, additional studies are necessary to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance.

This sequence change replaces alanine, which is neutral and non-polar, with glycine, which is neutral and non-polar, at codon 57 of the MYL3 protein (p.Ala57Gly). This variant is present in population databases (rs139794067, gnomAD 0.03%). This missense change has been observed in individual(s) with hypertrophic cardiomyopathy (PMID: 11174330, 20641121, 27532257, 28193612, 29121657, 31513939, 32380161, 33407484). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 31780). 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, 23748425). In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance.

This missense variant replaces alanine with glycine at codon 57 of the MYL3 protein. Computational prediction 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 lowers the binding capacity of the MYL3 protein to the myosin lever-arm in vitro (PMID: 22131351). In addition, transgenic mice expressing this variant showed decreased maximal force generation, high levels of heart fibrosis, and hypertrophy compared to wild-type (PMID: 23748425, 32034976). This variant has been reported in a three-generation Korean family affected with hypertrophic cardiomyopathy (PMID: 11174330, 20641121). Among 12 carriers in this family, 5 individuals were affected with late-onset hypertrophic cardiomyopathy, 6 individuals were affected with late-onset atrial fibrillation, heart failure and sudden cardiac death, and one adult individual had normal ECG and echocardiographic findings. This variant has also been reported to show 50% penetrance in a small family affected with hypertrophic cardiomyopathy (PMID: 29121657). This variant has been reported in another five unrelated individuals affected with hypertrophic cardiomyopathy (PMID: 30105547, 32492895, 33495596, 35626289, Irie et al. 2011, doi:10.1016/j.fsigss.2011.08.072). However, this variant has also been identified in 18/251470 chromosomes in the general population by the Genome Aggregation Database (gnomAD). In addition, in multiple case-control studies recently conducted, this variant has not shown a significant association with hypertrophic cardiomyopathy (communication with an external laboratory; ClinVar SCV000199362.6). Although there is a suspicion that this variant may be associated with disease, additional studies are necessary to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance.

The p.Ala57Gly variant in MYL3 has been identified in at least 17 individuals with HCM (Lee 2001 PMID: 11174330, Choi 2010 PMID: 20641121, Murakami 2014 (no PMID), Robyns 2020 PMID: 31513939, Chung 2020 PMID: 32380161, Kim 2020 PMID: 32492895, GeneDx pers. comm., Ambry pers. comm., Invitae pers. comm., LMM data) and segregated with disease in 5 affected family members from 2 families (Lee 2001 PMID: 11174330, Choi 2010 PMID: 20641121). It has also been reported by other clinical laboratories in ClinVar (Variation ID 31780) and has been identified in 0.03% (5/18394) of East Asian chromosomes and 0.01% (12/113750) of European chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org), which is higher than the maximum expected allele frequency for a pathogenic variant in the MYL3 gene associated with autosomal dominant HCM. In vivo and in vitro functional studies provide some evidence that this variant impacts protein function; however, these types of assays may not accurately represent biological function (Muthu 2011 PMID: 21885653, Lossie 2012 PMID: 22131351, Kazmierczak 2013 PMID: 23748425, Ma 2018 PMID: 29914921). Computational prediction tools and conservation analysis do not provide strong support for or against an impact to the protein. In summary, while there is some suspicion for a pathogenic role, based on the high allele frequency of this variant in the gnomAD population database the clinical significance of the p.Ala57Gly variant is uncertain. ACMG/AMP Criteria applied: PS3_Moderate; PP1_Moderate, BS1_Supporting.