ClinVar Genomic variation as it relates to human health

The MYL3 Ala57Asp has been previously identified in HCM cases (Jaafar N, et al., 2016; Almaas VM, et al., 2013; Vazquez-Alvarez MC, et al., 2012; Fokstuen S et al., 2011; Calore C, et al., 2011; Choi JO, et al., 2010). The cases reported by Vazquez-Alvarez MC, et al. (2012) included a prenatal HCM case, however the patient was also diagnosed with Noonan syndrome. An iPSC-CM model showed that both heterozygous and homozygous cardiac cells did not produce an HCM phenotype (Ma N, et al., 2018). Interestingly a different change at this position (Ala57Gly) has been reported in multiple HCM cases, suggesting that an amino acid substitution at this site may not be tolerated. Computational tools SIFT, PolyPhen2 and MutationTaster predict this variant to have a deleterious effect. The variant is present in the Genome Aggregation Database (http://gnomad.broadinstitute.org/), at an allele frequency of 0.0001588, which is higher then expected for HCM. We identified this variant in an HCM patient with a family history of HCM. A second variant (ACTC1 Ala323Val) was also identified in this patient. The ACTC1 variant was found to segregate to an affected fourth degree relative but MYL3 Ala57Asp did not segregate. The variant has been found to segregate with disease in two homozygous siblings reported by LMM (ClinVar: SCV000208872). In summary, based on the lack of phenotype in iPSC-CM, high allele frequency and non-segregation in affected family members, we classify MYL3 Ala57Asp (in the heterozygous form) as "likely benign".

The p.A57D variant (also known as c.170C>A), located in coding exon 3 of the MYL3 gene, results from a C to A substitution at nucleotide position 170. The alanine at codon 57 is replaced by aspartic acid, an amino acid with dissimilar properties. This alteration has been detected in individuals from hypertrophic cardiomyopathy (HCM) cohorts (Fokstuen S et al. J Med Genet. 2011;48:572-6; Almaas VM et al. Europace. 2013;15:1319-27; Berge KE et al. Clin Genet. 2014;86:355-60; Rubattu S et al. Int J Mol Sci, 2016 Jul;17:1239). This variant has also been identified in two unrelated homozygous individuals with HCM, both of whom had consanguineous parents and family histories of sudden death; heterozygous family members were reportedly unaffected, and one proband had three homozygous young children who had normal echocardiograms at the time of evaluation (Jaafar N et al. Genet Test Mol Biomarkers. 2016;20:674-679; Osborn DPS et al. Genet Med, 2021 Apr;23:787-792). In addition, multiple probands with this variant have had additional pathogenic variants in other HCM-associated genes (Ambry internal data; Broendberg AK et al. Eur J Hum Genet, 2018 03;26:303-313). Furthermore, this variant did not demonstrate a deleterious impact in several functional assays performed in human induced pluripotent stem cells heterozygous or homozygous for this alteration, although zebrafish models showed only partial rescue; given that the molecular mechanisms underlying HCM are not fully understood, the physiological significance of these results is unclear (Ma N et al. Circulation. 2018;138:2666-2681; Osborn DPS et al. Genet Med, 2021 Apr;23:787-792). 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). This amino acid position is well conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Since supporting evidence is conflicting at this time, the clinical significance of this alteration remains unclear.

This variant was observed as part of a predisposition screen in an ostensibly healthy population. A literature search was performed for the gene, cDNA change, and amino acid change (where applicable). Publications were found based on this search. However, the evidence from the literature, in combination with allele frequency data from public databases where available, was not sufficient to rule this variant in or out of causing disease. Therefore, this variant is classified as a variant of unknown significance.

In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; Functional studies using human induced pluripotent stem cell (iPSC) lines demonstrated that p.(A57D) expressed in the heterozygous and homozygous state did not produce an HCM phenotype (PMID: 29914921); This variant is associated with the following publications: (PMID: 29687901, 24111713, 27483260, 21239446, 27574918, 28971120, 28518168, 29914921, 30624779, 31006259, 23426552, 31019283, 33288880, 34636345, 34509299, 34137518, 33935716, 29710196, 26779504, 29343803, 35653365)

BS1, PP3

Criteria: PP2, PP3, BS3

This sequence change replaces alanine, which is neutral and non-polar, with aspartic acid, which is acidic and polar, at codon 57 of the MYL3 protein (p.Ala57Asp). This variant is present in population databases (rs139794067, gnomAD 0.06%). This missense change has been observed in individual(s) with hypertrophic cardiomyopathy (PMID: 21239446, 23426552, 24111713, 27483260). ClinVar contains an entry for this variant (Variation ID: 43121). 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 does not substantially affect MYL3 function (PMID: 29914921). 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 aspartic acid at codon 57 of the MYL3 protein. Computational prediction tools indicate that this variant has a deleterious impact on protein structure and function. A functional study with human-induced pluripotent stem cells has shown that this variant has no significant impact on MYL3 protein as determined by gene expression, sarcomere structure, cell size, contractility, action potentials and calcium handling (PMID: 29914921). However, another study with zebrafish knockdown model has shown that this variant is unable to rescue the compromised cardiac function as wild-type MYL3 does (PMID: 33288880). This variant has been reported in heterozygous multiple individuals affected with hypertrophic cardiomyopathy (PMID: 21239446, 23426552, 24111713, 27483260, 27574918, 33288880, 34137518, 37431535), as well as in five unrelated homozygous individuals (PMID: 27574918, 33288880, 34137518, 37431535). This variant has also been identified in 46/282810 chromosomes in the general population by the Genome Aggregation Database (gnomAD). In summary, this is a variant with uncertain functional impact that has been observed in the control population as well as in affected individuals. The available evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance.

The p.Ala57Asp variant in MYL3 has been reported in heterozygous state in at least 4 individuals with hypertrophic cardiomyopathy (Fokstuen 2011 PMID: 21239446, Almaas 2013 PMID: 23426552, Berge 2014 PMID: 24111713, Norrish 2019 PMID: 31006259, LMM data); two of these individuals were heterozygous for pathogenic variants in another gene. This variant has been identified in homozygous state in at least 2 individuals with cardiomyopathy (Osborn 2021 PMID: 33288880, LMM data). It has also been identified in 0.06% (17/30616) of South Asian chromosomes by gnomAD (http://gnomad.broadinstitute.org). This variant has also been reported in ClinVar (Variation ID 43121). Computational prediction tools and conservation analyses suggest that this variant may impact the protein, though this information is not predictive enough to determine pathogenicity. In vitro functional studies provide conflicting evidence regarding the impact of this variant on protein function (Ma 2018 PMID: 29914921 , Osborn 2021 PMID: 33288880); however, these types of assays may not accurately represent biological function. Another variant involving this codon (p.Ala57Gly) has been identified in individuals with hypertrophic cardiomyopathy and is classified as uncertain significance by this laboratory. In summary, the clinical significance of this variant is uncertain. ACMG/AMP Criteria applied: PP3, BP5.

MYL3 NM_000258.2 exon 3 p.Ala57Asp (c.170C>A): This variant has been reported in the literature in several individuals with HCM (Fokstuen 2011 PMID:21239446, Almaas 2013 PMID:23426552, Berge 2014 PMID:24111713, Jaafar 2015 PMID:26779504, Rubatta 2016 PMID:27483260, Dejgaard 2017 PMID:28971120). This variant is present in 0.05% (17/30616) of South Asian alleles in the Genome Aggregation Database (http://gnomad.broadinstitute.org/variant/3-46902303-G-T) and is present in ClinVar (Variation ID:43121). Please note, disease causing variants may be present in control databases at low frequencies, reflective of the general population, autosomal recessive inheritance carrier status, and/or variable expressivity. Evolutionary conservation and computational predictive tools suggest that this variant may impact the protein. Additionally, another variant at the same amino acid (p.Ala57Gly) has been reported in association with disease. 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 missense variant replaces alanine with aspartic acid at codon 57 of the MYL3 protein. Computational prediction suggests that this variant may have deleterious impact on protein structure and function (internally defined REVEL score threshold >= 0.7, PMID: 27666373). A functional study with human-induced pluripotent stem cells has shown that this variant has no significant impact on MYL3 protein as determined by gene expression, sarcomere structure, cell size, contractility, action potentials and calcium handling (PMID: 29914921). However, another study with zebrafish knockdown model has shown that this variant is unable to rescue the compromised cardiac function as wild-type MYL3 does (PMID: 33288880). This variant has been reported in multiple individuals affected with hypertrophic cardiomyopathy (PMID: 21239446, 23426552, 24111713, 27483260, 27574918, 33288880, 34137518), including three unrelated homozygotes (PMID: 27574918, 33288880, 34137518). This variant has also been identified in 46/282810 chromosomes in the general population by the Genome Aggregation Database (gnomAD). In summary, this is a variant with uncertain functional impact that has been observed in the control population as well as in affected individuals. The available evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance.

MYL3 p.Ala57Asp (c.170C>A) (NM_000258.1) There is moderate case data with some segregation demonstrated, but the variant is also seen at relatively high frequency in population databases and has been seen in combination with a pathogenic variant and in a homozygous state in an unaffected individual. We consider this conflicting data and deem the variant to be a variant of uncertain significance. We do not feel that this variant is suitable for assessing risk in healthy relatives ("predictive genetic testing"). We have seen this variant in an individual with HCM. Testing was performed by Familion. The variant has been seen in at least 5 unrelated cases of HCM (not including this patient's family). There is conflicting case data. This variant has been reported in 1 individual with HCM by Fokstuen et al., 2011, and in 1 individual with HCM by Almaas et al., 2013. It has also been reported in a Korean family, segregating with disease in 5 individuals with HCM by Choi et al., 2010. Controls/Population data: The variant has not been seen in 400 Familion laboratory controls. The variant was reported online in 14 of 60,669 (0.023%) individuals 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 6/25/16). Specifically, the variant was observed in following ethnicities European (Non-Finnish) 5/33,342; East Asian 3/4,323; Latino 1/5,787; South Asian 5/8,256. The phenotype of those individuals is not publicly available. The dataset is comprised of multiple cohorts, some of which were recruited from the general population, others were enriched for common cardiovascular disease.

The MYL3 c.170C>A variant is predicted to result in the amino acid substitution p.Ala57Asp. This variant has been reported in multiple individuals with hypertrophic cardiomyopathy (HCM) (Fokstuen et al. 2011. PubMed ID: 21239446; Berge et al. 2014. PubMed ID: 24111713; Almaas et al. 2013. PubMed ID: 23426552; Jaafar et al. 2015. PubMed ID: 26779504; Rubattu et al. 2016. PubMed ID: 27483260; Dejgaard et al. 2017. PubMed ID: 28971120; Table S10, Stava et al. 2022. PubMed ID: 35653365). This variant was also reported in the homozygous state to be associated with autosomal recessive HCM (Osborn et al. 2021. PubMed ID: 33288880). However, some individuals with this variant also carried additional variants in other HCM-related genes that are likely contributing to a HCM phenotype (Berge et al. 2014. PubMed ID: 24111713; Rubattu et al. 2016. PubMed ID: 27483260). Functional studies using human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) showed that both heterozygous and homozygous MYL3(170C>A)-iPSC-CMs did not reproduce a HCM phenotype (Ma et al. 2018. PubMed ID: 29914921). However, in another study, this variant failed to rescue the compromised cardiac function in a zebrafish knockdown model (Osborn et al. 2021. PubMed ID: 33288880). This variant is reported in 0.056% of alleles in individuals of South Asian descent in gnomAD. At this time, the clinical significance of this variant is uncertain due to the absence of conclusive functional and genetic evidence.