homozygous case; clinical and carrier status of parents are unkown

ACMG criterias used: PS3, PS4, PM2 and PP3.

PP3, PM2, PM3_supporting, PS3_supporting, PS4_moderate

This missense variant replaces glycine with arginine at codon 531 of the MYBPC3 protein. Computational prediction tools and conservation analyses suggest that this variant may have a deleterious impact on the protein function. In heart tissue engineered from mouse cardiac cells, this variant in heterozygous state was associated with a hypercontractile phenotype (PMID: 27108529). This variant has been reported in over ten unrelated individuals affected with hypertrophic cardiomyopathy (PMID: 16858239, 18533079, 20173211, 20624503, 21835320, 23508784, 23690394, 27483260, 27532257, 27600940, 28193612, 28241245, 28986452, 31941943, 32228044, 32369506, 36291626). Two of these individuals carried a second pathogenic variant either in the same gene (PMID: 20624503) or in another gene (PMID: 32228044). This variant has also been reported in an individual affected with left ventricular non-compaction (PMID: 28798025). This variant has been identified in 5/278462 chromosomes in the general population by the Genome Aggregation Database (gnomAD). A different variant causing the same amino acid change, c.1591G>A p.Gly531Arg, is also considered to be disease-causing (ClinVar variation ID: 164109). In summary, this variant has shown a relevant phenotype in an experimental study and has been observed in multiple individuals affected with hypertrophic cardiomyopathy. Based on available evidence, this variant is classified as Likely Pathogenic.

PS1, PS4, PS3_mod, PM2, PP3, PP5

This is a nonsynonymous variant in the MYBPC3 gene (OMIM: 600958). Pathogenic variants in this gene have been associated with autosomal dominant hypertrophic cardiomyopathy 4. This variant has been reported in at least 4 unrelated affected individuals (PMID: 16858239, 21835320, 35753512, 36252119). Functional studies have shown that this variant alters MYBPC3 protein function (PMID: 27108529) (PS3_Moderate), and mulltiple computational algorithms predict a deleterious effect for this variant (REVEL score: 0.854) (PP3). Furthermore, an alternate nucleotide substitution resulting in the same amino acid change (c.1591G>A) has been previously reported as pathogenic (PMID: 27108529) (PS1). This variant has a 0.0033% maximum allele frequency in non-founder control populations (https://gnomad.broadinstitute.org/) (PM2). Based on the current evidence, this variant is classified as likely pathogenic for autosomal dominant hypertrophic cardiomyopathy 4.

PS1, PM2, PM1, PP3, PP5

This variant was identified as compound heterozygous with NM_000256.3:c.1591G>C._x000D_ Criteria applied: PS1, PS4, PM2_SUP, PP3

The p.Gly531Arg variant in MYBPC3 has been reported in association with two distinct nucleotide changes: c.1591G>C and c.1591G>A. Considering both nucleotide changes, this variant has been reported in 1 individual with dilated cardiomyopathy (DCM; c.1591G>A; Waldmuller 2011 PMID: 21750094) and in over 10 individuals with hypertrophic cardiomyopathy (HCM; c.1591G>A: Girolami 2006 PMID: 16858239, Olivotto 2008 PMID: 18533079, Garcia-Castro 2009 PMID: 19150014, Michels 2011, Waldmuller 2011 PMID: 21750094, Walsh 2017 PMID: 27532257, LMM data; c.1591G>C: Millat 2010 PMID: 20624503, Olivotto 2011 PMID: 21835320, Coto 2012 PMID: 22765922, Rubattu 2016 PMID: 27483260, LMM data). Of the individuals with HCM, at least 4 individuals carried a second pathogenic MYBPC3 variant and had early onset disease (LMM data, Millat 2010 PMID: 20624503, Rubattu 2016 PMID: 27483260). Furthermore, the c.1591G>C variant was found by our laboratory to segregate with disease in 2 affected relatives from 1 family. This variant has also been reported by other clinical laboratories in ClinVar (Variation IDs: 164109 (c.1591G>A) and 42550 (c.1591G>C)). Additionally, the c.1591G>A variant has been identified in 0.01% (2/15288) of Latino and in 0.002% (1/41450) of African/African American chromosomes by gnomAD, while the c.1591G>C variant has been identified in 0.004% (3/68034) of European chromosomes in gnomAD (https://gnomad.broadinstitute.org, v.3.1.2). A functional study in which the c.1591G>C (p.Gly531Arg) variant was transfected into MYBPC3 knockout mouse cardiomyocytes alone and in combination with wild-type MYBPC3 (to represent homozygous and heterozygous disease states) revealed abnormal contractile force in both scenarios compared to wild-type (Wijnker 2016 PMID: 27108529). Computational prediction tools and conservation analysis also suggest that the variant may impact the protein. 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. ACMG/AMP Criteria applied: PS4_Moderate, PM2_Supporting, PS3_Moderate, PP3.

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Likely pathogenic. Following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with hypertrophic cardiomyopathy 4 (HCM; MIM#115197). (I) 0108 - This gene is associated with both recessive and dominant disease. Dominant inheritance is frequently reported in adult-onset conditions and recessive inheritance results in a more severe early onset phenotype (OMIM). (I) 0115 - Variants in this gene are known to have variable expressivity (PMID: 32841044). (I) 0200 - Variant is predicted to result in a missense amino acid change from glycine to arginine. (I) 0252 - This variant is homozygous. (I) 0304 - Variant is present in gnomAD (v2 and v3 (non-v2)) <0.01 for a recessive condition (8 heterozygotes, 0 homozygotes). A different nucleotide substitution resulting in the same amino acid change has also been observed in gnomAD (v2 and v3 (non-v2): 12 heterozygotes, 0 homozygotes). (SP) 0309 - An alternative amino acid change at the same position has been observed in gnomAD (v3) (p.(Gly531Trp): 1 heterozygote, 0 homozygotes). (I) 0501 - Missense variant consistently predicted to be damaging by multiple in silico tools or highly conserved with a major amino acid change. (SP) 0600 - Variant is located in the annotated Immunoglobulin I-set domain 3 domain (DECIPHER; PMID: 32841044). (I) 0710 - Another missense variant comparable to the one identified in this case has inconclusive previous evidence for pathogenicity. The p.(Gly531Trp) variant has been classified as a VUS by multiple clinical diagnostic laboratories (ClinVar). (I) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant (G>C) and a different nucleotide substitution (G>A), which result in the same amino acid change, have been classified as likely pathogenic and pathogenic by multiple clinical diagnostic laboratories and have been reported in multiple individuals with HCM (ClinVar; Atlas of Cardiac Genetic Variation; PMIDs: 21750094, 24793961, 28356264, 31941943, 32841044, 36291626). Additionally, this amino acid change has also been classified as a VUS by two clinical diagnostic laboratories and has also been reported in one individual with left ventricular noncompaction cardiomyopathy (ClinVar; PMID: 28798025). (SP) 1002 - This variant has moderate functional evidence supporting abnormal protein function. Mutant constructs transfected into knockout engineered heart cells mice tissues and expressed either as heterozygous or homozygous demonstrated abnormal force and contraction velocity compared to WT (PMID: 27108529). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

MYBPC3 Gly531Arg variant has been reported as two nucleotide changes: c.1591G>C and c.1591G>A. When considered together this variant has been identified in 1 DCM proband (Waldmuller S, et al., 2011) and more than 10 individuals with HCM and no other pathogenic variants (Girolami F, et al., 2006; Ho CY, et al., 2009; Michels M, 2011; Waldmuller S, et al., 2011; Olivotto I, et al., 2011; Coto E, et al., 2012; Bos JM, et al., 2014; Lopes LR, et al., 2015; Rubattu S, et al., 2016; Walsh R, et al., 2017; LMM, Pers. Comm.; GeneDx, Pers. Comm.) The variant has also been found to segregate with disease in at least 1 family (LMM, Pers. Comm.). We have identified the c.1591G>C in 2 HCM probands. One proband has family history of disease in their sibling, but segregation was not possible. In the other family, the proband also harbors a second likely pathogenic TNNT2 Phe110Leu. This variant is present at a low frequency in the Genome Aggregation Database (MAF= 0.000022; http://gnomad.broadinstitute.org/). In vitro functional studies show that p.Gly531Arg causes hypercontractility in engineered heart tissue (Wijnker PJ, et al., 2016). In silico tools SIFT, PolyPhen-2 and MutationTaster predict the variant to be deleterious. In summary, the variant has been reported in numerous cases, it is rare in the general population, functional studies and in silico tools predict the variant to impact protein function, therefore we classify MYBPC3 Gly531Arg as 'Likely Pathogenic'.

The p.G531R variant (also known as c.1591G>C), located in coding exon 17 of the MYBPC3 gene, results from a G to C substitution at nucleotide position 1591. The glycine at codon 531 is replaced by arginine, an amino acid with dissimilar properties. This variant, and another variant leading to the same amino acid substitution (c.1591G>A), have been reported in individual(s) with features consistent with hypertrophic cardiomyopathy (HCM) (Girolami F et al. J Cardiovasc Med (Hagerstown), 2006 Aug;7:601-7; Olivotto I et al. Mayo Clin Proc, 2008 Jun;83:630-8; Garc&iacute;a-Castro M et al. Rev Esp Cardiol, 2009 Jan;62:48-56; Ho CY et al. Circ Cardiovasc Genet, 2009 Aug;2:314-21; Millat G et al. Eur J Med Genet, 2010 Jul;53:261-7; Waldm&uuml;ller S et al. Eur J Heart Fail, 2011 Nov;13:1185-92; Lopes LR et al. Heart, 2015 Feb;101:294-301; Rubattu S et al. Int J Mol Sci, 2016 Jul;17; Walsh R et al. Genet Med, 2017 Feb;19:192-203; external communication; Ambry internal data). In a functional study utilizing engineered heart tissue derived from MYBPC3 knockout cardiomyocytes, c.1591G>C p.G531R was unable to completely rescue the hypercontractile phenotype of the knockout tissue; however, the physiological relevance of this result is unclear (Wijnker PJ et al. J. Mol. Cell. Cardiol. 2016;97:82-92). 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.

Identified in patients with cardiomyopathy referred for genetic testing at GeneDx and in published literature, although clinical details were often limited (PMID: 21835320, 25351510, 27483260, 27532257, 22765922, 16858239, 32841044, 35208637, 36291626, 20624503, 36252119, 36264615, 37652022); Not observed at significant frequency in large population cohorts (gnomAD); Functional studies examining the effect of c.1591 G>C in mouse-derived engineered heart tissue demonstrated that tissue expressing the variant is associated with a hypercontractile phenotype compared to wild type cardiac tissue (PMID: 27108529); 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: 28241245, 25351510, 23690394, 19150014, 27532257, 23508784, 27483260, 28193612, 16858239, 22765922, 21835320, 28798025, 34426522, 32841044, 35208637, 35753512, 35433691, 36291626, 35130036, 20624503, 36264615, 36252119, 37652022, 36243179, 39486665, 40679974, 40970488, 27108529)

Variant summary: MYBPC3 c.1591G>C (p.Gly531Arg) results in a non-conservative amino acid change located in the Immunoglobulin subtype 2 domain (IPR003598) of the encoded protein sequence. Algorithms developed to predict the effect of missense changes on protein structure and function all suggest that this variant is likely to be disruptive. The variant allele was found at a frequency of 1.2e-05 in 247086 control chromosomes. c.1591G>C has been observed in individual(s) affected with Hypertrophic Cardiomyopathy (e.g. Girolami_2006, Olivotto_2011, Coppini_2014, Rubattu_2016, Ho_2018, Bagnall_2022, Kurzlechner_2022, Preveden_2022, Mazzaccara_2022, Hagge_2024). These data indicate that the variant is likely to be associated with disease. At least one publication reports experimental evidence evaluating an impact on protein function by measuring contraction forces in an engineered heart tissue assay from MYBPC3-knock out mice. Human wild-type and variant MYBPC3 was transduced into the engineered heart tissue and contraction was assayed at varying Ca2+ levels. The variant failed to rescue the hypercontractile phenotype seen in non-transduced (MYBPC3 null) samples, when expressed either alone or jointly with wild-type MYBPC3, demonstrating the variant may also act in a dominant-negative manner (Wijnker_2016). The following publications have been ascertained in the context of this evaluation (PMID: 36252119, 25524337, 16858239, 39260623, 30297972, 35629155, 36291626, 21835320, 35208637, 27483260, 27108529). ClinVar contains an entry for this variant (Variation ID: 42550). Based on the evidence outlined above, the variant was classified as likely pathogenic.

This sequence change replaces glycine, which is neutral and non-polar, with arginine, which is basic and polar, at codon 531 of the MYBPC3 protein (p.Gly531Arg). This variant is present in population databases (rs397515912, gnomAD 0.004%). This missense change has been observed in individuals with hypertrophic cardiomyopathy (PMID: 18533079, 20624503, 21750094, 23508784, 24793961, 25524337, 27483260, 27532257, 27600940, 28356264, 30297972). ClinVar contains an entry for this variant (Variation ID: 42550). 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. In summary, the currently available evidence indicates that the variant is pathogenic, but additional data are needed to prove that conclusively. Therefore, this variant has been classified as Likely Pathogenic.

This missense variant replaces glycine with arginine at codon 531 of the MYBPC3 protein. Computational prediction suggests that this variant may have a deleterious impact on protein structure and function. In heart tissue engineered from mouse cardiac cells, this variant in heterozygous state was associated with a hypercontractile phenotype (PMID: 27108529). This variant has been reported in over ten unrelated individuals affected with hypertrophic cardiomyopathy (PMID: 16858239, 18533079, 20173211, 20624503, 21835320, 23508784, 23690394, 27483260, 27532257, 27600940, 28193612, 28241245, 28986452, 31941943, 32228044, 32369506, 36252119, 36291626). Three of these individuals carried a second pathogenic variant either in the same gene (PMID: 20624503) or in another gene (PMID: 32228044, 36252119). This variant has also been reported in an individual affected with left ventricular non-compaction (PMID: 28798025). This variant has been identified in 5/278462 chromosomes in the general population by the Genome Aggregation Database (gnomAD). A different variant causing the same amino acid change, c.1591G>A p.Gly531Arg, is also considered to be disease-causing (ClinVar variation ID: 164109). In summary, this variant has shown a relevant phenotype in an experimental study and has been observed in multiple individuals affected with hypertrophic cardiomyopathy. Based on available evidence, this variant is classified as Likely Pathogenic.

MYBPC3: PS1, PS4, PS3:Supporting