The NM_000256.3(MYBPC3):c.1484G>A (p.Arg495Gln). This variant has been reported in individuals with HCM and other cardiomyopathies (ClinVar Variation ID: 164113) and has also been identified in 5 out of 112974 (0.009% FAF 95% CI) of European chromosomes in gnomAD (https://gnomad.broadinstitute.org/; v.2.1). The variant is statistically increased in individuals with HCM compared to controls (OR lower 95% CI>10), therefore, the PS4 criterion has been applied at moderate strength (PS4_Moderate) and the PM2_Supporting criterion has not been applied. This variant segregated with disease in >7 affected individuals with HCM from at least 4 families (PP1_Strong; Maron 2011 PMID: 21185001, Agarwal 2015 PMID: 26271555, Mattos 2016 Year PMID: 27737317, Ross 2017 PMID: 28615295). This variant lies in a region of the protein where variants are statistically more likely to be disease-associated (PM1_Strength; Walsh 2019 PMID: 30696458). Computational prediction tools and conservation analyses do not provide strong support for or against an impact to the protein (REVEL score <0.7). In summary, this variant meets criteria to be classified as likely pathogenic for hypertrophic cardiomyopathy in an autosomal dominant manner based on PS4_Moderate, PP1_Strong and PM1.

ACMG Criteria: PS3, PM1, PM2_P, PM5, PP1, PP3, PP5; Variant was found in heterozygous state.

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as 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, however 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 arginine to glutamine. (I) 0251 - This variant is heterozygous. (I) 0302 - Variant is present in gnomAD (v3) <0.001 for a dominant condition (7 heterozygotes, 0 homozygotes). (SP) 0309 - An alternative amino acid change at the same position has been observed in gnomAD (v2, v3) (1 heterozygote, 0 homozygotes). (I) 0502 - Missense variant with conflicting in silico predictions and uninformative conservation. (I) 0600 - Variant is located in the annotated immunoglobulin I-set domain (DECIPHER). (I) 0701 - Other missense variants comparable to the one identified in this case have very strong previous evidence for pathogenicity. These alternative changes (p.(Arg495Gly), p.(Arg495Leu), p.(Arg495Trp)) have been reported many times as pathogenic and likely pathogenic, and observed in at least twenty individuals with hypertrophic cardiomyopathy (HCM) (ClinVar). (SP) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant has been reported over twenty times as pathogenic, and observed in many individuals with HCM (ClinVar, cardiodb.org). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

This missense variant replaces arginine with glutamine at codon 495 in the Ig-like domain C3 of the MYBPC3 protein. Computational prediction suggests that this variant may not impact protein structure and function (internally defined REVEL score threshold <= 0.5, PMID: 27666373). An experimental functional study using a transgenic pig model heterozygous for this variant has shown a phenotype of myocardial fibrosis as well as reduced MYBPC3 expression levels in cardiac fibroblasts (PMID: 36357371). This variant has been reported in over 20 unrelated individuals affected with hypertrophic cardiomyopathy (PMID: 11499718, 20019025, 22857948, 23396983, 24093860, 28024942, 28615295, 29121657, 29907873, 32746448, 35626289). This variant has been shown to segregate with hypertrophic cardiomyopathy in three different families (PMID: 9562578, 27737317, 29121657). This variant has been identified in 6/249188 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Different variants occurring at the same codon, p.Arg495Gly and p.Arg495Trp, are well documented pathogenic mutations (Clinvar variation ID: 42537 and 164114), indicating that arginine at this position is important for MYBPC3 protein function. Based on the available evidence, this variant is classified as Pathogenic.

The c.1484G>A (p.R495Q) alteration is located in exon 17 (coding exon 17) of the MYBPC3 gene. This alteration results from a G to A substitution at nucleotide position 1484, causing the arginine (R) at amino acid position 495 to be replaced by a glutamine (Q). Based on data from gnomAD, the A allele has an overall frequency of 0.002% (6/249188) total alleles studied. The highest observed frequency was 0.006% (1/17974) of East Asian alleles. This alteration has been reported in multiple unrelated individuals with hypertrophic cardiomyopathy (HCM) and has been reported to segregate with disease in families (Niimura, 1998; Maron, 2011; Brito, 2012; Kapplinger, 2014; Mattos, 2016). This variant has also been detected in noncompaction and dilated cardiomyopathy cohorts (van Waning, 2018; Sousa, 2019). In addition, alterations affecting the same amino acid, c.1483C>T (p.R495W) and c.1483C>G (p.R495G), have also been reported in association with HCM (Page, 2012; Garc&iacute;a-Castro, 2009). This amino acid position is highly conserved in available vertebrate species. This alteration is predicted to be deleterious by in silico analysis. Based on the available evidence, this alteration is classified as pathogenic.

The MYBPC3 Arg495Gln variant has been described in many HCM probands and has been reported to segregate with disease in several families (see literature). A study analysing the transcript and protein levels in two septal myectomy patients carrying the MYBPC3 Arg495Gln variant revealed that not only did the patients exhibit a 2-fold increase in MYBPC3 protein expression but were also found to predominately express the mutant peptide (Helms AS, et al., 2014), which is supportive of the pathogenic role of the variant. The variant is present at a low frequency in the Genome Aggregation Database (http://gnomad.broadinstitute.org/). We identified this variant in 2 HCM probands. One proband was diagnosed after they suffered resuscitated cardiac arrest in childhood, family screening identified an additional 3 affected family members in which the variant segregated (Ross SB, et al., 2017). The second proband does not have a family history of disease (Ingles et al., 2017). Interestingly, different rare variants at this position (Arg495Trp, Arg495Gly) have also been reported in 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. Based on the adapted ACMG guidelines (Kelly MA, et al., 2018) this variant has been reported in more than 15 HCM probands (PS4), segregates with disease in multiple families (PP1_strong), is rare in the general population (PM2) and in silico tools predict it to be deleterious (PP3), therefore we classify MYBPC3 Arg495Gln as "pathogenic".

This sequence change replaces arginine, which is basic and polar, with glutamine, which is neutral and polar, at codon 495 of the MYBPC3 protein (p.Arg495Gln). This variant is present in population databases (rs200411226, gnomAD 0.006%). This missense change has been observed in individuals with hypertrophic cardiomyopathy (PMID: 11499718, 20019025, 22857948, 23396983, 24093860). ClinVar contains an entry for this variant (Variation ID: 164113). 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. This variant disrupts the p.Arg495 amino acid residue in MYBPC3. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 18403758, 19659763, 20624503). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic.

The missense variant (chr11:47342718C>T), located in exon 17 (of 35) and absent in ClinVar, is reported in gnomAD v4.1 non-UKB with an allele frequency of 0.0036%, and in the scientific literature, also segregating with the phenotype, in individuals with hypertrophic cardiomyopathy (PMID: 39125703, 23861362, 27737317, 11499718, 20019025, 22857948, 23396983, 24093860, 38999502, 39554508). In silico analysis is inconclusive regarding the impact of this variant. This variant is located in a known mutational hotspot. Based on the currently available evidence and the specific ClinGen criteria for the gene (PMID: 29300372), this variant has been classified as pathogenic (PS4, PM1, PM2_P, PM5, PP1_S).

The c.1484G>A (p.Arg495Gln) variant is a known Pathogenic variant that has been previously reported as a heterozygous change in patients with MYBPC3-related cardiomyopathies (PMID: 9562578, 27737317, 38254962, 32746448, 33407484, 36252119). The c.1484G>A (p.Arg495Gln) variant affects a highly conserved amino acid and is located in a mutational hotspot for pathogenic variations associated with MYBPC3-related cardiomyopathies (PMID: 30696458). Different amino acid changes at the same residue (p.Arg495Gly, p.Arg495Trp) have been previously reported in individuals with MYBPC3-related cardiomyopathies (PMID: 18403758, 34400558, 19150014, 39160446). The c.1484G>A (p.Arg495Gln) variant is present in the latest version of the gnomAD population database at an allele frequency of 0.002% (39/1613896), and is absent in the homozygous state. Based on the available evidence, c.1484G>A (p.Arg495Gln) is classified as Pathogenic.

Across a selection of the available literature, the c.1484G>A (p.Arg495Gln) variant has been reported in a heterozygous state in 18 patients with hypertrophic cardiomyopathy, one patient with left ventricular noncompaction cardiomyopathy, two individuals with an indeterminate status, and in a compound heterozygous state in one individual with hypertrophic cardiomyopathy (Niimura et al. 1998; Zeller et al. 2006; Ehlermann et al. 2008; Millat et al. 2010; Antonio et al. 2011; Marsiglia et al. 2013; Helms et al. 2014). The p.Arg495Gln was also found in a heterozygous state in one unaffected individual but was absent from 536 control chromosomes. The variant is reported at a frequency of 0.00001 in the European (Finnish) population of the Exome Aggregation Consortium but this is based on one allele so is presumed to be rare. Helms et al. (2014) demonstrated that heart tissue samples carrying the p.Arg495Gln variant exhibited higher MYBPC3 expression and transcript levels but no increase in protein abundance when compared to control heart tissues. MYBPC3 variants have displayed reduced penetrance and variable expressivity. Based on the collective evidence, the p.Arg495Gln variant is classified as pathogenic for MYBPC3-related disorders.

Variant summary: The variant, MYBPC3 c.1484G>A (p.Arg495Gln) results in a conservative amino acid change located in the Immunoglobulin-like domain (Immunoglobulin subtype 2) and Immunoglobulin I-set domain of the encoded protein sequence. Three of five in-silico tools predict a benign effect of the variant on protein function. The variant allele was found at a frequency of 2.4e-05 in 253236 control chromosomes (gnomAD and publications) and has been reported in the literature in multiple individuals affected with Hypertrophic Cardiomyopathy (example, Helms_2014, Marsiglia_2013, Ng_2013, Lopes_2013, Millat_2010, Morita_2008, VanDriest_2004, Niimura_1998, Viswanathan_2017). In one family, this variant cosegregates with HCM in three affected family members, however, one unaffected family member whose age was above the age of onset carried this variant implying reduced penetrance or later onset (Niimura_1998). Two co-occurrences with other likely pathogenic variants (MYBPC3 p.Tyr1172fs, TNNI3 Arg141Gln) have been reported in two HCM patients, respectively (Millat_2010, Morita_2008). These data indicate that the variant is very likely to be associated with disease. Experimental evidence evaluating the impact of the variant on protein function, does not allow convincing conclusions about the variant effect (Helms_2014). However, myocardial tissue obtained from a human HCM subject that harbors the variant show molecular signatures of aberrant calcium signaling (increased Ca2+/Calmodulin dependent protein kinase II (CaMKII) activation and reduced SERCA2 (Sarcoplasmic/endoplasmic reticulum calcuium ATPase 2) mRNA levels, (Helms_2016). Experimental evidence suggest that mutations causative of HCM in both thick and thin sarcomere filaments may lead to abnormal calcium handling and increase the risk of arrhythmia in HCM (Helms_2016). Multiple ClinVar submissions from clinical diagnostic laboratories (evaluation after 2014) cites the variant as likely pathogenic/pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

The p.Arg495Gln variant in MYBPC3 has been reported in >20 individuals with HCM and segregated with disease in 5 affected relatives from 3 families (Niimura 1998 PMID: 9562578, Maron 2001 PMID: 11499718, Van Driest 2004 PMID: 15519027, Ehlermann 2008 PMID: 18957093, Fokstuen 2008 PMID: 18409188, Morita 2008 PMID: 18403758, Millat 2010 PMID: 20624503, Fokstuen 2011 PMID: 21239446, Brito 2012 PMID: 22857948, Lopes 2013 PMID: 23396983, Marsiglia 2013 PMID: 24093860, Kapplinger 2014 PMID: 24510615, Walsh 2017 PMID: 27532257, LMM data). However, multiple unaffected relatives from different families also carried this variant, at least 6 of whom were over the age of 50 (Brito 2012 PMID: 22857948, LMM data), suggesting reduced penetrance. This variant has also been reported by other clinical laboratories in ClinVar (Variation ID 164113) and has been identified in 0.005% (1/17974) of East Asian chromosomes and 0.004% (5/112974) of European chromosomes by gnomAD (http://gnomad.broadinstitute.org). Please note that for diseases with clinical variability or reduced penetrance, pathogenic variants may be present at a low frequency in the general population. In vitro functional studies suggest that carriers of this variant exhibit heightened expression of MYBPC3 as compared to controls, leading to an abundance of the mutant protein in cardiac tissue (Helms 2014 PMID: 25031304). However, it is unclear how this would impact cardiac function. Computation tools and conservation analysis are consistent with pathogenicity. In addition, 2 different amino acid changes at this position (p.Arg495Gly and p.Arg495Trp) have been reported in multiple individuals with HCM (Niimura 1998 PMID: 9562578, Maron 2001 PMID: 11499718, García-Castro 2009 PMID: 19150014, Martín 2009 PMID: 18713777, Rodríguez-García 2010 PMID: 20433692, Brito 2012 PMID: 22857948, Coto 2012 PMID: 22765922, Lopes 2013 PMID: 23396983), suggesting that changes at this position are not tolerated. In summary, this variant meets criteria to be classified as pathogenic for autosomal dominant HCM; however, it should be noted that penetrance may be reduced. ACMG/AMP Criteria applied: PS4, PM2_Supporting, PP1_Moderate, PP3, PM5_supporting.

ACMG categories: PS5,PM1,PM2,PM5,PP3,PP5

This missense variant replaces arginine with glutamine at codon 495 in the Ig-like domain C3 of the MYBPC3 protein. Computational prediction tools indicate that this variant's impact on protein structure and function is inconclusive. An experimental functional study using a transgenic pig model heterozygous for this variant has shown a phenotype of myocardial fibrosis as well as reduced MYBPC3 expression levels in cardiac fibroblasts (PMID: 36357371). This variant has been reported in over 70 unrelated individuals affected with hypertrophic cardiomyopathy (PMID: 11499718, 20019025, 22857948, 23396983, 24093860, 27532257, 28024942, 28615295, 29121657, 29907873, 32746448, 33782553, 35626289, 38104429). This variant has been shown to segregate with hypertrophic cardiomyopathy in three different families (PMID: 9562578, 27737317, 29121657). This variant has been identified in 6/249188 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Different variants occurring at the same codon, p.Arg495Gly and p.Arg495Trp, are well documented pathogenic mutations (Clinvar variation ID: 42537 and 164114), indicating that arginine at this position is important for MYBPC3 protein function. Based on the available evidence, this variant is classified as Pathogenic.

The variant is observed at an extremely low frequency in the gnomAD v4.0.0 dataset (total allele frequency: 0.002%). Predicted Consequence/Location: Missense variant The same nucleotide change resulting in the same amino acid change has been previously reported as pathogenic/likely pathogenic with strong evidence (ClinVar ID: VCV000164113 /PMID: 9562578 /3billion dataset). The variant has been observed in multiple (>3) similarly affected unrelated individuals (PMID: 24093860, 28024942, 32746448, 35626289). Different missense changes at the same codon (p.Arg495Gly, p.Arg495Leu, p.Arg495Trp) have been reported as pathogenic/likely pathogenic with strong evidence (ClinVar ID: VCV000042537, VCV000164114, VCV000915779 /PMID: 18403758, 19150014 /3billion dataset). Therefore, this variant is classified as Pathogenic according to the recommendation of ACMG/AMP guideline.

The p.(R495Q) mutant peptide was predominantly expressed compared to wild-type protein in heart tissue specimens from two HCM patients, though the precise correlation to phenotype is yet to be determined (PMID: 25031304); In silico analysis suggests that this missense variant does not alter protein structure/function; This variant is associated with the following publications: (PMID: 34598319, 31308319, 31006259, 39605252, 40225148, 34542152, 15519027, 11499718, 15115610, 16715312, 18403758, 18409188, 18957093, 20031618, 20019025, 20624503, 21415409, 22857948, 23396983, 23861362, 24093860, 21310275, 24510615, 18761664, 18803133, 9562578, 26671970, 27532257, 28166811, 28024942, 28797094, 29447731, 29121657, 29907873, 30871747, 31737537, 30847666, 31447099, 32480058, 33500567, 34540771, 33673806, 27737317, 32746448, 33407484, 33087929, 36252119, 36264615, 37652022, 36243179, 38104429, 34400558, 35626289, 35653365, 35535697, 25031304, 38456273, 34714385, 39472908, 39096151, 39160446, 38254962)

NM_000256.3(MYBPC3):c.1484G>A (p.Arg495Gln) is a missense variant that results in the substitution of arginine with glutamine. This variant has been recurrently observed in individuals with related phenotype (PMID: 11499718; PMID: 20019025; PMID: 22857948; PMID: 23396983; PMID: 24093860). Segregation evidence has been reported in affected families. The variant is present at low frequency in population datasets. Based on the available data, this variant is classified as pathogenic.

MYBPC3: PP1:Strong, PS4, PM2, PM5, PS3:Supporting

The MYBPC3 c.1484G>A variant is predicted to result in the amino acid substitution p.Arg495Gln. This variant has been reported to be causative in multiple unrelated patients for hypertrophic cardiomyopathy (HCM) (Niimura et al.1998. PubMed ID: 9562578; Van Driest et al. 2004. PubMed ID: 15519027; Jordan et al. 2011. PubMed ID: 21310275; Lopes et al. 2013. PubMed ID: 23396983; Ng et al. 2013. PubMed ID: 23861362; Helms et al. 2014. PubMed ID: 25031304; Mendes de Almeida et al. 2017. PubMed ID: 28797094; Walsh et al. 2017. PubMed ID: 27532257; McGurk et al. 2023. PubMed ID: 37652022). Two different missense variants affecting the same amino acid (p.Arg495Gly and p.Arg495Trp) have also been reported in association with HCM (Morita et al. 2008. PubMed ID: 18403758; García-Castro et al. 2009. PubMed ID: 19150014). This variant is reported in 0.0056% of alleles in individuals of East Asian descent in gnomAD. This variant is interpreted as pathogenic.