ClinVar Genomic variation as it relates to human health

The p.Val219Leu variant in MYBPC3 has been reported in >15 individuals with HCM and segregated with disease in one affected relative (Van Driest 2004, Pan 2012, Kaski 2012, LMM data). It has not been identified in large population studies. This variant is located in the first base of the exon, which is part of the 3? splice region. Computational splicing prediction tools do not predict altered sp licing. In vitro studies showed that this variant results in skipping of exon 6 (Crehalet 2012), which is predicted to cause a frameshift and a premature termin ation codon, leading to a truncated or absent protein. However, these types of o f prediction tools and assays may not accurately represent biological function. In summary, although additional segregation and in vivo studies are required to fully establish its clinical significance, the p.Val219Leu variant is likely pat hogenic.

The c.655G>C (p.Val219Leu) variant in the MYBPC3 gene has been reported in multiple unrelated patients with hypertrophic cardiomyopathy (HCM) (PMID: 15519027, 20624503, 20031618, 20031602, 23782526, 24510615, 26914223). This variant is absent from general population databases. It is located at the first nucleotide of exon 6 of the MYBPC3 gene and predicted to affect the RNA splicing. Functional studies have showed that the c.655G>C change causes skipping of exon 6, which is predicted to result in a truncated protein or absence of the protein (Crehalet et al., 2012). In summary, this c.655G>C (p.Val219Leu) in the MYBPC3 gene is classified as pathogenic.

The c.655G>C pathogenic mutation (also known as p.V219L) is located in coding exon 6 of the MYBPC3 gene. This variant results from a G to C substitution at nucleotide position 655. The valine at codon 219 is replaced by leucine, an amino acid with highly similar properties. This change occurs in the first base pair of coding exon 6. This mutation has been reported in a number of individuals in hypertrophic cardiomyopathy (HCM) cohorts (e.g., Van Driest SL et al. J. Am. Coll. Cardiol. 2004;44:1903-10; Kapplinger JD et al. J Cardiovasc Transl Res. 2014;7:347-61; Lopes LR et al. J. Med. Genet. 2013;50:228-39; Walsh R et al. Genet. Med. 2017;19:192-203). In addition, this alteration causes complete skipping of exon 6 in an in vitro minigene assay (Crehalet H et al. Cardiogenetics. 2012;2:e6; Ito K et al. Proc. Natl. Acad. Sci. U.S.A., 2017 07;114:7689-7694). Skipping of exon 6 is expected to lead to a frameshift with a predicted alternative stop (p.V219Rfs*42). A likely pathogenic variant in the same nucleotide, c.655G>T, reported to lead to skipping of exon 6 has also been associated with HCM (Millat G et al. Eur J Med Genet. 2010;53:261-7; Crehalet H et al. Cardiogenetics. 2012;2:e6). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

Variant summary: MYBPC3 c.655G>C (p.Val219Leu) results in a conservative amino acid change located in the Immunoglobulin-like domain (IPR007110) of the encoded protein sequence. Three of five in-silico tools predict a damaging effect of the variant on protein function. 4/4 computational tools predict no significant impact on normal splicing. However, experimental evidence suggests that the variant causes complete skipping of exon 6 (Crehalet_2012). The variant was absent in 246060 control chromosomes (gnomAD). c.655G>C has been reported in the literature in multiple individuals affected with Hypertrophic Cardiomyopathy (example: VanDriest_2004, Lopes_2013, Seidelmann_2017, Miller_2019). These data indicate that the variant is very likely to be associated with disease. Eight other ClinVar submitters (evaluation after 2014) cite the variant as pathogenic/likely pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

This sequence change replaces valine, which is neutral and non-polar, with leucine, which is neutral and non-polar, at codon 219 of the MYBPC3 protein (p.Val219Leu). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individuals with hypertrophic cardiomyopathy (PMID: 15519027, 20031602, 20031618, 20624503, 26914223). ClinVar contains an entry for this variant (Variation ID: 42784). 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. Studies have shown that this missense change alters mRNA splicing and is expected to lead to the loss of protein expression (Crehalet 2012; http://dx.doi.org/10.4081/cardiogenetics.2012.e6). For these reasons, this variant has been classified as Pathogenic.

This missense variant replaces valine with leucine at codon 219 in the Ig-like domain C1 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). Splice site prediction tools suggest that this variant may impact RNA splicing. In-vitro functional mini-gene assays have shown that this variant is expected to cause skipping of exon 6; this 118 base pair deletion is expected to create a frameshift and premature translation stop signal, expected to result in an absent or non-functional protein product (PMID: 28679633; DOI:10.4081/cardiogenetics.2012.e6). This variant has been reported in over 20 individuals affected with hypertrophic cardiomyopathy (PMID: 15519027, 20031602, 20031618, 23396983, 23690394, 23782526, 26914223, 27532257, 28087566, 28790153, 30645170, 31199839, 35199016). It has been shown that this variant segregates with disease in multiple affected individuals across two families (PMID: 28790153, 35199016). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). A different variant affecting the same codon, p.Val219Phe, is considered to be disease-causing (ClinVar variation ID: 188531), suggesting that valine at this position is important for MYBPC3 protein function. Loss of MYBPC3 function is a known mechanism of disease (clinicalgenome.org). Based on the available evidence, this variant is classified as Pathogenic.

Identified in a patient with HCM and fasciculoventricular bypass tract (FVBT) in published literature (PMID: 35199016); RNA studies demonstrate a damaging effect as a majority of reads result in no splicing occurring (PMID: 34461741); In silico analysis indicates that this missense variant does not alter protein structure/function; Not observed at significant frequency in large population cohorts (gnomAD); This variant is associated with the following publications: (PMID: 21415409, 23782526, 27532257, 15519027, 23074333, 24510615, 23690394, 23396983, 20031602, 26914223, 20031618, 20624503, 29540445, 22589294, 25351510, 28790153, 31199839, 34400558, 35208637, 30645170, 33190526, 28679633, 28087566, 34758253, 34363016, 36264615, 36136372, 36252119, 36243179, 35199016, 37652022, Crehalet2012[FunctionalStudy], 34461741)

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) 0209 - Splice site variant proven to affect splicing of the transcript with uncertain effect on protein sequence. This variant has been functionally proven to cause skipping of exon 6, which is an out-of-frame exon. The protein consequence is unknown however, a frameshift and nonsense-mediated decay (NMD) is expected (ClinVar, Crehalet, H. et al. (2012)). (SP) 0251 - This variant is heterozygous. (I) 0301 - Variant is absent from gnomAD (both v2 and v3). (SP) 0311 - An alternative nucleotide change at the same splice site is present in gnomAD (v2 and v3) (2 heterozygotes, 0 homozygotes). (I) 0704 - Another splice variant comparable to the one identified in this case has limited previous evidence for pathogenicity. c.655G>T; p.(Val219Phe) has been reported as pathogenic (ClinVar), and has also been functionally proven to result in exon 6 skipping (Crehalet, H. et al. (2012)). (SP) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant has been reported multiple times as pathogenic in patients with hypertrophic cardiomyopathy (ClinVar, PMID: 28087566). (SP) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

The c.655G>C (p.Val219Leu) variant in the MYBPC3 gene has been reported in multiple unrelated patients with hypertrophic cardiomyopathy (HCM) (PMID: 15519027, 20624503, 20031618, 20031602, 23782526, 24510615, 26914223). This variant is absent from general population databases. It is located at the first nucleotide of exon 6 of the MYBPC3 gene and predicted to affect the RNA splicing. Functional studies have showed that the c.655G>C change causes skipping of exon 6, which is predicted to result in a truncated protein or absence of the protein (Crehalet et al., 2012). In summary, this c.655G>C (p.Val219Leu) in the MYBPC3 gene is classified as pathogenic.

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. Val219Leu (c.655G>C) in exon 6 of the MYBPC3 gene (NM_000256.3) Given case data and functional studies we consider this variant likely disease causing. The variant has been seen in at least 15 unrelated cases of HCM (not including this patient). None of the studies below reported segregation data. The variant has been reported in multiple cases in the literature (Van Driest et al 2004, Kaski et al 2009, Ho et al 2009, Nunez et al 2013). Crehalet et al (2012) demonstrated that p.Val219Leu leads to complete skipping of exon 6. Many other variants in MYBPC3 that affect splicing have been implicated in cardiomyopathy. This is a conservative amino acid change with a nonpolar Valine replaced with a nonpolar Leucine at residue 219. Two variants at nearby residues of the variant (p.Asp228Asn and p.Gln208His) and another variant at the same residue (p.Val219Phe, Crehalet et al 2012) have been reported in association with HCM (CardioGenomics http://genepath.med.harvard.edu/ ). The Valine at codon 219 is highly, though not completely conserved (there is an Isoleucine in frogs). Of note, the variant affects the first base of exon 6, which is part of the splicing consensus sequence (and is most frequently a G). The Laboratory for Molecular Medicine performed in silico analysis with the program NNSplice and found the variant is predicted to affect splicing. In total this variant has not been seen in ~7500 published controls, laboratory controls, and publicly available general population samples. The variant was not observed in an internal sample of 400 presumably healthy controls of various ancestries at Familion Labs. Van Driest et al (2004) did not observe the variant in 100 Caucasian and 100 African American controls. Kaski et al (2009) did not identify the variant in 200 presumably healthy individuals of unspecified racial descent. GeneDx did not observe the variant in 400 Caucasian and African American controls. The variant is not currently listed in dbSNP or 1000 genomes and the variant is not listed in the NHLBI exome sequencing project, which currently includes data on ~4200 European Americans and ~2100 African Americans (as of March 10, 2015). There is one variation at codon 219 listed in the Exome Aggregation Consortium dataset (http://exac.broadinstitute.org/), which currently includes variant calls on ~47,500 individuals of European, African, Latino and Asian descent (as of March 10, 2015). The amino acid substitution reported in exac was different, p.Val219Phe.