ClinVar Genomic variation as it relates to human health

This TNNT2 Arg278Cys variant has been described in multiple HCM cohorts (see references) and in genetic screening of one DCM cohort (Millat G. et al., 2011). Segregation with disease has been shown where familial screening was available, though incomplete disease penetrance was observed (Gimeno JR, et al., 2009; García-Castro M, et al., 2007; Miliou A. et al., 2005; Theopistou A. et al., 2004; Watkins H, et al. 1995). The disease phenotype is variable. This mutation has been characterised by late-onset disease with mild hypertrophy but with a high incidence of sudden death. However, severe disease presentation including cardiac arrest events have been reported in young patients (Miliou A. et al., 2005; Theopistou A. et al., 2004; Watkins H. et al., 1995), including one paediatric HCM case (Kaski JP. et al., 2009). It should be noted that genetic analysis of the majority of these studies was limited to a small number of genes, and that additional mutations in other disease causing genes cannot be excluded in these patients. Studies by Gimeno JR. et al (2009) and Kaski JP. et al (2009) report carriers of the TNNT2 Arg278Cys mutation who also have a secondary mutation in another sarcomere gene present with moderate to severe phenotypes. Transgenic mouse models of this mutation did not develop significant hypertrophy or fibrosis (Hernandez OM. et al., (2005), however in vitro functional assays indicate that the mutation affects muscle contraction (Takahashi-Yanaga F. et al., 2001; Morimoto S. et al., 1999). We have detected this variant in 3 unrelated families in our cohort, though 2 families carry an additional "likely pathogenic" or "pathogenic variant" in another known HCM causing gene. Based on the current literature and our data, this variant in isolation may cause a mild phenotype, however further evidence is required to fully establish its role in disease pathogenicity.

Converted during submission to Uncertain significance.

One variant of uncertain significance, c.832C>T; p.Arg278Cys, was detected in the TNNT2 gene by massively parallel sequencing and confirmed by Sanger sequencing. The p.Arg278Cys variant (rs121964857) has been extensively reported in association with hypertrophic cardiomyopathy (HCM; first reported in Watkins 1995). However, the exact contribution of this variant to the development of HCM is currently under debate. For example, this variant is listed in the ClinVar database with conflicting interpretations of pathogenicity (Variation ID: 12411), with multiple clinical labs noting incomplete segregation/penetrance of this variants in familial studies (Garcia-Castro 2003, Theopistou 2004, Gimeno 2009, Ripoll-Vera 2016), and that multiple patients analyzed have additional clinically relevant variants in other HCM-related genes; an observation that has as has also been reported in the literature (Gimeno 2009 and Garcia-Castro 2009). Additionally, this variant is listed in the Genome Aggregation Database (gnomAD) browser with a frequency in non-Finnish Europeans of 0.06% (identified in 76 out of 125,670 chromosomes). However, this relatively high population frequency, while disqualifying this variant as a high penetrance, early onset disease allele, would be consistent with a late onset/low penetrance model of disease etiology (see case report in Elliott 1999 for example of late onset disease in a carrier). Functional studies using isolated muscle fibers show subtle, but reproducible, changes in force generation caused by the p.Arg278Cys variant (Yanaga 199, Szczesna 2000, and Hernandez 2005). However, whether these defects in vitro are relevant to disease manifestation in human patients is not completely understood. Taken together, the clinical significance of the p.Arg278Cys variant cannot be determined with certainty. And at most, the genetic evidence is consistent with this variant being a low penetrance risk factor or genetic modifier of more penetrant alleles. References: Elliott et al. Late-onset hypertrophic cardiomyopathy caused by a mutation in the cardiac troponin T gene. N Engl J Med. 1999; 341(24): 1855-1856. PMID: 10610467. Hernandez et al. F110I and R278C troponin T mutations that cause familial hypertrophic cardiomyopathy affect muscle contraction in transgenic mice and reconstituted human cardiac fibers. J Biol Chem. 2005; 280(44): 37183-37194. PMID: 16115869. Garcia-Castro et al. Hypertrophic cardiomyopathy: low frequency of mutations in the beta-myosin heavy chain (MYH7) and cardiac troponin T (TNNT2) genes among Spanish patients. Clin Chem. 2003; 49(8): 1279-1285. PMID: 12881443 Garcia-Castro et al. Mutations in sarcomeric genes MYH7, MYBPC3, TNNT2, TNNI3, and TPM1 in patients with hypertrophic cardiomyopathy. Rev Esp Cardiol. 2009; 62(1): 48-56. PMID: 19150014 Gimeno et al. Hypertrophic cardiomyopathy. A study of the troponin-T gene in 127 Spanish families. Rev Esp Cardiol. 2009; 62(12): 1473-1477. PMID: 20038417 Ripoll-Vera et al. Clinical and Prognostic Profiles of Cardiomyopathies Caused by Mutations in the Troponin T Gene. Rev Esp Cardiol (Engl Ed). 2016; 69(2): 149-158. PMID: 26507537 Szczesna et al. Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy. J Biol Chem. 2000; 275(1): 624-630. PMID: 10617660 Theopistou et al. Clinical features of hypertrophic cardiomyopathy caused by an Arg278Cys missense mutation in the cardiac troponin T gene. Am J Cardiol. 2004; 94(2): 246-249. PMID: 15246915 Watkins et al. Mutations in the genes for cardiac troponin T and alpha-tropomyosin in hypertrophic cardiomyopathy. N Engl J Med. 1995; 332(16): 1058-1064. PMID: 7898523 Yanaga et al. Ca2+ sensitization and potentiation of the maximum level of myofibrillar ATPase activity caused by mutations of troponin T found in familial hypertrophic cardiomyopathy. J Biol Chem. 1999; 274(13): 8806-8812. PMID: 10085122

The inherited heterozygous c.832C>T p.(Arg278Cys) variant in TNNT2 has previously been reported in multiple individuals affected with hypertrophic cardiomyopathy and/or dilated cardiomyopathy [PMID: 7898523, 10610467, 12860912, 14636924, 15246915, 16199542, 16715312, 20031618, 25611685,30645170, 33148509, 21846512, 24503780], though a subset of those individuals also carried a variant in MYH7 and/or MYBPC3 genes that may explain their cardiac phenotype [PMID: 20038417, 20031618, 26507537, 19150014]. There are also multiple familial studies in which this variant didn’t not co-segregate with disease and exhibited reduced penetrance [PMID:26507537, 15958377, 7898523, 20038417, 19150014, 32731933]. The c.832C>T variant has been deposited in ClinVar [ClinVarID: 12411] with conflicting interpretations of pathogenicity; Pathogenic (1 entry), Likely pathogenic (6 entries), and Uncertain significance (16 entries). The c.832C>Tvariant is observed in 219 alleles (0.00037 minor allele frequency with 0 homozygotes) in population databases (gnomAD v2.1.1 and v3.1.2, TOPMed Freeze 8). The c.832C>T variant is located in the last exon of this 16-exon gene and is predicted to replace a moderately conserved arginine amino acid with cysteine at position 278in the alpha tropomyosin and actin-binding domain of the encoded protein [PMID:20624503]. Functional studies have revealed conflicting results about potential pathogenicity of this variant [PMID:10405326, 10085122, 10617660, 11432788, 21683708, 22500102, 24418317, 33148509]. Given the lack of compelling evidence for its pathogenicity, this inherited c.832C>T p.(Arg278Cys) variant identified in TNNT2 is classified as a Variant of UncertainSignificance.

This sequence change replaces arginine, which is basic and polar, with cysteine, which is neutral and slightly polar, at codon 278 of the TNNT2 protein (p.Arg278Cys). This variant is present in population databases (rs121964857, gnomAD 0.06%), and has an allele count higher than expected for a pathogenic variant. This missense change has been observed in individual(s) with hypertrophic cardiomyopathy (PMID: 7898523, 15958377, 25524337, 26507537). This variant is also known as c.862C>T (p.Arg288Cys). ClinVar contains an entry for this variant (Variation ID: 12411). Advanced modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) performed at Invitae indicates that this missense variant is expected to disrupt TNNT2 protein function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects TNNT2 function (PMID: 10085122, 10405326, 10617660, 11432788, 16115869, 16777946, 19033660, 21683708, 22500102, 24418317). This variant disrupts the p.Arg278 amino acid residue in TNNT2. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 12974739, 15958377, 20057144, 23283745, 24793961). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. 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.

The p.Arg278Cys variant in TNNT2 has been reported in >30 individuals with clinical features of HCM and segregated with disease in at least 5 affected individuals (including one affected obligate carrier) from multiple families (Watkins 1995 PMID: 7898523, Elliott 1999 PMID: 10610467, Garcia-Castro 2003 PMID: 12881443, Torricelli 2003 PMID: 14636924, Van Driest 2003 PMID: 12860912, Theopistou 2004 PMID: 15246915, Ingles 2005 PMID: 16199542, Zeller 2006 PMID: 16715312, Kaski 2009 PMID: 20031618, Gimeno 2009 PMID: 20038417, Millat 2010 PMID: 20624503, Brito 2012 PMID: 22857948, Rubattu 2016 PMID: 27483260, LMM data). This variant has been identified by our laboratory in several individuals with HCM, though approximately half of them carried a second clinically significant variant in another gene. Many affected individuals carrying the p.Arg278Cys variant present at age 50 or older, suggesting a milder effect (LMM data). This variant has also been reported by other clinical laboratories in ClinVar (Variation ID: 12411) and has been identified in 0.066% (45/68030) European chromosomes by gnomAD (http://gnomad.broadinstitute.org, v.3.1.2). Please note that for diseases with clinical variability, reduced penetrance, or recessive inheritance, pathogenic variants may be present at a low frequency in the general population. Experimental studies evaluating the functional impact of this variant are conflicting: Animal models have shown that this variant does not cause significant hypertrophy or ventricular fibrosis even after chronic exercise challenge, and no difference in either ATPase calcium sensitivity or contractile force in muscle fibers ex vivo and in vitro (Hernandez 2005 PMID: 16115869), while in vitro functional studies show that this variant increases Ca2+ sensitivity and leads to abnormal myosin cross-bridges (Morimoto 1999 PMID 10405326, Yanaga 1999 PMID 10085122, Szczesna 2000 PMID 10617660, Schuldt 2021 PMID 33148509). Computational prediction tools do not provide evidence for or against an impact to the protein. In summary, while there is suspicion of a pathogenic role, likely with a milder effect when present in isolation, the clinical significance of this variant is uncertain. This variant should be interpreted carefully in the context of the individual’s age at onset. The ACMG/AMP Criteria applied: PS4_Supporting, PP1_Moderate, BP5.

Variant summary: TNNT2 c.832C>T (p.Arg278Cys) results in a non-conservative amino acid change in the encoded protein sequence. Three of three in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 0.00036 in 245204 control chromosomes, predominantly at a frequency of 0.00063 within the Non-Finnish European subpopulation in the gnomAD database. The observed variant frequency within Non-Finnish European control individuals in the gnomAD database is approximately 3 fold of the estimated maximal expected allele frequency for a pathogenic variant in TNNT2 causing Hypertrophic Cardiomyopathy With Sudden Cardiac Death phenotype (0.00025), strongly suggesting that the variant is a benign polymorphism found primarily in populations of Non-Finnish European origin. However, these occurrences do need to be cautiously considered due to the cohort harboring individuals that could have a TNNT2 phenotype. The variant has been reported to cause a high risk of sudden death even in cases without HCM, and has been found in patients with a family history of sudden death, both early and late in life (Miliou_2005, Gruner_2011, Brito_2012, Theopsitou_2004, Millat_2011, and Elliot_1999). In the literature, this variant has been found to co-occur with other variants that may explain the phenotype or confer a modifying affect (MYH7 c.2782G>A, MYH7 c.2167C>T, MYBPC3 c.1505G>A, MYBPC3 c.2198G>A, MYBPC3 c.1828G>C). While there are many published studies finding the variant in HCM patients, most do not screen multiple genes or report co-occurrence information, and thus the presence of another variant causing the phenotype cannot be ruled out. In addition, many family studies indicate the variant does not cosegregate with disease and/or has low penetrance (Ripoll-Soler_2017, Gimeno_2009, Garcio-Castro_2009). Functional studies have reported the variant to lead to increased Ca2+ sensitivity of ATP in rabbit cardiac myofibrils (Yanaga_1999) but not for skinned preparations from transgenic mouse hearts (Hernandez_2005). In addition, although transgenic mice carrying the variant neither exhibited extensive ventricular fibrosis nor developed significant hypertrophy (Hernandez_2005), they did exhibit significant impairment due to diastolic function (Sirenko_2006). While these functional studies may suggest the variant plays a role in muscle function, the biological relevance of these studies for humans is unclear. The following publications have been ascertained in the context of this evaluation (PMID: 26743238, 22857948, 24418317, 22500102, 10610467, 17101185, 19150014, 20038417, 21511876, 16115869, 28241245, 16199542, 20031618, 28771489, 21683708, 15958377, 20624503, 21846512, 10405326, 26507537, 16777946, 10617660, 15246915, 14636924, 12860912, 29121657, 7898523, 28193612, 10085122, 16715312). ClinVar contains an entry for this variant (Variation ID: 12411). Based on the evidence outlined above, the variant was classified as uncertain significance.

PM5, PP2, PP3, PP5

This missense variant replaces arginine with cysteine at codon 278 of the TNNT2 protein. Computational prediction is inconclusive regarding the impact of this variant on protein structure and function (internally defined REVEL score threshold 0.5 < inconclusive < 0.7, PMID: 27666373). Functional studies have shown that this variant increases Ca2+ sensitivity and leads to abnormal myosin cross-bridges (PMID: 10085122, 10405326, 10617660, 11432788, 21683708, 22500102, 24418317, 33148509). However, these results were not reproduced in transgenic mice, which did not show Ca2+ sensitivity, ventricular fibrosis or significant hypertrophy (PMID: 19033660, 16115869, 16777946). This variant has been reported in over 30 individuals affected with hypertrophic cardiomyopathy (PMID: 7898523, 10610467, 12860912, 14636924, 15246915, 15958377, 16199542, 16715312, 20031618, 25611685, 26507537, 30645170, 30762279, 32815737, 35514357, 37431535). Four of these patients also carried pathogenic variants in MYBPC3 and MYH7, respectively (PMID: 26507537, 37431535). Segregation analysis in families has yielded inconclusive results (PMID: 14563299, 15958377, 25524337). This variant has been reported in individuals affected with dilated cardiomyopathy (PMID: 21846512, 24503780) and arrhythmia (PMID: 26743238). This variant has been identified in over 200 individuals in the UK Biobank; of these individuals, four were affected with heart failure and one was affected with hypertrophic cardiomyopathy, while the majority of individuals were unaffected (PMID: 36264615). This variant has been identified in 98/275570 chromosomes in the general population by the Genome Aggregation Database (gnomAD). In summary, although this variant has been reported in many individuals affected with cardiovascular disorders, clinical significance of this variant is not clear due to inconclusive segregation analysis, co-occurrence with other disease-causing variants, elevated allele frequency in the general population and conflicting experimental data. Therefore, this variant is classified as a Variant of Uncertain Significance.

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as VUS-3C. Following criteria are met: 0105 - The mechanism of disease for this gene is not clearly established. Functional studies have suggested loss-of-function, gain-of-function and dominant-negative mechanisms based on calcium sensitivity, contractibility and mouse models (PMIDs: 18612386, 32098556, 33025817). (I) 0107 - This gene is associated with autosomal dominant disease. (I) 0115 - Variants in this gene are known to have variable expressivity, e.g., the variant, p.(Arg92Gln), has been reported to cause both DCM and HCM, even within the same family (PMID: 26507537). (I) 0200 - Variant is predicted to result in a missense amino acid change from arginine to cysteine. (I) 0251 - This variant is heterozygous. (I) 0302 - Variant is present in gnomAD <0.001 for a dominant condition (v2 & v3: 150 heterozygotes, 0 homozygotes). (I) 0309 - An alternative amino acid change at the same position has been observed in gnomAD (p.(Arg285His), v2 & v3: 11 heterozygotes, 0 homozygotes). (I) 0504 - Same amino acid change has been observed in placental mammals. (SB) 0600 - Variant is located in the annotated troponin domain (DECIPHER). (I) 0704 - Another missense variant comparable to the one identified in this case has limited previous evidence for pathogenicity. p.(Arg285Pro) has been reported in at least 7 individuals with HCM and has been more recently reported as likely pathogenic/pathogenic (cardiodb, ClinVar, PMID: 24793961). p.(Arg285His), a minor amino acid change, has been reported multiple times as a VUS, including in individuals with HCM (cardiodb, ClinVar, PMID: 33297573). (SP) 0809 - Previous evidence of pathogenicity for this variant is inconclusive. This variant has fourteen VUS, six likely pathogenic and two pathogenic submissions in ClinVar. This variant has been identified in many individuals with HCM however, some of them also have pathogenic variants in other sarcomeric genes (cardiodb, VCGS, PMIDs: 26507537, 12860912, 32228044, 35514357). (I) 0906 - Segregation evidence for this variant is inconclusive. This variant did not segregate with HCM in the 58-year old mother of a HCM patient tested at VCGS. This variant has been reported to have incomplete penetrance and to cause mild and late onset HCM (PMIDs: 20038417, 19150014, 15246915). (I) 1010 - Functional evidence for this variant is inconclusive. Three transgenic mice studies showed that this variant did not cause ventricular tachycardia, significant hypertrophy or ventricular fibrosis. However, transgenic mice cardiac muscle fibres did display significant increases in energy cost, a decrease in maximal force, and an increased Ca2+ sensitivity of force development (PMIDs: 19033660, 16115869, 16777946). A more recent study of transgenic mice showed the variant did not affect Ca2+ sensitivity in myocytes and there was no prolonged action potential in the heart (PMID: 22647877). (I) 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.832C>T (p.R278C) alteration is located in exon 16 (coding exon 15) of the TNNT2 gene. This alteration results from a C to T substitution at nucleotide position 832, causing the arginine (R) at amino acid position 278 to be replaced by a cysteine (C). Alteration conclusion: Based on the available evidence, the TNNT2 c.832C>T (p.R278C) alteration is classified as likely pathogenic; however, it may represent a milder allele that presents with later onset and incomplete penetrance. Based on data from gnomAD, the T allele has an overall frequency of 0.036% (98/275570) total alleles studied. The highest observed frequency was 0.061% (76/125670) of European (non-Finnish) alleles. The frequency for this variant is above the maximum credible frequency for a disease-causing variant in this gene based on internally established thresholds (Whiffin, 2017; Karczewski, 2020). However, this alteration is enriched in HCM cohorts compared with the general population (Alfares, 2015; Ambry internal data). This alteration has been reported in several individuals with hypertrophic cardiomyopathy (HCM) and has segregated with disease in multiple small families, although it has also been detected in unaffected relatives (Watkins, 1995; Garc&iacute;a-Castro, 2003; Theopistou, 2004; Brito, 2012; Alfares, 2015; Ripoll-Vera, 2016). This alteration was also identified in an individual with dilated cardiomyopathy (DCM), but clinical details were limited (Millat, 2011). Variants in other cardiac-related genes have co-occurred with this variant in a number of individuals, at least some of whom exhibited a severe HCM phenotype (Gimeno, 2009; Millat, 2010; Ripoll-Vera, 2016; Cecconi, 2016; Sanchez, 2016). This amino acid position is not well conserved in available vertebrate species. Functional studies have suggested that this alteration may affect muscle contraction through decreased maximal force, increased Ca2+ sensitivity, and/or altered myosin cross-bridges, but the physiological relevance of these effects is unclear (Yanaga, 1999; Morimoto, 1999; Szczesna, 2000; Hernandez, 2005; Midde, 2011; Brunet, 2012). Internal structural analysis suggests that this variant disrupts protein-protein interactions within the troponin complex (Ambry internal data). The in silico prediction for this alteration is inconclusive. Based on the available evidence, this alteration is classified as likely pathogenic.

Identified in a patient with HCM and prolonged QT interval; this patient also harbored a pathogenic variant in the KCNQ1 gene and a variant in the ANK2 gene (PMID: 37089884); Published functional studies suggest this variant may alter normal calcium sensitivity and force contractility, however it is not clear how well these studies reproduce in vivo conditions in humans (PMID: 16115869, 10085122, 10617660); In silico analysis indicates that this missense variant does not alter protein structure/function; This variant is associated with the following publications: (PMID: 16199542, 33025817, 20038417, 25637381, 33148509, 14636924, 7898523, 23074333, 15958377, 10617660, 30847666, 31737537, 31980526, 32731933, 32290750, 31513939, 24418317, 22500102, 21846512, 27022107, 24055113, 21683708, 23299917, 22857948, 12881443, 12860912, 27930701, 26774798, 26914223, 27483260, 26681313, 28518168, 29121657, 17101185, 28193612, 25524337, 11432788, 19033660, 24503780, 26743238, 10405326, 14563299, 16777946, 20800588, 20624503, 20031618, 16715312, 19150014, 10610467, 10085122, 30762279, 31028938, 31006259, 31019283, 27600940, 25611685, 25668678, 25351510, 22144547, 21511876, 18533079, 26183555, 21310275, 20031602, 30645170, 31323898, 30972196, 34426522, 24704860, 35626289, 26507537, 28255936, 34008892, 28771489, 35514357, 36293497, 35441061, 34088630, 12974739, 36698941, 35679367, Pisklova2023, 37180798, 2946667, 24793961, 36264615, 23283745, 37029482, Shafaattalab2023, 34400558, Cao2023, 15246915, 16115869, 37937776, 37652022, 37432431, 37089884)

TNNT2: PM5, PP1, PP4, PS3:Supporting

ACMG Criteria: PS3_M, PM5, BS1, BP5; Variant was found in heterozygous state.

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. p.Arg278Cys in TNNT2 This variant was recently reviewed in detail for one of our research projects. Based on the presence in the general population and the fact that many cases carry an additional pathogenic variant, we would consider this variant a variant of uncertain significance, possibly a modifier. TNNT2 gene p.Arg278Cys (c.832C>T) Summary: -Seen in at least 51 presumably unrelated cases of HCM (26 published, >25 unpublished). -most had European ancestry (at least 30 out of 51); ancestry for others was not reported. -Also seen in DCM cases. -14 of 47 cases who had sequencing of at least MYH7 and TNNT2 had another variant. -5/14 are likely pathogenic/pathogenic, while 4/14 are VUS; 5/14 variant details were not available to confirm the classifications. -60-year-old European (Spain) female with HCM had additional variant in MYBPC3 gene (p.Arg733His) (ClinVar: VUS by GeneDx) -50-year-old European (Spain) female with HCM (LVWT of 22 mm) and her 2 affected family members carried additional variant in MYH7 (p.Asp928Asn) (ClinVar: likely pathogenic by LMM; pathogenic by GeneDx) -14-year-old European (France) individual carried a second variant in MYBPC3 (p.Asp610His) (ClinVar: VUS by LMM) -30.5-year-old HCM case (LVWT of 24 mm) had an additional variant in MYBPC3 (p.Asp560Thrfs*19) (classified as likely pathogenic by LMM) -49-year-old female of unreported ancestry with HCM (LVWT of 35) had an additional variant in MYBPC3 (p.Arg1781His) (ClinVar: VUS by LMM) -46.5-year-old European (Italy) proband with HCM (LVWT of 28 mm) and one affected family member carried a second variant in MYBPC3 gene (p.Thr1095Met) (ClinVar: not found; likely pathogenic by Florence). -56-year-old European (Italy) proband with HCM (LVWT of 18 mm) carried a second variant in MYBPC3 (p.Lys814del) (ClinVar: not found; pathogenic by Florence) -34-year-old female of unreported ancestry with HCM carried an additional variant in the MYBPC3 gene (IVS11-9G>A; c.927-9G>A) (Helms et al (2014): pathogenic based on splice site functional studies) -15-year-old of unreported ancestry with HCM (LVWT of 38) carried an additional variant in the in MYBPC3 gene (p.Ala848Gly) (ClinVar: VUS by GeneDx) -There is some segregation data. In three families, four affected relatives (in addition to probands) carried this variant and one had SCD. In two other families, four affected relatives (in addition to probands) carried p.Arg278Cys in addition to another variant. -In total, the variant has been seen in 40 of ~48,482 individuals (0.08%) from published controls and publicly available datasets that approximate the general population. Most were European ancestry (33 out of 28,255 individuals). In ExAC the highest MAF was 0.06% (33/26987 Europeans). Published cases: Watkins et al (1995) first reported the variant in a 17-year-old female who had normal left ventricular thickness yet had suffered a cardiac arrest; she was resuscitated. The ethnicity of the proband was not reported. Probands included 16 from Europe, 4 from North America, 3 from Japan, and 1 each from China, Southeast Asia, and Pakistan. While this one case is somewhat concerning for an increased risk of sudden death conferred by this variant, other reported cases have had more typical HCM courses, consistent with the marked variable expressivity often seen in primary cardiomyopathies. Elliott et al (1999) reported the variant in a 57-year-old male of unreported ancestry with HCM who was treated at St. George’s Hospital Medical School, London. The genes analyzed were not reported. The proband presented with syncope and dyspnea at the age of 54 years, and was found to have a septum of 12 mm, a left ventricular outflow tract (LVOT) gradient, and systolic anterior motion (SAM) of the mitral valve at 57 years. His grandfather had died suddenly at the age of 60 years. No additional variants were reported. Van Driest et al (2003) observed the variant in three of 389 unrelated patients of unreported ancestry diagnosed with HCM that were cared for at Mayo Clinic’s HCM Clinic in Rochester, Minnesota. The cohort underwent an analysis of TNNT2, TNNI3, TPM1, and ACTC genes. The first proband, a male diagnosed with HCM at age of 54 years with no family history of HCM, presented with LVWT of 20 mm. The second proband, a male diagnosed with HCM at 31 years with family history of HCM, presented with angina, dyspnea, atrial fibrillation, LVWT of 15 mm, and needed a pacemaker. The third proband, a male diagnosed at age 69 year with no family history of HCM, presented with dyspnea, LVWT of 23 mm, and required a septal ablation. No additional variants were reported. Garcia-Castro et al (2003) reported this variant in a 60-year-old female, one of 30 European (Spanish) HCM cases who underwent analysis of the MYH7 and TNNT2 genes. She was diagnosed at age 49 with asymmetric septal hypertrophy and had a history of syncope, dyspnea, dizziness, palpitations, and an LVWT of 22 mm. In 2009, Garcia-Castro et al reported an additional variation in this proband in the MYBPC3 gene (c.2198G>A; p.Arg733His), which is reported as a VUS by GeneDx in ClinVar (as of Aug 29, 2011). They reported that her double heterozygote daughter (40 years old) and granddaughter (6 years old) were “asymptomatic” (no mention of echo phenotyping). Proband’s older 52-year-old sister had mild hypertrophy (LVWT of 13 mm) and carried only p.Arg278Cys. Torriceli et al (2003) reported this variant in one of 150 unrelated HCM patients with European ancestry (Italian) that were cared for at Azienda Ospedaliera Careggi, who underwent the analysis of the MYH7, MYBPC3, TNNT2, and MYL2 genes. This was a 62-year-old male who had an LV thickness of 24 mm, needed percutaneous septal ablation and had no family history of hypertrophic cardiomyopathy. No additional variants were reported. Theopistou et al (2004) reported this variant in two probands with HCM from two families of European ancestry (Greek), who underwent analysis of the TNNT2 gene. In one family, the proband was diagnosed with HCM and a septum of 22 mm at 13 years of age. He died suddenly at 15. His sibling and parents all had normal echocardiograms, and did not have genetics evaluation. In the other family, the proband, 40-year-old male diagnosed at the age of 33 years with LVWT of 22, had family history of sudden death and HCM. His mother had sudden death (no genetics evaluation), and his maternal aunt, who was diagnosed with HCM at 64 years with LVWT of 15 mm, tested positive for this variant. Three members of his family with the variant had normal echocardiograms at 14, 38, and 42 years of ages. No additional variants were reported for either family. Ingles et al (2005) reported this variant in one out of 80 Australian HCM cases. MYH7, MYBPC3, TNNT2, TNNI3, ACTC, MYL2, and MYL3 were evaluated and no other variants were found for this proband. No phenotypic information specific to this proband was provided by the authors. Zeller et al (2006) reported this variant in one of 30 European (German) HCM cases. No additional variants were found in any of the following genes: ACTC, ALP, CAPZB, CARP, DES, DMN, FKRP, FLT1, GJA1, JUP, LDB3, LMNA, MYBPC3, MYH7, MYOZ2, MYPN, NCK2, PLCG1, PXN, SGCD, TNNT2, TPM1, TPM2, TTID, and VEGF. No phenotypic information specific to this proband or additional variants were reported by the authors. Kaski et al (2009) reported this variant in one out of 79 cases with HCM. The ethnicity of the proband was not reported. No additional variants were found in any of the following analyzed genes: MYH7, MYBPC3, TNNI3, TNNT2, TPM1, MYL2, MYL3, ACTC, TNNC1, DES, and PRKAG2. The cohort comprised of 89.9% white, 3.8% Asian, 2.5% black, 2.5% Middle Eastern cases. All of the HCM cases were diagnosed under the age of 13 years. No phenotypic information specific to this proband was provided by the authors. Gimeno et al (2009) reported this variant in two probands with HCM from two families of European ancestry (Spanish). Of note, this report appears to be distinct from those by Garcia-Castro et al. The first proband, female diagnosed at the age of 50 years with LVWT of 22 mm, had two affected family members that carried this variant. Her brother was diagnosed at 18 years with LVWT of 22 mm who presented with palpitations and dyspnea, and her nephew (affected brother’s son) was diagnosed at the age of 18 with LVWT of 40 mm who presented with presyncope. The following genes were analyzed in this family: TNNT2, MYH7, MYBPC3, TPM1, ACTC, TNNI2, TNNC1, MYL2, MYL3. All three of the affected members of this family carried an additional variation in MYH7 gene (c.2782G>A; p.Asp928Asn), which is classified as likely pathogenic and pathogenic by LMM (Aug 26, 2014) and GeneDx (Mar 15, 2014) in ClinVar, respectively. Of note, the proband’s affected son who carried the MYH7 gene variant, but did not carry the TNNT2 variant. The second proband was a male diagnosed at the age of 59 years with 26 mm LVWT, persistent atrial fibrillation, and palpitations. No additional variants were reported for this proband. They report that his 33yo son carries the variant, has LVWT of 1.1 cm and a Brugada pattern on ECG. Millat et al (2010) reported this variant in 4 out of 192 unrelated HCM cases of European ancestry (French) who underwent analysis of MYH7, MYBPC3, TNNT2, and TNNI3 genes. One of the 4 variant carriers, diagnosed at the age of 14, also carried a second variant in the MYBPC3 gene (p.Asp610His), which is classified as a VUS by LMM in ClinVar (Sep 28, 2011). No phenotypic information specific to this proband was provided by the authors. Gruner et al (2011) reported this variant in 1 out of 61 unrelated patients with apical HCM and 3 out of 365 unrelated nonapical HCM cases. Ethnicity of these probands was not reported, however the majority of the cohort had European ancestry (312/425). The proband with apical HCM was a 50-year-old male with maximal wall thickness of 16 mm and syncope. He has no family history of HCM or SCD. One of the 3 nonapical HCM probands had a family history of SCD. No additional variants were reported for these probands after analyzing the following genes: MYBPC3, MYH7, MYL2, MYL3, TNNT2, TNNI3, TPM1, ACTC, GLA, LAMP2, PRKAG2, and PRKAG2. Millat et al (2011) reported this variant in 1 out of 105 unrelated European (French) cases with dilated cardiomyopathy, who underwent analysis of the MYH7, TNNT2, TNNI3 and LMNA genes. The proband was a 69-year-old female with LVEDD of 65 mm, syncope, atrial fibrillation, and NYHA class III (LVWT was not reported). She has at least two family members diagnosed with DCM but no molecular data available on them. Brito et al (2011) reported this variant in a mother and daughter out of 77 unrelated European (Portuguese) cases with HCM, who underwent analysis of MYBPC3, MYH7, TNNT2, TNNI3 and MYL2 genes. In addition, there was a family history of sudden death in a first-degree relative (aged <50 years). No additional variants were reported in this family. Nunez et al (2013) reported this variant in one out of 104 European (Spanish and English) patients with sporadic HCM who underwent analysis of MYH7, MYBPC3, TPM1, TNNT2 and TNNI3 genes. No additional variants were reported in this proband. No phenotypic information specific to this proband was provided by the authors. The variant Additional data: In-silico analysis with PolyPhen-2 predicts the variant to be probably damaging. The arginine at codon 278 is not well conserved in evolution and 2 species (elephant, manatee) carry a cysteine at this position. Other variants have been reported in association with disease at this codon: p.Arg278Pro (Van Driest et al, 2003). In addition, the following variants in nearby codons have been associated with HCM: p.Lys273Glu (Fujino, 2002), p.Arg286Cys (Richard, 2003), p.Arg286His (Van Driest, 2003). Functional studies by Yanaga et al (1999) indicate that p.Arg278Cys causes an increase in Ca2+ sensitivity therefore increasing the contractility of the cell and inducing hypertrophy. Frequency in controls, large cohorts unselected for HCM: In total the variant has been seen in 40 of 48,482 (0.08%) laboratory controls, published controls and individuals from publicly available population datasets. The variant was reported online in 33 of 26,987 European ancestry individuals (0.12%) and 3 of 4,095 African-American individuals in the ExAC Browser dataset (as of 5/1/15). None were selected for rare inherited cardiomyopathies and in some cases those phenotypes were excluded. However, the cohorts that were merged to create this dataset were all either general population samples or samples recruited for common cardiovascular disease such as hypertension. The variant was not observed in 1818 European and 100 African ancestry individuals across the following published studies in presumably healthy controls: 100 in Watkins (1995), 200 in Van Driest (2003), 200 in García-Castro (2003), 150 in Torriceli (2003), 100 in Miliou (2005), 150 in Ingles (2005), 168 in Zeller (2006), 200 in Kaski (2009), 200 in Millat (2010), 200 in Millat (2011), and 200 in Nunez (2013).