ClinVar Genomic variation as it relates to human health

TNNI3 Arg162Gln has been identified in multiple HCM probands (Walsh R, et al., 2017; Cecconi M, et al., 2016; Mouton JM, et al., 2015; Coppini R, et la., 2014; Kapplinger JD, et al., 2014; Rani DS, et al., 2012; Bos JM, et al., 2006; Mogensen J, et al., 2004; Van Driest SL, et al., 2003) and has been found to segregate with disease in 2 HCM families (Rani DS, et al., 2012; Mogensen J, et al., 2004). The variant is present at a low frequency in the Exome Aggregation Consortium dataset (MAF=0.00003; http://exac.broadinstitute.org/). We have identified this variant in 3 HCM probands (Burns et al., 2017), one of their families have been previously described (Ingles J, et al., 2005; Doolan A, et al., 2005). Computational tools CADD, MutationTaster, and PolyPhen-2 predict this variant to have a deleterious effect, however SIFT predicts this variant to be "tolerated". A mammalian two-hybrid system has shown that this missense change decreases troponin T and troponin C interaction (Doolan A, et al., 2005), whereas crystal structure modelling suggests that the Arg162Gln affects troponin C stability (Ramachandran G, et al., 2013). In summary, the TNNI3 Arg162Gln is a rare variant which has been described in multiple HCM probands around the world and has been found to segregate strongly in at least 2 families, therefore we classify this variant as "pathogenic".

The p.R162Q variant (also known as c.485G>A), located in coding exon 7 of the TNNI3 gene, results from a G to A substitution at nucleotide position 485. The arginine at codon 162 is replaced by glutamine, an amino acid with highly similar properties. This variant has been reported in multiple individuals with hypertrophic cardiomyopathy (HCM) (e.g., Van Driest SL et al. Circulation. 2003;108(4):445-51; Ingles J et al. J Med Genet. 2005;42(10):e59; Gruner C et al. Circ Cardiovasc Genet. 2011;4(3):288-95; Coppini R et al. J Am Coll Cardiol. 2014;64(24):2589-600; Lopes LR et al. Heart. 2015; 101(4):294-301; Mouton JM et al. Cardiovasc J Afr. 2015; 26(2):63-9; Walsh R et al. Genet. Med. 2017;19:192-203). This variant co-segregated with disease in three families; however, multiple unaffected family members also carried the variant (Mogensen J et al. J Am Coll Cardiol. 2004; 44(12):2315-25; Doolan A et al. J Mol Cell Cardiol. 2005; 38(2):387-93; Rani DS et al. BMC Med Genet. 2012;13:69). This amino acid position is not well conserved in available vertebrate species. In addition, this alteration is predicted to be tolerated by in silico analysis. Based on the majority of available evidence to date, this variant is likely to be pathogenic.

This sequence change replaces arginine, which is basic and polar, with glutamine, which is neutral and polar, at codon 162 of the TNNI3 protein (p.Arg162Gln). This variant is present in population databases (rs397516354, gnomAD 0.006%). This missense change has been observed in individuals with hypertrophic cardiomyopathy (PMID: 12860912, 15607392, 15698845, 15992656, 16352453, 22876777). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 43389). 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 tolerated. Experimental studies have shown that this missense change affects TNNI3 function (PMID: 15698845). For these reasons, this variant has been classified as Pathogenic.

PS3, PM5, PM1, PS4_moderate, PP3, PP1

Same nucleotide change resulting in same amino acid change has been previously reported as pathogenic/likely pathogenic with strong evidence (ClinVar ID: VCV000043389, PMID:12860912, PS1_S).The variant was co-segregated with Cardiomyopathy, hypertrophic, 7, associated with TNNI3 gene in multiple affected family members with additional meioses meeting strong evidence levels (PMID: 15607392, 22876777) (PP1_S). The variant has been observed in at least two similarly affected unrelated individuals (PMID: 12860912, 15607392, 16352453, PS4_M). Different missense changes at the same codon have been reported as pathogenic/likely pathogenic with strong evidence (ClinVar ID: VCV000043390,VCV000626844, PMID:9241277,12707239, PM5_M). In silico tool predictions suggest damaging effect of the variant on gene or gene product (3CNET: 0.883, PP3_P). A missense variant is a common mechanism associated with Cardiomyopathy (PP2_P). Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

Reported in multiple unrelated patients with HCM (Van Driest et al., 2003; Mogensen et al., 2004; Doolan et al., 2005; Ingles et al., 2005; Rani et al., 2012; Coppini et al., 2014; Kapplinger et al., 2014; Mouton et al., 2015; Restrepo-Cordoba et al., 2017); Not observed at significant frequency in large population cohorts (gnomAD); Published in vitro functional studies using HEK293 cells showed that the R162Q variant results in a 50% reduction of the troponin I and troponin C interaction (Doolan et al., 2005); In silico analysis supports that this missense variant does not alter protein structure/function; This variant is associated with the following publications: (PMID: 16020591, 9241277, 23967088, 25524337, 25940119, 21310275, 16199542, 22876777, 16352453, 15992656, 23396983, 25351510, 12707239, 24510615, 18227814, 27600940, 12860912, 27532257, 28138913, 15607392, 31006259, 23270746, 21511876, 10806205, 11735257, 31877599, 25228707, 24113344, 31447099, 24704860, 34426522, 32731933, 32686758, 31534214, 33087929, 15698845, 28790153, 28771489, 28166811, 29907799, 27840609, 33673806)

Variant summary: TNNI3 c.485G>A (p.Arg162Gln) results in a conservative amino acid change in the encoded protein sequence. Four of five in-silico tools predict a benign effect of the variant on protein function. The variant allele was found at a frequency of 4e-05 in 249030 control chromosomes (gnomAD). This frequency is not significantly higher than estimated for a pathogenic variant in TNNI3 causing Hypertrophic Cardiomyopathy (4e-05 vs 0.00013), allowing no conclusion about variant significance. c.485G>A has been reported in the literature in multiple individuals affected with Hypertrophic Cardiomyopathy (examples: Bos_2006, Cheng_2005, Doolan_2005,Mogensen_2004, VanDriest_2003). These data indicate that the variant is very likely to be associated with disease. The following publications have been ascertained in the context of this evaluation (PMID: 15698845, 15607392, 12860912, 15992656, 16352453). Thirteen clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 and all classified the variant as pathogenic/likely pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

This missense variant replaces arginine with glutamine at codon 162 of the TNNI3 protein. Computational prediction suggests that this variant may not impact protein structure and function (internally defined REVEL score threshold <= 0.5, PMID: 27666373). A functional study has shown that this variant disrupts interactions with troponin C and T (PMID: 15698845). This variant has been reported in over 15 unrelated individuals affected with hypertrophic cardiomyopathy (PMID: 12860912, 15607392, 15698845, 15992656, 16352453, 25940119, 31877599, 33029862, 3349559, 33673806, 35470680, 36291626). This variant has been shown to segregate with hypertrophic cardiomyopathy in a family study (PMID: 22876777). This variant has also been reported in the homozygous state in an individual affected with severe, early-onset hypertrophic cardiomyopathy (PMID: 31877599). This individual's four relatives were asymptomatic heterozygous carriers. This variant has been identified in 10/249030 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Different missense variants occurring at the same codon, p.Arg162Pro and p.Arg162Trp, are known to cause disease (ClinVar variation ID: 43390, 161396), indicating that arginine at this position is important for TNNI3 protein function. Based on the available evidence, this variant is classified as Likely Pathogenic.

The p.Arg162Gln variant in TNNI3 has been reported in >20 individuals with HCM, one of whom was homozygous and 3 of whom also carried other pathogenic variants in HCM-associated genes. This variant also segregated with disease in >10 affected relatives from multiple families (Van Driest 2003 PMID:12860912, Mogensen 2004 PMID:15607392, Doolan 2005 PMID:15698845, Cheng 2005 PMID:15992656, Ingles 2005 PMID:16199542, Bos 2006 PMID:16352453, Gruner 2011 PMID:21511876, Rani 2012 PMID:22876777, Kapplinger 2014 PMID:24510615, Mouton 2015 PMID:25940119, Cecconi 2016 PMID 27600940, Burns 2017 PMID 28790153, Walsh 2017 PMID 27532257, Lorenzini 2020 PMID 32731933, LMM data). However, the p.Arg162Gln variant was also present in several unaffected relatives, many of whom were older than 50, suggesting reduced penetrance or a milder role (Mogensen 2004 PMID: 15607392, LMM data). This variant did not segregate with disease in 1 affected relative, although this discrepancy may be explained by the presence of more than 1 pathogenic variant in this family or an environmental origin of disease. This variant has also been reported by other clinical laboratories in ClinVar (Variation ID 43389) and was also identified in 0.005% (6/113172) of European chromosomes by gnomAD (http://gnomad.broadinstitute.org). In vitro functional studies using a mammalian two-hybrid system suggests that this variant may impact protein function by affecting the interaction with other binding partners (troponin T and troponin C; Doolan 2005 PMID:15698845). Computational prediction tools and conservation analyses suggest that this variant may not impact the protein, though this information is not predictive enough to rule out pathogenicity. Additionally, two different variants at this position (p.Arg162Pro and p.Arg162Trp) were identified in multiple individuals with HCM (Kimura 1997 PMID:9241277, Richard 2005 PMID:12707239, Doolan 2005 PMID:15698845, Ingles 2005 PMID:16199542, Gray 2013 PMID:23270746, LMM data), suggesting that a change of this amino acid residue is not tolerated. In summary, the p.Arg162Gln variant meets criteria to be classified as likely pathogenic for autosomal dominant HCM; however, this variant may have a milder role suggested by the incomplete penetrance seen in some family members and the individuals who were homozygous or double heterozygous. ACMG/AMP Criteria applied: PS4_Moderate, PP1_Strong, PS3_Supporting, BP4.

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as Likely pathogenic. Following criteria are met: 0103 - Gain of function and loss of function are reported mechanisms of disease in this gene. The former is associated with familial restrictive cardiomyopathy 1 (MIM#115210) and hypertrophic cardiomyopathy 7 (MIM#613690), while the latter is associated with dilated cardiomyopathy 1FF (MIM#613286) (PMID: 19914256, PMID:21533915). (I) 0108 - This gene is associated with both recessive and dominant disease. The recessive form of inheritance is the exception and has only been reported in a small number of families (PMID: 15070570, PMID:23270746). (I) 0112 - The condition associated with this gene has incomplete penetrance (PMID: 15607392). (I) 0115 - Variants in this gene are known to have variable expressivity (PMID: 23270746). (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 (v2) <0.001 for a dominant condition (10 heterozygotes, 0 homozygotes). (SP) 0309 - An alternative amino acid change at the same position has been observed in gnomAD (v2) (10 heterozygotes, 0 homozygotes). (I) 0502 - Missense variant with conflicting in silico predictions and uninformative conservation. (I) 0600 - Variant is located in the annotated troponin domain (PDB). (I) 0705 - No comparable missense variants have previous evidence for pathogenicity. Multiple alternative amino acid changes have been reported at amino acid position 162, however, they are all major amino acid changes and are not comparable to p.(Arg162Gln) which is a minor change. (I) 0801 - This variant has strong previous evidence of pathogenicity in unrelated individuals. This variant has been reported in at least 20 HCM probands (ClinVar, PMID: 27532257, PMID: 28615295, PMID 15607392, VCGS). 0903 - This variant has limited evidence for segregation with disease. This variant has shown incomplete penetrance in multiple HCM families (PMID: 15607392, PMID: 28615295). (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 162 of the TNNI3 protein. Computational prediction suggests that this variant may not impact protein structure and function (internally defined REVEL score threshold <= 0.5, PMID: 27666373). A functional study has shown that this variant disrupts interactions with troponin C and T (PMID: 15698845). However, clinical relevance of this observation is not known. This variant has been reported in over ten unrelated individuals affected with hypertrophic cardiomyopathy (PMID: 12860912, 15607392, 15698845, 15992656, 16352453, 25940119, 31877599, 33029862, 3349559, Yang et al 2018). This variant has been shown to segregate with hypertrophic cardiomyopathy in a family study (PMID: 22876777). This variant has also been reported in the homozygous state in an individual affected with severe, early-onset hypertrophic cardiomyopathy (PMID: 31877599). The proband's four relatives were asymptomatic heterozygous carriers. This variant has been identified in 10/249030 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Different missense variants occurring at the same codon, p.Arg162Pro and p.Arg162Trp, are known to cause disease (Clinvar variation ID: 43390, 161396), indicating that arginine at this position is important for the protein function. Based on the available evidence, this variant is classified as Likely Pathogenic.

TNNI3: PP1:Strong, PM2, PM5, PS4:Moderate, PS3:Supporting, BP4

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. The variant has been seen in at least 11 unrelated cases of HCM (not including this patient's family). There is supporting segregation data available. We have seen this variant in one other person with HCM in our clinic. Per the LMM ClinVar summary "the Arg162Gln variant in TNNI3 has been reported in >10 individuals with HCM and segregated with disease in 11 affected family members across several families (Van Driest 2003, Doolan 2005, Mogensen 2004, Ingles 2005, Rani 2012, LMM unpublished data)." In ClinVar GeneDx notes that they have observed the variant in several unrelated individuals tested for HCM (phenotyeps not given). LMM notes "in 1 family tested at our laboratory, the variant was absent in an affected individual, raising additional concerns, although this discrepancy can have multiple explanations including the presence of more than 1 pathogenic variant or an environmental origin of disease." Other variants have been reported in association with disease at this codon (p.Arg162Pro and p. Arg162Gly) . The variant is located within a functionally significant troponin C binding site domain. In silico analysis of R162Q mutant protein demonstrated a decrease in protein stability (Ramachandran et al., 2013). There is also expertimental evidence tha tsuggests this variant dirupts protein-protein interactions. Per the LMM ClinVar submission "arginine (Arg) at position 162 is not conserved in evolutionarily distant species (two bat species, birds, and fish), supporting that a change at this position may be tolerated; however, not all pathogenic variants are conserved in lower species." The variant was reported online in 3 of 60,827 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 July 24, 2015). Specifically, the variant was observed in 2/33619 Europeans and 1/8314 South Asian. Another variant at the same codon, p.Arg162Trp, is present in 4 of 60,876 individuals in ExAC. 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. Note that other variants with strong evidence for pathogenicity have been seen at similar frequencies in a dataset like this so this does not necessarily rule out pathogenicity (Pan et al 2012).

The TNNI3 c.485G>A variant is predicted to result in the amino acid substitution p.Arg162Gln. This variant has been frequently reported and found to segregate with disease in individuals with hypertrophic cardiomyopathy (see, for example, Van Driest et al. 2003. PubMed ID: 12860912; Mogensen et al. 2004. PubMed ID: 15607392; Ingles et al. 2005. PubMed ID: 16199542; Supplementary Table 1, Coppini et al. 2014. PubMed ID: 25524337). This variant is reported in 0.0065% of alleles in individuals of South Asian descent in gnomAD. This variant is interpreted as pathogenic.