ClinVar Genomic variation as it relates to human health

The A226T variant of uncertain significance has been identified in the MYH7 gene. A226T has previously been reported in association with HCM (Bottillo et al., 2016a). In addition, this variant has been identified in one other unrelated individual referred for HCM genetic testing at GeneDx. This variant was not observed in approximately 6,500 individuals of European and African American ancestry in the NHLBI Exome Sequencing Project, indicating it is not a common benign variant in these populations. The A226T variant is a non-conservative amino acid substitution, which is likely to impact secondary protein structure as these residues differ in polarity, charge, size and/or other properties. This substitution occurs at a position that is conserved in mammals. Missense variants in nearby residues (Q222K, A223T, L227V) have been reported in the Human Gene Mutation Database in association with cardiomyopathy (Stenson et al., 2014); however, the pathogenicity of these variants has not been definitively determined. Furthermore, in silico analysis is inconsistent in its predictions as to whether or not the variant is damaging to the protein structure/function. Nevertheless, computational analysis by Bottillo et al. (2016b) predicted that A226T causes steric hindrance, which may lead to functional consequences.Therefore, based on the currently available information, it is unclear whether this variant is pathogenic or benign.

This sequence change replaces alanine, which is neutral and non-polar, with threonine, which is neutral and polar, at codon 226 of the MYH7 protein (p.Ala226Thr). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individuals with clinical features of hypertrophic cardiomyopathy (Invitae). ClinVar contains an entry for this variant (Variation ID: 372429). 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 MYH7 protein function with a positive predictive value of 95%. This variant disrupts the p.Ala226 amino acid residue in MYH7. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 27247418, 27532257; Invitae). 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 p.A226T variant (also known as c.676G>A), located in coding exon 6 of the MYH7 gene, results from a G to A substitution at nucleotide position 676. The alanine at codon 226 is replaced by threonine, an amino acid with similar properties. This alteration is located in the myosin head domain, which contains a statistically significant clustering of pathogenic missense variants (Homburger JR et al. Proc Natl Acad Sci U S A, 2016 06;113:6701-6; Walsh R et al. Genet Med, 2017 02;19:192-203; Ambry internal data). In addition, this alteration has been described in multiple individuals with hypertrophic cardiomyopathy (HCM) (Bottillo I et al. Gene. 2016;577:227-35; Rubattu S et al. Int J Mol Sci, 2016 Jul;17; Ambry internal data; external communication). Another alteration involving the same amino acid, p.A226V (c.677C>T), has also been reported in a HCM cohort (Homburger JR et al. Proc Natl Acad Sci U.S.A. 2016 06;113:6701-6). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). This amino acid position is well conserved in available vertebrate species. In addition, the in silico prediction for this alteration is inconclusive. Based on the majority of available evidence to date, this variant is likely to be pathogenic.

We have seen this variant in one case of HCM at our center. Testing was done at Invitae. Given it has only been published in one case, is rare, and the data on tolerance at this location is conflicted, we consider this variant a variant of uncertain significance, and we do not feel it is suitable for assessing risk in healthy relatives ("predictive genetic testing"). The variant has been seen in at least one unrelated case of HCM (not including this patient's family). There is no case data. Bottillo et al (2016) observed the variant in 1/44 patients with HCM in their cohort from Rome, Italy (authors are from “Sapienza University of Rome”). Cecconi et al (2016) and Rubattu et al (2016) also report a case of this variant. Given overlap in institution and at least one author for Bottillo et al and Rubattu et al, it seems reasonable to suspect they are reporting the same case (and the same cohort). It is more difficult to determine whether the Cecconi et al case is redundant. Unfortunately minimal to no case data is reported in these papers so it is difficult to assess whether the cases are distinct. The variant is not listed in ClinVar (as of 3 January 2016). The variant has not been observed in the SHaRe consortium and was not reviewed for the ClinGen MYH7 classification project. The variant was not reviewed for the ClinGen MYH7 pilot project. A different missense substitution at this codon (p.Ala226Val) has been reported with HCM and a different group has been determined to be pathogenic based on internal cases, in silico predictions, and absence in control populations. p.Ala226Val has been in seen in two patients with HCM in the SHaRe consortium, both cared for at the University of Michigan. One proband has an affected family member who also carries the variant. These individuals were tested at GeneDx and LMM so their case data overlaps with that of those two labs. These cases are also redundant with Homburger et al 2016 (PMID 27247418). Walsh et al (2016, PMID: 27532257) report p.Ala226Val was seen in 1 patient with HCM of 3200 patients with cardiomyopathy included in their analysis. The variant lies within the myosin head, which is less tolerant to variation (Walsh et al 2016). However, a study that further dissected the regions of the head that are enriched for variation in cases vs. reference samples did not see enrichment at this residue (Homburger et al 2016). There is no variation at 226 codon listed in the Genome Aggregation Consortium Dataset (gnomAD; http://gnomad.broadinstitute.org/), which currently includes variant calls on >140,000 unrelated individuals of African, Asian, European, Latino, and Ashkenazi descent. I could not find coverage data in gnomAD for this site, however a nearby site appears to be well covered. Note that at this time the gnomAD interface and data are still in beta, with this warning listed “All data on this website should be treated with caution”. There is no variation at codon 226 listed in ExAC, which currently includes variant calls on ~60,000 unrelated individuals of African, Asian, European, Latino, and Ashkenazi descent. The average coverage at that site in ExAC is 100x.