ClinVar Genomic variation as it relates to human health

This sequence change replaces aspartic acid, which is acidic and polar, with asparagine, which is neutral and polar, at codon 753 of the CAPN3 protein (p.Asp753Asn). This variant is present in population databases (rs146923842, gnomAD 0.1%), and has an allele count higher than expected for a pathogenic variant. This missense change has been observed in individuals with limb girdle muscular dystrophy (PMID: 16141003, 18854869). ClinVar contains an entry for this variant (Variation ID: 281081). 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 not expected to disrupt CAPN3 protein function. 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.

Reported, without a second identifiable CAPN3 variant, in an individual with LGMD2A who had complete calpain-3 deficiency on Western blot (PMID: 15221789); Reported in the heterozygous and compound heterozygous state in association with LGMD2A; however, parental testing was not completed and functional characterization of the variant was not performed (PMID: 16141003, 15689361, 17318636); Reported previously in five patients with LGMD and two of these patients harbored a second variant (phase unknown). No further clinical or segregation information was provided (PMID: 18854869, 30564623); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 17994539, 15689361, 31937337, 17318636, 27884173, 32403337, 31931849, 30564623, Koken_2022[Poster], 32528171, 34720847, 35157181, 38391941, 16141003, 18854869, 36575883, 15221789)

Across a selection of the available literature, the CAPN3 c.2257G>A (p.Arg753Asn) is identified in six probands described to have limb girdle muscular dystrophy in a heterozygous state and five probands in a compound heterozygous state (Fanin et al. 2004; Piluso et al. 2005; Saenz et al. 2005; Todorova et al. 2007; Fanin et al. 2009). The p.Arg753Asn variant absent from at least 400 controls and is reported at a frequency of 0.00118 in the European (non-Finnish) population of the Exome Aggregation Consortium. Todorova et al. (2007) noted that the variant, when observed in a heterozygous state in probands with limb girdle muscular dystrophy, seemed to confer a later onset and milder phenotype. Based on the evidence, the p.Asp753Asn variant is classified as likely pathogenic for calpainopathy. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.

NM_000070.2(CAPN3):c.2257G>A(D753N) is classified as a variant of uncertain significance in the context of calpainopathy. Sources cited for classification include the following: PMID 17318636, 17994539, 17897828, 18854869, 20044116, 16141003, 27884173 and 30564623. Classification of NM_000070.2(CAPN3):c.2257G>A(D753N) is based on the following criteria: At this time, there is insufficient or conflicting evidence to classify this variant as pathogenic or benign. Please note: this variant was assessed in the context of healthy population screening.

The c.2257G>A;p.(Asp753Asn) missense variant has been observed in affected individual(s) and ClinVar contains an entry for this variant (PMID: 31517061; 18854869) - PS4_moderate. The variant is present at low allele frequencies population databases (rs146923842 – gnomAD 0.007563%; ABraOM 0.000854 frequency - http://abraom.ib.usp.br/) - PM2_supporting. The p.(Asp753Asn) was detected in trans with a pathogenic variant (PMID: 31517061; 18854869) - PM3. Multiple lines of computational evidence support a deleterious effect on the gene or gene product - PP3. In summary, the currently available evidence indicates that the variant is likely pathogenic.

Variant summary: CAPN3 c.2257G>A (p.Asp753Asn) results in a conservative amino acid change located in the EF-hand domain of the encoded protein sequence. Three of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 0.00079 in 250800 control chromosomes (gnomAD). This frequency is not significantly higher than estimated for a pathogenic variant in CAPN3 causing Limb-Girdle Muscular Dystrophy, Autosomal Recessive (0.00079 vs 0.0032), allowing no conclusion about variant significance. c.2257G>A has been reported in the literature in the heterozygous or compound heterozygous state in individuals affected with Limb-Girdle Muscular Dystrophy without strong evidence for causality (eg. Fanin_2004, Piluso_2005, Saenz_2005, Todorova_2007, Guglieri_2008, Fanin_2009, Nallamilli_2018, Macias_2021, Ozyilmaz_2022). These reports do not provide unequivocal conclusions about association of the variant with autosomal recessive Limb-Girdle Muscular Dystrophy. To our knowledge, no experimental evidence demonstrating an impact on protein function has been reported. The following publications have been ascertained in the context of this evaluation (PMID: 30564623, 17994539, 31931849, 18854869, 16141003, 15221789, 32528171, 15689361, 32403337, 34720847, 31517061, 17318636, 35157181). Ten submitters have provided clinical-significance assessments for this variant to ClinVar after 2014 and classified the variant as either VUS (n=8) or likely pathogenic (n=2). Based on the evidence outlined above, the variant was classified as uncertain significance.

PM3_supporting

PM3, BS1

The CAPN3 p.D753N variant was identified in the heterozygous or compound heterozygous state in 9 of 2194 proband chromosomes (frequency: 0.004) from individuals with Limb girdle muscular dystrophy (LGMD) type 2A (LGMD2A) and unspecified muscular dystrophy (Todorova_2007_PMID:17318636; Piluso_2005_PMID:16141003; Fanin_2009_PMID:18854869). The variant was identified in dbSNP (ID: rs146923842), ClinVar (classified as uncertain significance by EGL Genetics, GeneDx and Invitae and as likely pathogenic by Counsyl and Illumina) and LOVD 3.0 (classified as pathogenic and a VUS). The variant was identified in control databases in 210 of 282180 chromosomes (0 homozygous) at a frequency of 0.0007442 increasing the likelihood this could be a low frequency benign variant (Genome Aggregation Database March 6, 2019, v2.1.1). The variant was observed in the following populations: Latino in 39 of 35324 chromosomes (freq: 0.001104), European (non-Finnish) in 142 of 128804 chromosomes (freq: 0.001102), Ashkenazi Jewish in 8 of 10354 chromosomes (freq: 0.000773), Other in 4 of 7212 chromosomes (freq: 0.000555), European (Finnish) in 6 of 25108 chromosomes (freq: 0.000239), East Asian in 4 of 19914 chromosomes (freq: 0.000201), South Asian in 4 of 30576 chromosomes (freq: 0.000131), and African in 3 of 24888 chromosomes (freq: 0.000121). The p.Asp661 residue is conserved in mammals and computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) provide inconsistent predictions regarding the impact to the protein; this information is not very predictive of pathogenicity. The variant occurs outside of the splicing consensus sequence and three of four in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) do not predict a greater than 10% difference in splicing; this is not very predictive of pathogenicity. Fanin et al. (2009) identified the p.D753N variant in two heterozygous and two compound heterozygous patients with Limb girdle muscular dystrophy; these patients had varying degrees of calpain levels and autolytic activity ranging from normal to absent (Fanin_2009_PMID:18854869). In summary, based on the above information the clinical significance of this variant cannot be determined with certainty at this time. This variant is classified as a variant of uncertain significance.