ClinVar Genomic variation as it relates to human health

Published functional studies demonstrate a damaging effect: Z-disc disruption and F-actin accumulation in mouse skeletal muscle, and disruption in the actin cytoskeleton in muscle cells (Lin et al., 2014); Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports that this missense variant does not alter protein structure/function; This variant is associated with the following publications: (PMID: 28269794, 19377068, 27546599, 27618136, 25208129, 27638134, 18765652, 15668942, 33742095, 31791368, 21676617, 32419263, 31589614, 17337483, 24668811)

The c.690-4678C>T intronic pathogenic mutation results from a C to T substitution 4678 nucleotides upstream from coding exon 5 in the LDB3 gene. This alteration, also known as c.494C>T p.A165V in isoform NM_001080116, has been detected in multiple individuals with myofibrillar myopathy and has been reported to segregate with disease (Selcen D et al. Ann. Neurol., 2005 Feb;57:269-76; Griggs R et al. Brain, 2007 Jun;130:1477-84; Olivé M et al. Neuromuscul. Disord., 2011 Aug;21:533-42; Semmler AL et al. Orphanet J Rare Dis, 2014 Aug;9:121; Vincent AE et al. Neuromuscul. Disord., 2016 10;26:691-701). Haplotype analysis has demonstrated that this variant is a European founder mutation (Griggs R et al. Brain, 2007 Jun;130:1477-84). Functional analyses indicate that this alteration may impact protein function, but the physiological relevance of the observed impacts is unclear (Lin X et al. J. Biol. Chem., 2014 May;289:13615-26). 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.

The c.690-4678C>T variant in LDB3 (also known as c.494C>T, p.Ala165Val with the alternate transcripts) is highly conserved. It was identified in 1/249596 (0.0004%) of alleles tested from presumed healthy individuals in the Genome Aggregation Database (gnomAD), and is considered to be a rare variant. It has been previously reported in multiple apparently unrelated individuals and families with myofibrillar myopathy, segregated with the disease, and has been reported to be a founder mutation (PMID; 15668942, 17337483, 25208129, and others). The majority of these affected individuals were not reported to have cardiomyopathy as a presenting or significant feature (reported using the alternate gene name ZASP, PMID 15668942, 17337483, 18765652, 27618136, doi: 10.17795/gct.34601, and others). Functional studies have shown conflicting results: one reported that this variant did not impact the binding between LDB3 and phosphoglucomutase 1, which is suspected to be the mechanism of development of DCM (PMID 19377068), and others showed that this variant disrupted the actin cytoskeleton of muscle cells, as well as the Ankrd2 binding (which creates the link between the sarcomere and the nucleus in skeletal muscle) (PMID 24668811, PMID 24647531). Further, heterozygous knock-in mice develop are found to develop myofibrillar myopathy, however, the cardiac muscle fibers of these mice showed normal histology (PMID 33742095). The p.Ala165 variant is present in exon 6 of alternate transcripts for LDB3; immunostaining of skeletal and cardiac muscle in mice with a knockout-validated LDB3 exon 6 antibody detected LDB3 in the skeletal muscle but not the cardiac muscle, suggesting that isoforms containing exon 6 are not the predominant isoforms expressed in the cardiac muscle (PMID 33742095). In silico splicing analyses do not predict this variant to have a significant effect on splicing. However, this prediction has not been confirmed by RNA functional studies. This variant is listed in ClinVar (VCV000004728). Based on the above evidence, we classify this variant as likely pathogenic for myofibrillar myopathy, which, in rare cases, could also produce cardiomyopathy.

LDB3: PM2, PP1:Moderate, PS3:Moderate, PS4:Moderate, PP3, PP4

The p.Ala165Val variant in LDB3 has been reported in at least 6 individuals with myofibrillar myopathy and segregated with disease in 9 affected individuals fro m 1 family (Selcen 2005, Griggs 2007, Fischer 2008, Vincent 2016, LMM data). It has also been identified in 1/113280 of European chromosomes by gnomAD (http://g nomad.broadinstitute.org) and reported in ClinVar (Variant ID #4728). An in vitr o function study showed that this variant disrupted Ankrd2 binding (Martinelli 2 014) and a study of mouse myoblasts transfected with the p.Ala165Val mutation sh owed a disrution of Z-disc structure and an accumulation of F-actin (Lin 2014). In summary, this variant meets criteria to be classified as pathogenic for autos omal dominant myofibrillar myopathy. ACMG/AMP criteria applied: PP1_Strong, PM2, PS3_Moderate, PS4_Moderate.

The LDB3 c.494C>T (p.Ala165Val) variant, also referred to as c.690-4678C>T, is a missense variant that has been reported in at least seven unrelated individuals with myofibrillar myopathy presenting with progressive muscle weakness, mainly distally, and myopathic motor unit potentials noted through electromyography. Muscle atrophy, tripping episodes, walking difficulty, peripheral neuropathy, decreased reflexes in the legs, tingling and numbness in the feet, and ankle movement problems were also noted in some individuals (Selcen and Engler 2005; Griggs et al. 2007; Fischer et al. 2008; Semmler et al. 2014). Only one individual had cardiac problems presenting as prolonged QT (Selec and Engler 2005). The p.Ala165Val variant segregated with disease in a large pedigree where six affected individuals carried the variant. Three additional family members who carried the variant were asymptomatic at the time of the study, but they ranged in age from 39-49 years (Griggs et al. 2007). In functional studies using a mouse model or cells derived from mice, the phenotype observed in humans was recapitulated when the p.Ala165Val variant was present in a heterozygous state. This included presence of muscle weakness, characteristic muscle fiber structural abnormalities, protein aggregation patterns, and Z-disc disruption generally observed in myofibrillar myopathy (Lin et al. 2014; Pathak et al. 2014). The p.Ala165Val variant is reported at a frequency of 0.000009 in the European (non-Finnish) population of the Genome Aggregation Database, but this is based on one allele in a region of good sequence coverage, so the variant is presumed to be rare. Based on the collective evidence, the p.Ala165Val variant is classified as pathogenic for myofibrillar myopathy.

This sequence change replaces alanine, which is neutral and non-polar, with valine, which is neutral and non-polar, at codon 165 of the LDB3 protein (p.Ala165Val). This variant is present in population databases (rs121908334, gnomAD 0.0009%). This missense change has been observed in individuals with myofibrillar myopathy (PMID: 15668942, 25208129). ClinVar contains an entry for this variant (Variation ID: 4728). 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 LDB3 function (PMID: 24647531, 24668811). For these reasons, this variant has been classified as Pathogenic.

The LDB3 c.494C>T variant is predicted to result in the amino acid substitution p.Ala165Val. This variant has been reported in individuals with myofibrillar myopathy and shown to segregate with disease in families (see, for example, Selcen and Engel. 2005. PubMed ID: 15668942; Griggs et al. 2007. PubMed ID: 17337483; Semmler et al. 2014. PubMed ID: 25208129). In vitro and in vivo experimental studies suggest this variant impacts protein function (Lin et al. 2014. PubMed ID: 24668811; Martinelli et al. 2014. PubMed ID: 24647531; Pathak et al. 2021. PubMed ID: 33742095). This variant is reported in 0.00088% of alleles in individuals of European (non-Finnish) descent in a large population database. This variant is interpreted as pathogenic.