ClinVar Genomic variation as it relates to human health

Not observed at significant frequency in large population cohorts (gnomAD); Canonical splice site variant expected to result in aberrant splicing, although in the absence of functional evidence the actual effect of this sequence change is unknown.; This variant is associated with the following publications: (PMID: 25525159, 27765764, 31447099, 32041611, 26582918, 32220565, 33303402, 11668627, 34037665)

This sequence change affects an acceptor splice site in intron 3 of the LDLR gene. It is expected to disrupt RNA splicing. Variants that disrupt the donor or acceptor splice site typically lead to a loss of protein function (PMID: 16199547), and loss-of-function variants in LDLR are known to be pathogenic (PMID: 20809525, 28645073). This variant is not present in population databases (gnomAD no frequency). Disruption of this splice site has been observed in individuals with familial hypercholesterolemia (PMID: 27765764, 27824480). This variant is also known as IVS3-2A>C inthe literature. ClinVar contains an entry for this variant (Variation ID: 251142). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site. For these reasons, this variant has been classified as Pathogenic.

This variant (also known as IVS3-2A>C) causes an A to C nucleotide substitution at the -2 canonical splice acceptor site of intron 3 of the LDLR gene. Splice site prediction tools suggest that this variant may have a significant impact on RNA splicing. Although this prediction has not been confirmed in published RNA studies, this variant is expected to result in an absent or disrupted protein product. This variant has been reported in at least 8 individuals affected with familial hypercholesterolemia (PMID: 11668627, 27765764, 27824480, 32220565). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Loss of LDLR function is a known mechanism of disease. Based on available evidence, this variant is classified as Likely Pathogenic.

The c.314-2A>C variant in LDLR has been reported in the heterozygous state in at least 5 individuals with familial hypercholesterolemia (FH; Wang 2001, Wang 2016. This variant has also been reported in ClinVar (Variation ID: 251142) and was absent from large population studies. This variant occurs in the invariant region (+/- 1,2) of the splice consensus sequence and is predicted to cause altered splicing leading to the activation of a downstream splice site that would result in the first 8 bases of the exon to be spliced out, causing a frameshift which alters the protein's amino acid sequence and leads to a premature termination codon 22 amino acids downstream. In summary, although additional studies are required to fully establish its clinical significance, the c.314-2A>C variant is likely pathogenic. ACMG/AMP Criteria applied (Richards 2015): PVS1_Strong; PM2; PS4_Supporting.

The c.314-2A>C intronic pathogenic mutation results from an A to C substitution two nucleotides upstream from coding exon 4 in the LDLR gene. Alterations that disrupt the canonical splice site are expected to result in aberrant splicing. In silico splice site analysis predicts that this alteration will weaken the native splice acceptor site and may result in the creation or strengthening of a novel splice acceptor site. The resulting transcript is predicted to be in-frame and is not expected to trigger nonsense-mediated mRNAdecay; however, direct evidence is unavailable. The exact functional effect of the altered amino acid sequence is unknown; however, the impacted region is critical for protein function (Ambry internal data). This alteration has been described in familial hypercholesterolemia (FH) cohorts (Wang J et al. Hum Mutat. 2001;18:359; Wang J et al. Arterioscler Thromb Vasc Biol, 2016 12;36:2439-2445; Sturm AC et al. JAMA Cardiol, 2021 08;6:902-909). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). Another alteration impacting the same acceptor site (c.314-1G>A) has also been detected in individuals with FH (Huijgen et al 2010; Hum Mut 31(6):752-60; Lombardi P et al. Hum Mutat, 1998;Suppl 1:S172-4; Luirink IK et al. Atherosclerosis, 2019 06;285:87-92). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

The c.314-2A>C intronic variant, also known as IVS3-2A>C is located at the canonical acceptor splice site of intron 3 of the LDLR gene, that encodes for low density lipoprotein receptor. This variant has been reported in at least ten individuals affected with familial hypercholesterolemia (FH) (PMID:11668627, 27765764, 34037665, 32220565). Another intronic variant affecting the same nucleotide, c.314-2A>G, has also been reported in individuals with FH (PMID: 27824480). Variants affecting the same splice site (c.314-1G>A and c.314-3C>T) have been observed in individuals diagnosed with FH (PMID 11810272, 16250003). In-silico computational prediction tools suggest that the c.314-2A>C variant likely leads to acceptor loss (SpliceAI: 0.99 [acceptor loss], 0.32 [acceptor gain at +10bp position]) and disturb normal splicing, resulting in aberrant or absence of protein product (PMID: 16199547). This variant is absent in the general population database, gnomAD. This variant has been interpreted as likely pathogenic/pathogenic by multiple submitters in the ClinVar database (ID: 251142). Therefore, the c.314-2A>C intronic variant in the LDLR gene is classified as pathogenic.