ClinVar Genomic variation as it relates to human health

Variant summary: POLR3A c.1771-7C>G alters a non-conserved nucleotide located close to a canonical splice site and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. 4/4 computational tools predict no significant impact on normal splicing. At least one publication reports experimental evidence that this variant affects mRNA splicing. The variant allele was found at a frequency of 0.00011 in 251416 control chromosomes. This frequency is not significantly higher than expected for a pathogenic variant in POLR3A causing Pol III-Related Leukodystrophy (0.00011 vs ND), allowing no conclusion about variant significance. c.1771-7C>G has been reported in the literature in multiple individuals affected with Pol III-Related Leukodystrophy. These data indicate that the variant is very likely to be associated with disease. Nine clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation (pathogenic/likely pathogenic n=8, VUS n=1). Based on the evidence outlined above, the variant was classified as pathogenic.

In silico analysis suggests this variant may impact gene splicing. In the absence of additional RNA/functional studies, the actual effect of this sequence change is unknown.; This variant is associated with the following publications: (PMID: 28459997, 31940116, 32582862, 33559318, 33597727, 32860008, 32214227, 33098801, 34440436, 34284285, 33726816)

ACMG classification criteria: PS3 supporting, PM3 very strong, PP1 supporting

The variant is observed at an extremely low frequency in the gnomAD v2.1.1 dataset (total allele frequency: 0.012%). In silico tools do not predict the variant to alter splicing and produce an abnormal transcript (SpliceAI: 0.04). The variant has been reported to be in trans with a pathogenic variant as either compound heterozygous or homozygous in at least 4 similarly affected unrelated individuals (PMID: 32582862). The variant has been reported at least twice as pathogenic with clinical assertions and evidence for the classification (ClinVar ID: VCV000449556 / PMID: 28459997). Therefore, this variant is classified as Pathogenic according to the recommendation of ACMG/AMP guideline.

This sequence change falls in intron 13 of the POLR3A gene. It does not directly change the encoded amino acid sequence of the POLR3A protein. RNA analysis indicates that this variant induces altered splicing and may result in an absent or disrupted protein product. This variant is present in population databases (rs201314157, gnomAD 0.02%). This variant has been observed in individuals with POLR3A-related conditions (PMID: 28459997, 31940116, 32214227). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 449556). Studies have shown that this variant results in skipping of exon 14 and introduces a premature termination codon (PMID: 28459997). The resulting mRNA is expected to undergo nonsense-mediated decay. For these reasons, this variant has been classified as Pathogenic.

POLR3A: PM3:Very Strong, PM2, PP4:Moderate, BP4

The POLR3A c.1771-7C>G variant is predicted to interfere with splicing. The variant is predicted to weaken the acceptor splice site based on available prediction programs (Alamut Visual v2.10). This variant has been reported in the homozygous state in multiple unrelated individuals with spastic ataxia (Minnerop et al. 2017. PubMed ID: 28459997). This variant has also been observed in the compound heterozygous state in multiple unrelated individuals with POLR3A-related leukodystrophy and movement disorder (Harting et al. 2020. PubMed ID: 31940116; Perrier et al. 2020. PubMed ID: 32582862). Analysis of cDNA derived from affected individuals who were homozygous for the c.1771-7C>G variant showed the presence of both wild-type transcript and two abnormal transcripts, either lacking exon 14 or lacking both exons 13 and 14 (Minnerop et al. 2017. PubMed ID: 28459997). This variant is reported in 0.017% of alleles in individuals of Latino descent in gnomAD (http://gnomad.broadinstitute.org/variant/10-79769440-G-C). This variant is interpreted as likely pathogenic.

The c.1771-7C>G variant in POLR3A has been reported in >10 individuals with POLR3A-related disorders (PMID: 28459997, 32214227, 31940116, 32582862, 33098801, 34440436, 32860008, 34234304, 33726816, 34284285, 33559318), segregated with disease in 3 affected relatives from 3 families (PMID: 28459997, 32582862), and has been identified in 0.01% (6/35434) of Latino/Admixed American chromosomes by the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org; dbSNP ID: rs201314157). Although this variant has been seen in the general population in a heterozygous state, its frequency is not high enough to rule out a pathogenic role. This variant has also been reported in ClinVar (Variation ID#: 449556) and has been interpreted as likely pathogenic or pathogenic by multiple laboratories. Of the many affected individuals, 5 of those were homozygotes, and at least 3 were compound heterozygotes that carried reported pathogenic and likely pathogenic variants in trans, which increases the likelihood that the c.1771-7C>G variant is pathogenic (VariationID: 684773, 430255, 684772; PMID: 28459997, 32214227, 31940116, 32582862, 33098801, 34440436, 32860008). In vitro functional studies provide some evidence that the c.1771-7C>G variant may impact protein function (PMID: 28459997, 32582862). However, these types of assays may not accurately represent biological function. This variant is located in the 5’ splice region. Computational tools do not suggest an impact to splicing. However, this information is not predictive enough to rule out pathogenicity. In summary, this variant meets criteria to be classified as pathogenic for autosomal recessive POLR3A-related disorders. ACMG/AMP Criteria applied: PS3_moderate, PM3_very-strong, PP1_moderate (Richards 2015).