ClinVar Genomic variation as it relates to human health

The c.8559-2A>G has been detected in >4 patients with Usher syndrome who were compound heterozygous for the this variant in trans with a truncating variant (PM3_VS; PMID: 25356976, 19737284, 26338283, 19023448). Segregation data was also available for two reported families with Usher syndrome. In one family, the variant was identified in trans with a frameshift variant in two affected siblings, and four unaffected siblings were either heterozygous for a single variant or wild-type for both variants. In the second family, an unaffected sibling was heterozygous for a single variant (PP1_Strong, PMID: 19023448). RT-PCR analysis of cells from a patient carrying the variant revealed that the variant causes skipping of exon 43, resulting in a 41 amino acid deletion of the USH2A protein (PM4, PMID: 20596040) The allele frequency of the c.8559-2A>G variant in the USH2A gene is 0.046% (8/17232) of East Asian chromosomes by the Genome Aggregation Database ( http://gnomad.broadinstitute.org), which is a low enough frequency to award PM2_supporting based on the thresholds defined by the ClinGen Hearing Loss Expert Panel for autosomal recessive hearing loss and Usher syndrome (PM2_Supporting). Several patients reported to harbor this variant displayed clinical features of Usher syndrome (PP4; PMID: 25356976, 19737284, 26338283, 19023448). In summary, this variant meets criteria to be classified as pathogenic for autosomal recessive Usher syndrome based on the ACMG/AMP criteria applied, as specified by the Hearing Loss Expert Panel: PM4, PM3_VS, PP1_S, PP4, PM2_P.

The 8559-2A>G variant in USH2A has been reported in 6 Asian individuals with Ush er type 2 and was absent in 470 Asian control chromosomes (Dai 2008, Nakanishi 2 009). All of these probands were compound heterozygous. This variant is predicte d to cause abnormal splicing because the nucleotide substitution occurs in the i nvariant region of the splice consensus sequence. Furthermore, RT-PCR analysis o f cells from a patient carrying the variant revealed that the variant causes ski pping of exon 43, resulting in a 41 amino acid deletion of the USH2A protein (Na kanishi 2010). In summary, this variant meets our criteria to be classified as p athogenic.

Canonical splice site variant expected to result in aberrant splicing, and published functional studies demonstrate skipping of exon 43 (PMID: 20596040); Reported as pathogenic by the ClinGen Hearing Loss Variant Curation Expert Panel (ClinVar SCV000840538.3; ClinVar); This variant is associated with the following publications: (PMID: 25356976, 21593743, 25252889, 28512305, 25078356, 34416374, 33629268, 30311386, 32531858, 35062939, 25133613, 25525159, 19023448, 26252086, 24853665, 19737284, 28968992, 26496393, 25558175, 29899460, 30029497, 29625443, 31872526, 31960602, 32093671, 31904091, 32100970, 31541171, 32188678, 33105608, 33124170, 34376197, 32675063, 32893482, 33090715, 33946315, 33691693, 34721897, 35870892, 35152177, 35052368, 34824372, 20596040, 26338283)

Canonical splice site: predicted to alter splicing and result in a loss or disruption of normal protein function through protein truncation. Multiple pathogenic variants are reported in the predicted truncated region (PVS1_VS).The variant has been reported multiple times as an established pathogenic/likely pathogenic variant (ClinVar ID: VCV000048604, 3billion dataset). The variant was co-segregated with Usher syndrome, type 2A in multiple affected family members with additional meioses meeting strong evidence levels (PMID: 19023448, PP1_S). It is observed at an extremely low frequency in the gnomAD v2.1.1 dataset (total allele frequency: 0.000032, PM2_M). The same variant was previously reported several times in trans with another pathogenic variant in this gene (PMID: 19737284, 26338283, 25356976, 19023448, PM3_VS). Therefore, this variant is classified as pathogenic according to the recommendation of ACMG/AMP guideline.

NM_206933.2(USH2A):c.8559-2A>G is a canonical splice site variant classified as pathogenic in the context of USH2A-related disorders. c.8559-2A>G has been observed in cases with relevant disease (PMID: 25324289, 20596040, 25356976). Functional assessments of this variant are available in the literature (PMID: 20596040). c.8559-2A>G has been observed in population frequency databases (gnomAD: EAS 0.05%). In summary, NM_206933.2(USH2A):c.8559-2A>G is a canonical splice site variant that has been observed more frequently in cases with the relevant disease than in healthy populations. Please note: this variant was assessed in the context of healthy population screening.

This sequence change affects an acceptor splice site in intron 42 of the USH2A 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 USH2A are known to be pathogenic (PMID: 10729113, 10909849, 20507924, 25649381). This variant is present in population databases (rs397518039, gnomAD 0.05%). Disruption of this splice site has been observed in individuals with Usher syndrome (PMID: 19023448, 19737284). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 48604). 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.

Variant summary: USH2A c.8559-2A>G is located in a canonical splice-site and is predicted to affect mRNA splicing resulting in a significantly altered protein due to either exon skipping, shortening, or inclusion of intronic material. Several computational tools predict a significant impact on normal splicing: Four predict the variant abolishes a 3' acceptor site. However, these predictions have yet to be confirmed by functional studies. The variant allele was found at a frequency of 3.2e-05 in 251134 control chromosomes (gnomAD). c.8559-2A>G has been reported in the literature in multiple individuals affected with Usher Syndrome (Dai_2008, Gao_2021). These data indicate that the variant is very likely to be associated with disease. 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: 19023448, 32188678). ClinVar contains an entry for this variant (Variation ID: 48604). Based on the evidence outlined above, the variant was classified as pathogenic.

Converted during submission to Pathogenic.