ClinVar Genomic variation as it relates to human health

Reduces the quality of the splice donor site in intron 4, and an in vitro functional study suggests a damaging effect with abnormal splicing and skipping of exon 4 (Tiwari et al., 2014); This variant is associated with the following publications: (PMID: 27353947, 23591405, 29192808, 30356807, 30656474, 32037395)

PS3, PS4_moderate, PM2, PP3

Clinical significance based on ACMG v2.0

Variant summary: FLVCR1 c.1092+5G>A alters a conserved nucleotide located close to a canonical splice site and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. Several computational tools predict a significant impact on normal splicing: Three predict the variant weakens a 5' donor site. At least one publication reports experimental evidence that this variant affects mRNA splicing resulting in skipping of exon 4 (Tiwari_2016). The variant allele was found at a frequency of 0.00024 in 238018 control chromosomes. c.1092+5G>A has been reported in the literature in multiple individuals affected with Retinitis Pigmentosa (e.g. Weisschuh_2020, Tiwari_2016, Glockle_2014). These data indicate that the variant is very likely to be associated with disease. The following publications have been ascertained in the context of this evaluation (PMID: 23591405, 27353947, 32531858). Ten submitters have cited clinical-significance assessments for this variant to ClinVar after 2014. All submitters classified the variant as pathogenic/likely pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

The frequency of this variant in the general population is consistent with pathogenicity (http://gnomad.broadinstitute.org). Experimental evidence suggests this variant results in abnormal RNA splicing and premature termination of the protein (PMID: 27353947). In multiple individuals, this variant has been seen with a single recessive pathogenic variant in the same gene, suggesting this variant may also be pathogenic. Computational tools yielded predictions that this variant may interfere with normal RNA splicing.

This variant has been observed in individuals with retinitis pigmentosa (PMID: 23591405, 27353947). This variant is present in population databases (rs556788423, gnomAD 0.04%). This sequence change falls in intron 4 of the FLVCR1 gene. It does not directly change the encoded amino acid sequence of the FLVCR1 protein. RNA analysis indicates that this variant induces altered splicing and may result in an absent or disrupted protein product. ClinVar contains an entry for this variant (Variation ID: 374768). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant results in skipping of exon 4 and introduces a premature termination codon (PMID: 27353947). The resulting mRNA is expected to undergo nonsense-mediated decay. For these reasons, this variant has been classified as Pathogenic.

The 1092+5G>A variant in FLVCR1 has been reported in >10 individuals with retinitis pigmentosa most of whom had no signs of ataxia. At least 4 individuals were homozygotes and at least 9 individuals were compound heterozygotes, and only 1 individual had signs of mild ataxia that was not progressive. This variant was also found in 1 unaffected homozygous adult sibling of one affected homozygous individual (Glockle 2014 PMID: 23591405, Tiwari 2016 PMID: 27353947, Yusuf 2018 PMID: 29192808, Kuehlewein 2019 PMID: 30656474, Weisschuh 2020 PMID: 32531858, Zampaglione 2020 PMID: 32037395). This variant has also been reported by other clinical laboratories in ClinVar (Variation ID 374768) and has been identified in 0.045% (511/1130502) of European chromosomes by gnomAD (http://gnomad.broadinstitute.org, v4.0.0). This variant is located in the 5' splice region. Computational tools do not predict a splicing impact, though this information is not predictive enough to rule out pathogenicity. In vitro splicing studies using homozygous patient RNA from blood show that this splice variant partially disrupts splicing, showing both normal transcript and misspliced transcript in which exon 4 is skipped, and is predicted to cause a frameshift and a premature termination codon that is predicted to undergo nonsense mediated decay. However, the proportion of misspliced products seemed to vary among the studies, as one showed a much weaker misspliced transcript (Tiwari 2016 PMID: 27353947, Kuehlewein 2019 PMID: 30656474). Additionally, another in vitro splicing study using mini gene assay did not reveal any misspliced products, suggesting this variant causes a splice defect that is most likely leaky and gives rise to variable amounts of correctly and misspliced transcripts that could potentially also be dependent on tissue or other modifying factors (Kuehlewein 2019 PMID: 30656474). It has been suggested that this variant may be hypomorphic or a mild allele as it was found in an unaffected homozygous individual and may only cause isolated retinopathy without the ataxia phenotype (Kuehlewein 2019 PMID: 30656474). In summary, although additional studies are required to fully establish its clinical significance, this variant is a mild allele that meets criteria to be classified as likely pathogenic for autosomal recessive FLVCR1-related retinopathy. ACMG/AMP Criteria applied: PM3_Strong, PS3_Moderate.

The FLVCR1 c.1092+5G>A variant is predicted to interfere with splicing. This variant has been reported in multiple individuals with retinal dystrophies (Table S1, Glöckle et al. 2014. PubMed ID: 23591405; Tiwari et al. 2016. PubMed ID: 27353947; Table S2, Zampaglione et al. 2020. PubMed ID: 32037395; Table S1, Weisschuh et al. 2020. PubMed ID: 32531858). A functional study using RT-PCR analysis in a patient’s sample showed that this variant causes skipping of exon 4 (Tiwari et al. 2016. PubMed ID: 27353947). This variant is reported in 0.044% of alleles in individuals of European (Non-Finnish) descent in gnomAD. This variant is interpreted as pathogenic.