ClinVar Genomic variation as it relates to human health

This variant was observed as part of a predisposition screen in an ostensibly healthy population. A literature search was performed for the gene, cDNA change, and amino acid change (where applicable). No publications were found based on this search. Allele frequency data from public databases did not allow this variant to be ruled in or out of causing disease. Therefore, this variant is classified as a variant of unknown significance.

This variant was classified as: Uncertain significance. The available evidence favors the pathogenic nature of this variant, however the currently available data is insufficient to conclusively support its pathogenic nature. Thus this variant is classified as Uncertain significance - favor pathogenic. The following ACMG criteria were applied in classifying this variant: PM5,PP3.

Identified in patients with ABCA4-related retinal dystrophy in published literature, although additional clinical information and familial segregation data were not provided for some cases (Kiratschky et al., 2008; Zaneveld et al., 2015; Sharon et al., 2020); Published functional studies demonstrate a damaging effect with aberrant splicing (Khan et al., 2019); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; In silico analysis supports that this missense variant has a deleterious effect on splicing; This variant is associated with the following publications: (PMID: 34426522, 15192030, 20852892, 18285826, 25474345, 31212395, 31456290, 29162642)

Variant summary: ABCA4 c.3608G>A (p.Gly1203Glu) results in a non-conservative amino acid change in the encoded protein sequence. Three of five in-silico tools predict a benign effect of the variant on protein function. Several computational tools predict a significant impact on normal splicing: Four predict the variant weakens the canonical 3' acceptor site. Three predict the variant creates cryptic 3' acceptor site. At-least one study has shown that this variant leads to skipping of exon 25. However, compared to altered mRNA product (lacking exon 25) the wild-type mRNA was produced in excess suggesting that this variant may not lead to a complete loss of function (Khan_2020). The variant allele was found at a frequency of 0.00031 in 250392 control chromosomes (gnomAD). This frequency is not significantly higher than estimated for a pathogenic variant in ABCA4 causing Retinitis Pigmentosa (0.00031 vs 0.0014), allowing no conclusion about variant significance. c.3608G>A has been reported in the literature in individuals affected with Stargardt disease and cone rod dystrophy (examples: Kitiratschky_2008, Zaneveld_2015, Sharon_2019, Khan_2020, and Mena_2021). At-least one of these reports described this variant as a hypomorphic variant (Hanany_2020). The following publications have been ascertained in the context of this evaluation (PMID: 31212395, 25474345, 31964843, 18285826, 31456290, 33841504, 35886001). Nine clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 and classified the variant as VUS (n=7) and pathogenic/likely pathogenic (n=2). Based on the evidence outlined above, the variant was classified as VUS-possibly pathogenic.

This sequence change replaces glycine, which is neutral and non-polar, with glutamic acid, which is acidic and polar, at codon 1203 of the ABCA4 protein (p.Gly1203Glu). This variant is present in population databases (rs146786552, gnomAD 0.06%). This missense change has been observed in individual(s) with clinical features of Stargardt disease (PMID: 25474345, 31212395, 33841504). ClinVar contains an entry for this variant (Variation ID: 497057). 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 disruptive. Studies have shown that this missense change does not significantly alter or has an unclear effect on ABCA4 gene expression (PMID: 31212395). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site. In summary, the currently available evidence indicates that the variant is pathogenic, but additional data are needed to prove that conclusively. Therefore, this variant has been classified as Likely Pathogenic.

Based on the classification scheme VCGS_Germline_v1.3.4, this variant is classified as VUS-3A. Following criteria are met: 0102 - Loss of function is a known mechanism of disease in this gene and is associated with ABCA4-related eye disease (OMIM). (I) 0106 - This gene is associated with autosomal recessive disease. (I) 0115 - Variants in this gene are known to have variable expressivity (PMID: 31522899). (I) 0209 - Splice site variant proven to affect splicing of the transcript with uncertain effect on protein sequence. Transfected HEK293 cells have shown that this variant results in exon 25 skipping, proven by Sanger sequencing of RT-PCR products (PMID: 31212395). (SP) 0251 - This variant is heterozygous. (I) 0304 - Variant is present in gnomAD <0.01 for a recessive condition (89 heterozygotes, 0 homozygotes). (SP) 0604 - Variant is predicted to cause exon 25 skipping, which does not contain an established domain, motif, hotspot or informative constraint region (PMID: 31212395). (I) 0705 - No comparable splice variants have previous evidence for pathogenicity. However, alternative missense changes at the same residue (p.Gly1203Arg, p.Gly1203Trp) have been reported. The arginine change has been reported several times as a VUS and once as likely pathogenic (ClinVar), and observed in a single patient with Stargardt disease who also had two additional missense variants of unknown phase. The change to tryptophan has been reported in a single compound heterozygous patient with Stardardt disease (PMID: 15192030; PMID: 30563929). (I) 0808 - Previous reports of pathogenicity for this variant are conflicting. This variant has been reported multiple times as a VUS (ClinVar), and observed in several heterozygous patients with cone rod dystrophy or Stardardt disease (PMID: 31212395, PMID: 18285826, PMID: 31456290). It has also been observed in a patient with Stargardt macular dystrophy with two additional variants, but phasing is unclear (PMID: 25474345). (I) 0905 - No published segregation evidence has been identified for this variant. (I) 1007 - No published functional evidence has been identified for this variant. (I) 1208 - Inheritance information for this variant is not currently available in this individual. (I) Legend: (SP) - Supporting pathogenic, (I) - Information, (SB) - Supporting benign

The ABCA4 c.3608G>A variant is predicted to result in the amino acid substitution p.Gly1203Glu. This variant has been reported in individuals with Stargardt disease, although in some cases a second ABCA4 variant was not detected (Kitiratschky et al. 2008. PubMed ID: 18285826; Table S2, Zaneveld et al. 2014. PubMed ID: 25474345; Table S2, Sharon et al. 2020. PubMed ID: 31456290). This variant affects the first nucleotide of exon 25 and is predicted to create a cryptic acceptor splice site two nucleotides away from the canonical acceptor splice site (SpliceAI, Jaganathan et al. 2019. PubMed ID: 30661751). Functional splicing studies have demonstrated that this variant indeed causes skipping of exon 25 in a small proportion of transcripts (Khan et al. 2019. PubMed ID: 31212395). This variant is reported in 0.057% of alleles in individuals of European (Non-Finnish) descent in gnomAD, including two homozygous individuals in dataset v4.1.0 (https://gnomad.broadinstitute.org/variant/1-94037350-C-T?dataset=gnomad_r4). Due to the conflicting evidence, the clinical significance of this variant is uncertain at this time.