This sequence change affects an acceptor splice site in intron 2 of the SLC46A1 gene. RNA analysis indicates that disruption of this splice site induces altered splicing and likely results in a shortened protein product. This variant is present in population databases (rs80338775, gnomAD 0.006%). Disruption of this splice site has been observed in individual(s) with hereditary folate malabsorption syndrome (PMID: 17129779, 19740703, 21489556). It has also been observed to segregate with disease in related individuals. This variant is also known as p.Tyr362_Gly389del. ClinVar contains an entry for this variant (Variation ID: 850). Algorithms developed to predict the effect of variants on gene product structure and function are not available or were not evaluated for this variant. Experimental studies have shown that disruption of this splice site affects SLC46A1 function (PMID: 17129779). Studies have shown that disruption of this splice site results in skipping of exon 3, but is expected to preserve the integrity of the reading-frame (PMID: 17129779). For these reasons, this variant has been classified as Pathogenic.

This variant was determined to be pathogenic according to ACMG Guidelines, 2015 [PMID:25741868].

Published functional studies demonstrate a damaging effect on transport function (Qiu et al., 2006); Not observed at significant frequency in large population cohorts (gnomAD); Canonical splice site variant with an unclear effect on protein function; This variant is associated with the following publications: (PMID: 17129779, 11807405, 20301716, 22454052, 23816405, 19740703, 20005757, 19176287, 21489556)

Variant summary: SLC46A1 c.1082-1G>A 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. Three predict the variant creates a 3' cryptic acceptor site. At least one publication reports experimental evidence that this variant affects mRNA splicing and results in skipping of exon 3 and consequent in-frame deletion (Qiu_2006). The variant allele was found at a frequency of 4.1e-06 in 244488 control chromosomes (gnomAD). c.1082-1G>A has been reported in the literature in multiple individuals affected with Congenital Defect Of Folate Absorption (Qiu_2006, Mahadeo_2011). These data indicate that the variant is very likely to be associated with disease. At least one publication reports experimental evidence evaluating an impact on protein function and this variant results in reduced protein expression and trapped intracellularly without detectable localization to the cell membrane (Qiu_2006). The following publications have been ascertained in the context of this evaluation (PMID: 21489556, 17129779). ClinVar contains an entry for this variant (Variation ID: 850). Based on the evidence outlined above, the variant was classified as pathogenic.

The c.1082-1G>A intronic alteration consists of a G to A substitution one nucleotide before exon 3 (coding exon 3) of the SLC46A1 gene. Alterations that disrupt the canonical splice site are expected to result in aberrant splicing. Based on data from gnomAD, the A allele has an overall frequency of 0.001% (2/275882) total alleles studied. This variant has been identified in the homozygous state in multiple individuals diagnosed with hereditary folate malabsorption (Mahadeo, 2011; Borzutzky, 2009; Geller, 2002). This nucleotide position is highly conserved in available vertebrate species. RNA studies demonstrate this alteration leads to abnormal splicing in the set of samples tested (Qiu, 2006). In silico splice site analysis predicts that this alteration will weaken the native splice acceptor site. Based on the available evidence, this alteration is classified as pathogenic.

The SLC46A1 c.1082-1G>A variant is predicted to disrupt the AG splice acceptor site and interfere with normal splicing. This is the most commonly reported causative variant in the SLC46A1 gene; it has been documented in many individuals of Puerto Rican descent (Mahadeo et al. 2011. PubMed ID: 21489556). This variant has been shown to lead to skipping of exon 3 and an in-frame deletion of 28 amino acids (p.Tyr362_Gly389del, Qiu et al. 2006. PubMed ID: 17129779, referred to as G>A at position 5882 in GenBank accession DQ496103). This variant is interpreted as pathogenic.

In-frame deletion resulting from skipping of exon 3 was detected in cDNA from transformed lymphocytes of individuals with HFM (Qiu et al 2006); the resulting transcript variant is BC01069.1. The same pathogenic variant identified in a total of ten unrelated families of Puerto Rican ancestry (Qiu et al 2006, Borzutzky et al 2009, Mahadeo et al 2011, Zhao et al 2017).