ClinVar Genomic variation as it relates to human health

The FANCA c.4199G>A (p.Arg1400His) variant has been reported in two studies and is found in two individuals with Fanconi anemia, including one in a compound heterozygous state with a missense variant and one in a heterozygous state in whom a second variant was not identified (Ameziane et al. 2008; De Rocco et al. 2014). Control data are unavailable for this variant, which is reported at a frequency of 0.000045 in the European (non-Finnish) population of the Exome Aggregation Consortium. Expression of the p.Arg1400His variant in lymphoblastoid cell lines lacking functional FANCA protein demonstrated an activity similar to wild type protein, however, compensation due to overexpression of the p.Arg1400His protein could not be ruled out (Ameziane et al. 2008). The evidence for this variant is limited. The variant is therefore classified as a variant of unknown significance but suspicious for pathogenicity for Fanconi anemia. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.

NM_000135.2(FANCA):c.4199G>A(R1400H) is a missense variant classified as a variant of uncertain significance in the context of Fanconi anemia complementation group A. R1400H has been observed in cases with relevant disease (PMID: 17924555, 29098742, 33172906). Functional assessments of this variant are available in the literature (PMID: 17924555, 33172906). R1400H has been observed in population frequency databases (gnomAD: AMR 0.01%). In summary, there is insufficient evidence to classify NM_000135.2(FANCA):c.4199G>A(R1400H) as pathogenic or benign. Please note: this variant was assessed in the context of healthy population screening.

Observed with a second FANCA variant in unrelated patients with Fanconi anemia in published literature, but it is not known whether the variants occurred on the same (in cis) or on different (in trans) chromosomes (Ameziane 2008, Kimble 2018); Published functional studies demonstrate decreased FANCA levels and impaired ubiquitination and foci formation upon MMC-induced damage; however, functional complementation assays show that overexpresson of this variant exhibited a level of activity similar to WT, suggesting that this may be a hypomorphic variant (Ameziane 2008, Lach 2020); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 29098742, 33172906, 17924555, 33822308)

Variant summary: FANCA c.4199G>A (p.Arg1400His) results in a non-conservative amino acid change in the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 3.6e-05 in 251486 control chromosomes (gnomAD). c.4199G>A has been reported in the literature in multiple compound heterozygous individuals diagnosed with Fanconi Anemia, where the diagnosis had been established by chromosomal breakage test (Ameziane_2008, Kimble_2018, Lach_2020), however 2 of these cases (brothers) had an atypical, delayed phenotype (i.e. presenting with esophageal malignancies at the age of 51y, with the lack of hematologic failure), and were subsequently diagnosed to have FA by chromosomal breakage and molecular testing (Lach_2020). Publications have also reported experimental evidence evaluating an impact on protein function. In an early study, expression of the R1400H variant in lymphoblastoid cell lines lacking functional FANCA protein demonstrated similar activity to the wild type, however due to overexpression of the construct, a potential hypomorphic effect couldn't be ruled out (Ameziane_2008). A later, more detailed study demonstrated a reduced protein level, and increased cytoplasmic localization, together with a mild decrease in Fancd2 ubiquitination and foci formation, and increased sensitivity to DNA cross-linking agents, providing evidence for a hypomorphic effect (Lach_2020). The following publications have been ascertained in the context of this evaluation (PMID: 17924555, 29098742, 33172906, 28717661, 33686268, 26580448). Six submitters have cited clinical-significance assessments for this variant to ClinVar after 2014 with conflicting assessments. Three submitters classified the variant as pathogenic/likely pathogenic and three classified it as uncertain significance. Based on the evidence outlined above, the variant was classified as likely pathogenic.

This sequence change replaces arginine, which is basic and polar, with histidine, which is basic and polar, at codon 1400 of the FANCA protein (p.Arg1400His). This variant is present in population databases (rs149851163, gnomAD 0.006%). This missense change has been observed in individuals with Fanconi anemia (PMID: 17924555, 29098742). ClinVar contains an entry for this variant (Variation ID: 408175). Advanced modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) performed at Invitae indicates that this missense variant is expected to disrupt FANCA protein function with a positive predictive value of 80%. This variant disrupts the p.Arg1400 amino acid residue in FANCA. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 15643609, 21273304, 24584348, 28102861, 28717661, 29098742). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic.

The c.4199G>A/p.R1400H variant was previously reported in an individual with Fanconi anemia, but determining pathogenicity was difficult because it was reported that overexpression of the mutant allele resulted in complementation comparable to that obtained with the wild type cDNA (Ameziane et al. 2008). This suggests that overexpression of the mutant protein is able to compensate for protein instability and/or defects in FANCA protein function. We have identified c.4199G>A/p.R1400H variant in another FA family (two affected brothers) in trans with a c.3788_3790delTCT, a known pathogenic variant. Our functional data support the variant as a most likely pathogenic allele. We observe increased mislocalization of mutant protein to the cytoplasm, decreased FANCD2 ubiquitination and foci formation, and cellular sensitivity to crosslinking agents. We have examined the variant by CRISPR/Cas9 mediated genome editing in otherwise wild type fibroblast cell lines and we showed that the R1400H variant in homozygous form had an incomplete loss of function (behaved as a hypomorph), and the cellular phenotype characteristic of Fanconi anemia was further exacerbated when it was present in trans to a loss-of-function allele, indicating a dose effect of a mutant protein.