ClinVar Genomic variation as it relates to human health

Canonical splice site variant with an unclear effect on protein function; a different variant at the same splice site, c.301+1G>C, demonstrated to result in an in-frame isoform that replaces 3 amino acids with 1 incorrect one in the critical RING domain (Borg 2010, Paul 2014, Leman 2018); Observed in individuals with breast, pancreatic, or prostate cancer (Rebbeck 2016, Na 2017, Alimirzaie 2018); Not observed at significant frequency in large population cohorts (gnomAD); Also known as c.420+1G>A and IVS6+1G>A; This variant is associated with the following publications: (PMID: 29940740, 25085752, 21769658, 27836010, 28526081, 29446198, 27989354, 24389207, 20104584, 30209399, 29750258)

This variant changes a G to A nucleotide at the +1 position of the intron 5 splice donor site in the BRCA1 gene. Splice prediction tools indicate a significant disruption to the native splice donor site and the possible activation of a cryptic donor site, resulting in an in-frame deletion at the end of exon 5. The detection of in-frame deletion in the RNA of carriers of this variant has been reported (ClinVar accession: SCV000185671.7, SCV000635877.6). An RNA study for a different variant at this splice donor site, c.301+6T>C, has shown a disruption at the native donor site that is rescued by the use of a cryptic donor site resulting in the in-frame deletion of 9 nucleotides from the end of exon 5 (PMID: 21769658). A functional study has reported that this variant c.301+1G>A impacts BRCA1 function in a haploid cell proliferation assay (PMID: 30209399). This variant has been reported in one individual affected with pancreatic cancer (PMID: 29940740). However, this variant has been detected in a breast cancer case-control meta-analysis in 1/53461 unaffected individuals and absent in 60466 cases (PMID: 33471991; Leiden Open Variation Database DB-ID BRCA1_005049). This variant has been detected in an individual affected with breast cancer who has a BRCA2 truncation variant that is expected to be loss-of-function and explains the disease (PMID: 27836010), and it also has been observed in trans with a pathogenic BRCA1 variant in an individual without features of Fanconi anemia (ClinVar accession: SCV000185671.7). Another commercial laboratory also has reported this variant as benign based in part on health history evidence (PMID: 28408614). This variant has been identified in 1/251180 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Although there is a suspicion that this variant may not be associated with disease, additional studies are necessary to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance.

The c.301+1G>A variant in BRCA1 has been reported in 1 Caucasian woman with brea st cancer who also carried another pathogenic variant in BRCA2 (Rebbeck 2016). T his variant has been identified in 1/111,504 European chromosomes by the genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org/; dbSNP rs5877821 73). This variant occurs in the invariant region (+/- 1,2) of the splice consens us sequence and variants in this region are predicted to cause altered splicing leading to an abnormal or absent protein. However, in vitro functional studies f or another variant at this exon-intron junction (301+6T>C) suggest the use of an alternative splice donor site resulting in an in-frame deletion of three amino acids (Thomassen 2012). Please note, these types of assays may not accurately re present biological function. In summary, due to conflicting and insufficient evi dence, the clinical significance of the c.301+1G>A variant is uncertain.

The c.301+1G>A intronic variant results from a G to A substitution one nucleotide after coding exon 4 of the BRCA1 gene. This variant has been reported in individuals with breast, prostate, and pancreatic cancer (Rebbeck TR et al. Breast Cancer Res, 2016 11;18(1):112; Na R et al. Eur Urol, 2017 05;71:740-747; Alimirzaie S et al. Arch Iran Med, 2018 06;21:228-233), as well as individuals from control groups in multiple studies (Hu C et al. JAMA, 2018 Jun;319:2401-2409; Dorling et al. N Engl J Med. 2021 02;384:428-439; Nguyen-Dumont T et al. Cancers (Basel), 2021 Mar;13:; Southey MC et al. NPJ Breast Cancer, 2021 Dec;7:153). This nucleotide position is highly conserved in available vertebrate species. In silico splice site analysis predicts that this alteration will weaken the native splice donor site. Alterations at this donor site are known to produce an in frame transcript impacting four amino acids in the BRCA1 RING domain (Ambry internal data). One functional study found that nucleotide substitutions at this donor site are non-functional in a high-throughput, genome editing, haploid cell survival assay (Findlay GM et al. Nature, 2018 10;562:217-222). However, this alteration has also been identified in trans with a pathogenic mutation in BRCA1 in an individual without features of Fanconi Anemia (Ambry internal data, personal communication). Since supporting evidence is conflicting at this time, the clinical significance of this alteration remains unclear.

PS3 Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. PM1 Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation. PM4 Protein length changes as a result of in-frame deletions/insertions in a nonrepeat region or stop-loss variants. PP4 Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology. BP2 Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.

This sequence change affects a donor splice site in intron 5 of the BRCA1 gene. RNA analysis indicates that disruption of this splice site induces altered splicing and likely results in the loss of 4 and insertion of 1 amino acid residue(s), but is expected to preserve the integrity of the reading-frame. This variant is present in population databases (rs587782173, gnomAD 0.0009%). Disruption of this splice site has been observed in individual(s) with BRCA1-related conditions (PMID: 27836010, 29446198). ClinVar contains an entry for this variant (Variation ID: 142004). Algorithms developed to predict the effect of variants on protein structure and function are not available or were not evaluated for this variant. Experimental studies have shown that disruption of this splice site affects BRCA1 function (PMID: 30209399). Studies have shown that disruption of this splice site results in the activation of a cryptic splice site in exon 5 (Invitae). In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance.