ClinVar Genomic variation as it relates to human health

This sequence change affects a donor splice site in intron 5 of the RB1 gene. RNA analysis indicates that disruption of this splice site induces altered splicing and likely results in a shortened protein product. This variant is not present in population databases (gnomAD no frequency). Disruption of this splice site has been observed in individual(s) with retinoblastoma as well as in unaffected family members, suggesting this may be a low penetrance variant (PMID: 10991691, 27582626, 33493472). ClinVar contains an entry for this variant (Variation ID: 579207). Studies have shown that disruption of this splice site results in skipping of exon 5, but is expected to preserve the integrity of the reading-frame (PMID: 10991691). 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.

The c.539+1G>A intronic variant results from a G to A substitution one nucleotide after coding exon 5 of the RB1 gene. This alteration has been identified in multiple probands from diverse ethnic backgrounds who have a personal history of unilateral retinoblastoma (Sánchez F et al. J. Med. Genet., 2000 Aug;37:615-20; Chai P et al. Exp Eye Res, 2021 Apr;205:108456; Singh J et al. Mol Vis, 2016 Aug;22:1036-47), and in two unaffected carriers from one family. This alteration was also identified as a somatic mutation in a patient with osteosarcoma which is a common second primary malignancy in patients with pathogenic RB1 mutations (Liu J et al. J Pediatr Hematol Oncol, 2017 05;39:302-305; Imbert-Bouteille M et al. Mol Genet Genomic Med, 2019 12;7:e913). In addition, this variant has been observed in individuals with no personal or family history of retinoblastoma and in two individuals with a family history of sarcoma (Ambry internal data) and one individual with personal history of osteosarcoma (personal communication). These data suggest that this variant may have reduced penetrance. This alteration has been shown to result in in-frame skipping of exon 5 (Sánchez F et al. J. Med. Genet., 2000 Aug;37:615-20; Ambry internal data). This nucleotide position is highly conserved in available vertebrate species. Alterations that disrupt the canonical splice site are expected to cause aberrant splicing, resulting in an abnormal protein or a transcript that is subject to nonsense-mediated mRNA decay. As such, this alteration is classified as likely pathogenic but is known to have reduced penetrance.

Variant summary: RB1 c.539+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: Three predict the variant abolishes the canonical 5' splicing donor site. However, these predictions have yet to be confirmed by functional studies. The variant was absent in 248194 control chromosomes (gnomAD). c.539+1G>A has been observed in a child affected with unilateral retinoblastoma and also in the unaffected father and paternal grandmother. In this case, the affected individual exhibited mosiasm comprising heterozygosity and homozygosity as the result of duplication of the paternal allele. RNA analysis demonstrated the in-frame skipping of exon 5 and the authors suggested c.539+1G>A is likely a variant with low penetrance (Sanchez_2000). It has been reported in the literature in other heterozygous individuals affected with retinoblastoma, also without strong evidence of high penetrance (example Singh_2016, Chai_2021). Two clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 and both classified the variant as likely pathogenic. Based on the evidence outlined above, the variant was classified as likely pathogenic.

PP4, PM2, PVS1_strong

The variant NM_000321.3:c.539+1G>A (chr13:48347864) in RB1 was detected in 1 heterozygote out of 58K WGS Icelanders (MAF= 0,001%). This variant has been reported in ClinVar previously as pathogenic. Based on ACMG criteria (PVS1, PM2) this variant classifies as likely pathogenic.