TP53 NM_000546.6:c.672+1G>A, Splice_Site

The c.672+1G>A intronic variant results from a G to A substitution one nucleotide after coding exon 5 of the TP53 gene. This variant has been reported in an individual with osteosarcoma diagnosed at age 15 (Sakurai N et al. Pediatr Int, 2013 Feb;55:107-11). Functional studies performed on this variant include RNA analysis which identified an 18 nucleotide insertion event and functional studies which demonstrated loss of function (Piao J et al. Mol Carcinog, 2013 Oct;52:770-6). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). 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 and will result in the creation or strengthening of a novel splice donor site; however, direct evidence is insufficient at this time (Ambry internal data). In addition to the clinical data presented in the literature, 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.

This sequence change affects a donor splice site in intron 6 of the TP53 gene. RNA analysis indicates that disruption of this splice site induces altered splicing and likely results in the gain of 6 amino acid residue(s), but is expected to preserve the integrity of the reading-frame. This variant is not present in population databases (gnomAD no frequency). Disruption of this splice site has been observed in individual(s) with Li-Fraumeni syndrome (PMID: 16494995, 23409989, 29070607; Invitae). In at least one individual the variant was observed to be de novo. ClinVar contains an entry for this variant (Variation ID: 216078). 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 TP53 function (PMID: 22495821). Studies have shown that disruption of this splice site results in the activation of a cryptic splice site in intron 6 (PMID: 22495821, 23409989). For these reasons, this variant has been classified as Pathogenic.

This variant causes a G>A nucleotide substitution at the +1 position of intron 6 of the TP53 gene. Splice site prediction tools suggest that this variant may have a significant impact on RNA splicing. A functional RNA study has shown that this variant may result in the use of a cryptic donor site 18 base pairs into intron 6, resulting in the insertion of 6 amino acids into the DNA binding domain (PMID: 23409989). Functional studies using this 6 amino acid construct showed impaired TP53 transcriptional transactivation, reduced cell cycle arrest at G0/G1 in response to DNA damage, and impaired ability to inhibit cell growth (PMID: 22495821). This variant has been reported in individuals affected with osteosarcoma and rhabdomyosarcoma, the latter of which was confirmed de novo (PMID: 22495821, 23409989, 29070607). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Loss of TP53 function is a known mechanism of disease. Based on the available evidence, this variant is classified as Pathogenic.