ClinVar Genomic variation as it relates to human health

The NM_000546.6: c.733G>A variant in TP53 is a missense variant predicted to cause substitution of glycine by serine at amino acid 245 (p.Gly245Ser). This variant has been identified as a de novo occurrence with unconfirmed parental relationships in 1 individual with a strongly LFS-associated cancer totaling 2 phenotype points (PS2_Moderate; Internal lab contributor: SCV000184981.8). This variant has been reported in an additional individual meeting Classical criteria and 11 individuals meeting Revised Chompret criteria. Based on this evidence, this variant scores 8 total points meeting the TP53 VCEP phenotype scoring criteria of ≥ 8 points. (PS4_Very Strong; PMIDs: 11370630, 32156018, 32888145, 33245408, 34670578, 35974385, 20522432, 24122735, Internal lab contributor: SCV000184981.8). At least two individuals with this variant were found to have a variant allele fraction of 5-25%, which is a significant predictor of variant pathogenicity (PP4_Moderate, PMID: 34906512, Internal lab contributors: SCV000184981.8). This variant has an allele frequency of 0.000002543 (3/1179926 alleles) in the European (non-Finnish) population in gnomAD v4.1.0 which is lower than the Clingen TP53 VCEP threshold (<0.00004) for PM2_Supporting, and therefore meets this criterion (PM2_Supporting). In vitro assays performed in yeast and/or human cell lines showed non-functional transactivation and loss of growth suppression activity indicating that this variant impacts protein function (PMIDs: 12826609, 30224644, 29979965) (PS3). This variant resides within a codon (NM_00546.4: 175, 245, 248, 249, 273, 282) of TP53 that is defined as a mutational hotspot by the ClinGen TP53 VCEP (PMID: 8023157 ) (PM1). Computational predictor scores (BayesDel = 0.5536; Align GVGD = Class C55) are above recommended thresholds (BayesDel > 0.16 and an Align GVGD Class of > 15), evidence that correlates with impact to TP53 via protein change (PP3). In summary, this variant meets the criteria to be classified as Pathogenic for Li Fraumeni Syndrome based on the ACMG/AMP criteria applied, as specified by the ClinGen TP53 VCEP: PS2_Moderate, PS4_Very Strong, PP4_Moderate, PM2_Supporting, PS3, PM1, PP3. (Bayesian Points: 20; VCEP specifications version 2.0; 7/24/2024)

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

c.733G>A, located in exon 7 of the TP53 gene, is predicted to result in the substitution of Glycine by Serine at codon 245, p.(Gly245Ser). It is located in a mutational hotspot (PM1). This variant is found in 1/268258 alleles at a frequency of 0.0003% in the gnomAD v2.1.1 database, non-cancer dataset. The SpliceAI algorithm predicts no significant impact on splicing. In-silico tools predict a pathogenic effect of the variant on protein function (aGVGD: C55; BayesDel: 0.55) (PP3). Transactivation assays show a non-functional allele according to Kato 2003 (PMID: 12826609) and there is evidence of a dominant negative effect and loss of function according to Giacomelli 2018 (PMID: 30224644) (PS3). At least, this variant has been reported in 7 individuals affected with a TP53-related phenotype, which awards 4.5 points to this variant as per ClinGen SVI Recommendation for LFS/Chompret Criterion (PMID: 11370630, 32156018, 32888145, 34670578, 20522432, 24122735) (PS4). It has been reported in ClinVar as a pathogenic variant, LOVD, CancerHotspots. Based on the currently available information, c.733G>A is classified as a pathogenic variant according to ClinGen-TP53 Guidelines version 1.4.

The p.Gly245Ser variant in TP53 has been reported in >15 individuals with TP53-a ssociated cancers. It also segregated with disease in at least 8 affected relati ves from multiple families (Varley 1997; Trkova 2003). This variant has also bee n identified in 1/66734 European chromosomes by the Exome Aggregation Consortium (ExAC, http://exac.broadinstitute.org; dbSNP rs29834575). In vitro functional s tudies demonstrate that the p.Gly245Ser variant has a dominant negative effect ( Marutani 1999). In summary, this variant meets criteria to be classified as path ogenic for Li-Fraumeni syndrome in an autosomal dominant manner based upon segre gation studies, low frequency in controls, and functional evidence.

Published functional studies demonstrate a damaging effect: impaired DNA binding, transcriptional activation, and growth suppression activities, with a dominant-negative effect observed (Kato 2003, Malcikova 2010, Monti 2011, Kotler 2018); Not observed at significant frequency in large population cohorts (Lek et al., 2016); 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: 23334668, 16401470, 27045317, 28222664, 30592087, 31081129, 20128691, 17606709, 21665182, 19367569, 1565143, 24573247, 25637381, 21343334, 25503501, 24122735, 26822949, 21761402, 17311302, 12676907, 26786923, 26641009, 24835218, 26225655, 27621308, 28369373, 28573494, 29602769, 28724667, 29025599, 29338689, 29979965, 28472496, 28975465, 23538418, 30076369, 29093764, 30720243, 30322717, 30093976, 30840781, 29263802, 30816478, 15951970, 31105275, 32000721, 27535533, 15510160, 29351919, 28929227, 32475984, 32156018, 33300245)

Variant summary: TP53 c.733G>A (p.Gly245Ser) results in a non-conservative amino acid change located in the DNA-binding domain (IPR011615) of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant was absent in 251472 control chromosomes. c.733G>A has been reported in the literature in multiple individuals affected with Li-Fraumeni Syndrome, osteosarcoma and other cancers (examples: Toguchida_1992, Giacomazzi_2013, Melhem-Bertrandt_2012, Bougeard_2001). 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. The most pronounced variant effect results in impaired DNA binding, transcriptional activation, and growth suppression activities, with a dominant-negative effect observed (Monti_2011, Kato_2003, etc). 13 clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All laboratories, including an expert panel, classified the variant as pathogenic/likely pathogenic. Based on the evidence outlined above, the variant was classified as pathogenic.

The TP53 c.733G>A (p.Gly245Ser) missense change has a maximum subpopulation frequency of 0.0040% in gnomAD v2.1.1 (http://gnomad.broadinstitute.org). This variant has been reported in individuals with LFS-associated cancers (PMID: 11370630, 24122735, 20522432, 12885464). Computational evidence supports a deleterious effect of this variant on protein function and transactivation assays show a low functioning allele according to Kato et al., and evidence of loss of function and a dominant negative effect according to Giacomelli et al. (PMID 12826609, 30224644). This variant is a somatic hotspot variant in tumors. In summary, this variant meets criteria to be classified as pathogenic.

This sequence change replaces glycine, which is neutral and non-polar, with serine, which is neutral and polar, at codon 245 of the TP53 protein (p.Gly245Ser). The frequency data for this variant in the population databases is considered unreliable, as metrics indicate poor data quality at this position in the gnomAD database. This missense change has been observed in individuals with Li-Fraumeni syndrome (LFS) or LFS-like syndrome and LFS-related cancers (PMID: 1565143, 11370630, 15925506, 16401470, 17311302, 20522432, 21761402, 24122735, 26225655, 27621308). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 12365). 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) expected to disrupt TP53 protein function. Experimental studies have shown that this missense change affects TP53 function (PMID: 12826609, 12917626, 15722483, 20128691, 21343334, 26205489). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may create or strengthen a splice site. For these reasons, this variant has been classified as Pathogenic.

PP3, PP5, PM1, PM2, PS3, PS4

This sequence change replaces glycine, which is neutral and non-polar, with serine, which is neutral and polar, at codon 245 of the TP53 protein (p.Gly245Ser). The frequency data for this variant in the population databases is considered unreliable, as metrics indicate poor data quality at this position in the gnomAD database. This missense change has been observed in individuals with Li-Fraumeni syndrome (LFS) or LFS-like syndrome and LFS-related cancers (PMID: 1565143, 11370630, 15925506, 16401470, 17311302, 20522432, 21761402, 24122735, 26225655, 27621308). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 12365). Advanced modeling performed at Invitae incorporating data from internal and/or published experimental studies (PMID: 12826609, 29979965, 30224644) indicates that this missense variant is expected to disrupt TP53 function with a positive predictive value of 97.5%. Experimental studies have shown that this missense change affects TP53 function (PMID: 12826609, 12917626, 15722483, 20128691, 21343334, 26205489). RNA analysis performed to evaluate the impact of this missense change on mRNA splicing indicates it does not significantly alter splicing (Invitae). For these reasons, this variant has been classified as Pathogenic.

This missense variant replaces glycine with serine at codon 245 in the DNA binding domain of the TP53 protein. Computational prediction suggests that this variant may have deleterious impact on protein structure and function (internally defined REVEL score threshold >= 0.7, PMID: 27666373). Functional studies have shown the mutant protein to be non-functional in transactivation assays (PMID: 11370630, 12826609, 15781620) and in cell growth assays (PMID: 29979965, 30224644). This variant has been reported in multiple individuals affected with either classic Li-Fraumeni syndrome (PMID: 11370630, 12885464 32156018, 32817165, 33163847, 33840814, 34709361, 35974385) or meeting the Chompret criteria for Li-Fraumeni syndrome, including individuals with early-onset breast cancer (PMID:20522432, 32888145, 32888145, 33245408, 34529667, 359743855; Color Health internal data). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Pathogenic.

The p.G245S pathogenic mutation (also known as c.733G>A), located in coding exon 6 of the TP53 gene, results from a G to A substitution at nucleotide position 733. The glycine at codon 245 is replaced by serine, an amino acid with similar properties. This variant has been reported in multiple individuals with clinical and family histories consistent with Li-Fraumeni syndrome (Toguchida J et al. N. Engl. J. Med. 1992 May;326:1301-8; Bougeard G et al. J Med Genet, 2001 Apr;38:253-7; Trkova M et al. Cancer Genet Cytogenet, 2003 Aug;145:60-4; Wong P et al. Gastroenterology. 2006 Jan;130:73-9; Ruijs MW et al. J Med Genet, 2010 Jun;47:421-8; Giacomazzi J et al. Cancer. 2013 Dec;119:4341-9). This variant has been determined to be the result of a de novo mutation or germline mosaicism in in one child with a choroid plexus carcinoma (Ambry internal data). This variant is in the DNA binding domain of the TP53 protein and is reported to have non-functional transactivation in yeast-based assays (Kato S et al. Proc. Natl. Acad. Sci. USA. 2003 Jul;100:8424-9). Studies conducted in human cell lines indicate this alteration is deficient at growth suppression and has a dominant negative effect (Kotler E et al. Mol.Cell. 2018 Jul;71:178-190.e8; Giacomelli AO et al. Nat. Genet. 2018 Oct;50:1381-1387). This variant has been detected in at least one individual at an allele fraction that is suggestive of clonal hematopoiesis, a predictor of TP53 pathogenicity (Ambry internal data; Fortuno C et al. Genet Med. 2022 03;24:673-680). This alteration has been observed numerous times as a somatic mutation in the cancerhotspots.org database (Chang MT et al. Cancer Discov. 2018 02;8:174-183). In addition, this alteration is predicted to be deleterious by in silico analysis. Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

The TP53 p.Gly245Ser variant was identified in 8 of 2224 proband chromosomes (frequency: 0.004) from Spanish, Dutch, British, and American individuals or families with Li-Fraumeni syndrome, and Li-Fraumeni syndrome with breast cancer (BRCA1/2 negative and early onset, or with a family history of multiple primary cancers, or HER-2 positive) or early onset CRC, and was not identified in 300 chromosomes from healthy individuals (Llovet 2017 , Martin 2003, Maxwell 2014, Ruijis 2009, Eccles 2016, Wong 2006). The variant falls within 1 of 5 conserved domains for which the majority of disease associated mutations (hotspots) occur (codons 175, 245, 248, 249 and 273); the variant is a structural mutant that is moderately destabilized when compared to wild-type p53 and has partial DNA binding thereby affecting the protein’s transcriptional activity, as evidenced through multiple studies using molecular dynamics simulations, crystallography/NMR Spectroscopy (Martin 2003, Lepre 2017, Wong 1999, Merabet 2010, Joerger 2006).The variant was also identified in dbSNP (ID: rs28934575) “With Likely pathogenic,Pathogenic allele”, ClinVar (classified pathogenic by Ambry Genetics, EGL Genetic Diagnostics (Eurofins Clinical Diagnostics), GeneDx, Invitae, Pathway Genetics and OMIM; and likely pathogenic by CSER_CC_NCGL (University of Washington Medical Center)), Clinvitae (1x), Cosmic (in numerous tumour tissue types: brain, breast, liver, lung, pancreatic, bladder, oesophageal, colon, stomach,ovarian, prostate, and uterine), UMD TP53 Mutation Database, and Database of germline p53 mutations. The variant was not identified in Genesight-COGR, LOVD 3.0, and IARC TP53 Database. The variant was identified in control databases in 1 of 277132 chromosomes at a frequency of 0.000004 (Genome Aggregation Database Feb 27, 2017), seen in the African population in 1 of 23998 chromosomes (freq: 0.00004), while not observed in the Other, Latino, European Non-Finnish, Ashkenazi Jewish, East Asian, European Finnish, and South Asian populations. The p.Gly245 residue is conserved across mammals and other organisms, and four out of five computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) suggest that the Ser variant may impact the protein; however, this information is not predictive enough to assume pathogenicity. The variant occurs outside of the splicing consensus sequence and 2 of 5 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) predict a greater than 10% difference in splicing; this is not very predictive of pathogenicity. In summary, based on the above information this variant meets our laboratory’s criteria to be classified as pathogenic.

The TP53 c.733G>A variant is predicted to result in the amino acid substitution p.Gly245Ser. This variant has been identified in individuals with a personal or family history of Li-Fraumeni syndrome and related cancers (Toguchida. 1992. PubMed ID: 1565143; Wong. 2006. PubMed ID: 16401470; Melhem-Bertrandt. 2012. PubMed ID: 21761402; Giacomazzi. 2013. PubMed ID: 24122735; Table S4, Bhai. 2021. PubMed ID: 34326862; Table S2, Guindalini. 2022. PubMed ID: 35264596). Functional analysis showed that this variant resulted in severe deficiency of transactivation activity and TP53 functionality (Table S1, Monti. 2011. PubMed ID: 21343334; Table 1, Zerdoumi. 2017. PubMed ID: 28472496). This variant is reported in 0.0040% of alleles in individuals of African descent in gnomAD and has been reported in ClinVar by multiple sources as pathogenic (https://www.ncbi.nlm.nih.gov/clinvar/variation/12365/). This variant is interpreted as pathogenic.