ClinVar Genomic variation as it relates to human health

Criteria changed for variants in last base of exon therefore downgrade classification

The p.Thr151Thr variant in MLH1 has been reported in one individual with colorec tal polyps and a family history of colorectal cancer (Shirts 2016). This variant has been identified in 1/16492 of South Asian chromosomes by the Exome Aggregat ion Consortium (ExAC, http://exac.broadinstitute.org; dbSNP rs369521379). Althou gh the p.Thr151Thr variant does not alter an amino acid residue, it is located i n the last base of the exon, which is part of the 5? splice region. Computationa l tools suggest some impact to splicing though this information is not predictiv e enough to determine pathogenicity. In contrast, this variant has been classifi ed as Likely Pathogenic on Sept 5, 2013 by the ClinGen-approved InSiGHT expert p anel (ClinVarSCV000106723.2), due a predicted splicing effect that has not been confirmed by published functional studies. In summary, the clinical significance of the p.Thr151Thr variant is uncertain.

This sequence change affects codon 151 of the MLH1 mRNA. It is a 'silent' change, meaning that it does not change the encoded amino acid sequence of the MLH1 protein. RNA analysis indicates that this variant induces altered splicing and likely results in the gain of 3 amino acid residue(s), but is expected to preserve the integrity of the reading-frame. This variant is present in population databases (rs369521379, gnomAD 0.004%). This variant has been observed in individual(s) with colon cancer and lung cancer (PMID: 26845104, 28449805, 31386297). ClinVar contains an entry for this variant (Variation ID: 90229). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant results in the activation of a cryptic splice site in intron 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.

Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis suggests this variant may impact gene splicing. In the absence of RNA/functional studies, the actual effect of this sequence change is unknown.; Observed in individuals with Lynch-related and other cancers as well as polyps, but also in cancer-free controls (Shirts et al., 2016; Sunga et al., 2017; Matejcic et al., 2020; Singh et al., 2020; Okawa et al., 2023); This variant is associated with the following publications: (PMID: 32832836, 28449805, 31386297, 12414824, 26845104, 30720243, 35451682, 36243179, 32634176, 33309985)

This synonymous variant does not change the amino acid sequence of the MLH1 protein but does change the last base of exon 5. Splice site prediction tools suggest that this variant may impact RNA splicing. However, this prediction has not been confirmed in published RNA studies. This variant has been reported in individuals affected with polyps (PMID: 26845104), lung cancer (PMID: 31386297), breast/ovarian cancer (PMID: 32068069), endometrial cancer (PMID: 32634176), and in a cohort of individuals affected with suspected Lynch syndrome (PMID: 28449805). However, case-control studies in Japan reported no association with colorectal (PMID: 33309985) or pancreatic cancer (PMID: 32980694). This variant has also been identified in 3/282760 chromosomes in the general population by the Genome Aggregation Database (gnomAD). The available evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance.

Variant summary: MLH1 c.453G>A (p.Thr151Thr) alters a conserved nucleotide located close to a canonical splice site and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. Several computational tools predict a significant impact on normal splicing: One predict the variant abolishes the canonical 5' splicing donor site. One predict the variant creates a 3' acceptor site. Three predict the variant weakens the canonical 5' splicing donor site. However, these predictions have yet to be confirmed by functional studies. The variant allele was found at a frequency of 8e-06 in 251372 control chromosomes. The available data on variant occurrences in the general population are insufficient to allow any conclusion about variant significance. c.453G>A has been reported in the literature in individuals affected with/undergoing testing for Hereditary Nonpolyposis Colorectal Cancer/Lynch syndrome or other cancers and also as a VUS in settings of multigene panel testing in individuals with colorectal cancer and Breast and/or ovarian cancer (example, Shirts_2016, Sunga_2017, Kiyozumi_2019, Molina-Zayas_2022). A large case-control study evaluating Biliary tract cancer reported the variant insignificantly distributed between cases and controls (Okawa_2023). These reports do not provide unequivocal conclusions about association of the variant with Hereditary Nonpolyposis Colorectal Cancer/Lynch syndrome. At-least one co-occurrence with another pathogenic variant has been observed at our laboratory (BRCA1 c.188T>A, p.Leu63Ter), providing supporting evidence for a benign role. To our knowledge, no experimental evidence demonstrating an impact on protein function has been reported. The following publications have been ascertained in the context of this evaluation (PMID: 31386297, 35451682, 36243179, 26845104, 28449805). ClinVar contains an entry for this variant (Variation ID: 90229). Based on the evidence outlined above, the variant was classified as uncertain significance.

MLH1 NM_000249.3:c.453G>A has a 75.7% probability of pathogenicity based on combining prior probability from public data with a likelihood ratio of 0.16 to 1, generated from evidence of seeing this as a somatic mutation in a tumor with loss of heterozygosity at the MLH1 locus. See Shirts et al 2018, PMID 29887214. This was also reported in Shirts et al 2016, PMID 26845104 in an individual with a family history of colorectal cancer.

The MLH1 c.453G>A (p.Thr151=) synonymous change has a maximum subpopulation frequency of 0.0040% in gnomAD v2.1.1 (https://gnomad.broadinstitute.org/variant/3-37048554-G-A). Algorithms that predict the impact of sequence changes on splicing indicate that this change may create or strengthen a splice site (PP3), but this prediction has not been confirmed by RNA studies. This variant has been reported in individuals with colorectal cancer and/or clinical features of Lynch syndrome (PMID: 26845104, 28449805, 33309985), individuals with other cancers (PMID: 31386297, 32634176), and in a tumor that demonstrated loss-of-heterozygosity of the wild-type allele (PMID: 29887214). It has also been reported in non-cancer controls (PMID: 33309985). In summary, this variant meets criteria to be classified as of uncertain significance based on the ACMG/AMP criteria: PP3.

The c.453G>A variant (also known as p.T151T) located in coding exon 5 of the MLH1 gene. This variant results from a G to A substitution at nucleotide position 453. This nucleotide substitution does not change the threonine at codon 151. However, this change occurs in the last base pair of exon 5 which makes it likely to have some effect on normal mRNA splicing. This alteration has been detected in numerous individuals who do not have a personal or family history that is consistent with or suggestive of hereditary nonpolyposis colorectal cancer (HNPCC)/Lynch syndrome (Ambry internal data). In one study, p.Thr151Thr was seen in 20/12503 unselected Japanese colorectal cancer patients and in 28/23705 controls (Fujita M et al. Clin Gastroenterol Hepatol, 2020 Dec). This alteration has been identified in an individual suspected of having Lynch syndrome, an individual with polyps, and a woman with endometrial cancer (Sunga AY et al. Cancer Genet, 2017 04;212-213:1-7; Shirts BH et al. Genet. Med., 2016 10;18:974-81; Singh AK et al. PLoS One, 2020 Jul;15:e0235613). This alteration was also detected in a study of 1,165 individuals with a history of colorectal cancer or colon polyps as well as 590 controls (Gordon AS et al. Am J Hum Genet, 2019 09;105:526-533). This nucleotide position is not well conserved in available vertebrate species. In silico splice site analysis predicts that this alteration may result in the creation or strengthening of a novel splice donor site. RNA studies have demonstrated an incomplete splice defect for this alteration resulting in a transcript predicted to lead to a protein with an in-frame insertion of 3 amino acids; however, the exact functional impact of the inserted amino acids is unknown at this time (Ambry internal data). Since supporting evidence is limited at this time, the clinical significance of this alteration remains unclear.