Submissions for variant NM_000249.4(MLH1):c.1693A>T (p.Ile565Phe)

gnomAD frequency: 0.00001  dbSNP: rs63750062

Total submissions: 7

Submitter	RCV	SCV	Clinical significance	Condition	Last evaluated	Review status	Method	Comment
GeneDx	RCV000160538	SCV000211112	uncertain significance	not provided	2022-07-22	criteria provided, single submitter	clinical testing	Not observed at significant frequency in large population cohorts (gnomAD); 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: 11093816, 21404117, 18383312, 19015241, 9833759, 15943554, 17510385, 21901500, 23741719, 34426522, 22753075, 12799449, 20533529, 31697235)
Color Diagnostics, LLC DBA Color Health	RCV000234862	SCV000292212	uncertain significance	Hereditary cancer-predisposing syndrome	2022-02-10	criteria provided, single submitter	clinical testing	This missense variant replaces isoleucine with phenylalanine at codon 565 of the MLH1 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). A functional study has shown that this variant causes a partial loss of mismatch repair activity of MLH1 protein (PMID: 17510385). This variant has been observed in several individuals affected with Lynch syndrome (PMID: 9833759, 15943554, 21404117, 21901500). However, a tumor sample from one of these individuals showed normal MLH1 protein expression but loss of MSH2 and MSH6 protein expression, indicating the presence of a pathogenic variant in the MSH2 gene (PMID: 15943554). In addition, one of the affected individuals carried a pathogenic MSH2 variant that could explain the observed phenotype (PMID: 21404117). This variant has been identified in 2/251154 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.
Invitae	RCV000627705	SCV000543656	uncertain significance	Hereditary nonpolyposis colorectal neoplasms	2024-01-03	criteria provided, single submitter	clinical testing	This sequence change replaces isoleucine, which is neutral and non-polar, with phenylalanine, which is neutral and non-polar, at codon 565 of the MLH1 protein (p.Ile565Phe). This variant is present in population databases (rs63750062, gnomAD 0.002%). This missense change has been observed in individual(s) with Lynch syndrome. However, in one of these individuals a pathogenic allele was also identified in MSH2, which suggests that this c.1693A>T variant was not the primary cause of disease. (PMID: 9833759, 21404117, 21901500). ClinVar contains an entry for this variant (Variation ID: 89842). 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) has been performed at Invitae for this missense variant, however the output from this modeling did not meet the statistical confidence thresholds required to predict the impact of this variant on MLH1 protein function. Experimental studies have shown that this missense change affects MLH1 function (PMID: 17510385). 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.
Ambry Genetics	RCV000234862	SCV000662094	uncertain significance	Hereditary cancer-predisposing syndrome	2023-11-16	criteria provided, single submitter	clinical testing	The p.I565F variant (also known as c.1693A>T), located in coding exon 15 of the MLH1 gene, results from an A to T substitution at nucleotide position 1693. The isoleucine at codon 565 is replaced by phenylalanine, an amino acid with highly similar properties. This alteration was detected in a proband with colorectal cancer diagnosed at age 44 as well as in another relative with colorectal cancer in a family meeting Amsterdam criteria (Hutter P et al. Int. J. Cancer. 1998 Dec;78:680-4). This alteration was detected in another individual who also carried a deletion in MSH2, and that proband's tumor that showed intact MLH1 protein expression but absent MSH2 and MSH6 proteins by IHC analysis (Hardt K et al. Fam. Cancer. 2011 Jun;10:273-84). Another study concluded that this variant was most likely not disease causing when it was detected in a Lynch-related tumor showing intact MLH1 and absent MSH2 and MSH6 proteins on IHC (Grabowski M et al. Genet. Test. 2005;9:138-46). Two Iranian patients with colorectal cancer meeting Amsterdam criteria were found to have this alteration; however, they were not tested for mutations in other MMR genes, and no IHC or MSI data was available on these tumors (Shahmoradi S et al. Fam. Cancer. 2012 Mar;11:13-7). In a study assessing the function of various MLH1 alterations, p.I565F demonstrated 52.5% of in vitro mismatch repair activity and >75% of relative MLH1 protein expression (Takahashi M et al. Cancer Res. 2007 May;67:4595-604). This amino acid position is well conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Since supporting evidence is limited at this time, the clinical significance of this alteration remains unclear.
CeGaT Center for Human Genetics Tuebingen	RCV000160538	SCV001153847	uncertain significance	not provided	2019-03-01	criteria provided, single submitter	clinical testing
All of Us Research Program, National Institutes of Health	RCV001354474	SCV004843196	uncertain significance	Lynch syndrome	2024-01-11	criteria provided, single submitter	clinical testing	This missense variant replaces isoleucine with phenylalanine at codon 565 of the MLH1 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). A functional study has shown that this variant causes a partial loss of mismatch repair activity of MLH1 protein (PMID: 17510385). This variant has been observed in several individuals affected with Lynch syndrome (PMID: 9833759, 15943554, 21404117, 21901500). However, a tumor sample from one of these individuals showed normal MLH1 protein expression but loss of MSH2 and MSH6 protein expression, indicating the presence of a pathogenic variant in the MSH2 gene (PMID: 15943554). In addition, one of the affected individuals carried a pathogenic MSH2 variant that could explain the observed phenotype (PMID: 21404117). This variant has been identified in 2/251154 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.
Department of Pathology and Laboratory Medicine, Sinai Health System	RCV001354474	SCV001549100	uncertain significance	Lynch syndrome		no assertion criteria provided	clinical testing	The MLH1 p.Ile565Phe variant was identified in 2 of 424 proband chromosomes (frequency: 0.005) from Swiss and German individuals or families with HNPCC (Hardt 2011, Hutter 1998). The variant was also identified in dbSNP (ID: rs63750062) as “With Uncertain significance allele” and ClinVar (classified as uncertain significance by an InSiGHT expert panel (2013), GeneDx, Color, Invitae and Ambry Genetics). The variant was not identified in UMD-LSDB. The variant was identified in control databases in 2 of 245948 chromosomes at a frequency of 0.000008 (Genome Aggregation Database Feb 27, 2017). It was observed in the following population: European Non-Finnish in 2 of 111450 chromosomes (freq: 0.00002) but not in the African, Other, Latino, Ashkenazi Jewish, East Asian, European Finnish, or South Asian populations. The p.Ile565 residue is conserved in mammals but not in more distantly related organisms, and four out of five computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) suggest that the Phe 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 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) do not predict a difference in splicing. One study using yeast assays, the variant showed decreased MLH1 expression at approximately 75% of wildtype and 53% in vitro MMR activity relative to wild type, but did not show a dominant mutator effect (Takahashi 2007). This variant was identified by our laboratory in a patient with an MLH1-deficient pancreatic tumour. In summary, based on the above information, the clinical significance of this variant cannot be determined with certainty at this time. This variant is classified as a variant of uncertain significance.