Submissions for variant NM_000249.4(MLH1):c.2041G>A (p.Ala681Thr)

dbSNP: rs63750217

Total submissions: 24

Submitter	RCV	SCV	Clinical significance	Condition	Last evaluated	Review status	Method	Comment
International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	RCV000075495	SCV000106491	pathogenic	Lynch syndrome	2013-09-05	reviewed by expert panel	research	Multifactorial likelihood analysis posterior probability >0.99
Invitae	RCV000524270	SCV000218745	pathogenic	Hereditary nonpolyposis colorectal neoplasms	2024-01-15	criteria provided, single submitter	clinical testing	This sequence change replaces alanine, which is neutral and non-polar, with threonine, which is neutral and polar, at codon 681 of the MLH1 protein (p.Ala681Thr). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with colorectal cancer and Lynch syndrome (PMID: 8880570, 12362047, 16083711, 18033691, 21642682, 22736432, 23354017, 23403630). It has also been observed to segregate with disease in related individuals. This variant is also known as 2062G>A. ClinVar contains an entry for this variant (Variation ID: 17099). 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) performed at Invitae indicates that this missense variant is expected to disrupt MLH1 protein function with a positive predictive value of 80%. Experimental studies have shown that this missense change affects MLH1 function (PMID: 9697702, 10037723, 12810663, 15864295, 16083711, 17510385, 21404117, 25477341). For these reasons, this variant has been classified as Pathogenic.
Ambry Genetics	RCV000213700	SCV000275301	pathogenic	Hereditary cancer-predisposing syndrome	2022-04-12	criteria provided, single submitter	clinical testing	The p.A681T pathogenic mutation (also known as c.2041G>A), located in coding exon 18 of the MLH1 gene, results from a G to A substitution at nucleotide position 2041. The alanine at codon 681 is replaced by threonine, an amino acid with similar properties. This alteration has been identified in multiple families either meeting Amsterdam II criteria or whose family history was suggestive of HNPCC/Lynch syndrome; it has also been shown to co-segregate with disease in several families (Froggatt NJ et al. J. Med. Genet. 1996 Sep;33:762-30; Shimodaira H et al. Nat. Genet. 1998 Aug;19:384-9; Kurzawski G et al. Clin. Genet. 2006 Jan;69:40-7; Barnetson RA et al. Hum. Mutat. 2008 Mar;29:367-74; Dymerska D et al. 2014 Clin. Genet. 2014 Aug;86:190-3; Rossi BM et al. BMC Cancer. 2017 Sep;17:623). Tumor analysis of two related individuals who carried this alteration and were diagnosed with colorectal cancer under the age of 55 years showed high microsatellite instability (MSI-H) and loss of MLH1 on immunohistochemistry (Barnetson RA et al. Hum. Mutat. 2008 Mar;29:367-74). In vitro studies have shown that while this alteration shows proficient MMR activity, it leads to decreased protein stability and thus, decreased protein expression (Raevaara TE et al. Gastroenterology. 2005 Aug;129:537-49; Takahashi M et al. Cancer Res. 2007 May;67:4595-604; Hinrichsen I et al. Clin. Cancer Res. 2013 May;19:2432-41). Another functional analysis of this variant has demonstrated that this amino acid change causes a reduction in MLH1 and PMS2 binding and a >80% reduction in the interaction between MLH1 and hExo1, a member of a family of conserved exonucleases which are implicated in many DNA metabolic processes, including DNA mismatch repair (MMR) and recombination (Schmutte CJ et al. J. Biol. Chem. 2001 Aug;276:33011-8). This variant was identified as germline in a patient with MSI-H colon cancer demonstrating MLH1-/PMS2- and LOH (Hampel H et al. JAMA Oncol. 2018 Jun;4(6):806-813), and somatic data also supports pathogenicity (Shirts BH et al. Am. J. Hum. Genet. 2018 Jul;103(1):19-29). Furthermore, this alteration has been classified as pathogenic using the following lines of evidence: in silico prediction models, segregation with disease, clinical phenotype including tumor characteristics, mutation co-occurrence, and functional studies (Thompson BA et al. Hum. Mutat. 2013 Jan;34:200-9; Thompson BA et al. Nat. Genet. 2014 Feb;46:107-15; available at [www.insight-group.org/variants/classifications/]). Based on the available evidence, this alteration is classified as a pathogenic mutation.
Counsyl	RCV000018632	SCV000488784	pathogenic	Colorectal cancer, hereditary nonpolyposis, type 2	2016-06-16	criteria provided, single submitter	clinical testing
GeneDx	RCV000519240	SCV000616784	pathogenic	not provided	2020-12-01	criteria provided, single submitter	clinical testing	Published functional studies are discordant: similar in vitro MMR activity compared to wild type, reduced MLH1 expression, reduced binding with PMS2, and significantly reduced binding with MRE11, increased cell survival in a methylation tolerance assay (Guerrette 1999, Vo 2005, Takahashi 2007, Hardt 2011, Drost 2018, Bouvet 2019); Not observed in large population cohorts (Lek et al., 2016); In silico analysis supports that this missense variant does not alter protein structure/function; This variant is associated with the following publications: (PMID: 29887214, 23403630, 12362047, 17054581, 21681552, 18561205, 15849733, 12624141, 22034109, 19669161, 19248199, 12810663, 30553995, 16083711, 11555625, 21120944, 22736432, 11879922, 16451135, 11139242, 18951440, 24333619, 24344984, 17510385, 21642682, 17473388, 15864295, 17210669, 10037723, 24362816, 23354017, 23760103, 16341804, 16276679, 17505997, 19731080, 14564042, 20007843, 16142001, 21404117, 16807412, 20305446, 9697702, 27629256, 8880570, 18033691, 17594722, 28449805, 28874130, 29785153, 30093976, 30504929, 30998989, 31159747, 31386297, 31660093, 31491536, 32587781, 32710294)
Color Diagnostics, LLC DBA Color Health	RCV000213700	SCV000689855	pathogenic	Hereditary cancer-predisposing syndrome	2020-04-22	criteria provided, single submitter	clinical testing	This missense variant replaces alanine with threonine at codon 681 in the PMS2/MLH3/PMS1 interacting domain 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). Functional studies have shown that this variant results in reduced protein expression and stability (PMID: 21404117, 23403630, 25477341, 29520894, 32076465) and disrupts binding to PMS2, MRE11, and EXO1 (PMID: 10037723, 11427529, 12810663, 15864295, 21404117). This variant has been shown to eliminate the dominant negative mutator effect (PMID: 9697702, 17510385, 21404117) and decrease MMR activity (PMID: 17210669, 17510385). However, some studies have contradicted these results and shown near normal expression and activity of the mutant protein (PMID: 12810663, 16083711, 17510385, 21120944, 23403630, 30504929). This variant has been reported in more than 60 families (>70 individuals) affected with colorectal cancer that meet either Amsterdam or Bethesda criteria (PMID: 8880570, 16083711, 16451135, 17054581, 17505997, 18033691, 21404117, 21642682, 22736432, 23354017, 24032978, 28874130, 29596542, 30324682, 31386297, 31491536, 31660093). Most of these individuals had tumors with high microsatellite instability and undetectable MLH1 expression by immunohistochemistry (PMID: 16083711, 17054581, 18033691, 21120944, 22736432, 29596542). It has been reported that this variant segregates with disease in at least 7 families and is thought to be a founder mutation in the Polish population (PMID: 8880570, 16451135, 18033691). 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.
GeneKor MSA	RCV000519240	SCV000821735	likely pathogenic	not provided	2020-01-01	criteria provided, single submitter	clinical testing	This sequence change replaces Alanine with Threonine at codon 681 of the MLH1 protein (p.Ala681Thr). The alanine residue is weakly conserved among species and is located in a domain of the protein that is known to be functionally important. There is a small physicochemical difference between Alanine and Threonine (Grantham Score 58). This sequence change has been reported in the literature and is not present in population databases (rs63750217). This variant was reported in several individuals affected with colorectal cancer and Lynch syndrome (PMID: 16083711, 23354017, 18033691, 8880570). The mutation database ClinVar contains entries for this variant (Variation ID: 17099).Algorithms developed to predict the effect of missense changes on protein structure and function suggest that this variant is likely to be damaging to the protein. Moreover, experimental studies have shown that this missense change disrupts MLH1 protein function (PMID: 9697702, 17510385, 21404117).
Fulgent Genetics, Fulgent Genetics	RCV000763105	SCV000893647	pathogenic	Mismatch repair cancer syndrome 1; Muir-Torré syndrome; Colorectal cancer, hereditary nonpolyposis, type 2	2018-10-31	criteria provided, single submitter	clinical testing
A.C.Camargo Cancer Center / LGBM, A.C.Camargo Cancer Center	RCV000075495	SCV000914326	pathogenic	Lynch syndrome	2019-01-30	criteria provided, single submitter	research
Women's Health and Genetics/Laboratory Corporation of America, LabCorp	RCV001328323	SCV001519395	pathogenic	Hereditary nonpolyposis colon cancer	2021-03-01	criteria provided, single submitter	clinical testing	Variant summary: MLH1 c.2041G>A (p.Ala681Thr) results in a non-conservative amino acid change located in the DNA mismatch repair protein Mlh1, C-terminal domain (IPR032189) 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 254122 control chromosomes (gnomAD and publication data). c.2041G>A has been reported in the literature in multiple individuals affected with Hereditary Nonpolyposis Colorectal Cancer and segregated with disease in several families (Barnetson_2008, Hardt_2011). These data indicate that the variant is very likely to be associated with disease. Although there are conflicting results, multiple functional studies report this variant has an impact on MLH1 protein function and results in reducing interacting with PMS2 and EXO1 proteins, decreasing in vitro MMR activity and MLH1 protein expression (Kondo_2003, Takahashi_2007, Hardt_2011, Koger_2018, PMID: 16083711). Nine ClinVar submitters (evaluation after 2014) cite the variant as pathogenic (n=7) and likely pathogenic (n=2). Based on the evidence outlined above, the variant was classified as pathogenic.
Revvity Omics, Revvity	RCV000519240	SCV002017488	pathogenic	not provided	2019-09-09	criteria provided, single submitter	clinical testing
Foundation for Research in Genetics and Endocrinology, FRIGE's Institute of Human Genetics	RCV000018632	SCV002102407	pathogenic	Colorectal cancer, hereditary nonpolyposis, type 2	2021-09-01	criteria provided, single submitter	research	A heterozygous missense variation in exon 18 of the MLH1 (Depth:125x) gene that results in the amino acid substitution of Threonine for Alanine at codon 681 was detected. The observed variant c.2041G>A (p.Ala681Thr) has not been reported in the 1000 genomes and gnomAD databases. The in-silico predictions of the variant are possibly damaging by PolyPhen-2 (HumDiv), damaging by SIFT, LRT and Mutation Taster2 tools. The reference codon is conserved across species.
Sema4, Sema4	RCV000213700	SCV002528707	pathogenic	Hereditary cancer-predisposing syndrome	2021-07-07	criteria provided, single submitter	curation
MGZ Medical Genetics Center	RCV002288511	SCV002581136	pathogenic	Muir-Torré syndrome	2022-07-26	criteria provided, single submitter	clinical testing
Laboratorio de Genetica e Diagnostico Molecular, Hospital Israelita Albert Einstein	RCV000018632	SCV003808038	pathogenic	Colorectal cancer, hereditary nonpolyposis, type 2	2022-06-30	criteria provided, single submitter	clinical testing	ACMG classification criteria: PS3 moderated, PS4 strong, PM2 moderated, PP1 strong, PP3 supporting
Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital	RCV000519240	SCV004024911	pathogenic	not provided	2023-08-15	criteria provided, single submitter	clinical testing
Baylor Genetics	RCV000018632	SCV004193068	pathogenic	Colorectal cancer, hereditary nonpolyposis, type 2	2021-07-30	criteria provided, single submitter	clinical testing
Quest Diagnostics Nichols Institute San Juan Capistrano	RCV000519240	SCV004220868	pathogenic	not provided	2023-01-27	criteria provided, single submitter	clinical testing	This variant has not been reported in large, multi-ethnic general populations (http://gnomad.broadinstitute.org). In the published literature, the variant has been reported in a large number of affected individuals with colorectal and Lynch syndrome-associated cancer (PMIDs: 31491536 (2020), 31660093 (2019), 31386297 (2019), 30093976 (2018), 28449805 (2017), 24344984 (2013), 18033691 (2008), 17054581 (2006), 12362047 (2002), 8880570 (1996)), and is described as a recurrent/founder variant in Poland (PMIDs: 16451135 (2006), 12362047 (2002)). Extensive functional studies indicate this variant has deleterious effects on MLH1 protein expression (PMID 32076465 (2020), 29520894 (2018), 25477341 (2015), 21404117 (2011)), PMS2 binding (PMID 21404117 (2011), 15864295 (2005), 12810663 (2003), 10037723 (1999)), and interactions with MRE11 and EXO1 proteins (PMID 15864295 (2005), 12810663 (2003), 11427529 (2001)). However, some studies showed the variant had neutral effects on DNA mismatch-repair activity and protein expression (PMIDs: 30504929 (2018), 23403630 (2013), 17510385 (2007), 16083711 (2005)). Additional analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded predictions that this variant is damaging. Based on the available information, this variant is classified as pathogenic.
OMIM	RCV000018632	SCV000038915	pathogenic	Colorectal cancer, hereditary nonpolyposis, type 2	2006-01-01	no assertion criteria provided	literature only
Mayo Clinic Laboratories, Mayo Clinic	RCV000202172	SCV000257082	uncertain significance	not specified		no assertion criteria provided	research
Bioscientia Institut fuer Medizinische Diagnostik GmbH, Sonic Healthcare	RCV000018632	SCV000484931	likely pathogenic	Colorectal cancer, hereditary nonpolyposis, type 2		no assertion criteria provided	clinical testing
Department of Pathology and Laboratory Medicine, Sinai Health System	RCV000519240	SCV000592438	likely pathogenic	not provided		no assertion criteria provided	clinical testing	The MLH1, c.2041G>A, p.Ala681Thr variant has been identified in our laboratory in one among the 688 individuals who have undergone MLH1 testing. It has also been reported in the literature in 17 out of 11502 proband chromosomes (frequency 0.001) of patients with HNPCC meeting the Amsterdam or Bethesda criteria; the variant was not reported in any of the 730 proband controls chromosomes (Wanat_2007, Barnetson_2006, Guerrette_1999, Shimodaira_1998, Kondo_2003, Raevaara_2005, Kansikas_2011, Betz_2010, Tournier_2008, de Leon_2007, Hardt_2011, Kurzawski_2006, Pedroni_2007, Takahashi_2007, Sheng_2006, Vo_2005). The p.Ala681 residue is conserved in mammals; however, computational analyses (PolyPhen, SIFT, AlignGVGD) provide inconsistent predictions regarding the impact to the protein and this information is not very predictive of pathogenicity. It is listed in dbSNP database as coming from a "clinical source" (ID#: rs.63750217) but no frequency information was provided therefore not very informative for assessing the population frequency. There is conflicting evidence in the literature regarding the effect of the variant on the function of MLH1. In two functional studies, the variant does not seem to have any appreciable effect on the function of the MLH1 protein (Raevaara_2005, Kansikas_2011), while another study classified it as a VUS due to inconclusive functional studies, even though the variant disrupted the interaction between MLH1 and MSH2 (Hardt_2011). However, other functional assays using site-directed mutagenesis and in vitro MMR assays found that the p.A681T variant conferred a defect in MMR function, and reduced the binding capacity of MLH1 to MSH2, increasing the likelihood that an alteration to this residue is pathogenic (Wanat_2007, Guerrette_1999, Hardt_2011, Takahashi_2007, Vo_2005). In at least two studies involving kindreds, the mutation segregated with the disease with (Shimodaira_1998, Wanat_2007). In addition, several studies have demonstrated MLH1 immunohistochemistry deficiency or MSI-high tumors in individuals with this deletion (Pedroni_2007, Barnetson_2006, Sheng_2006, Takahashi_2007) increasing the likelihood this variant is pathogenic. In summary, based on the above information, this variant is classified as predicted pathogenic.
Joint Genome Diagnostic Labs from Nijmegen and Maastricht, Radboudumc and MUMC+	RCV000519240	SCV001951498	pathogenic	not provided		no assertion criteria provided	clinical testing
Clinical Genetics DNA and cytogenetics Diagnostics Lab, Erasmus MC, Erasmus Medical Center	RCV000519240	SCV001971028	pathogenic	not provided		no assertion criteria provided	clinical testing