rs63750217

Positions:

• chr3-37048955-G-A
• chr3-37048955-G-C

Variant summary

Our verdict is Pathogenic. Variant got 14 ACMG points: 14P and 0B. PM2 PP3_Strong PP5_Very_Strong

The NM_000249.4(MLH1):​c.2041G>A​(p.Ala681Thr) variant causes a missense change. The variant allele was found at a frequency of 0.00000205 in 1,461,836 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a pathogenic outcome for this variant. Variant has been reported in ClinVar as Pathogenic (★★★).

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 0.0000021 (0 hom.)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Pathogenic reviewed by expert panel P:23 U:1
• Conservation: PhyloP100: 4.68

Genes affected

MLH1 (HGNC:7127): (mutL homolog 1) The protein encoded by this gene can heterodimerize with mismatch repair endonuclease PMS2 to form MutL alpha, part of the DNA mismatch repair system. When MutL alpha is bound by MutS beta and some accessory proteins, the PMS2 subunit of MutL alpha introduces a single-strand break near DNA mismatches, providing an entry point for exonuclease degradation. The encoded protein is also involved in DNA damage signaling and can heterodimerize with DNA mismatch repair protein MLH3 to form MutL gamma, which is involved in meiosis. This gene was identified as a locus frequently mutated in hereditary nonpolyposis colon cancer (HNPCC). [provided by RefSeq, Aug 2017]

ACMG classification

Verdict is Pathogenic. Variant got 14 ACMG points.

• PM2: Very rare variant in population databases, with high coverage
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.96
• PP5: Variant 3-37048955-G-A is Pathogenic according to our data. Variant chr3-37048955-G-A is described in ClinVar as [Pathogenic]. Clinvar id is 17099.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-37048955-G-A is described in Lovd as [Pathogenic]. Variant chr3-37048955-G-A is described in Lovd as [Benign]. Variant chr3-37048955-G-A is described in Lovd as [Likely_pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
MLH1	NM_000249.4	c.2041G>A	p.Ala681Thr	missense_variant	18/19	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.2041G>A	p.Ala681Thr	missense_variant	18/19	1	NM_000249.4	ENSP00000231790	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome AF: 0.00000205 AC: 3 AN: 1461836 Hom.: 0 Cov.: 31 AF XY: 0.00000138 AC XY: 1 AN XY: 727226

Gnomad4 AFR exome AF: 0.00

Gnomad4 AMR exome AF: 0.0000224

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.00

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.00000180

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:23 Uncertain:1

Revision: reviewed by expert panel

Submissions by phenotype

not provided Pathogenic:8

Pathogenic, criteria provided, single submitter	clinical testing	Revvity Omics, Revvity	Sep 09, 2019	- -
Likely pathogenic, no assertion criteria provided	clinical testing	Department of Pathology and Laboratory Medicine, Sinai Health System	-	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. -
Likely pathogenic, criteria provided, single submitter	clinical testing	GeneKor MSA	Jan 01, 2020	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). -
Pathogenic, criteria provided, single submitter	clinical testing	Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital	Aug 15, 2023	- -
Pathogenic, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Jan 27, 2023	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. -
Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Dec 01, 2020	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) -
Pathogenic, no assertion criteria provided	clinical testing	Clinical Genetics DNA and cytogenetics Diagnostics Lab, Erasmus MC, Erasmus Medical Center	-	- -
Pathogenic, no assertion criteria provided	clinical testing	Joint Genome Diagnostic Labs from Nijmegen and Maastricht, Radboudumc and MUMC+	-	- -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:6

Pathogenic, criteria provided, single submitter	research	Foundation for Research in Genetics and Endocrinology, FRIGE's Institute of Human Genetics	Sep 01, 2021	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. -
Pathogenic, no assertion criteria provided	literature only	OMIM	Jan 01, 2006	- -
Likely pathogenic, no assertion criteria provided	clinical testing	Bioscientia Institut fuer Medizinische Diagnostik GmbH, Sonic Healthcare	-	- -
Pathogenic, criteria provided, single submitter	clinical testing	Counsyl	Jun 16, 2016	- -
Pathogenic, criteria provided, single submitter	clinical testing	Baylor Genetics	Jan 17, 2024	- -
Pathogenic, criteria provided, single submitter	clinical testing	Laboratorio de Genetica e Diagnostico Molecular, Hospital Israelita Albert Einstein	Jun 30, 2022	ACMG classification criteria: PS3 moderated, PS4 strong, PM2 moderated, PP1 strong, PP3 supporting -

Hereditary cancer-predisposing syndrome Pathogenic:3

Pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Apr 12, 2022	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. -
Pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Apr 22, 2020	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. -
Pathogenic, criteria provided, single submitter	curation	Sema4, Sema4	Jul 07, 2021	- -

Lynch syndrome Pathogenic:2

Pathogenic, criteria provided, single submitter	research	A.C.Camargo Cancer Center / LGBM, A.C.Camargo Cancer Center	Jan 30, 2019	- -
Pathogenic, reviewed by expert panel	research	International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	Sep 05, 2013	Multifactorial likelihood analysis posterior probability >0.99 -

Hereditary nonpolyposis colon cancer Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Women's Health and Genetics/Laboratory Corporation of America, LabCorp

Mar 01, 2021

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. -

Muir-Torré syndrome;C1333991:Colorectal cancer, hereditary nonpolyposis, type 2;C5399763:Mismatch repair cancer syndrome 1 Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Fulgent Genetics, Fulgent Genetics

Oct 31, 2018

- -

Muir-Torré syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

MGZ Medical Genetics Center

Jul 26, 2022

- -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Jan 15, 2024

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. -

not specified Uncertain:1

Uncertain significance, no assertion criteria provided

research

Mayo Clinic Laboratories, Mayo Clinic

-

- -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.74
BayesDel_addAF	Pathogenic	0.49	D
BayesDel_noAF	Pathogenic	0.47
CADD	Pathogenic	28
DANN	Pathogenic	1.0
DEOGEN2	Pathogenic	0.92	D;.;.;.;.;.
Eigen	Pathogenic	0.77
Eigen_PC	Pathogenic	0.77
FATHMM_MKL	Uncertain	0.86	D
LIST_S2	Benign	0.84	T;D;.;.;.;T
M_CAP	Pathogenic	0.42	D
MetaRNN	Pathogenic	0.96	D;D;D;D;D;D
MetaSVM	Pathogenic	1.0	D
MutationAssessor	Pathogenic	3.3	M;.;.;.;.;.
MutationTaster	Benign	0.98	A;A;A;A;A;A
PrimateAI	Uncertain	0.49	T
PROVEAN	Benign	-2.2	N;N;N;N;N;N
REVEL	Pathogenic	0.83
Sift	Uncertain	0.0030	D;D;D;D;D;D
Sift4G	Uncertain	0.0070	D;D;D;D;D;D
Polyphen		0.99	D;.;.;.;.;.
Vest4		0.87
MutPred		0.82		Gain of phosphorylation at A681 (P = 0.0412);.;.;.;.;.;
MVP		0.98
MPC		0.29
ClinPred		0.91	D
GERP RS		5.9
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7
Varity_R		0.72
gMVP		0.77

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.0		Details are displayed if max score is > 0.2

Find out detailed SpliceAI scores and Pangolin per-transcript scores at spliceailookup.broadinstitute.org

Publications

LitVar

Other links and lift over

dbSNP: rs63750217; hg19: chr3-37090446;