NM_000249.4:c.114C>G

Variant names:

• chr3-36993661-C-G
• rs267607706
• NM_000249.4:c.114C>G

Variant summary

Our verdict is Pathogenic. The variant received 18 ACMG points: 18P and 0B. PM1 PM2 PM5 PP3_Strong PP5_Very_Strong

The NM_000249.4(MLH1):​c.114C>G​(p.Asn38Lys) variant causes a missense, splice region change involving the alteration of a non-conserved nucleotide. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a pathogenic outcome for this variant. 2/3 splice prediction tools predict no significant impact on normal splicing. Variant has been reported in ClinVar as Pathogenic (★★★). Another nucleotide change resulting in the same amino acid substitution has been previously reported as Likely pathogenic in ClinVar. Another variant affecting the same amino acid position, but resulting in a different missense (i.e. N38T) has been classified as Likely pathogenic.

• Frequency: Genomes: not found (cov: 33)
• Consequence: MLH1 NM_000249.4 missense, splice_region
• Scores: Splicing: ADA: 0.1989
• Clinical Significance: Pathogenic reviewed by expert panel P:5
• Conservation: PhyloP100: 2.48
• Publications: 7 publications found

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]

MLH1 Gene-Disease associations (from GenCC):

• Lynch syndrome

  Inheritance: AD Classification: DEFINITIVE, SUPPORTIVE Submitted by: G2P, ClinGen, Orphanet
• Lynch syndrome 2

  Inheritance: AD Classification: DEFINITIVE, STRONG Submitted by: Ambry Genetics, Genomics England PanelApp
• Muir-Torre syndrome

  Inheritance: AD Classification: DEFINITIVE, STRONG, MODERATE, SUPPORTIVE Submitted by: Genomics England PanelApp, Ambry Genetics, G2P, Orphanet
• mismatch repair cancer syndrome 1

  Inheritance: AR Classification: DEFINITIVE, STRONG, SUPPORTIVE Submitted by: Ambry Genetics, G2P, Labcorp Genetics (formerly Invitae), Orphanet, ClinGen
• Lynch syndrome 1

  Inheritance: AD Classification: STRONG Submitted by: Labcorp Genetics (formerly Invitae)
• ovarian cancer

  Inheritance: AD Classification: STRONG Submitted by: Genomics England PanelApp
• malignant pancreatic neoplasm

  Inheritance: AD Classification: MODERATE Submitted by: Genomics England PanelApp
• rhabdomyosarcoma

  Inheritance: AR Classification: MODERATE Submitted by: Genomics England PanelApp
• prostate cancer

  Inheritance: AD Classification: LIMITED Submitted by: Ambry Genetics
• breast cancer

  Inheritance: AD Classification: NO_KNOWN Submitted by: Ambry Genetics
• hereditary breast carcinoma

  Inheritance: AD Classification: NO_KNOWN Submitted by: ClinGen

ACMG classification

Our verdict: Pathogenic. The variant received 18 ACMG points.

• PM1: In a hotspot region, there are 22 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 1 benign, 50 uncertain in NM_000249.4
• PM2: Very rare variant in population databases, with high coverage
• PM5: Other missense variant is known to change same aminoacid residue: Variant chr3-36993660-A-C is described in ClinVar as Conflicting_classifications_of_pathogenicity. ClinVar VariationId is 619503.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.995
• PP5: Variant 3-36993661-C-G is Pathogenic according to our data. Variant chr3-36993661-C-G is described in ClinVar as Pathogenic. ClinVar VariationId is 89651.Status of the report is reviewed_by_expert_panel, 3 stars.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	MANE	Protein	UniProt
MLH1	NM_000249.4	c.114C>G	p.Asn38Lys	missense_variant, splice_region_variant	Exon 1 of 19	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.114C>G	p.Asn38Lys	missense_variant, splice_region_variant	Exon 1 of 19	1	NM_000249.4	ENSP00000231790.3

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 33

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:5

Revision: reviewed by expert panel

Submissions by phenotype

Hereditary cancer-predisposing syndrome Pathogenic:2

Sep 24, 2020

Color Diagnostics, LLC DBA Color Health

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This missense variant replaces asparagine with lysine at codon 38 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). Different variants affecting the same position (p.Asn38His, p.Asn38Ser, p.Asn38Thr) are considered to be disease-causing (ClinVar variation ID: 89645, 89648, 619503), suggesting that asparagine at this position is important for protein structure and function. Splice site prediction tools suggest that this variant may not impact RNA splicing. Functional studies have shown that this variant is abnormal for complementation of mismatch repair activity in MLH1-deficient HCT116 nuclear extract (PMID: 20020535) and the expression of the variant in mouse cells resulted in DNA damage tolerance (PMID: 31784484). This variant has been reported in an individual and a family affected with Lynch syndrome-associated cancer who met Bethesda and Amsterdam I criteria (PMID: 20020535, 18415027, InSiGHT database). The variant segregates with disease in the carrier family with segregation likelihood ratio of 95:1 or LOD score of approximately 2 (InSiGHT database). 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 Likely Pathogenic. -

Jun 07, 2018

Ambry Genetics

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The p.N38K pathogenic mutation (also known as c.114C>G), located in coding exon 1 of the MLH1 gene, results from a C to G substitution at nucleotide position 114. The asparagine at codon 38 is replaced by lysine, an amino acid with similar properties. One in vitro assay indicated that when compared to wild type MLH1, this alteration lead to a significant decrease in mismatch repair efficiency (Drost M et al. Hum. Mutat. 2010 Mar;31:247-53). Another study found that this alteration localized to the nucleus and retained interaction with PMS2 through an in vitro GST pull-down assay (Andersen SD et al. Hum. Mutat. 2012 Dec;33:1647-55). This mutation was identified in an individual with colon cancer diagnosed at 36 whose tumor exhibited microsatellite instability and copy-neutral loss of heterozygosity (van Puijenbroek M et al. Fam. Cancer 2008 Apr;7:319-30) and was detected in a patient with colon and endometrial cancers diagnosed before age 50 where the tumor tested was MSI-H and demonstrated isolated loss of PMS2 by IHC analysis (Ambry internal data). 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/]). This variant was not reported in population-based cohorts in the Genome Aggregation Database (gnomAD). This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. In addition, this alteration is predicted to be deleterious by MAPP-MMR in silico analyses (Chao EC et al. Hum. Mutat. 2008 Jun;29:852-60). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation. -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:1

Jul 10, 2023

Myriad Genetics, Inc.

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant is considered likely pathogenic. Functional studies indicate this variant impacts protein function [PMID: 20020535]. This variant is expected to disrupt protein structure [Myriad internal data]. -

Lynch syndrome Pathogenic:1

Sep 05, 2013

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Significance: Pathogenic

Review Status: reviewed by expert panel

Collection Method: research

Multifactorial likelihood analysis posterior probability >0.99 -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Aug 22, 2022

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

In summary, the currently available evidence indicates that the variant is pathogenic, but additional data are needed to prove that conclusively. Therefore, this variant has been classified as Likely Pathogenic. This variant disrupts the p.Asn38 amino acid residue in MLH1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 12373605, 15713769, 20704743, 26895986, 27435373, 28514183). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site. Experimental studies have shown that this missense change affects MLH1 function (PMID: 20020535, 22753075). Based on a multifactorial likelihood algorithm using genetic, in silico, and/or statistical data, this variant has been determined to have a high probability of being pathogenic (PMID: 24362816). ClinVar contains an entry for this variant (Variation ID: 89651). This missense change has been observed in individual(s) with clinical features of Lynch syndrome (PMID: 18415027). This variant is not present in population databases (gnomAD no frequency). This sequence change replaces asparagine, which is neutral and polar, with lysine, which is basic and polar, at codon 38 of the MLH1 protein (p.Asn38Lys). -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	1.0
BayesDel_addAF	Pathogenic	0.37	D
BayesDel_noAF	Pathogenic	0.30
CADD	Pathogenic	28
DANN	Uncertain	1.0
DEOGEN2	Pathogenic	0.97	D
Eigen	Pathogenic	0.83
Eigen_PC	Pathogenic	0.67
FATHMM_MKL	Uncertain	0.94	D
LIST_S2	Pathogenic	0.98	D
M_CAP	Pathogenic	0.84	D
MetaRNN	Pathogenic	1.0	D
MetaSVM	Uncertain	0.62	D
MutationAssessor	Pathogenic	5.0	H
PhyloP100		2.5
PrimateAI	Pathogenic	0.82	D
PROVEAN	Pathogenic	-4.8	D
REVEL	Pathogenic	0.89
Sift	Pathogenic	0.0	D
Sift4G	Pathogenic	0.0	D
Polyphen		1.0	D
Vest4		0.98
MutPred		0.97		Gain of methylation at N38 (P = 0.0088);
MVP		0.97
MPC		0.43
ClinPred		1.0	D
GERP RS		4.2
PromoterAI		-0.084	Neutral
Varity_R		0.97
gMVP		0.96
Mutation Taster		=4/96	disease causing (ClinVar)

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Benign	0.20
dbscSNV1_RF	Benign	0.41
SpliceAI score (max)		0.22		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.22		Position offset: 2

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

Other links and lift over

dbSNP: rs267607706; hg19: chr3-37035152;