3-37048563-C-T

Variant names:

• chr3-37048563-C-T
• rs63750610
• NM_000249.4:c.1943C>T

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.1943C>T​(p.Pro648Leu) variant causes a missense change. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a pathogenic outcome for this variant. Variant has been reported in ClinVar as Likely pathogenic (★★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. P648A) has been classified as Likely pathogenic.

• Frequency: Genomes: not found (cov: 31)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Likely pathogenic reviewed by expert panel P:8
• Conservation: PhyloP100: 5.88
• Publications: 20 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 13 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 4 benign, 31 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 chr3-37048562-C-G is described in ClinVar as Conflicting_classifications_of_pathogenicity. ClinVar VariationId is 525804.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.96
• PP5: Variant 3-37048563-C-T is Pathogenic according to our data. Variant chr3-37048563-C-T is described in ClinVar as Likely_pathogenic. ClinVar VariationId is 89953.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.1943C>T	p.Pro648Leu	missense_variant	Exon 17 of 19	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.1943C>T	p.Pro648Leu	missense_variant	Exon 17 of 19	1	NM_000249.4	ENSP00000231790.3

Frequencies

GnomAD3 genomes Cov.: 31

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 31

Alfa AF: 0.00000535 Hom.: 0

ClinVar

Significance: Likely pathogenic

Submissions summary: Pathogenic:8

Revision: reviewed by expert panel

Submissions by phenotype

not provided Pathogenic:2

Feb 26, 2021

Quest Diagnostics Nichols Institute San Juan Capistrano

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The variant has been reported in several symptomatic Lynch Syndrome patients in the published literature (PMIDs: 11726306 (2001), 16083711 (2005), 21404117 (2011), 25871621 (2015), and 27601186 (2016)). Functional studies demonstrated decreased protein expression, localization, and MMR function (PMIDs: 16083711 (2005), 17510385 (2007), 20533529 (2010), 21404117 (2011), and 22753075 (2012)). This variant has not been reported in large, multi-ethnic general populations (http://gnomad.broadinstitute.org) and is located in a potentially critical protein domain. Variants affecting the same amino acid position have been described as pathogenic. Based on the available information, this variant is classified as pathogenic. -

Oct 13, 2016

Clinical Genetics and Genomics, Karolinska University Hospital

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Hereditary cancer-predisposing syndrome Pathogenic:2

Jul 19, 2022

Ambry Genetics

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The p.P648L pathogenic mutation (also known as c.1943C>T) is located in exon 17 of the MLH1 gene. This alteration results from a C to T substitution at nucleotide position 1943. The proline at codon 648 is replaced by leucine, an amino acid with similar properties. This alteration was detected in an individual diagnosed with microsatellite unstable colorectal cancer at 43 (Raevaara et al. Gastroenterology 2005 Aug; 129(2): 537-49) as well as in cohorts of Lynch syndrome patients and families (Lagerstedt-Robinson K et al. Oncol. Rep., 2016 Nov;36:2823-2835). (Dudley B et al. Am. J. Surg. Pathol., 2015 Aug;39:1114-20). Functional studies demonstrated decreased protein expression and localization, but intact MMR function. In another study, this alteration showed a negative dominant mutator effect in yeast with MMR activity of 39.2% and MLH1 expression of 25-75% compared to wild type (Takahashi et al. Cancer Res 2007 May 15; 67(10): 4595-604). Another alteration in the same codon, p.P648S, has also been identified in HNPCC/Lynch syndrome families and shown to disrupt interaction between MLH1 and PMS2 in vitro (Bisgaard et al. Hum Mut 2002 Jul; 20(1): 20-7; Raevaara et al. Genes Chromosomes Cancer 2004 Jul; 40(3): 261-5; Belvederesi et al. Eur J Hum Genet 2006 Jul; 14(7): 853-9; Ou et al. Hum Mut 2007 Nov; 28(11): 1047-54). 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 B et al. Hum Mutat. 2013 Jan;34(1):200-9; Thompson B et al. Nat Genet. 2014 Feb;46(2):107-15; available at [www.insight-group.org/variants/classifications/]). Based on protein sequence alignment, this amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation. -

Aug 23, 2023

Color Diagnostics, LLC DBA Color Health

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This missense variant replaces proline with leucine at codon 648 of the MLH1 protein. The reference amino acid proline 648 is conserved across species (PMID: 12519945, 22908213) and computational prediction suggests that this variant may have deleterious impact on protein structure and function (internally defined REVEL score threshold >= 0.7, PMID: 27666373). Multiple functional studies have shown that this variant causes reduced MLH1 protein expression and decreased binding to PMS2, although the variant impact on MMR activity is inconclusive (PMID: 16083711, 17510385, 20020535, 20533529, 21404117, 22753075, 23403630). This variant has been reported in three individuals from two families affected with colorectal cancer whose tumors showed features of loss of DNA mismatch repair function (PMID: 16083711, 20020535, 21404117, 23403630, 25871621), and it has been observed in an additional individual with suspected Lynch syndrome (PMID: 27601186). Different variants affecting the same position (p.Pro648Ser and p.Pro648Arg) are considered to be disease-causing (ClinVar variation ID: 17097, 479696) suggesting that this position is important for the protein function. 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. -

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

Feb 22, 2016

Division of Human Genetics, Children's Hospital of Philadelphia

Significance: Likely pathogenic

Review Status: no assertion criteria provided

Collection Method: research

- -

Lynch syndrome 1 Pathogenic:1

Jun 13, 2018

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Significance: Likely pathogenic

Review Status: reviewed by expert panel

Collection Method: curation

Variant reclassification due to new tumour Likelihood Ratios: Multifactorial likelihood analysis posterior probability > 0.95 (0.989) -

Lynch syndrome Pathogenic:1

Oct 02, 2023

All of Us Research Program, National Institutes of Health

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This missense variant replaces proline with leucine at codon 648 of the MLH1 protein. The reference amino acid proline 648 is conserved across species (PMID: 12519945, 22908213) and computational prediction suggests that this variant may have deleterious impact on protein structure and function (internally defined REVEL score threshold >= 0.7, PMID: 27666373). Multiple functional studies have shown that this variant causes reduced MLH1 protein expression and decreased binding to PMS2, although the variant impact on MMR activity is inconclusive (PMID: 16083711, 17510385, 20020535, 20533529, 21404117, 22753075, 23403630). This variant has been reported in three individuals from two families affected with colorectal cancer whose tumors showed features of loss of DNA mismatch repair function (PMID: 16083711, 20020535, 21404117, 23403630, 25871621), and it has been observed in an additional individual with suspected Lynch syndrome (PMID: 27601186). Different variants affecting the same position (p.Pro648Ser and p.Pro648Arg) are considered to be disease-causing (ClinVar variation ID: 17097, 479696) suggesting that this position is important for the protein function. 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. -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Jan 22, 2023

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

For these reasons, this variant has been classified as Pathogenic. This variant disrupts the p.Pro648 amino acid residue in MLH1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 15139004, 16083711, 16724012, 18307539, 21404117). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. Experimental studies have shown that this missense change affects MLH1 function (PMID: 16083711, 17510385, 20533529, 21404117, 22753075). 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. ClinVar contains an entry for this variant (Variation ID: 89953). This missense change has been observed in individual(s) with Lynch syndrome (PMID: 11726306, 16083711, 21404117; Invitae). It has also been observed to segregate with disease in related individuals. This variant is not present in population databases (gnomAD no frequency). This sequence change replaces proline, which is neutral and non-polar, with leucine, which is neutral and non-polar, at codon 648 of the MLH1 protein (p.Pro648Leu). -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.69
BayesDel_addAF	Pathogenic	0.49	D
BayesDel_noAF	Pathogenic	0.47
CADD	Uncertain	25
DANN	Uncertain	1.0
DEOGEN2	Pathogenic	0.94	D;.;.;.;.;.
Eigen	Pathogenic	1.0
Eigen_PC	Pathogenic	0.94
FATHMM_MKL	Pathogenic	0.98	D
LIST_S2	Uncertain	0.95	D;D;.;.;.;D
M_CAP	Pathogenic	0.38	D
MetaRNN	Pathogenic	0.96	D;D;D;D;D;D
MetaSVM	Pathogenic	1.0	D
MutationAssessor	Pathogenic	3.4	M;.;.;.;.;.
PhyloP100		5.9
PrimateAI	Uncertain	0.70	T
PROVEAN	Pathogenic	-9.4	D;D;D;D;D;D
REVEL	Pathogenic	0.94
Sift	Pathogenic	0.0	D;D;D;D;D;D
Sift4G	Pathogenic	0.0	D;D;D;D;D;D
Polyphen		1.0	D;.;.;.;.;.
Vest4		0.89
MutPred		0.89		Gain of helix (P = 0.062);.;.;.;.;.;
MVP		0.97
MPC		0.41
ClinPred		1.0	D
GERP RS		5.8
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7
Varity_R		0.95
gMVP		0.93
Mutation Taster		=0/100	disease causing (ClinVar)

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.010		Details are displayed if max score is > 0.2

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

Other links and lift over

dbSNP: rs63750610; hg19: chr3-37090054; COSMIC: COSV51620534;