3-37048609-G-C

Variant names:

• chr3-37048609-G-C
• rs63751662
• NM_000249.4:c.1989G>C

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.1989G>C​(p.Glu663Asp) variant causes a missense, splice region change involving the alteration of a conserved nucleotide. The variant was absent in control chromosomes in GnomAD project. 3/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Likely pathogenic (★★). Another nucleotide change resulting in the same amino acid substitution has been previously reported as Pathogenic in ClinVar. Another variant affecting the same amino acid position, but resulting in a different missense (i.e. E663G) has been classified as Likely pathogenic.

• Frequency: Genomes: not found (cov: 31)
• Consequence: MLH1 NM_000249.4 missense, splice_region
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Pathogenic/Likely pathogenic criteria provided, multiple submitters, no conflicts P:3
• Conservation: PhyloP100: 7.39
• Publications: 19 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 7 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 2 benign, 43 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 chr3-37048608-A-G is described in ClinVar as Conflicting_classifications_of_pathogenicity. ClinVar VariationId is 89971.
• PP3: Splicing predictors support a deleterious effect. Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF, max_spliceai. No scorers claiming Uncertain. No scorers claiming Benign.
• PP5: Variant 3-37048609-G-C is Pathogenic according to our data. Variant chr3-37048609-G-C is described in ClinVar as Pathogenic/Likely_pathogenic. ClinVar VariationId is 639057.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars.

Variant Effect in Transcripts

ACMG analysis was done for transcript: NM_000249.4. You can select a different transcript below to see updated ACMG assignments.

RefSeq Transcripts

Selected	Gene	Transcript	Tags	HGVSc	HGVSp	Effect	Exon Rank	Protein	UniProt
	MLH1	NM_000249.4	MANE Select	c.1989G>C	p.Glu663Asp	missense splice_region	Exon 17 of 19	NP_000240.1
	MLH1	NM_001354629.2		c.1890G>C	p.Glu630Asp	missense splice_region	Exon 16 of 18	NP_001341558.1
	MLH1	NM_001354630.2		c.1824G>C	p.Glu608Asp	missense splice_region	Exon 16 of 18	NP_001341559.1

Ensembl Transcripts

Selected	Gene	Transcript	Tags	HGVSc	HGVSp	Effect	Exon Rank	Protein	UniProt
	MLH1	ENST00000231790.8	TSL:1 MANE Select	c.1989G>C	p.Glu663Asp	missense splice_region	Exon 17 of 19	ENSP00000231790.3
	MLH1	ENST00000458205.6	TSL:1	c.1266G>C	p.Glu422Asp	missense splice_region	Exon 18 of 20	ENSP00000402667.2
	MLH1	ENST00000432299.6	TSL:1	n.*1821G>C		splice_region non_coding_transcript_exon	Exon 15 of 17	ENSP00000416783.1

Frequencies

GnomAD3 genomes Cov.: 31

GnomAD4 exome Cov.: 30

GnomAD4 genome Cov.: 31

ClinVar

Significance: Pathogenic/Likely pathogenic

Submissions summary: Pathogenic:3

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Lynch syndrome Pathogenic:1

Jun 26, 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 glutamic acid with aspartic acid at codon 663 of the MLH1 protein. Computational prediction is inconclusive regarding the impact of this variant on protein structure and function (internally defined REVEL score threshold 0.5 < inconclusive < 0.7, PMID: 27666373). Splice site prediction tools suggest that this variant may have a significant impact on RNA splicing. To our knowledge, functional studies have not been reported for this variant. This variant has been reported in an individual affected with colorectal cancer that exhibited loss of MLH1 and PMS2 proteins by immunohistochemistry analyses (PMID: 33259954), and an individual affected with bile duct cancer (PMID: 29345684). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). A different nucleotide change resulting in the same missense amino acid change, c.1989G>T (p.Glu663Asp), has been reported in multiple individuals and families affected with hereditary non-polyposis colorectal cancer (PMID: 10480359, 16395668, 17510385, 18561205, 24278394), and RNA studies have shown that this variant results in cryptic donor site activation and exon 17 skipping (PMID: 10480359, 16395668, 18561205). The c.1989G>T (p.Glu663Asp) variant is considered to be disease-causing (ClinVar variation ID: 89980). Based on the available evidence, this variant is classified as Likely Pathogenic.

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Nov 06, 2021

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This missense change has been observed in individuals with clinical features of Lynch syndrome and/or bile duct cancer (PMID: 29345684; Invitae). It has also been observed to segregate with disease in related individuals. For these reasons, this variant has been classified as Pathogenic. This variant disrupts the c.1989G nucleotide in the MLH1 gene. Other variant(s) that disrupt this nucleotide have been determined to be pathogenic (PMID: 10480359, 16395668, 18561205, 24278394). This suggests that this nucleotide is clinically significant, and that variants that disrupt this position are likely to be disease-causing. Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site. Experimental studies are conflicting or provide insufficient evidence to determine the effect of this variant on MLH1 function (PMID: 17510385). Algorithms developed to predict the effect of missense changes on protein structure and function are either unavailable or do not agree on the potential impact of this missense change (SIFT: "Deleterious"; PolyPhen-2: "Benign"; Align-GVGD: "Class C35"). ClinVar contains an entry for this variant (Variation ID: 639057). This variant is not present in population databases (gnomAD no frequency). This sequence change replaces glutamic acid, which is acidic and polar, with aspartic acid, which is acidic and polar, at codon 663 of the MLH1 protein (p.Glu663Asp). This variant also falls at the last nucleotide of exon 17, which is part of the consensus splice site for this exon.

Hereditary cancer-predisposing syndrome Pathogenic:1

Jul 06, 2020

Ambry Genetics

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The p.E663D variant (also known as c.1989G>C), located in coding exon 17 of the MLH1 gene, results from a G to C substitution at nucleotide position 1989. The glutamic acid at codon 663 is replaced by aspartic acid, an amino acid with highly similar properties. However, this change occurs in the last base pair of coding exon 17, which makes it likely to have some effect on normal mRNA splicing. Using the BDGP and ESEfinder splice site prediction tools, this alteration is predicted to weaken the efficiency of the native splice donor site; however, direct evidence is unavailable. Another alteration at this position (c.1989G>T) was shown to cause an in-frame deletion of exon 17 based on several studies, and has been detected in multiple Lynch syndrome families (Wang Q et al. Hum. Genet. 1999 July;105:79-85; Auclair J et al. Hum. Mutat., 2006 Feb;27:145-54; De Lellis L et al. PLoS ONE, 2013 Nov;8:e81194; Magnani G et al. Gastroenterol Res Pract, 2015 Oct;2015:132190). MLH1 c.1989G>C was not reported in population-based cohorts in the Genome Aggregation Database (gnomAD). This amino acid position is highly conserved in available vertebrate species. Based on the majority of available evidence to date, this variant is likely to be pathogenic.

Computational scores

Source: dbNSFP v4.9

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.82
BayesDel_addAF	Pathogenic	0.45	D
BayesDel_noAF	Pathogenic	0.41
CADD	Pathogenic	35
DANN	Uncertain	0.99
DEOGEN2	Uncertain	0.73	D
Eigen	Benign	-0.067
Eigen_PC	Benign	0.033
FATHMM_MKL	Pathogenic	0.97	D
LIST_S2	Uncertain	0.95	D
M_CAP	Uncertain	0.13	D
MetaRNN	Uncertain	0.49	T
MetaSVM	Uncertain	0.15	D
MutationAssessor	Benign	1.8	L
PhyloP100		7.4
PrimateAI	Uncertain	0.77	T
PROVEAN	Benign	-2.3	N
REVEL	Pathogenic	0.67
Sift	Benign	0.066	T
Sift4G	Benign	0.12	T
Polyphen		0.094	B
Vest4		0.68
MutPred		0.39		Gain of sheet (P = 0.1451)
MVP		1.0
MPC		0.084
ClinPred		0.83	D
GERP RS		3.7
Varity_R		0.58
gMVP		0.67
Mutation Taster		=0/100	disease causing (ClinVar)

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.94
SpliceAI score (max)		0.95		Details are displayed if max score is > 0.2
DS_DG_spliceai		0.95		Position offset: 31
DS_DL_spliceai		0.66		Position offset: 0

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

Other links and lift over

dbSNP: rs63751662; hg19: chr3-37090100;