3-37048609-G-T

Position:

• chr3-37048609-G-T

Variant summary

Our verdict is Pathogenic. Variant got 14 ACMG points: 14P and 0B. PM1 PM2 PM5 PP5_Very_Strong

The ENST00000231790.8(MLH1):​c.1989G>T​(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 Pathogenic (★★★). Another nucleotide change resulting in same amino acid change has been previously reported as Likely pathogenicin ClinVar. Another variant affecting the same amino acid position, but resulting in a different missense (i.e. E663A) has been classified as Uncertain significance.

• Frequency: Genomes: not found (cov: 31)
• Consequence: MLH1 ENST00000231790.8 missense, splice_region
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Pathogenic reviewed by expert panel P:7
• Conservation: PhyloP100: 7.39

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.

• PM1: In a hotspot region, there are 5 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 2 benign, 16 uncertain in ENST00000231790.8
• 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 id is 89971.We mark this variant Likely_pathogenic, oryginal submissions are: {Pathogenic=2, Uncertain_significance=1}.
• PP5: Variant 3-37048609-G-T is Pathogenic according to our data. Variant chr3-37048609-G-T is described in ClinVar as [Pathogenic]. Clinvar id is 89980.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-37048609-G-T is described in Lovd as [Pathogenic]. Variant chr3-37048609-G-T is described in Lovd as [Likely_pathogenic]. Variant chr3-37048609-G-T is described in Lovd as [Benign].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
MLH1	NM_000249.4	c.1989G>T	p.Glu663Asp	missense_variant, splice_region_variant	17/19	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.1989G>T	p.Glu663Asp	missense_variant, splice_region_variant	17/19	1	NM_000249.4	ENSP00000231790	P1

Frequencies

GnomAD3 genomes Cov.: 31

GnomAD4 exome Cov.: 30

GnomAD4 genome Cov.: 31

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:7

Revision: reviewed by expert panel

Submissions by phenotype

Lynch syndrome Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Aug 15, 2023	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). RNA studies using patient RNA have shown that this variant causes in-frame skipping of exon 17 (PMID: 16395668, 18561205). This exon contains several domains including the PMS2/MLH3/PMS1 and Exo1 interacting domains and the NES domain (PMID: 10037723, 11427529, 15754314). A functional study demonstrated this variant had 68.5% mismatch repair activity compared to wild type (PMID: 17510385). This variant has been reported in individuals affected with Lynch syndrome and Lynch syndrome-associated disease (PMID: 10480359, 17510385, 18561205, 24278394, 33259954). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Loss of MLH1 function is a known mechanism of disease. Therefore, this variant is classified as Pathogenic. -
Pathogenic, reviewed by expert panel	research	International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	Sep 05, 2013	Variant causes splicing aberration, interrupting functional domain (full inactivation of variant allele) -

not provided Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Jan 03, 2018	This variant is denoted MLH1 c.1989G>T at the cDNA level. Located in the last nucleotide of exon 17, this variant is predicted by multiple slicing based models to damage the natural splice donor site and cause abnormal splicing. Both Auclair et al. (2006) and Tournier et al. (2008) confirm, via RNA based studies, that MLH1 c.1989G>T causes skipping of exon 17 by activating a cryptic donor splice site. The resulting skipping of exon 17 is expected to be in-frame, however, this interrupts the domain of interaction with PMS2/MLH3/PMS1 (Raevaara 2005). This variant has been reported in multiple individuals with colon and/or other Lynch syndrome-related cancers, many of whose tumors are reported to show absence of the MLH1 protein via mismatch repair immunohistochemistry (MMR IHC) and/or have been found to be microsatellite (MSI) unstable (Wang 1999 Bonadona 2011, Hardt 2011, De Lellis 2013, Magnani 2015). The International Society for Gastrointestinal Hereditary Tumours Incorporated (InSiGHT) classifies this variant as pathogenic (Thompson 2014). Although the nucleotide substitution results in the change of a Glutamic acid to an Aspartic acid at codon 663, and is called Glu663Asp in the literature, we are using only the nucleotide nomenclature to refer to the variant since the defect is determined to be one of splicing rather than a resulting missense variant. MLH1 c.1989G>T was not observed in large population cohorts (Lek 2016). The nucleotide which is altered, a guanine (G) at base 1989, is conserved across species. Based on the current evidence, we consider this variant to be pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Jan 17, 2017	- -

Carcinoma of colon Pathogenic:1

Likely pathogenic, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

-

The MLH1 p.Glu663Asp variant was identified in 2 of 496 proband chromosomes (frequency: 0.004) from individuals with Lynch syndrome (Auclair 2006, Hardt 2011). This variant was also identified dbSNP (ID: rs63751662) “With untested allele”, HGMD, UMD (15X as a causal variant), “Mismatch Repair Variants Database”, “InSIGHT Colon Cancer Database” and “MMR Gene Unclassified Variants Database”. The p.Glu663 residue is conserved in mammals and lower organisms; however, computational analyses (PolyPhen2, SIFT, AlignGVGD, BLOSUM) provide inconsistent predictions regarding the impact to the protein and this information is not very predictive of pathogenicity. The variant occurs at the last base of the exon, a position which has been shown to be part of the splicing consensus sequence, and variants involving this position sometimes affect splicing. In-silico or computational prediction software (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) predicts a greater than 10% difference in splicing in 2 of 5 different programs. Auclair (2006) and Tournier (2008) determined that the variant is associated with aberrant splicing using both ex vivo functional splicing assays and in vivo analysis of patient RNA transcripts. These studies showed that the variant resulted in exon 17 skipping and cryptic donor splice site activation. However, a study by Takahasi (2007) determined that MLH1 expression of the variant was >75% that of wild type, while assays evaluating dominant mutator effect and mismatch repair activity of the variant yielded inconclusive results. In summary, based on the above information, the clinical significance of this variant cannot be determined with certainty at this time although we would lean towards a more pathogenic role for this variant. This variant is classified as predicted pathogenic. -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Jan 16, 2024

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). RNA analysis indicates that this missense change induces altered splicing and likely results in a shortened protein product. This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individuals with Lynch syndrome (PMID: 10480359, 16395668, 21404117, 21642682, 24278394). ClinVar contains an entry for this variant (Variation ID: 89980). An algorithm developed to predict the effect of missense changes on protein structure and function (PolyPhen-2) suggests that this variant is likely to be tolerated. Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this missense change results in skipping of exon 17, but is expected to preserve the integrity of the reading-frame (PMID: 10480359, 16395668, 18561205; Invitae). For these reasons, this variant has been classified as Pathogenic. -

Hereditary cancer-predisposing syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Nov 21, 2023

The c.1989G>T pathogenic mutation (also known as p.E663D), located in coding exon 17 of the MLH1 gene, results from a G to T substitution at nucleotide position 1989. The amino acid change results in glutamic acid to aspartic acid at codon 663, 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. This alteration has been identified in multiple individuals who met Bethesda and/or Amsterdam criteria for HNPCC/Lynch syndrome and several of these individuals had loss of MLH1/PMS2 staining in their tumors on immunohistochemistry (Ambry internal data; Wang Q et al. Hum. Genet. 1999 July;105:79-85; Hardt K et al. Fam. Cancer 2011 Jun;10:273-84; Magnani G et al. Gastroenterol Res Pract 2015 Oct;2015:132190; De Lellis L et al. PLoS ONE 2013 Nov;8:e81194). In silico splice site analysis predicts that this alteration may weaken the native splice donor site and will result in the creation or strengthening of a novel splice donor site. Functional RNA studies have reported cryptic donor site activation and/or in-frame deletion of exon 17 for this mutation based on RT-PCR using patient RNA or minigene assay (Wang Q et al. Hum. Genet. 1999 July;105:79-85; Auclair J et al. Hum Mutat, 2006 Feb;27:145-54; Tournier I et al. Hum Mutat, 2008 Dec;29:1412-24; Ambry internal data). Of note, this alteration is designated as "663 GAG>GAT" and p.Glu663Asp in published literature. Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation. -

Computational scores

Source: dbNSFP v4.3

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;D;.;.;.;D
M_CAP	Uncertain	0.13	D
MetaRNN	Uncertain	0.49	T;T;T;T;T;T
MetaSVM	Uncertain	0.15	D
MutationAssessor	Benign	1.8	L;.;.;.;.;.
MutationTaster	Benign	1.0	D;D;D;D;D;D
PrimateAI	Uncertain	0.77	T
PROVEAN	Benign	-2.3	N;N;N;N;N;N
REVEL	Pathogenic	0.67
Sift	Benign	0.066	T;D;D;D;D;T
Sift4G	Benign	0.12	T;T;T;T;T;T
Polyphen		0.094	B;.;.;.;.;.
Vest4		0.68
MutPred		0.39		Gain of sheet (P = 0.1451);.;.;.;.;.;
MVP		1.0
MPC		0.084
ClinPred		0.82	D
GERP RS		3.7
Varity_R		0.58
gMVP		0.67

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.97
SpliceAI score (max)		0.92		Details are displayed if max score is > 0.2
DS_DG_spliceai		0.92		Position offset: 31
DS_DL_spliceai		0.39		Position offset: 0

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

Publications

LitVar

Other links and lift over

dbSNP: rs63751662; hg19: chr3-37090100;