3-36996706-C-G

Variant summary

Our verdict is Pathogenic. Variant got 10 ACMG points: 10P and 0B. PM1 PM2 PM5 PP3_Strong

The NM_000249.4(MLH1):c.204C>G(p.Ile68Met) variant causes a missense change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.00000274 in 1,458,640 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 Uncertain significance (★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. I68N) has been classified as Likely pathogenic.

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 0.0000027 (0 hom.)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Uncertain significance criteria provided, multiple submitters, no conflicts U:7
• Conservation: PhyloP100: 0.261

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 10 ACMG points.

• PM1: In a hotspot region, there are 6 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 0 benign, 15 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 chr3-36996705-T-A is described in ClinVar as [Likely_pathogenic]. Clinvar id is 90008.Status of the report is reviewed_by_expert_panel, 3 stars.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.977

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.204C>G	p.Ile68Met	missense_variant	2/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.204C>G	p.Ile68Met	missense_variant	2/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD3 exomes AF: 0.00000398 AC: 1 AN: 251396 Hom.: 0 AF XY: 0.00000736 AC XY: 1 AN XY: 135872

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.00

Gnomad SAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.00000880

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.00000274 AC: 4 AN: 1458640 Hom.: 0 Cov.: 30 AF XY: 0.00000551 AC XY: 4 AN XY: 725930

Gnomad4 AFR exome AF: 0.00

Gnomad4 AMR exome AF: 0.00

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: 0.00000361

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 32

ExAC AF: 0.00000824 AC: 1

EpiCase AF: 0.000109

EpiControl AF: 0.0000593

ClinVar

Significance: Uncertain significance

Submissions summary: Uncertain:7

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Lynch syndrome Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Apr 03, 2023	This missense variant replaces isoleucine with methionine at codon 68 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). To our knowledge, functional studies have not been reported for this variant. This variant has been reported in an individual affected with colorectal cancer but without the clinical features of Lynch syndrome (PMID: 25435955). This variant has been identified in 1/251396 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Different variants affecting the same codon, c.203T>G (p.Ile68Ser) and c.203T>A (p.Ile68Asn), are considered to be disease-causing (ClinVar variation ID: 820585, 90008), suggesting that this position is important for the protein function. The available evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance. -
Uncertain significance, criteria provided, single submitter	clinical testing	Mendelics	Jul 02, 2018	- -

not provided Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Oct 31, 2022	The frequency of this variant in the general population, 0.000004 (1/251396 chromosomes, http://gnomad.broadinstitute.org), is uninformative in assessment of its pathogenicity. In the published literature, the variant has been reported in individuals with rectal cancer (PMID: 25435955 (2015)) and thyroid cancer (PMID: 25576899 (2015)). 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 benign. Based on the available information, we are unable to determine the clinical significance of this variant. -
Uncertain significance, criteria provided, single submitter	clinical testing	GeneDx	Dec 01, 2022	Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; Observed in individuals with colorectal cancer (Vodicka et al., 2015); This variant is associated with the following publications: (PMID: 25576899, 22753075, 16083711, 21120944, 25435955) -

Hereditary cancer-predisposing syndrome Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Mar 17, 2023	This missense variant replaces isoleucine with methionine at codon 68 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). To our knowledge, functional studies have not been reported for this variant. This variant has been reported in an individual affected with colorectal cancer but without the clinical features of Lynch syndrome (PMID: 25435955). This variant has been identified in 1/251396 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Different variants affecting the same codon, c.203T>G (p.Ile68Ser) and c.203T>A (p.Ile68Asn), are considered to be disease-causing (ClinVar variation ID: 820585, 90008), suggesting that this position is important for the protein function. The available evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance. -
Uncertain significance, criteria provided, single submitter	clinical testing	Ambry Genetics	May 01, 2023	The p.I68M variant (also known as c.204C>G), located in coding exon 2 of the MLH1 gene, results from a C to G substitution at nucleotide position 204. The isoleucine at codon 68 is replaced by methionine, an amino acid with highly similar properties. This alteration has been reported in both germline and tumor DNA of a 59 year old female with rectal cancer; however, the expression of MLH1 was not altered in the tumor. Authors also report that the amino acid residue 68 is located within the enzymatic core on the MLH1 protein which interacts with ATP; they conclude that a substitution by methionine may decrease MLH1 activity (Vodicka P et al, Oncol Lett 2015 Jan; 9(1):183-186). This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Since supporting evidence is limited at this time, the clinical significance of this alteration remains unclear. -

Hereditary nonpolyposis colorectal neoplasms Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Invitae

Jan 21, 2024

This sequence change replaces isoleucine, which is neutral and non-polar, with methionine, which is neutral and non-polar, at codon 68 of the MLH1 protein (p.Ile68Met). This variant is present in population databases (rs780141938, gnomAD 0.0009%). This missense change has been observed in individual(s) with colorectal cancer (PMID: 25435955). ClinVar contains an entry for this variant (Variation ID: 486852). 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) has been performed at Invitae for this missense variant, however the output from this modeling did not meet the statistical confidence thresholds required to predict the impact of this variant on MLH1 protein function. This variant disrupts the p.Ile68 amino acid residue in MLH1. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 10082584, 11304573, 11555625, 12810663, 14961575, 17510385, 24362816). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.72
BayesDel_addAF	Pathogenic	0.39	D
BayesDel_noAF	Pathogenic	0.32
Cadd	Uncertain	23
Dann	Uncertain	1.0
DEOGEN2	Uncertain	0.79	D
Eigen	Benign	0.11
Eigen_PC	Benign	0.14
FATHMM_MKL	Uncertain	0.85	D
LIST_S2	Pathogenic	0.99	D
M_CAP	Pathogenic	0.42	D
MetaRNN	Pathogenic	0.98	D
MetaSVM	Benign	-0.78	T
MutationAssessor	Benign	2.0	M
MutationTaster	Benign	1.0	D;D;D;D;D;D
PrimateAI	Uncertain	0.79	T
PROVEAN	Uncertain	-2.7	D
REVEL	Pathogenic	0.81
Sift	Pathogenic	0.0	D
Sift4G	Pathogenic	0.0	D
Polyphen		1.0	D
Vest4		0.81
MutPred		0.90		Loss of catalytic residue at L73 (P = 0.039);
MVP		0.96
MPC		0.41
ClinPred		0.96	D
GERP RS		3.1
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.1
Varity_R		0.96
gMVP		0.84

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: rs780141938; hg19: chr3-37038197;