3-37048555-C-G

Position:

• chr3-37048555-C-G

Variant summary

Our verdict is Uncertain significance. Variant got 3 ACMG points: 4P and 1B. PM1 PM2 BP4

The NM_000249.4(MLH1):​c.1935C>G​(p.Asn645Lys) variant causes a missense change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.00000137 in 1,461,766 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a benign outcome for this variant. 13/21 in silico tools predict a benign outcome for this variant. Variant has been reported in ClinVar as Uncertain significance (★★). Another nucleotide change resulting in same amino acid change has been previously reported as Uncertain significancein ClinVar. Another variant affecting the same amino acid position, but resulting in a different missense (i.e. N645S) has been classified as Uncertain significance.

• Frequency:
  • Genomes: not found (cov: 31)
  • Exomes 𝑓: 0.0000014 (0 hom.)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Uncertain significance criteria provided, multiple submitters, no conflicts U:5
• Conservation: PhyloP100: 0.781

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 Uncertain_significance. Variant got 3 ACMG points.

• PM1: In a hotspot region, there are 6 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 2 benign, 14 uncertain in NM_000249.4
• PM2: Very rare variant in population databases, with high coverage
• BP4: Computational evidence support a benign effect (MetaRNN=0.41658816).

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.1935C>G	p.Asn645Lys	missense_variant	17/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.1935C>G	p.Asn645Lys	missense_variant	17/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 31

GnomAD4 exome AF: 0.00000137 AC: 2 AN: 1461766 Hom.: 0 Cov.: 31 AF XY: 0.00000138 AC XY: 1 AN XY: 727194

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: 0.00000180

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 31

Alfa AF: 0.0000468 Hom.: 0

ClinVar

Significance: Uncertain significance

Submissions summary: Uncertain:5

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Hereditary cancer-predisposing syndrome Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Aug 09, 2023	This missense variant replaces asparagine with lysine at codon 645 of the MLH1 protein. Computational prediction suggests that this variant may not impact protein structure and function (internally defined REVEL score threshold <= 0.5, PMID: 27666373). To our knowledge, functional studies have not been reported for this variant. This variant has not been reported in individuals affected with MLH1-related disorders in the literature. This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). 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	Sep 11, 2023	The p.N645K variant (also known as c.1935C>G), located in coding exon 17 of the MLH1 gene, results from a C to G substitution at nucleotide position 1935. The asparagine at codon 645 is replaced by lysine, an amino acid with similar properties. This amino acid position is well conserved in available vertebrate species. In addition, the in silico prediction for this alteration is inconclusive. Since supporting evidence is limited at this time, the clinical significance of this alteration remains unclear. -

Lynch syndrome Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

All of Us Research Program, National Institutes of Health

Sep 05, 2023

This missense variant replaces asparagine with lysine at codon 645 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). To our knowledge, functional studies have not been reported for this variant. This variant has not been reported in individuals affected with hereditary cancer in the literature. This variant has been reported as a secondary finding in a non-cancer study (PMID: 31422574). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). 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. -

not provided Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

GeneDx

Oct 19, 2023

Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports that this missense variant does not alter protein structure/function; Identified via WES in an individual with a neurodevelopmental disorder and no personal history of cancer (Kraemer et al., 2019); This variant is associated with the following publications: (PMID: 31857677, 12799449, 20533529, 22753075, 31422574) -

Hereditary nonpolyposis colorectal neoplasms Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Invitae

Jan 22, 2024

This sequence change replaces asparagine, which is neutral and polar, with lysine, which is basic and polar, at codon 645 of the MLH1 protein (p.Asn645Lys). This variant is not present in population databases (gnomAD no frequency). This variant has not been reported in the literature in individuals affected with MLH1-related conditions. ClinVar contains an entry for this variant (Variation ID: 216335). 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 not expected to disrupt MLH1 protein function with a negative predictive value of 95%. RNA analysis performed to evaluate the impact of this missense change on mRNA splicing indicates it does not significantly alter splicing (Invitae). 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	Benign	0.19
BayesDel_addAF	Uncertain	0.054	T
BayesDel_noAF	Benign	-0.16
CADD	Benign	17
DANN	Benign	0.94
DEOGEN2	Uncertain	0.74	D;.;.;.;.;.
Eigen	Benign	-0.52
Eigen_PC	Benign	-0.36
FATHMM_MKL	Uncertain	0.87	D
LIST_S2	Uncertain	0.89	D;D;.;.;.;D
M_CAP	Benign	0.037	D
MetaRNN	Benign	0.42	T;T;T;T;T;T
MetaSVM	Benign	-0.56	T
MutationAssessor	Benign	1.2	L;.;.;.;.;.
MutationTaster	Benign	1.0	D;D;D;D;D;D
PrimateAI	Uncertain	0.57	T
PROVEAN	Benign	-2.0	N;N;N;N;N;N
REVEL	Uncertain	0.36
Sift	Benign	0.83	T;T;T;T;T;T
Sift4G	Benign	1.0	T;T;T;T;T;T
Polyphen		0.0030	B;.;.;.;.;.
Vest4		0.57
MutPred		0.56		Gain of methylation at N645 (P = 0.018);.;.;.;.;.;
MVP		0.87
MPC		0.077
ClinPred		0.15	T
GERP RS		2.1
Varity_R		0.43
gMVP		0.46

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.040		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: rs863224636; hg19: chr3-37090046;