3-37025851-A-G

Variant summary

Our verdict is Uncertain significance. Variant got 4 ACMG points: 4P and 0B. PM1 PM2

The NM_000249.4(MLH1):c.1253A>G(p.Asp418Gly) variant causes a missense change involving the alteration of a non-conserved nucleotide. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a benign outcome for this variant. 13/23 in silico tools predict a benign 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. D418N) has been classified as Uncertain significance.

• Frequency: Genomes: not found (cov: 31)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Uncertain significance criteria provided, multiple submitters, no conflicts U:6
• Conservation: PhyloP100: 0.372

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 4 ACMG points.

• PM1: In a hotspot region, there are 3 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 4 benign, 17 uncertain in NM_000249.4
• PM2: Very rare variant in population databases, with high coverage

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.1253A>G	p.Asp418Gly	missense_variant	12/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.1253A>G	p.Asp418Gly	missense_variant	12/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 31

GnomAD3 exomes AF: 0.00000398 AC: 1 AN: 251348 Hom.: 0 AF XY: 0.00000736 AC XY: 1 AN XY: 135842

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 Cov.: 34

GnomAD4 genome Cov.: 31

ExAC AF: 0.00000824 AC: 1

ClinVar

Significance: Uncertain significance

Submissions summary: Uncertain:6

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

not provided Uncertain:2

Uncertain significance, no assertion criteria provided	clinical testing	Department of Pathology and Laboratory Medicine, Sinai Health System	-	The MLH1 p.Asp418Gly variant was not identified in the literature nor was it identified in the UMD-LSDB databases. The variant was identified in dbSNP (rs754898711) as “with uncertain significance allele” and ClinVar (classified as uncertain significance by Ambry Genetics, GeneDx and Color). The variant was identified in control databases in 1 of 251,348 chromosomes at a frequency of 0.000004 (Genome Aggregation Database Feb 27, 2017). The variant was observed in the following populations: European in 1 of 113,640 chromosomes (freq: 0.000009), while the variant was not observed in the African, Latino, Ashkenazi Jewish, East Asian, Finnish, Other and South Asian populations. The p.Asp418 residue is not conserved in mammals and computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) do not suggest a high likelihood of impact to the protein; however, this information is not predictive enough to rule out pathogenicity. The variant occurs outside of the splicing consensus sequence and 4 of 4 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) predict the creation of a new 5’ splice donor site. However, this information is not predictive enough to assume pathogenicity. In summary, based on the above information the clinical significance of this variant cannot be determined with certainty at this time. This variant is classified as a variant of uncertain significance. -
Uncertain significance, criteria provided, single submitter	clinical testing	GeneDx	Sep 13, 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; In silico analysis suggests this variant may impact gene splicing. In the absence of RNA/functional studies, the actual effect of this sequence change is unknown.; Observed in at least one individual with a personal history of colon cancer who underwent multi-gene panel testing (Yurgelun et al., 2017); This variant is associated with the following publications: (PMID: 22753075, 27121310, 28135145, 32885271) -

Hereditary cancer-predisposing syndrome Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	May 06, 2019	- -
Uncertain significance, criteria provided, single submitter	clinical testing	Ambry Genetics	Jun 14, 2023	The p.D418G variant (also known as c.1253A>G), located in coding exon 12 of the MLH1 gene, results from an A to G substitution at nucleotide position 1253. The aspartic acid at codon 418 is replaced by glycine, an amino acid with similar properties. This variant has been previously reported in an individual diagnosed with colorectal cancer (Yurgelun MB et al. J Clin Oncol, 2017 Apr;35:1086-1095). This variant was also observed in 1/3251 individuals who met eligibility criteria for hereditary breast and ovarian cancer syndrome (Lerner-Ellis J et al. J Cancer Res Clin Oncol, 2021 Mar;147:871-879). This amino acid position is not 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. -

Colorectal cancer, hereditary nonpolyposis, type 2 Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Mendelics

May 28, 2019

- -

Hereditary nonpolyposis colorectal neoplasms Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Invitae

Mar 23, 2023

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. Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may create or strengthen a splice site. 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. ClinVar contains an entry for this variant (Variation ID: 423761). This missense change has been observed in individual(s) with colorectal cancer (PMID: 28135145). This variant is not present in population databases (gnomAD no frequency). This sequence change replaces aspartic acid, which is acidic and polar, with glycine, which is neutral and non-polar, at codon 418 of the MLH1 protein (p.Asp418Gly). -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Benign	0.064
BayesDel_addAF	Uncertain	0.071	D
BayesDel_noAF	Benign	-0.14
Cadd	Benign	16
Dann	Benign	0.90
DEOGEN2	Uncertain	0.51	D;.;.;.;.;.
Eigen	Benign	-0.56
Eigen_PC	Benign	-0.60
FATHMM_MKL	Benign	0.18	N
LIST_S2	Benign	0.64	T;T;.;.;.;T
M_CAP	Benign	0.015	T
MetaRNN	Benign	0.10	T;T;T;T;T;T
MetaSVM	Benign	-1.0	T
MutationAssessor	Benign	-0.46	N;.;.;.;.;.
MutationTaster	Benign	1.0	N;N;N;N;N;N
PrimateAI	Benign	0.29	T
PROVEAN	Benign	-0.59	N;N;N;N;N;N
REVEL	Uncertain	0.58
Sift	Benign	0.32	T;T;T;T;T;T
Sift4G	Benign	0.47	T;T;T;T;T;T
Polyphen		0.0	B;.;.;.;.;.
Vest4		0.29
MutPred		0.21		Gain of catalytic residue at D418 (P = 0.0251);.;.;.;.;.;
MVP		0.97
MPC		0.086
ClinPred		0.048	T
GERP RS		1.0
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.1
Varity_R		0.038
gMVP		0.22

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.24		Details are displayed if max score is > 0.2
DS_DG_spliceai		0.24		Position offset: -1

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

Publications

LitVar

Other links and lift over

dbSNP: rs754898711; hg19: chr3-37067342; COSMIC: COSV104371918; COSMIC: COSV104371918;