3-36993602-A-T

Variant summary

Our verdict is Pathogenic. Variant got 16 ACMG points: 16P and 0B. PM1 PM2 PP3_Strong PP5_Very_Strong

The NM_000249.4(MLH1):c.55A>T(p.Ile19Phe) variant causes a missense change involving the alteration of a conserved nucleotide. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a pathogenic outcome for this variant. Variant has been reported in ClinVar as Pathogenic (★★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. I19L) has been classified as Uncertain significance.

• Frequency: Genomes: not found (cov: 33)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Pathogenic reviewed by expert panel P:5
• Conservation: PhyloP100: 8.85

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 16 ACMG points.

• PM1: In a hotspot region, there are 4 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 3 benign, 17 uncertain in NM_000249.4
• PM2: Very rare variant in population databases, with high coverage
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.945
• PP5: Variant 3-36993602-A-T is Pathogenic according to our data. Variant chr3-36993602-A-T is described in ClinVar as [Pathogenic]. Clinvar id is 90274.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-36993602-A-T is described in Lovd as [Pathogenic]. Variant chr3-36993602-A-T is described in Lovd as [Benign]. Variant chr3-36993602-A-T is described in Lovd as [Likely_pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.55A>T	p.Ile19Phe	missense_variant	1/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.55A>T	p.Ile19Phe	missense_variant	1/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 33

Bravo AF: 0.00000756

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:5

Revision: reviewed by expert panel

Submissions by phenotype

Carcinoma of colon Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

-

The p.Ile19Phe variant has been previously identified in 3 of 102 probands with colorectal or endometrial cancer (Kurzawski 2002, Andrew 2002, Perera 2010). In one study, this variant was demonstrated to segregate with colon cancer in two individuals, increasing the likelihood this variant is pathogenic (Andrew 2002). In addition, this variant was previously identified in 1 family from our laboratory with a striking history of Lynch syndrome related tumors, three affected individuals with the variant, and in 5 other affected individuals who were not tested for the variant (but at least two are obligate carriers). MSI-H tumor was demonstrated in several of these individuals but MLH1 was shown to be intact by IHC. A second family was also identified by our laboratory and this variant was shown to segregate with disease in 2 affected individuals and the tumours analyzed were demonstrated to be MSI high. In addition, the p.Ile19 residue is conserved across mammals and lower species and in-silico data (AlignGVGD, SIFT) suggest this alteration may impact the protein. However, this information is not predictive enough to determine pathogenicity. This variant is listed in dbSNP (ID#: rs63750648) as coming from a "Clinical Source" with no frequency information and is not informative for assessing its frequency in the population. In summary, based on the above evidence this variant meets our laboratory's criteria to be classified as pathogenic. -

Lynch syndrome 1 Pathogenic:1

Pathogenic, reviewed by expert panel

curation

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Jun 13, 2018

Multifactorial likelihood analysis posterior probability > 0.95 (0.997) -

not provided Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

GeneDx

Sep 01, 2020

Observed in individuals with colorectal or other Lynch-associated cancers meeting revised Bethesda or Amsterdam II criteria (Andrew 2002, Kurzawski 2002, Perera 2010, Yurgleun 2017); Not observed in large population cohorts (Lek 2016); Published functional studies demonstrate partial loss of mismatch repair function (Ellison 2004); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 18383312, 30212499, 15475387, 29478780, 20864636, 12362047, 12537657, 28135145, 26333163, 26248088, 31857677, 31391288) -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Invitae

Jun 29, 2023

For these reasons, this variant has been classified as Pathogenic. Experimental studies have shown that this missense change affects MLH1 function (PMID: 15475387). 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 expected to disrupt MLH1 protein function. ClinVar contains an entry for this variant (Variation ID: 90274). This missense change has been observed in individuals with clinical features of Lynch syndrome and/or colorectal cancer (PMID: 12362047, 12537657, 20864636, 28135145, 28514183, 31857677). It has also been observed to segregate with disease in related individuals. This variant is not present in population databases (gnomAD no frequency). This sequence change replaces isoleucine, which is neutral and non-polar, with phenylalanine, which is neutral and non-polar, at codon 19 of the MLH1 protein (p.Ile19Phe). -

Hereditary cancer-predisposing syndrome Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Feb 10, 2023

The p.I19F variant (also known as c.55A>T), located in coding exon 1 of the MLH1 gene, results from an A to T substitution at nucleotide position 55. The isoleucine at codon 19 is replaced by phenylalanine, an amino acid with similar properties. This alteration has been identified in multiple individuals with Lynch syndrome-related cancers whose families were either suspicious for Lynch syndrome or met Amsterdam criteria (Andrew SE et al. Genet.Test. 2002;6(4):319-22; Kurzawski G et al. J. Med. Genet. 2002 Oct;39:e65; Perera S et al. J. Molec. Diagnost. 2010 Nov;12(6):757-764; Yurgelun MB et al. J. Clin. Oncol. 2017 Apr;35(10):1086-1095). Protein functional analysis using a yeast-human hybrid assay demonstrated reduced mismatch repair function for this variant (Ellison AR et al. Nucleic Acids Res. 2004 Oct;32(18):5321-38). A family study conducted in our laboratory demonstrated segregation with disease for four affected individuals across two generations. This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). Based on the majority of available evidence to date, this variant is likely to be pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.99
BayesDel_addAF	Pathogenic	0.51	D
BayesDel_noAF	Pathogenic	0.49
Cadd	Pathogenic	30
Dann	Uncertain	0.99
DEOGEN2	Pathogenic	0.82	D
Eigen	Pathogenic	1.0
Eigen_PC	Pathogenic	0.94
FATHMM_MKL	Pathogenic	0.99	D
LIST_S2	Pathogenic	1.0	D
M_CAP	Pathogenic	0.51	D
MetaRNN	Pathogenic	0.94	D
MetaSVM	Pathogenic	1.0	D
MutationAssessor	Pathogenic	4.9	H
MutationTaster	Benign	1.0	D
PrimateAI	Pathogenic	0.85	D
PROVEAN	Uncertain	-3.1	D
REVEL	Pathogenic	0.93
Sift	Pathogenic	0.0	D
Sift4G	Uncertain	0.0020	D
Polyphen		1.0	D
Vest4		0.95
MVP		0.98
MPC		0.50
ClinPred		1.0	D
GERP RS		6.0
Varity_R		0.98
gMVP		0.94

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: rs63750648; hg19: chr3-37035093;