3-37014544-C-T

Variant summary

Our verdict is Uncertain significance. Variant got 5 ACMG points: 5P and 0B. PM1 PM5 PP3

The NM_000249.4(MLH1):c.790C>T(p.His264Tyr) variant causes a missense, splice region change. The variant allele was found at a frequency of 0.0000141 in 1,558,108 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a pathogenic outcome for this variant. 2/3 splice prediction tools predicting alterations to normal splicing. 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. H264D) has been classified as Uncertain significance.

• Frequency:
  • Genomes: 𝑓 0.000020 (0 hom., cov: 33)
  • Exomes 𝑓: 0.000014 (0 hom.)
• Consequence: MLH1 NM_000249.4 missense, splice_region
• Scores: Splicing: ADA: 0.9880
• Clinical Significance: Uncertain significance criteria provided, multiple submitters, no conflicts U:9
• Conservation: PhyloP100: 5.77

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 5 ACMG points.

• PM1: In a hotspot region, there are 3 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 3 benign, 17 uncertain in NM_000249.4
• PM5: Other missense variant is known to change same aminoacid residue: Variant chr3-37017506-A-G is described in Lovd as [Pathogenic].
• PP3: Splicing scoreres supports a deletorius effect: Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF. No scorers claiming Uncertain. Scorers claiming Benign: max_spliceai.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.790C>T	p.His264Tyr	missense_variant, splice_region_variant	9/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.790C>T	p.His264Tyr	missense_variant, splice_region_variant	9/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes AF: 0.0000197 AC: 3 AN: 152106 Hom.: 0 Cov.: 33

Gnomad AFR AF: 0.0000483

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.00

Gnomad ASJ AF: 0.00

Gnomad EAS AF: 0.00

Gnomad SAS AF: 0.00

Gnomad FIN AF: 0.00

Gnomad MID AF: 0.00

Gnomad NFE AF: 0.0000147

Gnomad OTH AF: 0.00

GnomAD3 exomes AF: 0.0000160 AC: 4 AN: 250062 Hom.: 0 AF XY: 0.0000222 AC XY: 3 AN XY: 135222

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: 0.0000984

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.00

Gnomad OTH exome AF: 0.000164

GnomAD4 exome AF: 0.0000135 AC: 19 AN: 1406002 Hom.: 0 Cov.: 24 AF XY: 0.0000228 AC XY: 16 AN XY: 702876

Gnomad4 AFR exome AF: 0.0000310

Gnomad4 AMR exome AF: 0.0000224

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.0000470

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.00000942

Gnomad4 OTH exome AF: 0.0000511

GnomAD4 genome AF: 0.0000197 AC: 3 AN: 152106 Hom.: 0 Cov.: 33 AF XY: 0.0000135 AC XY: 1 AN XY: 74290

Gnomad4 AFR AF: 0.0000483

Gnomad4 AMR AF: 0.00

Gnomad4 ASJ AF: 0.00

Gnomad4 EAS AF: 0.00

Gnomad4 SAS AF: 0.00

Gnomad4 FIN AF: 0.00

Gnomad4 NFE AF: 0.0000147

Gnomad4 OTH AF: 0.00

Bravo AF: 0.0000264

ExAC AF: 0.00000824 AC: 1

ClinVar

Significance: Uncertain significance

Submissions summary: Uncertain:9

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Hereditary cancer-predisposing syndrome Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	Ambry Genetics	Dec 19, 2023	The p.H264Y variant (also known as c.790C>T), located in coding exon 9 of the MLH1 gene, results from a C to T substitution at nucleotide position 790. The amino acid change results in histidine to tyrosine at codon 264, an amino acid with similar properties. However, this change occurs in the last base pair of coding exon 9, which makes it likely to have some effect on normal mRNA splicing. RNA studies have demonstrated that this alteration does not result in abnormal splicing in the set of samples tested (Ambry internal data). In one functional study, the p.H264Y variant demonstrated restored cytotoxicity to methylation damage in a human colorectal cancer cell line; however, the mean survival score was between those of known damaging and neutral alterations leading to the tentative classification of "potentially neutral" (Bouvet D et al. Gastroenterology. 2019 08;157(2):421-431). This alteration has been reported in a Chinese male diagnosed with a MSI-H colorectal cancer at age 30y which demonstrated absent MLH1 protein expression on IHC. However, authors note that this alteration was detected in conjunction with the MLH1 p.T117M mutation, presumably on the same chromosome (in cis) as this patient's affected mother was also found to carry both alterations (Chan TL et al. J. Natl. Cancer Inst., 1999 Jul;91:1221-6; Lucci-Cordisco E et al. Cancer Biomark, 2006;2:11-27; Yuen ST et al. Oncogene, 2002 Oct;21:7585-92). 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. -
Uncertain significance, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Oct 17, 2022	This missense variant replaces histidine with tyrosine at codon 264 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). Functional studies report conflicting results for this variant. One study assaying DNA damage tolerance found this variant to be functional (PMID: 30998989), while another study assaying DNA repair activity in mouse cells reported this variant as pathogenic (PMID: 31784484). This variant has been reported in individuals affected with colorectal cancer (PMID: 10413423, 30998989). This variant has been identified in 5/281464 chromosomes 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 specified Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Women's Health and Genetics/Laboratory Corporation of America, LabCorp

Aug 23, 2019

Variant summary: MLH1 c.790C>T (p.His264Tyr) results in a conservative amino acid change located in the DNA mismatch repair protein, S5 domain 2-like domain (IPR013507) of the encoded protein sequence. Four of five in-silico tools predict a damaging effect of the variant on protein function. Despite the variant being located at the last nucleotide position in exon 9, 5/5 computational tools predict no significant impact on normal splicing. Additionally, no truncated protein product was detected in a patient sample carrying the variant, supporting the lack of impact of this variant on splicing (Chan_1999). The variant allele was found at a frequency of 1.6e-05 in 250062 control chromosomes (gnomAD). The available data on variant occurrences in the general population are insufficient to allow any conclusion about variant significance. c.790C>T has been reported in the literature in a mother and son with colorectal cancer who also carried another pathogenic variant MLH1 c.350C>T (p.T117M), providing supporting evidence for a benign role (Chan_1999, Yuen_2002). One publication reports experimental evidence evaluating an impact on protein function, however, does not allow convincing conclusions about the variant effect (Bouvet_2019). Three submitters have provided clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All laboratories classified the variant as uncertain significance. Based on the evidence outlined above, the variant was classified as uncertain significance. -

Colorectal cancer, hereditary nonpolyposis, type 2 Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Johns Hopkins Genomics, Johns Hopkins University

Jul 27, 2023

This MLH1 missense variant has been reported in a mother and son who were both affected with colorectal cancer, and both of them had a second MLH1 variant (c.350C>T; p.Thr117Met) that is known to be pathogenic. c.790C>T (rs63751597) is rare (<0.1%) in a large population dataset (gnomAD v2.1.1: 5/281464 total alleles; 0.002%; no homozygotes). It has been reported in ClinVar (Variation ID 90367). Two bioinformatic tools queried predict that this substitution would be damaging, and the histidine residue at this position is evolutionarily conserved across most of the species assessed. However, assays studying the effect of this variant on protein function have been inconclusive, with one study suggesting the variant is neutral and another suggesting a pathogenic role. While this variant changes the last nucleotide of exon 9, it is not predicted to affect normal exon 9 splicing, and at least one experimental study supports the lack of an effect on splicing. We consider the clinical significance of c.790C>T in MLH1 to be uncertain at this time. -

Lynch syndrome 1 Uncertain:1

Uncertain significance, no assertion criteria provided

clinical testing

Ding PR Lab, Sun Yat-sen University Cancer Center

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Lynch syndrome Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

All of Us Research Program, National Institutes of Health

Apr 15, 2023

This missense variant replaces histidine with tyrosine at codon 264 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). Functional studies report conflicting results for this variant. One study assaying DNA damage tolerance found this variant to be functional (PMID: 30998989), while another study assaying DNA repair activity in mouse cells reported this variant as pathogenic (PMID: 31784484). This variant has been reported in individuals affected colorectal cancer (PMID: 10413423, 30998989). This variant has been identified in 5/281464 chromosomes 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 17, 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 an individual and parent both with colorectal cancer who both also carried a pathogenic MLH1 variant (Chan et al., 1999); This variant is associated with the following publications: (PMID: 27498913, 23760103, 17192056, 12386821, 18383312, 34426522, 22753075, 21153778, 35031544, 31784484, 23403630, 10956410, 10413423, 30998989) -

Hereditary nonpolyposis colorectal neoplasms Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Invitae

Oct 10, 2022

This sequence change replaces histidine, which is basic and polar, with tyrosine, which is neutral and polar, at codon 264 of the MLH1 protein (p.His264Tyr). This variant is present in population databases (rs63751597, gnomAD 0.01%). This missense change has been observed in individual(s) with colorectal cancer (PMID: 10413423, 30998989). ClinVar contains an entry for this variant (Variation ID: 90367). Algorithms developed to predict the effect of missense changes on protein structure and function are either unavailable or do not agree on the potential impact of this missense change (SIFT: "Deleterious"; PolyPhen-2: "Probably Damaging"; Align-GVGD: "Class C0"). Experimental studies are conflicting or provide insufficient evidence to determine the effect of this variant on MLH1 function (PMID: 30998989, 31784484). RNA analysis performed to evaluate the impact of this missense change on mRNA splicing indicates it does not significantly alter splicing (PMID: 10413423). 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. -

Hereditary breast ovarian cancer syndrome Uncertain:1

Uncertain significance, criteria provided, single submitter

curation

German Consortium for Hereditary Breast and Ovarian Cancer, University Hospital Cologne

Jan 09, 2024

. According to the ACMG standard criteria we chose these criteria: PM2 (supporting pathogenic): popmax:AFR popmax AF:4.82905e-05 popmax AC:2, PP3 (supporting pathogenic): CADD:29.9 REVEL: 0.911 BayesDEL:0.409449 -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Uncertain	0.43
BayesDel_addAF	Pathogenic	0.31	D
BayesDel_noAF	Pathogenic	0.41
Cadd	Pathogenic	32
Dann	Uncertain	1.0
DEOGEN2	Pathogenic	0.85	D;.;.;.;.;.;.
Eigen	Pathogenic	0.92
Eigen_PC	Pathogenic	0.84
FATHMM_MKL	Pathogenic	0.98	D
LIST_S2	Uncertain	0.97	D;D;.;.;.;D;D
M_CAP	Uncertain	0.12	D
MetaRNN	Pathogenic	0.97	D;D;D;D;D;D;D
MetaSVM	Pathogenic	0.95	D
MutationAssessor	Pathogenic	3.8	H;.;.;.;.;.;.
MutationTaster	Benign	1.0	D;D;D;D;D;D
PrimateAI	Uncertain	0.68	T
PROVEAN	Pathogenic	-4.6	D;D;D;D;D;D;D
REVEL	Pathogenic	0.91
Sift	Uncertain	0.0040	D;D;D;D;D;D;D
Sift4G	Uncertain	0.018	D;D;D;D;D;T;D
Polyphen		1.0	D;.;.;.;.;.;.
Vest4		0.83
MutPred		0.92		Gain of catalytic residue at L259 (P = 0.0603);.;.;.;.;.;.;
MVP		0.95
MPC		0.42
ClinPred		0.90	D
GERP RS		5.4
Varity_R		0.90
gMVP		0.67

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	0.99
dbscSNV1_RF	Pathogenic	0.81
SpliceAI score (max)		0.050		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: rs63751597; hg19: chr3-37056035; COSMIC: COSV99212436; COSMIC: COSV99212436;