3-36993572-C-T

Position:

• chr3-36993572-C-T

Variant summary

Our verdict is Uncertain significance. Variant got 4 ACMG points: 4P and 0B. PM2 PP3_Moderate

The ENST00000231790.8(MLH1):​c.25C>T​(p.Arg9Trp) variant causes a missense change. The variant allele was found at a frequency of 0.000000684 in 1,461,882 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. R9G) has been classified as Uncertain significance.

• Frequency:
  • Genomes: not found (cov: 33)
  • Exomes 𝑓: 6.8e-7 (0 hom.)
• Consequence: MLH1 ENST00000231790.8 missense
• Clinical Significance: Uncertain significance criteria provided, multiple submitters, no conflicts U:5
• Conservation: PhyloP100: 4.51

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.

• PM2: Very rare variant in population databases, with high coverage
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.91

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
MLH1	NM_000249.4	c.25C>T	p.Arg9Trp	missense_variant	1/19	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.25C>T	p.Arg9Trp	missense_variant	1/19	1	NM_000249.4	ENSP00000231790	P1

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD4 exome AF: 6.84e-7 AC: 1 AN: 1461882 Hom.: 0 Cov.: 31 AF XY: 0.00000138 AC XY: 1 AN XY: 727244

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: 8.99e-7

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 33

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	Mar 03, 2023	This missense variant replaces arginine with tryptophan at codon 9 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). There is conflicting functional evidence for the variant. A functional study has reported this variant to cause an intermediate reduction in vitro mismatch repair activity and significantly reduce MLH1 and PMS2 protein expression compared to wild type in transfected cells (PMID: 22736432). Another functional study reported it to have protein stability, damage response signaling, and DNA repair function similar to wild type (PMID: 36054288). This variant has been reported in two related individuals affected with colorectal cancer and/or polyps, both of whom also had a pathogenic APC co-variant (PMID: 22736432). 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	Jun 17, 2024	The p.R9W variant (also known as c.25C>T), located in coding exon 1 of the MLH1 gene, results from a C to T substitution at nucleotide position 25. The arginine at codon 9 is replaced by tryptophan, an amino acid with dissimilar properties. This alteration has been identified in an individual with a personal history of MSI colorectal cancer associated with multiple colon polyps; however, this individual was also positive for a pathogenic mutation in the APC gene (Borràs E et al. Hum. Mutat., 2012 Nov;33:1576-88). This alteration was also identified in an individual diagnosed with colorectal cancer (Dámaso E et al. Cancers (Basel), 2020 Jul;12:). This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Based on the available evidence, the clinical significance of this variant remains unclear. -

not specified Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

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

Feb 20, 2023

Variant summary: MLH1 c.25C>T (p.Arg9Trp) results in a non-conservative amino acid change located in the N-terminal domain (IPR002099) of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant was absent in 251406 control chromosomes (gnomAD). c.25C>T has been reported in the literature within a family in two individuals affected with colorectal cancer and/or adenomatous polyposis (Borras_2012), however both of these patients carried a co-occurring pathogenic variant (APC c.1958+3A>G), providing supporting evidence for a benign role. On the other hand, authors of this study also reported experimental evidence evaluating an impact on protein function, and demonstrated intermediate level of MMR activity for the variant protein (Borras_2012). Later functional studies resulted in conflicting outcomes (Houlleberghs_2020, Rath_2022). Three clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014, and all of them 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

Baylor Genetics

Oct 03, 2023

- -

Hereditary nonpolyposis colorectal neoplasms Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

May 09, 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. Experimental studies are conflicting or provide insufficient evidence to determine the effect of this variant on MLH1 function (PMID: 22736432, 36054288). 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. ClinVar contains an entry for this variant (Variation ID: 90122). This missense change has been observed in individual(s) with colorectal cancer and adenomatous polyposis (PMID: 22736432, 32635641). This variant is not present in population databases (gnomAD no frequency). This sequence change replaces arginine, which is basic and polar, with tryptophan, which is neutral and slightly polar, at codon 9 of the MLH1 protein (p.Arg9Trp). -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Uncertain	0.43
BayesDel_addAF	Pathogenic	0.47	D
BayesDel_noAF	Pathogenic	0.43
CADD	Pathogenic	34
DANN	Pathogenic	1.0
DEOGEN2	Pathogenic	0.84	D
Eigen	Pathogenic	0.71
Eigen_PC	Uncertain	0.63
FATHMM_MKL	Pathogenic	0.98	D
LIST_S2	Pathogenic	0.98	D
M_CAP	Pathogenic	0.63	D
MetaRNN	Pathogenic	0.91	D
MetaSVM	Pathogenic	0.95	D
MutationAssessor	Pathogenic	3.9	H
MutationTaster	Benign	1.0	D
PrimateAI	Pathogenic	0.80	T
PROVEAN	Pathogenic	-4.6	D
REVEL	Pathogenic	0.86
Sift	Uncertain	0.0020	D
Sift4G	Uncertain	0.014	D
Polyphen		1.0	D
Vest4		0.83
MutPred		0.54		Loss of disorder (P = 0.0292);
MVP		0.93
MPC		0.40
ClinPred		1.0	D
GERP RS		4.0
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		2.6
Varity_R		0.84
gMVP		0.72

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: rs587779000; hg19: chr3-37035063;