3-37028891-T-C

Variant summary

Our verdict is Likely pathogenic. Variant got 9 ACMG points: 9P and 0B. PM1 PM2 PP3_Strong PP5

The NM_000249.4(MLH1):c.1517T>C(p.Val506Ala) variant causes a missense change. The variant allele was found at a frequency of 0.00000658 in 152,072 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 Conflicting classifications of pathogenicity (no stars). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. V506D) has been classified as Uncertain significance.

• Frequency: Genomes: 𝑓 0.0000066 (0 hom., cov: 32)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:10 U:1
• Conservation: PhyloP100: 6.20

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 Likely_pathogenic. Variant got 9 ACMG points.

• PM1: In a hotspot region, there are 2 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 1 benign, 15 uncertain in NM_000249.4
• PM2: Very rare variant in population databases, with high coverage
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.941
• PP5: Variant 3-37028891-T-C is Pathogenic according to our data. Variant chr3-37028891-T-C is described in ClinVar as [Conflicting_classifications_of_pathogenicity]. Clinvar id is 89757.We mark this variant Likely_pathogenic, oryginal submissions are: {Likely_pathogenic=9, Pathogenic=1, Uncertain_significance=1}. Variant chr3-37028891-T-C is described in Lovd as [Pathogenic]. Variant chr3-37028891-T-C is described in Lovd as [Benign]. Variant chr3-37028891-T-C is described in Lovd as [Likely_pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.1517T>C	p.Val506Ala	missense_variant	13/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.1517T>C	p.Val506Ala	missense_variant	13/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes AF: 0.00000658 AC: 1 AN: 152072 Hom.: 0 Cov.: 32

Gnomad AFR AF: 0.00

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

GnomAD4 exome Cov.: 32

GnomAD4 genome AF: 0.00000658 AC: 1 AN: 152072 Hom.: 0 Cov.: 32 AF XY: 0.0000135 AC XY: 1 AN XY: 74294

Gnomad4 AFR AF: 0.00

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: 0.00000378

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:10 Uncertain:1

Revision: criteria provided, conflicting classifications

Submissions by phenotype

not provided Pathogenic:3 Uncertain:1

Likely pathogenic, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Apr 22, 2023	The frequency of this variant in the general population, 0.000032 (1/31390 chromosomes, http://gnomad.broadinstitute.org), is uninformative in assessment of its pathogenicity. In the published literature, the variant has been reported in individuals with Lynch syndrome (PMID: 8574961 (1996), 19690142 (2009), 22949387 (2013), 23403630 (2013), 25980754 (2015)). Multiple studies have reported on the functional effect of this variant, some indicating a detrimental effect and others indicating no detrimental effect of the variant on protein function. It has been identified in tumors with high microsatellite instability, as well as associated with reduced interaction with PMS2 and decreased/defective mismatch repair activity (PMID: 9697702 (1998), PMID: 10037723 (1999), and PMID: 22949387 (2013)). In contrast, it has been claimed to be repair-proficient despite observed reduced stability and expression of MLH1 (PMID: 23403630 (2013)). Analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded predictions that this variant is damaging. Based on the available information, this variant is classified as likely pathogenic. -
Uncertain significance, criteria provided, single submitter	clinical testing	Eurofins Ntd Llc (ga)	Sep 15, 2017	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	Revvity Omics, Revvity	Aug 04, 2022	- -
Likely pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Sep 05, 2023	Observed in multiple individuals with personal and family history consistent with Lynch syndrome (Liu et al., 1996; Syngal et al., 1999; Chao et al., 2008); Published functional studies demonstrate a damaging effect with respect to a dominant mutator effect, MLH1 expression, and PMS2 and EXO1 binding, while studies assessing mismatch repair activity show results comparable to wild-type (Shimodaira et al., 1998; Guerrette et al., 1999; Kondo et al., 2003; Takahashi et al., 2007; Hinrichsen et al., 2013); 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; This variant is associated with the following publications: (PMID: 8574961, 17594722, 10037723, 22949387, 10422993, 12810663, 19015241, 25980754, 17192056, 10874307, 18383312, 22290698, 19690142, 22788692, 22658618, 22426235, 19250818, 11292842, 31697235, 31391288, 32719484, 32427313, 30787465, 17510385, 23403630, 9697702, 36054288, 31784484, 12799449, 20533529, 22753075) -

Hereditary cancer-predisposing syndrome Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Apr 11, 2023	This missense variant replaces valine with alanine at codon 506 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 have shown that this variant reduced MLH1 protein expression (PMID: 9697702, 17510385, 23403630), disrupted PMS2 and EXO1 binding activity (PMID: 10037723, 12810663), and reduced dominant mutator effect (PMID: 9697702, 17510385). However, the impact of this variant on DNA mismatch repair activity remains unclear (PMID: 17510385, 23403630, 36054288). This variant has been reported in individuals and families affected with Lynch Syndrome (PMID: 8574961, 18383312, 19250818, 22426235, 25980754, 31391288). This variant has been identified in 1/31390 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Likely Pathogenic. -
Likely pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Apr 26, 2023	The p.V506A variant (also known as c.1517T>C), located in coding exon 13 of the MLH1 gene, results from a T to C substitution at nucleotide position 1517. The valine at codon 506 is replaced by alanine, an amino acid with similar properties. This alteration was identified in several individuals whose family history met Amsterdam I/II criteria for Lynch syndrome and/or tumors showed loss of MLH1 and/or PMS2 protein expression on immunohistochemistry (IHC) as well as high microsatellite instability (MSI-H) (Ambry internal data; Liu B et al. Nat. Med. 1996 Feb;2:169-74; ). This alteration has also been observed in an individual whose colorectal tumor demonstrated normal mismatch repair protein expression on IHC (Ambry internal data). Multiple yeast-based functional studies of this variant have demonstrated reduced binding of the V506A hMLH1 protein with hPMS2 when compared to wild-type hMLH1 (Guerrette S et al. J. Biol. Chem. 1999 Mar 5;274:6336-41; Kondo E et al. Cancer Res. 2003 Jun;63:3302-8; Shimodaira H et al. Nat. Genet. 1998 Aug;19:384-9). Further, multiple functional studies where cell lines were transfected with this alteration have been shown to exhibit reduced MLH1 expression; however, in vitro functional assays showed that mismatch repair activity was comparable to wild-type (Hinrichsen I et al. Clin. Cancer Res. 2013 May;19:2432-41; Takahashi M et al. Cancer Res. 2007 May;67:4595-604). This amino acid position is well conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Based on the majority of available evidence to date, this variant is likely to be pathogenic. -

not specified Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

ARUP Laboratories, Molecular Genetics and Genomics, ARUP Laboratories

Jun 06, 2019

The MLH1 c.1517T>C; p.Val506Ala variant has been described in individuals and families that met diagnostic criteria for Lynch syndrome (Chao 2008, Liu 1996, Syngal 1999), and segregates with disease in several unrelated families (internal data, personal communication with GeneDx and Invitae). In vitro functional analyses demonstrate reduced protein expression, but close to wild-type mismatch repair activity (Hinrichsen 2013, Takahashi 2007). Additionally, yeast two hybrid assays have demonstrated reduced functional outputs (Kondo 2003, Shimodaira 1998). This variant is reported in ClinVar (Variation ID: 89757), and is only observed on one allele in the Genome Aggregation Database, indicating it is not a common polymorphism. The valine at codon 506 is moderately conserved, and computational analyses (SIFT, PolyPhen-2) predict that this variant is deleterious. Based on available information, this variant is considered to be likely pathogenic. References: Chao EC et al. Accurate classification of MLH1/MSH2 missense variants with multivariate analysis of protein polymorphisms-mismatch repair (MAPP-MMR). Hum Mutat. 2008 Jun;29(6):852-60. Hinrichsen I et al. Expression defect size among unclassified MLH1 variants determines pathogenicity in Lynch syndrome diagnosis. Clin Cancer Res. 2013 May 1;19(9):2432-41. Kondo E et al. A yeast two-hybrid assay provides a simple way to evaluate the vast majority of hMLH1 germ-line mutations. Cancer Res. 2003 Jun 15;63(12):3302-8. Liu B et al. Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients. Nat Med. 1996 Feb;2(2):169-74. Shimodaira H et al. Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae. Nat Genet. 1998 Aug;19(4):384-9. Syngal S et al. Interpretation of genetic test results for hereditary nonpolyposis colorectal cancer: implications for clinical predisposition testing. JAMA. 1999 Jul 21;282(3):247-53. Takahashi M et al. Functional analysis of human MLH1 variants using yeast and in vitro mismatch repair assays. Cancer Res. 2007 May 15;67(10):4595-604. -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Baylor Genetics

Sep 05, 2023

- -

Hereditary nonpolyposis colon cancer Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

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

Mar 27, 2023

Variant summary: MLH1 c.1517T>C (p.Val506Ala) results in a non-conservative amino acid change located in the C-terminal domain (IPR032189) of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 3.8e-06 in 261636 control chromosomes (gnomAD). c.1517T>C has been reported in multiple colorectal cancer patients from families fulfilling the Amsterdam and the revised Bethesda criteria (e.g. Liu_1996, Chao_2008, Yurgelun_2015, Syngal_1999, Medeiros_2012). These data indicate that the variant is likely to be associated with disease. In in vitro functional studies, the variant showed reduced MLH1 expression and altered binding to PMS2 and EXO1, but mismatch repair (MMR) activity close to wild type (Shimodaira_1998, Guerrette_1999, Takahashi_2007, Hinrichsen_2013). Eight (other) submitters have provided clinical-significance assessments for this variant to ClinVar after 2014, and classified it as pathogenic/likely pathogenic (n=7) or uncertain significance (n=1). Based on the evidence outlined above, the variant was classified as likely pathogenic. -

MLH1-related disorder Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

PreventionGenetics, part of Exact Sciences

Mar 01, 2023

The MLH1 c.1517T>C variant is predicted to result in the amino acid substitution p.Val506Ala. This variant has been reported in families with hereditary non-polyposis colorectal cancer (Table 2, Liu et al. 1996. PubMed ID: 8574961; Table 3, Syngal et al. 1999. PubMed ID: 10422993; Table S1, Chao et al. 2008. PubMed ID: 18383312). It has been reported in individuals undergoing Lynch syndrome population screening, as well as an unaffected individual from a breast cancer cohort study (Table S3, Grzymski et al. 2020. PubMed ID: 32719484; Table S3, Palmer et al. 2020. PubMed ID: 32427313). In vitro experimental studies suggest this variant leads to reduced binding to hPMS2, reduced MMR activity, and reduced protein expression (Guerrette et al. 1999. PubMed ID: 10037723; Figure 1, Hinrichsen et al. 2013. PubMed ID: 23403630; Table S1, Ou et al. 2007. PubMed ID: 17594722; Table 1, Takahashi et al. 2007. PubMed ID: 17510385). In vivo experimental studies suggest this variant reduces binding to EXO1 and inactivates the dominant negative phenotype of hMLH1 in some strains of yeast (Table 1, Figure 3b, Shimodaira et al. 1998. PubMed ID: 9697702; Table 1, Kondo et al 2003. PubMed ID: 12810663; Table 1, Takahashi et al. 2007. PubMed ID: 17510385). This variant is reported in 1 of ~31,000 alleles in gnomAD (http://gnomad.broadinstitute.org/variant/3-37070382-T-C). It is interpreted as likely pathogenic by the vast majority of submitters in ClinVar (https://preview.ncbi.nlm.nih.gov/clinvar/variation/89757/). At this time, the clinical significance of this variant is uncertain due to the absence of conclusive functional and genetic evidence. -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Invitae

Jan 27, 2024

This sequence change replaces valine, which is neutral and non-polar, with alanine, which is neutral and non-polar, at codon 506 of the MLH1 protein (p.Val506Ala). This variant is present in population databases (rs63749909, gnomAD 0.007%). This missense change has been observed in individuals with Lynch syndrome (PMID: 8574961, 10422993, 18383312; Invitae). ClinVar contains an entry for this variant (Variation ID: 89757). An algorithm developed specifically for the MLH1 gene suggests that this missense change is likely to be deleterious (PMID: 18383312). Experimental studies have shown that this missense change affects MLH1 function (PMID: 9697702, 10037723, 12810663, 17510385, 23403630). For these reasons, this variant has been classified as Pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.86
BayesDel_addAF	Pathogenic	0.41	D
BayesDel_noAF	Pathogenic	0.35
Cadd	Uncertain	25
Dann	Uncertain	1.0
DEOGEN2	Pathogenic	0.92	D;.;.;.;.;.;T;D
Eigen	Pathogenic	0.73
Eigen_PC	Pathogenic	0.71
FATHMM_MKL	Uncertain	0.90	D
LIST_S2	Uncertain	0.88	D;D;.;.;.;D;T;D
M_CAP	Uncertain	0.095	D
MetaRNN	Pathogenic	0.94	D;D;D;D;D;D;D;D
MetaSVM	Pathogenic	0.95	D
MutationAssessor	Uncertain	2.8	M;.;.;.;.;.;.;.
MutationTaster	Benign	0.95	D;D;D;D;D;D
PrimateAI	Benign	0.47	T
PROVEAN	Uncertain	-3.7	D;D;D;D;D;D;D;D
REVEL	Pathogenic	0.76
Sift	Uncertain	0.0010	D;D;D;D;D;D;D;D
Sift4G	Uncertain	0.0020	D;D;D;D;D;D;D;D
Polyphen		0.96	D;.;.;.;.;.;.;.
Vest4		0.56
MutPred		0.81		Gain of loop (P = 0.002);.;.;.;.;.;.;.;
MVP		0.97
MPC		0.25
ClinPred		0.98	D
GERP RS		5.9
Varity_R		0.85
gMVP		0.65

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: rs63749909; hg19: chr3-37070382;