3-36993632-G-T

Variant names:

• chr3-36993632-G-T
• rs63750656
• NM_000249.4:c.85G>T

Variant summary

Our verdict is Likely pathogenic. The variant received 8 ACMG points: 8P and 0B. PM1 PM2 PM5 PP3_Moderate

The NM_000249.4(MLH1):​c.85G>T​(p.Ala29Ser) 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 Conflicting classifications of pathogenicity (no stars). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. A29G) has been classified as Pathogenic.

• Frequency: Genomes: not found (cov: 33)
• Consequence: MLH1 NM_000249.4 missense
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:1 U:6 B:1
• Conservation: PhyloP100: 9.49
• Publications: 17 publications found

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]

MLH1 Gene-Disease associations (from GenCC):

• Lynch syndrome

  Inheritance: AD Classification: DEFINITIVE, SUPPORTIVE Submitted by: G2P, ClinGen, Orphanet
• Lynch syndrome 2

  Inheritance: AD Classification: DEFINITIVE, STRONG Submitted by: Ambry Genetics, Genomics England PanelApp
• Muir-Torre syndrome

  Inheritance: AD Classification: DEFINITIVE, STRONG, MODERATE, SUPPORTIVE Submitted by: Genomics England PanelApp, Ambry Genetics, G2P, Orphanet
• mismatch repair cancer syndrome 1

  Inheritance: AR Classification: DEFINITIVE, STRONG, SUPPORTIVE Submitted by: Ambry Genetics, G2P, Labcorp Genetics (formerly Invitae), Orphanet, ClinGen
• Lynch syndrome 1

  Inheritance: AD Classification: STRONG Submitted by: Labcorp Genetics (formerly Invitae)
• ovarian cancer

  Inheritance: AD Classification: STRONG Submitted by: Genomics England PanelApp
• malignant pancreatic neoplasm

  Inheritance: AD Classification: MODERATE Submitted by: Genomics England PanelApp
• rhabdomyosarcoma

  Inheritance: AR Classification: MODERATE Submitted by: Genomics England PanelApp
• prostate cancer

  Inheritance: AD Classification: LIMITED Submitted by: Ambry Genetics
• breast cancer

  Inheritance: AD Classification: NO_KNOWN Submitted by: Ambry Genetics
• hereditary breast carcinoma

  Inheritance: AD Classification: NO_KNOWN Submitted by: ClinGen

ACMG classification

Our verdict: Likely_pathogenic. The variant received 8 ACMG points.

• PM1: In a hotspot region, there are 24 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 3 benign, 54 uncertain in NM_000249.4
• PM2: Very rare variant in population databases, with high coverage
• PM5: Other missense variant is known to change same aminoacid residue: Variant chr3-36993633-C-G is described in ClinVar as Pathogenic. ClinVar VariationId is 90403.Status of the report is reviewed_by_expert_panel, 3 stars.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.905

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	MANE	Protein	UniProt
MLH1	NM_000249.4	c.85G>T	p.Ala29Ser	missense_variant	Exon 1 of 19	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.85G>T	p.Ala29Ser	missense_variant	Exon 1 of 19	1	NM_000249.4	ENSP00000231790.3

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD4 exome Cov.: 31

GnomAD4 genome Cov.: 33

Alfa AF: 0.00 Hom.: 0

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:1 Uncertain:6 Benign:1

Revision: criteria provided, conflicting classifications

Submissions by phenotype

not provided Uncertain:2 Benign:1

Jun 03, 2020

GeneDx

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

MLH1 c.-27C>A has been reported in cis with MLH1 c.85G>T (p.Ala29Ser) in patients with Lynch-related cancers and tumor studies consistent with pathogenic variants in this gene (Raevaara 2005, Hitchins 2011, Ward 2013, Kwok 2014); Published functional studies, when MLH1 c.85G>T (p.Ala29Ser) is evaluated in isolation, demonstrate no damaging effect; however, the MLH1 c.[-27C>A;85G>T] haplotype results in constitutional MLH1 promoter methylation and reduced allelic expression (Raevaara 2005, Takahashi 2007, Hitchins 2011, Kwok 2014); While segregation data of MLH1 c.-27C>A alone is not available, the MLH1 c.[-27C>A;85G>T] haplotype segregates with disease in affected individuals from several unrelated families in published literature (Raevaara 2005, Hitchins 2011, Ward 2013, Kwok 2014); Not observed in large population cohorts (Lek 2016); In silico analysis supports that this missense variant does not alter protein structure/function; This variant is associated with the following publications: (PMID: 24084575, 18383312, 12658575, 16083711, 17594722, 21120944, 22949387, 17370310, 17192056, 16995940, 22878509, 17510385, 21840485, 25345868, 27435373, 28152038, 29341452, 30283143) -

Nov 05, 2020

ARUP Laboratories, Molecular Genetics and Genomics, ARUP Laboratories

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Oct 02, 2024

Quest Diagnostics Nichols Institute San Juan Capistrano

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The MLH1 complex allele c.[-27C>A;85G>T] commonly occurs in cis in a haplotype that has been reported in multiple individuals with Lynch Syndrome or a Lynch Syndrome associated phenotype (PMIDs: 21840485 (2011), 21120944 (2011), 22878509 (2013), 24084575 (2014), and 27435373 (2016)). In the affected families, the haplotype was associated with differing levels of MLH1 promoter constitutional methylation, microsatellite instability and loss of PMS2 and/or MLH1 staining on immunohistochemistry in tumor samples (PMID: 21840485 (2011)), 22878509 (2013), 24084575 (2014)). In addition, an in vitro study has shown that the MLH1 c.[-27C>A;85G>T] haplotype results in diminished promotor activity and reduced MLH1 transcriptional activity (PMID: 21840485 (2011)). The haplotype has not been reported in large, multi-ethnic general populations (http://gnomad.broadinstitute.org). The MLH1 c.[-27C>A;85G>T] haplotype has been shown to cause MLH1 promoter methylation and affect allelic expression (PMID: 21840485 (2011)). Based on the available information, the MLH1 c.[-27C>A;85G>T] complex allele is classified as pathogenic. -

Colorectal cancer, hereditary nonpolyposis, type 2 Uncertain:2

Mar 14, 2023

Myriad Genetics, Inc.

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant is classified as a variant of uncertain significance as there is insufficient evidence to determine its impact on protein function and/or cancer risk. -

Sep 26, 2017

Counsyl

Significance: Uncertain significance

Review Status: no assertion criteria provided

Collection Method: clinical testing

This submission and the accompanying classification are no longer maintained by the submitter. For more information on current observations and classification, please contact variantquestions@myriad.com. -

Hereditary cancer-predisposing syndrome Pathogenic:1

Aug 15, 2024

Ambry Genetics

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

****************HAPTOTYPE RD************************ The p.A29S alteration (also known as c.85G>T) is located in coding exon 1 of the MLH1 gene. This alteration results from a G to T substitution at nucleotide position 85. The alanine at codon 29 is replaced by serine, an amino acid with similar properties. The c.-27C>A alteration is located in the 5' untranslated region (5’UTR) of the MLH1 gene. This variant results from a C to A substitution 27 nucleotides upstream from the first translated codon. The c.-27C>A and c.85G>T alterations are linked in cis, as both are part of a European ancestral haplotype. The c.[-27C>A;85G>T] haplotype has been reported to be linked to a constitutional MLH1 epimutation in several unrelated families that either met Amsterdam I/II criteria or were suspected to have HNPCC/Lynch syndrome (Kwok CT et al. Eur. J. Hum. Genet. 2014 May;22(5):617-24; Hitchins MP et al. Cancer Cell 2011;20:200-13; Ward RL et al. Genet. Med. 2013;15:25-35). Within these families, the c.[-27C>A;85G>T] haplotype was associated with differing levels of MLH1 promoter constitutional methylation and with loss of PMS2 and/or MLH1 staining on immunohistochemistry in tumor samples (Kwok CT et al. Eur. J. Hum. Genet. 2014 May;22(5):617-24; Hitchins MP et al. Cancer Cell 2011;20:200-13; Ward RL et al. Genet. Med. 2013;15:25-35). Luciferase reporter assays performed for the c.-27C>A alteration demonstrated reduced promoter activity (Hitchins MP et al. Cancer Cell 2011;20:200-13; Ward RL et al. Genet. Med. 2013;15:25-35). In contrast, luciferase reporter assays performed for the c.85G>T alteration showed little to no reduction in promoter activity compared to wild type suggesting the c.-27C>A alteration accounts for allelic repression of MLH1 transcription resulting in loss of MLH1 expression; however, a direct causal relationship has yet to be clarified (Hitchins MP et al. Cancer Cell 2011;20:200-13). Based on the supporting evidence, the c.[-27C>A;85G>T] haplotype is interpreted as a disease-causing mutation. -

not specified Uncertain:1

-

Mayo Clinic Laboratories, Mayo Clinic

Significance: Uncertain significance

Review Status: no assertion criteria provided

Collection Method: clinical testing

- -

Hereditary nonpolyposis colorectal neoplasms Uncertain:1

Dec 31, 2023

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This sequence change replaces alanine, which is neutral and non-polar, with serine, which is neutral and polar, at codon 29 of the MLH1 protein (p.Ala29Ser). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individuals with Lynch syndrome, and in many individuals the variant has been reported on the same chromosome with the c.-27C>A promoter MLH1 variant. (PMID: 2408575, 16083711, 21840485, 22878509). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 90397). 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. Experimental studies have shown that this missense change does not substantially affect MLH1 function (PMID: 16083711, 17510385, 21840485). 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. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Benign	0.098
BayesDel_addAF	Pathogenic	0.43	D
BayesDel_noAF	Pathogenic	0.37
CADD	Pathogenic	26
DANN	Uncertain	1.0
DEOGEN2	Uncertain	0.46	T
Eigen	Uncertain	0.62
Eigen_PC	Pathogenic	0.69
FATHMM_MKL	Pathogenic	0.98	D
LIST_S2	Pathogenic	0.97	D
M_CAP	Pathogenic	0.30	D
MetaRNN	Pathogenic	0.91	D
MetaSVM	Uncertain	-0.26	T
MutationAssessor	Benign	1.4	L
PhyloP100		9.5
PrimateAI	Uncertain	0.65	T
PROVEAN	Benign	-1.5	N
REVEL	Pathogenic	0.77
Sift	Benign	0.036	D
Sift4G	Benign	0.17	T
Polyphen		0.88	P
Vest4		0.66
MutPred		0.86		Gain of disorder (P = 0.0732);
MVP		0.98
MPC		0.31
ClinPred		0.96	D
GERP RS		6.0
PromoterAI		0.0080	Neutral
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.6
Varity_R		0.69
gMVP		0.57
Mutation Taster		=41/59	disease causing (ClinVar)

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.010		Details are displayed if max score is > 0.2

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

Other links and lift over

dbSNP: rs63750656; hg19: chr3-37035123;