3-37001053-G-C

Variant names:

• chr3-37001053-G-C
• rs63751665
• NM_000249.4:c.306G>C

Variant summary

Our verdict is Pathogenic. The variant received 18 ACMG points: 18P and 0B. PM1 PM2 PM5 PP3_Strong PP5_Very_Strong

The NM_000249.4(MLH1):​c.306G>C​(p.Glu102Asp) variant causes a missense, splice region change. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a pathogenic outcome for this variant. 3/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Pathogenic (★★★). Another nucleotide change resulting in the same amino acid substitution has been previously reported as Uncertain significance in ClinVar. Another variant affecting the same amino acid position, but resulting in a different missense (i.e. E102A) has been classified as Likely pathogenic.

• Frequency: Genomes: not found (cov: 32)
• Consequence: MLH1 NM_000249.4 missense, splice_region
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Pathogenic reviewed by expert panel P:5
• Conservation: PhyloP100: 6.33
• 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: Pathogenic. The variant received 18 ACMG points.

• PM1: In a hotspot region, there are 15 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 1 benign, 36 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 chr3-37001052-A-C is described in ClinVar as Conflicting_classifications_of_pathogenicity. ClinVar VariationId is 486866.
• PP3: Splicing predictors support a deleterious effect. Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF, max_spliceai. No scorers claiming Uncertain. No scorers claiming Benign.
• PP5: Variant 3-37001053-G-C is Pathogenic according to our data. Variant chr3-37001053-G-C is described in ClinVar as Pathogenic. ClinVar VariationId is 90150.Status of the report is reviewed_by_expert_panel, 3 stars.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	MANE	Protein	UniProt
MLH1	NM_000249.4	c.306G>C	p.Glu102Asp	missense_variant, splice_region_variant	Exon 3 of 19	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.306G>C	p.Glu102Asp	missense_variant, splice_region_variant	Exon 3 of 19	1	NM_000249.4	ENSP00000231790.3

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 26

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:5

Revision: reviewed by expert panel

Submissions by phenotype

Hereditary cancer-predisposing syndrome Pathogenic:2

Jun 07, 2022

Ambry Genetics

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The c.306G>C pathogenic mutation (also known as p.E102D), located in coding exon 3 of the MLH1 gene, results from a G to C substitution at nucleotide position 306. The amino acid change results in glutamic acid to aspartic acid at codon 102, an amino acid with highly similar properties. However, this change occurs in the last base pair of coding exon 3, which makes it likely to have some effect on normal mRNA splicing. In one study, this alteration, which was also detected with a MLH1 variant with no predicted effect on splicing (c.248G>T), demonstrated in-frame exon 3 skipping upon RT-PCR analysis using patient RNA and no full-length transcript was expressed by the variant allele (Borràs E et al. Hum. Mutat., 2012 Nov;33:1576-88). Another alteration at the same nucleotide position, c.306G>A, which also results in exon 3 skipping, has been classified as pathogenic based on identification in individuals with constitutional mismatch repair deficiency syndrome and Lynch syndrome (Ambry internal data; external communication with outside laboratory). In a functional study, MLH1 p.E102D demonstrated reduced mismatch repair activity (56%) compared to wild type MLH1 (79.7%) in a complementation assay using a human colon cancer cell line, but relative MLH1 protein expression was >75% in the same cell line upon transient expression (Takahashi et al. Cancer Res. 2007. 67(10): 4595-4604). RNA studies have demonstrated that this alteration results in abnormal splicing in the set of samples tested (Ambry internal data). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.

Oct 07, 2024

Molecular Diagnostics Laboratory, Catalan Institute of Oncology

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

PVS1 (RNA), PM2_Supporting, PP4 c.306G>C is located in the last nucleotide of MLH1 exon 3. Functional RNA studies have demonstrated complete abnormal splicing for this variant causing the skipping of exon 3 (r.208_306del, p.Lys70_102del) (PVS1)(PMID: 22736432). It is not present in the population database gnomAD v2.1.1, non cancer dataset (PM2_supporting). This variant has been identified in a patient with colorrectal cancer with microsatellite instability and MLH1 protein expression lost with no promoter methylation (PMID: 22736432, internal case)(PP4). In adition, this variant has been reported in the ClinVar database (1x as likely pathogenic, 2x pathogenic) and in the LOVD database (1x pathogenic, 1x uncertain significance). In addition, the variant has been also reported in InSiGHT database 2013/09/05 v1.9 as pathogenic variant (Summary Justification Variant causes splicing aberration: full inactivation of variant allele). Based on currently available information, the variant c.306G>C is classified as a pathogenic variant according to ACMG guidelines.

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:1

Jul 12, 2023

Myriad Genetics, Inc.

Significance: Likely pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant is considered likely pathogenic. mRNA analysis has demonstrated abnormal mRNA splicing occurs [PMID: 22736432, Myriad internal data].

Lynch syndrome Pathogenic:1

Sep 05, 2013

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Significance: Pathogenic

Review Status: reviewed by expert panel

Collection Method: research

Variant causing splicing aberration predicted to interrupt known functional domains: full inactivation of variant allele

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Jul 17, 2023

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

An algorithm developed to predict the effect of missense changes on protein structure and function (PolyPhen-2) suggests that this variant is likely to be disruptive. ClinVar contains an entry for this variant (Variation ID: 90150). This variant has been observed in individual(s) with clinical features of Lynch syndrome (PMID: 22736432). This variant is not present in population databases (gnomAD no frequency). This sequence change affects codon 102 of the MLH1 mRNA. It is a 'silent' change, meaning that it does not change the encoded amino acid sequence of the MLH1 protein. RNA analysis indicates that this variant induces altered splicing and likely results in a shortened protein product. Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant results in skipping of 3, but is expected to preserve the integrity of the reading-frame (PMID: 22736432). For these reasons, this variant has been classified as Pathogenic. This variant disrupts a region of the MLH1 protein in which other variant(s) (p.Thr82Ala) have been determined to be pathogenic (PMID: 10422993, 17510385, 19690142, 20587412, 21239990, 21404117, 22736432, 23403630, 26300997, 28514183). This suggests that this is a clinically significant region of the protein, and that variants that disrupt it are likely to be disease-causing.

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	1.0
BayesDel_addAF	Pathogenic	0.54	D
BayesDel_noAF	Pathogenic	0.54
CADD	Pathogenic	34
DANN	Uncertain	1.0
DEOGEN2	Pathogenic	0.95	D
Eigen	Pathogenic	1.1
Eigen_PC	Pathogenic	0.97
FATHMM_MKL	Pathogenic	0.99	D
LIST_S2	Pathogenic	0.99	D
M_CAP	Pathogenic	0.34	D
MetaRNN	Pathogenic	0.99	D
MetaSVM	Uncertain	0.59	D
MutationAssessor	Pathogenic	4.9	H
PhyloP100		6.3
PrimateAI	Uncertain	0.68	T
PROVEAN	Uncertain	-2.8	D
REVEL	Pathogenic	0.88
Sift	Pathogenic	0.0	D
Sift4G	Pathogenic	0.0	D
Vest4		0.93
ClinPred		1.0	D
GERP RS		5.6
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		2.7
Varity_R		0.96
gMVP		0.93
Mutation Taster		=2/98	disease causing (ClinVar)

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.96
SpliceAI score (max)		0.93		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.93		Position offset: 0

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

Other links and lift over

dbSNP: rs63751665; hg19: chr3-37042544;