3-37012102-A-G

Variant names:

• chr3-37012102-A-G
• rs267607780
• NM_000249.4:c.677+3A>G

Variant summary

Our verdict is Pathogenic. The variant received 11 ACMG points: 11P and 0B. PM2 PP3 PP5_Very_Strong

The NM_000249.4(MLH1):​c.677+3A>G variant causes a splice region, intron change. The variant allele was found at a frequency of 0.00000069 in 1,449,200 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a benign outcome for this variant. 2/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Pathogenic (★★★).

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 6.9e-7 (0 hom.)
• Consequence: MLH1 NM_000249.4 splice_region, intron
• Scores: Splicing: ADA: 0.9988
• Clinical Significance: Pathogenic reviewed by expert panel P:11
• Conservation: PhyloP100: 5.58
• Publications: 10 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 11 ACMG points.

• PM2: Very rare variant in population databases, with high coverage
• PP3: Splicing predictors support a deleterious effect. Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF. Scorers claiming Uncertain: max_spliceai. No scorers claiming Benign.
• PP5: Variant 3-37012102-A-G is Pathogenic according to our data. Variant chr3-37012102-A-G is described in ClinVar as Pathogenic. ClinVar VariationId is 90315.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.677+3A>G		splice_region_variant, intron_variant	Intron 8 of 18	ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.677+3A>G		splice_region_variant, intron_variant	Intron 8 of 18	1	NM_000249.4	ENSP00000231790.3

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome AF: 6.90e-7 AC: 1 AN: 1449200 Hom.: 0 Cov.: 29 AF XY: 0.00000139 AC XY: 1 AN XY: 721734

African (AFR) AF: 0.00 AC: 0 AN: 33312

American (AMR) AF: 0.00 AC: 0 AN: 44714

Ashkenazi Jewish (ASJ) AF: 0.00 AC: 0 AN: 26070

East Asian (EAS) AF: 0.00 AC: 0 AN: 39668

South Asian (SAS) AF: 0.00 AC: 0 AN: 86070

European-Finnish (FIN) AF: 0.00 AC: 0 AN: 53394

Middle Eastern (MID) AF: 0.00 AC: 0 AN: 5750

European-Non Finnish (NFE) AF: 9.09e-7 AC: 1 AN: 1100188

Other (OTH) AF: 0.00 AC: 0 AN: 60034

GnomAD4 genome Cov.: 32

Alfa AF: 0.00 Hom.: 0

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:11

Revision: reviewed by expert panel

Submissions by phenotype

not provided Pathogenic:4

Dec 07, 2022

Revvity Omics, Revvity

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Nov 04, 2016

Eurofins Ntd Llc (ga)

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Mar 30, 2020

Mayo Clinic Laboratories, Mayo Clinic

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

PVS1, PP4, PP5 -

Jul 28, 2020

GeneDx

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Non-canonical splice site variant demonstrated to cause exon 8 skipping, which would be predicted to result in a null allele (Krger 2003, Naruse 2009, Betz 2010) Not observed in large population cohorts (Lek 2016) Classified as pathogenic by a well-established clinical consortium and/or database Observed in several individuals with colorectal cancer, including those with tumor studies consistent with pathogenic variants in MLH1 (Montera 2000, Krger 2003, Casey 2005, Mangold 2005, Rosty 2016) This variant is associated with the following publications: (PMID: 30719162, 31433215, 28248820, 19685281, 12655562, 19669161, 15713769, 24090359, 15365996, 25479140, 16216036, 15849733, 26895986, 10882759, 25525159, 23741719) -

Lynch syndrome Pathogenic:2

Oct 23, 2023

All of Us Research Program, National Institutes of Health

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant causes an A>G nucleotide substitution at the +3 position of intron 8 of the MLH1 gene . Functional RNA studies have shown that this variant causes an out-of-frame skipping of exon 8, creating a frameshift and premature translation stop signal and expected to result in an absent or non-functional protein product (PMID: 12655562, 15713769, 19685281, 19669161, 24090359). This variant has been reported in at least eight individuals affected with Lynch syndrome (PMID: 10882759, 12655562, 15849733, 15713769, 19685281, 19669161, 26895986). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Loss of MLH1 function is a known mechanism of disease (clinicalgenome.org). Based on the available evidence, this variant is classified as Pathogenic. -

Sep 05, 2013

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Significance: Pathogenic

Review Status: reviewed by expert panel

Collection Method: research

Variant causes splicing aberration: full inactivation of variant allele -

Hereditary cancer-predisposing syndrome Pathogenic:2

Jul 17, 2023

Ambry Genetics

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The c.677+3A>G pathogenic mutation, results from an A to G substitution three nucleotides downstream from coding exon 8 in the MLH1 gene. This mutation has been identified in a family meeting Amsterdam criteria for HNPCC/Lynch syndrome as well as a 44 year-old patient diagnosed with rectal cancer whose tumor analysis showed microsatellite instability and absence of MLH1 protein on immunohistochemistry (Casey G et al JAMA. 2005 Feb 16;293(7):799-809; Kruger S et al Hum Mutat. 2003 Apr;21(4):445-6). This alteration has been associated with exon-skipping, premature protein truncation, and a lack of full-length transcript production from the variant allele (Betz B et al J Cancer Res Clin Oncol. 2010 Jan;136(1):123-34; Naruse H et al Fam Cancer. 2009;8(4):509-17). This alteration has been classified as pathogenic by multifactorial analysis, which integrates the following lines of evidence to produce a quantitative likelihood of pathogenicity: in silico prediction models, segregation with disease, tumor characteristics, mutation co-occurrence, and functional assay results (Thompson B et al. Nat Genet. 2014 Feb;46(2):107-15; available at [www.insight-group.org/variants/classifications/]). Of note, this mutation is also designated as IVS8+3 A>G in published literature. Based on the available evidence, c.677+3A>G is classified as a pathogenic mutation. -

Sep 06, 2023

Color Diagnostics, LLC DBA Color Health

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant causes an A>G nucleotide substitution at the +3 position of intron 8 of the MLH1 gene . Functional RNA studies have shown that this variant causes an out-of-frame skipping of exon 8, creating a frameshift and premature translation stop signal and expected to result in an absent or non-functional protein product (PMID: 12655562, 15713769, 19685281, 19669161, 24090359). This variant has been reported in at least eight individuals affected with Lynch syndrome (PMID: 10882759, 12655562, 15849733, 15713769, 19685281, 19669161, 26895986). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Loss of MLH1 function is a known mechanism of disease (clinicalgenome.org). Based on the available evidence, this variant is classified as Pathogenic. -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:1

Mar 09, 2023

Baylor Genetics

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Carcinoma of colon Pathogenic:1

-

Department of Pathology and Laboratory Medicine, Sinai Health System

Significance: Pathogenic

Review Status: no assertion criteria provided

Collection Method: clinical testing

The MLH1 c.677+3A>G variant was identified in 5 of 4150 proband chromosomes (frequency: 0.001) from individuals or families with Lynch Syndrome, and was not identified in 146 control chromosomes from healthy individuals (Casey 2005, Mangold 2005, Kruger 2003, Montera 2000). The variant was also identified in dbSNP (ID: rs267607780) as “likely pathogenic” “pathogenic” and “uncertain significance”, Clinvitae database (as pathogenic), “Mismatch Repair Genes Variant Database”, “MMR Gene Unclassified Variants Database”, InSiGHT Colon Cancer Gene Variant Database (LOVD) (11x as pathogenic), ClinVar database (as pathogenic, by InSIGHT, Ambry Genetics, Mayo Clinic). The variant was not found in COSMIC, Zhejiang Colon Cancer Database (LOVD), GeneInsight - COGR database, and UMD. The c.677+3A>G variant is located in the 5' splice region but does not affect the invariant +1 and +2 positions. However, positions +3 to +6 are part of the splicing consensus sequence and variants involving these positions sometimes affect splicing. In addition, 3 of 5 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) predict a greater than 10% difference in splicing. Mulitple studies found patients with the variant to be MSI-H and in both in silico prediction models and in vitro RT-PCR studies, the variant was found to cause aberrant splicing leading to exon 8 skipping and complete loss of expression of the MLH1 protein (Betz 2010, Casey 2005, Mangold 2005, Kruger 2003, Montera 2000, Mangold 2005). In summary, based on the above information, this variant meets our laboratory’s criteria to be classified as pathogenic. -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Dec 02, 2024

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Pathogenic

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This sequence change falls in intron 8 of the MLH1 gene. It does not directly change the encoded amino acid sequence of the MLH1 protein. RNA analysis indicates that this variant induces altered splicing and may result in an absent or altered protein product. This variant is not present in population databases (gnomAD no frequency). This variant has been observed in individuals with Lynch syndrome (PMID: 12655562, 15289847, 15365996, 15713769, 19669161, 26895986). Invitae Evidence Modeling of clinical and family history, age, sex, and reported ancestry of multiple individuals with this MLH1 variant has been performed. This variant is expected to be pathogenic with a positive predictive value of at least 99%. This is a validated machine learning model that incorporates the clinical features of 1,370,736 individuals referred to our laboratory for MLH1 testing. This variant is also known as IVS8+3A>G. ClinVar contains an entry for this variant (Variation ID: 90315). 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 exon 8 and retention of intron 7, and produces a non-functional protein and/or introduces a premature termination codon (PMID: 12655562, 15365996, 19669161; internal data). For these reasons, this variant has been classified as Pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.19
CADD	Uncertain	25
DANN	Benign	0.96
PhyloP100		5.6
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.1
Mutation Taster		=0/100	disease causing (ClinVar)

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.96
SpliceAI score (max)		0.26		Details are displayed if max score is > 0.2
DS_DG_spliceai		0.26		Position offset: -11
DS_DL_spliceai		0.25		Position offset: -3

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

Other links and lift over

dbSNP: rs267607780; hg19: chr3-37053593;