chr3-37014428-A-G

Variant names:

• chr3-37014428-A-G
• rs766711342
• NM_000249.4:c.678-4A>G

Variant summary

Our verdict is Uncertain significance. The variant received 2 ACMG points: 2P and 0B. PP3_Moderate

The NM_000249.4(MLH1):​c.678-4A>G variant causes a splice region, intron change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.0000165 in 1,333,212 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a benign outcome for this variant. 3/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Uncertain significance (★★).

• Frequency:
  • Genomes: not found (cov: 32)
  • Exomes 𝑓: 0.000017 (0 hom.)
• Consequence: MLH1 NM_000249.4 splice_region, intron
• Scores: Splicing: ADA: 0.9998
• Clinical Significance: Uncertain significance criteria provided, multiple submitters, no conflicts U:7
• Conservation: PhyloP100: 1.69
• Publications: 2 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: Uncertain_significance. The variant received 2 ACMG points.

• PP3: Splicing predictors support a deleterious effect. Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF, max_spliceai. No scorers claiming Uncertain. No scorers claiming Benign.

Variant Effect in Transcripts

ACMG analysis was done for transcript: NM_000249.4. You can select a different transcript below to see updated ACMG assignments.

RefSeq Transcripts

Selected	Gene	Transcript	Tags	HGVSc	HGVSp	Effect	Exon Rank	Protein	UniProt
	MLH1	NM_000249.4	MANE Select	c.678-4A>G		splice_region intron	N/A	NP_000240.1
	MLH1	NM_001354628.2		c.678-4A>G		splice_region intron	N/A	NP_001341557.1
	MLH1	NM_001354629.2		c.579-4A>G		splice_region intron	N/A	NP_001341558.1

Ensembl Transcripts

Selected	Gene	Transcript	Tags	HGVSc	HGVSp	Effect	Exon Rank	Protein	UniProt
	MLH1	ENST00000231790.8	TSL:1 MANE Select	c.678-4A>G		splice_region intron	N/A	ENSP00000231790.3
	MLH1	ENST00000456676.7	TSL:1	c.678-4A>G		splice_region intron	N/A	ENSP00000416687.3
	MLH1	ENST00000413740.2	TSL:1	c.678-4A>G		splice_region intron	N/A	ENSP00000416476.2

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD2 exomes AF: 0.0000120 AC: 3 AN: 250318 AF XY: 0.00000739

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.0000265

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.0000165 AC: 22 AN: 1333212 Hom.: 0 Cov.: 21 AF XY: 0.0000179 AC XY: 12 AN XY: 670364

African (AFR) AF: 0.00 AC: 0 AN: 31002

American (AMR) AF: 0.00 AC: 0 AN: 44482

Ashkenazi Jewish (ASJ) AF: 0.00 AC: 0 AN: 25356

East Asian (EAS) AF: 0.00 AC: 0 AN: 39092

South Asian (SAS) AF: 0.00 AC: 0 AN: 83478

European-Finnish (FIN) AF: 0.00 AC: 0 AN: 53362

Middle Eastern (MID) AF: 0.00 AC: 0 AN: 5480

European-Non Finnish (NFE) AF: 0.0000221 AC: 22 AN: 994904

Other (OTH) AF: 0.00 AC: 0 AN: 56056

GnomAD4 genome Cov.: 32

Alfa AF: 0.00 Hom.: 0

ClinVar

Significance: Uncertain significance

Submissions summary: Uncertain:7

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Hereditary cancer-predisposing syndrome Uncertain:2

Jun 29, 2025

Ambry Genetics

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The c.678-4A>G intronic variant results from an A to G substitution 4 nucleotides upstream from coding exon 9 in the MLH1 gene. This nucleotide position is highly conserved in available vertebrate species. This variant was detected in 1/104 cases of familial cancer patients in an Italian cohort (Paduano F et al. Genes (Basel), 2022 Jul;13). In silico splice site analysis predicts that this alteration may weaken the native splice acceptor site and will result in the creation or strengthening of a novel splice acceptor site. RNA studies have demonstrated this alteration results in the in-frame insertion of one amino acid; however, the exact functional impact of this inserted amino acid is unknown at this time (Ambry internal data). Since supporting evidence is limited at this time, the clinical significance of this alteration remains unclear.

Apr 06, 2023

Color Diagnostics, LLC DBA Color Health

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant causes an A to G nucleotide substitution at the -4 position of intron 8 of the MLH1 gene. Splice site prediction tools predict that this variant may have a significant impact on RNA splicing. Although this prediction has not been confirmed in published RNA studies. To our knowledge, functional studies have not been reported for this variant. This variant has been reported in an individual affected with pancreatic cancer (PMID: 35886069). This variant has been identified in 3/250318 chromosomes 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.

Colorectal cancer, hereditary nonpolyposis, type 2 Uncertain:1

Sep 04, 2023

Baylor Genetics

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Muir-Torré syndrome;C1333991:Colorectal cancer, hereditary nonpolyposis, type 2;C5399763:Mismatch repair cancer syndrome 1 Uncertain:1

May 23, 2024

Fulgent Genetics, Fulgent Genetics

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Lynch syndrome Uncertain:1

May 04, 2023

All of Us Research Program, National Institutes of Health

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant causes an A to G nucleotide substitution at the -4 position of intron 8 of the MLH1 gene. Splice site prediction tools predict that this variant may have a significant impact on RNA splicing. Although this prediction has not been confirmed in published RNA studies. To our knowledge, functional studies have not been reported for this variant. This variant has been reported in an individual affected with pancreatic cancer (PMID: 35886069). This variant has been identified in 3/250318 chromosomes 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.

not provided Uncertain:1

Aug 27, 2023

GeneDx

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports a deleterious effect on splicing; Observed in an individual with pancreatic cancer (Paduano et al., 2022); This variant is associated with the following publications: (PMID: 35886069)

Hereditary nonpolyposis colorectal neoplasms Uncertain:1

Oct 08, 2024

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Uncertain significance

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 likely results in the gain of 1 amino acid residue(s), but is expected to preserve the integrity of the reading-frame. This variant is present in population databases (rs766711342, gnomAD 0.003%). This variant has not been reported in the literature in individuals affected with MLH1-related conditions. ClinVar contains an entry for this variant (Variation ID: 234545). Studies have shown that this variant results in the activation of a cryptic splice site in intron 8 (internal data). 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 v4.9

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.23
CADD	Benign	22
DANN	Benign	0.92
PhyloP100		1.7

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.92
SpliceAI score (max)		0.62		Details are displayed if max score is > 0.2
DS_AG_spliceai		0.62		Position offset: 1
DS_AL_spliceai		0.39		Position offset: 4

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

Other links and lift over

dbSNP: rs766711342; hg19: chr3-37055919;