rs267607733

chr3-37001057-A-G

Variant summary

Our verdict is Uncertain significance. Variant got 1 ACMG points: 1P and 0B. PP3

The NM_000249.4(MLH1):c.306+4A>G variant causes a splice donor region, intron change. The variant allele was found at a frequency of 0.0000332 in 1,564,326 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a benign outcome for this variant. Variant has been reported in ClinVar as Uncertain significance (★★★).

• Frequency:
  • Genomes: 𝑓 0.000013 (0 hom., cov: 32)
  • Exomes 𝑓: 0.000035 (0 hom.)
• Consequence: MLH1 NM_000249.4 splice_donor_region, intron
• Scores: Splicing: ADA: 0.9950
• Clinical Significance: Uncertain significance reviewed by expert panel U:11 B:1
• Conservation: PhyloP100: 6.51

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 Uncertain_significance. Variant got 1 ACMG points.

• PP3: Splicing scoreres supports a deletorius effect: Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF, max_spliceai. No scorers claiming Uncertain. No scorers claiming Benign.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.306+4A>G		splice_donor_region_variant, intron_variant		ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.306+4A>G		splice_donor_region_variant, intron_variant		1	NM_000249.4	P1

Frequencies

GnomAD3 genomes AF: 0.0000131 AC: 2 AN: 152192 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: 0.0000294

Gnomad OTH AF: 0.00

GnomAD3 exomes AF: 0.0000119 AC: 3 AN: 251334 Hom.: 0 AF XY: 0.00 AC XY: 0 AN XY: 135850

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 SAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.0000264

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.0000354 AC: 50 AN: 1412134 Hom.: 0 Cov.: 25 AF XY: 0.0000241 AC XY: 17 AN XY: 705532

Gnomad4 AFR exome AF: 0.00

Gnomad4 AMR exome AF: 0.00

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.00

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.0000469

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome AF: 0.0000131 AC: 2 AN: 152192 Hom.: 0 Cov.: 32 AF XY: 0.0000134 AC XY: 1 AN XY: 74356

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: 0.0000294

Gnomad4 OTH AF: 0.00

Bravo AF: 0.0000113

EpiCase AF: 0.00

EpiControl AF: 0.000119

ClinVar

Significance: Uncertain significance

Submissions summary: Uncertain:11 Benign:1

Revision: reviewed by expert panel

Submissions by phenotype

Hereditary cancer-predisposing syndrome Uncertain:3

Uncertain significance, criteria provided, single submitter	clinical testing	Ambry Genetics	Jul 04, 2023	The c.306+4A>G intronic variant results from an A to G substitution 4 nucleotides after coding exon 3 in the MLH1 gene. This alteration has been reported in 1/1197 individuals from Greece, Romania, and Turkey undergoing evaluation for inherited cancer predisposition (Tsaousis GN et al. BMC Cancer, 2019 Jun;19:535). This variant was also observed in 1/287 patients with hereditary breast and/or ovarian cancer (Caminsky NG et al. Hum Mutat, 2016 07;37:640-52). Furthermore, this variant was identified in 1/199 endometrial cancer patients (Singh AK et al. PLoS One, 2020 Jul;15(7):e0235613). In a functional RNA study, this variant was associated with in-frame exon 3 skipping and activation of a cryptic donor site in exon 3; however, splicing was tested using a mini gene assay and patient RNA was not analyzed (Tournier I et al. Hum. Mutat. 2008 Dec;29:1412-24). RNA studies have demonstrated that this alteration results in abnormal splicing in the set of samples tested (Ambry internal data). However, this alteration has been detected in many probands who do not have a personal or family history that is consistent with or suggestive of hereditary nonpolyposis colorectal cancer (HNPCC)/Lynch syndrome and one of these individuals was diagnosed with microsatellite stable colorectal cancer that demonstrated normal mismatch repair protein expression by immunohistochemistry (Ambry internal data). This nucleotide position is highly conserved in available vertebrate species. In silico splice site analysis predicts that this alteration will weaken the native splice donor site. Since supporting evidence is conflicting at this time, the clinical significance of this alteration remains unclear. -
Uncertain significance, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Apr 28, 2023	This variant causes an A to G nucleotide substitution at the +4 position of intron 3 of the MLH1 gene. To our knowledge, functional studies have not been reported for this variant. Splice site prediction tools are inconclusive regarding the impact of this variant on RNA splicing. This variant has been reported in individuals affected with endometrial cancer and breast cancer (PMID: 26898890, 32634176). This variant has been identified in 3/251334 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. -
Uncertain significance, criteria provided, single submitter	clinical testing	GeneKor MSA	Aug 01, 2018	- -

Lynch syndrome Uncertain:2

Uncertain significance, reviewed by expert panel	curation	International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	Oct 18, 2018	Insufficient evidence: splicing not tested in patient RNA -
Uncertain significance, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	May 16, 2023	This variant causes an A to G nucleotide substitution at the +4 position of intron 3 of the MLH1 gene. To our knowledge, functional studies have not been reported for this variant. Splice site prediction tools are inconclusive regarding the impact of this variant on RNA splicing. This variant has been reported in individuals affected with endometrial cancer and breast cancer (PMID: 26898890, 32634176). This variant has been identified in 3/251334 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:2

Uncertain significance, criteria provided, single submitter	clinical testing	GeneDx	Nov 14, 2022	Not observed at significant frequency in large population cohorts (gnomAD); In silico analysis supports a deleterious effect on splicing; Observed in individuals with suspected Lynch syndrome or familial breast/ovarian cancer (Tournier et al., 2008; Caminsky et al., 2016; Singh et al., 2020); Published functional studies demonstrate an impact on splicing in an ex vivo assay; however, these findings were not confirmed in patient RNA (Tournier et al., 2008); This variant is associated with the following publications: (PMID: 25525159, 26898890, 20858721, 31159747, 18561205, 32634176) -
Uncertain significance, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Sep 09, 2020	- -

Colorectal cancer, hereditary nonpolyposis, type 2 Uncertain:1 Benign:1

Likely benign, criteria provided, single submitter	clinical testing	Myriad Genetics, Inc.	Nov 13, 2023	This variant is considered likely benign. This variant is intronic and is not expected to impact mRNA splicing. This variant is strongly associated with less severe personal and family histories of cancer, typical for individuals without pathogenic variants in this gene [PMID: 27363726]. -
Uncertain significance, criteria provided, single submitter	clinical testing	Baylor Genetics	Oct 26, 2023	- -

not specified Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

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

Dec 11, 2023

Variant summary: MLH1 c.306+4A>G alters a conserved nucleotide located close to a canonical splice site and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. Consensus agreement among computation tools predict no significant impact on normal splicing. At least one publication reports experimental evidence that this variant affects mRNA splicing, resulting in activation of a cryptic donor site in an ex vivo assay (Tournier_2008). However, this was not confirmed in patient RNA. The variant allele was found at a frequency of 1.2e-05 in 251334 control chromosomes (gnomAD). The available data on variant occurrences in the general population are insufficient to allow any conclusion about variant significance. c.306+4A>G has been reported in the literature in individuals affected with a personal or family history of cancer without strong evidence of causality (e.g. Grant_2015, Caminsky_2016, Ring_2016, Tsoaousis_2019, Singh_2020, Bhai_2021). These reports do not provide unequivocal conclusions about association of the variant with Lynch Syndrome. The following publications have been ascertained in the context of this evaluation (PMID: 18561205, 25479140, 26898890, 27443514, 31159747, 32634176, 34326862). Seven submitters, including an expert panel, have cited clinical-significance assessments for this variant to ClinVar after 2014. All submitters classified the variant as uncertain significance. Based on the evidence outlined above, the variant was classified as uncertain significance. -

Breast and/or ovarian cancer Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

CHEO Genetics Diagnostic Laboratory, Children's Hospital of Eastern Ontario

Jan 06, 2023

- -

Hereditary nonpolyposis colorectal neoplasms Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Invitae

Jan 01, 2024

This sequence change falls in intron 3 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 a shortened protein product. This variant is present in population databases (rs267607733, gnomAD 0.003%). This variant has been observed in individual(s) with breast and/or ovarian cancer, endometiral cancer, and/or clinical features of Lynch syndrome (PMID: 18561205, 31159747). ClinVar contains an entry for this variant (Variation ID: 90147). 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 3, but is expected to preserve the integrity of the reading-frame (PMID: 18561205; Invitae). 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
BayesDel_noAF	Benign	-0.22
Cadd	Benign	16
Dann	Benign	0.96

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	0.99
dbscSNV1_RF	Pathogenic	0.78
SpliceAI score (max)		0.61		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.61		Position offset: -4

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

Publications

LitVar

Other links and lift over

dbSNP: rs267607733; hg19: chr3-37042548;