Submissions for variant NM_000249.4(MLH1):c.677+3A>G

dbSNP: rs267607780

Total submissions: 11

Submitter	RCV	SCV	Clinical significance	Condition	Last evaluated	Review status	Method	Comment
International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	RCV000075806	SCV000106820	pathogenic	Lynch syndrome	2013-09-05	reviewed by expert panel	research	Variant causes splicing aberration: full inactivation of variant allele
Mayo Clinic Laboratories, Mayo Clinic	RCV000201996	SCV000257112	pathogenic	not provided	2020-03-30	criteria provided, single submitter	clinical testing	PVS1, PP4, PP5
Ambry Genetics	RCV000222833	SCV000276798	pathogenic	Hereditary cancer-predisposing syndrome	2023-07-17	criteria provided, single submitter	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.
GeneDx	RCV000201996	SCV000568565	pathogenic	not provided	2020-07-28	criteria provided, single submitter	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)
Eurofins Ntd Llc (ga)	RCV000201996	SCV000702138	pathogenic	not provided	2016-11-04	criteria provided, single submitter	clinical testing
Color Diagnostics, LLC DBA Color Health	RCV000222833	SCV000905466	pathogenic	Hereditary cancer-predisposing syndrome	2023-09-06	criteria provided, single submitter	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.
Invitae	RCV000812851	SCV000953179	pathogenic	Hereditary nonpolyposis colorectal neoplasms	2023-11-08	criteria provided, single submitter	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 disrupted 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). 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 introduces a premature termination codon (PMID: 12655562, 15365996, 19669161; Invitae). The resulting mRNA is expected to undergo nonsense-mediated decay. For these reasons, this variant has been classified as Pathogenic.
Revvity Omics, Revvity	RCV000201996	SCV003821595	pathogenic	not provided	2022-12-07	criteria provided, single submitter	clinical testing
Baylor Genetics	RCV003466960	SCV004193018	pathogenic	Colorectal cancer, hereditary nonpolyposis, type 2	2023-03-09	criteria provided, single submitter	clinical testing
All of Us Research Program, National Institutes of Health	RCV000075806	SCV004822619	pathogenic	Lynch syndrome	2023-10-23	criteria provided, single submitter	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.
Department of Pathology and Laboratory Medicine, Sinai Health System	RCV001353985	SCV000592368	pathogenic	Carcinoma of colon		no assertion criteria provided	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.