Submissions for variant NM_000249.4(MLH1):c.306+4A>G

gnomAD frequency: 0.00001  dbSNP: rs267607733

Total submissions: 12

Submitter	RCV	SCV	Clinical significance	Condition	Last evaluated	Review status	Method	Comment
International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	RCV000075633	SCV000106635	uncertain significance	Lynch syndrome	2018-10-18	reviewed by expert panel	curation	Insufficient evidence: splicing not tested in patient RNA
Ambry Genetics	RCV000130593	SCV000185466	uncertain significance	Hereditary cancer-predisposing syndrome	2023-07-04	criteria provided, single submitter	clinical testing	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.
Invitae	RCV000524290	SCV000543612	uncertain significance	Hereditary nonpolyposis colorectal neoplasms	2024-01-01	criteria provided, single submitter	clinical testing	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.
GeneDx	RCV000482840	SCV000565142	uncertain significance	not provided	2022-11-14	criteria provided, single submitter	clinical testing	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)
Color Diagnostics, LLC DBA Color Health	RCV000130593	SCV000684817	uncertain significance	Hereditary cancer-predisposing syndrome	2023-04-28	criteria provided, single submitter	clinical testing	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.
GeneKor MSA	RCV000130593	SCV000822023	uncertain significance	Hereditary cancer-predisposing syndrome	2018-08-01	criteria provided, single submitter	clinical testing
Quest Diagnostics Nichols Institute San Juan Capistrano	RCV000482840	SCV001470335	uncertain significance	not provided	2020-09-09	criteria provided, single submitter	clinical testing
Myriad Genetics, Inc.	RCV003451129	SCV004186079	likely benign	Colorectal cancer, hereditary nonpolyposis, type 2	2023-11-13	criteria provided, single submitter	clinical testing	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].
Baylor Genetics	RCV003451129	SCV004195027	uncertain significance	Colorectal cancer, hereditary nonpolyposis, type 2	2023-10-26	criteria provided, single submitter	clinical testing
CHEO Genetics Diagnostic Laboratory, Children's Hospital of Eastern Ontario	RCV003492415	SCV004240754	uncertain significance	Breast and/or ovarian cancer	2023-01-06	criteria provided, single submitter	clinical testing
Women's Health and Genetics/Laboratory Corporation of America, LabCorp	RCV003488375	SCV004240801	uncertain significance	not specified	2023-12-11	criteria provided, single submitter	clinical testing	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.
All of Us Research Program, National Institutes of Health	RCV000075633	SCV004824905	uncertain significance	Lynch syndrome	2023-05-16	criteria provided, single submitter	clinical testing	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.