3-37012099-G-T
Variant summary
Our verdict is Pathogenic. Variant got 16 ACMG points: 16P and 0B. PM2PM5PP3_StrongPP5_Very_Strong
The NM_000249.4(MLH1):c.677G>T(p.Arg226Leu) variant causes a missense, splice region change involving the alteration of a conserved nucleotide. The variant allele was found at a frequency of 0.000000689 in 1,452,078 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a pathogenic outcome for this variant. 2/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Likely pathogenic (★★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. R226?) has been classified as Likely pathogenic.
Frequency
Consequence
NM_000249.4 missense, splice_region
Scores
Clinical Significance
Conservation
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ACMG classification
Verdict is Pathogenic. Variant got 16 ACMG points.
Transcripts
RefSeq
Ensembl
Frequencies
GnomAD3 genomes
GnomAD4 exome AF: 6.89e-7 AC: 1AN: 1452078Hom.: 0 Cov.: 30 AF XY: 0.00 AC XY: 0AN XY: 722968
GnomAD4 genome
ClinVar
Submissions by phenotype
not provided Pathogenic:4Uncertain:1
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This variant has been reported in individuals affected with Lynch syndrome or related cancers in the published literature (PMID: 18772310 (2008), 20223024 (2006), 16830052 (2006), 12655568 (2003), 8566964 (1996)). Additionally, functional studies have determined that this variant interferes with normal MLH1 mRNA splicing and is damaging to the mismatch repair (MMR) activity of the MLH1 protein (PMID: 29505604 (2018), 18561205 (2008), 17510385 (2007)). Based on the available information, the variant is classified as pathogenic. -
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This variant is denoted MLH1 c.677G>T at the cDNA level. Located in the last nucleotide of exon 8, it destroys a natural splice site and causes abnormal splicing. Multiple studies report, but do not quantify, aberrant splicing caused by this variant that results in skipping of exon 8 (Maliaka 1996, Kurzawski 2006, Hardt 2011). This is concordant with multiple protein and RNA analyses of an alternate variant at this position, MLH1 c.677G>A, which have consistently demonstrated that it causes complete skipping of exon 8 (Leung 1998, Sharp 2004, Pagenstecher 2006, Tournier 2008). In addition, in vitro functional assays of MLH1 c.677G>T showed reduced mismatch repair activity and possibly reduced protein expression (Takahashi 2007). MLH1 c.677G>T has been observed in multiple individuals with early-onset colorectal, endometrial, or gastric cancer whose family histories fulfilled Bethesda or Amsterdam Criteria for Hereditary Nonpolyposis Colorectal Cancer, but also in a sporadic gastric tumor (Maliaka 1996, Evans 2001, Kurzawski 2002, Bartosova 2003, Wagner 2003, Kurzawski 2006, Zavodna 2006, Alemayehu 2008, Hardt 2011, Schofield 2012, Raskin 2017, Martin-Morales 2018). Tumor testing in many of these individuals showed microsatellite instability and loss of MLH1 protein expression, and Bujalkova et al. (2008) found tumoral loss of heterozygosity. In addition, MLH1 c. 677G>T co-occurred with two other pathogenic variants, one in BRCA2 and one in MSH6, in an individual with early-onset endometrial cancer and bilinear family history (Gong 2012). Although the nucleotide substitution results in the change of an Arginine to a Leucine at codon 226, and is called Arg226Leu in the literature, we are using only the nucleotide nomenclature to refer to the variant since the defect is determined to be one of splicing rather than a resulting missense variant. MLH1 c.677G>T was not observed in large population cohorts (Lek 2016). The nucleotide which is altered, a guanine (G) at base 677, is conserved across species. Based on the current evidence, we consider this variant to be pathogenic. -
Hereditary cancer-predisposing syndrome Pathogenic:4
This missense variant replaces arginine with leucine at codon 226 of the MLH1 protein. Computational prediction suggests that this variant may have deleterious impact on protein structure and function (internally defined REVEL score threshold >= 0.7, PMID: 27666373). Functional RNA studies or assays of patient RNA or cDNA have shown that this variant causes exon 8 deletion predicted to result in a premature truncation (PMID: 15300854,16341550, 18561205, 29505604). The variant protein in HCT116 cells was reported to have 32% of wild-type DNA mismatch repair (PMID: 17510385). This variant has been reported in individuals affected with Lynch syndrome (PMID: 8566964, 15300854, 16341550 16830052, 20223024, 21034533, 30256826). 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. -
This variation is a single nucleotide substitution, resulting in the replacement of Arginine with Leucine at codon 226 of the MLH1 protein. The arginine residue is conserved among species and is located in a functional domain of the protein. There is a large physiochemical difference between leucine and leucine (Grantham Score 102).This variant has been reported in the literature in in multiple individuals affected with Lynch syndrome (PMID: 18383312, PMID: 20223024, PMID: 16830052, PMID: 12655568). Loss of MLH1 protein and microsatellite instability has been detected in many of these cases in the tumor tissue (PMID: 18772310). Algorithms developed to predict the effect of missense changes on protein structure and function suggest that this variant may be damaging to the protein. In vitro functional assays showed reduced mismatch repair activity and possibly reduced protein expression (PMID: 17510385 ).The mutation databases (ClinVar, InSiGHT) contain entries for this variant. -
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The c.677G>T pathogenic mutation (also known as p.R226L), located in coding exon 8 of the MLH1 gene, results from a G to T substitution at nucleotide position 677. This change occurs in the last base pair of coding exon 8, which makes it likely to have some effect on normal mRNA splicing. This mutation has been reported in multiple families meeting either Amsterdam diagnostic criteria or Bethesda guidelines for Lynch syndrome (Maliaka YK et al. Hum. Genet., 1996 Feb;97:251-5; Kurzawski G et al. Clin. Genet., 2006 Jan;69:40-7; Alemayehu A et al. Genes Chromosomes Cancer. 2008 Oct;47(10):906-14; Wagner A et al. Am J Hum Genet. 2003 May;72(5):1088-100; Bartosova Z et al. Hum. Mutat. 2003 Apr;21(4):449; Zavodna K et al. Neoplasma 2006 ;53(4):269-76; Evans DG et al. J. Natl. Cancer Inst. 2001 May;93(9):716-7). Functional RNA studies have demonstrated abnormal splicing for this variant with out-of-frame exon 8 skipping (Ambry internal data; Kurzawski G et al. Clin. Genet., 2006 Jan;69:40-7). In addition, in an in vitro complementation assay, the p.R226L variant showed decreased MMR activity (Takahashi et al. Cancer Res. 2007. 67(10):4595-4604). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation. -
Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:2
A heterozygous c. 677G>T (p.Arg226Leu) pathogenic variant in the MLH1 gene was detected in this individual. This variant has been previously described as disease-causing (PMID: 18383312, 16830052, 8566964, 12655568). Therefore, we consider this variant to be likely pathogenic. [alaimo, 2018-01-24] -
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Lynch syndrome Pathogenic:1
G>non-G at last base of exon with first 6 bases of the intron not GTRRGT (splicing aberration reported, but not quantified) -
Lynch-like syndrome Pathogenic:1
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Hereditary nonpolyposis colorectal neoplasms Pathogenic:1
This sequence change replaces arginine, which is basic and polar, with leucine, which is neutral and non-polar, at codon 226 of the MLH1 protein (p.Arg226Leu). RNA analysis indicates that this missense change 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 missense change has been observed in individual(s) with clinical features of Lynch syndrome (PMID: 8566964, 12655568, 16830052, 17510385, 18383312, 20223024; internal data). It has also been observed to segregate with disease in related individuals. 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. ClinVar contains an entry for this variant (Variation ID: 90319). An algorithm developed specifically for the MLH1 gene suggests that this missense change is likely to be deleterious (PMID: 18383312). Experimental studies have shown that this missense change affects MLH1 function (PMID: 17510385). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this missense change results in skipping of exon 8, and produces a non-functional protein and/or introduces a premature termination codon (internal data). This variant disrupts the c.677G nucleotide in the MLH1 gene. Other variant(s) that disrupt this nucleotide have been determined to be pathogenic (PMID: 12547705, 15300854, 16341550, 18561205, 21034533). This suggests that this nucleotide is clinically significant, and that variants that disrupt this position are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic. -
Computational scores
Source:
Splicing
Find out detailed SpliceAI scores and Pangolin per-transcript scores at