Submissions for variant NM_000249.4(MLH1):c.2246T>C (p.Leu749Pro)

dbSNP: rs267607894

Total submissions: 11

Submitter	RCV	SCV	Clinical significance	Condition	Last evaluated	Review status	Method	Comment
International Society for Gastrointestinal Hereditary Tumours (InSiGHT)	RCV000075583	SCV000106580	pathogenic	Lynch syndrome	2013-09-05	reviewed by expert panel	research	Multifactorial likelihood analysis posterior probability >0.99
Ambry Genetics	RCV000216146	SCV000278167	pathogenic	Hereditary cancer-predisposing syndrome	2022-05-11	criteria provided, single submitter	clinical testing	The p.L749P pathogenic mutation (also known as c.2246T>C) is located in coding exon 19 of the MLH1 gene. This results from a T to C substitution at nucleotide position 2246 which is 25 nucleotides from the end of the protein. The leucine at codon 749 is replaced by proline, an amino acid with a few similar properties. This pathogenic mutation has been identified in multiple patients with Lynch syndrome whose tumors all exhibited MSI instability, but the absence of MLH1 staining on IHC was not always noted (Dudley B et al. Am. J. Surg. Pathol. 2015 Aug;39(8):1114-20; Perera S et al. J Mol Diagn. 2010 Nov; 12(6): 757-64; Colombino M et al. Ann Oncol. 2003 Oct; 14(10):1530-6; Losi L et al. Am J Gastroenterol. 2005 Oct; 100(10):2280-7; Pedroni M et. al. Dis. Markers. 2007; 23(3):179-87). In one study, authors performed multiple independent transfection experiments and reproducibly showed MLH1 expression levels similar to wild type but corresponding PMS2 levels were similar to those achieved when PMS2 was expressed in the absence of MLH1 (reduction was statistically significant p>0.05). They demonstrated this alteration disturbs MLH1-PMS2 dimerization, as it is located within the proposed dimerization interface, a conserved hydrophobic surface area. Authors suggest the pathogenic effect of this mutation is due to direct interference with dimerization and severely compromised mismatch repair activity (Kosinski J et al. Hum Mutat. 2010 Aug; 31(8):975-82). In another study, cells with the MLH1 p.L749P pathogenic mutation had protein expression levels of greater than 70% when compared to wild-type; however the repair activity was less than 30% when compared to wild-type (Hinrichsen I et al. Clin Cancer Res. 2013 May; 19(9):2432-41). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). This amino acid position is highly conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Based on the available evidence, this alteration is classified as a disease-causing mutation.
Invitae	RCV000627694	SCV000543529	pathogenic	Hereditary nonpolyposis colorectal neoplasms	2023-12-15	criteria provided, single submitter	clinical testing	This sequence change replaces leucine, which is neutral and non-polar, with proline, which is neutral and non-polar, at codon 749 of the MLH1 protein (p.Leu749Pro). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individuals with clinical features of Lynch syndrome and/or Lynch syndrome (PMID: 14504054, 16181381, 20864636, 21286667, 23752102, 26557847; Invitae). ClinVar contains an entry for this variant (Variation ID: 90097). Advanced modeling of protein sequence and biophysical properties (such as structural, functional, and spatial information, amino acid conservation, physicochemical variation, residue mobility, and thermodynamic stability) performed at Invitae indicates that this missense variant is expected to disrupt MLH1 protein function with a positive predictive value of 95%. Experimental studies have shown that this missense change affects MLH1 function (PMID: 20533529, 22736432, 23403630). For these reasons, this variant has been classified as Pathogenic.
GeneDx	RCV000478074	SCV000565172	pathogenic	not provided	2018-01-31	criteria provided, single submitter	clinical testing	This pathogenic variant is denoted MLH1 c.2246T>C at the cDNA level, p.Leu749Pro (L749P) at the protein level, and results in the change of a Leucine to a Proline (CTA>CCA). This variant has been reported in individuals with Lynch-associated cancers, including several who met Amsterdam criteria and/or whose tumor testing demonstrated absence of MLH1 on immunohistochemistry (IHC) and/or microsatellite instability (Colombino 2003, Losi 2005, de Leon 2007, Pedroni 2007, Perera 2010, Colombino 2011, Raymond 2013, Dudley 2015, Magnani 2015). In vitro functional studies revealed this variant to have reduced mismatch repair activity compared to wild-type (Kosinki 2010, Borras 2012, Hinrichsen 2013). Protein expression assays by Kosinski et al. (2010) and Hinrichsen et al. (2013) demonstrate MLH1 Leu749Pro to have MLH1 levels similar to wild-type, but decreased PMS2 levels suggesting a defect in the MLH1-PMS2 heterodimer. Other analyses show significantly decreased expression levels of both MLH1 and PMS2, supporting a defect in stability (Borras 2012). MLH1 Leu749Pro was not observed in large population cohorts (Lek 2016). This variant is not located in a known functional domain. In silico analysis, which includes protein predictors and evolutionary conservation, supports a deleterious effect. Based on currently available evidence, we consider MLH1 Leu749Pro to be pathogenic.
Quest Diagnostics Nichols Institute San Juan Capistrano	RCV000478074	SCV000888178	pathogenic	not provided	2016-06-01	criteria provided, single submitter	clinical testing
Color Diagnostics, LLC DBA Color Health	RCV000216146	SCV000905472	pathogenic	Hereditary cancer-predisposing syndrome	2022-08-22	criteria provided, single submitter	clinical testing	This missense variant replaces leucine with proline at codon 749 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 studies have shown that this variant decreases protein stability and impairs DNA mismatch repair activity (PMID: 20533529, 22736432, 23403630, 28767177). This variant has been reported in individuals affected with Lynch Syndrome and colorectal cancer (PMID: 14504054, 16181381, 20864636, 21286667, 23752102, 25871621, 26557847). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Pathogenic.
Revvity Omics, Revvity	RCV000478074	SCV002024362	pathogenic	not provided	2019-10-04	criteria provided, single submitter	clinical testing
Neuberg Centre For Genomic Medicine, NCGM	RCV001823108	SCV002072850	pathogenic	Colorectal cancer, hereditary nonpolyposis, type 2		criteria provided, single submitter	clinical testing	The missense variant p.L749P in MLH1 (NM_000249.3) has been previously reported in patients with Lynch syndrome (Raymond V et al, 2013).In vitro functional studies revealed this variant to have reduced mismatch repair activity compared to wild-type (Borras 2012; Hinrichsen 2013).Protein expression assays show MLH1 levels similar to wild-type, but decreased PMS2 levels (Hinrichsen 2013). It has been submiited to ClinVar as Pathogenic. The p.L749P variant is novel (not in any individuals) in gnomAD Exomes and is novel (not in any individuals) in 1000 Genomes. There is a moderate physicochemical difference between leucine and proline. The p.L749P missense variant is predicted to be damaging by both SIFT and PolyPhen2. The leucine residue at codon 749 of MLH1 is conserved in all mammalian species. The nucleotide c.2246 in MLH1 is predicted conserved by GERP++ and PhyloP across 100 vertebrates. For these reasons, this variant has been classified as Pathogenic.
Myriad Genetics, Inc.	RCV001823108	SCV004186498	pathogenic	Colorectal cancer, hereditary nonpolyposis, type 2	2023-07-25	criteria provided, single submitter	clinical testing	This variant is considered pathogenic. Functional studies indicate this variant impacts protein function [PMID: 23403630, 22736432, 20533529]. This variant is strongly associated with more severe personal and family histories of cancer, typical for individuals with pathogenic variants in this gene [PMID: 27363726].
Baylor Genetics	RCV001823108	SCV004195107	pathogenic	Colorectal cancer, hereditary nonpolyposis, type 2	2023-09-12	criteria provided, single submitter	clinical testing
Department of Pathology and Laboratory Medicine, Sinai Health System	RCV001355258	SCV001550087	likely pathogenic	Carcinoma of colon		no assertion criteria provided	clinical testing	The MLH1 p.Leu749Pro variant was identified in 5 of 7124 proband chromosomes (frequency: 0.001) from individuals or families with colorectal cancer, or adrenocortical carcinoma (Colombino 2003, Dudley 2015, Pedroni 2007, Raymond 2013). The variant was also identified in dbSNP (ID: rs267607894) as “With Pathogenic allele”, in ClinVar (classified as pathogenic by Ambry Genetics, GeneDx, InSight; as likely pathogenic by Invitae), Mismatch Repair Genes Variant Database, and Insight Hereditary Tumors Database (9x pathogenic). The variant was not identified in Cosmic, MutDB, UMD-LSDB, Zhejiang University Database, or the MMR Gene Unclassified Variants Database. The variant was not identified in the 1000 Genomes Project, the NHLBI GO Exome Sequencing Project, the Exome Aggregation Consortium (August 8th 2016), or the Genome Aggregation Database (Feb 27, 2017). The p.Leu749 residue is conserved across mammals and other organisms, and computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) suggest that the variant may impact the protein; however, this information is not predictive enough to assume pathogenicity. The variant occurs outside of the splicing consensus sequence and in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) do not predict a difference in splicing. The variant caused decreased co-expression of PMS2, which is unstable in the absence of interaction with MLH1, suggesting that this alteration may confer a pathogenic effect (Kosinski 2010, Borras 2012, Dudley 2015). The variant also severely compromised mismatch repair activity (Kosinski 2010). However, the study on allelic expression by Perera (2010) discovered that this variant is not associated with an allelic imbalance; it is likely that this variant affects heterodimerization of MLH1 rather than its expression or stability. In summary, based on the above information the clinical significance of this variant cannot be determined with certainty at this time although we would lean towards a more pathogenic role for this variant. This variant is classified as likely pathogenic.