Submissions for variant NM_000249.4(MLH1):c.1988A>G (p.Glu663Gly)

dbSNP: rs63751682

Total submissions: 4

Submitter	RCV	SCV	Clinical significance	Condition	Last evaluated	Review status	Method	Comment
GeneDx	RCV000218482	SCV000279599	uncertain significance	not provided	2018-02-27	criteria provided, single submitter	clinical testing	This variant is denoted MLH1 c.1988A>G at the cDNA level. Although the nucleotide substitution results in the change of a Glutamic Acid to a Glycine at codon 663 and is called Glu663Gly in the literature, we are using only the nucleotide nomenclature to refer to the variant since the defect is likely to be one of splicing rather than a resulting missense variant. Located in the second to last nucleotide of exon 17, this variant is predicted to weaken the natural splice donor site. An RT-PCR study reported skipping of exon 17 in an individual with MLH1 c.1988A>G who had colon cancer showing microsatellite instability, absence of MLH1 protein, and loss of the wild type allele; however, the relative amount of MLH1 del17 transcript compared to full-length transcript was not quantified in this patient (Dieumegard 2000). Because MLH1 del17 is a known naturally occurring isoform of MLH1, the contribution of MLH1 c.1988A>G to the presence of the aberrant transcript in Dieumegard et al.?s report is not clear (Genuardi 1998, Thompson 2015). MLH1 c.1988A>G has also been reported in another individual fulfilling Amsterdam criteria for Lynch syndrome, but no additional splicing studies were performed in this individual (Salehi 2009). Although in vitro functional studies show mismatch repair activity, MLH1 protein expression, and PMS2 stabilization comparable to wild-type, these studies would not assess an effect on splicing (Takahashi 2007, Wanat 2007, Kosinski 2010). MLH1 c.1988A>G was not observed in large population cohorts (Lek 2016). The nucleotide which is altered, an adenine (A) at base 1988, is conserved across species. This position and MLH1 exon 17 is located in the region of interaction with PMS2, MLH3, and PMS1 (Raevaara 2005, Kansikas 2011, Andersen 2012). According to the International Society for Gastrointestinal Hereditary Tumours (InSiGHT) database, MLH1 c.1988A>G is an uncertain variant due to insufficient evidence (Thompson 2014). Based on currently available evidence, it is unclear whether MLH1 c.1988A>G is a pathogenic or benign variant. We consider it to be a variant of uncertain significance.
Ambry Genetics	RCV001013918	SCV001174562	pathogenic	Hereditary cancer-predisposing syndrome	2023-03-30	criteria provided, single submitter	clinical testing	The c.1988A>G pathogenic mutation (also known as p.E663G), located in coding exon 17 of the MLH1 gene, results from an A to G substitution at nucleotide position 1988. The glutamic acid at codon 663 is replaced by glycine, an amino acid with similar properties. This variant has been identified in multiple probands who met Amsterdam I/II criteria for Lynch syndrome and tumor demonstrated high microsatellite instability with loss of MLH1 and/or PMS2 expression by immunohistochemistry (Ambry internal data). This nucleotide position is highly conserved in available vertebrate species. This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). At the protein level, in vitro studies showed MMR activity of 69.7% and relative MLH1 expression of >75%. It also showed a positive mutator effect in all three yeast assays similar to wildtype MLH1 (Takahashi M et al. Cancer Res, 2007 May;67:4595-604). Additional studies have also shown this alteration to have an insignificant impact on MLH1 expression and MMR activity (Wanat JJ et al. Hum. Mol. Genet., 2007 Feb;16:445-52; Kosinski J et al. Hum Mutat, 2010 Aug;31:975-82; Abildgaard AB et al. Elife, 2019 11;8). At the RNA level, in silico splice site analysis predicts that this alteration will result in the creation or strengthening of a novel splice donor site. RNA studies have demonstrated that this alteration results in abnormal splicing in the set of samples tested (Dieumegard B et al. Br. J. Cancer, 2000 Feb;82:871-80, Ambry internal data). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation.
Labcorp Genetics (formerly Invitae), Labcorp	RCV001360197	SCV001556102	pathogenic	Hereditary nonpolyposis colorectal neoplasms	2023-09-21	criteria provided, single submitter	clinical testing	For these reasons, this variant has been classified as Pathogenic. This variant disrupts a region of the MLH1 protein in which other variant(s) (p.Pro654Leu) have been determined to be pathogenic (PMID: 16083711, 17510385, 20533529, 21404117). This suggests that this is a clinically significant region of the protein, and that variants that disrupt it are likely to be disease-causing. Studies have shown that this missense change results in skipping of exon 17 , but is expected to preserve the integrity of the reading-frame (Invitae). Experimental studies have shown that this missense change does not substantially affect MLH1 function (PMID: 17210669, 17510385, 20533529, 31697235). An algorithm developed to predict the effect of missense changes on protein structure and function (PolyPhen-2) suggests that this variant is likely to be disruptive. ClinVar contains an entry for this variant (Variation ID: 89971). This missense change has been observed in individual(s) with clinical features of Lynch syndrome (PMID: 10732761; Invitae). This variant is not present in population databases (gnomAD no frequency). This sequence change replaces glutamic acid, which is acidic and polar, with glycine, which is neutral and non-polar, at codon 663 of the MLH1 protein (p.Glu663Gly). RNA analysis indicates that this missense change induces altered splicing and likely results in a shortened protein product.
Myriad Genetics, Inc.	RCV005246622	SCV005897253	likely pathogenic	Colorectal cancer, hereditary nonpolyposis, type 2	2024-10-14	criteria provided, single submitter	clinical testing	This variant is considered likely pathogenic. mRNA analysis has demonstrated abnormal mRNA splicing occurs [Myriad internal data].