In this work, we have analyzed alterations observed in (suspected) Lynch syndrome patients in the region of the MLH1 gene corresponding to the CTD in the MLH1 protein, in order to test if defects in MLH1-PMS2 dimerization may underlie their (confirmed or questionable) pathogenicity. We have selected 19 alterations () that fall inside and outside the two predicted dimerization interfaces ().
From the six variants located within or in proximity of the predicted dimerization interface, three (p.Gln542Leu, p.Leu749Pro, p.Tyr750X) showed a defect in PMS2 stabilization, suggesting that they confer a pathogenic effect due to direct interference with dimerization. These also severely compromised mismatch repair activity. Therefore, our data confirm that p.Leu749Pro is deleterious (). The unclassified p.Tyr750X variant, which lacks seven residues at the MLH1 C-terminus, has been identified in one Lynch syndrome patient in the United Kingdom (Syngal, et al., 1999
) and in further patients without clinical confirmation of Lynch syndrome in the United Kingdom and China (Stone, et al., 2001
; Wang, et al., 2006
). Co-segregation data, which can provide the most reliable clinical information on pathogenicity, is unavailable for this mutation. However, the current findings confirm that p.Tyr750X is pathogenic. The p.Gln542Leu variant has been identified in Korean kindreds with confirmed Lynch syndrome but without co-segregation information (Han, et al., 1995
; Shin, et al., 2004
). It has been classified as VUS due to incongruent biochemical data: it has either been found to have no effect on MLH1 function (Guerrette, et al., 1999
; Kondo, et al., 2003
; Shimodaira, et al., 1998
) or be deleterious (Ellison, et al., 2001
; Takahashi, et al., 2007
). Even excess amounts of MLH1 Gln542Leu repaired mismatches less than half as efficiently as wildtype MLH1 in our experiments, and the significant reduction of MLH1-PMS2 heterodimer formation in cells will decrease the repair efficiency even more. The repair activity of this mutant (44%) is clearly below the limit that previous comprehensive analyses have established as non-pathogenic even for variants which have no defect in expression of MLH1 or stabilization of PMS2: this minimum repair efficiency has been 70% (Raevaara, et al., 2005
) or 75% (Takahashi, et al., 2007
). Therefore, p.Gln542Leu must be considered deleterious.
Altogether, the current study included seven alterations with uncertain effect (VUS, , third column). Pathogenicity of a variant can be caused by compromised repair activity as well as by low stability (Raevaara, et al., 2005
). According to our analysis, five of the VUS must be considered deleterious (, last column): p.Ala586Pro, p.Leu636Pro and p.Thr662Pro were severely compromised in expression, while p.Gln542Leu and p.Arg755Ser were defective in repair activity (, and ). Both p.Lys618Ala and p.Arg659Gln, classified as VUS and neutral, respectively, displayed mild reductions of repair or expression; although these alterations therefore seem largely neutral, a final judgement on their pathogenicity is not possible from the current data. Therefore, we classified both as VUS. Interestingly, one variant classified as deleterious was indistinguishable from the wild-type in our analyses: p.Asp601Gly has been identified in an Arab kindred with microsatellite-unstable carcinoma but without co-segregation information (Chen-Shtoyerman, et al., 2003
). As yet, this alteration has not been tested experimentally. Since the current study has not found any evidence of a repair defect, application of the criteria suggested by Chao et al
. would result in re-classification of this alteration to a VUS.
The data of the current study also confirmed that eight further MLH1 alterations have a deleterious effect, mostly due to defects in expression ().
Our bioinformatic analyses showed that the interaction interface in the Ex subdomain is conserved in MutL and in MLH1 and PMS2 families (), and that it contains a hydrophobic patch (). Both these features are typical hallmarks of strong protein-protein interaction sites (Tsai, et al., 1997
; Valdar and Thornton, 2001
; Young, et al., 1994
). In contrast, the originally suggested dimerization interface in the In subdomain is not conserved in all MutL subfamilies and is not hydrophobic (). The three variants that affected PMS2 stabilization and mismatch repair (p.Gln542Leu, p.Leu749Pro, and p.Tyr750X) are located in the dimerization interface of the Ex subdomain (). Conversely, two alterations located within or in proximity of the alternative dimer interface (p.Arg659Gln and p.Glu663Gly) affected neither PMS2 stabilization nor repair activity. These observations confirm that the dimerization interface is located in the Ex subdomain (Kosinski, et al., 2008
; Kosinski, et al., 2005
) and not in the In subdomain (Cutalo, et al., 2006
; Guarne, et al., 2004
Mapping of amino acid substitutions onto the structural model of MutLα-CTD with their effect on protein expression and dimerization
The major biochemical evidence that dimerization occurs by the In subdomain has come from investigations of yMutLα using chemical surface modification experiments (Cutalo, et al., 2006
). In that study, three lysine residues that become buried only in the yMutLα dimer (as opposed to monomer) were identified. However, these results can be also explained by additional interactions of CTD with NTD or the linker in the dimeric state. Importantly, these lysine residues are near the hinge region between CTD subdomains (Kosinski, et al., 2005
), so they could become buried after dimerization solely due to conformational changes.
Previous analyses have also investigated the effect of small MLH1 alterations (missense type and deletions of few residues) on its dimerization with PMS2 by affinity methods, yeast two-hybrid analyses and coimmunoprecipitation (). Only one study has as yet used the stabilization of PMS2 as a measure of interaction (Mohd, et al., 2006
). While all methods seemed to work acceptably well in deletion studies, many results concerning missense alterations are conflicting. For example, p.Arg659Pro was frequently found to disturb dimerization (), seemingly supporting that dimerization occurs via the In domain. However, our data and other investigations showed that this is a very destabilizing alteration (introducing the helix-breaking Pro residue into an α-helix, ), therefore a (partial) unfolding of MLH1 probably causes the loss of dimerization without p.Arg659 being actually within the interaction interface. This is corroborated by two other substitutions of this residue (p.Arg659Leu and p.Arg659Gln) which showed no effect on dimerization in our analysis.
Data gained with affinity methods frequently are conflicting with other methods (). Both MLH1 and PMS2 have been routinely expressed in bacteria for this investigation, therefore PMS2 will lack its extensive post-translational modifications (Raschle, et al., 2002
), which may affect its interacting properties. Additionally, it has been observed that suitable, stringent washing conditions may be required to detect a decrease in affinity in vitro
(Mohd, et al., 2006
). For these reasons, detecting the stabilization of PMS2 after MLH1-PMS2 expression in mammalian cells seems to be the most reliable method for identifying a defect of dimerization, and it is probably the test giving best biological (and diagnostic) information.
In conclusion, the current work demonstrates that three MLH1 variants (p.Gln542Leu, p.Leu749Pro, and p.Tyr750X) observed in Lynch syndrome patients disturb MLH1-PMS2 dimerization. They are all located within a conserved hydrophobic surface area suggested as dimerization interface based on our bioinformatic analysis using the recently constructed model of MutLα-CTD. These alterations also severely affected mismatch repair, confirming that they are pathogenic and suggesting that defective dimerization underlies their deleterious effect. Moreover, the current work provides strong evidence that five MLH1 variants with uncertain significance (VUS) are deleterious and confirms the deleterious effect of eight further alterations, suggesting that all 13 variants can be causative for Lynch syndrome.