A cell-free assay for the functional analysis of variants of the mismatch repair protein MLH1

Abstract

The hereditary colon and endometrium cancer predisposition Lynch Syndrome (also called HNPCC) is caused by a germ-line mutation in one of the DNA mismatch repair (MMR) genes. A significant fraction of the gene alterations detected in suspected Lynch Syndrome patients is comprised of amino acid substitutions. The relevance for cancer risk of these variants is difficult to assess, as currently no time- and cost-effective, validated, and widely applicable functional assays for the measurement of MMR activity are available. Here we describe a rapid, cell-free, and easily quantifiable MMR activity assay for the diagnostic assessment of variants of the MLH1 MMR protein. This assay allows the parallel generation and functional analysis of a series of variants of the MLH1 protein in vitro using readily available, or preprepared, reagents. Using this assay we have tested 26 MLH1 variants and of these, 15 had lost activity. These results are in concordance with those obtained from first-generation assays and with in silico and pathology data. After its multifocal technical and clinical validation this assay could have great impact for the diagnosis and counseling of carriers of an MLH1 variant and their relatives.

Three novel germline mutations in MLH1 and MSH2 in families with Lynch syndrome living on Jeju island, Korea

“…However, amino acid alterations comprise a significant proportion of these mutations (~15% in MSH2, ~30% in MLH1, and ~40% in MSH6) (19), the consequences of which remain unclear regarding DNA repair. A single amino acid change does not necessarily result in a dysfunctional protein.…”

Classification of mismatch repair gene missense variants with PON-MMR

“…The numbers are too E23D I25F P28L M35R E37K N38D N38H S44F S46I G54E D63E N64S G65D G67R G67W I68N C77R C77Y F80V T82I K84E R100P E102K E102D I107R H109P A111V A111P T117R T117M A128P L155R V185G S193P R226L R226Q G244D G244V S247P R265C R265S L292P D304V V326A H329P Q346H D485E Q542L L549P L550P I565F L574P P581L L582F A586P A589D K618T P648S P648L L653R [ [Fan et al, 2007;Takahashi et al, 2007] [ Takahashi et al, 2007] [ Kansikas et al, 2011;Kariola et al, 2004] [ Takahashi et al, 2007] [ Takahashi et al, 2007] [ Fan et al, 2007] [ Drost et al, 2010] [ Takahashi et al, 2007] [ Kansikas et al, 2011;Kariola et al, 2004] [ Kansikas et al, 2011;Takahashi et al, 2007] [ Kansikas et al, 2011;Raevaara et al, 2004] [ Kansikas et al, 2011;Kariola et al, 2004] [ Takahashi et al, 2007] I25T …”

“…The experimentally verified functional effects of MMR missense variants were collected from articles. The most widely applied methods in these studies included in vitro MMR activity [Christensen et al, 2009;Drost et al, 2010;Jäger et al, 2001;Kansikas et al, 2011;Kariola et al, 2004;Korhonen et al, 2008;Nyström-Lahti et al, 2002;Ollila et al, 2006a, b;Raevaara et al, 2004Raevaara et al, , 2005Takahashi et al, 2007;Trojan et al, 2002]. Additional methods were in vivo DNA MMR assays in yeast [Ellison et al, 2001], yeast twohybrid system [Fan et al, 2007;Ou et al, 2009], and RNA expression [Pagenstecher et al, 2006].…”

Interpreting the clinical significance of mismatch repair gene sequence variants

“…Whereas, cell-based assays may measure spontaneous mutation rates or response to methylating agents using human expression systems in mammalian cell lines 295,296 . Recently a rapid next-generation in vitro assay for testing the integral activity of missense substitutions encoded by sequence variants in MLH1, MSH2, MSH6, and PMS2 has been developed for use in a clinical setting [297][298][299] . In the assays variant proteins derived using a mutagenic PCR procedure are expressed in an in vitro expression kit (under development for commercial use), and repair efficiency is quantified fluorescently in a complementation assay.…”

“…Our experiences with functional assay interpretation in chapter 4, demonstrate the importance of combining results for different assays to get a comprehensive representation of the variants effect on protein function. As a starting point, a rapid cell-free "complete in vitro MMR assay" (CIMRA) [297][298][299]396 will be validated against 159 non-synonymous sequence alterations that have been shown to be benign or disease causing through: presence in the general population (allele frequency >1%), or a high (≥0.95) or low (<0.05) probability of pathogenicity as determined by multifactorial likelihood analysis. Also, ensuring that variants classified using functional assay data or shown to cause aberrant splicing are excluded from the validation set.…”