Variants of unknown significance

Variants of uncertain significance challenge Lynch syndrome diagnosis

Knowing that inherited defects in mismatch repair (MMR) genes predispose to Lynch syndrome (LS), the identification of these mutations in suspected LS families is of prime importance. However, a major problem in the diagnosis and management of LS is the frequent occurrence of variants of uncertain significance (VUS) in the MMR genes. Where some variations, such as frameshift mutations or gross deletions are almost always disease causing, the consequences of non-truncating mutations can vary from none to complete dysfunction of the protein and the identification of LS mutation carriers prior to tumor formation is challenging due to variable degrees of pathogenicity and penetrance of VUS.

MMR gene variant pathogenicity indications of unique single nucleotide substitutions reported in the LOVD database (2013)

MMR gene variant pathogenicity indications of unique single nucleotide substitutions reported in the LOVD database (2013)

Recently, we participated in the enormous collaborative effort accomplished by The International Society for Gastrointestinal Hereditary Tumours (InSiGHT) to gather all available clinical and functional data of over 2300 unique constitutional MMR gene variants listed in the LOVD database. Here, the data was used to develop, test and apply a standardized five-tiered system for VUS classification, which will help in their pathogenicity interpretation and will greatly facilitate the consistent management of families carrying VUS (Thompson et al, 2014. Nature Genetics).

One of the functional assays applied to the pathogenicity assessments is our in vitro MMR assay (Nyström-Lahti et al, 2002; Kansikas et al, 2011). The assay investigates how a non-truncating mutation affects the quantity and repair efficiency of the protein variant as compared to the wild-type protein. Of a total of 86 MMR gene variants, we have functionally assayed 38 MLHI, 31 MSH2, 16 MSH6, 7 MLH3 and 1 MSH3 variants individually (Kariola et al, 2002, 2003, 2004; Raevaara et al, 2005; Ollila et al, 2006, 2008; Korhonen et al, 2008; Christensen et al, 2009; Kantelinen et al, 2010, 2011, 2012; Baas et al, 2013), and 9 variation pairs affecting the MuSα heterodimer complex protein MSH2 and/or MSH6 (Kantelinen et al, 2012) with the in vitro MMR assay. Of these, approximately 45% of MLH1, 50% of MSH2 and 25% or MSH6 were shown to be MMR deficient.

The in vitro MMR assay evaluates the repair efficiency of a GT heteroduplex DNA substrate, which upon repair reveals a unique restriction site detectable by standard restriction analysis.

The in vitro MMR assay evaluates the repair efficiency of a GT heteroduplex DNA substrate, which upon repair reveals a unique restriction site detectable by standard restriction analysis.

Furthermore, our recent findings demonstrate that the in vitro MMR assay can also be used to detect partial and quantitative MMR deficiency caused by MLH1, MSH2 or MSH6 heterozygosity in normal cells (Kansikas et al, 2014). Unlike existing strategies, this approach allows the recognition of individuals with increased cancer susceptibility due to deficient MMR, even in cases where no family member has, yet, developed cancer, where mutation tests result in no detectable change, and where the underlying change is not genetic but epigenetic. These findings contribute towards the goal of Lynch syndrome identification prior to tumor formation and have been supported by ERC proof of concept and TEKES grants.

The in vitro MMR assessment of MMR gene knockdown fibroblasts demonstrates that the reduced mRNA expression of MLH1, MSH2 or MSH6 can be detected by reduced in vitro MMR efficiencies.

The in vitro MMR assessment of MMR gene knockdown fibroblasts demonstrates that the reduced mRNA expression of MLH1, MSH2 or MSH6 can be detected by reduced in vitro MMR efficiencies.