A Novel and Rapid Method of Determining the Effect of Unclassified MLH1 Genetic Variants on Differential Allelic Expression

Abstract

Germline mutations in mismatch repair genes predispose patients to Lynch Syndrome and the majority of these mutations have been detected in two key genes, MLH1 and MSH2. In particular, about a third of the missense variants identified in MLH1 are of unknown clinical significance. Using the PeakPicker software program, we have conducted a proof-of-principle study to investigate whether missense variants in MLH1 lead to allelic imbalances. Lymphocyte RNA extracted from patients harboring known MLH1 variants was used to quantify the ratio of variant to wild-type transcript, while patient lymphocyte DNA was used to establish baseline allelic expression levels. Our analysis indicated that the missense variants c.350C>T, c.793C>T, and c.1852_1853AA>GC, as well as the truncating variant c.1528C>T were all associated with significantly unbalanced allelic expression. However, the variants c.55A>T and c.2246T>C did not demonstrate an allelic imbalance. These results illustrate a novel and efficient method to investigate the pathogenicity of unclassified genetic variants discovered in mismatch repair genes, as well as genes implicated in other inherited diseases. In addition, the PeakPicker methodology has the potential to be applied in the diagnostic setting, which, in conjunction with results from other assays, will help increase both the accuracy and efficiency of genetic testing of colorectal cancer, as well as other inherited diseases.

Assessment of the InSiGHT Interpretation Criteria for the Clinical Classification of 24MLH1andMSH2Gene Variants

“…Total RNA was extracted from blood samples collected into PAXgene Blood RNA tubes (PreAnalytiX; Qiagen), using the PAXgene Blood RNA Kit (Qiagen) according to the manufacturer's instructions. Samples from heterozygotes for the same SNPs who had no additional MLH1 and MSH2 sequence change were used as controls [Perera et al., ; Pastrello et al., ]. ASE was calculated after measuring peak heights in heterozygous samples [Castellsagué et al., ].…”

“…ASE was calculated after measuring peak heights in heterozygous samples [Castellsagué et al., ]. Values included in the 0.8–1.2 range were assumed as a cut‐off for normal ASE according to previous studies [Renkonen et al., ; Castellsagué et al., ; Perera et al., ]. All experiments were carried out in triplicate and two independent replicates of all experiments were performed.…”

Refining the role ofpms2in Lynch syndrome: germline mutational analysis improved by comprehensive assessment of variants

“…ASE was calculated by dividing the proportion of variant/wild-type allele in cDNA by the proportion of variant/wild-type allele in gDNA. We used ≤0.5 as a threshold value for ASE definition 43. Experiments were performed in triplicate.…”

Comprehensive functional assessment ofMLH1variants of unknown significance

“…ASE was calculated by dividing the proportion of variant/wild‐type allele in cDNA by the proportion of variant/wild‐type allele in gDNA. We used ≤0.5 as a threshold value for ASE definition [Perera et al, 2010; Renkonen et al, 2003, 2005]. Experiments were performed in triplicate.…”