Lynch syndrome (hereditary non-polyposis colorectal cancer) is an inherited disease caused by germ-line mutation in mismatch repair genes such as MLH1, MSH2, and MSH6. The mutations include missense and nonsense mutations, small insertions and deletions, and gross genetic alterations including large deletions and duplications. In addition to these genetic changes, mutations in introns are also involved in the pathogenesis. However, it is sometimes difficult to interpret correctly the pathogenicity of variants in exons as well as introns. To evaluate the effect of splice-site mutations in two Lynch syndrome patients, we carried out a functional splicing assay using minigenes. Consequently, this assay showed that the mutation of c.1731+5G>A in MLH1 led to exon15 skipping, and that the mutation of c.211+1G>C in MSH2 created an activated cryptic splice-site 17-nucleotides upstream in exon1. These aberrant splicing patterns were not observed when wild type sequence was used for the assay. We also obtained concordant results by RT-PCR experiments with transcripts from the patients. Furthermore, additional functional splicing assays using two different intronic mutations described in earlier studies revealed splicing alterations that were in complete agreement with the reports. Therefore, functional splicing assay is helpful for evaluating the effects of genetic variants on splicing.
Supplementary Fig. 1. (A) A schematic representation of plasmids containing wild type or mutant DNA fragment of case 1. (B) RT-PCR analysis of transcripts in cells transfected with wild type or mutant plasmids using murine Gapdh-specific (upper panel) or human GAPDH-specific (lower panel) primers. RT-PCR products from the cells transfected with pcDNAmGapdh1 (lane1), pcDNAmGapdh1-MLH1-ex8wt (lane2), pcDNAmGapdh1-MLH1-ex8mut(G) (lane3), pcDNAmGapdh1-MLH1-ex8mut(C) (lane4), and mock (lane5) were separated on an agarose gel. Negative PCR experiment without DNA is shown in lane6
Supplementary Fig. 2. (A) A schematic representation of plasmids containing wild type and mutant DNA fragment of case 2. (B) RT-PCR analysis of transcripts from the cells transfected with wild type or mutant plasmids using MLH1 exon1- and murine Gapdh exon3-specific primers (upper panel) or human GAPDH-specific primers (lower panel). RT-PCR products from the cells transfected with pcDNAmGapdh1 (lane1), pcDNAmGapdh1-MLH1-ex1wt (lane2), pcDNAmGapdh1-MLH1-ex1mut (lane3), and mock (lane4) were separated on an agarose gel. Negative PCR experiment without DNA is shown in lane5. The products of pcDNAmGapdh1-MLH1-ex1mut revealed aberrant bands in addition to the band corresponding to the wild type products