title: Determination of splice-site mutations in Lynch syndrome (hereditary non-polyposis colorectal cancer) patients using functional splicing assay.
abstract: 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.
authors.affiliation: Division of Clinical Genome Research, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
authors.forename: Hiromu
authors.initials: H
authors.lastname: Naruse
authors.forename: Noriko
authors.initials: N
authors.lastname: Ikawa
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yamaguchi
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Masami
authors.initials: M
authors.lastname: Arai
authors.forename: Chikashi
authors.initials: C
authors.lastname: Ishioka
authors.forename: Kokichi
authors.initials: K
authors.lastname: Sugano
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tamura
authors.forename: Naohiro
authors.initials: N
authors.lastname: Tomita
authors.forename: Nagahide
authors.initials: N
authors.lastname: Matsubara
authors.forename: Teruhiko
authors.initials: T
authors.lastname: Yoshida
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Moriya
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Furukawa
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D000328:Adult; D001483:Base Sequence; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D005260:Female; D020022:Genetic Predisposition to Disease; D005821:Genetic Techniques; D006801:Humans; D008297:Male; D008875:Middle Aged; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000235:genetics*; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D022821:RNA Splice Sites; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein
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