Familial Cancer

, 8:509

Determination of splice-site mutations in Lynch syndrome (hereditary non-polyposis colorectal cancer) patients using functional splicing assay

Authors

  • Hiromu Naruse
    • Division of Clinical Genome Research, Institute of Medical ScienceThe University of Tokyo
    • Health Science Research Institute Inc.
  • Noriko Ikawa
    • Division of Clinical Genome Research, Institute of Medical ScienceThe University of Tokyo
  • Kiyoshi Yamaguchi
    • Division of Clinical Genome Research, Institute of Medical ScienceThe University of Tokyo
  • Yusuke Nakamura
    • Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical ScienceThe University of Tokyo
  • Masami Arai
    • HNPCC Registry and Genetic Testing Project of the Japanese Society for Cancer of the Colon and Rectum (JSCCR)
  • Chikashi Ishioka
    • HNPCC Registry and Genetic Testing Project of the Japanese Society for Cancer of the Colon and Rectum (JSCCR)
  • Kokichi Sugano
    • HNPCC Registry and Genetic Testing Project of the Japanese Society for Cancer of the Colon and Rectum (JSCCR)
  • Kazuo Tamura
    • HNPCC Registry and Genetic Testing Project of the Japanese Society for Cancer of the Colon and Rectum (JSCCR)
  • Naohiro Tomita
    • HNPCC Registry and Genetic Testing Project of the Japanese Society for Cancer of the Colon and Rectum (JSCCR)
  • Nagahide Matsubara
    • HNPCC Registry and Genetic Testing Project of the Japanese Society for Cancer of the Colon and Rectum (JSCCR)
  • Teruhiko Yoshida
    • HNPCC Registry and Genetic Testing Project of the Japanese Society for Cancer of the Colon and Rectum (JSCCR)
  • Yoshihiro Moriya
    • HNPCC Registry and Genetic Testing Project of the Japanese Society for Cancer of the Colon and Rectum (JSCCR)
    • Division of Clinical Genome Research, Institute of Medical ScienceThe University of Tokyo
    • HNPCC Registry and Genetic Testing Project of the Japanese Society for Cancer of the Colon and Rectum (JSCCR)
Article

DOI: 10.1007/s10689-009-9280-6

Cite this article as:
Naruse, H., Ikawa, N., Yamaguchi, K. et al. Familial Cancer (2009) 8: 509. doi:10.1007/s10689-009-9280-6

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.

Keywords

Lynch syndromeHNPCCMLH1MSH2MutationSplicingMinigene

Abbreviations

HNPCC

Hereditary non-polyposis colorectal cancer

MLH1

mutL homolog 1 gene

MSH2

mutS homolog 2 gene

RT-PCR

Reverse transcription-PCR

NMD

Nonsense-mediated RNA decay

ESE

Exonic splicing enhancer

UV

Uncharacterized variant

Supplementary material

10689_2009_9280_MOESM1_ESM.pdf (137 kb)
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
10689_2009_9280_MOESM2_ESM.pdf (116 kb)
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
10689_2009_9280_MOESM3_ESM.xls (22 kb)
Supplementary material 3 (XLS 21 kb)

Copyright information

© Springer Science+Business Media B.V. 2009