Division of Clinical Genome Research, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
Fam Cancer. 2009;8(4):509-17. doi: 10.1007/s10689-009-9280-6. Epub 2009 Aug 15.
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.
林奇综合征（遗传性非息肉病性结直肠癌）是一种由错配修复基因（如 MLH1、MSH2 和 MSH6）的种系突变引起的遗传性疾病。突变包括错义突变和无义突变、小插入和缺失以及包括大片段缺失和重复在内的大的遗传改变。除了这些遗传变化外，内含子中的突变也参与了发病机制。然而，有时很难正确解释外显子和内含子中变异的致病性。为了评估两个林奇综合征患者中剪接位点突变的影响，我们使用小基因进行了功能剪接测定。结果表明，MLH1 中的 c.1731+5G>A 突变导致外显子 15 跳跃，MSH2 中的 c.211+1G>C 突变在外显子 1 上游的 17 个核苷酸处创建了激活的隐式剪接位点。当使用野生型序列进行测定时，未观察到这些异常剪接模式。我们还通过来自患者的转录本的 RT-PCR 实验获得了一致的结果。此外，使用先前研究中描述的两种不同内含子突变进行的其他功能剪接测定揭示了与报告完全一致的剪接改变。因此，功能剪接测定有助于评估遗传变异对剪接的影响。
