Department of Neurosciences, University of Padova, Italy.
Hum Mutat. 2010 Sep;31(9):E1658-69. doi: 10.1002/humu.21320.
Variants of unknown significance in the CAPN3 gene constitute a significant challenge for genetic counselling. Despite the frequency of intronic nucleotide changes in this gene (15-25% of all mutations), so far their pathogenicity has only been inferred by in-silico analysis, and occasionally, proven by RNA analysis. In this study, 5 different intronic variants (one novel) that bioinformatic tools predicted would affect RNA splicing, underwent comprehensive studies which were designed to prove they are disease-causing. Muscle mRNA from 15 calpainopathy patients was analyzed by RT-PCR and splicing-specific-PCR tests. We established the previously unrecognized pathogenicity of these mutations, which caused aberrant splicing, most frequently by the activation of cryptic splicing sites or, occasionally, by exon skipping. The absence or severe reduction of protein demonstrated their deleterious effect at translational level. We concluded that bioinformatic tools are valuable to suggest the potential effects of intronic variants; however, the experimental demonstration of the pathogenicity is not always easy to do even when using RNA analysis (low abundance, degradation mechanisms), and it might not be successful unless splicing-specific-PCR tests are used. A comprehensive approach is therefore recommended to identify and describe unclassified variants in order to offer essential data for basic and clinical geneticists.
CAPN3 基因中的意义不明变异是遗传咨询的重大挑战。尽管该基因的内含子核苷酸变化频率很高（占所有突变的 15-25%），但到目前为止，它们的致病性仅通过计算机分析推断得出，偶尔也通过 RNA 分析证明。在这项研究中，我们对 5 种不同的内含子变异体（一种新的）进行了综合研究，这些变异体被生物信息学工具预测会影响 RNA 剪接，旨在证明它们是致病的。通过 RT-PCR 和剪接特异性-PCR 测试分析了 15 名钙蛋白酶病患者的肌肉 mRNA。我们确定了这些突变的先前未被认识的致病性，这些突变导致异常剪接，最常见的是通过激活隐匿剪接位点，或者偶尔通过外显子跳跃。缺失或严重减少的蛋白质证明了它们在翻译水平上的有害影响。我们得出结论，生物信息学工具对于预测内含子变异的潜在影响是有价值的；然而，即使使用 RNA 分析（低丰度、降解机制），也并非总是容易证明其致病性，除非使用剪接特异性-PCR 测试。因此，建议采用综合方法来识别和描述未分类的变异体，以便为基础和临床遗传学家提供必要的数据。
