Zou Wen-Bin, Wu Hao, Boulling Arnaud, Cooper David N, Li Zhao-Shen, Liao Zhuan, Chen Jian-Min, Férec Claude
Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai, China.
Institut National de la Santé et de la Recherche Médicale (INSERM), U1078, Brest, France.
Hum Genomics. 2017 May 4;11(1):7. doi: 10.1186/s40246-017-0103-9.
SPINK1 (serine protease inhibitor, kazal-type, 1), which encodes human pancreatic secretory trypsin inhibitor, is one of the most extensively studied genes underlying chronic pancreatitis. Recently, based upon data from qualitative reverse transcription-PCR (RT-PCR) analyses of transfected HEK293T cells, we concluded that 24 studied SPINK1 intronic variants were not of pathological significance, the sole exceptions being two canonical splice site variants (i.e., c.87 + 1G > A and c.194 + 2T > C). Herein, we employed the splicing prediction tools included within the Alamut software suite to prioritize the 'non-pathological' SPINK1 intronic variants for further quantitative RT-PCR analysis.
Although our results demonstrated the utility of in silico prediction in classifying and prioritizing intronic variants, we made two observations worth noting. First, we established that most of the prediction tools employed ignored the general rule that GC is a weaker donor splice site than the canonical GT site. This finding is potentially important because for a given disease gene, a GC variant donor splice site may be associated with a milder clinical manifestation. Second, the non-pathological c.194 + 13T > G variant was consistently predicted by different programs to generate a new and viable donor splice site, the prediction scores being comparable to those for the physiological c.194 + 2T donor splice site and even higher than those for the physiological c.87 + 1G donor splice site. We do however provide convincing in vitro evidence that the predicted donor splice site was not entirely spurious.
Our findings, taken together, serve to emphasize the importance of functional analysis in helping to establish or refute the pathogenicity of specific intronic variants.
SPINK1（丝氨酸蛋白酶抑制剂，Kazal 型，1）编码人胰腺分泌性胰蛋白酶抑制剂，是慢性胰腺炎相关研究最为广泛的基因之一。最近，基于对转染的 HEK293T 细胞进行的定性逆转录聚合酶链反应（RT-PCR）分析数据，我们得出结论，所研究的 24 个 SPINK1 内含子变异不具有病理学意义，唯一的例外是两个典型剪接位点变异（即 c.87+1G>A 和 c.194+2T>C）。在此，我们使用 Alamut 软件套件中包含的剪接预测工具，对“非病理性”SPINK1 内含子变异进行优先级排序，以便进一步进行定量 RT-PCR 分析。
尽管我们的结果证明了计算机预测在对内含子变异进行分类和排序方面的实用性，但我们有两点值得注意的观察结果。首先，我们发现所使用的大多数预测工具都忽略了 GC 作为供体剪接位点比典型的 GT 位点弱这一普遍规则。这一发现可能具有重要意义，因为对于给定的疾病基因，GC 变异供体剪接位点可能与较轻的临床表现相关。其次，不同程序一致预测非病理性的 c.194+13T>G 变异会产生一个新的可行供体剪接位点，预测分数与生理性 c.194+2T 供体剪接位点相当，甚至高于生理性 c.87+1G 供体剪接位点。然而，我们确实提供了令人信服的体外证据，表明预测的供体剪接位点并非完全虚假。
综合我们的研究结果，强调了功能分析在帮助确定或反驳特定内含子变异致病性方面的重要性。
