Ketterling R P, Drost J B, Scaringe W A, Liao D Z, Liu J Z, Kasper C K, Sommer S S
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, Minnesota, USA.
Hum Mutat. 1999;13(3):221-31. doi: 10.1002/(SICI)1098-1004(1999)13:3<221::AID-HUMU6>3.0.CO;2-U.
Small consensus sequences have been defined for RNA splicing, but questions about splicing in humans remain unanswered. Analysis of germline mutations in the factor IX gene offers a highly advantageous system for studying the mutational process in humans. In a sample of 860 families with hemophilia B, 9% of independent mutations are likely to disrupt splicing as their primary mode of action. This includes 26 splicing mutations reported herein. When combined with the factor IX splice mutations reported by others, at least 104 independent mutations have been observed, 80 of which are single base substitutions within the splice donor and splice acceptor consensus sequences. After analysis of these mutations, the following inferences emerge: (1) the susceptibility of a splice donor sequence to deleterious mutation depends on the degree of similarity with the donor consensus sequence, suggesting a simple "5-6 hypothesis" for predicting deleterious vs. neutral mutations; (2) the great majority of mutations that disrupt the splice donor or splice acceptor sequences result in at least a 100-fold decrement in factor IX coagulant activity, indicating that the mutations at these sites generally function as an on/off switch; (3) mutations that create cryptic splice junctions or that shorten but do not interrupt the polypyrimidine tract in the splice acceptor sequence can reduce splicing by a variable amount; and (4) there are thousands of potential donor-acceptor consensus sequence combinations in the 38-kb factor IX gene region apparently not reduced by evolutionary selective pressure, presenting an apparent paradox; i.e., mutations in the donor and acceptor consensus sequences at intron/exon splice junctions can dramatically alter normal splicing, yet, appropriately spaced, good matches to the consensus sequences do not predispose to significant amounts of alternative splicing.
RNA剪接的小共有序列已被定义，但关于人类剪接的问题仍未得到解答。对凝血因子IX基因种系突变的分析为研究人类的突变过程提供了一个非常有利的系统。在860个B型血友病家庭的样本中，9%的独立突变可能以破坏剪接作为其主要作用方式。这包括本文报道的26个剪接突变。当与其他人报道的凝血因子IX剪接突变相结合时，至少观察到104个独立突变，其中80个是剪接供体和剪接受体共有序列内的单碱基替换。对这些突变进行分析后，得出以下推论：(1) 剪接供体序列对有害突变的敏感性取决于与供体共有序列的相似程度，这提示了一个预测有害突变与中性突变的简单 “5-6假说”；(2) 绝大多数破坏剪接供体或剪接受体序列的突变会导致凝血因子IX凝血活性至少下降100倍，这表明这些位点的突变通常起开/关开关的作用；(3) 产生隐蔽剪接位点或缩短但不中断剪接受体序列中多嘧啶序列的突变可不同程度地减少剪接；(4) 在38 kb的凝血因子IX基因区域内有成千上万种潜在的供体-受体共有序列组合，显然未因进化选择压力而减少，这呈现出一个明显的悖论；即内含子/外显子剪接位点处供体和受体共有序列的突变可显著改变正常剪接，然而，适当间隔的、与共有序列良好匹配的序列并不会导致大量的可变剪接。
