Naylor J, Brinke A, Hassock S, Green P M, Giannelli F
Division of Medical and Molecular Genetics, United Medical School, Guy's Hospital, London, UK.
Hum Mol Genet. 1993 Nov;2(11):1773-8. doi: 10.1093/hmg/2.11.1773.
Surprisingly half of all severe haemophilia A patients have no mutation in the promoter, coding sequences and normal RNA processing signals of the factor VIII gene. Instead they manifest a unique mRNA defect that prevents the amplification of the message across the boundary between exon 22 and 23. This locates the defect to internal regions of intron 22. Novel sequences 3' to exon 22 were isolated from the 9 available patients with the above abnormality by combining RACE and vectorette amplifications on trace amounts of mRNA. This showed that exons 1-22 of the factor VIII mRNA had become part of a hybrid message containing new multi exonic sequences expressed in normal cells. The novel sequences were not located in a YAC covering the whole factor VIII gene. Southern blots from patients probed by novel sequences and clones covering intron 22 showed no obvious abnormalities. This suggested inversions involving intron 22 repeated sequences. Screening of 3 YAC libraries with the novel sequences located them at least 200 kb telomeric (5') to factor VIII and pulsed field gel analysis detected abnormal bands in patients. This demonstrates that the mutations in the patients are inversions of long DNA regions possibly involving the repeated sequences and occurring at the surprising rate of approximately 4 x 10(-6) per gene per gamete per generation.
令人惊讶的是，所有重度甲型血友病患者中有一半在凝血因子VIII基因的启动子、编码序列和正常RNA加工信号中没有突变。相反，他们表现出一种独特的mRNA缺陷，这种缺陷阻止了外显子22和23之间边界处信息的扩增。这将缺陷定位到内含子22的内部区域。通过对微量mRNA进行RACE和载体扩增相结合的方法，从9例患有上述异常的患者中分离出了外显子22下游的新序列。这表明凝血因子VIII mRNA的外显子1 - 22已成为一种杂合信息的一部分，该杂合信息包含在正常细胞中表达的新的多外显子序列。这些新序列并不位于覆盖整个凝血因子VIII基因的酵母人工染色体（YAC）中。用新序列和覆盖内含子22的克隆对患者进行Southern杂交，未显示明显异常。这提示涉及内含子22重复序列的倒位。用新序列筛选3个YAC文库，将它们定位在凝血因子VIII端粒（5'）至少200 kb处，脉冲场凝胶分析在患者中检测到异常条带。这表明患者中的突变是长DNA区域的倒位，可能涉及重复序列，且以每代每个基因每个配子约4×10⁻⁶的惊人速率发生。
