Analysis of factor VIII mRNA reveals defects in everyone of 28 haemophilia A patients.
Analysis of factor VIII mRNA reveals defects in everyone of 28 haemophilia A patients.
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对 VIII 因子 mRNA 的分析揭示了 28 名 A 型血友病患者中所有人的缺陷。
DOI：
10.1093/hmg/2.1.11
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发表时间：
1993
影响因子：
3.5
通讯作者：
Francesco Giannelli
中科院分区：
文献类型：
--
作者：
J. Naylor;P. Green;C. Rizza;Francesco Giannelli
血友病A是一种突变异质性疾病，由大而复杂的因子VIII基因缺陷引起。最近的研究检查推定的启动子，所有外显子和大多数内含子/外显子边界未能检测到突变的一半患者严重的疾病，导致假设，如突变的远程控制区域，甚至在基因以外的因子VIII。我们从反转录的mRNA和基因组DNA中扩增了8个片段的因子VIII基因（推定的启动子、编码区和多聚腺苷酸化/切割信号区）。然后通过化学错配检测定位任何突变，并通过直接测序进行表征。这种快速而有效的方法已经完全成功，并揭示了一组导致严重疾病的不寻常的突变。在我们报道的28例患者中，5例有轻度或中度疾病，并且都有错义突变。23名患者受到严重影响，其中13名患者存在不同的有害突变，这些突变在基因组DNA水平上得到了充分表征。其余10名患者的mRNA均含有第22号外显子与第23号外显子不连续的mRNA。由于所有的外显子都是正常的，内含子22的剪接位点也是正常的，这些患者的突变应该在内含子22的未被筛选的区域。这些结果证明，所有血友病A病例都是由于因子VIII基因突变所致，其中，出乎意料的是，内含子22似乎是导致重度血友病A的约40%突变的靶点。
Haemophilia A is a mutationally heterogeneous disease caused by defects in the large and complex factor VIII gene. Recent studies examining the putative promoter, all exons and most intron/exon boundaries have failed to detect mutations in half the patients with severe disease leading to hypotheses such as mutations in remote controlling regions or even in genes other than factor VIII. We have amplified the factor VIII gene (putative promotor, coding region and polyadenylation/cleavage signal region) in 8 fragments from reverse transcribed mRNA and genomic DNA. Any mutation is then located by chemical mismatch detection and characterised by direct sequencing. This rapid and efficient method has been fully successful and has revealed an unusual cluster of mutations causing severe disease. Of the 28 patients we have reported, 5 had mild or moderate disease and all had a missense mutation. Twenty-three patients were severely affected and 13 of these had different detrimental mutations that were fully characterised at the genomic DNA level. The remaining 10 patients all had mRNA with exon 22 not contiguous to exon 23. Since all exons were normal and so were the splice sites of intron 22, the mutation in these patients should be in the regions of intron 22 that were not screened. These results prove that all haemophilia A cases are due to mutations of the factor VIII gene where, unexpectedly, intron 22 seems to be the target of approximately 40% of the mutations causing severe haemophilia A.