Collier S, Sinnott P J, Dyer P A, Price D A, Harris R, Strachan T
University Department of Medical Genetics, St Mary's Hospital, Manchester, UK.
EMBO J. 1989 May;8(5):1393-402. doi: 10.1002/j.1460-2075.1989.tb03520.x.
The human steroid 21-hydroxylase gene, CYP21B, and its closely homologous pseudogene, CYP21A, are each normally located centromeric to a complement C4 gene C4B and C4A respectively, in an organization suggesting tandem duplication of a CYP21 + C4 unit. Such an organization has been considered to facilitate gene deletion and addition events by unequal crossover between the tandem repeats. However, the large size (approximately 30 kb) of the individual CYP21 + C4 repeat units together with the difficulty in identifying reliable CYP21A- and CYP21B-specific markers has prevented direct monitoring of gene organization on individual haplotypes by conventional Southern analyses. In the present investigation we have sought to clarify the CYP21 and C4 gene organization in members of 32 British 21-hydroxylase deficiency families by employing additional experimental approaches, notably a long-range restriction mapping approach, which permits assessment through a VNTR type of analysis, of the number of CYP21 and C4 units on individual haplotypes. Our results show that there is a very high frequency (33%) of 21-hydroxylase deficiency haplotypes where functional CYP21B gene sequence has been removed as a consequence of CYP21 + C4 gene deletion while several haplotypes show evidence of gene addition. In each case that we have investigated the gene deletion and gene addition haplotypes differ in length from conventional haplotypes by integral multiples of approximately 30 kb, which strongly supports the involvement of unequal crossover mechanisms. Additionally, the comparatively frequent occurrence of CYP21 fusion genes which contain both CYP21A- and CYP21B-associated markers is suggested by the combined data from Southern analyses, long-range restriction mapping and characterization of selected regions of CYP21 genes which have been amplified in vitro.
人类类固醇21-羟化酶基因CYP21B及其紧密同源的假基因CYP21A，通常分别位于补体C4基因C4B和C4A的着丝粒侧，其排列方式提示CYP21 + C4单元发生了串联重复。这种排列方式被认为可通过串联重复序列之间的不等交换促进基因缺失和添加事件。然而，单个CYP21 + C4重复单元的大尺寸（约30 kb）以及难以鉴定可靠的CYP21A和CYP21B特异性标记，使得无法通过传统的Southern分析直接监测个体单倍型上的基因排列。在本研究中，我们试图通过采用其他实验方法，特别是长距离限制性图谱分析方法，来阐明32个英国21-羟化酶缺乏症家族成员中的CYP21和C4基因排列情况。该方法允许通过VNTR类型的分析评估个体单倍型上CYP21和C4单元的数量。我们的结果表明，21-羟化酶缺乏症单倍型的频率非常高（33%），其中功能性CYP21B基因序列因CYP21 + C4基因缺失而被去除，而几个单倍型显示出基因添加的证据。在我们研究的每一个案例中，基因缺失和基因添加单倍型的长度与传统单倍型相差约30 kb的整数倍，这有力地支持了不等交换机制的参与。此外，Southern分析、长距离限制性图谱分析以及体外扩增的CYP21基因选定区域的特征分析的综合数据表明，含有CYP21A和CYP21B相关标记的CYP21融合基因相对频繁出现。
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