Mutational Analysis of CYP21A2 Gene and CYP21A1P Pseudogene: Long-range PCR on Genomic DNA
Mutational Analysis of CYP21A2 Gene and CYP21A1P Pseudogene: Long-range PCR on Genomic DNA
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DOI：
10.1007/978-1-4939-0835-6_19
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发表时间：
2014-01-01
期刊：
影响因子：
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通讯作者：
Lee, Hsien-Hsiung
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文献类型：
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作者：
Lee, Hsien-Hsiung
CYP 21 A2，编码P450 c21（类固醇21-羟化酶）的基因，有一个重复的假基因CYP 21 A1 P。该基因和假基因在外显子和非编码序列中分别具有98%和96%的序列同源性，并且位于染色体6p21.3上的HLA III类人组织相容性复合体基因座内相距30 kb。CYP 21 A1 P由于其编码区存在11个恶化的突变而无活性。这些突变可在减数分裂或有丝分裂过程中通过基因间重组转移到功能性CYP 21 A2上，导致21-羟化酶缺乏引起的先天性肾上腺皮质增生（CAH）。相反，部分CYP 21 A2序列可以转移到CYP 21 A1 P，修饰单倍型。在这里，我们描述了一个成熟的方案，可用于明确研究CYP 21 A2基因和CYP 21 A1 P假基因的突变谱。该方案是基于长距离PCR扩增等位基因特异性引物，然后通过DNA测序的较小片段。
CYP21A2, the gene that codes for P450c21 (Steroid 21-hydroxylase), has a duplicated pseudogene called CYP21A1P. The gene and the pseudogene share 98 % and 96 % sequence homology in exons and in non-coding sequences, respectively, and are located 30 kb apart within the HLA class III human histocompatibility complex locus on chromosome 6p21.3. CYP21A1P is inactive due to the presence of 11 deteriorated mutations in its coding region. These mutations can be transferred to the functional CYP21A2 through intergenic recombination during meiosis or mitosis and lead to the congenital adrenal hyperplasia (CAH) resulting from 21-hydroxylase deficiency. Conversely, portions of CYP21A2 sequence can be transferred to CYP21A1P, modifying the haplotype. Here, we describe a well-established protocol that can be used to unambiguously study the mutational profile of CYP21A2 gene and CYP21A1P pseudogene. The protocol is based on long-range PCR amplification with allele-specific primers, followed by DNA sequencing of smaller fragments.