21-羟化酶缺陷症基因诊断方法的建立及应用

Establishment and application of a method for gene diagnosis of 21-hydroxylase deficiency

目的建立一种荧光偏振检测技术联合直接测序法诊断先天性肾上腺皮质增生症中21-羟化酶缺陷症的新方法,并对中国人群中最常见的2种基因缺失和8种点突变进行检测。方法收集40例健康者及2例患者外周血,提取基因组DNA。设计通用引物,同时扩增CYP21A2和CYP21A1P等位基因片段,然后用序列特异的TAMRA荧光标记探针对扩增产物进行杂交检测,利用荧光偏振仪检测扩增杂交的荧光偏振值,确定是否存在该基因的缺失,同时用直接测序法验证检测结果;对Exon3Del 8bp(GAGACTAC)缺失进行检测时,将缺失的8个碱基设计在引物上,确保引物的特异性。同时根据CYP21A2基因与其假基因序列差异,设计5对特异性引物扩增CYP21A2基因,PCR后直接测序检测中国人群中最常见的8种点突变,包括P30L、i2g、I172N、E6Cluster、V281L、920-921insT、Q218X、R356W。结果荧光偏振法未检测到CYP21A2基因大片段缺失,检测结果与直接测序法结果对比差异无统计学意义(P>0.05),一致率达100%,40例健康者未检测到点突变,1例患者检测出Exon1G56R,Exon7c.920-921InsT插入杂合突变,1例患者检测出Exon4I172N、Exon8Q318X杂合突变。结论荧光偏振检测法联合直接测序法诊断21-OHD是一种高效的检测手段,可以广泛用于临床,不仅能对患者基因突变类型进行分析,在指导遗传咨询及产前诊断方面亦发挥重要作用。

Objective To establish a new method for detecting 21‐hydroxylase deficiency in congenital adrenal hyperplasia（CAH） by using the fluorescence polarization technology combined with direct sequencing ,and to detect 2 most common gene deletions and 8 point mutations of CYP21A2 gene in Chinese population .Methods The peripher‐al blood samples in 40 normal people and 2 patients were collected and genomic DNA were extracted .To detect the gene deletion of CYP21A2 ,at first ,universal primers were designed to amplifly CYP21A2 and CYP21A1P allele frag‐ments ,and then the specific TAMRA fluorescence labeled probe was used to hybrid with amplification products ,at last ,the fluorescence polarization instrument was adopted to test FP value for determining whether the gene deletion was happened .The sequence GAGACTAC was designed on the primer which was specific to CYP21A2 to detect the Exon3 Del 8 bp（GAGACTAC） deletion .To detect point mutations ,5 pairs highly specific primers for CYP21A2 gene were designed according to the sequence differences between CYP21A2 and its pseudogene .The whole CYP21A2 gene was amplified and sequenced to detect the most common type of 8 kinds of point mutations in Chinese people ,in‐cluding P30L ,i2g ,I172N ,E6Cluster ,V281L ,920‐921insT ,Q218X and R356W .Results CYP21A2 gene deletion was not detected by the fluorescence polarization method ,the results had no statistical differences compared with the di‐rect sequencing method ,the consistency rate reached up to 100% .No point mutation of CYP21A2 gene was found in the 40 healthy people .The two patients ,one case was detected with Exon1 G56R ,Exon7 c .920‐921 insert T hetero‐zygous mutations ,another patient carried Exon4 I172N ,Exon8 Q318X heterozygous mutations .Conclusion The flu‐orescence polarization method combined with direct sequencing in the diagnosis of 21‐OHD is an effective way ,which can be widely used in clinic ,not only can analyze the type of gene mutation ,but also can play an important role in ge‐netic counseling and prenatal diagnosis .