Pitfalls of PCR-based Genotyping in Patients with 21-hydroxylase Deficiency

以聚合酶反應爲基礎(PCR-based genotyping)的基因分析是現行診斷21-羥化酶缺管症突變的常用方法。利用此法，我們完成了七名先天性腎上腺肥大症的基因分析，確認了所有的突變，包括一個缺損突變，三個傳訊核酸黏合(splicing)突變，四個lle172Asn突變及三個Arg356Trp突變。分析過程中我們發現PCR-based genotyping的陷阱，當病人之兩個染色分體(chromatid)均爲21-羥化酶基因之缺損(deletion)突變，且其中至少一個染色分體帶有CYP21A1P-CYP21A2融合基因時，容易發生誤診。些外，我們也發現一個“染色分體複製遺漏”(”allele dropout”)情況，導致可能錯惡的基因分型。我們在此呼顛以聚合酶反應爲基礎(PCR-baded genotyping)分析21-羥化酶缺乏症的産前診斷時，應輔以微衛星聯鎖分析(microsatellite linkage analysis)及/或直接酸定序分析以確保其準確率。

關鍵字

21-羥化酶缺管症；先天性腎上腺肥大症； CYP21A2基因；染色分體複製遺漏

並列摘要

Mutation analysis of CYP2IA2 gene was performed in seven patients with congenital adrenal hyperplasia (CAH) by combining differential long template polymerase chain reaction (PCR) amplification and amplified created restriction site (ACRS) methods. All mutations were identified, including five alleles of deletions, three alleles of splicing (IVS2-12[C/A]＞G), four alleles of lie] 72Asn, and two alleles of Arg356Trp. During the course of genetic analysis of CYP2IA2, we found that misgenotyping of CAH by PCR-based method is possible if both alleles of a CAH patient were deletion mutations and at least one of them carried a CYP2/AIP-CYP2IA2 fusion gene. We also found a patients mother was misgenotyping as IVS2-12[CIA]＞G homozygous due to allele dropout” in the PCR amplification process. We present in this article evidences of mis-genotyping by PCR-based amplification method. Due to the pitfalls observed in this study, we recommend that more methods, including microsatellite linkage analysis and direct sequencing, should be performed with direct amplification of known mutations in prenatal diagnosis of CAH to avoid misdiagnosis.

並列關鍵字

congenital adrenal hyperplasia (CAH) ； 21-hydroxylase ； CYP21A2 ； fusion gene ； allele dropout

國際替代計量

Pitfalls of PCR-based Genotyping in Patients with 21-hydroxylase Deficiency

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