Ezquieta B, Beneyto M, Muñoz-Pacheco R, Barrio R, Oyarzabal M, Lechuga J L, Luzuriaga C, Hermoso F, Quinteiro S, Martinez S
Servicio de Bioquímica, Laboratorio Diagnóstico Molecular, Hospital General Universitario Gregorio Marañón, Madrid, Spain.
Prenat Diagn. 2006 Dec;26(12):1172-8. doi: 10.1002/pd.1584.
The detection of 21-OH deficiency (21OHD) carriers in the general population requires that misinterpretations of apparently severe mutations in alleles carrying duplicated genes be avoided. Prenatal treatment prevents virilization in female fetuses and genetic counseling may be offered to couples in which one partner is either a patient or a carrier. This paper proposes a semiquantitative PCR method involving primer extension that distinguishes the severe point mutation Q318X in single gene copy alleles from the normal/nondeficient variant in gene-duplicated alleles.
DNA from 65 individuals carrying Q318X variants, that of 85 partners of 21OHD carriers or patients, and one fetal sample (as well as the DNA of his family) were analyzed. 21OHD alleles were studied by gene-specific PCR/allele-specific oligonucleotides hybridization for common mutations, Southern analysis, complementary direct sequencing and microsatellite typing. Primer extension analysis of the Q318X variants using fluorescent dideoxynucleotides was performed on CYP21A2 gene-specific PCR-amplified DNA samples from controls, patients, potential carriers and prenatal samples.
Different fluorescence patterns were seen for the severe mutation (single gene copy) and the nondeficient (gene-duplicated) alleles carrying Q318X. The normal/mutant fluorescence peak (N/M) ratio was < 1 in all heterozygous carriers (mean 0.83; min. 0.70; max. 0.95). In all normal individuals carrying the gene-duplicated Q318X normal variant, the N/M ratio was > 1 (mean 1.69; min. 1.44; max. 2.02).
The proposed method discriminated between the severe Q318X mutation and the normal Q318X variant in gene duplication, and could be a useful complementary tool in prenatal diagnosis and carrier detection.
在普通人群中检测21-羟化酶缺乏症（21OHD）携带者时，需要避免对携带重复基因的等位基因中明显严重突变的错误解读。产前治疗可防止女性胎儿男性化，对于夫妻双方中有一方是患者或携带者的情况，可提供遗传咨询。本文提出了一种涉及引物延伸的半定量PCR方法，该方法可区分单基因拷贝等位基因中的严重点突变Q318X与基因重复等位基因中的正常/非缺陷变体。
分析了65名携带Q318X变体的个体、85名21OHD携带者或患者的配偶以及一份胎儿样本（及其家族的DNA）的DNA。通过基因特异性PCR/等位基因特异性寡核苷酸杂交研究21OHD等位基因的常见突变，采用Southern分析、互补直接测序和微卫星分型。使用荧光双脱氧核苷酸对Q318X变体进行引物延伸分析，该分析在来自对照、患者、潜在携带者和产前样本的CYP21A2基因特异性PCR扩增的DNA样本上进行。
携带Q318X的严重突变（单基因拷贝）和非缺陷（基因重复）等位基因呈现出不同的荧光模式。在所有杂合携带者中，正常/突变荧光峰（N/M）比值均<1（平均值0.83；最小值0.70；最大值0.95）。在所有携带基因重复的Q318X正常变体的正常个体中，N/M比值>1（平均值1.69；最小值1.44；最大值2.02）。
所提出的方法能够区分严重的Q318X突变和基因重复中的正常Q318X变体，可作为产前诊断和携带者检测的有用补充工具。
