Guldberg P, Güttler F
Danish Center for Human Genome Research, John F Kennedy Institute, Glostrup.
Acta Paediatr Suppl. 1994 Dec;407:27-33. doi: 10.1111/j.1651-2227.1994.tb13443.x.
Mutations in the phenylalanine hydroxylase (PAH) gene represent the root cause of PAH-deficient hyperphenylalaninemia. To date, more than 160 different mutations have been reported. Single-base substitutions and microdeletions account for the majority of molecular defects. This review provides a brief general introduction to various strategies for detection of PAH mutations, and summarizes our own methodological developments. We have established a method based on PCR in combination with denaturing gradient gel electrophoresis (DGGE) for mutation scanning of the entire coding sequence and all exon/intron boundaries of the PAH. Systematic application of this method to the study of a large number of mutant chromosomes from hyperphenylalaninemic patients demonstrated a 98% diagnostic efficiency and a 100% mutation detection efficiency. We have created compromised PCR and DGGE conditions for simultaneous amplification and simultaneous mutation scanning of all PAH-coding fragments. This technique is convenient in a diagnostic setting and allows "same-day" DNA-based diagnosis of newborns with hyperphenylalaninemia. A further modification of the method allows unambiguous identification of known mutations, circumventing the cumbersome step of nucleotide sequencing.
苯丙氨酸羟化酶（PAH）基因突变是PAH缺乏型高苯丙氨酸血症的根本原因。迄今为止，已报道了160多种不同的突变。单碱基替换和微缺失占分子缺陷的大部分。本综述简要介绍了检测PAH突变的各种策略，并总结了我们自己的方法学进展。我们建立了一种基于聚合酶链反应（PCR）结合变性梯度凝胶电泳（DGGE）的方法，用于对PAH的整个编码序列以及所有外显子/内含子边界进行突变扫描。将该方法系统应用于对大量高苯丙氨酸血症患者突变染色体的研究，结果显示诊断效率为98%，突变检测效率为100%。我们创建了优化的PCR和DGGE条件，用于同时扩增和同时对所有PAH编码片段进行突变扫描。该技术在诊断环境中很方便，能够对高苯丙氨酸血症新生儿进行基于DNA的“当日”诊断。对该方法的进一步改进可以明确鉴定已知突变，避免了繁琐的核苷酸测序步骤。
