Okano Y, Wang T, Eisensmith R C, Steinmann B, Gitzelmann R, Woo S L
Howard Hughes Medical Institute, Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
Am J Hum Genet. 1990 Jan;46(1):18-25.
We report missense mutations associated with haplotype 1 and haplotype 4 alleles of the human phenylalanine hydroxylase (PAH) gene. Individual exon-containing regions were amplified by polymerase chain reaction from genomic DNA of a PKU patient who was a haplotype 1/4 compound heterozygote. The amplified DNA fragments were subcloned into M13 for sequence analysis. Missense mutations were observed in exons 5 and 7, resulting in the substitution of Arg by Gln at residues 158 and 261 of the enzyme, respectively. Expression analysis in heterozygous mammalian cells after site-directed mutagenesis demonstrated that the Arg158-to-Gln158 mutation is a PKU mutation, whereas the Arg261-to-Gln261 mutation is apparently silent in the assay system. Hybridization analysis using allele-specific oligonucleotide probes demonstrated that the Arg158-to-Gln158 mutation is present in two of six mutant haplotype 4 alleles among the Swiss and constitutes about 40% of all mutant haplotype 4 alleles in the European population. The mutation is not present in normal alleles or in any mutant alleles of other haplotypes. The results provide conclusive evidence that there is linkage disequilibrium between mutation and haplotype in the PAH gene and that multiple mutations have occurred in the PAH gene of a prevalent haplotype among Caucasians.
我们报告了与人类苯丙氨酸羟化酶（PAH）基因单倍型1和单倍型4等位基因相关的错义突变。通过聚合酶链反应从一名苯丙酮尿症（PKU）患者的基因组DNA中扩增出各个含外显子区域，该患者为单倍型1/4复合杂合子。将扩增的DNA片段亚克隆到M13中进行序列分析。在外显子5和7中观察到错义突变，分别导致该酶第158位和第261位的精氨酸被谷氨酰胺取代。定点诱变后在杂合哺乳动物细胞中的表达分析表明，精氨酸158突变为谷氨酰胺158的突变是一种PKU突变，而精氨酸261突变为谷氨酰胺261的突变在检测系统中显然是沉默的。使用等位基因特异性寡核苷酸探针的杂交分析表明，精氨酸158突变为谷氨酰胺158的突变存在于瑞士人群中六个突变单倍型4等位基因中的两个，约占欧洲人群所有突变单倍型4等位基因的40%。该突变不存在于正常等位基因或其他单倍型的任何突变等位基因中。这些结果提供了确凿的证据，证明PAH基因中突变与单倍型之间存在连锁不平衡，并且在高加索人中一种常见单倍型的PAH基因中发生了多个突变。
