Eiken H G, Knappskog P M, Apold J, Flatmark T
Department of Medical Genetics, Haukeland Hospital, University of Bergen, Norway.
Hum Mutat. 1996;7(3):228-38. doi: 10.1002/(SICI)1098-1004(1996)7:3<228::AID-HUMU7>3.0.CO;2-6.
The G46S mutation in the phenylalanine hydroxylase (PAH) gene was identified by fluorescence-based single-strand conformation polymorphism (F-SSCP) analysis on phenylketonuria (PKU) haplotype 5.9 alleles. DNA sequencing of PAH exon 2 revealed a G-to-A transition in cDNA position 136. G46S mutations were present on 17 of 236 Norwegian PKU alleles (7.2%) and on 8 of 176 Swedish PKU alleles (4.5%). Analysis of all 13 exons with the flanking regions further detected a 1316-35c > t polymorphism (PAH intron 12), associated with both G46S and haplotype 5.9. Three patients were homozygous for the G46S mutation, two were untreated and had mild and severe mental retardation, respectively. The G46S mutation was introduced in the PAH cDNA by site-directed mutagenesis and expressed in three different systems (the pMAL/Escherichia coli system, the pcDNA3/human embryonic kidney (A293) cells, and the pcDNA3/TnT coupled in vitro transcription-translation system). The mutant recombinant E. coli fusion protein was recovered in high yield and with a specific activity of the purified tetrameric form, which was higher than the wild-type activity. After transient expression in A293 cells, the amount of the G46S protein was only about 3% of the wild type at equal PAH mRNA levels. The fusion protein cleaved by restriction protease factor Xa, as well as the enzyme produced by in vitro transcription-translation, revealed an abnormal susceptibility to form catalytically inactive high-molecular-mass aggregates of the enzyme. This aggregation, followed by an increased cellular degradation of the G46S mutant enzyme, is compatible with the clinical/metabolic phenotype of the affected homozygous and compound heterozygous patients.
通过对苯丙酮尿症（PKU）单倍型5.9等位基因进行基于荧光的单链构象多态性（F-SSCP）分析，鉴定出苯丙氨酸羟化酶（PAH）基因中的G46S突变。PAH外显子2的DNA测序显示，cDNA位置136处发生了G到A的转变。236个挪威PKU等位基因中有17个（7.2%）存在G46S突变，176个瑞典PKU等位基因中有8个（4.5%）存在该突变。对所有13个外显子及其侧翼区域的分析进一步检测到一个1316-35c>t多态性（PAH内含子12），它与G46S和单倍型5.9均相关。三名患者为G46S突变纯合子，其中两名未经治疗，分别患有轻度和重度智力发育迟缓。通过定点诱变将G46S突变引入PAH cDNA，并在三种不同系统（pMAL/大肠杆菌系统、pcDNA3/人胚肾（A293）细胞以及pcDNA3/TnT偶联的体外转录-翻译系统）中表达。突变的重组大肠杆菌融合蛋白以高产率回收，纯化的四聚体形式具有比野生型更高的比活性。在A293细胞中瞬时表达后，在PAH mRNA水平相等的情况下，G46S蛋白的量仅约为野生型的3%。经限制性蛋白酶因子Xa切割的融合蛋白以及体外转录-翻译产生的酶，显示出对形成酶的无催化活性高分子量聚集体的异常敏感性。这种聚集，随后G46S突变酶的细胞降解增加，与受影响的纯合子和复合杂合子患者的临床/代谢表型相符。
