宁夏地区苯丙酮尿症患儿苯丙氨酸羟化酶基因突变分析

Mutational spectrum of phenylalanine hydroxylase gene in Ningxia patients with phenylketonuria

目的：分析宁夏地区苯丙酮尿症（PKU）患者苯丙氨酸羟化酶（PAH）基因突变分布特征，了解其热点突变位点及区域。方法采用直接测序的方法，首先对30例宁夏地区 PKU 患者 PAH 基因6个热点突变外显子3，5，6，7，11，12进行测序，对未检出突变的患者再进行外显子1，2，4，8，9，10，13测序。采用多重连接探针扩增（MLPA）方法对于通过测序方法未能明确突变基因型的患者进行外显子大片段缺失突变检测。结果通过对30例患者进行全部外显子序列突变分析，在60个等位基因中明确了58个突变等位，检出率为96.7％。其中在外显子3、5、6、7、11、12检出了49个突变，检出率为81.6％。这58个突变等位分属于23个突变位点，包括错义突变9种、剪接突变9种、无义突变2种、微小缺失突变2种、大片段缺失突变1种。其中 R243Q 突变频率最高（18.3％），其次是 IVS4-1G ﹥ A（11.7％）和 R111X（11.7％）。在外显子11的测序中发现了一个新的缺失突变 N393del。采用 MLPA 技术在3例患者中检测到了大片段缺失突变 c.-1932＋3402del，其中1例为纯合缺失，2例为杂合缺失突变。结论宁夏地区 PKU 患者 PAH 基因突变具有明显的热点突变和突变热点区域。宁夏地区 PKU 患者存在 PAH 基因大片段缺失突变，MLPA 技术是检测 PAH 基因大片段缺失的有效手段。

Objective To analyze the mutation distribution of phenylalanine hydroxylase（PAH）gene in pa-tients with phenylketonuria（PKU）in Ningxia,and understand the hot spots and regions of PAH gene. Methods First-ly,6 hot mutation exons including 3,5,6,7,11,12 and their surrounding introns of the PAH gene in 30 patients with PKU in Ningxia were directly sequenced. And then the last 7 exons of 1,2,4,8,9,10,13 were sequenced for the patients in which 2 mutant alleles were not characterized. Multiplex ligation - dependent probe amplification （MLPA）was performed for the identification of uncharacterized mutant alleles after PAH sequence analysis of patients with PKU. Results Among 60 alleles,there were 58 mutant alleles（96. 7% ）. Forty - six（81. 6% ）mutant alleles were found in the exons 3,5,6,7,11,12. A total of 23 various mutations were detected,including missense（n = 9）, splicing（n = 9）,nonsense（n = 2）,small deletion（n = 2）and large deletion（n = 1）. The most common mutations were R243Q（18. 3% ）,IVS4 - 1G ﹥ A（11. 7% ）and R111X（11. 7% ）. Among them,a novel mutation N393del was detec-ted in exon 11. MLPA identified a large deletion（c. - 1932 ＋ 3402del）in 3 patients,1 of them was homoallelic muta-tion,and the others were heteroallelic mutation. Conclusions There are obvious hotspots and hot spot areas of PAH gene in PKU patients in Ningxia. There is a large deletion mutation in PKU patients in Ningxia. The MLPA is an effec-tive assay to detect large deletion in PAH gene.