苯丙酮尿症患儿苯丙氨酸羟化酶基因突变的研究

Mutation spectrum of the PAH gene in phenylketonuria children in Ningxia

目的了解宁夏地区苯丙酮尿症(PKU)儿童苯丙氨酸羟化酶(PAH)基因突变的特征。方法以经新生儿疾病筛查及气相色谱-质谱联用技术确诊的30例宁夏PKU儿童为病例组,30例正常儿童为对照组,应用PCR技术扩增PAH基因的3、5、6、7、11和12,六个外显子,再经单链构象多态性分析和DNA测序分析PCR扩增产物。结果在60个等位基因中检出51个突变基因,检出率85%;六个外显子共检出16种致病突变,包括8种错义突变(R241C、R243Q、R252Q、G 257 V、R359K*、R408Q、R 413 P、Q419R),3种剪接突变(IVS 4-1 G>A、Y 204 C、IVS 7+2 T>A),3种无义突变(R 111 X、Q160X、Y356X),1种同义突变(V399V)和1种缺失突变(N183del);R243Q突变频率最高,检出率为18.3%,其次是Y 204 C(11.7%)、IVS 4-1 G>A(10.0%)、R 111 X(6.7%)和IVS 7+2 T>A(6.7%)。病例组中发现Exon 6的N183del(C.547-549del GAA)缺失突变和Exon 11的R359K(C.1078G>A)错义突变,为国内首次发现;病例组和对照组中均检出V245V(C.735G>A)和Q232Q(C.696A>G)两种静止突变,且差异无统计学意义(P>0.05)。结论宁夏PKU儿童PAH基因六个外显子最常见的突变类型是错义突变,特别是R243Q;发现中国人群PAH基因的2种新的突变。

Objective To determine the mutation spectrum of the PAH gene in PKU children in Ningxia, six exons of PAH gene were sequenced in each of the 30 phenylketonuria （PKU） children. Methods 30 children diagnosed as PKU by the neonatal screening and/or GC/MS analysis in Ningxia were enrolled in this study. Meanwhile, 30 normal children were served as controls. The exons 3, 5, 6, 7, 11 and 12 of the PAH gene were amplified by polymerase chain reaction. The amplicons were analyzed by single strand conformation polymorphism and sequencing. Results Mutations were identified for 51 of 60 alleles in this study, representing a mutation detection rate of 85 %. A total of 16 different causative mutations were detected, including 8 missense mutations （R241 C, R243 Q, R252Q, G257V, R359K, R408 Q, R413 P, Q419R）, 3 splicing mutations （IVS4- 1G〉A, Y204C, IVST＋2T〉A）, 3 nonsense mutations （Rlll X, Q 160x, Y356X）, 1 synonymous mutation （v399v） and 1 deletion （N 183 del）. R 243 Q （ 18.3 %） had the highest frequency of PAH mutations, and then Y 204 C （ 11.7 %）, IVS 4 - 1 G 〉 A （ 10.0 %）, R 111 X （ 6.7 %） and IVS 7 ＋ 2 T 〉 A （ 6.7 %）. For the first time in China, two novel mutations, deletion mutation N 183 del （C. 547 - 549 delGAA） in exon 6 and missense mutation R 359 K （C. 1078 G〉A） in exon 11, were identified in PKU children. Two silent mutations, V245 V （C. 735 G〉A） and Q232 Q （C. 696A〉G）, were observed in PKU children and the controls, but there were no significant difference between them （P〉0.05）. Conclusions The most common mutations were missense and R243 Q had the highest frequency of mutation. The identification of 2 novel mutations expands the spectrum of Chinese PAH mutations.