中国回族首次检出苯丙酮尿症R176X突变型纯合子:1例报告

背景：苯丙酮尿症是由于苯丙氨酸羟化酶基因突变引起，苯丙氨酸羟化酶基因突变主要是因为碱基的置换，短片段、插入等。目的：鉴定回族苯丙酮尿症家系中苯丙氨酸羟化酶基因突变。设计：开放性实验。单位：解放军兰州军区乌鲁木齐总医院和北京首都儿科研究所。对象：患儿，男，回族，就诊时3岁1个月。1岁左右时发现智力滞后，近3岁时到医院就医，诊断为脑性瘫痪，反复治疗无效后于2004—12-13转入解放军兰州军区乌鲁木齐总医院就诊。尿液三氯化铁试验呈强阳性，血苯丙氨酸浓度测定1680μmol／L，遂确诊患儿为经典型苯丙酮尿症。方法：抽取患儿及父母静脉血各5mL，EDTA-Na2抗凝。基因组DNA提取采用经典的酚，氯仿法进行。PAH基因外显子7，6，11，3，12，5的PCR引物序列参照文献设计。PCR产物用2％琼脂糖凝胶电泳检测。取5μL PCR产物与等体积的变性缓冲液混匀，97℃变性5min，冰浴并迅速上样于80g／L非变性聚丙烯酰胺凝胶中电泳。电泳结束后常规方法银染，分析并记录单链DNA带型。采用PCR产物直接测序的方法，由上海博亚生物技术公司应用AB1377全自动序列分析仪（PE公司）完成样品纯化及序列分析。主要观察指标：尿液三氯化铁试验，血液血苯丙氨酸浓度和苯丙氨酸羟化酶突变基因类型。结果：分析患者及其父母PAH第7，6，11，3，12，5外显子基因，发现在外显子6中患者及父母的SSCP电泳行为与正常对照均不相同，其中父亲和母亲的电泳条带位置一致，而患儿的电泳条带位置相异。测序结果显示，父亲和母亲的苯丙氨酸羟化酶基因cDNA第526位发生了胞嘧啶被胸腺嘧啶替代的点突变，是R176X突变型杂合子，而患儿的两条染色体都在同一位点发生了突变，为R176X突变型纯合子。结论：在中国回族首次检出苯丙酮尿症R176X突变型纯合予。

R176X mutant homozygote in phenylketonuria firstly detected in Hui nationality in China: One case report

BACKGROUND: Phenylketonuria is caused by gene mutation of phenylalanine hydroxylasel (PAH), which is mainly induced by permutation, short segments and insertion of base.OBJECTIVE: To evaluate the gene mutation of phenylalanine hydroxylasel in phenylketonuria in Hui nationality.DESIGN: Open study.SETTING: Urumqi General Hospital of Lanzhou Military Area Command of Chinese PLA; Capital Pediatrics Institute.PARTICIPANTS: A boy of Hui nationality in China and aged 3.1 years was selected in this study. The boy had intellect hysteresis in his one year and received medical treatment in his three years, while he was diagnosed as cerebral paralysis. After repeatedly inefficient treatment, he was hospitalized in our hospital on December 13, 2004. Iron sesquichloride in urine was strongly positive and concentration of serum phenylalanine was 1 680 μmol/L; therefore, he was diagnosed as the typical phenylketonuria.METHODS: 5 mL venous blood was selected from the boy and his parents, respectively, and anticoagulated with EDTA-Na2. DNA in gene group was extracted by using typical phenol/chloroform method. In addition, polymerase chain reaction (PCR) primer sequence of extron 7, 6, 11, 3, 12 and 5 of PAH gene was designed based on references. And then, PCR products were detected with 2% agarose gel electrophoresis. 5 μL PCR products were mixed with the same volume of degenerated buffer solution, degenerated at 97 ℃ for 5 minutes, put in iced bath and performed with 80 g/Lnon-degenerated polyacrylamide gel electrophoresis. After that, the products were dealt with sliver staining routinely, and single strand DNA banding patterns were analyzed and recorded. ABI377 automatic sequenator (PE Company) was used to detect PCR sequence and purify PCR product in Shanghai Boya Biotechnology Company.MAIN OUTCOME MEASURES: Iron sesquichloride in urine, concentration of serum phenylalanine and mutant gene types of phenylalanine hydroxylase.RESULTS: Extron 7, 6, 11, 3, 12 and 5 of PAH gene were analyzed in the boy and his parents. The results demonstrated that SSCP electrophoresis in extron 6 was different from that in the normal control group. Site of electrophoresis strip of his father was coincident with that of his mother, but different from that of the boy. Sequencing results indicated that point mutation (cytosine replaced by thymine), which was a R176X mutant heterozygote, occurred at the 526th site of cDNA of phenylalanine hydroxylase gene in his parents; however, two chromosomes of the boy had mutation at the same site, which was R176X mutant homozygote.CONCLUSION: Mutation of R176X homozygote of phenylketonurea is firstly reported in Hui nationality in China.