论文部分内容阅读
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5μLPCR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ABI377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突变型纯合子。
Background: Phenylketonuria is caused by a mutation in the phenylalanine hydroxylase gene. Mutations in the phenylalanine hydroxylase gene are mainly due to base substitutions, short fragments, and insertions. Objective: To identify the mutation of phenylalanine hydroxylase gene in the family of phenylketonuria in the Hui ethnic group. Design: Open experiment. Unit: People's Liberation Army Lanzhou Military Region Urumqi General Hospital and Beijing Capital Institute of Pediatrics. Subjects: children, male and Hui nationality, at the age of 3 years and 1 month. About 1 year old found that mental retardation, nearly 3 years old to the hospital for medical treatment, diagnosed as cerebral palsy, repeated treatment ineffective in 2004-12-13 into the PLA General Hospital of Urumqi, Lanzhou. Urine ferric chloride test was strongly positive, blood phenylalanine concentration of 1680μmol / L, then the diagnosis of children with classic phenylketonuria. Methods: Children and their parents were drawn venous blood 5mL, EDTA-Na2 anticoagulation. Genomic DNA extraction was performed using the classical phenol / chloroform method. PAH gene exons 7,6,11,3,12,5 PCR primer sequence reference design. PCR products were detected by 2% agarose gel electrophoresis. Take 5μL PCR products with equal volume of denaturation buffer mix, denaturation at 97 ℃ 5min, ice bath and quickly loaded on 80g / L non-denaturing polyacrylamide gel electrophoresis. After the electrophoresis silver staining of conventional methods, analysis and record single-stranded DNA banding. The PCR products were directly sequenced and the samples were purified and sequenced by Shanghai Bea Biotech using the ABI377 Automated Sequence Analyzer (PE). MAIN OUTCOME MEASURES: Urine ferric chloride test, blood paraheine concentration, and phenylalanine hydroxylase mutant genotypes. Results: The PAH genes 7, 6, 11, 3, 12, and 5 were analyzed in patients and their parents. The SSCP electrophoresis behavior of patients and parents in exon 6 was different from that of the normal controls, in which the father and mother The electrophoresis bands are in the same position, while the electrophoresis bands of the children are in different positions. Sequencing results showed that point mutation at the 526th position of the phenylalanine hydroxylase gene cDNA of the father and mother occurred when the cytosine was replaced by thymine, which was a R176X mutant heterozygote, while both chromosomes of the two children were in the same place Point mutation occurred, as R176X mutant homozygotes. Conclusions: The R 176X mutant homozygote of phenylketonuria was first detected in Hui nationality in China.