CBS突变是维生素B6反应性的良好预测指标：一项基于对35例巴西经典型同型胱氨酸尿症患者分析的研究。
CBS mutations are good predictors for B6-responsiveness: A study based on the analysis of 35 Brazilian Classical Homocystinuria patients.
作者信息
Poloni Soraia, Sperb-Ludwig Fernanda, Borsatto Taciane, Weber Hoss Giovana, Doriqui Maria Juliana R, Embiruçu Emília K, Boa-Sorte Ney, Marques Charles, Kim Chong A, Fischinger Moura de Souza Carolina, Rocha Helio, Ribeiro Marcia, Steiner Carlos E, Moreno Carolina A, Bernardi Pricila, Valadares Eugenia, Artigalas Osvaldo, Carvalho Gerson, Wanderley Hector Y C, Kugele Johanna, Walter Melanie, Gallego-Villar Lorena, Blom Henk J, Schwartz Ida Vanessa D
机构信息
Post-Graduation Program in Genetics and Molecular Biology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
Laboratory of Basic Research and Advanced Investigations in Neurosciences (BRAIN), Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil.
出版信息
Mol Genet Genomic Med. 2018 Mar;6(2):160-170. doi: 10.1002/mgg3.342. Epub 2018 Jan 20.
BACKGROUND
Classical homocystinuria (HCU) is a monogenic disease caused by the deficient activity of cystathionine β-synthase (CβS). The objective of this study was to identify the CBS mutations in Brazilian patients with HCU.
METHODS
gDNA samples were obtained for 35 patients (30 families) with biochemically confirmed diagnosis of HCU. All exons and exon-intron boundaries of CBS gene were sequenced. Gene expression analysis by qRT-PCR was performed in six patients. Novel missense point mutations were expressed in E. coli by site-directed mutagenesis.
RESULTS
Parental consanguinity was reported in 16 families, and pyridoxine responsiveness in five (15%) patients. Among individuals from the same family, all presented the same phenotype. Both pathogenic mutations were identified in 29/30 patients. Twenty-one different mutations were detected in nine exons and three introns; being six common mutations. Most prevalent were p.Ile278Thr (18.2%), p.Trp323Ter (11.3%), p.Thr191Met (11.3%), and c.828+1G>A (11.3%). Eight novel mutations were found [c.2T>C, c.209+1delG, c.284T>C, c.329A>T, c.444delG, c.864_868delGAG c.989_991delAGG, and c.1223+5G>T]. Enzyme activity in E. coli-expressed mutations was 1.5% for c.329A>T and 17.5% for c.284T>C. qRT-PCR analysis revealed reduced gene expression in all evaluated genotypes: [c.209+1delG; c.572C>T]; [c.2T>C; c.828+1G>A]; [c.828+1G>A; c.1126G>A]; [c.833T>C; c.989_991delAGG]; [c.1058C>T; c.146C>T]; and [c.444delG; c.444delG]. The expected phenotype according to the genotype (pyridoxine responsiveness) matched in all cases.
CONCLUSIONS
Most patients studied were pyridoxine nonresponsive and presented early manifestations, suggesting severe phenotypes. Many private mutations were observed, but the four most prevalent mutations together accounted for over 50% of mutated alleles. A good genotype-phenotype relationship was observed within families and for the four most common mutations.
背景
经典型同型胱氨酸尿症（HCU）是一种由胱硫醚β-合酶（CβS）活性缺乏引起的单基因疾病。本研究的目的是鉴定巴西HCU患者中的CBS突变。
方法
获取了35例（30个家系）经生化确诊为HCU的患者的基因组DNA样本。对CBS基因的所有外显子和外显子-内含子边界进行测序。对6例患者进行了qRT-PCR基因表达分析。通过定点诱变在大肠杆菌中表达新的错义点突变。
结果
16个家系报告有父母近亲结婚情况，5例（15%）患者对吡哆醇有反应。在同一家系的个体中，均表现出相同的表型。在29/30例患者中鉴定出两种致病突变。在9个外显子和3个内含子中检测到21种不同的突变；其中6种为常见突变。最常见的是p.Ile278Thr（18.2%）、p.Trp323Ter（11.3%）、p.Thr191Met（11.3%）和c.828+1G>A（11.3%）。发现了8种新突变[c.2T>C、c.209+1delG、c.284T>C、c.329A>T、c.444delG、c.864_868delGAG、c.989_991delAGG和c.1223+5G>T]。在大肠杆菌中表达的c.329A>T突变的酶活性为1.5%，c.284T>C突变的酶活性为17.5%。qRT-PCR分析显示，在所有评估的基因型中基因表达均降低：[c.209+1delG；c.572C>T]；[c.2T>C；c.828+1G>A]；[c.828+1G>A；c.1126G>A]；[c.833T>C；c.989_991delAGG]；[c.1058C>T；c.146C>T]；以及[c.444delG；c.444delG]。在所有病例中，根据基因型预期的表型（吡哆醇反应性）均相符。
结论
大多数研究患者对吡哆醇无反应且有早期表现，提示为严重表型。观察到许多私人突变，但四种最常见的突变共占突变等位基因的50%以上。在家族内部以及四种最常见的突变中观察到良好的基因型-表型关系。
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