Gene analysis of Δ^4-3-oxosteroid 5β-reductase in neonatal hepatitis patients with predominant urinary Δ^4-3-oxo bile acids

尿Δ^4-3-含氧胆汁酸为主的新生儿肝炎患者Δ^4-3-氧代类固醇5β-还原酶基因分析

• 批准号: 11671257
• 负责人: KONDO Kazuhiro
• 金额: $ 1.73万
• 依托单位: Miyazaki Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671257/
• 关键词: Δ^4-3-oxosteroid 5β-reductase; Δ^4-3-oxo bile acids; neonatal hepatitis

Background - Numbers of neonatal hepatitis patients exhibiting predominant urinary excretion of Δ^4-3-oxo bile acids have been reported since 1988. There has been some controversy whether this phenomenon is due to primary genetic deficiency or secondary impairment of Δ^4-3-oxosteroid 5β-reductase (5β-reductase) in bile acids' biosynthetic pathway.Aim - To study such patients genetically using the techniques of molecular biology.Patients and Methods - Liver specimens obtained from four neonatal hepatitis patients exhibiting predominant amounts of urinary Δ^4-3-oxo bile acids (48.8 to 88.9 %) were examined. All had chronic cholestasis with hepatic failure of unclear etiologies. Immunoblot analysis, RNA blot hybridization analysis, and analysis of the cDNA sequences amplified by the reverse transcriptase-polymerase chain reaction method were employed for 5β-reductase. Bile acids profiles of serum as well as urine were also analyzed by gas chromatography-mass spectrometry.Results - In all patients immunoblot analysis showed a weak band of the enzyme though the amounts were smaller than those in the control group. In one patient, RNA blot hybridization analysis also revealed an indistinct band of the enzyme. The full length of cDNA of the enzyme was amplified in all patients, and none exhibited a mutation in the DNA sequence. Despite Δ^4-3-oxo bile acids' predominancy in urine, the amounts of these acids in serum were extremely small compared to normal bile acids.Conclusions - Predominant excretion of urinary Δ^4-3-oxo bile acids in our patients was not caused by genetic defect of 5β-reductase. Even predominancy of urinary Δ^4-3-oxo bile acids does not lead to the diagnosis of 5β-reductase deficiency, for which combined evaltuations of the steroid metabolism in biological fluids including urine, serum, and bile are necessary.

背景 - 自 1988 年以来，已有大量新生儿肝炎患者表现出以 Δ^4-3-含氧胆汁酸为主的尿排泄的报道。这种现象是由于胆汁酸生物合成途径中 Δ^4-3-氧类固醇 5β-还原酶 (5β-reductase) 的原发性遗传缺陷还是继发性损伤所致，一直存在一些争议。 使用分子生物学技术对患者进行遗传学分析。患者和方法-检查了从四名新生儿肝炎患者获得的肝脏样本，这些患者表现出主要量的尿Δ^4-3-含氧胆汁酸（48.8%至88.9%）。所有患者均患有慢性胆汁淤积并伴有病因不明的肝功能衰竭。对于5β-还原酶，采用免疫印迹分析、RNA印迹杂交分析以及逆转录酶-聚合酶链式反应法扩增的cDNA序列分析。还通过气相色谱-质谱法分析了血清和尿液的胆汁酸谱。结果-在所有患者中，免疫印迹分析显示酶的弱条带，尽管其量小于对照组。在一名患者中，RNA 印迹杂交分析还显示出一条模糊的酶带。所有患者的酶 cDNA 全长均得到扩增，没有人出现 DNA 序列突变。尽管尿液中以Δ^4-3-含氧胆汁酸为主，但与正常胆汁酸相比，血清中这些酸的含量极少。结论-我们患者中尿液中主要排泄的Δ^4-3-含氧胆汁酸并不是由5β-还原酶的遗传缺陷引起的。即使尿中 Δ^4-3-含氧胆汁酸占主导地位，也不会导致 5β-还原酶缺乏症的诊断，为此需要对生物体液（包括尿液、血清和胆汁）中的类固醇代谢进行综合评估。