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

背景-自1988年以来，新生儿肝炎患者主要表现为Δ^4-3-氧胆汁酸尿排泄。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-还原酶）的原发性遗传缺陷还是继发性损伤引起的，一直存在争议。目的：利用分子生物学技术对此类患者进行遗传学研究。患者和方法：对4例新生儿肝炎患者的肝脏标本进行了检查，结果显示尿中Δ^4-3-氧胆汁酸含量显著（48.8%至88.9%）。所有患者均有慢性胆汁淤积伴肝功能衰竭，病因不明。5β-还原酶采用免疫印迹分析、RNA印迹杂交分析和逆转录-聚合酶链反应扩增cDNA序列分析。用气相色谱-质谱法分析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带，尽管量比对照组小。在一名患者中，RNA印迹杂交分析也显示了酶的模糊带。所有患者的酶cDNA全长扩增，没有表现出突变的DNA序列。尽管Δ^4-3-氧胆汁酸在尿液中占主导地位，但与正常胆汁酸相比，血清中这些酸的含量极低。结论：我们的患者尿液中Δ^4-3-氧胆汁酸的主要排泄不是由5β-还原酶的遗传缺陷引起的。即使尿液Δ^4-3-氧胆酸占优势也不能导致5β-还原酶缺乏症的诊断，因此需要对包括尿液、血清和胆汁在内的生物液体中的类固醇代谢进行综合评估。

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.