Clinical Features And Molecular Genetics Of HSD3B7 Deficiency And AKR1D1 Deficiency

Part One:Clinical features and mutation spectrum of HSD3B7 deficiency and AKR1D1 deficiency in ChinaAims:To explore the significance of HSD3B7 dificiency and AKR1D1 deficiency in Chinese patients with intrahepatic cholestasis. To further the knowledge of clinical features and mutation spectrum.Methods:Patients with intrahepatic cholestasis of unknown cause after exclusion of other main causes of cholestasis from January 2009 to January 2014 were enrolled. Four patients did gene analysis, then patiens with genetic diagnosis and 34 new patients did FAB-MS anlysis of urinary bile acids. Addionally, two patients with idiopathic cholestasis did whole exome sequencing. The clinical features, biochemical parameters and response to therapy were compared with patients with or without inborn errors of bile acid synthesis.Results:10 patients with 14 mutations in HSD3B7 gene and 5 patients with 6 mutations in AKRID1 gene were identified. Ratio of abnormal kidney structure was higher in patients with HSD3B7 or AKR1D1 deficiency than patients with ATP8B1 or ABCB11 dificency (P=0.005). The serum gamma-glutamyl-transpeptidase (GGT) and total bile acid (TBA) levels were lower in patients with HSD3B7 or AKR1D1 deficiency than those in patients without bile acid synthetic defects (P=0.016,0.000). UDCA or CDCA therapy normalized serum biochemical parameters of 11 patiens. For 8 patients, CDCA therapy appears to be effective in accomplishing adequate down-regulation in bile acid synthesis.Conclusion:Some cases of intrahepatic cholestasis with normal GGT and TBA could be attributed to HSD3B7 or AKR1D1 deficiency in China. Normal GGT and TBA are biochemical features and abnormal kidney structure is another clinical feature. CDCA therary could normalized clinical symptoms, serum and urinary biochemical parameters.Part Two:Functional analysis of missense mutations in AKR1D1 geneAims:To investgate causal roles of these missense mutation in AKR1D1 in 5β-reductase deficiency.Methods:(1)AKR1D1 cDNA was obtainedfrom liver tissue which was the waste of liver surgery; (2) Construction of wild-type expression plasmid pcDNA4-AKRlDl; (3) Site directed mutagenesis to construct four kinds of expression vectors containing novel missense mutation; (4) To identified best conditon of transient transfection, using plasmid pcDNA4-EGFP transfected HEK293 cells and observing the amount of fluorescence 24 and 48 hours later; (5)Use wild-type and mutated-type expression vectors transfected HEK293 cells; (6) 48 hours later, extracting total protein; (7) AKR1D1 expression was detected by immunoblotting; (8) Protein expression weas compared among wild-type AKR1D1 and mutated-type AKR1D1.Results:We successfully constructed expression vector:pcDNA4-AKRlDl, pcDNA4.0-AKR1D1-D53G, pcDNA4.0-AKR1D1-D241V, pcDNA4.0-AKR1D1-R266Q and pcDNA4.0-AKR1D1-R307C; (2) When wild-type and all mutants were over-expressed in HEK 293 cells, protein expression levels of D53G and D241V were elevated, protein expression levels of R266Q and R307C showed no difference.Conclusion:For all four missense mutation identified in Chinese patients with functional bile acid deficiency, D53G and D241V affected AKR1D1 enzyme expression and therefore may be causal for this disease, R266Q and R307C show no affection on enzyme expression. We need further purify mutant enzymes.Part Three:Gene analysis and prenatal diagnosis for a family of primary AKR1D1 deficiencyAims:For an earnet request of parents of an proband with CBAS2, we present a report on prenatal diagnosis using direct AKRIDI gene analysis in a family with the proband with A4-3-oxosteroid 5β-reductase (AKRIDI) deficiency.Methods:DNA was obtained from parents and fetal DNA from the amniotic fluid by amniocentesis in 20 weeks of pregnacy and cultured fetal amniotic fluid cell. Exon 4 and exon7 of AKR1D1 gene were sequencing. Third gene sequecing, serum biochemical parameters and FAB-MS analysis of urinary bile acids were did one month after the second child born.Results:Gene sequencing revealed C.396C>A from father and c.722A>T from mother. Neither mutations was not found in the fetal DNA. One month after the birth, gene sequencing, serum and urine biochemical parameters indicated the second child was normal.Discussion:This is the first report on direct prenatal diagnosis of AKR1D1 deficiency. The second child is heathy.