The Influence Of Common Exon Variant(P364L) On Unconjugated Bilirubin Glucuronidation By UDP-glucuronosyltransferase 1A1

A genetic disease with hyperbilirubinemia which is based on the elevation of unconjugated bilirubin is not rare in clinical treatmeat.The most common disease is Gilbert syndrome(GS) which was first studied by Gilbert and Lereboullet in 1901 and the next most common disease is Crigler-Najjar sydrome Ⅱ(CNS-Ⅱ) which was first studied by Arias in 1952.The least common disease is Crigler-Najjar sydrome Ⅰ(CNS-Ⅰ) which is the most lethal type.GS, CNS-Ⅰ and CNS-Ⅱ have the same pathogenesis that is decrease of activity in UDP-glucuronosyltransferase 1A1,being caused by the mutation in UGT1A1 gene.To date 130 mutations in UGT1A1 gene had been found,most of which were located in exons 1,2 and 5,however mutations in exons 3 and 4 were few. Our team’s research has found that a patient with CNS-Ⅱ was carrying a homozygotic mutation of c.1091C>T in exon 4.This missense mutation led to amino acid substitute at 364 th locus,namely, leucine substitute to proline.The report of missense mutation(P364L) was few in past research and there was no report in non-Asians.So far,the patient diagnosed with CNS-Ⅱ,being found by our team,is the first report.Therefore,we created plasmid with mutant gene to research the catalytic function of mutant enzyme on unconjugated bilirubin.Objective: Validate the corollary that mutation of c.1091C>T in exon 4,leading to leucine substitute to proline at 364 th locus,reduces the catalytic function of UDP-glucuronosyltransferase 1A1 on unconjugated bilirubin.Methods: Use plasmid with normol UGT1A1 gene as a template to create plasmid with mutant gene(c.1091C>T) by Site-directed mutagenesis.Sequenced the mutant plasmid and blast the consequence with the normal plasmid.Validate if the mutant plasmid is created rightly.Transfect HEK293 cells with normal and mutant plasmid respectively and express normal enzyme and mutant enzyme.Extract total RNA from the HEK293 cells being transfected by normal and mutant plasmid respectively and synthesize cDNA by reverse transcription. Real-time PCR was used to detect mutant and normol plasmid gene-expression differences.Ultransound was used to lyse HEK293 cells and collect supernatant after being centrifugated under low temperature by high speed.Western blot was used to detect the expression of normal and mutant enzyme.Draw the standard curve of unconjugated bilirubin.Combine unconjugated bilirubin with uridine diphosphate glucuronic acid catalyzed by normal and mutant enzyme respectively. High performance liquid chromatograph was used to analyze quantitatively the concentration of unconjugated bilirubin.Compare catalytic activity of normal enzyme with mutant enzyme on unconjugated bilirubin.Results:1. The consequence showed that 1091 th base in exon 4 was substituted for guanine by adenine. That was substituted for cytosine by thymine in the complementary strand.The construction of mutant plasmid was very success.2. The consequence of WB showed that protein expressed by HEK293 cells, being transfected by normal or mutant plasmid, were both located at 55 KD.3. Extract total RNA from the HEK293 cells which were transfected by normal plasmid or mutant plasmid and the consequence of FQ-PCR analysis showed that there was no difference in gene expression between normal and mutant plasmid.4. Draw the standard curve of unconjugated bilirubin and obtain regression equation(y=0.0571x+0.1011, R2=0.9946).X represents peak area of unconjugated bilirubin.Y represents concentration of unconjugated bilirubin(μg/m L). Concentration of unconjugated bilirubin showed a liner relationship among 0.31255μg/m L to 5μg/m L.5. Combine unconjugated bilirubin with uridine diphosphate glucuronic acid catalyzed by normal and mutant enzyme( P364L) respectively. Concentration of unconjugated bilirubin decreased most quickly in first 15 minutes in both two reactions.Overall,the concentration of unconjugated bilirubin decreased more slowly, being catalyzed by mutant enzyme(P364L).Conclusion:Catalytic activity of mutant enzyme(P364L)on unconjugated bilirubin is lower than normal enzyme.