INHERITED DISORDERS OF BILIRUBIN GLUCURONIDATION

该项目的具体目的是（A）在Crigler-Najjar综合征I型和II型患者中进行基因型-表型相关性研究，（B）确定吉尔伯特综合征的分子基础。 除了在这些目标上取得进展外，我们还在一个密切相关的领域取得了额外的进展，即I型Crigler-Najjar综合征基因治疗方法的临床前开发。基因型-表型相关性：我们继续分析UGT 1A 1基因编码区的序列。 这些研究已经证实，编码UGT 1A 1（UGT 1A 1）的五个外显子中的任何一个突变都可以显着降低酶活性，导致Crigler-Najjar综合征I型和II型。 以下突变是特别感兴趣的。(i)在编码UDP-葡萄糖醛酸结合区和推定膜锚定区序列下游的外显子5中发现的一种新突变。 这表明，在没有指定特定功能的结构域中缺失一些氨基酸残基也可能对酶活性至关重要。(ii)推测的胆红素结合位点上游区域的突变与严重的高胆红素血症相关，与1型Crigler-Najjar综合征一致。 然而，在这种情况下，胆汁中含有少量胆红素葡萄糖醛酸苷。 该病例强调了Crigler-Najjar综合征I型和II型表型之间的重叠。 我们的假设是胆红素结合域附近的突变将增加胆红素酶的Km，从而增加血清胆红素水平。 然而，在这些情况下，Vmax可能相对不受影响，因此，胆汁可能含有胆红素葡萄糖醛酸苷。 这一假设正在通过表达构建体的定点诱变进行测试。 由于我们最近在体内定点基因修复方面的工作，Crigler-Najjar综合征基因型诊断的意义有所增加。 这种策略需要合成的RNA-DNA嵌合体的产生和递送，该嵌合体能够在突变位点对齐并触发细胞的错配修复系统。 与其他基因治疗方法相比，这种方法需要鉴定特定的遗传病变。吉尔伯特综合征的分子基础：我们继续对大量轻度非结合型高胆红素血症患者进行基因分析。 这些研究揭示了以下有趣的发现：（i）吉尔伯特综合征的临床诊断与UGT 1A 1外显子1上游比正常TATAA元件更长的TATAA元件之间存在极高的相关性。 事实上，在高加索人、黑人和（亚洲）印第安人中，我们没有发现这一规则的例外。 在某些吉尔伯特综合征病例中，TATAA元件延长了四个核苷酸（TATA），而不是两个（TA）。然而，TATAA元件比正常短两个核苷酸（TA）与吉尔伯特综合征无关。 (ii)据报道，UGT 1A 1外显子1的结构突变与许多日本患者的吉尔伯特综合征相关。 我们广泛的研究没有发现这种突变在高加索人，黑人和（亚洲）印度人。 我们将通过定点诱变，然后在COS细胞中表达，确定日本报道的突变的意义。

The specific aims of this project were (A) to perform a genotype-phenotype correlation in patients with Crigler-Najjar syndrome types I and II, and (B) to define the molecular basis of Gilbert syndrome. In addition to making progress on these aims, we have made additional advances in a closely related area, preclinical development of gene therapy methods for Crigler-Najjar syndrome type I. Genotype-phenotype correlation: We have continued to analyze the sequence of the coding region for the UGT1A1 gene. These studies have confirmed that mutations in any of the five exons encoding UGT1A1 (bilirubin-UGT) can markedly reduce the enzyme activity, causing Crigler-Najjar syndrome types I and II. The following mutations were of specific interest. (i) A novel mutation found in exon 5, downstream to sequences encoding the UDP-glucuronic acid-binding and the putative membrane-anchoring regions. This shows that deletion of a few aminoacid residues in the domains for which no specific function have been assigned can also be critical for enzyme activity. (ii) A mutation upstream to the putative region of the bilirubin-binding site was associated with severe hyperbilirubinemia, consistent with Crigler-Najjar syndrome type 1. However, in this case, the bile contained a small amount of bilirubin glucuronides. This case highlights the overlap between phenotypes of Crigler-Najjar syndrome types I and II. Our hypothesis is that mutations in the vicinity of the bilirubin-binding domain will increase the Km of the enzyme for bilirubin, thereby increasing serum bilirubin levels. However, in these cases, the Vmax may be relatively unaffected and, therefore, the bile may contain bilirubin glucuronides. This hypothesis is being tested by site-directed mutagenesis of the expression constructs. The significance of genotypic diagnosis of Crigler-Najjar syndrome has increased as a result of our recent work on site-directed gene repair in vivo. This strategy requires the generation and delivery of a synthetic RNA-DNA chimera that is capable of aligning at the site of mutation and triggering the cell's mismatch repair system. In contrast to other methods of gene therapy, this method requires identification of the specific genetic lesion. Molecular basis of Gilbert syndrome: We have continued to perform genetic analysis in a large number of patients with mild unconjugated hyperbilirubinemia. These studies have revealed the following interesting findings: (i) There is an extremely high correlation between the clinical diagnosis of Gilbert syndrome and a longer than normal TATAA element upstream to exon 1 of UGT1A1. In fact, among Caucasians, blacks and (Asian) Indians, we have found no exceptions to this rule. In some cases of Gilbert syndrome, the TATAA element is elongated by four nucleotides (TATA), rather than two (TA). However, TATAA elements that were shorter than normal by two nucleotides (TA) were not associated with Gilbert syndrome. (ii) A structural mutation in exon 1 of UGT1A1 has been reported to be associated with Gilbert syndrome in many Japanese patients. Our extensive studies have not revealed this mutation in Caucasians, blacks and (Asian) Indians. We will determine the significance of the mutation reported from Japan by site-directed mutagenesis followed by expression in COS cells.