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，以及(B)确定Gilbert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UGT1A1基因编码区的序列。这些研究证实，编码UGT1A1（胆红素- ugt）的5个外显子中的任何一个突变都能显著降低酶活性，导致I型和II型Crigler-Najjar综合征。下面的突变是我们特别感兴趣的。(i)在编码udp -葡萄糖醛酸结合区和假定的膜锚定区序列的下游外显子5中发现了一个新的突变。这表明，在没有特定功能的结构域中，一些氨基酸残基的缺失也可能对酶的活性至关重要。（ii）胆红素结合位点上游的一个突变与严重的高胆红素血症有关，与Crigler-Najjar综合征1型一致。然而，在这种情况下，胆汁含有少量胆红素葡萄糖醛酸盐。该病例突出了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区域附近的突变会增加胆红素酶的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盐。这一假设正在通过表达结构的定点诱变进行验证。由于我们最近在体内定点基因修复方面的工作，基因型诊断克里格勒-纳贾尔综合征的意义有所增加。这种策略需要合成RNA-DNA嵌合体的产生和传递，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，这种方法需要识别特定的遗传病变。吉尔伯特综合征的分子基础：我们继续对大量轻度非共轭高胆红素血症患者进行遗传分析。这些研究揭示了以下有趣的发现：(i) Gilbert综合征的临床诊断与UGT1A1外显子1上游比正常更长的TATAA元件之间存在极高的相关性。事实上，在白种人、黑人和（亚洲）印度人中，我们没有发现例外。在吉尔伯特综合征的一些病例中，TATAA元件被拉长了四个核苷酸（TATA），而不是两个核苷酸（TA）。然而，比正常短两个核苷酸（TA）的TATAA元件与Gilbert综合征无关。（ii）据报道，UGT1A1外显子1的结构突变与许多日本患者的吉尔伯特综合征有关。我们的广泛研究尚未发现这种突变在白种人、黑人和（亚洲）印第安人中。我们将通过位点定向诱变，然后在COS细胞中表达来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.