Genetic factors determining the severity of hepatitis in patients receiving hepatitis virus infection : Significance of osteopontin promoter SNPs

骨桥蛋白是一种具有RGD序列的细胞外基质，它也是启动Th1免疫反应的关键细胞因子。最近，我们在骨桥蛋白基因启动子区域发现了4个SNP(-155、-433、-616、-1,748)，证明在NT-433处的SNP是反映慢性丙型肝炎患者肝炎活动性的一个指标。在ALT持续正常的患者中，该SNP中T/T的频率为12%，低于ALT水平升高的患者(42%)。因此，肝脏中骨桥蛋白的表达可能是通过由NT-443的SNP决定的，从而调节肝炎的活动性。因此，在本项目中，骨桥蛋白启动子SNPs的功能在体外进行了评估。在日本人中存在3种单倍型，分别为缺失、T、缺失、C和3型：G、T。每种单倍型的DNA样品通过聚合酶链式反应在基因启动子区域0-658之间进行扩增。将所得到的片段导入HepG2细胞，进行双荧光素酶报告基因检测。启动子活性在1型高于2型，而在3型明显低于1型和2型。这些结果表明，在SNP中，等位基因C的启动子活性高于等位基因T，而在缺失突变的SNPs中，等位基因G的启动子活性比缺失突变的减弱。考虑到Y染色体上编码的翻译因子SRY可以与骨桥蛋白启动子NT-155附近的寡核苷酸结合，NT-155上的SNP和NT-443上的SNP可能是判断肝炎病毒感染后肝炎严重程度的指标，而且NT-155上的SNP的意义可能因患者性别而异。

Osteopontin is an extracellular matrix with RGD sequence, which also acts as a cytokine crucial for the initiation of Th1 immune reaction. Recently, we identified 4 SNPs (nt-155, -433, -616, -1,748) in promoter region of osteopontin gene, and demonstrated that SNP at nt -433 is a marker to reflect hepatitis activity in patients with chronic hepatitis C. The frequency of T/T in this SNP was 12% in the patients with sustained normal ALT levels, and this ratio was smaller than in those with elevated ALT levels (42%). Thus, it seems that hepatitis activity may be regulated in the patients through osteopontin expression in the liver determined by SNP at nt -443. Thus, in the present project, the function of osteopontin promoter SNPs was evaluated in vitro. Three haplotypes existed in Japanese showing homozygotes both in SNPs at nt -155 and nt -443 ; Type 1 : deletion, T, type 2 : deletion, C and type 3 : G, T, respectively. DNA samples of each haplotype were amplified by PCR between nt 0 and -658 in promoter region of the gene. The resultant fragments were subjected to dual-luciferase reporter assay following transfection into HepG2cells. Promoter activity was greater in cDNA of type 1 than in that of type 2, while the activity was markedly reduced in cDNA of type 3 compared to those of types 1 and 2. These data suggest that promoter activities were greater in cDNA with allele C in SNP at nt -443 than in those with allele T, and the activities were attenuated in cDNA with allele G compared to that with deletion mutation in SNPs at nt -155. Considering that SRY, a translation factor coded on Y chromosome, can bind to the oligonucleotide around nt -155 of osteopomtin promoter, SNP at nt -155 as well as SNP at nt -443 may be responsible for determination of hepatitis severity in patients following hepatitis virus infection and the significance of SNP at nt -155 might be different depending on sex of the patients.