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，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发现了4个SNP (nt-155, -433, -616, - 1748)，证明了nt -433位点的SNP是反映慢性丙型肝炎患者肝炎活动的一个标记。在ALT水平持续正常的患者中，该SNP的T/T频率为12%，比ALT水平升高的患者（42%）要小。因此，似乎肝炎的活动性可能是通过由SNP决定的nt -443位点骨桥蛋白在肝脏中的表达来调节的。因此，在本项目中，我们在体外评估骨桥蛋白启动子snp的功能。日本人有3个单倍型，在nt -155和nt -443位点均有纯合子；类型1：缺失，T，类型2：缺失，C，类型3:G， T。每个单倍型的DNA样本在基因启动子区nt 0 ~ -658之间进行PCR扩增。将所得片段转染hepg2细胞后进行双荧光素酶报告基因检测。1型cDNA的启动子活性高于2型cDNA，而3型cDNA的启动子活性明显低于1型和2型cDNA。这些数据表明，在nt -443位点SNP中，等位基因C的启动子活性比等位基因T的启动子活性高，而在nt -155位点SNP中，等位基因G的启动子活性比缺失突变的启动子活性低。考虑到编码在Y染色体上的翻译因子SRY可以结合骨脂素启动子nt -155周围的寡核苷酸，nt -155和nt -443 SNP可能决定肝炎病毒感染后患者肝炎的严重程度，nt -155 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.