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

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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.