Analysis of human interferon-gamma-inducible protein 10 (IP-10)/CXCL10 promoter polymorphism at position -938

Introduction - Interferon-γ inducible protein 10 (IP-10)/CXCLl10 was shown to be an indicator of disease progress for severe acute respiratory syndrome (SARS); a high plasma level in the early clinical stage was associated with subsequent adverse outcome. The mechanism that triggers CXCL10 expression in SARS-CoV infection is still unknown.
Method - We conducted a genetic epidemiological study to identify the single nucleotide polymorphism (SNP) of CXCL10 that might be associated with severe SARS clinical outcomes. With luciferase assay and electromobility shift assay (EMSA), we conducted in vitro functional study of the polymorphic alleles of CXCL10 promoter with the attempt to identify the regulatory factors for CXCL10 expression.
Results - Five SNPs of CXCL10 were typed for 108 SARS patients along with 242 healthy control DNAs. A genotype TT at the CXCL10(-938) SNP locus was identified to correlate with severity of SARS-CoV infected patients, especially among SARS patients with a detectably higher nasopharyngeal virus load.
DNA fragment of the 996 bp upstream of the CXCL10 start codon containing either (-938C) or (-938T) SNP was cloned into the luciferase reporter pGL3 vector along with a series of 5’ end truncated CXCL10 promoter DNA fragments. With IFN-γ stimulation in A549 cell and HMEC-1 cells, the shortest two fragments (-704, and -413) showed a high luciferase activity, which dropped with each increment of the 5’ end DNA length; stimulation with IFN-γ and TNF-α in combination induced a higher luciferase activity, but the drop of activity was reversed with the fragment of -704 and -996, suggesting possibly IFN-γ associated negative regulation factors and TNF-α associated positive regulation factors could bind to this region. The difference of luciferase activity between the two alleles of CXCL10(-996C) and CXCL10(-996T) could not be consistently demonstrated, however.
We used nuclear extracts from IFN-γ induced THP-1 cells and the 32P-labeled probes of CXCL10(-928~-948) promoter sequence containing (-938C) or (-938T) and antibodies against a number of TFs antibodies to perform EMSA. The (-938C) probe consistently binds to more nuclear proteins than the (-938T) probe, and three putative binding proteins, YY-1, MZF and Pax-6, of CXCL10 (-938) were found to reduce the shifted band in EMSA and supershift assay. The activation functions of YY-1 and MZF on CXCL10 expression were demonstrated by luciferase assay and the results showed YY-1 and MZF could trigger the activation of CXCL10, however, YY-1 and MZF induced activity were not different between the two alleles.
Conclusion - The genotype TT of CXCL10 (-938) SNP was associated with adverse outcome of SARS patient. The DNA sequence flanking the CXCL10 (-938) SNP locus possibly contain binding motifs of YY-1, MZF and Pax-6. However, the functional difference between these two alleles of CXCL10 (-938) could not be demonstrated in vitro by luciferase assay and EMSA in the study.