A Functional CATTn Microsatellite In The Promoter Of Macrophage Migration Inhibitory Factor In Sepsis

Background and ObjectiveMacrophage migration inhibitory factor (MIF) is a potent pro-inflammatory cytokine. An important role for MIF within the cytokine cascade is to act in concert with endogenous glucocorticoids to control the set-point and magnitude of the inflammatory response. MIF plays a pivatol role in the pathogenesis of sepsis. An elevated expression of MIF in the circulation has been documented to be associated with the high severity and fatal outcome of sepsis. A novel CATT-tetranucleotide microsatellite repeat at position -794 of the human MIF gene has been discovered. It functionally affects the activity of the MIF promoter using gene reporter assays. This microsatellite consists of four genotypes which comprise diverse CATT repeat units, the allele with 5-CATT repeat has the lowest level of basaland stimulated MIF promoter activity in vitro. The presence of the lower expressing, 5-CATT repeat allele correlated with less disease severity in a cohort of rheumatoid arthritis patients, and associated with higher susceptibility to or severity of ulcerative colitis, atopy or sarcoidosis.Genetic polymorphisms of the innate immunity, pro- and anti-inflammatory cytokines, and coagulation mediators have been shown to contribute to the pathogenesis of sepsis, a systemic inflammatory response syndrome to infection. Whether this functional CATTn microsatellite in the promoter of MIF affects the susceptibility to and outcome of sepsis has not yet been examined. Analyses of MIF microsatellite genotypes in patients with sepsis may help to classify patients into risk categories and to identify those patients who may benefit from anti-MIF therapeutic strategies.Materials and MethodsAfter approval by the hospital and national ethics committee, written informed consent was obtained from the patient or a first degree relative. Blood samples were obtained from 98 consecutive postoperative patients with the diagnosis of severe sepsis (sepsis group) and 73 patients without sepsis (controls) in the surgical intensive care unit. The diagnosis of severe sepsis met the criteria recommended by the American College of Chest Physicians/Society of Critical CareMedicine Consensus Conference Committee. Acute Physiologic and Chronic Health Evaluation (APACHE II) score was obtained within the first 12 hours in intensive care. Genomic DNA was obtained by a standard phenol-chloroform extraction procedure from peripheral blood. The DNA fragment containing the CATT-tetranucleotide microsatellite repeat at position -794 of the human MIF gene was amplified using polymerase chain reaction (PCR) with a FAM-labeled upstream primer. The amplified DNA fragment was analyzed by the capillary electrophoresis (CE) instead of conventional slab gel electrophoresis. Statistical analysis of the genotype distribution and allele frequencies between sepsis individuals and controls, between survivors and non-survivors were done by Fisher's exact test. A p < 0.05 was regarded as statistically significant.ResultsThe underlying diseases for the severe sepsis patients were peritonitis, pancreatitis, pneumonia and multiple trauma. The APACH II score in sepsis was 22.5±2.1, the mortality in sepsis patients reached 51%. There is no significant difference in the race, age, sexual. Four kinds of alleles with 5, 6, or 7 or 8-CATT repeat units and seven kinds of genotypes with 5/5 > 5/6 > 5^ 5/8 > 6/6 > 6/7 > 7/7 CATT repeat units were detected among these patients either with or without sepsis. The genotype distribution and allele frequencies between sepsis individualsand controls, between survivors and non-survivors didn't differ (p > 0.05).Discussion and conclusionThis study doesn't demonstrate difference in the genotype distribution and allele frequencies between sepsis individuals and controls, between survivors and non-survivors.Microsatellite repeats is abundant sources of genetic variation, the variable number of CATT repeat units in the promoter of MIF has been shown to correlate with the level of MIF gene expression and the severity of inflammatory diseases. The association between CATTn microsatellite in the promoter of MIF gene and the incidence, outcome of severe sepsis needs to be taken into account.In vitro and in vivo study showed that the lower number of CATT repeats correlated with the less production of MIF, and the less severity in rheumatoid arthritis, ulcerative colitis, et al. However, the functional aspect of transcriptional regulation by nuclear factors binding to sites within this microsatellite regions is unclear and still to be addressed. This study shows that the functional CATTn microsatellite in the promoter of macrophage migration inhibitory factor is neither related to the incidence of severe sepsis nor to the fatal outcome of sepsis. Thus the variable number of CATT tetranucleotide repeat at position -794 of the human MIF, which was previously related to the level ofMIF gene expression, is not to be regarded as a genomic marker for susceptibility and prognosis of sepsis. This knowledge may contribute to the genetic analysis of sepsis, and further study will be focus on the functional SNPs within the MIF gene in sepsis and a larger and diverse population genetic study.