Study On The Association Of Polymorphisms In HLA-DRB1, TLR9 With Acute Pancreatitis

[Backgroud] Acute pancreatitis is a relatively common disease with variable involvement of peripancreatic tissues and remote organ systems. In the past decade, our understanding of the causes, pathogenesis, natural history, and treatment of acute pancreatitis has grown strikingly. However, in severe disease, the mortality rate remains to be as high as 30% to 40%. Acute pancreatitis continues to be among the most mysterious human diseases that are difficult to study.The latest evidence demonstrates that acute pancreatitis are most often complex disorders that occur in individuals having multiple risk factors for susceptibility and severity, both of which together contribute to the aetiology through pathological pathways that are activated by triggering factors. One of the fascinating questions inattempts to understand the genetics of pancreatitis is what makes the disease expression so variable. Recent breakthroughs in understanding the origin and pathways toward acute pancreatitis has revealed that specific variation in the genomic DNA sequence of individuals strongly influence their susceptibility to pancreatitis, the severity and nature of the inflammatory process and, the likelihood of various complications.In the future, early use of genetic testing will be likely to play a critical role in early diagnosis and prognosis of pancreatic diseases, and willhopefully guide new and effective preventative and therapeutic interventions. The remarkable advance was possible because of the availability of new genetic resources such as the human genome, high throughput DNA sequencing and mutation detection. We are now on the verge of a new era in testing and treating acute pancreatitis.It is increasingly recognized that both innate and acquired immune system play a major role in acute pancreatitis and suggested that inappropriate activation of the immune system might increase the severity of the local disease and the systemic complications.HLA is the most polymorphic gene complex of all human genetic systems. It plays a key role in specific immune response and determines the heterogeneity of an individual associated with diseases. Analyses of the distribution of its polymorphism have been used in fields of transplantation, anthropology and disease association study.The gene product HLA-DR of the major histocompatibility complex (MHC) class II antigens is a vital component of the antigen-presenting mechanism in human immune system. HLA-DR acts as regulator and effector and is capable of influencing the immune response by releasing mediators of inflammation. The HLA-DRB1 gene, encoding HLA-DR molecule, bears intensive polymorphism. This polymorphism has been associated with the susceptibility and the severity to certain pancreatic diseases including pancreatic cancer, diabetes mellitus and chronic pancreatitis. Discovering the distribution of its polymorphism is necessary for medical researches and could be helpful to revealing the mysteries of acute pancreastitis.An ancestral conserved family of Toll-like receptors (TLRs) plays a vital role in the innate immune response. Pathogen recognition by TLRs provokes rapid activation of innate immunity by inducing production of proinflammatory cytokines and upregulation of costimulatory molecules.Activated innate immunity subsequently leads to effective adaptive immunity. TLRs sense not only foreign structures but also endogenous molecules and stimulate immune cells such as B and T lymphocytes, natural killer cells, monocytes, macrophages and dendritic cells.Toll-like receptor 9 (TLR9) recognizes CpG DNA and activates NF-kB signaling cascade resulting in the secretion of a number of pro-inflammatory cytokines and chemokines. Over the past few years, significant evidence has been accumulated that synthesis and release of proinflammatory cytokines and chemokines are responsible for the local injury and the systemic dispersion of the inflammation. If there is an upregulated and inappropriate response to the initial injury, the systemic inflammatory response syndrome (SIRS) will supervene. The systemic manifestations are responsible for the majority of pancreatitis-associated morbidity and mortality and are due to the actions of specific inflammatory cytokines.Under selective pressure from infectious microorganisms, multicellular organisms have evolved immunological defense mechanisms, broadly categorized as innate or adaptive. Recent insights into the complex mechanisms of human innate immunity suggested that genetic variability in genes encoding its components might play a role in determining susceptibility to a range of common human diseases that have an inflammatory component. Although highly conserved across a wide range of species, TLR9 gene, demonstrated recently, has substantial interindividual variability predominantly in the form of single nucleotide polymorphisms (SNPs) that might be associated with altered innate immune responses to many diseases, such as hypernomic inflammation (asthma). These genetic discoveries have provided new insights into the pathobiologic mechanisms of pancreatic inflammation.[ Objective ] The aim of this study was to investigate whether polymorphisms in the HLA-DRBl and TLR9 genes differentially affect the incidence and the clinical course of acute pancreatitis.[Methods] Following informed consent from all patients and healthy volunteers, 2 ml of peripheral blood was collected from each of 90 unrelated healthy Chengdu Han individuals and 131 Chengdu Han acute pancreatitis patients admitted to West China Hospital of Sichuan University. The patients were divided into groups in terms of severity (mild/severe), according to Atlanta classification. Whole DNA was prepared from all subjects. Two assays were carried out: (a) HLA-DRBl polymorphism, and (b) TLR9 single-nucleotide polymorphisms (SNP) at -1237.HLA-DRBl polymorphic site within exon 2 of the gene was amplified with only a single PCR amplification by using a novel high throughput technique (PCR-ST Amp), which improved PCR-SBT gene typing methods. PCR conditions consisted of 95 °C for 15 minutes and then 30 cycles of 95 °C for 20 seconds, 62 °C for 10 seconds, and 72 °C for 90 seconds. The PCR products were separated by electrophoresis on a 2% agarose gel, and then, after purified, sequenced by using automated PE Biosystims 373 DNA sequencers. TLR9 nucleotide polymorphisms (SNP) at -1237 was amplified by using the Real-time fluorescence quantitative PCR with SYBR green I.Statistical analysis: The occurrence of each allele in the populations was expressed as a percentage of the total number of alleles and presented to give a frequency. The genotype frequency (i.e., the number of individuals carrying at least one copy of a specific allele as a proportion of the total number of individuals) also was calculated. The x 2 test with contingency tables was performed to determine the p values, which were then corrected for the number of variables tested (Pc).[Results] The rf tests showed that HLA-DRB1 loci and TLR9 SNP at -1237 of Chengdu Han Ethnic Group both accorded with Hardy-Weinberg equilibrium. The frequency of HLA-DRBl*0406 and HLA-DRB1*14O5 alleles were significantly increased in patients compared with controls (HLA-DRB 1*0406 : P = 0.03 < 0.05 , RR = 3.05 > 1 , 95%CI > 1 ; HLA-DRB 1*1405: P=0.04<0.05, RR=7.84>1, 95%CI>1), while the frequency of HLA-DRB 1*0411 allele was found to be significantly decreased between the two groups (P=0.03<0.05, RR=O.17<1, 95%CI < 1) .When the patients were analyzed according to severity, there was a significantly increased frequency of HLA-DRBl*1101 allele (P = 0.03< 0.05, RR=0.45< 1, 95%CI< 1) in the severe group compared with the mild group.The frequency of C allele at TLR9 -1237 was found to be increased in the patients group compared with the controls group (15.3% vs. 10.6%; P>0.05). When patients were categorised according to severity, there was an increased frequency of the C allele at TLR9 -1237 in the severe group compared with the mild group (18.9% vs. 11.9%; P>0.05), however, there were no significant differences between the frequencies of C allele at TLR9 -1237 in the patients group and the controls group or between the severe group and the mild group. When patients were categorised according to complication, there was an increased frequency in C allele at TLR9 -1237 (23.9% vs. 15.8%; P >0.05) in the infection group compared with the no infection groups, however, there was no significant difference between the two groups. Whereas, there was a significantly increased frequency of C allele at TLR9 -1237 (23.9% vs. 7.9%; P<0.05) in the SIRS group compared with the no SIRS group.[Conclusions] Our study shows that the novel PCR-ST Amp method is accurate, sensitive, reproducible, and of high efficiency. As far as we can determine this approach is the first to describe a high throughput protocol for the SBT of HLA-DRB 1. According to our data, it was suggested that in Chengdu Han ethnic group, HLA-DRB 1*0406 and HLA-DRB1*14O5 alleles may be associated with susceptibility to acute pancreatitis, HLA-DRB1*O411 allele may be associated with resistance to acute pancreastitis, and HLA-DRBl*1101 allele may be a risk factor associated with severe acute pancreatitis. This study also suggests that increased risk for SIRS in severe acute pancreatitis patients with C allele at TLR9 -1237. In addition, data of HLA-DRB 1 and TLR9 polymorphism in Chengdu Han Ethnic Group will contribute much to the improvement of research on disease association and basic immunology. In a word, to our knowledge, this is the first report of the association between HLA-DRB 1, TLR9 gene polymorphisms and acute pancreatitis.