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THE ROLE OF IDENTIFICATION OF SPINK1 GENE MUTATION STATUS IN PATIENTS WITH ACUTE PANCREATITIS OF ALIMENTARY GENESIS FOR THE PREDICTING THE SEVERITY OF CLINICAL COURSE AND FORMATION OF COMPLICATIONS

Abstract
Purpose of the study. To determine the rate of SPINK1 mutation status in patients with acute pancreatitis of alimentary genesis and to determine the prognostic value of allele status of the gene for the assessment of severity and the formation of complicated course.
Materials and methods. 70 patients with acute alimentary pancreatitis underwent examination, 48 (68,57%) men and 22 (31,43%) women. Average age made 45,4 ± 13,87 years. Severe course of acute pancreatitis was diagnosed in 34 (48,57 %) persons, in 25 (35,72%) – moderate severity, in 11 (15,71%) – mild cases. Complicated clinical course of acute pancreatitis was recorded in 59 (84,29%) patients of the group, in the rest patients – 11 (15,71%) clinical course had uncomplicated nature. The severity of clinical course and structure of complications were assessed according to the classification of Atlanta (2012). In order to predict the severity of acute pancreatitis repeated measurements related to the level of band neutrophils, amylase and glucose were taken. Statistical analysis was performed by means of program STATISTICA (StatSoft Statisticа v.10). Outcomes. Mutation of SPINK1 gene was statistically found in most cases in patients with acute alimentary pancreatitis with heavy – 16 (47,06%) and moderate severity level – 8 (32,0%) (р = 0,02). The presence of SPINK1 mutation status is related to statistically higher chances of severe clinical course (OR=3,11, CI(1,08–8,92), р = 0,03). In patients with heterozygotic mutations of SPINK1 we have established statistically higher chances for the formation of pancreatic aggregation (OR = 4,5, CI (1,36–14,93), p = 0,01), pseudocysts (OR = 3,58, CI (1,01–12,74), p = 0,04) and pleural empyema (OR = 15,0, CI (1,56–143,83), p = 0,004).The carriers of homozygotic mutations of SPINK1 have higher risks for the development of peritonitis (OR = 12,89, CI (1,01–164,48), p = 0,04), pleurisy (OR = 12,89, CI (1,01–164,48),p = 0,04) and systemic complications (OR = 2,61, CI (2,14–13,14), p = 0,02).
Conclusion. Consequently, we have established a high informative value of identification of SPINK1 mutation status in patients with acute pancreatitis of alimentary genesis regarding the prediction of severity level of inflammatory process and the formation of complicated clinical
course.
Keywords: acute alimentary pancreatitis, SPINK1 mutation status, SPINK1 polymorphism, pancreatitis, gene of pancreatictrypsin inhibitor.

according to the classification of Atlanta (2012). In order to predict the severity of acute pancreatitis repeated measurements related to the level of band neutrophils, amylase and glucose were taken. Statistical analysis was performed by means of program STATISTICA (StatSoft Statisticа v.10).

INTRODUCTION
The problem of diagnosis and management of acute pancreatitis is one of the most complex and relevant in the field of modern surgery of the gastrointestinal tract. In view of the variety and variability of clinical manifestations, early diagnosis of the disease remains problematic regardless the wide range of modern laboratory and testing tools. Besides, the lack of specific therapy is a factor of high incidence of adverse effects of medical management and the formation of a number of comorbid conditions that lower the living standards of persons, preferably of working age.
Multifactorial etiology of acute pancreatitis has been proved. The main factors are ill-balanced diet, excessive consumption of hardened fats, protein deficiency in the diet, starvation, alcohol abuse, etc. The role of genetic factors in the pathogenesis of acute pancreatitis is promising. At present time, a number of mutations have been identified that determine the tendency to acute pancreatitis. Thus, the development activity of autocatalytic process in the pancreas is related to the neutralizing effect of secretory pancreatic trypsin inhibitor, the synthesis of which is encoded by SPINK1 gene. In case of mutation in the pancreatic trypsin inhibitor gene, trypsin inactivation of the gland tissue is impaired, which leads to activation of pancreatic enzymes, proteolytic necrosis of the pancreatic tissues, and venous lysis. The presence of SPINK1 homozygous mutation is related to the processes of intra-acinar trypsin inactivation and is a leading factor in the formation of the inflammatory process in the pancreas.
The importance of genetic factors in the development of the inflammatory process in the pancreas has been described in numerous papers; however the relation of mutational status taking into account the severity of the clinical course and the development of complications remains unstudied. Studying the role of N34S polymorphism of SPINK1 gene in the prediction of severity level and the risk assessment of the complicated clinical course of acute pancreatitis is a pressing problem and requires further study.

RESEARCH OBJECTIVE
To determine the rate of SPINK1 mutation status in patients with acute pancreatitis of alimentary genesis and to determine the prognostic value of allele status of the gene for the assessment of severity level and the formation of complicated clinical course. To identify the alleles of SPINK1 gene we used amplification of the appropriate gene segment using the method of allele-specific polymerase chain reaction (two amplification reactions were performed in parallel with two pairs of allele-specific primers) in real time using a set of reagents using the method of SNP-express-PB (Lytech, Russian Federation). Amplification was performed using the device iCycler IQTM5 instrument (Bio-Rad, USA). Amplification mode: 930C, 1 min; 35 cycles: 930C, 10 s.; 640C, 10s, 720C, 20 s.

MATERIALS AND METHODS
According to Kolmagorov-Smirnov test, the distribution of the study group differed from normal with a high degree of probability (p < 0,05). Non-parametric criteria of Kraskell-Wallis and Mann-Whitney were used to compare the indices of the independent groups. The severity level of acute pancreatitis was predicted on the basis of laboratory indicators using a statistical model of logistic regression. By calculation of odds ratio (OR) and 95% confidence intervals (CI), the relation between the studied laboratory parameters with the severity of acute alimentary pancreatitis was assessed in terms of quantity. Each factor was separately assessed. The probability of an error-free prediction was determined at p < 0,05. STATISTICA (StatSoft Statisticа v.10) software was used for statistical analysis of the resulting array.
The rate of SPINK1 mutation status in patients with complicated clinical course of acute pancreatitis significantly differed from the indices of group with uncomplicated clinical course (р = 0,03) (table 2). Mutations of SPINK1 gene were not found in patients with uncomplicated clinical course of acute pancreatitis -11 (100%) and 35 (59,32%) patients had complications. When comparing frequency indices we have determined significant difference in the indices (р = 0,03).

CONCLUSIONS
We have recorded a significantly higher rate of SPINK1 mutation status identification in patients with severe forms of acute alimentary pancreatitis and in case of a complicated course.
We have proved a high prognostic value of SPINK1 mutation status identification for assessing the severity of the inflammatory process of the pancreas. We have determined that the identification of SPINK1 mutation status is a reliable and accurate criterion for assessing the risk of complications in case of acute pancreatitis of alimentary genesis.