Ivashchuk and Sydorchuk: Analysis of polymorphic variants of CFTR (rs 113993960), IL-4 (rs 2243250), PRSS1 (rs 111033565), SPINK1 (rs ID 6690) and TNF-α (rs 1800629) Genes in Patients with Edematous Pancreatitis Living in Northern Bukovyna region



Problem statement and analysis of the recent research

The range of a number of genes associated with congenital or acquired pathology (disorders) of pancreas has ethnic and population specificity [3, 7, 13] which determines their separate value for the study in a particular region and provides prerequisites for the development of recommendations concerning early diagnosis and prophylaxis of hereditary and acquired diseases of pancreas. One of the most important risk factors of pancreatitis is the excessive consumption of alcohol and cholelithiasis [10]. However, it should be noted that chronic pancreatitis (CP), for example, develops only in 10% of patients who abuse alcohol [17]. At the same time, CP can develop without visible or triggering factors. It is called idiopathic pancreatitis constituting 10-30% of all CP cases [1, 15, 16]. In addition, genetic mutations are the recognized risk factor of pancreatitis initiation. They lead to enzymopathy and, hypothetically, are the cause of acute or chronic recurrent inflammation of pancreas. The basic mutations in genes often studied in association with pancreatitis are cystic fibrosis gene (CFTR), cationic tripsinogen gene (PRSS1) and pancreatic secretory trypsin inhibitor (SPINK1) [6, 20]. The mutations in other genes affecting the pancreas state were also described including the ones responsible for the synthesis of alpha-1-antitrypsin [9], cathepsin B [2]. The mutations in PPAR-g genes family which are responsible for carbohydrate and lipid metabolism [19] may affect diabetes mellitus development in patients with pancreatitis, especially with CP [4]. Mutations occurrence of the abovementioned genes influencing acute pancreatitis (AP) or CP phenotype formation are substantially different in different populations and ethnic groups and are also associated with hereditary factors [5, 11].

Resulting from the above mentioned data, it was necessary to study the incidence of CFTR (rs 113993960), IL-4 (rs 2243250), PRSS1 (rs 111033565), SPINK1 (rs ID6690) and TNF-α (rs 1800629) genes mutations in the Northern Bukovyna region to determine their role in the pathogenesis of acute edematous pancreatitis in order to identify the patterns and mechanisms of AP development and the exacerbation of chronic pancreatitis (ECP).

The study of this genes combination in case of pancreas pathology at the moment of this study beginning has not been conducted in Ukraine yet. Although the performance of some not numerous studies of mononucleotide polymorphisms of PRSS1 and SPINK1 genes started during the last five years in Ukraine (Chernivtsi) for AP, mostly its destructive forms [12].

The objective of the research was to study the incidence of CFTR (rs 113993960), IL-4 (rs 2243250), PRSS1 (rs 111033565), SPINK1 (rs ID6690) and TNF-α (rs 1800629) genes mutations in Northern Bukovyna region and their dependence on etiological factor, sex and type of edematous pancreatitis.

Materials and methods

The study included 123 patients with AP and ECP (edematous form). They were admitted to the Local emergency hospital (Chernivtsi, Ukraine) during the last four years. The diagnosis of AP and ECP was made on the basis of the existing national and international recommendations criteria [14, 18]. All patients signed an informed consent and underwent a complex of examinations: clinical, laboratory and instrumental ones according to protocol recommendations. The patients included 23 (18.7%) women and 100 (81.3%) men. The patients’ average age constituted 45.1±5.19 years in men, 53.2±7.07 years in women (23 to 77). The control group included 40 apparently healthy individuals who were not relatives of the patients, of corresponding sex and age.

Molecular genetic testing which included the determination of polymorphic variants of five genes: IL-4 (C-590T), TNF-α (G-308A), PRSS1 (R122H), SPINK1 (N34S) and CFTR (delF508), were performed at the laboratory of the State institution “Reference centre of molecular diagnostics of the Ministry of Health of Ukraine” (Kyiv) and at the laboratory of Medical Biology and Genetics Department of Bukovyna State Medical University. The polymorphic variants of IL-4 (C-590T), TNF-α (G-308A), PRSS1 (R122H), SPINK1 (N34S) and CFTR (delF508) genes were studied using polymerase chain reaction (PCR) method with the use of oligonucleotide primers manufactured by “Metabion” (Germany) according to the modified protocols [8]. Amplification products of DNA fragments of genes were digested in hydrolysis using restriction enzyme (“Thermo Scientific”, USA): enzyme PmlI (Eco72I) for PRSS1 gene, AvaII - for IL-4 gene, NcoI - for TNFα gene. The resulting fragments were analysed in agarose gel with the addition of ethidium bromide, molecular weight marker GeneRuler 50 bp (DNA Ladder, “Thermo Scientific”, USA), and further visualization by using transilluminator and Vitran software.

Compliance of the genotypes distribution with gene polymorphism according to Hardy-Weinberg law in the control group was tested with the chi-square test with 1 degree of freedom, without Yates correction, and the difference in the distribution of genotypes in the control group and among patients was checked with the chi-square test with 2 degrees of freedom.

Results of the research and their discussion

The distribution of analyzed genes genotypes depending on the type of pancreatitis detected the following: the relative incidence of polymorphic variants of PRSS1 (rs 111033565), CFTR (rs 113993960), SPINK1 (rs ID 6690) and TNF-α (rs 1800629) genes between patients with AP and ECP did not significantly differ. However, the carriers of CC-genotype of IL-4 gene were noted among AP patients and in the control group, than among those with ECP by 22.39% (χ2=4.80; p=0.028) and 21.76% (χ2=3.67; p=0.055) more often. The favorable alleles dominated the mutant ones in all groups. The expected population balance of genotypes corresponded to the Hardy-Weinberg law and certified normal population distribution.

The genotypes distribution of CFTR (rs 113993960), IL-4 (rs 2243250), PRSS1 (rs 111033565), SPINK1 (rs ID6690) and TNF-α (rs 1800629) genes depending on the etiology of edematous pancreatitis is shown in Table 1. The relative incidence of GG-genotype of PRSS1 gene predominated in patients with alcohol-related AP than in those with gallstone one by 9.09% [OR=8.60; p=0.033], while GA-genotype, on the contrary, was more often observed in patients with gallstone AP than alcohol-related one. Significant difference in the genotypes distribution taking into consideration etiological factor for other analyzed genes was not established.

Table 1

Distribution of polymorphic variants of CFTR (rs 113993960), IL-4 (rs 2243250), PRSS1 (rs 111033565), SPINK1 (rs ID 6690) and TNF-α (rs 1800629) genes depending on the edematous pancreatitis etiology

The studied genes, n (%) Control group, n=40 (%) Acute pancreatitis OR [95% CI] χ2 р
Alcohol-related, n (%) Gallstone, n (%)
PRSS1 365G>A (p.R122H), n=123 (%) GG 38 (95.0) 74 (98.67) 43 (89.58) 8.60 [0.97-76.1] р=0.033
GA 2 (5.0) 1 (1.33) 5 (10.42) 0,12 [0.01-1.03]
χ2 р χ2=64.80 р<0.001 χ2=142.11 р<0.001 χ2 =60.17 р<0.001 - -
IL-4 (C-590T), n=101 (%) CC 26 (65.0) 39 (60.94) 19 (51.35) 1.48 [0.65-3.35] χ2<1.0 р>0.05
CT 11 (27.50) 21 (32.81) 13 (35.14) 0.90 [0.38-2.12] χ2<1.0 р>0.05
TT 3 (7.50) 4 (6.25) 5 (13.51) 0.43 [0.11-1.70] χ2<1.0 р>0.05
χ2 р χ2 =36.48 р<0.001 χ2 =56.98 р<0.001 χ2 =16.33 р<0.001 - -
CFTR (delF508), n=101 (%) NN 39 (97.50) 61 (95.31) 37 (100.0) 0.95 [0.90-1.01] р>0.05
NM 1 (2.50) 3 (4.69) 0 - -
χ2 р χ2 =72.20 р<0.001 χ2 =105.13 р<0.001 - - -
SPINK1 -215G-A (N34S), n=63 (%) GG 40 (100.0) 37 (97.37) 25 (100.0) 0.97 [0.92-1.03] р>0.05
GA 0 1 (2.63) 0 - -
χ2 р - χ2=68.21 р<0.001 - - -
TNF-α (G-308A), n=11 (%) GG 27 (67.50) 5 (71.43) 4 (100.0) 0.71 [0.45-1.14] р>0.05
GA 13 (32.50) 2 (28.57) 0 - -
χ2 р χ2 =9.80 р=0.002 р>0.05 - - -

Note.

OR – odds ratio;

CI – confidence interval;

n – absolute number.

According to sex distribution men dominated among the examined individuals generally by 4.35 times: 81.3% (n=100) versus 18.7% (n=23) of women (p<0.001). Wild CC-genotype of IL-4 gene significantly more often occurred in men with AP (χ2=3.59, p=0.045) than in those with ECP. However, TC-genotype of this gene was detected relatively more often in women with ECP than in those with AP by 13.10% [OR=6.0; 95% CI: 1.14-31.47; p=0.027] (Table 2). The genotypes distribution of PRSS1 gene between groups (AP vs. ECP) depending on sex did not differ.

Table 2

Distribution of polymorphic variants of PRSS1 (rs 111033565) and IL-4 (rs 2243250) genes in patients with pancreatitis depending on sex and order of appearance

The studied genes, n (%) Acute pancreatitis, n (%) Exacerbation of chronic pancreatitis, n (%) OR [95% CI] χ2 р
PRSS1 (365G>A), n=123 (%) GG M 57 (78.08) 37 (74.0) 1.25 [0.54-2.90] χ2<1.0 p>0.05
W 11 (15.07) 12 (24.0) 0.56 [0.23-1.40] χ2=1.56 р>0.05
GA M 5 (6.85) 1 (2.0) 3.60 [0.41-31.82] p>0.05
W 0 0 - -
IL-4 (C-590T), n=101 (%) CC M 35 (54.69) 13 (35.14) 2.23 [0.97-5.14] χ2=3.59 р=0.045
W 7 (10.94) 3 (8.11) 1.39 [0.34-5.74] p>0.05
TC M 16 (25.0) 10 (27.03) 0.90 [0.36-2.26] χ2<1.0 p>0.05
W 2 (3.12) 6 (16.22) 0.17 [0.03-0.87] р=0.027
TT M 4 (6.25) 4 (10.81) 0.55 [0.13-2.34] p>0.05
W 0 1 (2.70) - -

Note.

M, W – men, women;

OR – odds ratio;

CI – confidence interval;

n – absolute number.

Acute alcohol-related pancreatitis was observed in men significantly more often than gallstone pancreatitis, namely by 53.58% (χ2=46.64; p<0.001) in carriers of “wild” GG-genotype of PRSS1 gene, by 29.64 % (χ2=8.25; p=0.004) and 19.30% (χ2=4.57; p=0.032) in carriers of CC TC-genotypes of IL-4 gene, by 42.40% (χ2=24.74; p<0.001) in carriers of NN-genotype of CFTR gene, and by 38.74% (χ2=13.82; p<0.001) in carriers of GG-genotype of SPINK1 gene

However, gallstone AP was observed in women significantly more frequently than alcoholic-related one, namely by 44.50% (χ2=38.12; p<0.001) in carriers of GG-genotype of PRSS1 gene, by 20.06% (p=0.001) in carriers of CC-genotype of IL-4 gene, by 47.09% (χ2=34.03; p<0.001) in carriers of NN-genotype of CFTR gene and by 41.37% (p<0.001) in carriers of GG-genotype of SPINK1 gene, respectively.

The priority of edematous pancreatitis occurrence depending on etiology taking into consideration the analyzed genes polymorphism is shown in Tables 3 and 4. Edematous AP was more often of alcohol-related etiology than of gallstone one, however only significantly in carriers of GG-genotype of PRSS1 gene by 18.92% (p=0.047). The analysis of polymorphic variants of other investigated genes did not detect the statistically significant dependence.

Table 3

Distribution of polymorphic variants of PRSS1 (rs 111033565) and IL-4 (rs2243250) genes in patients with pancreatitis depending on the order of appearance and etiology

The studied genes, n (%) Alcohol-related, n (%) Gallstone, n (%) OR [95% CI] χ2 р
PRSS1 (365G>A), n=123 (%) GG AP 47 (62.67) 21 (43.75) 2.01 [0.96-4.19] χ2=3.46 p=0.047
ECP 27 (36.0) 22 (45.83) 0.66 [0.32-1.39] χ2=1.40 p>0.05
GA AP 1 (1.33) 4 (8.33) 0.15 [0.02-1.37] χ2<1.0 p>0.05
ECP 0 1 (2.08) - -
IL-4 (C-590T), n=101 (%) CC AP 27 (42.19) 15 (40.54) 1.07 [0.47-2.44] χ2<1.0 p>0.05
ECP 12 (18.75) 4 (10.81) 1.90 [0.57-6.40] χ2=1.11 p>0.05
TC AP 14 (21.88) 4 (10.81) 2.31 [0.70-7.63] χ2=1.96 p>0.05
ECP 7 (10.94) 9 (24.32) 0.38 [0.13-1.13] χ2=3.15 p=0.069
TT AP 2 (3.125) 2 (5.41) 0.56 [0.08-4.18] p>0.05
ECP 2 (3.125) 3 (8.11) 0.37 [0.06-2.30] p>0.05

Note.

AP – acute pancreatitis;

ECP – exacerbation of chronic pancreatitis;

OR – odds ratio;

CI – confidence interval,

n – absolute number.

Table 4

Distribution of polymorphic variants of CFTR (rs 113993960), SPINK1 (rs ID 6690) and TNF-α (rs 1800629) genes in patients with pancreatitis depending on the order of appearance and etiology

The studied genes, n (%) Alcoholic, n (%) Biliary, n (%) OR [95% CI] χ2 р
CFTR (delF508), n=101 (%) NN AP 41 (64.06) 20 (54.05) 1.52 [0.66-3.45] χ2<1.0 p>0.05
ECP 20 (31.25) 17 (45.95) 0.53 [0.23-1.23] χ2=2.18 р>0.05
NM AP 3 (4.69) 0 - -
ECP 0 0 - -
SPINK1 -215G-A, n=63 (%) GG AP 24 (63.16) 11 (44.0) 2.18 [0.78-6.10] χ2=2.24 p>0.05
ECP 13 (34.21) 14 (56.0) 0.41 [0.14-1.15] χ2=2.92 p>0.05
GA AP 1 (2.63) 0 - -
ECP 0 0 - -
TNF-α (G-308A), n=11 (%) GG AP 3 (42.86) 1 (25.0) 2.25 [0.15-33.93] p>0.05
ECP 2 (28.57) 3 (75.0) 0.13 [0.01-2.18] p>0.05
GA AP 1 (14.29) 0 - -
ECP 1 (14.29) 0 - -

Note.

AP – acute pancreatitis;

ECP – exacerbation of chronic pancreatitis;

OR – odds ratio;

CI – confidence interval,

n – absolute number.

Conclusions

1. The mutation of CFTR (rs 113993960), PRSS1 (rs 111033565), SPINK1 (rs ID6690) and TNF-α (rs 1800629) genes in the homozygous state was not detected in the adults of Northern Bukovyna region. According to the nature of the allelic distribution of genes mentioned above in all groups the wild allele dominated over the mutant one. The expected population genotype balance corresponded to the Hardy-Weinberg law and certified normal population distribution.

2. Alcohol-related AP was observed in men significantly more often than gallstone pancreatitis, namely by 53.58% in carriers of “wild” GG-genotype of PRSS1 gene, by 42.40% in carriers of NN-genotype of CFTR gene and by 38.74% in carriers of GG-genotype of SPINK1 gene, respectively. However, was observed in women significantly more frequently than alcoholic-related one, namely by 44.50% in carriers of GG-genotype of PRSS1 gene, by 47.09% in carriers of NN-genotype of CFTR gene and by 41.37% in carriers of GG-genotype of SPINK1 gene, respectively.

3. The mutation of IL-4 gene (rs 2243250) in the homozygous state occurred with the frequency of 7.5% among the generally healthy population of Bukovina and 8.91% among patients with edematous pancreatitis. The wild CC-genotype of IL-4 gene was significantly more often detected in men with AP than in those with ECP. However, TC-genotype of this gene was relatively more often present in women with ECP than in those with AP by 13.10%. Alcohol-related AP was by 29.64% and 19.30% significantly more often observed in men, the carriers of the C-allele of gene IL-4 than the gallstone one. However, gallstone AP was by 20.06% more frequently observed in women carriers of CC-genotype of gene IL-4 than alcohol-related one.

Prospects for further research

Prospects for further research involve analysis of the possible models of edematous pancreatitis inheritance (dominant, recessive, over-dominant and additive) taking into account the polymorphism of selected genes.

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