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 Table of Contents  
ORIGINAL ARTICLE
Year : 2023  |  Volume : 3  |  Issue : 1  |  Page : 24-28

Association of Heat-Shock Protein Gene Polymorphisms with Disease Severity in Acute Pancreatitis


Department of Gastrointestinal Sciences, Christian Medical College, Vellore, Tamil Nadu, India

Date of Submission27-Nov-2022
Date of Decision29-Nov-2022
Date of Acceptance30-Nov-2022
Date of Web Publication28-Dec-2022

Correspondence Address:
L S Unnikrishnan
Department of Gastrointestinal Sciences, Christian Medical College, Vellore - 632 002, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ghep.ghep_32_22

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  Abstract 


Background: Severe acute pancreatitis is the result of a cytokine storm induced by pancreatic inflammation. Mutations in the heat-shock protein 70 (HSP70) gene can induce excessive cytokine secretion in the context of inflammation. We undertook this study to determine whether common single-nucleotide polymorphisms (SNPs) in the HSP70 gene were associated with severe acute pancreatitis. Methods: One hundred and twenty-seven consecutive patients with acute pancreatitis admitted to the hospital were evaluated and followed up for clinical outcomes. DNA from venous blood was extracted, and the following SNPs were genotyped – rs1061581 (HSPA1B), rs2227956 (HSPA1 L), rs1008438 (HSPA1A), and rs1043618 (HSPA1A). Polymerase chain reaction-restriction fragment length polymorphism was used for genotyping at the first two loci, while allele-specific polymerase chain reaction was used to genotype the two latter SNPs. Results: None of the four hP70 gene polymorphisms that were studied showed any significant difference between acute mild and acute severe pancreatitis. However, the T allele in rs2227956 showed a trend to association with severe pancreatitis (P = 0.08), as did the TT genotype (0.09). Conclusion: HSP70 gene polymorphisms did not significantly associate with the severity of illness in acute pancreatitis.

Keywords: Acute pancreatitis, cytokine storm, disease severity, heat-shock protein 70 gene, organ failure


How to cite this article:
Unnikrishnan L S, Pugazhendhi S, Chowdhury SD, Ramakrishna BS. Association of Heat-Shock Protein Gene Polymorphisms with Disease Severity in Acute Pancreatitis. Gastroenterol Hepatol Endosc Pract 2023;3:24-8

How to cite this URL:
Unnikrishnan L S, Pugazhendhi S, Chowdhury SD, Ramakrishna BS. Association of Heat-Shock Protein Gene Polymorphisms with Disease Severity in Acute Pancreatitis. Gastroenterol Hepatol Endosc Pract [serial online] 2023 [cited 2023 Jan 27];3:24-8. Available from: http://www.ghepjournal.com/text.asp?2023/3/1/24/365724




  Introduction Top


Acute pancreatitis, due to a variety of causes, including alcoholism and gallstones, is a disease of variable severity. The Atlanta classification (in the revised form currently) is widely used in classifying disease severity based on organ failure and local complications.[1],[2] Mild pancreatitis is usually interstitial pancreatitis, while severe pancreatitis is characterized by pancreatic necrosis of varying extents. Between 10% and 20% of patients develop necrotizing pancreatitis, which is severe and characterized by significant morbidity, mortality, and long-term consequences, including endocrine insufficiency. On the other hand, patients with acute mild pancreatitis usually recover early without complications.

Acute pancreatitis is characterized by the activation of precursor enzymes within the pancreatic acinar cell, leading to the destruction of pancreatic parenchyma and the systemic activation of coagulation, kinin, complement, and fibrinolytic cascades.[3] This leads to the liberation of cytokines such as tumor necrosis factor-alpha (TNF-α) interleukins 1, 6, and 8 (IL-1, IL-6, IL-8) and platelet-activating factor, as well as reactive oxygen metabolites, all of which are responsible for the systemic manifestations of pancreatitis (e.g., increased capillary permeability, vasodilation, and the reduced cardiac contractility) leading to shock, acute renal failure, and the acute respiratory distress syndrome. The organ failure which defines severe pancreatitis is the result of a cytokine storm.[4] The release of inflammatory cytokines in response to a particular stimulus is likely to be determined by the genetic makeup of the individual. A number of genes associated with inflammatory cytokine responses have been investigated for associations with the severity of acute pancreatitis.

Heat-shock proteins (HSPs) are chaperone proteins that protect living cells against injury-inducing stimuli. HSP70 is a 70 kDa protein that is an integral part of innate immune pathways that modulate inflammatory reactions in response to infections and cell damage. In an experimental animal model of pancreatitis, it has been shown that HSP70 protects against cell injury and acinar necrosis.[5] In humans, three members of the HSP70 gene family cluster on the short arm of chromosome 6; they are HSPA1 L (HSP70-hom), HSPA1A (HSP70-1), and HSPA1B (HSP70-2). Certain genotypes of HSP70 are associated with a higher cytokine output in response to any inflammatory stimulus. Single-nucleotide polymorphisms (SNP) in the HSP70 genes have been associated with clinical outcomes in a variety of diseases, including diabetic foot ulcer and sepsis, presumably by modulating inflammation.[6],[7]

Genotype assessments are important prognostic tools to predict disease severity and the course of acute pancreatitis. Thus, genotype assessments may also be used to guide treatment or to identify populations at risk for severe acute pancreatitis. This pilot study was undertaken to determine whether common SNPs in the HSP70 gene were associated with the disease severity in acute pancreatitis.


  Methods Top


This was a prospective cohort study performed at the Christian Medical College (CMC), Vellore. Consecutive cases of acute pancreatitis admitted to the hospital were included in the study if they met the study criteria. Inclusion criteria were a history of acute abdominal pain supported by radiological (ultrasound or computed tomography) evidence of acute pancreatitis with the laboratory evidence of serum amylase >3 times the upper limit of normal. Patients with evidence of chronic pancreatitis or pancreatic neoplasia (ductal dilatation, ductal or parenchymal calcification, or pancreatic mass) were excluded, as those patients were unwilling to give written informed consent. Patients were investigated according to the standard clinical protocols to identify the cause of pancreatitis, including significant alcohol use, presence of gallstones, hypercalcemia, or hypertriglyceridemia. The original Atlanta classification was used to classify the severity and complications of the disease. The Human Ethics Committee of the CMC, Vellore, reviewed and approved the proposal and consent forms. The study was conducted in the Department of Gastrointestinal Sciences in CMC, Vellore, from February 2011 to December 2012. Informed written consent was obtained from all participating patients.

Nine milliliters of venous blood was collected in ethylenediaminetetraacetic acid-coated vacutainer tubes after obtaining informed written consent. Genomic DNA was isolated using the standard salting-out procedure. Genomic DNA was extracted from the mononuclear cells in peripheral blood by phenol–chloroform extraction and stored at −20°C. The primers and restriction enzymes used to genotype the HSPA1B A1538G, HSPA1 L C2437T, rs1008438, and rs1043618 loci have been described earlier [Table 1].[6] The HSP70 polymorphisms rs1061581 and rs2227956 were detected by polymerase chain reaction (PCR)-restriction fragment length polymorphism, while rs1008438 and rs1043618 were detected by allele-specific PCR. The PCR reactions were of 20 μl volume, and each reaction mix contained 1× Taq DNA Polymerase Master Mix Red (Ampliqon, USA) and 250nM of forward and reverse primers (Sigma-Genosys, India). The thermal cycling protocol for rs1061581, rs2227956, rs1008438, and rs1043618 comprised initial denaturation at 95°C for 5 min, cycle denaturation at 94°C for 30 s, annealing temperature at 58°C and 62°C for 30 s, extension at 72°C for 30 s, and the cycle was repeated for 34 more times, final extension at 72°C for 5 min. The PCR products were checked for amplification by resolving on 2% agarose gel electrophoresis and checked with UV transilluminator (Vilber Lourmat, France). The amplified samples were digested with 2 units of restriction enzymes, PstI and NcoI (MBI Fermentas), respectively, for rs1061581 and rs2227956 analysis at 37°C for 16 h. The digested PCR products were resolved on 2% agarose gel electrophoresis, and the gel patterns were documented using a gel documentation system (Vilber Lourmat, France) [Figure 1] and [Figure 2]. Genotypes were assigned as earlier described.[6]
Table 1: Primers used to genotype the single-nucleotide polymorphisms in this study

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Figure 1: PCR-RFLP for a polymorphism in the HSP gene. For rs1061581, the wild-type gene product size is 1117 base pairs, while the mutant gene gets split into 936 and 181 base pair fragments. PCR-RFLP: Polymerase chain reaction-restriction fragment length polymorphism, HSP: Heat shock protein

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Figure 2: Allele-specific PCR for two polymorphisms in the HSP gene. In the allele-specific PCR, products get amplified differentially in two different PCRs. One allele (e.g. C) gets amplified in one PCR, while the other allele (e.g., G) gets amplified in PCR using the second allele-specific primer set. If a patient's sample shows amplification in one of the PCRs only, then he/she is homozygous for that particular allele at that locus, while the amplification in both PCRs indicate heterozygosity at that particular locus. Product sizes remain the same. PCR: Polymerase chain reaction, HSP: Heat shock protein

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Patients were followed until death or discharge, and relevant clinical and laboratory data were recorded in pro forma and transferred to an Excel sheet later. Since this study was carried out before the revised Atlanta classification of the severity of acute pancreatitis, disease severity and complications were defined as per the original Atlanta classification.[1] Patients who were discharged at request in moribund condition were also clubbed with those who had died for the purpose of statistical analysis. Apart from seeing the relationship between polymorphism with the severity of pancreatitis, we also looked into the possible association between HSP70 gene polymorphism and the occurrence of organ failure, length of hospital stay, length of stay in the intensive care unit, and mortality.

Sample size

From previous studies, we determined that the inflammatory HSPA1B AG genotype was present in approximately half of our population.[6] Our hypothesis was that HSP70 genotypes would have modified the course of acute pancreatitis rather than having a causal link with acute pancreatitis. Therefore, we expected that the percentage of acute pancreatitis patients having inflammatory HSP70 genotypes would be similar to that in the general population. We assumed that one-third of hospitalized patients with acute pancreatitis would have a severe illness, while two-thirds would have mild illness. Assuming a relative risk of 3 for the development of severe pancreatitis in someone with an inflammatory genotype compared to a noninflammatory genotype, we calculated the sample size of 124 patients to disprove the null hypothesis with a study power of 80% and type I error of 0.05.

Statistics

The data were rechecked after entry for errors, coded, and analyzed using the software SPSS 11.5 (SPSS Inc, Chicago, USA) for Windows. Categorical data were compared using the Pearson's Chi-square test, while continuous data were compared using two-tailed independent t-tests. Continuous variables were expressed as mean ± standard deviation.


  Results Top


One hundred and twenty-seven consecutively admitted patients were enrolled in the study. Of these patients, 61 had mild pancreatitis and 66 had severe pancreatitis. The characteristics of these patients are shown in [Table 2]. Alcohol was the most common etiological factor (62 patients, 48.8%), second, being biliary (30 patients, 24%), followed by idiopathic (25 patients, 20%), postendoscopic retrograde cholangiopancreatography (eight patients, 6.2%), and drugs (NeoMercazole in one patient).
Table 2: Characteristics of the participants in the study

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Organ failure was noted in 30 of 127 patients (24.0%), of which 24 had respiratory complications, 14 had cardiovascular, eight had renal, and one had gastrointestinal bleed-related complications. The chest X-ray at the time of admission showed bilateral pleural effusion in 28, left-sided pleural effusion in 22, and acute respiratory distress syndrome features in two. Seventeen patients (13.3%) required a ventilatory support, of which 15 required invasive ventilatory support. Seventeen patients (13.3%) required pressor support during their hospital stay and 5 (4%) required dialysis. Forty-nine patients required nasojejunal feeding, three required total parenteral nutrition, and the rest were managed by oral feeding. Fifty patients (41.7%) had local complications, of which acute fluid collection was noted in 47, pseudocyst in nine, and abscess in one.

Seventeen had organisms grown on blood culture, including Klebsiella (8), Escherichia coli (7), Acinetobacter (1), Enterococcus (1), and Candida (1). Ascitic fluid culture grew Klebsiella in three and E. coli in one, while urine culture grew E. coli, Klebsiella, and Enterococcus in one patient each. Sputum culture grew Klebsiella in two patients.

Six patients (4.7%) required surgery. Of the 127 patients, 114 (89.7%) were alive at the time of discharge, 7 (5.5%) patients were discharged against medical advice in moribund condition, and 6 (4.7%) died during the hospital stay.

None of the four HSP70 gene polymorphisms was significantly associated with the severity of pancreatitis. However, the T allele in rs2227956 showed a trend to association with severe pancreatitis (P = 0.08) [Table 3], as did the TT genotype (0.09) [Table 4]. HSP70 gene polymorphism did not associate with the occurrence of individual organ failure, length of hospital stay, length of the intensive care unit stay, or mortality.
Table 3: Allele frequencies in the four single-nucleotide polymorphisms studied, with odds ratio and confidence intervals

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Table 4: HSP70 genotype frequencies in acute mild and acute severe pancreatitis

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  Discussion Top


HSPs are a group of proteins whose expression is increased when cells are exposed to elevated temperatures or other forms of stress. They help in maintaining the metabolic and structural integrity of cells. They are designated according to their molecular weight, for example, HSP70 refers to a family of HSPs of approximately 70 kDa in size. HSP70 is the major HSP expressed in humans and is involved in antigen binding and presentation to the immune system. HSP70 has both anti-inflammatory and proinflammatory effects, depending on the cell type and context, and whether it is intracellular or extracellular in the location.[8] The intracellular effects are often anti-inflammatory, by inhibiting signaling through nuclear factor kappa B.[9] The extracellular effects can lead to inflammatory cytokine production or the induction of regulatory immune cells and reduced inflammation.

Certain genotypes of HSP70 are associated with a higher output of cytokines in response to inflammatory stimuli. For example, HSPA1 L gene polymorphisms were associated with the magnitude of the HSP70 response in monocytes and lymphocytes, with the CC genotype being associated with lower levels of inducible HSP70 than the TT genotype.[10] The HSPA1B G allele has been shown to be associated with higher levels of HSP70 in the serum of patients with chronic heart failure.[11] In patients with severe multiple trauma, the genotypes HSPA1B AG or HSPA1 L CT were associated with significantly higher plasma concentrations of TNF-α and IL-6 than those with GG or TT genotypes.[12]

HSPs are upregulated in acute pancreatitis and show a protective effect in experimental pancreatitis.[13] HSP 70 protects against cellular injury and acinar necrosis, thereby reducing the severity of acute pancreatitis.[8] In a small study of 15 patients, low levels of HSP 70 protein in the serum were associated with the severity and death.[14] These patients also had higher serum levels of TNF-α, IL-6, and IL-8. The HSPA1B + 1267 A/G polymorphism has previously been studied in relation to acute pancreatitis. Tukiainen et al., in a case–control study, showed that this polymorphism did not show a significant association with the disease severity in patients with acute pancreatitis.[15] On the other hand, Balog et al. concluded that the G allele and AG genotype in this polymorphism were significantly associated with the severity of acute pancreatitis, although the overall prevalence in patients with acute pancreatitis was similar to that in controls.[16] The third study concluded that the AG allele of this polymorphism was significantly more common in patients with acute pancreatitis than controls and additionally found a trend toward a significant association (P = 0.071) with the severity grade of pancreatitis.[17] The last study included only 50 patients with acute pancreatitis.

The present study showed that although there was no significant association between three of the studied polymorphisms with disease severity, the HPSA1 L polymorphism showed a trend to significance when evaluating its association with disease severity in acute pancreatitis. The numbers included in the present study were small, and probably, the study was underpowered to detect a statistically significant difference. We did not include a normal control population in the study because our aim was to evaluate whether this polymorphism predisposed to more severe pancreatitis. Like many common inflammatory disorders, the occurrence of disease may not depend on genetic factors; however, genetic factors related to the immune and inflammatory cascades may determine who develops the mild disease and who develops the severe disease.[18],[19]


  Conclusion Top


Larger studies will probably be necessary to identify the genetic markers that determine the disease severity and outcomes in acute pancreatitis, and their identification may lead to interventions designed to reduce morbidity and mortality in this disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Bradley EL 3rd. A clinically based classification system for acute pancreatitis. Summary of the International symposium on acute pancreatitis, Atlanta, Ga, September 11 through 13, 1992. Arch Surg 1993;128:586-90.  Back to cited text no. 1
    
2.
Banks PA, Bollen TL, Dervenis C, Gooszen HG, Johnson CD, Sarr MG, et al. Classification of acute pancreatitis – 2012: Revision of the Atlanta classification and definitions by international consensus. Gut 2013;62:102-11.  Back to cited text no. 2
    
3.
Raraty MG, Connor S, Criddle DN, Sutton R, Neoptolemos JP. Acute pancreatitis and organ failure: Pathophysiology, natural history, and management strategies. Curr Gastroenterol Rep 2004;6:99-103.  Back to cited text no. 3
    
4.
Makhija R, Kingsnorth AN. Cytokine storm in acute pancreatitis. J Hepatobiliary Pancreat Surg 2002;9:401-10.  Back to cited text no. 4
    
5.
Saluja A, Dudeja V. Heat shock proteins in pancreatic diseases. J Gastroenterol Hepatol 2008;23 Suppl 1:S42-5.  Back to cited text no. 5
    
6.
Mir KA, Pugazhendhi S, Paul MJ, Nair A, Ramakrishna BS. Heat-shock protein 70 gene polymorphism is associated with the severity of diabetic foot ulcer and the outcome of surgical treatment. Br J Surg 2009;96:1205-9.  Back to cited text no. 6
    
7.
Ramakrishna K, Pugazhendhi S, Kabeerdoss J, Peter JV. Association between heat shock protein 70 gene polymorphisms and clinical outcomes in intensive care unit patients with sepsis. Indian J Crit Care Med 2014;18:205-11.  Back to cited text no. 7
[PUBMED]  [Full text]  
8.
Bhagat L, Singh VP, Hietaranta AJ, Agrawal S, Steer ML, Saluja AK. Heat shock protein 70 prevents secretagogue-induced cell injury in the pancreas by preventing intracellular trypsinogen activation. J Clin Invest 2000;106:81-9.  Back to cited text no. 8
    
9.
Venkatraman A, Ramakrishna BS, Shaji RV, Kumar NS, Pulimood A, Patra S. Amelioration of dextran sulfate colitis by butyrate: Role of heat shock protein 70 and NF-kappaB. Am J Physiol Gastrointest Liver Physiol 2003;285:G177-84.  Back to cited text no. 9
    
10.
Singh R, Kølvraa S, Bross P, Jensen UB, Gregersen N, Tan Q, et al. Reduced heat shock response in human mononuclear cells during aging and its association with polymorphisms in HSP70 genes. Cell Stress Chaperones 2006;11:208-15.  Back to cited text no. 10
    
11.
Jenei ZM, Gombos T, Förhécz Z, Pozsonyi Z, Karádi I, Jánoskuti L, et al. Elevated extracellular HSP70 (HSPA1A) level as an independent prognostic marker of mortality in patients with heart failure. Cell Stress Chaperones 2013;18:809-13.  Back to cited text no. 11
    
12.
Schröder O, Schulte KM, Ostermann P, Röher HD, Ekkernkamp A, Laun RA. Heat shock protein 70 genotypes HSPA1B and HSPA1L influence cytokine concentrations and interfere with outcome after major injury. Crit Care Med 2003;31:73-9.  Back to cited text no. 12
    
13.
Rakonczay Z Jr, Takács T, Boros I, Lonovics J. Heat shock proteins and the pancreas. J Cell Physiol 2003;195:383-91.  Back to cited text no. 13
    
14.
Arriaga-Pizano L, Boscó-Gárate I, Martínez-Ordaz JL, Wong-Baeza I, Gutiérrez-Mendoza M, Sánchez-Fernandez P, et al. High serum levels of high-mobility group Box 1 (HMGB1) and low levels of heat shock protein 70 (Hsp70) are associated with poor prognosis in patients with acute pancreatitis. Arch Med Res 2018;49:504-11.  Back to cited text no. 14
    
15.
Tukiainen E, Kylänpää ML, Puolakkainen P, Kemppainen E, Halonen K, Orpana A, et al. Polymorphisms of the TNF, CD14, and HSPA1B genes in patients with acute alcohol-induced pancreatitis. Pancreas 2008;37:56-61.  Back to cited text no. 15
    
16.
Balog A, Gyulai Z, Boros LG, Farkas G, Takács T, Lonovics J, et al. Polymorphism of the TNF-alpha, HSP70-2, and CD14 genes increases susceptibility to severe acute pancreatitis. Pancreas 2005;30:e46-50.  Back to cited text no. 16
    
17.
Srivastava P, Shafiq N, Bhasin DK, Rana SS, Pandhi P, Behera A, et al. Differential expression of heat shock protein (HSP) 70-2 gene polymorphism in benign and malignant pancreatic disorders and its relationship with disease severity and complications. JOP 2012;13:414-9.  Back to cited text no. 17
    
18.
van den Berg FF, Kempeneers MA, van Santvoort HC, Zwinderman AH, Issa Y, Boermeester MA. Meta-analysis and field synopsis of genetic variants associated with the risk and severity of acute pancreatitis. BJS Open 2020;4:3-15.  Back to cited text no. 18
    
19.
Rodriguez-Nicolas A, Jiménez P, Carmona FD, Martín J, Matas Cobos AM, Ruiz-Cabello F, et al. Association between genetic polymorphisms of inflammatory response genes and acute pancreatitis. Immunol Invest 2019;48:585-96.  Back to cited text no. 19
    


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