AIM: To investigate whether the heat shock protein 70-2 (HSP70-2) polymorphism is associated with enterocutaneous fistulas in a Chinese population.

METHODS: This study included 131 patients with enterocutaneous/enteroatmospheric fistulas. Patients with inflammatory bowel disease or other autoimmune diseases were excluded from this study. All patients with enterocutaneous/enteroatmospheric fistulas were followed up for three months to observe disease recurrence. In addition, a total of 140 healthy controls were also recruited from the Jinling Hospital, matched according to the sex and age of the patient population. Genomic DNA was extracted from peripheral blood from each participant. The HSP70-2 restriction fragment length polymorphism related to the polymorphic PstI site at position 1267 was characterized by polymerase chain reaction (PCR). First PCR amplification was carried out, and then PCR products were digested with PstI restriction enzyme. The DNA lacking the polymorphic PstI site within HSP70-2 generates a product of 1117 bp in size (allele A), whereas the HSP70-2 PstI polymorphism produces two fragments of 936 bp and 181 bp in size (allele B).

RESULTS: The frequency of the HSP70-2 PstI polymorphism did not differ between patients and controls; however, the A allele was more predominant in patients with enterocutaneous fistulas than in controls (60.7% vs 51.4%, P = 0.038, OR = 1.425, 95%CI: 1.019-1.994). Sixty-one patients were cured by a definitive operation, drainage operation, or percutaneous drainage while 52 patients were cured by nonsurgical treatment. There was no significant difference in the frequency of the HSP70-2 PstI polymorphism between the patients who had surgery compared to those who did not (P = 0.437, OR = 1.237, 95%CI: 0.723-2.117). Moreover, 11 patients refused any treatment for economic reasons or tumor burden, and 7 patients with enterocutaneous fistulas (5.8%) died during the follow-up period. However, there was no significant difference in the frequency of the HSP70-2 PstI polymorphism between the patients who survived compared to those who died (P = 0.403, OR = 0.604, 95%CI: 0.184-1.986).

CONCLUSION: The A allele of the HSP70-2 PstI polymorphism was associated with enterocutaneous fistulas in this Chinese population.

Postoperative enterocutaneous fistulas (ECF) are one of the most devastating abdominal complications after surgery, which are defined as an abnormal communication between the intra-abdominal gastrointestinal tract (i.e., the stomach, duodenum, small bowel, and colon) and skin[1-3]. Postoperative ECF result from a wide variety of conditions and circumstances but caused a high mortality rate of 50%-60% before the 1970s[4,5]. Treatment of patients with postoperative ECF requires a greater understanding of their metabolic and anatomic derangements. To date, with the development of nutritional support, interventional radiology, and wound management, the overall mortality rate due to postoperative ECF has declined to 3%-12%[6]. However, with the introduction of damage control principles in surgery, open abdominal management has been used in patients undergoing complex surgery, who may have increased risk for the development of postoperative ECF[7]. Surgical techniques may also cause a new type of enteric fistulas, enteroatmospheric fistulas, which is defined as a fistula between the lumen of the bowel or other hollow viscus and the atmosphere (open abdomen or chest)[8,9]. Currently, enterocutaneous/enteroatmospheric fistulas may be caused by trauma or surgery as a result of traumatic or iatrogenic bowel injury, infection, or anastomotic leakage[1,2,6,10-12]. The genomic composite score can be rapidly used to predict clinical outcomes in trauma patients[13], and genome-wide expression studies have identified 12 plasma proteins as candidates for biomarker-based risk stratification in adults with septic shock[14]. So, genetic or epigenetic factors may increase the risk for individual patients to develop enterocutaneous/enteroatmospheric fistulas.

Heat shock proteins (HSPs) are highly conserved proteins in most mammalian and prokaryotic cells, and act as chaperones to other unfolded proteins and peptides during elevated temperatures or other stress, such as heat, ischemia-reperfusion, inflammation, and microbial infection[15-17]. As the most highly inducible HSP, the HSP70 gene family contains HSP70-1, HSP70-2, and HSP70-Hom. This family has been mapped to the class III region of the human genome, which is the region in chromosome 6 that contains human leukocyte antigen (HLA) genes[17,18]. The HSP70-2 gene polymorphism generates a PstI site due to an A to G substitution at position 1267. Previous studies have demonstrated that this HSP70-2 polymorphism is associated with Crohn’s disease (CD), especially with the perforating form, abscess formation, fistulas, and requirement for surgery in Japanese[19], Caucasian[20], Korean[21], and Chinese[22] populations. Thus, the aim of this study was to investigate whether the HSP70-2 PstI polymorphism is associated with enterocutaneous/ enteroatmospheric fistulas in a Chinese population.

Heat shock protein was discovered by Ritossa et al[16] in 1962; subsequently, HSPs have been detected in almost all organisms[17]. They are divided into different classes according to their molecular weights, i.e., HSP100 (approximately 100 kDa), HSP90, HSP70, and so on[17]. In humans, the HSP70 family of genes is mapped to the short arm of the sixth chromosome, close to HLA; thus, they may play a role in the regulation of immune responses. For example, HSP70 expression can be induced by a variety of infectious and inflammatory stimuli[24,25] (Christians, 2002 #26) and functions to protect cell homeostasis[26,27]. The extracellular HSP70 protein can induce a proinflammatory response through toll-like receptor-2 and toll-like receptor-4[28]. However, the HSP70-2 PstI polymorphism is a nonfunctional single nucleotide polymorphism (SNP) that does not cause a change in the derived amino acid[29,30], although HSP70-2 SNPs have been considered as candidates for increased susceptibility to inflammatory diseases. Tahara et al[31] have found that the HSP70-2 BB genotype is associated with a reduced risk of gastric premalignant conditions in H. pylori-infected older individuals. In addition, Partida-Rodríguez et al[32] have shown that an HSP70-2 SNP is significantly associated with both gastric cancer and duodenal ulcers. Thus, in the current study, we determined the association between the HSP70-2 PstI polymorphism and a patient’s susceptibility to enterocutaneous/enteroatmospheric fistulas. We found that the genotype frequency of the HSP70-2 PstI polymorphism did not differ between patients and controls, but the A allele was more prevalent in patients with enterocutaneous fistulas than in controls, which may indicate that the HSP70-2 A allele is more susceptible to enterocutaneous/enteroatmospheric fistula development.

Indeed, other studies have shown that HSP70 allele frequencies are associated with the pathogenesis of autoimmunity[33]. Their data show that different HSP70-2 allele frequencies are associated with spondylarthropathies[34], rheumatoid arthritis[35], systemic lupus erythematous[33], celiac disease[18], insulin-dependent diabetes (IDDM)[36,37], (Caplen, 1990 #37;Pociot, 1993 #36) type 2 diabetes and obesity[38], and autoimmune thyroid disease[39]. Moreover, recent studies have associated an HSP70-2 SNP with IBD[19-21,29,30]. In these studies, the HSP70-2 genotype BB of the PstI polymorphism or the HSP70-2 allele B was associated with the perforating form of CD or an indication for surgical treatment of CD, respectively, although the role of HSP70-2 in the pathogenesis of CD is still unknown. However, another study[15] did not confirm these data. In our previous study[22], we showed that the HSP70-2 A allele of the PstI polymorphism is associated with susceptibility to CD and surgical treatment of perforating CD. Therefore, in the current study, we excluded patients with CD, which may comprise up to 20% of patients with fistulas[2,10].

ECF are an uncommon but not well-studied postoperative complication, but they may have been first described as early as 450 BC[1,2]. The “modern” history of ECF began with William Beaumont and his patient, Alexis St. Martin in the 18th century; during this time period, ECF were a challenging surgical complication with a high mortality rate because of a combination of factors, including sepsis, electrolyte imbalance, and malnutrition[1,2,40]. In 1969, Dudrick et al[41] reported the advent of parenteral nutrition, which was a “game changer” for surgeons to prevent malnutrition and to provide patients with time to heal; therefore, the mortality rate due to fistulas has decreased ever since. In 1993, Rotondo et al[42] described the term “damage control laparotomy.” Next, an “open abdomen” has become more and more common in current clinical practice[8,9,11,43]. Moreover, enteroatmospheric fistulas are one of the most common and potentially devastating complications after surgery[3]. But in our current study, there were not many cases of patients with enteroatmospheric fistulas.

In conclusion, in our current study, we found that the HSP70-2 A allele of the PstI polymorphism was associated with ECF, while the HSP70-2 BB genotype was slightly more prevalent in low output patients and the HSP70-2 AB genotype was more prevalent in moderate output patients. In high output patients, the HSP70-2 AA genotype was more common than the others. To the best of our knowledge, the current study is the first report on an HSP70-2 PstI polymorphism in patients with ECF. However, our current study does have some limitations; for example, the small sample size, lack of a validation cohort, and the loss of statistical significance of the PstI polymorphism after the multivariate analysis. Thus, future studies with a larger sample size are needed to verify our current findings.