Genetic Risk for Alcoholic Chronic Pancreatitis

In recent years many studies have examined the genetic predisposition to pancreatic diseases. Pancreatic disease of an alcoholic etiology was determined to be a multi-factorial disease, where environmental factors interact with the genetic profile of the individual. In this review we discuss the main results from studies examining the frequency of genetic mutations in alcoholic chronic pancreatitis.


The Role of Genetics
In this sense, molecular biology has contributed greatly to the understanding of the pathogenesis of pancreatitis. In 1996, Whitcomb et al. found the R122H mutation in the cationic trypsinogen gene (PRSS1) was associated with hereditary pancreatitis [15]. This study also showed inflammation was definitively linked to pancreatic enzyme activation and autodigestion of the parenchyma, as proposed by Chiari in 1896 [16]. Evidence suggests mutations in PRSS1 cause the prematurely activated trypsin to be resistant to inactivation through autolysis, since arginine residues in the peptide chain are known sites of hydrolysis by trypsin itself. In the R122H mutation, arginine is substituted with histidine at codon 122, located on the external surface of the cationic trypsinogen molecule; this leads to inappropriate activity of trypsin, which damages the acinar cells and leads to ductal and interstitial injury through direct activation of other digestive zymogens and immunogenic stimuli [17].
Hereditary pancreatitis is characterized by an early onset in childhood, repeated bouts of acute pancreatitis, progression to fibrosis and pancreatic insufficiency, and high risk of adenocarcinoma of the pancreas, about 50 times higher than the general population [18,19]. It is a rare disease transmitted as an autosomal dominant trait, with a penetrance of about 80%. The diagnostic criteria includes a family history of the patient; for the diagnosis, there must be recurrent acute pancreatitis or chronic pancreatitis in at least two first degree relatives or three or more second degree relatives in two or more generations, for whom there were no identified precipitating factors. Since they are rigorous, these criteria have been questioned; some institutions define hereditary pancreatitis as any patient with no other detectable cause for the disease and a relative of first or second degree with chronic pancreatitis. The search for genetic mutations has also been adopted as a diagnostic tool [20,21]. In addition to R122H, which is the most frequent mutation in these patients, the N29I mutation was found to be the second most frequent; others were also identified, including A16V, D22G, K23R, N29T, E79K, and R122C [22][23][24][25][26][27][28][29].
Surveys were performed to attempt to correlate the development of alcoholic chronic pancreatitis with PRSS1 gene mutations. Monaghan et al. in the USA, Perri et al. in Italy, and Chandak et al. in India studied these populations, but found no mutations [30][31][32]. In Brazil, Bernardino et al. [33] identified the E79K mutation in exon 3 of the PRSS1 gene in only one out of 64 patients with alcoholic pancreatitis and there was no statistically significant difference when compared to the control group. Therefore, as expected, a significant association between alcoholic pancreatitis and PRSS1 gene mutations was not demonstrated since the presence of these mutations in affected families causes early presenting pancreatic disease, where its manifestation depends little on the interference of other factors [1,34].
Moreover, there are protection mechanisms directed against pancreatic activation of trypsinogen and prevention of sustained activity of trypsin [10]. The SPINK1 (serine protease inhibitor Kazal type 1) gene encodes a protein inhibitor of trypsin, which is synthesized in acinar cells and is able to inhibit 20% of intracellular activity of trypsin. It is considered the first line of defense against accidental premature activation of trypsinogen within acinar cells [35]. In 2000, the N34S mutation in exon 3 of SPINK1 was identified in an analysis of patients with pancreatitis and no mutations in the PRSS1 gene [36]. Further studies determined there is an association between this mutation and pancreatitis. A meta-analysis performed by Aoun et al. [37] included 24 case-control studies that investigated the frequency of this mutation in their populations; it was concluded that the N34S mutation is strongly associated with idiopathic, tropical, and alcoholic chronic pancreatitis, but with less impact on the latter. Other mutations, in addition to N34S, were found in the SPINK1 gene, such as P55S, M1T, L14P, and others, but at much lower frequencies [38].
Chymotrypsin C (CTRC) is also responsible for the degradation of trypsin and trypsinogen isoforms with high specificity. Mutations in the CTRC encoding gene are potentially new candidates associated with pancreatic diseases [39]. In the study conducted by Rosendahl et al. [39] in 2008, the R254W mutation in exon 7 of the CTRC gene was found in 2.3% of 348 patients with alcoholic pancreatitis and in 0.5% of 432 patients with chronic liver disease caused by ethanol consumption, the difference being statistically significant (P = 0.03, OR = 5.1, 95% CI = 1.1-24.0). Chang et al. [40] identified other variants and haplotypes of the CTRC gene in a population of 126 patients with chronic pancreatitis of different etiologies, also occurring at a rate of 2.3%.
The protection of ductal cells depends on maintaining an alkaline pH and a rapid enzyme flow into the duodenum, which requires normal functioning of CFTR (cystic fibrosis transmembrane conductance regulator or transmembrane conductance regulator Cystic Fibrosis) [10]. This protein is present on the surface of the majority of human epithelial cells and functions as a chloride channel also responsible for the secretion of bicarbonate to pancreatic juices. In 1989, cystic fibrosis, an autosomal recessive disease characterized by severe lung injury, liver disease, atresia of the vas deferens, and pancreatic insufficiency manifested since childhood was attributed to mutations in this gene. Since then, more than 1000 mutations have been described in the CFTR gene and the disease was shown to have a much more varied phenotypic spectrum that can occur as monosymptomatic or atypical forms, such as the development of isolated pancreatic disease, secondary to mutations that reduce but do not completely eliminate the function of the protein [41]. It has also been suggested that mutations in the CFTR gene could predispose individuals to alcoholic chronic pancreatitis. It is difficult to test this hypothesis because it is a long gene comprising 24 exons that together encode a single protein of 1480 amino acids [42]. Table 1 lists some of the research completed so far [43][44][45][46][47][48][49][50][51][52][53][54][55][56]. Despite the high number of studies performed, it is difficult to reach a definitive conclusion about the real impact of carrying mutations in this gene on determining the susceptibility to alcoholic pancreatitis. This is due to several factors, including the variability in the methodologies, the number of mutations studied, and the number of patients included, which was most often inadequate and did not include a control population. In two studies conducted in Brazil, the methodology used was different. In the first study, Bernardino et al. [33] included patients with pancreatitis of other etiologies in addition to patients with pancreatic diseases due to alcohol and used a technique known as SSCP (Single Strand Conformational Polymorphism) to screen for mutations. Regions with found abnormalities were subsequently sequenced. This allowed the detection of mutations in the CFTR gene in five of 16 patients with idiopathic pancreatitis and in three of 64 patients with chronic pancreatitis caused by ethanol abuse. Yet, the presence of mutations in the CFTR gene was not investigated in the control population.
We performed the second study (da Costa et al. [56]). Intron 8 of the CFTR gene was studied by sequencing in chronic alcoholics diagnosed with pancreatic disease (group A), alcoholics without pancreatitis (group B), and blood donor volunteers who served as healthy controls (Group C). Different genetic profiles between the populations were found, with the 5T/7T genotype found more frequently (11.8% of cases, P < 0.05) in group A than in alcoholics without pancreatitis (group B, 2.9% of cases). The presence of a combination of haplotypes, (TG) 10-T7/(TG) 11-T7, occurred more frequently (P < 0.05) in controls (groups B and C; 23.5% and 20.2%, respectively) than in patients (group A; 7.3%).
In addition to the previously mentioned studies, other research was conducted on the occurrence of mutations in several other genes. In patients with pancreatitis of alcoholic etiology, the hypothesis remained that mutations would act as cofactors that influence an individual's susceptibility, predisposing them to develop the disease in the presence of the aggressive environmental factor, alcohol [2]. Other genes that may be involved in the pathogenesis of alcoholic pancreatitis include those that code for enzymes that act in the metabolism of ethanol which, once absorbed, is catalyzed in the liver initially to acetaldehyde and then to acetate. The enzymes predominantly involved in this process include alcohol dehydrogenase and aldehyde dehydrogenase [13]. Kimura et al. [57]; Shimosegawa, Kume, and Masamune [58]; and Cichoz-Lach et al. [59] found different polymorphisms that varied between the studies. However, no definite conclusions were drawn about the participation of these genes in the development of alcoholic pancreatitis.
Another candidate gene is CASR (calcium sensing receptor). Hypercalcemia is associated with pancreatitis, possibly through the activation of trypsinogen and stabilization of trypsin [60]. CASR has a role in the homeostasis of calcium and was identified in both ductal and acinar cells [61]. In a study directed by Muddana et al. [62], an association was found (P = 0.018) between the R990G mutation and pancreatic diseases in patients who reported moderate or high ethanol consumption. Moreover, studies investigated the frequency of polymorphisms in genes encoding cytokines such as interleukins, TNFα (tumor necrosis factor-alpha), TGFβ1 (transforming growth factor-beta-1), INFδ (interferon-gamma), VEGF (vascular endothelial growth factor), and ICAM-1 (inter-cellular adhesion molecule 1). These cytokines are important in the development of inflammation and establishment of fibrosis; yet, results were variable and a no clear role was found for determining predisposition to the disease [63][64][65]. Other studies evaluated genes encoding detoxified enzymes such as glutathione S transferase [66][67][68], UDP glucuronosyltransferase [69], manganese -superoxide dismutase [66], and methylenetetrahydrofolate reductase [70]. These enzymes provide protection against products of oxidative stress, which apparently contribute to the development of pancreatitis [71]. While some researchers [66,67] found no association with chronic pancreatic disease, others [68][69][70] suggest the presence of low levels of detoxification may be a risk factor. The relevance of genetic variations leading to deficiencies of serum antiproteases, such as alpha 1 antitrypsin and alpha 2 macroglobulin, remains speculative [72,73].

Conclusions
In summary, we conclude that alcoholic pancreatitis is a complex disorder. The combination of, or interaction between, alcohol consumption and genetic factors results in the disease; yet, further studies are needed to improve the understanding of the mechanisms governing these overlapping causes. Furthermore, it is crucial to understand the profile of the population receiving health care. Brazil is characterized by multiple ethnic and racial mixes and is highly variable between regions. This may have implications in the approach for diagnosing patients, possibly leading to individualized follow-up and treatment.

Conflict of Interest
The authors declare no conflict of interest.