|Year : 2015 | Volume
| Issue : 4 | Page : 239-244
|Bactericidal permeability increasing protein gene polymorphism is associated with inflammatory bowel diseases in the Turkish population
Güray Can1, Hakan Akın2, Filiz T. Özdemir3, Hatice Can4, Bülent Yılmaz5, Fatih Eren3, Özlen Atuğ2, Belkıs Ünsal6, Hülya O. Hamzaoğlu7
1 Department of Gastroenterology, Abant İzzet Baysal University, Faculty of Medicine, Bolu, Turkey
2 Department of Gastroenterology, Marmara University, Faculty of Medicine, Istanbul, Turkey
3 Department of Genetics, Marmara University Institute of Gastroenterology, Istanbul, Turkey
4 Department of Internal Medicine, Abant İzzet Baysal University, Faculty of Medicine, Bolu, Turkey
5 Department of Gastroenterology, Bolu İzzet Baysal State Hospital, Bolu, Turkey
6 Department of Gastroenterology, Katip Çelebi University, Faculty of Medicine, İzmir, Turkey
7 Department of Gastroenterology, İstanbul Acıbadem Fulya Hospital, Istanbul, Turkey
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|Date of Submission||01-Oct-2014|
|Date of Acceptance||28-Oct-2014|
|Date of Web Publication||29-Jul-2015|
| Abstract|| |
Background/Aims: Inflammatory bowel disease, a chronic inflammatory disease with unknown etiology, affects the small and large bowel at different levels. It is increasingly considered that innate immune system may have a central position in the pathogenesis of the disease. As a part of the innate immune system, bactericidal permeability increasing protein has an important role in the recognition and neutralization of gram-negative bacteria. The aim of our study was to investigate the involvement of bactericidal permeability increasing protein gene polymorphism (bactericidal permeability increasing protein Lys216Glu) in inflammatory bowel disease in a large group of Turkish patients. Patients and Methods: The present study included 528 inflammatory bowel disease patients, 224 with Crohn’s disease and 304 with ulcerative colitis, and 339 healthy controls. Results: Bactericidal permeability increasing protein Lys216Glu polymorphism was found to be associated with both Crohn’s disease and ulcerative colitis (P = 0.0001). The frequency of the Glu/Glu genotype was significantly lower in patients using steroids and in those with steroid dependence (P = 0.012, OR, 0.80; 95% confidence interval [CI]: 0.68-0.94; P = 0.0286, OR, 0.75; 95% CI: 0.66-0.86, respectively). There was no other association between bactericidal permeability increasing protein gene polymorphism and phenotypes of inflammatory bowel disease. Conclusions: Bactericidal permeability increasing protein Lys216Glu polymorphism is associated with both Crohn’s disease and ulcerative colitis. This is the first study reporting the association of bactericidal permeability increasing protein gene polymorphism with steroid use and dependence in Crohn’s disease.
Keywords: Bactericidal permeability increasing protein, inflammatory bowel disease, single nucleotide polymorphism
|How to cite this article:|
Can G, Akın H, Özdemir FT, Can H, Yılmaz B, Eren F, Atuğ &, Ünsal B, Hamzaoğlu HO. Bactericidal permeability increasing protein gene polymorphism is associated with inflammatory bowel diseases in the Turkish population. Saudi J Gastroenterol 2015;21:239-44
|How to cite this URL:|
Can G, Akın H, Özdemir FT, Can H, Yılmaz B, Eren F, Atuğ &, Ünsal B, Hamzaoğlu HO. Bactericidal permeability increasing protein gene polymorphism is associated with inflammatory bowel diseases in the Turkish population. Saudi J Gastroenterol [serial online] 2015 [cited 2022 Nov 29];21:239-44. Available from: https://www.saudijgastro.com/text.asp?2015/21/4/239/161642
Inflammatory bowel disease (IBD) is a chronic inflammatory disease with unknown etiology, which affects the different layers of small and large intestine at different levels. It is known that a lot of environmental and genetic factors have a role in the development of the disease. Immunologic response is excessively increased against intestinal microorganisms in genetically susceptible persons. Healthy people have an immune tolerance against commensal bacteria; however, there is a loss of immune tolerance in patients with IBD. It is not clear what mechanisms are responsible for this situation. Luminal antigens not contacting the immune system normally may trigger the immune response by crossing the epithelial barrier as a result of increased permeability of the intestinal epithelium., On the other hand, improper activation of pathogen recognition receptors that identify the pathogen microorganisms may be responsible for the excessive immune response.
As a part of the innate immune system, nucleotide-binding oligomerization domain-2 (NOD2) protein, whose function is to identify the bacterial motifs, was found to have three different mutations associated with susceptibility to Crohn’s disease (CD). Thereafter, two different single nucleotide polymorphisms (SNP) associated with IBD were identified in toll-like receptor-4 (TLR4) recognizing lipopolysaccharide (LPS), an integral part of the gram-negative bacterial membrane. It is increasingly recognized that innate immune system may have a central position in the pathogenesis of the disease. Proteins that play a role in the recognition of pathogen microorganisms are believed to be associated with IBD pathogenesis.
Various antimicrobial peptides are secreted by the host against invasive pathogens. These peptides selectively identify and eradicate pathogen microorganisms before spreading all over the body. Bactericidal permeability increasing protein (BPI), one of the most important of these peptides, prevents inflammatory response by binding to LPS. Normally, it has an opposite effect to LPS binding protein (LBP) that induces acute inflammation. It is effective in the elimination of bacteria besides limiting inflammation at a focal area without progression to systemic inflammation.
Some authors have asserted that any impairment in the functions of BPI protein by virtue of BPI gene polymorphism may change the pathogen microorganism recognition ability of the innate immune system and eventually give rise to undesirable outcomes in IBD. The results of the studies so far, on the association between IBD and BPI polymorphism are not satisfactory, besides not compatible with each other. Akın et al. found that BPI gene polymorphism was significantly associated with UC and CD; however, their results conflicted with the current literature. It was thought that these conflicting results might be due to being a single-centre study and the small sample size of their study. The aim of our study was to investigate the involvement of protein (BPI) gene polymorphism (Lys216Glu) in a large group of Turkish IBD patients recruited from three geographically different regions, as a continuation of the previous study in Turkish population.
| Patients and Methods|| |
The patients with IBD were recruited from three geographically different regions including Marmara University Faculty of Medicine Hospital, Balıkesir State Hospital and İzmir Katip Çelebi University Atatürk Training and Research Hospital. IBD patients who were admitted to the gastroenterology clinics of these centers between November 2009 and March 2010 were included in our study. CD and UC were defined using conventional clinical, radiological, endoscopic and histological criteria. The patients who had indeterminate colitis and those younger than 18 years were excluded from the study. Cases were phenotyped according to the Montreal classification systems that allow genotype-phenotype analysis. The study population consisted of 528 IBD patients (304 UC, 224 CD) and 339 healthy controls. Clinical and demographic data of the study population were recorded. Informed consent was taken from all participants before participating.
Venous blood samples taken from all patients and healthy controls via venipuncture were collected into sterile, ethylenediaminetetraacetic acid (K3EDTA) containing tubes. Genomic DNA was isolated from collected blood samples by phenol-chloroform method. High-Pure polymerase chain reaction (PCR) Template Preparation Kit (Cat. No: 11 796 828 001, Roche, Mannheim, Germany) was used for DNA extraction.
The Lys216Glu (645 A/G) (rs4358188) SNP in BPI gene was genotyped with PCR and restriction fragment length polymorphism procedure (RFLP). PCR solution was prepared by using 100 ng genomic DNA, 1 × PCR buffer (Fermentas, Burlington, Ontario, Canada), 0.2 mM of each dNTP (Fermentas), 0.5 U FastStart Taq DNA polymerase (Fermentas), 5 pmol primer and 2.5 mM MgCl2. The final volume of solution was increased to 20 mL with 12.5 mL distilled water. The solution prepared for PCR was heated for 15 minutes at 95°C, then after denaturation for 35 cycles at 94°C for 30seconds, and a gap of 30 seconds at 60°C, it was followed by 30 seconds at 72°C for annealing, and finally 10 minutes at 72°C for extension. After the PCR reaction was completed, amplification products were digested with HindIII restriction enzyme (Fermentas) for overnight at 37°C. The length of PCR products, primers and the heat of annealing are demonstrated in [Table 1]. For electrophoresis, 2.5% standard agarose gel was prepared with 100 cc 0.5X Tris Borate EDTA (TBE) and 20 mL (10 mg/mL) ethidium bromide. The resulting fragments were placed on agarose gel after staining with bromophenol blue, and were run on agarose gel at 80 volts for 20 minutes by electrophoresis. The resulting bands were read and compared with representative samples for BPI [Figure 1]. The length of restriction fragments and the restriction’s heat are given in [Table 1].
|Figure 1: The resulting bands from polymerase chain reaction and restriction fragment length polymorphism procedure for bactericidal permeability increasing protein in study population. m: Marker, k: Control group, -k: Negative control|
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|Table 1: Polymerase chain reaction and restriction fragment length polymorphism procedure conditions, primers and resulting products|
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Chi-square test with Yates correction was used for the comparison of allele and genotype frequencies of disease and control groups. The Fisher’s exact test was used at the appropriate conditions. Bonferroni correction was applied for multiple comparisons. Comparison of observed and predicted frequencies was analyzed by Hardy-Weinberg equilibrium. A P value < 0.05 was considered to be statistically significant. Statistical Package for the Social Sciences (SPSS) for Windows (version 16.0; SPSS Inc., Chicago, IL, USA) was used for all statistical analysis in this study.
The Ethics Committee of Marmara University Faculty of Medicine, Istanbul, Turkey approved this study (Istanbul 9. Clinical Research Ethics Committee; project no: 09.2009.0018; acceptance date: 07 Jan 2010).
| Results|| |
The present study included 528 patients with IBD and 339 healthy controls. The demographic and clinical characteristics of the study groups are given in [Table 2]. UC, CD and control groups were equally distributed in terms of age and gender. Statistical analysis revealed that BPI Lys216Glu (645 A > G) SNP was found to be associated with both CD and UC (P = 0.0001) as compared with healthy controls [Table 3]. It was demonstrated that both Glu/Glu genotype and Glu allele frequencies were significantly increased in the UC group (P = 0.0006, OR = 1.862 95% CI = 1.302-2.662; P = 0.0003, OR = 1.504 95% CI = 1.207-1.875, respectively), however only Glu allele frequencies were shown to be increased in the CD group as compared with healthy controls (P = 0.0013, OR = 1.491 95% CI = 1.172-1.895). On the other hand, Lys/Lys genotype and Lys allele frequencies were significantly decreased both in the UC (P = 0.0113, OR = 0.686 95% CI = 0.422-0.897; P = 0.0003, OR = 0.664 95% CI = 0.533-0.828, respectively), and CD groups (P = 0.0001, OR = 0.405 95% CI = 0.256-0.640; P = 0.0013, OR = 0.670 95% CI = 0.527-0.852, respectively). Genotype and allele frequencies of patient and control groups are shown in [Table 3]. When the association of corticosteroid use and corticosteroid dependence in IBD with BPI gene polymorphism were investigated separately, the frequency of Glu/Glu genotype was found to be significantly lower in CD patients using corticosteroids and in those with corticosteroid dependence (P = 0.0127, OR = 0.80 95% CI = 0.68-0.94; P = 0.0286, OR = 0.75 95% CI = 0.66-0.86, respectively) [Table 4]. No association was found for other drug usage, disease phenotype and localization, duration of inflammation, family history, extraintestinal manifestations, bowel resection and smoking.
|Table 3: Genotype and Allele frequencies of Lys216Glu (645 A>G) polymorphism in bactericidal permeability increasing protein gene|
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|Table 4: The association between corticosteroid use and dependence in inflammatory bowel disease and bactericidal permeability increasing protein gene polymorphism|
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| Discussion|| |
To the best of our knowledge, four studies were performed on the association between IBD and BPI gene polymorphism [Table 5]. The first study was performed in a German population in 2004 by Török et al. This study found no significant association between IBD and BPI gene polymorphism. However, it was determined that concurrent presence of TLR4, which is another pathogen recognition molecule, and BPI (Lys/Glu genotype) gene polymorphisms significantly increased the risk of CD. Another study in German population was conducted by Klein et al. in 2005. In variance with the results of Török et al., he found that Glu/Glu genotype was protective against CD. The discrepancy between the results of the two studies performed in the same population can be attributed to the number of patients participated in the studies. In another study, performed by Petermann et al. in a New Zealand population, a significant association was shown only between Lys allele and ileocolonic CD. On the other hand, it was found that the combination of CD14 and BPI gene polymorphisms was higher in UC. Lastly, Akın et al. investigated BPI gene polymorphism in Turkish IBD patients in 2008 and found BPI gene polymorphism to be significantly associated not only with CD but also with UC, as compared with the other studies performed in different populations. Contrary to the study by Klein et al., Akın et al. found that both Glu allele and Glu/Glu genotype were risk factors for both UC and CD. Moreover, the association between BPI polymorphism and UC was reported for the first time in the Turkish population. The number of patients included in our study was much greater than that in the study by Akın et al. and we showed a stronger association in the same population. Contrary to the results of the study by Akın et al., we found that both Lys allele and Lys/Lys genotype were protective against UC and CD, but weak association between Glu/Glu genotype and CD in Akın’s study lost its significance. It can be related to the number of patients involved in the studies. While Glu/Glu genotype was protective against CD with regard to the study by Klein et al., it was found to be a risk factor for both UC and CD in the study of Akın et al. As for our study, it is only a risk factor for UC. On the other hand, Petermann et al. studied the polymorphism in patients of European origin. The results of two studies performed in the German population were neither similar to each other nor to those of Petermann et al. The results of the Turkish studies partially resemble each other, but they are quite different from the results of the other studies. Differences in the results of these studies may be explained by ethnic differences rather than methodological reasons, as all the studies used the same methodological procedure. As a result, it brings to mind that different mechanisms may play a role in the etiology of IBD in different populations. Because we showed the association between BPI and IBD in our population, we can say that BPI seems to have a role in the etiology of UC in Turkish population.
|Table 5: Genotype and allele frequencies of BPI (Lys216Glu) polymorphism studies in IBD patients from different population|
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In addition, our study is the only report, which determined an association between corticosteroid use and dependence in CD, and BPI gene polymorphism. The frequency of Glu/Glu genotype was significantly lower both in CD patients who were using steroids and in those with steroid dependence [Table 4]. We infer that Glu/Glu genotype is associated with mild CD. This finding may help us plan the treatment strategy in patients carrying this polymorphism.
In a recent study, the concentration of BPI protein was demonstrated to be increased in the intestinal tissue specimens of patients with ulcerative colitis (UC). In addition, it was shown that the level of autoantibodies for BPI protein was increased in direct proportion to increase of the severity of IBD. Although it was shown that BPI was associated with IBD, we do not know what are the modifications, what effect BPI gene polymorphism has on BPI molecule, and which mechanisms are involved in this process. Further research is required to to explain this obscurity.
In conclusion, our study provided a larger data to evaluate the association between BPI and IBD in the Turkish population, and it demonstrated that there was a strong association between BPI and UC, while the previously shown association between BPI and CD was weakened. BPI may be more important in the etiology of UC in the Turkish population than the other populations. It was shown for the first time that BPI is associated with drug usage and with mild CD. Ethnic differences may play a major role in the conflicting results obtained in different studies. Further large-scale studies in different populations are necessary to explore this association.
| References|| |
Hendrickson BA, Gokhale R, Cho JH. Clinical aspects and pathophysiology of inflammatory bowel disease. Clin Microbiol Rev 2002;15:79-94.
Lakatos PL, Fischer S, Lakatos L, Gal I, Papp J. Current concept on the pathogenesis of inflammatory bowel disease-crosstalk between genetic and microbial factors: Pathogenic bacteria and altered bacterial sensing or changes in mucosal integrity take “toll”? World J Gastroenterol 2006;12:1829-41.
Hilsden RJ, Meddings JB, Sutherland LR. Intestinal permeability changes in response to acetylsalicylic acid in relatives of patients with Crohn’s disease. Gastroenterology 1996;110:1395-403.
Macpherson A, Khoo UY, Forgacs I, Philpott-Howard J, Bjarnason I. Mucosal antibodies in inflammatory bowel disease are directed against intestinal bacteria. Gut 1996;38:365-75.
Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, et al
. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 2001;411:603-6.
Hugot JP, Chamaillard M, Zouali H, Lesage S, Cézard JP, Belaiche J, et al
. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 2001;411:599-603.
Hampe J, Cuthbert A, Croucher PJ, Mirza MM, Mascheretti S, Fisher S, et al
. Association between insertion mutation in NOD2 gene and Crohn’s disease in German and British populations. Lancet 2001;357:1925-8.
Uyar FA, Hamzaoğlu HO, Türe F, Gül A, Tözün N, Direskeneli GS. Distribution of common CARD15 variants in patients with sporadic Crohn’s disease: Cases from Turkey. Dig Dis Sci 2006;51:706-10.
Arbour NC, Lorenz E, Schutte BC, Zabner J, Kline JN, Jones M, et al
. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet 2000;25:187-91.
Ooi CE, Weiss J, Elsbach P, Frangione B, Mannion B. A 25-kDa NH2-terminal fragment carries all the antibacterial activities of the human neutrophil 60-kDa bactericidal/permeability-increasing protein. J Biol Chem 1987;262:14891-4.
Ooi CE, Weiss J, Doerfler ME, Elsbach P. Endotoxin-neutralizing properties of the 25 kD N-terminal fragment and a newly isolated 30 kD C-terminal fragment of the 55-60 kD bactericidal/permeability-increasing protein of human neutrophils. J Exp Med 1991;174:649-55.
van der Schaft DW, Toebes EA, Haseman JR, Mayo KH, Griffioen AW. Bactericidal/permeability-increasing protein (BPI) inhibits angiogenesis via induction of apoptosis in vascular endothelial cells. Blood 2000;96:176-81.
Klein W, Tromm A, Folwaczny C, Hagedorn M, Duerig N, Epplen J, et al
. A polymorphism of the bactericidal/permeability increasing protein (BPI) gene is associated with Crohn’s disease. J Clin Gastroenterol 2005;39:282-3.
Akin H, Tahan G, Türe F, Eren F, Atuğ O, Tahan V, et al
. Association between bactericidal/permeability-increasing protein (BPI) gene polymorphism (Lys216Glu) and inflammatory bowel disease. J Crohns Colitis 2011;5:14-8.
John SW, Weitzner G, Rozen R, Scriver CR. A rapid procedure for extracting genomic DNA from leukocytes. Nucleic Acids Res 1991;19:408.
Török HP, Glas J, Tonenchi L, Mussack T, Folwaczny C. Polymorphisms of the lipopolysaccharide-signaling complex in inflammatory bowel disease: Association of a mutation in the Toll-like receptor 4 gene with ulcerative colitis. Clin Immunol 2004;112:85-91.
Petermann I, Huebner C, Browning BL, Gearry RB, Barclay ML, Kennedy M, et al
. Interactions among genes influencing bacterial recognition increase IBD risk in a population-based New Zealand cohort. Hum Immunol 2009;70:440-6.
Levy O, Canny G, Serhan CN, Colgan SP. Expression of BPI (bactericidal/permeability-increasing protein) in human mucosal epithelia. Biochem Soc Trans 2003;31:795-800.
Haapamäki MM, Häggblom JO, Grönroos JM, Pekkala E, Alanen K, Nevalainen TJ. Bactericidal/permeability-increasing protein in colonic mucosa in ulcerative colitis. Hepatogastroenterology 1999;46:2273-7.
Monajemi H, Meenan J, Lamping R, Obradov DO, Radema SA, Trown PW, et al
. Inflammatory bowel disease is associated with increased mucosal levels of bactericidal/permeability-increasing protein, Gastroenterology 1996;110:733-9.
Walmsley RS, Zhao MH, Hamilton MI, Brownlee A, Chapman P, Pounder RE, et al
. Antineutrophil cytoplasm autoantibodies against bactericidal/permeability-increasing protein in inflammatory bowel disease. Gut 1997;40:105-9.
Department of Gastroenterology, Abant ?zzet Baysal University, Faculty of Medicine, Gölköy Campus, 14280, Bolu
Source of Support: Nil, Conflict of Interest: None declared.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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