Contents lists available atScienceDirect
Infection, Genetics and Evolution
journal homepage:www.elsevier.com/locate/meegidResearch Paper
Association between polymorphisms in HLA-A, HLA-B, HLA-DR, and DQ
genes from gastric cancer and duodenal ulcer patients and cagL among
cagA-positive Helicobacter pylori strains: The
first study in a Turkish
population
Banu Tufan Kocak
a, Suat Saribas
b, Suleyman Demiryas
c, Erkan Yilmaz
d, Omer Uysal
e,
Nuray Kepil
f, Mehmet Demirci
g, Harika Oyku D
ınc
b, Seher Akkus
b, Reyhan Gülergün
b,
Nesrin Gareayaghi
h, Hüseyin Emre Da
ğdeviren
b, Dogukan Ozbey
b, Hamit Harun Da
ğ
b,
Hrisi Bahar Tokman
b, Ihsan Tasci
c, Bekir Kocazeybek
b,⁎aT.C. Health Ministry Erenkoy Mental Health, Neurology Training and Research Hospital, Istanbul, Turkey bIstanbul University-Cerrahpasa, Cerrahpasa Medical Faculty, Department of Medical Microbiology, Istanbul, Turkey cIstanbul University-Cerrahpasa, Cerrahpasa Medical Faculty, Department of General Surgery, Istanbul, Turkey
dIstanbul University-Cerrahpasa, Cerrahpasa Medical Faculty, Department of Organ Transplantation, HLA Laboratory, Istanbul, Turkey eMedical School of Bezmialem, Vakif University, Deparment of Biostatistics, Istanbul, Turkey
fIstanbul University-Cerrahpasa, Cerrahpasa Medical Faculty, Department of Pathology, Istanbul, Turkey gBeykent University Medical Faculty, Department of Medical Microbiology, Istanbul, Turkey
hIstanbul Sisli Hamidiye Etfal Training and Research Hospital, Blood Center, University of Health Sciences, Istanbul, Turkey
A R T I C L E I N F O Keywords: H. pylori CagL HLA CagA gastric cancer duodenal ulcer A B S T R A C T
Colonization of the human gastric mucosa by H. pylori may cause peptic and duodenal ulcers (DUs), gastric lymphomas, and gastric cancers. The cagL gene is a component of cag T4SS and is involved in cagA translocation into host. An association between the risk of gastric cancer and the type of HLA class II (DR and/or DQ) was suggested in different populations. The aim of this study was to investigate, the clinical association of the cagL gene with host HLA alleles in H. pylori strains that were isolated from patients with gastric cancer, DU, and non-ulcer dyspepsia (NUD) and to determine the HLA allele that confers susceptibility or resistance for the risk of gastric cancer and DU development in Turkish patients.
A total of 94 patients (44 gastric cancer and 50 DU patients; 58 male, 36 female; mean age, 49.6 years), and 86 individuals (50 NUD patients and 36 persons with normal gastrointestinal system [NGIS]; 30 male, 56 female; mean age, 47.3 years) were included as the patient and the control groups, respectively. CagA and cagL were determined by PCR method. DNA from peripheral blood samples was obtained by EZ-DNA extraction kit. For HLA SSO typing, LIFECODES SSO Typing kits (HLA-A, HLA-B HLA-C, HLA-DRB1 and HLA-DQA1/B1 kits) were used.
The CagL/CagA positivity distribution in the groups were as follows: 42 (95.4%) gastric cancer, 46 (92%) DU and, 34 (68%) NUD and no NGIS cases. The HLA-DQA1*01 (OR: 3.82) allele was significantly different, sug-gesting that these individuals with H. pylori strains harbouring the CagL/CagA positivity are susceptible to the risk of gastric cancer and DU, and the HLA-DQA1*05 (OR, 0.318) allele was suggested as a protective allele for the risk of gastric cancer and DU using univariate analyses. HLA-DQA1*01 (OR, 2.21), HLA-DQB1*06 (OR, 2.67), sex (male, OR, 2.27), and CagL/CagA/(< 2) EPIYA C repeats (OR, 5.72) were detected independent risk factors that increased the risk of gastric cancer and DU using multivariate analyses. However, the HLA-DRB1*04 (OR, 0.28) allele was shown to be a protective allele, which decreased the risk of gastric cancer and DU.
Gastric pathologies result from an interaction between bacterial virulence factors, host epigenetic and en-vironmental factors, and H. pylori strain heterogeneity, such as genotypic variation among strains and variations in H. pylori populations within an individual host.
https://doi.org/10.1016/j.meegid.2020.104288
Received 27 January 2020; Received in revised form 8 March 2020; Accepted 12 March 2020
⁎Corresponding author at: Istanbul University, Cerrahpasa Faculty of Medicine, Department of Medical Microbiology, Cerrahpasa Street, 34098 Istanbul, Turkey. E-mail address:bzeybek@Istanbul.edu.tr(B. Kocazeybek).
Available online 13 March 2020
1567-1348/ © 2020 Elsevier B.V. All rights reserved.
1. Introduction
Helicobacter pylori infects approximately half of the world’s popu-lation; it is usually acquired in childhood and persists throughout the patient’s life. Persistent colonization of the human gastric mucosa by H. pylori may cause inevitable peptic and duodenal ulcers (DUs), gastric lymphomas, and gastric cancers (Román-Román et al., 2019). The cagL gene is a component of cag Type IV secretion systems (T4SSs). T4SSs are membrane-associated transporter complexes of bacteria to deliver effector molecules such as, virulence factors, toxins and DNAs transfer to other bacteria and eukaryotic cells and the delivered effector mole-cules may affect and alter host cellular processes, resulting in disease development. The T4SS forms a needle-like surface appendage called the T4SS pilus, and CagA is translocated into the cytoplasm of the gastric epithelial cells through the T4SS pilus. One of the genes that encode the T4SS is cagL, and that this protein is localized on the surface of the T4SS of H. pylori and is essential for the formation of cagPAI-associated pili between H. pylori andα5β1 integrin receptor on gastric epithelial cells for CagA translocation. (Román-Román et al., 2019; Backert and Meyer, 2006).
CagL is a pilus protein that interacts with host cellular a5b1 in-tegrins through its arginine–glycine–aspartate (RGD) motif. (Kwok et al., 2007) By binding to integrin receptors, CagL may cause various cellular alterations such as stimulation of cell spreading, formation of focal adhesion, and activation of focal adhesion kinase and epidermal growth factor receptor (EGFR). These cellular changes are also trig-gered by purified recombinant cagL alone, which indicates the im-portance of CagL as a component of T4SS. Additionally, its activity varies according to the RGD motif polymorphisms (Tegtmeyer et al., 2010). CagL also causes transient hypochlorhydria by disrupting the interaction between the metalloprotease ADAM17 and the integrin α5β1(Saha et al., 2010). CagA is an oncoprotein that may induce hy-permethylation of tumor suppressor genes by injecting them into host gastric epithelial cells, and it may trigger some signal transduction events, such as proliferation and inflammation, induction of pro-in-flammatory responses that lead to chronic inflammation of gastric mucosa, and induction of gastric carcinogenesis (Yamaoka, 2010;Lai et al., 2006).
HLAs exhibiting polymorphisms bind antigen peptides and present them to T cells, which differentiate into cytotoxic or helper T cells. An association between the risk of gastric cancer and the type of HLA class II (DR and/or DQ) was suggested in different populations (Quintero et al., 2005;Li et al., 2005). Polymorphisms in HLA genes in the host are suggested to enhance the risk of developing gastric cancer, especially if the infected strain also contains virulence factors that are associated with an enhanced inflammatory response. Most studies have focused on screening of class II antigens—especially in gastric cancer cases (Li et al., 2005;Magnusson et al., 2001;Wu et al., 2002). HLA-DRB1*0405, DRB1*04051, HLA-DQA1*0102, *0301, and DQB1*0301 alleles of class II antigens were associated with gastric cancer. (Yoshitake et al., 1999;Ohtani et al., 2003)
In this study, we evaluated the association of the H. pylori CagL protein with the HLA alleles in patients with gastric cancer and DU. Many studies have evaluated the association between HLA alleles and gastric pathologies in H. pylori-positive patients, but there has been no similar study that included the association between H. pylori virulence factors, such as CagL, CagA, and multiple EPIYA repeats, and the host’s genetic susceptibility. We designed this case-control study to determine plausible explanations in the absence of studies that evaluated the as-sociation between HLA alleles in the host and H. pylori virulence factors such as CagL, CagA, and multiple EPIYA C repeats in patients with gastrointestinal pathologies. The aim of this study was to investigate, for thefirst time, the clinical association of the cagL gene with host HLA alleles in H. pylori strains that were isolated from patients with gastric cancer, DU, and non-ulcer dyspepsia (NUD) and to determine the HLA allele that confers susceptibility or resistance for the risk of gastric
cancer and DU development in Turkish patients. 2. Material and methods
2.1. Study design and patients
This study was designed as a case-control study between 10.09.2014 and 09.03.2018. All participants were diagnosed with H. pylori infec-tion. The study groups were matched according to the age and gender distribution (p > 0.05). A total of 94 patients (44 gastric cancer and 50 DU patients; 58 male, 36 female; mean age, 49.6 years; age range, 19–79 years), and 86 individuals (50 NUD patients and 36 persons with normal gastrointestinal system [NGIS]; 30 male, 56 female; mean age, 47.3 years; age range, 18–86 years) were included as the patient and the control groups, respectively.
The blood samples for HLA genotyping of HLA alleles were collected on the same day that the biopsy samples (corpus and antrum) were taken. The antrum and corpus biopsy specimens were stored and used in molecular studies. We excluded patients who were under 18 years of age, had previous gastric surgery and H. pylori eradication treatment, or who had a history of therapy with antibiotics, antisecretory drugs, bismuth salts, or sucralfate in the month before the samples were taken. Two biopsies from the antrum and the corpus were collected and transferred immediately to Brucella broth. The study was approved by the Clinical Research Ethics Board of Istanbul University, Cerrahpasa Faculty of Medicine (Ethical approval; Ethical approval No: A-15/2014) and recognized standards of the Declaration of Helsinki. All participants provided informed consent to participate in the study.
2.2. PCR
H. pylori DNA extractions were isolated from the antrum and corpus biopsy specimens. Biopsy samples were homogenized using a Magna Lyser Homogenizer (Roche Diagnostic, Basel, Switzerland). DNA was extracted using the QIAamp DNA Mini Kit (QiagenGmbH, Hilden, Germany), according to the manufacturer’s instructions.
2.2.1. ureC gene detection in H. pylori
H. pylori ureC gene concentration was assessed using the H. pylori-QLS 1.0 kit (Fluorion) to detect 156 bp from the ureC gene in H. pylori DNA extractions. (He et al., 2002)
2.2.2. Amplification of the H. pylori cagA gene
Primers described in other studies were used to detect the H. pylori cagA gene (349 bp) (Table 1) (van Doorn et al., 1998;Erzin et al., 2006) . The study protocol was as follows: initial denaturation at 95°C for 2 min, followed by 45 cycles of 95°C for 30 s, 53°C for 45 s, and 72°C for 45 s. Thefinal elongation was at 5 min at 72°C.
2.2.3. Amplification and typing of EPIYA motifs in the cagA 3′ variable region
Primers (forward [cagA28F] and reverse [cagAP1C, cagA-P2CG, cagA-P2TA, and cagA-P3E]) were used to amplify DNA for EPIYA-A, -B, -C, and -D, respectively (Table 1). The PCR protocol was based on a previously described method (Argent et al., 2005).
2.2.4. Empty-site PCR
All strains that were negative for EPIYA PCR were confirmed as CagA-negative using an empty-site-positive PCR assay (Table 1, cag empty PCR), as previously described (Occhialini et al., 2001). 2.2.5. CagL amplification by PCR
A fragment of 651 bp from gene cagL (hp0539) were amplified by PCR using primers cagL forward and reverse (Table 1). The reaction mixture had afinal volume of 25 μL and contained 2.5 mM MgCl2, 0.25 mM dNTP’s, 10x PCR Buffer, 5 pmol of each oligonucleotide, 1 U of Taq
recombinant DNA polymerase (Invitrogen, Massachusetts, USA), and 50 ng of DNA. The conditions used were: 1 cycle at 95 °C for 10 min; 40 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min; and 1 cycle at 72 °C for 15 min in a thermal cycler (Biorad T100, USA). Each re-action included a positive (DNA from strain 26695) and a negative (DNA was substituted with water) control. All reactions were performed in a Mastercycler Ep gradient thermocycler (Eppendorf, Hamburg, Germany). PCR products were analyzed by agarose gel electrophoresis at 2% and stained with ethidium bromide and were visualized. 2.3. Blood collection, DNA extraction and HLA SSO typing
The DNA isolation was performed from 3-ml blood samples by using an automated DNA isolation device Bio Robot EZ1 (Qiagen-Germany) and EZ-DNA extraction kit (Qiagen-Germany). HLA typing at low level (2 digits) of HLA-A, HLA-B, HLA-C and HLA-DQ alleles were de-termined with Luminex 100/200 Instrument technology that uses se-quence-specific oligonucleotide (SSO) probes bound to color-coded microbeads in order to identify HLA alleles encoded by the DNA sample (Luminex Corp., USA). LIFECODES SSO Typing kits (HLA-A-cat628911, B-cat628915, C-cat628921, DRB1-cat628923 and HLA-DQA1/B1-cat628930 SSO Typing Kits) were used for the HLA typing (Lifecodes, Immucor, Germany). This tests is a reverse sequence-specific oligonucleotide (rSSO) DNA typing assay in which SSO probes and color-coded microspheres are used in order to identify HLA alleles.
PCR mixture included 15μL of the lifecodes Master Mix (Immucor), 200 ng of genomic DNA, and 2.5 U Taq polymerase for a 50μL final volume. The cycles were as following; denaturation at 95°C for 5 min; 40 cycles of amplification (8 cycles: 95°C for 30 sec, 60°C for 45 sec, 72°C for 45 sec, and 32 cycles: 95°C for 30 sec, 63°C for 45 sec, 72°C for 45 sec); and extension at 72°C for 15 min. Hybridization was performed under the following conditions: 97°C for 5 min, 47°C for 30 min, and 56°C for 10 min with 15 μL probe mix and 5 μL PCR product. The samples were diluted with 170μL of the 1:200 pre-diluted streptavidin-phycoerythrin solution and analyzed within 30 min by using the Luminex 200 system (Luminex Corp.). Lot-specific background control value was subtracted from the raw medianfluorescence intensity (MFI) value of the sample to produce the background-corrected data. The background-corrected data were divided by the background-corrected values for the corresponding consensus probe producing the normalized data (adjusted MFI value). The probe-hit pattern was compared with the common and well-documented (CWD) HLA alleles Probe Hit Tables (IMGT/HLA Sequence Database Release 3.11.0) by using the MatchIT DNA program (Immucor).
2.4. Statistical analyses
The HLA allele frequencies were calculated by taking the ratio of each HLA allele count, which was achieved by dividing the total allele counts inside each HLA group (Table 2). The frequencies of alleles in
the patient group, control group, and subgroups were compared using the Chi-squared test and Fisher’s exact test with contingency tables and odds ratios (OR) (Tables 3 and 4A-C). Results were defined as the Benjamini–Hochberg-adjusted P-value. Multivariate logistic regression with an enter method was used to assess the association of HLA alleles and the likelihood of gastric cancer or DU based on the cagL/cagA/ EPIYA C repeats/(≥2) (Table 5A-C). The significance value was defined as P < 0.05. All statistical analysis was performed using SPSS v.25 (IBM Corp., Armonk, NY, USA).
3. Results
Among the 94 tested HLA alleles, 64 HLA alleles were detected in the patient groups and the control group (41 for class I and 23 for class II). The allele frequencies of HLA-A, -B, -DRB1, -DQA1, and -DQB1 are shown in Table 2. A*02, B*35, DRB1*13, HLA-DQA1*01, and HLA-DQB1*06 were detected as the highest alleles in the patient groups. However, HLA-A*03, HLA-B*50, HLA-DRB1*04, HLA-DQA1*05, and HLA-DQB1*03 alleles were significantly different compared to CG (Table 3).
Multiple (≥2) EPIYA-C repeats together with cagA positivity were detected in 40 (42.52%) cases in the patient group. Additionally 26 (59%) multiple EPIYA-C repeats for gastric cancer and 14 (28%) mul-tiple EPIYA-C repeats for DU cases were detected in patient group, but only two (2.3%) multiple EPIYA-C repeats for NUD cases—and no EPIYA-C repeats for NGIS cases—were detected. The CagL/CagA posi-tivity distribution in the groups were as follows: 42 (95.4%) gastric cancer, 46 (92%) DU and, 34 (68%) NUD and no NGIS cases. When we compared the selected highest detected HLA alleles between the patient and control groups, the HLA-DQA1*01 (OR: 3.82) allele was sig-nificantly different, suggesting that these individuals with H. pylori strains harbouring the CagL/CagA positivity are susceptible to the risk of gastric cancer and DU, and the HLA-DQA1*05 (OR, 0.318) allele was suggested as a protective allele using univariate analyses (Table 4a). However, when HLA alleles from patients with gastric cancer and DU were compared (subgroups of patient group) based on CagL/CagA po-sitivity, no HLA allele for the risk of gastric cancer or DU was detected using univariate analyses (Table 4b). When we compared the gastric cancer and DU subgroups based on CagL/CagA//(≥2) EPIYA-C, only the HLA-DRB1*04 (OR, 0.047) allele was suggested that these dividuals are susceptible to the risk of DU than gastric cancer in in-dividuals with H. pylori strains harbouring CagL/CagA//(≥2) EPIYA-C (Table 4c).
HLA-DQA1*01 (OR, 2.21), HLA-DQB1*06 (OR, 2.67), sex (male, OR, 2.27), and CagL/CagA/(< 2) EPIYA C repeats (OR, 5.72) were independent risk factors that increased the risk of gastric cancer and DU based on the multivariate logistic regression analyses. However, the HLA-DRB1*04 (OR, 0.28) allele was shown to be a protective allele, which decreased the risk of gastric cancer and DU (Table 5).
Table 1
PCR primers for amplification of cagA, vacA, babA2, EPIYA repeats and cagL gene sequences
Gene Primer Primer Sequence (5’➔3’) References
cagA Forward GAT AAC AGG CAA GCT TTT GAG G CTG (Caliskan et al., 2015) Reverse CAA AAG ATT GTT TGG CAG A
cagL Forward AGC CAA TTT TGA AGC GAA TG (Yeh et al., 2011a, b) Reverse CAA GCG TCT GTG GAA GCA GTG
cagA28F Forward TCT CAA AGG AGC AAT TGG C (Kocazeybek et al., 2015) Reverse GTC CTG CTT TCT TTT TAT TAA CTT KAG C
cagA-P1C Reverse TTT AGC AAC TTG AGC GTA AAT GGG (Kocazeybek et al., 2015) cagA-P2TA Reverse TTT AGC AAC TTG AGT ATA AAT GGG (Kocazeybek et al., 2015) cagA-P3E Reverse ATC AAT TGT AGC GTA AAT GGG (Kocazeybek et al., 2015)
cag: cytotoxin-associated gene vac: vacuolating cytotoxin gene bab: blood group antigen-binding adhesin
Table 2 Frequency of detected HLA class I alleles and the class II alleles in the cancer and ulcer patients (cases) and in the control population HLA-A Allele frequency HLA-B Allele frequency HLA-DRB1 Allele Frequency HLA-DQA1 Allele Frequency HLA-DQB1 Allele Frequency Alleles Cases (n=94, %) Control (n=86, %) Alleles Cases (n=94, %) Control (n=86, %) Alleles Cases (n=94, %) Control (n=86, %) Alleles Cases (n=94, %) Control (n=86, %) Alleles Cases (n=94, %) Control (n=86, %) 01 22 (11.4) 24 (13.9) 07 18 (9.5) 22 (12.8) 01 14 (7.4) 8 (4.6) 01 76 (40) 30 (17.4) 02 20 (10.6) 26 (15.1) 02 52 (27.6) 38 (22) 08 6 (3.2) 6 (3.5) 03 10 (5.3) 10 (5.8) 02 20 (10.6) 20 (11.6) 03 42 (23) 64 (37.2) 03 14 (7.4) 20 (11.6) 13 6 (3.2) 4 (2.3) 04 16 (8.5) 24 (14) 03 12 (6.4) 22 (12.8) 04 22 (11.7) 28 (16.2) 11 18 (9.5) 18 (10.4) 14 6 (3.2) 4 (2.3) 07 10 (5.3) 20 (11.6) 04 16 (8.5) 20 (11.6) 05 48 (25.5) 34 (19.8) 23 10 (5.3) 4 (2.3) 15 2 (1) 6 (3.5) 08 4 (2.1) 6 (3.5) 05 28 (14.9) 54 (31.4) 06 52 (27.6) 20 (11.6) 24 26 (13.8) 24 (13.9) 18 10 (5.3) 10 (5.8) 10 6 (3.2) 4 (2.3) 06 36 (19.1) 24 (13.9) 25 0 (0) 2 (1.1) 27 6 (3.2) 4 (2.3) 11 34 (18) 42 (24.4) 26 4 (2.1) 4 (2.3) 35 40 (21.3) 26 (15.1) 12 6 (3.2) 2 (1.2) 29 8 (4.2) 8 (4.6) 37 4 (2.1) 0 (0) 13 36 (19) 24 (14) 30 4 (2.1) 6 (3.4) 38 8 (4.2) 2 (1.2) 14 18 (9.5) 8 (4.6) 31 2 (1) 4 (2.3) 39 0 (0) 4 (2.3) 15 28 (14.9) 22 (12.8) 32 16 (8.5) 10 (5.8) 40 6 (3.2) 6 (3.5) 16 6 (3.2) 4 (2.3) 33 6 (3.1) 4 (2.3) 41 4 (2.1) 2 (1.2) 68 4 (2.1) 6 (3.4) 44 8 (4.2) 14 (8.1) 69 2 (1) 0 (0) 45 0 (0) 2 (1.2) 48 2 (1) 2 (1.2) 49 6 (3.2) 4 (2.3) 50 0 (0) 10 (5.8) 51 24 (12) 30 (17.4) 52 14 (7.4) 2 (1.2) 53 0 (0) 2 (1.2) 54 2 (1) 0 (0) 55 6 (3.2) 8 (4.6) 57 2 (1) 2 (1.2) 58 8 (4.2) 0 (0) 78 2 (1) 0 (0)
4. Discussion
Gastric pathologies such as gastric cancer, peptic ulcer, or DU are suggested to be caused by the combined effects of the host’s genetic, epigenetic, and environmental factors along with H. pylori infection. Various clinical outcomes of H. pylori infection may result from dif-ferent host responses in individuals based on the host’s genetic sus-ceptibility, which makes them susceptible or resistant to the gastro-intestinal pathologies (Zabalata, 2012). HLA gene polymorphisms are caused from genetically variability in the coding loci of DP, HLA-DQ, and HLA-DR genes that encode HLA class II molecules in the
human genome. Moreover, some specific HLA class II alleles were suggested to be associated with the risk of developing various gastro-intestinal pathologies such as gastric cancer and duodenal development in individuals infected with H. pylori (Magnusson et al., 2001). In H. pylori-positive gastric cancer studies, class II HLA-DRB1 and HLA-DQB1 alleles were extensively studied (Lee et al., 2009; Magnusson et al., 2001).
In the literature, there were only classical comparison studies but no studies exist that involve various H. pylori virulence factors such as CagL, CagA, and multiple EPIYA C repeats. To the best of our knowl-edge, this is thefirst study that evaluated the association between the Table 3
Comparison of HLA alleles which makes susceptible and resistant to the GC/DU in patient and control groups.
HLA Alleles Patient Group H. pylori (+) (n=94, alleles=198) Control Group H. pylori (+) (n=86, alleles=172) OR 95% CI p value Minimum Maximum HLAs increasing susceptibility to GC/DU
HLA-A*02 52 (26%) 38 (22%) 1.34 0.833 2.182 0.2239
HLA-DQA1*01 40 (20%) 26 (15%) 1.51 0.880 2.615 0.1329
HLA-B*35 36 (19%) 24 (14%) 1.46 0.831 2.567 0.1880
HLA-DQB1*06 76 (40%) 30 (17%) 3.21 1.968 5.242 0.0001 HLA-DRB1*13 52 (26%) 20 (11%) 2.90 1.652 5.139 0.0002 HLAs making resistant to GC/DU
HLA-A*03 14 (7%) 20 (11%) 0.61 0.298 1.252 0.1787
HLA-B*50 0 (0%) 10 (6%) 0.08 0.010 0.680 0.0201
HLA-DRB1*04 16 (8%) 24 (14%) 0.57 0.293 1.120 0.1038 HLA-DQA1*05 28 (14%) 54 (31%) 0.38 0.228 0.639 0.0003 HLA-DQB1*03 42 (22%) 64 (37%) 0.48 0.305 0.770 0.0022
GC: Gastric Cancer, DU: Duodenal Ulcer. Table 4
The comparison of HLA alleles which makes susceptible and resistant to the risk of GC/DU between patient and control groups (4a), between gastric cancer and duodenal ulcer cases, due to the cagL/cagA criteria (4b) and between gastric cancer and duodenal ulcer cases due to CagLCcagA (≥2) EPIYA-C criteria (4c)
(4a) PG cagL/cagA (n=88, alleles:172, %) CG cagL/cagA (n=34, alleles:68, %) OR 95% CI p value Minimum Maximum HLA-A*02 44 (25.5%) 12 (17.6%) 1.55 0.56 4.25 0.387 HLA-B*35 34 (19.7%) 10 (14.7%) 1.38 0.46 4.11 0.552 HLA-DRB1*13 32 (18.6%) 16 (23.5%) 0.72 0.27 1.88 0.505 HLA-DQA1*01 70 (40.6%) 12 (17.6%) 3.82 1.15 8.20 0.020 HLA-DQB1*06 48 (27.9%) 12 (17.6%) 1.75 0.64 4.75 0.26 HLA-DQA1*05 26 (15.1%) 24 (35.2%) 0.318 0.12 0.79 0.012 HLA-A*03 14 (8%) 6 (8.8%) 0.89 0.21 3.67 0.87 HLA-B*50 0 (0%) 4 (5.8%) - - - 0.022 HLA-DRB1*04 16 (9.30%) 14 (20.5%) 0.38 0.12 1.16 0.08 HLA-DQB1*03 40 (23.2%) 24 (35.2%) 0.53 0.22 1.27 0.157 (4b) GC cagL/cagA (n=42, alleles: 84, %) DU cagL/cagA (n=46, alleles: 92, %) OR 95% CI p value Minimum Maximum HLA-A*02 18 (21.4%) 26 (28.2%) 0.69 0.26 1.84 0.460 HLA-B*35 20 (23.8%) 14 (15.2%) 1.74 0.59 5.09 0.308 HLA-DRB1*13 20 (23.8%) 12 (13.1%) 2.08 0.68 6.34 0.191 HLA-DQA1*01 38 (45.2%) 32 (34.8%) 1.54 0.65 3.65 0.317 HLA-DQB1*06 26 (30.9%) 22 (23.9%) 1.42 0.55 3.65 0.459 (4c) GC cagL/cagA/(≥2) EPIYA-C (n=42,alleles: 84, %) DU cagL/cagA/(≥2) EPIYA-C (n=26, alleles: 52, %) OR 95% CI p value Minimum Maximum HLA-A*02 12 (14.2%) 8 (15.3%) 0.67 0.15 2.99 0.60 HLA-B*35 12 (14.2%) 4 (7.7%) 1.65 0.28 9.60 0.575 HLA-DRB1*13 12 (14.2%) 6 (11.5%) 1.00 0.20 4.85 0.8663 HLA-DQA1*01 24 (28.5%) 10 (19.2%) 1.37 0.35 5.33 0.648 HLA-DRB1*06 12 (14.2%) 8 (15.3%) 0.67 0.15 2.99 0.60 HLA-DRB1*04 2 (2.3%) 12 (23%) 0.047 0.005 0.455 0.03
host’s genetic factors and H. pylori virulence factors in gastric pathol-ogies. The CagL/CagA positivity distribution in the groups was as fol-lows: patient group, 88 (93.6%); and control group, 34 (68%). HLA-DQA1*01 (OR, 2.21), HLA-DQB1*06 (OR, 2.67), sex (male, OR, 2.27), and CagL/CagA/ < 2) EPIYA C repeats (OR, 5.72) were considered to be independent risk factors that increased the risk of gastric cancer and DU in CagL/CagA-positive H. pylori strains based on multivariate lo-gistic regression analyses. However, the HLA-DRB1*04 (OR, 0.28) allele was shown to be a protective allele, which decreased the risk of gastric cancer and DU. As mentioned above, univariate and multivariate ana-lyses showed similar results, except HLA-DQB1*06 (OR, 2.67), which was significant for the risk of gastric cancer and DU based on the multivariate analyses.
Other studies byYadegar et al. (2014),Yeh et al. (2011a, 2011b), Shukla et al. (2013)andRaei et al. (2015)showed a high prevalence (96.7 %, 98.6%, 86.6%, and 98.1%, respectively) of cagL genotypes in peptic ulcer and gastric cancer patients with virulent H. pylori strains, which are in accordance with the present study (93.6%). A meta-ana-lysis of Asian and European populations byWang et al. (2015)showed that HLA-DQA1*01 was positively associated with gastric cancer and DU, which is similar to this study. However, no association was found for any of the HLA-II alleles to be associated with H. pylori infection among the European population in this meta-analysis (Wang et al., 2015). This suggests that the association between HLA alleles, gastric pathologies, and H. pylori infections in the Turkish population is similar to that of the Asian populations.Quintero et al. (2005)detected that CagA-positive patients with the DQB1*0602 allele have an increased risk for distal gastric cancer, which is similar to our results. Ad-ditionally, HLA-DQA1*01 and HLA-DQB1*06 alleles were positively associated with gastric cancer and DU with H. pylori infections in the studies by Herrera-Goepfert et al. (2004), Wu et al. (2002), and Quintero et al. (2005), which are similar to our results.Ando et al. (2009)showed that HLA DRB1*0405 allele expression was significantly higher in H. pylori-infected patients with intestinal-type gastric cancer than in H. pylori-infected NUD patients but the DRB1*04 allele was shown to be a protective HLA allele in this study, which is contrary to their results. There was a negative relationship between the HLA DRB1*04 alleles and intestinal metaplasia in the antrum with H. pylori infection, which is similar to our results (Ryberg et al., 2013). Garza-González et al. (2004)found that DQA1*0503 was significantly lower in the GC group (OR: 0.13) with H. pylori infection. In a Turkish study, the HLA-CW5 allele was more prevalent in patients with GC (Bilici et al., 2010). The results of these studies may be different because of the different populations; differences in research design, environmental factors, bacterial virulence factors; and/or other host genetic factors (Ando et al., 2009). There are a few study for the association of HLA alleles with the GC development in different geographical areas.Lee et al. (2009)reported that the DRB1*0404 allele was suggested to be significantly associated with GC incidence. They concluded that the diverse results for the association of HLA alleles with GC in different
populations may be caused by differences in the ethnic backgrounds of the HLA allelic variations and heterogeneity in the tumorigenesis of GC. Magnusson et al. (2001)suggested a stronger association with H. pylori for the intestinal as compared with the diffuse type of gastric cancer. They suggested that diffuse gastric cancers are less dependent on H. pylori infection unlike intestinal type gastric cancers. When they in-cluded only H. pylori -positive subjects, the OR was 5, but when only the negative subjects were included, the OR was as high as 15.6 and they suggested that the DRB1*1601 allele is associated mainly with cancer development, rather than infection. The pathway which is responsible for gastric cancer development in these patients may be less dependent on H. pylori infection. HLA DR-DQ alleles are commonly associated with gastric cancer risk through other mechanisms than an increased sus-ceptibility to H. pylori infection. In a literature survey, we met only a few of studies that detected significant alleles for HLA class I alleles such as HLA-CW*03 was found to be significantly higher for GC risk in H. pylori-positive individuals byLi et al. (2005).The reason behind the scarcity of studies for HLA class I alleles is that HLA class I molecules on tumor cells have been regarded as crucial sites where cytotoxic T lymphocytes (CTLs) can recognize tumor-specific antigens and are strongly associated with antitumor activity other than bacterial pep-tides (Kaneko et al., 2011).
Human major histocompatibility complex (MHC) class II molecules are known to be important host factors that affect the type and intensity of immune responses to bacteria and toxins (Llewelyn et al., 2004). The HLA-D region is known to be responsible for 50% of the heritability in hosts (Azuma et al., 1995) and it may be responsible for the variations and degrees of the immune responses in different individuals to various exogenous antigens. Individuals with various HLA allele types may show different degrees of immune response. Allele-specific antigenic peptides to T-cells may contribute to the differences between HLA-DQ genotypes and susceptibility or resistance to H. pylori infection (Herrera-Goepfere et al., 2006). In the host, helper T cells may re-cognize peptides that are presented by HLA class II molecules leading to T cell activation and an immune response. For the immune response variation in different individuals, HLA polymorphism is suggested to be responsible for making individuals susceptible or resistant to infections and autoimmune diseases (Zhao et al., 2012). It is generally accepted that in the epithelial cells of normal gastric mucosa, class II antigens are not expressed but this expression may be seen in the inflamed mucosa in patients with chronic gastritis (Lopes et al., 2006). Supporting this idea, epithelial cells were shown to behave as antigen-presenting cells for CD4+ cells (Hoang et al., 1992).
H. pylori has also been reported to upregulate expression of MHC class II on gastric epithelial cells and this is mediated by activation of lamina propria T cells and macrophages with subsequent release of interferon-γ and other cytokines (Lopes et al., 2006). Adhesion of H. pylori to gastric epithelial cells that were transferred with HLA genes led to different degrees of apoptosis (Fan et al., 1998). As shown in the above studies, H. pylori and its virulence factors are closely associated Table 5
Results of logistic regressions according to the variables in GC/DU cases
Variable(s) B S.E. Wald df p value Exp(B) 95% CI for Exp(B)
Gender 0.822 0.368 5.003 1 0.025 2.275 1.107 4.677 HLA-DQA1*01 0.793 0.338 5.518 1 0.019 2.211 1.140 4.286 HLA-DQA1*05 -0.534 0.319 2.810 1 0.094 0.586 0.314 1.095 HLA-DQB1*06 0.984 0.366 7.234 1 0.007 2.674 1.306 5.476 HLA-B*50 -10.964 8404.392 0.000 1 0.999 0.000 HLA-DRB1*04 -1.256 0.600 4.374 1 0.037 0.285 0.088 0.924 CagL/CagA/ < 2 EPIYA-C 1.744 0.644 7.345 1 0.007 5.721 1.621 20.195 Constant -10.107 8404.392 0.000 1 0.999 0.000
B: beta regression coefficient; Wald, test statistics used for the determination of the meaning of variables; df: degrees of freedom; exp(B): exponential; GC; Gastric Cancer, DU: Duodenal Ulcer. Variable(s) entered on step 1: Gender, Age, DQA1*01, DQA1*05, DQB1*03, DQB1*06, A*02, A*03, HLA-B*35, HLA-B*50, HLA-DRB1*04, HLA-DRB1*13, CagA+CagL+EPIYA
with MHC class II molecules. In this context, HLA molecule poly-morphism may change the affinity for H. pylori peptides and result in an increase in T lymphocyte activation and an increase in the in-flammatory response. Otherwise, different HLA molecules may change their affinity for tumor antigens and compromise tumor cell removal and tumor progression in gastric malignancies. HLA allele poly-morphism may maintain peptides that are derived from H. pylori, which are selectively bound by HLA allotypes (Pérez-Rodríguez et al., 2017). The H. pylori cagT4SS protein CagL interacts with the integrin α5β1 receptor by its RGD motif to inject CagA into gastric epithelial cells and the RGD motif is also involved in upregulation of gastrin expression on gastric epithelial cells, resulting in hypergastrinemia and gastric cancer. The CagL protein is also responsible for H. pylori-induced interleukin (IL)-8 expression, which is related to gastric inflammation (Koelblen et al., 2017;Bonsor et al., 2015;Wiedemann et al., 2012). Consequent host signaling pathway activation by CagA is known to stimulate the transcription factor NF-kB and upregulate host cell proinflammatory responses (Cherati et al., 2017). Polymorphisms at these amino acid sequences may affect the CagL-binding affinity to the α5β1 integrin and sequence variations of CagL correlate with gastroduodenal disorders in different geographic regions (Gorrell et al., 2018).
CagL is also thought to be a good vaccine target because it is ex-pressed on the cell surface and it is well conserved among the H. pylori strains with T4SS (Guo et al., 2017). To understand the importance of CagL in the pathogenesis of H. pylori infections, we must look at the distinct properties of CagL. An increased risk of gastric cancer and ulcer, which is associated with the presence of cagPAI, has largely been attributed to CagA, but CagL may be an equally important effector for these gastric pathologies (Salama et al., 2013). The CagL protein in H. pylori strains binds and activates toll-like receptor TLR5 in the host. TLR5 binds a conserved domain, termed D1, which is present in fla-gellins of several bacteria but not in H. pylori. However, virulent H. pylori strains have a type IV secretion system (T4SS) for delivery of virulence factors into gastric epithelial cells. CagL contains a D1-like motif for binding and activation of TLR5+ epithelial cells and it was suggested that CagL may modulate immune responses to H. pylori by activating TLR5. Long-term infection with CagL-positive strains in-creased the risk of peptic ulcer disease and gastric cancer (Pachathundikandi et al., 2019).
Additionally, the presence of the adhesive RGD sequence within the HLA-DQ suggests that this HLA-DQ molecule may interact with some integrin receptors (Kastin, 2006). Considering this to be a hypothesis, some HLA-DQ alleles may also interact with the same integrin receptors and present CagL peptides to T cells to induce a host cell’s proin-flammatory responses leading to gastric cancers. This possible asso-ciation should be investigated in molecular studies. Some HLA-DR molecules may also have a band with a molecular weight that is similar to that of β1 integrin (Altomonte et al., 2003). If this is true, HLA molecules may directly bind the CagL protein, which may make some individuals susceptible to the risk of gastric cancer by presenting its peptides to T lymphocytes. It is difficult for the host to evade the effects of the CagL protein because CagL can also trigger intracellular signaling pathways via RGD-dependent binding to integrins and, thus, it can in-duce cell proinflammatory responses independent of CagA transloca-tion. Polymorphisms at amino acid residues 58–62 upstream of the critical RGD motif (CagL hypervariable motif) may correlate with gas-tric pathologies that differ in geographical regions. This study had some limitations such as not including HLA-C alleles and the low resolution of the HLA kits (Yadegar et al., 2019).
5. Conclusions
Thus, HLA-DQA1*01 (OR, 2.21), HLA-DQB1*06 (OR, 2.67), sex (male, OR, 2.27), and CagL/CagA/(< 2) EPIYA C repeats (OR, 5.72) were shown to be independent risk factors that increase the risk of gastric cancer and DU based on multivariate logistic regression results.
However, the HLA-DRB1*04 (OR, 0.28) allele was suggested to be a protective allele, which decreased the risk of gastric cancer and DU. The role of H. pylori-specific CagA, CagL, and EPIYA-C repeats are important factors leading to gastric pathologies and H. pylori is known to cause many gastroduodenal diseases such as peptic ulcer disease and gastric adenocarcinoma. These diseases result from an interaction between bacterial virulence factors, host epigenetic and environmental factors, and H. pylori strain heterogeneity, such as genotypic variation among strains and variations in H. pylori populations within an individual host. This study sheds light on the relationship between H. pylori cagL protein and HLA alleles, but comprehensive, prospective, large-scale studies should be undertaken in the future to further clarify this relationship. Declaration of Competing Interest
The author(s) declare that there are no conflicts of interest Acknowledgments
This work was supported by the Istanbul University Research Fund under project number 45151.
References
Altomonte, M., Fonsatti, E., Visintin, A., Maio, M., 2003. Targeted therapy of solid ma-lignancies via HLA class II antigens: a new biotherapeutic approach? Oncogene. 22, 6564–6569. Review. https://doi.org/10.1038/sj.onc.1206960.
Ando, T., Ishikawa, T., Kato, H., Yoshida, N., Naito, Y., Kokura, S., Yagi, N., Takagi, T., Handa, O., Kitawaki, J., Nakamura, N., Hasegawa, G., Fukui, M., Imamoto, E., Nakamura, C., Oyamada, H., Isozaki, Y., Matsumoto, N., Nagao, Y., Okita, M., Nakajima, Y., Kurokawa, M., Nukina, M., Ohta, M., Mizuno, S., Ogata, M., Obayashi, H., Park, H., Kitagawa, Y., Nakano, K., Yoshikawa, T., 2009. Synergistic effect of HLA class II loci and cytokine gene polymorphisms on the risk of gastric cancer in Japanese patients with Helicobacter pylori infection. Int. J. Cancer. 125, 2595–2602.
https://doi.org/10.1002/ijc.24666.
Argent, R.H., Zhang, Y., Atherton, J.C., 2005. Simple method for determination of the number of Helicobacter pylori CagA variable-region EPIYA tyrosine phosphorylation motifs by PCR. J. Clin. Microbiol. 43, 791–795.https://doi.org/10.1128/JCM.43.2. 791-795.2005.
Azuma, T., Ito, Y., Miyaji, H., Dojyo, M., Tanaka, Y., Hirai, M., Ito, S., Kato, T., Kohli, Y., 1995. Immunogenetic analysis of the human leukocyte antigen DQA1 locus in pa-tients with duodenal ulcer or chronic atrophic gastritis harbouring Helicobacter pylori. Eur. J. Gastroerol. Hepatol. 7, 71–73.8574742.
Backert, S., Meyer, T.F., 2006. Type IV secretion systems and their effectors in bacterial pathogenesis. Curr. Opin. Microbio. 9, 207–217.https://doi.org/10.1016/j.mib. 2006.02.008.
Bilici, M., Okcu, N., Cayır, K., Pirim, I., Tekin, S.B., Gundogdu, C., 2010. Distribution of HLA tissue groups in patients with gastric cancer. Eurasian. J. Med. 42, 9–11.https:// doi.org/10.5152/eajm.2010.03.
Bonsor, D.A., Pham, K.T., Beadenkopf, R., Diederichs, K., Haas, R., Beckett, D., Fischer, W., Sundberg, E.J., 2015. Integrin engagement by the helical RGD motif of the Helicobacter pylori CagL protein is regulated by pH induced displacement of a neighboring helix. J. Biol. Chem. 290, 12929–12940.https://doi.org/10.1074/jbc. M115.641829.
Çalışkan, R., Sayı, Yazgan A., Tokman, H.B., Sofyalı, E., Erzin, Y.Z., Akgul, O., Kurt, A., Kalaycı, F., Ziver, T., Yuksel, P., Bal, K., Kocazeybek, B., 2015. The cytokine response in THP-1 (monocyte) and HL-60 (neutrophil-differentiated) cells infected with dif-ferent genotypes of Helicobacter pylori strains. Turk. J. Gastroenterol. 26, 297–303.
https://doi.org/10.5152/tjg.2015.8058.
Cherati, M.R., Shokri-Shirvani, J., Karkhah, A., Rajabnia, R., Nouri, H.R., 2017. Helicobacter pylori cagL amino acid polymorphism D58E59 pave the way toward peptic ulcer disease while N58E59 is associated with gastric cancer in north of Iran. Microb. Pathog. 107, 413–418.https://doi.org/10.1016/j.micpath.2017.04.025. Erzin, Y., Koksal, V., Altun, S., Dobrucali, A., Aslan, M., Erdamar, S., Dirican, A.,
Kocazeybek, B., 2006. Prevalence of Helicobacter pylori vacA, cagA, cagE, iceA, babA2 genotypes and correlation with clinical outcome in Turkish patients with dyspepsia. Helicobacter. 11, 574–580.https://doi.org/10.1111/j.1523-5378.2006.00461.x. Fan, X., Crowe, S.E., Behar, S., Gunasena, H., Ye, G., Haeberle, H., Van Houten, N., Gourley, W.K., Ernst, P.B., Reyes, V.E., 1998. The effect of class II major histo-compatibility complex expression on adherence of Helicobacter pylori and induction of apoptosis in gastric epithelial cells: a mechanism for T helper cell type 1-mediated damage. J. Exp. Med. 187, 1659–1669.https://doi.org/10.1084/jem.187.10.1659. Garza-González, E., Bosques-Padilla, F.J., Pérez-Pérez, G.I., Flores-Gutiérrez, J.P.,
Tijerina-Menchaca, R., 2004. Association of gastric cancer, HLA-DQA1, and infection with Helicobacter pylori CagA+ and VacA+ in a Mexican population. J.
Gastroenterol. 39, 1138–1142.https://doi.org/10.1007/s00535-004-1462-2. Gorrell, R.J., Zwickel, N., Reynolds, J., Bulach, D., Kwok, T., 2018. Helicobacter pylori
with gastric cancer. J. Infect. Dis. 213, 1927–1931.https://doi.org/10.1093/infdis/ jiw060.
Guo, L., Yin, R., Xu, G., Gong, X., Chang, Z., Hong, D., Liu, H., Ding, S., Han, X., Li, Y., Tang, F., Liu, K., 2017. Immunologic properties and therapeutic efficacy of a multi-valent epitope-based vaccine against four Helicobacter pylori adhesins (urease, Lpp20, HpaA, and CagL) in Mongolian gerbils. Helicobacter. 22, 6.https://doi.org/10.1111/ hel.12428.
He, Q., Wang, J.P., Osato, M., Lachman, L.B., 2002. Real-time quantitative PCR for de-tection of Helicobacter pylori. J. Clin. Microbiol. 40, 3720–3728.https://doi.org/10. 1128/jcm.40.10.3720-3728.2002.
Herrera-Goepfere, R., Yamamoto-Furusho, J.K., Onate-Ocana, L.F., Camorlinga-Ponce, M., Munoz, L., Ruiz-Morales, J.A., Vargas-Alarcon, G., Granados, J., 2006. Role of the HLA-DQ locus in the development of chronic gastritis and gastric carcinoma in Mexican patients. World. J. Gastroenterol. 12, 7762–7767.https://doi.org/10.3748/ wjg.v12.i48.7762.
Herrera-Goepfert, R., Zúñiga, J., Hernández-Guerrero, A., Rodríguez-Reyna, T., Osnalla, N., Ruíz-Morales, J., Vargas-Alarcón, G., Yamamoto-Furusho, J.K., Mohar-Betancourt, A., Hernández-Pando, R., Granados, J., 2004. Association of the HLA-DQB*0501, allele of the major histocompatibility complex with gastric cancer in Mexico. Gac. Med. Mex. 140, 299–303 PMID: 15259342.
Hoang, P., Crotty, B., Dalton, H.R., Jewell, D.P., 1992. Epithelial cells bearing class II molecules stimulate allogeneic human colonic intraepithelial lymphocytes. Gut. 33, 1089–1093.https://doi.org/10.1136/gut.33.8.1089.
Kaneko, K., Ishigami, S., Kijima, Y., Funasako, Y., Hirata, M., Okumura, H., Shinchi, H., Koriyama, C., Ueno, S., Yoshinaka, H., Natsugoe, S., 2011. Clinical implication of HLA class I expression in breast cancer. BMC. Cancer. 11, 454.https://doi.org/10. 1186/1471-2407-11-454.
Kastin, A., 2006. RGD-Peptides and some immunological problems. In: Kastin, A.J. (Ed.), Handbook of Biologically Active Peptides. Academic press, Publisher: Elsevier, pp. 576.
Kocazeybek, B.S., Caliskan, R., Erdamar, C.S., Ergin, S., Kuskucu, M., Kepil, N., Oyku, D.H., Ziya, Erzin, Saribas, Y., Bahar, S., Tokman, H., Kalayci, F., Akgul, O., Yuksel, P., Karakullukcu, A., Ziver, T., Sirekbasan, S., Caglar, E., Bal, K., 2015. Patterns of EPIYA motifs among cagA-positive Helicobacter pylori strains: a case-control study in a Turkish population with Eurasian geographical features. J. Med. Microbiol. 64, 1117–1123.https://doi.org/10.1099/jmm.0.000141.
Koelblen, T., Bergé, C., Cherrier, M.V., Brillet, K., Jimenez-Soto, L., Ballut, L., Takagi, J., Montserret, R., Rousselle, P., Fischer, W., Haas, R., Fronzes, R., Terradot, L., 2017. Molecular dissection of protein-protein interactions between integrinα5β1 and the Helicobacter pylori Cag type IV secretion system. FEBS. J. 284, 4143–4157.https:// doi.org/10.1111/febs.14299.
Kwok, T., Zabler, D., Urman, S., Rohde, M., Hartig, R., Wessler, S., Misselwitz, R., Berger, J., Sewald, N., König, W., Backert, S., 2007. Helicobacter exploits integrin for type IV secretion and kinase activation. Nature. 449, 856–862.https://doi.org/10.1038/ nature06187.
Lai, Y.P., Yang, J.C., Lin, T.Z., Lin, J.T., Wang, J.T., 2006. Helicobacter pylori infection and CagA protein translocation in human primary gastric epithelial cell culture. Helicobacter. 11, 451–459.https://doi.org/10.1111/j.1523-5378.2006.00438.x. Lee, H.W., Hahm, K.B., Lee, J.S., Ju, Y.S., Lee, K.M., Lee, K.W., 2009. Association of the
human leukocyte antigen class II alleles with chronic atrophic gastritis and gastric carcinoma in Koreans. J. Dig. Dis. 10, 265–271. https://doi.org/10.1111/j.1751-2980.2009.00395.x.
Li, Z., Chen, D., Zhang, C., Li, Y., Cao, B., Ning, T., Zhao, Y., You, W., Ke, Y., 2005. HLA polymorphisms are associated with Helicobacter pylori infected gastric cancer in a high risk population. China. Immunogenetics 56, 781–787.https://doi.org/10.1007/ s00251-004-0723-9.
Llewelyn, M., Sriskandan, S., Peakman, M., Ambrozak, D.R., Douek, D.C., Kwok, W.W., Cohen, J., Altmann, D.M., 2004. HLA class II polymorphisms determine responses to bacterial superantigens. J. Immunol. 172, 1719–1726.https://doi.org/10.4049/ jimmunol.172.3.1719.
Lopes, A.I., Victorino, R.M., Palha, A.M., Ruivo, J., Fernandes, A., 2006. Mucosal lym-phocyte subsets and HLA-DR antigen expression in paediatric Helicobacter pylori-as-sociated gastritis. Clin. Exp. Immunol. 14, 13–20. https://doi.org/10.1111/j.1365-2249.2006.03100.x.
Magnusson, P.K.E., Enroth, H., Eriksson, I., Held, M., Nyrén, O., Engstrand, L., Hansson, L.E., Gyllensten, U.B., 2001. Gastric cancer and human leukocyte antigens: distinct DQ and DR alleles are associated with development of gastric cancer and infection by Helicobacter pylori. Cancer. Res. 61, 2684–2689 PMID: 11289148.
Occhialini, A., Marais, A., Urdaci, M., Sierra, R., Muñoz, N., Covacci, A., Mégraud, F., 2001. Composition and gene expression of the cag pathogenicity island in Helicobacter pylori strains isolated from gastric carcinoma and gastritis patients in Costa Rica. Infect. Immun. 69, 1902–1908. https://doi.org/10.1128/IAI.69.3.1902-1908.2001.
Ohtani, M., Azuma, T., Yamazaki, S., Yamakawa, A., Ito, Y., Muramatsu, A., Dojo, M., Yamazaki, Y., Kuriyama, M., 2003. Association of the HLADRB1 gene locus with gastric adenocarcinoma in Japan. Dig. Liver. Dis. 35, 468–472.https://doi.org/10. 1016/s1590-8658(03)00218-4.
Pachathundikandi, S.K., Tegtmeyer, N., Arnold, I.C., Lind, J., Neddermann, M., Falkeis-Veits, C., Chattopadhyay, S., Brönstrup, M., Tegge, W., Hong, M., Sticht, H., Vieth, M., Müller, A., Backert, S., 2019. T4SS-dependent TLR5 activation by Helicobacter pylori infection. Nat. Commun. 10, 5717. https://doi.org/10.1038/s41467-019-13506-6.
Pérez-Rodríguez, M., Partida-Rodríguez, O., Camorlinga-Ponce, M., Flores-Luna, L.,
Lazcano, E., Gómez, A., Herrera-Goepfert, R., Medrano-Guzmán, R., Torres, J., 2017. Polymorphisms in HLA-DQ genes, together with age, sex, and Helicobacter pylori in-fection, as potential biomarkers for the early diagnosis of gastric cancer. Helicobacter. 22, 1.https://doi.org/10.1111/hel.12326.
Quintero, E., Pizarro, M.A., Rodrigo, L., Piqué, J.M., Lanas, A., Ponce, J., Miño, G., Gisbert, J., Jurado, A., Herrero, M.J., Jiménez, A., Torrado, J., Ponte, A., Díaz-de-Rojas, F., Salido, E., 2005. Association of Helicobacter pylori-related distal gastric cancer with the HLA class II gene DQB10602 and cagA strains in a southern European population. Helicobacter. 10, 12–21.https://doi.org/10.1111/j.1523-5378.2005. 00287.x.
Raei, N., Latifi-Navid, S., Zahri, S., 2015. Helicobacter pylori cag pathogenicity island cagL and orf17 genotypes predict risk of peptic ulcerations but not gastric cancer in Iran. Asian. Pac. J. Cancer. Prev. 16, 6645–6650.https://doi.org/10.7314/apjcp.2015.16. 15.6645.
Román-Román, A., Martínez-Santos, V.I., Castañón-Sánchez, C.A., Albañil-Muñoz, A.J., González-Mendoza, P., Soto-Flores, D.G., Martínez-Carrillo, D.N., Fernández-Tilapa, G., 2019. CagL polymorphisms D58/K59 are predominant in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis. Gut. Pathog. 11, 5.
https://doi.org/10.1186/s13099-019-0286-9.
Ryberg, A., Petersson, F., Redeen, S., Eriksson, O., Borch, K., 2013. Host gene poly-morphisms in relation to Helicobacter pylori infection and associated diseases in a population based cohort. Gastroenterology. Res. 6, 207–218.https://doi.org/10. 4021/gr578w.
Saha, A., Backert, S., Hammond, C.E., Gooz, M., Smolka, A.J., 2010. Helicobacter pylori CagL activates ADAM17 to induce repression of the gastric H, K-ATPase alpha sub-unit. Gastroenterology 139, 239–248.https://doi.org/10.1053/j.gastro.2010.03.036. Salama, N.R., Hartung, M.L., Müller, A., 2013. Life in the human stomach: persistence
strategies of the bacterial pathogen Helicobacter pylori. Nat. Rev. Microbiol. 11, 385–399.https://doi.org/10.1038/nrmicro3016.
Shukla, S.K., Prasad, K.N., Tripathi, A., Jaiswal, V., Khatoon, J., Ghsohal, U.C., Krishnani, N., Husain, N., 2013. Helicobacter pylori cagL amino acid polymorphisms and its as-sociation with gastroduodenal diseases. Gastric. Cancer. 16, 435–439.https://doi. org/10.1007/s10120-012-0189-7.
Tegtmeyer, N., Hartig, R., Delahay, R.M., Rohde, M., Brandt, S., Conradi, J., Takahashi, S., Smolka, A.J., Sewald, N., Backert, S.A., 2010. smallfibronectin-mimicking protein from bacteria induces cell spreading and focal adhesion formation. J. Biol. Chem. 285, 23515–23526.https://doi.org/10.1074/jbc.M109.096214.
van Doorn, L.J., Figueiredo, C., Rossau, R., Jannes, G., van Asbroek, M., Sousa, J.C., Carneiro, F., Quint, W.G., 1998. Typing of Helicobacter pylori vacA gene and detection of cagA gene by PCR and reverse hybridization. J. Clin. Microbiol. 36, 1271–1276.
9574690.
Wang, J., Zhang, Q., Liu, Y., Han, J., Ma, X., Luo, Y., Liang, Y., Zhang, L., Hu, Y., 2015. Association between HLA-IIgene polymorphism and Helicobacter pylori infection in Asian and European population: A meta-analysis. Microb. Pathog. 82, 15–26.https:// doi.org/10.1016/j.micpath.2015.03.011.
Wiedemann, T., Hofbaur, S., Tegtmeyer, N., Huber, S., Sewald, N., Wessler, S., Backert, S., Rieder, G., 2012. Helicobacter pylori CagL dependent induction of gastrin expression via a novel alpha-beta5-integrin-integrin linked kinase signalling complex. Gut. 61, 986–996.https://doi.org/10.1136/gutjnl-2011-300525.
Wu, M.S., Hsieh, R.P., Huang, S.P., Chang, Y.T., Lin, M.T., Chang, M.C., Shun, C.T., Sheu, J.C., Lin, J.T., 2002. Association of HLA-DQB1*0301 and HLA-DQB1*0602 with different subtypes of gastric cancer in Taiwan. Jpn. J. Cancer. Res. 93, 404–410.
https://doi.org/10.1111/j.1349-7006.2002.tb01271.x.
Yadegar, A., Mobarez, A.M., Alebouyeh, M., Mirzaei, T., Kwok, T., Zali, M.R., 2014. Clinical relevance of cagL gene and virulence genotypes with disease outcomes in a Helicobacter pylori infected population from Iran. World. J. Microbiol. Biotechnol. 30, 2481–2490.https://doi.org/10.1007/s11274-014-1673-5.
Yadegar, A., Mobarez, A.M., Zali, M.R., 2019. Genetic diversity and amino acid sequence polymorphism in Helicobacter pylori CagL hypervariable motif and its association with virulence markers and gastroduodenal diseases. Cancer. Med. 8, 1619–1632.https:// doi.org/10.1002/cam4.1941.
Yamaoka, Y., 2010. Mechanisms of disease: Helicobacter pylori virulence factors. Nat. Rev. Gastroenterol. Hepatol. 7, 629–641.https://doi.org/10.1038/nrgastro.2010.154. Yeh, Y.C., Chang, W.L., Yang, H.B., Cheng, H.C., Wu, J.J., Sheu, B.S., 2011a. H. pylori
cagL amino acid sequence polymorphism Y58E59 induces a corpus shift of gastric integrinα5β1 related with gastric carcinogenesis. Mol. Carcinog. 50, 751–759.
https://doi.org/10.1002/mc.20753.
Yeh, Y.C., Chang, W.L., Yang, H.B., Cheng, H.C., Wu, J.J., Sheu, B.S., 2011b. H. pylori cagL amino acid sequence polymorphism Y58E59 induces a corpus shift of gastric integrin a5b1 related with gastric carcinogenesis. Mol. Carcinog. 50, 751–759.
https://doi.org/10.1002/mc.20753.
Yoshitake, S., Okada, M., Kimura, A., Sasazuki, T., 1999. Contribution of major histo-compatibility complex genes to susceptibility and resistance in Helicobacter pylori related diseases. Eur. J. Gastroenterol. Hepatol. 11, 875–880.https://doi.org/10. 1097/00042737-199908000-00011.
Zabaleta, J., 2012. Multifactorial etiology of gastric cancer. Methods. Mol. Biol. 863, 411–435.https://doi.org/10.1007/978-1-61779-612-8_26.
Zhao, Y., Wang, J., Tanaka, T., Hosono, A., Ando, R., Soeripto, S., Ediati Triningsih, F.X., Triono, T., Sumoharjo, S., Astuti, E.Y., Gunawan, S., Tokudome, S., 2012. Association between HLA-DQ genotypes and haplotypes vs Helicobacter pylori infection in an Indonesian population. Asian. Pac. J. Cancer. Prev. 13, 1247–1251.https://doi.org/ 10.7314/apjcp.2012.13.4.1247.
