Functionally significant polymorphisms of the MMP-9 gene are associated with peptic ulcer disease in the Caucasian population of Central Russia

doi:10.21203/rs.3.rs-464509/v1

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Functionally significant polymorphisms of the MMP-9 gene are associated with peptic ulcer disease in the Caucasian population of Central Russia

https://doi.org/10.21203/rs.3.rs-464509/v1

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This study analyzed the association of functionally significant SNPs of matrix metalloproteinase (MMP) genes in the development of peptic ulcer disease (PUD) in Caucasians from Central Russia. Ten SNPs of the MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 genes were analyzed for association with PUD in a cohort of 798 patients with PUD (including 404 H. pylori-positive and 394 H. pylori-negative) and 347 H. pylori-negative controls using logistic regression and assuming the additive, recessive, and dominant genetic models. Allele G of the rs17576 MMP9 locus conferred a higher risk for PUD (OR_adj = 1.31, p_perm=0.016), haplotype AACG of loci rs17576-rs3787268-rs2250889-rs17577 of the MMP9 gene decreased risk for PUD (OR_adj=0.17, p_perm=0.003). Also, allele C of rs3918249, allele G of rs17576 and haplotype CG of rs3918249-rs17576 of the MMP9 gene increased risk for H. pylori-positive PUD (OR_adj = 1.82, p_perm=0.002; OR_adj = 1.53–1.95 p_perm=0.001–0.013 and OR_adj = 1.49 p_perm=0.009 respectively). The above loci and 50 linked to them possess significant regulatory effects and may affect the alternative splicing of four genes and the expression of 17 genes in various organs and tissues related to the PUD pathogenesis.

Epigenetics & Genomics

MMP

Peptic ulcer disease

SNP

Association.

Peptic ulcer is the cyclical appearance of a limited mucosal defect in the digestive tract (usually the stomach or duodenum) extending deeply beyond the muscular plate of the mucous membrane, with inflammatory infiltration and thrombotic necrosis in adjacent tissues¹. The prevalence of peptic ulcer disease (PUD) in the general population is estimated at 5–10%². Symptoms of PUD are variable and may include epigastric pain on an empty stomach and after eating, heartburn, nausea, vomiting, weight loss, and bleeding or perforation with the complicated disease^1–3. Identifying the risk factors and mechanisms of PUD helps to develop efficient strategies for diagnostics and treatment¹.

Mucosal defects in patients with the acid peptic disease have been traditionally considered as a result of increased gastric acid secretion in the stomach and degradation of the mucus barrier^2,4. Risk factors for PUD, including gastric and duodenal ulcers, are infection by H. pylori, alcohol and tobacco consumption, use of non-steroidal anti-inflammatory drugs (NSAIDs) and aspirin, stress, etc.^2–6. However, only a relatively small proportion of people infected by H. pylori or using NSAIDs develop PUD that suggests variation in individual susceptibility to the beginning of mucosal damage⁷. On the other hand, about one-fifth of cases include H. pylori-negative, NSAID-negative, and aspirin-negative PUD collectively classified as an idiopathic ulcer⁸. This type of ulcer is thought to occur due to the imbalance between factors important for mucosal integrity and aggressive insults, but the exact pathogenic mechanisms of idiopathic peptic ulcer remain unknown³.

Matrix metalloproteinases (MMPs) are endopeptidases playing an important role in the extracellular matrix (ECM) remodeling, cell proliferation, and inflammation. MMPs are synthesized and secreted by gastric and duodenal epithelial cells, macrophages, and neutrophils¹³. Since ECM degradation is an important factor of gastric and duodenal mucosal damage and subsequent PUD, MMPs play a key role in this process^12,13,14. There is evidence that cleaving and remodeling of the ECM by MMPs is one of the factors contributing to gastric ulceration (GU)^15,16. The role of several MMPs (MMP-1, MMP-2, MMP-3, MMP-9, and MMP-13) in GU was studied using animal models^16–19. MMP-9 was shown to be important in the early phase of chronic GU¹⁹.

Several genes have been reported for their association with peptic ulcers^7,9−11. Polymorphisms of the MMPs genes (MMP-9, MMP-7, MMP-3) may contribute to a genetic risk profile for gastric and duodenal ulcers in chronic H. pylori infection^9–11. H. pylori infection can induce the expression of MMP-3, MMP-7, and MMP-9 in the gastric mucosa and sera^10,20,21. MMP-9 was significantly up-regulated in H. pylori-positive as compared to H. pylori-negative GU²².

Despite the apparently significant role of MMPs in PUD pathogenesis, associations of MMP genetic variants with PUD have been poorly analyzed: only a few studies of this problem have been published so far^{9–11, 23}. This prompts for filling in this gap.

The present study analyzed polymorphisms of MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 genes for their association with PUD and possible role in the susceptibility to the disease in the Caucasian sample from the Central Region of Russia.

The phenotypic data of the study participants are shown in Table 1. The PUD patients had a more common family history of peptic ulcer (p = 0.0005), alcohol (p = 0.0005) and tobacco (p = 0.0005) consumption, stress (p = 0.0005), the presence of cardiovascular pathology (p = 0.0005) versus control group. These parameters were used as confounding factors (covariates) in the regression association analyses.

Table 1

Phenotypic characteristics of the study participants.
Parameters	Control mean ± SD, % (n)	PUD mean ± SD, % (n)	p
N	347	798	-
Age, years (min–max)	48.47 ± 13.69 (22–79)	48.54 ± 14.28 (20–79)	0.92
Gender ratio, f/m	66.28/33.72 (230/117)	67.42/32.58 (538/260)	0.76
BMI, kg/m2	26.83 ± 5.09	26.94 ± 5.30	0.78
Age of developing peptic ulcer, years	-	41.12 ± 12.87	-
Family history of peptic ulcer	4.32 (15)	18.29 (146)	0.0005
Current smoking	14.99 (52)	33.08 (264)	0.0005
Alcohol consumption	32.28 (112)	51.13 (408)	0.0005
Stress	37.17 (129)	77.19 (616)	0.0005
Positivity H. pylori test (endoscopic biopsy and histological identification)	-	50.63 (404)	-
PUD characteristics
Location Stomach: Body Pylorus Antrum Duodenum: Bulb	- - - -	2.76 (22) 3.01 (24) 48.62 (388) 45.61 (364)	- - - -
Sizes ulcer (diameter) (cm)	-	0.61 ± 0.40	-
Sizes ulcer: Small (< 0.5 cm) Medium (0.5-1.0 cm) Large (> 1.0 cm)	- - -	45.37 (362) 44.86 (358) 9.77 (78)	- - -
PUD associated complications
Bleeding	-	3.51 (28)	-
Perforation	-	8.27 (66)	-
Stenosis	-	6.52 (52)	-
Malignancy	-	2.26 (18)	-
Other somatic pathologies
Cardiovascular pathology	26.80 (93)	48.37 (386)	0.0005
Endocrine pathology	3.17 (11)	5.01 (40)	0.22
Kidney pathology	2.59 (9)	3.76 (30)	0.41
Respiratory system pathology	4.32 (15)	5.76 (46)	0.39
Nervous system pathology	7.78 (27)	9.52 (76)	0.40
Musculoskeletal system pathology	6.91 (24)	8.02 (64)	0.60
p values < 0.05 are shown in bold.

Supplementary Table S1 shows distributions of genotypes and alleles of the ten studied SNPs in the PUD patients and control groups. All analyzed SNPs were in the HWE (p > 0.005, p_bonf>0.05). After the Bonferroni correction, only polymorphisms of the MMP9 gene manifested association with PUD (Table 2). Specifically, the increased risk of PUD was associated with allele G of SNP rs17576 (additive model, the odds ratio adjusted for confounding factors OR_adj = 1.31, p_perm = 0.016, power − 82.98%) (Table 2). Two loci were associated with H. pylori-positive PUD (rs3918249 and rs17576) (Table 3). Allele C of SNP rs3918249 showed a significant association with the increased risk of H. pylori-positive PUD (dominant model, OR_adj = 1.82, p_perm = 0.002, power − 96.43%). The increased risk of H. pylori-positive PUD was also associated with a carriage of allele G of loci rs17576 according to the all three genetic models: additive (OR_adj = 1.53, p_perm = 0.001, power − 98.14%), dominant (OR_adj = 1.67, p_perm = 0.013, power − 90.21%), recessive (OR_adj = 1.95, p_perm = 0.007, power − 94.75%).

Table 2

Associations of the *MMP* gene polymorphisms with PUD.
SNP	Gene	MAF	n	Additive model				Dominant model					Recessive model
				OR	95% CI		Р	OR		95% CI		Р	OR	95% CI		Р
				OR	L95	U95	Р	OR		L95	U95	Р	OR	L95	U95	Р
rs1940475	MMP-8	T	1136	0.96	0.79	1.18	0.708	0.91	0.66		1.26	0.573	0.99	0.71	1.39	0.960
rs1799750	MMP-1	2G	1107	0.89	0.73	1.09	0.263	0.86	0.62		1.19	0.362	0.84	0.59	1.02	0.345
rs679620	MMP-3	T	1133	0.97	0.79	1.20	0.797	0.93	0.66		1.30	0.655	1.01	0.72	1.41	0.979
rs243865	MMP-2	T	1121	0.96	0.76	1.22	0.749	0.94	0.69		1.27	0.672	1.01	0.57	1.80	0.969
rs3918242	MMP-9	T	1127	1.00	0.75	1.32	0.973	1.06	0.77		1.46	0.733	0.58	0.22	1.52	0.266
rs3918249	MMP-9	C	1125	1.16	0.93	1.43	0.181	1.45	1.07		1.97	0.018	0.88	0.59	1.33	0.549
rs17576	MMP-9	G	1140	1.31	1.05	1.60	0.016	1.35	0.99		1.83	0.054	1.51	1.00	2.27	0.048
rs3787268	MMP-9	A	1133	1.12	0.87	1.45	0.384	1.17	0.86		1.58	0.315	1.02	0.48	2.14	0.968
rs2250889	MMP-9	G	1128	0.79	0.57	1.09	0.148	0.77	0.53		1.12	0.172	0.63	0.22	1.80	0.388
rs17577	MMP-9	A	1112	1.00	0.75	1.32	0.988	1.01	0.80		1.52	0.563	0.46	0.18	1.17	0.102
All results were obtained after adjustment for covariates.
ОR, odds ratio.
95% CI, 95% confidence interval.
p values < 0.017 are shown in bold.

Table 3

Associations of the *MMP* gene polymorphisms with *H. pylori*-positive and *H. pylori*-negative PUD.
SNP	Gene	MAF	n	Additive model				Dominant model					Recessive model
				OR	95% CI		Р	OR		95% CI		Р	OR	95% CI		Р
				OR	L95	U95	Р	OR		L95	U95	Р	OR	L95	U95	Р
H. pylori-positive PUD
rs1940475	MMP-8	T	744	0.97	0.76	1.23	0.774	0.91	0.62		1.36	0.656	0.99	0.66	1.49	0.979
rs1799750	MMP-1	2G	725	0.88	0.69	1.13	0.313	0.83	0.56		1.23	0.361	0.85	0.55	1.31	0.452
rs679620	MMP-3	T	743	0.92	0.71	1.18	0.505	0.85	0.57		1.28	0.447	0.93	0.62	1.42	0.744
rs243865	MMP-2	T	735	0.98	0.74	1.30	0.879	0.90	0.63		1.30	0.588	1.26	0.64	2.46	0.509
rs3918242	MMP-9	T	739	1.17	0.83	1.63	0.376	1.34	0.92		1.96	0.127	0.30	0.06	1.39	0.123
rs3918249	MMP-9	C	737	1.33	1.03	1.72	0.031	1.82	1.23		2.67	0.002	1.03	0.63	1.67	0.914
rs17576	MMP-9	G	746	1.53	1.19	1.98	0.001	1.67	1.14		2.43	0.008	1.95	1.22	3.11	0.005
rs3787268	MMP-9	A	745	1.23	0.91	1.67	0.181	1.26	0.87		1.81	0.219	1.43	0.62	3.30	0.396
rs2250889	MMP-9	G	736	0.77	0.51	1.15	0.203	0.78	0.49		1.23	0.282	0.42	0.09	2.01	0.280
rs17577	MMP-9	A	728	1.20	0.86	1.68	0.271	1.43	0.98		2.09	0.067	0.37	0.10	1.35	0.132
H. pylori-negative PUD
rs1940475	MMP-8	T	738	0.98	0.77	1.25	0.893	0.94	0.63		1.39	0.752	1.02	0.68	1.53	0.920
rs1799750	MMP-1	2G	721	0.91	0.71	1.17	0.459	0.90	0.61		1.33	0.596	0.86	0.56	1.32	0.483
rs679620	MMP-3	T	735	0.86	0.67	1.10	0.235	0.79	0.53		1.19	0.258	0.84	0.55	1.28	0.419
rs243865	MMP-2	T	729	0.94	0.70	1.26	0.696	0.97	0.67		1.39	0.849	0.80	0.39	1.67	0.558
rs3918242	MMP-9	T	731	0.83	0.59	1.18	0.296	0.80	0.53		1.20	0.276	0.83	0.28	2.46	0.739
rs3918249	MMP-9	C	733	1.00	0.78	1.30	0.975	1.15	0.80		1.66	0.448	0.77	0.46	1.30	0.323
rs17576	MMP-9	G	740	1.08	0.83	1.40	0.569	1.09	0.76		1.57	0.626	1.12	0.67	1.88	0.660
rs3787268	MMP-9	A	733	1.01	0.74	1.40	0.930	1.09	0.75		1.57	0.656	0.62	0.21	1.77	0.367
rs2250889	MMP-9	G	734	0.81	0.55	1.20	0.293	0.78	0.49		1.22	0.268	0.82	0.25	2.07	0.739
rs17577	MMP-9	A	724	0.80	0.57	1.14	0.217	0.81	0.54		1.22	0.310	0.53	0.17	1.65	0.270
All results were obtained after adjustment for covariates.
ОR, odds ratio.
95% CI, 95% confidence interval.
p values < 0.017 are shown in bold.

Haplotype AACG defined by rs17576-rs3787268-rs2250889-rs17577 was associated with PUD (OR_adj=0.17, p = 0.001, р_perm=0.003), haplotype CG defined by rs3918249-rs17576 of the MMP9 gene was associated with H. pylori-positive PUD (OR_adj=1.49, p = 0.004, р_perm=0.009) (Fig. 1). Thus, in total five polymorphisms of the MMP9 gene were associated with PUD (two individually and three within haplotypes).

Functional SNP

Non-synonymous SNPs. Among the PUD-associated SNPs, three polymorphisms (rs17576, rs2250889, and rs17577) were missense (Supplementary Table S2).

Regulatory effects. The data on the regulatory effects of the PUD-associated loci of the MMP9 gene are presented in Supplementary Table S3. According to the HaploReg database, three SNPs were located in evolutionarily conserved regions, all five polymorphisms - in the region of DNA binding with modified histone (H3K4me3, H3K9ac) marking promoters and hypersensitivity region to DNAse-1 in various tissues, four SNPs – in the region of DNA binding with modified histone (H3K4me1, H3K27ac) marking enhancers and two polymorphisms - in the protein-bound region. Importantly, the PUD-associated SNPs manifest their regulatory effects in the tissues and organs related to the pathogenesis of the disease (fetal stomach and small intestine, adult gastric and small intestine, adult stomach and duodenum mucosa, etc.).

In addition to the five PUD-associated SNPs, regulatory significance was estimated for 50 polymorphisms linked to them (Supplementary Table S3). Three synonymous SNPs were located in exons of the MMP9 gene, 28 SNPs were in 5'-UTR of the MMP9, ZNF335, and SLC12A5 genes, 19 were in introns. Ten loci were located in evolutionarily conserved regions. The in silico analysis of the linked SNPs suggested several polymorphisms with pronounced regulatory effects (Supplementary Table S3). For example, rs3848722 (was in linkage disequilibrium with SNPs rs3918249 and rs17576) is located in the hypersensitive region to DNAase-I (19 tissues), in the region of DNA binding with modified histone marking promoters and enhancers (5 and 14 tissues respectively), and a putative transcription factor binding sites (Pax-6, HNF4, ZID, NRSF).

Expression QTLs. In silico analysis for the eQTL impact of the PUD-associated SNPs shows their might affect the expression of 17 genes (MMP9, CD40, NTTIP1, NEURL2, PCIF1, PLTP, RP11-465L10.10, RP3-337O18.9, RPL13P2, SLC12A5, SNX21, SPATA25, SYS1, WFDC10B, WFDC3, ZNF335, ZSWIM1) in more than 20 tissues and organs (Supplementary Table S4). The PUD-associated loci were also in strong LD with the 48 SNPs affecting the expression of the above 17 genes in various organs and tissues (Supplementary Table S5).

Splicing QTLs. The PUD-associated SNPs had possess the potential impact on the genes alternative splicing and might sQTL affect for 4 genes (PLTP, ACOT8, SNX21, SLC12A5) (Supplementary Table S6). These loci were tightly linked to 48 polymorphisms affecting sQTL of the above four genes in more than 20 tissues and organs (Supplementary Table S7).

The present study reports for the first time the association of MMP-9 gene polymorphisms with PUD in Caucasians from Central Russia: allele G of SNPs rs17576 locus increased risk for PUD (OR_adj = 1.31) whereas haplotype AACG of rs17576-rs3787268-rs2250889-rs17577 decreased the risk (OR_adj=0.17). Also, allele C of rs3918249, allele G of the rs17576 and haplotype CG of rs3918249-rs17576 increased risk for the H. pylori-positive PUD (OR_adj = 1.82, OR_adj = 1.53–1.95 and OR_adj = 1.49 respectively). The PUD-associated loci appeared to possess a significant regulatory effects and influence the expression of 17 genes and alternative splicing of four genes.

One of the PUD-associated loci, rs17576, was previously shown as a candidate for H. pylori-positive gastric ulcer¹¹, peptic ulcer, and H. pylori-positive peptic ulcer⁹. However, the data about the risk alleles of this locus were contradictory, Specifically, Shaimardanova et al.⁹ reported allele G (i.e., the same as determined in the present study) as the risk factor for PUD and H. pylori-positive PUD in Tatars from the Bashkortostan Republic of Russia, whereas Hellmig et al.¹¹ determined allele A as the risk factor for H. pylori-positive gastric ulcer in Germans. On the other hand, Yeh Y.C. et al.¹⁰ did not find any association of rs17576 with either gastric or duodenal ulcer after H. pylori infection in Taiwanese. Okada R. et al.²⁴ ported the association of rs17576 MMP9 с gastric cancer both individually and within haplotype CAA rs3918242-rs17576-rs17577 of the MMP-9 gene.

The MMP-9 protein (gelatinase B) cleaves denatured collagen and plays a significant role in ECM modification²⁵. MMPs can be induced by both H. pylori bacterial products and proinflammatory cytokines²⁶. Overexpression of MMPs may result in extracellular matrix breakdown and tissue disintegration. Li et al.²² reported higher MMP-9 expression in the gastric mucosa at the boundary of the gastric ulcer. Significantly elevated expression of pro-MMP9 (about 12-fold) was documented in the indomethacin-induced gastric ulcer as compared to unaffected tissues. Ethanol produced an even stronger effect and increased pro-MMP-9 expression in rat gastric tissues up to 22-fold¹⁷. Overexpression of MMP-9 in indomethacin-induced gastric ulcer in mice correlated with up-regulation of activator protein-1 and preceded oxidative stress27.

During PUD, the gastric and duodenal mucosa is infiltrated by monocytes, lymphocytes, neutrophils, and plasma cells. Inflammatory cells produce multiple pro-inflammatory cytokines and growth factors (e.g., epidermal growth factor, transforming growth factor-β, platelet-derived growth factor, vascular endothelial growth factor, etc.)^2,22. Pro-inflammatory cytokines can elevate the expression of MMPs²⁶. Chronic inflammation precedes oxidative stress and increases the expression of MMP-9²².

We determined associations of the MMP-9 gene polymorphisms with H. pylori-positive PUD but did not find the association of any of the analyzed MMP genes with H. pylori-negative PUD. The polymorphisms of the MMP-9 gene may contribute to a complex genetic risk profile of PUD in chronic H. pylori infection^9,11. Our results are in agreement with the previous reports about more significant contribution of MMP-9 to the development of H. pylori-positive gastric ulcer and gastritis as compared to the other MMP genes^22,28−30. Li et al.²² showed that MMP-9 expression levels in the gastric mucosa were significantly elevated in H. pylori-positive gastric ulcer patients as compared to the H. pylori-negative ones and correlated with the histologically determined activity level and inflammation at the boundary of the ulcer. Epithelium of the H. pylori-induced gastric ulcer manifested higher MMP-9 expression than that of the NSAID-related gastric ulcer²⁸. Significantly higher serum levels of MMP-9 were determined in patients with H. pylori-positive gastritis as compared to H. pylori-negative controls²⁹. Antral mucosa of H. pylori-infected patients with gastritis demonstrated a 19-fold higher MMP-9 protein activity and 10-fold increase of MMP-9 gene expression of than that of uninfected individuals³⁰. Successful treatment of the H. pylori infection lowered the MMP-9 expression levels, whereas the elevated levels remain unchanged when the treatment failed³¹.

It should be noted that the current study is somewhat limited because only one ethnic population was analyzed. The well-known ethnic disparities in the prevalence of complex diseases warrant validation studies of the determined associations of the MMP genes and PUD in other ethnic populations.

Genetic variants of the gene are associated with PUD in a population of Central Russia. However, the data about possible role of the MMP genes polymorphic variants in the susceptibility to PUD in different ethnic populations remain inconsistent that warrants further studies to identify possible causative variants for the disease.

Study subjects

In total, 1145 participants of Russian origin and born in Central Russia^32,22, including 798 patients with PUD (434 with gastric ulcer and 364 with duodenal ulcer), and 347 controls, were recruited for the study. PUD and complications (if any) were determined on the basis of conventional clinical and endoscopic findings. The control group consisted of healthy individuals with no symptoms of gastrointestinal disease³⁴. They were not examined by endoscopy because, apart from ethical reasons, the chance of finding an active ulcer in patients without symptoms was very low³⁵. Patients and control group volunteers that used NSAIDs, corticosteroids, and aspirin for a long-term treatment were excluded.

The H. pylori infection in patients was diagnosed by detection of the pathogen in biopsies obtained during endoscopy. Among 798 patients with PUD, 404 were H. pylori-positive and 394 were H. pylori-negative. In the controls, the presence of H. pylori was diagnosed by the serological test using a commercial IgG ELISA kit (Plate Helicobacter IgG, Roche). Control group volunteers diagnosed with H. pylori infection were excluded from the study.

The study protocol was approved by the Medical Institution Ethics Committee of Belgorod State University. All participants signed an informed consent prior to enrolment in the study. All methods were performed in accordance with the relevant guidelines and regulations. The participants took the medical examination at the Department of Gastroenterology of St. Joasaph Belgorod Regional Clinical Hospital.

Isolation of DNA and genotyping

A blood sample (4–5 ml) was collected by venipuncture from all study participants in EDTA-coated tubes (Vacutainer®). Genomic DNA was isolated from the buffy coat using a standard phenol/chloroform procedure (as described earlier³⁶).

Ten SNPs of the MMP genes (rs1799750 MMP-1, rs243865 MMP-2, rs679620 MMP-3, rs1940475 MMP-8, rs3918242, rs3918249, rs3787268, rs2250889, rs17576, and rs17577 MMP-9) were selected for the analysis according to the following criteria^37,38: previously reported associations with digestive diseases (PUD, gastric cancer, etc.), regulatory potential, and MAF > 0.05.

All selected SNPs had significant regulatory potential as evidenced by the HaploReg online tools³⁹ (Supplementary Table S8); eight polymorphisms were associated with digestive diseases (PUD, gastric and esophageal cancer, digestive cancers, gastritis) (including two SNPs associated with PUD) in previously published candidate gene association studies (Supplementary Table S9). Two SNPs (rs3918249 and rs3787268 MMP-9) did not demonstrate a significant association with digestive diseases but had significant regulatory potential (according to HaploReg).

The polymorphisms were genotyped using the MALDI-TOF mass spectrometry iPLEX platform (Agena Bioscience Inc, San Diego, CA). The quality was controlled by genotyping of blind replicates⁴⁰. Regenotyping of 5% of the studied samples, selected on a random basis, showed 100% reproducibility of the original results.

Statistical analysis

The observed allele and genotype frequencies were assessed for correspondence to the Hardy-Weinberg equilibrium using the chi-square test. Associations of the SNPs with PUD were analyzed by logistic regression according to three main genetic models, additive, recessive, and dominant⁴¹. The regression analysis was adjusted for covariates: family history of peptic ulcer, alcohol and tobacco consumption, stress, the presence of cardiovascular pathology were used as qualitative variables (Table 1). The haplotype blocks were constructed for MMP-9 gene variants using the «Solid Spine» algorithm (D’>0.8) by HaploView program⁴². The logistic regression analyses and adaptive permutation test to adjust for multiple comparisons⁴³ were calculate by using the PLINK software⁴⁴. P_perm-value ≤ 0.017 was set to be statistically significant (after the Bonferroni correction based on the numbers of paired comparisons, n = 3: PUD – control, H. pylori-positive PUD - control, and H. pylori-negative PUD - control).

Functional SNPs

The polymorphisms associated with PUD and those strongly linked to them (r² ≥ 0.8) were analyzed for their functional significance (non-synonymous SNPs, regulatory potential, eQTLs, and sQTLs)⁴⁵. SNPs in strong linkage disequilibrium (LD) with the PUD-associated variants were identified using HaploReg³⁹. Non-synonymous SNPs and their functional predictions were analyzed using the SIFT online tool⁴⁶. The regulatory impact of the candidate MMP loci for PUD was evaluated by using HaploReg³⁹. The effects of the investigated SNPs on the mRNA levels and splicing QTLs was estimated using the GTEx project data⁴⁷ and the FDR ≤ 0.05 as the significance level. Likewise, eQTL and sQTL values of polymorphisms in strong LD (r² ≥ 0.8) with the PUD-associated loci were estimated⁴⁸.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Author contributions

OM, VD, MC substantial contributions to conception and design. OM, ER acquisition of data. IP, VD analysis and interpretation of data. OM, IP, drafting the article. ER, VD, MC revising it critically for important intellectual content. All authors final approval of the version to be published.

All authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Competing Interests

The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.

Narayanan, M., Reddy K. M., Marsicano E. Peptic ulcer disease and Helicobacter pyloriMo. Med. 115, 219–224 (2018).
Lanas, A., Chan, F. K. L. Peptic ulcer disease. Lancet. 390, 613–624. doi: 10.1016/S0140-6736(16)32404-7 (2017).
Søreide, K. et al. Perforated peptic ulcer. Lancet. 386, 1288–1298. doi: 10.1016/S0140-6736(15)00276-7 (2015).
Kuna, L. et al. Peptic Ulcer Disease: A Brief Review of Conventional Therapy and Herbal Treatment Options. Clin. Med. 8, 179. doi: 10.3390/jcm8020179 (2019).
Levenstein, S., Rosenstock, S., Jacobsen, R. K., Jorgensen, T. Psychological stress increases risk for peptic ulcer, regardless of Helicobacter pylori infection or use of nonsteroidal anti-inflammatory drugs. Gastroenterol. Hepatol. 13, 498–506.e1. doi: 10.1016/j.cgh.2014.07.052 (2015).
Huang, J. Q., Sridhar, S., Hunt, R. H. Role of helicobacter pylori infection and non-steroidal anti-inflammatory drugs in peptic-ulcer disease: A meta-analysis. Lancet. 359, 14–22. doi: 10.1016/S0140-6736(02)07273-2 (2002).
Datta, De D., Roychoudhury, S. To be or not to be: The host genetic factor and beyond in Helicobacter pylori mediated gastro-duodenal diseases. World J. Gastroenterol. 21, 2883–2895. doi: 10.3748/wjg.v21.i10.2883 (2015).
Charpignon, C. et al. Peptic ulcer disease: One in five is related to neither Helicobacter pylori nor aspirin/NSAID intake. Aliment. Ther. 38, 946–954. doi: 10.1111/apt.12465 (2013).
Shaymardanova, E. Kh. et al. Role of Allelic Genes of Matrix Metalloproteinases and Their Tissue Inhibitors in the Peptic Ulcer Disease Development. Genetika. 52, 364-375. Russian (2016 ).
Yeh, Y. C., Cheng, H. C., Chang, W. L., Yang, H. B., Sheu, B. S. Matrix metalloproteinase-3 promoter polymorphisms but not dupA-H. pylori correlate to duodenal ulcers in H. pylori-infected females. BMC Microbiol. 10, 218. doi: 10.1186/1471-2180-10-218 (2010).
Hellmig, S. et al. Genetic variants in matrix metalloproteinase genes are associated with development of gastric ulcer in H. Pylori infection. J. Gastroenterol. 101, 29–35 (2006).
Tarnawski, A. S., Ahluwalia, A. Molecular mechanisms of epithelial regeneration and neovascularization during healing of gastric and esophageal ulcers. Curr. Med. Chem. 19, 16–27 (2012).
Shahin, M. et al. Remodeling of extracellular matrix in gastric ulceration. Microsc. Res. Tech.53, 396–408 (2001).
Ganguly, K., Kundu, P., Banerjee, A., Reiter, R. J., Swarnakar, S. Hydrogen peroxide-mediated downregulation of matrix metalloprotease-2 in indomethacin-induced acute gastric ulceration is blocked by melatonin and other antioxidants. Free. Radic. Biol. Med. 41, 911–925 (2006).
Chakraborty, S. et al. The use of nano-quercetin to arrest mitochondrial damage and MMP-9 upregulation during prevention of gastric inflammation induced by ethanol in rat. Biomaterials. 33, 2991–3001 (2012).
Kim, S. J., Park, Y. S., Paik, H. D., Chang, H. I. Effect of anthocyanins on expression of matrix metalloproteinase-2 in naproxen-induced gastric ulcers. Br. J. Nutr. 106, 1792–1801 (2011).
Singh, L. P., Mishra, A., Saha, D., Swarnakar, S. Doxycycline blocks gastric ulcer by regulating matrix metalloproteinase-2 activity and oxidative stress. World J. Gastroenterol. 17, 3310–3321 (2011).
Pradeepkumar Singh, L., Vivek Sharma, A., Swarnakar, S. Upregulation of collagenase-1 and -3 in indomethacin-induced gastric ulcer in diabetic rats: role of melatonin. J. Pineal. Res. 51, 61–74 (2011).
Kim, S. J. et al. Antiulcer activity of anthocyanins from Rubus coreanus via association with regulation of the activity of matrix metalloproteinase-2. J. Agric. Food Chem. 59, 11786–11793 (2011).
Mori, N. et al. Helicobacter pylori induces matrix metalloproteinase-9 through activation of nuclear factor kappaB. Gastroenterology. 124, 983–992. doi: 10.1053/gast.2003.50152 (2003).
Crawford, H. C. et al. Helicobacter pylori strain-selective induction of matrix metalloproteinase-7 in vitro and within gastric mucosa. Gastroenterology. 125, 1125–1136. doi: 10.1016/S0016-5085(03)01206-X (2003).
Li, S. L. et al. Increased expression of matrix metalloproteinase-9 associated with gastric ulcer recurrence. World J. Gastroenterol. 19, 4590-4595. doi: 10.3748/wjg.v19.i28.4590 (2013).
Shan, Q. W. et al. Relationship between gene polymorphisms in MMP-9 and Helicobacter pylori-related upper gastrointestinal disease in children. Zhongguo Dang Dai Er Ke Za Zhi. 12, 262-266. (2010).
Okada, R. et al. Matrix metalloproteinase 9 gene polymorphisms are associated with a multiple family history of gastric cancer. Gastric Cancer. 20, 246-253. doi: 10.1007/s10120-016-0608-2 (2017).
Cui, N., Hu, M., Khalil, R. A. Biochemical and Biological Attributes of Matrix Metalloproteinases. Mol. Biol. Transl. Sci. 147, 1‐73. doi:10.1016/bs.pmbts.2017.02.005 (2017).
Bergin, P. J. et al. Gastric gelatinase B/matrix metalloproteinase-9 is rapidly increased in Helicobacter felis-induced gastritis. FEMS Immunol. Med. Microbiol. 52, 88-98. doi: 10.1111/j.1574-695X.2007.00349.x (2008 ).
Ganguly, K., Swarnakar, S. Chronic gastric ulceration causes matrix metalloproteinases-9 and -3 augmentation: alleviation by melatonin. Biochimie. 94, 2687–2698 (2012).
Cheng, H. C. et al. Expressions of MMPs and TIMP-1 in gastric ulcers may differentiate H. pylori-infected from NSAID-related ulcers. Scientific World Journal. 2012, 539316 (2012).
Rautelin, H. I. et al. Enhanced systemic matrix metalloproteinase response in Helicobacter pylori gastritis. Med. 41, 208–215 (2009).
Bergin, P. J. et al. Increased production of matrix metalloproteinases in Helicobacter pylori-associated human gastritis. Helicobacter. 9, 201-210. doi: 10.1111/j.1083-4389.2004.00232.x (2004 ).
Kubben, F. J. et al. Eradication of Helicobacter pylori infection favourably affects altered gastric mucosal MMP-9 levels. 12, 498–504 (2007).
Litovkina O. et al. Genes involved in the regulation of vascular homeostasis determine renal survival rate in patients with chronic glomerulonephritis. Gene. 546, 112-116. doi: 10.1016/j.gene.2014.04.020 (2014).
Reshetnikov, E. A. et al. The insertion-deletion polymorphism of the ACE gene is associated with increased blood pressure in women at the end of pregnancy. J. Renin Angiotensin Aldosterone Syst. 16, 623-632. doi: 10.1177/1470320313501217 (2015).
Minyaylo, O. N. Allele distribution and haploblock structure of matrix metalloproteinase gene polymorphism in patients with H. pylori-negative gastric ulcer and duodenal ulcer. Research Results in Biomedicine. 6, 488-502. Russian. DOI: 10.18413/2658-6533-2020-6-4-0-5 (2020).
García-González, M. A. et al. Association of interleukin 1 gene family polymorphisms with duodenal ulcer disease. Clin. Exp. Immunol. 134, 525–531 (2003).
Ponomarenko, I. et al. Candidate genes for age at menarche are associated with endometriosis. Reprod. Biomed. Online. 41, 943-956. doi:10.1016/j.rbmo.2020.04.016 (2020).
Starikova, D., Ponomarenko, I., Reshetnikov, E., Dvornyk, V., Churnosov, M. Novel data about association of the functionally significant polymorphisms of the MMP-9 gene with exfoliation glaucoma in the Caucasian population of Central Russia. Ophthalmic Res. preprint doi:10.1159/000512507 (2020).
Ponomarenko, I.V. et al. Association of genetic polymorphisms with age at menarche in Russian women. Gene. 686, 228-236 (2019).
Ward, L. D., Kellis, M. HaploReg v4: systematic mining of putative causal variants, cell types, regulators and target genes for human complex traits and disease. Nucleic Acids Res. D1, D877-D881 (2016).
Golovchenko, O. et al. Functionally significant polymorphisms of ESR1and PGR and risk of intrauterine growth restriction in population of Central Russia. Eur. J. Obstet. Gynecol. Reprod. Biol. 253, 52-57. doi:10.1016/j.ejogrb.2020.07.045 (2020).
Ponomarenko, I. et al. Candidate genes for age at menarche are associated with endometrial hyperplasia. Gene. 757, 144933. doi:10.1016/j.gene.2020.144933 (2020).
Barrett, J. C., Fry, B., Maller, J., Daly, M. J. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 21, 263-265. doi: 10.1093/bioinformatics/bth457 (2005).
Che, R., Jack, J. R., Motsinger-Reif, A. A., Brown, C. C. An adaptive permutation approach for genome-wide association study: evaluation and recommendations for use. BioData Min. 7, 9. doi:10.1186/1756-0381-7-9 (2014).
Purcell, S. et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am. J. Hum. Genet. 81, 559-575. doi: 10.1086/519795 (2007).
Ponomarenko, I. et al. Candidate genes for age at menarche are associated with uterine leiomyoma. Front. Genet. 11, 512940. doi: 10.3389/fgene.2020.512940 (2021).
Kumar, P., Henikoff, S., Ng, P. C. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat. Protoc. 7, 1073-1081 (2009).
The GTEx Consortium. Genetic effects on gene expression across human tissues. Nature. 550, 204–213 (2017).
Moskalenko, M., Ponomarenko, I., Reshetnikov, E., Dvornyk, V., Churnosov, M. Polymorphisms of the matrix metalloproteinase genes are associated with essential hypertension in a Caucasian population of Central Russia. Rep. 11, 5224. doi: 10.1038/s41598-021-84645-4 (2021).

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Functionally significant polymorphisms of the MMP-9 gene are associated with peptic ulcer disease in the Caucasian population of Central Russia

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