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Autophagy and inflammatory bowel disease: Association between variants of the autophagy-related IRGM gene and susceptibility to Crohn's disease

Digestive and Liver Disease, Volume 47, Issue 9, September 2015, Pages 744 - 750

Abstract

Background

Crohn's disease and ulcerative colitis are inflammatory bowel diseases involving a genetically determined inappropriate mucosal immune response towards luminal antigens, including resident bacterial flora. Recent studies identified susceptibility genes involved in autophagy.

Aims

We analyzed known autophagic loci (IRGM, ULK1 and AMBRA1) previously described as associated with inflammatory bowel diseases or with other autoimmune and/or inflammatory disorders in a sample of Italian inflammatory bowel diseases patients in order to confirm their possible involvement and relative contribution in the disease.

Methods

We performed a case–control association study, a sub-phenotype correlation and a haplotype analysis. The analysis included 263 Crohn's disease, 206 ulcerative colitis patients and 245 matched healthy controls. Five polymorphisms were genotyped by allelic discrimination assays.

Results

IRGM was the most strongly associated with Crohn's disease susceptibility [rs13361189: P = 0.011, OR = 1.66 [95% CI: (1.12–2.45)]; rs4958847: P = 0.05, OR = 1.43 [95% CI: (1–2.03)]. The SNP rs13361189 was also found to increase the risk of Crohn's disease clinical sub-phenotype (fibrostricturing behaviour, ileal disease, perianal disease, intestinal resection). These findings suggest that IRGM variants may modulate clinical characteristics of Crohn's disease.

Conclusions

Our study confirms IRGM rs13361189 and rs4958847 polymorphisms to be important for Crohn's disease susceptibility and phenotype modulation, in accordance with previous findings.

Keywords: Autophagy, Genetics, IBD.

1. Introduction

Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) of unknown aetiology characterized by a chronic relapsing course [1], [2], and [3]. Current evidence suggests that IBD pathogenesis involves a genetically determined inappropriate mucosal immune response towards luminal antigens, including the resident bacterial flora [4], [5], [6], [7], [8], and [9]. Environmental factors (i.e. appendectomy, smoking habits) also appear to be involved in the development of IBD. Identification of possible genetic factors contributing to the development of IBD will help decode the pathways involved in the pathogenesis of IBD.

Recent genome-wide association (GWA) studies and meta-analyses have been highly successful in identifying genes contributing to susceptibility of CD and/or UC. To date, the number of known risk loci has expanded to 163 [10] . Some of them are specific for the single disease, whereas others are shared between CD and UC. Approximately 30% of IBD-related genetic loci are shared [3] and [11]. These loci contain genes involved in different biological pathways such as innate pattern recognition, epithelial barrier homeostasis and maintenance of epithelial barrier integrity, lymphocyte differentiation and autophagy.

Autophagy represents a characteristic example of a genetically mediated pathway, which is abnormal in IBD [12] . This highly evolutionary conserved mechanism is a self-degrading process in which targeted cytoplasmic constituents are isolated from the rest of the cell within the autophagosome, which are then fused with lysosomes and degraded or recycled [13] .

Autophagy has several roles in innate and adaptive immunity including pattern recognition, receptor signalling, regulation of cell death, elimination of bacteria and viruses, and immune cell homeostasis [14] and [15]. Thus it is thought that defects in autophagy – through autophagy gene mutation and/or microbial antagonism – may result in IBD pathogenesis causing an impaired antibacterial response and so an ineffective control of bacterial infection and chronic inflammation.

The initial identification of a link between autophagy and IBD arose from discovery of the association between CD and two genes variants, ATG16L1 and IRGM. ATG16L1 encodes a protein that functions as a molecular scaffold, mediating protein–protein interactions necessary to autophagosome formation in response to both classical and pathogen-related autophagy stimuli [16] and [17]. IRGM gene encodes a GTP-binding protein with a key function in the innate immune response by regulating autophagy formation in response to intracellular pathogens [19], [20], [21], and [22]. Experiments in mice have shown that expression of the murine homologue LRG-47 is induced by IFNγ and stimulates macroautophagy to generate large autolysosomal organelles as a mechanism for elimination of intracellular organisms [23] and [24]. Human IRGM lacks the IFNγ response element but has been observed to play a similar role in clearing intracellular pathogens [25] .

Additional CD risk loci have since been identified in autophagy genes such as NOD2, VDR, ULK1 and LRKK. ULK1 is involved in the autophagy initiation [18] , VDR is involved in the detection of autophagic triggers [26] ; others orchestrate autophagosomal maturation (LRRK2) [27] . In addition, the autophagy-related gene DAP1 has been implicated in UC [28] . It is interesting to note that NOD2, the major gene associated with CD susceptibility, is also involved in autophagic triggers [29] and [30]. AMBRA1 is an autophagic gene regulating ULK1 stability and kinase activity, and a crucial factor for autophagy regulation in vertebrates [31] . In fact, in the autophagic process the ULK1-dependent AMBRA1 phosphorylation leads to the release of the BECLIN 1 core complex from dynein and the translocation of the core complex to the ER, where it can promote the autophagosome formation [32] .

Given the important role of autophagy in down regulating the growth of microorganisms [33] , genetic variations involved in autophagy may result in reduced pathogen clearance.

Due to the link between the innate immune pathway, the autophagy pathway and IBD, in the present study we aimed to evaluate the role of some of the genes involved in autophagy in IBD. The possible association with clinical characteristics of IBD was also investigated.

2. Materials and methods

2.1. Study population

The IBD population included 263 subjects with CD and 206 with UC, diagnosed according to current guidelines [34] and [35] under regular follow up at the tertiary referral IBD Centre of Tor Vergata University Rome, Italy, where patients were retrospectively enrolled. As controls, 245 healthy unrelated blood donors (53.4% females and 46.6% was males) were included in the analysis.

For each patient, actively followed in the study, a detailed demographic, familial, and clinical history was obtained. Patients with CD were assessed based on the Montreal classification including age at diagnosis, location and behaviour of disease. In patients with UC, anatomic location was also assessed following the Montreal classification: proctitis, left-sided UC and extensive UC (pancolitis) [36] . The study protocol was approved by the local ethics committee, and written informed consent was obtained from each patient.

2.2. DNA extraction and genotyping

DNA was extracted using a Qiagen blood DNA mini kit. A total of 5 Single Nucleotide Polymorphisms (SNPs) at or near genes involved in autophagy were selected for this study. The selected SNPs are the following: rs12303764 and rs3923716 (ULK1), rs13361189 and rs4958847 (IRGM) and rs11819869 (AMBRA1).

All polymorphisms were analyzed by allelic discrimination assay by TaqMan® technology, using MGB specific allelic probes and ABI PRISM 7500 FAST. Each assay was run with positive (sample previously confirmed by direct sequencing as heterozygous and/or variant homozygous) and negative controls.

2.3. Statistical analysis

The Hardy–Weinberg equilibrium was verified, in patients and control groups by the Pearson test, for all SNPs except for the rs3923716 SNP (ULK1) only in Crohn's patients.

Differences in allelic, genotypic and haplotypic frequencies between cases and controls and differences between genotypes distribution and the clinical patients characteristics were evaluated by Pearson χ2 test. Clinical characteristics considered included: CD localization, CD behaviour, UC extent, age at time of diagnosis of IBD and presence of extra intestinal manifestations (cutaneous, articular, ocular) in both CD and UC. Odds ratios (ORs) with 95% CI were calculated; ORs for genotypes were calculated considering together the heterozygotes and variant homozygotes genotypes. The cut off for statistical significance was P < 0.05.

Considering the results obtained through the previous analysis, a log-linear analysis (by a 3-way contingency table) was performed in order to assess whether the association of IRGM is dependent or independent from NOD2.

The case/control association analysis, the sub-phenotypes analysis and the log-linear analysis were performed by the SPSS program (SSPS Inc., IL, USA), while the analysis of haplotypes was performed by using the Arlequin software, version 3.5 [37] .

3. Results

3.1. Case/control association analysis

Clinical characteristics of the IBD patients are summarized in Table 1 . We analyzed five polymorphisms located in autophagic genes, (AMBRA1, ULK1 and IRGM) by allelic discrimination assay. Deviation from the Hardy–Weinberg equilibrium was detected for the rs3923716 SNP (ULK1) in Crohn's patients. In Table 2a and Table 2b, we reported the comparisons between genotypes and allele frequencies in cases and controls, for all studied polymorphisms. The relative statistical significance and ORs are also reported.

Table 1 Clinical characteristics of enrolled patients with Crohn's disease and ulcerative colitis.

  CD UC
N 263 206
Females (%) 139 (52.7) 87 (42.1)
Mean IBD duration (years) 16 ± 10 10 ± 8.6
Mean age at diagnosis of IBD (years) 30 ± 11 37.4 ± 15
Familial history of IBD (%) 55 (20.8) 29 (14.1)
CD localization
 Ileum (%) 156 (59.5) n.a
 Colon (%) 24 (9.1) n.a
 Ileo-colon (%) 75 (28.6) n.a
CD behaviour
 Fibrostricturing (%) 122 (46.4) n.a
 Penetrating (%) 65 (24.6) n.a
 Inflammatory (%) 76 (29.0) n.a
UC extent
 Proctitis (%) n.a 105 (50.8)
 Left-sided (%) n.a 32 (15.4)
 Pancolitis (%) n.a 68 (32.8)
Perianal disease (%) 82 (31.1) 0
Extraintestinal manifestations
 Total (%) 151 (57.5) 65 (31.6)
 Articular (%) 123 (46.8) 48 (23.5)
 Cutaneous (%) 45 (17.2) 25 (12.3)
 Ocular (%) 17 (6.3) 7 (3.6)
Appendectomy (%) 99 (37.7) 11 (5.2)
Current smokers (%) 82 (31) 27 (13)
Non-smokers (%) 121 (46) 129 (62.5)
Ex-smokers (%) 60 (23) 50 (24.5)

IBD, Inflammatory Bowel Disease; CD, Crohn's disease; UC, Ulcerative colitis; n.a., not applicable.

Table 2a Case–control association analysis in Crohn's disease patients.

Gene SNPs   N Genotypes a Alleles
        wt hz var P value b OR (95% CI) wt var P value OR (95% CI)
IRGM rs13361189 T>C CD 262 180 (68.7%) 75 (28.6%) 7 (2.7%) 0.009 1.78 (1.15–2.75) 435 (83%) 89 (17%) 0.011 1.66 (1.12–2.45)
Controls 191 152 (79.6%) 36 (18.8%) 3 (1.6%) 340 (89%) 42 (11%)
rs4958847 G>A CD 263 165 (62.7%) 86 (32.7%) 12 (4.6%) 0.09 1.41 (0.94–2.12) 416 (79.1%) 110 (20.9%) 0.05 1.43 (1–2.03)
Controls 179 126 (70.4%) 50 (27.9%) 3 (1.7%)     302 (84.4%) 56 (15.6%)    
 
AMBRA1 rs11819869 C>T CD 218 146 (67%) 68 (31.2%) 4 (1.8%) 0.28 0.79 (0.52–1.21) 360 (82.6%) 76 (17.4%) 0.26 0.81 (0.56–1.17)
Controls 167 103 (61.7%) 59 (35.3%) 5 (3%)     265 (79.3%) 69 (20.7%)    
 
ULK1 rs12303764 T>G CD 247 105 (42.5%) 110 (44.5%) 32 (13%) 0.7 0.93 (0.65–1.33) 320 (64.8%) 174 (35.2%) 1 1 (0.77–1.31)
Controls 245 100 (40.8%) 118 (48.2%) 27 (11%)     318 (64.9) 172 (35.1%)    
rs3923716 C>A CD 245 189 (77.1%) 48 (19.6%) 8 (3.3%) 0.6 0.89 (0.58–1.37) 426 (86.9%) 64 (13.1%) 0.89 0.98 (0.67–1.43)
Controls 225 169 (75.1%) 52 (23.1%) 4 (1.8%)     390 (86.7%) 60 (13.3%)    

a wt” indicates the homozygous genotype for the wild-type allele; “hz” indicates the heterozygous genotype; “var” indicates the homozygous genotype for the variant allele.

b Heterozygotes and variant homozygotes were considered together (1 df) in the comparisons between genotypes by Chi square test

CD, Crohn's disease.

Table 2b Case–control association analysis in ulcerative colitis patients.

Gene SNPs   N Genotypes a Alleles
        wt hz var P value b OR (95% CI) wt var P value OR (95% CI)
IRGM rs13361189 T>C UC 205 149 (72.7%) 52 (25.4%) 4 (1.9%) 0.11 1.46 (0.92–2.34) 350 (85.4%) 60 (14.6%) 0.13 1.39 (0.91–2.12)
Controls 191 152 (79.6%) 36 (18.8%) 3 (1.6%)     340 (89%) 42 (11%)    
rs4958847 G>A UC 186 126 (67.7%) 51 (27.4%) 9 (4.9%) 0.58 1.13 (0.73–1.77) 303 (81.5%) 69 (18.5%) 0.3 1.23 (0.83–1.81)
Controls 179 126 (70.4%) 50 (27.9%) 3 (1.7%)     302 (84.4%) 56 (15.6%)    
 
AMBRA1 rs11819869 C>T UC 206 133 (64.6%) 67 (32.5%) 6 (2.9%) 0.57 0.88 (0.58–1.35) 333 (80.8%) 79 (19.2%) 0.61 0.91 (0.64–1.31)
Controls 167 103 (61.7%) 59 (35.3%) 5 (3%)     265 (79.3%) 69 (20.7%)    
 
ULK1 rs12303764 T>G UC 206 85 (41.3%) 90 (43.7%) 31 (15%) 0.92 0.98 (0.67–1.43) 260 (63.1%) 152 (36.9%) 0.58 1.08 (0.82–1.42)
Controls 245 100 (40.8%) 118 (48.2%) 27 (11%)     318 (64.9%) 172 (35.1%)    
rs3923716 C>A UC 206 155 (75.2%) 46 (22.3%) 5 (2.5%) 1 0.99 (0.64–1.54) 356 (86.4%) 56 (13.6%) 0.92 1.02 (0.69–1.51)
Controls 225 169 (75.1%) 52 (23.1%) 4 (1.8%)     390 (86.7%) 60 (13.3%)    

a wt” indicates the homozygous genotype for the wild-type allele; “hz” indicates the heterozygous genotype; “var” indicates the homozygous genotype for the variant allele

b Heterozygotes and variant homozygotes were considered together (1 df) in the comparisons between genotypes by Chi square test

UC, ulcerative colitis.

The two analyzed IRGM SNPs were associated with CD susceptibility. In particular, the variant allele of the rs13361189 (T>C) showed a significantly risk effect (OR = 1.66 and P = 0.011) whereas the variant allele of the rs4958847 (G>A) showed only a borderline association with the disease (OR = 1.43 and P = 0.05). No statistically significant associations were observed between IRGM alleles and UC. However, in females we observed a borderline association between the variant allele of rs13361189 (T>C) SNP and susceptibility to UC (OR = 2.11, P = 0.04 for the genotypes and OR = 1.84, P = 0.06 for the alleles). Regarding the other genes, no significant association in CD nor UC was observed.

3.2. Sub-phenotype correlation

A sub-phenotype correlation analysis was performed by considering subgroups of CD and UC patients showing specific clinical characteristics vs controls. Significant correlations were observed between IRGM genotypes and the following CD clinical phenotypes: fibrostricturing behaviour, ileal localization, perianal disease and previous intestinal resection ( Table 3 ). In particular, a significantly increased risk of ileal localization of CD was observed for the rs13361189 SNP, with an OR equal to 2.09 (95%CI 1.28–3.41; P = 0.003), and for the rs4958847 SNP (OR = 1.64 [95% CI 1.04–2.60]; P = 0.03). The rs13361189 variant allele also increases the risk of fibrostricturing disease (OR = 2.1 [95% CI 1.25–3.55]; P = 0.005), perianal disease (OR = 1.91 [95% CI 1.05–3.46]; P = 0.03) and previous intestinal resection (OR = 1.93 [95% CI 1.17–3.17]; P = 0.009). Moreover, in CD, a correlation was also observed with the rs11819869 SNP in AMBRA1 gene: the variant allele of this SNP seems to reduce the risk of inflammatory behaviour (OR = 0.49 [95% CI 0.25–0.96]; P = 0.04), (Supplementary Table S1). No correlations were observed between the different SNPs and UC subgroups (Supplementary Table S2).

Table 3 Crohn's disease subgroups associated with IRGM gene variants.

SNP Sub-phenotype Genotypes P value (1df) OR (95% CI) Alleles P value (1df) OR (95% CI)
    TT TC + CC     T C    
Rs13361189 Fibrostricturing behaviour 74 (64.9%) 40 (35.1) 0.005 a 2.1 (1.25–3.55) 186 (81.6%) 42 (18.4%) 0.01 1.83 (1.15–2.91)
Ileal disease 95 (65.1%) 51 (34.9%) 0.003 a 2.09 (1.28–3.41) 237 (81.2%) 55 (18.8%) 0.004 1.88 (1.22–2.90)
Perianal disease 51 (67.1%) 25 (32.9%) 0.03 a 1.91 (1.05–3.46) 127 (83.6%) 25 (16.4%) 0.009 1.60 (0.93–2.72)
Intestinal resection 95 (66.9%) 47 (33.1%) 0.009 a 1.93 (1.17–3.17) 234 (82.4%) 50 (17.6%) 0.014 1.73 (1.11–2.70)
Controls 152 (79.6%) 39 (20.4%)     340 (89%) 42 (11%)    
SNP Sub-phenotype Genotypes P value (1df) OR (95% CI) Alleles P value (1df) OR (95% CI)
    GG GA + AA     G A    
Rs4958847 Ileal disease 87 (59.2%) 60 (40.8) 0.03 a 1.64 (1.04–2.60) 176 (72.1%) 68 (27.9%) 0.0003 2.08 (1.40–3.11)
Controls 126 (70.4%) 53 (29.6%)     302 (84.4%) 56 (15.6%)    

a Heterozygotes and variant homozygotes were considered together (1 df) in the comparisons between genotypes by Chi square test.

3.3. Haplotypes analysis

Given that the IRGM SNPs analyzed show a difference in the distribution of allelic and genotyping frequencies between CD patients and controls, we performed a detailed haplotype analysis investigating the role of IRGM haplotypes on CD susceptibility. A significant difference was observed between CD patients and controls (P = 0.04, Table 4 ). The TG haplotype, characterized by the presence of the rs13361189 and rs4958847 wild type alleles, showed a protective effect respect to CD (OR = 0.68, 95% CI 0.47–0.96; P = 0.03) while the CA haplotype, carrying the rs13361189 and rs4958847 variant alleles, showed an increased risk to develop CD (OR = 1.56, 95% CI 1.04–2.34; P = 0.03). As shown in Table 4 , these data were confirmed also by comparison of haplotypes in the subgroup of patients with ileal localization and fibrostricturing behaviour (P = 0.008 and OR = 0.59, P = 0.068 and OR = 0.67 for the TG haplotype respectively; P = 0.012 and OR = 1.76, P = 0.058 and OR = 1.59 for the CA haplotype respectively).

Table 4 Comparison of IRGM haplotypes distribution between: Crohn's disease patients vs. controls; Crohn's disease patients with ileal localization vs. controls; Crohn's disease patients with fibrostricturing behaviour vs. controls.

Haplotype CD Controls P value (3df) Comparisons between each haplotype versus others
        P value OR (95% CI)
TG 409 (57.7%) 300 (42.3%) 0.04 0.03 0.68 (0.47–0.96)
TA 25 (59.5%) 17 (40.5%) 1 1 (0.53–1.89)
CG 4 (100%) 0 (0%) 0.15 ND
CA 84 (68.3%) 39 (31.7%) 0.03 1.56 (1.04–2.34)
Haplotype Ileal CD Controls P value (3df) Comparisons between each haplotype versus others
        P value OR (95% CI)
TG 222 (42.5%) 300 (57.5%) 0.015 0.008 0.59 (0.40–0.88)
TA 15 (46.9%) 17 (53.1%) 0.84 1.08 (0.53–2.20)
CG 3 (100%) 0 (0%) 0.03 ND
CA 52 (57.1%) 39 (42.9%) 0.012 1.76 (1.13–2.76)
Haplotype Fibrostricturing CD Controls P value (3df) Comparisons between each haplotype versus others
        P value OR (95% CI)
TG 177 (37.1%) 300 (62.9%) 0.015 0.068 0.67 (0.44–1.03)
TA 8 (32%) 17 (68%) 0.48 0.73 (0.31–1.72)
CG 4 (100%) 0 (0%) 0.022 ND
CA 37 (48.7%) 39 (51.3%) 0.058 1.59 (0.98–2.58)

CD, Crohn's disease.

The haplotypes correspond to the following order of SNPs: rs13361189 T>C–rs4958847 G>A.

However, these findings suggest that specific allele combinations do not increase the risk of CD respect to the single variant allele.

3.4. IRGM–NOD2 interaction

Since NOD2 gene is considered the major genetic determinant for CD risk, possible interactions between IRGM SNPs and the three major NOD2 SNPs in determining CD risk were searched by log-linear model. Given the incomplete linkage disequilibrium between the two IRGM SNPs analyzed (r2 = 65%) we performed a log-linear interaction considering only the most strongly associated IRGM SNPs.

Table 5 shows the 3 way-contingency table with the combined distribution of the rs13361189 IRGM SNP and the compound NOD2 genotype (rs2066844, rs2066845 and rs2066847) in CD subjects and in controls. The log-linear analysis shows a significant interaction among the three factors (P = 0.0001), between IRGM and the disease (P = 0.02) and between NOD2 and the disease (P < 0.0001).

Table 5 Interaction among rs13361189 (IRGM), the three SNPs rs2066844, rs2066845 and rs2066847 compound NOD2 genotype, and Crohn's disease/control status: three way contingency table analysis by log-linear model.

CD case/controls   Compound NOD2 genotype
    0 1 2
    Cases Controls Cases Controls Cases Controls
rs13361189 IRGM TT 135 (55.3%) 109 (44.7%) 28 (75.7%) 9 (24.3%) 12 (100%) 0 (0%)
TC 48 (68.6%) 22 (31.4%) 20 (87%) 3 (13%) 3 (100%) 0 (0%)
CC 6 (75%) 2 (25%) 1 (100%) 0 (0%) 0 (0%) 0 (0%)

CD, Crohn's disease.

XYZ interaction P = 0.0001; XZ interaction P = 0.02 and YZ interaction P < 0.0001.

X = rs2066844 + rs2066845 + rs2066847, compound NOD2 genotype; Y = rs13361189 IRGM SNP; Z = cases/controls.

0 = wild-type for all three NOD2 SNPs; 1 = hz heterozygous for at least one of the three NOD2 SNPs; 2= heterozygous for at least two of the three NOD2 SNPs.

4. Discussion

GWA studies have opened a window into the complex biology of IBD, revealing which specific genes in known adaptive and innate immune system pathways are involved in IBD pathogenesis. GWA studies also identified entirely unexpected paths to disease, such as autophagy.

Autophagy has a central function in physiological and pathological processes, being involved in innate and adaptive immunity by delivering intracellular pathogens and other antigens. Different genes involved in this process, such as ATG16L1, IRGM, ULK1 and NOD2, were already found to be significantly associated with IBD, especially with CD susceptibility [16], [17], [18], [19], [20], [29], and [30].

Based on these observations, we performed a replication study on the Italian population to test the association of autophagic polymorphisms with CD/UC and with particular clinical phenotypes.

Our study showed significant association of a key autophagy gene – the IRGM gene – with CD susceptibility. Particularly, from the case/control association analysis we found that the variant allele of the IRGM SNP rs13361189 was significantly associated with CD susceptibility, whereas the variant allele of the rs4958847 showed only a borderline association with the disease ( Table 2 ). However, by considering the different CD subgroups and the haplotypes analysis, present findings support that both polymorphisms are significantly associated with CD subgroups, thus suggesting that these two polymorphisms may modulate clinical characteristics of CD.

Present observations are largely consistent with previous findings. Several studies in various ethnic cohorts, indeed already reported the association of this two noncoding IRGM SNPs (rs13361189 and rs4958847) with CD in various ethnic cohorts [2], [20], [27], [38], [39], [40], [41], and [42].

These results are consistent with the IRGM gene function. In fact, IRGM gene encodes a GTP-binding protein, which has a key function in the innate immune response by regulating autophagy formation in response to intracellular pathogens [22] and [43]. Thus, reduced function and/or activity of this gene could lead to the persistence of intracellular bacteria, resulting in tissue damage and chronic intestinal inflammation [44] and [45]. McCarroll et al. have indeed shown that the most significant CD-associated SNP (rs13361189) is in perfect linkage disequilibrium with a 20 kb deletion polymorphism upstream of IRGM, which is considered the causal variant responsible for IRGM association with CD [42] . McCarroll et al. reported that the deletion (CD risk) and CD protective haplotypes differ in their ability to activate IRGM expression, thus modulating the cellular autophagy of internalized bacteria. Moreover, interestingly, Brest et al. recently showed that a synonymous variant in IRGM (the rs10065172), in perfect linkage disequilibrium with the 20 kb deletion polymorphism upstream of IRGM and with the rs13361189, alters a binding site for mir196 and causes deregulation of IRGM-dependent xenophagy in CD [46] . These results suggest that the CD association of IRGM arises from an alteration in IRGM regulation affecting the efficacy of autophagy.

In the present study, no association was observed with other genes, not even for the ULK1 gene, an excellent candidate for CD due to its role in the initial stage of autophagy. Located on chromosome 12q24.3, ULK1 is a serine/threonine kinase, relevant for recruitment and membrane trafficking of Atg proteins and regulatory molecules and subsequent autophagosome formation [47] . Therefore, similarly to IRGM, reduced function and/or activity of the ULK1 gene could lead to the persistence of intracellular bacteria, resulting in tissue damage and chronic intestinal inflammation. Two studies have already provided evidence for the involvement of ULK1 in the pathogenesis and phenotypes of CD [18] and [47]. However, in our study we failed to replicate this association. This lack of association may be related both to the SNPs selection and to the limited sample size of our study.

In contrast, for UC, no significant association was found for the studied SNPs. Nevertheless, for the IRGM gene, a borderline association in females between the variant allele of rs13361189 SNP and susceptibility to UC (OR = 2.11, P = 0.04 for the genotypes and OR = 1.84, P = 0.06 for the alleles) was observed. This is in line with the literature data describing autophagy genes mostly involved with CD and not with UC [16], [17], [18], [19], [20], [29], and [30].

Finally, the significant statistical interaction among the IRGM studied SNPs and the three major NOD2 SNPs in determining CD susceptibility (3 way log-linear analysis) could also suggest a possible epistatic relationship between the two genes determining the development of CD. The nature of this possible interaction needs to be confirmed and verified at functional level.

It is becoming increasingly evident that variants in the autophagy genes influence the antimicrobial seroreactivity in CD. A number of studies have suggested a positive association between NOD2 or IRGM and cumulative seroreactivity against a panel of anti-microbial antibodies including anti-I2, anti-flagellin, anti-ompC and especially ASCA [48], [49], [50], [51], [52], and [53]. Furthermore, CD patients with a higher antibody levels and stronger antibody responses had a greater frequency of ileal involvement, a higher risk for fibrostenosing/perforating disease and had the need for abdominal surgery [54] .

In conclusion, present findings confirm IRGM rs13361189 and rs4958847 polymorphisms as important markers for CD susceptibility and phenotype modulation. Additional independent multicentre studies including a higher number of IBD patients from different study populations are however required in order to confirm the reported observations. The possible contribution of tested genes to IBD susceptibility and their interactions is relevant for exploring possible therapeutic options in the autophagy pathway.

Conflict of interest

None declared.

Appendix A. Supplementary data

The following are the supplementary data to this article:

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Supplementary Table S1 Crohn's disease: non-statistically significant comparisons between each phenotype vs controls.

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Supplementary Table S2 Ulcerative colitis: non-statistically significant comparisons between each phenotype vs controls.

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Footnotes

a Department of Biomedicine and Prevention, Genetics Section, University of Rome “Tor Vergata”, Italy

b Gastroenterology Unit, Department of Systems Medicine, University of Rome “Tor Vergata”, Rome, Italy

Corresponding author at: Department of Biomedicine and Prevention, Genetics Section, University of Rome “Tor Vergata”, Via Montpellier, 1, 00133 Rome, Italy. Tel.: +39 06 72596090; fax: +39 06 20427313.