Abstract
The present study was performed to determine what proportion of people in Sardinia with or without Crohn's disease were infected with Mycobacterium avium subspecies paratuberculosis and had a preponderance of allelic variants of Nod2, an intracellular protein involved in Crohn's disease susceptibility. Genetic analysis of the alleles of the NOD2/CARD15 gene (insC3020, G908R, and R702W alleles), linked to susceptibility or genetic predisposition to Crohn's disease in humans, was carried out on specimens from 37 Crohn's disease patients and 34 patients without Crohn's disease. Our results show that more than 70 percent of people in Sardinia with Crohn's disease carry at least one of the susceptibility-associated NOD2/CARD15 alleles and were infected with Mycobacterium avium subspecies paratuberculosis.
Recent genetic and epidemiological studies provide suggestive evidence for the presence of genetic determinants of susceptibility to Crohn's disease (CD) and its clinical progression. In a recent report, Behr and colleagues documented the coexistence of Mycobacterium avium subsp. paratuberculosis infection and a permissive NOD2/CARD15 mutant in one individual with CD (1). CD is a disease of unknown etiology linked to immune dysregulation (10). An increasing amount of evidence supports the idea that CD is caused by Mycobacterium avium subsp. paratuberculosis (8, 11, 12). In 2001, three independent mutations within the NOD2/CARD15 gene were discovered to be strongly linked to CD in Europeans (3, 4, 9). According to the observation of Behr et al., the presence of a gene that is associated with an increased susceptibility to develop CD does not preclude the possibility that the disease may be infectious in etiology (1). It is possible that genetically identifiable subpopulations may have different tendencies to develop CD when exposed to the same infectious agent (3, 6). The following case study suggests an exploitation of these considerations in a group of Mediterranean patients with CD.
Endoscopic mucosal biopsy specimens were obtained from 37 patients with CD and 34 individuals without it who were attending the Clinic of Surgery, University of Sassari, for ileocolonoscopy. Biopsy specimens were taken both from the visibly inflamed regions in the patients with inflammatory bowel disease and from several regions throughout the intestines of control subjects. Informed consent for sampling and publication of the results was obtained. Biopsy specimens were examined for the presence of Mycobacterium avium subsp. paratuberculosis and the carriage of NOD2/CARD15 mutations. All the individuals examined were living in Sardinia and were of Sardinian origin. Tissue samples were placed into liquid 7H9 medium, transported directly to the laboratory, and processed within 1 h. One biopsy specimen was used to make a smear and examined by Ziehl-Neelsen and rhodamine-auramine staining as previously described (12).
Samples for PCR analysis for bacterial presence were processed as previously described (7). Primers p89 and p92 described in our previous work (11, 12) were used to amplify a 284-bp fragment specific for Mycobacterium avium subsp. paratuberculosis. The reaction mixture (final volume of 50 μl) comprised primers at a concentration of 0.5 μM, Expand High Fidelity reaction buffer (1×), 200 μM (each) dNTPs, and 3 U of Expand High-Fidelity Taq polymerase (Expand High-fidelity PCR system; Roche, Lewes, United Kingdom). Cycling conditions were 1 cycle of 94°C for 3 min and 36 cycles of 94°C for 40 s, 62°C for 40 s, and 72°C for 40 s, followed by a final step of 72°C for 5 min. Amplified fragments were visualized with ethidium bromide on 2.5% agarose-1000 gel (Life Technologies, Grand Island, NY) and purified with a QIAquick gel extraction kit (QIAGEN, Crawley, United Kingdom). Each amplicon was then sequenced in both directions by using p89 and p92 primers, and the sequences obtained had confirmed 100% homology to the IS900 locus (GenBank accession no. AF416985).
Mycobacterium avium subsp. paratuberculosis DNA was detected in 25 CD patients (67.6%) and in 7 controls (20.6%) (Table 1). The results indicated that the presence of Mycobacterium avium subsp. paratuberculosis is significantly associated with CD (P = 0.001; odds ratio [OR], 4.94 [95% confidence interval {CI95 }, 1.85 to 13.29]).
TABLE 1.
Patient detailsa
| Patient | Sex | Disease or group | PCR result (presence of M. avium subsp. paratuberculosis) | Genetic analysis result for indicated allele
|
Patient | Sex | Disease or group | PCR result (presence of M. avium subsp. paratuberculosis) | Genetic analysis result for indicated allele
|
|||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| insC3020 | R702W | G908R | insC3020 | R702W | G908R | |||||||||
| 4 | F | Crohn's disease | Pos | WT | WT | WT | ||||||||
| 6 | F | Crohn's disease | Pos | HET | WT | WT | ||||||||
| 9 | M | Crohn's disease | Pos | WT | WT | WT | ||||||||
| 10 | M | Crohn's disease | Pos | WT | HET | WT | ||||||||
| 15 | M | Crohn's disease | Pos | HET | WT | HET | ||||||||
| 16 | M | Crohn's disease | Pos | WT | WT | WT | ||||||||
| 19 | F | Crohn's disease | Pos | WT | HET | WT | ||||||||
| 24 | F | Crohn's disease | Pos | WT | WT | WT | ||||||||
| 25 | F | Crohn's disease | Pos | HOM | WT | HOM | ||||||||
| 28 | F | Crohn's disease | Pos | WT | WT | WT | ||||||||
| 29 | M | Crohn's disease | Neg | WT | WT | WT | ||||||||
| 55 | F | Crohn's disease | Pos | WT | HET | HET | ||||||||
| 62 | M | Crohn's disease | Neg | HET | WT | WT | ||||||||
| 63 | M | Crohn's disease | Pos | WT | WT | WT | ||||||||
| 66 | F | Crohn's disease | Pos | WT | WT | WT | ||||||||
| 67 | M | Crohn's disease | Pos | WT | WT | HET | ||||||||
| 69 | F | Crohn's disease | Neg | WT | WT | WT | ||||||||
| 72 | M | Crohn's disease | Neg | WT | WT | WT | ||||||||
| 74 | M | Crohn's disease | Pos | HET | HET | WT | ||||||||
| 78 | F | Crohn's disease | Pos | WT | WT | WT | ||||||||
| 79 | F | Crohn's disease | Neg | WT | WT | HET | ||||||||
| 81 | M | Crohn's disease | Neg | WT | WT | WT | ||||||||
| 82 | F | Crohn's disease | Pos | HET | WT | WT | ||||||||
| 83 | M | Crohn's disease | Pos | WT | WT | WT | ||||||||
| 91 | F | Crohn's disease | Neg | WT | WT | WT | ||||||||
| 107 | M | Crohn's disease | Neg | WT | WT | WT | ||||||||
| 110 | F | Crohn's disease | Neg | WT | WT | HET | ||||||||
| 111 | F | Crohn's disease | Pos | WT | WT | WT | ||||||||
| 113 | F | Crohn's disease | Pos | HET | WT | WT | ||||||||
| 117 | F | Crohn's disease | Pos | WT | WT | WT | ||||||||
| 120 | F | Crohn's disease | Neg | HET | WT | WT | ||||||||
| 125 | M | Crohn's disease | Neg | WT | HET | WT | ||||||||
| 127 | F | Crohn's disease | Neg | WT | WT | WT | ||||||||
| 130 | F | Crohn's disease | Pos | HET | HET | WT | ||||||||
| 132 | F | Crohn's disease | Pos | WT | HET | WT | ||||||||
| 133 | F | Crohn's disease | Pos | HET | WT | WT | ||||||||
| 135 | R | Crohn's disease | Pos | WT | HET | WT | ||||||||
| 5 | M | Control | Pos | WT | HET | WT | ||||||||
| 26 | F | Control | Neg | WT | WT | WT | ||||||||
| 54 | M | Control | Pos | WT | WT | WT | ||||||||
| 56 | M | Control | Pos | WT | WT | WT | ||||||||
| 57 | M | Control | Neg | WT | WT | WT | ||||||||
| 58 | M | Control | Neg | HET | WT | WT | ||||||||
| 59 | M | Control | Neg | WT | WT | WT | ||||||||
| 60 | F | Control | Neg | WT | WT | WT | ||||||||
| 64 | F | Control | Neg | WT | WT | WT | ||||||||
| 65 | F | Control | Neg | WT | WT | WT | ||||||||
| 68 | F | Control | Neg | WT | WT | WT | ||||||||
| 70 | M | Control | Neg | WT | WT | WT | ||||||||
| 71 | M | Control | Neg | WT | WT | WT | ||||||||
| 73 | M | Control | Neg | WT | WT | HET | ||||||||
| 75 | M | Control | Neg | WT | WT | WT | ||||||||
| 77 | F | Control | Neg | WT | WT | WT | ||||||||
| 84 | M | Control | Neg | WT | WT | WT | ||||||||
| 85 | F | Control | Neg | WT | WT | WT | ||||||||
| 87 | F | Control | Neg | WT | WT | WT | ||||||||
| 88 | F | Control | Neg | HET | WT | WT | ||||||||
| 89 | F | Control | Neg | WT | WT | HET | ||||||||
| 93 | F | Control | Neg | WT | WT | WT | ||||||||
| 103 | F | Control | Neg | WT | WT | WT | ||||||||
| 105 | M | Control | Neg | WT | WT | WT | ||||||||
| 108 | M | Control | Neg | WT | WT | WT | ||||||||
| 114 | F | Control | Pos | WT | WT | WT | ||||||||
| 116 | M | Control | Pos | WT | WT | WT | ||||||||
| 118 | M | Control | Pos | WT | WT | WT | ||||||||
| 128 | F | Control | Neg | WT | WT | WT | ||||||||
| 131 | F | Control | Pos | HET | WT | WT | ||||||||
| 137 | M | Control | Neg | WT | WT | WT | ||||||||
| 138 | F | Control | Neg | WT | HET | WT | ||||||||
| 139 | F | Control | Neg | WT | WT | WT | ||||||||
| 140 | F | Control | Neg | WT | WT | WT | ||||||||
Abbreviations: M, male; F, female. Results of genetic analysis of the alleles of the NOD2/CARD15 gene are reported as wild type (WT), heterozygous for the mutation (HET), and homozygous for the mutation (HOM). Results of PCR for M. avium subsp. paratuberculosis DNA are shown as positive (Pos) or negative (Neg).
Because of the recently published data showing genetic susceptibility to CD, we also performed genetic testing, aimed at susceptibility-linked alleles of the NOD2/CARD15 gene (insC3020, G908R, and R702W alleles), as previously described (2). Our results indicated that 19 (51.35%) of the CD patients were carriers of at least one of the susceptibility-associated NOD2/CARD15 alleles (10 carried the insC3020 mutation, 8 the R702W mutation, and 6 the G908R mutation), whereas only 7 (20.6%) of the healthy controls carried at least one of the mutated NOD2/CARD15 alleles. A significant association between the carriage of at least one NOD2/CARD15 mutation and development of CD was observed (P = 0.007; OR = 4.07 [CI95, 1.42 to 11.66]). The results confirm for the first time the associations of NOD2/CARD15 mutations and CD in the Sardinian population.
Among the CD patients who carried at least one of the susceptibility-associated NOD2/CARD15 alleles, 14 (73.7%) had evidence for the presence of Mycobacterium avium subsp. paratuberculosis DNA. This association was marginally significant (P < 0.04; OR = 1.7 [CI95, 0.92 to 3.2]) in our study group. Since several findings indicate that the presence of certain bacteria in the face of permissive NOD2/CARD15 mutations is necessary for development of CD (5, 7), it is possible that in individuals with NOD2/CARD15 mutations, the innate response to mycobacterial exposure would be inadequate, permitting establishment of a persistent mycobacterial infection. Chronic infection could then activate the inflammatory response which is characteristic of CD..
Collectively, our findings support and substantiate the suggestions of Behr et al. that these cases may illustrate a potential conceptual approach to CD etiology, mostly based on tandem searches for bacterial trigger and host susceptibility (1). However, further studies involving a larger number of cases and controls on a global scale are clearly needed to elucidate the mechanism behind the etiology of CD.
Acknowledgments
We thank The Sardinian Region for supporting the research.
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