Figures
Abstract
Background
Recent evidence suggests a crucial role of the endocannabinoid system, including the cannabinoid 1 receptor (CNR1), in intestinal inflammation. We therefore investigated the influence of the CNR1 1359 G/A (p.Thr453Thr; rs1049353) single nucleotide polymorphism (SNP) on disease susceptibility and phenotype in patients with ulcerative colitis (UC) and Crohn's disease (CD).
Methods
Genomic DNA from 579 phenotypically well-characterized individuals was analyzed for the CNR1 1359 G/A SNP. Amongst these were 166 patients with UC, 216 patients with CD, and 197 healthy controls.
Results
Compared to healthy controls, subjects A/A homozygous for the CNR1 1359 G/A SNP had a reduced risk to develop UC (p = 0.01, OR 0.30, 95% CI 0.12–0.78). The polymorphism did not modulate CD susceptibility, but carriers of the minor A allele had a lower body mass index than G/G wildtype carriers (p = 0.0005). In addition, homozygous carriers of the G allele were more likely to develop CD before 40 years of age (p = 5.9×10−7) than carriers of the A allele.
Citation: Storr M, Emmerdinger D, Diegelmann J, Pfennig S, Ochsenkühn T, Göke B, et al. (2010) The Cannabinoid 1 Receptor (CNR1) 1359 G/A Polymorphism Modulates Susceptibility to Ulcerative Colitis and the Phenotype in Crohn's Disease. PLoS ONE 5(2): e9453. https://doi.org/10.1371/journal.pone.0009453
Editor: Syed A. Aziz, Health Canada, Canada
Received: January 15, 2010; Accepted: January 27, 2010; Published: February 26, 2010
Copyright: © 2010 Storr et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: M. Storr is supported by grants from the Deutsche Forschungsgemeinschaft (DFG STO645/2-2), the Crohn's and Colitis Foundation of Canada (CCFC), and a grant from the University of Calgary (URGC-1011592). J. Diegelmann received a grant from the University of Munich (Promotionsstipendium). S. Brand is supported by grants from the DFG (BR 1912/5-1), the Else Kraner-Fresenius-Stiftung (Else Kraner Fresenius Memorial Stipendium 2005; P50/05/EKMS05/62), by the Ludwig-Demling Grant 2007 from DCCV e.V., and grants from the Ludwig-Maximilians University Munich (Excellence Initiative - Investment Fund 2008 and FaFoLe program). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Anecdotal reports suggest that marijuana- or tetrahydrocannabinol-containing products may be effective in alleviating symptoms in patients with ulcerative colitis (UC) and Crohn's disease (CD). [1], [2] This is supported by recent studies of our group and others suggesting that pharmacological activation of the cannabinoid 1 (CB1) receptor with selective receptor agonists decreases the inflammatory response in various murine models of colonic inflammation including dinitrobenzene sulphonic acid (DNBS)-, trinitrobenzene sulphonic acid (TNBS)- and dextran sodium sulfate (DSS)-induced colitis. [3]–[7] Interestingly, pharmacological blockade of CB1 receptors or genetic ablation of CB1 receptors (CNR1−/− mice) aggravates intestinal inflammation in these models, [3], [7] emphasizing the physiological relevance of the CB1 receptor in the protection against intestinal inflammation. Increased mucosal levels of the endocannabinoid anandamide during intestinal inflammation in humans further stress the role of the CB1 receptor and the endocannabinoid system in intestinal inflammation. [4] Thus, present knowledge suggests up-regulation of endocannabinoids as an important protective mechanism in intestinal inflammation.
The endocannabinoid system and the CB1 and CB2 receptors seem to be crucially involved in the regulation of multiple physiological functions, e.g. in the heart, where they relax coronary arteries and decrease cardiac work, [8] in organ perfusion, [9] in metabolic homeostasis, [10], [11] and in the regulation of bone mass by osteoclasts, [12] as well as in the protection against stress responses, inflammation, and associated repair mechanisms. [13], [14] Although recent evidence suggests that the endocannabinoid system is involved in many physiological and pathophysiological functions of the gastrointestinal tract such as intestinal motility, secretion, and intestinal inflammation [3], [15]–[20], the exact mechanisms underlying these findings are not yet known. It was recently suggested that CB1 signaling may be up-regulated during colitis, [3] but it is unknown whether this is a specific feature of the colitis model or a general response to intestinal inflammation.
Moreover, the role of the CB1 receptor in human inflammatory bowel disease (IBD) has not been clarified. Increased anandamide levels were found in mucosal biopsies from UC patients, suggesting a role of the endocannabinoid system in UC. [4] In contrast, the colonic expression of the endocannabinoid 2-acyl-glycerol (2-AG) is not increased in UC. [4] So far, however, no other studies analyzing the endocannabinoid system or the pharmacological effects of cannabinoids in human IBD have been published.
Gastrointestinal inflammation is likely the result of multiple factors, e.g., increased pro-inflammatory stimuli and reduced protective capability. The overall balance between pro- and anti-inflammatory mechanisms may determine the progression and severity of intestinal inflammation. [21], [22] Given the results of recent genome-wide association studies, [23] genetic susceptibility is an important factor contributing to IBD development. Moreover, knowledge of genetic susceptibility factors could provide important pathophysiologic insights for the generation of novel IBD therapeutics.
Considering our previous work on the endocannabinoid system in murine intestinal inflammation, [3], [6], [7], [24] we hypothesized that genetic variants in the CNR1 gene, which may modulate CB1 receptor function, could be associated with an increased susceptibility to IBD. To test our hypothesis, we genotyped a cohort of more than 550 individuals including 382 IBD patients and analyzed whether the 1359 G/A (p.Thr453Thr; rs1049353) single nucleotide polymorphism (SNP) within the CNR1 gene encoding the CB1 receptor modulates the susceptibility to CD and UC or results in a certain IBD phenotype. The selection of the CNR1 1359 G/A SNP was based on previous studies reporting that this polymorphism is associated with other disorders modulated by the endocannabinoid system such as alcohol dependence and hebephrenic schizophrenia. [25], [26]
Methods
Ethics Statement
The study was approved by the Ethics Committee of the Medical Faculty of the University of Munich. All participating subjects gave their written, informed consent prior to the genetic analysis.
Human Study Population
The study population comprised 579 individuals, including 216 patients with CD, 166 with UC, and 197 healthy, unrelated controls. Patients and controls were recruited at the IBD center of the Ludwig-Maximilians-University Munich, Campus Grosshadern, from September 2002 to December 2006. The diagnoses of CD and UC were established following clinical guidelines, using endoscopic, radiological, and histopathologic criteria. Table 1 shows the baseline characteristics of the study population. All 197 controls were unrelated, healthy individuals of Caucasian origin and sex-matched (by frequency) to the CD group. Controls were healthy blood donors without a history or family history of IBD. Demographics and routine clinical data (including location and behavior of IBD, disease-related complications, and prescription data of immunosuppressive and immunomodulatory therapy e.g., azathioprine, 6-mercaptopurine, methotrexate, infliximab) were recorded by retrospective analysis of the clinical charts by two independent investigators and an interview including a questionnaire at the time of enrollment. All data were collected blind to the CNR1 genotype. Patients with CD or UC were grouped according to age at diagnosis, disease localization, and behavior status of the Vienna classification, [27] and the recent modifications suggested by the Montreal classification. [28]
DNA Extraction and Genotyping of the CNR1 Polymorphism
Recently, a guanosine-to-adenine substitution at nucleotide position 1359 has been identified in the CNR1 gene (rs1049353) [25], [29]. Thus, three genotypes (GG, GA, AA) are possible. Genomic DNA was isolated from peripheral blood leukocytes by standard procedures using the DNA blood mini kit from Qiagen (Hilden, Germany). Genotyping was done as previously described [7], [30]. Briefly, a single 20-µl PCR was performed to genotype this site using approximately 200 ng of genomic DNA and 20 pmol each of the following primers 5′-GAAAGCTGCATCAAGAGCCC-3′ (forward) and 5′-TTTTCCTGTGCTGCCAGGG-3′ (reverse). Other conditions were as follows: 1.5 mM MgCl2, 400 µM of each dNTP, 1.25 U Taq polymerase, and 1× reaction buffer (Life Technologies, Rockville, MD). DNA amplification was performed with 40 cycles of 94°C, 60°C, and 72°C for 30 seconds each, preceded by a single cycle of 95°C for 15 minutes and followed by a single cycle of 72°C for 5 minutes. Five µl of the resulting 111 bp PCR product were then digested overnight with 10 U of Mspl (New England Biolabs, Beverly, MA) at 37°C. This resulted in fragments of 92 and 19 bp, when a G was present at nucleotide position 385, while the fragment remained uncut, when an A was present. Restriction digests were analyzed by electrophoresis of the digestion mixture in a 2% agarose gel stained with ethidium bromide. The assay was verified by sequencing the PCR product and the digested PCR fragments of all possible genotypes.
Results
The CNR1 1359 G/A (p.Thr453Thr) Polymorphism Modulates UC but Not CD Susceptibility
Given the above reported increased CB1 receptor expression in several models of intestinal inflammation and previous studies implicating the CNR1 1359 G/A (p.Thr453Thr) SNP in endocannabinoid-mediated diseases, [25], [31] we investigated whether this polymorphism modulates susceptibility and phenotype of CD and UC. The demographic characteristics of the IBD and control population analyzed are given in Table 1. The CD patients were classified with regard to their disease phenotype, considering disease location, the age at diagnosis, and disease behaviour by using the Montreal classification. [27], [28] The majority of patients had an onset of the disease in their mid20s (mean age at first diagnosis of CD: 28.1±11.4 years). The mean age at first diagnosis of UC was 31.8±13.7 years. 13.9% of the CD patients and 12.7% of the UC patients had a positive family history of IBD.
The results of the CNR1 p.Thr453Thr genotyping analysis in 216 CD patients, 166 UC patients, and 197 controls are shown in Table 2. The frequencies of heterozygous and homozygous carriers of this polymorphism did not differ significantly from the expected ratio according to the Hardy-Weinberg law. Patients with UC were less likely to be 1359 A/A homozygous (p = 0.01, OR 0.30, 95% CI 0.12–0.78). In contrast, this polymorphism did not influence susceptibility to CD (Table 2).
The CNR1 1359 G/A (p.Thr453Thr) Polymorphism Modulates Disease Onset and Body Mass Index in CD
In Table 3, we provide a detailed genotype-phenotype analysis of the CNR1 1359 G/A polymorphism in CD patients. Carriers of the 1359 A/A genotype were likely to have a lower body weight (p = 0.0005). In addition, homozygous carriers of the major G allele were more likely to develop CD before 40 years of age (p = 5.9×10−7) than carriers of the minor A allele. There was no association between the CNR1 p.Thr453Thr polymorphism and disease location, use of immunosuppressive drugs, family history of IBD, CD-related surgery, stenoses, and abscesses (Table 3). In addition, the CNR1 p.Thr453Thr polymorphism did not influence the UC phenotype (Table 4).
Discussion
We analyzed the effect of the CNR1 p.Thr453Thr polymorphism on IBD susceptibility and disease phenotype. This study was based on our previous results which suggested that CB1 receptor signaling is involved in defense mechanisms in response to acute intestinal inflammation in animal models. [3], [7] Based on this knowledge, we hypothesized that differential CB1 receptor expression, e.g., modulated by genetic factors, may contribute to IBD susceptibility. In the present study, we focused on the 1359 G/A polymorphism within the CNR1 gene encoding the CB1 receptor, given the importance of this nucleotide substitution in other endocannabinoid-mediated disorders such as alcohol dependence [25] and schizophrenia [32]. Although the CNR1 1359 G/A (p.Thr453Thr) SNP is a silent mutation, which does not result in an amino acid exchange, it might be associated with alterations e.g. in RNA splicing. [33]
Our study demonstrated an association with UC susceptibility but not with CD susceptibility. The prevalence of 1359 A/A homozygous carriers was 10.7% in the control population, 6.9% in CD patients, and only 3.6% in UC patients. The genotype frequencies found for our control population were within the values expected from Hardy-Weinberg equilibrium and were similar to a previous German control cohort. [29] However, given the limited size of the study population and the low prevalence of 1359 A/A homozygous carriers among UC patients, this finding has to be confirmed in larger replication cohorts. Furthermore, given that the study was primarily designed to detect differences in the frequency of IBD risk alleles, the control population was selected only regarding absence of IBD and other chronic diseases as well as being negative for a family history of IBD. Therefore a selection bias resulting in differences e.g. in BMI can not be excluded though it is intriguing that a lower BMI was found with CD and not with UC.
The human CNR1 gene is localized on chromosome 6q14–q15. [34] Interestingly, an earlier genome-wide family-based linkage study found an association of this region with celiac disease [35]. We recently demonstrated that celiac disease and UC (but not CD) share another common susceptibility locus on chromosome 4q27. [36] Although none of the recent genome-wide association studies demonstrated the CNR1 gene as a major IBD susceptibility gene, a previous genome scan in 260 IBD-affected relative pairs found nominal evidence for linkage of IBD to loci on chromosome 6q (lod = 2.21 between D6S2436/D6S305). [37]
We recently confirmed a number of CD susceptibility genes found in genome-wide associations studies such as NOD2, [38], [39] IL23R, [40] and ATG16L1 [41], but we also demonstrated differences in the genetic susceptibility to CD [42], suggesting that there are differences in the genetic susceptibility to IBD even between different Caucasian populations. In addition, other genetic associations such as those of TLR4 SNPs with CD susceptibility shown by our group [43] were not among the major CD susceptibility genes in a recent meta-analysis of genome-wide scans, although this gene has been confirmed as a CD susceptibility gene. [44]
Currently, it is unknown if the CNR1 1359 G/A (p.Thr453Thr) SNP modulates CB1 receptor expression or function. Particularly changes at amino acid positions 418–439 seem to be associated with a lack of receptor desensitization [45], and allelic variation in the CNR1 gene was suggested to be associated with a lower rather than a higher receptor activity, [46] but detailed studies investigating receptor activity based on different CNR1 genotypes are lacking. The assumption that allelic variation is associated with reduced CB1 activity would also explain the low BMI found in the 1359 A/A homozygous CD patients. Consistent with these data, the 1359 G/G wildtype genotype has been shown to be associated with an increased BMI and overweight in an Italian study of a healthy population. [47] A low BMI in CD patients is also considered to be an indicator of high disease activity. Therefore, 1359 A/A homozygosity could contribute to a more severe disease phenotype. This would be consistent with our results in CNR1−/− mice, demonstrating that these knockout mice take a more fulminate course in DNBS and DSS colitis. [3] However, functional experiments have to analyze if CNR1 signaling is indeed decreased in 1359 A/A homozygous patients.
Our findings add evidence that targeting the CB1 receptor system may modulate intestinal inflammation, suggesting this receptor as a potential target for future treatments. Similarly, animal models suggest that CB1 receptor activation with exogenous CB1 receptor agonists induces protection against intestinal inflammation. [3], [5] Therefore, the increased CB1 receptor expression seen in murine colitis models is likely an intrinsic protective mechanism to counter-regulate the deleterious effects of intestinal inflammation. The physiological importance of the CB1 receptor and the endocannabinoid system becomes obvious when endocannabinoid levels are increased by blocking their degradation. Under these circumstances, intestinal inflammation is reduced and the CB1 receptor is involved in this protection, emphasizing the important pathophysiological role of this system in intestinal inflammation. [7] Whether monitoring of CB1 receptor function or genotyping can identify responders of future treatments targeting the CB1 receptor remains speculative and has to be clarified in clinical trials.
In summary, we demonstrate that the CNR1 1359 G/A polymorphism modulates IBD susceptibility and phenotype. Specifically, we show that 1359 A/A homozygosity protects against UC and that CD patients carrying the minor A allele have a later disease onset and a lower BMI. These findings have to be confirmed in a larger replication study. Given the low prevalence of 1359 A/A homozygous carriers, this likely can be achieved only in a large multicenter trial. Nevertheless, our findings provide further evidence that endocannabinoids modulate intestinal inflammation, suggesting that this system could act as a target for future therapeutic interventions.
Author Contributions
Conceived and designed the experiments: MS JD TO BG PL SB. Performed the experiments: DE. Analyzed the data: MS DE JD SP PL SB. Contributed reagents/materials/analysis tools: MS TO BG PL. Wrote the paper: MS SB.
References
- 1. Grinspoon L, Bakalar JB (1995) Marihuana as medicine. A plea for reconsideration. JAMA 273: 1875–1876.
- 2. Grinspoon L (1999) The future of medical marijuana. Forsch Komplementärmed 6: Suppl 340–43.
- 3. Massa F, Marsicano G, Hermann H, Cannich A, Monory K, et al. (2004) The endogenous cannabinoid system protects against colonic inflammation. J Clin Invest 113: 1202–1209.
- 4. D'Argenio G, Valenti M, Scaglione G, Cosenza V, Sorrentini I, et al. (2006) Up-regulation of anandamide levels as an endogenous mechanism and a pharmacological strategy to limit colon inflammation. FASEB J 20: 568–570.
- 5. Kimball ES, Schneider CR, Wallace NH, Hornby PJ (2006) Agonists of cannabinoid receptor 1 and 2 inhibit experimental colitis induced by oil of mustard and by dextran sulfate sodium. Am J Physiol Gastrointest Liver Physiol 291: G364–G371.
- 6. Sibaev A, Massa F, Yuce B, Marsicano G, Lehr HA, et al. (2006) CB1 and TRPV1 receptors mediate protective effects on colonic electrophysiological properties in mice. J Mol Med 84: 513–520.
- 7. Storr M, Keenan CM, Emmerdinger D, Zhang H, Yuce B, et al. (2008) Targeting endocannabinoid degradation protects against experimental colitis in mice: involvement of CB1 and CB2 receptors. J Mol Med 86: 925–936.
- 8. Hiley CR (2009) Endocannabinoids and the Heart. J Cardiovasc Pharmacol.
- 9. Caraceni P, Pertosa A, Giannone F, Domenicali M, Grattagliano I, et al. (2009) Antagonism of the cannabinoid CB-1 receptor protects rat liver against ischemia-reperfusion injury complicated by endotoxemia. Gut 58: 1135–1143.
- 10. Sarzani R, Bordicchia M, Marcucci P, Bedetta S, Santini S, et al. (2009) Altered pattern of cannabinoid type 1 receptor expression in adipose tissue of dysmetabolic and overweight patients. Metabolism 58: 361–367.
- 11. Scheen AJ (2009) The endocannabinoid system: a promising target for the management of type 2 diabetes. Curr Protein Pept Sci 10: 56–74.
- 12. Idris AI, van 't Hof RJ, Greig IR, Ridge SA, Baker D, et al. (2005) Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors. Nat Med 11: 774–779.
- 13. Maresz K, Pryce G, Ponomarev ED, Marsicano G, Croxford JL, et al. (2007) Direct suppression of CNS autoimmune inflammation via the cannabinoid receptor CB1 on neurons and CB2 on autoreactive T cells. Nat Med 13: 492–497.
- 14. Teixeira-Clerc F, Julien B, Grenard P, Tran Van NJ, Deveaux V, et al. (2006) CB1 cannabinoid receptor antagonism: a new strategy for the treatment of liver fibrosis. Nat Med 12: 671–676.
- 15. De Petrocellis L, Cascio MG, Di Marzo V (2004) The endocannabinoid system: a general view and latest additions. Br J Pharmacol 141: 765–774.
- 16. Storr M, Yuce B, Goeke B (2006) Perspectives of cannabinoids in gastroenterology. Z Gastroenterol 44: 185–191.
- 17. Storr M, Yuce B, Andrews C, Sharkey KA (2008) The role of the endocannabinoid system in the pathophysiology and treatment of irritable bowel syndrome. Neurogastroenterol Motil 20: 857–868.
- 18. Izzo AA, Camilleri M (2008) Emerging role of cannabinoids in gastrointestinal and liver diseases: basic and clinical aspects. Gut 57: 1140–1155.
- 19. Di Carlo G, Izzo AA (2003) Cannabinoids for gastrointestinal diseases: potential therapeutic applications. Expert Opin Investig Drugs 12: 39–49.
- 20. Izzo AA, Camilleri M (2008) Emerging Role of Cannabinoids in Gastrointestinal and Liver Diseases: Basic and Clinical Aspects. Gut 57: 1140–1155.
- 21. Mayer EA, Collins SM (2002) Evolving pathophysiologic models of functional gastrointestinal disorders. Gastroenterology 122: 2032–2048.
- 22. Barbara G, De Giorgio R, Stanghellini V, Cremon C, Corinaldesi R (2002) A role for inflammation in irritable bowel syndrome? Gut 51: Suppl 1i41–i44.
- 23. Barrett JC, Hansoul S, Nicolae DL, Cho JH, Duerr RH, et al. (2008) Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease. Nat Genet 40: 955–962.
- 24. Storr MA, Keenan CM, Zhang H, Patel KD, Makriyannis A, et al. (2009) Activation of the cannabinoid 2 receptor (CB(2)) protects against experimental colitis. Inflamm Bowel Dis 15: 1678–1685.
- 25. Schmidt LG, Samochowiec J, Finckh U, Fiszer-Piosik E, Horodnicki J, et al. (2002) Association of a CB1 cannabinoid receptor gene (CNR1) polymorphism with severe alcohol dependence. Drug Alcohol Depend 65: 221–224.
- 26. Ujike H, Takaki M, Nakata K, Tanaka Y, Takeda T, et al. (2002) CNR1, central cannabinoid receptor gene, associated with susceptibility to hebephrenic schizophrenia. Mol Psychiatry 7: 515–518.
- 27. Gasche C, Scholmerich J, Brynskov J, D'Haens G, Hanauer SB, et al. (2000) A simple classification of Crohn's disease: report of the Working Party for the World Congresses of Gastroenterology, Vienna 1998. Inflamm Bowel Dis 6: 8–15.
- 28. Silverberg MS, Satsangi J, Ahmad T, Arnott ID, Bernstein CN, et al. (2005) Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: Report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol 19: Suppl A5–36.
- 29. Gadzicki D, Muller-Vahl K, Stuhrmann M (1999) A frequent polymorphism in the coding exon of the human cannabinoid receptor (CNR1) gene. Mol Cell Probes 13: 321–323.
- 30. Storr M, Emmerdinger D, Diegelmann J, Yuece B, Pfennig S, et al. (2008) The role of fatty acid hydrolase (FAAH) gene variants in inflammatory bowel disease. Aliment Pharmacol Ther 29: 542–551.
- 31. Horne AW, Phillips JA III, Kane N, Lourenco PC, McDonald SE, et al. (2008) CB1 expression is attenuated in Fallopian tube and decidua of women with ectopic pregnancy. PLoS ONE 3: e3969.
- 32. Leroy S, Griffon N, Bourdel MC, Olie JP, Poirier MF, et al. (2001) Schizophrenia and the cannabinoid receptor type 1 (CB1): association study using a single-base polymorphism in coding exon 1. Am J Med Genet 105: 749–752.
- 33. Komar AA (2007) Silent SNPs: impact on gene function and phenotype. Pharmacogenomics 8: 1075–1080.
- 34. Hoehe MR, Caenazzo L, Martinez MM, Hsieh WT, Modi WS, et al. (1991) Genetic and physical mapping of the human cannabinoid receptor gene to chromosome 6q14–q15. New Biol 3: 880–885.
- 35. King AL, Yiannakou JY, Brett PM, Curtis D, Morris MA, et al. (2000) A genome-wide family-based linkage study of coeliac disease. Ann Hum Genet 64: 479–490.
- 36. Glas J, Stallhofer J, Ripke S, Wetzke M, Pfennig S, et al. (2009) Novel genetic risk markers for ulcerative colitis in the chromosome 4q27 region harboring IL2/IL21 region are in epistasis with IL23R and suggest a common genetic background for ulcerative colitis and celiac disease. Am J Gastroenterol 104: 1737–1744.
- 37. Barmada MM, Brant SR, Nicolae DL, Achkar JP, Panhuysen CI, et al. (2004) A genome scan in 260 inflammatory bowel disease-affected relative pairs. Inflamm Bowel Dis 10: 513–520.
- 38. Schnitzler F, Brand S, Staudinger T, Pfennig S, Hofbauer K, et al. (2006) Eight novel CARD15 variants detected by DNA sequence analysis of the CARD15 gene in 111 patients with inflammatory bowel disease. Immunogenetics 58: 99–106.
- 39. Seiderer J, Schnitzler F, Brand S, Staudinger T, Pfennig S, et al. (2006) Homozygosity for the CARD15 frameshift mutation 1007fs is predictive of early onset of Crohn's disease with ileal stenosis, entero-enteral fistulas, and frequent need for surgical intervention with high risk of re-stenosis. Scand J Gastroenterol 41: 1421–1432.
- 40. Glas J, Seiderer J, Wetzke M, Konrad A, Torok HP, et al. (2007) rs1004819 is the main disease-associated IL23R variant in German Crohn's disease patients: combined analysis of IL23R, CARD15, and OCTN1/2 variants. PLoS ONE 2: e819.
- 41. Glas J, Konrad A, Schmechel S, Dambacher J, Seiderer J, et al. (2007) The ATG16L1 gene variants rs2241879 and rs2241880 (T300A) are strongly associated with susceptibility to Crohn's disease in the German population. Am J Gastroenterol 103: 682–691.
- 42. Glas J, Seiderer J, Pasciuto G, Tillack C, Diegelmann J, et al. (2009) rs224136 on chromosome 10q21.1 and variants in PHOX2B, NCF4, and FAM92B are not major genetic risk factors for susceptibility to Crohn's disease in the German population. Am J Gastroenterol 104: 665–672.
- 43. Brand S, Staudinger T, Schnitzler F, Pfennig S, Hofbauer K, et al. (2005) The role of Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms and CARD15/NOD2 mutations in the susceptibility and phenotype of Crohn's disease. Inflamm Bowel Dis 11: 645–652.
- 44. De Jager PL, Franchimont D, Waliszewska A, Bitton A, Cohen A, et al. (2007) The role of the Toll receptor pathway in susceptibility to inflammatory bowel diseases. Genes Immun 8: 387–397.
- 45. Jin W, Brown S, Roche JP, Hsieh C, Celver JP, et al. (1999) Distinct domains of the CB1 cannabinoid receptor mediate desensitization and internalization. J Neurosci 19: 3773–3780.
- 46. Siegfried Z, Kanyas K, Latzer Y, Karni O, Bloch M, et al. (2004) Association study of cannabinoid receptor gene (CNR1) alleles and anorexia nervosa: differences between restricting and binging/purging subtypes. Am J Med Genet B Neuropsychiatr Genet 125B: 126–130.
- 47. Gazzerro P, Caruso MG, Notarnicola M, Misciagna G, Guerra V, et al. (2007) Association between cannabinoid type-1 receptor polymorphism and body mass index in a southern Italian population. Int J Obes (Lond) 31: 908–912.