Deciphering the Role of CARD15/NOD2 in Inflammatory Diseases: Insights into Crohn's Disease and Blau Syndrome

Posted on January 2, 2024 • 7 minutes • 1456 words • Other languages: Русский

Table of contents

References

CARD15 /NOD2 is the most prominent gene linked to Inflammatory Bowel Disease (IBD). Its connection to Crohn’s Disease (CD) was simultaneously identified by two research groups and has since been further validated by numerous studies. Located on chromosome 16q12 , corresponding to the IBD1 locus, the CARD15/NOD2 gene exhibits around 60 polymorphisms, including 30 mutations leading to significant amino acid changes in the protein. Most mutations in this gene are unique, with over 80% of the altered chromosomes displaying one of three primary variants: R702W, G908R, and 1007fs. In an examination of over 300 IBD families, none had chromosomes carrying two of these variants simultaneously, indicating that these mutations are independently related to the disease.

Surprisingly, these three variants share a common ancestral haplotype, which in itself is not directly linked to CD. The discovery that families with either common or unique mutations accounted for the entire linkage at the IBD1 locus provided strong evidence of CARD15/NOD2’s role as a CD susceptibility gene. The gene’s relatively straightforward mutation pattern has facilitated the development of genetic testing procedures for CD diagnosis. However, carrying a CARD15/NOD2 mutation is neither a necessary nor a sufficient condition for developing CD. Approximately 50% of CD patients and 20% of healthy individuals have one or more mutations in this gene, but the frequency of mutations does not differ significantly between Ulcerative Colitis (UC) patients and healthy controls. Notably, 17% of CD patients carry mutations on both of their chromosome 16s, a higher percentage than expected by chance, suggesting a dosage effect and a potential recessive model for CD associated with CARD15/NOD2 mutations.

Despite these findings, genetic testing for CARD15/NOD2 mutations is not routinely used in CD diagnosis. This is partly because no preventive measures can be recommended for individuals genetically predisposed to CD, making pre-symptomatic diagnosis questionable in value. Additionally, the odds ratio for developing the disease is 2 to 3 for carriers of a single mutation and 20 to 40 for those with double mutations. Given the relatively low prevalence of CD (about 1 in 1000), the increased risk associated with these genotypes is only a few percent, making the information too limited for practical use.

To assess the significance of genotype testing in diagnostic processes, various research groups have explored the relationship between genotype and phenotype. It’s been observed that individuals with mutations, particularly those with double mutations, often experience earlier onset of symptoms, ileal disease, and more local complications like stenoses and fistulas. However, this data has limited practical application in clinical settings. The usefulness of genetic testing in choosing treatments is also constrained, leading to its current exclusion from routine clinical practice. Nevertheless, this stance may change soon due to the discovery of other CD susceptibility genes like DLG5 and OCTN, with the anticipation of a molecular diagnostics kit in the coming years.

The CARD15/NOD2 protein comprises three parts: a leucine-rich repeat domain (LRR) with a horseshoe shape, a nucleotide-binding domain (NBD) for protein self-oligomerization, and two caspase recruitment domains (CARDs) for homophilic CARD/CARD interactions (Ogura et al., 2001b). Structurally similar to innate immunity proteins, including those in plants, CARD15/NOD2 is primarily found in white blood cells like monocytes, macrophages, and dendritic cells. It can also be expressed in enterocytes after activation by inflammatory cytokines such as TNFa or IFNg. Paneth cells, which play a role in local immunity and secrete lysozyme and defensins, exhibit high levels of CARD15/NOD2 expression. Thus, CARD15/NOD2 is a key candidate for explaining the interaction between the host and intestinal flora.

CARD15/NOD2’s initial association with bacterial lipopolysaccharide (LPS) response was later revised by discoveries revealing its sensitivity to bacterial cell wall peptidoglycan (PG), particularly identifying muropeptides such as muramyl dipeptide (MDP), commonly utilized in vaccine formulations. The challenge lies in understanding how MDP, a hydrophilic molecule, penetrates cellular barriers, suggesting the likelihood of other proteins playing a crucial role in this interaction with CARD15/NOD2. The activation of CARD15/NOD2 by muropeptides leads to the triggering of the NF-kB pathway, potentially through the self-assembly of NOD domains and subsequent engagement of the Rick/Rip2 protein in CARD/CARD interactions. This activation process of NF-kB is thought to contribute to an increased inflammatory response, evidenced by the production of cytokines like TNFa and IL10, thereby establishing a connection between intestinal microbiota and inflammatory conditions.

Research efforts aiming to confirm the theory that malfunctions in the CARD15/NOD2 pathway contribute to the development of Crohn’s Disease (CD) have predominantly utilized basic NF-kB activation experiments in transiently transfected cell lines. Such studies should be interpreted carefully due to their preliminary nature. Nonetheless, the prevailing consensus in the scientific community is that a significant number of mutations linked to CD, though not universally, are characterized by diminished NF-kB activation when exposed to muramyl dipeptide (MDP). This observation implies that the disruption of the pro-inflammatory process involving CARD15/NOD2 could play a role in the pathogenesis of CD, an inflammatory disease.

The unresolved conundrum in inflammatory diseases where a reduction in a pro-inflammatory mechanism leads to inflammation is a topic of ongoing debate. Many in the scientific community believe that such a reduction alters the immune response, switching it from innate to adaptive, which may intensify reactions and disrupt gut tolerance. This idea, however, remains unverified. A different line of thought suggests that the CARD15/NOD2 pathway might play a role in dampening inflammation. Emerging research shows that CARD15/NOD2 has the capability to affect other pathways that activate NF-kB. For example, it’s been found to suppress TAK1 and alter the response of Toll-like Receptor 2 (TLR2) to peptidoglycan, consequently diminishing the production of IFNg and IL12 (Netea et al., 2004; Watanabe et al., 2004). These insights underline the importance of more comprehensive research before making conclusive judgments. Doubts have also been cast on the loss-of-function theory related to CARD15/NOD2, especially since not all mutations linked to Crohn’s Disease show identical functional defects, calling into question the applicability of existing research models. Furthermore, experiments with CARD15/NOD2-deficient mice have not shown any definitive disease phenotype, particularly in relation to gastrointestinal disorders.

Variations in the CARD15/NOD2 gene have been associated with a range of complex genetic diseases beyond Crohn’s Disease (CD), including asthma and graft versus host disease. This emphasizes the gene’s broad impact across different disorders and the necessity to explore the entire range of phenotypes linked to a single gene. Notably, no connections have been established between CARD15/NOD2 and certain diseases like spondyloarthropathies, rheumatoid arthritis, or Wegener’s disease.

Additionally, CARD15/NOD2 plays a role in Blau syndrome (BS), an inflammatory disease characterized by granuloma formation (Miceli-Richard et al., 2001). BS is an uncommon autosomal dominant Mendelian disorder, manifesting with repetitive inflammatory episodes, including skin rashes, uveitis, and arthritis, but without gastrointestinal involvement. Studies show that BS involves specific mutations in the NBD region of CARD15/NOD2, particularly at codons 334 and 469 (Miceli-Richard et al., 2001). These mutations lead to a functional increase, enabling the protein to activate NF-kB without muramyl dipeptide (MDP) stimulation, a contrast to the loss-of-function mutations observed in CD. The gain-of-function mutations in BS align with its auto-inflammatory nature.

The mutations in BS also share similarities with those in the PYPAF1 gene, linked to Muckle–Wells syndrome, familial cold autoinflammatory syndrome, and chronic infantile neurological cutaneous and articular syndrome.

Recent theories propose that bacterial components might inhibit CARD15/NOD2, blending the findings from CD and BS research. This underscores the complexity of studying IBD and similar genetic disorders, which involve multiple interacting factors. Even after identifying three genes susceptible to IBD using positional cloning techniques, the challenge remains to connect these genetic insights to a comprehensive disease mechanism model.

References

Brant, S. R., Panhuysen, C. I., Nicolae, D. et al. (2003). MDR1 Ala893 polymorphism is associated with inflammatory bowel disease. AmJ Hum Genet, 73, 1282–92.
Bridger, S., Lee, J. C., Bjarnason, I., Jones, J. E. and Macpherson, A. J. (2002). In siblings with similar genetic susceptibility for inflammatory bowel disease, smokers tend to develop Crohn’s disease and non-smokers develop ulcerative colitis. Gut, 51, 21–5.
Bull, T. J., McMinn, E. J., Sidi-Boumedine, K. et al. (2003). Detection and verification of Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn’s disease. J Clin Microbiol, 41, 2915–23.
Calkins, B. M. (1989). A meta-analysis of the role of smoking in inflammatory bowel disease. Dig Dis Sci, 34, 1841–54.
Chamaillard, M., Philpott, D., Girardin, S. E. et al. (2003).Gene-environment interaction modulated by allelic heterogeneity in inflammatory diseases. Proc Natl Acad Sci USA, 100, 3455–60.
Hampe, J., Grebe, J., Nikolaus, S. et al. (2002). Association of NOD2 (CARD 15) genotype with clinical course of Crohn’s disease: a cohort study. Lancet, 359, 1661–5.
Hugot, J. P., Alberti, C., Berrebi, D., Bingen, E. and Cezard, J. P. (2003a). Crohn’s disease: the cold chain hypothesis. Lancet, 362, 2012–15.