Genes and Microbes Linked at Last

Association ofNOD2Leucine-Rich Repeat Variants With Susceptibility to Crohn's Disease. Hugot J-P, Chamaillard M, Zouali H, et al. Nature 2001;411:599–603.A Frameshift Mutation inNOD2Associated With Susceptibility to Crohn's Disease. Ogura Y, Bonen D, Inohara N, et al. Nature 2001;411:603–6.Association Between Insertion Mutation inNOD2Gene and Crohn's Disease in German and British Populations. Hampe J, Cuthbert A, Croucher PJP, et al. Lancet 2001;357:1925–8. Summary: Three independent groups have recently reported mutations in the chromosome 16 pericentromeric NOD2 gene that are associated with Crohn disease. Hugot et al. used classic positional cloning methods to identify NOD2 as a Crohn disease susceptibility gene. The NOD2 gene encodes a 12 exon, 1040 amino acid polypeptide, containing two caspase recruitment domains (CARP1, CARP2), a nucleotide-binding domain (NBP) and, at the carboxyl terminus, ten 27-aminoacid leucine-rich repeats (LRR). Two mutations that changed a single amino acid, Arg702Trp and Gly908Arg had allele frequencies of 11% and 6%, respectively, in patients with Crohn disease and 4% and 1%, respectively, in healthy controls. A frameshift mutation, 3020insC, which produces a truncated protein missing the last 33 amino acids in the LRR region, had allele frequencies of 12% and 2% in patients and controls, respectively. Overall, 29% of Crohn disease patients carried at least one NOD2 variant allele. Providing evidence of a gene dosage effect, the relative risk of development of Crohn disease was 3 among heterozygotes, 38 among homozygotes, and 44 among compound heterozygotes, i.e., those with two different NOD2 variant alleles. NOD2 mutations were not associated with ulcerative colitis. At the same time, Ogura et al. were successful in their use of the candidate gene approach to gene identification. They selected NOD2 as a potential Crohn disease gene, based on knowledge of its role in the recognition of bacterial components, and on the gene location within the IBD1 region. The NOD2 protein sequenced in Crohn disease patients was truncated, similarly to the one described by Hugot et al., because of a frameshift mutation in the LRR region of the gene. The association between the 3020insC frameshift mutation and Crohn disease was confirmed through transmission disequilibrium testing among trios (affected patient and their two unaffected parents) and case–control analysis. Frequency of the mutant allele among all Crohn disease groups was 8.2%. The genotype relative risk for heterozygous and homozygous 3020insC was 1.5 and 17.6, respectively, as compared with wild-type controls. To investigate the relation between NOD2 and bacteria, an embryonic kidney-cell line was cotransfected with an NF-κB (nuclear transforming factor κB) reporter construct and plasmids, containing either the wild-type NOD2 or the 3020insC frameshift mutation. Although LPS from different bacteria evoked widely varying responses through the NOD2 pathway, NF-κB activity was consistently lower in strains containing the NOD2 mutation than in strains with the wild-type NOD2 gene. Subsequent to the publication on an electronic database of the association between Crohn disease and the 3020insC NOD2 frameshift mutation, Hampe et al. corroborated this association in British and German families. In 512 individuals from families with two or more first-degree relatives affected with inflammatory bowel disease (IBD) and in 369 trios (affected child of nonaffected parents), the NOD2 polymorphism was highly associated with the presence of Crohn disease. A gene–dosage relation was again observed, with the highest risk in homozygotes (42.1; 95% confidence interval: 4.3–∞) and an intermediate risk in heterozygotes of 2.6 (95% confidence interval: 1.5–4.5). The lack of association with ulcerative colitis was confirmed. Comment: Positional cloning of genes responsible for Mendelian disorders is commonplace, but the application of genome scanning techniques to traits not attributable to a single gene is relatively new. Hugot et al. reported the first genome scan in families with two or more siblings affected with Crohn disease, and thereby identified the pericentromeric region of chromosome 16 as a possible locus of an inflammatory bowel disease gene (Nature 1996;379:821–3). Linkage to this region, named IBD1, was subsequently confirmed in a multicenter replication study undertaken by the International Inflammatory Bowel Disease Genetics Consortium (Am J Hum Genet 2001;68:1165–71). The research achievement of Hugot et al. in identifying mutations in NOD2 represents the first time a susceptibility gene in any complex disorder has been first localized by linkage studies (Nature 1996;379:821–3) and then positionally cloned (Nature 2001;411:599–603). The simultaneously reported work of Ogura et al. is a demonstration of the success of candidate gene studies, when guided by prior gene localization. The discovery of NOD2 mutations in patients with Crohn disease clearly links genetic susceptibility and enteric bacteria, two factors long suspected as important in etiopathogenesis. NOD2 participates in the innate immune system, which regulates the immediate response to microbial pathogens. It is expressed solely in monocytes and acts as an intracellular receptor for bacterial lipopolysaccharide (LPS). Lipopolysaccharide binds to LRR of the carboxyl terminus; such stimulation results in NOD2 activation of the NF-κB signaling cascade (J Biol Chem 2001; 276: 4812–8). Lipopolysaccharide can also activate NF-κB through interactions with toll-like receptor 4 (TLR4) on the cell surface. Identification of the NOD2 gene is a major clue to etiopathogenesis of Crohn disease, but the mechanisms by which NOD2 variants predispose to Crohn disease must be further elucidated. All three NOD2 mutations identified by Hugot et al. alter the structure of either the LRR domain of the protein or the adjacent region. The investigators postulate, therefore, that the LRR domain of the Crohn disease-associated variants is likely to be impaired in its recognition of microbial components. Whether only one specific pathogen or multiple bacterial antigens are capable of initiating the intestinal inflammation in individuals with abnormal innate immunity remains unanswered, but can now be intensively explored. The observation by Ogura et al. of in vitro reduced activation of NF-κB by truncated NOD2 is surprising because deletion of the entire LRR region has been previously shown to lead to enhanced NF-κB activity (J Biol Chem 2001;276:4812–8). Moreover, NF-κB is activated in mononuclear cells of the intestinal lamina propria in Crohn disease and has a pivotal role in IBD (Gut 1998;42:477–84). Possible explanations include hyperresponsiveness of the truncated NOD2 to LPS from bacteria other than those tested, or an overactivation of the adaptive immune system caused by an incomplete innate immune response. The associations and prevalences of NOD2 mutations observed by the three groups of investigators are remarkably consistent. NOD2 appears to be associated with familial and sporadic Crohn disease. The lack of association between NOD2 mutations and ulcerative colitis is consistent with its negative linkage studies at the IBD1 locus. Further specific phenotypic correlations with NOD2 genotypes are awaited with interest. The IBD1 was the first of several loci that have been identified via genome-wide scanning in multiplex families (Nature Genet 1996;14:199–202;Am J Hum Genet 1999;64:808–16;Am J Hum Genet 2000;66:1863–70;Proc Natl Acad Sci U S A 1998;95:7502–7). Although a locus on chromosome 12 (IBD2) (Nature Genet 1996;14:199–202) is the only other linkage so well documented, it is very likely that several genes confer susceptibility to IBD and that different genes may have similar phenotypic expression. The existence of other genes is strongly suggested by the fact that NOD2 mutations have only been found in 20% to 30% of patients with Crohn disease. Other proteins involved in the innate response to microbial flora will undoubtedly be studied as additional candidate genes and be identified in ongoing linkage studies. However, this first successful identification of a major gene associated with IBD that directs the synthesis of a protein product that appears to have a real role in pathogenesis is an outstanding milestone in unraveling this complex disease.

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