DLG5 and OCTN

Two Gold Medals for the Genetics of Inflammatory Bowel Disease? Peltekova VD, Wintle RF, Rubin LA, et al. Funcitonal variants of OCTN cation transporter genes are associated with Crohn disease. Nat Genet. 2004;36:471–475. Stoll M, Corneliussen B, Costello CM, et al. Genetic variation in DLG5 is associated with inflammatory bowel disease. Nat Genet. 2004;36:476–480. It appears as if the genetics of inflammatory bowel disease (IBD) is holding its Olympic games every 3 years: after the publication of NOD2/CARD15, the first gene for Crohn’s disease, by Hugot et al 1 and Ogura et al 2 in May 2001 in Nature, Nature Genetics released two new important articles in the field of IBD genetics in May 2004. 3,4 The first study by Peltekova et al 3 from the University of Toronto, Canada, reported on two functional mutations in the carnitine/organic cation transporter (OCTN) cluster on 5q31 (the IBD5 locus) that were associated with Crohn’s disease (CD). 3 Rioux et al 5 first reported linkage for CD on 5q31 in the Canadian population. This region has been designated as the IBD5 locus. IBD5 is very attractive as a candidate region for IBD, since it harbors the cytokine gene cluster. Fine mapping of this locus refined the region to a 250-kilobase (kb) risk haplotype (surrounding the OCTN genes), but precise identification of the underlying causal genetic variants was impossible due to strong linkage disequilibrium (LD) across the region. 6 At least three independent groups subsequently confirmed the association of the IBD5-risk haplotype with CD, 7,–9 and also with UC. 7,10 Peltekova et al 3 resequenced the five known genes in the IBD5 region and identified 10 new single nucleotide polymorphisms (SNPs), including two novel SNPs in OCTN that were predicted to have functional effects. The first SNP located in OCTN1 is a missense mutation consisting of a C → T change at Position 1672 leading to a change in amino acids from leucine (L) to phenylalanine (F) at Position 503 in Exon 9. The second variant consists of a G → C change in the promoter region of OCTN2 at Position –207. 3 Both mutations are, as expected from the previous studies on IBD5, in strong LD and create a two-allele risk haplotype, TC, which in the study by Peltekova et al, 3 had a frequency of 54% in CD patients compared with 42% in control subjects (p = 0.0003). The risk for the disease was even greater in the presence of both the OCTN-TC haplotype, and at least one NOD2/CARD15 variant, confirming previously reported interactions between IBD5 and NOD2/CARD15. The OCTN-TC haplotype was not significantly increased in UC in the study by Peltekova et al, 3 a contrast that some studies on IBD5 have shown in the past. 7,10 In a subsequent step, the authors carried out preliminary functional experiments and showed that fibroblasts expressing the 503F variant had a 2.7 times lower carnitine uptake rate and an increased transport of xenobiotics compared with the cells expressing the conservative 503L. The OCTN2 promoter variant was shown to disrupt a heat shock element 207 bp upstream of the start codon. Now, what are these OCTNs, and do they fit into our current ideas on IBD, and CD in particular? To answer this question, we need to understand more about the carnitine/OCTN subfamily. The first OCTN (OCT1) was cloned 10 years ago by Gründemann et al. 11 Three members of this subfamily (OCT1, OCT2, and OCT3) have been described in the meanwhile. 11,–13 Many drugs, xenobiotics, and endogenous metabolites are positively charged (ie, organic cations [OCTs]), and therefore need membrane transporters for their absorption, distribution, and elimination. As membrane transporters of OCTs, OCTNs are therefore important in the maintenance of intracellular homeostasis. In addition to the classic subfamily of OCTs, a n ovel OCT subfamily (OCT N) has recently been reported. 14,15 Like the OCTs, the OCTNs also play a role in the elimination of cationic drugs. In addition, however, the OCTN subfamily may also transport carnitine, an ability that has not been reported for OCTs. Carnitine is an endogenous amine that is an essential cofactor of the metabolism of lipids. Carnitine is involved in the transport of long-chain fatty acids into mitochondria, where fatty acids will undergo β-oxidation. Hence, carnitine plays an important role in the energy production of the cell. In humans, OCTN1 and OCTN2 map to IBD5 on 5q31. An OCTN3 has recently been described in humans (in analogy to three known OCTNs in mice), but the corresponding gene has not yet been identified nor has its sequence been retrieved from any DNA sequence database. 16 OCTNs are widely expressed in human tissue (ie, heart, skeletal muscle, kidney, placenta, intestine, and brain). The OCTN1 protein contains 551 amino acids, and is a multispecific and pH-dependent OCTN. OCTN2 consists of 557 amino acids and has 75.8% homology with OCTN1. OCTN2 functions as a Na+-dependent carnitine transporter as well as a Na+-independent transporter for other organic cations. Examples of cationic drugs that are transported by OCTN2 include verapamil, quinidine, valproate, and pyrilamine. The affinity of OCTN2 for carnitine is markedly higher than that of OCTN1. Mutations in OCTN2 have been associated with systemic carnitine deficiency, an autosomal-recessive disorder of fatty acid oxidation that is characterized by cardiomyopathy, muscle weakness, hyperammonemia, and hypoglycemia, as a result of insufficient fatty acid β-oxidation and insufficient energy for muscle and cardiac functions. 17 Now, given this background on OCTs and OCTNs, how do we make the link to IBD? There is some evidence that the inhibition of fatty acid oxidation in the epithelium of the colonic mucosa is associated with the development of ulcerative colitis. Rectal instillation of sodium 2-bromo-octanoate, a specific inhibitor of β-oxidation, produces weight loss and bloody diarrhea in rats with histologic signs of ulcers, mucus cell depletion, vessel dilatation, and increases of acute inflammatory cells. 18 The second article in the May issue of Nature Genetics was by Stoll et al 4 from the University of Kiel, Germany. This group of authors reported the association of IBD with mutations in the DLG5 gene on the long arm of chromosome 10 (10q23) using positional cloning techniques and fine-mapping of the susceptibility region identified by Hampe et al 19 in 1999. The DLG5 gene (ie, Drosophila Discs Large Homolog 5) is a 180-kb protein containing 1900 amino acids. 20 DLG5 is a member of the MAGUK (Membrane-Associated Guanylate Kinase) family. MAGUKs are known to form scaffolds for proteins involved in intracellular signal transduction. Scaffolding proteins organize protein complexes at cellular junctions to integrate the tethering of adhesion molecules, receptors, and intracellular signaling enzymes. MAGUKs bind to transmembrane proteins at the cytoplasmic side and to other signaling proteins, and ensure efficient signal interactions between the different components of the signaling pathways. 21 DLG5, as a MAGUK member, is important in maintaining the epithelial structure, and the genetic variants in DLG5 could therefore interfere with the epithelial barrier. The MAGUK family of proteins all share characteristic domains, namely, the PDZ, SH3, and GK domains. DLG5 has four PDZ domains. These are important for protein–protein interactions. The SH3 domain (Scr homology 3) was originally discovered as a homologous region present in the tyrosine kinase product of the v-Src oncogene. This region is important for coupling transmembrane receptors to signaling molecules. Finally, the guanylate kinase (GK) domain interacts with other proteins, and aggregates adhesion molecules and receptors. The GK domains of MAGUKs share 40% sequence homology with authentic GK enzymes, which phosphorylate guanosine monophosphate to form guanosine diphosphate. Genetic analyses have revealed that lethal mutations of MAGUKs often occur in the SH3 and GK domains, indicating a critical role for these domains. Stoll et al 4 identified two haplotypes in DLG5 with a significant transmission distortion to affected offspring. The haplotype D, characterized by a G → A change at nucleotide 113 leading to a change from arginine to glutamine at amino acid Position 30 (R30Q), was overtransmitted to individuals with IBD and CD. In an independent case-control sample, 25% of IBD patients carried at least one risk allele, compared with 17% of healthy control subjects (p = 0.001). The overall risk for IBD associated with the 113A variant was, however, moderate (odds ratio 1.6), but this is not unexpected in a polygenic disease model. Using in silico analysis, the R30Q variant probably impedes the scaffolding of DLG5, although functional experiments to prove this are not provided in the article. More difficult to explain is that in the case-control sample, the mutated allele appears to be overrepresented only in the heterozygote patients, and not in the homozygote patients. The second haplotype A, tagged by eight equally informative haplotype-tagging SNPs, was under-transmitted to affected offspring with IBD and CD. The authors also examined gene–gene interactions between DLG5 and CARD15, and showed the interaction between the 113A variant on haplotype D and the presence of CARD15 variants. Although both studies are exciting, not only for the field of IBD genetics but also for understanding the pathogenesis of IBD, they clearly leave us with a lot of questions. First of all, as for all studies, confirmation of the data in independent cohorts is needed. Since the OCTN SNPs are located on the previously reported IBD5-risk haplotype, they are very likely to be in complete LD. Therefore, the LD results of these OCTN1 and OCTN2 variants with the IBD5 haplotype-tagged SNPs are needed before definitive conclusions on OCTN as a CD gene can be drawn. For the DLG5 gene, it is unknown whether it explains all of the linkage peak observed at 10q, and also whether it is a gene predisposing to both CD and UC, or to CD alone? Second, the prevalence of these variants among different populations is not known and needs to be studied to understand their importance in various subgroups. We have learned from the NOD2/CARD15 gene that major differences in prevalence exist among populations. CARD15 variants are found in 35% to 45% of white CD patients, with the exception of Scandinavian, Irish, and Scottish patients, in whom the prevalence is much lower. In Japanese and Chinese patients, as well as in African-American populations, CARD15 variants cannot be found. Variants in the IBD5 risk haplotype appear to be very rare in the Japanese population. 7 Another intriguing question concerns how these variants translate into the clinical phenotype. The IBD5 haplotype has been associated with perianal disease in a cohort from the United Kingdom. 22 We will have to see whether this is confirmed for the OCTN SNPs. Also, given the positive interaction between the IBD5 region and CARD15, 7,9,10 the question arises of whether the OCTN variants are also associated with ileal disease and fibroste-nosing behavior, as has unmistakably been shown for the NOD2/CARD15 gene. Other questions arise for DLG5, which is expressed widely in human tissue including the placenta, heart, skeletal muscle, liver, pancreas, small bowel, and colon, and in intestinal epithelial cells. How can mutations in a gene that is widely expressed be associated only with an intestinal disorder? There have been seven splice variants described in DLG5, but it is not known whether certain splice variants have unique expressions. Finally, functional studies, including animal studies are greatly needed for both genes to understand their role in the pathophysiology of IBD. To conclude, the era of molecular classification of CD has started, and the field of IBD genetics has again earned two Olympic gold medals. Now, with more and more genes being identified in CD, a challenge for the future will lie in unraveling the functional defects in these genes and in understanding the various gene–gene and gene–environment interactions. Let us meet again in 3 years time . . . or sooner?