Roesler, Joachim; Thürigen, Anett; Sun, Liping; Koch, Rainer; Winkler, Ulf; Laass, Martin W; Gahr, Manfred; Rösen-Wolff, Angela; Henker, Jobst
Crohn disease (CD [OMIM 266600]) is a chronic relapsing intestinal inflammatory disorder with peak incidence occurring between 15 and 30 years of age (1). However, the incidence of CD has been steadily increasing in the pediatric age group (2), suggesting that environmental factors associated with modern life influence the pathogenesis of this disorder (3). Nevertheless, genetic susceptibility plays a crucial role in the etiology of CD.
With the use of genome-wide linkage analyses and positional cloning methods, a gene on chromosome 16 (16q12, CARD15) has been identified to be associated with CD (4,5). It is a member of the Apaf-1/Ced4 superfamily that is expressed in monocytes and intestinal epithelial cells (6-9) and important for inflammatory signal transduction (10,11). Many patients with CD have the insertion of a cytosine (3020insC) in this gene, which leads to an early stop codon and the deletion of 33 amino acids at the C terminal end of the leucine-rich region. A good hypothetic model of how the CARD15 gene product altered by this and other mutations could lead to enhanced inflammation has been provided recently (12,13). In this model, which is supported by good evidence, the altered CARD15 does not downregulate the Toll-like receptor 2-mediated T helper type 1 proinflammatory responses as the normal CARD15 does. CD susceptibility loci other than CARD15 have also been detected and shown to influence the clinical phenotype of this disorder in a different way (14,15). As an environmental factor, an enhanced inflammatory response to bacteria that can selectively survive or replicate in the cold (in refrigerators) such as Yersinia spp and Listeria spp has been hypothesized (3).
It has been found that genetic factors are particularly important in early onset CD (16,17). The cytosine insertion mutation 3020insC was significantly more common in a pediatric CD population than in patients with a majority of adults (26% versus 11% of the alleles) (17). Furthermore, the pediatric patients with at least one of six CARD15 disease-associated mutations had a high risk of inflammation located in the terminal ileum and in 10 of 19 patients with two mutations intestinal resection surgery was necessary because of stricturing. We analyzed the association of CARD15 mutations with overall disease activity and therapy that was applied without knowledge of these mutations in an entirely pediatric CD patient population and, for the first time, the association with the therapeutic success after 2 years of treatment.
MATERIALS AND METHODS
Of 154 patients with inflammatory bowel disease that we care for in our outpatient department, 65 patients (58% boys and 42% girls) with CD could be included in this study. The diagnosis of CD was confirmed by clinical, radiologic, endoscopic and histologic analysis according to the criteria of Lennard-Jones (18). We excluded patients in cases of uncertainty or indeterminate colitis. The cohort of the patients of this study partially overlaps with that of a first study from Saxony (17). Patients were only analyzed if all relevant data were available. Therefore, the number of patients is given separately for all statistical evaluations if fewer than 65 patients were included.
The Pediatric Crohn Disease Activity Index (PCDAI) was calculated according to Harms et al. (19). This score evaluates both clinical symptoms and laboratory findings. A score lower than 150 indicates a low activity, between 150 to 220 indicates a moderate activity and more than 220 points indicates a severe activity.
Body mass index (BMI) and height percentiles were calculated with the Genotropin Auxology Calculator (Pharmacia and Upjohn, Stockholm, Sweden). Therapy was performed according to the published current guidelines of the German Society of Pediatric Gastroenterology and Nutrition, which are similar to international recommendations (20).
The oligopeptide fluid formula Modulen (Nestle, Glendale, CA) was used as alimentary supplementation. For isolation of DNA the Invisorb® Spin Blood Kit (Berlin-Buch, Germany) was used according to the protocol provided.
Polymerase Chain Reaction Conditions
A pair of primers was designed for exons 4, 8 and 11 of the CARD15 gene (OMIM 605956), according to Sun et al. (17). Thermostable DNA polymerase, Ampli Taq Gold™ and 10× buffer containing 15 mM MgCl2 were purchased from Perkin Elmer (Weiterstadt, Germany). 5 μL buffer was mixed with 2.5 μL dNTPs (10 mmol/L of each), 2.5 μL of each primer (250 ng), 5 μL genomic DNA solution, 0.5 μL polymerase (2.5 U) and 37 μL ddH2O. The cycle program included 10 minutes at 94°C to denature the DNA, 35 cycles of 45 seconds at 94°C, 45 seconds at annealing temperatures adapted to the primer pairs (54°C to 62°C), 90 seconds at 72°C and 5 minutes final extension at 72°C. Synthesis of appropriately sized polymerase chain reaction products was confirmed by agarose gel electrophoresis. The polymerase chain reaction product was cleaned from primers and other low molecular weight compounds by Microcon-50 filter devices (Amicon, Witten, Germany).
The BigDye™ Terminator Cycle Sequencing Reaction was purchased from Perkin Elmer (Weiterstadt, Germany) to determine mutations in the CARD15 gene according to the protocol provided. Briefly, after mixing 0.5 μL of the above polymerase chain reaction products (approximately 50 ng DNA) with 1.0 μL primer (50 ng), 4 μL terminator ready reaction mix (containing Mg2+, pyrophosphatase and DNA polymerase) and ddH2O to a final reaction volume of 20 μL, the following cycle conditions were applied: 25 cycles at 96°C for 30 seconds, 15 seconds at annealing temperatures adapted to the primer pairs (54°C to 62°C), and 60°C for 4 minutes (Trio Thermoblock, Biometra). The sequence was determined using the ABI 310 or 3100 automatic sequencer (Applied Biosystems, Foster City, CA). The sequences of the exons 4, 8 and 11, including adjacent intronic regions, were determined in each patient sample. The DNA sequence of each patient was compared with the published CARD15 gene sequence (OMIM 605956) and the sequence of a healthy control using the ABI 310 sequence navigator software, version 1.0.1 (Applied Biosystems).
Data were evaluated with the SPSS 11.5 and Epi Info 5.0 (SPSS Inc., Chicago, IL and WHO, Gent, Switzerland) software. The chi-squared method (cross-table analyses) and the Fisher exact test were used to analyze possible associations with mutations.
Among 65 CD patients with the first disease manifestation at an age ranging from 3 to 18 years (median, 12 years) we found 19 (29.2%) individuals with one and 16 (24.6%) individuals with two mutations in the CARD15 gene, whereas 30 (46.2%) patients had none of the genetic alterations searched for (R702W, G908R and 3020insC) (Table 1). At the time of diagnosis there were no significant differences in the mean BMI percentiles (Fig. 1, Table 2, A) or mean height percentiles (Fig. 2) between these groups. However, a nonsignificant trend was found: the number of patients below the third percentile of BMI was somewhat higher in the group with mutations than in the group without (Table 2, A, before therapy).
Physicians seeing these patients regularly assessed their degree of severity subjectively and referred them to the categories mild, moderate and severe (Fig. 3). Afterwards the PCDAI (Fig. 4) had been calculated as a more objective assessment before the patients' mutations were evaluated. Accentuated trends could be observed; more patients with mutations than patients without mutations were subjectively rated as severe (Fig. 3) and the degree of PCDAI depended on the number of mutations found (Fig. 4). However, these trends did not reach significance. Therapy had been performed according to the best available evidence (20). In keeping with these guidelines the therapy had been adapted to disease activity before the mutations were known. More long term and intermittent prednisolone and budesonide were applied to patients with mutations than to patients with normal CARD15 sequences (Table 2, B). These differences were close to significance for intermittent prednisolone and reached significance for budesonide. Oral alimentary supplementation and feeding of such extra nutrition via gastric tubes were significantly more often applied to patients with mutations (Table 2, D). Furthermore, these patients were more often treated with the tumor necrosis factor alpha-antagonist Remicade (non-significant trend, Table 2, C).
After 2 years of therapy according to Escher et al. (20) most patients of all groups had profited from the treatment. The PCDAI had decreased (Fig. 4, significantly within the group A2, and the BMI percentiles had increased considerably (Fig. 1). Furthermore, the differences in PCDAI (Fig. 4) and BMI (Fig. 1, Table 2, A) between groups of patients with different numbers of mutations had decreased remarkably or even disappeared after therapy. A minor and nonsignificant slowdown of growth was seen in all patients groups over the 2-year treatment period (Fig. 2), possibly caused by steroid medication.
Several studies confirm that the three CARD15 mutations R702W, G908R, and 3020insC are associated with CD, preferentially showing certain characteristics such as main inflammatory activity in the terminal ileum, intestinal stricturing and onset early in life (4,5,16,17,21). We here demonstrate a trend towards enhanced overall disease activity (Figs. 1, 3, 4, Table 2, A) in pediatric patients with these mutations at the time of diagnosis. Typically, the range of scatter was considerable for all data collected and therefore significance was difficult to achieve.
In our study, the effect of CARD15 mutations on body weight is much weaker than the effect described in the study of Tomer et al. (21). We only found a small BMI median difference between the patients with CARD15 mutations and those without (Fig. 1). However, we saw a nonsignificant trend towards a greater number of patients with a BMI below the third percentile in the group with mutations before therapy (Table 2, A). In contrast Tomer et al. (21) found 44% of their patients with CARD15 mutations below the fifth percentile for weight at diagnosis, whereas only 15% of CD children without mutations were below this percentile (P = 0.003). There are several possible explanation for the difference between the two studies: Patients in our study could have been diagnosed before a large difference in BMI evolved or additional genetic or environmental factors may account for the difference. In any case, our study shows that CARD15 mutations do not completely determine a stronger decrease in BMI compared with patients without such mutations but are only one influencing factor on BMI among others.
The higher degree of disease activity in patients with CARD15 mutations is further emphasized by the finding that these patients got more therapy from physicians who were unaware of their mutations (Table 2, B, C). Especially steroids were prescribed significantly more frequent (budesonide and intermittent prednisolone application). Furthermore, these patients received more oral alimentary supplementation and feeding via gastric tubes (Table 2, D). The initial and long-term more intense therapy for patients with mutations is the most striking finding of our study.
Importantly, the enforced treatment efforts for these patients led to a satisfying improvement of the clinical condition in many of them after 2 years of therapy. The average PDCAI decreased and the average BMI increased to levels that were in the same ranges as for patients without mutations (Figs. 1,4). However, few patients of any group reached the BMI 50% percentile or the range above. Taken together, inflammatory activity in pediatric CD patients with CARD15 mutations is higher than in patients without mutations. Former patients need more therapy but, with some exceptions, show a satisfying response in terms of clinical improvement.
Treatment of patients according to disease activity and response to medication appears to be sufficient for an appropriate management in many patients. Knowledge of CARD15 genetic variants is probably not advantageous for choosing an optimal therapy so far. However, this may change in the future. Disease activity in a few patients was almost resistant to therapy or relapsed rapidly after tempering. Most of such patients seem to have CARD15 mutations; these patients may profit from future therapies designed to interfere with proinflammatory activity induced by the altered CARD15.
The authors thank the Falk Foundation for financial support.
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