Reference
Complete the bibliographic reference for the article according to AJE format.

Hampe J, Grebe J, Nikolaus S, Solberg C, Croucher PJ, Mascheretti S, et al. Association of NOD2 (CARD 15) with clinical course of Crohn’s disease: a cohort study. Lancet 2002 May 11;359(9318):1661-5.

Category of HuGE information
Specify the types of information (from the list below) available in the article:

1. Prevalence of gene variant
2. Gene-disease association
3. Gene-environment interaction
4. Gene-gene interaction
5. Genetic test evaluation/monitoring

1. Gene prevalence
2. Gene-disease association

Study hypotheses or purpose
The authors study hypotheses or main purpose for conducting the study.

Investigation of the importance of three major functional variants of NOD2 – SNP8, SNP12, and SNP13 - on the clinical course of Crohn’s disease, including the presence of stenoses, fistulae, arthritis, or other extraintestinal complications; resections; frequency of relapse; and location of disease (ileal, right colon, or left colon)

Gene(s)
Identification of the following:

1. Gene name
2. Chromosome location
3. Gene product/function
4. Alleles
5. OMIM #

1. Gene: NOD2 / CARD15
2. Chromosome location:  16q12
3. Gene product/function:   Mediates inflammatory response to bacterial antigens. The NOD family of proteins is mainly expressed in macrophages, monocytes, and B-cells and therefore could be a part of innate immunity for maintenance of the intestinal barrier (a). 
   Ogura et al. (2001) proposed that NOD2 serves as an intracellular receptor for bacterial products in monocytes and transduces signals leading to NFkB activation (b).
4. Alleles: C14772T (SNP8), 625386C (SNP12), 32629insC (SNP13)
5. OMIM #: 605956

Environmental factor(s)
Identification of the major environmental factors studied (infectious, chemical, physical, nutritional, and behavioral)

Health outcome(s)
Identification of the major health outcome(s) studied

1. Crohn's disease

1. Case-control
2. Cohort 
3. Cross-sectional
4. Descriptive or case series
5. Clinical trial
6. Population screening

1. Case-control
2. Case-series

Case definition
For study designs 2, 3, and 6, the following are defined, where available:

1. Case selection criteria
2. Exclusion criteria
3. Gender
4. Race/ethnicity
5. Age
6. Time period
7. Geographic location
8. Number of participants

RETROSPECTIVE CASE SERIES

1. Disease case definition: Diagnosis of Crohn’s disease using standard clinical criteria based on endoscopy, radiology, or both
2. Exclusion criteria: Patients whose diagnosis changed during the course of illness were said to have indeterminate colitis and were excluded.
3. Gender: Both (33 % male)
4. Race/ethnicity: German ; not specified
5. Age: Range not specified; mean = 23.5 (8.0)
6. Time period: July 1996 – January 1998
7. Geographic location: Germany
8. Number of participants: 446

GERMAN PROSPECTIVE CASE SERIES

1. Disease case definition: Diagnosis of Crohn’s disease made using standard clinical, radiological, and endoscopic criteria, with compatible histologic findings
2. Exclusion criteria: Patients with discordant clinical diagnosis and histologic findings; patients whose last endoscopy had been performed more than 24 months ago
3. Gender: Both (34 % male)
4. Race/ethnicity: German; not specified
5. Age: Range not specified; mean = 28.3 (11.9)
6. Time period: Not specified
7. Geographic location: Germany
8. Number of participants: 106

NORWEGIAN PROSPECTIVE CASE SERIES

1. Disease case definition: Diagnostic and clinical data reviewed independently by two gastroenterologists; first 55 patients who entered 10-year follow-up were included
2. Exclusion criteria: Not specified
3. Gender: Both (47 % male)
4. Race/ethnicity: Norwegian; not specified
5. Age: Range not specified; mean = 28.3 (16.0)
6. Time period: Registered January 1, 1990 – December 31, 1993
7. Geographic location: Four counties in southeast Norway
8. Number of participants: 55

Control definition
For study design 1, define the following if available:

1. Control selection criteria
2. Matching variables
3. Exclusion criteria
4. Gender
5. Race/ethnicity
6. Age
7. Time period
8. Geographic location
9. Number of participants

1. Control selection criteria: Recruited from the blood-donor system at the university hospitals of Kiel and Berlin (for German controls) and the Rikshospitalet, Oslo (for Norwegian controls)
2. Matching variables: None
3. Exclusion criteria: Not specified
4. Gender: Both
5. Race/ethnicity: German and Norwegian; not specified
6. Age: Not specified
7. Time period: Not specified
8. Geographic location: Germany and Norway
9. Number of participants: German n=373; Norwegian n=202

1. Environmental factor
2. Exposure assessment
3. Exposure definition
4. Number of participants with exposure data (%
   of total eligible)

1. Gene
2. DNA source
3. Methodology
4. Number of participants genotyped (% of total eligible) 

1. Gene: NOD2 (CARD15)
2. DNA source : Fresh or frozen blood
3. Methodology: Method described by Hampe et al. (a)
4. Number of participants genotyped: 925 German, 257 Norwegian (100% of total eligible)

1. Prevalence of gene variant
2. Gene-disease association
3. Gene-environment interaction
4. Gene-gene interaction
5. Genetic test evaluation/monitoring

1. Prevalence of Gene Variant:

1=Mutant; 2=Wild-type
* Retrospective and prospective groups combined
NOTE: See attached Table 1 for frequencies in other populations

2. Gene-disease association
Analysis of genotype-phenotype relation of three NOD2 polymorphisms

* Only seven haplotypes considered in likelihood maximization
† Only six haplotypes considered in likelihood maximization
NOTE: See attached Tables 2, 3, and 4 for comparisons from other studies.

Measures of association comparing the presence of at least one SNP to “wild type” individuals in the two case series*

1. Prevalence of gene variant
CI = confidence intervals

2. Gene-disease association
Ileal Disease

CI = confidence intervals
*Calculations completed by the reviewer

In the retrospective case series, six clinical characteristics showed noteworthy haplotype associations: fistulae, all three locations of disease (ileal, left colonic and right colonic disease), stenosis, and resection. In the German prospective case series, these haplotype associations could be replicated for ileal (p=0.006) and right colonic disease (p≤0.001). A similar trend was noted in the Norwegian patients.

Comments
Provide additional insight, including methodologic issues and/or concerns about the study

Selection of subjects

• The controls were recruited from blood donor systems at several hospitals in Germany and Norway . These individuals are by no means representative of the same population that gives rise to Crohn’s disease patients

• Retrospective cases were recruited consecutively from an invited group of more than 9,000 CD patients from the German Crohn’s and Colitis Foundation. The researchers received more than 6,000 responses, and only included first 446 in the case series. This limited number of cases creates the potential for selection bias by disease severity and patient motivation. Since we are including clinical characteristics like frequency of relapse, which is a measure of disease severity, this selection bias could be a very important source of bias in the study.

Nature of the disease

• CD is an especially heterogeneous disorder, and clinical assessment varies drastically, with no standard, accepted diagnostic criteria. CD encompasses a wide rage of phenotypes or clinical presentations that are subsumed under the same diagnosis. This lack of diagnostic specificity possibly reduces the power of the analyses to detect potentially relevant clinical associations.

Sample size and other concerns

• Due to the small size of the Norwegian prospective case series (n=55), the researchers noted that some associations seen in the German case series could not be replicated. This finding, in addition to the concern stated below, leads one to question the advantages of including this case series in any of the analyses.

• In the Results and Discussion sections, no mention was made of likelihood maximization results for the Norwegian case series. However, the researchers mention that “similar trends” were noted. Since haplotype-clinical characteristic associations were analyzed separately for the two case series, it would have been advantageous to present these results as well.

Consistency with other studies

• Previous studies of stenoses, fistulae, extraintestinal involvement, and relapse frequency have also failed to demonstrate significant associations with any of the NOD2 mutant haplotypes(c-e). However, the association of ileal disease is similar to the findings of Ahmad et al., in that possession of a NOD2 variant was significantly associated with ileal disease (RR=3.3 for an single variant to RR=23.1 for more than one variant, with the greatest RR for the carriage of SNP13) (f). Additionally, Cuthbert et al. found the frequency of each mutant NOD2 haplotype was significantly increased in ileum-specific disease (OR=2.04, 95% CI=1.23-3.38), indicating a twofold increased risk of ileal disease in patients who carry a NOD2 mutation (g). Finally, most previous studies did not differentiate between right and left colonic disease, so the association with right colonic disease is novel and needs to be replicated.

References

1. Hampe J, Grebe J, Nikolaus S, Solberg C, Croucher PJ, Mascheretti S, et al. Association of NOD2 (CARD 15) with clinical course of Crohn’s disease: a cohort study. Lancet 2002 May 11;359(9318):1661-5.
2. Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature 2001 May 31;411(6837):603-6.
3. Lesage S, Zouali H, Cezard JP, Colombel JF, Belaiche J, Almer S, et al. CARD15/NOD2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease. Am J Hum Genet 2002 Apr;70(4):845-57.
4. Vermeire S, Wild G, Kocher K, Cousineau J, Dufresne L, Bitton A , et al. CARD15 genetic variation in a Quebec population: prevalence, genotype-phenotype relationship, and haplotype structure. Am J Hum Genet 2002 Jul;71(1):74-83.
5. Louis E, Collard A, Oger AF, Degroote E, Aboul Nasr El Yafi FA, Belaiche J. Behaviour of Crohn's disease according to the Vienna classification: changing pattern over the course of the disease. Gut 2001 Dec;49(6):777-82.
6. Ahmad T, Armuzzi A, Bunce M, Mulcahy-Hawes K, Marshall SE, Orchard TR , et al. The molecular classification of the clinical manifestations of Crohn's disease. Gastroenterology 2002 Apr;122(4):854-66.
7. Cuthbert AP, Fisher SA, Mirza MM, King K, Hampe J, Croucher PJ, Mascheretti S , et al. The contribution of NOD2 gene mutations to the risk and site of disease in inflammatory bowel disease. Gastroenterology 2002 Apr;122(4):867-74.
8. Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, Belaiche J, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature 2001 May 31;411(6837):599-603.
9. Bonen DK, Ogura Y, Nicolae DL, Inohara N, Saab L, Tanabe T . Crohn's disease-associated NOD2 variants share a signaling defect in response to lipopolysaccharide and peptidoglycan. Gastroenterology 2003 Jan;124(1):140-6.
10. Vavassori P, Borgiani P, D'Apice MR, De Negris F, Del Vecchio Blanco G, Monteleone I, et al. 3020insC mutation within the NOD2 gene in Crohn's disease: frequency and association with clinical pattern in an Italian population. Dig Liver Dis 2002 Feb;34(2):153.
11. Hampe J, Cuthbert A, Croucher PJ, Mirza MM, Mascheretti S, Fisher S, et al Association between insertion mutation in NOD2 gene and Crohn's disease in German and British populations. Lancet 2001 Jun 16;357(9272):1925-8.

Supplementary Tables 

• Table 1 : Allele Frequencies of NOD2 (CARD15) Variants
• Table 2 : Crohn’s Disease Phenotypes (Location) and Composite NOD2 Genotypes and Allele Frequencies from Cuthbert, et al (g)  
• Table 3 : Crohn’s Disease Phenotypes (Location and Behavior) and Allele Frequencies of NOD2 Mutations from Ahmad, et al (f)
• Table 4 : Crohn’s Disease Phenotypes (Location and Behavior) and Percentages of CD Patients Carrying a CARD15 variant from Vermeire, et al (d)