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Human Molecular Genetics Advance Access originally published online on July 5, 2007
Human Molecular Genetics 2007 16(18):2175-2186; doi:10.1093/hmg/ddm169
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Investigation of NOD1/CARD4 variation in inflammatory bowel disease using a haplotype-tagging strategy

Johan Van Limbergen1,4, Elaine R. Nimmo1, Richard K. Russell4, Hazel E. Drummond1, Linda Smith1, Niall H. Anderson2, Gail Davies1, Ian D. Arnott1, David C. Wilson3,4 and Jack Satsangi1,*

1 Gastrointestinal Unit, Molecular Medicine Centre, Western General Hospital, 2 Public Health Sciences, 3 Child Life and Health, University of Edinburgh, Edinburgh, UK and 4 Department of Paediatric Gastroenterology and Nutrition, Royal Hospital for Sick Children, Edinburgh, UK

* To whom correspondence should be addressed at: Gastrointestinal Unit, Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh EH4 2XU, UK. Tel: +44 1316511807; Fax: +44 1316511085; Email: j.satsangi{at}ed.ac.uk

Received April 6, 2007; Accepted June 28, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Both NOD1/CARD4 and NOD2/CARD15 are intracellular pattern-recognition receptors involved in the innate immune response. Germline NOD2/CARD15 variation has a definite effect on susceptibility to Crohn's disease (CD) and phenotype, although this contribution is weak in Scotland and Scandinavia. The NOD1/CARD4 gene lies within the putative inflammatory bowel disease (IBD) locus at 7p14.3. We have assessed, in detail, the influence of germline NOD1/CARD4 variation on IBD susceptibility and phenotype in the Scottish population. Two thousand two hundred and ninety-six subjects, including 356 children with IBD, were involved in parallel case–control and family-based association studies. Nine tagging single-nucleotide polymorphisms (SNPs) were selected based on HapMap data spanning the whole of the NOD1/CARD4 gene. Our case–control SNP analysis was powered to detect an effect size with OR 1.5 for IBD and OR 1.6 for CD. No significant associations were observed between any of nine the NOD1/CARD4 SNPs studied and IBD, CD or ulcerative colitis (UC) (P > 0.05 for all). Haplotype case–control analysis was also negative (P > 0.05 in IBD, CD and UC). Multimarker transmission disequilibrium testing analysis was negative (P > 0.05 in IBD, CD and UC). NOD2/CARD15 variant carriage had no influence on NOD1/CARD4 effect on IBD susceptibility. This study represents the first application of a gene -wide haplotype-tagging approach to assess, in detail, the contribution of NOD1/CARD4 in IBD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The inflammatory bowel diseases (IBDs), Crohn's disease (CD) and ulcerative colitis (UC) are common relapsing conditions characterized by both gastrointestinal and systemic manifestations and are responsible for significant morbidity in adults and children. In Northern Europe, these illnesses affect one in 250 of the adult population (1). Up to 25% of IBD first presents during childhood and adolescence, often with serious consequences for growth, puberty, education and quality of life (2). The incidence of childhood-onset IBD in Northern Europe is among the highest reported worldwide (3–7). An increasing body of evidence has emerged in recent years to support the hypothesis that a germline predisposition towards a dysregulation of the immune response against environmental factors, notably the intestinal microflora, is critical in IBD pathogenesis (8–10).

Strong genetic epidemiological evidence from concordance data in twins and multiplex IBD families have catalysed the application of non-parametric linkage analysis in genome-wide searches for sub-chromosomal regions involved in IBD susceptibility, a decade ago (11). These studies were successful in identifying a number of confirmed linkage regions (IBDs 1–9) as well as other putative IBD loci, with less definite evidence for linkage (8,12).

Fine mapping of the IBD1 region on chromosome 16 led to the identification of the NOD2 (nucleotide-binding oligomerization domain 2)/CARD15 (caspase activation recruitment domain 15) gene (13–15). In the index studies, more than 40% of CD patients carried at least one NOD2/CARD15 variant (13,16). Meta-analysis suggests the strongest sub-phenotypic association is with ileal CD (17). In Northern European populations, the contribution of NOD2/CARD15 variants to CD susceptibility is smaller than in other populations studied to date (16,18–25). NOD2/CARD15 is a pathogen-associated molecular pattern receptor for muramyl dipeptide, a degradation product of bacterial peptidoglycan of both Gram-negative and -positive bacteria (26,27). NOD2/CARD15 is characterized by a modular domain organization of a C-terminal leucine-rich repeat (LRR) domain, a central nucleotide-binding NOD domain and an N-terminal protein–protein interaction domain composed of two CARDs (caspase activation and recruitment domain) (28).

Like NOD2/CARD15, NOD1/CARD4 is a member of the phylogenetically conserved Nod-like receptor family (10,28). The NOD1/CARD4 gene contains 14 exons (54 kb) and is located within the putative IBD susceptibility locus on chromosome 7p14.3 which was described in the only UK genome sib-pair analysis (29). Structurally, the NOD1/CARD4 protein is similar to NOD2/CARD15, but only has one CARD domain (30). At its LRR domain NOD1/CARD4 binds intracellular {gamma}-D-glutamyl-meso-diaminopimelic acid (iE-DAP), a breakdown product of peptidoglycan from Gram-negative bacteria and a limited number of Gram-positive bacteria (31–34). In contrast to NOD2/CARD15, NOD1/CARD4 is constitutively expressed in epithelial cells throughout the gastrointestinal tract (35–37). Recent work has implicated NOD1/CARD4 in the response against Helicobacter pylori and Gram-negative enteric pathogens which avoid recognition by Toll-like receptors (TLRs) (36,38,39). These observations make NOD1/CARD4 a strong functional and positional IBD candidate gene.

A number of groups have investigated whether germline variation of NOD1/CARD4 is associated with increased susceptibility to IBD (40–45). Of these, the only positive study has received the greatest attention. McGovern et al. (42) initially suggested the association of the deletion variant of a complex intronic insertion*2/deletion*1 polymorphism (32656) of NOD1/CARD4 with susceptibility to IBD using a combination of transmission disequilibrium testing (TDT) and case–control analysis. In a cohort of 556 trios, these investigators observed distortion of transmission of this NOD1/CARD4 variant in IBD and UC, but not CD. Single-nucleotide polymorphism (SNP) case–control analysis (664 IBD patients and 335 controls) showed significant associations with IBD and CD (especially onset < 25 years), but not UC. They went on to perform a sliding haplotype TDT analysis, a new approach in IBD genetics, obtaining significance for a two-marker haplotype spanning a large section of the LRR domain. In addition, a gene-dosage effect of the deletion variant on age of onset in CD and IBD was demonstrated (42). The same group also described an association of the insertion allele of this complex insertion/deletion with high IgE levels and asthma (37).

However, well-designed replication studies involving a combined total of over 10 000 subjects have been negative, thereby shedding doubt on the contribution of this single NOD1/CARD4 SNP (43–45). Tremelling et al. (43) assessed 1370 IBD patients and 760 controls from the Cambridge area and did not find evidence for association of this NOD1/CARD4 variant with IBD (P = 0.74). Franke et al. (44) were also unable to replicate the suggested association in a German case–control analysis of 1015 IBD patients and 886 controls (P = 0.51) and a TDT of 775 IBD trios (P = 0.24). Our own studies on 3962 subjects from Scotland and Sweden demonstrated that the insertion/deletion variant of NOD1/CARD4 did not contribute to IBD susceptibility in adults nor in our large collection of children with IBD (n = 313) (45). Using a combination of case–control and TDT analyses, this study excluded an effect size of odds ratio > 1.2 for IBD. Indeed, all three replication studies had > 95% power to replicate the effect size (odds ratio 2.0 for CD), as calculated in the initial study by the Oxford group.

In this study, our aim was to assess the overall contribution of NOD1/CARD4 to IBD susceptibility, using a robust gene-wide haplotype-tagging approach. In view of the suggested effect of NOD1/CARD4 on age at diagnosis of IBD (CD), it is noteworthy that we have access to more than 350 children diagnosed with IBD before the age of 17. We examined the influence of NOD1/CARD4 haplotypic variation on IBD phenotype and investigated the interaction between germline variants of NOD1/CARD4 and NOD2/CARD15 in determining IBD susceptibility and phenotype. The results provide strong evidence that inherited variation of NOD1/CARD4 is not a strong determinant of disease susceptibility in the Scottish population, based on detailed power calculations for SNP and haplotype analyses.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The haplotype structure of the genomic region spanning NOD1/CARD4 with the position of identified tagging SNPs is provided in Figure 1 (based on HapMap data Release 21). Nine SNPs were genotyped in 2296 subjects of which 1323 were IBD patients, including 356 children with IBD and their parents. We assessed association with IBD, CD and UC using single- and multiple-marker case–control analysis, TDT and log-likelihood analysis.


Figure 1
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  		Figure 1. View of the genomic region containing NOD1/CARD4 generated from HapMap (Release 21) project data using Haploview software. The haplotypic variation of the NOD1/CARD4 is tagged by nine SNPs shown in the lower panel, covering three haplotype blocks (blocks 3–5).

 
Power of the study
We have first analysed the power associated with the analysis of individual SNPs, as the power of each analysis is dependent upon the allelic frequency. We estimated the population risk to IBD as 0.004 (0.002 for CD and UC), based on population-based data by Rubin et al. (1). In this study, the age–sex-adjusted point prevalence for UC on 1st January 1995 was 243.4/100 000 (confidence interval, CI 217.4–269.4) and for CD 144.8/100 000 (CI 124.8–168.8). The most recent childhood-onset IBD prevalence data in the Scottish population were derived from hospital discharges and date back to the period before 1997 (symptom onset 1981–1995) (4). Armitage et al. estimated the combined (CD + UC) crude prevalence of childhood-onset IBD in Scotland to be 22.9/100 000 (13.7/100 000 CD, 9.2/100 000 UC) of the population aged 0–16 years. It is pertinent to point out that both the Rubin and Armitage studies reflect reported disease prevalence more than a decade ago. Since then, the incidence of IBD has continued to rise, and more up-to-date data from the Scottish Executive are awaited.

In comparison with dominant and recessive risk models, we assessed the multiplicative risk model to be more appropriate for the investigation of a complex polygenic disease. Using a multiplicative risk model, our case–control analysis had 90% power to detect an OR of 1.5 to IBD and 1.6 for CD based on case–control analysis of the tagging SNPs with {alpha} = 0.005 to allow for multiple testing (Table 2). By comparison, the index paper by McGovern et al. (42) quoted an OR of 2.0 for CD. Effect sizes excluded (calculated as OR) are given in Tables 2 and 3 for the IBD population overall, as well as for the early-onset IBD cohort.


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  		Table 1. NOD1/CARD4-tagging SNPs: rs-number, genomic location and flanking sequences

 


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  		Table 2. Genotype frequencies of nine NOD1/CARD4 haplotype-tagging SNPs in healthy controls and patients with IBD, CD and UC

 


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  		Table 3. Genotype frequencies of nine NOD1/CARD4 haplotype-tagging SNPs in healthy controls and children/adolescents with IBD, CD and UC, diagnosed before age 17

 
In addition, we have revised the power calculation concerning the haplotype analysis and are able to demonstrate that for the nine-marker haplotypes with frequency > 5% (77% of haplotypes), our study was adequately powered to exclude OR of 1.8 for IBD with 90% power. For this calculation, {alpha} was again set at 0.005 to stringently correct for multiple testing of the nine most common haplotypes (Table 4).


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  		Table 4. Haplotypes consisting of nine NOD1/CARD4-tagging SNPs (haplotype frequency > 1%) in Scottish IBD patients (adult + paediatric combined) and controls

 
For all analyses, the 99.5% CIs are given to ensure compatibility with the statistical threshold of significance, {alpha} = 0.005, as obtained after Bonferroni correction.

The true power of this study to demonstrate an effect of each SNP on disease susceptibility is best estimated by integrating case–control analysis and TDT analysis. Results of this analysis, which was first described by Kazeem and Farall, are given in Table 7.

Case–control analysis in Scottish IBD patients
No significant associations were observed between any of the nine NOD1/CARD4 SNPs studied and IBD, CD or UC when assessing allelic and genotype frequencies (after Bonferroni correction P > 0.05 for all) (Table 2). Although, rs932272 showed weak association with IBD and CD (P = 0.04 in combined adults + paediatric IBD/CD, P = 0.02, P = 0.01 in paediatric IBD and CD, respectively), these findings did not retain significance after Bonferroni correction.

Similarly, when disease susceptibility was studied in case–control analysis of haplotype frequencies, no significant effect of NOD1/CARD4 on IBD susceptibility was observed (corrected P > 0.05 for IBD, CD and UC) (Table 4).

After stratification of CD patients and controls for carriage of the three common NOD2/CARD15-variant alleles (R702W, G908R and Leu1007fsinsC), none of the haplotype variants of NOD1/CARD4 was associated with CD (corrected P > 0.05).

The two-marker haplotype rs2970500/rs2075820 overlaps with the area of strongest association in the report by McGovern et al. (rs6958571/rs2907748) (42). Case–control analysis of this two-marker haplotype was non-significant in IBD, CD and UC (P > 0.16, P > 0.33 and P > 0.11, respectively).

We also assessed whether germline variation of NOD1/CARD4 influences the phenotype of IBD. We used the Montreal guidelines for classification of disease behaviour and disease location of CD and UC in our analyses (Table 5). No significant influence of NOD1/CARD4 haplotype variants on phenotype was evident (46). Stratification for NOD2/CARD15-variant carriage (R702W, G908R or Leu1007fsinsC) did not influence the observed lack of effect of NOD1/CARD4 on phenotype in CD or UC.


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  		Table 5. Phenotypic classification of adult- and childhood-onset IBD cohorts according to the Montreal classification

 
Childhood-onset IBD
We assessed in detail whether the haplotypic variants of NOD1/CARD4 were associated with increased susceptibility in our cohort of 356 children (42). Sub-group susceptibility analysis of patients with early-onset IBD (diagnosed at < 17 years of age) did not show any of the tagging SNPs (either assessed as single marker or in haplotypes) to be associated significantly with IBD, CD or UC (corrected P > 0.05 for all) (Table 3). Also in early-onset CD patients, NOD2/CARD15-variant carriage did not play a significant role in determining the contribution of NOD1/CARD4 to disease susceptibility (corrected P > 0.05).

Genotype–phenotype analysis in childhood-onset CD and UC, based on the Montreal classification, did not show any significant effect of NOD1/CARD4 haplotypic variation on disease phenotype at diagnosis.

Transmission disequilibrium test
Results of single-marker TDT analysis for IBD, CD and UC are shown in Table 6. No significant distortion of transmission was observed for any of the markers, after correction for multiple analyses. Multiple-marker TDT, followed by permutation analysis, in IBD, CD and UC trios yielded no significant distortion of transmission of any of the haplotypes studied (P = 0.74 for IBD, P = 0.77 for CD and P = 0.68 for UC—haplotype frequencies > 2% were studied comprising all nine tagging SNPs). The two-marker haplotype rs2970500/rs2075820 which overlaps with the area of strongest association in the report by McGovern et al. (rs6958571/rs2907748) showed no distortion of transmission in IBD (P = 0.25), CD (P = 0.47) and UC (P = 0.42) (42).


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  		Table 6. Results of TDT analysis in families with early-onset IBD analysis using FBAT software package

 
Log-likelihood analysis
Using log-likelihood analysis (heterogeneity model—1000 permutations), set {chi}2 statistics (degrees of freedom = 511) were reached 103, 396 and 35 times for IBD, CD and UC, respectively (corresponding uncorrected P-values 0.10, 0.39 and 0.03).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We present the first study of a gene-wide haplotype-tagging approach to assess the contribution of germline variation of NOD1/CARD4 to IBD susceptibility. We have made use of data of the second phase of the HapMap project to select SNPs tagging the haplotypic variation of the NOD1/CARD4 gene (47). This has allowed us to assess the contribution of NOD1/CARD4 to IBD in a more robust manner compared with previously performed SNP analysis. In the present study comprising 2296 subjects, we have used three different methods [case–control analysis (single marker and haplotype), TDT (single marker and haplotype) and log-likelihood analysis] to provide robust support for a lack of association between germline variation of NOD1/CARD4 and IBD. Detailed power calculations, both for SNP and haplotype analyses, have demonstrated that our study was adequately powered to detect an OR of 1.5 and 1.6 for IBD and CD, respectively, after applying stringent criteria for multiple testing ({alpha} = 0.005). This compares favourably with the calculated OR for CD of 2.0 in the index paper from Oxford, reporting association of NOD1/CARD4 + 32656 with IBD/CD (42). Furthermore, access to our large cohort of patients with childhood-onset IBD (n = 356), characterized by a small but definite contribution of NOD2/CARD15, has permitted us to conclude that common haplotypic variation of NOD1/CARD4 is not an important genetic determinant of early-onset disease.

There have been a number of notable successes in IBD genetics in recent years, which have been recently reviewed (8).

However, the study of the contribution of NOD1/CARD4 to IBD has seen replication studies unable to confirm a positive index study, mirroring the experience in other complex diseases (48). Whether true genetic heterogeneity, phenotypic differences between IBD cohorts or population admixture underlie these differences has often been difficult to elucidate. In IBD, only for NOD2/CARD15, true genetic heterogeneity has been demonstrated with certainty (8,19,49).

As described, in the index study from investigators from Oxford the NOD1/CARD4 + 32656 deletion allele was associated with IBD, using a combination of sliding-haplotype TDT and case–control analysis of a SNP (42). The detailed results of this study are worthy of consideration. Using TDT of the NOD1/CARD4 + 32656 deletion*1 allele in 556 trios, McGovern et al. demonstrated significant distortion of transmission for IBD and UC, although not CD (allelic frequency: 73.4%; IBD P = 0.02, CD non-significant, UC P = 0.01). Using a case–control analysis (664 IBD patients and 335 controls), associations with IBD and CD (especially onset < 25 years) were demonstrated (IBD P = 0.01, CD P = 0.003, IBD < 25 years P = 3 x 10–4, CD < 25years P = 4 x 10–5, UC P = 0.34). In a sliding-haplotype analysis, significant transmission distortion in IBD, CD and UC was found for a haplotype spanning a large section of the LRR domain (+32656*2/+45343: IBD P = 3 x 10–7, CD P = 0.007, UC P = 7x 10–5). This technique does not provide an assessment of the gene-wide contribution of haplotypic variation of NOD1/CARD4 to IBD.

Our own data on IBD patients from Scotland and Sweden, as well as studies from East Anglia (UK) and Germany were, however, unable to confirm these findings, in spite of adequate power (43–45). The significant differences between frequencies of the deletion allele in control populations, in the UK studies, are noteworthy, as it seems that these differences, rather than differences between allelic frequencies in patients, underlie the discrepancies between centres (45). The difference (68.2% versus 74.3%, P = 0.003) between the control allelic frequency of the NOD1/CARD4 + 32656 deletion allele in the Oxford and Cambridge studies seems more likely to be explained by a statistical false-positive result or population stratification rather than by genetic heterogeneity. Although true heterogeneity between different populations has been demonstrated for NOD2/CARD15, considering the short geographical distance between the two centres, this possibility seems highly unlikely compared with admixture of the control population (19). Indeed, the single variant TDT analysis, not subject to this confounding element, was negative for CD in the Oxford study. It is noteworthy that in the most recent regression analysis by McGovern et al. from Oxford, carriage of the deletion allele of NOD1/CARD4 + 32656 did not contribute significantly to the diagnosis of CD (50). Our own detailed analysis of a two-marker haplotype overlapping with the haplotype of strongest association in the Oxford study did not show any association with IBD, CD or UC.

Notwithstanding these discrepancies, NOD1/CARD4 has remained a strong functional IBD candidate gene in view of its important role in regulating the innate immune response. The expression pattern of NOD1/CARD4 across the gastrointestinal tract and the presence of different splice variants with altered binding properties of the LRR domain illustrate the importance of NOD1/CARD4 in the ability of the mucosa to sense the microbial content of the gut lumen (37,51). Functional work has also provided evidence for interaction with Toll-like receptor pathways, again suggestive of a crucial role for NOD1/CARD4 in the innate immune response against bacteria (38,52). Furthermore, the presence of an altered mucosa-associated microflora in IBD patients is indicative of an innate immune response which is either unable to recognize these microorganisms appropriately (due to altered expression or altered ligand binding) or unable to orchestrate an effective second-line immune reaction to clear them (10,53,54).

The problems associated with the analysis of SNPs have stimulated researchers to turn to haplotype-tagging-based approaches to assess the gene-wide contribution to the disease under study. The use of a gene-wide haplotype-tagging strategy has already proven successful in the analysis of other IBD candidate genes, in which equivocal evidence had been provided by SNP analyses in different populations.

Applying this approach to capture the haplotypic variations of the Multi Drug Resistance 1 (MDR1) gene, Ho et al. (55) observed a highly significant association in the Scottish adult population between the common haplotypes of MDR1 and UC (P = 4.22x10–7) but not CD (P = 0.22). These data provided really clear evidence of an important contribution to susceptibility and phenotype, in the face of inconsistent reports from other populations and have been supported by meta-analysis (56). Ho et al. (57) also confidently refuted the role of the pregnane X receptor gene (PXR/NR1I2) in IBD in the same population after application of a gene-wide haplotype-tagging approach.

In the present study, a tagging approach based on HapMap data available for this region has excluded an important role of common germline NOD1/CARD4 variation in our high-incidence population. Increasing data support the validity of this approach.

There is now good evidence that tagging SNP identification based on sample sizes like those of the HapMap project is appropriate to capture common variants (mean allelic frequency > 5%) with little loss of power if a causal variant is represented by tagging rather than by direct genotyping (58,59). The transferability of tagging SNPs in genetic association studies across different populations has also been studied (60). In recent reports by de Bakker et al. (59) and Conrad et al. (61), the portability of tagging SNP selection based on HapMap data across multiple populations was demonstrated, in particular, for Caucasian populations.

To date, analysis of candidate genes in our early-onset Scottish IBD population has demonstrated only weak effects of the IBD5 locus (OCTN1&2 genes), DLG5 and NOD2/CARD15 on disease susceptibility (18,62,63). In view of the high incidence of disease, it is likely that other important determinants are present, which underlie the susceptibility of our and other populations. Most recently, the application of genome-wide association studies in IBD genetics has led to the discovery of several new determinants. The multicentre North-American consortium described an association between ileal CD and the common allele of a rare non-synonymous variant of the IL23-R (interleukin-23 receptor) gene on chromosome 1p31 (64). This has been unequivocally replicated in four independent studies, including our own paediatric cohort (65–68). Subsequently, Hampe et al. (69) reported on the association of a coding variant of ATG16L1 (Autophagy-related 16-like 1) gene, on chromosome 2q37.1. This finding has also been replicated recently (66,70–72). In addition to describing the association between CD and another autophagy gene (IRGM), the British Wellcome Trust Case Control Consortium (WTCCC) also replicated the association with a gene desert on chromosome 5p13.1, putatively regulating the expression of the prostaglandin receptor PTGER4 as first reported by Libioulle et al. (66,73).

Relevant to our study, none of the genome-wide scans to date report association with NOD1/CARD4 in CD. Thus, the North-American genome-wide association study did not find any evidence for association between ileal CD and any of the marker SNPs in the region of NOD1/CARD4 (1991 cases and 1214 controls were studied) (Judy Cho, Personal communication). Our own detailed analysis of WTCCC data does not reveal association with CD in the British population (1746 cases versus 10 603 controls) (http://www.wtccc.org.uk/). Although we did not have access to the primary data, the studies by Hampe et al. and Libioulle et al. also do not report any association between CD and NOD1/CARD4 SNPs.

In summary, we have examined in detail, both in adult- and childhood-onset IBD, the role of germline variation of NOD1/CARD4. Although the importance of NOD1/CARD4 in maintaining intestinal epithelial homeostasis through adequate recognition of mucosa-associated bacterial flora is undeniable, the present data argue strongly against a contribution of common haplotypic variation to inherited susceptibility to IBD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Study population and phenotypic classification
Two thousand two hundred and ninety-six subjects comprising 1323 IBD patients (662 CD, 628 UC and 33 IBDU), 603 parents and 370 controls were genotyped. IBD patients [624 males/699 females; median age at diagnosis (quartile 1—quartile 3) 26.08 years (14.83–39.91 years)] were recruited from the Western General Hospital and the three tertiary paediatric gastroenterology centres in Scotland (Edinburgh, Glasgow and Aberdeen). The cohort of children with IBD used in the TDT analysis was also used for the Scottish IBD case–control analysis (combined and childhood onset). Control subjects consisted of Scottish blood donors (n = 260) and healthy volunteers (as assessed by questionnaire) recruited as part of the local study of IBD genetics (n = 110). More than 97% of the study population (patients and controls) was Caucasian. There were 246 complete family trios (69%).

The diagnosis of IBD was based on standard criteria (74). Data were collected retrospectively by review of patient files and questionnaire as previously described (18,75). The Montreal classification was used to classify disease location and behaviour for CD and UC (46) (Table 5). In this recently revised classification system, CD location can be ileal (L1), colonic (L2), ileocolonic (L3) or upper gastrointestinal tract only (L4) with the added possibility of upper gastrointestinal tract disease acting as a modifier for the L1, L2 or L3 categories. CD behaviour is classified as non-stricturing/non-penetrating (B1), stricturing (B2) or penetrating (intestinal) (B3). Penetrating perianal disease can be scored as a suffix p, added to B1, B2 or B3. UC extent is classified as extensive (E3), left-sided colitis (E2) or proctitis only (E1). Local ethical committees at all the participating centres approved the study protocol. Informed, written consent was obtained from all participating patients, families and controls.

SNP selection
The NOD1/CARD4 gene is located between positions 30,430,672 and 30,484,833 (Ensembl Release 43—ENSG00000106100). Haplotype-tagging SNPs were selected using genotypic data from the CEU study group (30 US trios with Northern and Western European ancestry, collected by the Centre d'Etude du Polymorphisme Humain (CEPH)), available from the HapMap project (released on 21 July 2006; http://www.hapmap.org/cgi-perl/gbrowse/hapmap_B35/) (Fig. 1 and Table 1). SNPs were selected using Haploview software (version 3.32, freely available from http://www.broad.mit.edu/mpg/haploview/). SNPs were identified using a haplotype-tagging strategy based on solid spine of linkage disequilibrium (r2>0.8, haplotype frequency > 5%, minor allele frequency > 10%). Tagging of the 5' region of NOD1/CARD4 was achieved by tagging the rs2529447 variant of (block 5—Fig. 1) at position 30,501,735. The 3' region was tagged to position 30,437,394 (rs10267377, block 3—Fig. 1).

For all patients and controls, all SNPs conformed to the Hardy–Weinberg equilibrium (P > 0.01).

DNA extraction and genotyping
DNA samples were extracted from peripheral venous blood by a modified salting out technique or using the Nucleon kit (Tepnel Life Sciences PLC, Manchester, UK) (76). All individuals were genotyped for the nine SNPs tagging the NOD1/CARD4 gene using TaqMan (7900HT sequence detection system; Applied Biosystems, Foster City, CA, USA). The three common NOD2/CARD15 polymorphisms (R702W, G908R and Leu1007fsinsC) were genotyped as previously described (18,19).

Statistics
Allelic, genotype and haplotype frequencies were compared between cases and controls using {chi}2 (Minitab Software, Release 13.20, Minitab, Inc., State College, PA, USA, and GraphPad Instat software version 3.06, San Diego, CA, USA) and Haploview software (version 3.32). Single- and multiple-marker TDT analyses were performed using FBAT software (version 1.7.3, available from http://www.biostat.harvard.edu/~fbat/fbat.htm) (77). ORs (99.5% CI) for each of the SNPs were calculated using TDTHAP, run in R 2.5.0 (78). Log-likelihood analysis was carried out using PM software (1000 permutations—random seeds) (79). Power calculations were performed using QUANTO version 1.2 (http://hydra.usc.edu/gxe) (80,81). The method for integrating results from case–control studies and TDT to provide a combined estimate of disease-marker association was described by Kazeem and Farall (82) (Table 7). As this approach is only useful when the samples of subjects used in the two analyses are independent of each other, yet share the same genealogy, we based this calculation on our TDT cohort of early-onset IBD and our samples of adult-onset IBD patients in the case–control analysis.


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  		Table 7. A combined estimate of disease-marker association by integration of TDT and case–control analysis is given

 


   ACKNOWLEDGEMENTS
 
The authors acknowledge the contribution of staff at the Medical Research Council Human Genetics Unit Edinburgh, the Wellcome Trust Clinical Research Facility Edinburgh and the referring physicians from Scottish Gastrointestinal Medicine and Surgery services. The authors also like to acknowledge the help of all patients and parents who participated in the study together with the specialist nurses, dieticians and secretaries in each of the Scottish paediatric teaching hospitals as well as the paediatricians, practice nurses and GP's throughout Scotland whose support was invaluable. J.V.L. is funded by a Research Training Fellowship from Action Medical Research, The Gay-Ramsay-Steel-Maitland or Stafford Trust and the Hazel M. Wood Charitable Trust. R.K.R. was funded by a University of Edinburgh Medical Faculty Fellowship. E.R.N was supported by a Wellcome Trust Programme Grant (072789/Z/03/Z). Financial assistance was also provided by Schering-Plough and the GI/Nutrition Research Fund, Child Life and Health, University of Edinburgh.

Conflict of Interest statement: None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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