How to increase our belief in discovered statistical interactions via large-scale association studies?

The understanding that differences in biological epistasis may impact disease risk, diagnosis, or disease management stands in wide contrast to the unavailability of widely accepted large-scale epistasis analysis protocols. Several choices in the analysis workflow will impact false-positive and false-negative rates. One of these choices relates to the exploitation of particular modelling or testing strategies. The strengths and limitations of these need to be well understood, as well as the contexts in which these hold. This will contribute to determining the potentially complementary value of epistasis detection workflows and is expected to increase replication success with biological relevance. In this contribution, we take a recently introduced regression-based epistasis detection tool as a leading example to review the key elements that need to be considered to fully appreciate the value of analytical epistasis detection performance assessments. We point out unresolved hurdles and give our perspectives towards overcoming these.

Behind the (impedance) baseline in children.

Impedance baseline is a new parameter recently related to esophageal integrity. The aim of this study was to assess the effect of different factors on impedance baseline in pediatric patients. We analyzed the impedance baseline of 800 children with symptoms of gastroesophageal reflux. Mean impedance baseline was automatically calculated throughout 24-hour tracings. The presence of different age groups and of esophagitis was evaluated. Unpaired t-test, Spearman rank correlation, polynomial, and regression plot were used for statistical analysis. Age-related percentile curves were created. We considered a P-value<0.05 as statistically significant. Impedance baseline was significantly (P<0.001) lower in younger compared to older children up to 48 months. The mean increase of baseline per month was much higher in the first 36 months of life (47.5 vs. 2.9 Ohm in Channel 1 and 29.9 vs. 2.3 Ohm in Channel 6, respectively) than in older ages. Patients with esophagitis showed significantly decreased impedance baseline (P<0.05). Infants (especially in the first months of life) and young children present a significantly lower impedance baseline compared to older children both in proximal and distal esophagus. The presence of esophagitis may also determine a decreased impedance baseline regardless of the age of the patients.

Effects of haptoglobin polymorphisms and deficiency on susceptibility to inflammatory bowel disease and on severity of murine colitis.

BACKGROUND: Haptoglobin (Hp) is a haemoglobin-binding protein with immunomodulatory properties. Its gene (16q22) harbours a common polymorphism with two different alleles: Hp1 and Hp2. Genotype Hp22 has been shown to be over-represented in different immune diseases. Results in Crohn's disease (CD) are contradictory. AIMS: To determine whether Hp plays a role in inflammatory bowel disease, both genetically and functionally. METHODS: 1061 patients with CD, 755 with ulcerative colitis (UC) and 152 with primary sclerosing cholangitis, as well as 452 healthy controls, were genotyped using touch-down PCR. To confirm association results, 464 CD trios and 151 UC trios were genotyped. Serum Hp concentrations were determined in 62 individuals of different genotype. Colitis was induced in mice with dextran sulphate sodium (DSS) and oxazolone (Oxa). Cytokine production was evaluated by mRNA quantification in colonic tissue and ELISA on supernatants of mesenteric lymph node cells. RESULTS: Prevalence of Hp2 was higher in CD and UC than in controls. In the confirmatory cohorts, Hp2 was over-transmitted to the affected offspring. Serum Hp concentrations were higher in individuals with genotypes Hp11 and Hp21 than in those with Hp22 (1.38 vs 0.89 g/l). DSS- and Oxa-induced colitis were more severe in Hp-deficient mice than in control mice and accompanied by higher concentrations (although not statistically significantly different) of tissue mRNA for cytokines. Interleukin-17 production was significantly higher in the presence of Hp-deficient serum compared with wild-type serum. CONCLUSIONS: The Hp gene may play a role in susceptibility to inflammatory bowel disease. Its implication in other immune diseases underscores the common pathways between these diseases. Experimental models of colitis showed that Hp has a protective role in inflammatory colitis, most likely by inhibiting the production of Th1 and Th17 cytokines.

Effects of domperidone on QTc interval in infants.

AIM: To prospectively evaluate the effects of oral domperidone on the QTc interval in infants. METHODS: Infants (0-1 year) with a diagnosis of gastro-oesophageal reflux (GOR) disease were included. A 12-lead electrocardiography (ECG) was performed in all infants at baseline and 1 h after the intake of domperidone after 7-14 days; the corrected QTc interval was calculated by one investigator (MV) according to Bazett's formula. RESULTS: Forty-five infants were enrolled in this study. The mean gestational age was of 38.6 weeks (35.5-42.0), and the mean age at the start of domperidone was 75.3 days (19-218 days). No statistically significant difference in corrected QTc was observed between baseline and the second ECG (0.389 ± 0.02 vs. 0.397 ± 0.31; p 0.130)). A trend was observed regarding gender: Although there was no difference in QTc change in girls (p 0.622), there was a strong trend in boys (p 0.051). Two infants (both boys) had a clinically significant QTc prolongation (> 460 msec) without symptoms. The Spearman correlation test showed no relation between the QTc change and age (r: -0.05822; p 0.7284). There was no relation between domperidone dosage and QTc change. CONCLUSION: Overall, the group-analysis showed no statistical significant difference in QTc duration induced by domperidone. However, 2/45 (4.4%) infants had a prolonged QTc interval (> 460 msec) induced by domperidone. As a consequence, QTc measurement should be recommended in routine in infants when domperidone is started.

Perspectives on genome-wide multi-stage family-based association studies.

With the establishment of large consortiums of researchers, genome-wide association (GWA) studies have become increasingly popular and feasible. Although most of these association studies focus on unrelated individuals, a lot of advantages can be exploited by including families in the analysis as well. To overcome the additional genotyping cost, multi-stage designs are particularly useful. In this article, I offer a perspective view on genome-wide family-based association analyses, both within a model-based and model-free paradigm. I highlight how multi-stage designs and analysis techniques, which are quite popular in clinical epidemiology, can enter GWA settings. I furthermore discuss how they have proven successful in reducing analysis complexity, and in overcoming one of the most cumbersome statistical hurdles in the genome-wide context, namely controlling increased false positives due to multiple testing.

Analysis of the high affinity IgE receptor genes reveals epistatic effects of FCER1A variants on eczema risk.

BACKGROUND: High levels of total and allergen-specific IgE levels are a key feature in allergic diseases. The high-affinity receptor for IgE, which is composed of one alpha (FCER1A), one beta (FCER1B), and two gamma (FCER1G) subunits, represents the central receptor of IgE-induced reactions. In a genome-wide association scan, we recently identified associations between functional FCER1A variants and total serum IgE levels. Previous studies had reported linkage and association of FCER1B variants with IgE and atopic traits. The FCER1G gene has not yet been investigated with regard to atopy. Filaggrin (FLG) is the strongest known risk gene for eczema, in particular the allergic subtype of eczema. METHODS: We investigated the association of FCER1A, FCER1B, and FCER1G variants with IgE in a large population-based cohort (n = 4261) and tested for epistatic effects using the model-based multifactor dimensionality reduction (MB-MDR) method. In addition, we investigated a potential interaction between FLG and FCER1A variants in a large collection of eczema cases (n = 1018) and population controls. RESULTS: Three strongly correlated FCER1A polymorphisms were significantly associated with total and specific IgE levels as well as allergic sensitization. No associations were seen for FCER1B and FCER1G. After adjustment for FLG effects, a significant epistatic effect of the FCER1A variants rs10489854 and rs2511211 on eczema risk was detected. CONCLUSIONS: These results suggest that FCER1A variants by themselves and in combination influence IgE levels and act synergistically to influence eczema risk.

Long-term outcome of treatment with infliximab in 614 patients with Crohn's disease: results from a single-centre cohort.

BACKGROUND AND AIMS: This observational study assessed the long-term clinical benefit of infliximab (IFX) in 614 consecutive patients with Crohn's disease (CD) from a single centre during a median follow-up of 55 months (interquartile range (IQR) 27-83). METHODS: The primary analysis looked at the proportion of patients with initial response to IFX who had sustained clinical benefit at the end of follow-up. The long-term effects of IFX on the course of CD as reflected by the rate of surgery and hospitalisations and need for corticosteroids were also analysed. RESULTS: 10.9% of patients were primary non-responders to IFX. Sustained benefit was observed in 347 of the 547 patients (63.4%) receiving long-term treatment. In 68.3% of these, treatment with IFX was ongoing and in 31.7% IFX was stopped, with the patient being in remission. Seventy patients (12.8%) had to stop IFX due to side effects and 118 (21.6%) due to loss of response. Although the yearly drop-out rates of IFX in patients with episodic (10.7%) and scheduled treatment (7.1%) were similar, the need for hospitalisations and surgery decreased less in the episodic than in the scheduled group. Steroid discontinuation also occurred in a higher proportion of patients in the scheduled group than in the episodic group. CONCLUSIONS: In this large real-life cohort of patients with CD, long-term treatment with IFX was very efficacious to maintain improvement during a median follow-up of almost 5 years and changed disease outcome by decreasing the rate of hospitalisations and surgery.

Mucosal gene signatures to predict response to infliximab in patients with ulcerative colitis.

BACKGROUND AND AIMS: Infliximab is an effective treatment for ulcerative colitis with over 60% of patients responding to treatment and up to 30% reaching remission. The mechanism of resistance to anti-tumour necrosis factor alpha (anti-TNFalpha) is unknown. This study used colonic mucosal gene expression to provide a predictive response signature for infliximab treatment in ulcerative colitis. METHODS: Two cohorts of patients who received their first treatment with infliximab for refractory ulcerative colitis were studied. Response to infliximab was defined as endoscopic and histological healing. Total RNA from pre-treatment colonic mucosal biopsies was analysed with Affymetrix Human Genome U133 Plus 2.0 Arrays. Quantitative RT-PCR was used to confirm microarray data. RESULTS: For predicting response to infliximab treatment, pre-treatment colonic mucosal expression profiles were compared for responders and non-responders. Comparative analysis identified 179 differentially expressed probe sets in cohort A and 361 in cohort B with an overlap of 74 probe sets, representing 53 known genes, between both analyses. Comparative analysis of both cohorts combined, yielded 212 differentially expressed probe sets. The top five differentially expressed genes in a combined analysis of both cohorts were osteoprotegerin, stanniocalcin-1, prostaglandin-endoperoxide synthase 2, interleukin 13 receptor alpha 2 and interleukin 11. All proteins encoded by these genes are involved in the adaptive immune response. These markers separated responders from non-responders with 95% sensitivity and 85% specificity. CONCLUSION: Gene array studies of ulcerative colitis mucosal biopsies identified predictive panels of genes for (non-)response to infliximab. Further study of the pathways involved should allow a better understanding of the mechanisms of resistance to infliximab therapy in ulcerative colitis. ClinicalTrials.gov number, NCT00639821.

TNFA -308G>A in two international population-based cohorts and risk of asthma.

Genetic association studies have related the tumour necrosis factor-alpha gene (TNFA) guanine to adenine substitution of nucleotide -308 (-308G>A) polymorphism to increased risk of asthma, but results are inconsistent. The aim of the present study was to test whether two single-nucleotide polymorphisms, of TNFA and of the lymphotoxin-alpha gene (LTA), are associated with asthma, bronchial hyperresponsiveness and atopy in adults, by combining the results of two large population-based multicentric studies and conducting a meta-analysis of previously published studies. The European Community Respiratory Health Survey (ECRHS) and Swiss Cohort Study on Air Pollution and Lung and Heart Diseases in Adults (SAPALDIA) used comparable protocols, including questionnaires for respiratory symptoms and measures of lung function and atopy. DNA samples from 11,136 participants were genotyped at TNFA -308 and LTA 252. Logistic regression employing fixed and random effects models and nonparametric techniques were used. The prevalence of asthma was 6%. The TNFA -308G>A polymorphism was associated with increased asthma prevalence and with bronchial hyperresponsiveness. No consistent association was found for atopy. The LTA 252A>G polymorphism was not associated with any of the outcomes. A meta-analysis of 17 studies showed an increased asthma risk for the TNFA -308 adenine allele. The tumour necrosis factor-alpha gene nucleotide -308 polymorphism is associated with a moderately increased risk of asthma and bronchial hyperresponsiveness, but not with atopy. These results are supported by a meta-analysis of previously published studies.

Anti-infliximab antibody concentrations can guide treatment intensification in patients with Crohn's disease who lose clinical response.

BACKGROUND: The presence of antibodies towards infliximab (ATI) is associated with lower infliximab (IFX) trough concentrations and loss of response. IFX treatment intensification is effective for restoring response in most, but not all patients with Crohn's disease (CD). AIM: To compare outcome, pharmacokinetics and immunogenicity of treatment intensification strategies in patients with CD who lost clinical response to IFX. METHODS: A retrospective cohort study was conducted, including 103 patients with CD who lost clinical response during IFX maintenance therapy and therefore received a double dose IFX (10 mg/kg) and/or a next infusion after a shortened interval. IFX and ATI concentrations were measured in consecutive trough samples, just before (T0) and after (T+1) treatment intensification. RESULTS: Clinical response (physicians' global assessment) and biological response and remission (CRP) were restored in 63%, 42% and 24% of patients (evaluated at T+1). Treatment intensification increased IFX trough concentrations from 1.2 µg/mL [0.3-3.6] at T0 to 3.6 µg/mL [0.5-10.2] at T+1 (P < .0001). Using a drug tolerant assay, ATI were detected in the T0 sample of 47% of patients. ATI negatively impacted the achieved IFX trough concentration (Spearman r -0.57, P < .0001) and the probability of clinical response (P = 0.034) at T+1. When ATI were quantifiable but <282 ng/mL eq. at T0, combined interval shortening and dose doubling was more effective for restoring therapeutic IFX trough concentrations (≥3 µg/mL at T+1) than dose doubling alone, which in turn was more effective than interval shortening alone (P < .001). CONCLUSION: Antibodies towards infliximab can guide clinical decision-making on treatment intensification.

Baseline health-related quality-of-life data as prognostic factors in a phase III multicentre study of women with metastatic breast cancer.

The potential value of baseline health-related quality-of-life (HRQOL) and clinical factors in predicting prognosis was examined using data from an international randomised phase III trial which compared doxorubicin and paclitaxel with doxorubicin and cylophosphamide as first line chemotherapy in 275 women with metastatic breast cancer. The European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 and the related breast module (QLQ-BR23) were used to assess baseline HRQOL data. The Cox proportional-hazards regression model was used for both univariate and multivariate analyses of survival. In the univariate analyses, performance status (P<0.001) and number of sites involved (P=0.001) were the most important clinical prognostic factors. The HRQOL variables at baseline most strongly associated with longer survival were better appetite, physical and role functioning, as well as less fatigue (P<0.001). The final multivariate model retained performance status (P<0.001) and appetite loss (P=0.005) as the variables best predicting survival. Substantial loss of appetite was the only independent HRQOL factor predicting poor survival and was strongly correlated (/r/>0.5) with fatigue, role and physical functioning. In addition to known clinical factors, appetite loss appears to be a significant prognostic factor for survival in women with metastatic breast cancer. However, the mechanism underlying this association remains to be precisely defined in future studies.

Genetic variations in toll-like receptor pathway and lung function decline in Cystic fibrosis patients.

The toll-like receptor (TLR) family maintains pulmonary homeostasis by pathogen recognition, clearance and regulation of inflammation. Genes affecting inflammation response play a key role in modifying Cystic fibrosis (CF) lung disease severity. We assessed the impact of single nucleotide polymorphisms (SNPs) of TLR genes (TLR1 to TLR10, CD14, lipopolyssacharide-binding protein (LBP)) on lung function in CF patients. Each SNP was tested for time-dependent effect on FEV1, using six genetic models. In addition, we investigated associations between SNP genotypes and extreme subject specific slopes of FEV1 decline. Variant alleles of polymorphisms of TLR2 rs1898830, rs5743708, and rs3804100 demonstrated a consistent association with lung disease severity (p = 0.008, p = 0.006 and p = 0.029 respectively). Patients homozygous for variant C allele of TLR5 polymorphism rs5744174 are more frequently associated with extreme fast FEV1 decline (OR: 20 (95% Confidence Interval:1.85-216.18)). Patients homozygous AA for TLR1 polymorphism rs5743551 are more frequently associated with faster decline of FEV1 compared to heterozygous genotype (OR:7.33 (95% CI:1.63-33.11). Our findings indicate that variations in TLR1, TLR2 and TLR5 genes may influence CF lung function decline. Further functional analysis is required to provide new insights into the pathogenesis of TLRs in CF lung disease severity.

EU Pancreas: an integrated European platform for pancreas cancer research--from basic science to clinical and public health interventions for a rare disease.

BACKGROUND: Large-scale international collaboration is essential to decipher relevant information in the context of omics-scale interrogations in cancer research. This is even more important for rare and fatal diseases like pancreas cancer (PC). METHODS: The COST Action BM1204 is a unique platform to facilitate the collaboration of a broad range of European and international PC multidisciplinary research groups in order to: (1) integrate knowledge and experience in a multidisciplinary way 'from cell to society', (2) promote the application of uniform study tools and protocols, (3) foster their optimal use by early-stage researchers, (4) enhance the mobility and training of researchers, and (5) disseminate the results produced to the broader society. RESULTS: This Action will develop novel interdisciplinary tools for collaborative research to improve our understanding of PC and its prevention, diagnosis and treatment. It also aims to answer questions related to the etiology, early detection, evidence-based and personalized treatment, and health management for PC. Furthermore, the Action will contribute to new insights into PC personalized medicine and beyond as well as to the understanding of complex and rare diseases taking PC as a best practice example. The Action aims at attracting young scholars across a range of disciplines in collaboration with more experienced researchers and enhancing active European participation in the international scenario of PC research. CONCLUSION: The ultimate aim is to foster PC research in Europe and to coordinate this effort with other international initiatives to reduce disease mortality.

Profile of pediatric Crohn's disease in Belgium.

AIM: A Belgian registry for pediatric Crohn's disease, BELCRO, was created. This first report aims at describing disease presentation and phenotype and determining associations between variables at diagnosis and registration in the database. METHODS: Through a collaborative network, children with previously established Crohn's disease and newly diagnosed children and adolescents (under 18 y of age) were recruited over a 2 year period. Data were collected by 23 centers and entered in a database. Statistical association tests analyzed relationships between variables of interest at diagnosis. RESULTS: Two hundred fifty-five patients were included. Median age at diagnosis was 12.5 y (range: 1.6-18 y); median duration of symptoms prior to diagnosis was 3 m (range: 1-12 m). Neonatal history and previous medical history did not influence disease onset nor disease behavior. Fifty three % of these patients presented with a BMI z-score < -1. CRP was an independent predictor of disease severity. Steroids were widely used as initial treatment in moderate to severe and extensive disease. Over time, immunomodulators and biological were prescribed more frequently, reflecting a lower prescription rate for steroids and 5-ASA. A positive family history was the sole significant determinant for earlier use of immunosuppression. CONCLUSION: In Belgium, the median age of children presenting with Crohn's disease is 12.5 y. Faltering growth, extensive disease and upper GI involvement are frequent. CRP is an independent predictive factor of disease activity. A positive family history appears to be the main determinant for initial treatment choice.

Polymorphisms in the lectin pathway genes as a possible cause of early chronic Pseudomonas aeruginosa colonization in cystic fibrosis patients.

Genes of innate immunity may be involved in early onset of chronic Pa (Pseudomonas aeruginosa) colonization (cPaC) in cystic fibrosis (CF) patients. We studied 19 single nucleotide polymorphisms (SNPs) in 5 genes coding for proteins of the lectin complement pathway: MBL2 (Mannose binding lectin 2), MASP 1, 2, 3 (MBL-associated serine Protease) and FCN 1, 2 (Ficolin) gene in 96 CF patients. Association survival analysis using different genetic models was performed looking for an association between SNPs and age at onset of cPaC. CF patients who are MBL deficient are earlier chronic Pa colonized compared to MBL sufficient patients. Also patients with MBL2 genotype YO/YO, YO/XA, XA/XA, YA/YO and YA/XA are earlier chronic Pa colonized. CF patients heterozygous or homozygous for mutant alleles of two linked SNPs in the FCN1 gene (rs2989727 and rs1071583) are earlier colonized with Pa. Similarly, earlier onset of Pa colonization is seen in CF patients heterozygous for linked SNPs of FCN2 gene (rs7865453 and rs7851696) and MASP3 gene (rs7851696). Variants in MBL2, FCN1, FCN2 and MASP3 genes are significantly associated with earlier onset of chronic P. aeruginosa colonization.

Safety and cost of infliximab for the treatment of Belgian pediatric patients with Crohn's disease.

Biologicals have become an important component in the treatment of Crohn's disease in children. Their increased and long term use raises safety concerns. We describe safety and cost of infliximab in Belgian pediatric Crohn's disease patients. All patients on infliximab as part of the present or past treatment for Crohn's Disease until January 1st 2011 were selected from an existing database. Information on disease phenotype, medication and adverse events were extracted. Adverse events occurred in 25.9% of patients exposed to infliximab of which 29.6% were severe. In total 31.7% of patients stopped infliximab therapy. The main reasons for discontinuation were adverse events in 45.4% and loss of response in 30.3%. No malignancies or lethal complications occurred over this 241 patient year observation period. Immunomodulators were concomitant medication in 75% of patients and were discontinued subsequently in 38.4% of them. The cost of infliximab infusions per treated patient per year in the Belgian health care setting is approximately 9 474 euro, including only medication and hospital related costs. Even though infliximab is relatively safe in pediatric CD on the short term, close follow-up and an increased awareness of the possible adverse reactions is highly recommended. Adverse reactions appeared in 25.9% of all patients and were the main reason for discontinuation. Treatment cost has to be balanced against efficacy and modifications in disease course. In the Belgian health care system, the medication is available to all patients with moderate to severe CD.

Approaches to handling incomplete data in family-based association testing.

The high throughput of data arising from the complete sequence of the human genome has left statistical geneticists with a rich and extensive information source. The wide availability of software and the increase in computing power has improved the possibilities to access and process such data. One problem is incompleteness of the data: unobserved or partially observed data points due to technical reasons or reasons associated with the patient's status or erroneous measurements of phenotype or genotype, to name a few. When not properly accounted for, these sources of incompleteness may seriously jeopardize the credibility of results from analyses. In this paper we provide some perspectives on the occurrence and analysis of different forms of incomplete data in family-based genetic association testing.

Sensitivity analysis of longitudinal binary quality of life data with drop-out: an example using the EORTC QLQ-C30.

Analysing quality of life data (QOL) may be complicated for several reasons. Quality of life data not only involves repeated measures but is also usually collected on ordered categorical responses. In addition, it is evident that not all patients provide the same number of assessments, due to attrition caused by death or other medical reasons. In the recent statistical literature, increasing attention is given to methods which can handle non-continuous outcomes in the presence of missing data. The aim of this paper is to investigate the effect on statistical conclusions of applying different modelling techniques to QOL data generated from an EORTC phase III trial. Treatment effects and treatment differences are of major concern. First, a random-effects model is fitted, relating a binary longitudinal response (derived from the physical functioning scale of the QLQ-C30) to several covariates. In a second approach, marginal models are fitted, retaining the response variable and the mean structure used before. The fitted marginal models only differ with respect to the considered estimation procedure: generalized estimating equations (GEE); weighted generalized estimating equations (WGEE), and maximum likelihood (ML).

Genome wide scan in a Flemish inflammatory bowel disease population: support for the IBD4 locus, population heterogeneity, and epistasis.

BACKGROUND AND AIMS: Genome wide scans in inflammatory bowel disease (IBD) have indicated various susceptibility regions with replication of 16cen (IBD1), 12q (IBD2), 6p (IBD3), 14q11 (IBD4), and 3p21. As no linkage was previously found on IBD regions 3, 7, 12, and 16 in Flemish IBD families, a genome wide scan was performed to detect other susceptibility regions in this population. METHODS: A cohort of 149 IBD affected relative pairs, all recruited from the Northern Flemish part of Belgium, were genotyped using microsatellite markers at 12 cM intervals, and analysed by Genehunter non-parametric linkage software. All families were further genotyped for the three main Crohn's disease associated variants in the NOD2/CARD15 gene. RESULTS: Nominal evidence for linkage was observed on chromosomes 1 (D1S197: multipoint non-parametric linkage (NPL) score 2.57, p = 0.004; and at D1S305-D1S252: NPL 2.97, p = 0.001), 4q (D4S406: NPL 1.95, p = 0.03), 6q16 (D6S314: NPL 2.44, p = 0.007), 10p12 (D10S197: NPL 2.05, p = 0.02), 11q22 (D11S35-D11S927: NPL 1.95, p = 0.02) 14q11-12 (D14S80: NPL 2.41, p = 0.008), 20p12 (D20S192: NPL 2.7, p = 0.003), and Xq (DXS990: NPL 1.70, p = 0.04). A total of 51.4% of patients carried at least one NOD2/CARD15 variant. Furthermore, epistasis was observed between susceptibility regions 6q/10p and 20p/10p. CONCLUSION: Genome scanning in a Flemish IBD population found nominal evidence for linkage on 1p, 4q, 10p12, and 14q11, overlapping with other genome scan results, with linkage on 14q11-12 supporting the IBD4 locus. The results further show that epistasis is contributing to the complex model of IBD and indicate that population heterogeneity is not to be underestimated. Finally, NOD2/CARD15 is clearly implicated in the Flemish IBD population.