Principal components analysis of Raman spectral data for screening of Hepatitis C infection.

In the current study, Raman spectroscopy is employed for the identification of the biochemical changes taking place during the development of Hepatitis C. The Raman spectral data acquired from the human blood plasma samples of infected and healthy individuals is analysed by Principal Components Analysis and the Raman spectral markers of the Hepatitis C Virus (HCV) infection are identified. Spectral changes include those associated with nucleic acidsat720 cm-1, 1077 cm-1 1678 (CO stretching mode of dGTP of RNA), 1778 cm-1 (RNA), with proteins at 1641 cm-1(amide-I), 1721 cm-1(CC stretching of proteins) and lipids at 1738 cm-1(CO of ester group in lipids). These differences in Raman spectral features of blood plasma samples of the patients and healthy volunteers can be associated with the development of the biochemical changes during HCV infection.

Raman spectroscopy of blood plasma samples from breast cancer patients at different stages.

Raman spectroscopy was employed for the characterization of blood plasma samples from patients at different stages of breast cancer. Blood plasma samples taken from clinically diagnosed breast cancer patients were compared with healthy controls using multivariate data analysis techniques (principal components analysis - PCA) to establish Raman spectral features which can be considered spectral markers of breast cancer development. All the stages of the disease can be differentiated from normal samples. It is also found that stage 2 and 3 are biochemically similar, but can be differentiated from each other by PCA. The Raman spectral data of the stage 4 is found to be biochemically distinct, but very variable between patients. Raman spectral features associated with DNA and proteins were identified, which are exclusive to patient plasma samples. Moreover, there are several other spectral features which are strikingly different in the blood plasma samples of different stages of breast cancer. In order to further explore the potential of Raman spectroscopy as the basis of a minimally invasive screening technique for breast cancer diagnosis and staging, PCA-Factorial Discriminant Analysis (FDA) was employed to classify the Raman spectral datasets of the blood plasma samples of the breast cancer patients, according to different stages of the disease, yielding promisingly high values of sensitivity and specificity for all stages.

Pooled sequencing of 531 genes in inflammatory bowel disease identifies an associated rare variant in BTNL2 and implicates other immune related genes.

The contribution of rare coding sequence variants to genetic susceptibility in complex disorders is an important but unresolved question. Most studies thus far have investigated a limited number of genes from regions which contain common disease associated variants. Here we investigate this in inflammatory bowel disease by sequencing the exons and proximal promoters of 531 genes selected from both genome-wide association studies and pathway analysis in pooled DNA panels from 474 cases of Crohn's disease and 480 controls. 80 variants with evidence of association in the sequencing experiment or with potential functional significance were selected for follow up genotyping in 6,507 IBD cases and 3,064 population controls. The top 5 disease associated variants were genotyped in an extension panel of 3,662 IBD cases and 3,639 controls, and tested for association in a combined analysis of 10,147 IBD cases and 7,008 controls. A rare coding variant p.G454C in the BTNL2 gene within the major histocompatibility complex was significantly associated with increased risk for IBD (p = 9.65x10-10, OR = 2.3[95% CI = 1.75-3.04]), but was independent of the known common associated CD and UC variants at this locus. Rare (<1%) and low frequency (1-5%) variants in 3 additional genes showed suggestive association (p<0.005) with either an increased risk (ARIH2 c.338-6C>T) or decreased risk (IL12B p.V298F, and NICN p.H191R) of IBD. These results provide additional insights into the involvement of the inhibition of T cell activation in the development of both sub-phenotypes of inflammatory bowel disease. We suggest that although rare coding variants may make a modest overall contribution to complex disease susceptibility, they can inform our understanding of the molecular pathways that contribute to pathogenesis.

Exome sequencing of 75 individuals from multiply affected coeliac families and large scale resequencing follow up.

Coeliac disease (CeD) is a highly heritable common autoimmune disease involving chronic small intestinal inflammation in response to dietary wheat. The human leukocyte antigen (HLA) region, and 40 newer regions identified by genome wide association studies (GWAS) and dense fine mapping, account for ∼40% of the disease heritability. We hypothesized that in pedigrees with multiple individuals with CeD rare [minor allele frequency (MAF) <0.5%] mutations of larger effect size (odds ratios of ∼2-5) might exist. We sequenced the exomes of 75 coeliac individuals of European ancestry from 55 multiply affected families. We selected interesting variants and genes for further follow up using a combination of: an assessment of shared variants between related subjects, a model-free linkage test, and gene burden tests for multiple, potentially causal, variants. We next performed highly multiplexed amplicon resequencing of all RefSeq exons from 24 candidate genes selected on the basis of the exome sequencing data in 2,248 unrelated coeliac cases and 2,230 controls. 1,335 variants with a 99.9% genotyping call rate were observed in 4,478 samples, of which 939 were present in coding regions of 24 genes (Ti/Tv 2.99). 91.7% of coding variants were rare (MAF <0.5%) and 60% were novel. Gene burden tests performed on rare functional variants identified no significant associations (p<1×10(-3)) in the resequenced candidate genes. Our strategy of sequencing multiply affected families with deep follow up of candidate genes has not identified any new CeD risk mutations.

Integrative biology approach identifies cytokine targeting strategies for psoriasis.

Cytokines are critical checkpoints of inflammation. The treatment of human autoimmune disease has been revolutionized by targeting inflammatory cytokines as key drivers of disease pathogenesis. Despite this, there exist numerous pitfalls when translating preclinical data into the clinic. We developed an integrative biology approach combining human disease transcriptome data sets with clinically relevant in vivo models in an attempt to bridge this translational gap. We chose interleukin-22 (IL-22) as a model cytokine because of its potentially important proinflammatory role in epithelial tissues. Injection of IL-22 into normal human skin grafts produced marked inflammatory skin changes resembling human psoriasis. Injection of anti-IL-22 monoclonal antibody in a human xenotransplant model of psoriasis, developed specifically to test potential therapeutic candidates, efficiently blocked skin inflammation. Bioinformatic analysis integrating both the IL-22 and anti-IL-22 cytokine transcriptomes and mapping them onto a psoriasis disease gene coexpression network identified key cytokine-dependent hub genes. Using knockout mice and small-molecule blockade, we show that one of these hub genes, the so far unexplored serine/threonine kinase PIM1, is a critical checkpoint for human skin inflammation and potential future therapeutic target in psoriasis. Using in silico integration of human data sets and biological models, we were able to identify a new target in the treatment of psoriasis.

Multiple common variants for celiac disease influencing immune gene expression.

We performed a second-generation genome-wide association study of 4,533 individuals with celiac disease (cases) and 10,750 control subjects. We genotyped 113 selected SNPs with P(GWAS) < 10(-4) and 18 SNPs from 14 known loci in a further 4,918 cases and 5,684 controls. Variants from 13 new regions reached genome-wide significance (P(combined) < 5 x 10(-8)); most contain genes with immune functions (BACH2, CCR4, CD80, CIITA-SOCS1-CLEC16A, ICOSLG and ZMIZ1), with ETS1, RUNX3, THEMIS and TNFRSF14 having key roles in thymic T-cell selection. There was evidence to suggest associations for a further 13 regions. In an expression quantitative trait meta-analysis of 1,469 whole blood samples, 20 of 38 (52.6%) tested loci had celiac risk variants correlated (P < 0.0028, FDR 5%) with cis gene expression.

Structural studies of B-type Aurora kinase inhibitors using computational methods.

AIM: To characterize the structural features of quinazoline-based Aurora B inhibitors that influence its inhibitor activity. METHODS: Two geometrical methods, Method 1 and Method 2, were used to develop the 3D-QSAR models. The most active ligand was used as the template for the alignment of all the ligands in Method 1, and a conformer of the cocrystal ligand was used as the template for the alignment of all the ligands in Method 2. RESULTS: The models suggest that highly active ligands can be designed by varying the R1 substituent at position 7 of the quinazoline ring with positively charged, bulky, hydrophobic groups, while bulky and hydrophobic groups around the thiazole ring are desirable for higher activity. CONCLUSION: This study emphasizes that the bioactive conformer is rather different from the minima. The steric, electrostatic, and hydrophobic field effects contribute to its inhibitory activity.

Pharmacophore and docking-based combined in-silico study of KDR inhibitors.

The growth and metastasis of solid tumors is dependent on angiogenesis. The vascular endothelial growth factor (VEGF) and its cell surface receptor in human KDR (kinase domain containing receptor or VEGFR-2) have particular interest because of their importance in angiogenesis. The development of novel inhibitors of VEGFR-2 would be helpful to check the growth of tumors. Quantitative structure activity relationship (QSAR) analyses used to understand the structural factors affecting inhibitory potency of thiazole-substituted pyrazolone derivatives. Several pharmacophore-based models indicated the importance of steric, hydrophobic and hydrogen bond acceptor groups to inhibitory activity. The comparative molecular field analyses (CoMFA) and comparative molecular similarity indices analyses (CoMSIA) based 3D-QSAR models were derived using pharmacophore-based alignment. Both CoMFA (q(2)=0.70, r(2)=0.97 and r(predictive)(2)=0.61) and CoMSIA (q(2)=0.54, r(2)=0.82 and r(predictive)(2)=0.66) gave reasonable results. The molecular docking (receptor-guided technique) with a recently reported receptor structure (PDB=1YWN) were performed. The docked alignment was subsequently used for 3D-QSAR (CoMFA; q(2)=0.56, r(2)=0.97, r(predictive)(2)=0.82, CoMSIA; q(2)=0.58 r(2)=0.91, r(predictive)(2)=0.69). The overall both studies were indicated, steric, electrostatic and hydrogen bond acceptor effects contribute to the inhibitory activity. CoMFA and CoMSIA models suggested that a positive bulk with hydrophobic effect is desirable around position 4 and 5 and hydrogen bond acceptor groups around pyrazolones ring will be helpful.

Elucidation of binding mode and three dimensional quantitative structure-activity relationship studies of a novel series of protein kinase B/Akt inhibitors.

Protein kinase B (PKB; also known as Akt kinase) is located downstream in the PI-3 kinase pathway. Overexpression and constitutive activation of PKB/Akt leads to human prostate, breast and ovarian carcinomas. A series of 69 PKB/Akt inhibitors were examined to explore their binding modes using FlexX, and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies based on comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed to provide structural insights into these compounds. CoMFA produced statistically significant results, with cross-validated q ( 2 ) and non-cross validated correlation r(2) coefficients of 0.53 and 0.95, respectively. For CoMSIA, steric, hydrophobic and hydrogen bond acceptor fields jointly yielded 'leave one out' q(2) = 0.51 and r(2) = 0.84. The predictive power of CoMFA and CoMSIA was determined using a test set of 13 molecules, which gave correlation coefficients, r(2)(predictive) of 0.58 and 0.62, respectively. Molecular docking revealed that the binding modes of these molecules in the ATP binding sites of the Akt kinase domain were very similar to those of the co-crystallized ligand. The information obtained from 3D contour maps will allow the design of more potent and selective Akt kinase inhibitors.

Receptor guided 3D-QSAR: a useful approach for designing of IGF-1R inhibitors.

Research by other investigators has established that insulin-like growth factor-1 receptor (IGF-1R) is a key oncological target, and that derivatives of 1, 3-disubstituted-imidazo[1,5-alpha] pyrazine are potent IGF-1R inhibitors. In this paper, we report on our three-dimensional quantitative structure activity relationship (3D-QSAR) studies for this series of compounds. We validated the 3D-QSAR models by the comparison of two major alignment schemes, namely, ligand-based (LB) and receptor-guided (RG) alignment schemes. The latter scheme yielded better 3D-QSAR models for both comparative molecular field analysis (CoMFA) (q(2) = 0.35, r(2) = 0.95) and comparative molecular similarity indices analysis (CoMSIA) (q(2) = 0.51, r(2) = 0.86). We submit that this might arise from the more accurate inhibitor alignment that results from using the structural information of the active site. We conclude that the receptor-guided 3D-QSAR may be helpful to design more potent IGF-1R inhibitors, as well as to understand their binding affinity with the receptor.

3D and quantum QSAR of non-benzodiazepine compounds.

A combined physicochemical and 3D technique is used to establish the QSAR of four series of non-benzodiazepines towards BzR. In physicochemical QSAR study, the semi-empirical PM3 based parameters like hardness, electronegativity, electrophilicity index, molar refractivity, heat of formation, solvent assessable surface area and logP were used as descriptors. The heat of formation (HF) and logP are recognized as the most important descriptors for binding affinity of such compounds to BzR. The 3D QSAR study reveals that beta-Carbolines of series "A" and Imidazo[1, 2-alpha]pyrimidines of series "C" show steric bulk interaction, the beta-Carbolines of series "B" and di-substituted purines of series "D" have dominance of electrostatic interaction with BzR. The CoMSIA also indicate the same trend in terms of steric and electrostatic interaction. The CoMSIA reveals that only in case of series "D", the hydrophobic field effect is important. Some possible candidates for all four series were designed and their probable activities were estimated by using different QSAR models. The activities of designed molecules are in better range and developed models might be helpful to design the potent ligands of BzR.

Novel isoforms of the CARD8 (TUCAN) gene evade a nonsense mutation.

CARD8 (TUCAN) is implicated in the regulation of apoptosis and inflammation, and is a positional and functional candidate gene for inflammatory bowel disease (IBD). Recent investigations have reported conflicting results of association between a CARD8 nonsynonymous SNP, rs2043211, and IBD. SNP rs2043211 results in an A>T transversion in the CARD8 template strand, which introduces a stop codon polymorphism (Cys10Stop), and genotyping of the Cys10Stop variant revealed that 9% of the control population was homozygous for the 'Stop' allele. The effect of the Stop allele on mRNA and protein expression of the two known isoforms of this gene was investigated. IBD patients homozygous for the Stop allele showed somewhat reduced expression of CARD8 mRNA, but, contrary to expectation, expressed a 48 kDa protein isoform. A search of the EST database and reverse transcription-PCR analysis revealed a novel coding exon and three novel CARD8 mRNA isoforms that are conserved in primates. The isoforms of CARD8 differ in their N-termini, resulting in diverse predicted molecular weights (47, 48, 51, 54 and 60 kDa) and multiple outcomes for the variant including Cys10Stop, Cys34Stop, Phe52Ile and Phe102Ile; one isoform may arise through transcription and translation initiated downstream of rs2043211 to yield a novel protein isoform of approximately 47 kDa. The multiple isoforms and differing consequences for a predicted stop codon polymorphism underline the importance of detailed analysis of the effects of proposed functional variants on gene expression.

Diverse effects of the CARD15 and IBD5 loci on clinical phenotype in 630 patients with Crohn's disease.

OBJECTIVES: Genetic variants at the CARD15 and IBD5 loci are strongly associated with Crohn's disease (CD), but evidence of the effect of these variants on the clinical expression of CD is conflicting and has often been hampered by small sample sizes. We studied 630 well-characterized patients to clarify the genotype/phenotype relationship in CD. METHODS: Patients and healthy controls were genotyped for three common mutations in CARD15 and a marker of the IBD5 risk haplotype. Allele frequencies were compared between phenotypic subgroups using chi2 or Fisher's exact tests. Genotype/phenotype analysis was carried out using multinomial logistic regression modelling allowing for adjustment for correlated or confounding factors. RESULTS: The mean age at diagnosis was significantly lower in carriers of the CARD15 or IBD5 risk alleles. After correction for age and smoking, CARD15 mutations were strongly associated with both ileal disease (P=8.8 x 10(-6)) and stenotic disease (P=0.003), but the association with stenotic disease appeared to be due to a confounding effect with ileal disease. CARD15 mutations were also associated with the presence of granulomas (P=5.7 x 10(-5)), which remained significant after adjustment for age at diagnosis and disease location (P=0.0047). The IBD5 risk haplotype frequency was significantly elevated in cases with perianal disease (P=0.028) and axial spondyloarthropathy (P=0.012). CONCLUSION: Genetic variants at the CARD15 and IBD5 loci have diverse effects on clinical expression in CD. CARD15 mutations are significantly correlated with the presence of granulomas.

Mechanism based QSAR studies of N-phenylbenzamides as antimicrobial agents.

N-Phenyl benzamides are potent antibacterial agents. They are active against both Gram-positive and Gram-negative bacteria. The Gram-positive bacteria have strong and thick cell wall while the Gram-negative bacterial have thin and permeable cell wall. The DFT based QSAR reveals that molecular weight and total energy significantly contribute to activity against both kinds of target. The electrophilicity index involved in QSAR models derived with anti-Gram-positive activity indicates the dominance of electrostatic interaction. The molar refractivity and logP is involved in QSAR model derived with anti-Gram-negative activity shows steric and hydrophobic interaction. The CoMFA and CoMSIA results also indicate that anti-Gram-positive bacterial activity is a function of electrostatic field effect but the anti-Gram-negative activity depends on hydrophobicity and steric field effect. The CoMFA and CoMSIA contour maps give an indication, the electropositive group around benzene "X" and an electronegative group around carbonyl oxygen is desirable for better anti-Gram-positive bacterial activity. A hydrophobic group around meta position of ring "X" with bulky group at ortho position and a small group at para position are desirable for better activity against Gram-negative target. The findings are reasonable and the mechanism might be different due to difference in composition of cell wall. The cell wall of Gram-positive target does not allow the permeability and only external electrostatic interaction is possible while the cell wall of Gram-negative target allows the permeability of molecules inside the cell for possible hydrophobic and steric bulk interaction.

A nonsynonymous SNP in ATG16L1 predisposes to ileal Crohn's disease and is independent of CARD15 and IBD5.

BACKGROUND & AIMS: A genome-wide association scan of nonsynonymous DNA polymorphisms identified association of a threonine-to-alanine substitution (T300A) in the autophagy-related 16-like gene ATG16L1 with Crohn's disease. We investigated this association in independent U.K. cohorts of Crohn's disease and ulcerative colitis. METHODS: The T300A variant (rs2241880) was genotyped in an independent sample of 727 Crohn's disease and 877 ulcerative colitis cases, and in 579 controls. We then performed an extension analysis combining these data with the U.K. data from the initial study to give a total of 1236 U.K. Crohn's disease cases and 1235 controls to estimate disease risk and test for interaction with the CARD15 and IBD5 risk loci and for association with disease subtypes. RESULTS: The association of T300A was replicated in the independent sample of 727 Crohn's disease cases (P = .001), and was strongly associated in the extended analysis of 1236 Crohn's cases (P = 2.4 x 10(-6)). The 300A/A genotype conferred a 1.65-fold risk of Crohn's disease, with a 2.2-fold risk of ileal disease. Analysis of the interaction of ATG16L1 with CARD15 and IBD5 indicated that all 3 loci contribute independently to disease risk. Homozygosity for the risk allele at all 3 loci conferred a combined risk of 20.4 (95% confidence interval: 8.71, 47.7) for Crohn's disease. The ATG16L1 risk genotype showed a modest but significant association with ulcerative colitis (P = .026). CONCLUSIONS: The association of ATG16L1 with Crohn's disease and possibly with ulcerative colitis supports a role for autophagy in the pathogenesis of inflammatory bowel disease.

Mutation, selection, and evolution of the Crohn disease susceptibility gene CARD15.

Three common mutations in the CARD15 (NOD2) gene are known to be associated with susceptibility to Crohn disease (CD), and genetic data suggest a gene dosage model with an increased risk of 2-4-fold in heterozygotes and 20-40-fold in homozygotes. However, the discovery of numerous rare variants of CARD15 indicates that some heterozygotes for the common mutations have a rare mutation on the other CARD15 allele, which would support a recessive model for CD. We addressed this issue by screening CARD15 for mutations in 100 CD patients who were heterozygous for one of the three common mutations. We also developed a strategy for evaluating potential disease susceptibility alleles (DSAs) that involves assessing the degree of evolutionary conservation of involved residues, predicted effects on protein structure and function, and genotyping in a large sample of cases and controls. The evolutionary analysis was aided by sequencing the entire coding region of CARD15 in three primates (chimp, gibbon, and tamarin) and aligning the human sequence with these and orthologs from other species. We found that 11 of the 100 CD patients screened had a second potential pathogenic mutation within the exonic and periexonic sequences examined. Assuming that there are no additional pathogenic mutations in noncoding regions, our study suggests that most carriers of the common DSAs are true heterozygotes, and supports previous evidence for a gene dosage model. Four novel nonsynonymous mutations were detected, one of which would produce premature termination of translation c.2686C>T (p.Arg896X). Two potential DSAs--c.2107C>T (p.Arg703Cys) and g.2238T>A (c.74-7T>A)--were significantly associated with CD in the case control sample. Analysis of the evolution of CARD15 revealed strong conservation of the encoded protein, with identity to the human sequence ranging from 99.1% in the chimp to 44.5% in fugu. Higher primates possess an open reading frame (ORF) upstream of the putative initiation site in other species that encodes a further 27 N-terminal amino acids, while four regions of high conservation are observed outside of the known domains of CARD15, indicative of additional residues of functional importance. The strategy developed here may have general application to the assessment of mutation pathogenicity and genetic models in other complex disorders.

Genetic evidence for interaction of the 5q31 cytokine locus and the CARD15 gene in Crohn disease.

A common haplotype spanning 250 kb in the cytokine gene cluster on chromosome 5q31 has recently been reported to be strongly associated with Crohn disease (CD) in Canadian families. We have replicated this finding by both the transmission-disequilibrium test (TDT) (P=.016) and in a case-control association study (P=.008) in a large European cohort of patients with CD, although the increase in disease risk was small (odds ratio 1.49 for homozygotes, 95% CI 1.11-2.0). No association was detected in families or individuals with ulcerative colitis (UC). Stratification of offspring with CD in the TDT sample by mutation status in the CD susceptibility gene CARD15 showed that the association with the 5q31 risk haplotype was present only in offspring with at least one of the known CARD15 disease susceptibility alleles (P=.044). The 5q31 risk haplotype frequency was 53.1% in unrelated individuals with CD who had one or two CARD15 mutations versus 43.7% in control subjects (P=.0001) but was not significantly elevated in individuals with CD who had no CARD15 mutations (45.4%, P=.41). Kaplan-Meier survival analysis of age at disease onset showed a significantly earlier onset in homozygotes for the 5q31 risk haplotype (P=.0019). These findings suggest that genetic variants at the 5q31 (IBD5) locus may hasten the onset of Crohn disease and cooperate with CARD15 in disease causation.

Association of NOD2 (CARD 15) genotype with clinical course of Crohn's disease: a cohort study.

BACKGROUND: Crohn's disease is a heterogeneous disorder for which NOD2 (CARD 15) has been identified as a susceptibility gene. We investigate the relation between NOD2 genotype and phenotypic characteristics of patients with Crohn's disease. METHODS: Hypotheses about the relation between NOD2 genotype and Crohn's disease phenotype were generated retrospectively from a group of 446 German patients with this disorder. Positive findings (p<0.10) were verified in prospectively established cohorts of 106 German and 55 Norwegian patients with Crohn's disease. All patients were genotyped for the main coding mutations in NOD2, denoted SNP8, SNP12, and SNP13, with Taqman technology. FINDINGS: In the retrospective cohort, six clinical characteristics showed noteworthy haplotype association: fistulising, ileal, left colonic and right colonic disease, stenosis, and resection. In the German prospective cohort, these haplotype associations could be replicated for ileal (p=0.006) and right colonic disease (p < or =0.001). A similar trend was noted in the Norwegian patients. INTERPRETATION: We recorded a distinct relation between NOD2 genotype and phenotype of Crohn's disease. Test strategies with NOD2 variations to predict the clinical course of Crohn's disease could lead to the development of new therapeutic paradigms.

The contribution of NOD2 gene mutations to the risk and site of disease in inflammatory bowel disease.

BACKGROUND & AIMS: Mutations in the NOD2 gene are strongly associated with susceptibility to Crohn's disease (CD). We analyzed a large cohort of European patients with inflammatory bowel disease to determine which mutations confer susceptibility, the degree of risk conferred, their prevalence in familial and sporadic forms of the disease, and whether they are associated with site of disease. METHODS: Individuals were genotyped for 4 NOD2 mutations: P268S, R702W, G908R, and 3020insC. Allelic transmission distortion to 531 CD- and 337 ulcerative colitis-affected offspring was assessed by the transmission disequilibrium test. Association was also tested in an independent cohort of 995 patients with inflammatory bowel disease and 290 controls. Cases were stratified by disease site and compared across NOD2 genotypes. RESULTS: R702W, G908R, and 3020insC were strongly associated with CD but not with ulcerative colitis. Linkage disequilibrium was observed between P268S and the other mutations, forming 3 independent disease haplotypes. Genotype relative risks were 3.0 for mutation heterozygotes and 23.4 for homozygotes or compound heterozygotes. The frequency of NOD2 mutations was higher in cases from families affected only with CD and was significantly increased in ileal-specific disease cases compared with colon-specific disease (26.9% vs. 12.7%, P = 0.0004). CONCLUSIONS: The R702W, G908R, and 3020insC mutations are strong independent risk factors for CD and are associated particularly with ileal disease.