Ling Mei

Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis

We present a genome-wide association study of ileal Crohn disease and two independent replication studies that identify several new regions of association to Crohn disease. Specifically, in addition to the previously established CARD15 and IL23R associations, we identified strong and significantly replicated associations (combined P < 10−10) with an intergenic region on 10q21.1 and a coding variant in ATG16L1, the latter of which was also recently reported by another group. We also report strong associations with independent replication to variation in the genomic regions encoding PHOX2B, NCF4 and a predicted gene on 16q24.1 (FAM92B). Finally, we demonstrate that ATG16L1 is expressed in intestinal epithelial cell lines and that functional knockdown of this gene abrogates autophagy of Salmonella typhimurium. Together, these findings suggest that autophagy and host cell responses to intracellular microbes are involved in the pathogenesis of Crohn disease.

Increased Immune Reactivity Predicts Aggressive Complicating Crohn’s disease in Children

BACKGROUND & AIMS The ability to identify children with CD who are at highest risk for rapid progression from uncomplicated to complicated phenotypes would be invaluable in guiding initial therapy. The aims of this study were to determine whether immune responses and/or CARD15 variants are associated with complicated disease phenotypes and predict disease progression. METHODS Sera were collected from 796 pediatric CD cases and tested for anti-Cbir1 (flagellin), anti-outer membrane protein C, anti-Saccharomyces cerevisiae, and perinuclear antineutrophil cytoplasmic antibody by using enzyme-linked immunosorbent assay. Genotyping (Taqman MGB) was performed for 3 CARD15 variants (single nucleotide polymorphisms 8, 12, and 13). Associations between immune responses (antibody sum and quartile sum score, CARD15, and clinical phenotype were evaluated. RESULTS Thirty-two percent of patients developed at least 1 disease complication within a median of 32 months, and 18% underwent surgery. The frequency of internal penetrating, stricturing, and surgery significantly increased (P trend < .0001 for all 3 outcomes) with increasing antibody sum and quartile sum score. Nine percent of seropositive groups had internal penetrating/stricturing versus 2.9% in the seronegative group (P = .01). Twelve percent of seropositive groups underwent surgery versus 2% in the seronegative group (P = .0001). The highest antibody sum group (3) and quartile sum score group (4) demonstrated the most rapid disease progression (P < .0001). Increased hazard ratio was observed for antibody sum group 3 (7.8; confidence interval, 2.2-28.7), P < .002 and quartile sum score group 4 (11.0; confidence interval, 1.5-83.0, P < .02). CONCLUSIONS The rate of complicated CD increases in children as the number and magnitude of immune reactivity increase. Disease progression is significantly faster in children expressing immune reactivity.

Does Infliximab Influence Surgical Morbidity of Ileal Pouch-Anal Anastomosis in Patients with Ulcerative Colitis?

PurposeSince infliximab has been approved for treatment of patients with refractory ulcerative colitis, surgeons will be increasingly faced with operating on patients who have failed therapy with this potent immunosuppressant. This study was designed to compare short-term complications in patients with ulcerative colitis who were treated with and without infliximab before colectomy.MethodsThe charts of patients undergoing ileal pouch-anal anastomosis or subtotal colectomy for refractory ulcerative colitis during the five-year period ending October 2005 were reviewed. Postoperative medical and surgical complications were assessed.ResultsSeventeen patients had failed infliximab treatment and 134 patients were never treated with infliximab. Ileal pouch-anal anastomosis was performed in 112 patients (74 percent) and subtotal colectomy in 39 patients (36 percent). There were no deaths. Postoperative complications were observed in 43 patients (28 percent), with no significant difference observed between infliximab-treated (37 percent) and infliximab-untreated patients (27 percent). Of 61 patients (40 percent) treated with preoperative cyclosporine A, 5 patients also had been treated with infliximab. The infliximab and cyclosporine A-treated patient group had an 80 percent complication rate, significantly higher than the 29 percent complication rate noted in the cyclosporine A only-treated group (P--.04).ConclusionsAlthough preoperative treatment with infliximab alone does not significantly increase the incidence of postoperative complications, using both inflixiamb and cyclosporine A before colectomy in refractory ulcerative colitis is associated with high surgical morbidity.

Both Preoperative Perinuclear Antineutrophil Cytoplasmic Antibody and Anti-CBir1 Expression in Ulcerative Colitis Patients Influence Pouchitis Development After Ileal Pouch-Anal Anastomosis

BACKGROUND & AIMS Acute pouchitis (AP) and chronic pouchitis (CP) are common after ileal pouch-anal anastomosis (IPAA) for ulcerative colitis. The aim of this study was to assess associations of preoperative perinuclear antineutrophil cytoplasmic antibody (pANCA) and anti-CBir1 flagellin on AP or CP development. METHODS Patients were assessed prospectively for clinically and endoscopically proven AP (antibiotic responsive) or CP (antibiotic-dependent or refractory to antibiotic therapy). Sera from 238 patients were analyzed for ANCA and anti-CBir1 using an enzyme-linked immunosorbent assay. pANCA(+) patients were substratified into high-level (>100 EU/mL) and low-level (<100 EU/mL) groups. RESULTS After a median follow-up period of 47 months, 72 patients (30%) developed pouchitis. Pouchitis developed in 36% of pANCA(+) patients versus 16% of pANCA(-) patients (P = .005), 46% of anti-CBir1(+) patients versus 26% of anti-CBir1(-) patients (P = .02), and 54% of 35 pANCA(+)/anti-CBir1(+) patients versus 31% of 136 pANCA(+)/anti-CBir1(-) patients (P = .02). AP developed in 37 pANCA(+) patients (22%) versus 6 pANCA(-) patients (9%) (P = .02), and 12 anti-CBir1(+) patients (26%) versus 31 anti-CBir1(-) patients (16%) (P = .1). Although AP was not influenced by pANCA level, AP was seen in 38% of low-level pANCA(+)/anti-CBir1(+) patients versus 18% low-level pANCA(+)/anti-CBir1(-) patients (P = .03). CP was seen in 29% of high-level pANCA(+) patients versus 11% of low-level pANCA(+) patients (P = .03). CONCLUSIONS Both pANCA and anti-CBir1 expression are associated with pouchitis after IPAA. Anti-CBir1 increases the incidence of AP only in patients who have low-level pANCA expression, and increases the incidence of CP only in patients who have high-level pANCA expression. Diverse patterns of reactivity to microbial antigens may manifest as different forms of pouchitis after IPAA.

Genome wide association (GWA) predictors of anti-TNFα therapeutic responsiveness in pediatric inflammatory bowel disease

Background: Interindividual variation in response to anti‐TNF&agr; therapy may be explained by genetic variability in disease pathogenesis or mechanism of action. Recent genome‐wide association studies (GWAS) in inflammatory bowel disease (IBD) have increased our understanding of the genetic susceptibility to IBD. The aim was to test associations of known IBD susceptibility loci and novel “pharmacogenetic” GWAS identified loci with primary nonresponse to anti‐TNF&agr; in pediatric IBD patients and develop a predictive model of primary nonresponse. Methods: Primary nonresponse was defined using the Harvey Bradshaw Index (HBI) for Crohns disease (CD) and partial Mayo score for ulcerative colitis (UC). Genotyping was performed using the Illumina Infinium platform. Chi‐square analysis tested associations of phenotype and genotype with primary nonresponse. Genetic associations were identified by testing known IBD susceptibility loci and by performing a GWAS for primary nonresponse. Stepwise multiple logistic regression was performed to build predictive models. Results: Nonresponse occurred in 22 of 94 subjects. Six known susceptibility loci were associated with primary nonresponse (P < 0.05). Only the 21q22.2/BRWDI loci remained significant in the predictive model. The most predictive model included 3 novel “pharmacogenetic” GWAS loci, the previously identified BRWD1, pANCA, and a UC diagnosis (R2 = 0.82 and area under the curve [AUC] = 0.98%). The relative risk of nonresponse increased 15‐fold when number of risk factors increased from 0–2 to ≥3. Conclusions: The combination of phenotype and genotype is most predictive of primary nonresponse to anti‐TNF&agr; in pediatric IBD. Defining predictors of response to anti‐TNF&agr; may allow the identification of patients who will not benefit from this class of therapy. Inflamm Bowel Dis 2010

Anti-Flagellin (CBir1) Phenotypic and Genetic Crohn's Disease Associations

Background Antibody reactivity to microbial antigens correlates with distinct Crohns disease (CD) phenotypes such as fistulizing or fibrostenosing disease. We examined the association between anti‐CBir1 and clinical phenotypes and NOD2 variants in a large cohort of adult CD patients. Methods Sera and genomic DNA were collected from 731 patients with CD and tested for immune responses to I2, CBir1, oligomannan, and outer membrane porin C (OmpC) and the 3 most common CD‐associated NOD2 variants. Results Anti‐CBir1 reactivity was significantly associated with fibrostenosis (FS), internal penetrating (IP) disease phenotypes, small bowel (SB) involvement, and SB surgery but negatively associated with ulcerative colitis (UC)‐like CD. Multivariate logistic regression analysis showed that anti‐CBir1 was independently associated with FS and UC‐like CD irrespective of the antibody reactivity to I2, oligomannan, or OmpC, but not with SB involvement or SB surgery. The magnitude of anti‐CBir1 reactivity, when added to the quantitative response toward the other 3 CD‐associated antigens, enhances the discrimination of FS, IP, UC‐like CD, and SB involvement, but not SB surgery. Finally, although the frequency of anti‐CBir1 was similar in patients with none versus at least 1 NOD2 variant, the quantitative response to CBir1 flagellin was significantly higher in patients with CD carrying at least 1 NOD2 variant versus those carrying no variants (median anti‐CBir1 titer 33.39 versus 28.36, respectively; P = 0.01). Conclusions Anti‐CBir1 serum reactivity in CD patients is independently associated with FS and complicated SB CD. Quantitative, but not qualitative, response to CBir1 is also significantly associated with the CD‐associated NOD2 variants. (Inflamm Bowel Dis 2007)

TNFSF15 is an Ethnic Specific IBD Gene

Background: Inflammatory bowel disease (IBD) is a clinically and, likely, genetically heterogeneous group of disorders. A recent report suggests that genetic variations in the TNFSF15 gene contribute to the susceptibility of IBD in both Japanese and Caucasian populations. The aim was to confirm the association between TNFSF15 high‐ and low‐risk haplotypes and IBD in a Caucasian population. Methods: Five single‐nucleotide polymorphisms (SNPs) that comprise the 2 common haplotypes were genotyped in 599 Caucasian patients with Crohns disease (CD), 382 Caucasian patients with ulcerative colitis (UC), and 230 ethnically matched healthy controls, including both Jews and non‐Jews. Results: The previously reported ‘risk’ haplotype was not associated with CD or UC (88.2% in CD cases versus 88.3% in controls, P = 0.96; 88.1% in UC cases versus 88.3% in controls, P = 0.78). We did, however, observe an increased frequency of the “protective” haplotype in non‐Jewish controls for both CD and UC (38.8% CD cases versus 50% controls, P = 0.01; 37.3% UC cases versus 50% controls, P = 0.01) with no such effect observed in the Jewish samples. There was an interactive effect between ethnicity and the protective haplotype in CD (P = 0.04). Conclusions: We observed a protective haplotype, consisting of the minor alleles for all 5 markers, to have a higher frequency in the non‐Jewish controls than in CD and UC. Of further interest, the haplotype frequency was in the opposite direction in our Jewish case‐control panels (both CD and UC), leading us to conclude 1) that TNFSF15 is indeed an IBD susceptibility gene, and 2) the disease susceptibility is ethnic‐specific.

Age of diagnosis influences serologic responses in children with Crohn's disease: a possible clue to etiology?

Background: Crohns disease (CD) is often associated with antibodies to microbial antigens. Differences in immune response may offer clues to the pathogenesis of the disease. The aim was to examine the influence of age at diagnosis on the serologic response in children with CD. Methods: Data were drawn from 3 North American multicenter pediatric inflammatory bowel disease (IBD) research consortia. At or shortly after diagnosis, pANCA, ASCA IgA, ASCA IgG, anti‐ompC, and anti‐CBir1 were assayed. The results were compared as a function of age at CD diagnosis (0–7 years versus 8–15 years). Results: In all, 705 children (79 <8 years of age at diagnosis, 626 ≥8 years) were studied. Small bowel CD was less frequent in the younger group (48.7% versus 72.6%; P < 0.0001), while colonic involvement was comparable (91.0% versus 86.5%). ASCA IgA and IgG were seen in <20% of those 0–7 years old compared to nearly 40% of those 8–15 years old (P < 0.001), while anti‐CBir1 was more frequent in the younger children (66% versus 54%, P < 0.05). Anti‐CBir1 detected a significant number of children in both age groups who otherwise were serologically negative. Both age at diagnosis and site of CD involvement were independently associated with expression of ASCA and anti‐CBir1. Conclusions: Compared to children 8–15 years of age at diagnosis, those 0–7 years are more likely to express anti‐CBir1 but only half as likely to express ASCA. These age‐associated differences in antimicrobial seropositivity suggest that there may be different, and as yet unrecognized, genetic, immunologic, and/or microbial factors leading to CD in the youngest children.

IBD-associated TL1A gene (TNFSF15) haplotypes determine increased expression of TL1A protein.

Background The recently identified member of the TNF superfamily TL1A (TNFSF15) increases IFN-γ production by T cells in peripheral and mucosal CCR9+ T cells. TL1A and its receptor DR3 are up-regulated during chronic intestinal inflammation in ulcerative colitis and Crohns disease (CD). TL1A gene haplotypes increase CD susceptibility in Japanese, European, and US cohorts. Methodology and Principal Findings Here we report that the presence of TL1A gene haplotype B increases risk in Jewish CD patients with antibody titers for the E. coli outer membrane porin C (OmpC+) (Haplotype B frequency in Jewish CD patients: 24.9% for OmpC negative and 41.9% for OmpC positive patients, respectively, P≤0.001). CD14+ monocytes isolated from Jewish OmpC+ patients homozygous for TL1A gene haplotype B express higher levels of TL1A in response to FcγR stimulation, a known inducing pathway of TL1A, as measured by ELISA. Furthermore, the membrane expression of TL1A is increased on peripheral monocytes from Jewish but not non-Jewish CD patients with the risk haplotype. Conclusions and Significance These findings suggest that TL1A gene variation exacerbates induction of TL1A in response to FcγR stimulation in Jewish CD patients and this may lead to chronic intestinal inflammation via overwhelming T cell responses. Thus, TL1A may provide an important target for therapeutic intervention in this subgroup of IBD patients.

Association between angiotensinogen, angiotensin II receptor genes, and blood pressure response to an angiotensin-converting enzyme inhibitor.

Background— To identify the genetic contribution to the variation in blood pressure (BP) response to angiotensin-converting enzyme inhibitors (ACEIs), single-nucleotide polymorphisms (SNPs) in the angiotensinogen (AGT), angiotensin receptor 1 (AGTR1), and angiotensin receptor 2 (AGTR2) genes were evaluated for their association with BP response to ACEI in Chinese patients with hypertension in a 2-stage design. Methods and Results— We selected 1447 hypertensive patients from a 3-year benazepril postmarket surveillance trial and genotyped them for 14 SNPs in the AGT, AGTR1, and AGTR2 genes. The AGT rs7079 (C/T) SNP (3′-untranslated region) was significantly associated with the response of diastolic BP to benazepril (diastolic BP response: −7.4 mm Hg for subjects with the CC genotype, −8.9 mm Hg for CA, and −10.1 mm Hg for AA; P=0.001). Although there was no association of individual SNPs in the AGTR1 gene, there was a graded response between common haplotypes and systolic BP reduction in the order of haplotype 2 (H2)/lack of haplotype 3 (non-H3) (−13.6 mm Hg) > non-H2/non-H3 (−10.9 mm Hg) > H3/non-H2 (−6.6 mm Hg) (P=0.004). The total variations in response to ACEI therapy that were explained by the AGT SNP and AGTR1 haplotype groups were 13% for systolic and 9% to 9.6% for diastolic BP, respectively. Conclusion— AGT SNP rs7079 and AGTR1 haplotypes were associated with BP reduction in response to ACEI therapy in hypertensive Chinese patients. This will be useful in future studies, providing genetic markers to predict the hypertensive response to ACEI therapy.