Janos Papp

Deletions removing the last exon of TACSTD1 constitute a distinct class of mutations predisposing to Lynch syndrome

Several different genetic alterations in the etiology of Lynch syndrome (hereditary nonpolyposis colorectal cancer [HNPCC]) are known, mostly point mutations and genomic rearrangements in 1 of at least 3 mismatch‐repair (MMR) genes. However, no susceptibility factor has yet been identified in a significant part (30–50%) of clinicopathologically well‐defined HNPCC families, suggesting the presence of other predisposing mechanisms. In a set of probands from 27 Lynch syndrome families who lacked evidence of a germline mutation in either the MSH2 or MLH1 gene, we performed genomic deletion screening with the use of multiplex ligation‐dependent probe amplification (MLPA) and sequencing. We used immunohistochemistry (IHC) and microsatellite instability (MSI) analyses on samples of the probands of all families. Comparative analysis of mRNA transcripts was performed on blood leukocyte–derived samples from mutation carriers and noncarrier controls. We report that large germline deletions encompassing the last exons of the TACSTD1 gene, upstream of MSH2, cosegregate with the HNPCC phenotype in 19% (5/27) of families tested. The tumors of the carriers show high‐level MSI and MSH2 protein loss. We show that these deletions, by removing the transcriptional termination sequences of the upstream gene, give rise to multiple TACSTD1/MSH2 fusion transcripts. Our results provide evidence that deletions removing the last exon of TACSTD1 constitute a distinct class of mutations predisposing to Lynch syndrome. Thus, analysis of the 3′ region of the TACSTD1 gene should be included in the routine mutation screening protocols for HNPCC. Hum Mutat 30, 197–203, 2009.

Seroreactivity to microbial components in Crohn's disease is associated with ileal involvement, noninflammatory disease behavior and NOD2/CARD15 genotype, but not with risk for surgery in a Hungarian cohort of IBD patients

Background: Antibodies directed against Saccharomyces cerevisiae (ASCA), perinuclear components of neutrophils (pANCA), and porin protein C of Escherichia coli (anti‐OmpC) are reported to be associated with disease phenotype and may be of diagnostic importance in inflammatory bowel disease (IBD). Since limited data are available from Eastern Europe, we assessed the above antibodies in Hungarian IBD patients. Methods: In all, 653 well‐characterized, unrelated consecutive IBD patients (Crohns disease [CD]: 558, m/f: 263/295, duration: 8.1 ± 10.7 years; ulcerative colitis [UC]: 95, m/f: 44/51, duration: 8.9 ± 9.8 years) and 100 healthy subjects were investigated. Sera were assayed for anti‐Omp and ASCA by enzyme‐linked immunosorbent assay (ELISA) and ANCA by indirect immunofluorescence assay (IIF). TLR4 and NOD2/CARD15 variants were tested by polymerase chain reaction/restriction fragment length polymorphism (PCR‐RFLP). Detailed clinical phenotypes were determined by reviewing the medical charts. Results: Anti‐Omp, ASCA, and atypical pANCA antibodies were present in 31.2%, 59.3%, and 13.8% of CD, 24.2%, 13.7%, and 48.5% of UC patients, and in 20%, 16%, and 5.6% of controls, respectively. ASCA and anti‐Omp positivity were associated with increased risk for CD (odds ratio [OR]ASCA = 7.65, 95% confidence interval [CI]: 4.37–13.4; OROmp = 1.81, 95% CI: 1.08–3.05). In a logistic regression analysis, anti‐Omp and ASCA were independently associated with ileal and noninflammatory disease, but not with a risk for surgery or response to steroids or infliximab. A serology dosage effect was also observed. ASCA and anti‐Omp antibodies were associated with NOD2/CARD15, in addition to a gene dosage effect. No associations were found in UC. Conclusions: Serological markers were useful in the differentiation between CD and UC in an Eastern European IBD cohort. Reactivity to microbial components was associated with disease phenotype and NOD2/CARD15 genotype, further supporting the role of altered microbial sensing in the pathogenesis of CD.

Association of beta-defensin 1 single nucleotide polymorphisms with Crohn's disease

Objective. It has been suggested that deficient defensin expression is associated with the chronic inflammation of Crohns disease. The regional localization of Crohns disease, ileal or colonic disease can be linked to different defensin profiles. As constitutive β-defensin 1 has a colonic expression, we considered it of interest to investigate single nucleotide polymorphisms (SNPs) of the β-defensin 1 gene (DEFB1) in Crohns disease. Material and methods. Three SNPs of the DEFB1 gene, DEFB1 G-20A (rs11362), DEFB1 C-44G (rs1800972) and DEFB1 G-52A (rs1799946), were genotyped either by Custom TaqMan® SNP genotyping assays or by restriction fragment length polymorphisms (RFLP) in 190 patients with Crohns disease and 95 Hungarian controls. Results. It was found that the G-20A and C-44G SNPs had a strong association with the colonic and ileocolonic localizations of the disease, respectively, but no association was detected for the ileal localization. A significantly higher frequency of the GA genotype of G-20A was observed among patients with colonic localization (60%) as compared with healthy controls (39%), with an odds ratio (OR) of 2.39. The GG genotype of C-44G SNP, which is regarded as a protective genotype, was much less frequent (4%) among patients than among controls (12%), OR 3.367. Conclusions. These results indicate that genetic variations in the DEFB1 gene encoding constitutive human β-defensin 1 may be associated with the risk for Crohns disease and may determine disease phenotype, e.g. colonic localization.

Early azathioprine/biological therapy is associated with decreased risk for first surgery and delays time to surgery but not reoperation in both smokers and nonsmokers with Crohn's disease, while smoking decreases the risk of colectomy in ulcerative colitis.

Background/aims Smoking may alter the natural course of Crohns disease (CD). Smokers are more likely to develop complications, relapses and have a greater risk for surgery. In contrast, in ulcerative colitis (UC), smoking might improve the disease course. Our aim was to assess the combined effect of disease phenotype, smoking, and immunomodulator [azathioprine (AZA), AZA/biological] treatment on the risk of intestinal resection/reoperation in CD and colectomy in UC. Patients/methods Six hundred and eighty-one inflammatory bowel disease patients were analyzed (CD: 340, male/female: 155/185, duration: 9.4±7.5 years; UC: 341, male/female: 174/164, duration: 11.5±9.7 years). Patients were interviewed on their smoking habits at the time of diagnosis and during the regular follow-up visits. Medical records were retrospectively analyzed. Results Smoking was present in 45.5% in CD and 15.8% in UC. CD patients who underwent at least one bowel resection comprised 46.5%. In an univariate analysis, disease location, behavior, AZA, or AZA/biological use before surgery [odds ratio (OR): 0.26 and 0.22, P<0.001] and smoking (OR: 1.61, P = 0.03) were associated with risk for first surgery. Smoking, AZA, or AZA/biological (P<0.001) use before first surgery and disease behavior were independently associated with risk for surgery in a proportional Cox-regression analysis. Perianal disease (OR: 3.2, P = 0.001) and frequent relapses (OR: 4.8, P<0.001) but not smoking, AZA, or AZA/biological use after first surgery were predictive for reoperation. In UC, the rate of colectomy was 5.6%. Disease location (P = 0.001) and smoking status (P = 0.02) were independently associated with risk for colectomy in a proportional Cox-regression analysis. Conclusion Our data suggest that early AZA/biological therapy reduces the risk for first operation but not reoperation in CD, in both smokers and nonsmokers. In contrast, smoking was associated with a decreased need for colectomy in UC.

Pancreatic autoantibodies are associated with reactivity to microbial antibodies, penetrating disease behavior, perianal disease, and extraintestinal manifestations, but not with NOD2/CARD15 or TLR4 genotype in a hungarian IBD cohort†

Background: Pancreatic autoantibodies (PAB) and goblet cell autoantibodies (GAB) are specific for Crohns disease (CD) and ulcerative colitis (UC), but the sensitivity alone is low. Conventional antibodies and carbohydrates (glycans) are associated with disease phenotype and may be of diagnostic importance in inflammatory bowel disease (IBD). Our aim was to determine the accuracy of PAB and GAB autoantibodies as well as to study relevant phenotype–serotype associations. Methods: A Hungarian study cohort of 1092 subjects, including 689 well‐characterized, unrelated IBD patients (CD: 579, m/f ratio: 274/305, duration: 7.9 ± 11.2 years; UC: 110, m/f ratio: 53/57, duration: 8.9 ± 9.8 years), 139 celiac patients, 100 healthy, and 64 non‐IBD gastrointestinal controls were investigated. Sera were assayed for PAB‐GAB IgA/IgG, anti‐Omp, anti‐Saccharomyces cerevisiae antibodies (ASCA), and anti‐glycans. TLR4 and NOD2/CARD15 was tested by polymerase chain reaction / restriction fragment length polymorphism (PCR‐RFLP). Detailed clinical phenotypes were determined. Results: The prevalence of PAB was significantly more frequent in CD (41.1%) versus UC (22.7%), celiac (22.3%), and controls (8% and 4.6%, P < 0.01 for each), while GAB detection was poor in all groups except UC (15.4%). In CD the combination of PAB and/or anti‐glycans/ASCA increased the sensitivity to 72% and 59%, respectively, for isolated colonic disease. PAB was associated to gylcans (odds ratio [OR] 1.74,P = 0.002), ASCA IgG/IgA (OR 1.75, P = 0.002), Omp (OR 1.86, P = 0.001) as well as perforating, perianal disease, arthritis, ocular, and cutaneous manifestations (P = 0.002–0.032). In contrast, PAB and GAB antibodies were not associated with NOD2/CARD15 or TLR4, response to medical therapy, or need for surgery. No associations were found in UC. Conclusions: PAB autoantibodies in combination with ASCA or anti‐glycan antibodies increase the sensitivity for detecting CD, especially isolated colonic CD. Antibody response to PAB was associated with complicated disease phenotype and extraintestinal manifestations in this Eastern European IBD cohort.

ATG16L1 and IL23 receptor (IL23R) genes are associated with disease susceptibility in Hungarian CD patients

BACKGROUND North American and European genome-wide association scans have identified ATG16L1 and IL23R as novel inflammatory bowel disease (IBD) susceptibility genes and subsequent reports confirmed these findings in large independent populations. The aims of this study were to investigate the association and examine genotype-phenotype relationships in a Hungarian IBD cohort. METHODS 415 unrelated IBD patients (CD: 266, age: 35.2+/-12.1 years, duration: 8.7+/-7.5 years and UC: 149, age: 44.4+/-15.4 years, duration: 10.7+/-8.9 years) and 149 healthy subjects were investigated. IL23R Arg381Gln (R381Q, rs11209026) and ATG16L1 Thr300Ala (T300A, rs2241880) polymorphisms were tested using LightCycler allele discrimination method. Detailed clinical phenotypes were determined by reviewing the medical charts. RESULTS The association between IL23R rs11209026, ATG16L1 rs2241880 and CD was confirmed (OR(IL23R381Q): 0.38, 95% CI: 0.16-0.87; OR(ATG16L1300AA): 1.86, 95% CI: 1.04-3.40). No difference was found between patients with UC and either controls or CD. In CD, IL23R 381Gln heterozygosity was associated with inflammatory disease (70% vs. 34%, p=0.037), while disease restricted to the colon was more prevalent in patients with the ATG16L1 300Ala/Ala homozygosity (33.3% vs. 21.1%, p=0.036). In addition, carriage of the variant alleles did not predict response to steroids, infliximab or need for surgery. CONCLUSIONS We confirmed that ATG16L1 and IL23R are susceptibility loci for CD in Hungarian CD patients. Further studies are needed to confirm the reported phenotype-genotype associations found in this study.

Claudin expression in Barrett's esophagus and adenocarcinoma

Claudins (CLDNs) are key molecules in cell adhesion, polarity, and control of paracellular solute transport. Several studies suggested that changes in claudin pattern have a role in cancer development. This study aimed to detect alterations in CLDN 1, 2, 3, 4, and 7 expression patterns in Barretts esophagus (BE) and adenocarcinoma (ACC) compared with that in foveolar epithelium (FOV), normal squamous epithelium (SQ), and squamous cell carcinoma (SQCC). One hundred twenty five surgically or endoscopically removed, paraffin-embedded cases were studied by immunohistochemistry and analyzed statistically. BE, ACC, and FOV were dissected from 30 paraffin-embedded samples for further mRNA expression analysis. CLDN 7 was the dominating type in all epithelia and carcinomas, but its expression did not differ in normal and altered tissues. CLDN 1 expression was significantly increased in SQCC compared with that in SQ. CLDNs 3 and 4 were significantly elevated both in BE and ACC compared with that in FOV. CLDN 2 expression increased significantly in ACCs compared with that in BE. This is the first report proving similarities and differences regarding claudin expression pattern in BE and ACC compared with that in FOV and SQ. Our data prove a close link in CLDN pattern between BE and ACC, adding further evidence that BE is an alteration preceding esophageal ACC.

The effect of long-acting somatostatin analogue on enzyme changes after endoscopic pancreatography

The effect of the long-acting somatostatin analogue, octreotide acetate (Sandostatin) on enzyme elevation after endoscopic pancreatography was studied in a prospective, randomized, double-blind trial. Sixty-three consecutive patients undergoing ERCP were randomly allocated to two group. In the control group, 34 patients received isotonic sodium-chloride, and in the treated group 29 patients received 0.1 mg of octreotide acetate subcutaneously before the pancreatography. After the endoscopy, amylase levels increased to pathological range in 15 of the controls and in 3 of the treated patients, whereas lipase levels showed a pathological rise in 17 of the controls and in 5 of the treated patients. A significant difference (p less than 0.01) was observed in the amylase and lipase changes between the two groups at 90 and 180 min after pancreatography. The enzyme levels showed at 90 min, mean +/- SD amylase: controls 540 +/- 185 units/liter, treated patients 261 +/- 108 units/liter; lipase: controls 304 +/- 98 units/liter, treated patients 198 +/- 88 units/liter. These findings suggest that the use of long-acting somatostatin analogue ameliorates the enzyme increases in the serum after endoscopic pancreatography.

New clinical aspects of pancreas divisum.

The clinical and pathogenetic features of pancreas divisum are surveyed on the basis of ERCP examination. This congenital anomaly of the pancreas can be distinguished from ductal obstruction on the basis of the radiographic configuration. In such patients the parenchyma of the pancreas can be properly evaluated only if the dorsal and ventral pancreatic ducts are visualized simultaneously. Pancreas divisum may be regarded as imposing an increased risk of supervening pancreatic diseases.

DLG5 R30Q Is Not Associated With IBD in Hungarian IBD Patients but Predicts Clinical Response to Steroids in Crohn's Disease

fragment length polymorphism. DNA was screened for NOD2/ CARD15 mutations by denaturing high-performance liquid chromatography. Detailed clinical phenotype was determined by reviewing the medical charts. Results: The carrier frequency of the R30Q variant allele was not significantly different in IBD (22.0%), CD (20.8%), and UC (27.6%) patients compared with healthy control subjects (28.0%). In CD, the 113A variant allele was associated with steroid resistance (16.3% vs noncarriers, 7.6%; odds ratio [OR], 2.4; 95% CI 1.3Y4.5; P = 0.013). In a logistic regression model carriage of DLG5 R30Q, perianal involvement and frequent relapses were independently associated with steroid resistance. No phenotype-genotype associations were found in UC patients, although a trend toward more extensive disease was observed in carriers of the variant allele (OR = 2.1; 95% CI 0.95Y4.4; P = 0.07). Conclusions: The present data strongly contrast previous data from Germany. DLG5 113A is not associated with disease susceptibility, but there was a tendency for this allele to confer resistance to steroids. Further studies are required to evaluate the significance of DLG5 in other populations from geographically diverse regions.