Evaluation of AGR2 and AGR3 as candidate genes for inflammatory bowel disease.

Linkage analyses have implicated chromosome 7p21.3 as a susceptibility region for inflammatory bowel disease (IBD). Recently, the mouse phenotype with diarrhea and goblet cell dysfunction caused by anterior gradient protein 2 dysfunction was reported (European patent WO2004056858). The genes encoding for the human homologues AGR2 and AGR3 are localized on chromosome 7p21.3. The gene structures were verified and mutation detection was performed in 47 IBD patients. A total of 30 single nucleotide polymorphisms (SNPs) were tested for association to ulcerative colitis (UC, N = 317) and Crohn's disease (CD, N = 631) in a German cohort and verified in a UK cohort of 384 CD and 311 UC patients. An association signal was identified in the 5' region of the AGR2 gene (most significant SNP hcv1702494, nominal P(TDT) = 0.011, P(case/control) = 0.0007, OR = 1.34, combined cohort). The risk haplotype carried an odds ratio of 1.43 in the German population (P = 0.002). AGR2 was downregulated in UC patients as compared to normal controls (P < 0.001) and a trend toward lower expression was seen in carriers of the risk alleles. Luciferase assays of the AGR2 promoter showed regulation by the goblet cell-specific transcription factors FOXA1 and FOXA2. In summary, AGR2 represents an interesting new avenue into the etiopathophysiology of IBD and the maintenance of epithelial integrity.

New strategies for efficient typing of HLA class-II loci DQB1 and DRB1 by using Pyrosequencing.

The characterization of genetic risk factors for complex diseases located on chromosome-6 frequently requires human leucocyte antigen (HLA) genotyping of large patient cohorts. Currently available methods do not support high-throughput HLA typing beyond the major allele group level. We, thus, developed a high-throughput approach for the HLA-DQB1 and HLA-DRB1 loci that is based on Pyrosequencing. Pyrosequencing offers a higher degree of automation than direct sequencing or oligotyping. Using a dispensation order optimized for the particular HLA locus, rapid group typing and fine resolution can be achieved. We implemented the method for two important HLA loci--DQB1 and DRB1. The HLA-DQB1 typing method comprises the following steps: splitting the potential alleles after a generic polymerase chain reaction (PCR) amplification into groups with a first Pyrosequencing reaction and resolving the split allele groups by means of five further Pyrosequencing reactions. The HLA-DR gene family is known to be the most polymorphic one in the HLA class-II region because of a large number of DRB1 alleles. Because of this complex nature, HLA-DRB1 typing was performed by means of a combination of sequence-specific PCR typing and Pyrosequencing. HLA-DQB1 typing and HLA-DRB1 typing were performed successfully by using standard DNA samples with the help of known HLA genotypes and in a blind study by using the samples from the Deutscher Zell Austausch 2002 and 2003. The approach was optimized and was practically tested for genotyping in disease association studies. Our well-elaborated Pyrosequencing-based protocols offer a new alternative to the existing HLA class-II typing methods and represent a convenient and economic solution, a unique combination of high accuracy with high-sample throughput.

CARD15 gene mutations in sarcoidosis.

Sarcoidosis, Blau syndrome and Crohn's disease are complex disorders, characterised by granulomatous inflammation affecting a variety of organs. Mutations of the CARD15 gene, on chromosome 16, have been shown to contribute significantly to Crohn's disease and to cause Blau syndrome. These factors prompted the current authors to study CARD15 mutations in sarcoidosis. A total of 138 families were genotyped, including 302 patients with sarcoidosis and 127 patients without a family history of sarcoidosis (together with their parents), for four main coding CARD15 polymorphisms associated with increased risk of Crohn's disease. Furthermore, the gene segment that harbours Blau syndrome mutations was sequenced in 39 selected patients from 39 families with affected siblings identical for one or two parental chromosomes 16s and in eight patients from multi-case families. None of the reported Blau syndrome mutations and no new sequence alterations were found. There was an increased frequency of transmission of the rare allele of the polymorphic sites 802C>T (SNP5) and 2722G>C (SNP12) in at least one of the two study groups. In conclusion, CARD15 mutations, which are important in Crohn's disease and Blau syndrome, play no major role in sarcoidosis in this study population. However, these mutations could be of limited importance, especially in patients without a family history of sarcoidosis.