IL-1 gene cluster polymorphisms and development of primary gastric B-cell lymphoma in Helicobacter pylori infection.
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Case-Control Studies↗
Biomedical subjects
Jochen Hampe
Publications and source records attributed to Jochen Hampe.
Widespread occurrence of alternative splicing at NAGNAG acceptors contributes to proteome plasticity.
Splice acceptors with the genomic NAGNAG motif may cause NAG insertion-deletions in transcripts, occur in 30% of human genes and are functional in at least 5% of human genes. We found five significant biases indicating that their distribution is nonrandom and that they are evolutionarily conserved and tissue-specific. Because of their subtle effects on mRNA and protein structures, these splice acceptors are often overlooked or underestimated, but they may have a great impact on biology and disease.
Alternative Splicing↗
Genetic variation in DLG5 is associated with inflammatory bowel disease.
Crohn disease and ulcerative colitis are two subphenotypes of inflammatory bowel disease (IBD), a complex disorder resulting from gene-environment interaction. We refined our previously defined linkage region for IBD on chromosome 10q23 and used positional cloning to identify genetic variants in DLG5 associated with IBD. DLG5 encodes a scaffolding protein involved in the maintenance of epithelial integrity. We identified two distinct haplotypes with a replicable distortion in transmission (P = 0.000023 and P = 0.004 for association with IBD, P = 0.00012 and P = 0.04 for association with Crohn disease). One of the risk-associated DLG5 haplotypes is distinguished from the common haplotype by a nonsynonymous single-nucleotide polymorphism 113G-->A, resulting in the amino acid substitution R30Q in the DUF622 domain of DLG5. This mutation probably impedes scaffolding of DLG5. We stratified the study sample according to the presence of risk-associated CARD15 variants to study potential gene-gene interaction. We found a significant difference in association of the 113A DLG5 variant with Crohn disease in affected individuals carrying the risk-associated CARD15 alleles versus those carrying non-risk-associated CARD15 alleles. This is suggestive of a complex pattern of gene-gene interaction between DLG5 and CARD15, reflecting the complex nature of polygenic diseases. Further functional studies will evaluate the biological significance of DLG5 variants.
Alleles↗
Patterns of linkage disequilibrium in the MHC region on human chromosome 6p.
Single nucleotide polymorphisms (SNPs) in the human genome are thought to be organised into blocks of high internal linkage disequilibrium (LD), separated by intermittent recombination hotspots. Since understanding haplotype structure is critical for an accurate assessment of inter-individual genetic differences, we investigated up to 968 SNPs from a 10-Mb region on chromosome 6p21, including the human major histocompatibility complex (MHC), in five different population samples (45-550 individuals). Regions of well-defined block structure were found to coexist alongside large areas lacking any clear structure; occasional long-range LD was observed in all five samples. The four white populations analysed were remarkably similar in terms of the extend and spatial distribution of local LD. In US African Americans, the distribution of LD was similar to that in the white populations but the observed haplotype diversity was higher. The existence of large regions without any clear block structure renders the systematic and thorough construction of SNP haplotype maps a crucial prerequisite for disease-association studies.
Black or African American↗
In vitro alterations of intestinal bacterial microbiota in fecal samples during storage.
The human gastrointestinal tract harbors an extremely diverse and complex microbial ecosystem. Most of the existent data about the enteric microflora have been generated using stool samples, but the collection and storage of fecal samples are often problematic. The influence of the storage of stool samples on the bacterial diversity and the degradation of bacterial DNA was analysed in this study. Stool samples from 5 healthy volunteers were exposed to different storage temperatures and durations. The bacterial diversity and the amount of intact bacterial DNA were analysed by single-stranded conformation polymorphism analysis (SSCP) and real-time polymerase chain reaction (PCR), both using a 16S rDNA approach. Additionally, biopsy specimens were taken from 3 of the 5 individuals to compare fecal and mucosal flora. The bacterial diversity of the fecal flora and the total number of bacteria were significantly reduced after 8 and 24 hours at both room temperature and 4 degrees C. The mucosa-associated bacterial microflora showed substantial differences compared with the fecal flora. The observed alterations of fecal flora during storage point to the difficulty of the molecular analysis of the bacterial diversity and the enumeration of bacterial cells in fecal samples.
Adult↗
Quantification of intestinal bacterial populations by real-time PCR with a universal primer set and minor groove binder probes: a global approach to the enteric flora.
The composition of the human intestinal flora is important for the health status of the host. The global composition and the presence of specific pathogens are relevant to the effects of the flora. Therefore, accurate quantification of all major bacterial populations of the enteric flora is needed. A TaqMan real-time PCR-based method for the quantification of 20 dominant bacterial species and groups of the intestinal flora has been established on the basis of 16S ribosomal DNA taxonomy. A PCR with conserved primers was used for all reactions. In each real-time PCR, a universal probe for quantification of total bacteria and a specific probe for the species in question were included. PCR with conserved primers and the universal probe for total bacteria allowed relative and absolute quantification. Minor groove binder probes increased the sensitivity of the assays 10- to 100-fold. The method was evaluated by cross-reaction experiments and quantification of bacteria in complex clinical samples from healthy patients. A sensitivity of 10(1) to 10(3) bacterial cells per sample was achieved. No significant cross-reaction was observed. The real-time PCR assays presented may facilitate understanding of the intestinal bacterial flora through a normalized global estimation of the major contributing species.
Bacteria↗
Nonelectrophoretic method for high-throughput HLA-DRB1 group genotyping.
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DNA Probes, HLA↗
Locking of 3' ends of single-stranded DNA templates for improved Pyrosequencing performance.
In Pyrosequencing, a DNA strand complementary to a single-stranded DNA (ssDNA) template is synthesized, whereby each incorporated nucleotide yields detectable light, and the light intensity is proportional to the incorporated nucleotides. Correct data interpretation (i.e., signal-to-noise ratio of light intensities) is hampered by artifacts due to the formation of secondary structures of single-stranded templates. Critical among these is the looping back of the template's nonbiotinylated 3' end to itself In the resulting structure, the 3' end functions as a primer, the extension of which results in background signals. We present two ways of preventing the self-priming of a template's 3' end: (i) the use of a modified oligonucleotide, called blOligo, which is complementary to the template's 3' end and (ii) the extension of the template's 3' end with a ddNMP. In contrast to unprotected 3' ends of ssDNA templates, causing inconsistent results, we show that protecting the 3' end of an ssDNA template using either blOligos or ddNMP enables the correct interpretation of signals and results in reliable quantification.
Alleles↗
Entropy-based SNP selection for genetic association studies.
Because of their abundance, density, and ease of practical use, single-nucleotide polymorphisms (SNPs) have become the major source of information for association gene mapping in humans. Sensible strategies for selecting practically useful SNPs are therefore required. Among the factors influencing the mapping utility of a given set of SNPs are (1). their individual diversity, (2). their haplotype structure in the population of interest, and (3). their physical distribution. We propose a strategy integrating these aspects into a single mapping utility measure, which is based upon Shannon entropy, and which maximizes the amount of information extracted from a genomic region under a Malecot model of linkage disequilibrium (LD) decay. The same utility measure has also been used to define a criterion guiding SNP discovery and rational decision-making about the continuation or termination of a mapping study. The proposed strategy performs consistently well in a data set comprising 549 German control individuals, genotyped for 136 SNPs from four genomic regions of different LD structure. Adoption of the method in practice is estimated to save up to 30% of genotyping load when compared with equidistant SNP localization or pair-wise LD minimization alone.
DNA↗
Stratification by CARD15 variant genotype in a genome-wide search for inflammatory bowel disease susceptibility loci.
Previously we have conducted a genome-wide search for inflammatory bowel disease susceptibility loci in a large European cohort. Results from this study demonstrated suggestive evidence of linkage to loci at chromosomes 1q, 6p, and 10p and replicated linkages on chromosomes 12 and 16. Recently, NOD2/CARD15 on chromosome 16q12 has been found to be strongly associated with Crohn's disease. In order to determine if there are other loci in the genome that interact with the three associated functional variants in CARD15 (R702W, G908R, 1007fs), we have stratified our large inflammatory bowel disease genome scan cohort by dividing pedigrees into two groups stratified by CARD15 variant genotype. The two pedigree groups were analysed using non-parametric allele sharing methods. The group of pedigrees that contained one of the three CARD15 variants had two suggestive linkage results occurring in 6p (lod = 3.06 at D6S197, IBD phenotype) and 10p (lod=2.29 at D10S197, CD phenotype). In addition, at 16q12 where CARD15 is located, the original genome scan had a peak lod score of 2.18 at D16S415 (CD phenotype). The stratified pedigree cohort containing one of three CARD15 variants had a peak lod score of 0.90 at D16S415 (CD phenotype), accounting for approximately less than half of the genetic evidence for linkage at this locus. This result is in agreement with the existence of a substantial number of private variants at the NOD2/CARD15 locus. Interaction with NOD2/CARD15 needs to be considered in future gene identification efforts on chromosomes 6 and 10.
Apoptosis Regulatory Proteins↗
IBD5 is a general risk factor for inflammatory bowel disease: replication of association with Crohn disease and identification of a novel association with ulcerative colitis.
Inflammatory bowel disease (IBD) refers to complex chronic relapsing autoimmune disorders of the gastrointestinal tract that have been traditionally classified into Crohn disease (CD) and ulcerative colitis (UC). We have previously reported that genetic variation within a 250-kb haplotype (IBD5) in the 5q31 cytokine gene cluster confers susceptibility to CD in a Canadian population. In the current study, we first replicated this association by examining 368 German trios with CD and demonstrating, by transmission/disequilibrium testing (TDT), that the same haplotype is associated with CD (chi2=5.97; P=.007). Our original association study focused on the role of IBD5 in CD; we next explored the potential contribution of this locus to UC susceptibility in 187 German trios. Given the TDT results in the present cohort with UC, IBD5 may also act as a susceptibility locus for UC (chi2=8.10; P=.002). We then examined locus-locus interactions between IBD5 and CARD15, a locus reported elsewhere to confer risk exclusively to CD. Our current results indicate that the two loci act independently to confer risk to CD but that these two loci may behave in an epistatic fashion to promote the development of UC. Moreover, IBD5 was not associated with particular clinical manifestations upon phenotypic stratification in the current cohort with CD. Taken together, our results suggest that IBD5 may act as a general risk factor for IBD, with loci such as CARD15 modifying the clinical characteristics of disease.
Colitis, Ulcerative↗
Association of inflammatory bowel disease with indicators for childhood antigen and infection exposure.
BACKGROUND AND AIMS: Genetic susceptibility plays an important role in the predisposition to inflammatory bowel disease (IBD), but genetics alone cannot explain the six- to eightfold rise in incidence accompanying postwar socioeconomic changes in developed countries. It is presently unclear how environmental factors either trigger or modify the risk for and course of IBD in the presence of genetic susceptibility. PATIENTS AND METHODS: We evaluated 2,351 consecutive responses from IBD patients and from 3,364 of their unaffected first-degree relatives (parents/siblings) who completed a multi-item questionnaire with their physicians as part of a study of IBD genetics. All single-patient families were excluded from the analysis to avoid ascertainment bias, resulting in 120-133 independent cases for the analysis of environmental factors and 1,685 patients to be included in the analysis of birth rank. RESULTS: No statistically significant association was observed between the presence of IBD and the availability of either (warm) tap water, water toilets, or central heating during childhood. However, higher birth rank (> or =3) was significantly associated with a lower risk of IBD (odds ratio 0.68). CONCLUSION: Lower birth rank as a possible indicator of increased childhood infection exposure was associated with a higher risk for IBD. Future studies need to address the interaction of known genetic variations (e.g., in the NOD2 gene) with environmental factors potentially mediating this effect.
Birth Order↗
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.
Aging↗
High-resolution SNP scan of chromosome 6p21 in pooled samples from patients with complex diseases.
We apply a high-throughput protocol of chip-based mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight; MALDI-TOF) as a method of screening for differences in single-nucleotide polymorphism (SNP) allele frequencies. Using pooled DNA from individuals with asthma, Crohn's disease (CD), schizophrenia, type 1 diabetes (T1D), and controls, we selected 534 SNPs from an initial set of 1435 SNPs spanning a 25-Mb region on chromosome 6p21. The standard deviations of measurements of time of flight at different dots, from different PCRs, and from different pools indicate reliable results on each analysis step. In 90% of the disease-control comparisons we found allelic differences of <10%. Of the T1D samples, which served as a positive control, 10 SNPs with significant differences were observed after taking into account multiple testing. Of these 10 SNPs, 5 are located between DQB1 and DRB1, confirming the known association with the DR3 and DR4 haplotypes whereas two additional SNPs also reproduced known associations of T1D with DOB and LTA. In the CD pool also, two earlier described associations were found with SNPs close to DRB1 and MICA. Additional associations were found in the schizophrenia and asthma pools. They should be confirmed in individual samples or can be used to develop further quality criteria for accepting true differences between pools. The determination of SNP allele frequencies in pooled DNA appears to be of value in assigning further genotyping priorities also in large linkage regions.
Asthma↗
Haplotype structure and association to Crohn's disease of CARD15 mutations in two ethnically divergent populations.
Current debate focuses on the relevance of linkage disequilibrium (LD), ethnicity and underlying haplotype structure to the search for genes involved in complex disorders. The recently described association between single nucleotide polymorphisms (SNPs) of the CARD15 (NOD2) gene and Crohn's disease (CD) in populations of north-European descent provides a test case that we have subjected to detailed SNP haplotype based analyses. We examined 23 SNPs spanning 290 kb, including CARD15, in large North-European and Korean samples of patients with Crohn's disease and normal controls. In Europeans we confirmed that the three disease-associated SNPs occur independently but share a common background haplotype. This suggests a common origin and the possibility of an undiscovered more strongly predisposing mutation. Korean CD patients present a phenotype identical to the European patients and have not previously been screened for CARD15. The three disease-associated SNPs were absent and there was no evidence of association between CARD15 and CD. Consequently, the disease-associated mutations in the Europeans, which are rare, have arisen recently (after the Asian-European split). Our results highlight important issues relevant to mapping the genes that predispose to complex disorders. First, although ethnically divergent populations may present identical phenotypes they do not necessarily share the same set of predisposing genes. Second, although single-locus tests of association showed consistent association with markers throughout the gene, pair-wise LD between markers (r(2) and D') yielded very little information about actual disease-association. Third, a population comparative approach allowed refining of the marker set through the examination of shared polymorphisms and common LD-groups. This approach, in conjunction with the examination of the mutational steps in a haplotype network, allows unambiguous identification of the potentially causative mutations.
Asian People↗
Lack of association between the C3435T MDR1 gene polymorphism and inflammatory bowel disease in two independent Northern European populations.
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Colitis, Ulcerative↗
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.
Carrier Proteins↗
Genomic structure, chromosome mapping and expression analysis of the human AVIL gene, and its exclusion as a candidate for locus for inflammatory bowel disease at 12q13-14 (IBD2).
Chronic inflammatory bowel disease is a multifactorial disorder with two major clinical forms, Crohn's disease and ulcerative colitis. One of the potential susceptibility loci for inflammatory bowel disease (IBD2) was localized at 12q13-14 in the vicinity of the deoxyribonucleic acid marker D12S83 by linkage analysis. A candidate susceptibility gene for IBD2 in this region is the AVIL gene. AVIL encodes a protein (advillin) which belongs to the gelsolin/villin family of proteins and might therefore be involved in morphogenesis of microvilli. We have determined the genomic organization of the AVIL gene, including the transcription start site and its localization with respect to D12S83. The 2457 bp coding region of AVIL consists of 19 exons and is localized to 12q14 proximal to D12S83. Primer extension analysis suggests two transcription start sites localized at -548 and -664 bp upstream to the ATG translation codon. We have evaluated AVIL as a candidate susceptibility gene for IBD2 in 24 unrelated patients with evidence of linkage to chromosome 12, as well as in 91 individuals from 19 affected IBD families for putative single nucleotide polymorphisms.
Chromosome Mapping↗