Genetic analysis of innate immunity in Crohn's disease and ulcerative colitis identifies two susceptibility loci harboring CARD9 and IL18RAP

The two main phenotypes of inflammatory bowel disease (IBD)--Crohn's disease (CD) and ulcerative colitis (UC)--are chronic intestinal inflammatory disorders with a complex genetic background. Using a three-stage design, we performed a functional candidate-gene analysis of innate immune pathway in IBD. In phase I, we typed 354 SNPs from 85 innate immunity genes in 520 Dutch IBD patients (284 CD, 236 UC) and 808 controls. In phase II, ten autosomal SNPs showing association at p < 0.006 in phase I were replicated in a second cohort of 545 IBD patients (326 CD, 219 UC) and 360 controls. In phase III, four SNPs with p < 0.01 in the combined phase I and phase II analysis were genotyped in an additional 786 IBD samples (452 CD, 334 UC) and 768 independent controls. Joint analysis of 1851 IBD patients (1062 CD, 789 UC) and 1936 controls demonstrated strong association to the IL18RAP rs917997 SNP for both CD and UC (p(IBD) 1.9 x 10(-8); OR 1.35). Association in CD is independently supported by the Crohn's disease dataset of the Wellcome Trust Case Control Consortium (imputed SNP rs917997, p = 9.19 x 10(-4)). In addition, an association of the CARD9 rs10870077 SNP to CD and UC was observed (p(IBD) = 3.25 x 10(-5); OR 1.21). Both genes are located in extended haplotype blocks on 2q11-2q12 and 9q34.3, respectively. Our results indicate two IBD loci and further support the importance of the innate immune system in the predisposition to both CD and UC.     

Analysis of single-nucleotide polymorphisms in the interleukin-4 receptor gene for association with inflammatory bowel disease

 Genetic linkage analysis in families with multiple cases of inflammatory bowel disease (IBD) has mapped a gene which confers susceptibility to IBD to the pericentromeric region of chromosome 16 (IBD1). The linked region includes the interleukin(IL)-4 receptor gene (IL4R). Since IL-4 regulation and expression are abnormal in IBD, the IL4R gene is thus both a positional and functional candidate for IBD1. We screened the gene for single-nucleotide polymorphisms (SNPs) by fluorescent chemical cleavage analysis, and tested a subset of known and novel SNPs for allelic association with IBD in 355 families, which included 435 cases of Crohn's disease and 329 cases of ulcerative colitis. No association was observed between a haplotype of four SNPs (val50ile, gln576arg, A3044G, G3289A) and either the Crohn's disease or ulcerative colitis phenotypes using the transmission disequilibrium test. There was also no evidence for association when the four markers were analyzed individually. The results indicate that these variants are not significant genetic determinants of IBD, and that the IL4R gene is unlikely to be IBD1. Linkage disequilibrium analyses showed that the val50ile and gln576arg variants are in complete equilibrium with each other, although they are separated by only about 21 kilobases of genomic DNA. This suggests that a very dense SNP map may be required to exclude or detect disease associations with some candidate genes. Received: 23 June 1999 / Revised: 18 August 1999     

A linkage map of mouse chromosome 19: definition of comparative mapping relationships with human chromosomes 10 and 11 including the MEN1 locus.

A linkage map of mouse Chromosome (Chr) 19 was constructed using an interspecific cross and markers defined by restriction fragment length variants. The map includes 20 markers, 9 of which had not been mapped previously in the mouse. The data further defined the relationship between genes on mouse Chr 19 and those on the long arm of human Chr 10 and the pericentric region of the long arm of human Chr 11. The comparative mapping analysis suggests that the proximal segment of mouse Chr 19 may contain the MEN1 locus and that the current study has identified additional genes that may be useful for positional cloning of this putative tumor suppressor gene.     

High-density genome scan in Crohn disease shows confirmed linkage to chromosome 14q11-12

Epidemiological studies have shown that genetic factors contribute to the pathogenesis of the idiopathic inflammatory bowel diseases (IBD), Crohn disease (CD) and ulcerative colitis (UC). Recent genome scans and replication studies have identified replicated linkage between CD and a locus on chromosome 16 (the IBD1 locus), replicated linkage between IBD (especially UC) and a locus on chromosome 12q (the IBD2 locus), and replicated linkage between IBD (especially CD) and a locus on chromosome 6p (the IBD3 locus). Since the estimated locus-specific lambdas values for the regions of replicated linkage do not account for the overall lambdas in CD, and since the published genome scans in IBD show at least nominal evidence for linkage to regions on all but two chromosomes, we performed an independent genome scan using 751 microsatellite loci in 127 CD-affected relative pairs from 62 families. Single-point nonparametric linkage analysis using the GENEHUNTER-PLUS program shows evidence for linkage to the adjacent D14S261 and D14S283 loci on chromosome 14q11-12 (LOD = 3.00 and 1.70, respectively), and the maximal multipoint LOD score is observed at D14S261 (LOD = 3.60). In the multipoint analysis, nominal evidence for linkage (P<.05) is observed near D2S117 (LOD = 1.25), near D3S3045 (LOD = 1.31), between D7S40 and D7S648 (LOD = 0.91), and near D18S61 (LOD = 1.15). Our finding of significant linkage to D14S261 and the finding of suggestive linkage to the same locus in an independent study (multipoint LOD = 2.8) satisfies criteria for confirmed linkage, so we propose that the region of interest on chromosome 14q11-12 should be designated the IBD4 locus.     

A genetic variant in the gene encoding the stress70 protein chaperone family member STCH is associated with gastric cancer in the Japanese population

Association analysis, based on linkage disequilibrium between specific alleles in the candidate loci and nearby genetic markers, has been proposed to identify genes conferring susceptibility to multifactorial diseases. Using the affected sib-pair method, we previously mapped four candidate chromosomal regions, 1p32, 2q33-q35, 11p13-p14, and 21q21, for gastric cancer by linkage analysis. To identify genes involved in the disease, we performed a gene-based association analysis of 66 genes, located on 21p11-21q22, using 126 single nucleotide polymorphisms (SNPs) as genetic markers in 373 patients with 250 controls. We found a significant association of five SNPs in the stress70 protein chaperon family member STCH gene with gastric cancer, especially with the non-cardia localization subgroup (P = 0.0005-0.02, odds ratio = 1.44-1.72). Comparisons of haplotype frequency showed significant association between TTGGC haplotype and gastric cancer (P = 0.0001, odds ratio = 1.59). These results suggest that, in the Japanese population, STCH might be a new candidate for conferring susceptibility to this disease.     

Biallelic expression of<Emphasis Type="Italic"> HRAS</Emphasis> and<Emphasis Type="Italic"> MUCDHL</Emphasis> in human and mouse

At least eight genes clustered in 1 Mb of DNA on human chromosome (Chr) 11p15.5 are subject to parental imprinting, with monoallelic expression in one or more tissues. Orthologues of these genes show conserved linkage and imprinting on distal Chr 7 of mice. The extended imprinted region has a bipartite structure, with at least two differentially methylated DNA elements (DMRs) controlling the imprinting of two sub-domains. We previously described three biallelically expressed genes ( MRPL23, 2G7 and TNNT3) in 100 kb of DNA immediately downstream of the imprinted H19 gene, suggesting that H19 marks one border of the imprinted region. Here we extend this analysis to two additional downstream genes, HRAS and MUCDHL (mu-protocadherin). We find that these genes are biallelically expressed in multiple fetal and adult tissues, both in humans and in mice. The mouse orthologue of a third gene, DUSP8, located between H19 and MUCDHL, is also expressed biallelically. The DMR immediately upstream of H19 frequently shows a net gain of methylation in Wilms tumors, either via Chr 11p15.5 loss of heterozygosity (LOH) or loss of imprinting (LOI), but changes in methylation in CpG-rich sequences upstream and within the MUCDHL gene are rare in these tumors and do not correlate with LOH or LOI. These findings are further evidence for a border of the imprinted region immediately downstream of H19, and the data allow the construction of an imprinting map that includes more than 20 genes, distributed over 3 Mb of DNA on Chr 11p15.5.     

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.     

Identification of a novel inflammation-protective locus in the Fischer rat

Inbred LEW/N rats are relatively susceptible, while histocompatible inbred F344/N rats are relatively resistant to development of a wide variety of inflammatory diseases in response to a range of pro-inflammatory stimuli. In a LEW/N vs. F344/N F2 intercross, we identified a quantitative trait locus (QTL) on Chr 10 that protects in a dominant fashion against the exudate volume component of innate inflammation in the F344/N rat, as well as a suggestive QTL on Chr 2 near the Fibrinogen cluster region. The exudate volume linkage region on Chr 10 may be similar to one of the multiple regions found to link to inflammatory arthritis phenotypes in other crosses. The suggestive linkage on Chr 2 has not been previously reported and does not seem to contribute to this phenotype in the same manner as the QTL on Chr 10. These findings are consistent with the hypothesis that the innate exudate volume trait is a sub-phenotype of more complex inflammatory phenotypes, such as arthritis, and genes within the Chr 10 linkage region could account for differences in this non-specific acute phase component of the inflammatory response. Since the rat Chr 10 exudate volume linkage region we have identified is syntenic with a region of human Chr 17 that has been shown to link to a variety of autoimmune/inflammatory diseases, including insulin-dependent diabetes mellitus, multiple sclerosis, and psoriasis, identification of genes within this linkage region will shed light on genes relevant to the earliest inflammatory component and to susceptibility and resistance to such human autoimmune/inflammatory diseases. Received: 4 August 1998 / Accepted: 4 December 1998     

Linkage and Association Studies of Prostate Cancer Susceptibility: Evidence for Linkage at 8p22-23

Multiple lines of evidence have implicated the short arm of chromosome 8 as harboring genes important in prostate carcinogenesis. Although most of this evidence comes from the identification of frequent somatic alterations of 8p loci in prostate cancer cells (e.g., loss of heterozygosity), studies have also suggested a role for 8p genes in mediation of inherited susceptibility to prostate cancer. To further examine this latter possibility, we performed linkage analyses, in 159 pedigrees affected by hereditary prostate cancer (HPC), using 24 markers on the short arm of chromosome 8. In the complete set of families, evidence for prostate cancer linkage was found at 8p22-23, with a peak HLOD of 1.84 (P=.004), and an estimate of the proportion of families linked (alpha) of 0.14, at D8S1130. In the 79 families with average age at diagnosis >65 years, an allele-sharing LOD score of 2.64 (P=.0005) was observed, and six markers spanning a distance of 10 cM had LOD scores >2.0. Interestingly, the small number of Ashkenazi Jewish pedigrees (n=11) analyzed in this study contributed disproportionately to this linkage. Mutation screening in HPC probands and association analyses in case subjects (a group that includes HPC probands and unrelated case subjects) and unaffected control subjects were carried out for the putative prostate cancer-susceptibility gene, PG1, previously localized to the 8p22-23 region. No statistical differences in the allele, genotype, or haplotype frequencies of the SNPs or other sequence variants in the PG1 gene were observed between case and control subjects. However, case subjects demonstrated a trend toward higher homozygous rates of less-frequent alleles in all three PG1 SNPs, and overtransmission of a PG1 variant to case subjects was observed. In summary, these results provide evidence for the existence of a prostate cancer-susceptibility gene at 8p22-23. Evaluation of the PG1 gene and other candidate genes in this area appears warranted.     

Extensive linkage disequilibrium,a common 16.7-kilobase deletion,and evidence of balancing selection in the human protocadherin alpha cluster

Regions of extensive linkage disequilibrium (LD) appear to be a common feature of the human genome. However, the mechanisms that maintain these regions are unknown. In an effort to understand whether gene density contributes to LD, we determined the degree of promoter sequence variation in a large tandem-arrayed gene family, the human protocadherin alpha cluster, on chromosome 5. These genes are expressed at synaptic junctions in the developing brain and the adult brain and may be involved in the determination of synaptic complexity. We sequenced the promoters of all 13 alpha protocadherin genes in 96 European Americans and identified polymorphisms in the promoters alpha 1, alpha 3, alpha 4, alpha 5, alpha 7, alpha 9, alpha 11, and alpha 13. In these promoters, 11 common SNPs are in extensive LD, forming two 48-kb haplotypes of equal frequency, in this population, that extend from the alpha1 through alpha 7 genes. We sequenced these promoters in East Asians and African Americans, and we estimated haplotype frequencies and calculated LD statistics for all three populations. Our results indicate that, although extensive LD is an ancient feature of the alpha cluster, it has eroded over time. SNPs 3' of alpha 7 are involved in ancestral recombination events in all populations, and overall alpha-cluster LD is reduced in African Americans. We obtained significant positive values for Tajima's D test for all alpha promoter SNPs in Europeans (D=3.03) and East Asians (D=2.64), indicating an excess of intermediate-frequency variants, which is a signature of balancing selection. We also discovered a 16.7-kb deletion that truncates the alpha 8 gene and completely removes the alpha 9 and alpha 10 genes. This deletion appears in unaffected individuals from multiple populations, suggesting that a reduction in protocadherin gene number is not obviously deleterious.     

Inflammatory bowel disease: the role of inflammatory cytokine gene polymorphisms

The mechanisms responsible for development of inflammatory bowel disease (IBD) have not been fully elucidated, although the main cause of disease pathology is attributed to up-regulated inflammatory processes. The aim of this study was to investigate frequencies of polymorphisms in genes encoding pro-inflammatory and anti-inflammatory markers in IBD patients and controls. We determined genotypes of patients with IBD (n= 172) and healthy controls (n= 389) for polymorphisms in genes encoding various cytokines (interleukin (IL)-1beta, IL-6, tumour necrosis factor (TNF), IL-10, IL-1 receptor antagonist). Association of these genotypes to disease incidence and pathophysiology was investigated. No strong association was found with occurrence of IBD. Variation was observed between the ulcerative colitis study group and the control population for the TNF-alpha-308 polymorphism (p= 0.0135). There was also variation in the frequency of IL-6-174 and TNF-alpha-308 genotypes in the ulcerative colitis group compared with the Crohn's disease group (p= 0.01). We concluded that polymorphisms in inflammatory genes are associated with variations in IBD phenotype and disease susceptibility. Whether the polymorphisms are directly involved in regulating cytokine production, and consequently pathophysiology of IBD, or serve merely as markers in linkage disequilibrium with susceptibility genes remains unclear.     

A second-generation genomewide screen for asthma-susceptibility alleles in a founder population

A genomewide screen for asthma- and atopy-susceptibility loci was conducted, using 563 markers, in 693 Hutterites who are members of a single 15-generation pedigree, nearly doubling the sample size from the authors' earlier studies. The resulting increase in power led to the identification of 23 loci in 18 chromosomal regions showing evidence for linkage that is, in general, 10-fold more significant (P<.001 vs. P<.01) than the linkages reported previously in this population. Moreover, linkages to loci in 11 chromosomal regions were identified for the first time in the Hutterites in this report, including five regions (5p, 5q, 8p, 14q, and 16q) showing evidence both of linkage, by the likelihood ratio (LR) chi(2), and of disequilibrium, by the transmission/disequilibrium test. A region on chromosome 19 continues to show evidence for linkage, by both tests, in this study. Studies of 17 candidate genes provide evidence for association with variation in the IL4RA gene (16p12), the HLA class II genes (6p21), and the interferon-alpha gene cluster (9p22), but the lack of evidence for linkage in these regions by the LR chi(2) test suggests that these are minor susceptibility loci. A polymorphism in the CD14 gene is in linkage disequilibrium with an as yet unidentified susceptibility allele in the 5q cytokine cluster, a region showing evidence for linkage among the Hutterites. Finally, 10 of the regions showing evidence for linkage in the Hutterites have shown evidence of linkage to related phenotypes in other genome screens, suggesting that these regions may contain common alleles that have relatively large effects on asthma and atopy phenotypes in diverse populations.     

Localization of the IGHG,PRKACB, and TNP2 genes in pigs by in situ hybridization

The porcine genes encoding the immunoglobulin gamma heavy chain (IGHG), cAMP-dependent protein kinase catalytic beta subunit (PRKACB), and transition protein 2 (TNP2) were mapped to Chromosomes (Chrs) 7 q25–q26, 6q31–q33, and 3p13-cent, respectively, by in situ hybridization. Localization of the IGHG gene confirms the assignment of linkage group III to Chr 7. Our results show that the IGHG locus in pigs, similar to the situation in other mammalian species, viz. humans, mouse, cattle, and river buffaloes, is located on the terminal region of the chromosome. The assignment of the PRKACB gene extends the homology observed between porcine Chr 6q and human Chr 1p. Mapping of the TNP2 gene provides the first marker assigned to the p arm of Chr 3 in pigs. The present study contributes to the development of the physical gene map in pigs and also bears significance in terms of comparative gene mapping.     

Differences between the radiation hydrid and genetic linkage maps of bovine Chromosome 5 resolved with a quasi-phylogenetic method of analysis

Two major differences were detected in gene order between the radiation hybrid map and the genetic linkage map of bovine Chromosome (Chr) 5, and these were resolved by analyzing the raw radiation hybrid data by a quasi-phylogenetic method. Seventeen loci were typed on the new cattle whole genome radiation hybrid panel. Most of these loci are framework loci and include AGLA293, BM315, BM6026, BP1, BZRP, CD9, CSSM22, CSSM34, CYP2D@, ETH2, ETH10, ETH152, IGF1, LALBA, SLC2A3, SYT1, and TPI1. BP1 was found to be closer to the centromere than either BM6026 or SYT1 with two standard computer software packages for analyzing radiation hybrid panel data. This is inconsistent with any of the genetic linkage maps as well as their consensus. CYP2D@ was placed between ETH2 and BZRP, and this is also inconsistent with the genetic linkage maps, since CYP2D@ should be the most telomeric of the loci tested in this study. Resolution was reached by analyzing the raw radiation hybrid data for clones that bind some but not all of the loci, and the binding pattern was more consistent with the linkage maps and less consistent with the software-generated radiation hybrid map. The comparative mapping data confirm the relative inversion of gene order of SYT1 compared with humans and mice. A non-polymorphic fragment for CD9 indicates the conservation of gene order for three loci located on human Chr 12p. The genes of bovine Chr 5 conserved on human Chr 12p are located separately from the genes conserved on human Chr 12q. It is recommended that the raw data for radiation hybrid maps be made publicly available so that conflicts in gene order can be evaluated explicity. Received: 19 February 1999 / Accepted: 3 January 2000     

Genomic organization and sequence variation of the human integrin subunit alpha8 gene (ITGA8).

The integrin alpha8 is highly expressed during kidney and lung development. alpha8-deficient mice display abnormal renal development suggesting that alpha8 plays a critical role in organogenesis. Therefore, it would be of considerable interest to understand the genomic structure, localization and sequence variation of the alpha8 gene. Using FISH and genomic database analysis, we show that alpha8 gene maps to chromosome 10p13 and consists of >200 kbp organized into 30 exons. Examination of 47 individuals from two different ethnic groups (European and African descent) identified 286 varying sites. The diversity of alpha8 is comparable to that of other regions within the human genome. Eight of the varying sites were located in the coding regions: six resulted in nonsynonymous substitutions of which two lead to non-conservative changes in protein. None of the sites showed significant deviation from Hardy-Weinberg equilibrium. We mapped the coding region single nucleotide polymorphisms (SNPs) onto a model of the predicted alpha8 structure and found all the SNPs were located in the "calf" of the extracellular domain. In the European population, the linkage disequilibrium statistic D' showed three blocks of relatively non-recombinant regions in the alpha8 gene while the African population showed more evidence of recombination. The observed patterns of the linkage disequilibrium statistic R2 suggest that a large number of sites will need to be genotyped to ensure coverage of the entire gene for genetic association studies. Identification of the sequence variation will allow genetic association studies of alpha8 in kidney and lung disease.     

DNA polymorphism haplotypes of the human apolipoprotein APOA1-APOC3-APOA4 gene cluster

Summary The genes coding for apolipoproteins A1, C3, and A4 (APOA1, APOC3, APOA4) are closely linked and tandemly organized within a 15-kilobase (kb) DNA segment on the long arm of human chromosome 11. The nucleotide variability of a 61-kb DNA segment containing these genes and their flanking sequences was studied by restriction analysis of a sample of 18 unrelated Northern Europeans using seven different genomic DNA probes. Eleven restriction site polymorphisms located within this DNA segment were used for haplotype analysis of 129 Mediterranean and 67 American black chromosomes. Estimation of the extent of nonrandom association between these polymorphisms indicated considerable linkage disequilibrium within the APOA1-APOC3-APOA4 gene cluster. Several haplotypes arose by recombination, and the rate of recombination within this gene cluster was estimated to be at least 4 times greater than that expected based on uniform recombination. The polymorphism information content of each of these polymorphisms, taken individually, ranges between 0.053 and 0.375, while that of their haplotypes ranges between 0.858 and 0.862. Therefore, DNA polymorphism haplotypes in the APOA1-APOC3-APOA4 gene cluster constitute a highly informative genetic marker on the long arm of human chromosome 11.