Genetic analysis of autoimmune and metabolic traits in chickens

Studying how changes in DNA affect organisms and gene functions, can give us an increased understanding of the genetic regulation of diseases and metabolic traits. Chickens and other farm animals have long been used as a tool to study human diseases and other complex traits since they provide an excellent opportunity to study these traits in a controlled environment. In this thesis I have applied new statistical methods to experimental data from two outbred chicken crosses to investigate the genetic architecture and regulation of autoimmune and metabolic traits. The first cross was generated from two chicken lines divergently selected for high and low body weight and was used to study how a four-QTL network affects metabolic traits (papers I-III). The second cross was generated to study the genetic regulation of autoimmune systemic sclerosis (paper IV). Our explorations of the effects of the four-QTL network on other metabolic traits indicate that the major contribution of the network is to increase body size through an overall increase in growth of all organs rather than by changing the relative size of different components. To be able to study the networks affect on metabolic traits further, we first generated and analysed an advanced intercross line to narrow down the regions. Once the regions were fine mapped, we designed and generated a so called introgression line. Three of the fine mapped QTL were transferred (introgressed) from the low weight line into a high weight line background. This introgression line can be used to further investigate the QTL network. In the second cross, three suggestive autosomal susceptibility QTL were mapped in the chicken genome, with several orthologues of human systemic sclerosis candidate genes located within two of these loci. We also observed less disease in females with a W chromosome inherited from the diseased founder line

Genetic analysis of metabolic traits in an intercross between 8-week body-weight selected chicken lines

Metabolic traits are of paramount importance in agricultural production, as this group includes most traits of economic interest in livestock improvement. Examples include growth rate, feed efficiency and fat deposition. An improved understanding of the genetic basis of these traits can both improve our understanding of the genes that have been under selection and identify genes and pathways to be included in future breeding programs. A novel genetic mechanism has been found to regulate growth in chicken lines divergently selected for body weight. A network of four interacting genes explains nearly half of the difference in body weight at 8-weeks of age between the two lines. The central locus in this network is located on chromosome 7 and it has a role in releasing the genetic effects of three other loci in the network located on chromosome 3, 4 and 20. Interestingly, the release of the genetic effects is also reciprocal as the loci on chromosome 3, 4 and 20 jointly release the genetic effect on growth for the QTL on chromosome 7. The original report by Carlborg et al report results on body weight and fat deposition, the study does, however not report results on other phenotypes collected on the F2 individuals. This thesis presents results from analyses to evaluate the effects of the four QTL network on other measured traits in the F2 population and to see which traits that are useful in further epistatic analyses (CARLBORG et al. 2006). Furthermore the study also serves as a replication of the original study by analysing data on a larger number of added genetic markers in the QTL regions. The four QTL network was shown to have significant effects on body weight at different ages, abdominal fat and body compositions. The effect of body composition is most likely the results of an increase in general body size as the effects were not significant after corrected for body weight in the analyses. The network do, however, appear to have an effect on abdominal fat deposition and breast weight even after correcting for body weight. When corrected for body weight at slaughter (10-weeks of age) there were no significant effects on shank weight. No effects could be shown for the gene pair 7 and 4, and for 7 and 20 for other traits than body weight. The regression analysis indicates that chromosome 3 in a chromosome 7-homozygous low-line (LL) background increases relative abdominal fat and decreases relative breast muscle going from LL to HH (Homozygous high-line). When abdominal fat is not corrected for body weight at slaughter, the increase in fat deposition is proportional with increased body weight. In a chromosome 7-HH background, absolute abdominal fat is increasing with increased body-weight but relative abdominal fat is not when chromosome 3 is going from LL to HH. Relative breast muscle is decreasing, while absolute breast muscle is proportional with an increase in body weight. These results suggest that there is a change in the chicken body composition when selected for higher body weight. Chickens tend to go from lean and muscular to fat and thin. Understanding the genetic regulation of metabolic traits for which the lines differ is of crucial interest. Therefore we collected information from the literature on the descriptive statistics for these traits. Statistics were used to explore the sample size needed in an experiment to detect genetic effects of various sizes in the high- and low- lines. From these it was concluded that there is a lack of power to detect genetic effects on the network for most tested metabolic traits on the cross and that another experimental strategy is needed to explore this further. Next step in this study will be to introgress the 4-QTLs from the low line chickens into a high line background

Targeted Analysis of Serum Proteins Encoded at Known Inflammatory Bowel Disease Risk Loci

Few studies have investigated the blood proteome of inflammatory bowel disease (IBD). We characterized the serum abundance of proteins encoded at 163 known IBD risk loci and tested these proteins for their biomarker discovery potential. Based on the Human Protein Atlas (HPA) antibody availability, 218 proteins from genes mapping at 163 IBD risk loci were selected. Targeted serum protein profiles from 49 Crohns disease (CD) patients, 51 ulcerative colitis (UC) patients, and 50 sex- and age-matched healthy individuals were obtained using multiplexed antibody suspension bead array assays. Differences in relative serum abundance levels between disease groups and controls were examined. Replication was attempted for CD-UC comparisons (including disease subtypes) by including 64 additional patients (33 CD and 31 UC). Antibodies targeting a potentially novel risk protein were validated by paired antibodies, Western blot, immuno-capture mass spectrometry, and epitope mapping. By univariate analysis, 13 proteins mostly related to neutrophil, T-cell, and B-cell activation and function were differentially expressed in IBD patients vs healthy controls, 3 in CD patients vs healthy controls and 2 in UC patients vs healthy controls (q <0.01). Multivariate analyses further differentiated disease groups from healthy controls and CD subtypes from UC (P <0.05). Extended characterization of an antibody targeting a novel, discriminative serum marker, the laccase (multicopper oxidoreductase) domain containing 1 (LACC1) protein, provided evidence for antibody on-target specificity. Using affinity proteomics, we identified a set of IBD-associated serum proteins encoded at IBD risk loci. These candidate proteins hold the potential to be exploited as diagnostic biomarkers of IBD.Peer reviewe

Exploring the genetics of irritable bowel syndrome: A GWA study in the general population and replication in multinational case-control cohorts

OBJECTIVE: IBS shows genetic predisposition, but adequately powered gene-hunting efforts have been scarce so far. We sought to identify true IBS genetic risk factors by means of genome-wide association (GWA) and independent replication studies. DESIGN: We conducted a GWA study (GWAS) of IBS in a general population sample of 11\u2005326 Swedish twins. IBS cases (N=534) and asymptomatic controls (N=4932) were identified based on questionnaire data. Suggestive association signals were followed-up in 3511 individuals from six case-control cohorts. We sought genotype-gene expression correlations through single nucleotide polymorphism (SNP)-expression quantitative trait loci interactions testing, and performed in silico prediction of gene function. We compared candidate gene expression by real-time qPCR in rectal mucosal biopsies of patients with IBS and controls. RESULTS: One locus at 7p22.1, which includes the genes KDELR2 (KDEL endoplasmic reticulum protein retention receptor 2) and GRID2IP (glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein), showed consistent IBS risk effects in the index GWAS and all replication cohorts and reached p=9.31 710(-6) in a meta-analysis of all datasets. Several SNPs in this region are associated with cis effects on KDELR2 expression, and a trend for increased mucosal KDLER2 mRNA expression was observed in IBS cases compared with controls. CONCLUSIONS: Our results demonstrate that general population-based studies combined with analyses of patient cohorts provide good opportunities for gene discovery in IBS. The 7p22.1 and other risk signals detected in this study constitute a good starting platform for hypothesis testing in future functional investigations. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions

Polymorphisms in Genes of Relevance for Oestrogen and Oxytocin Pathways and Risk of Barrett's Oesophagus and Oesophageal Adenocarcinoma: A Pooled Analysis from the BEACON Consortium.

BACKGROUND: The strong male predominance in oesophageal adenocarcinoma (OAC) and Barrett's oesophagus (BO) continues to puzzle. Hormonal influence, e.g. oestrogen or oxytocin, might contribute. METHODS: This genetic-epidemiological study pooled 14 studies from three continents, Australia, Europe, and North America. Polymorphisms in 3 key genes coding for the oestrogen pathway (receptor alpha (ESR1), receptor beta (ESR2), and aromatase (CYP19A1)), and 3 key genes of the oxytocin pathway (the oxytocin receptor (OXTR), oxytocin protein (OXT), and cyclic ADP ribose hydrolase glycoprotein (CD38)), were analysed using a gene-based approach, versatile gene-based test association study (VEGAS). RESULTS: Among 1508 OAC patients, 2383 BO patients, and 2170 controls, genetic variants within ESR1 were associated with BO in males (p = 0.0058) and an increased risk of OAC and BO combined in males (p = 0.0023). Genetic variants within OXTR were associated with an increased risk of BO in both sexes combined (p = 0.0035) and in males (p = 0.0012). We followed up these suggestive findings in a further smaller data set, but found no replication. There were no significant associations between the other 4 genes studied and risk of OAC, BO, separately on in combination, in males and females combined or in males only. CONCLUSION: Genetic variants in the oestrogen receptor alpha and the oxytocin receptor may be associated with an increased risk of BO or OAC, but replication in other large samples are needed

Fine mapping and replication of QTL in outbred chicken advanced intercross lines

Background: Linkage mapping is used to identify genomic regions affecting the expression of complex traits. However, when experimental crosses such as F2 populations or backcrosses are used to map regions containing a Quantitative Trait Locus (QTL), the size of the regions identified remains quite large, i.e. 10 or more Mb. Thus, other experimental strategies are needed to refine the QTL locations. Advanced Intercross Lines (AIL) are produced by repeated intercrossing of F2 animals and successive generations, which decrease linkage disequilibrium in a controlled manner. Although this approach is seen as promising, both to replicate QTL analyses and fine-map QTL, only a few AIL datasets, all originating from inbred founders, have been reported in the literature. Methods: We have produced a nine-generation AIL pedigree (n = 1529) from two outbred chicken lines divergently selected for body weight at eight weeks of age. All animals were weighed at eight weeks of age and genotyped for SNP located in nine genomic regions where significant or suggestive QTL had previously been detected in the F2 population. In parallel, we have developed a novel strategy to analyse the data that uses both genotype and pedigree information of all AIL individuals to replicate the detection of and fine-map QTL affecting juvenile body weight. Results: Five of the nine QTL detected with the original F2 population were confirmed and fine-mapped with the AIL, while for the remaining four, only suggestive evidence of their existence was obtained. All original QTL were confirmed as a single locus, except for one, which split into two linked QTL. Conclusions: Our results indicate that many of the QTL, which are genome-wide significant or suggestive in the analyses of large intercross populations, are true effects that can be replicated and fine-mapped using AIL. Key factors for success are the use of large populations and powerful statistical tools. Moreover, we believe that the statistical methods we have developed to efficiently study outbred AIL populations will increase the number of organisms for which in-depth complex traits can be analyzed

Obesity and Risk of Esophageal Adenocarcinoma and Barrett’s Esophagus: A Mendelian Randomization Study

Data from observational studies suggest that body mass index (BMI) is causally related to esophageal adenocarcinoma (EAC) and its precursor, Barrett’s esophagus (BE). However, the relationships may be affected by bias and confounding

A genome-wide association study identifies new susceptibility loci for esophageal adenocarcinoma and Barrett's esophagus.

Esophageal adenocarcinoma is a cancer with rising incidence and poor survival. Most such cancers arise in a specialized intestinal metaplastic epithelium, which is diagnostic of Barrett's esophagus. In a genome-wide association study, we compared esophageal adenocarcinoma cases (n = 2,390) and individuals with precancerous Barrett's esophagus (n = 3,175) with 10,120 controls in 2 phases. For the combined case group, we identified three new associations. The first is at 19p13 (rs10419226: P = 3.6 × 10(-10)) in CRTC1 (encoding CREB-regulated transcription coactivator), whose aberrant activation has been associated with oncogenic activity. A second is at 9q22 (rs11789015: P = 1.0 × 10(-9)) in BARX1, which encodes a transcription factor important in esophageal specification. A third is at 3p14 (rs2687201: P = 5.5 × 10(-9)) near the transcription factor FOXP1, which regulates esophageal development. We also refine a previously reported association with Barrett's esophagus near the putative tumor suppressor gene FOXF1 at 16q24 and extend our findings to now include esophageal adenocarcinoma