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Objective: To identify and characterize genetic loci associated with the risk of developing ANCA-associated vasculitides (AAV). Methods: Genetic association analyses were performed after Illumina sequencing of 1853 genes and subsequent replication with genotyping of selected single nucleotide polymorphisms in a total cohort of 1110 Scandinavian cases with granulomatosis with polyangiitis or microscopic polyangiitis, and 1589 controls. A novel AAV-associated single nucleotide polymorphism was analysed for allele-specific effects on gene expression using luciferase reporter assay. Results: PR3-ANCA+ AAV was significantly associated with two independent loci in the HLA-DPB1/HLA-DPA1 region [rs1042335, P = 6.3 × 10−61, odds ratio (OR) 0.10; rs9277341, P = 1.5 × 10−44, OR 0.22] and with rs28929474 in the SERPINA1 gene (P = 2.7 × 10−10, OR 2.9). MPO-ANCA+ AAV was significantly associated with the HLA-DQB1/HLA-DQA2 locus (rs9274619, P = 5.4 × 10−25, OR 3.7) and with a rare variant in the BACH2 gene (rs78275221, P = 7.9 × 10−7, OR 3.0), the latter a novel susceptibility locus for MPO-ANCA+ granulomatosis with polyangiitis/microscopic polyangiitis. The rs78275221-A risk allele reduced luciferase gene expression in endothelial cells, specifically, as compared with the non-risk allele. Conclusion: We identified a novel susceptibility locus for MPO-ANCA+ AAV and propose that the associated variant is of mechanistic importance, exerting a regulatory function on gene expression in specific cell types.

Objective Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of complex autoimmune conditions characterized by inflammation in skeletal muscle and extramuscular compartments, and interferon (IFN) system activation. We undertook this study to examine the contribution of genetic variation to disease susceptibility and to identify novel avenues for research in IIMs. Methods Targeted DNA sequencing was used to mine coding and potentially regulatory single nucleotide variants from ~1,900 immune-related genes in a Scandinavian case–control cohort of 454 IIM patients and 1,024 healthy controls. Gene-based aggregate testing, together with rare variant– and gene-level enrichment analyses, was implemented to explore genotype–phenotype relations. Results Gene-based aggregate tests of all variants, including rare variants, identified IFI35 as a potential genetic risk locus for IIMs, suggesting a genetic signature of type I IFN pathway activation. Functional annotation of the IFI35 locus highlighted a regulatory network linked to the skeletal muscle–specific gene PTGES3L, as a potential candidate for IIM pathogenesis. Aggregate genetic associations with AGER and PSMB8 in the major histocompatibility complex locus were detected in the antisynthetase syndrome subgroup, which also showed a less marked genetic signature of the type I IFN pathway. Enrichment analyses indicated a burden of synonymous and noncoding rare variants in IIM patients, suggesting increased disease predisposition associated with these classes of rare variants. Conclusion Our study suggests the contribution of rare genetic variation to disease susceptibility in IIM and specific patient subgroups, and pinpoints genetic associations consistent with previous findings by gene expression profiling. These features highlight genetic profiles that are potentially relevant to disease pathogenesis.

Objectives: To further elucidate the role of the MHC in ankylosing spondylitis by typing 17 genes, searching for HLA-B∗27 independent associations and assessing the impact of sex on this male biased disease. Methods: High-confidence two-field resolution genotyping was performed on 310 cases and 2196 controls using an n-1 concordance method. Protein-coding variants were called from next-generation sequencing reads using up to four software programs and the consensus result recorded. Logistic regression tests were applied to the dataset as a whole, and also in stratified sets based on sex or HLA-B∗27 status. The amino acids driving association were also examined. Results: Twenty-five HLA protein-coding variants were significantly associated to disease in the population. Three novel protective associations were found in a HLA-B∗27 positive population, HLA-A∗24:02 (OR = 0.4, CI = 0.2–0.7), and HLA-A amino acids Leu95 and Gln156. We identified a key set of seven loci that were common to both sexes, and robust to change in sample size. Stratifying by sex uncovered three novel risk variants restricted to the female population (HLA-DQA1∗04.01, -DQB1∗04:02, -DRB1∗08:01; OR = 2.4–3.1). We also uncovered a set of neutral variants in the female population, which in turn conferred strong effects in the male set, highlighting how population composition can lead to the masking of true associations. Conclusion: Population stratification allowed for a nuanced investigation into the tightly linked MHC region, revealing novel HLA-B∗27 signals as well as replicating previous HLA-B∗27 dependent results. This dissection of signals may help to elucidate sex biased disease predisposition and clinical progression.

Objectives. Clinical presentation of primary Sjogrens syndrome (pSS) varies considerably. A shortage of evidence-based objective markers hinders efficient drug development and most clinical trials have failed to reach primary endpoints. Methods. We performed a multicentre study to identify patient subgroups based on clinical, immunological and genetic features. Targeted DNA sequencing of 1853 autoimmune-related loci was performed. After quality control, 918 patients with pSS, 1264 controls and 107 045 single nucleotide variants remained for analysis. Replication was performed in 177 patients with pSS and 7672 controls. Results. We found strong signals of association with pSS in the HLA region. Principal component analysis of clinical data distinguished two patient subgroups defined by the presence of SSA/SSB antibodies. We observed an unprecedented high risk of pSS for an association in the HLA-DQA1 locus of odds ratio 6.10 (95% CI: 4.93, 7.54, P=2.2 x 10(-62)) in the SSA/SSB-positive subgroup, while absent in the antibody negative group. Three independent signals within the MHC were observed. The two most significant variants in MHC class I and II respectively, identified patients with a higher risk of hypergammaglobulinaemia, leukopenia, anaemia, purpura, major salivary gland swelling and lymphadenopathy. Replication confirmed the association with both MHC class I and II signals confined to SSA/SSB antibody positive pSS. Conclusion. Two subgroups of patients with pSS with distinct clinical manifestations can be defined by the presence or absence of SSA/SSB antibodies and genetic markers in the HLA locus. These subgroups should be considered in clinical follow-up, drug development and trial outcomes, for the benefit of both subgroups., Funding Agencies|Swedish Research Council for Medicine and Health; Swedish Rheumatism Association; King Gustav Vs 80-year Foundation; Knut and Alice Wallenberg FoundationKnut & Alice Wallenberg Foundation; AstraZeneca-Science for Life Laboratory Research Collaboration grant (DISSECT)

Objectives: Systemic lupus erythematosus (SLE) is an autoimmune disease with extensive heterogeneity in disease presentation between patients, which is likely due to an underlying molecular diversity. Here, we aimed at elucidating the genetic aetiology of SLE from the immunity pathway level to the single variant level, and stratify patients with SLE into distinguishable molecular subgroups, which could inform treatment choices in SLE. Methods: We undertook a pathway-centred approach, using sequencing of immunological pathway genes. Altogether 1832 candidate genes were analysed in 958 Swedish patients with SLE and 1026 healthy individuals. Aggregate and single variant association testing was performed, and we generated pathway polygenic risk scores (PRS). Results: We identified two main independent pathways involved in SLE susceptibility: T lymphocyte differentiation and innate immunity, characterised by HLA and interferon, respectively. Pathway PRS defined pathways in individual patients, who on average were positive for seven pathways. We found that SLE organ damage was more pronounced in patients positive for the T or B cell receptor signalling pathways. Further, pathway PRS-based clustering allowed stratification of patients into four groups with different risk score profiles. Studying sets of genes with priors for involvement in SLE, we observed an aggregate common variant contribution to SLE at genes previously reported for monogenic SLE as well as at interferonopathy genes. Conclusions: Our results show that pathway risk scores have the potential to stratify patients with SLE beyond clinical manifestations into molecular subsets, which may have implications for clinical follow-up and therapy selection.

BackgroundAutoimmune disease is one of the leading causes of morbidity and mortality worldwide. In Addison's disease, the adrenal glands are targeted by destructive autoimmunity. Despite being the most common cause of primary adrenal failure, little is known about its aetiology. MethodsTo understand the genetic background of Addison's disease, we utilized the extensively characterized patients of the Swedish Addison Registry. We developed an extended exome capture array comprising a selected set of 1853 genes and their potential regulatory elements, for the purpose of sequencing 479 patients with Addison's disease and 1394 controls. ResultsWe identified BACH2 (rs62408233-A, OR = 2.01 (1.71-2.37), P = 1.66 x 10(-15), MAF 0.46/0.29 in cases/controls) as a novel gene associated with Addison's disease development. We also confirmed the previously known associations with the HLA complex. ConclusionWhilst BACH2 has been previously reported to associate with organ-specific autoimmune diseases co-inherited with Addison's disease, we have identified BACH2 as a major risk locus in Addison's disease, independent of concomitant autoimmune diseases. Our results may enable future research towards preventive disease treatment.

About one-third of oestrogen receptor alpha-positive breast cancer patients treated with tamoxifen relapse. Here we identify the nuclear receptor retinoic acid receptor alpha as a marker of tamoxifen resistance. Using quantitative mass spectrometry-based proteomics, we show that retinoic acid receptor alpha protein networks and levels differ in a tamoxifen-sensitive (MCF7) and a tamoxifen-resistant (LCC2) cell line. High intratumoural retinoic acid receptor alpha protein levels also correlate with reduced relapse-free survival in oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen solely. A similar retinoic acid receptor alpha expression pattern is seen in a comparable independent patient cohort. An oestrogen receptor alpha and retinoic acid receptor alpha ligand screening reveals that tamoxifen-resistant LCC2 cells have increased sensitivity to retinoic acid receptor alpha ligands and are less sensitive to oestrogen receptor alpha ligands compared with MCF7 cells. Our data indicate that retinoic acid receptor alpha may be a novel therapeutic target and a predictive factor for oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen.

Important aims of cancer proteomics include gaining better understanding of cancer biology and identifying cancer biomarkers. Mass spectrometry (MS) based shotgun proteomics allow for identification and quantification of thousands of proteins in complex human samples. However, proteomics discovery research in clinical material faces many challenges. The biological differences between groups are often expected to be rather small, at the same time the human proteome is highly complex and there is large biological variation between clinical samples. To be able to extract meaningful results from proteomics data derived from biological and clinical material, care has to be taken to all the critical steps in the data analysis workflow. First of all we need to have robust methods to extract good quality data. A proper statistical analysis is then of outmost importance, taking into account risks of over- fitting and false positives. In addition, we also need system based approaches to relate the data to clinical and biological questions. The main goal of this thesis was to generate robust methods for selection of key proteins, networks and pathways relevant for answering biological and clinical questions. The work includes development and evaluation of workflows for quantitative analysis of proteomics data. In paper I, a multivariate meta-analysis workflow was developed to link existing proteomics data from human colon and prostate tumours. The aim was to identify proteins distinguishing between normal and tumour samples independent of tissue origin, as well as to find unique markers. The bioinformatics workflow for meta- analysis developed in this study enabled the finding of a common protein profile for the two malign tumour types, which was not possible when analysing the data sets separately. The purpose of paper II was to generate a basis for the decision of what protein quantities are reliable and find a way for accurate and precise protein quantification. We develope

Objective: Thyroid proteomics is a new direction in thyroid cancer research aiming at etiological understanding and biomarker identification for improved diagnosis. Methods: Two-dimensional electrophoresis was applied to cytosolic protein extracts from frozen thyroid samples (ten follicular adenomas, nine follicular carcinomas, ten papillary carcinomas, and ten reference thyroids). Spots with differential expression were revealed by image and multivariate statistical analyses, and identified by mass spectrometry. Results: A set of 25 protein spots significant for discriminating between the sample groups was identified. Proteins identified for nine of these spots were studied further including 14-3-3 protein beta/alpha, epsilon, and zeta/delta, peroxiredoxin 6, selenium-binding protein 1, protein disulfide-isomerase precursor, annexin A5 (ANXA5), tubulin alpha-1B chain, and alpha 1-antitrypsin precursor. This subset of protein spots carried the same predictive power in differentiating between follicular carcinoma and adenoma or between follicular and papillary carcinoma, as compared with the larger set of 25 spots. Protein expression in the sample groups was demonstrated by western blot analyses. For ANXA5 and the 14-3-3 proteins, expression in tumor cell cytoplasm was demonstrated by immunohistochemistry both in the sample groups and an independent series of papillary thyroid carcinomas. Conclusion: The proteins identified confirm previous findings in thyroid proteomics, and suggest additional proteins as dysregulated in thyroid tumors.

Background: In order to develop a framework for the analysis of sex-biased genes, we present a characterization of microarray data comparing male and female gene expression in 18 day chicken embryos for brain, gonad, and heart tissue. Results: From the 15982 significantly expressed coding regions that have been assigned to either the autosomes or the Z chromosome ( 12979 in brain, 13301 in gonad, and 12372 in heart), roughly 18% were significantly sex- biased in any one tissue, though only 4 gene targets were biased in all tissues. The gonad was the most sex- biased tissue, followed by the brain. Sex- biased autosomal genes tended to be expressed at lower levels and in fewer tissues than unbiased gene targets, and autosomal somatic sex- biased genes had more expression noise than similar unbiased genes. Sex-biased genes linked to the Z- chromosome showed reduced expression in females, but not in males, when compared to unbiased Z- linked genes, and sex- biased Z- linked genes were also expressed in fewer tissues than unbiased Z coding regions. Third position GC content, and codon usage bias showed some sex- biased effects, primarily for autosomal genes expressed in the gonad. Finally, there were several over-represented Gene Ontology terms in the sex- biased gene sets. Conclusion: On the whole, this analysis suggests that sex- biased genes have unique genomic and organismal properties that delineate them from genes that are expressed equally in males and females.

The evolution of birds from theropod dinosaurs took place approximately 150 million years ago, and was associated with a number of specific adaptations that are still evident among extant birds, including feathers, song and extravagant secondary sexual characteristics. Knowledge about the molecular evolutionary background to such adaptations is lacking. Here, we analyse the evolution of > 5000 protein-coding gene sequences expressed in zebra finch brain by comparison to orthologous sequences in chicken. Mean d(N)/d(S) is 0.085 and genes with their maximal expression in the eye and central nervous system have the lowest mean d(N)/d(S) value, while those expressed in digestive and reproductive tissues exhibit the highest. We find that fast-evolving genes (those which have higher than expected rate of nonsynonymous substitution, indicative of adaptive evolution) are enriched for biological functions such as fertilization, muscle contraction, defence response, response to stress, wounding and endogenous stimulus, and cell death. After alignment to mammalian orthologues, we identify a catalogue of 228 genes that show a significantly higher rate of protein evolution in the two bird lineages than in mammals. These accelerated bird genes, representing candidates for avian-specific adaptations, include genes implicated in vocal learning and other cognitive processes. Moreover, colouration genes evolve faster in birds than in mammals, which may have been driven by sexual selection for extravagant plumage characteristics.

The numerous physiological and phenotypic differences between the sexes, as well as the disparity between male and female reproductive interests, result in sexual conflicts, which are often manifested at the genomic level. Sexually antagonistic genes benefit one sex at the expense of the other and experience strong pressure to evolve male- and female-specific expression patterns to resolve sexual conflicts and maximize fitness for both sexes. Sex-biased gene expression has recently been demonstrated for much of the metazoan transcriptome, suggesting that many loci are sexually antagonistic. However, many coding regions function in multiple processes throughout the organism. This pleiotropy increases the complexity of selection for any given gene, which in turn may obscure sex-specific selective pressures and hamper the evolution of sex-biased gene expression. Here we use microarray gene expression data, in conjunction with data on transcript abundance from expressed sequence tag libraries, to demonstrate that loci with sex-biased gene expression are significantly less pleiotropic than unbiased genes. This relationship was independent of sex chromosome gene dosage effects, and the results were concordant across two study organisms, chicken and mouse. These results suggest that the resolution of sexually antagonistic gene expression is determined by the evolutionary constraints acting on any given antagonistic locus.

Background: The contrasting dose of sex chromosomes in males and females potentially introduces a large-scale imbalance in levels of gene expression between sexes, and between sex chromosomes and autosomes. In many organisms, dosage compensation has thus evolved to equalize sex-linked gene expression in males and females. In mammals this is achieved by X chromosome inactivation and in flies and worms by up- or down-regulation of X-linked expression, respectively. While otherwise widespread in systems with heteromorphic sex chromosomes, the case of dosage compensation in birds (males ZZ, females ZW) remains an unsolved enigma. Results: Here, we use a microarray approach to show that male chicken embryos generally express higher levels of Z-linked genes than female birds, both in soma and in gonads. The distribution of male-to-female fold-change values for Z chromosome genes is wide and has a mean of 1.4-1.6, which is consistent with absence of dosage compensation and sex-specific feedback regulation of gene expression at individual loci. Intriguingly, without global dosage compensation, the female chicken has significantly lower expression levels of Z-linked compared to autosomal genes, which is not the case in male birds. Conclusion: The pronounced sex difference in gene expression is likely to contribute to sexual dimorphism among birds, and potentially has implication to avian sex determination. Importantly, this report, together with a recent study of sex-biased expression in somatic tissue of chicken, demonstrates the first example of an organism with a lack of global dosage compensation, providing an unexpected case of a viable system with large-scale imbalance in gene expression between sexes.

Regional biases in substitution pattern are likely to be responsible for the large-scale variation in base composition observed in vertebrate genomes. However, the evolutionary forces responsible for these biases are still not clearly defined. In order to study the processes of mutation and fixation across the entire human genome, we analyzed patterns of substitution in Alu repeats since their insertion. We also studied patterns of human polymorphism within the repeats. There is a highly significant effect of recombination rate on the pattern of substitution, whereas no such effect is seen on the pattern of polymorphism. These results suggest that regional biases in substitution are caused by biased gene conversion, a process that increases the probability of fixation of mutations that increase GC content. Furthermore, the strongest correlate of substitution patterns is found to be male recombination rates rather than female or sex-averaged recombination rates. This indicates that in addition to sexual dimorphism in recombination rates, the sexes also differ in the relative rates of crossover and gene conversion., Fullt text article at mbe.oupjournals.org

Sexual dimorphism is an emerging feature of ankylosing spondylitis (AS), a chronic rheumatic condition affecting up to three times more men than women. Using 691 individuals from a Swedish case-control cohort, we revealed that sex biases are also a hallmark of AS genetic predisposition, and that this multifactorial disease is in part driven by both rare and common variants. We identified SNPs via the targeted re-sequencing of 7 270 coding and non-coding loci, and assessed novel patterns of association with both single marker and aggregate loci SKAT tests. The male specific RUNX3 locus (including rs7414934, OR=2.58, p=1.7x10-5) and female specific MICB SKAT locus (27 variants, p=1.2x10-6; rs3828903, OR=4.62, p=6.2x10-13) exceeded discovery thresholds. Multiple risk variants from each locus were shown to be functionally active in immune (Jurkat), skin (HaCat) and bone (SaOS-2) cell lines. Differential patterns of genetic predisposition may point to alternative disease mechanisms in male and female patients. Genetic and functional analyses demonstrated that risk alleles should not be considered in isolation and that associated variants would likely affect gene regulation across multiple tissues. This work illustrates the need to consider the contribution of sex to the genetics of AS and the duality that individual loci may play in the key clinical outcomes of disease.