Contribution of Rare Genetic Variation to Disease Susceptibility in a Large Scandinavian Myositis Cohort.

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.

Identification and functional characterization of a novel susceptibility locus for small vessel vasculitis with MPO-ANCA.

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.

Molecular pathways in patients with systemic lupus erythematosus revealed by gene-centred DNA sequencing.

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.

Genetic and clinical basis for two distinct subtypes of primary Sjögren's syndrome.

OBJECTIVES: Clinical presentation of primary Sjögren's 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=2.2×10-62) 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.

Common genetic variation in the autoimmune regulator (AIRE) locus is associated with autoimmune Addison's disease in Sweden.

Autoimmune Addison's disease (AAD) is the predominating cause of primary adrenal failure. Despite its high heritability, the rarity of disease has long made candidate-gene studies the only feasible methodology for genetic studies. Here we conducted a comprehensive reinvestigation of suggested AAD risk loci and more than 1800 candidate genes with associated regulatory elements in 479 patients with AAD and 2394 controls. Our analysis enabled us to replicate many risk variants, but several other previously suggested risk variants failed confirmation. By exploring the full set of 1800 candidate genes, we further identified common variation in the autoimmune regulator (AIRE) as a novel risk locus associated to sporadic AAD in our study. Our findings not only confirm that multiple loci are associated with disease risk, but also show to what extent the multiple risk loci jointly associate to AAD. In total, risk loci discovered to date only explain about 7% of variance in liability to AAD in our study population.

SpliceVista, a tool for splice variant identification and visualization in shotgun proteomics data.

Alternative splicing is a pervasive process in eukaryotic organisms. More than 90% of human genes have alternatively spliced products, and aberrant splicing has been shown to be associated with many diseases. Current methods employed in the detection of splice variants include prediction by clustering of expressed sequence tags, exon microarray, and mRNA sequencing, all methods focusing on RNA-level information. There is a lack of tools for analyzing splice variants at the protein level. Here, we present SpliceVista, a tool for splice variant identification and visualization based on mass spectrometry proteomics data. SpliceVista retrieves gene structure and translated sequences from alternative splicing databases and maps MS-identified peptides to splice variants. The visualization module plots the exon composition of each splice variant and aligns identified peptides with transcript positions. If quantitative mass spectrometry data are used, SpliceVista plots the quantitative patterns for each peptide and provides users with the option to cluster peptides based on their quantitative patterns. SpliceVista can identify splice-variant-specific peptides, providing the possibility for variant-specific analysis. The tool was tested on two experimental datasets (PXD000065 and PXD000134). In A431 cells treated with gefitinib, 2983 splice-variant-specific peptides corresponding to 939 splice variants were identified. Through comparison of splice-variant-centric, protein-centric, and gene-centric quantification, several genes (e.g. EIF4H) were found to have differentially regulated splice variants after gefitinib treatment. The same discrepancy between protein-centric and splice-centric quantification was detected in the other dataset, in which induced pluripotent stem cells were compared with parental fibroblast and human embryotic stem cells. In addition, SpliceVista can be used to visualize novel splice variants inferred from peptide-level evidence. In summary, SpliceVista enables visualization, detection, and differential quantification of protein splice variants that are often missed in current proteomics pipelines.

Retinoic acid receptor alpha is associated with tamoxifen resistance in breast cancer.

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.

Defining, comparing, and improving iTRAQ quantification in mass spectrometry proteomics data.

The purpose of this study was to generate a basis for the decision of what protein quantities are reliable and find a way for accurate and precise protein quantification. To investigate this we have used thousands of peptide measurements to estimate variance and bias for quantification by iTRAQ (isobaric tags for relative and absolute quantification) mass spectrometry in complex human samples. A549 cell lysate was mixed in the proportions 2:2:1:1:2:2:1:1, fractionated by high resolution isoelectric focusing and liquid chromatography and analyzed by three mass spectrometry platforms; LTQ Orbitrap Velos, 4800 MALDI-TOF/TOF and 6530 Q-TOF. We have investigated how variance and bias in the iTRAQ reporter ions data are affected by common experimental variables such as sample amount, sample fractionation, fragmentation energy, and instrument platform. Based on this, we have suggested a concept for experimental design and a methodology for protein quantification. By using duplicate samples in each run, each experiment is validated based on its internal experimental variation. The duplicates are used for calculating peptide weights, unique to the experiment, which is used in the protein quantification. By weighting the peptides depending on reporter ion intensity, we can decrease the relative error in quantification at the protein level and assign a total weight to each protein that reflects the protein quantitation confidence. We also demonstrate the usability of this methodology in a cancer cell line experiment as well as in a clinical data set of lung cancer tissue samples. In conclusion, we have in this study developed a methodology for improved protein quantification in shotgun proteomics and introduced a way to assess quantification for proteins with few peptides. The experimental design and developed algorithms decreased the relative protein quantification error in the analysis of complex biological samples.

Proteomic profiling of follicular and papillary thyroid tumors.

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 α1-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.

Natural selection in avian protein-coding genes expressed in brain.

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 unique genomic properties of sex-biased genes: insights from avian microarray data.

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.

Pleiotropic constraint hampers the resolution of sexual antagonism in vertebrate gene expression.

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.

Faced with inequality: chicken do not have a general dosage compensation of sex-linked genes.

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.

Rapid evolution of female-biased, but not male-biased, genes expressed in the avian brain.

The powerful pressures of sexual and natural selection associated with species recognition and reproduction are thought to manifest in a faster rate of evolution in sex-biased genes, an effect that has been documented particularly for male-biased genes expressed in the reproductive tract. However, little is known about the rate of evolution for genes involved in sexually dimorphic behaviors, which often form the neurological basis of intrasexual competition and mate choice. We used microarray data, designed to uncover sex-biased expression patterns in embryonic chicken brain, in conjunction with data on the rate of sequence evolution for >4,000 coding regions aligned between chicken and zebra finch in order to study the role of selection in governing the molecular evolution for sex-biased and unbiased genes. Surprisingly, we found that female-biased genes, defined across a range of cutoff values, show a higher rate of functional evolution than both male-biased and unbiased genes. Autosomal male-biased genes evolve at a similar rate as unbiased genes. Sex-specific genomic properties, such as heterogeneity in genomic distribution and GC content, and codon usage bias for sex-biased classes fail to explain this surprising result, suggesting that selective pressures may be acting differently on the male and female brain.

Male-driven biased gene conversion governs the evolution of base composition in human alu repeats.

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.