Katelin Haynes

Quantitative gene profiling of long noncoding RNAs with targeted RNA sequencing

We compared quantitative RT-PCR (qRT-PCR), RNA-seq and capture sequencing (CaptureSeq) in terms of their ability to assemble and quantify long noncoding RNAs and novel coding exons across 20 human tissues. CaptureSeq was superior for the detection and quantification of genes with low expression, showed little technical variation and accurately measured differential expression. This approach expands and refines previous annotations and simultaneously generates an expression atlas.

Excessive bone formation in a mouse model of ankylosing spondylitis is associated with decreases in Wnt pathway inhibitors.

IntroductionAnkylosing spondylitis (AS) is unique in its pathology where inflammation commences at the entheses before progressing to an osteoproliferative phenotype generating excessive bone formation that can result in joint fusion. The underlying mechanisms of this progression are poorly understood. Recent work has suggested that changes in Wnt signalling, a key bone regulatory pathway, may contribute to joint ankylosis in AS. Using the proteoglycan-induced spondylitis (PGISp) mouse model which displays spondylitis and eventual joint fusion following an initial inflammatory stimulus, we have characterised the structural and molecular changes that underlie disease progression.MethodsPGISp mice were characterised 12 weeks after initiation of inflammation using histology, immunohistochemistry (IHC) and expression profiling.ResultsInflammation initiated at the periphery of the intervertebral discs progressing to disc destruction followed by massively excessive cartilage and bone matrix formation, as demonstrated by toluidine blue staining and IHC for collagen type I and osteocalcin, leading to syndesmophyte formation. Expression levels of DKK1 and SOST, Wnt signalling inhibitors highly expressed in joints, were reduced by 49% and 63% respectively in the spine PGISp compared with control mice (P < 0.05) with SOST inhibition confirmed by IHC. Microarray profiling showed genes involved in inflammation and immune-regulation were altered. Further, a number of genes specifically involved in bone regulation including other members of the Wnt pathway were also dysregulated.ConclusionsThis study implicates the Wnt pathway as a likely mediator of the mechanism by which inflammation induces bony ankylosis in spondyloarthritis, raising the potential that therapies targeting this pathway may be effective in preventing this process.

Genetic Variants in ERAP1 and ERAP2 Associated With Immune-Mediated Diseases Influence Protein Expression and the Isoform Profile.

Endoplasmic reticulum aminopeptidase 1 (ERAP‐1) and ERAP‐2, encoded on chromosome 5q15, trim endogenous peptides for HLA‐mediated presentation to the immune system. Polymorphisms in ERAP1 and/or ERAP2 are strongly associated with several immune‐mediated diseases with specific HLA backgrounds, implicating altered peptide handling and presentation as prerequisites for autoreactivity against an arthritogenic peptide. Given the thorough characterization of disease risk–associated polymorphisms that alter ERAP activity, this study aimed instead to interrogate the expression effect of chromosome 5q15 polymorphisms to determine their effect on ERAP isoform and protein expression.

Epigenetic and gene expression analysis of ankylosing spondylitis-associated loci implicate immune cells and the gut in the disease pathogenesis

Ankylosing spondylitis (AS) is a common immune-mediated arthropathy primarily affecting the spine and pelvis. Most AS patients have subclinical intestinal inflammation, suggesting the gut microbiome and the immune response play a role in pathogenesis. Susceptibility to AS is primarily genetic, and at least 114 susceptibility variants have been identified to date. We applied bioinformatic methods utilizing epigenetic and gene and protein expression data to identify the cell types through which AS-associated variants operate. Variants were enriched in transcriptionally regulated regions in monocytes, CD4+ and CD8+ T cells, natural killer cells, regulatory T cells and B cells and mucosa from the small intestine, sigmoid colon and rectum. Weak signals were detected in bone cells, consistent with bone disease being a secondary manifestation. RNA sequencing of blood cells from AS patients and controls identified differentially expressed genes. Interrogation of expression databases showed that the upregulated genes were enriched in monocytes and downregulated genes were enriched in CD8+ T cells and natural killer cells. Gene Ontology term enrichment analysis identified microbes and the gut in the aetiology of AS. These findings identify the key immune cell types that drive the disease, and further highlight the involvement of the gut microbiome in the pathogenesis of AS.

A Novel Monocyte-Specific Transcript Underlies the Chromosome 21Q22 Intergenic Genetic Association in Ankylosing Spondylitis

Robinson P1, Leo P2, Pointon J3, Harris J4, Cremin K4, Bradbury L4, Stebbings S5, Harrison A6, Duncan E4, Wordsworth P3, Brown M4 1Centre for Neurogenetics and Statistical Genomics, Queensland Brain Institute, University of Queensland 2University of Queensland Diamantina Insititute, University of Queensland, Brisbane, Australia 3National Institute for Health Research (NIHR) Oxford Musculoskeletal Biomedical Research Unit, Nuffield Orthopaedic Centre, Headington, Oxford, UK 4University of Queensland Diamantina Institute, Translational Research Institute, Princess Alexandra Hospital, Brisbane, Queensland, Australia 5University of Otago, Dunedin, New Zealand 6University of Otago, Wellington, New Zealand