Variants at multiple loci implicated in both innate and adaptive immune responses are associated with Sjögren’s syndrome

Sjögren’s syndrome is a common autoimmune disease (~0.7% of European Americans) typically presenting as keratoconjunctivitis sicca and xerostomia. In addition to strong association within the HLA region at 6p21 (Pmeta=7.65×10−114), we establish associations with IRF5-TNPO3 (Pmeta=2.73×10−19), STAT4 (Pmeta=6.80×10−15), IL12A (Pmeta =1.17×10−10), FAM167A-BLK (Pmeta=4.97×10−10), DDX6-CXCR5 (Pmeta=1.10×10−8), and TNIP1 (Pmeta=3.30×10−8). Suggestive associations with Pmeta<5×10−5 were observed with 29 regions including TNFAIP3, PTTG1, PRDM1, DGKQ, FCGR2A, IRAK1BP1, ITSN2, and PHIP amongst others. These results highlight the importance of genes involved in both innate and adaptive immunity in Sjögren’s syndrome

Genome-wide association study identifies Sjögren’s risk loci with functional implications in immune and glandular cells

Sjögren’s disease is a complex autoimmune disease with twelve established susceptibility loci. This genome-wide association study (GWAS) identifies ten novel genome-wide significant (GWS) regions in Sjögren’s cases of European ancestry: CD247, NAB1, PTTG1-MIR146A, PRDM1-ATG5, TNFAIP3, XKR6, MAPT-CRHR1, RPTOR-CHMP6-BAIAP6, TYK2, SYNGR1. Polygenic risk scores yield predictability (AUROC = 0.71) and relative risk of 12.08. Interrogation of bioinformatics databases refine the associations, define local regulatory networks of GWS SNPs from the 95% credible set, and expand the implicated gene list to >40. Many GWS SNPs are eQTLs for genes within topologically associated domains in immune cells and/or eQTLs in the main target tissue, salivary glands.Research reported in this publication was supported by the National Institutes of Health (NIH): R01AR073855 (C.J.L.), R01AR065953 (C.J.L.), R01AR074310 (A.D.F.), P50AR060804 (K.L.S.), R01AR050782 (K.L.S), R01DE018209 (K.L.S.), R33AR076803 (I.A.), R21AR079089 (I.A.); NIDCR Sjögren’s Syndrome Clinic and Salivary Disorders Unit were supported by NIDCR Division of Intramural Research at the National Institutes of Health funds - Z01-DE000704 (B.W.); Birmingham NIHR Biomedical Research Centre (S.J.B.); Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy – EXC 2155 – Projektnummer 390874280 (T.W.); Research Council of Norway (Oslo, Norway) – Grant 240421 (TR.R.), 316120 (M.W-H.); Western Norway Regional Health Authority (Helse Vest) – 911807, 912043 (R.O.); Swedish Research Council for Medicine and Health (L.R., G.N., M.W-H.); Swedish Rheumatism Association (L.R., G.N., M.W-H.); King Gustav V’s 80-year Foundation (G.N.); Swedish Society of Medicine (L.R., G.N., M.W-H.); Swedish Cancer Society (E.B.); Sjögren’s Syndrome Foundation (K.L.S.); Phileona Foundation (K.L.S.). The Stockholm County Council (M.W-H.); The Swedish Twin Registry is managed through the Swedish Research Council - Grant 2017-000641. The French ASSESS (Atteinte Systémique et Evolution des patients atteints de Syndrome de Sjögren primitive) was sponsored by Assistance Publique-Hôpitaux de Paris (Ministry of Health, PHRC 2006 P060228) and the French society of Rheumatology (X.M.).publishedVersio

Genome-wide association study identifies Sjögren's risk loci with functional implications in immune and glandular cells.

Sjögren’s disease is a complex autoimmune disease with twelve established susceptibility loci. This genome-wide association study (GWAS) identifies ten novel genome-wide significant (GWS) regions in Sjögren’s cases of European ancestry: CD247, NAB1, PTTG1-MIR146A, PRDM1-ATG5, TNFAIP3, XKR6, MAPT-CRHR1, RPTOR-CHMP6-BAIAP6, TYK2, SYNGR1. Polygenic risk scores yield predictability (AUROC = 0.71) and relative risk of 12.08. Interrogation of bioinformatics databases refine the associations, define local regulatory networks of GWS SNPs from the 95% credible set, and expand the implicated gene list to >40. Many GWS SNPs are eQTLs for genes within topologically associated domains in immune cells and/or eQTLs in the main target tissue, salivary glands.We thank all the research and clinical staff, consortium investigators, and study participants (detailed in Supplementary Information), and funding agencies who made this study possible. The content of this publication is solely the responsibility of the authors and does not represent the official views of the funding agencies listed below. Research reported in this publication was supported by the National Institutes of Health (NIH): R01AR073855 (C.J.L.), R01AR065953 (C.J.L.), R01AR074310 (A.D.F.), P50AR060804 (K.L.S.), R01AR050782 (K.L.S), R01DE018209 (K.L.S.), R33AR076803 (I.A.), R21AR079089 (I.A.); NIDCR Sjögren’s Syndrome Clinic and Salivary Disorders Unit were supported by NIDCR Division of Intramural Research at the National Institutes of Health funds - Z01-DE000704 (B.W.); Birmingham NIHR Biomedical Research Centre (S.J.B.); Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy – EXC 2155 – Projektnummer 390874280 (T.W.); Research Council of Norway (Oslo, Norway) – Grant 240421 (TR.R.), 316120 (M.W-H.); Western Norway Regional Health Authority (Helse Vest) – 911807, 912043 (R.O.); Swedish Research Council for Medicine and Health (L.R., G.N., M.W-H.); Swedish Rheumatism Association (L.R., G.N., M.W-H.); King Gustav V’s 80-year Foundation (G.N.); Swedish Society of Medicine (L.R., G.N., M.W-H.); Swedish Cancer Society (E.B.); Sjögren’s Syndrome Foundation (K.L.S.); Phileona Foundation (K.L.S.). The Stockholm County Council (M.W-H.); FOREUM Foundation for Research in Rheumatology (R.J., M.W-H). The Swedish Twin Registry is managed through the Swedish Research Council - Grant 2017-000641. The French ASSESS (Atteinte Systémique et Evolution des patients atteints de Syndrome de Sjögren primitive) was sponsored by Assistance Publique-Hôpitaux de Paris (Ministry of Health, PHRC 2006 P060228) and the French society of Rheumatology (X.M.). We want to acknowledge the following invesigators who recruited patients: Jacques-Eric Gottenberg, Valerie Devauchelle-Pensec, Jean Jacques Dubost, Anne-Laure Fauchais, Vincent Goeb, Eric Hachulla, Claire Larroche, Véronique Le Guern, Jacques Morel, Aleth Perdriger, Emmanuelle Dernis, Stéphanie Rist, Damien Sene, Olivier Vittecoq. We also thank Sarah Tubiana and all staff members of the Bichat Hospital Biological Resource Center (Paris) for centralizing and managing biological collection. We also thank Rezvan Kiani Dehkordi, Karolina Tandre, Käth Nilsson, Marianne Eidsheim, Kjerstin Jacobsen, Ingeborg Kvivik and Kjetil Bårdsen for collecting patient blood samples. We acknowledge the SNP&SEQ Technology Platform, Uppsala, part of National Genomics Infrastructure (NGI) Sweden, for genotyping of Scandinavian samples, and the Swedish Twin Registry for access to data. The SNP&SEQ Technology Platform was supported by Science for Life Laboratory, Uppsala University, the Knut and Alice Wallenberg Foundation and the Swedish Research Council. Last, we thank the investigators for the following dbGaP studies: Phs000428.v2.p2: This study used control data from the Health and Retirement Study in dbGaP (phs000428.v2.p2) submitted by David Weir, PhD at the University of Michigan and funded by the National Institute of Aging RC2 AG036495 and RC4 AG039029. Phs000672.v1.p1: Genotype data from the Sjögren’s International Collaborative Clinical Alliance (SICCA) Registry was obtained through dbGAP accession number phs000672.v1.p1. This study was supported by the National Institute of Dental and Craniofacial Research (NIDCR), the National Eye Institute, and the Office of Research on Women’s Health through contract number N01-DE-32636. Genotyping services were provided by the Center for Inherited Disease Research (CIDR). CIDR is fully funded through a federal contract from the National Institutes of Health (NIH) to the Johns Hopkins University (contract numbers HHSN268200782096C, HHSN268201100011I, HHSN268201200008I). Funds for genotyping were provided by the NIDCR through CIDR’s NIH contract. Assistance with data cleaning and imputation was provided by the University of Washington. We thank investigators from the following studies that provided DNA samples for genotyping: the Genetic Architecture of Smoking and Smoking Cessation, Collaborative Genetic Study of Nicotine Dependence (phs000404.v1.p1); Age-Related Eye Disease Study (AREDS) - Genetic Variation in Refractive Error Substudy (phs000429.v1.p1); and National Institute of Mental Health’s Human Genetics Initiative (phs000021.v3.p2, phs000167.v1.p1). We thank the many clinical collaborators and research participants who contributed to this research. Phs000196.v3.p1: Investigators and Parkinson Disease patients that contributed to this Genome-wide Association Study of Parkinson Disease. phs000187.v1.p1: Research support to collect data and develop an application to support the High Density SNP Association Analysis of Melanoma project was provided by 3P50CA093459, 5P50CA097007, 5R01ES011740, and 5R01CA133996

Massively parallel reporter assay confirms regulatory potential of hQTLs and reveals important variants in lupus and other autoimmune diseases

Summary: We designed a massively parallel reporter assay (MPRA) in an Epstein-Barr virus transformed B cell line to directly characterize the potential for histone post-translational modifications, i.e., histone quantitative trait loci (hQTLs), expression QTLs (eQTLs), and variants on systemic lupus erythematosus (SLE) and autoimmune (AI) disease risk haplotypes to modulate regulatory activity in an allele-dependent manner. Our study demonstrates that hQTLs, as a group, are more likely to modulate regulatory activity in an MPRA compared with other variant classes tested, including a set of eQTLs previously shown to interact with hQTLs and tested AI risk variants. In addition, we nominate 17 variants (including 11 previously unreported) as putative causal variants for SLE and another 14 for various other AI diseases, prioritizing these variants for future functional studies in primary and immortalized B cells. Thus, we uncover important insights into the mechanistic relationships among genotype, epigenetics, and gene expression in SLE and AI disease phenotypes

UNC-41/Stonin Functions with AP2 to Recycle Synaptic Vesicles in <em>Caenorhabditis elegans</em>

<div><p>The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (<em>Drosophila</em> Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the <em>unc-41</em> gene, which encodes the stonin ortholog in the nematode <em>Caenorhabditis elegans</em>. Transgenic expression of <em>Drosophila stonedB</em> rescues <em>unc-41</em> mutant phenotypes, demonstrating that UNC-41 is a <em>bona fide</em> member of the stonin family. In <em>unc-41</em> mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of <em>snt-1 unc-41</em> double mutants is stronger than <em>snt-1</em> mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that <em>unc-41</em> mutants have defects in synaptic vesicle membrane endocytosis, including a ∼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in <em>apm-2</em> mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in <em>unc-41 apm-2</em> double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin.</p> </div

Overexpression of synaptotagmin does not rescue <i>unc-41</i> mutants.

<p>(A) Images of synaptotagmin (SNT-1::GFP) under different overexpression conditions. All images were taken at identical settings. High levels of tagged synaptotagmin were observed in the nerve rings of <i>snt-1(md290)</i> or <i>unc-41(e268)</i> mutants when injected at 5 ng/µl. Scale bar is 2 µm. (B) Swimming assays. Details are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#s4" target="_blank">Materials and Methods</a>. Error bars indicate SEM. Rescue of <i>unc-41</i> mutant phenotypes was not observed with the <i>snt-1::GFP</i> construct injected at 1 or 5 ng/µl.</p

Fatty infiltration of the minor salivary glands is a selective feature of aging but not Sjögren’s syndrome

Objective: Determine the presence and assess the extent of fatty infiltration of the minor salivary glands (SG) of primary SS patients (pSS) as compared to those with non-SS sicca (nSS). Methods: Minor SG biopsy samples from 134 subjects with pSS (n = 72) or nSS (n = 62) were imaged. Total area and fatty replacement area for each glandular cross-section (n = 4–6 cross-sections per subject) were measured using Image J (National Institutes of Health, Bethesda, MD). The observer was blinded to subject classification status. The average area of fatty infiltration calculated per subject was evaluated by logistic regression and general linearized models (GLM) to assess relationships between fatty infiltration and clinical exam results, extent of fibrosis and age. Results: The average area of fatty infiltration for subjects with pSS (median% (range) 4.97 (0.05–30.2)) was not significantly different from that of those with nSS (3.75 (0.087–41.9). Infiltration severity varied widely, and subjects with fatty replacement greater than 6% were equivalently distributed between pSS and nSS participants (χ2 p = .50). Age accounted for all apparent relationships between fatty infiltration and fibrosis or reduced saliva flow. The all-inclusive GLM for prediction of pSS versus non-SS classification including fibrosis, age, fatty replacement, and focus score was not significantly different from any desaturated model. In no iteration of the model did fatty replacement exert a significant effect on the capacity to predict pSS classification. Conclusions: Fatty infiltration is an age-associated phenomenon and not a selective feature of Sjögren’s syndrome. Sicca patients who do not fulfil pSS criteria have similar rates of fatty infiltration of the minor SG

Phenotypic rescue of <i>unc-41</i> mutants by transgenic expression of <i>Drosophila</i> stoned B (STNB).

<p>(A) A YFP::STNB fusion protein is correctly trafficked and localized to synaptic regions. Shown is the head of a young adult hermaphrodite; anterior is to the left, and the scale bar is 10 µm. STNB is specifically associated with synaptic regions, including the nerve ring (nr) and dorsal (dnc) and ventral (vnc) nerve cords. (B) Behavioral analyses of young adult hermaphrodites expressing STNB or YFP::STNB transgenes. The strains analyzed were (from left to right) RM2086, RM2655, RM2683, and RM3644; genotypes are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#pone.0040095.s006" target="_blank">List S1</a>. Details of swimming and expulsion assays are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#s4" target="_blank">Materials and Methods</a>. For swimming assays, each bar represents the mean number of body bends/min for 20 individuals of each strain. For expulsion assays, each bar represents the percentage of defecation cycles ending with an enteric muscle contraction (EMC, %) for 10 individuals of each strain. Error bars indicate SEM. Asterisks denote significant difference from <i>unc-41</i> (P<0.0001).</p

The <i>unc-41</i> gene products are differentially expressed in the <i>C. elegans</i> nervous system.

<p>Animals expressing the <i>Punc-41A::</i>NLS-CFP (green) and <i>Punc-41B::</i>NLS-YFP (red) transgenes were imaged on a confocal microscope. An L1 larva is shown in the left column (panels A, C, and E). One of the six GABA motor neurons (DD) is indicated. The animal’s head is in the upper left of the image, and the animal is positioned with its ventral surface on the inside of the arc (scale bar is 10 µm). Shown in the right column (panels B, D, and F) is the head region of an adult hermaphrodite. The nerve ring (nr), dorsal nerve cord (dnc), and ventral nerve cord (vnc) are indicated for reference. Anterior is to the left, ventral is down, and the bar is 10 µm.</p

The <i>unc-41</i> gene and UNC-41 proteins.

<p>(A) The <i>unc-41</i> gene consists of 12 exons spanning ∼9 kb of genomic DNA. The two promoter regions are shown in green, and several <i>unc-41</i> mutations are indicated. (B) The <i>unc-41</i> gene products are members of the stonin family. Shown are features of the UNC-41A (∼188 kDa) and B (∼160 kDa) proteins, as well as the <i>Drosophila</i> stoned B and human stonin 2 proteins. Each of these proteins possesses a central stonin-homology domain (SHD) and a C-terminal µ-homology domain (µHD); significant sequence similarity among the proteins is limited to these domains. The brown rectangles indicate proline-rich domains (defined as a sequence of ≥21 amino acids containing ≥33% prolines). Blue circles indicate NPF motifs. Three motifs, shown as triangles, interact with the α-ear domains of the AP2 complex: red triangles indicate WxxF motifs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#pone.0040095-Jha1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#pone.0040095-Ritter1" target="_blank">[30]</a>, orange triangles indicate DPF motifs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#pone.0040095-Brett1" target="_blank">[28]</a>, and the green triangle indicates an FxDxF motif <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#pone.0040095-Brett1" target="_blank">[28]</a>. The pink diamonds indicate C-terminal PDZ domain-binding motifs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#pone.0040095-Sheng1" target="_blank">[31]</a>. Asterisks indicate that the marked site is not conserved even among closely related species.</p