Katherine Anne Siminovitch

Objective: To identify risk alleles relevant to the causal and biologic mechanisms of antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV).Methods: A genome‐wide association study and subsequent replication study were conducted in a total cohort of 1,986 cases of AAV (patients with granulomatosis with polyangiitis [Wegener's] [GPA] or microscopic polyangiitis [MPA]) and 4,723 healthy controls. Meta‐analysis of these data sets and functional annotation of identified risk loci were performed, and candidate disease variants with unknown functional effects were investigated for their impact on gene expression and/or protein function.Results: Among the genome‐wide significant associations identified, the largest effect on risk of AAV came from the single‐nucleotide polymorphism variants rs141530233 and rs1042169 at the HLA–DPB1 locus (odds ratio [OR] 2.99 and OR 2.82, respectively) which, together with a third variant, rs386699872, constitute a triallelic risk haplotype associated with reduced expression of the HLA–DPB1 gene and HLA–DP protein in B cells and monocytes and with increased frequency of complementary proteinase 3 (PR3)–reactive T cells relative to that in carriers of the protective haplotype. Significant associations were also observed at the SERPINA1 and PTPN22 loci, the peak signals arising from functionally relevant missense variants, and at PRTN3, in which the top‐scoring variant correlated with increased PRTN3 expression in neutrophils. Effects of individual loci on AAV risk differed between patients with GPA and those with MPA or between patients with PR3‐ANCAs and those with myeloperoxidase‐ANCAs, but the collective population attributable fraction for these variants was substantive, at 77%.Conclusion: This study reveals the association of susceptibility to GPA and MPA with functional gene variants that explain much of the genetic etiology of AAV, could influence and possibly be predictors of the clinical presentation, and appear to alter immune cell proteins and responses likely to be key factors in the pathogenesis of AAV.

Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery data sets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4,261 controls). We discover and validate six previously unknown risk loci for PBC (P_combined−8) and used pathway analysis to identify JAK-STAT/IL12/IL27 signalling and cytokine–cytokine pathways, for which relevant therapies exist.

TNFα is one of the initial and important mediators to activate downstream signaling pathways by binding to trimerized TNFα receptors (TNFR), and thus is an ideal drug target for cancer therapy. Taking advantage of intrinsic homotimerization of the globular domain of adiponectin (gAD), we have developed a novel TNFα antagonist, the trimerized fusion protein named sTNFRII-gAD. However, our previously-used CHO expression system yielded less than 10 mg/L of sTNFRII-gAD. To produce large quantities of sTNFRII-gAD efficiently, we used a modified CHO-S cell expression system, which is based on a pMH3 vector with non-coding GC-rich DNA fragments for high-level gene expression. We obtained stable clones that produced 75 mg/L of sTNFRII-gAD in the 96-well plate, adapted the clones to 40 ml suspension serum-free batch culture, then optimized the culturing conditions to scale up the fed-batch culture in a 3 L shake-flask and finally in a 5 L AP30 bioreactor. We achieved a final yield of 52 mg/L of sTNFRII-gAD. The trimerized sTNFRII-gAD exhibited the higher affinity to TNFα with a dissociation constant (Kd) of 5.63 nM than the dimerized sTNFRII-Fc with a Kd of 13.4 nM, and further displayed the higher TNFα-neutralizing activity than sTNFRII-Fc (p<0.05) in a L929 cytotoxicity assay. Therefore, the strategy employed in this study may provide an efficient avenue for large-scale production of other recombinant proteins by use of the modified CHO-S cell expression system.

Objective: Associations have been reported between candidate genes and the response to methotrexate (MTX) in rheumatoid arthritis (RA) patients, but most of the studies have been small and have yielded conflicting results. This study was undertaken to provide a systematic review of all genetic variant associations with MTX efficacy and toxicity, and to conduct a meta‐analysis evaluating the most commonly studied single‐nucleotide polymorphism for which prior cumulative analysis has been lacking.Methods: A systematic review and meta‐analysis were performed to identify genetic variant associations with MTX efficacy and toxicity. Studies were identified from the Medline, EMBase, HuGENet Navigator, and Cochrane Library databases through December 2012, and from the 2009–2011 abstracts of the American College of Rheumatology and the European League Against Rheumatism annual meeting proceedings. Additional unpublished genotype data from a Canadian cohort of patients with early RA were also included.Results: Among the 87 identified studies examining genetic associations with MTX efficacy and toxicity, the reduced folate carrier 1 gene (RFC1) variant 80G>A (Arg²⁷His, rs1051266) was selected for random‐effects meta‐analysis. RFC1 80G>A was associated with MTX efficacy in both the recessive model (odds ratio [OR] 1.42, 95% confidence interval [95% CI] 1.04–1.93) and the additive model (OR 1.28, 95% CI 1.10–1.49). Restriction of the sensitivity analyses to studies that involved Caucasian subjects only and that used similar outcome measures (MTX failure versus nonfailure) maintained and improved the associations in both models. No significant association between RFC1 80G>A and MTX toxicity was detected.Conclusion: In these analyses of available data from observational studies, RFC1 80G>A was found to be associated with MTX efficacy, but not toxicity, in RA patients. This variant merits further prospective analysis as a potential predictor of MTX efficacy. Variability in the definitions of response in pharmacogenetic studies is a source of data heterogeneity that should be addressed.

The mammalian diaphanous-related formin (mDia1), a Rho-regulated cytoskeletal modulator, has been shown to promote T lymphocyte chemotaxis and interaction with antigen presenting cells, but the mechanisms underpinning mDia1 roles in these processes have not been defined. Here we show that mDia1^-/- T cells exhibit impaired lymphocyte function-associated antigen 1 (LFA-1)-mediated T cell adhesion, migration and in vivo trafficking. These defects are associated with impaired microtubule (MT) polarization and stabilization, altered MT dynamics and reduced peripheral clustering of the MT plus-end-protein, adenomatous polyposis coli (APC) in migrating T cells following LFA-1-engagement. Loss of mDia1 also leads to impaired inducible inactivation of the glycogen synthase kinase (GSK) 3β as well as hyperphosphorylation and reduced levels of APC in migrating T cells. These findings identify essential roles for the mDia1 formin in modulating GSK3β-dependent MT contributions to induction of T-cell polarity, adhesion and motility.

Objective To fine-map the NF-κB activating protein-like (NKAPL) locus identified in a prior genome-wide study as a possible rheumatoid arthritis (RA) risk locus and thereby delineate additional variants with stronger and/or independent disease association. Methods Genotypes for 101 SNPs across the NKAPL locus on chromosome 6p22.1 were obtained on 1368 Canadian RA cases and 1471 controls. Single marker associations were examined using logistic regression and the most strongly associated NKAPL locus SNPs then typed in another Canadian and a US-based RA case/control cohort. Results Fine-mapping analyses identified six NKAPL locus variants in a single haplotype block showing association with p≤5.6×10⁻⁸ in the combined Canadian cohort. Among these SNPs, rs35656932 in the zinc finger 193 gene and rs13208096 in the NKAPL gene remained significant after conditional logistic regression, contributed independently to risk for disease, and were replicated in the US cohort (P_comb=4.24×10⁻¹⁰ and 2.44×10⁻⁹, respectively). These associations remained significant after conditioning on SNPs tagging the HLA-shared epitope (SE) DRB1*0401 allele and were significantly stronger in the HLA-SE negative versus positive subgroup, with a significant negative interaction apparent between HLA-DRB1 SE and NKAPL risk alleles. Conclusions By illuminating additional NKAPL variants with highly significant effects on risk that are distinct from, but interactive with those arising from the HLA-DRB1 locus, our data conclusively identify NKAPL as an RA susceptibility locus.

Objective: To identify genetic determinants of granulomatosis with polyangiitis (Wegener's) (GPA).Methods: We carried out a genome‐wide association study (GWAS) of 492 GPA cases and 1,506 healthy controls (white subjects of European descent), followed by replication analysis of the most strongly associated signals in an independent cohort of 528 GPA cases and 1,228 controls.Results: Genome‐wide significant associations were identified in 32 single‐nucleotide polymorphic (SNP) markers across the HLA region, the majority of which were located in the HLA–DPB1 and HLA–DPA1 genes encoding the class II major histocompatibility complex (MHC) DPβ chain 1 and DPα chain 1 proteins, respectively. Peak association signals in these 2 genes, emanating from SNPs rs9277554 (for DPβ chain 1) and rs9277341 (DPα chain 1) were strongly replicated in an independent cohort (in the combined analysis of the initial cohort and the replication cohort, P = 1.92 × 10⁻⁵⁰ and 2.18 × 10⁻³⁹, respectively). Imputation of classic HLA alleles and conditional analyses revealed that the SNP association signal was fully accounted for by the classic HLA–DPB1*04 allele. An independent single SNP, rs26595, near SEMA6A (the gene for semaphorin 6A) on chromosome 5, was also associated with GPA, reaching genome‐wide significance in a combined analysis of the GWAS and replication cohorts (P = 2.09 × 10⁻⁸).Conclusion: We identified the SEMA6A and HLA–DP loci as significant contributors to risk for GPA, with the HLA–DPB1*04 allele almost completely accounting for the MHC association. These two associations confirm the critical role of immunogenetic factors in the development of GPA.

To further characterize the genetic basis of primary biliary cirrhosis (PBC), we genotyped 2426 PBC patients and 5731 unaffected controls from three independent cohorts using a single nucleotide polymorphism (SNP) array (Immunochip) enriched for autoimmune disease risk loci. Meta-analysis of the genotype data sets identified a novel disease-associated locus near the TNFSF11 gene at 13q14, provided evidence for association at six additional immune-related loci not previously implicated in PBC and confirmed associations at 19 of 22 established risk loci. Results of conditional analyses also provided evidence for multiple independent association signals at four risk loci, with haplotype analyses suggesting independent SNP effects at the 2q32 and 16p13 loci, but complex haplotype driven effects at the 3q25 and 6p21 loci. By imputing classical HLA alleles from this data set, four class II alleles independently contributing to the association signal from this region were identified. Imputation of genotypes at the non-HLA loci also provided additional associations, but none with stronger effects than the genotyped variants. An epistatic interaction between the IL12RB2 risk locus at 1p31and the IRF5 risk locus at 7q32 was also identified and suggests a complementary effect of these loci in predisposing to disease. These data expand the repertoire of genes with potential roles in PBC pathogenesis that need to be explored by follow-up biological studies.

We fine mapped two primary biliary cirrhosis (PBC) risk loci, CLEC16A (C-type lectin domain family 16 member A)–suppressor of cytokine signaling 1 (SOCS1) and Spi-B protein (SPIB) and sequenced a locus, sialic acid acetylesterase (SIAE), proposed to harbor autoimmunity-associated mutations. In all, 1450 PBC cases and 2957 healthy controls were genotyped for 84 single-nucleotide polymorphisms (SNPs) across the CLEC16A-SOCS1 and SPIB loci. All 10 exons of the SIAE gene were resequenced in 381 cases and point substitutions of unknown significance assayed for activity and secretion. Fine mapping identified 26 SNPs across the CLEC16A-SOCS1 and 11 SNPs across the SPIB locus with significant association to PBC, the strongest signals at the CLEC16A-SOCS1 locus emanating from a SOCS1 intergenic SNP (rs243325; P=9.91 × 10⁻⁹) and at the SPIB locus from a SPIB intronic SNP (rs34944112; P=3.65 × 10⁻⁹). Among the associated SNPs at the CLEC16A-SOCS1 locus, two within the CLEC16A gene as well as one SOCS1 SNP (rs243325) remained significant after conditional logistic regression and contributed independently to risk. Sequencing of the SIAE gene and functional assays of newly identified variants revealed six patients with functional non-synonymous SIAE mutations (Fisher's P=9 × 10⁻⁴ vs controls) We demonstrate independent effects on risk of PBC for CLEC16A, SOCS1 and SPIB variants, while identifying functionally defective SIAE variants as potential factors in risk for PBC.

Schwann cells elaborate myelin sheaths around axons by spirally wrapping and compacting their plasma membranes. Although actin remodeling plays a crucial role in this process, the effectors that modulate the Schwann cell cytoskeleton are poorly defined. Here, we show that the actin cytoskeletal regulator, neural Wiskott-Aldrich syndrome protein (N-WASp), is upregulated in myelinating Schwann cells coincident with myelin elaboration. When N-WASp is conditionally deleted in Schwann cells at the onset of myelination, the cells continue to ensheath axons but fail to extend processes circumferentially to elaborate myelin. Myelin-related gene expression is also severely reduced in the N-WASp-deficient cells and in vitro process and lamellipodia formation are disrupted. Although affected mice demonstrate obvious motor deficits these do not appear to progress, the mutant animals achieving normal body weights and living to advanced age. Our observations demonstrate that N-WASp plays an essential role in Schwann cell maturation and myelin formation.