IgA vasculitis (IgAV) is a pediatric disease with skin and systemic manifestations. Here, we conducted genome, transcriptome, and proteome-wide association studies in 2,170 IgAV cases and 5,928 controls, generated IgAV-specific maps of gene expression and splicing from blood of 255 pediatric cases, and reconstructed myeloid-specific regulatory networks to define disease master regulators modulated by the newly identified disease driver genes. We observed significant association at the HLA-DRB1 (OR=1.55, P=1.1×10-25) and fine-mapped specific amino-acid risk substitutions in DRβ1. We discovered two novel non-HLA loci: FCAR (OR=1.51, P=1.0×10-20) encoding a myeloid IgA receptor FcαR, and INPP5D (OR=1.34, P=2.2×10-9) encoding a known inhibitor of FcαR signaling. The FCAR risk locus co-localized with a cis-eQTL increasing FCAR expression; the risk alleles disrupted a PRDM1 binding motif within a myeloid enhancer of FCAR. Another risk locus was associated with a higher genetically predicted levels of plasma IL6R. The IL6R risk haplotype carried a missense variant contributing to accelerated cleavage of IL6R into a soluble form. Using systems biology approaches, we prioritized IgAV master regulators co-modulated by FCAR, INPP5D and IL6R in myeloid cells. We additionally identified 21 shared loci in a cross-phenotype analysis of IgAV with IgA nephropathy, including novel loci PAID4, WLS, and ANKRD55.

• Publikační typ
• časopisecké články MeSH
• preprinty MeSH

IgA nephropathy (IgAN) is a progressive form of kidney disease defined by glomerular deposition of IgA. Here we performed a genome-wide association study of 10,146 kidney-biopsy-diagnosed IgAN cases and 28,751 controls across 17 international cohorts. We defined 30 genome-wide significant risk loci explaining 11% of disease risk. A total of 16 loci were new, including TNFSF4/TNFSF18, REL, CD28, PF4V1, LY86, LYN, ANXA3, TNFSF8/TNFSF15, REEP3, ZMIZ1, OVOL1/RELA, ETS1, IGH, IRF8, TNFRSF13B and FCAR. The risk loci were enriched in gene orthologs causing abnormal IgA levels when genetically manipulated in mice. We also observed a positive genetic correlation between IgAN and serum IgA levels. High polygenic score for IgAN was associated with earlier onset of kidney failure. In a comprehensive functional annotation analysis of candidate causal genes, we observed convergence of biological candidates on a common set of inflammatory signaling pathways and cytokine ligand-receptor pairs, prioritizing potential new drug targets.

• MeSH
• celogenomová asociační studie MeSH
• IgA nefropatie * farmakoterapie   genetika   diagnóza   MeSH
• imunoglobulin A genetika   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• imunoglobulin A MeSH

INTRODUCTION: Immunoglobulin A1 (IgA1) with galactose-deficient O-glycans (Gd-IgA1) play a key role in the pathogenesis of IgA nephropathy (IgAN). Mucosal-tissue infections increase IL-6 production and, in patients with IgAN, are often associated with macroscopic hematuria. IgA1-secreting cell lines derived from the circulation of patients with IgAN, compared to those of healthy controls (HCs), produce more IgA1 that has O-glycans with terminal or sialylated N-acetylgalactosamine (GalNAc). GalNAc residues are added to IgA1 hinge region by some of the 20 GalNAc transferases, the O-glycosylation-initiating enzymes. Expression of GALNT2, encoding GalNAc-T2, the main enzyme initiating IgA1 O-glycosylation, is similar in cells derived from patients with IgAN and HCs. In this report, we extend our observations of GALNT14 overexpression in IgA1-producing cell lines from patients with IgAN. METHODS: GALNT14 expression was analyzed in peripheral blood mononuclear cells (PBMCs) from patients with IgAN and from HCs. Moreover, the effect of GALNT14 overexpression or knock-down on Gd-IgA1 production in Dakiki cells was assessed. RESULTS: GALNT14 was overexpressed in PBMCs from patients with IgAN. IL-6 increased GALNT14 expression in PBMCs from patients with IgAN and HCs. We used IgA1-producing cell line Dakiki, a previously reported model of Gd-IgA1-producing cells, and showed that overexpression of GalNAc-T14 enhanced galactose deficiency of IgA1, whereas siRNA-mediated GalNAc-T14 knock-down reduced it. GalNAc-T14 was localized in trans-Golgi network, as expected. CONCLUSIONS: Overexpression of GALNT14 due to inflammatory signals during mucosal infections may contribute to overproduction of Gd-IgA1 in patients with IgAN.

• Klíčová slova
• GalNAc-T14, IL-6 cytokine, IgA nephropathy, inflammation,
• Publikační typ
• časopisecké články MeSH

Mucin-type O-glycosylation occurs on many proteins that transit the Golgi apparatus. These glycans impact structure and function of many proteins and have important roles in cellular biosynthetic processes, signaling and differentiation. Although recent technological advances have enhanced our ability to profile glycosylation of glycoproteins, limitations in the understanding of the biosynthesis of these glycan structures remain. Some of these limitations stem from the difficulty to track the biosynthetic process of mucin-type O-glycosylation, especially when glycans occur in dense clusters in repeat regions of proteins, such as the mucins or immunoglobulin A1 (IgA1). Here, we describe a series of nano-liquid chromatography (LC)-mass spectrometry (MS) analyses that demonstrate the range of glycosyltransferase enzymatic activities involved in the biosynthesis of clustered O-glycans on IgA1. By utilizing nano-LC-MS relative quantitation of in vitro reaction products, our results provide unique insights into the biosynthesis of clustered IgA1 O-glycans. We have developed a workflow to determine glycoform-specific apparent rates of a human UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltrasnfersase (GalNAc-T EC 2.4.1.41) and demonstrated how pre-existing glycans affect subsequent activity of glycosyltransferases, such as core 1 galactosyltransferase and α2,3- and α2,6-specific sialyltransferases, in successive additions in the biosynthesis of clustered O-glycans. In the context of IgA1, these results have potential to provide insight into the molecular mechanisms implicated in the pathogenesis of IgA nephropathy, an autoimmune renal disease involving aberrant IgA1 O-glycosylation. In a broader sense, these methods and workflows are applicable to the studies of the concerted and competing functions of other glycosyltransferases that initiate and extend mucin-type core 1 clustered O-glycosylation.

• Klíčová slova
• IgA1 hinge region, polypeptide GalNAc-transferase, LC–MS, clustered glycosylation, mucin-type glycosylation,
• MeSH
• glykosylace MeSH
• glykosyltransferasy metabolismus   MeSH
• imunoglobulin A metabolismus   MeSH
• lidé MeSH
• polysacharidy analýza   biosyntéza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• glykosyltransferasy MeSH
• imunoglobulin A MeSH
• polysacharidy MeSH

The onset of IgA nephropathy (IgAN), characterized by glomerular deposition of IgA-containing immune complexes, is often associated with synpharyngitic hematuria. Innate immune responses mediated by Toll-like receptors (TLR) may play a role in IgAN onset and/or progression. Here, we assessed the expression of TLR 4, 7, 8, and 9 in renal-biopsy specimens from patients with IgAN, with different degree of proteinuria and eGFR, compared with normal-kidney and disease-control tissues (ANCA-associated vasculitis). Renal-biopsy specimens from 34 patients with IgAN and 7 patients with ANCA-associated vasculitis were used. In addition, we used 15 healthy portions of renal-tissue specimens from kidneys after nephrectomy for cancer as control specimens. Expression of TLR 4, 7, 8, and 9 was assessed using immunohistochemical staining of paraffin-embedded renal-biopsy tissue specimens with specific antibodies and evaluated semiquantitatively by light microscopy. Linear discriminant analysis (LDA) was used to test whether intrarenal staining of TLR 4, 7, 8, and 9 distinguished patients with IgAN from controls or correlated with eGFR and/or proteinuria. eGFR was calculated using the creatinine-based formula. Moreover, the biopsies from patients with IgAN were scored according to the Oxford Classification. LDA showed that staining for TLR 4, 7, 8, and 9 was more intense in specimens from IgAN patients compared to normal kidney tissues. The intensity of intrarenal staining of TLRs discriminated four groups of IgAN patients with different eGFR and proteinuria and MEST scoring.

• Klíčová slova
• ANCA-associated vasculitis, IgA nephropathy, Renal biopsy, Renal insufficiency, Toll-like receptors,
• MeSH
• hodnoty glomerulární filtrace MeSH
• IgA nefropatie komplikace   metabolismus   patologie   MeSH
• lidé MeSH
• studie případů a kontrol MeSH
• stupeň závažnosti nemoci MeSH
• toll-like receptory metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• toll-like receptory MeSH

GalNAc-type O-glycans are often added to proteins post-translationally in a clustered manner in repeat regions of proteins, such as mucins and IgA1. Observed IgA1 glycosylation patterns show that glycans occur at similar sites with similar structures. It is not clear how the sites and number of glycans added to IgA1, or other proteins, can follow a conservative process. GalNAc-transferases initiate GalNAc-type glycosylation. In IgA nephropathy, an autoimmune disease, the sites and O-glycan structures of IgA1 hinge-region are altered, giving rise to a glycan autoantigen. To better understand how GalNAc-transferases determine sites and densities of clustered O-glycans, we used IgA1 hinge-region (HR) segment as a probe. Using LC-MS, we demonstrated a semi-ordered process of glycosylation by GalNAc-T2 towards the IgA1 HR. The catalytic domain was responsible for selection of four initial sites based on amino-acid sequence recognition. Both catalytic and lectin domains were involved in multiple second site-selections, each dependent on initial site-selection. Our data demonstrated that multiple start-sites and follow-up pathways were key to increasing the number of glycans added. The lectin domain predominately enhanced IgA1 HR glycan density by increasing synthesis pathway exploration by GalNAc-T2. Our data indicated a link between site-specific glycan addition and clustered glycan density that defines a mechanism of how conserved clustered O-glycosylation patterns and glycoform populations of IgA1 can be controlled by GalNAc-T2. Together, these findings characterized a correlation between glycosylation pathway diversity and glycosylation density, revealing mechanisms by which a single GalNAc-T isozyme can limit and define glycan heterogeneity in a disease-relevant context.

• Klíčová slova
• O-glycosylation, GalNAc-transferase, IgA1 hinge region, clustered glycosylation, restricted glycan heterogeneity,
• MeSH
• biokatalýza MeSH
• glykosylace MeSH
• imunoglobulin A metabolismus   MeSH
• lidé MeSH
• N-acetylgalaktosaminyltransferasy metabolismus   MeSH
• polypeptid-N-acetylgalaktosaminyltransferasa MeSH
• polysacharidy biosyntéza   chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• imunoglobulin A MeSH
• N-acetylgalaktosaminyltransferasy MeSH
• polysacharidy MeSH

IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide and a common cause of end-stage renal disease. Evaluation of a kidney biopsy is necessary for diagnosis, with routine immunofluorescence microscopy revealing dominant or co-dominant IgA immunodeposits usually with complement C3 and sometimes IgG and/or IgM. IgA nephropathy reduces life expectancy by more than 10 years and leads to kidney failure in 20-40% of patients within 20 years of diagnosis. There is accumulating clinical, genetic, and biochemical evidence that complement plays an important role in the pathogenesis of IgA nephropathy. The presence of C3 differentiates the diagnosis of IgA nephropathy from the subclinical deposition of glomerular IgA. Markers for the activation of the alternative and mannan-binding lectin (MBL) pathways in renal-biopsy specimens are associated with disease activity and portend a worse renal outcome. Complement proteins in the circulation have also been evaluated in IgA nephropathy and found to be of prognostic value. Recently, genetic studies have identified IgA nephropathy-associated loci. Within these loci are genes encoding products involved in complement regulation and interaction with immune complexes. Put together, these data identify the complement cascade as a rational treatment target for this chronic kidney disease. Recent case reports on the successful use of humanized anti-C5 monoclonal antibody eculizumab are consistent with this hypothesis, but a better understanding of the role of complement in IgA nephropathy is needed to guide future therapeutic interventions.

• Klíčová slova
• IgA nephropathy, IgAN, IgAN pathogenesis, IgAN treatment, alternative complement pathway, complement, mannan binding lectin complement pathway,
• MeSH
• chronická renální insuficience imunologie   MeSH
• glomerulonefritida imunologie   MeSH
• IgA nefropatie imunologie   MeSH
• imunoglobulin A imunologie   MeSH
• komplement C3 imunologie   MeSH
• komplement C5 imunologie   MeSH
• ledviny imunologie   MeSH
• lidé MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• imunoglobulin A MeSH
• komplement C3 MeSH
• komplement C5 MeSH

BACKGROUND: IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, has serious outcomes with end-stage renal disease developing in 30-50% of patients. The diagnosis requires renal biopsy. Due to its inherent risks, non-invasive approaches are needed. METHODS: We evaluated 91 Czech patients with biopsy-proven IgAN who were assessed at time of diagnosis for estimated glomerular filtration rate (eGFR), proteinuria, microscopic hematuria, and hypertension, and then followed prospectively. Serum samples collected at diagnosis were analyzed for galactose-deficient IgA1 (Gd-IgA1) using new native-IgA1 and established neuraminidase-treated-IgA1 tests, Gd-IgA1-specific IgG autoantibodies, discriminant analysis and logistic regression model assessed correlations with renal function and Oxford classification (MEST score). RESULTS: Serum levels of native (P <0.005) and neuraminidase-treated (P <0.005) Gd-IgA1 were associated with the rate of eGFR decline. A higher relative degree of galactose deficiency in native serum IgA1 predicted a faster eGFR decline and poor renal survival (P <0.005). However, Gd-IgA1 has not differentiated patients with low vs. high baseline eGFR. Furthermore, patients with high baseline eGFR that was maintained during follow-up were characterized by low serum levels of Gd-IgA1-specific IgG autoantibodies (P = 0.003). CONCLUSIONS: Including levels of native and neuraminidase-treated Gd-IgA1 and Gd-IgA1-specific autoantibodies at diagnosis may aid in the prognostication of disease progression in Czech patients with IgAN. Future tests will assess utility of these biomarkers in larger patients cohorts from geographically distinct areas.

• MeSH
• autoprotilátky krev   imunologie   MeSH
• biologické markery krev   MeSH
• dospělí MeSH
• galaktosa krev   imunologie   MeSH
• IgA nefropatie krev   diagnóza   imunologie   mortalita   MeSH
• imunoglobulin A krev   imunologie   MeSH
• lidé MeSH
• následné studie MeSH
• progrese nemoci MeSH
• prospektivní studie MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• autoprotilátky MeSH
• biologické markery MeSH
• galaktosa MeSH
• imunoglobulin A MeSH

INTRODUCTION: IgA nephropathy is a chronic renal disease characterized by mesangial immunodeposits that contain autoantigen, which is aberrantly glycosylated IgA1 with some hinge-region O-glycans deficient in galactose. Macroscopic hematuria during an upper respiratory tract infection is common among patients with IgA nephropathy, which suggests a connection between inflammation and disease activity. Interleukin-6 (IL-6) is an inflammatory cytokine involved in IgA immune response. We previously showed that IL-6 selectively increases production of galactose-deficient IgA1 in IgA1-secreting cells from patients with IgA nephropathy. METHODS: We characterized IL-6 signaling pathways involved in the overproduction of galactose-deficient IgA1. To understand molecular mechanisms, IL-6 signaling was analyzed by kinomic activity profiling and Western blotting, followed by confirmation assays using siRNA knock-down and small-molecule inhibitors. RESULTS: STAT3 was differentially activated by IL-6 in IgA1-secreting cells from patients with IgA nephropathy compared with those from healthy control subjects. Specifically, IL-6 induced enhanced and prolonged phosphorylation of STAT3 in the cells from patients with IgA nephropathy, which resulted in overproduction of galactose-deficient IgA1. This IL-6-mediated overproduction of galactose-deficient IgA1 could be blocked by small molecule inhibitors of JAK/STAT signaling. DISCUSSION: Our results revealed that IL-6-induced aberrant activation of STAT3-mediated overproduction of galactose-deficient IgA1. STAT3 signaling pathway may thus represent a new target for disease-specific therapy of IgA nephropathy.

• Klíčová slova
• IgA nephropathy, IgA1, O-glycans, aberrant glycosylation, autoantigen,
• Publikační typ
• časopisecké články MeSH

Autoantibodies against galactose-deficient IgA1 drive formation of pathogenic immune complexes in IgA nephropathy. IgG autoantibodies against galactose-deficient IgA1 in patients with IgA nephropathy have a specific amino-acid sequence, Y1CS3, in the complementarity-determining region 3 of the heavy chain variable region compared with a Y1CA3 sequence in similar isotype-matched IgG from healthy controls. We previously found that the S3 residue is critical for binding galactose-deficient IgA1. To determine whether this difference is due to a rare germline sequence, we amplified and sequenced the corresponding germline variable region genes from peripheral blood mononuclear cells of seven patients with IgA nephropathy and six healthy controls from whom we had cloned single-cell lines secreting monoclonal IgG specific for galactose-deficient IgA1. Sanger DNA sequencing revealed that complementarity-determining region 3 in the variable region of the germline genes encoded the Y1C(A/V)3 amino-acid sequence. Thus, the A/V>S substitution in the complementarity-determining region 3 of anti-galactose-deficient-IgA1 autoantibodies of the patients with IgA nephropathy is not a rare germline gene variant. Modeling analyses indicated that the S3 hydroxyl group spans the complementarity-determining region 3 loop stem, stabilizing the adjacent β-sheet and stem structure, important features for effective binding to galactose-deficient IgA1. Understanding processes leading to production of the autoantibodies may offer new approaches to treat IgA nephropathy.

• Klíčová slova
• IgA nephropathy, immune complexes, immunology,
• MeSH
• autoprotilátky genetika   MeSH
• galaktosa nedostatek   MeSH
• IgA nefropatie enzymologie   genetika   imunologie   MeSH
• imunoglobulin A * MeSH
• lidé MeSH
• mutace * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• autoprotilátky MeSH
• galaktosa MeSH
• imunoglobulin A * MeSH