Genetic biomarkers for intravenous immunoglobulin response in chronic inflammatory demyelinating polyradiculoneuropathy

Background and purpose: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a clinical and electrophysiological heterogeneous immune-mediated polyneuropathy. In

Extensive Ethnic Variation and Linkage Disequilibrium at the FCGR2/3 Locus: Different Genetic Associations Revealed in Kawasaki Disease

The human Fc-gamma receptors (FcγRs) link adaptive and innate immunity by binding immunoglobulin G (IgG). All human low-affinity FcγRs are encoded by the FCGR2/3 locus containing functional single nucleotide polymorphisms (SNPs) and gene copy number variants. This locus is notoriously difficult to genotype and high-throughput methods commonly used focus on only a few SNPs. We performed multiplex ligation-dependent probe amplification for all relevant genetic variations at the FCGR2/3 locus in >4,000 individuals to define linkage disequilibrium (LD) and allele frequencies in different populations. Strong LD and extensive ethnic variation in allele frequencies was found across the locus. LD was strongest for the FCGR2C-ORF haplotype (rs759550223+rs76277413), which leads to expression of FcγRIIc. In Europeans, the FCGR2C-ORF haplotype showed strong LD with, among others, rs201218628 (FCGR2A-Q27W, r2 = 0.63). LD between these two variants was weaker (r2 = 0.17) in Africans, whereas the FCGR2C-ORF haplotype was nearly absent in Asians (minor allele frequency <0.005%). The FCGR2C-ORF haplotype and rs1801274 (FCGR2A-H131R) were in weak LD (r2 = 0.08) in Europeans. We evaluated the importance of ethnic variation and LD in Kawasaki Disease (KD), an acute vasculitis in children with increased incidence in Asians. An association of rs1801274 with KD was previously shown in ethnically diverse genome-wide association studies. Now, we show in 1,028 European KD patients that the FCGR2C-ORF haplotype, although nearly absent in Asians, was more strongly associated with susceptibility to KD than rs1801274 in Europeans. Our data illustrate the importance of interpreting findings of association studies concerning the FCGR2/3 locus with knowledge of LD and ethnic variation

Evaluation of high-throughput genomic assays for the Fc gamma receptor locus

Cancer immunotherapy has been revolutionised by the use of monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics

Extensive Ethnic Variation and Linkage Disequilibrium at the FCGR2/3 Locus: Different Genetic Associations Revealed in Kawasaki Disease.

The human Fc-gamma receptors (FcγRs) link adaptive and innate immunity by binding immunoglobulin G (IgG). All human low-affinity FcγRs are encoded by the FCGR2/3 locus containing functional single nucleotide polymorphisms (SNPs) and gene copy number variants. This locus is notoriously difficult to genotype and high-throughput methods commonly used focus on only a few SNPs. We performed multiplex ligation-dependent probe amplification for all relevant genetic variations at the FCGR2/3 locus in >4,000 individuals to define linkage disequilibrium (LD) and allele frequencies in different populations. Strong LD and extensive ethnic variation in allele frequencies was found across the locus. LD was strongest for the FCGR2C-ORF haplotype (rs759550223+rs76277413), which leads to expression of FcγRIIc. In Europeans, the FCGR2C-ORF haplotype showed strong LD with, among others, rs201218628 (FCGR2A-Q27W, r 2 = 0.63). LD between these two variants was weaker (r 2 = 0.17) in Africans, whereas the FCGR2C-ORF haplotype was nearly absent in Asians (minor allele frequency <0.005%). The FCGR2C-ORF haplotype and rs1801274 (FCGR2A-H131R) were in weak LD (r 2 = 0.08) in Europeans. We evaluated the importance of ethnic variation and LD in Kawasaki Disease (KD), an acute vasculitis in children with increased incidence in Asians. An association of rs1801274 with KD was previously shown in ethnically diverse genome-wide association studies. Now, we show in 1,028 European KD patients that the FCGR2C-ORF haplotype, although nearly absent in Asians, was more strongly associated with susceptibility to KD than rs1801274 in Europeans. Our data illustrate the importance of interpreting findings of association studies concerning the FCGR2/3 locus with knowledge of LD and ethnic variation

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

A field guide to human Fc-gamma receptors: Genetics, cellular expression and interaction with immunoglobulins

In many autoimmune diseases, antibodies (immunoglobulins) play a central role in the pathophysiology. These auto-antibodies are often of the Immunoglobulin G (IgG) class and can cause damage to tissues in various ways. In many cases this process occurs via cellular receptors for IgG on immune cells, the Fc-gamma receptors (FcγRs). These receptors, upon binding IgG, instruct the cell to attack. For instance, auto-antibodies directed against a patient’s own blood cells cause destruction of these cells via FcγRs on macrophages in the spleen. This thesis describes our research on human FcγRs and (patho)physiology of IgG–FcγR interactions in autoimmune diseases. We have thoroughly studied the complex genetics of the different FCGR genes, describing novel variants and a high degree of linkage disequilibrium and ethnic variation. These findings are crucial for genetic association studies, and we used this knowledge to show an association of FCGR genetic variation with various diseases (Systemic Lupus Erythematosus and Kawasaki Disease). Furthermore, we studied cellular expression of the FcγRs on immune cells in relation to FCGR genetic variation, including an analysis of human splenic macrophages, which had not been characterized in detail before. Finally, we investigated the poorly understood working mechanism of intravenous immunoglobulins (IVIg), a treatment for autoimmune diseases that consists of a multitude of normal IgG molecules, aiming for improvement of this therapy. We showed that sialylation of glycans in the IVIg is not important, whereas the binding avidity of the IgG to FcγRs seems to be very important for the efficacy of IVIg

Immunomodulation by IVIg and the Role of Fc-Gamma Receptors: Classic Mechanisms of Action after all?

Intravenous IgG (IVIg) contains polyclonal immunoglobulin G (IgG) from thousands of donors. It is administered at a low dose at regular intervals as antibody replacement therapy and at a higher dose as immunomodulatory treatment in various auto-immune or auto-inflammatory diseases. The working mechanism of immunomodulation is not well understood. Many different explanations have been given. During the last decade, we have focused on classical antibody binding via the Fc-domain of the IgG molecules to the common IgG receptors, i.e. the Fcγ receptors (FcγRs). Variation in the genes encoding human FcγRs determines function as well as expression among immune cells. As described here, NK cells and myeloid cells, including macrophages, can express different FcγR variants, depending on the individual's genotype, copy number variation (CNV), and promoter polymorphisms. B-cells seem to only express the single inhibitory receptor. Although these inhibitory FcγRIIb receptors are also expressed by monocytes, macrophages, and only rarely by NK cells or neutrophils, their presence is unlikely to explain the immunomodulatory capacity of IVIg, nor does the sialylation of IgG. Direct IVIg effects at the level of the activating FcγRs, including the more recently described FcγRIIc, deserve renewed attention to describe IVIg-related immunomodulatio

Immunomodulation by IVIg and the role of Fc-gamma receptors: classic mechanisms of action after all?

Intravenous IgG (IVIg) contains polyclonal IgG from thousands of donors. It is administered at a low dose at regular intervals as antibody replacement therapy and at a higher dose as immunomodulatory treatment in various auto-immune or auto-inflammatory diseases. The working mechanism of immunomodulation is not well understood. Many different explanations have been given. During the last decade we have focused on classical antibody binding via the Fc-domain of the IgG molecules to the common IgG receptors, i.e. the Fcγ receptors (FcγRs). Variation in the genes encoding human FcγRs determines function as well as expression among immune cells. As described here, NK cells and myeloid cells, including macrophages, can express different FcγR variants, depending on the individual’s genotype, copy number variation (CNV) and promoter polymorphisms. B cells seem to only express the single inhibitory receptor. Although these inhibitory FcγRIIb receptors are also expressed by monocytes, macrophages, and only rarely by NK cells or neutrophils, their presence is unlikely to explain the immunomodulatory capacity of IVIg, nor does the sialylation of IgG. Direct IVIg effects at the level of the activating FcγRs, including the more recently described FcγRIIc, deserve renewed attention to describe IVIg-related immunomodulation