Judy Geissler

Copy number variation at the FCGR locus includes FCGR3A, FCGR2C and FCGR3B but not FCGR2A and FCGR2B.

Human Fcã receptors (FcãRs) are glycoproteins that bind the Fc region of IgG. The genes encoding the low‐affinity FcãRs are located on chromosome 1q23‐24. Beside single nucleotide polymorphisms (SNPs), gene copy number variation (CNV) is now being recognized as an important indicator for inter‐individual differences. Recent studies on identifying CNV in the human genome suggest large areas at chromosome 1q23‐24 to be involved, and CNV in this region has been associated with manifestations of systemic autoimmune disease. To study both SNPs and CNV of the low‐affinity FcãRs in one assay, we have developed a Multiplex Ligation‐dependent Probe Amplification (MLPA) assay. A novel CNV for FCGR3A was observed. Similar to FCGR3B and FCGR2C, a gene‐dosage effect of FCGR3A was found, that seemed to correlate nicely with the FcãRIIIa expression on NK cells. Next, we delineated the approximate boundaries of CNV at the FCGR locus. Variation in co‐segregation of neighboring FCGR genes was limited to four variants, with patterns of Mendelian inheritance. No CNV of the FCGR2A and FCGR2B genes was observed in over 600 individuals. In conclusion, we report a novel CNV of the FCGR3A gene that correlates with FcãRIIIa expression and function on NK cells. Only FCGR3A, FCGR2C and FCGR3B show CNV, in contrast to FCGR2A and FCGR2B.

Association of mannose-binding lectin genotype with cardiovascular abnormalities in Kawasaki disease

Kawasaki disease is an acute vasculitis of possible infectious cause, which in particular affects the coronary arteries. Young children rely mostly on their innate immune system for protection against invading microorganisms, of which mannose-binding lectin is an important component. We aimed to investigate the possible role of the gene for this molecule (MBL) in white Dutch patients with Kawasaki disease. In 90 patients, frequency of mutations in the MBL gene was higher than in healthy children. In children younger than 1 year, those with mutations were at higher risk of development of coronary artery lesions than were those without (odds ratio 15.7, 95% CI 1.4-176.5, p=0.026). Our findings suggest that the innate immune system contributes differently to pathophysiology of Kawasaki disease at various ages.

Phenotypic Variation in IgG Receptors by Nonclassical FCGR2C Alleles

The balance between activating and inhibitory signals from the different FcγRs for IgG ensures homeostasis of many inflammatory responses. FCGR2C is the product of an unequal crossover of the FCGR2A and FCGR2B genes encoding the activating FcγRIIa (CD32a) and inhibitory FcγRIIb (CD32b), respectively. A single nucleotide polymorphism (SNP) in exon 3 of FCGR2C results in either expression of the activating FcγRIIc (CD32c) (FCGR2C-open reading frame [ORF]) or its absence because of a stop codon (FCGR2C-Stop). Two additional variations in FcγRIIb/c expression on leukocytes have now been identified. In case of “nonclassical” FCGR2C-ORF alleles, FcγRIIc expression was unexpectedly absent, because of novel splice site mutations near exon 7 leading to another stop codon. In some individuals with FCGR2C-Stop alleles FcγRIIb was detected on NK cells, which normally are devoid of this protein. Individuals with these nonclassical FCGR2C-Stop alleles carried a deletion of FCGR2C-FCGR3B that extends into the promoter region of the adjacent FCGR2B gene and probably deletes a negative regulatory element in the FCGR2B promoter in NK cells. FcγRIIb expression on NK cells effectively inhibited killing mediated by FcγRIIIa (CD16a) in Ab-dependent cytotoxicity tests. Our findings demonstrate a more extensive and previously unnoticed variation in FcγR expression with relevance to immunity and inflammation.

Polymorphisms in chemokine receptor genes and susceptibility to Kawasaki disease.

Kawasaki disease (KD) is an acute vasculitis occurring in young children. Its aetiology is unknown, but an infectious agent is assumed. Increased levels of proinflammatory cytokines and chemokines have been reported in KD. Genetic variation in these genes and the receptors for these genes could influence the regulation of cytokines and chemokines. In a case–control study of 170 Dutch Caucasian KD patients and 300 healthy Dutch Caucasian controls, common genetic variants in chemokine receptor genes CCR3, CCR2, CCR5, CX3CR1, CXCR1 and CXCR2 were analysed. Of the eight studied single nucleotide polymorphisms (SNPs) in the CCR3–CCR2–CCR5 gene cluster, four showed a significant association with susceptibility to KD. Moreover the CCR5‐Δ32 was observed with an allele frequency of 10·7% in the control population compared to 6·5% in the KD patients (P = 0·04). Two haplotypes of the CCR3–CCR2–CCR5 gene‐cluster appear to be at risk haplotypes for KD and one a protective haplotype. No association was observed with the studied SNPs in CX3CR1, CXCR1 and CXCR2. In conclusion, in a Dutch cohort of KD patients an association of KD occurrence with common genetic variants in the chemokine receptor gene‐cluster CCR3–CCR2–CCR5 was observed.

Toll-like receptor responses in IRAK-4-deficient neutrophils.

Human neutrophils were found to express all known Toll-like receptors (TLRs) except TLR3 and TLR7. IRAK-4-deficient neutrophils were tested for their responsiveness to various TLR ligands. Essentially all TLR responses in neutrophils, including the induction of reactive oxygen species generation, adhesion, chemotaxis and IL-8 secretion, were found to be dependent on IRAK-4. Surprisingly, the reactivity towards certain established TLR ligands, imiquimod and ODN-CpG, was unaffected by IRAK-4 deficiency, demonstrating their activity is independent of TLR. TLR-4-dependent signaling in neutrophils was totally dependent on IRAK-4 without any major TRIF-mediated contribution. We did not observe any defects in killing capacity of IRAK-4-deficient neutrophils for Staphylococcus aureus, Escherichia coli and Candida albicans, suggesting that microbial killing is primarily TLR independent.

The involvement of Fc gamma receptor gene polymorphisms in Kawasaki disease.

Kawasaki disease is an acute febrile syndrome in infancy, characterized by vasculitis of medium‐sized arteries. Without treatment the disease can lead to coronary artery lesions (CAL) in approximately 25% of the children. Therapy consists of intravenous immunoglobulins (IVIG), leading to a decrease of complications to 5–16%. Little is known about the working mechanisms of IVIG. In this study we evaluated the involvement of Fcγ receptors (FcγRs) in Kawasaki disease by the determination of the frequency of known single nucleotide polymorphisms (SNPs) in the genes coding for the FcγRs and compared this with frequencies in a cohort of healthy controls. There was no difference in the distribution of the functionally relevant genotypes for FcγRIIa‐131H/R, FcγRIIb‐232I/T, FcγRIIIa‐158 V/F and FcγRIIIb‐NA1/NA2 between the patient group and the healthy controls. Furthermore, there were no polymorphisms linked to the disease severity as indicated by the absence or development of CAL during the disease. Altered transcription or expression of FcγR on specific cell types of the immune system may still play a role in susceptibility and treatment success, but at a level different from the functional SNPs in FcγR genes tested in this study.

Changes in gene expression of granulocytes during in vivo granulocyte colony-stimulating factor/dexamethasone mobilization for transfusion purposes

The treatment of healthy donors with granulocyte colony-stimulating factor (G-CSF) and dexamethasone results in sufficient numbers of circulating granulocytes to prepare granulocyte concentrates for clinical purposes. Granulocytes obtained in this way demonstrate relatively normal functional behavior combined with a prolonged life span. To study the influence of mobilizing agents on granulocytes, we used oligonucleotide microarrays to identify genes that are differentially expressed in mobilized granulocytes compared with control granulocytes. More than 1000 genes displayed a differential expression pattern, with at least a 3-fold difference. Among these, a large number of genes was induced that encode proteins involved in inflammation and the immune response, such as C-type lectins and leukocyte immunoglobulin-like receptors. Because mobilized granulocytes have a prolonged life span, we focused on genes involved in the regulation of apoptosis. One of the most prominent among these was CAST, the gene encoding calpastatin. Calpastatins are the endogenous inhibitors of calpains, a family of calcium-dependent cysteine proteases recently shown to be involved in neutrophil apoptosis. Transcriptional activity of the CAST gene was induced by G-CSF/dexamethasone treatment both in vivo and in vitro, whereas the protein expression of CAST was stabilized during culture. These studies provide new insight in the genotypic changes as well as in the regulation of the immunologic functions and viability of mobilized granulocytes used for clinical transfusion purposes.

Primary Immunodeficiency Caused by an Exonized Retroposed Gene Copy Inserted in the CYBB Gene

Retrotransposon‐mediated insertion of a long interspersed nuclear element (LINE)‐1 or an Alu element into a human gene is a well‐known pathogenic mechanism. We report a novel LINE‐1‐mediated insertion of a transcript from the TMF1 gene on chromosome 3 into the CYBB gene on the X‐chromosome. In a Dutch male patient with chronic granulomatous disease, a 5.8‐kb, incomplete and partly exonized TMF1 transcript was identified in intron 1 of CYBB, in opposite orientation to the host gene. The sequence of the insertion showed the hallmarks of a retrotransposition event, with an antisense poly(A) tail, target site duplication, and a consensus LINE‐1 endonuclease cleavage site. This insertion induced aberrant CYBB mRNA splicing, with inclusion of an extra 117‐bp exon between exons 1 and 2 of CYBB. This extra exon contained a premature stop codon. The retrotransposition took place in an early stage of fetal development in the mother of the patient, because she showed a somatic mosaicism for the mutation that was not present in the DNA of her parents. However, the mutated allele was not expressed in the patients mother because the insertion was found only in the methylated fraction of her DNA.

SBDS Expression and Localization at the Mitotic Spindle in Human Myeloid Progenitors

Background Shwachman-Diamond Syndrome (SDS) is a hereditary disease caused by mutations in the SBDS gene. SDS is clinically characterized by pancreatic insufficiency, skeletal abnormalities and bone marrow dysfunction. The hematologic abnormalities include neutropenia, neutrophil chemotaxis defects, and an increased risk of developing Acute Myeloid Leukemia (AML). Although several studies have suggested that SBDS as a protein plays a role in ribosome processing/maturation, its impact on human neutrophil development and function remains to be clarified. Methodology/Principal Findings We observed that SBDS RNA and protein are expressed in the human myeloid leukemia PLB-985 cell line and in human hematopoietic progenitor cells by quantitative RT-PCR and Western blot analysis. SBDS expression is downregulated during neutrophil differentiation. Additionally, we observed that the differentiation and proliferation capacity of SDS-patient bone marrow hematopoietic progenitor cells in a liquid differentiation system was reduced as compared to control cultures. Immunofluorescence analysis showed that SBDS co-localizes with the mitotic spindle and in vitro binding studies reveal a direct interaction of SBDS with microtubules. In interphase cells a perinuclear enrichment of SBDS protein which co-localized with the microtubule organizing center (MTOC) was observed. Also, we observed that transiently expressed SDS patient-derived SBDS-K62 or SBDS-C84 mutant proteins could co-localize with the MTOC and mitotic spindle. Conclusions/Significance SBDS co-localizes with the mitotic spindle, suggesting a role for SBDS in the cell division process, which corresponds to the decreased proliferation capacity of SDS-patient bone marrow CD34+ hematopoietic progenitor cells in our culture system and also to the neutropenia in SDS patients. A role in chromosome missegregation has not been clarified, since similar spatial and time-dependent localization is observed when patient-derived SBDS mutant proteins are studied. Thus, the increased risk of myeloid malignancy in SDS remains unexplained.

Fc-gamma receptor polymorphisms differentially influence susceptibility to systemic lupus erythematosus and lupus nephritis

OBJECTIVE To determine relevant Fc-gamma receptor (FcγR) polymorphisms in relation to susceptibility to SLE and LN, and to determine the functional consequences of genetic associations found. METHODS Using multiplex ligation-dependent probe amplification, copy number regions (CNRs) and relevant known functional single nucleotide polymorphisms of FcγRII and FcγRIII were determined in a LN-enriched cohort of 266 Dutch Caucasian SLE patients and 919 healthy Caucasian controls. Expression of FcγRs on leukocytes was assessed using flow cytometry. RESULTS In multivariable analysis, low copy number of CNR1 (including FCGR3B; odds ratio (OR) 2.04; 95% CI: 1.29, 3.23), FCGR2A-131RR (OR 2.00; 95% CI: 1.33, 2.99), and the 2B.4 haplotype of FCGR2B (OR 1.59; 95% CI: 1.13, 2.24), but not FCGR2C open reading frame, were significantly (all P < 0.01) and independently associated with susceptibility to SLE. The 2B.4 haplotype was negatively associated with LN and led to surface expression of FcγRIIb on neutrophils and monocytes. CONCLUSION This study is the first to investigate the most relevant and functional single nucleotide polymorphisms and copy number variations of FcγRII and FcγRIII polymorphisms in one study population, enabling the determination of the individual contribution of each polymorphism in multivariable analysis. Three polymorphisms were shown to be independently associated with susceptibility to SLE. The novel findings of a negative association of the 2B.4 haplotype with LN, and increased expression of FcγRIIb on neutrophils and monocytes as a result of this 2B.4 haplotype warrant future research in the role of these cells and FcγRs in the pathogenesis of SLE and LN.