Berthold Hoppe

Detailed analysis of the variability of peptidylarginine deiminase type 4 in German patients with rheumatoid arthritis: a case–control study

Peptidylarginine deiminase type 4 (PADI4) genotypes were shown to influence susceptibility to rheumatoid arthritis (RA) in the Japanese population. Such an association could not previously be confirmed in different European populations. In the present study, we analysed exons 2–4 of PADI4 in 102 German RA patients and 102 healthy individuals to study the influence of PADI4 variability on RA susceptibility by means of haplotype-specific DNA sequencing. Analyses of the influence of PADI4 and HLA-DRB1 genotypes on disease activity and on levels of anti-cyclic citrullinated peptide antibodies were performed.Comparing the frequencies of PADI4 haplotype 4 (padi4_89*G, padi4_90*T, padi4_92*G, padi4_94*T, padi4_104*C, padi4_95*G, padi4_96*T) (patients, 14.7%; controls, 7.8%; odds ratio = 2.0, 95% confidence interval = 1.1–3.8) and carriers of this haplotype (patients, 27.5%; controls, 13.7%; odds ratio = 2.4, 95% confidence interval = 1.2–4.8), a significant positive association of PADI4 haplotype 4 with RA could be demonstrated. Other PADI4 haplotypes did not differ significantly between patients and controls. Regarding the individual PADI4 variants, padi4_89 (A→G), padi4_90 (C→T), and padi4_94 (C→T) were significantly associated with RA (patients, 49.5%; controls, 38.7%; odds ratio = 1.6, 95% confidence interval = 1.1–2.3). Considering novel PADI4 variants located in or near to exons 2, 3, and 4, no quantitative or qualitative differences between RA patients (8.8%) and healthy controls (10.8%) could be demonstrated. While the PADI4 genotype did not influence disease activity and the anti-cyclic citrullinated peptide antibody level, the presence of the HLA-DRB1 shared epitope was significantly associated with higher anti-cyclic citrullinated peptide antibody levels (P = 0.033).The results of this small case–control study support the hypothesis that variability of the PADI4 gene may influence susceptibility to RA in the German population. Quantitative or qualitative differences in previously undefined PADI4 variants between patients and controls could not be demonstrated.

Gene polymorphisms implicated in influencing susceptibility to venous and arterial thromboembolism: Frequency distribution in a healthy German population

Evolvement and progression of cardiovascular diseases affecting the venous and arterial system are influenced by a multitude of environmental and hereditary factors. Many of these hereditary factors consist of defined gene polymorphisms, such as single nucleotide polymorphisms (SNPs) or insertion-deletion polymorphisms, which directly or indirectly affect the hemostatic system. The frequencies of individual hemostatic gene polymorphisms in different normal populations are well defined. However, descriptions of patterns of genetic variability of a larger extent of different factors of hereditary hypercoagulability in single populations are scarce. The aim of this study was i) to give a detailed description of the frequencies of factors of hereditary thrombophilia and their combinations in a German population (n = 282) and ii) to compare their distributions with those reported for other regions. Variants of coagulation factors [factor V 1691G>A (factor V Leiden), factor V 4070A>G (factor V HR2 haplotype), factor VII Arg353Gln, factor XIII Val34Leu, beta-fibrinogen -455G>A, prothrombin 20210G>A], coagulation inhibitors [tissue factor pathway inhibitor 536C>T, thrombomodulin 127G>A], fibrinolytic factors [angiotensin converting enzyme intron 16 insertion/deletion, factor VII-activating protease 1601G>A (FSAP Marburg I), plasminogen activator inhibitor 1-675 insertion/deletion (5G/4G), tissue plasminogen activator intron h deletion/insertion], and other factors implicated in influencing susceptibility to thromboembolic diseases [apolipoprotein E2/E3/E4, glycoprotein Ia 807C>T, methylenetetrahydrofolate reductase 677C>T] were included. The distribution of glycoprotein Ia 807C>T deviated significantly from the Hardy-Weinberg equilibrium, and a comparison with previously published data indicates marked region and ethnicity dependent differences in the genotype distributions of some other factors.

Fibrinogen and factor XIII at the intersection of coagulation, fibrinolysis and inflammation

Fibrinogen and factor XIII are two essential proteins that are involved directly in fibrin gel formation as the final step of a sequence of reactions triggered by a procoagulant stimulus. Haemostasis is the most obvious function of the resulting fibrin clot. Different variables affect the conversion of fibrinogen to fibrin as well as the mode of fibrin polymerisation and fibrin crosslinking, hereby, critically influencing the architecture of the resulting fibrin network and consequently determining its mechanical strength and resistance against fibrinolysis. Due to fibrinogens structure with a multitude of domains and binding motifs the fibrin gel allows for complex interactions with other coagulation factors, with profibrinolytic as well as antifibrinolyic proteins, with complement factors and with various cellular receptors. These interactions enable the fibrin network to control its own further state (i. e. expansion or degradation), to influence innate immunity, and to function as a scaffold for cell migration processes. During the whole process of fibrin gel formation biologically active peptides and protein fragments are released that additionally influence cellular processes via chemotaxis or by modulating cell-cell interactions. Thus, it is not surprising that fibrinogen and factor XIII in addition to their haemostatic function influence innate immunity as well as cell-mediated reactions like wound healing, response to tissue injury or inflammatory processes. The present review summarises current knowledge of fibrinogens and factor XIIIs function in coagulation and fibrinolysis giving special emphasis on their relation to inflammation control.

Influence of peptidylarginine deiminase type 4 genotype and shared epitope on clinical characteristics and autoantibody profile of rheumatoid arthritis

Background: Recent evidence suggests that distinction of subsets of rheumatoid arthritis (RA) depending on anti-cyclic citrullinated peptide antibody (anti-CCP) status may be helpful in distinguishing distinct aetiopathologies and in predicting the course of disease. HLA-DRB1 shared epitope (SE) and peptidylarginine deiminase type 4 (PADI4) genotype, both of which have been implicated in anti-CCP generation, are assumed to be associated with RA. Objectives: To elucidate whether PADI4 affects the clinical characteristics of RA, and whether it would modulate the effect of anti-CCPs on clinical course. The combined effect of SE and PADI4 on autoantibody profile was also analysed. Methods: 373 patients with RA were studied. SE, padi4_94C>T, rheumatoid factor, anti-CCPs and antinuclear antibodies (ANAs) were determined. Disease severity was characterised by cumulative therapy intensity classified into ordinal categories (CTI-1 to CTI-3) and by Steinbrocker score. Results: CTI was significantly associated with disease duration, erosive disease, disease activity score (DAS) 28 and anti-CCPs. The association of anti-CCPs with CTI was considerably influenced by padi4_94C>T genotype (C/C: ORadj = 0.93, padj = 0.92; C/T: ORadj = 2.92, padj = 0.093; T/T: ORadj = 15.3, padj = 0.002). Carriage of padi4_94T exhibited a significant trend towards higher Steinbrocker scores in univariate and multivariate analyses. An association of padi4_94C>T with ANAs was observed, with noteworthy differences depending on SE status (SE−: ORadj = 6.20, padj<0.04; SE+: ORadj = 0.36, padj = 0.02) and significant heterogeneity between the two SE strata (p = 0.006). Conclusions: PADI4 genotype in combination with anti-CCPs and SE modulates clinical and serological characteristics of RA.

Heparin or aspirin or both in the treatment of recurrent abortions in women with antiphospholipid antibody (syndrome).

Purpose of reviewPresence of antiphospholipid antibodies (aPL) is associated with unsuccessful pregnancy outcome. Based on an early concept of aPL-induced thrombophilia and placental thrombosis, antithrombotic interventions have been used to reduce incidence of miscarriage in antiphospholipid antibody syndrome (APS). The aim of this review is to summarize current knowledge on pathogenesis of miscarriage in APS and the impact of different antithrombotic therapy strategies. Recent findingsPathogenetic concepts on miscarriage in APS comprise aPL-mediated cell activation, disturbance of coagulation along with increased complement activation. There is increasing evidence that heparin exerts its effect by inhibiting complement activation rather than by its anticoagulation capacity. In this regard, the outcome of pregnancies in APS has considerably improved by the invention of therapies using combinations of aspirin, unfractionated heparin (UFH) and/or low molecular weight heparin (LMWH). However, there is no clear evidence as to which treatment regimen should be preferred. Some studies indicate superiority of aspirin plus heparin over aspirin-only. Whether heparin-only treatment would confer equal effects and whether UFH and LMWH were of comparable efficacy currently is unknown. SummaryTreatment with aspirin and heparin decreases the risk of miscarriages in APS. Well designed trials as well as better patients-at-risk profiling are warranted that identify which treatment strategy should be favored and whether detailed characterization of aPL specificities could help in individualizing therapy.

Increased RBC autoantibody production in pregnancy.

BACKGROUND: Autoimmunization against RBCs is generally believed to occur very rarely during pregnancy and to represent a high risk for those affected. The occurrence of benign RBC autoantibodies in pregnancy is reported.

Coagulation and the fibrin network in rheumatic disease: a role beyond haemostasis

Activation of the immune system has been increasingly recognised to be associated with procoagulatory status in patients with inflammatory rheumatic disease. Changes in endothelial cell and platelet activation, blood flow, expression and activity of different coagulation factors, and impaired fibrinolysis serve as pathophysiological basis for enhanced risk of venous thromboembolism in inflammatory rheumatic diseases, such as rheumatoid arthritis (RA), connective tissue diseases and vasculitides. Recent studies identifying mechanisms for a functional role of coagulation factors beyond haemostasis have provided examples of interesting links between the coagulation system and innate immune activation. Furthermore, citrullinated fibrinogen is an important and early autoantigen in patients with RA carrying the HLA-DRβ1 shared epitope allele, which demonstrates an adaptive immune response to a coagulation factor in an inflammatory rheumatic disease. Additional studies have provided strong evidence that a multitude of different components of the haemostatic system (such as thrombin, fibrinogen, coagulation factor XIII and factors of the fibrinolytic system) are relevant mediators of inflammatory processes as well as of inflammatory control. Understanding the interactions between coagulation and the immune system in inflammatory rheumatic diseases will not only improve our knowledge of disease mechanisms, but could also permit the development of innovative therapeutic interventions.

High variability of peptidylarginine deiminase 4 (PADI4) in a healthy white population: characterization of six new variants of PADI4 exons 2–4 by a novel haplotype-specific sequencing-based approach

Seven single nucleotide polymorphisms (SNPs) of the peptidylarginine deiminase 4 (PADI4) gene have recently been reported to be strongly associated with rheumatoid arthritis in Japanese individuals. These SNPs are located in or close to exons 2–4 of PADI4 and are organized in at least four different haplotypes. However, a detailed sequencing-based characterization of the PADI4 gene in other populations is still lacking. We therefore analyzed exons 2–4 of the PADI4 gene in 102 healthy white Germans individuals by DNA sequencing and characterized new variants and haplotypes by a novel haplotype-specific sequencing-based approach. The haplotypes 2/3 (padi4_89*G, padi4_90*T, padi4_92*G, padi4_94*T, padi4_104*T, padi4_95*C, padi4_96*C), and haplotype 4 (padi4_89*G, padi4_90*T, padi4_92*G, padi4_94*T, padi4_104*C, padi4_95*G, padi4_96*T) conferring susceptibility to rheumatoid arthritis were detected at frequencies of 30.9% and 7.8%, respectively. In addition, three novel coding SNPs in exons 2, 3, and 4, and three SNPs in introns 2 and 3 located near the exon-intron boundaries were identified in 11 individuals (10.8%). The so-called nonsusceptibility haplotype 1 (padi4_89*A, padi4_90*C, padi4_92*C, padi4_94*C, padi4_104*C, padi4_95*G, padi4_96*T) occurred at a frequency of 58.3%. Additionally, we identified a closely related novel haplotype, haplotype 1B (2.9%), that differs from haplotype 1 only by padi4_92*G/padi4_96*C. This haplotype was not described in the Japanese population. Our results indicate that the PADI4 gene exhibits a remarkable variability and a rather complex haplotypic organization. Further studies on disease association of PADI4 should be performed by haplotype-specific sequencing-based approaches to identify the exact genotype of the PADI4 fragment of interest.

Identification of 34 N-glycan isomers in human serum by capillary electrophoresis coupled with laser-induced fluorescence allows improving glycan biomarker discovery

AbstractAlterations in glycosylation have been observed in many human diseases and specific changes in glycosylation have been proposed as relevant diagnostic information. Capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) is a robust method to quantify desialylated N-glycans that are labeled with 8-aminopyrene-1,3,6-trisulfonic acid prior to analysis. To date, only a maximum of 12 glycan structures, the most abundant ones, have been identified by CE-LIF to characterize glycome modulations of total serum in the course of the diseases. In most forms of cancer, findings using CE-LIF were limited to the increase of triantennary structures carrying a Lewisx epitope. In this work, we identified 32 linkage and positional glycan isomers in healthy human serum using exoglycosidase digestions as well as standard glycoproteins, for which we report the assignment of novel structures. It was possible to identify and quantify 34 glycan isomers in the serum of primary epithelial ovarian cancer patients (EOC). Reduced levels of diantennary structures and of high-mannose 5 were statistically significant in the EOC samples, and also, elevated branching as well as increased antennary fucosylation were observed. For the first time, we could demonstrate that not only antennary fucosylation was of relevance in tetraantennary structures but also core-fucosylated tetraantennary N-glycans were statistically increased in EOC patients. The results of the current study provide an improved dataset to be used in glycan biomarker discovery. Graphical abstractᅟ

Association of angiotensin-converting enzyme intron 16 insertion/deletion polymorphism with history of foetal loss

Introduction. The angiotensin-converting enzyme (ACE) intron 16 insertion/deletion (I/D) polymorphism is associated with ACE activity and has been discussed as a risk factor for pre-eclampsia. Disturbances of uteroplacental circulation are involved in the pathogenesis of pre-eclampsia. In this study, we tested whether the ACE I/D genotype is associated with history of foetal loss (FL) or uteroplacental dysfunction (UPD). Patients and methods. ACE I/D genotype was determined in 312 women presenting with a history of FL and 112 women admitted because of UPD. The association of the ACE I/D genotype with FL or UPD was assessed in a case-control study using 527 patients with diagnoses other than FL or UPD. To exclude potential biases due to associations of this genotype with other diagnoses, we additionally performed a case-control study using 553 healthy controls. Results. ACE I/D genotype was significantly associated with history of FL in both case-control studies (patient controls: odds ratio 1.52, p<0.02; healthy controls: odds ratio 1.48, p=0.02). There was no evidence for allele-dose dependency. No association of the ACE I/D genotype with UPD could be detected. Conclusions. The ACE I/D genotype exhibits a statistically significant association with a history of FL. These results corroborate an involvement of the renin-angiotensin system in pregnancy complications.