Ágnes Szilágyi

Real-time PCR quantification of human complement C4A and C4B genes

BackgroundThe fourth component of human complement (C4), an essential factor of the innate immunity, is represented as two isoforms (C4A and C4B) in the genome. Although these genes differ only in 5 nucleotides, the encoded C4A and C4B proteins are functionally different. Based on phenotypic determination, unbalanced production of C4A and C4B is associated with several diseases, such as systemic lupus erythematosus, type 1 diabetes, several autoimmune diseases, moreover with higher morbidity and mortality of myocardial infarction and increased susceptibility for bacterial infections. Despite of this major clinical relevance, only low throughput, time and labor intensive methods have been used so far for the quantification of C4A and C4B genes.ResultsA novel quantitative real-time PCR (qPCR) technique was developed for rapid and accurate quantification of the C4A and C4B genes applying a duplex, TaqMan based methodology. The reliable, single-step analysis provides the determination of the copy number of the C4A and C4B genes applying a wide range of DNA template concentration (0.3–300 ng genomic DNA). The developed qPCR was applied to determine C4A and C4B gene dosages in a healthy Hungarian population (N = 118). The obtained data were compared to the results of an earlier study of the same population. Moreover a set of 33 samples were analyzed by two independent methods. No significant difference was observed between the gene dosages determined by the employed techniques demonstrating the reliability of the novel qPCR methodology. A Microsoft Excel worksheet and a DOS executable are also provided for simple and automated evaluation of the measured data.ConclusionThis report describes a novel real-time PCR method for single-step quantification of C4A and C4B genes. The developed technique could facilitate studies investigating disease association of different C4 isotypes.

4G/5G polymorphism of PAI-1 gene is associated with multiple organ dysfunction and septic shock in pneumonia induced severe sepsis: prospective, observational, genetic study

IntroductionActivation of inflammation and coagulation are closely related and mutually interdependent in sepsis. The acute-phase protein, plasminogen activator inhibitor-1 (PAI-1) is a key element in the inhibition of fibrinolysis. Elevated levels of PAI-1 have been related to worse outcome in pneumonia. We aimed to evaluate the effect of functionally relevant 4G/5G polymorphism of PAI-1 gene in pneumonia induced sepsis.MethodsWe enrolled 208 Caucasian patients with severe sepsis due to pneumonia admitted to an intensive care unit (ICU). Patients were followed up until ICU discharge or death. Clinical data were collected prospectively and the PAI-1 4G/5G polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism technique. Patients were stratified according to the occurrence of multiple organ dysfunction syndrome, septic shock or death.ResultsWe found that carriers of the PAI-1 4G/4G and 4G/5G genotypes have a 2.74-fold higher risk for multiple organ dysfunction syndrome (odds ratio [OR] 95% confidence interval [CI] = 1.335 - 5.604; p = 0.006) and a 2.57-fold higher risk for septic shock (OR 95%CI = 1.180 - 5.615; p = 0.018) than 5G/5G carriers. The multivariate logistic regression analysis adjusted for independent predictors, such as age, nosocomial pneumonia and positive microbiological culture also supported that carriers of the 4G allele have a higher prevalence of multiple organ dysfunction syndrome (adjusted odds ratio [aOR] = 2.957; 95%CI = 1.306 -6.698; p = 0.009) and septic shock (aOR = 2.603; 95%CI = 1.137 - 5.959; p = 0.024). However, genotype and allele analyses have not shown any significant difference regarding mortality in models non-adjusted or adjusted for acute physiology and chronic health evaluation (APACHE) II. Patients bearing the 4G allele had higher disseminated intravascular coagulation (DIC) score at admission (p = 0.007) than 5G/5G carriers. Moreover, in 4G allele carriers the length of ICU stay of non-survivors was longer (p = 0.091), fewer ventilation-free days (p = 0.008) and days without septic shock (p = 0.095) were observed during the first 28 days.ConclusionsIn Caucasian patients with severe sepsis due to pneumonia carriers of the 4G allele of PAI-1 polymorphism have higher risk for multiple organ dysfunction syndrome and septic shock and in agreement they showed more fulminant disease progression based on continuous clinical variables.

Novel duplication in the F12 gene in a patient with recurrent angioedema.

Edema formation is mediated by histamine or bradykinin release and may have several hereditary and acquired causes. In hereditary forms of bradykinin-mediated angioedemas, mutations in the genes encoding C1-inhibitor (SERPING1) as well as coagulation factor XII (F12) have been described. We present a novel F12 gene mutation, a duplication of 18 base pairs (c.892_909dup) in a 37-year-old woman with recurrent angioedema and normal C1-inhibitor level. A single episode of facial edema in the family of the patient showed co-segregation with the mutation. This duplication is causing the repeated presence of 6 amino acids (p.298-303) in the same region of factor XII, as those three mutations described previously in cases of hereditary angioedema with normal C1-INH function. These results may confirm the importance of the proline-rich region of factor XII protein in edema formation.

Multivariate analysis of dopaminergic gene variants as risk factors of heroin dependence.

Background Heroin dependence is a debilitating psychiatric disorder with complex inheritance. Since the dopaminergic system has a key role in rewarding mechanism of the brain, which is directly or indirectly targeted by most drugs of abuse, we focus on the effects and interactions among dopaminergic gene variants. Objective To study the potential association between allelic variants of dopamine D2 receptor (DRD2), ANKK1 (ankyrin repeat and kinase domain containing 1), dopamine D4 receptor (DRD4), catechol-O-methyl transferase (COMT) and dopamine transporter (SLC6A3) genes and heroin dependence in Hungarian patients. Methods 303 heroin dependent subjects and 555 healthy controls were genotyped for 7 single nucleotide polymorphisms (SNPs) rs4680 of the COMT gene; rs1079597 and rs1800498 of the DRD2 gene; rs1800497 of the ANKK1 gene; rs1800955, rs936462 and rs747302 of the DRD4 gene. Four variable number of tandem repeats (VNTRs) were also genotyped: 120 bp duplication and 48 bp VNTR in exon 3 of DRD4 and 40 bp VNTR and intron 8 VNTR of SLC6A3. We also perform a multivariate analysis of associations using Bayesian networks in Bayesian multilevel analysis (BN-BMLA). Findings and conclusions In single marker analysis the TaqIA (rs1800497) and TaqIB (rs1079597) variants were associated with heroin dependence. Moreover, –521 C/T SNP (rs1800955) of the DRD4 gene showed nominal association with a possible protective effect of the C allele. After applying the Bonferroni correction TaqIB was still significant suggesting that the minor (A) allele of the TaqIB SNP is a risk component in the genetic background of heroin dependence. The findings of the additional multiple marker analysis are consistent with the results of the single marker analysis, but this method was able to reveal an indirect effect of a promoter polymorphism (rs936462) of the DRD4 gene and this effect is mediated through the –521 C/T (rs1800955) polymorphism in the promoter.

The Major Autoantibody Epitope on Factor H in Atypical Hemolytic Uremic Syndrome Is Structurally Different from Its Homologous Site in Factor H-related Protein 1, Supporting a Novel Model for Induction of Autoimmunity in This Disease.

Background: It is unknown why patients with autoantibodies against complement factor H (CFH) lack homologous CFHR1 protein. Results: The autoantibody epitope on CFH was identified, and the structure of the corresponding part of CFHR1 was solved. Conclusion: The autoantigenic epitope of CFH and its homologous site in CFHR1 are structurally different. Significance: A plausible explanation for formation of autoantibodies due to CFHR1 deficiency in autoimmune atypical hemolytic uremic syndrome was obtained. Atypical hemolytic uremic syndrome (aHUS) is characterized by complement attack against host cells due to mutations in complement proteins or autoantibodies against complement factor H (CFH). It is unknown why nearly all patients with autoimmune aHUS lack CFHR1 (CFH-related protein-1). These patients have autoantibodies against CFH domains 19 and 20 (CFH19–20), which are nearly identical to CFHR1 domains 4 and 5 (CFHR14–5). Here, binding site mapping of autoantibodies from 17 patients using mutant CFH19–20 constructs revealed an autoantibody epitope cluster within a loop on domain 20, next to the two buried residues that are different in CFH19–20 and CFHR14–5. The crystal structure of CFHR14–5 revealed a difference in conformation of the autoantigenic loop in the C-terminal domains of CFH and CFHR1, explaining the variation in binding of autoantibodies from some aHUS patients to CFH19–20 and CFHR14–5. The autoantigenic loop on CFH seems to be generally flexible, as its conformation in previously published structures of CFH19–20 bound to the microbial protein OspE and a sialic acid glycan is somewhat altered. Cumulatively, our data suggest that association of CFHR1 deficiency with autoimmune aHUS could be due to the structural difference between CFHR1 and the autoantigenic CFH epitope, suggesting a novel explanation for CFHR1 deficiency in the pathogenesis of autoimmune aHUS.

The role of complement in Streptococcus pneumoniae-associated haemolytic uraemic syndrome

BACKGROUND Atypical forms of haemolytic uraemic syndrome (aHUS) include HUS caused by defects in the regulation of alternative complement pathway and HUS linked to neuraminidase-producing pathogens, such as Streptococcus pneumoniae. Increasing data support a pathogenic role of neuraminidase in the development of S. pneumoniae-associated haemolytic uraemic syndrome (SP-HUS), but the role of complement has never been clarified in detail. Therefore, we aimed to investigate whether the pathologic complement profile and genetic risk factors of aHUS are present in patients with SP-HUS. METHODS Enrolling five patients with SP-HUS classical and alternative pathway activity, besides C3, C4, factors H, B, I and anti-factor H autoantibody levels were determined. The coding regions of CFH, CFI, CD46 (MCP), THBD, C3 and CFB genes were sequenced and the copy number of CFI, CD46, CFH and related genes were also analyzed. RESULTS We found that in the acute phase samples of SP-HUS patients, complement components C4, C3 and activity of the classical and alternative pathways were decreased, indicating severe activation and complement consumption, but most of these alterations normalized later in remission. Three of the patients carried mutations and risk haplotypes in complement-mediated aHUS associated genes. The identified mutations include a previously published CFI variant (P50A) and two novel ones in CFH (R1149X) and THBD (T44I) genes. CONCLUSIONS Our results suggest that severe complement dysregulation and consumption accompany the progress of invasive pneumococcal disease (IPD)-associated SP-HUS and genetic variations of complement genes may contribute to the development of this complication in a proportion of the affected patients.

Contribution of Serotonin Transporter Gene Polymorphisms to Pediatric Migraine

Background.—The serotonin transporter gene is a promising candidate locus for the genetic susceptibility of migraine.

Polymorphisms of TNF-alpha and LT-alpha genes in multiple myeloma

Allelic distribution of -308 G>A (TNF 1/2) polymorphism of the TNF-alpha, and the +252 A>G promoter polymorphism of the LT-alpha gene, the 1267 A>G polymorphism of the HSP70-2 gene as well as the -429 T>C promoter polymorphism of the RAGE gene were tested in 94 MM cases and 141 controls. Significantly less MM patients than controls carried the TNF2 allele (p=0.018) and the TNF2-LTA 252G haplotype (p=0.025). The difference was, however, restricted to the females, as well as the relatively young (<69 years) subjects. By contrast, we did not find differences with the other SNPs tested.

Structural Basis for the Function of Complement Component C4 within the Classical and Lectin Pathways of Complement.

Complement component C4 is a central protein in the classical and lectin pathways within the complement system. During activation of complement, its major fragment C4b becomes covalently attached to the surface of pathogens and altered self-tissue, where it acts as an opsonin marking the surface for removal. Moreover, C4b provides a platform for assembly of the proteolytically active convertases that mediate downstream complement activation by cleavage of C3 and C5. In this article, we present the crystal and solution structures of the 195-kDa C4b. Our results provide the molecular details of the rearrangement accompanying C4 cleavage and suggest intramolecular flexibility of C4b. The conformations of C4b and its paralogue C3b are shown to be remarkably conserved, suggesting that the convertases from the classical and alternative pathways are likely to share their overall architecture and mode of substrate recognition. We propose an overall molecular model for the classical pathway C5 convertase in complex with C5, suggesting that C3b increases the affinity for the substrate by inducing conformational changes in C4b rather than a direct interaction with C5. C4b-specific features revealed by our structural studies are probably involved in the assembly of the classical pathway C3/C5 convertases and C4b binding to regulators.

Comparative analysis of IL6 promoter and receptor polymorphisms in myelodysplasia and multiple myeloma.

The serum levels of interleukin 6 (IL6) are known to be elevated in two diseases of the elderly age, myelodysplastic syndrome (MDS) and multiple myeloma (MM). Authors suppose that one of the possible causes of this elevation could be a difference between these patients and healthy subjects in the frequency of polymorphic variants of the genes regulating IL6 levels. Scarce and contradictory comparative data are available for MM and to our best knowledge this is the first study on IL6 promoter and IL6 receptor (IL6R) polymorphism in MDS. Therefore we determined the Asp358Ala polymorphism of the IL6 receptor gene and the -174 G>C promoter polymorphism of the IL6 gene in blood samples of 102 MDS and 100 MM patients and 99 age- and sex-matched hospitalized controls had been tested for this purpose as well. There was no significant difference between patients with either disease and controls regarding IL6 promoter/L-6R. Authors therefore assume other mechanisms causing high IL6 levels are not related to either of these polymorphisms. Moreover authors consider important to propose a hypothesis how elements of signal transduction in iron metabolism might be involved in the development of MM and MDS in elderly age.