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1.  Both Positive and Negative Selection Pressures Contribute to the Polymorphism Pattern of the Duplicated Human CYP21A2 Gene 
PLoS ONE  2013;8(11):e81977.
The human steroid 21-hydroxylase gene (CYP21A2) participates in cortisol and aldosterone biosynthesis, and resides together with its paralogous (duplicated) pseudogene in a multiallelic copy number variation (CNV), called RCCX CNV. Concerted evolution caused by non-allelic gene conversion has been described in great ape CYP21 genes, and the same conversion activity is responsible for a serious genetic disorder of CYP21A2, congenital adrenal hyperplasia (CAH). In the current study, 33 CYP21A2 haplotype variants encoding 6 protein variants were determined from a European population. CYP21A2 was shown to be one of the most diverse human genes (HHe=0.949), but the diversity of intron 2 was greater still. Contrary to previous findings, the evolution of intron 2 did not follow concerted evolution, although the remaining part of the gene did. Fixed sites (different fixed alleles of sites in human CYP21 paralogues) significantly accumulated in intron 2, indicating that the excess of fixed sites was connected to the lack of effective non-allelic conversion and concerted evolution. Furthermore, positive selection was presumably focused on intron 2, and possibly associated with the previous genetic features. However, the positive selection detected by several neutrality tests was discerned along the whole gene. In addition, the clear signature of negative selection was observed in the coding sequence. The maintenance of the CYP21 enzyme function is critical, and could lead to negative selection, whereas the presumed gene regulation altering steroid hormone levels via intron 2 might help fast adaptation, which broadly characterizes the genes of human CNVs responding to the environment.
PMCID: PMC3843699  PMID: 24312389
2.  Association of Ficolin-3 with Severity and Outcome of Chronic Heart Failure 
PLoS ONE  2013;8(4):e60976.
Inflammatory mechanisms involving complement activation has been shown to take part in the pathophysiology of congestive heart failure, but the initiating mechanisms are unknown. We hypothesized that the main initiator molecules of the lectin complement pathway mannose-binding lectin (MBL), ficolin-2 and ficolin-3 were related to disease severity and outcome in chronic heart failure.
Methods and Results
MBL, ficolin-2 and ficolin-3 plasma concentrations were determined in two consecutive cohorts comprising 190 patients from Hungary and 183 patients from Norway as well as controls. Disease severity and clinical parameters were determined at baseline, and all-cause mortality was registered after 5-years follow-up. In univariate analysis a low level of ficolin-3, but not that of MBL or ficolin-2, was significantly associated with advanced heart failure (New York Heart Association Class IV, p<0.001 for both cohorts) and showed inverse correlation with B- type natriuretic peptide (BNP) levels (r = −0.609, p<0.001 and r = −0.467, p<0.001, respectively). In multivariable Cox regression analysis, adjusted for age, gender and BNP, decreased plasma ficolin-3 was a significant predictor of mortality (HR 1.368, 95% CI 1.052–6.210; and HR 1.426, 95% CI 1.013–2.008, respectively). Low ficolin-3 levels were associated with increased complement activation product C3a and correspondingly decreased concentrations of complement factor C3.
This study provides evidence for an association of low ficolin-3 levels with advanced heart failure. Concordant results from two cohorts show that low levels of ficolin-3 are associated with advanced heart failure and outcome. The decrease of ficolin-3 was associated with increased complement activation.
PMCID: PMC3626638  PMID: 23596511
3.  Intraspecific Evolution of Human RCCX Copy Number Variation Traced by Haplotypes of the CYP21A2 Gene 
Genome Biology and Evolution  2012;5(1):98-112.
The RCCX region is a complex, multiallelic, tandem copy number variation (CNV). Two complete genes, complement component 4 (C4) and steroid 21-hydroxylase (CYP21A2, formerly CYP21B), reside in its variable region. RCCX is prone to nonallelic homologous recombination (NAHR) such as unequal crossover, generating duplications and deletions of RCCX modules, and gene conversion. A series of allele-specific long-range polymerase chain reaction coupled to the whole-gene sequencing of CYP21A2 was developed for molecular haplotyping. By means of the developed techniques, 35 different kinds of CYP21A2 haplotype variant were experimentally determined from 112 unrelated European subjects. The number of the resolved CYP21A2 haplotype variants was increased to 61 by bioinformatic haplotype reconstruction. The CYP21A2 haplotype variants could be assigned to the haplotypic RCCX CNV structures (the copy number of RCCX modules) in most cases. The genealogy network constructed from the CYP21A2 haplotype variants delineated the origin of RCCX structures. The different RCCX structures were located in tight groups. The minority of groups with identical RCCX structure occurred once in the network, implying monophyletic origin, but the majority of groups occurred several times and in different locations, indicating polyphyletic origin. The monophyletic groups were often created by single unequal crossover, whereas recurrent unequal crossover events generated some of the polyphyletic groups. As a result of recurrent NAHR events, more CYP21A2 haplotype variants with different allele patterns belonged to the same RCCX structure. The intraspecific evolution of RCCX CNV described here has provided a reasonable expectation for that of complex, multiallelic, tandem CNVs in humans.
PMCID: PMC3595039  PMID: 23241443
allele-specific long-range PCR; CNV; genealogy network; nonallelic homologous recombination
4.  Comparison of epitope specificity of anti-heat shock protein 60/65 IgG type antibodies in the sera of healthy subjects, patients with coronary heart disease and inflammatory bowel disease 
Cell Stress & Chaperones  2011;17(2):215-227.
Previously, we reported on the presence of antibodies to linear epitopes of human and mycobacterial 60 kD heat shock proteins (HSP) in the sera of healthy blood donors. Since many recent findings indicate that the levels of these antibodies may be altered in coronary heart disease (CHD) and also inflammatory bowel diseases (IBD), it seemed worthwhile to compare the epitope specificity of the anti-HSP60 and anti-HSP65 antibodies in the sera of patients with these diseases to those in healthy subjects. The multipin enzyme-linked immunosorbent assay method was applied with a large overlapping set of synthetic 10-mer peptides covering selected regions of human HSP60 and Mycobacterium bovis HSP65. Sera of 12 healthy persons (HP), 14 CHD, and 14 IBD patients with the same concentration of total anti-HSP60 and HSP65 IgG antibodies were tested. We have identified CHD-specific epitopes in the equatorial domain of the HSP60 protein but in neither region of the HSP65 molecule, indicating that the formation of anti-HSP60 antibodies is not or only partially due to the cross-reaction between human HSP60 and bacterial HSP65. IBD-specific epitopes were found in many regions of the HSP60 and in even more regions of the HSP65 molecule including an IBD-specific T cell epitope in region X as well. These findings indicate that the epitope specificity of the anti-human and anti-mycobacterial HSP60 antibodies associated with various diseases is different.
Electronic supplementary material
The online version of this article (doi:10.1007/s12192-011-0301-7) contains supplementary material, which is available to authorized users.
PMCID: PMC3273563  PMID: 22038196
Coronary heart disease; Inflammatory bowel disease; Human HSP60; Mycobacterial HSP65; Epitope analysis
5.  458 Differences of Complement Activation Profile between Type I and Type II of Hereditary Angioedema Due to C1-inhibitor Deficiency 
The World Allergy Organization Journal  2012;5(Suppl 2):S162-S163.
In hereditary angioedema (HAE), diverse mutations in the C1-inhibitor gene may produce either normal C1-inhibitor protein in insufficient quantities (HAE type I), or a dysfunctional protein in normal or even excessive amounts (HAE type II). Previously, we have found strong association between baseline level of functional C1-inhibitor and severity of HAE. Our aim was to investigate complement activation products in HAE, during a follow-up period, and to analyze the relationship between these products and severity of disease.
107 HAE patients (96 HAE type I, 11 type II) and 113 healthy control subjects were included. C1rC1sC1-INH, C3bBbP, and SC5b-9 levels were determined using ELISA methods in single EDTA-plasma samples of controls and in 4 samples from patients taken in 4 subsequent years. Between-group differences were evaluated with the Mann-Whitney test, and correlations were calculated using non-parametric Spearman's correlation coefficients.
Median levels of C1rC1sC1-INH (60 U/mL [40–113] vs 8 U/mL [4–10]; P < 0.0001) and SC5b-9 (0.6 U/mL [0.4–1.2] vs 1.8[0.9–2.8]; P < 0.0001) differed between patients and controls. Significant differences were found between HAE type I and type II as regards median C1rC1sC1-INH level (54 U/mL [33–97] vs 31 U/mL [21–49], P < 0.0001), and in an opposite manner with C3bBbP median levels (6 U/mL [4–12] vs 10 U/mL [8–17], P = 0.0002). Level of C1rC1sC1-INH correlated with the number of attacks (r = 0.3546, P = 0.0004) in HAE type I, but not in HAE type II. Dividing the patients into 2 subgroups based on danazol therapy, significant association (r = 0.3705, P = 0.0026) was found between level of C1rC1sC1-INH and annual attack number only in patients not treated with danazol. Similar results were found as regards the number of C1-inhibitor vials administered, which correlated with the level of C1rC1sC1-INH only in HAE type I (r = 0.4288, P < 0.0001) and in patients not treated with danazol (r = 0.4783, P < 0.0001).
Monitoring the level of C1rC1sC1-INH is mostly informative in HAE type I patients, who are not treated with danazol. No correlation was found in HAE type II patients between level of C1rC1sC1-INH and disease severity markers. As this observation may be explained by the small number of HAE type II patients, multicenter studies are needed, including more HAE type II patients.
PMCID: PMC3513152
6.  Low ficolin-3 levels in early follow-up serum samples are associated with the severity and unfavorable outcome of acute ischemic stroke 
A number of data indicate that the lectin pathway of complement activation contributes to the pathophysiology of ischemic stroke. The lectin pathway may be triggered by the binding of mannose-binding lectin (MBL), ficolin-2 or ficolin-3 to different ligands. Although several papers demonstrated the significance of MBL in ischemic stroke, the role of ficolins has not been examined.
Sera were obtained within 12 hours after the onset of ischemic stroke (admission samples) and 3-4 days later (follow-up samples) from 65 patients. The control group comprised 100 healthy individuals and 135 patients with significant carotid stenosis (patient controls). The concentrations of ficolin-2 and ficolin-3, initiator molecules of the lectin complement pathway, were measured by ELISA methods. Concentration of C-reactive protein (CRP) was also determined by a particle-enhanced immunturbidimetric assay.
Concentrations of both ficolin-2 and ficolin-3 were significantly (p < 0.001) decreased in both the admission and in the follow-up samples of patients with definite ischemic stroke as compared to healthy subjects. Concentrations of ficolin-2 and ficolin-3 were even higher in patient controls than in healthy subjects, indicating that the decreased levels in sera during the acute phase of stroke are related to the acute ischemic event. Ficolin-3 levels in the follow-up samples inversely correlated with the severity of stroke indicated by NIH scale on admission. In follow-up samples an inverse correlation was observed between ficolin-3 levels and concentration of S100β, an indicator of the size of cerebral infarct. Patients with low ficolin-3 levels and high CRP levels in the follow up samples had a significantly worse outcome (adjusted ORs 5.6 and 3.9, respectively) as measured by the modified Rankin scale compared to patients with higher ficolin-3 and lower CRP concentrations. High CRP concentrations were similarly predictive for worse outcome, and the effects of low ficolin-3 and high CRP were independent.
Our findings indicate that ficolin-mediated lectin pathways of complement activation contribute to the pathogenesis of ischemic stroke and may be additive to complement-independent inflammatory processes.
PMCID: PMC3314397  PMID: 22206485
stroke; ischemic stroke; outcome; complement; lectin pathway; ficolins; ficolin-2; ficolin-3; CRP
7.  High levels of acute phase proteins and soluble 70 kDa heat shock proteins are independent and additive risk factors for mortality in colorectal cancer 
Cell Stress & Chaperones  2010;16(1):49-55.
Recently, we reported that high soluble Hsp70 (sHsp70) level was a significant predictor of mortality during an almost 3-year-long follow-up period in patients with colorectal cancer. This association was the strongest in the group of <70-year-old female patients as well as in those who were in a less advanced stage of the disease at baseline. According to these observations, measurement of the serum level of sHsp70 is a useful, stage-independent prognostic marker in colorectal cancer, especially in patients without distant metastasis. Since many literature data indicated that measurement of C-reactive protein (CRP) and other acute phase proteins (APPs) may also be suitable for predicting the mortality of patients with colorectal cancer, it seemed reasonable to study whether the effect of sHsp70 and other APPs are related or independent. In order to answer this question, we measured the concentrations of CRP as well as of other complement-related APPs (C1 inhibitor, C3, and C9) along with that of the MASP-2 complement component in the sera of 175 patients with colorectal cancer and known levels of sHsp70, which have been used in our previous study. High (above median) levels of CRP, C1 esterase inhibitor (C1-INH), and sHsp70 were found to be independently associated with poor patient survival, whereas no such association was observed with the other proteins tested. According to the adjusted Cox proportional hazards analysis, the additive effect of high sHsp70, CRP, and C1-INH levels on the survival of patients exceeded that of high sHsp70 alone, with a hazard ratio (HR) of 2.83 (1.13–70.9). In some subgroups of patients, such as in females [HR 4.80 (1.07–21.60)] or in ≤70-year-old patients [HR 11.53 (2.78–47.70)], even greater differences were obtained. These findings indicate that the clinical mortality–prediction value of combined measurements of sHsp70, CRP, and C1-INH with inexpensive methods can be very high, especially in specific subgroups of patients with colorectal cancer.
PMCID: PMC3024085  PMID: 20730518
Colorectal cancer; Heat shock proteins; HSP70; Soluble HSP70; Surival; CRP; C3; C9; MASP-2; C1-inhbitor; Acute phase proteins; APP
8.  Serum level of soluble 70-kD heat shock protein is associated with high mortality in patients with colorectal cancer without distant metastasis 
Cell Stress & Chaperones  2009;15(2):143-151.
Many findings indicate that measuring the serum concentration of soluble 70-kD heat shock protein (soluble HSP70) may provide important information in cardiovascular, inflammatory, and pregnancy-related diseases; however, only scarce data are available in cancer. Therefore, using a commercial ELISA kit, we measured soluble HSP70 concentration in the sera of 179 patients with colorectal cancer. We investigated the relationship between soluble HSP70 concentration and mortality, during 33.0 (24.4–44.0) months long follow-up. High (>1.65 pg/ml, median concentration) soluble HSP70 level was a significant (hazard ratio: 1.88 (1.20–2.96, p = 0.005) predictor of mortality during the follow-up period. When we compared the soluble HSP70 levels in patients with non-resected primary tumors as compared to those who were recruited into the study 4–6 weeks after the tumor resection they were found to be significantly (p = 0.020) higher in the former group. Since the patients with non-resected primary tumors had also distant metastasis and died early, we limited the further analysis to 142 patients with no distant metastasis at the beginning of the follow-up. This association remained significant even after multiple Cox-regression analysis had been performed to adjust the data for age and sex (p = 0.028); age, sex, and TNM-T stage (p = 0.041); age, sex, and TNM-N stage (p = 0.021); age, sex, and histological grade (p = 0.023); or age, sex, and tumor localization (p = 0.029). Further analysis showed that the significant association between high HSP70 levels and poor survival is in the strongest in the group of <70-year-old female patients (HR: 5.52 (2.02-15.15), p = 0.001), as well as in those who were in a less advanced stage of the disease at baseline. These novel findings indicate that the serum level of soluble HSP70 might prove a useful, stage-independent prognostic marker in colorectal cancer without distant metastasis.
PMCID: PMC2866989  PMID: 19578980
Colorectal cancer; Mortality; Heat shock proteins; HSP70; Predictive marker
9.  Association of Smoking Behavior with an Odorant Receptor Allele Telomeric to the Human Major Histocompatibility Complex 
Genetic testing  2008;12(4):481-486.
Smoking behavior has been associated in two independent European cohorts with the most common Caucasian human leukocyte antigen (HLA) haplotype (A1-B8-DR3). We aimed to test whether polymorphic members of the two odorant receptor (OR) clusters within the extended HLA complex might be responsible for the observed association, by genotyping a cohort of Hungarian women in which the mentioned association had been found. One hundred and eighty HLA haplotypes from Centre d’Etude du Polymorphisme Humain families were analyzed in silico to identify single-nucleotide polymorphisms (SNPs) within OR genes that are in linkage disequilibrium with the A1-B8-DR3 haplotype, as well as with two other haplotypes indirectly linked to smoking behavior. A nonsynonymous SNP within the OR12D3 gene (rs3749971T) was found to be linked to the A1-B8-DR3 haplotype. This polymorphism leads to a 97Thr → Ile exchange that affects a putative ligand binding region of the OR12D3 protein. Smoking was found to be associated in the Hungarian cohort with the rs3749971T allele (p = 1.05×10−2), with higher significance than with A1-B8-DR3 (p = 2.38×10−2). Our results link smoking to a distinct OR allele, and demonstrate that the rs3749971T polymorphism is associated with the HLA haplotype-dependent differential recognition of cigarette smoke components, at least among Caucasian women.
PMCID: PMC2635552  PMID: 18939942
10.  Antibodies against C-Reactive Protein Cross-React with 60-Kilodalton Heat Shock Proteins▿  
Clinical and Vaccine Immunology  2007;14(4):335-341.
C-reactive protein (CRP) is an acute-phase reactant frequently used in histochemistry as a marker of ongoing inflammation. Furthermore, CRP is a powerful biomarker for the prediction of coronary artery disease risk. Heat-shock protein 60 (Hsp60) and CRP are complement-activating molecules, and the effect of their interactions on the regulation of complement activation was studied. However, during the first experiments, we learned that polyclonal anti-CRP antibodies cross-react with Hsp60. Therefore, the aim of our present study was to analyze the cross-reactivity of anti-CRP antibodies (Ab) with Hsp60 in solid-phase enzyme immune assays, in epitope studies using a series of overlapping synthetic peptides, and in Ouchterlony analyses. We found that three different commercial rabbit polyclonal antibodies and two monoclonal (9C9 and CRP-8) anti-CRP antibodies specifically recognize recombinant human Hsp60 and recombinant Mycobacterium tuberculosis Hsp65, respectively. Hsp60 was found to inhibit the binding of anti-CRP polyclonal Ab to Hsp60. Six epitope regions of Hsp60 were recognized by the anti-CRP antibodies, and one region (amino acids [AA] 218 to 232) was recognized by monoclonal antibodies CRP-8 and 9C9. This epitope region of Hsp60 displays 26.6% amino acid identity to CRP AA region 77 to 90. These data suggest that the B-cell epitopes shared between CRP and Hsp60 give rise to a true mimicry-based cross-reaction and the induction of cross-reactive antibodies. Our study underlines the importance of thorough study design and careful interpretation of results while using polyclonal anti-CRP antibodies for histochemistry, especially at low dilutions. Furthermore, analytical interference with Hsp60 in CRP assays should also be tested.
PMCID: PMC1865608  PMID: 17301219
11.  Human serum fetuin A/α2HS-glycoprotein level is associated with long-term survival in patients with alcoholic liver cirrhosis, comparison with the Child-Pugh and MELD scores 
BMC Gastroenterology  2007;7:15.
Serum concentration of fetuin A/α2HS-glycoprotein (AHSG) is a good indicator of liver cell function and 1-month mortality in patients with alcoholic liver cirrhosis and liver cancer. We intended to determine whether decreased serum AHSG levels are associated with long-term mortality and whether the follow-up of serum AHSG levels can add to the predictive value of the Child-Pugh (CP) and MELD scores.
We determined serum AHSG concentrations in 89 patients by radial immunodiffusion. Samples were taken at the time of enrolment and in the 1st, 3rd, 6th, and the 12th month thereafter.
Forty-one patients died during the 1-year follow-up period, 37 of them had liver failure. Data of these patients were analysed further. Deceased patients had lower baseline AHSG levels than the 52 patients who survived (293 ± 77 vs. 490 ± 106 μg/ml, mean ± SD, p < 0.001). Of all laboratory parameters serum AHSG level, CP and MELD scores showed the greatest difference between deceased and survived patients. The cutoff AHSG level 365 μg/ml could differentiate between deceased and survived patients (AUC: 0.937 ± 0.025, p < 0.001, sensitivity: 0.865, specificity: 0.942) better than the MELD score of 20 (AUC: 0.739 ± 0.052, p < 0.001, sensitivity: 0.595, specificity: 0.729). Initial AHSG concentrations < 365 μg/ml were associated with high mortality rate (91.4%, relative risk: 9.874, 95% C.I.: 4.258–22.898, p < 0.001) compared to those with ≥ 365 μg/ml (9.3%). Fourteen out of these 37 fatalities occurred during the first month of observation. During months 1–12 low AHSG concentration proved to be a strong indicator of mortality (relative risk: 9.257, 95% C.I.: 3.945–21.724, p < 0.001). Multiple logistic regression analysis indicated that decrease of serum AHSG concentration was independent of all variables that differed between survived and deceased patients during univariate analysis. Multivariate analysis showed that correlation of low serum AHSG levels with mortality was stronger than that with CP and MELD scores. Patients with AHSG < 365 μg/ml had significantly shortened survival both in groups with MELD < 20 and MELD ≥ 20 (p < 0.0001 and p = 0.0014, respectively).
Serum AHSG concentration is a reliable and sensitive indicator of 1-year mortality in patients with alcoholic liver cirrhosis that compares well to the predictive value of CP score and may further improve that of MELD score.
PMCID: PMC1852564  PMID: 17394649
12.  Heat shock protein 70 is a potent activator of the human complement system 
Cell Stress & Chaperones  2002;7(1):17-22.
According to new hypotheses, extracellular heat shock proteins (Hsps) may represent an ancestral danger signal of cellular death or lysis-activating innate immunity. Recent studies demonstrating a dual role for Hsp70 as both a chaperone and cytokine, inducing potent proinflammatory response in human monocytes, provided support for the hypothesis that extracellular Hsp is a messenger of stress. Our previous work focused on the complement-activating ability of human Hsp60. We demonstrated that Hsp60 complexed with specific antibodies induces a strong classical pathway (CP) activation. Here, we show that another chaperone molecule also possesses complement-activating ability. Solid-phase enzyme-linked immunosorbent assay was applied for the experiments. Human Hsp70 activated the CP independently of antibodies. No complement activation was found in the case of human Hsp90. Our data further support the hypothesis that chaperones may messenger stress to other cells. Complement-like molecules and primitive immune cells appeared together early in evolution. A joint action of these arms of innate immunity in response to free chaperones, the most abundant cellular proteins displaying a stress signal, may further strengthen the effectiveness of immune reactions.
PMCID: PMC514798  PMID: 11892984

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