Enhanced Allergic Inflammation of Der p 2 Affected by Polymorphisms of MD-2 Promoter

Purpose

Myeloid differentiation-2 (MD-2) has been associated with endotoxin and inflammatory disorders because it can recognize lipopolysaccharide (LPS) binding and attenuate Toll-like receptor 4 (TLR4)-mediated signaling. However, its role in allergic inflammation has yet to be clarified. We examined whether single nucleotide polymorphisms (SNPs) in MD-2 promoter can affect MD-2 expression and aimed to clarify the relationship between Der p 2 allergy and SNPs of MD-2 promoter.

Methods

The function of SNPs of MD-2 promoter and the effects of cytokines and immunoglobulin on the secretion and mRNA expression were investigated in 73 allergic subjects with different MD-2 gene promoter variants. Peripheral blood mononuclear cells were cultured with or without LPS in the presence of Dermatophagoides pteronyssinus group 2 allergen (Der p 2), followed by mRNA extraction and cytokine expression analysis. The culture supernatants were collected for cytokine measurement.

Results

Patients with the MD-2 promoter SNPs (rs1809441/rs1809442) had increased mRNA expressions of MD-2, ε heavy chain of IgE (Cε), and interleukin (IL)-8; however, only MD-2 and IL-8 were further up-regulated after Der p 2 stimulation. Patients with SNPs of MD-2 promoter tended to have high levels of IL-1β, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α after Der p 2 and LPS stimulation. Increased secretions of IL-6, IL-8, and IL-10 were found to be up-regulated by Der p 2 stimulation, and an increased secretion of IFN-γ and decreased secretion of IL-4 were noted after LPS stimulation.

Conclusions

The high levels of proinflammatory cytokines secreted by Der p 2 were predetermined by MD-2 promoter SNPs (rs1809441/rs1809442). Through cytokine secretion by Der p 2 and LPS, these SNPs may serve as an indicator of the pathological phenotype of Der p 2-induced allergic inflammation.     

Effect of Amino Acid Polymorphisms of House Dust Mite Der p 2 Variants on Allergic Sensitization

Purpose

The sequence variations of the Der p 2 allergen of Dermatophagoides pteronyssinus diverge along 2 pathways with particular amino acid substitutions at positions 40,47,111, and 114. The environmental prevalence and IgE binding to Der p 2 variants differ among regions. To compare IgE binding to Der p 2 variants between sera from Bangkok, Thailand and Perth, Western Australia with different variants and to determine the variant-specificity of antibodies induced by vaccination with recombinant variants.

Methods

The structures of recombinant variants produced in yeast were compared by circular dichroism and 1-anilinonaphthalene 8-sulfonic acid staining of their lipid-binding cavity. Sera from subjects in Bangkok and Perth where different variants are found were compared by the affinity (IC₅₀) of IgE cross-reactivity to different variants and by direct IgE binding. Mice were immunized with the variants Der p 2.0101 and Der p 2.0110, and their IgG binding to Der p 2.0103, 2.0104, and 2.0109 was measured.

Results

The secondary structures of the recombinant variants resembled the natural allergen but with differences in ANS binding. The IC₅₀ of Der p 2.0101 required 7-fold higher concentrations to inhibit IgE binding to the high-IgE-binding Der p 2.0104 than for homologous inhibition in sera from Bangkok where it is absent, while in sera from Perth that have both variants the IC₅₀ was the same and low. Reciprocal results were obtained for Der p 2.0110 not found in Perth. Direct binding revealed that Der p 2.0104 was best for detecting IgE in both regions, followed by Der p 2.0101 with binding to other variants showing larger differences. Mouse anti-Der p 2.0101 antibodies had a high affinity of cross-reactivity but bound poorly to other variants.

Conclusions

The affinity of IgE antibody cross-reactivity, the direct IgE binding, and the specificities of antibodies induced by vaccination show that measures of allergic sensitization and therapeutic strategies could be optimized with knowledge of Der p 2 variants.     

The association of SERPINE2 gene with COPD in a Chinese Han population

Purpose

Polymorphisms of several candidate genes have been studied and associated with the development of chronic obstructive pulmonary disease (COPD). One such candidate is the SERPINE2 (Serpin peptidase inhibitor, clade E member 2) gene.

Materials and Methods

To assess whether the SERPINE2 gene is associated with COPD in a Chinese Han population. Samples were collected from a Chinese Han population and analyzed for the association of single nucleotide polymor phisms (SNPs) or haplotypes of SERPINE2 gene with COPD in a case-control study. Three SNPs including rs840088 G/A in intron 1, rs1438831 A/G in 5'' upstream sequence and rs3795879 G/A in intron 3 were detected using the polymerase chain reaction (PCR)-based restriction fragment length polymorphism technique in 409 COPD subjects and 411 controls. Genotyping of the SREPINE2 polymorphisms at positions rs840088, rs1438831and rs3795879 was performed.

Results

We found that none of the rs840088G/A, rs1438831G/A and rs3795879 G/A polymorphisms were associated with the disease. The p-values were 0.630, 0.208 and 0.398 respectively.

Conclusion

Our data suggested that there was no significant association between SERPINE2 polymorphism and COPD susceptibility in the Chinese Han population.     

Production of Egg Yolk Antibodies Specific to House Dust Mite Proteins

Purpose

House dust mites (HDMs) are an important source of indoor allergens associated with asthma, rhinitis and atopic dermatitis. Chicken immunoglobulin (Ig) Y is known to be a good alternative to mice and rabbit antibody production. In this study, we produced IgYs specific to HDMs and investigated their IgE immunoreactivities.

Materials and Methods

Total IgYs were isolated from the yolks of White Leghorn hens immunized with either Dermatophagoides pteronyssinus or D. farinae protein extract. Control antibodies were separated from the yolks of immunized hens with phosphate buffered saline. IgYs specific to HDMs were analyzed using enzyme-linked immunosorbent assay and Western blotting analysis.

Results

The concentration of egg IgY specific to D. farinae in an immunized hen increased and the highest achieved was 661.3 ug/mg (per an egg) on day 47, compared with 760 ug/mg IgY specific to D. pteronyssinus on day 16. The D. pteronyssinus or D. farinae-specific IgY was detected by binding of each mite proteins, and their immunoreactivities were elevated dependent of the specific IgY concentration.

Conclusion

IgY specific to HDMs may be a promising antibody for immunological diagnosis as well as identification of possible resistance relating to HDM allergy.     

Profiles of IgE Sensitization to Der f 1, Der f 2, Der f 6, Der f 8, Der f 10, and Der f 20 in Korean House Dust Mite Allergy Patients

Purpose

Measurement of IgE specific to purified house dust mite (HDM) allergens may improve allergy diagnosis. This study aimed to investigate the sensitization profiles of Korean HDM allergic subjects suffering from respiratory allergy and atopic dermatitis (AD) to Der f 1, Der f 2, Der f 6, Der f 8, Der f 10, and Der f 20.

Methods

Recombinant HDM allergens were produced in Pichia pastoris (Der f 1) or Escherichia coli (5 allergens). IgE reactivity to the individual recombinant allergens and total extract of mite was assessed by ELISA.

Results

Der f 1 was recognized by 79.1%, Der f 2 by 79.1%, Der f 6 by 9.3%, Der f 8 by 6.2%, Der f 10 by 6.2%, and Der f 20 by 6.6% of the patients'' sera tested, while the prevalence of IgE reactivity to total mite extract was 94.7%. Combination of Der f 1 and Der f 2 had a sensitivity of 87.6%. Specific IgE to Der f 2 alone was detected from 89.4% of HDM-sensitized respiratory allergy subjects and 92.3% to the combination of the 2 major allergens Der f 1 and Der f 2. However, sera from fewer patients with AD, namely 72.4% and 71.0%, recognized Der f 1 and Der f 2, respectively. The combination of 2 major allergens allowed diagnosis of 84.5% of the AD patients. No correlation between sensitization to specific allergens and HDM allergy entity was found.

Conclusions

Der f 2 was the most frequently sensitized allergen among the HDM-sensitized respiratory and AD patients in Korea, and the combination of the group 1 and 2 major allergens increased the diagnostic sensitivity. Minor allergens did not significantly improve diagnostic sensitivity. However, further studies are needed to analyze the relationship between sensitization to other HDM allergens and the disease entity of the HDM allergy.     

Association between CDH13 Variants and Cardiometabolic and Vascular Phenotypes in a Korean Population

Purpose

Although some CDH13 single nucleotide polymorphisms (SNPs) have been shown to be determinants of blood adiponectin levels, the clinical implications of CDH13 variants are not yet completely understood. The purpose of this study was to evaluate the effects of SNPs of CDH13 on metabolic and vascular phenotypes.

Materials and Methods

We included 238 hypertensive subjects and 260 age- and sex-matched controls. Seven tagging-SNPs were identified in the CDH13 gene by whole gene sequencing. The association between these SNP variants and the risk of hypertension, metabolic traits, and carotid intima-media thickness (IMT) was examined.

Results

Minor allele carriers of rs12444338 had a lower risk of hypertension, but the association turned out just marginal after adjusting confoudners. Blood glucose levels were higher in the minor allele carriers of c.1407C>T (p=0.01), whereas low-density lipoprotein-cholesterol levels were greater in those of rs6565105 (p=0.02). The minor allele of rs1048612 was associated with a higher body mass index (p=0.01). In addition, the mean carotid IMT was significantly associated with rs12444338 (p=0.02) and rs1048612 (p=0.02).

Conclusion

These results provide evidence that CDH13 variants are associated with metabolic traits and carotid atherosclerosis in Koreans. This study shows the multifaceted effects of CDH13 variants on cardiometabolic risk.     

Probable role of Beta 2-adrenergic receptor gene haplotype in toluene diisocyanate-induced asthma

Purpose

A genetic polymorphism of the beta 2-adrenergic receptor is a major factor associated with the asthmatic phenotype. The association of this polymorphism with toluene diisocyanate (TDI)-induced asthma has not been investigated. We examined 103 TDI-induced asthma patients (TDI-OA), 60 asymptomatic exposed controls (AEC), and 263 unexposed healthy controls (NC) in order to identify beta 2-adrenergic receptor gene (ADRB2) polymorphisms and the possible association with TDI-induced asthma.

Methods

Single nucleotide polymorphisms (SNPs) of ADRB2 were genotyped by direct sequencing. Serum-specific IgE and IgG levels were measured using an enzyme-linked immunosorbent assay. Phenotypes and clinical patient parameters were compared.

Results

SNPs were identified (-47 T>C, -20 T>C, Arg16Gly A>G, Gln27Glu C>G, Leu134Leu G>A, Arg175Arg C>A) during ADRB2 screening (from -231 to 793 bp). No significant differences in allelic and genotypic frequencies were noted for any of the six ADRB2 SNPs. The Arg16Gly A>G, Leu134Leu G>A, and Arg175Arg C>A SNPs and haplotype 1 [TTACGC] were significantly associated with specific IgE antibodies to the TDI-human serum albumin (HSA) conjugate in TDI-exposed subjects (P<0.05). Exposed workers with the ADRB2 ht1/ht1 homozygote had a significantly higher TDI-HSA conjugate-specific IgE sensitization rate than did those with the null ht1 haplotype (odds ratio, 15.40; 95% confidence interval, 1.81-131.06).

Conclusions

ADRB2 polymorphisms may affect IgE-specific sensitization to TDI-HSA conjugate in TDI-exposed workers.     

Association Between Serum IgE Levels and the CTLA4 +49A/G and FCER1B -654C/T Polymorphisms in Korean Children With Asthma

Purpose

T cells play a central role in cell-mediated immunity, atopic disease, and asthma. The balance of CD28/cytotoxic T-lymphocyte antigen 4 (CTLA4)-derived signal transduction plays an important role in the activation of T cells and an increased immunoglobulin E (IgE) response. The aim of the current study was to investigate the association between polymorphisms in the genes encoding both CTLA4 and the high-affinity IgE receptor 1B (FCER1B) and serum IgE levels in Korean children with asthma.

Methods

We enrolled 238 controls and 742 children with asthma. The CTLA4 +49A/G and FCER1B -654C/T polymorphisms were genotyped by PCR-restriction fragment length polymorphism analysis.

Results

We observed no difference in the distribution of CTLA4 +49A/G among controls, children with asthma, and those with atopic asthma. In contrast, the GA genotype of CTLA4 +49A/G in children with atopic asthma was significantly higher compared to that in those with non-atopic asthma. Moreover, significantly higher log Dp/Df-specific IgE levels were found in children with asthma and those with atopic asthma carrying one or two copies of the CTLA4 +49A versus those homozygous for +49G. Gene-gene interactions between CTLA4 and FCER1B with the heterozygote and homozygote of variant genotypes were associated with the log Dp/Df-specific IgE levels, but not asthma development. In addition, children with Dp/Df (+) asthma carried an elevated combined genotype of risk allele compared to those with Dp/Df (-) asthma.

Conclusions

The CTLA4 +49A/G polymorphism may contribute to the production of IgE in Korean children with asthma, especially in Dp/Df-specific IgE levels, but not in the direct development of asthma. In addition, Dp/Df-specific IgE levels with a FCER1B -654C/T polymorphism may involve additive effects.     

Molecular mimicry as the key to the dominance of the house dust mite allergen Der p 2

Evaluation of: Trompette A, Divanovic S, Visintin A et al. Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein. Nature 457, 585–588 (2009).

The majority of complex sources of allergen contain a small number of dominant allergens that bind at least half of the IgE antibodies of allergic subjects. For the house dust mite Dermatophagoides pteronyssinus, these allergens are Der p 1 and Der p 2. There is evidence that the cysteine-protease activity of Der p 1 imparts adjuvant activity to the allergen. It has now been shown that Der p 2 mimics the activity of its fellow ML-domain protein, MD-2, by presenting lipopolysaccharide to Toll-like receptor-4 for the activation of inflammatory genes. In accord with this, Der p 2 presented with lipopolysaccharide also induced enhanced type 1 allergic sensitization of mice, even when they were deficient in MD-2. The mimicry of MD-2 can thus also self adjuvant Der p 2 to enhance it allergenicity. This not only describes an intriguing mechanism for enhancing allergenicity but also, since both of the dominant mite allergens have intrinsic adjuvant activity, exemplifies an important principle for driving allergic sensitization.     

Perinatal Immunization With Vaccine-Grade Listeria monocytogenes Provides Protection Against Murine Th2 Airway Inflammation

Purpose

Asthma is a chronic respiratory disorder that leads to inflammation and narrowing of the airways. Its global prevalence has attained epidemic levels and treatment options that reach beyond temporary relief of symptoms are urgently needed. Since the processes leading to clinically symptomatic asthma start early in life, we set out to systematically evaluate a neonatal immunotherapeutic based on Listeria monocytogenes (Lm) for the control of allergic sensitization.

Methods

We modified Lm to express the model allergen, ovalbumin (OVA), and tested the ability of neonatal immunization with this strain to control allergic sensitization in a mouse model of OVA-induced asthma. Mice were immunized as newborns with live or heat killed LmOVA or live Lm, followed 6 weeks later by allergic sensitization with OVA. In order to determine whether the T_H1-polarizing effect of this vaccine vector inadvertently may exacerbate development of certain T_H1-driven allergic diseases, mice immunized as newborns were assessed in a model of adult hypersensitivity pneumonitis (HP).

Results

Both LmOVA and Lm-control vaccines were highly effective in providing long-lasting protection from airway inflammation after only one immunization given perinatally. Serum antibody levels and lung cytokine production suggest that this prophylactic strategy is associated with an allergen specific T_H1-dominated response. Specifically, LmOVA vaccinated mice displayed significantly elevated OVA-specific serum IgG2a, but no difference in anti-OVA IgE antibodies and only slightly decreased anti-OVA IgG1 antibodies. Importantly, Lm-based neonatal vaccination did not exacerbate Th1/Th17 driven HP, arguing against broad spectrum immune skewing.

Conclusions

Our findings highlight the promise of early life Lm-based immunomodulatory interventions as a prophylactic strategy for allergic asthma.     

PTK2 and PTPN11 expression in myelodysplastic syndromes

OBJECTIVE:

The aim of this study was to evaluate the expression of protein tyrosine kinase 2 and protein tyrosine phosphatase non-receptor type 11, which respectively encode focal adhesion kinase protein and src homology 2 domain-containing protein-tyrosine phosphatase 2, in hematopoietic cells from patients with myelodysplastic syndromes.

METHODS:

Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions were analyzed by quantitative polymerase chain reaction in bone marrow cells from patients with myelodysplastic syndromes and healthy donors.

RESULTS:

Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions did not significantly differ between normal cells and myelodysplastic cells.

CONCLUSIONS:

Our data suggest that despite the relevance of focal adhesion kinase and src homology 2 domain-containing protein-tyrosine phosphatase 2 in hematopoietic disorders, their mRNA expression do not significantly differ between total bone marrow cells from patients with myelodysplastic syndromes and healthy donors.     

Novel nonsense mutation of BRCA2 gene in a Moroccan man with familial breast cancer

Background

Breast cancer is the most common cancer in women worldwide. About 5 to 10% of cases are due to an inherited predisposition in two major genes, BRCA1 and BRCA2, transmitted as an autosomal dominant form. Male breast cancer is rare and is mainly due to BRCA2 than BRCA1 germline mutations.

Objective

Molecular study of BRCA2 gene in man with familial breast cancer.

Methods

PCR and direct sequencing of BRCA2 gene.

Results

Identification of novel heterozygous germline mutation c.6428C>A ; p.Ser2143Stop of BRCA2 gene.     

Whole-Genome Analysis in Korean Patients with Autoimmune Myasthenia Gravis

Purpose

The underlying cause of myasthenia gravis (MG) is unknown, although it likely involves a genetic component. However, no common genetic variants have been unequivocally linked to autoimmune MG. We sought to identify the genetic variants associated with an increased or decreased risk of developing MG in samples from a Korean Multicenter MG Cohort.

Materials and Methods

To determine new genetic targets related to autoimmune MG, a whole genome-based single nucleotide polymorphisms (SNP) analysis was conducted using an Axiom™ Genome-Wide ASI 1 Array, comprising 598375 SNPs and samples from 109 MG patients and 150 neurologically normal controls.

Results

In total, 641 SNPs from five case-control associations showed p-values of less than 10^-5. From regional analysis, we selected seven candidate genes (RYR3, CACNA1S, SLAMF1, SOX5, FHOD3, GABRB1, and SACS) for further analysis.

Conclusion

The present study suggests that a few genetic polymorphisms, such as in RYR3, CACNA1S, and SLAMF1, might be related to autoimmune MG. Our findings also encourage further studies, particularly confirmatory studies with larger samples, to validate and analyze the association between these SNPs and autoimmune MG.     

Association Analysis Between FILIP1 Polymorphisms and Aspirin Hypersensitivity in Korean Asthmatics

Purpose

Aspirin exacerbated respiratory disease (AERD) results in a severe asthma attack after aspirin ingestion in asthmatics. The filamin A interacting protein 1 (FILIP1) may play a crucial role in AERD pathogenesis by mediating T cell activation and membrane rearrangement. We investigated the association of FILIP1 variations with AERD and the fall rate of forced expiratory volume in one second (FEV1).

Methods

A total of 34 common FILIP1 single nucleotide polymorphisms (SNPs) were genotyped in 592 Korean asthmatic subjects that included 163 AERD patients and 429 aspirin-tolerant asthma (ATA) controls.

Results

This study found that 5 SNPs (P=0.006-0.01) and 2 haplotypes (P=0.01-0.03) of FILIP1 showed nominal signals; however, corrections for the multiple testing revealed no significant associations with the development of AERD (P_corr>0.05). In addition, association analysis of the genetic variants with the fall rate of FEV1, an important diagnostic marker of AERD, revealed no significant evidence (P_corr>0.05).

Conclusions

Although further replications and functional evaluations are needed, our preliminary findings suggest that genetic variants of FILIP1 might be not associated with the onset of AERD.     

The prevalence of aminoglycoside-modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in Pseudomonas aeruginosa

INTRODUCTION:

Pseudomonas aeruginosa (P. aeruginosa) is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an important component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance.

OBJECTIVES:

The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investigate the occurrence of aminoglycoside resistance and the prevalence of four important modifying enzyme genes (aac (6′)-I, aac (6′)-II, ant (2″)-I, aph (3′)-VI) in P. aeruginosa in Iran.

METHODS:

A total of 250 clinical isolates of P. aeruginosa were collected from several hospitals in seven cities in Iran. Antimicrobial susceptibility tests (using the disk diffusion method and E-tests) were performed for all 250 isolates. In addition, all isolates were screened for the presence of modifying enzyme genes by polymerase chain reaction.

RESULTS:

The resistance rates, as determined by the disk diffusion method, were as follows: gentamicin 43%, tobramycin 38%, and amikacin 24%. Of the genes examined, aac (6′)-II (36%) was the most frequently identified gene in phenotypic resistant isolates, followed by ant (2″)-I, aph (3′)-VI, and aac (6′)-I.

CONCLUSIONS:

Aminoglycoside resistance in P. aeruginosa remains a significant problem in Iran. Therefore, there is considerable local surveillance of aminoglycoside resistance.     

Evaluation of the Essential Oil of Foeniculum Vulgare Mill (Fennel) Fruits Extracted by Three Different Extraction Methods by GC/MS

Background

Hydrodistillation (HD) and steam-distillation, or solvent extraction methods of essential oils have some disadvantages like thermal decomposition of extracts, its contamination with solvent or solvent residues and the pollution of residual vegetal material with solvent which can be also an environmental problem. Thus, new green techniques, such as supercritical fluid extraction and microwave assisted techniques, are potential solutions to overcome these disadvantages.

Materials and Methods

The aim of this study was to evaluate the essential oil of Foeniculum vulgare subsp. Piperitum fruits extracted by three different extraction methods viz. Supercritical fluid extraction (SFE) using CO₂, microwave-assisted extraction (MAE) and hydro-distillation (HD) using gas chromatography-mass spectrometry (GC/MS).

Results

The results revealed that both MAE and SFE enhanced the extraction efficiency of the interested components. MAE gave the highest yield of oil as well as higher percentage of Fenchone (28%), whereas SFE gave the highest percentage of anethol (72%).

Conclusion

Microwave-assisted extraction (MAE) and supercritical fluid extraction (SFE) not only enhanced the essential oil extraction but also saved time, reduced the solvents use and produced, ecologically, green technologies.     

Receptor Interacting Protein 2 (RIP2) Is Dispensable for OVA-Induced Airway Inflammation in Mice

Purpose

Asthma is a pulmonary chronic inflammatory disease characterized by airway obstruction and hyperresponsiveness. Pattern recognition receptors are known to play a key role in the development of allergic diseases as well as host defenses against microbial infection. Receptor interacting protein 2 (RIP2), a serine/threonine kinase, is an adaptor molecule of NOD1 and NOD2, and genetic variation in this receptor is known to be associated with the severity of allergic asthma in children. In this study, we examined the role of RIP2 in the development of allergic airway inflammation in a mouse model.

Methods

Airway inflammation was induced in mice through intranasal administration of ovalbumin (OVA) after 2 intraperitoneal immunizations with OVA. Lung inflammation and mucus hypersecretion were examined histologically and total cell infiltration in bronchoalveolar (BAL) fluids was determined. Levels of the Th2-related cytokines, IL-5 and IL-13, in lung extracts were measured by ELISA. Serum antigen-specific IgE and IgG1 levels were also assessed.

Results

OVA-induced lung inflammation and mucus hypersecretion were not different between WT and RIP2-deficient mice. The IL-5 and IL-13 levels in the bronchoalveolar (BAL) fluids were also not impaired in RIP2-deficient mice compared to WT mice. Moreover, RIP2 deficiency did not affect serum OVA-specific IgG1 and IgE levels.

Conclusions

Our results suggest that RIP2 is not associated with the development of allergic airway inflammation.     

Hypermethylation of p16 and DAPK promoter gene regions in patients with non-invasive urinary bladder cancer

Introduction

The aim of the study was to examine the frequency of methylation status in promoter regions of p16 and DAPK genes in patients with non-invasive bladder cancer.

Material and methods

Forty-two patients (92.9% men, 73.8% smokers, 71.4% T1G1, 19.1% T1G2, 9.5% T1G3) and 36 healthy controls were studied. Isolation of genomic DNA from blood serum and methylation-specific PCR (MSP) were applied. Methylation status – methylated and unmethylated promoter regions of p16 and DAPK genes were analysed.

Results

Seventeen out of 42 patients (40.5%) had the methylated p16 gene, while methylation of the DAPK gene was seen in 27 of 42 cases (64.3%). In 12 patients (28.6%) both analysed genes were methylated. A statistically significant (p = 0.046) higher frequency of DAPK gene methylation (71.4%) was observed in patients with lower grade (G1) bladder cancer.

Conclusions

Detection of the aberrant hypermethylation of DAPK and p16 genes in blood DNA from non-invasive bladder cancer patients might offer an effective means for earlier auxiliary diagnosis of the malignancy.