HLA profile of the donors in the Macedonian Bone Marrow Donor Registry.

The importance of HLA alleles in the process of haematopoietic stem cell transplantation, especially the process of unrelated donor search, is enormous. Macedonian Bone Marrow Donor Registry was established in 2010 and has registered volunteer donors from different nationalities that live in the Republic of Macedonia. The aim of this study was to determine the HLA allele and haplotype frequencies of the volunteer donors from the Macedonian Bone Marrow Donor Registry and to compare this results with the Macedonians from a family study. We analyzed 1,541 donors, with different nationalities, Macedonian, Albanian and Macedonian Muslims that were most numerous in MBMDR, and typed them for HLA-A, -B, -C, -DRB1, whereas Macedonian also for HLA-DQA1 and HLA-DQB1 by SSO method (One Lambda, CA, USA). The most frequent alleles in Macedonians were HLA-A*02, 01, 24; HLA-B*35, 18, 51; HLA-C*07, 04, 12; HLA-DRB1*11, 16, 13; HLA-DQA1*01, 05 and HLA-DQB1*05, 03, 06; in Albanians they were HLA-A*02, 24,01; HLA-B*51, 18, 35; HLA-C*07, 04, 12, HLA-DRB1*11, 13,16; and in Macedonian Muslims they were HLA-A*02, 01, 24; HLA-B*18, 51, 35, HLA-C*07, 04, 02 and HLA-DRB1*11, 16, 14. The most common haplotype in Macedonian was HLA-A*01-B*08-C*07-DRB1*03, whilst in Albanian and Macedonian Muslims HLA-A*02-B*18-C*07-DRB1*11. The comparison of the HLA allele groups between Macedonian from MBMDR and family study showed similar distribution. This study confirmed the close relationship between the populations that live in the Balkan Peninsula.

Latent Tuberculosis Infection - Diagnosis and Treatment.

INTRODUCTION: Latent tuberculosis infection (LTBI) is defined as a state of persistent immune response to stimulation by Mycobacterium tuberculosis antigens without evidence of clinically manifested active tuberculosis (TB). Diagnosis and treatment for LTBI are important for TB, especially in high-risk populations. Tuberculin skin test (TST) and interferon-gamma release assays (IGRAs) are used to diagnose LTBI. AIM: The study aims to present the first results with IGRA test compared with TST in the screening of LTBI and the treatment results in the cases with LTBI in Macedonia. MATERIAL AND METHODS: In this study 73 cases diagnosed and treated with LTBI in 2016 were included. For diagnosis of LTBI, we used TST RT -23 5T.U. and commercial IGRA test such as QuantiFERON-TB Gold In-Tube (QFT-IT). RESULTS: Out of 73 cases with LTBI, 61.64% were men, and 38.36% were women. Among all age groups, the most frequent were cases between 5 and 14 years old (54.79%). Among the evaluated risk groups for LTBI, the most frequent were children household contacts with pulmonary TB cases (61-83.65%), followed by people living with HIV (9-12.33%) and only 3 cases with other medical reasons. Positive TST had 34 cases (46.57%) and positive IGRA test 25 cases (34.25%). Regarding the treatment regimes, we use two regimes: 50 cases (68.44%) received 6 months daily regime with Isoniazid, and 23 cases (31.51%) received 3 months daily regime with Isoniazid and Rifampicin. Treatment outcomes showed that the most patients completed treatment regimes: 55 (75.34%) and only 10 (13.09%) interrupted the treatment. CONCLUSION: Despite the progress made in the last few years, several challenges remain to be addressed for better management of LTBI which will contribute to strength TB control in the country.

Relationship between Vitamin D, Inflammation and Lung Function In Patients with Severe Uncontrolled Asthma.

BACKGROUND: Recently epidemiological studies showed that low vitamin D is linked to airway hyperresponsiveness, decreased lung function, poor asthma control, and steroid-resistant asthma. AIM: We investigated the relationship between Vitamin D, inflammation with circulating IL-33 and lung function in 30 patients with severe uncontrolled asthma. MATERIALS AND METHODS: The study included 30 patients with severe uncontrolled asthma. In each of them were measured serum levels of IL-33 and Vitamin D by the ELISA method. The pulmonary function is measured by basic spirometry parameters, FEV1. The results were statistically elaborated according to the Pearson's Correlation Tests. RESULTS: The results showed statistically insignificant correlation between Vitamin D and IL-33, and Vitamin D with FEV1 (Vit.D/IL-33; r = 0.11323, p = 0.551); (Vit.D/FEV1; r = -0.1005; p = 0.597) Correlation between IL-33 and FEV1 is negative but statistically significant (IL-33/FEV1; r = -0.5248; p = 0.003). CONCLUSION: Because there are little studies about the link between vitamin D and asthma, further research to clarify the mechanism how vitamin D control the activity of CD4+ T cells and the related Th2-type cytokines in the parthenogenesis of asthma.

Total IgE Distribution in Food Allergy Suspected Patients in Republic of Macedonia (2001-2011).

BACKGROUND: IgE may be considered the hallmark of allergic disorders. It is easily detected in serum and can be measured as total IgE and as allergen-specific IgE. In fact, the serum IgE assay is used to diagnose an allergy. AIM: The aim of this study is to evaluate, investigate and present the distribution of total serum IgE levels, determined with UniCap system, in food-allergy suspected patients in a Republic of Macedonia. MATERIAL AND METHODS: In this study we analyzed retrospectively 8898 consecutive patients that were admitted for allergy testing at the Institute of Immunobiology and Human Genetics during the ten year period between 01.01.2001 and 01.01.2011. Total IgE levels in patient sera were detected with the in vitro system UniCAP100 (Pharmacia, Uppsala, Sweden). RESULTS: When we analyzed the number of patients according to the total IgE groups, we noted that most of the patients have normal levels of total IgE in serum. However, we also discovered a group of patients with elevated levels of total IgE that are greater than 200 kU/L. The average concentration of total serum IgE is higher in women in the age group 6 (6-7 years), followed by a steep decrease in the age group 9 (9-10 years), and after that the average concentrations of total IgE were mostly constant with the exception of a partial increase in the age group 21 (65-69 years). For men, the average serum concentrations of total IgE were highest in the age group of 6 (6-7 years), which was significantly higher than the average concentrations of total IgE in all other age groups. CONCLUSION: The large number of enrolled patients, a particular strength of this study, revealed that average concentrations of total IgE in men are higher than in women and that total IgE did not decrease with age. On the contrary, increased total IgE levels were found in patients aged 65 and 69 of both genders. We continue our work with analyses of the specific IgE antibodies values toward food and the correlation with total IgE values.

Distribution of 22 cytokine gene polymorphisms in Roma from the Republic of Macedonia.

The aim of this study was to analyze 22 cytokine polymorphisms in the Roma population from the Republic of Macedonia. The Roma population consists of 77 healthy unrelated individuals, residents of different geographical regions of the Republic of Macedonia (Skopje, Gostivar, and Kochani). Blood samples were collected after obtaining written consent. DNA was isolated from peripheral blood and 22 polymorphisms were typed: IL1A -889, IL1B -511, IL1B +3962, IL1R pst1 1970, IL1RN mspa11100, IL4RA +1902, IL12 -1188, IFNG utr5644, TGF-ß1 cdn10, TGF-ß1 cdn25, TNF-α -308, TNF-α -238, IL-2 -330, IL-2 +166, IL-4 -1098, IL-4 -590, IL-4 -33, IL-6 -174, IL-6 565, IL-10 -1082, IL-10 -819, and IL-10 -592. Cytokine genotyping was performed by PCR-SSP. The population genetics analysis package, PyPop, was used for analysis of the cytokine data. Fnd was negative and significantly different from 0 for IL-4 -590 (p of F=0.006), IL-10 -1082 (p of F=0.010), IFN utr5644 (p of F=0.024), IL-4 -1098 (p of F=0.026) and TGF-1 cdn25 (p of F=0.001) alleles, as well as for IL-2 haplotypes (p=0.025). Several SNPs (IL-12B -1188, IL-2 -330, IL-4 -1098, IL-4 -590, and IL-10 -1082) were not in HWP (p<0.05). A few SNPs (IL-12B -1188, IL-2 -330, IL-4 -1098, IL-4 -590, and IL-10 -1082) and several observed frequencies of cytokine diplotypes (IL-2/GG:TG, IL-2/TG:TG, IL-4/GCC:GCC, IL-4/TTC:TTC, IL-4/TTT:TTC, IL-10/GCC:GCC, IL-10/ATA:GCC, IL-10/ACC:GCC, and IL-10/ACC:ATA) were not in HWP and were significantly different from the expectations. Hardy Weinberg proportion could not be calculated for TNF genotypes and diplotypes because nearly all genotypes and diplotypes belong to GG genotype or GG:GG diplotype. The results of cytokine polymorphisms in Roma population can be used for characterization of the current genetic profile of the Gypsies, anthropological comparisons, as well as for the association studies with different diseases.

IL10 -1082, IL10 -819 and IL10 -592 polymorphisms are associated with chronic periodontitis in a Macedonian population.

Genetic polymorphisms in the interleukin 10 (IL10) gene have been reported to influence the host response to microbial challenge by altering levels of cytokine expression. We analyzed nucleotide polymorphisms in the promoter region of the IL10 gene and its relation with periodontal disease in a Macedonian population. The study population consisted of 111 unrelated subjects with chronic periodontitis and 299 healthy controls. DNA was isolated and IL10 genotyping performed by PCR-SSP (Heidelberg kit) for the alleles and genotypes of IL10 -1082, IL10 -819 and IL10 -592. Frequencies of IL10 haplotypes and the haplotype zygotes were also examined. Comparisons between groups were tested using the Pearson's p-value. After Bonferroni adjustment, significant associations were detected between subjects with chronic periodontitis and IL10 genotypes (IL10 -1082/A:G was negative or protective and IL10 -1082/G:G was positive or susceptible). Cytokine polymorphism on the IL10 gene appears to be associated with susceptibility to chronic periodontitis in Macedonians.

Gene polymorphisms of 22 cytokines in Macedonian children with atopic dermatitis.

Atopic dermatitis (AD) is a common chronically relapsing skin disease associated with abnormal cytokine production, and activation of T-helper 2 cells. The aim if this study was to determine whether cytokine gene polymorphisms might influence the development of AD. Single nucleotide polymorphisms in the genes for I-L1alpha, IL-1beta, IL-1R, IL-2, IL-4, IL-6, IL-10, IL-12, TGF beta, TNF and IFNgamma were investigated by PCR and sequence specific primers in Macedonian patients with AD (67 children, age of 6 months to 5 years) and 301 normal unrelated individuals. Susceptible cytokine polymorphisms for AD for eleven genotypes (IL-4 -33/T:T IL-4 -1098/G:G, TGFbeta cdn25C:G, IL-4 -1098/T:T, IL-1alpha -889/C:T, IL-2 +166/T:T, IL-1beta -511/C:T, IL-12 -1188/C:T, IL-10 -1082/A:G, IL-1beta +3962/C:T, IFNgamma +874/A:T), five diplotypes, six haplotypes, and for alleles were found. Protective cytokine polymorphisms for AD for seven cytokine genotypes (IL-4 -1098/G:T, TGFbeta cdn25/G:G, IL-4 -33/C:C, IL-1alpha -889/C:C, IFNgamma +874/A:A, IL-10 -1082/A:A, IL-1beta -511/C:C), one cytokine diplotypes, two cytokine haplotypes, and four cytokine alleles were also found. We concluded that several cytokine polymorphisms are protective, or susceptible associated with AD in population of Macedonians.

IL4 gene polymorphisms and their relation to periodontal disease in a Macedonian population.

Genetic polymorphisms in the interleukin-4 (IL4) gene have been reported to influence the host response to microbial challenge by altering levels of cytokine expression. We analyzed nucleotide polymorphisms in the promoter region of the IL4 gene and its relation with periodontal disease in a Macedonian population. The study population consisted of 92 unrelated subjects with chronic periodontitis and 286 healthy controls. DNA was isolated and IL4 genotyping performed by polymerase chain reaction-single-strand polymorphism (Heidelberg kit) for the alleles and genotypes of IL4 -1098, IL4 -590, and IL4 -33. Frequencies of IL4 haplotypes and the haplotype zygotes were also examined. Comparisons between groups were tested using the Pearson's p value. After Bonferroni adjustment, significant associations were detected between subjects with periodontitis and the following: (1) cytokine alleles IL4 -1098 and IL4 -33; (2) cytokine genotypes IL4 -1098/G:T; IL4 -1098/T:T, and IL4 -33/T:T, (3) cytokine haplotypes IL4/GCC, IL4/TCC, and IL4/TTC; and (4) cytokine haplotype zygotes IL4/TTC: TCC, IL4/TCT:TTT, and IL4/GCC:TTC. Cytokine polymorphism on the IL4 gene appears to be associated with susceptibility to chronic periodontitis in Macedonians.

Distribution of killer cell immunoglobulinlike receptors in the Macedonian population.

The aim of this study was to analyze killer immunoglobulinlike receptor (KIR) gene polymorphism in the Macedonian population. The study sample consists of 214 healthy unrelated individuals, aged 20-35 years. All individuals are of Macedonian origin and nationality, and residents of different geographic regions. The population genetics analysis package, Arlequin, was used for analysis of the data. We found that all 16 KIR genes were observed in the Macedonian population and framework genes KIR3DL3, KIR2DL4, and KIR3DL2 were present in all individuals. A total of 56 different KIR genotypes were found in the Macedonian population, based on the presence of 16 KIR genes. Neighbor-joining phylogenetic tree, constructed on the basis of standard genetic distances of KIR genes, shows that Macedonian population is in the same cluster with England West Midlands Indian Asian, Brazil SouthEast Caucasian, Romania Caucasians, Spain Basque, England West Midlands Caucasian, France Reunion, and Spain Granada populations. The frequency of KIR loci in Macedonian population shares several general features with other Caucasoid populations studied before.

Family analysis of immunoglobulin classes and subclasses in children with autistic disorder.

Autistic disorder is a severe neurodevelopment disorder characterized by a triad of impairments in reciprocal social interaction, verbal and nonverbal communication, and a pattern of repetitive stereotyped activities, behaviours and interests. There are strong lines of evidence to suggest that the immune system plays an important role in the pathogenesis of autistic disorder. The aim of this study was to analyze quantitative plasma concentration of immunoglobulin classes, and subclasses in autistic patients and their families. The investigation was performed retrospectively in 50 persons with autistic disorder in the Republic of Macedonia. Infantile autistic disorder was diagnosed by DSM-IV and ICD-10 criteria. Plasma immunoglobulin classes (IgM, IgA, and IgG) and subclasses (IgG1, IgG2, IgG3, and IgG4) were determined using Nephelometer Analyzer BN-100. Multiple comparisons for the IgA variable have shown statistically significant differences between three pairs: male autistic from the fathers (p = 0,001), female autistic from the mothers (p = 0,008), as well as healthy sisters from the fathers (p = 0,011). Statistically significant differences found between three groups regarding autistic disorder (person with autistic disorder, father/mother of a person with autistic disorder, and brother/sister) independent of sex belongs to IgA, IgG2, and IgG3 variables. Multiple comparisons for the IgA variable have shown statistically significant differences between children with autistic disorder from the fathers and mothers (p < 0,001), and healthy brothers and sisters from the fathers and mothers (p < 0,001). Comparison between healthy children and children with autistic disorder from the same family should be tested for immunoglobulin classes and subclasses in order to avoid differences between generations.

Investigation of SERPINE1 genetic polymorphism in Macedonian patients with occlusive artery disease and deep vein thrombosis.

BACKGROUND: Raised SERPINE1 plasma levels are related to a 1-bp guanine deletion/insertion (4G5G) polymorphism in the promoter of the SERPINE1 (plasminogen activator inhibitor 1 - PAI1) gene. Evidence suggested that the plasma levels of SERPINE1 modulate the risk of coronary artery disease; furthermore, that the 4G5G polymorphism affects the expression of the SERPINE1 gene. AIM: To analyse association of SERPINE1 polymorphism with occlusive artery disease (OAD) and deep venous thrombosis (DVT) in Macedonians in order to investigate its role as a part of candidate genes in different vascular diseases in Macedonians. METHODS: Investigated groups consisted of 82 healthy patients, 75 with OAD, and 66 with DVT. Blood samples were collected after written informed consent was obtained, and DNA was isolated from peripheral blood leukocytes. Identification of SERPINE1 polymorphism was done with CVD StripAssay (ViennaLab, Labordiagnostica GmbH, Austria). The population genetics analysis package, PyPop, was used for analysis of the SERPINE1 data. Pearson's P-values, crude odds ratio and Wald's 95% CI were calculated with Bonferroni corrected p value. RESULTS: The frequency of 4G allele for SERPINE1 was 0.538 for DVT, 0.555 for healthy participants, and 0.607 for OAD. The frequency of 5G allele for SERPINE1 was the smallest in patients with OAD (0.393) and was higher in healthy participants (0.445), and patients with DVT (0.462). Test of neutrality (Fnd) showed negative value, but was significantly different from 0 for SERPINE1 in healthy participants (p of F = 0.041) and in patients with DVT (p of F = 0.030). SERPINE1 genotypes in healthy participants and patients with OAD were not in Hardy Weinberg proportions (p = 0.019 and 0.001, respectively). No association between SERPINE1 polymorphisms and OAD or DVT was found. CONCLUSION: There is no significant relationship between SERPINE1 polymorphisms and occlusive artery disease or deep venous thrombosis in Macedonian population.

Association between 22 cytokine gene polymorphisms and dilated cardiomyopathy in Macedonian patients.

BACKGROUND: Inflammation is an important component in the pathogenesis of many cardiovascular diseases and one of the commonest mechanisms in cardiomyopathy. There have been several studies on the cytokine polymorphism and dilated cardiomyopathy (DCM), but the results obtained were contradictory. AIM: To examine a possible role of 22 cytokine gene polymorphisms in host susceptibility to or protection against DCM in Macedonians. METHODS: In this study 301 healthy unrelated individuals and 52 patients with DCM were studied. Cytokine genotyping was performed by PCR with sequence-specific priming (PCR-SSP) (Heidelberg kit). RESULTS: After the Bonferroni adjustment, the IL-4 -1098/T, IL-4 -1098/T:T, IL-4/TCC, and IL-4/TCC:TTC cytokine genes were positively associated with DCM, while a negative association was identified for IL-4 -1098/G, IL-4 -1098/G:T, IL-1B +3962/C:C, IL-4/GCC, and IL-4/GCC:TTC. CONCLUSIONS: These results suggest that some cytokine gene polymorphisms are significantly associated and affect host susceptibility/resistance to DCM in Macedonians.

Association of 22 cytokine gene polymorphisms with rheumatoid arthritis in population of ethnic Macedonians.

To examine the possible role of 22 cytokine gene polymorphisms in host susceptibility to or protection against RA in Macedonians. In this study, 301 healthy unrelated individuals and 85 patients with RA were studied. Cytokine genotyping was performed by PCR with sequence-specific priming (PCR-SSP) (Heidelberg kit). Results showed susceptible association for four cytokine alleles, six cytokine genotypes, one haplotype, and four combinations of haplotypes, while protective associations were found for four cytokine alleles, three cytokine genotypes, three haplotypes, and only one combination of haplotypes. These results suggest that IL-4 -1098, IL-4 -590, IL-10 -1082, IL-10 -819, IL-2 -330, IL-6 -174, and TNF-alpha -238 cytokine gene polymorphisms might be significantly associated and affect host susceptibility and/or resistance to RA in Macedonians.

Association of cytokine gene polymorphisms with chronic obstructive pulmonary disease in Macedonians.

The aim of this study was to examine the association of 22 cytokine gene polymorphism in Macedonians with chronic obstructive pulmonary disease (COPD). The sample of the population comprised of 301 normal respondents and 62 patients with COPD. Cytokine genotyping was performed by polymerase chain reaction with sequence-specific priming (PCR-SSP). Positive (susceptible) association was found between patient with COPD and IL-1alpha -889/C allele; where as negative (protective) association among was found for the following alleles IL-1beta +3962/C; IL-12B -1188/A; IFNgamma +874/T; IL-2 -330/G; IL-4 -1098/G and IL-4-33/C. We found positive (susceptible) association between patients with COPD and following genotypes: IL4 -33/T:T; IFNgamma +874/A:A; IL-4 -1098/T:T ; IL-1alpha -889/C:C; IL-1beta +3962/C:T; IL-12B -1188/C:C; IL-4Ralpha +1902/G:G; IL-10 -1082/G:G; IL-2 -330/T:T; IL-4 -590/C:C; and IL-1alpha -889/C:T. Negative (protective) association between patients with COPD and following genotypes was found: IFNgamma +874/A:T; IL-4 -33/C:T; IL-4 -1098/G:T; IL-2 -330/G:T; IL-1beta +3962/C:T; IL-4 -590/C:T; IL-10 -1082/A:G; and IL-4 -33/C:C. Positive (susceptible) association between patients with COPD and following haplotypes was found: IL-4/TCT; IL-10/ATC; and IL-2/TG, and negative (protective) association was found between the patients with COPD and haplotypes for: IL-4/TTC; and IL-4/GCC. It could be concluded that several cytokine polymorphisms are positively (susceptible), or negatively (protective) associated with COPD in Macedonians.

Association of 22 cytokine gene polymorphisms with tuberculosis in Macedonians.

OBJECTIVE: To examine the possible role of 22 cytokine gene polymorphisms in host susceptibility to or protection against tuberculosis (TB) in Macedonians. METHOD: 301 healthy unrelated individuals and 75 patients with pulmonary TB were studied. Cytokine genotyping was performed by PCR with sequence-specific priming (PCR-SSP) (Heidelberg kit). RESULTS: TNF-alpha-238/G, IL-1R psti1970/C, IL-1beta + 3962/T:T, IL-4-1098/T:T, IFNgamma utr5644/A:A, IL-10-1082/G:G, IL-4-590/C:C, IL-10/ATC, IL-4/TCT, IL-4/TCC, IL-10/ATC:GCC, IL-4/TCT:TTT, IL-4/TCC:TTC, IL-10/GCC:GCC and IL-4/TCC:TCC were positively associated with TB, while protective association was identified for IL-4-098/G, IL-1beta + 3962/C, IFNgamma utr5644/T, IL-1beta + 3962/C:T, IL-4-1098/G:T, IL-4-590/C:T, IFNgamma utr5644/A:T, IL-4/GCC, IL-4/TTC and IL-4/GCC:TTC. CONCLUSION: These results suggest that some cytokine polymorphisms are significantly associated and affect host susceptibility/resistance to TB in Macedonians.

Methylenetetrahydrofolate reductase (MTHFR-677 and MTHFR-1298) genotypes and haplotypes and plasma homocysteine levels in patients with occlusive artery disease and deep venous thrombosis.

The aim was to investigate different genotypes and haplotypes of methylenetetrahydrofolate reductase (MTHFR-677, -1298) and plasma concentration of total homocysteine (tHcy) in Macedonian patients with occlusive artery disease (OAD) and deep venous thrombosis (DVT). Investigated groups consists of 80 healthy, 74 patients with OAD, and 63 patients with DVT. Plasma tHcy was measured with Microplate Enzyme Immunoassay. Identification of MTHFR genotypes and haplotypes was done with CVD StripAssay. The probability level (P-value) was evaluated by the Student's t-test. Plasma concentration of tHcy in CC and CT genotypes of MTHFR C677T was significantly increased in patients with OAD and in patients with DVT. Plasma concentration of tHcy in AC genotype of MTHFR A1298C was increased in patients with OAD and in patients with DVT. Plasma concentration of tHcy was significantly increased in AA genotype of patients with OAD, but not in patients with DVT. We found a significant increase of plasma tHcy in patients with OAD in comparison with healthy respondents for normal:heterozygote (CC:AC), heterozygote:normal (CT:AA), and heterozygote:heterozygote (CT:AC) haplotypes. Plasma concentration of tHcy in patients with DVT in comparison with healthy respondents was significantly increased for normal:normal (CC:AA), normal heterozygote (CC:AC), and heterozygote:heterozygote (CT:AC) haplotypes. We conclude that MTHFR C677T and MTHFR A1289C genotypes and haplotypes are connected with tHcy plasma levels in Macedonian patients with OAD and DVT.

Association of cytokine gene polymorphisms with bronchial asthma in Macedonians.

Bronchial asthma is a multifactorial disease whereby both environmental and genetic factors contribute to its aetiology and/or clinical severity. The aim of this study was to examine the association of 22 cytokine gene polymorphism in the Macedonian population with bronchial asthma (BA). The sample of the population comprised of 301 normal unrelated individuals and 74 patients with BA. Cytokine genotyping was performed by PCR. Susceptible cytokine polymorphisms for BA for ten genotypes (IL-4 -1098/T:T, TNF-alpha -238/A:G, IL-4 -590/C:C, IL-2 +166/T:T, IL-2 -330/T:T, IL-10 -1082/G:G, IFNgamma utr5644/T:T, IL-10 -1082/A:A, IL-1beta +3962/T:T, IL-6 -174/G:G), six diplotypes, four haplotypes, and two alleles were found. Protective cytokine polymorphisms for BA for seven cytokine genotypes (IL-4 -1098/G:T, TNF- alpha -238/G:G, IL-2 -330/G:T, IL-4 -590/C:T, IFNgamma utr5644/A:T, IL-1beta +3962/C:T, IL-10 -1082/A:G), six cytokine diplotypes, four cytokine haplotypes, and four cytokine alleles were found. We concluded that several cytokine polymorphisms are protective, or susceptible associated with BA in population of Macedonians.

Ambiguous allele combinations at group level of HLA-A, -C, and -B genes in Macedonian population using reverse line strip typing method.

The aim of this study was to investigate the occurrence of ambiguous allele combinations at the allele group level of HLA-A, -C and -B loci in the Macedonian population. The DNA samples of 214 healthy unrelated Macedonian volunteers were obtained from our DNA Bank. HLA typing was performed using the IHWG-RLS method (Reverse Line Strip, Roche Molecular Systems, USA) consisting of PCR amplification of exon 2 and 3 of HLA-A, -B and -C genes, followed by hybridization. The statistical analysis of the observed ambiguity frequency was performed by using the Arleqin Software. At the HLA-A locus only one ambiguous allele combination at the allele group level in 214 samples was observed with a frequency of 0.467% (1/214 = 0.467%). A total of 6 different HLA-C ambiguous allele combinations at the allele group level in twelve samples with a frequency of 5.607% (12/214 = 5.607%) and 11 different for HLA-B locus in nineteen samples with a frequency of 8.879% (19/214 = 8.879%) were observed in 214 samples. In conclusion we can say that analysis of the frequency of allele ambiguities revealed that the ambiguities involved some of the most common alleles in our population, obviating the need to introduce ambiguity resolution technique(s)/strategies in the HLA laboratory.

Association of methylenetetrahydrofolate reductase (MTHFR-677 and MTHFR-1298) genetic polymorphisms with occlusive artery disease and deep venous thrombosis in Macedonians.

AIM: To analyze the association of methylenetetrahydrofolate reductase polymorphisms (MTHFR-677 and MTHFR-1298) with occlusive artery disease and deep venous thrombosis in Macedonians. METHODS: We examined 83 healthy respondents, 76 patients with occlusive artery disease, and 67 patients with deep venous thrombosis. Blood samples were collected and DNA was isolated from peripheral blood leukocytes. Identification of MTHFR mutations was done with CVD StripAssay (ViennaLab, Labordiagnostika GmbH, Vienna, Austria) and the population genetics analysis package, PyPop, was used for the analysis. Pearson P values, crude odds ratio, and Wald's 95% confidence intervals were calculated. RESULTS: The frequency of C alleles of MTHFR-677 was 0.575 in patients with deep venous thrombosis, 0.612 in patients with occlusive artery disease, and 0.645 in healthy participants. The frequency of T allele of MTHFR-677 was lower in healthy participants (0.355) than in patients with occlusive artery disease (0.388) and deep venous thrombosis (0.425). The frequency of A allele for MTHFR-1298 was 0.729 in healthy participants, 0.770 in patients with occlusive artery disease, and 0.746 in patients with deep venous thrombosis. The frequency of C allele of MTHFR-1298 was 0.271 in healthy participants, 0.230 in patients with occlusive artery disease, and 0.425 in patients with deep venous thrombosis. No association of MTHFR-677 and MTHFR-1289 polymorphisms with occlusive artery disease and deep venous thrombosis was found, except for the protective effect of MTHFR/CA:CC diplotype for occlusive artery disease. CONCLUSION: We could not confirm a significant association of MTHFR-677 and MTHFR-1289 polymorphisms with occlusive artery disease or deep venous thrombosis in Macedonians, except for the protective effect of MTHFR/CA:CC diplotype against occlusive artery disease.

Cytokine gene polymorphisms in population of ethnic Macedonians.

AIM: To genotype cytokine polymorphisms in the Macedonian population as a part of the international project Cytokine Polymorphism Component (CPC). METHODS: The sample consisted of 125 healthy unrelated individuals, 46 men and 79 women, aged 20-35 years. All individuals were of Macedonian origin and nationality, Christian Orthodox religion, and residents of different regions of the Republic of Macedonia. Blood samples were collected after written consent was obtained, DNA was isolated from peripheral blood leukocytes by the phenol-chloroform extraction method, and the samples were stored in the Anthropology section of the Macedonian Human DNA Bank (hDNAMKD). Fourteen cytokine genes were identified as candidates: gamma-interferon (IFNgamma); interleukin (IL) 1 alpha (IL-1alpha); IL-1 beta (IL-1beta); IL-1 receptor (IL-1R); IL-1R antagonist (IL-1RA); IL-2; IL-4; IL-4 receptor alpha (IL-4Ralpha); IL-6; IL-10; IL-12B; TGF beta 1 (TGF-beta1); and TNF alpha (TNF-alpha). Cytokine genotyping for the anthropology samples was performed by polymerase chain reaction with sequence-specific priming (PCR-SSP) (Heidelberg kit). The population genetics analysis package, PyPop, was used for the analysis of the cytokine data for this report. RESULTS: The frequency of alleles for some single nucleotide polymorphisms (SNP) varied from 0.967 for TGF-beta1 cdn25/G and 0.920 for TNF-alpha -238/G, to 0.914 for IL-1alpha -889/C, indicating common "wild type" allele in those cytokines. For the most SNPs, the test of neutrality showed negative value for F(nd) statistic, which indicated balancing selection operating on the alleles at that locus. F(nd) was negative and significantly different from 0 for IFNgamma UTR5644 and TGF-beta1 cdn10 (P=0.006 and P=0.007, respectively). Most of SNPs showed a good fit with HWP expectations. A few SNPs (IL-1alpha -889, IL-1beta -511, IL-1beta+3962, and IFNgammaUTR5644) were not in HWP (P< or =0.005), and Guo and Thompson Hardy Weinberg Output (GTHWO) was significant (P< or =0.005). The most frequent haplotypes for TGF-beta1 were TG (0.491) and CG (0.476), with the absence of TGF-beta1/TC haplotype in the sample from the Macedonian population. Test of neutrality showed negative value for F(nd) statistic (Ewens-Watterson test of neutrality) which indicated balancing selection operating on the haplotypes at that locus, except for the IL-4 haplotypes, where it showed a positive value for F(nd) statistic, but without significance. F(nd) was negative and significantly different from 0 for IL-10 haplotypes (P=0.002). In the sample from the Macedonian population, D' was equal to 1 in all haplotypes with P values <0.0001, except for TNF-alpha (P< or =0.012), which indicated that one or more haplotypes were missing. CONCLUSION: The analysis of cytokine alleles, genotypes, and haplotypes in the sample from the Macedonian population showed a good fit with Hardy Weinberg equilibrium for most of SNPs and can be used for anthropological comparisons, as well as for association studies with different diseases.