HLA profile of the Macedonian, Albanian and Macedonian Muslim donors in the Macedonian Bone Marrow Donor Registry.

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 (MBMDR). We analyzed 1541 donors, from different nationalities and presented the HLA allele and haplotype frequencies for Macedonian, Albanian and Macedonian Muslims, most numerous nationalities in MBMDR. Difference between the three groups was observed for allele frequencies in HLA-C and HLA-DRB1 loci. The most common haplotype in Macedonian was HLA-A*01-B*08-C*07-DRB1*03, while in Albanian and Macedonian Muslims HLA-A*02-B*18-C*07-DRB1*11. This study confirmed the close relationship between the populations that live in the Balkan Peninsula.

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.

HLA-A, -B, -C and -DRB1 allele and haplotype frequencies in the Macedonian population based on a family study.

AIM: The aim of this study was to determine HLA allele and 2-, 3- and 4-loci haplotype frequencies in a sample from Macedonian population with defined haplotypes based on family history. MATERIAL AND METHODS: We analysed 286 unrelated individuals with Macedonian origin, parents of patients who needed stem cell transplantation, in the period of 01.01.2003 till 31.12.2016. Allele and haplotype frequencies, as well as Hardy-Weinberg equilibrium were calculated using the Arlequin3.5 software. Population comparison was calculated using the PHYLIP software. RESULTS: We identified 18 HLA-A, 26 HLA-B, 13 HLA-C and 13 HLA-DRB1 allele group families. The most frequent allele groups in our population were HLA-A*02 (29.0%), HLA-A*24 (13.8%), HLA-B*35 (16.1%), HLA-B*51 (14.7%), HLA-B*18 (14.7%), HLA-C*07 (27.9%), HLA-DRB1*11 (25.5%) and HLA-DRB1*16 (14.8%). The most frequent four loci haplotype was HLA-A*01-B*08-C*07-DRB1*03 (2.7%). Our comparison showed that the Macedonian population is closely related to the neighbouring countries in the Balkan Peninsula. CONCLUSION: This study provides data about the HLA diversity in the Macedonian population, which can be very important in the process of unrelated donor search, and in addition yields control group for future disease association studies in our population.

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.

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.

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.

Influence of the elevated ambient temperature on immunoglobulin G and immunoglobulin G subclasses in sera of Wistar rats.

The aim of our research was to examine changes in the immune system of the rats influenced by the elevated ambient temperature. Male Wistar rats were divided, into 2 groups and housed at 20 +/- 2 degrees C (n = 64, control group) and 35 +/- 1 degrees C (n = 74, experimental group), during precise timing of 1, 4, 7, 14, 21, and 30 days. All the animals were given food and water ad libitum, and were lighted during 12 hours per day. We have measured IgG, IgG1, IgG2a, IgG2b and IgG2c. The obtained results showed significant elevation in the level of IgG after 4 and 7 days (+32%), IgG2a after 7th (+88%), 14th and 21nd day (+110%), IgG2b after 14 days (+60%) at 35 +/- 1 degrees C compared with the control group at 20 +/- 2 degrees C. IgG1 level was not affected and IgG2c showed significant decrease after 21st day at 35 +/- 1 degrees C. In conclusion, during the elevated ambient temperature the immune system is activated as one of the regulation mechanisms in homeostasis and survival of the population.