Search

The medical and socio-economic consequences that stemmed from the COVID-19 pandemic, forced the healthcare policymakers in Republic of North Macedonia to rely on five different vaccines against the SARS-CoV-2 virus, in order to reach a satisfactory level of herd immunity. It is here where we got the idea to compare the heterologous Gam-COVID-Vac/BNT162b2 regimen to the homologous BNT162b2 regimen, with our main focus being the immunogenicity differences between the two of them. Additionally, we researched the variation in humoral immune response relative to age strata; the reactogenicity differences; and discrepancies in SARS-CoV-2 infection incidence between the two regimens. To achieve this, antibody titers in sera samples from fifty-three (53) healthcare workers, divided in heterologous and homologous group, were analysed at six different time checkpoints. Our results showed robust immunogenic response after the administration of the booster dose (4. 2-fold increase in antibody titers), followed by a slower-waning humoral immune protection in the heterologous regimen, compared to the homologous BNT162b2 schedule, furthermore confirmed by non-inferiority testing (Geometric Mean Ratio=0,98) at the final checkpoint. That, coupled with the similar reactogenicity (p=0,767) of both regimens, imply that the Gam-COVID-Vac/BNT162b2 combination might be a feasible approach in the effort to contain the COVID-19 pandemic.

Background: Genetic factors play an important role in deep vein thrombosis (DVT). The duration of anticoagulation therapy in patients with verified genetic inheritance and previous events of DVT is still questionable. Case reports: We present three cases of siblings (two brothers and one sister) with verified Venous thromboembolism (VTE) and genetic inheritance. The first case is a 33 y.o. male who was admitted with bilateral massive pulmonary thromboembolism and DVT of the right femoral vein. He had an episode of DVT 4 years ago. Fibrinolytic therapy was introduced immediately. Afterwards, unfractionated heparin was introduced, and then switched to enoxaparin and acenocoumarol. Because of inappropriate INR, it was switched then to rivaroxaban. The imaging methods showed significant improvement, and the patient was discharged from the hospital with rivaroxaban at 2x15 mg/day for another 2 weeks and was instructed to continue 20 mg/day until his next control. In the meantime, the second case, a 36 y.o. male, brother to the first patient, came with vein thrombosis of vena saphena magna of the left leg. Treatment with Acenocoumarol was started and continued for 2 years until complete resolution of the thrombi, and then it was changed to Aspirin. The third case is the sister of the first 2 cases, a 38 y.o female with symptoms and findings almost similar to those in the second case. She was treated with Acenocoumarol for 6 months. Doppler ultrasound showed complete resolution of the thrombosis and anticoagulation therapy was stopped. Genetic investigations for mutation showed presence of homozygous gene mutation for Prothrombin (PTB G20210A) in the first patient, his brother (the second case) was compound heterozygote for PTB and for MTHFR C677T, and his sister (third case) was heterozygous only for the PTB mutation. According to the clinical (recurrent unprovoked DVT with thromboembolic complications) and genetic testing (homozygous gene mutation for PTB) in the first patient, we decided to continue the secondary thromboprophylaxis with rivaroxaban 10 mg/day indefinitely. Conclusion: Testing for genetically inherited thrombophilia should be included in the risk assessment for recurrence, and performed in all patients under 50 y.o. who have a first, non-provoked episode of thrombosis, in order to determine the duration of anticoagulation therapy.

Background 21-hydroxylase deficiency (21OHD) is an autosomal recessive disorder with an incidence of 1:10,000-1:20,000 and is the result of various mutations in the CYP21A2 gene. 21OHD has been described in many different populations, but it has not been studied in Roma individuals so far. The aim of the study was to analyse the genotype in Roma patients with 21OHD and the prevalence of the disease in the Roma population of North Macedonia. Methods Molecular analysis of the nine most frequent CYP21A2 mutations in all known Roma patients with CAH in North Macedonia, relatives and healthy individuals of Roma ancestry, using the PCR/ACRS method. Results Ten Roma patients with 21OHD were identified, of which nine had the salt-wasting and one had the simple virilizing form. Calculated incidence of 21OHD in the North Macedonian Roma population was 1:3375. Interestingly, 9/10 patients (90%) were homozygous for the In2G splicing mutation (293-13A/C > G). Standard therapy with hydrocortisone and fludrocortisone had been introduced according to the guidelines. In 16 healthy relatives investigated for CYP21A2 mutations, heterozygosity for the In2G mutation was detected in 13/32 (40.6%) alleles. In 100 healthy Roma individuals, none related to the analysed families, no CYP21A2 mutations were detected. Conclusion The Roma population in North Macedonia had a very high incidence of classic 21OHD. Almost all patients had the severe salt-wasting form and the In2G/In2G genotype.

Leptin, as a major adipokine, positively correlates with the body’s fat, while atopy is an important feature in the development of childhood asthma. We aimed to evaluate the relationship between leptin, parameters of obesity, and atopy in children with asthma. The study included 112 children (73 boys, 39 girls, mean age 11.1±2.4). 41 were overweight, 38 had asthma and a normal body mass index (BMI), and 33 were overweight asthmatics. Serum leptin levels, BMI, waist circumference (WC), and waist to hips ratio (WHR) were measured. Skin prick test (SPT)/CAP, total serum IgE, fractional exhaled nitric oxide (FeNO), and pulmonary function tests were performed. In asthmatic children, serum leptin median level was 9.2±16.2 ng/ml, in overweight children was 30.6±21.6 ng/ml, and in overweight asthmatics was 31.1±20.3 ng/ml with a significant difference between the groups (p=0.0374), yet with a significantly lower median level in the group of children with asthma compared to the overweight children: with asthma (p=0.00001) and without asthma (p=0.00001). In the three groups of patients, BMI and WC displayed a significant positive correlation with leptin (for BMI r=0.652 vs. r=0.530 vs. r=0.563, respectively and for WC r=0.508 vs. r=0.426 vs. r=0.527, respectively). No significant correlations of leptin within atopy parameters (Eo, IgE, SPT/CAP, FeNO) in all three analyzed groups (p>0.05) was detected. Conclusion: Atopy was not confirmed as an underlying mechanism of the association between asthma and being overweight. Leptin had a significant linear correlation as a parameter of central obesity with BMI and WC in all three groups, but not with WHR.

De novo variants in the WDR26 gene leading to haploinsufficiency have recently been associated with Skraban-Deardorff syndrome. This condition is an ultra-rare autosomal dominant neurodevelopmental disorder characterized by a broad range of clinical signs, including intellectual disability (ID), developmental delay (DD), seizures, abnormal facial features, feeding difficulties, and minor skeletal anomalies. Currently, 18 cases have been reported in the literature and for only 15 of them a clinical description is available. Here, we describe a child with Skraban-Deardorff syndrome associated with the WDR26 pathogenic de novo variant NM_025160.6:c.69dupC, p.(Gly24ArgfsTer48), and an adult associated with the pathogenic de novo variant c.1076G > A, p.(Trp359Ter). The adult patient was a 29-year-old female with detailed information on clinical history and pharmacological treatments since birth, providing an opportunity to map disease progression and patient management. By comparing our cases with published reports of Skraban-Deardorff syndrome, we provide a genetic and clinical summary of this ultrarare condition, describe the clinical management from childhood to adult age, and further expand on the clinical phenotype.

Noonan syndrome (NS) is a genetic autosomal dominant condition, caused by mutations in PTPN11 and other genes. The aim of this report is to highlight a finding of a rare mutation in the RAF1 gene in a six-year-old child evaluated for Noonan Syndrome. An Ampliseq Research Panel covering A2ML1, BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NRAS, PTPN11, RAF1, RIT1, SHOC2, SOS1 and SPRED1 genes was used on the Ion Torrent platform. Out of 54 variants detected, a single nucleotide missense mutation c.483T>G in the RAF1 gene was classified as likely pathogenic, based on a single previous submission to Clinvar. Further investigations may shed light on the possible role of this variant in the pathogenesis of Noonan Syndrome and other RASopathies.

Placental growth factor (PlGF) is crucial during placental development in early pregnancy. Several studies in pregnancies with complications such as preeclampsia or small for gestational age neonates find that PlGF levels are significantly lower in the first trimester, which implies that the concentration of PlGF could be used as an early screening biomarker for these conditions. This study aimed to compare the performance of chemiluminescence immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) for the quantification of human PlGF in serum. This is a comparative study on 88 pregnant women in the first trimester subjected to measurement of PlGF in serum using two commercially available kits: Human PlGF Quantikine HS ELISA (R&D Systems) and PlGF CLIA (Snibe). The overall coefficient of correlation between the tests was 0.93. When the cut-off value of 40 pg/mL was applied, it dropped significantly to 0.50 towards the lower values, while remaining an excellent 0.91 in the group with higher concentrations of PlGF. While R&D Systems’s ELISA seems to have better sensitivity, it is not very convenient to use for a small number of samples. Snibe’s CLIA automated method is user-friendly, fast and powerful. Both tests show excellent performance when indicating risk-free pregnancies.

Genetics in Macedonia-Following the international trends.

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.

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.

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.

BACKGROUND: With a frequency of 1:1600, the alpha-1-antitrypsin deficiency is one of the most frequent hereditary diseases and can be recessively inherited. AAT deficiency is most often caused by inheritance of the so-called PiZ allele. Inheritance of this allele increases the risk of developing chronic obstructive pulmonary diseases (COPD) and liver disease. AIM: The aim of this study was to present immunonephelometry and reverse hybridization genotyping in diagnosis of alpha-1-antitrypsin deficiency in Republic of Macedonia. MATERIAL AND METHODS: At the Institute of Immunobiology and Human Genetics, part of the Faculty of Medicine in Skopje, in the previous 7 years, total of 361 patients with suspected alpha-1-antitrypsin (AAT) deficiency were referred for analysis of AAT concentration using nephelometry (Dade Behring) and subsequent AAT genotyping of individuals with alpha-1-antytripsin deficiency at protein level, based on reverse hybridization technique. RESULTS: Measurement of AAT concentration (g/l) by nephelometry have shown normal level in the range of 1.37-1.41 g/l (88%), lower than normal AAT levels in the range of 0.70-0.83 g/l (8.03%), and concentration above the normal levels in the range of 2.28-2.4 g/l (3.88%). CONCLUSION: Diagnosis in the case of a suspicion of AAT deficiency is carried out by measuring the alpha-1-antitrypsin level in blood and by genotyping of alpha-1-antytripsin allele.

Introduction: Natural killer (NK) cells are the predominant lymphocyte population in the decidua. Being the most abundant leucocytes, the activity of NK cells is important in different immuno-pathological conditions, such as recurrent spontaneous abortions, infertility and problems in implantation. The NK cells recognize HLA class I molecules on trophoblasts trough killer immunoglobulin-like receptors (KIRs) found on their surface. The KIRs are classified as either activating or inhibitory, regarding the effect they produce on NK cells upon interaction with corresponding ligand. Since KIR genes exhibit extensive polymorphism and individuals differ in both the number and kind (activating vs. inhibitory) of KIR genes, it is hypothesized that the KIR gene content might influence the pregnancy outcome. Aim: The aim of this pilot study is to analyze the frequency of different KIR genes in women with infertility problems, and compare them to healthy women. Material and Methods: Total of 122 healthy women (Control) and 25 women with reproductive problems (MISSC) participated in this study. After signing of written consent DNA was isolated from peripheral blood using phenol/chloroform method. The genotyping of 16 KIR genes was performed using commercially available kit from Dynal Biotech, (Pel-Freez Clinical Systems, Brown Deer, WI, USA), based on SSP method. Results: We found that inhibitory KIR are present in similar observed frequency in both control and patients with MISSC, except KIR2DL5 which was found in lower frequency in patients with MISSC. There are no significant differences of all noninhibitory KIR between control and patients with MISSC. The number of inhibitory KIR genes in patients with MISSC was lover, except for seven inhibitory KIR genes which was almost doubled. The number of noninhibiotry (stimulatory) KIR genes was lower in patients with MISSC, except for those with three KIR genes which were almost four times more frequent. We found significantly bigger percentage of 0,34 – 0,60 activating/inhibitory KIR gene number ratio in the patients with MISSC. Conclusion: In conclusion, there are differences in the KIR gene distribution, gene number, and activating/inhibitory KIR gene number ratio between control and Macedonian patients with MISSC. Further analysis of frequencies of corresponding KIR genotypes or in the ratio of activating/inhibiting genes content in two groups are needed.

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.

Immune thrombocytopenia (ITP) is an autoimmune blood disease of unknown etiology. The aim of our study was to investigate a possible role of FCGR2A and FCGR3A polymorphisms in the development of primary ITP.We analyzed 125 adult patients with ITP and 120 healthy controls. Genotyping was performed by using PCR-RFLP methods.Our results showed significantly higher frequency of high-affinity FCGR3A-158V allele in patients with ITP compared with control subjects (47.2% versus 37.5%; p = 0.037). We did not find significant differences in the genotype distribution or allele frequencies for FCGR2A-131H/R between patients and controls, p = 0.652 and p = 0.478. In the groups of patients with unresponsive and responsive ITP we found significantly different genotype distribution and allele frequencies for FCGR3A, p = 0.036 and p = 0.008 respectively. There was no significant difference in genotype and allele frequencies for FCGR2A between these two groups of patients. Our results confirmed that the combination of high-affinity FCGR2A-131H and FCGR3A-158V allele was more common in patients with ITP than in controls (55% versus 40%; p = 0.024).Our results suggest possible role of FCGR3A polymorphism in the etiology, development and clinical outcome of ITP, but larger prospective studies are needed to confirm these results.

Background: Distribution of CYP2C9 and VKORC1 gene polymorphisms may vary significantly among different ethnic groups, and eventually influence the variation in drug metabolism or even failure.Objective: The aim of this study was to evaluate the prevalence of CYP2C9 and VKORC1 alleles in the healthy population of Republic of Macedonia compared to the global geographic data reported from different ethnic populations. Also, to genotype CYP2C9 and VKORC1 genes and eventually to divide individuals in poor, extensive, or intermediate metabolizer.Material and Methods: Blood samples were collected after signing written consent, DNA was isolated from peripheral blood, and CYP2C9 and VKORC1 genes were typed (n=124). Genotyping was performed by commercially available kits (GeneID GmbH, Strassberg, Germany, AID Diagnostica), based on the method of polymerase chain reaction with a subsequent hybridization. The population genetics analysis package, PyPop ver. 0.6.0, was used for analysis of the data.Results: The frequency of alleles varies from 0.931 for CYP2C9*3 to 0.109 for CYP2C9*2 indicating common “wild type” allele in those genes. The frequency ranges spanned ~50% for each allele of VKORC1 gene, indicating no common “wild type” allele in this gene. Test of neutrality showed significant negative value for VKORC1 polymorphism that indicates balancing selection operating on the alleles at that locus. All polymorphisms of CYP2C9*2, CYP2C9*3 and VKORC1 showed a good fit with Hardy-Weinberg expectations.Conclusion: The results of polymorphic alleles of CYP2C9 and VKORC1 genes in Macedonian population can be used for the variation in drug metabolism studies as well for adapting dosage regimes for oral anticoagulant therapies.

Rationale: Sputum examination is a valuable research tool to study airway diseases, but the requirement to process the samples within 2 hours of their collection poses limitations to its wider applicability. A way to bypass this hurdle would be to freeze the sample at the time of collection and to examine it at a later stage. Methods and Subjects: We developed a protocol for freezing of sputum upon collection by adding dimethylsulfoxide to it. We tested the reproducibility of cell counts in frozen samples and in fresh portions from the same sputum specimens. We took sputum from 41 asthmatics (18 males) with different levels of control of their disease: 19 of the samples were spontaneously produced and 22 were induced with hypertonic saline. Results: Significant correlations (p

West Nile virus (WNV) is a neurotropic, arthropod-borne flavivirus that is maintained in an enzootic cycle between mosquitoes and birds, but can also infect and cause disease in horses and humans. The aim of this study was to examine KIR gene polymorphisms by determining the frequencies of 16 KIR genes and pseudogenes and KIR genotypes in Macedonian patients with West Nile virus infection, and to compare with healthy Macedonians. The studied sample consists Republic of Macedonia, hospitalized at the University Clinic of Infective Diseases between September 2011 and October 2011, and reported through WHO. For KIR genotyping, commercially available PEL-FREEZ KIR genotyping SSP kit (Dynal Biotech, Brown Deer, WI) was used. The population genetics analysis package, Arlequin, was used for analysis of the data. We found that all 16 KIR genes were observed in the studied individuals and framework genes (KIR3DL3, KIR3DP1, KIR2DL4, and KIR3DL2) were present in all individuals. Comparison of KIR frequencies between Macedonian patients with West Nile virus infection and healthy Macedonian population reveals several significant differences in the inhibitory group (KIR2DL2), and in the non inhibitory group (KIR2DS1, KIR2DS2, KIR2DS5, and KIR3DS1). The single most frequent genotypes in the Bx group were genotypes ID71 and ID89 with statistically significant difference compared to healthy Macedonians. Our results suggest that specific KIR genotypes could be connected with West Nile virus infection.

Aim: The aim of this paper was to genotype HLA DQB1 locus in healthy unrelated Macedonian population. Material and Methods: Reverse Line Strip typing for HLA DQB1 locus was performed on a population of 217 samples from healthy individuals. The results were obtained as alleles, and as NMDP Codes. Alleles were genotyped and expressed as high resolution with more than four digits with slash between ambiguous alleles. NMDP Codes were expressed as four digits for unambiguous results, and with combination of numbers and characters for the certain ambiguous combination. We did not found any genotypic ambiguities in HLA-DQB1. Results: We have found 33 different alleles of HLA DQB1 in Macedonian population, 12 of which were unambiguous with the frequency of 40.55%, and 21 were ambmiguous with the frequency of 58.53%. The biggest frequency (33.64%) was found for HLA DQB1*05 unambiguous groups, and for HLA DQB1*03 ambiguous groups (36.40%). The biggest allele frequency in Macedonian population for HLA DQB1 was found for HLA DQB1*03NX (0301/01/01/02/09/13) with 27.88%, followed with HLA DQB1*0502 with 13.82%, and HLA DQB1*02MN (0201/02/03) with 10.37%. Conclusion: Allele frequency of HLA DQB1 in Macedonian population is similar with that found in other European populations and can be used for anthropology and disease association studies.

Summary The aim of this study was to analyze Killer Ig-Like Receptor (KIR) gene polymorphisms in Roma people from Republic of Macedonia. The studied sample consists of 103 healthy unrelated individuals, aged 20–45 years. All individuals are of Roma origin, residents of different geographical regions (Gostivar, Skopje, and Kochani). The population genetics analysis package, Arlequin, was used for analysis of the data. We found that all 16 KIR genes were observed in the Roma individuals and framework genes (KIR3DL3, KIR3DP1, KIR2DL4, and KIR3DL2) were present in all individuals. The frequencies of other KIR genes were: KIR2DP1 (1), KIR2DL1 (0.961), KIR2DL2 (0.544), KIR2DL3 (0.874), KIR2DL5 (0.311), KIR3DL1 (0.990), KIR2DS1 (0.330), KIR2DS2 (0.553), KIR2DS3 (0.359), KIR2DS4 (0.981), KIR2DS5 (0.291), and KIR3DS1 (0.379). The results of tested linkage disequilibrium (LD) amongKIR genes demonstrated that KIR genes present a wide range of linkage disequilibrium. The obtained results for KIR genes and genotype frequencies in Macedonian Roma individuals can be used for anthropological comparisons.

Background: Glycoprotein (GP) Ia/IIa or α2β1 integrin is a platelet receptor for collagen and it mediates platelet adhesion to vascular subendothelium and is involved in thromb formation. Genetic polymorphism of α2β1 known as Bgl II affects the density of platelet GP Ia/IIa receptor on the platelet surface. Recent studies had shown relationship between this polymorphism and the risk of myocardial infarction, stroke, as well as diabetic retinopathy. Aim: The aim of this study was to determine the frequency of this polymorphism in Macedonian healthy population. Materials and methods: We genotyped 217 healthy Macedonian individuals using the PCR and RFLP (restriction fragment length polymorphism) method. Results: The allele frequencies in this study were 0.32 for Bgl II (+) allele and 0.67 for Bgl II (-). Distribution of Bgl II genotypes in Macedonian population was Bgl II (+/+) =16/217 (7.3%), Bgl II (+/-) =107/217 (49.3%) and Bgl II (-/-) =94/217 (43.3%). Conclusion: Our results showed a slightly lower proportion of the Bgl II (+) allele (0.32) in Macedonian population, but not significantly different from other Caucasian 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.

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.

Aim. To investigate the effect of the peroxisome proliferators-activator receptor gamma agonist, rosiglitazone, on metabolic parameters and adiponectin levels in an animal model of the metabolic syndrome. Material and methods. Metabolic syndrome was induced in 32 male Wistar rats by adding a fructose in drinking water for 12 weeks. During the last 4 weeks, 16 rats were treated with rosiglitazone (5 mg/kg/day), while the remaining 16 did not receive any medication (fructose group). Another control group consumed standard rat chow and water for 12 weeks. Results. Chronic fructose administration induced a significant increase in systolic blood pressure (SBP), body weight, serum triglycerides (TG), free fatty acids (FFA), insulin, glucose AUC 0-120 (during oral glucose tolerance test) and decreased serum high density lipoprotein (HDL) cholesterol and adiponectin concentrations compared with the control group. Treatment with rosiglitazone over the final 4 weeks reversed these effects and significantly reduced SBP, TG, FFA, insulin concentrations and glucose AUC 0-120 compared with the fructose group. In addition, rosiglitazone increased serum levels of adiponectin twofold from 3.44 ± 0.46 to 7.03 ± 1.30 μg/ ml. Conclusion. This study indicates that rosiglitazone treatment improves the components of the metabolic syndrome, which is accompanied with an increase in adiponectin concentrations. OPEN ACCESS

Background. In this paper we present the initial results of introducing the method of multiparameter flow cytometry (MPF) in patients with acute leukemia in the Republic of Macedonia.

This year the monographic book “60 Years Macedonian Medical Association 19452005” was published, denoting more then six decades successful existence of Macedonian Medical Association (MMA). In the period behind us, the MMA has been bearer of the continuous development and growth of the health provision system. Starting its activities back in 1945, with only 123 doctors and dentists working in Republic of Macedonia, the MMA today represents more then 5000 doctors, of whom 3025 are specialists in various fields. The doctors in the MMA are organized into 22 regional or local associations and 70 specialist and sub-specialist associations. The MMA was the predecessor of the Faculty of Medicine in Macedonia, with the vast majority of the staff being recruited from MMA. A major contribution to the professional and scientific development of the medical profession in Macedonia was achieved through the introduction of the scientific journal and organization of scientific congresses under auspice of MMA. Today MMA is an active participant, initiator and consultant in the creation and improvement of the laws regulating the health service provision in the country. The highest state honour, the 11th October Award was awarded to MMA, as recognition for its continuous commitment to progress of Macedonian medicine.

Alpha-1-antitrypsin (AAT) is a protease inhibitor which plays an important role of protector of the lung tissues against the proteolytic effect of elastase secreted from neutrophils. Its deficiency is associated with liver disease in children and emphysema in adults. So far, more than 75 variants of this protein are defined, but most of the cases of AAT deficiency are caused by homozygosis for the deficient allele PIZ or by heterozygous combination of the 2 most common deficient alleles, PIS and PIZ. A diagnosis in the case of a suspicion of AAT deficiency is carried out by measuring the alpha-1 antitrypsin level in blood and by genotyping the alpha-1 antitrypsin alleles. The importance of early diagnosis resides in the possibility of undergoing a lifestyle modification (such as vigorous smoking cessation, for example) and treatment of pulmonary disease thus significantly decreasing the morbidity. The family studying is important to identify individuals at high risk, and initiation of AAT replacement therapy in individuals. Having in mind the underdiagnosis of this disease, we hope to contribute with this case report to the medical community in Republic of Macedonia to raise the awareness of this disease, and also of the possibilities of exact diagnosis.

Faculty of Medicine in Skopje, Republic of Macedonia was founded in 1947 year with its educational and health activities which should lead to improvement of the health care in the country. In the past sixty years Faculty of Medicine changed its structure, position, and the role several times in order to fulfill the needs of the society for continous increase of quantity and quality of tertiary health care, education of medical students, as well as research in the biomedical fields. Today, the organizational structure of the Medical Faculty consists of 12 institutes and 3 teaching bases in the pre-clinical department and 25 clinics, 3 institutes and 7 teaching bases which take active part in both, education and scientific activities. Altogether there are 33 departments which perform the entire educational (undergraduate, postgraduate, specialization and sub-specialization), scientific and providing health activities. These 33 departments are actively involved in the realization of the study programmes, not only at the Medical Faculty, but the Faculty of Stomatology, Pharmaceutical Faculty, the Faculty of Natural Sciences, and the Faculty of Physical Culture as well. Medical Faculty is a member of the European and the global family of institutions of this kind.

The aim of this study was to genotype IL-1 gene cluster polymorphisms in the Macedonian population. A group composed of 301 healthy unrelated individuals was selected. IL-1 genotyping of the samples was performed by the PCR-SSP procedure followed by analysis of the IL-1 data with the PyPop population genetic analysis software package. The frequency of IL-1 alleles varies from 0.814 for IL1A -889/C, 0.729 for IL-1B +3962/C, 0.698 for IL-1RN mspa 11100/T, 0.671 for IL-1B -511/C, followed by 0.663 for IL-1R psti 1970/C indicating common “wild type” allele. We determined 27 different haplotypes from the total number of 32. The most frequent haplotypes for IL-1 are CCCCT (0.191), CTCCT (0.127), CCCTT (0.098), CCCCC (0.085), CTCTT (0.082). All pairs of loci for IL-1, except for IL-1R psti 1970, are in linkage disequilibrium, with p

Specific IgA, IgG, and IgE antibodies to food antigens in 35 participants with autistic disorder and 21 of their siblings in the Republic of Macedonia were examined. Statistically significant higher plasma concentration of IgA antibodies against alpha-lactalbumin, beta-lactoglobulin, casein, and gliadin were found in the children with autistic disorder. Plasma concentrations of IgG antibodies against alpha-lactalbumin, beta-lactoglobulin, and casein in participants with autistic disorder were significantly higher. IgE-specific antibodies (alpha-lactalbumin, beta-lactoglobulin, casein, and gluten), as well as plasma concentration of total IgE, also were statistically significantly higher in the participants with autistic disorder. Gender differences were found for select IgA, IgG, and IgE (but not for total IgE) food-specific antibodies (kU/L) in the participants with autistic disorder and their siblings.

The Familial Mediterranean fever (FMF, MIM249100) is an autoimflammatory genetic disease characterized with recurrent painful attacks in the abdomen, chest or joints, usually accompanied with high body temperature. It is classically inherited in an autosomal recessive manner. It is associated with mutations of the MEFV gene, coding for the protein pyrin. More than 140 mutations of the MEFV gene are defined worldwide. Despite the progress in establishing reliable tests practical for routine use, as much as 20% of the patients with FMF remain without a detectable mutation in the MEFV gene. This is the main reason why the diagnosis of FMF remains still a clinical one, according to Tel Hashomer criteria. A 10-year old girl admitted to the Clinic of Pediatrics at the Faculty of Medicine in Skopje for unexplained fever. After numerous laboratory analyses and specialist consultations were done, genetic testing for FMF was requested. The presence of an heterozygous mutation E148Q was confirmed at the Institute for Immunobiology and Human Genetics using a PCR based, reverse hybridization method. Administration of colchicine, the therapy of choice, in a dose of 1.5 mg/day, lead to complete resolution of the symptoms within some days following commencement. Although the disease is classically inherited in a recessive manner, some atypical cases of autosomal dominant inheritance are described. Our patient may be another example supporting the unusual dominant inheritance, since the heterozygous state for the E148Q mutation was the only positive finding in the genotyping of the 12 most frequent MEFV mutations.

AIM: The aim of this study was to analyze Killer Ig-Like Receptor ( KIR ) gene polymorphisms in Albanians from Republic of Macedonia. MATERIAL NA METHODS: The studied sample consists of 104 healthy unrelated individuals, aged 20-45 years. All individuals are of Albanian nationality, residents of different geographical regions (Skopje, Gostivar, and Tetovo) in Republic of Macedonia. The population genetics analysis package, Arlequin, was used for analysis of the data. RESULTS: All 16 KIR genes known were observed in the Albanian individuals and framework genes (KIR3DL3, KIR 3DP1, KIR 2DL4, and KIR3 DL2) were present in all individuals. The frequencies of other KIR genes were: KIR 2DP1 (0.981) , KIR 2DL1 (1), KIR 2DL2 (0.615), KIR 2DL3 (0.865), KIR 2DL5 (0.414), KIR 3DL1 (0.933), KIR 2DS1 (0.462), KIR 2DS2 (0.606), KIR 2DS3 (0.327), KIR2DS4 (0.875) , KIR 2DS5 (0.298), and KIR 3DS1 (0.442). Tested linkage disequilibrium (LD) among KIR genes demonstrated that KIR genes present a wide range of linkage disequilibrium. CONCLUSION: This is the first study analyzing the polymorphism of KIR genes and genotype frequencies in Albanian individuals in the world. The results can be used for anthropological comparisons.

A total of 850 persons from the Republic of Macedonia, divided in the four groups, were DNA-HLA genotyped: 1. Donors of the Macedonian Donor Registry (220); 2. Patients for bone marrow transplantation and related potential donors (350); 3. Patients with rheumatoid arthritis, tuberculosis, cardiomyopaties and other diseases (150); and 4. Ambiguity resolution of the genotypes (130). Class I HLA genes (A, C, B, and DQB1) were typed by Reverse Line Strip Typing (RLS); Class II HLA gene (DRB1) were typed by Sequence Based Typing (SBT). The main results of the project are: a) Creation and legislation of Macedonian Donor Registry, and Macedonian Human DNA Bank; b) HLA typing of unrelated healthy persons for class I HLAA, -C, and –B, and class II HLA-DRB1; c) HLA genotyping of blood related samples for Bone Marrow Transplantation; d) Association analysis of HLA alleles with Rheumatoid Arthritis (HLA-DRB1, and HLA-B27), and with Pulmonary Tuberculosis (HLA-DQB1); e) Evaluation of allele and genotype ambiguities for HLA class I, and HLA class II, as well as resolution of HLA class I and class II ambiguities; f)

The aim of this study was to examine the gene frequencies of 16 KIR genes and pseudogenes ( KIR2DL1 , KIR2DL2 , KIR2DL3 , KIR2DL4 , KIR2DL5 , KIR3DL1 , KIR3DL2 , KIR3DL3 , KIR2DS1 , KIR2DS2 , KIR2DS3 , KIR2DS4 , KIR2DS5 , KIR3DS1 , KIR2DP1 , and KIR3DP1 ) and KIR genotypes in Macedonian patients with haematological malignancies, to compare them with the corresponding frequencies of healthy Macedonians and to analyse eventual association of specific genes or genotypes with the studied haematological diseases. The study included 63 patients of which 40 had acute myeloblastic leukaemia (63.5%), 11 (17.5%) had chronic myeloid leukaemia, 8 (12.7%) acute lymphoblastic leukaemia, 2 (3.17%) non-Hodgkin Lymphoma, 1 (1.59%) aplastic anemia, and 1 (1.59%) chronic lymphocytic leukaemia. Comparison of KIR gene frequencies between the 63 patients and healthy Macedonians reveals statistically significant difference for KIR3DL2 (F= 1 in the control group, and 0.95 in the patients group, p=0.001). Another statistically significant difference was observed for the frequency of Bx3 and Bx439 genotypes both found more often in patients group (P=0.017 and P= 0.009, respectively). Further analysis, involving larger series of patients and targeted at the ligands of the KIRs are needed in order to determine a certain KIR gene and/or genotype as either predisposing, or protecting factor for haematological malignancy in patients from Republic of Macedonia.

Aim: The aim of this study was to examine the gene frequencies of 16 KIR genes and pseudogenes and KIR genotypes in Macedonian patients with transplanted bone marrow and their sibling donors in treatment of haematological malignancy, and to analyse eventual association of the gene content with the occurrence of a graft versus host disease (GVHD). Material and Methods: The study was performed on 24 patients and their HLA-matched sibling donors. Results: Comparison of KIR gene frequencies between the total 24 donors and healthy Macedonians reveals statistically significant difference for KIR2DS1 (F= 0.481 in the controls group, and 0.76 in the patients group, p=0.004). This significance is even higher when the frequency of KIR2DS1 in controls is compared with the frequency in donors from pairs with GVHD (F= 0.923, P= 0.002). Another significant difference was observed for the frequency of the full-length allele of KIR2DS4*001-002, present in 25.2% of the control individuals, but in as much as 81.8% of the recipients of haematopoietic stem cell (P=0.0005). We did not see any statistically significant difference in distribution of the C1/C1, C1/C2 and C2/C2 groups among GVHD pairs. Conclusion: Our results address the difference between the haematopoietic stem cell transplantation settings with sibling and unrelated donors and suggest that the KIR2DS4*001/002 might be a predisposing factor for severe GVHD in sibling HSCT.

Background: Infection by the pandemic influenza A (H1N1)pdm09 virus results in significant pathology disease in many cases in different populations worldwide. The natural killer (NK) cells are among the major effectors important in early innate immune responses to viral infections, interacting with host cells through their activating or inhibiting receptors. Aim: The aim of this study was to analyze Killer Ig-Like Receptor ( KIR ) gene polymorphisms in critically ill Macedonian patients with pandemic influenza A (H1N1)pdm09 infection. Material and Methods: The studied sample consists of 63 critically ill Macedonian patients with pandemic influenza A (H1N1)pdm09 infection. The population genetics analysis package, Arlequin, was used for analysis of the data. Results: We found that all 16 KIR genes were observed in the studied individuals and framework genes ( KIR3DL3, KIR3DP1, KIR2DL4, and KIR3DL2 ) were present in all individuals. The results of tested linkage disequilibrium (LD) among KIR genes demonstrated that KIR genes present a wide range of linkage disequilibrium. Comparison of KIR gene frequencies between critically ill H1N1/09 Macedonian patients and healthy subjects reveals statistically significant difference for frequency of KIR2DL1 (F=1 in the patients group, and 0.94 in the control group, p=0.045). Conclusion: We did not found any significant association of all 16 KIR genes or KIR genotypes with critically ill (H1N1)pdm09 Macedonian patients, except for the KIR2DL1 .

Aim: To analyze the influence of the hyperthermic environmental temperature (35 ± 1°C) on the adenosine deaminase activity (ADA) in tissues rich with lymphoid cells in Wistar rats. Material and Methods: The activity of ADA was examined in thymus, spleen, Payer’s patches, mesenteric lymph nodes and serum. The experimental animals (n = 11 male rats) were exposed on hyperthermic environment (35 ± 1°C) and relative air humidity of 20 - 30% within a period of 30 days. The control group of animals (n=10) were kept on room temperature (18 - 22°C). Results: The body weight was decreased, spleen mass, and the thymus mass were reduced. Activity of ADA in the thymus was significantly increased from 10.58 ± 1.57 U/g in the control group of rats to 13.38 ± 2.7 U/g in rats exposed on 35 ± 1°C (p

To examine the possible role of 22 cytokine gene polymorphisms in host susceptibility to or protection against tuberculosis (TB) in Macedonians.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).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.These results suggest that some cytokine polymorphisms are significantly associated and affect host susceptibility/resistance to TB in Macedonians.

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.To examine a possible role of 22 cytokine gene polymorphisms in host susceptibility to or protection against DCM in Macedonians.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).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.These results suggest that some cytokine gene polymorphisms are significantly associated and affect host susceptibility/resistance to DCM in Macedonians.

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.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.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.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.There is no significant relationship between SERPINE1 polymorphisms and occlusive artery disease or deep venous thrombosis in Macedonian population.