DNA typing of HLA-A, -C, -B, AND -DRB1 in the children with autism in the Republic of Macedonia.

In the present study, we report the first DNA analysis of HLA class I and class II alleles in Macedonian autistic subjects. We have analyzed the HLA-A, -C, -B, DRB1 genotypes of 35 autistic patients, and 98 healthy unrelated Macedonians (control group). HLA DNA typing of class I genes was performed using a Reverse Line Strip method (RLS), and the Sequencing Based Typing method (SBT) was used for typing of class II genes. In the autistic subjects for HLA-A locus 14 alleles have been identified with 2 being predominant *02 (25.7 %), and *24 (18.6 %). Among the 11 identified HLA-C alleles, 3 were predominant such as *12 (20.0 %), *07 (17.1 %), and *03 (12.9 %). Among the 18 identified HLA-B alleles, 2 were predominant: *51 (18.6 %), and *18 (11.4 %). For HLA-DRB1 locus, 10 alleles have been identified with 2 of them predominant such as: *11 (21.4 %), and *01 (14.3 %). The allele and haplotype frequencies in the patients group were compared to those of 98 control subjects. Our results showed significantly increased frequencies of HLA-C*03 (OR = 2.74*; χ2 = 4.68; p = 0.03), and HLA-DRB1*01 (OR = 3.10*; χ2 = 6.26; p = 0.012) alleles in autistic patients when compared to the controls. The most frequent haplotype frequencies in autistic sample were A*11-C*12-B*52-DRB1*15 (2.9 %), A*24-C*03-B*55-DRB1*16 (2.9 %), and A*24-C*03-B*55-DRB1*16 (2.9 %), but they were not statistically significant when compared to the control group. None of our patients carried allele or haplotype, which were protective in our population. Hardy-Weinberg equilibrium in autistic group showed that HLA-A (p < 0.03), HLA-C (p < 0.04), and HLA-DRB1 (p < 0.002) loci were in linkage disequilibria. In the control group, we found only for the HLA-DRB1 locus linkage disequilibrium (p < 0.002). Our results demonstrated the association of HLA-C*03 and HLA-DRB1*01 alleles with Macedonian autistic patients (Tab. 7, Ref. 37).

IL1 cluster gene polymorphisms in Macedonian patients with chronic periodontitis.

Several studies have investigated the genetic polymorphisms for cytokines as potential genetic markers for periodontitis. The aim of this study was to determine the prevalence of IL1 cluster genes polymorphisms and their association with chronic periodontitis in the Macedonian population. The group of 114 unrelated Macedonian subjects with chronic periodontitis and 301 periodontitis-free Macedonian subjects were studied. DNA was isolated from peripheral blood leukocytes by phenol-chloroform extraction method. Cytokine genotyping was performed by PCR-SSP. The population genetics analysis package (PyPop) was used for analysis of the cytokine data for this report. Crude odds ratio (OR) was calculated as estimates of the relative risk with 95 % confidence interval (CI). Genotype frequency of IL1B -511/C:T was significantly higher in patients with periodontitis than in controls (OR=2.11, 95 % CI=1.35-3.32, p=0.001). IL1 cluster gene haplotype frequencies of TTTCT and TCTTT were associated with higher risk for periodontitis (OR=5.06, 95 % CI=1.68-15.26, p<0.0014 and OR=8.35, 95 % CI=1.67-41.69, p<0.002, respectively). No significant association of IL1 composite genotype (IL1 -889A:IL1B +3962) with periodontitis in Macedonians was found. The latter association was found to be significant in genotype IL1B -511/C:T, haplotype TTTCT, and haplotype TCTTT, but without significant association in IL1 composite genotype (Tab. 5, Ref. 43).

Low density lipoprotein size in relation to carotid intima-media thickness in coronary artery disease.

OBJECTIVE: With increasing interest in the role of non-traditional lipid risk factors in coronary artery disease, we undertook this study to relate LDL subclass size and carotid intima-media thickness of the common carotid artery in coronary artery disease patients. METHODS: The study was conducted in 106 patients during their first visit (study group I) and after 12 months (study group II). Intima-media thickness of the common carotid artery was determined using B-mode ultrasound. Separation of LDL subclasses was preformed by 3-31 % polyacrylamide gradient gel electrophoresis. RESULTS: LDL3 was the dominant subclass in both study groups, but there was statistically significant difference in the distribution of dominant LDL subclasses (p<0.01). The mean carotid intima- media thickness was significantly increased (p<0.001) in coronary artery disease patients after 12 months period. There was significant negative correlation between intima-media thickness and LDL size in both study groups (p<0.05). Intima-media thickness was not significantly correlated with plasma lipid concentrations. Multiple regression analyses show that strongest independent predictor of the intima-media thickness variation was diastolic blood pressure, followed by LDL size and age, and accounted for 29 % of the observed variability in intima-media thickness. CONCLUSION: LDL particle size is independently associated with carotid intima-media thickness in coronary artery disease patients with normal levels of traditional lipid risk. These results imply that small, dense LDL subclasses are an important indicator for assessing atherosclerosis and its progression (Tab. 4, Ref. 39).

Distribution of killer cell immunoglobulin-like receptor genes in Roma from Republic of Macedonia.

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, KIR- 2DL4, 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), KIR- 2DS1 (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) among KIR 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.

Analysis of the HLA population data (AHPD) submitted to the 15th International Histocompatibility/Immunogenetics Workshop by using the Gene[rate] computer tools accommodating ambiguous data (AHPD project report).

During the 15th International Histocompatibility and Immunogenetics Workshop (IHIWS), 14 human leukocyte antigen (HLA) laboratories participated in the Analysis of HLA Population Data (AHPD) project where 18 new population samples were analyzed statistically and compared with data available from previous workshops. To that aim, an original methodology was developed and used (i) to estimate frequencies by taking into account ambiguous genotypic data, (ii) to test for Hardy-Weinberg equilibrium (HWE) by using a nested likelihood ratio test involving a parameter accounting for HWE deviations, (iii) to test for selective neutrality by using a resampling algorithm, and (iv) to provide explicit graphical representations including allele frequencies and basic statistics for each series of data. A total of 66 data series (1-7 loci per population) were analyzed with this standard approach. Frequency estimates were compliant with HWE in all but one population of mixed stem cell donors. Neutrality testing confirmed the observation of heterozygote excess at all HLA loci, although a significant deviation was established in only a few cases. Population comparisons showed that HLA genetic patterns were mostly shaped by geographic and/or linguistic differentiations in Africa and Europe, but not in America where both genetic drift in isolated populations and gene flow in admixed populations led to a more complex genetic structure. Overall, a fruitful collaboration between HLA typing laboratories and population geneticists allowed finding useful solutions to the problem of estimating gene frequencies and testing basic population diversity statistics on highly complex HLA data (high numbers of alleles and ambiguities), with promising applications in either anthropological, epidemiological, or transplantation studies.

Distribution of the 22 cytokine gene polymorphisms in healthy Macedonian population.

BACKGROUND: Distribution of cytokine gene polymorphisms may vary significantly among different ethnic groups, and eventually contribute to observed differences in disease frequencies. OBJECTIVES: To genotype 22 cytokine polymorphisms in the Macedonian population. The Macedonian population consists of 301 healthy unrelated individuals. METHODS: Blood samples were collected after written consent, DNA was isolated from peripheral blood, and 22 polymorphisms were typed: IL-1alpha -889, IL-1beta -511, IL-1beta +3962, IL-1R psti1970, IL-1RN mspa11100, IL-4Ralpha +1902, IL-12 -1188, IFNgamma utr5644, TGF-beta1 cdn10, TGF-beta1 cdn25, TNF-alpha -308, TNF-alpha -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 (Heidelberg kit). The population genetics analysis package, PyPop, was used for analysis of the cytokine data. RESULTS: Test of neutrality (Fnd) showed negative value, but was significantly different from 0 for TGF-beta1 1 cdn10 and IFNgamma utr5644 (p of F = 0.001, and 0.012 respectively). Several SNPs (IL-1alpha -889, IL-1beta +3962, IL-2 + 166, IL-4 -1098, IL-4 -590, IL-4 -33, and IL-10 -592) were not in HWP (p 0.005). Test of neutrality for cytokine haplotypes (TGF-beta1, TNFalpha, IL-2, IL-4, IL-6, and IL-10) showed significantly difference from 0 only for IL-2 haplotypes (p=0.020). CONCLUSION: The results of cytokine polymorphisms in Macedonian population can be used for anthropological comparisons, as well as for association studies with different diseases (Tab. 6, Ref. 34). Full Text (Free, PDF) www. bmj. sk.

Low density lipoprotein subclass distribution in children with diabetes mellitus.

BACKGROUND: The role of small dense low-density lipoprotein (sLDL) subclasses in atherosclerosis has been demonstrated in many studies. Among other metabolic changes, the alteration in LDL lipoprotein subclass distribution and size has been proved in diabetic adults. Because there is not enough literature data presenting LDL subclass distribution in childhood, the aim of this study was to examine LDL subclass profile in diabetic children compared with healthy control. MATERIAL AND METHODS: Plasma LDL subclasses in 30 children with type I diabetes mellitus and 100 healthy children aged 9-18 years were analyzed using non-denaturing polyacrilamide gradient (3-31%) gel electrophoresis. Conventional plasma lipid and apoprotein parameters which are thought to affect LDL size were determined as well. RESULTS: Analysis of LDL phenotype has shown that a great percentage of healthy children (89%) yield bigger LDL1 with LDL2 subclasses being dominant (phenotype A), whereas 11 % of the children belong to phenotype B characterized by the presence of small, atherogenic LDL3 and LDL4 subclasses. In diabetic children despite no significant differences in their plasma lipid profile when compared with healthy control, the frequency of LDL phenotype B was increased (86.7 %), and the mean LDL diameter was smaller (p < 0.0001). LDL size was inversely correlated with plasma levels of triglycerides, and positively correlated with plasma HDL cholesterol and BMI. CONCLUSION: Although plasma levels for lipid and apoprotein were within the normal range, the increased frequency of LDL phenotype B confirms a grater risk of atherosclerosis development in children with diabetes mellitus. LDL size measurement may potentially help to assess cardiovascular risk and adapt the treatment goals thereafter (Tab. 3, Ref. 38). Full Text (Free, PDF) www.bmj.sk.

Lipoprotein (a) and apolipoprotein (a) phenotypes in healthy Macedonian children.

BACKGROUND: Plasma levels of lipoprotein(a) [Lp(a)] are determined largely by genetic variation in the gene encoding apolipoprotein(a) [apo(a)], the unique protein component of Lp(a). High plasma levels of Lp(a) increase the risk of premature atherosclerosis. However, the association of apo(a) phenotypes with the development of these diseases remains largely unexplored. OBJECTIVES: Determination of Lp(a) levels and apo(a) isoforms (phenotypes) in 100 (51 boys, 49 girls) Macedonian healthy children aged 9-18. METHODS: We used 3-15 % gradient SDS-PAGE for separation of apo(a) isoforms. According to the different apo(a) electrophoretic mobilities, Apo(a) was classified into five single and respective double-band phenotypes. RESULTS: Each individual expressed a single (homozygotic), double-band (heterozygotic) or no band (null phenotype). The apo(a) phenotype frequencies revealed that the frequency of single-band phenotype expression (64 %) was higher than that of double bands (32 %) and that the frequency of phenotypes representative of low molecular weight was very low (4%). The most frequent phenotype was S4 (42.65%). The distribution of plasma Lp(a) levels was skewed, with the highest frequencies at low levels. The mean Lp(a) concentration was 11.95 (SD of 5.98 and median of 9.62 mg/dL). We did not find differences in the mean and median plasma Lp(a) levels between boys and girls (p > 0.05). A strong inverse relationship was found between the apparent molecular weight of apo(a) phenotypes and plasma Lp(a) concentration (r = -0.4257). CONCLUSIONS: Determination of Lp(a) levels and apo(a) phenotypes in children, may help in preventing and reducing the risk of atherosclerotic development (Tab. 6, Ref 32). Full Text (Free, PDF) www.bmj.sk.

Association of polymorphisms in human platelet antigens with idiopathic thrombocytopenic purpura in Macedonians.

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease characterized by thrombocytopenia due to the presence of platelet autoantibodies specific for platelet membrane glycoproteins, such as GPIIb/IIIa, GPIb/IX and GPIa/IIa. These autoantibodies cause an accelerated clearance of opsonized platelets by phagocytes and inhibition of platelet production. Human platelet antigen (HPA) systems HPA-1, HPA-2, HPA-3 and HPA-5 are components of platelet GP complexes GPIIb/IIIa, GPIb/IX and GPIa/IIa. The HPA system consists of more than 12 bi-allelic antigen polymorphisms in which a base-pair substitution leads to change in an amino acid sequence of a membrane glycoprotein expressed on the platelet surface. The aim of this study was to examine the association of HPA-1, HPA-2, HPA-3 and HPA-5 polymorphisms with idiopathic thrombocytopenic purpura. We performed genotyping of HPA-1, HPA-2, HPA-3, and HPA-5 systems in 60 patients with ITP and 120 healthy participants. Genotyping of HPA-1, -2, -3, and -5 alleles were performed by PCR and RFLP methods by using specific primers and restriction enzymes. Allele and genotype frequencies of HPA-1, HPA-3, and HPA-5 were not significantly different between patients and healthy participants. After Bonferroni adjustment a significant association in ITP patients with HPA-2 alleles (P=0.015, OR=1.923, CI=1.126-3.284) was found. Allele frequencies for HPA-2a were 0.852 in healthy participants and 0.750 in patients, and for HPA-2b 0.148 and 0.250 respectively. These results suggests that HPA-2b allele was more frequent in patients with ITP and may be involved in the formation of a specific autoepitope.

The effect of a small dose of ketamine on postoperative analgesia and cytokine changes after laparoscopic cholecystectomy.

BACKGROUND AND OBJECTIVES: In this study we assesed the effect of a small dose of ketamine on the production of TNFα, IL-1ß and IL-6 and the postoperative pain in patients undergoing laparoscopic cholecystectomy. METHODS: Fifty patients undergoing laparoscopic cholecystectomy were randomized in two equal groups. Patients in the ketamine group after induction in anesthesia received ketamine--025 mg/kg(-1). At the same time patients from the control group received sodium chloride. Postoperatively, the pain was assessed with VAS at periods of 30 min at 1, 2, 4, 8, 18, 24 and 48 hours. TNFα, IL-1ß and IL-6 were evaluated before surgery at 4, 18 and 24h after the operation. RESULTS: Differences of mean values of TNFα and IL-1ß between the two groups in the postoperative period were not significant. Mean values of IL-6 in the investigated group A were significantly lower than the mean values of IL-6 in the investigated group B after the 4th hour (p=0.00990), after the 18th hour (p=0.00133) and as after the 24th hour following surgery (p=000860). the difference in pain intensity according to the VAS scale was also statistically significantly smaller in group A after 30 min, 1,2,8 and 12 hours after surgery. CONCLUSIONS: The addition of a small-dose of ketamine in patiens undergoing laparoscopic cholecystectomy resulted in attenuation of secretion of TNFα, IL-1ß, IL-6 and reduction of postoperative pain.

A case report of aggressive adult neuroblastoma mimicking acute leukemia with fulminant course and fatal outcome.

A case of aggressive adult neuroblastoma mimicking acute leukemia with fulminant course and fatal outcome is described. Pancytopenia and circulating blasts cells at presentation suggested the diagnosis of acute leukemia in the previously healthy 38 years old Caucasian male patient, but flow-cytometry analysis of the bone marrow disclosed the correct diagnosis of neuroblastoma. The immunophenotype was CD45-/CD56+/CD9+ in around 50% of the mononuclear cells, indicating neuroectodermal origin of the malignant cells. Subsequently, the diagnosis was confirmed by immunohistochemical staining of a bone marrow biopsy. A review of the reported cases of neuroblastoma with leukemic features showed that several of them were misdiagnosed as having leukemia and that the diagnosis of neuroblastoma was made at autopsy examination, indicating that misdiagnosis may happen more often than is appreciated. It is in our opinion that the diagnosis of neuroblastoma should be considered in all cases of acute leukemia and pancytopenia, regardless of the age group of the patients.

Gene frequencies of human platelet antigens in the Macedonian population.

Human platelet antigen (HPA) systems consist of more than 12 bi-allelic antigen polymorphisms. Due to these polymorphisms, platelet-membrane glycoproteins can be recognized as alloantigens or autoantigens and can cause conditions such as fetomaternal alloimmune thrombocytopenia, post-transfusion refractoriness to platelets, and post-transfusion throbocytopenic purpura. The purpose of this study was to investigate the distribution of HPA-1, -2, -3, and -5 in Macedonian population by using the polymerase chain reaction and restriction fragment length polymorphism. The allele frequencies were 0.865 for HPA-1a, 0.135 for HPA-1b, 0.852 for HPA-2a, 0.148 for HPA-2b, 0.578 for HPA-3a, 0.422 for HPA-3b, 0.909 for HPA-5a, and 0.091 for HPA-5b. Results of our study were not significantly different from those reported in the other European studies. Our population displayed the highest frequency for HPA-2b allele (0.148) reported among European population.

High-resolution typing of HLA-DRB1 locus in the Macedonian population.

The Macedonian population is of special interest for HLA anthropological study in the light of unanswered questions regarding its origin and relationship with other populations, especially the neighbouring Balkanians. Two studies have been performed to examine HLA molecular polymorphism in the Macedonian population, so far. The present study is the first to be performed in Macedonia using high-resolution sequence-based method for direct HLA typing. The study included 158 unrelated healthy volunteers of Macedonian origin and nationality, having a Christian Orthodox religion. After the simultaneous amplification of exon-2 on both HLA-DRB1 alleles, DNA sequencing was used for genotype assignment. In the 158 samples analysed, all 316 alleles were typed and a total of 29 different DRB1 alleles were detected, with DRB1*1601 being the most frequent allele (14.9%), followed by DRB1*1104 (13.9%). A phylogenetic tree constructed on the basis of the high-resolution data deriving from other populations revealed the clustering of Macedonians together with other Balkan populations (Greeks, Croats, Turks and Romanians) and Sardinians, close to another "European" cluster consisting of the Italian, French, Danish, Polish and Spanish populations. The included African populations grouped on the opposite side of the tree.