Goran Brajušković

Molecular genetics and genetic testing in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is the most common adult onset muscular dystrophy, presenting as a multisystemic disorder with extremely variable clinical manifestation, from asymptomatic adults to severely affected neonates. A striking anticipation and parental-gender effect upon transmission are distinguishing genetic features in DM1 pedigrees. It is an autosomal dominant hereditary disease associated with an unstable expansion of CTG repeats in the 3′-UTR of the DMPK gene, with the number of repeats ranging from 50 to several thousand. The number of CTG repeats broadly correlates with both the age-at-onset and overall severity of the disease. Expanded DM1 alleles are characterized by a remarkable expansion-biased and gender-specific germline instability, and tissue-specific, expansion-biased, age-dependent, and individual-specific somatic instability. Mutational dynamics in male and female germline account for observed anticipation and parental-gender effect in DM1 pedigrees, while mutational dynamics in somatic tissues contribute toward the tissue-specificity and progressive nature of the disease. Genetic test is routinely used in diagnostic procedure for DM1 for symptomatic, asymptomatic, and prenatal testing, accompanied with appropriate genetic counseling and, as recommended, without predictive information about the disease course. We review molecular genetics of DM1 with focus on those issues important for genetic testing and counseling.

Endothelial nitric oxide synthase gene polymorphisms and prostate cancer risk in Serbian population

Genome‐wide association studies (GWAS) have identified over 46 SNPs associated with human prostate cancer (PCa). Some studies have shown correlation of the nitric oxide synthase (NOS) NOS3 gene polymorphisms with the risk and/or progression of PCa. This study aimed to evaluate the association of NOS3 gene polymorphisms (−786T>C, −764A>G, −714G>T, −690C>T, −649G>A and 894G>T) with PCa risk and progression. 150 patients with PCa, 150 patients with BPH and 100 age‐matched healthy controls were recruited in this study. Genotyping of promoter polymorphisms was performed by bi‐directional DNA sequencing, and for 894G>T by RFLP analysis. There was no significant association between the alleles and genotypes of these genetic variants and PCa risk. For −786T>C polymorphism, we found that C allele is associated with absence of metastases, assuming dominant genetic model (P = 0.049; OR, 0.50; 95% CI, 0.25–1.00). It was found that, compared with NOS3 −690C>T variant CC genotype, CT and TT genotypes confer decreased risk of developing metastases (dominant model, P = 0.015, OR, 0.24; 95% CI, 0.07–0.88) and show association with low clinical tumour stage, compared with stages T3 and T4 (dominant model, P = 0.046, OR, 0.20; 95% CI, 0.04–1.02). Genetic variants −764A>G, −714G>T, −649G>A were not detected in our study group. There is evidence of an inverse correlation of the NOS3 894G>T minor allele with high serum PSA (>20 ng/ml) (dominant model, P = 0.013, OR, 0.37; 95% CI, 0.17–0.82). Our results suggest that NOS3 gene polymorphisms are genetic susceptibility factors for the progression of PCa and patient outcome.

Assessment of association between genetic variants in microRNA genes hsa-miR-499, hsa-miR-196a2 and hsa-miR-27a and prostate cancer risk in Serbian population.

Due to their potentially functional significance, genetic variants within microRNA genes have been recognized as candidates for cancer-related genetic biomarkers. Among the most extensively studied so far are rs3746444, rs11614913 and rs895819. Nevertheless, only few previous studies in Asian population analyzed the association of rs3746444 and rs11614913 with prostate cancer (PCa) risk, while rs895819 was not evaluated in relation to this issue. The aim of this study was to assess the possible association between these genetic variants and PCa risk and progression in Serbian population. 355 samples of peripheral blood were obtained from the patients with PCa and 353 samples from patients with benign prostatic hyperplasia (BPH). 312 volunteers derived from general population who gave samples of buccal swabs were included in the control group. Genotyping of rs3746444, rs11614913 and rs895819 was performed by using PCR-RFLP method, HRM analysis and allele-specific PCR, respectively. Allelic and genotypic associations were evaluated by unconditional linear (for serum PSA level in PCa patients) and logistic regression method with adjustment for age. Minor allele C of rs895819 was found to be associated with the increased risk of developing PCa under dominant (P=0.035; OR=1.38, 95%CI 1.02-1.86) and overdominant (P=0.04; OR=1.37, 95%CI 1.01-1.85) genetic model. Same genetic variant was found to be associated with the clinical stage of localized PCa, as well as with the presence of distant metastases. Allele G of rs3746444 was also shown to be associated with the decreased risk of PCa progression. According to our data, rs3746444 qualifies for a genetic variant potentially associated with PCa aggressiveness in Serbian population. Furthermore, our study provided the first evidence of association between rs895819 and PCa risk, as well as for its genetic association with the presence of distant metastases among PCa patients.

Joint effect of ADARB1 gene, HTR2C gene and stressful life events on suicide attempt risk in patients with major psychiatric disorders

Abstract Objectives. Adenosine to inosine RNA editing, serotonin 2C receptor (HTR2C), and stressful life events (SLEs) have all been implicated in suicidal behaviour. We examined the main and moderating effects of RNA editing (ADAR, ADARB1) and HTR2C genes, childhood trauma (CT), recent SLEs and psychiatric disorders as contributors to suicide attempt (SA) vulnerability. Methods. Study included 165 suicide attempters and 188 suicide non-attempters, all diagnosed with one of major psychiatric disorders. CT and recent SLEs were assessed using Early Trauma Inventory–Self Report and List of Threatening Experiences Questionnaire, respectively. Selected ADAR and ADARB1 tag-variants, and HTR2C rs6318 were pre-screened for association with SA, while generalized linear models and backward selection were applied to identify individual and interacting SA risk factors. Results. ADARB1 rs9983925 and rs4819035 and HTR2C rs6318 were associated with SA. The best minimal model found emotional abuse, recent SLEs, rs9983925 and rs6318 as independent SA risk factors, and general traumas as a factor moderating the effect of psychiatric disorders and emotional abuse. Conclusions. SA vulnerability in psychiatric patients is related to the joint effect of ADARB1 and HTR2C variants, the existing mood disorder and the cumulative exposures to a various childhood and recent stressful experiences.

Bcl-2 and Bax interaction in B-lymphocytes of peripheral blood in patients with chronic lymphocytic leukemia

Background. Chronic lymphocytic leukemia (CLL) is a neoplastic disease characterized by the accumulation of morphologically mature monoclonal CD5+ B cells in the early phase (G0/G1) of the cell cycle. The accumulation of neoplastically transformed B-lymphocytes (CLL cells) is primarily the consequence of apoptosis blocking in these cells. Bcl-2 proteins are well-known modulators of this process. Some of these proteins are anti-apoptotic while the others are pro-apoptotic. All contain at least one of the four conserved regions called the Bcl-2 homologous domains (BH1-BH4). Evidence indicates that Bcl-2 and Bax form homo- and heterodimers. The anti-apoptotic effect of Bcl-2 protein is based on its ability to bind Bax protein in the heterodimer form, and thus to block the forming of Bax/Bax proapoptotic homodimers. The ratio of Bcl-2/Bax represents the cell autonomous rheostat which determinates the type of the cell reaction to an apoptotic stimulus. Methods. The aim of this study was to determine the level of interaction between these two proteins in CLL cells using the co-immunoprecipition method. The study included the analysis of 20 peripheral blood specimens from 20 patients with CLL, and 20 peripheral blood specimens from healthy persons, who were in the control group. Specimens were precipitated with the monoclonal antibody for Bcl-2 protein, and immunoblotted with the palyclonal antibody for Bax protein (IP: Bcl-2/WB:Bax). At the same time, specimens were precipitated with the polyclonal antibody for Bax protein, and immunoblotted with the monoclonal antibody for Bcl-2 protein (IP: Bax/WB:Bcl-2). The intensity of Bcl-2 and Bax proteins binding compared to the control samples of the peripheral blood from healthy persons, was increased in CLL cells. Results. IP: Bax/WB: Bcl-2 showed a high level of “free“ Bcl-2 protein which was not bound in the heterodimer form to Bax protein. Simultaneously IP: Bcl- 2/WB: Bax showed that a higher quantity of Bax protein was bound in the heterodimer form to Bcl-2 protein as opposed to the quantity of pro-apoptotic Bax protein potentially bound in the homodimer form. Conclusion. Further studies involving larger groups of patients are necessary to explore the potential significance of the Bcl-2/Bax protein ratio as a prognostic parameter in the CLL treatment.

Association between a Genetic Variant in the hsa-miR-146a Gene and Cancer Risk: An Updated Meta-Analysis

Background and Aims: A large number of studies have investigated the association between the potentially functional genetic variant rs2910164 located in the hsa-miR-146a gene and susceptibility to various types of cancer. Nevertheless, the results obtained in these studies are contradictory. Therefore, we conducted a meta-analysis of data from eligible reports comprising a total of 28,359 cases and 41,678 controls. Methods: The literature included in this meta-analysis was selected from the PubMed database. Quantitative data synthesis was performed by using the OpenMeta-analyst software. Results: The meta-analysis yielded no evidence of an association between rs2910164 and the overall cancer risk. Conversely, the C allele of this genetic variant was found to be associated with a decreased risk of developing bladder and cervical cancer in multiple genetic models. The same direction of association was found for the C allele and liver cancer, gastric cancer and oral squamous cell carcinoma risk. In contrast to these results, the same allelic variant of rs2910164 was found to confer an increased risk of developing lung cancer. The stratified meta-analysis based on ethnicity did not show significant differences in the association between rs2910164 and cancer risk in populations with different ethnic backgrounds. Conclusion: We conclude that rs2910164 may represent a valuable biomarker associated with the risk of developing specific types of cancer.

Genetic Variants within Endothelial Nitric Oxide Synthase Gene and Prostate Cancer: A Meta‐Analysis

Several variants within gene‐encoding endothelial isoform of nitric oxide synthase have been reported to confer prostate cancer (PCa) susceptibility and/or progression. Nevertheless, studies referring to this issue have yielded inconsistent results. In order to elucidate the involvement of these variants in prostate carcinogenesis, we have conducted a meta‐analysis of previously published case‐control and relevant case‐only studies. Eleven studies comprising in total 3,806 cases and 4,466 controls were included in the meta‐analysis which yielded evidence of association of rs2070744 (ORCC = 1.43, 95% CI 1.04–1.97; p = 0.03) and intron 4a/b variant (ORab+aa = 1.47, 95% CI 1.00–2.14; p = 0.05) with PCa risk under recessive and dominant model, respectively. Furthermore, PCa patients carrying 4a/b a allele were found to have an increased risk of cancer progression to a less differentiated form, characterized by a high Gleason score (OR = 2.29, 95% CI 1.51–3.49; p < 0.01) and to higher TNM stage (OR = 2.55, 95% CI 1.71–3.81; p < 0.01). These results support the involvement of NOS3 variants in molecular pathogenesis of PCa.

Assessment of association between common variants at 17q12 and prostate cancer risk-evidence from Serbian population and meta--analysis.

This study aimed to evaluate possible association between genotypes and alleles of two 17q12 polymorphisms (rs3760511 and rs7501939) and prostate cancer (PCa) risk and progression. Two hundred seventy‐one patients with PCa, 261 patients with benign prostatic hyperplasia (BPH), and 171 controls were included in the study. Single nucleotide polymorphisms (SNPs) were genotyped by using PCR followed by restriction fragment length (PCR‐RFLP) analysis. We conducted meta‐analysis of published studies regarding association of these SNPs with PCa risk. Evidence of positive association between the AC genotype of the SNP rs3760511 and BPH risk for the best‐fitting overdominant model of association (BPH vs. controls comparison, p = 0.026; odds ratio [OR] = 1.58; 95% confidence interval [95%CI] 1.05–2.36) were obtained. The association between T allele of rs7501939 and PCa risk was determined in PCa versus controls comparison (p = 0.0032; OR = 0.66, 95%CI 0.50–0.87) with the best‐fitting model of inheritance being log‐additive. This variant was also found to be associated with the risk of BPH (p = 0.0023; OR = 0.65, 95%CI 0.49–0.86). We found no association between parameters of PCa progression and the analyzed SNPs. Meta‐analysis showed strong association between these variants and PCa risk. Our study shows association between SNPs at locus 17q12 and the risk of prostatic diseases in Serbian population. At the same time, results of meta‐analysis suggest the association of these SNPs with PCa risk.

[Expression of Bcl-2 protein and the amplification of c-myc gene in patients with chronic myeloid leukemia].

BACKGROUND/AIM Chronic myeloid leukemia (CML) represents a malignant myeloproliferative disease developed out of pluripotent hematopoietic stem cell that contains the fusion bcr-abl gene. Disorders that occur in the process of apoptosis represent one of the possible molecular mechanisms that bring about the disease progress. The aim of our study was to carry out the analysis of the presence of the amplification of the c-myc oncogene, as well as the analysis of the changes in the expression of Bcl-2 in the patients with CML. METHODS Our study included 25 patients with CML (18 in chronic phase, 7 in blast transformation). Using an immunohistochemical alkaline phosphatase-anti-alkaline phosphatase (APAAP) method, we analyzed the expression of cell death protein in the mononuclear bone marrow cells of 25 CML patients. By a differential PCR (polymerase chain reaction) method, we followed the presence of amplified c-myc gene in mononuclear peripheral blood cells. RESULTS The level of the expression of Bcl-2 protein was considerably higher in the bone marrow samples of the patients undergoing blast transformation of the disease. The amplification of c-myc gene was detected in 30% of the patients in blast transformation of the disease. CONCLUSION The expression of Bcl-2 protein and the amplification of c-myc gene are in correlation with the disease progression.

Tryptophan Hydroxylase 1 Variant rs1800532 is Associated with Suicide Attempt in Serbian Psychiatric Patients but does not Moderate the Effect of Recent Stressful Life Events

Tryptophan hydroxylase 1 (TPH1) gene, coding for serotonin synthesizing enzyme, and recent stressful life events (SLEs) have been commonly associated with suicidal behavior. TPH1 has been also hypothesized to be involved in stress-response mechanisms. The aim of this study was to assess TPH1 variant rs1800532 and its possible interaction with recent SLEs as risk factors for suicide attempt (SA) in Serbian psychiatric patients, including 165 suicide attempters and 188 suicide nonattempters. rs1800532 and recent SLEs were independently associated with SA, while rs1800532 did not moderate the effect of recent SLEs on SA vulnerability among Serbian psychiatric patients.