Keratitis-ichthyosis-deafness Syndrome with Heterozygous p.D50N in the GJB2 Gene in Two Serbian Adult Patients.

Purpose: Keratitis-ichthyosis-deafness (KID) syndrome is a rare congenital ectodermal dysplastic syndrome presenting with keratitis, ichthyosis and sensorineural hearing loss. The most common causes of KID syndrome are heterozygous missense mutations in the GJB2 gene that codes for connexin 26. Case report: During the ophthalmological examination, two adult females complained of recent worsening of visual acuity in both eyes. Anamnesis revealed that their eyes were red and irritated from early childhood onwards. Both of them had thickening and keratinisation of eyelid margins, lash loss, diffuse opacification of cornea and conjunctiva caused by keratinisation of eye surface, superficial and deep corneal vascularisation and corneal oedema. Partial sensorineural hearing loss and difficulties in speech were also noted along with typical ichthyosiform erythroderma. Genetic testing of the GJB2 gene revealed a heterozygous p.D50N mutation in both patients.Patients were treated with a combined topical corticosteroid and artificial tears therapy, with steroid therapy being intensified during the last month. The therapy increased the visual acuity by decreasing corneal oedema and by forming a more regular air-tear interface during the six months follow up. Subsequently, the disease progressed despite the continuation of the therapy. Conclusion: This is the first report of Serbian patients with KID syndrome. Despite the administration of the combined topical corticosteroid and artificial tears therapy the disease is relentlessly progressive and therapeutic success of ophthalmological signs with local therapeutic modalities used so far had been disappointing.

NOS3 gene variants and male infertility: Association of 4a/4b with oligoasthenozoospermia.

Results of recent studies confirmed that oxidative stress negatively affects sperm motility and causes sperm DNA damage. Produced by nitric oxide synthase 3 (NOS3), nitric oxide is considered to be one of the important mediators of oxidative stress in testis tissue. The aim of this study was to assess the possible association of three genetic variants (rs2070744, rs1799983 and intron variant 4a/4b) in NOS3 gene and infertility occurrence in two groups of infertile men (idiopathic azoospermia and oligoasthenozoospermia) and fertile controls. Genotypes for the single-nucleotide genetic variants rs1799983 and rs2070744 were determined by PCR-RFLP, while genotyping of intron 4 variant 4a/4b was performed by gel electrophoresis of PCR products. Statistical analysis was performed by SNPStats software. No significant association between the three genetic variants of the NOS3 gene and infertility risk was determined comparing allele and genotype frequencies among group of patients diagnosed with azoospermia and the control group. Nevertheless, there was a significant positive association between 4a/4b and infertility in the group of males diagnosed with oligoasthenozoospermia, under overdominant genetic model. Our findings suggest that tandem repeat variant within intron 4 of the NOS3 gene is associated with an increased risk of infertility in men diagnosed with idiopathic oligoasthenozoospermia.

Molecular genetic and clinical characterization of myotonic dystrophy type 1 patients carrying variant repeats within DMPK expansions.

Myotonic dystrophy type 1 (DM1) is caused by a highly unstable expansion of CTG repeats in the DMPK gene. Its huge phenotypic variability cannot be explained solely by the repeat number. Recently, variant repeats within the DMPK expansions have emerged as potential disease modifiers. The frequency of variant expanded alleles was estimated in 242 DM1 patients from 174 Serbian families using repeat-primed PCR (RP-PCR). The patterns of variant repeats were determined by direct sequencing of RP-PCR or PCR products. PCR-based southern blot was performed to get insight into the intergenerational mutational dynamics of variant expanded alleles. All patients carrying variant repeats were clinically re-examined. Variant repeats were observed in eight patients from five families (2.9%). They were detected only at the 3' end of DMPK expansions. CCG variant repeats were present in seven patients, either as a part of regular runs of CCGCTG hexamer, individual repeats, or CCG blocks. Analyses of three intergenerational transmissions revealed a considerable stability or likely a contraction of variant expanded alleles. Intriguingly, a decrease in age at onset accompanied these transmissions. Overall, patients were characterized by a milder phenotype and/or some atypical symptoms that could be rather clinically suggestive of myotonic dystrophy type 2. In addition, the first case of de novo CTC variant repeat was observed. Variant repeats might explain a part of the phenotypic variability in a small percent of DM1 patients and likely display a stabilizing effect on the meiotic instability of DMPK expanded alleles.

Genetic variants in RNA-induced silencing complex genes and prostate cancer.

PURPOSE: The purpose of this study is to evaluate the potential association between genetic variants in genes encoding the components of RNA-induced silencing complex and prostate cancer (PCa) risk. Genetic variants chosen for this study are rs3742330 in DICER1, rs4961280 in AGO2, rs784567 in TARBP2, rs7813 in GEMIN4 and rs197414 in GEMIN3. METHODS: The study involved 355 PCa patients, 360 patients with benign prostatic hyperplasia and 318 healthy controls. For individuals diagnosed with PCa, clinicopathological characteristics including serum prostate-specific antigen level at diagnosis, Gleason score (GS) and clinical stage were determined. Genotyping was performed using high-resolution melting analysis, PCR-RFLP, TaqMan SNP Genotyping Assay and real-time PCR-based genotyping assay using specific probes. Allelic and genotypic associations were evaluated by unconditional linear and logistic regression methods. RESULTS: The study provided no evidence of association between the analyzed genetic variants and PCa risk. Nevertheless, allele A of rs784567 was found to confer the reduced risk of higher serum PSA level at diagnosis (P = 0.046; Difference = -66.64, 95 % CI -131.93 to 1.35, for log-additive model). Furthermore, rs4961280, as well as rs3742330, were shown to be associated with GS. These variants, together with rs7813, were found to be associated with the lower clinical stage of PCa. Also, rs3742330 minor allele G was found to be associated with lower PCa aggressiveness (P = 0.036; OR 0.14, 95 % CI 0.023-1.22, for recessive model). CONCLUSIONS: According to our data, rs3742330, rs4961280 and rs7813 qualify for potentially protective genetic variants against PCa progression. These variants were not shown to be associated with PCa risk.

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.

Expression of programmed cell death proteins in patients with chronic myeloid leukemia.

PURPOSE: Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease developing out of pluripotent hematopoietic stem cells that contain the fusion Bcr-Abl gene. The mechanisms that lead to these changes at molecular level are still unknown as are the mechanisms that increase the proliferative capacity of these cells. Disorders that occur in the process of apoptosis represent one of the possible molecular mechanisms that bring about disease progress. In our study we analyzed the presence of mutated (mut) p53 gene and the amplification of Bax proteins in patients with CML. PATIENTS AND METHODS: This study included 30 patients with CML (23 in chronic phase, 7 in blast transformation). Using immunohistochemistry with alkaline phosphatase / anti-alkaline phosphatase (APAAP) method we analyzed the expression of cell death proteins p53 and Bax in mononuclear bone marrow cells. Polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) method was used to analyze the presence of mut p53 gene in mononuclear peripheral blood cells. Reverse transcription-polymerase chain reaction (RT-PCR) method was used to analyze the presence of Bcr-Abl in peripheral blood cells. RESULTS: High expression of Bax protein was detected in all analyzed patients, but no significant differences were noticed among them. No mut p53 gene was detected in any of the analyzed samples. Bcr-Abl b3a2 protein form was detected in all patients with variant translocations. CONCLUSION: Lack of mut p53 product in the peripheral blood and bone marrow cells in patients with CML suggests that this gene plays no important role in disease pathology. Increased level of Bax protein expression is an essential characteristic of CML cells but it is not related with the clinical stage of disease.

[In vivo study of spontaneous and therapy-induced apoptosis in patients with chronic lymphocytic leukemia treated with chlorambucil]. / In vivo pracenje spontane i terapijom indukovane apoptoze kod bolesnika sa HLL lecenih hlorambucilom.

The efficiency of Chlorambucil in the induction of apoptosis was investigated in the study, and measurable apoptosis parameters were compared to the other prognostic factors with the aim of possible prediction of clinical response to the therapy in the patients with CD5 + B-cell chronic lymphocytic leukemia (B-CLL). Seven newly diagnosed patients, initially treated with daily high-doses of Chlorambucil (HD-CLB) were analyzed. Quantitative analysis of apoptosis parameters on semi-fine sections obtained from peripheral blood was performed prior and during the first five days of therapy. The level of spontaneous apoptosis (SA) was determined, as well as the maximal response by apoptosis (MAR), and the time needed to establish maximal response by apoptosis (TMAR), respectively. The results revealed that the level of SA in the studied group of patients was 11.39%-20.50%. In three patients with achieved criteria for complete remission (CR) was observed high level of SA, TMAR 2-4 days and MAR 23.42-26.36%, respectively. All patients with CR were with negative LDT, non-diffuse involvement of bone marrow and clinical stage B. Criteria for partial remission (PR) were achieved in 4 patients. Within this group, all three measurable parameters of apoptosis could have been determined in only one patient, while in the rest was noticed the increased percentage of apoptotic cells on the last day of follow-up. In all patients was observed negative LDT, diffuse bone marrow involvement, and 2 out of 4 patients had CLPL of cytomorphological type and clinical stage B. By comparing the obtained values of measurable apoptotic parameters with the clinical response to the applied therapy with HD-CLB, it is possible to divide our patients into two groups: patients who have achieved CR have the highest percentage of cells dying due to the therapy-induced apoptosis, as well as the higher values of measurable parameters compared to the certain parameters of the patients with the criteria for PR. Our preliminary results of therapeutic response to the apoptosis might be useful for the timely decision upon the duration of therapy and change of modality of treatment for every patient during the follow-up period.

Fungus-like hyphochytrids associated with human disease.

We report two cases, with liver and brain abscess, respectively, where fungus-like organisms belonging to the Hyphochytriomycota were found at the site of inflammation together with Peptococcus in the first and Cysticercus cellulosae in the second case. This is the first time these groups of organisms have been reported in human material. The role of hyphochytrids in human pathology remained uncertain as they were found together with already known human pathogens.