Quantitative analysis of the dystrophin gene by real-time PCR

Duchenne and Becker muscular dystrophy (DMD/BMD) are severe X-linked neuromuscular disorders caused by mutations in the dystrophin gene. Our aim was to optimize a quantitative real-time PCR method based on SYBR® Green I chemistry for routine diagnostics of DMD/BMD deletion carriers. Twenty female relatives of DMD/BMD patients with previously detected partial gene deletions were studied. The relative quantity of the target exons was calculated by a comparative threshold cycle method (ΔΔCt). The carrier status of all subjects was successfully determined. The gene dosage ratio for non-carriers was 1.07±0.20, and for carriers 0.56±0.11. This assay proved to be simple, rapid, reliable and cost-effective

Single institute study of FLT3 mutation in acute myeloid leukemia with near tetraploidy in Serbia

Patients with de novo acute myeloid leukemia (AML) and near-tetraploid or completely tetraploid karyotype at presentation are rare. We present four patients with near-tetraploidy/tetraploidy in a cohort of 426 consecutive AML patients (0.98%) in respect to their cytogenetic findings, immunophenotype pattern, response to chemotherapy, course of disease and molecular analyses including tyrosine kinase receptor FLT3 gene, NRAS gene, and tumour suppressor gene, p53. We have found FLT3/ITD mutation only in one patient among the four with near-tetraploidy. The main finding is that these patients had a variable clinical course, with two having a long period of remission (36 and 12 months) and two died, not having achieved remission

Y chromosome microdeletions in infertile male candidates for microfertilization

Uvod. Mikrodelecije Y hromozoma su, posle Klinefelterovog (Klinefelter) sindroma, najčešći genetski uzrok neplodnosti muškaraca. Cilj rada. Cilj istraživanja je bio da se utvrdi učestalost mikrodelecija Y hromozoma kod muškaraca s idiopatskom neplodnošću koji su kandidati za mikrofertilizaciju (Intracytoplasmic Sperm Injection - ICSI), kao i da se ispita korelacija genotipa i fenotipa kod bolesnika sa delecijama Y hromozoma. Metod rada. U studiju je prvobitno uključeno 160 odabranih muškaraca s malim brojem spermatozoida (manje od 5×106 spermatozoida po mililitru ejakulata), međutim, 40 muškaraca je isključeno iz daljeg ispitivanja (kod 10 ispitanika su utvrđeni citogenetski poremećaji, a kod 30 je dijagnostikovan jedan od poznatih uzroka neplodnosti). Kontrolnu grupu ispitanika činilo je 150 muškaraca koji su ostvarili očinstvo bar jednom u poslednje dve godine. Genomska DNK je izolovana iz periferne krvi 120 odabranih ispitanika, a postojanje ili izostanak delecija Y hromozoma analizirano je u dve multipleks reakcije lančanog umnožavanja (Polimerase Chain Reaction - PCR) korišćenjem odgovarajućih prajmera. Rezultati. Izostanak amplifikacije korišćenjem bar jednog para prajmera dokazan je kod 12 ispitanika (10%), dok u kontrolnoj grupi ispitanika nije dokazana nijedna delecija Y hromozoma. Od ukupno 12 otkrivenih delecija, kod devet ispitanika delecija je lokalizovana u regionu AZFc (75%), kod jednog ispitanika je otkrivena u regionu AZFa (8%), dok su kod dva ispitanika delecije zabeležene u regionu AZFbc (17%). Zaključak. Ispitivanje mikrodelecija Y hromozoma treba razmotriti kao važan elemenat genetskog savetovanja infertilnih parova u Srbiji. Odluka o primeni tehnika asistirane reprodukcije treba da bude zasnovana na rezultatima kliničkog pregleda, spermograma, kariotipa, endokrinih ispitivanja, kao i na rezultatima analize mikrodelecija Y hromozoma.Introduction Y chromosome microdeletions are the second most frequent genetic cause of male infertility after Klinefelter's syndrome. Objective The aim of the study was to determine the frequency of Y chromosome microdeletions in a group of infertile men with an idiophatic cause of infertility, candidates for microfertilization (Intra-cytoplasmic Sperm Injection - ICSI) in Serbia and to correlate genotype-phenotype in patients with Y chromosome microdeletions. METHOD One hundred and sixty patients with low sperm count (less than 5x106 spermatozoa/ml) were enrolled in the study. Forty patients were excluded from the study: ten because they were diagnosed with cytogenetic abnormality and thirty patients were diagnosed with other known causes of infertility. The control group consisted of 150 men who fathered at least one child in the last two years. Genomic DNA was extracted from peripheral blood samples and two multiplex polymerase chain reactions (PCR) analyses were performed using specific primers to confirm the presence or absence of Y chromosome microdeletions. Results Microdeletions were detected in 12 of 120 (10%) cases, while no deletions were detected in the control group. Of total number of 12 deletions, nine were detected in AZFc region (75%), one in AZFa (8%), and two in AZFbc (17%). Conclusion Testing for Y chromosome microdeletions should be considered as an important element in diagnosis and genetic counselling of infertile couples in Serbia. Decisions regarding the assisted reproduction should be made based on the detailed clinical, endocrinological and cytogenetic examinations, spermogram, presence or absence and type of AZF microdeletions and CFTR gene mutations

Analysis of association between polymorphisms of MTHFR, MTHFD1 and RFC1 genes and efficacy and toxicity of methotrexate in rheumatoid arthritis patients

A folate analogue methotrexate (MTX) is the most commonly used disease-modifying drug in the treatment of rheumatoid arthritis. However, the clinical response of RA patients treated with MTX shows interindividual differences and 30% of patients discontinue therapy due to the side effects. In a group of 184 RA patients treated with MTX we have investigated whether polymorphisms in MTHFR (rs1801133, rs1801131), MTHFD1 (rs2236225) and RFC1 (rs144320551) genes may have impact on MTX efficacy and/or adverse drugs effects (ADEs). The efficacy of the MTX therapy has been estimated using the disease activity score in 28 joints (DAS28-ESR) based on EULAR criteria and relative DAS28 values (rDAS28) and all adverse drug events were recorded. Patients were genotyped for selected polymorphism by PCR-RFLP method. According to the EULAR response criteria after 6 months of MTX therapy 146 (79.3%) patients were classified as responders, (17 patients (11.6%) were good and 129 patients (88.4%) were moderate responders) and 38 patients (20.7%) as non-responders. ADEs were observed in 53 (28.8%) patients. The majority of ADEs were mild (36 (19.56%) patients) to moderate (12 (6.25%) patients). Five patients (2.7%) had serious ADEs. Association studies have been conducted between obtained genotypes and the efficacy and toxicity of MTX. We have observed no association between polymorphisms and efficacy or toxicity of MTX in RA patients. [Projekat Ministarstva nauke Republike Srbije, br. 175091

Low frequency of NRAS and KRAS2 gene mutations in childhood myelodysplastic syndromes

In children, myelodysplastic syndromes (MDS) represent less then 10% of all hematological malignancies; consequently, molecular genetic studies dealing with this group of patients are scarce. We have analyzed 35 archival bone marrow samples of children with MDS for the presence of mutations in the first and second exons of the NRAS and KRAS2 genes. Mutations were detected with single-strand conformation polymorphism analysis in three patients. One patient harbored a mutation in the second exon of NRAS and two patients in the second exon of KRAS2. Sequencing was performed in two samples and novel mutations were found in both. One patient had a missense mutation in codon 45 of NRAS; the other had a silent mutation in codon 53 and a missense mutation in codon 55 of KRAS2