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Kvantitativna fluorescentna verižna reakcija s polimerazo (QF-PCR) je uveljavljena metoda za usmerjeno, hitro, zanesljivo in poceni določanje najpomembnejših kromosomskih anomalij v prenatalni diagnostiki. To so trisomije kromosomov 13, 18 in 21, ki jih v Laboratoriju za medicinsko genetiko UKC Maribor rutinsko določamo od leta 2008 z lastno razvitim testom. Določanje številčnih sprememb kromosomov X in Y je del QF-PCR testa, ko se ta izvaja kot samostojni test. Trenutno veljavne smernice britanskega združenja za klinično citogenetiko priporočajo usmerjeno diagnostiko oz. preiskovanje prenatalnih vzorcev z metodo QF-PCR ob utemeljenem sumu na številčne anomalije spolnih kromosomov. V prispevku predstavljamo metodologijo in verifikacijo določanja številčnih anomalij kromosomov X in Y z uporabo mikrosatelitnih genetskih označevalcev z metodo QF-PCR. Gre za razširitev zmožnosti QF-PCR analize lastnega razvoja, ki vključuje pripravo optimiziranih kombinacij dodatnih devet začetnih oligonukleotidov, specifičnih za kromosoma X in/ali Y za izvajanje hkratne verižne reakcije s polimerazo. Za verifikacijo smo uporabili 23 predhodno citogenetsko opredeljenih vzorcev s prisotnimi različnimi oblikami odstopanj v številu kromosomov X in Y. Predstavljeni so tudi rezultati verifikacije metode, ki je pokazala 100% ujemanje rezultatov.

Kvantitativna verižna reakcija s polimerazo (QF-PCR) z uporabo markerjev specifičnih za kratka ponavljajoča se zaporedja (STR-short tandem repeat), značilna za določen kromosom, je uspešna metoda za hitro diagnosticiranje najpogostejših kromosomskih aneuploidij v prenatalnem obdobju. Namen našega prispevka je predstaviti omenjeno metodo in analizirati naše rezultate po treh letih uporabe QF-PCR kot hitrega testa pri iskanju najpogostejših prenatalnih kromosomskih sprememb: trisomije kromosoma 21, 18, in 13. Genomsko DNA za QF-PCR analizo smo pripravili iz vzorcev amnijskih celic, horionskih resic ali placentarnih celic, ki smo jih pridobili od nosečnic s povišanim tveganjem za rojstvo otroka z katero od najpogostejših prenatalnih kromosomopatij. Za testiranje s QF-PCR metodo smo uporabili 20 različnih STR začetnih oligonukleotidov, specifičnih za kromosome 13, 18, 21, Y ter amelogeninski X/Y alel. Pri 63.8% vzorcev je obenem bila opravljena še citogenetska analiza v našem laboratoriju zato imamo možnost primerjave obojih rezultatov. V triletnem obdobju smo opravili 243 analiz prenatalnih vzorcev: iz amnijskih celic 72.3%, horionskih resic 27.2%, v dveh primerih smo kot vzorec uporabili celice placente. Pri 7.8% vzorcev smo s QF-PCR našli kromosomsko anevploidijo, najpogostejša je bila trisomija kromosoma 21 (3.7%).

Purpose: The CYP21A2 gene encodes the enzyme 21–hydroxilase, which is responsible for the production of steroids. These hormones are key mediators of sexual development and conception. Patients with 21–hydroxilase deficiency tend to be affected in different stages of life. The purpose of this study was to compare the genetic profiles of women with unexplained infertility problems with the genetic profiles of healthy controls. Furthermore, were analyzed associations between mutations of the CYP21A2 gene and various clinical and laboratory parameters. Methods: We enrolled 300 women, diagnosed with unexplained infertility problems, into this retrospective study. For each subject, we recorded important clinical and laboratory parameters during different phases of the menstrual cycle. In the control group, we enrolled 100 samples. Each subject provided a blood sample, which was used to isolate DNA for subsequent polymerase chain reaction (PCR). Results: In total, 11.6% of the test subjects exhibited significant hormonal deviations (HD) (estrogens and/ or progestins and/or total testosterone) and 5.3% were diagnosed with polycystic ovarian syndrome (PCOS). We identified a significant association between the p.V281L mutation and the frequency of test subjects with significant HD (x2=6.99, p=0.01). A similar association was also observed between p.V281L mutation and the frequency of test subjects with PCOS (x2=16.78, p=0.00). However, we did not establish any associations between the frequency of mutations in test subjects when compared with controls. In addition, we did not find any significance in the frequency of CYP21A2 gene mutations and any of the laboratory parameters tested. Conclusion: Our results identify a significant association of the p.V281L mutation in the CYP21A2 gene with the frequencies of both PCOS and significant HD.

Purpose: The VKORC1 polymorphism is an important genetic factor affecting warfarin dose requirement. Patients require different warfarin doses in order to achieve the target therapeutic anticoagulation. The aim of our study was to determine the frequency of single nucleotide polymorphisms (SNP) in the VKORC1 gene in the general population, using a simple, rapid, and economical method. Methods: For genotyping, the restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)–amplified DNA was used and compared to allele– specific polymerase chain reaction. We genotyped 441 DNA samples obtained from the healthy general population in North–Eastern Slovenia. Genotypes for the tested group were evaluated to determine whether the population followed the Hardy–Weinberg equilibrium. The genotypes and allele frequencies were calculated. Results: The results obtained using the allele–specific polymerase chain reaction were consistent with those obtained using the PCR + RFLP method. The G allele frequency (0.62) was higher than the A allele frequency (0.38) in the general population from North–Eastern Slovenia. Conclusions: The PCR+RFLP method involved additional manipulation of the PCR products at the expense of analysis time, consumption of reagents and equipment. The allele–specific polymerase chain reaction was a simple and rapid method for the detection of SNP in the VKORC1 gene, and is available in any laboratory with the minimum of equipment and reagents required.

Purpose: About 10-15% of all pregnancies terminate as spontaneous miscarriages. In the first trimester, ≈50% of spontaneous miscarriages are the result of chromosomal aberrations, mostly chromosomal aneuploidies. Cytogenetic analyses are used to confirm aneuploidy in failed pregnancies. Culture failure or poor-quality chromosomes are often problems in those cases. In such situations, methods that are independent of tissue culture are used, and we employed multiplex ligation-dependent probe amplification (MLPA). We determined if MLPA is an appropriate and compatible method compared with classical cytogenetic analyses on fetal tissues. Methods: All fetal samples received from spontaneous abortions were cultured, karyotyped (if possible) and genomic DNA extracted. MLPA analyses were undertaken using subtelomeric probe kits. Additionally, comparative genomic hybridization (CGH) was used to confirm aneuploidy detected by MLPA in cases of failed culture growth. Results: MLPA analyses confirmed an unbalanced chromosome abnormality identified by cytogenetic analyses in all cases in which tissue culture was successful, and provided data in cases of failed culture growth. Several common numeric chromosome aberrations were detected, as well as rare trisomies and other unbalanced chromosome rearrangements. Conclusions: MLPA analyses can provide information about the karyotype of a DNA sample if cytogenetic analyses are not possible because of a lack of viable cells or if only a small amount of genomic DNA is available. Th

Purpose: The objective of this study was to introduce the GeneXpert Ultra cartridge-based BCR-ABL diagnostic test into routine molecular monitoring of minimal residual disease for chronic myeloid leukemia (CML) patients who are on tyrosine kinase inhibitors (TKIs).Methods: BCR-ABL1 transcript quantification was performed on 100 peripheral blood samples from 33 CMLpatients at different stages of the disease using automated GeneXpert BCR-ABL Ultra assay and following a standardized laboratory protocol (SP).Results: Analysis results were expressed as % on the International Scale (IS) for BCR-ABL1 real-time PCR quantification and estimated using molecular response (MR) according to the laboratory recommendations for scoring deep MRs following CML treatment. A comparison of the two methods showed that there was no statistically significant difference in the results expressed as % on the IS (p = 0.098). Samples were divided into six groups (MR1, MR2, MR3, MR4, MR4.5, and MR5) according to the estimated MR. A total of 74/100 samples had a completely identical MR with both methods, 12/100 samples had a higher MR obtained with GeneXpert, and 14/100 samples had a lower MR compared to the SP. In addition, 2/74 samples had a different MR for two classes obtained with GeneXpert compared to the SP, while the others 72/74 samples differed in one MR class. Analysis of the major molecular response resulted in 82% agreement. Minimal amounts of BCR-ABL1 copies were detected with GeneExpert in eight samples while the SP did not detect them. None of the GeneXpert results had any effect on the clinical decision for treatment with TKIsobtained using the SP.Conclusion: The Xpert BCR-ABL Ultra assay can be considered a useful and very fast clinical tool for molecular follow up of patients on TKIs.

Purpose: To evaluate the diagnostic accuracy of the UroVysion FISH test for detecting transitional cell bladder cancer on a mixed sample of urological patients. Methods: Urine samples were collected from patients before cystoscopy. In the case of tumor identification, transurethral resection and histological verification were performed. The fluorescent in situ hybridisation (FISH), using a commercial kit (UroVysion) containing hybridization probes for chromosomes 3, 7, 17, and 9p21, was used. At least 25 morphologically abnormal cells or all cells present on the slides were analyzed. Results: Of 179 samples, 1 was infected and in 35 (20%), no cells were identified. Test sensitivity was 76.2% (95% CI 52.8–91.8) and specificity 93.6% (95% CI 88.6–96.9); as compared to the data in the literature, this result was considered significant. Most tumors had numerical chromosome aberrations. The prevalence of histologically, which verified tumors, was 11.8%. Conclusion: Price and time–consuming procedures are major obstacles for use of the UroVysion FISH test. However, neither cytology nor UroVysion FISH are 100% specific. Higher sensitivity compared to cytology, which is evident not only in case of superficial, but also of invasive tumors (like T1) and its ability to provide numerical results, are advantages which may make UroVysion FISH test useful. Thus, this test has potential for future use. cells were identified. Test sensitivity was 76.2% (95% CI 52.8–91.8) and specificity

Objectives To evaluate the impact of prenatal screening and genetic testing for trisomy 21 (T21) on the prevalence of T21 in Slovenia. Design and setting Data about all prenatally and postnatally confirmed cases of T21 in Slovenia between 1981 and 2012 were collected retrospectively from all genetic laboratories in Slovenia. The expected number of babies with T21 according to maternal age was calculated. Main outcome measures The primary outcomes measures were number of fetuses and newborn infants with T21 diagnosed prenatally and postnatally and the impact of advances in screening and genetic diagnostics on the prevalence of newborns with T21 in Slovenia. Results Despite a significantly increased mean maternal age from 25.4 years in year 1981 to 30.3 years in year 2012 the prevalence of newborn infants with T21 was 0.51 per 1000 births compared to 0.55 per 1000 births, respectively. The prevalence of prenatally diagnosed cases increased from 0.03 per 1000 births to 2.06 per 1000. The detection rate of T21 in year 2012 was 78,9%. The total number of prenatal invasive procedures (chorionic villous sampling and amniocenteses) carried out during that period was rising until 2002, since when it is stable at around 7%. Conclusion The advancement and implementation of screening tests and prenatal diagnostic procedures in Slovenia caused an important improvement in the efficiency of the prenatal detection of T21.

Objective To evaluate the rate and type of aneuploidies of chromosomes 13, 16, 18, 21 and 22, with respect to the length of in vitro maturation (IVM) period, and to compare the results to previously published studies on aneuploidy rates of unfertilized, uninseminated mature oocytes and first polar bodies. Study design Two hundred and twelve immature germinal vesicle stage oocytes were assigned to two groups. After successful IVM, depending on their maturational period of 24 h (Group A) or 36 h (Group B), chromosomal analysis was performed by five color fluorescence in situ hybridization (FISH). In Groups A and B the rates of aneuploid oocytes were calculated and compared by chi-square test. Also the rates of hyperhaploidy, hypohaploidy, disomy and nullisomy were determined and compared by chi-square test. The difference was considered statistically significant at p -value of Results The prolonged IVM did not significantly affect the aneuploidy rate compared to the shorter maturation period (48.1% and 45.0%, respectively). Regarding the unbalanced premature chromatid separation, no statistically significant difference was found between hyperhaploidy and hypohaploidy (14.8% versus 8.3%). For chromosome nondisjunction, higher frequency of disomy than nullisomy was observed (30.6% versus 14.8%; p Conclusions The aneuploidy rate of IVM GV-oocytes is comparable to the aneuploidy rate of in vivo matured oocytes and first polar bodies, regardless of the length of maturation period. This suggests that the immature oocytes can be used in infertility treatment after they complete maturation.

Aim To identify cryptic subtelomeric rearrangement, a possible cause of idiopathic mental retardation by means of multiprobe telomere fluorescent in situ hybridization (T-FISH). Methods Hundred patients (median age 3.0 years) with mental retardation and dysmorphic features were screened using specific T-FISH probes. Multiplex ligation-dependent probe amplification and comparative genomic hybridization were used for the confirmation of results. Results Telomere fluorescent in situ hybridization revealed 11 subtelomeric abnormalities in 10 patients (10%; 95% CI, 5.0-17.5). Four of these had only a deletion of subtelomere 2q, which was apparently a normal variant. Among 6 true aberrations (6%; 95% CI, 2.5-12.5) we found 2 de novo subtelomeric deletions and 4 unbalanced subtelomeric rearrangements (one de novo). All clinically significant subtelomeric rearrangements were confirmed by multiplex ligation-dependent probe amplification. Comparative genomic hybridization was used to investigate the whole genome of patients in whom a subtelomeric anomaly was found, confirming some, but not all subtelomeric rearrangements. Conclusion Telomere fluorescent in situ hybridization and multiplex ligation-dependent probe amplification are both very useful and interchangeable methods for the detection of unbalanced chromosome rearrangements, but T-FISH also detects balanced rearrangements. In our experiment, the resolution power of comparative genomic hybridization was too low for subtelomeric screening compared with T-FISH and multiplex ligation-dependent probe amplification.