[Diagnosis of tuberous sclerosis complex focusing on prenatal period]. / Diagnostika komplexu tuberózní sklerózy se zamerením na prenatální období.

UNLABELLED: Tuberous sclerosis is a disease with an autosomal dominant pattern of inheritance which is characterized by the development of benign tumours in many tissues and organs. Clinical signs are extremely variable, causing mutations in the gene TSC1 or TSC2. Complex formed by the products of the TSC genes regulates cell growth and proliferation by inhibition of mTORC1 signalling. Early diagnosis of TSC is very important to plan appropriate perinatal care. Using ultrasound and eventually MRI it is possible in the prenatal period to capture the following major features of tuberous sclerosis: cardiac rhabdomyo-ma, subependymal nodules, cortical tubers and renal angiomyolipomas. In connection with the syndrome of contiguous genes TSC2 / PKD1 can also be detect foetal renal cysts. Often these TSC-associated lesions represent an incidental finding during a routine ultrasound. In the period from the 20th week of pregnancy it is most often found cardiac rhabdomyoma/s as the first marker suggestive of tuberous sclerosis. In the case, where one of the parents is a carrier of already identified mutation in the TSC gene, it is possible to carry out targeted genetic testing of a sample of DNA isolated from cells of chorionic villi, amniocytes or tissue from aborted foe-tuses. Significantly more time consuming is to perform molecular analysis of the TSC genes in foetuses with suspected tuberous sclerosis without the occurrence of illness in the family. After finding a causal mutation and its confirmation, it is possible to offer genetic testing for other persons at risk, prenatal (eventually preimplantation) diagnosis for future pregnancies. It is also necessary to consider the possibility of gonadal mosaicism. DESIGN: Review of the literature.

[RHD genotyping from cell-free fetal DNA circulating in pregnant women peripheral blood and sensitivity assessment of innovated diagnostic approaches for introduction into the clinical practice]. / Stanovení RHD genotypu plodu z plazmy periferní krve tehotné zeny a posouzení citlivosti nových diagnostických postupu pro zavedení do klinické praxe.

OBJECTIVE: Introduction of fetal RHD genotyping from cell-free fetal DNA circulating in the peripheral blood of pregnant women to clinical practice. Sensitivity assessment of innovated method using range of dilution series and internal control of amplification. DESIGN: Procedure creating of noninvasive determination of fetal RHD genotyping from blood plasma of pregnant women. Detection of limit of minority representation RHD+/- sample in the RHD-/- sample. SETTING: University Hospital Olomouc, Institute of Medical Genetics and Fetal Medicine, Clinic of Obstetrics and Gynecology, Transfusion Department. METHODS: TaqMan Real-Time PCR without an internal amplification controls. Optimization and calibration of RHD genotyping using RHD multiplex by TaqMan Real-Time PCR with an internal amplification control and by minisequencing (Snapshot - multiplex) with an internal amplification controls. RESULTS: RHD positive or negative fetuses were determined by amplification curves from Real-Time PCR system that matches the parameters for the evaluation of the output data using series of amplification and contamination parallel controls. TaqMan based Real-Time PCR and minisequencing (SNaPshot) based quantification were able to detect 0.22% of artificial RHD+/- sample diluted in RHD-/- sample. In addition, SNaPshot assay is suitable for heterozygozity and homozygozity recognition. CONCLUSION: Current established and routinely used procedure is based on the detection of exon 7 of the RHD gene and on the series of parallel amplification and contamination controls. Both newly developed methods could be, after validation of the larger set of control samples, introduced into clinical practice.

[Analysis of cell free fetal DNA fragmentation rate in pregnant women during the course of gravidit]. / Analýza fragmentu volné fetální DNA v plazme tehotných zen v prubehu tehotenství.

AIM OF STUDY: To assess cell free fetal DNA (cffDNA) fragmentation rate in pregnant women during the course of gravidity. STUDY DESIGN: QF PCR efficiency in cffDNA and quantitative analyses in particular cffDNA molecular size fractions. SETTING: The study was performed at Department of Medical Genetics and Fetal Medicine, University Hospital Olomouc. METHOD: 1. 363 plasma DNA samples from women in different week of pregnancy (from 4th w.g. to 37th w.g.) were tested for QF PCR efficiency in particular STRs and AMELX/Y. 2. Size fractionated cff DNA (150-300 bp, 300-500 bp, 500-760 bp) was quantified by QF PCR in 91 pregnant women (from 9th w.g. to 40th w.g.). 3. Size fractionated cff DNA from male fetuses was quantified by real time PCR (SRY/internal control) in 22 pregnant women (from 9th w.g. to 36th w.g.). RESULTS: 1. QF PCR efficiency decreased from longer to shorter molecules. 2. The only 500 -760 bp fraction showed cffDNA increase in relation to week of gravidity. 3. Indirect relation between amount of cffDNA and week of gravidity was found in 150-300 bp fraction by Real-time PCR. CONCLUSION: Assembling of all 3 approaches indicates increase of longer cffDNA molecules during the gravidity while level of the short cffDNA molecule fragments probably remains from the approximately 9th w.g. the same.

[Noninvasive fetal sex detection from maternal plasma in pregnant women]. / Neinvazivní detekce gonozomálních DNA sekvencí v plazme gravidních zen s vyuzitím kapilární elektroforézy.

OBJECTIVE: Objective of our study was optimization of noninvasive fetal sex detection from maternal plasma in pregnant women. STUDY DESIGN: Molecular DNA quantitative analyses in gonosomal loci. SETTING: The study was performed at Department of Medical Genetics and Fetal Medicine, University Hospital Olomouc. METHODS: Together 475 DNA samples isolated from maternal plasma in different weeks of pregnancy ranging from 4th w.g. to 37th w.g. Y chromosomal sequences in AMELY and TSPY were tested by refined quantitative fluorescent PCR using capillary electrophoresis. RESULTS: The method is able to distinguish 1% of Y chromosomal sequences of artificial mixtures. Investigation and assessment in cell free fetal DNA samples achieved 4.05% of false positivity and 7.15% of false negativity in Y sequence detection. CONCLUSION: Established method allows detecting fetal sex with high sensitivity and specificity. The method is possible to use also for quantitative purposes.

[Paternity testing based on the analysis of DNA and its revision]. / Paternitní posudek zalozený na analýze DNA a jeho revize.

Paternity testing is nowadays mostly based on the analysis of DNA short tandem repeats (STR). Number and selection of STR loci differ according to identification kit producers and also particular laboratories. This article refers a revision of formerly excluded paternity. The cause of the mismatch was revealed by the enlargement of STR loci panel and reciprocal evaluation of samples and paternity was on the contrary confirmed. Different possibilities of failures, their consequences and preventions are also discussed.

Tumors overexpressing RNF168 show altered DNA repair and responses to genotoxic treatments, genomic instability and resistance to proteotoxic stress.

Chromatin DNA damage response (DDR) is orchestrated by the E3 ubiquitin ligase ring finger protein 168 (RNF168), resulting in ubiquitin-dependent recruitment of DDR factors and tumor suppressors breast cancer 1 (BRCA1) and p53 binding protein 1 (53BP1). This ubiquitin signaling regulates pathway choice for repair of DNA double-strand breaks (DSB), toxic lesions whose frequency increases during tumorigenesis. Recruitment of 53BP1 curbs DNA end resection, thereby limiting homologous recombination (HR) and directing DSB repair toward error-prone non-homologous end joining (NHEJ). Under cancer-associated ubiquitin starvation conditions reflecting endogenous or treatment-evoked proteotoxic stress, the ubiquitin-dependent accrual of 53BP1 and BRCA1 at the DNA damage sites is attenuated or lost. Challenging this current paradigm, here we identified diverse human cancer cell lines that display 53BP1 recruitment to DSB sites even under proteasome inhibitor-induced proteotoxic stress, that is, under substantial depletion of free ubiquitin. We show that central to this unexpected phenotype is overabundance of RNF168 that enables more efficient exploitation of the residual-free ubiquitin. Cells with elevated RNF168 are more resistant to combined treatment by ionizing radiation and proteasome inhibition, suggesting that such aberrant RNF168-mediated signaling might reflect adaptation to chronic proteotoxic and genotoxic stresses experienced by tumor cells. Moreover, the overabundant RNF168 and the ensuing unorthodox recruitment patterns of 53BP1, RIF1 and REV7 (monitored on laser micro-irradiation-induced DNA damage) shift the DSB repair balance from HR toward NHEJ, a scenario accompanied by enhanced chromosomal instability/micronuclei formation and sensitivity under replication stress-inducing treatments with camptothecin or poly(ADP-ribose) polymerase (PARP) inhibitor. Overall, our data suggest that the deregulated RNF168/53BP1 pathway could promote tumorigenesis by selecting for a more robust, better stress-adapted cancer cell phenotype, through altered DNA repair, fueling genomic instability and tumor heterogeneity. Apart from providing insights into cancer (patho)biology, the elevated RNF168, documented here also by immunohistochemistry on human clinical tumor specimens, may impact responses to standard-of-care and some emerging targeted cancer therapies.

[Rapid detection of most frequent chromosomal aneuploidies by the multiplex QF PCR method in the first trimester of pregnancy]. / Rychlá detekce nejcastejsích chromozomálních aneuploidií metodou multiplex QF PCR v prvním trimestru gravidity.

OBJECTIVE: Rapid detection of most frequent aneuploidies by the multiplex QF PCR method in non-cultured samples of chorial tissue. Summarized results of QF PCR method applied in the management of care of pregnant women in the first trimester of pregnancy. TYPE OF STUDY: An original contribution. SETTING: Institute of Medical Genetics and Fetal Medicine, Faculty Hospital and Medical Faculty, Palacky University Olomouc. METHODS: The samples of chorial tissue were obtained from 101 pregnant women. Non-cultured samples were processed by the multiplex QF PCR method. STR loci of chromosomes 13, 18, 21 and X and Y were analyzed. These markers were amplified in two separate multiplex PCR reactions under the same conditions and subjected to fragmentation analysis in capillary electrophoresis. RESULTS: All 101 analyzed samples of chorial tissue were successfully amplified. In this group, 16 pathologies of the fetuses were detected by the multiplex QF PCR method. Triploidy was detected in two cases, trisomy of chromosome 21--Down syndrome was found in seven cases, and trisomy of chromosome 18--Edwards syndrome was found in six cases and monosomy of gonosome X--the Turner' s syndrome was revealed once. CONCLUSIONS: The multiplex QF PCR method is an indispensable part of the screening of the first trimester and provides a rapidly available and reliable result in the examined patients.

[Prenatal diagnostics of tuberous sclerosis based on causal mutation knowledge]. / Prenatální diagnostika tuberózní sklerózy zalozená na znalosti kauzální mutace.

BACKGROUND: Tuberous sclerosis is an autosomal-dominant disease characterised by development of benign growth - hamartomas in different organs. Disorder is caused by mutations affecting either of the tumor-suppressor genes, TSC1 or TSC2. Quest for causing mutations is very difficult due to their random distribution over the genes. METHODS AND RESULTS: Article refers on accomplishment of the first tuberous sclerosis prenatal diagnostics in Czech Republic based on knowledge of causing mutation. Foetal DNA sample, obtained in 13th week from Q435X family pregnant woman, was analyzed by DGGE method. CONCLUSIONS: Examination excluded presence of tested TSC1 gene defect in an offspring.

[Analysis of free foetal DNA in maternal plasma using STR loci]. / Analýza volné fetální DNA v maternální plazme s vyuzitím STR lokusu.

BACKGROUND: Problems of maternal and foetal genotype differentiation of maternal plasma in pregnant women are solved generally by real-time systems. In this case the specific probes are used to distinguish particular genotype. Mostly gonosomal sequences are utilised to recognise the male foetus. This work describes possibilities in free foetal DNA detection and quantification by STR. METHODS AND RESULTS: Artificial genotype mixtures ranging from 0,2 % to 100 % to simulate maternal and paternal genotypes and 27 DNA samples from pregnant women in different stage of pregnancy were used for DNA quantification and detection. Foetal genotype was confirmed by biological father genotyping. The detection was performed in STR from 21st chromosome Down syndrome (DS) responsible region by innovated (I) QF PCR which allows to reveal and quantify even very rare DNA mosaics. The STR quantification was assessed in artificial mixtures of genotypes and discriminability of particular genotypes was on the level of few percent. Foetal DNA was detected in 74 % of tested samples. CONCLUSIONS: The IQF PCR application in quantification and differentiation between maternal and foetal genotypes by STR loci could have importance in non-invasive prenatal diagnostics as another possible marker for DS risk assessment.

[Frequency view on genome changes testing]. / Frekvencní pohled na vysetrení odchylek genomu.

Laboratories dealing with human genome, both inherited and acquired changes, dispose with similar methods and technology. The spectrum of genetic tests is relatively broad and the number of mutations or variants tested differs substantially. Also the number of examinations carried out in individual laboratories varies. Data presented in the tables come from the year 2004 and indicate the number of examinations requested and number of positive results. Many laboratories mentioned in the registry CZDDNAL (http://www.uhkt.cz/lab_a_vysetreni/nr lab_dna_diag/dna_lab_db) perform the same tests but there is also a great number of tests carried out by only one laboratory. Reasons of the request, cost-effectiveness and clinical utility of genetic testing is being discussed.

[Molecular genetic study of causes of the Prader-Willi and Angelman syndrome]. / Molekulárne-genetická studie pricin Prader-Williho a Angelmanova syndromu.

BACKGROUND: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinctive diseases with severe impairment of psychomotoric development and behaviour. Both syndromes are caused by the loss of paternal (PWS) or maternal (AS) gene expression of chromosomal region 15q11-13. The work reveals the various causes of this loss. The choice of the most suitable method for screening of the genome mutations in the patients suspected of PWS and AS is another purpose of the work. METHODS AND RESULTS: The methyl specific analysis (MS PCR) in locus SNRPN, short tandem repeat (STR) analysis and fluorescent in situ hybridization (FISH) were used. In the group of 55 patients tested for PWS and AS only maternal allele was present in 11 patients and only paternal allele was present in 1 patient in the locus SNRPN: 10 microdeletions 15q11-13, 1 UPD(15)mat and 1 UPD(15)pat. CONCLUSIONS: MS PCR seems to be the most profitable method for the first step of selection of PWS patients. In positive cases is inevitable to use also additional tests of molecular diagnosis to distinguish the particular mechanism leading to the disorders. In AS patients is also MSPCR recommended as the first step although it is necessary to exclude mutation in UBE3A gene in case of MS PCR negativity.

[DNA analysis of chromosome Y in the area of the azoospermia factor (AZF) in infertile men]. / DNA analýza chromozomu Y v oblasti AZF u sterilních muzu.

OBJECTIVE: Establishment of investigation of sterile male DNA in AZF region--choice of loci and primers for investigation, optimization of PCR conditions (polymerase chain reaction). DESIGN: For practice. SETTING: Department of Medical Genetics and Foetal Medicine, Faculty of Medicine, Palacky University and Faculty Hospital Olomouc. METHODS: PCR amplification of DNA isolated from blood of sterile men and consequential electrophoresis of synthesized DNA fragments to appoint microdeletions in AZF. RESULTS: From January to June 2000 were detected the microdeletions in AZF of 3 out of 79 sterile men (3.80%) by means of the Experteam firm kit. From July to December 2000 were tested and optimized conditions of amplification of 10 AZF loci to substitute the kit and they were used for examination of the first 20 sterile men of our collection. CONCLUSION: In our laboratory was established routine examination male sterility related to microdeletions in AZF. With our collection of loci was substituted the original Experteam firm kit and was widened spectrum of observed loci.

[Angiomyolipoma--its role in prenatal diagnosis of tuberous sclerosis]. / Angiomyolipomy--prínos jejich vysetrení v prenatální diagnostice tuberózní sklerózy.

Tuberous sclerosis (TSC) is a frequent hereditary autosomal-dominant disease characterised by hamartomas developing in many organs. The disorder is caused by mutations affecting either of the tumor-suppressor genes, TSC1 and TSC2. Tumorogenesis is triggered by the loss of second functional gene copy, mostly accompanied by loss of heterozygosity (LOH) of flanking polymorphic markers. Search for causing mutations is very laborious, time consuming and low effective. Prenatal diagnosis is often hampered by lack of detection of causing mutation. Detection of LOH in hamartomatous tissue suggests which gene is involved in particular case of disease and specifies which of homologous chromosomes carries germinal mutation. Examination of LOH is useful for prenatal diagnostics especially when time is lacking due to patient's pregnancy or in case of mutation screening failure.

[Microdeletion of the azoospermia factor as one of the causes of male infertility]. / Mikrodelece faktoru azoospermie jako jedna z prícin muzské infertility.

One of the possible causes of male infertility is microdeletion of the Y chromosome in the Yq11.23 region--named the azoospermia factor. These deletions are associated with azoospermia or severe oligozoospermia. In these cases, testicular histopathological findings comprise a wide spectrum, from total absence of germ cells, through arrest of their maturation to decreased sperm production. Most Y-chromosome microdeletions arise de novo but transmission from the father is also possible, either by the natural way or by assisted reproduction. In relation to the assisted reproduction, the relationship between the Y-deletions and presence of spermatozoa in testis, fertilization capability and embryo quality were examined. Heredity of the deleted Y chromosome is holandric and therefore all sons of males with deletions will carry the same defect and will probably have fertility problems. Another negative influence of deletions on a man's health has not yet been identified.

[DNA diagnosis in Czech patients with tuberous sclerosis]. / DNA diagnostika u Ceských pacientu s tuberózní sklerózou.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterised by mental retardation, epilepsy, facial angiofibromas, lesions in the CNS and the occurrence of hamartomata in different tissues. There are two genes responsible (TSC1 and TSC2). The aim of the study is to adopt a diagnosis of the disease on DNA level. METHODS AND RESULTS: 111 DNA samples were collected from 45 families with 54 clinically diagnosed patients. Three families with multiple incidence are linked to chromosome 9. In one case a large deletion was found using TSC2 specific cDNA probes. Most TSC mutations are supposed to be small mutations, identifiable by SSCP method. Using this method we examined all 23 TSC1 exons and 20 out of 41 TSC2 exons. In the TSC1 gene we found 1 nonsense and 2 frameshift mutations and 2 intragenic polymorphisms useful for linkage. In the TSC2 exons was identified so far 6 aberrations, cause of which is being checked by sequencing. CONCLUSIONS: DNA mutation analysis is effective namely in families with multiple incidence. In such families the linkage can be used and there is more TSC1 cases which are easily identifiable. Analysis is not economical for differential diagnosis. Total number of revealed mutations (6.7%) matches last reports. No correlation between phenotype and type of mutation was found.

[Prospects and applications of innovated quantitative fluorescent PCR (IQF PCR) in analyses of genetic mosaics using gonosomal sequences]. / Moznosti a vyuzití inovované kvantitativní fluorescencní PCR (IQF PCR) v analýze genetického mozaicizmu pomocí gonozomálních sekvencí.

BACKGROUND: Quantification of fluorescence labelled PCR products on capillary electrophoresis (QF PCR) has limits primarily in the possibility of more sensitive analyses to detect minority cell lines and small time related variations. PCR efficiency and human factor affect measuring error and reproducibility of results in these cases. The aim of this work was to assess and optimise of innovated (I)QF PCR in quantification of Y sequences in gonosomal mosaics. METHODS AND RESULTS: Artificially prepared Y/X mosaics were tested and quantified by IQF PCR, which replaces real-time PCR. Comparison of relative fluorescence to PCR cycles in different Y/X dilutions was plotted on the graphs. Calibration curve for Y sequences quantification was set by the analyses of ratio of Y/X fluorescent signals. An empirical formula was created for the rare mosaic calculation. CONCLUSIONS: QF PCR refined by manual real-time PCR eliminates limits of QF PCR and specifies quantitative analyses based on PCR. The outstanding feature of IQF PCR is its high sensitivity and accuracy in quantification of Y/X gonosomal mosaics.

[Increased sensitivity for detection of mutations in exon 15 of the APC gene in patients with familial adenomatous polyposis]. / Zvýsení citlivosti pro detekci mutací v 15. exonu APC genu u pacientu s familiární adenomatózní polypózou.

BACKGROUND: Protein Truncation Test (PTT) was used to detect mutations in exon 15 of the APC gene in patients with Familial Adenomatous Polyposis. This method is limited by its ability to detect polypetide chains up to a certain minimum length. The aim of this study was to increase the sensitivity of detection of mutations in this region by using the technique of Denaturing Gradient Gel Electrophoresis (DGGE). METHODS AND RESULTS: Study were performed on 122 patients without detected mutations in the APC gene. The patients were divided into two independent groups 15A and 15A+15B (with 51 and 71 patients respectively). All the patients were tested with the DGGE and the positive findings were confirmed with sequencing. No mutation was detected in the group 15A (0%). In group 15A+15B one (1.4%) polymorphism and four (5.63%) patients with nonsense mutations were detected. CONCLUSIONS: DGGE is an effective method for detecting mutations in the first part of exon 15 of APC gene. It allows detecting any change in DNA strand. DGGE complements PTT in scanning of the whole exon 15 of APC gene.

[Analysis of specific sequences in female patients with Turner syndrome--initial study]. / Analýza y specifických sekvencí u pacientek s turnerovým syndromem--úvodní studie.

BACKGROUND: DNA sequences from chromosome Y can cause gonadoblastoma development in patients with Turner syndrome (TS). Estimated risk is about 30%. The aim of the study is detection of Y-sequences of DNA level, calculation of mosaicism and its cytogenetic location. Clinical result of the study is the recommendation to gonadectomy of proved positive patients. METHODS AND RESULTS: Samples from 110 patients were collected. The PCR method and analysis of products on agarose gel was compared with analysis of DNA fragments from quantitative fluorescent (QF) PCR on capillary electrophoresis. The loci DYZ3, AMGX/Y and SRY were used for detection. The method QF PCR was effected for DYZ3 and AMGX/Y loci. The positive cases were examined by FISH method. Five (4.5%) and 3 (2.7%) positive cases were detected in DYZ3 and SRY resp. loci by electrophoresis on agarose gel. Seventeen (15.5%) and 7 (6.4%) positive cases were detected in DYZ3 and AMGX/Y resp. by capillary electrophoresis. The estimated mosaicism ranged from 1:5 to 1:100,000. CONCLUSIONS: QG PCR is the most sensitive method for diagnostics of Y-sequences. Simultaneously the incidence of Y-positive cells can be estimated. The positive cases with marker in karyotype were confirmed by FISH.

Identification of a large insertion and two novel point mutations (3671del8 and S1221X) in tuberous sclerosis complex (TSC) patients. Mutations in brief no. 119. Online.

Important symptoms of tuberous sclerosis complex (TSC), an autosomal dominant disorder, are hamartomata in several organs, mental retardation and epilepsy. Either one of two loci can be involved (TSC1 and TSC2), of which the TSC2 gene has been cloned. To date, only 35 mutations in the TSC2 gene have been described ranging from large deletions to point mutations. Southern blot analysis using cDNA clones of the TSC2 gene was performed on a cohort of 160 unrelated TSC patients and revealed a 10 kb insertion. The insertion was also present in DNA of the affected father. Both patients showed renal angiomyolipoma, hypomelanotic macules and epilepsy. SSCP analysis of exons 1,2,3,9,12,14,30a and 36 identified two mutations in exon 30a: 3671del8 and S1221X. Symptoms of the sporadic patient with the 3671del8 mutation are cortical tubers, subependymal nodules, facial angiofibroma, ungual fibroma, renal angiomyolipoma, hypomelanotic macules, epilepsy and mental retardation. Clinical symptoms of the patient with the S1221X mutation are facial angiofibroma, ungual fibroma, hypomelanotic macules, epilepsy and mental retardation. His parents were negative for the S1221X mutation, although a germline mosaicism can not be excluded. Besides the previously described polymorphism 1596C->T, two rare variants were observed, a substitution of C->T at position 1294 and at position 1299 C->A.