[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.

[Chronic benign exsudative pericarditis--a case of familiar incidence]. / Chronická exsudativní benigní perikarditida--popis familiárního výskytu.

Pericarditis is a common disease caused by a number of factors. Chronic pericarditis is defined as the presence of pericardial effusion for more than 3 or 6 months. The case study reports a case of familiar incidence of chronic exsudative pericarditis in a young woman, her sister and her mother, with an analysis of diagnostic and therapeutic options. According to available literature, this is the second case described of such form of familiar incidence.

[Inherited metabolic disorders and pregnancy]. / Puvodní práce Dedicné metabolické choroby a tehotenství.

OBJECTIVE: Review of contemporary knowledge about inherited metabolic disorders in pregnant women and fetuses. DESIGN: A review of literature. SETTING: Laboratory for inherited metabolic disorders, Departament of Clinical Biochemistry and Childern's Clinic, University Hospital and Palacky University, Olomouc. METHOD: Review of literature and publications using medical databases. CONCLUSION: Metabolic examination of mother or child is recommended if any clinical problems during or immediately after pregnancy occur. Nowadays most of inherited errors of metabolism can be diagnosed prenataly in first or second trimester of pregnancy.

[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.

[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.

[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.

[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.

[Importance of genetic testing in couples with reproductive disorders]. / Význam genetického vysetrení u páru s poruchami reprodukce.

OBJECTIVE: To determine the prevalence of chromosomal aberrations in infertile couples undergoing in vitro fertilization (IVF). DESIGN: Cytogenetic analysis of peripheral blood lymphocytes in the group of patients undergoing IVF. Detection of chromosomal aberrations in the fetuses after IVF. SETTING: Department of Medical Genetics and Fetal Medicine, Medical Faculty, Palacký University and the University Hospital, Olomouc. METHODS: Cultivation of peripheral blood lymphocytes or fibroblasts of amniotic fluid. Using fluorescent in situ hybridization in cases of mosaicism. RESULTS: Out of 638 patients undergoing treatment for male or female infertility, 595 had normal karyotype and 43 (6.8%) had abnormal karyotype. There were detected 9 (1.4%) cases of balanced chromosomal rearrangements, 2 (0.31%) cases of deletion of Y chromosome, 2 (0.31%) cases of inversion, 2 (0.31%) cases of marker chromosome, 5 (0.78%) cases of gonosomal aneuploidy (47,XXY) and 23 (3.65%) cases of gonosomal mosaicism--out of the 22 (3.5%) cases of low-level mosaicism. In the small group of pregnant patients after IVF investigated for the risk of genetic disorders included in our study (n = 60) the frequency of chromosomal abnormalities was 9 (15%). CONCLUSIONS: Our data show that a high number of infertile couples is affected by chromosomal aberrations which occur more frequently in females than in males. It is caused by high frequency of low-level gonosomal mosaicism in the group of infertile women. Chromosomal analyses are highly recommended before each IVF procedure.

[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.

[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.

[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.

Manifestations and treatment of Schimke immuno-osseous dysplasia: 14 new cases and a review of the literature.

UNLABELLED: Schimke immuno-osseous dysplasia (SIOD) is a rare autosomal recessive spondylo-epiphyseal dysplasia. The characteristic features of SIOD include 1) short stature with hyperpigmented macules and an unusual facies, 2) proteinuria with progressive renal failure, 3) lymphopenia with recurrent infections, and 4) cerebral ischaemia. Although 25 patients have been reported with this disorder, the clinical course and phenotype of SIOD are not well characterized. This report summarizes the clinical findings, course and treatment of reported patients and includes 14 additional patients with SIOD. We emphasize the high incidence of cerebral ischaemia and ocular abnormalities, define the high incidence of thyroid dysfunction and blood cytopenia, and confirm the absence of effective and durable medical therapies. CONCLUSION: Schimke immuno-osseous dysplasia is a multi-system autosomal recessive disorder with variable expression that affects the skeletal, renal, immune, vascular, and haematopoietic systems. Medical therapy is limited especially for more severely affected individuals.

[The importance of ultrasonography in the 1st and 2nd trimester of pregnancy in prenatal diagnosis of chromosome aberrations]. / Význam ultrazvuku v I. a II. trimestru gravidity pro prenatální diagnostiku chromosomálních aberací.

Indications for examination of the foetal karyotype on the basis of ultrasonic atypies or detection of specific US minor-markers is an important screening method for prenatal detection of chromosomal aneuploidies of the foetus. It is associated with possible detailed evaluation of certain factors by more perfect ultrasonographic equipment. In the group investigated by the authors this indication was of greatest importance for detection of chromosomal aneuploidies in-8.52%. Isada et al. [18] reports 16%; Donner et al. [11] found chromosomal aberrations in 9.5% abnormal sonograms. The highest sensitivity is obtained by assessment of the "nuchal translucency" (NT) which can be investigated during the first and second trimester of pregnancy. In the author's group it was recorded in 47% foetuses with Down's syndrome. Risk assessment of chromosomal trisomies based on the assessed NT during the first trimester was done by means of the Fetal Database programme. The assessment calls for special training. A reduced length of the femur is a significant marker of chromosomal aberrations if the disproportion with BPD between the 15th and 22nd week of gestation is 10 days or more. The importance of other ultrasonographic markers such as a reduced femur, hyperechogenity of the gut, pyelectasia etc. requires further studies. In the author's investigation the risk of chromosomal aberrations increases significantly only after detection of two or more of these atypical features. Chorionic and amniotic atypies in the first trimester and placental dysmorphias (partial moles) in the second trimester of gestation are frequently associated with chromosomal triploidy. The prenatal diagnosis of these abnormalities is possible by the vaginal and transabdominal route. It calls for considerable enlargement of the picture similarly as investigations of "NT".

Familial hydronephrosis unlinked to the HLA complex.

Clinical findings, management, and possible linkage of congenital hydronephrosis caused by ureteropelvic junction stenosis to the HLA complex were studied in four families. These families provide evidence of possible autosomal dominant inheritance. HLA class I antigen studies in all four and class II (HLA-DR) in three families were performed. These studies failed to show close linkage to the chromosome 6 markers in two families but there was consistent inheritance in the other two. Although formal linkage calculations are not presented, it is apparent that in some families HLA haplotyping is not useful in predicting prevence of renal obstruction.

[Dysplastic gangliocytoma of the cerebellum (Lhermitte-Duclos)]. / Dysplastický gangliocytom mozecku (Lhermitte-Duclos).

In a surgical specimen from a 33-year old woman with features of expansive cerebellar symptomatology, dysplastic gangliocytoma of the cerebellum (Lhermitte-Duclos) was diagnosed by histology. The lesion was characterized by typical proliferation of dysplastic neuronal elements in the cortical granular and Purkinje cell layers. Immunohistology showed focal positivity of synaptophysin in some cells, thus confirming their neuronal origin. In electron microscopy, peculiar features of ER in some of dysplastic cells was also noted. Genetic examination proved familiar incidence of Cowden's disease in several members. Chromosomal examination of the patient (karyotype 46, XX) was without abnormalities.

Spondyloepiphyseal dysplasia with nephrotic syndrome (Schimke immunoosseous dysplasia).

The first symptoms of immunooseous dysplasia were growth retardation and myopia. Nephrotic syndrome was diagnosed at the age of 8 years. Skeletal roentgenograms showed spondyloepiphyseal dysplasia. In the renal biopsy there was nodular accumulations of PAS-positive hyaline material at the base of the granular stalks. There was lymphopenia with decreased CD4 and CD8 subpopulations. The condition of the patient gradually worsened until she died unexpectedly at 10 years with clinical symptoms of encephalitis. Autopsy documented cytomegaloviral pneumonia and advanced mesangioproliferative glomerulonephritis. In the spleen there was PAS-positive hyaline material massively infiltrating the walls of the central arterioles of the splenic follicles. There was marked depletion of lymphocytes in the spleen and in lymph nodes. The differential diagnosis of immunooseous dysplasia in the framework of spondyloepiphyseal dysplasia is discussed.

[The basal cell nevus syndrome. Case report of a 14-year-old girl]. / "Basal cell nevus syndrom". Kasuistika výskytu u 14letého devcete.

"Basal cell naevus syndrome" (Gorlin's syndrome) is a sporadic autosomal dominant hereditary precancerous condition which affects several organ systems. The dominating clinical manifestations are multiple basal cell naevi which develop into malignant basocellular carcinoma. Further abnormalities include abnormalities of the vertebrae and ribs, odontogenic keratocysts, calcification of the falx cerebri, a special facial appearance with progeny and macrocephaly. Affected patients may develop also ovarian fibromas, fibrosarcomas, cardiac fibromas, medulloblastomas and meningiomas. Lymphatic and chylous mesenteric cysts are also frequent. In the submitted paper the authors present the case-history of a 14-year-old girl with multiple naevi, histologically specified as solid, superficial and tricho-epithelial basalioma. Phenotypic manifestations, multiple keratocysts, bone abnormalities and calcifications of the falx cerebri which are detected in the girl led to the diagnosis of Gorlin's syndrome. The authors discuss the problem of cytogenetic findings (structural abnormalities, markers of mutagenicity) and possible therapy.

[Present possibilities of prenatal diagnosis of Fabry's disease]. / Soucasné moznosti prenatální diagnostiky Fabryho choroby.

Fabry's disease (angiokeratoma corporis diffusum universale) is an inborn error of metabolism, which is based on a shortage in the cumulation of glycosphingolipids in endothelial and epithelial cells of glomeruli, vascular endothelia and in ganglion cells with subsequent severe organ damage. Heredity is X-linked recessive. In the submitted paper the authors present a case-history of a family where three men were affected and carriership was confirmed in five women. During the first pregnancy one of them a successful prenatal diagnosis was made from cultivated amniotic fluid cells. Chromosomal examination revealed female sex and the levels of the alpha-galactosidase A were established in cultivated cells. A healthy girl was delivered.