Publications by authors named "Radek Vrtel"

28 Publications

  • Page 1 of 1

Risk Minimization of Hemolytic Disease of the Fetus and Newborn Using Droplet Digital PCR Method for Accurate Fetal Genotype Assessment of , , and from Cell-Free Fetal DNA of Maternal Plasma.

Diagnostics (Basel) 2021 Apr 28;11(5). Epub 2021 Apr 28.

Department of Medical Genetics, University Hospital and Palacky University Olomouc, 775 20 Olomouc, Czech Republic.

The molecular pathology of hemolytic disease of the fetus and newborn (HDFN) is determined by different , , and genotypes and by blood group incompatibility between the mother and fetus that is caused by erythrocyte antigen presence/absence on the cell surface. In the Czech Republic, clinically significant antierythrocyte alloantibodies include anti-D, anti-K, anti C/c, and anti-E. Deletion of the gene and then three single nucleotide polymorphisms in the and genes (rs676785, rs609320, and rs8176058) are the most common. The aim of this study is to develop effective and precise monitoring of fetal genotypes from maternal plasma of these polymorphisms using droplet digital (dd)PCR. Fifty-three plasma DNA samples (from 10 to 18 weeks of gestation) were analyzed (10 , 33 , and 10 ). The ddPCR methodology was validated on the basis of the already elaborated and established method of minisequencing and real-time PCR and with newborn phenotype confirmation. The results of ddPCR were in 100% agreement with minisequencing and real-time PCR and also with newborn phenotype. ddPCR can fully replace the reliable but more time-consuming method of minisequencing and real-time PCR examination. Accurate and rapid noninvasive fetal genotyping minimizes the possibility of HDFN developing.
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http://dx.doi.org/10.3390/diagnostics11050803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8146004PMC
April 2021

Haplotype analysis of the X chromosome in patients with Turner syndrome in order to verify the possible effect of imprinting on selected symptoms.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2021 Jan 12. Epub 2021 Jan 12.

Department of Medical Genetics, Faculty of Medicine and Dentistry, Palacky University Olomouc and University Hospital Olomouc, Czech Republic.

Aims: Turner syndrome is the only chromosome monosomy that is postnatally compatible with life. The reported incidence of TS is 1 in 2500 liveborn girls. The phenotype of these girls is highly variable, with cardiac abnormalities being life-threatening defects. The aim of the study was to reveal the possible influence of the parental origin of the X chromosome in these patients on a selected phenotype that is associated with Turner syndrome. Selected symptoms and parameters were: a bicuspid aortic valve, aortic coarctation, lymphoedema, pterygium colli, coeliac disease, thyroiditis, otitis media, diabetes mellitus 2, renal abnormalities, spontaneous puberty, and IVF.

Methods: The X chromosome haplotype was determined for a group of 45,X patients verified by native FISH. A molecular diagnostic method based on the detection of different lengths of X chromosome-linked STR markers using the Argus X-12 QS kit was used to determine the X haplotype.

Results: Our results, analysed by Fisher's exact (factorial) test, suggest independence between the maternal/paternal origin of the inherited X chromosome and the presence of the anomalies that were studied (P=1 to P=0.34).

Conclusion: In the group of 45,X patients, who were precisely selected by means of the native FISH method, no correlation was demonstrated with the parental origin of the X chromosome and the observed symptom.
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http://dx.doi.org/10.5507/bp.2020.060DOI Listing
January 2021

Multigene Panel Germline Testing of 1333 Czech Patients with Ovarian Cancer.

Cancers (Basel) 2020 Apr 13;12(4). Epub 2020 Apr 13.

Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, 128 53 Prague, Czech Republic.

Ovarian cancer (OC) is the deadliest gynecologic malignancy with a substantial proportion of hereditary cases and a frequent association with breast cancer (BC). Genetic testing facilitates treatment and preventive strategies reducing OC mortality in mutation carriers. However, the prevalence of germline mutations varies among populations and many rarely mutated OC predisposition genes remain to be identified. We aimed to analyze 219 genes in 1333 Czech OC patients and 2278 population-matched controls using next-generation sequencing. We revealed germline mutations in 18 OC/BC predisposition genes in 32.0% of patients and in 2.5% of controls. Mutations in , , , and mismatch repair genes conferred high OC risk (OR > 5). Mutations in and were associated with moderate risk (both OR = 3.5). mutations dominated in almost all clinicopathological subgroups including sporadic borderline tumors of ovary (BTO). Analysis of remaining 201 genes revealed somatic mosaics in and germline mutations in and associating with a high/moderate OC risk significantly; however, further studies are warranted to delineate their contribution to OC development in other populations. Our findings demonstrate the high proportion of patients with hereditary OC in Slavic population justifying genetic testing in all patients with OC, including BTO.
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http://dx.doi.org/10.3390/cancers12040956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226062PMC
April 2020

Searching for genetic variants associated with thrombophilia.

Cas Lek Cesk 2019 ;158(1):28-32

Thrombotic states are inherited or acquired predisposition for thrombosis in the human vascular system. Nowadays Leiden mutation and mutation in prothrombin G20210A contributing to congenital thrombophilia are routinely tested. These mutations have a high prevalence in the population. Congenital deficiencies of protein S, protein C and antithrombin III are rare thrombophilia with lower population frequency, but higher risk of thromboembolic event. The genetic causes are mutations in the genes, which encode these proteins. The choice of proper molecular genetic testing depends on the difference in the detection of well-known single nucleotide polymorphism or unknown/rare variant. For the detection of causative variant FV Leiden and prothrombin G20210A are mostly used PCR-RFLP, reverse Strip Assay®, allele-specific PCR, TaqMan real-time PCR and SNaPshot®. Precise patient selection should precede the genetic testing of rare variants in anticoagulant proteins. It is appropriate to use methodology of massive parallel sequencing supplemented by a methodology for the detection of larger gene rearrangements - MLPA. We are successfully employing this approach in our institute. This methodology is faster with larger analytic capacity compared to commonly used direct sequencing by Sanger method.
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June 2019

Fetal heart rhabdomyomatosis: a single-center experience.

J Matern Fetal Neonatal Med 2021 Mar 15;34(5):701-707. Epub 2019 May 15.

Biomedical Research Centre, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic.

The primary aim of the study was to evaluate the prevalence of fetal heart tumors in a single tertiary referral center over a period of 15 years. The secondary aim was to confirm the presence of tuberous sclerosis complex (TSC) through the evaluation of germline mutation in / and assess the outcomes in affected fetuses and newborns. A retrospective study was conducted between 2003 and 2017. Fetal echocardiography was performed in the second trimester of pregnancy in the study population. The identification of heart tumors and further follow-up were performed by a pediatric cardiologist. Molecular genetic analysis was conducted on fetuses and children in cases where TSC was suspected. In total, 39,018 fetuses were examined between 2003 and 2017. Heart tumors were detected in nine fetuses and were diagnosed as rhabdomyoma in all cases. We identified mutations in one of the or genes in all cases with multiple rhabdomyomas (8/9). In all born children (5/9), the genetically confirmed diagnosis of TSC was established, and clinically pathological deposits in the brain were found. Fetal heart tumors are usually represented by rhabdomyomas having a good cardiac prognosis. However, rhabdomyoma is usually the first symptom of TSC with a subsequent brain disorder and impaired neurological development.
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http://dx.doi.org/10.1080/14767058.2019.1613365DOI Listing
March 2021

Generation of human iPSCs from fetal prostate fibroblasts HPrF.

Stem Cell Res 2019 03 7;35:101405. Epub 2019 Feb 7.

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic; International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic. Electronic address:

Human induced pluripotent stem cell line was generated from commercially available primary human prostate fibroblasts HPrF derived from a fetus, aged 18-24 weeks of gestation. The fibroblast cell line was reprogrammed with Yamanaka factors (OCT4, SOX2, c-MYC, KLF4) using CytoTune™-iPS 2.0 Sendai Reprogramming Kit. Pluripotency of the derived transgene-free iPS cell line was confirmed both in vitro by detecting the expression of factors of pluripotency on a single-cell level, and in vivo using teratoma formation assay. This iPS cell line will be a useful tool for studying both normal prostate development and prostate cancer disease.
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http://dx.doi.org/10.1016/j.scr.2019.101405DOI Listing
March 2019

Generation of human iPSCs from human prostate cancer-associated fibroblasts IBPi002-A.

Stem Cell Res 2018 12 16;33:255-259. Epub 2018 Nov 16.

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic; International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic. Electronic address:

A human induced pluripotent stem cell line was generated from cancer-associated fibroblasts of a 68-years old patient with diagnosed prostate adenocarcinoma (PCa). The fibroblast cell line was reprogrammed with Epi5™ Episomal iPSC Reprogramming Kit. Pluripotency of the derived transgene-free iPS cell line was confirmed both in vitro by detecting expression of factors of pluripotency on a single-cell level, and also in vivo using teratoma formation assay. This new iPS cell line may be used for differentiation into different prostate-specific cell types in differentiation studies.
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http://dx.doi.org/10.1016/j.scr.2018.11.006DOI Listing
December 2018

New endemic familial parkinsonism in south Moravia, Czech Republic and its genetical background.

Medicine (Baltimore) 2018 Sep;97(38):e12313

Department of Neurology Department of Medical Genetics, Faculty of Medicine and Dentistry, Palacký University, University Hospital, Olomouc General Practitioner, Lipov General Practitioner, Velká nad Veličkou General Practitioner, Blatnice pod Svatým Antonínkem Neurology Outpatient Clinic, Veselí nad Moravou First Department of Neurology, Masaryk University Medical School, St. Anne University Hospital, Brno Department of Epidemiology and Public Health, Faculty of Medicine and Dentistry, Palacky University, University Hospital, Olomouc, Czech Republic.

An increased prevalence of familial neurodegenerative parkinsonism or cognitive deterioration was recently found in a small region of southeastern Moravia.The aim of the study was to assess the genetic background of this familial disease.Variants in the ADH1C, EIF4G1, FBXO7, GBA + GBAP1, GIGYF2, HTRA2, LRRK2, MAPT, PRKN, DJ-1, PINK1, PLA2G6, SNCA, UCHL1, VPS35 genes were examined in 12 clinically positive probands of the pedigree in which familial atypical neurodegenerative parkinsonism was identified in previous epidemiological studies. Libraries were sequenced by massive parallel sequencing (MPS) on the Personal Genome Machine (PGM; Ion Torrent). Data were analyzed using Torrent Suite and IonReporter software. All variants were then verified and confirmed by Sanger sequencing.We identified 31 rare heterozygous variants: 11 missense variants, 3 synonymous variants, 8 variants in the UTR region, and 9 intronic variants. Six variants (rs1801334, rs33995883, rs35507033, rs781737269, rs779760087, and rs63750072) were evaluated as pathogenic by at least one in-silico predictor.No single "founder" pathogenic variant associated with parkinsonism has been found in any of the probands from researched pedigree. It may rather be assumed that the familial occurrence of this disease is caused by the combined influence of several "small-effect" genetic variants that accumulate in the population with long-lasting inbreeding behavior.
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http://dx.doi.org/10.1097/MD.0000000000012313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160209PMC
September 2018

Mutational analysis of TSC1 and TSC2 genes in Tuberous Sclerosis Complex patients from Greece.

Sci Rep 2017 12 1;7(1):16697. Epub 2017 Dec 1.

Laboratory of Environmental Mutagenesis and Carcinogenesis, Institute of Biosciences and Applications, National Centre for Scientific Research "Demokritos", Athens, Greece.

Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder causing benign tumors in the brain and other vital organs. The genes implicated in disease development are TSC1 and TSC2. Here, we have performed mutational analysis followed by a genotype-phenotype correlation study based on the clinical characteristics of the affected individuals. Twenty unrelated probands or families from Greece have been analyzed, of whom 13 had definite TSC, whereas another 7 had a possible TSC diagnosis. Using direct sequencing, we have identified pathogenic mutations in 13 patients/families (6 in TSC1 and 7 in TSC2), 5 of which were novel. The mutation identification rate for patients with definite TSC was 85%, but only 29% for the ones with a possible TSC diagnosis. Multiplex ligation-dependent probe amplification (MLPA) did not reveal any genomic rearrangements in TSC1 and TSC2 in the samples with no mutations identified. In general, TSC2 disease was more severe than TSC1, with more subependymal giant cell astrocytomas and angiomyolipomas, higher incidence of pharmacoresistant epileptic seizures, and more severe neuropsychiatric disorders. To our knowledge, this is the first comprehensive TSC1 and TSC2 mutational analysis carried out in TSC patients in Greece.
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http://dx.doi.org/10.1038/s41598-017-16988-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711901PMC
December 2017

Single giant mediastinal rhabdomyoma as a sole manifestation of TSC in foetus.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2017 Sep 12;161(3):326-329. Epub 2017 Jun 12.

Outpatient Cardiology Department, Pediatric Clinic, The University Hospital Brno, Czech Republic.

Background: Presence of multiple cardiac rhabdomyomas is one of the major features of Tuberous sclerosis (TSC), but isolated progressing single giant rhabdomyoma is very rare and not typical of TSC.

Case Report: This report presents family without obvious history of TSC with occurrence of giant mediastinal rhabdomyoma affecting the haemodynamics in male foetus, without other TSC symptoms. Girl from the next gravidity had prenatally detected multiple rhabdomyomas and small subcortical tuber of brain detected after birth. DNA analysis found novel c.4861A>T TSC2 variant and large deletion in TSC2 in tumour tissue from male foetus. The novel TSC2 variant was also present in the girl and her healthy father, in silico analysis suggested its functional effect on TSC2. Brain MRI of the father detected mild TSC specific abnormality.

Conclusion: We suggest the novel TSC2 mutation is a cause of mild TSC in this family and has reduced expression. The clinical and molecular findings in this family also emphasize that TSC diagnosis should be also evaluated in case of single giant foetal cardiac rhabdomyoma.
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http://dx.doi.org/10.5507/bp.2017.023DOI Listing
September 2017

Familial atypical parkinsonism with rare variant in VPS35 and FBXO7 genes: A case report.

Medicine (Baltimore) 2016 Nov;95(46):e5398

Department of Neurology Department of Medical Genetics and Fetal Medicine, Faculty of Medicine and Dentistry, Palacky University, University Hospital, Olomouc Department of Nanomaterials in Natural Sciences, Technical University, Liberec, Czech Republic.

Background: A higher prevalence of parkinsonism was recently identified in southeastern Moravia (Czech Republic). Further research confirmed 3 large pedigrees with familial autosomal-dominant parkinsonism spanning 5 generations.

Methods: This case report concerns a patient belonging to one of these 3 pedigrees, in whom motor and oculomotor symptoms were accompanied by frontal-type dementia, who finally developed a clinical phenotype of progressive supranuclear palsy. Molecular genetic examinations were performed due to the positive family history.

Results: No previously described causal mutation was found. After filtering against common variants (minor allele frequency (MAF) < 0.01), 2 noncoding and 1 synonymous rare mutation potentially associable with parkinsonism were identified: GIGYF2-GRB10 Interacting GYF Protein 2, PARK11 (c.*2030G > A, rs115669549); VPS35 gene-vacuolar protein sorting 35, PARK17 (c.102 + 33G > A, rs192115886); and FBXO7-F-box only protein 7 gene, PARK15 (c.540A > G, rs41311141).

Conclusion: As to the changes in the FBXO7 and VPS35 genes (despite phylogenetic conservation in primates), probably neither the FBXO7 nor the VPS35 variants will be direct causal mutations. Both described variants, and possibly the influence of their combination, could increase the risk of the disease.
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http://dx.doi.org/10.1097/MD.0000000000005398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120934PMC
November 2016

DNA damage signalling barrier, oxidative stress and treatment-relevant DNA repair factor alterations during progression of human prostate cancer.

Mol Oncol 2016 06 3;10(6):879-94. Epub 2016 Mar 3.

Danish Cancer Society Research Center, Copenhagen, Denmark; Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden; Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic. Electronic address:

The DNA damage checkpoints provide an anti-cancer barrier in diverse tumour types, however this concept has remained unexplored in prostate cancer (CaP). Furthermore, targeting DNA repair defects by PARP1 inhibitors (PARPi) as a cancer treatment strategy is emerging yet requires suitable predictive biomarkers. To address these issues, we performed immunohistochemical analysis of multiple markers of DNA damage signalling, oxidative stress, DNA repair and cell cycle control pathways during progression of human prostate disease from benign hyperplasia, through intraepithelial neoplasia to CaP, complemented by genetic analyses of TMPRSS2-ERG rearrangement and NQO1, an anti-oxidant factor and p53 protector. The DNA damage checkpoint barrier (γH2AX, pATM, p53) mechanism was activated during CaP tumorigenesis, albeit less and with delayed culmination compared to other cancers, possibly reflecting lower replication stress (slow proliferation despite cases of Rb loss and cyclin D1 overexpression) and progressive loss of ATM activator NKX3.1. Oxidative stress (8-oxoguanine lesions) and NQO1 increased during disease progression. NQO1 genotypes of 390 men did not indicate predisposition to CaP, yet loss of NQO1 in CaP suggested potential progression-opposing tumour suppressor role. TMPRSS2-ERG rearrangement and PTEN loss, events sensitizing to PARPi, occurred frequently along with heterogeneous loss of DNA repair factors 53BP1, JMJD1C and Rev7 (all studied here for the first time in CaP) whose defects may cause resistance to PARPi. Overall, our results reveal an unorthodox DNA damage checkpoint barrier scenario in CaP tumorigenesis, and provide novel insights into oxidative stress and DNA repair, with implications for biomarker guidance of future targeted therapy of CaP.
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http://dx.doi.org/10.1016/j.molonc.2016.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423169PMC
June 2016

Clinical Potential of Effective Noninvasive Exclusion of KEL1-Positive Fetuses in KEL1-Negative Pregnant Women.

Fetal Diagn Ther 2016 23;40(1):48-53. Epub 2015 Oct 23.

University Hospital and Palacky University Olomouc, Olomouc, Czech Republic.

Background: The clinical importance of assessing the fetal KEL genotype is to exclude 'K'-positive fetuses (genotype KEL1/KEL2) in 'K'-alloimmunized pregnant women (genotype KEL2/KEL2). Noninvasive assessment of the fetal KEL genotype is not yet available in the Czech Republic.

Objective: The aim of this study was to assess the fetal KEL1/KEL2 genotype from cell-free fetal DNA in the plasma of KEL2/KEL2 pregnant women.

Methods: The fetal genotype was assessed by minisequencing (a dilution series including control samples). A total of 138 pregnant women (between the 8th and 23rd gestational week) were tested by minisequencing. The fetal genotype was further verified by analysis of a buccal swab from the newborn.

Results: Minisequencing proved to be a reliable method. In 2.2% (3/138) of the examined women, plasma sample testing failed; 94.8% (128/135) had the KEL2/KEL2 genotype, and a total of 3.1% of fetuses (4/128) had the KEL1/KEL2 genotype. Sensitivity and specificity reached 100% (p < 0.0001).

Conclusion: Minisequencing is a reliable method for the assessment of the fetal KEL1 allele from the plasma of KEL2/KEL2 pregnant women.
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http://dx.doi.org/10.1159/000441296DOI Listing
February 2017

Familial, autosomal-dominant neurodegenerative parkinsonism with cognitive deterioration spanning five generations in a genetically isolated population of south-eastern Moravia, Czech Republic.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2016 Mar 15;160(1):158-60. Epub 2015 Apr 15.

General Practicioner, Velka nad Velickou-Javornik, Czech Republic.

Background: An epidemiological study conducted over four years revealed increased prevalence of neurodegenerative parkinsonism in a small, isolated region (10 villages, with a combined population of 8664, with approx. 2927 over 50 years of age) of south-eastern Moravia, Czech Republic. The aim of this study was to obtain more detailed information on the medical history of the relatives of individuals with confirmed parkinsonism in an isolated rural population in south-eastern Moravia, Czech Republic.

Methods: We did detailed genealogical research on the families of all inhabitants with confirmed parkinsonism and compiled the pedigrees. These were modified on the basis of information from a consecutive door-to-door survey and local municipal and church registers.

Results: In the first stage, three large pedigrees with a familial occurrence of parkinsonism were found; two originated in one of the region's villages. In the second stage, these two pedigrees were combined into one large family tree.

Conclusions: The high prevalence of parkinsonism in the researched area is caused by the familial aggregation of parkinsonism that was found in two large family trees. This is probably the result of the genetic isolation of the regional population due to the very low migration rate of its inhabitants to neighboring regions in the last two centuries.
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http://dx.doi.org/10.5507/bp.2015.013DOI Listing
March 2016

STAT6 - polymorphisms, haplotypes and epistasis in relation to atopy and asthma.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2013 Jun 6;157(2):172-80. Epub 2013 Jun 6.

Department of Medical Genetics and Fetal Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc and University Hospital Olomouc, Czech Republic.

Background: STAT6 has an important role in the IL-4 / IL-13 signalling pathway. Genome - wide association studies have shown that particular polymorphism (SNP) or haplotype variants of STAT6 as well as epigenetic gene modifications are associated with IgE level and asthma in childhood.

Methods: A review of the available literature was performed to map out the function and signalling pathway of STAT6, studies of STAT6 SNPs association with susceptibility to asthma and atopy, covering the years 1997 - 2012 were summarized, and the value of epigenetic and epistatic influences on STAT6 and their relevance to the development of the studied phenotype (atopy or asthma) were determined.

Results: There are 2 SNPs (rs71802646 and rs320411) with clinical association and proven functional effect on STAT6 expression. The effect of STAT6 SNPs cumulates in haplotypes and more potently during interaction with SNPs in the genes from the signalling pathway (IL4, IL4Ra, and IL13). Expression of STAT6 is also influenced by DNA methylation. Atopy is traditionally believed to be maternally inherited but there is one report about paternally overtransmitted STAT6 haplotype (TCA haplotype, built from rs324011, rs3024974 and rs4559 SNPs).

Conclusions: STAT6 polymorphisms and their combinations have an important influence on IgE level and development of asthma. However, the interaction between SNPs in the IL-4 / IL-13 signalling pathway is of greater impact. Hypermethylation of the STAT6 promoter is also significant in the regulation of STAT6 expression and this fact opens possibilities for targeting therapy in asthma.
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http://dx.doi.org/10.5507/bp.2013.043DOI Listing
June 2013

Possible control of paternal imprinting of polymorphisms of the ADAM33 gene by epigenetic mechanisms and association with level of airway hyperresponsiveness in asthmatic children.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2013 Dec 23;157(4):367-73. Epub 2013 Apr 23.

Department of Paediatrics, University Hospital Olomouc and Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic.

Introduction: ADAM33 is the candidate gene most commonly associated with asthma and airway hyperreactivity (AHR).

Aim: The aim of this study was to determine whether level of AHR is associated with certain alleles or haplotypes of the ADAM33 gene in asthmatic children.

Methods: One hundred and nine asthmatic children and 46 controls from the general population were examined with spirometry before and after histamine and methacholine inhalation. All subjects were genotyped for single-nucleotide polymorphisms (SNPs) of the ADAM33 gene. Haplotypes were determined according to genotypes of the patient's parents.

Results: We found the three most frequent ADAM33 haplotypes (a1-3) were associated with the highest level of AHR to methacholine and histamine in 66% of asthmatic children. The paternally transmitted GGGCTTTCGCA haplotype was seen in 73.3% asthmatic children with serious AHR to methacholine challenge (paternal and maternal origin of haplotype 73.3% to 37.5, P=0.046) Significant differences in the relative frequency of paternal haplotypes with high levels of AHR to histamine were found (P=0.013).

Conclusion: ADAM33 haplotypes (a1, a2, a3) are associated with severity of AHR and are significantly more often transmitted in the paternal line.
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http://dx.doi.org/10.5507/bp.2013.025DOI Listing
December 2013

A transgenic minipig model of Huntington's Disease.

J Huntingtons Dis 2013 ;2(1):47-68

Laboratory of Cell Regeneration and Plasticity, Institute of Animal Physiology and Genetics, v.v.i., AS CR, Libechov, Czech Republic.

Background: Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan.

Objective: To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1-548 under the control of human HTT promoter.

Methods: Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted.

Results: One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old sibling pairs showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa.

Conclusions: The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study.
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http://dx.doi.org/10.3233/JHD-130001DOI Listing
September 2014

Association of STAT6 and ADAM33 single nucleotide polymorphisms with asthma bronchiale and IgE level and its possible epigenetic background.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2012 Sep 30;156(3):236-47. Epub 2012 Jan 30.

Department of Medical Genetics and Fetal Medicine, University Hospital Olomouc and Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic.

Background: ADAM33 and STAT6 belong to the candidate genes that have been commonly associated with asthma, bronchial hyperresponsiveness or IgE levels. Our objective was to assess the association of 11 SNPs of the ADAM33 and 6 of the STAT6 and their haplotypes with IgE levels and asthma. We also evaluated the possible role of parental origin of haplotypes on IgE levels.

Methods: We enrolled 109 children with asthma and 45 healthy controls. Genotyping was performed by TaqMan probes and confirmed by sequencing. Haplotype construction was based on the knowledge of parental genotypes and also inferred by using the EM algorithm and Bayes' theorem.

Results: None of the SNPs were associated with elevated IgE level or asthma. We found that the most frequent STAT6 haplotype ATTCAA (built from rs324012, rs324011, rs841718, rs3024974, rs3024974, rs4559 SNPs, respectively) was associated with elevated total IgE levels (P=0.01) and this haplotype was predominantly transmitted paternally (P<0.001). We compared our results with those of studies performed on German and Australian Caucasian populations and found that rs324011, rs3024974 and rs4559 SNPs in STAT6 should have a major effect on IgE levels. Therefore, we suggest the TCA haplotype alone (built from rs324011, rs3024974 and rs4559 SNPs, respectively) in STAT6 is associated with total IgE elevation.

Conclusions: The influence of paternal origin of the STAT6 haplotype on IgE levels is surprising but the exact role of possible paternal imprinting in STAT6 regulation should be investigated and confirmed in future studies.
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http://dx.doi.org/10.5507/bp.2012.009DOI Listing
September 2012

Giant angiofibromas in tuberous sclerosis complex: a possible role for localized lymphedema in their pathogenesis.

J Am Acad Dermatol 2012 Dec 30;67(6):1319-26. Epub 2012 Apr 30.

Sikl's Department of Pathology, Charles University in Prague, Faculty of Medicine in Pilsen, Pilsen, Czech Republic.

Background: Giant angiofibromas in patients with tuberous sclerosis complex (TSC) are rare.

Objective: We sought to report two patients who had TSC with unusually large and disfiguring facial angiofibromas and to identify underlying histopathologic changes that may possibly explain the clinical features.

Methods: We performed a clinicopathologic, immunohistochemical, and molecular biologic study using 42 lesional specimens and peripheral blood from one of the two patients. The immunohistochemical investigations were mainly focused on the vascular moiety of the lesions. TSC1 and TSC2 alterations were studied using multiplex ligation-dependent probe amplification for large deletion/duplication mutations, whereas screening for small mutations was performed using polymerase chain reaction amplification of individual coding exons and exon-intron junctions of both genes followed by an analysis on denaturation gradient gel electrophoresis.

Results: Histopathologic examination revealed, in addition to findings typical of angiofibroma, several unusual features including multinucleated giant cells containing multiple intracytoplasmic vacuoles, Touton-like cells, emperipolesis, pagetoid dyskeratosis, vacuolar alteration at the dermoepidermal junction, Civatte bodies, and melanophages in the subjacent dermis. Numerous dilated lymphatic vessels were detected indicating localized lymphostasis, probably caused by secondary lymphedema. The lymphatic nature of the vessels was confirmed by immunohistochemical study. Genetic testing for TSC1 and TSC2 gene mutations revealed a substitution on position c.2251C>T resulting in a nonsense mutation R751X in fragment 20.2.

Limitations: Histopathologic specimens and peripheral blood were available from only one patient.

Conclusion: Localized lymphedema may contribute to the formation of large disfiguring angiofibromas in patients with TSC.
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http://dx.doi.org/10.1016/j.jaad.2012.03.021DOI Listing
December 2012

Sequence recombination in exon 1 of the TSPY gene in men with impaired fertility.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2011 Sep;155(3):287-98

Department of Medical Genetics and Foetal Medicine, Palacky University Olomouc and University Hospital Olomouc, Czech Republic.

Aim: The aim of this study was to evaluate TSPY (testis specific protein on the Y chromosome) gene and 5'UTR (UnTranslated Region) polymorphisms in men with impaired fertility compared to fertile controls.

Methods: We analyzed 72 infertile men and 31 fertile controls usingconventional sequencing analysis to find crucial SNPs (single nucleotide polymorphism) and other changes.

Results: The most remarkable changes were found in the 1(st) exon only. In one half of the both infertile men and fertile controls, the most frequent finding was 26 SNPs with a similar pattern. In the other half we found highly relevant changes, generating a stop codon in the first third of exon 1. Early termination cut down the protein by 78.5%. This kind of change was not found in the fertile controls. No correlation was found between the spermiogram and the changes leading to the stop codon. The distribution of men with deletions, insertion and higher gene copy number was not statistically different.

Conclusion: The changes found in exon 1 in infertile men could fundamentally affect the process of spermatogenesis. These findings could significantly enhance our understanding of the molecular-genetic causes of male infertility.
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http://dx.doi.org/10.5507/bp.2011.034DOI Listing
September 2011

NQO1 expression correlates inversely with NFκB activation in human breast cancer.

Breast Cancer Res Treat 2012 Apr 25;132(3):955-68. Epub 2011 Jun 25.

Department of Obstetrics and Gynecology, Biomedicum Helsinki, Helsinki University Central Hospital, PO Box 700, 00029 Helsinki, Finland.

NQO1 participates in cellular defense against oxidative stress and regulates apoptosis via p53- and NFκB-mediated pathways. We have previously found that homozygous missense variant NQO1*2 (rs1800566) predicts poor survival among breast cancer patients, particularly after anthracycline-based adjuvant chemotherapy. Here, we investigated NQO1 and NFκB protein expression and global gene expression profiles in breast tumors with correlation to tumor characteristics and survival after adjuvant chemotherapy. We used immunohistochemical analysis of tissue microarrays to study NQO1 and NFκB expression in two series of tumors: 1000 breast tumors unselected for treatment and 113 from a clinical trial comparing chemotherapy regimens after anthracycline treatment in advanced breast cancer. We used gene expression arrays to define genes co-expressed with NQO1 and NFκB. NQO1 and nuclear NFκB were expressed in 83% and 11% of breast tumors, and correlated inversely (P = 0.012). NQO1 protein expression was associated with estrogen receptor (ER) expression (P = 0.011), whereas 34.5% of NFκB-nuclear/activated tumors were ER negative (P = 0.001). NQO1 protein expression and NFκB activation showed only trends, but no statistical significance for patient survival or outcome after anthracycline treatment. Gene expression analysis highlighted 193 genes that significantly correlated with both NQO1 and NFκB in opposite directions, consistent with the expression patterns of the two proteins. Inverse correlation was found with genes related to oxidation/reduction, lipid biosynthesis and steroid metabolism, immune response, lymphocyte activation, Jak-STAT signaling and apoptosis. The inverse relationship between NQO1 protein expression and NFκB activation, underlined also by inverse patterns of association with ER and gene expression profiles of tumors, suggests that NQO1-NFκB interaction in breast cancer is different from several other tissue types, possibly due to estrogen receptor signaling in breast cancer. Neither NQO1 nor NFκB protein expression appear as significant prognostic or predictive markers in breast cancer.
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http://dx.doi.org/10.1007/s10549-011-1629-5DOI Listing
April 2012

TSC2/PKD1 contiguous gene syndrome: a report of 2 cases with emphasis on dermatopathologic findings.

Am J Dermatopathol 2009 Aug;31(6):532-41

Sikl's Department of Pathology, Charles University, Medical Faculty Hospital, Pilsen, Czech Republic.

The association of tuberous sclerosis complex (TSC) and autosomal dominant polycystic kidney disease (ADPKD), termed TSC2/PKD1 contiguous gene syndrome, is a result of molecular defect demonstrating by deletion disrupting TSC2 and PKD1. Dermatopathology of this syndrome has never been addressed. We report 2 sporadic cases form of TSC2/PKD1 contiguous gene syndrome, with emphasis on dermatopathologic findings. Both patients presented with a typical phenotype of TSC and early-onset renal polycystic requiring kidney transplantation in one of the patients. Of a total of 13 cutaneous lesions studied, there were 7 facial angiofibromas, 2 shagreen patches, 1 periungual fibroma, 1 hypopigmented macule, 1 epidermoid cyst, and 1 intradermal melanocytic nevus. The histological features were basically similar to those occurring in TSC, but some unusual features were identified. In both patients, deletions in the region of TSC2 and PKD1 were revealed performing by multiplex ligation probe amplification test. It is concluded that the histopathological features of skin lesions in this syndrome are similar to those encountered in TSC. Clinical awareness and appropriate molecular investigation of TSC2/PKD1 contiguous gene syndrome is necessary in all patients with a typical phenotype of TSC in infancy, adolescence, or adult age, because of severity of the renal alterations.
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http://dx.doi.org/10.1097/DAD.0b013e3181970e44DOI Listing
August 2009

Modulation of the E2F1-driven cancer cell fate by the DNA damage response machinery and potential novel E2F1 targets in osteosarcomas.

Am J Pathol 2009 Jul 18;175(1):376-91. Epub 2009 Jun 18.

Department of Histology and Embryology, School of Medicine, University of Athens, Athens, Greece.

Osteosarcoma is the most common primary bone cancer. Mutations of the RB gene represent the most frequent molecular defect in this malignancy. A major consequence of this alteration is that the activity of the key cell cycle regulator E2F1 is unleashed from the inhibitory effects of pRb. Studies in animal models and in human cancers have shown that deregulated E2F1 overexpression possesses either "oncogenic" or "oncosuppressor" properties, depending on the cellular context. To address this issue in osteosarcomas, we examined the status of E2F1 relative to cell proliferation and apoptosis in a clinical setting of human primary osteosarcomas and in E2F1-inducible osteosarcoma cell line models that are wild-type and deficient for p53. Collectively, our data demonstrated that high E2F1 levels exerted a growth-suppressing effect that relied on the integrity of the DNA damage response network. Surprisingly, induction of p73, an established E2F1 target, was also DNA damage response-dependent. Furthermore, a global proteome analysis associated with bioinformatics revealed novel E2F1-regulated genes and potential E2F1-driven signaling networks that could provide useful targets in challenging this aggressive neoplasm by innovative therapies.
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http://dx.doi.org/10.2353/ajpath.2009.081160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708823PMC
July 2009

NAD(P)H:quinone oxidoreductase 1 NQO1*2 genotype (P187S) is a strong prognostic and predictive factor in breast cancer.

Nat Genet 2008 Jul 30;40(7):844-53. Epub 2008 May 30.

Department of Obstetrics and Gynecology, Helsinki University Central Hospital, FI-00029 Helsinki, Finland.

NQO1 guards against oxidative stress and carcinogenesis and stabilizes p53. We find that a homozygous common missense variant (NQO1(*)2, rs1800566(T), NM_000903.2:c.558C>T) that disables NQO1 strongly predicts poor survival among two independent series of women with breast cancer (P = 0.002, N = 1,005; P = 0.005, N = 1,162), an effect particularly evident after anthracycline-based adjuvant chemotherapy with epirubicin (P = 7.52 x 10(-6)) and in p53-aberrant tumors (P = 6.15 x 10(-5)). Survival after metastasis was reduced among NQO1(*)2 homozygotes, further implicating NQO1 deficiency in cancer progression and treatment resistance. Consistently, response to epirubicin was impaired in NQO1(*)2-homozygous breast carcinoma cells in vitro, reflecting both p53-linked and p53-independent roles of NQO1. We propose a model of defective anthracycline response in NQO1-deficient breast tumors, along with increased genomic instability promoted by elevated reactive oxygen species (ROS), and suggest that the NQO1 genotype is a prognostic and predictive marker for breast cancer.
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http://dx.doi.org/10.1038/ng.155DOI Listing
July 2008

Refined fluorescent STR quantification of cell-free fetal DNA during pregnancy in physiological and Down syndrome fetuses.

Prenat Diagn 2008 May;28(5):425-33

Department of Clinical Genetics and Fetal Medicine, University Hospital Olomouc, I.P Pavlova 6, 775 20 Olomouc, Czech Republic.

Background: Cell-free fetal (cff) DNA analysis by short tandem repeats (STR) has the advantage of better recognizing the different genotypes. However, quantitative examination by quantitative fluorescent (QF) polymerase chain reaction (PCR) by STRs is limited to only a rough approximation. This project focuses on a more precise calculation of the relative cff DNA amount tested in the STRs' loci.

Methods: The cff DNA was analyzed in 363 samples from 258 pregnant women with physiological fetuses in different stages of pregnancy (from 4-37 gestational weeks) separately in three STRs [D21S1435, D21S1446 and PentaD (pD)] and also by gonosomal sequences amelogenin gene, X/Y-linked/testis specific protein, Y-linked (AMELX/Y/TSPY). Seventeen samples of cff DNA from fetuses with Down syndrome (DS) were compared. We optimized the refined quantitative fluorescent (RQF) PCR for STRs in a particular locus.

Results And Conclusions: The cff DNA detection rate was 74% in at least one of the STRs. The efficiency decreased from shorter to longer PCR fragments. All three STR and gonosomal loci proved an increase in cff DNA during pregnancy. The stutter variability rate is greatest in short STR fragments and decreases as the STR fragments increase in length. Results showed that DS samples had a significantly higher amount of cff DNA.
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http://dx.doi.org/10.1002/pd.1996DOI Listing
May 2008

TSPY gene copy number as a potential new risk factor for male infertility.

Reprod Biomed Online 2007 May;14(5):579-87

Department of Medical Genetics and Fetal Medicine, University Hospital Olomouc, IP Pavlova 6, 775 20 Olomouc, Czech Republic.

The human TSPY (testis-specific protein, Y-linked) gene family (30-60 copies) is situated in the MSY (male-specific) region of the Y chromosome. Testis-specific expression indicates that the gene plays a role in spermatogenesis. Refined quantitative fluorescence PCR (polymerase chain reaction) was applied to evaluate the relative number of TSPY copies compared with AMELY/X (amelogenin gene, Y-linked) genes in 84 stratified infertile men and in 40 controls. A significantly higher number of TSPY copies was found in infertile men compared with the controls (P = 0.002). The diagnostic discrimination potential of the relative number of TSPY copies was evaluated by receiver operating characteristic curve analysis. TSPY/AMELY was unambiguously found to be powerful in the diagnostic separation of both the control samples and the infertile men, reaching a good level of specificity (0.642) and sensitivity (0.732) at a cut-off point of 0.46. The findings were supported by independently repeated studies of randomly selected positive samples and controls. Evaluation of the TSPY copy number offers a completely new diagnostic approach in relation to the genetic cause of male infertility. The possible effect of the copy number of TSPY genes on spermatogenesis may explain indiscrete pathological alterations of spermatid quality and quantity.
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http://dx.doi.org/10.1016/s1472-6483(10)61049-8DOI Listing
May 2007

Refined quantitative fluorescent PCR of Y-chromosome DNA sequences mosaics in Turner's syndrome patients--alternative to real-time PCR.

J Biochem Biophys Methods 2004 Aug;60(2):151-62

Department of Clinical Genetics and Foetal Medicine, Palacky University, University Hospital Olomouc, I.P Pavlova 6, 775 20 Olomouc, Czech Republic,

Background: Real-time polymerase chain reactions (PCRs) are the most frequently used techniques for gonosomal mosaics quantification. The primary aim of this work is to assess and optimize the refined technique of quantitative fluorescent polymerase chain reaction (RQF PCR) in the quantification of Y-chromosome sequences in gonosomal mosaics. The method was applied to the analysis of Y-chromosome sequences (amelogenin gene, AMELX/Y-loci) in peripheral lymphocytes and gonadal tissues in Y-positive Turner's syndrome (TS) patients.

Methods: RQF PCR was used for molecular quantification, and fluorescent in situ hybridization (FISH) technique was used for comparison.

Results: Based on a formulated calibration curve, DNA mosaics from six Y-positive patients and gonads from one patient were deducted. For calculation of rare mosaics, it is possible to take advantage of a new empirical formula. FISH results were comparable to RQF PCR.

Conclusion: The sensitivity of RQF PCR brings significant progress in the analysis of gonosomal aberrations. RQF PCR also finds applications in prenatal diagnostics of maternal contaminations of amniotic fluid and foetal DNA in maternal blood and analysis of chimerism in patients after bone marrow transplantation. The method is very convenient for determining the number of testis-specific protein, Y-linked (TSPY) gene repetitions.
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http://dx.doi.org/10.1016/j.jbbm.2004.05.004DOI Listing
August 2004
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