Screening of RHD fetal genotype in RhD negative women.

OBJECTIVE: In the Czech Republic in all women within a first trimester screening a laboratory testing for RhD blood group from the peripheral blood should be performed. The aim of the screening is to diagnose RhD negative pregnant women, who may be at risk of developing RhD alloimmunization if the fetus is RhD positive. Currently, the prevention of RhD alloimmunization is carried out regardless of the knowledge of RHD fetal status. Already at the beginning of pregnancy it is possible to determine the RHD genotype of the fetus non-invasively due to cell free fetal DNA circulating in maternal peripheral blood detection. The issue of screening examination of fetal RHD genotype is solved worldwide. In some European countries, the examination is routinely established and thus contributes to the optimization of prenatal care for RhD negative pregnant women, immunoglobulin administration is targeted only in pregnancies with RhD positive fetus. The aim of our study is to evaluate clinical and laboratory effectiveness of fetal RHD genotype screening in RhD negative women by TaqMan Real-time PCR method. Designe: Prospective cohort study. SETTING: Obstetrics and Gynecology Clinic of the Faculty of Medicine University Palacky and the University Hospital Olomouc; Institute of Medical Genetics of the Faculty of Medicine UP and the University Hospital Olomouc; Transfusion Department of the University Hospital Olomouc; Institute of Biophysics of the Faculty of Medicine UP Olomouc. MATERIAL AND METHODS: In 2011-2015 at the University Hospital Olomouc 337 examinations of RHD fetal genotype were performed in pregnant women in first and second trimester and evaluated by TaqMan Real-time PCR, followed by verification of the newborn RHD genotype. RESULTS: Methodology of fetal RHD genotype examination is accurate, reliable and useful in clinical practice. The sensitivity was 97.8%. The specificity was 98.7%. When assessing the effectiveness of the introduction of non- -invasive fetal RHD genotype screening in RhD negative women, it is necessary to assess the medical, organizational and economic aspects. More consistent prevention of RhD alloimmunization in the cases actually indicated may reduce the incidence of RhD alloimmunization. CONCLUSION: From the medical point of view the RHD genotype determination in all RhD negative women at the beginning of pregnancy seems effective. It allows to diagnose about 40% of pregnancies with RhD negative fetuses that do not require administration of IgG anti-D. IgG anti-D should be administered only in indicated cases. Determination of fetal RHD genotype by using TaqMan Real-time PCR is useful in clinical practice.

The effectiveness of KEL and RHCE fetal genotype assessment in alloimmunized women by minisequencing.

OBJECTIVE: To evaluate the effectiveness of the fetal KEL and RHCE genotype assessment in alloimmunized pregnant women by minisequencing. DESIGN: Prospective cohort study. SETTING: Obstetrics and Gynecology Clinic of the Faculty of Medicine UP and the University Hospital Olomouc; Institute of Medical Genetics of the Faculty of Medicine UP and the University Hospital Olomouc; Transfusion Department of the University Hospital Olomouc; Institute of Biophysics of the Faculty of Medicine UP Olomouc. SUBJECT AND METHOD: In the years 2001-2019, 366 samples of pregnant women in the first and second trimester were assessed KEL (n = 327) or RHCE (n = 39) genotype from the free fetal DNA circulating in the peripheral blood by minisequencing. The genotype of the fetus was verified from the buccal smear of the newborn. RESULTS: The KEL genotype was assessed in 327 women (the presence of a variant of the KEL1 alele, which corresponds to the presence of the erythrocyte antigen “K“. The analysis failed in 2 cases (2/327), 16 heterozygote women (KEL1/KEL2) were excluded and in the case of 309 homozygote women (KEL2/KEL2) the fetal KEL genotype was assessed. In the case of 95.8% of the fetuses (296/309) and 95.5% of the newborns (295/309), the KEL2/KEL2 genotype was assessed. In the case of 4.2 % of the fetuses (13/309) and 4.5% of the newborns (14/309), the KEL1/KEL2 genotype was assessed. The sensitivity was 92.86%. The specificity was 100%. The RHCE genotype was assessed in 39 women. In the case of 22 women, the presence of a variant of the RHCE gene, which corresponds to the presence of the erythrocyte antigen “C“/“c“, was assessed. 5 heterozygote women (C/c) were excluded. In the case of 11 homozygote women (C/C), the RHCE genotype was assessed. In the case of 64% (7/11) of the fetuses and newborns, the C/c genotype was assessed, in the case of 36% (4/11) the C/C genotype was assessed. In the case of 6 homozygote women (c/c), the RHCE genotype was assessed. In the case of 67% (4/6) of the fetuses and newborns, the C/c genotype was assessed, in the case of 33% (2/6) the c/c genotype was assessed. The sensitivity and specificity were 100%. In the case of 17 women, the presence of the variant of the RHCE gene, which corresponds to the presence of the erythrocyte antigen “E“/“e“, was assessed. 1 heterozygote woman (E/e) was excluded. In the case of 16 homozygote women (e/e), the RHCE genotype was assessed. In the case of 75% (12/16) of the fetuses and newborns, the e/e genotype was assessed, in the case of 25% (4/16) the E/e genotype was assessed. The sensitivity and specificity were 100%. CONCLUSION: The minisequencing method using the capillary electrophoresis enabled a reliable detection of the fetal KEL and RHCE genotype from the peripheral blood of pregnant women.

Twelve toll-like receptor (TLR) genes in the family Equidae - comparative genomics, selection and evolution.

Toll-like receptors (TLRs) represent an important part of the innate immune system. While human and murine TLRs have been intensively studied, little is known about TLRs in non-model species. The order Perissodactyla comprises a variety of free-living and domesticated species exposed to different pathogens in different habitats and is therefore suitable for analyzing the diversity and evolution of immunity-related genes. We analyzed TLR genes in the order Perissodactyla with a focus on the family Equidae. Twelve TLRs were identified by bioinformatic analyses of online genomic resources; their sequences were confirmed in equids by genomic DNA re-sequencing of a panel of nine species. The expression of TLR11 and TLR12 was confirmed in the domestic horse by cDNA sequencing. Phylogenetic reconstruction of the TLR gene family in Perissodactyla identified six sub-families. TLR4 clustered together with TLR5; the TLR1-6-10 subfamily showed a high degree of sequence identity. The average estimated evolutionary divergence of all twelve TLRs studied was 0.3% among the Equidae; the most divergent CDS were those of Equus caballus and Equus hemionus kulan (1.34%) in the TLR3, and Equus africanus somaliensis and Equus quagga antiquorum (2.1%) in the TLR1 protein. In each TLR gene, there were haplotypes shared between equid species, most extensively in TLR3 and TLR9 CDS, and TLR6 amino acid sequence. All twelve TLR genes were under strong negative overall selection. Signatures of diversifying selection in specific codon sites were detected in all TLRs except TLR8. Differences in the selection patterns between virus-sensing and non-viral TLRs were observed.

[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.

[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.

[Early fetal karyotyping and its role in prenatal diagnosis]. / Vcasná karyotypizace fetu a její prínos pro prenatální diagnostiku.

OBJECTIVE: Shift of indicated invasive examination in prenatal diagnostics towards the earlier phases of pregnancy with preservation of quality of cytogenetic detection. DESIGN: Cytogenetic and molecular-cytogenetic analysis of the chorionic villi after long term culture. SETTING: Department of Medical Genetics and Foetal Medicine, Faculty of Medicine, Palacky University Olomouc, Faculty Hospital in Olomouc. METHODS: Cultivation of fibroblasts developing from chorionic villi after enzymatic or mechanical disintegration and their karyotyping. Using fluorescent in situ hybridisation to identify the most common chromosomal aneuploidies and to determine gonosomes in indicated cases. RESULTS: Testing and optimisation of long term culture method and its routine use. Method was utilised so far in 12 patients and successfulness was 83%. Additional fluorescent in situ hybridisation was performed in 6 cases. CONCLUSION: Using long term culture method of chorionic villi as reliable and routine tool in prenatal diagnostics.

[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.

[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.

[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.

[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.