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

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.

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

The molecular pathology of hemolytic disease of the fetus and newborn (HDFN) is determined by different RHD, RHCE, and KEL 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 RHD gene and then three single nucleotide polymorphisms in the RHCE and KEL 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 RHD, 33 RHCE, and 10 KEL). 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 RHD examination. Accurate and rapid noninvasive fetal genotyping minimizes the possibility of HDFN developing.

Two Reliable Methodical Approaches for Non-Invasive RHD Genotyping of a Fetus from Maternal Plasma.

Noninvasive fetal RHD genotyping is an important tool for predicting RhD incompatibility between a pregnant woman and a fetus. This study aimed to assess a methodological approach other than the commonly used one for noninvasive fetal RHD genotyping on a representative set of RhD-negative pregnant women. The methodology must be accurate, reliable, and broadly available for implementation into routine clinical practice. A total of 337 RhD-negative pregnant women from the Czech Republic region were tested in this study. The fetal RHD genotype was assessed using two methods: real-time PCR and endpoint quantitative fluorescent (QF) PCR. We used exon-7-specific primers from the RHD gene, along with internal controls. Plasma samples were analyzed and measured in four/two parallel reactions to determine the accuracy of the RHD genotyping. The RHD genotype was verified using DNA analysis from a newborn buccal swab. Both methods showed an excellent ability to predict the RHD genotype. Real-time PCR achieved its greatest accuracy of 98.6% (97.1% sensitivity and 100% specificity (95% CI)) if all four PCRs were positive/negative. The QF PCR method also achieved its greatest accuracy of 99.4% (100% sensitivity and 98.6% specificity (95% CI)) if all the measurements were positive/negative. Both real-time PCR and QF PCR were reliable methods for precisely assessing the fetal RHD allele from the plasma of RhD-negative pregnant women.

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

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.

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

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.

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

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.