• MeSH
• Anemia, Hemolytic, Autoimmune drug therapy   genetics   MeSH
• beta-Globins genetics   isolation & purification   MeSH
• Asthma genetics   MeSH
• Cell Differentiation MeSH
• Iron Chelating Agents therapeutic use   MeSH
• Child MeSH
• Erythropoietin metabolism   MeSH
• Erythrocytes physiology   MeSH
• Erythropoiesis genetics   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Cell Proliferation MeSH
• Receptors, Erythropoietin metabolism   MeSH
• Gene Expression Regulation MeSH
• Exome Sequencing MeSH
• Signal Transduction MeSH
• Severity of Illness Index MeSH
• STAT3 Transcription Factor genetics   MeSH
• STAT5 Transcription Factor metabolism   MeSH
• Germ-Line Mutation genetics   MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Publication type
• Letter MeSH
• Research Support, Non-U.S. Gov't MeSH

Wear testing of total joint replacement (TJR) is mandatory in preclinical testing before implantation of TJR into the human body. Testing is governed by current international standards that recommend bovine serum (BS) as a lubricating fluid to replace synovial fluid (SF). Recently, the use of BS has been criticized because of differences in content, fluid characteristics, and nonhuman origin. As a result, a more realistic lubricant mimicking SF is needed. To define SF composition, we analyzed SF obtained during revisions of total hip and knee arthroplasties and compared it with SF obtained during primary arthroplasties and from patients without TJR. Samples were acquired from 152 patients. We found that the median total protein concentration for all SF was 36.8 mg/mL, which is significantly higher than concentrations currently recommended by the ISO standards. The γ-globulin concentration was significantly higher and the phospholipid concentration significantly lower in patients with revision of TJR compared with patients without TJR. No significant difference was found in hyaluronic acid concentration and viscosity among the groups. Our results support the need to improve the definition of a more clinically relevant wear testing lubricant in the ISO standards. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1422-1431, 2017.

• MeSH
• Biomimetic Materials chemistry   MeSH
• Adult MeSH
• Phospholipids analysis   metabolism   MeSH
• gamma-Globins analysis   metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Lubricants chemistry   MeSH
• Arthroplasty, Replacement, Hip MeSH
• Aged MeSH
• Synovial Fluid chemistry   metabolism   MeSH
• Arthroplasty, Replacement, Knee MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

We report an infant with sickle cell disease phenotype by biochemical analysis whose β-globin gene (HBB) sequencing showed sickle cell mutation (HBBS ) heterozygosity. The proband has a unique head-to-tail duplication of the β-globin gene cluster having wild-type (HBBA ) and HBBS alleles inherited from her father; constituting her HBBS /HBBS -HBBA genotype. Further analyses revealed that proband's duplicated β-globin gene cluster (∼650 kb) encompassing HBBA does not include the immediate upstream locus control region (LCR) or 3' DNase I hypersensitivity (HS) element. The LCR interacts with β-globin gene cluster involving long range DNA interactions mediated by various transcription factors to drive the regulation of globin genes expression. However, a low level of HBBA transcript was clearly detected by digital PCR. In this patient, the observed transcription from the duplicated, distally displaced HBBA cluster demonstrates that the loss of LCR and flanking 3'HS sites do not lead to complete silencing of HBB transcription.

• MeSH
• 3' Flanking Region MeSH
• beta-Globins genetics   MeSH
• Genes, Duplicate * MeSH
• Transcription, Genetic MeSH
• Infant MeSH
• Humans MeSH
• Mutation MeSH
• Locus Control Region MeSH
• Anemia, Sickle Cell genetics   MeSH
• Gene Silencing MeSH
• Check Tag
• Infant MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH

β-Thalassemia (β-thal) is considered rare in Central Europe. As in other malaria-free regions, the presence of β-thal in Central Europe reflects historical and recent immigration, and demographic changes that have influenced the genetic variability of the current populations living in this area. This study assesses the frequency and spectrum of mutations on the β-globin gene in Czech and Slovak subjects with clinical symptoms of thalassemia. The results of the initial part of this research were published more than two decades ago; the aim of this study was to update these original reports. During the period from 2002 to 2015, 400 cases from Czech and Slovak hematological centers were analyzed. Twenty-nine β-thal mutations, identified in 356 heterozygotes from 218 unrelated families, involve five unique mutations including a recently described insertion of a transposable L1 element into the β-globin gene. One mutation described here is reported for the first time. Most of the mutations were of Mediterranean origin and accounted for 82.0% of cases. All but one case studied were heterozygous carriers, manifesting β-thal minor, with rare exceptions represented by the rare (β(0)) codons 46/47 (+G) (HBB: c.142_142dupG) mutation associated with an α-globin gene quadruplication and by dominantly inherited β-thal with a more severe phenotype. One double heterozygous β-thal patient was a recent immigrant from Moldavia. The list of δβ-thal alleles (26 carriers, 16 families) contains Hb Lepore and two types of δβ(0)-thal deletions. In the past, genetic drift and migration as well as recent immigrations were responsible for the introduction of Mediterranean alleles, while several mutations described in single families were of local origin.

• MeSH
• Asian People ethnology   genetics   MeSH
• White People ethnology   genetics   MeSH
• beta-Globins genetics   MeSH
• beta-Thalassemia epidemiology   genetics   MeSH
• Emigration and Immigration MeSH
• Gene Frequency MeSH
• Genetic Drift MeSH
• Heterozygote MeSH
• Humans MeSH
• Molecular Epidemiology * MeSH
• Mutation * MeSH
• Pedigree MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Slovakia epidemiology   MeSH

Mutations in SNRP200 gene cause autosomal-dominant retinal disorder retinitis pigmentosa (RP). The protein product of SNRNP200 is BRR2, a DExD/H box RNA helicase crucial for pre-mRNA splicing. In this study, we prepared p.S1087L and p.R1090L mutations of human BRR2 using bacterial artificial chromosome recombineering and stably expressed them in human cell culture. Mutations in BRR2 did not compromise snRNP assembly and both mutants were incorporated into the spliceosome just as the wild-type (wt) protein. Surprisingly, cells expressing RP mutants exhibited increased splicing efficiency of the LDHA gene. Next, we found that depletion of endogenous BRR2 enhanced usage of a β-globin cryptic splice site while splicing at the correct splice site was inhibited. Proper splicing of optimal and cryptic splice sites was restored in cells expressing BRR2-wt but not in cells expressing RP mutants. Taken together, our data suggest that BRR2 is an important factor in 5'-splice-site recognition and that the RP-linked mutations c.3260C>T (p.S1087L) and c.3269G>T (p.R1090L) affect this BRR2 function.

• MeSH
• Alternative Splicing MeSH
• beta-Globins genetics   metabolism   MeSH
• HeLa Cells MeSH
• Cloning, Molecular MeSH
• Humans MeSH
• RNA Splice Sites genetics   MeSH
• Mutation * MeSH
• RNA Precursors genetics   metabolism   MeSH
• Genes, Reporter MeSH
• Retinitis Pigmentosa genetics   MeSH
• Ribonucleoproteins, Small Nuclear genetics   MeSH
• RNA Helicases genetics   MeSH
• Spliceosomes MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

We describe the molecular etiology of β(+)-thalassemia that is caused by the insertion of the full-length transposable element LINE-1 (L1) into the intron-2 of the β-globin gene (HBB). The transcript level of the affected β-globin gene was severely reduced. The remaining transcripts consisted of full-length, correctly processed β-globin mRNA and a minute amount of three aberrantly spliced transcripts with a decreased half-life due to activation of the nonsense-mediated decay pathway. The lower steady-state amount of mRNA produced by the β-globin(L1) allele also resulted from a reduced rate of transcription and decreased production of full-length β-globin primary transcripts. The promoter and enhancer sequences of the β-globin(L1) allele were hypermethylated; however, treatment with a demethylating agent did not restore the impaired transcription. A histone deacetylase inhibitor partially reactivated the β-globin(L1) transcription despite permanent β-globin(L1) promoter CpG methylation. This result indicates that the decreased rate of transcription from the β-globin(L1) allele is associated with an altered chromatin structure. Therefore, the molecular defect caused by intronic L1 insertion in the β-globin gene represents a novel etiology of β-thalassemia.

• MeSH
• Alleles MeSH
• Alternative Splicing MeSH
• beta-Globins genetics   MeSH
• beta-Thalassemia genetics   MeSH
• CpG Islands MeSH
• Long Interspersed Nucleotide Elements * MeSH
• Adult MeSH
• Transcription, Genetic MeSH
• Introns * MeSH
• Mutagenesis, Insertional * MeSH
• Humans MeSH
• DNA Methylation MeSH
• Gene Order MeSH
• Promoter Regions, Genetic MeSH
• Gene Expression Regulation MeSH
• RNA Stability MeSH
• Gene Silencing MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Beta-thalassaemia is a congenital disorder caused by point mutations in a haemoglobin beta-globin chain. The heterozygous form produces microcytosis and normal iron levels, however, haemoglobin electrophoresis shows elevated amounts of haemoglobin A2 and eventually foetal haemoglobin F as well. METHODS: Between 2005-2011, in three centres in Slovakia, carriers of beta-thalassaemic genes or other haemoglobinopathies were searched for. Diagnosis was performed by haematologists whereby the family history was evaluated, together with the overall clinical condition, blood count and blood smear, iron parameters, haemolysis and haemoglobin electrophoresis testing. A proportion of patients was examined by molecular genetic methods. RESULTS: A clinical suspicion of the heterozygous form of beta-thalassaemia was documented in 402 patients (21.9%) out of a total of 1,834 examinations. From these patients, 87 underwent molecular genetic testing and mutations of beta globin genes were identified in 70 of them, where the most frequent mutations were IVS 2.1 (28.5%), IVS 1.110 (25.6%) and IVS 1.1 (11.3%). Evidence of haemoglobin S (sickle cell anaemia) was also notable in one case (patient of African origin). Unusually high levels of haemoglobin F (6-21%) were found in 23 adult subjects. CONCLUSION: The study showed that there is a higher number of heterozygotes for beta-thalassaemia and rarely haemoglobinopathies. It is necessary to continue in search of pathological gene carriers in Slovakia.

• MeSH
• beta-Globins genetics   MeSH
• beta-Thalassemia diagnosis   epidemiology   genetics   MeSH
• Child MeSH
• Adult MeSH
• Hemoglobinopathies diagnosis   epidemiology   genetics   MeSH
• Heterozygote MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Molecular Biology MeSH
• Mutation MeSH
• Child, Preschool MeSH
• Aged MeSH
• Anemia, Sickle Cell diagnosis   epidemiology   genetics   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Slovakia MeSH

Changes in nuclear architecture play an important role in the regulation of gene expression. The importance of epigenetic changes is observed during granulopoiesis, when changes in the nuclear architecture are considered a major factor that influences the downregulation of genes. We aimed to assess the influence of chromatin condensation on the regulation of gene expression during granulopoiesis. Based on a previously published microarray analysis, we chose loci with different levels of transcriptional activity during granulopoiesis. Fluorescent in situ hybridisation (FISH) and immunofluorescent labelling of RNA polymerase II were used to determine the relationship between the transcriptional activity of gene clusters and their localisation within areas with different levels of chromatin condensation. Although active loci were positioned outside of areas of condensed chromatin, downregulation of genes during granulopoiesis was not accompanied by a shift of the downregulated loci to condensed areas. Only the beta-globin cluster was subjected to chromatin condensation and localised to condensed areas. Our results indicate that granulopoiesis is accompanied by a non-random, tissue-specific pattern of chromatin condensation. Furthermore, we observed that the decrease in the quantity of RNA polymerase II correlates with the differentiation process and likely acts in synergy with chromatin condensation to downregulate total gene expression.

• MeSH
• beta-Globins biosynthesis   MeSH
• Down-Regulation physiology   MeSH
• HL-60 Cells MeSH
• Leukopoiesis physiology   MeSH
• Humans MeSH
• Multigene Family physiology   MeSH
• Chromatin Assembly and Disassembly physiology   MeSH
• Oligonucleotide Array Sequence Analysis MeSH
• Gene Expression Profiling MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

This study evaluated quantification of fetal extracellular DNA in maternal plasma for differentiation between cases at risk of onset of placental-insufficiency-related complications and normal pregnancies. Using real-time polymerase chain reaction, fetal (sex-determining region Y [SRY] and hypermethylated RASSF1A sequence) and total (beta-globin [GLO] gene) extracellular DNA was examined in 70 normal pregnancies, 18 at risk of placental-insufficiency-related pregnancy complications, 24 preeclampsia with or without (w or w/o) intrauterine growth retardation (IUGR) (median 34.0 week), and 11 IUGR (median 28.5 week). IUGR was diagnosed when estimated fetal weight was below the 10th percentile for evaluated gestational age. Although increased levels of extracellular DNA were detected in pregnancies with preeclampsia w or w/o IUGR relative to controls (RASSF1A, p < 0.001; SRY, p = 0.009; GLO, p < 0.001), quantities of fetal extracellular DNA in IUGR were not statistically significant (RASSF1A, p = 0.21; SRY, p = 0.2). RASSF1A, SRY, and GLO achieved 93.1%, 93.6%, and 92.1% accuracy for differentiation between normal pregnancy and preeclampsia w or w/o IUGR. Lower sensitivity was observed for pregnancies with onset of IUGR (RASSF1A, 60.0%; SRY, 80.0%; GLO, 72.7%), but did not influence final accuracy (RASSF1A, 91.6%; SRY, 92.5%; GLO, 89.5%). Among 18 patients at risk, 8 pregnancies involving 3 female and 5 male fetuses developed preeclampsia (n = 4), IUGR (n = 3), and chronic placentopathy causing hypoxia (n = 1). Elevation of extracellular DNA was demonstrated in 3/5 (SRY), 1/8 (hypermethylated RASSF1A), and 4/8 (GLO) patients at the earliest 26 weeks and at the latest 2 weeks before the onset of symptoms. These data indicate that fetal and total extracellular DNA concentrations can be significantly elevated in plasma of patients who later developed placental-insufficiency-related pregnancy complications. However, this is strongly individualized, and not a rule for all cases, and probably depends on the actual occurrence of excessive placental trophoblast apoptosis.

• MeSH
• beta-Globins genetics   metabolism   MeSH
• Time Factors MeSH
• DNA genetics   MeSH
• Extracellular Space genetics   MeSH
• Genetic Markers genetics   MeSH
• Humans MeSH
• Mothers MeSH
• DNA Methylation MeSH
• Tumor Suppressor Proteins blood   genetics   MeSH
• Placental Insufficiency blood   diagnosis   genetics   MeSH
• Fetus cytology   MeSH
• Sex-Determining Region Y Protein blood   genetics   MeSH
• Risk MeSH
• Case-Control Studies MeSH
• Pregnancy MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH