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BACKGROUND AND OBJECTIVES: The aim of the study was to optimize routine non-invasive prenatal detection of fetal RHD gene from plasma of RhD-negative pregnant women (the median of gestational age was 25 weeks, range 10-38) to detect RhD materno-fetal incompatibility and to avoid the redundant immunoprophylaxis. MATERIALS AND METHODS: Initially only one exon of RHD gene (exon 10) was investigated in 281 plasma samples (144 verified after delivery), in the second phase three RHD exons (5, 7, 10) were analyzed in 246 samples of plasma and maternal genomic DNA (204 verified) by real-time PCR method. Detection of Y-chromosomal sequence DYS-14 and five X-chromosomal insertion/deletion polymorphisms was used to confirm the fetal cfDNA detectability in plasma. Specific polymorphisms in RHD gene were detected by sequence-specific primer PCR in nine samples. RESULTS: When only the RHD exon 10 was tested, 2·8% of verified samples were false positive and 3·5% false negative. With three RHD exons (5, 7, 10) and maternal genomic DNA testing, only one case was false negative (0·5%). Nine samples were inconclusive due to RHD-positive results in maternal genomic DNA. These samples were analyzed for specific mutations in RHD gene. Combination of both methods for fetal cfDNA verification succeeded in 75% of tested group. CONCLUSION: Implementation of analysis of three RHD exons and maternal genomic DNA to routine practice lowers dramatically the ratio of false positive and negative results. This method enables more accurate determination of fetal RHD status with the reduction of unnecessary medical care and RhD immunoprophylaxis.
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Diagnóstico Prenatal , Sistema del Grupo Sanguíneo Rh-Hr , ADN , Femenino , Feto , Genotipo , Humanos , Lactante , Embarazo , Reacción en Cadena en Tiempo Real de la Polimerasa , Sistema del Grupo Sanguíneo Rh-Hr/genéticaRESUMEN
Autoimmune hemolytic anemia (AIHA) is caused by auto-antibodies directed against self red blood cell (RBC) surface antigens. A consequence may be an intravascular hemolysis mediated by activated complement or extravascular hemolysis caused by destruction of complex of RBC with autoantibody in spleen and liver. The basic classification subdivides AIHA in primary/idiopathic and secondary with known underlying disease. A classification according to the thermal range of antibody recognizes warm AIHA, cold aglutinin disease (CAD), mixed AIHA and paroxysmal cold hemoglobinuria. Pathogenesis of AIHA consists of a defective antigen presentation to immunocompetent cells, insufficient process of T-lymphocyte tolerance to autoantigens and induction of autoantibody production by B-lymphocytes. For the diagnosis of AIHA are essential direct and indirect antiglobulin tests. The first-line therapy for warm AIHA is still administration of corticosteroids. For non-responding patients, second-line treatment includes rituximab or splenectomy. Combination of other immunosuppressive drugs represents a third-line treatment for resistant/relapsing patients. Rituximab is a treatment of choice for patients with CAD. Key words: anemia hemolytic - autoimmunity - corticosteroids - diagnosis - pathogenesis - rituximab - splenectomy - treatment.
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Anemia Hemolítica Autoinmune , Corticoesteroides , Anemia Hemolítica Autoinmune/diagnóstico , Anemia Hemolítica Autoinmune/tratamiento farmacológico , Anemia Hemolítica Autoinmune/inmunología , Autoanticuerpos , Humanos , Rituximab/uso terapéutico , EsplenectomíaRESUMEN
BACKGROUND: The c.1-67C variant polymorphism in a GATA motif of the FY promoter is known to result in erythroid-specific FY silencing, that is, in Fy(a-) and Fy(b-) phenotypes. A Caucasian donor presented with the very rare Fy(a-b-) phenotype and was further investigated. STUDY DESIGN AND METHODS: Genomic DNA was analyzed by sequencing to identify the cause of the Fy(a-b-) phenotype. Samples were collected from some of his relatives to establish a correlation between the serology and genotyping results. Red blood cells were analyzed by gel column agglutination and flow cytometry. Genomic DNA was analyzed on genotyping microarrays, by DNA sequencing and by allele-specific PCR. RESULTS: In the donor, a single-nucleotide polymorphism T>C within the GATA motif was found at Position c.1-69 of the FY promoter and shown to occur in the FY*A allele. His genotype was found to be FY*A(-69C), FY*BW.01. In six FY*A/FY*B heterozygous members of the family, a perfect correlation was found between the presence vs. absence of the FY*A(-69C) variant allele and a Fy(a-) vs. Fy(a+) phenotype. CONCLUSION: The location of the c.1-69C polymorphism in a GATA motif whose disruption is known to result in a Fy null phenotype, together with the perfect correlation between the presence of the FY*A(-69C) allele and the Fy(a-) phenotype support a cause-effect relationship between the two.
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Sistema del Grupo Sanguíneo Duffy/genética , Alelos , Femenino , Genotipo , Humanos , Masculino , Linaje , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Población BlancaRESUMEN
BACKGROUND: One branch of the RHD phylogenetic tree is represented by the weak D type 4 cluster of alleles with F223V as the primordial amino acid substitution. F223V as well as a large number of further substitutions causing D variants are located at the extracellular RhD protein vestibule, which represents the entrance to the transmembraneous channel of the RhD protein. STUDY DESIGN AND METHODS: RHD and RHCE nucleotide sequences were determined from genomic DNA and cDNA. D epitope patterns were established with commercial monoclonal anti-D panels. RESULTS: The RHD alleles DOL-1 and DOL-2 had the two amino acid substitutions M170T (509T>C) and F223V (667T>G) in common. DOL-2 harbored the additional substitution L378V (1132C>G). Both alleles were observed in Africans and are probably evolutionary related. DMI carried M170I (510G>A), which differed from the DOL-typical substitution. DFW and DFL harbored the substitutions H166P (497A>C) and Y165C (494A>G). The antigen densities of DOL-1, DFL, and DFW were only moderately reduced. CONCLUSION: DOL-1 and DOL-2 belong to the weak D type 4 cluster of RHD alleles. Together with DMI, DFL, and DFW they represent D variants with amino acid substitutions located at extracellular loops 3 or 4 lining the RhD protein vestibule. These substitutions were of minor influence on antigen density while adjacent substitutions in the transmembraneous section caused weak D antigen expression. All these D variants were partial D and alloanti-D immunizations have been observed in DOL-1, DMI, and DFL carriers. The substitution at position 170 causes partial D although located deep in the vestibule.
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Sistema del Grupo Sanguíneo Rh-Hr/genética , Alelos , Humanos , Isoanticuerpos/inmunología , Familia de Multigenes , Filogenia , Globulina Inmune rho(D)RESUMEN
The Bloodgen project was funded by the European Commission between 2003 and 2006, and involved academic blood centres, universities, and Progenika Biopharma S.A., a commercial supplier of genotyping platforms that incorporate glass arrays. The project has led to the development of a commercially available product, BLOODchip, that can be used to comprehensively genotype an individual for all clinically significant blood groups. The intention of making this system available is that blood services and perhaps even hospital blood banks would be able to obtain extended information concerning the blood group of routine blood donors and vulnerable patient groups. This may be of significant use in the current management of multi-transfused patients who become alloimmunised due to incomplete matching of blood groups. In the future it can be envisaged that better matching of donor-patient blood could be achieved by comprehensive genotyping of every blood donor, especially regular ones. This situation could even be extended to genotyping every individual at birth, which may prove to have significant long-term health economic benefits as it may be coupled with detection of inborn errors of metabolism.
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A 45-year-old man developed chorea, behavioural changes, moderate amyotrophy and polyneuropathy. Hypertrophic cardiomyopathy and increased serum lactate dehydrogenase and creatine kinase (CK) were found. Acanthocytes were not detected. The absence of XK protein and faintly expressed Kell antigens on erythrocytes were found. Genetic test revealed a R133X mutation of the XK gene, confirming the McLeod syndrome. After 7 years he suddenly developed delirium followed by severe hypoglycaemia, hyperthermia, rhabdomyolysis, hepatic and renal failure. Malignant arrhythmia caused death.
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Acantocitos/patología , Corea/genética , Corea/metabolismo , Trastornos de los Cromosomas Sexuales , Acantocitos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Corea/complicaciones , Creatina Quinasa/sangre , Humanos , Hidroliasas/sangre , Sistema del Grupo Sanguíneo de Kell/sangre , Masculino , Persona de Mediana Edad , MutaciónRESUMEN
BACKGROUND: RhD and RhCE are structurally related to ammonium transporter proteins, yet their physiologic function remains unclear. Recent three-dimensional homology modeling with Escherichia coli AmtB as a template defined a putative transmembraneous channel. Three RhD variants with amino acid substitutions located at the extracellular channel aperture are described. STUDY DESIGN AND METHODS: Blood samples were selected because of serologic abnormalities. RHD, RHCE, and LW nucleotide sequences were determined from genomic DNA. D epitope patterns were established with monoclonal anti-D panels. Three-dimensional Rh structures were calculated by alignment to AmtB. RESULTS: The RHD allele DCS-1 was found to carry the two amino acid substitutions F223V (667T > G) and A226P (676G > C) caused by missense mutations in RHD exon 5. This study compared DCS-1 with the D variants DFV (F223V) and DCS-2 (A226P), harboring solely one or the other of the two substitutions. All three D variants were associated with a cDE haplotype. The antigen densities were approximately 3,000 D antigens per red blood cell for DCS-1, 800 for DCS-2, and 9,300 for DFV. DCS-1 and DCS-2 were partial D, because they lacked distinct epitopes. DFV presented as an almost normal D phenotype; the sample contained allo-anti-LW(a). The D(w) antigen was absent from DCS-1, DFV, and DAU-4, but expressed by DAU-5. CONCLUSION: DCS-1, DCS-2, and DFV carry amino acid substitutions at the extracellular vestibule, visualized by 3-dimensional modeling. Proline at position 226 greatly influenced the D antigen density and may reduce the RhD membrane integration. Although the F223V substitution is regarded as the initial event in the evolution of the weak D Type 4 cluster, the current DFV allele probably evolved independently, as evident from different RHCE haplotypes.