RESUMEN
Denaturing gradient gel electrophoresis (DGGE) was carried out on PCR products amplified from exons 2 and 5 of RHD and RHCE. Exon 2 of RHD and exon 2 of the C allele of RHCE have an identical sequence, which differs from that of the c allele of RHCE. One band representing D and/or C, and another representing c, could be distinguished by DGGE of exon 2 amplifications of genomic DNA from individuals with the appropriate Rh phenotype. C and c could only be distinguished in D-negative samples. Exon 5 of RHD and exon 5 of the E and e alleles of RHCE all have different nucleotide sequences. Bands representing D, E and e could be distinguished following DGGE of the products of exon 5 amplification of genomic DNA from individuals with red cells of the appropriate Rh phenotype. In samples from individuals with VS+ red cells (V+ or V-) there was a shift of the band representing e. Sequencing demonstrated that VS is associated with a RHCE e sequence with a single base change predicting a Leu245 --> Val substitution in the Rh polypeptide. This substitution may be responsible for the VS and e5 antigens.
Asunto(s)
ADN/genética , Electroforesis/métodos , Sistema del Grupo Sanguíneo Rh-Hr/genética , Secuencia de Bases , Análisis Mutacional de ADN , Exones , Humanos , Datos de Secuencia Molecular , Mutación , Ácidos Nucleicos Heterodúplex , Fenotipo , Reacción en Cadena de la Polimerasa , Sistema del Grupo Sanguíneo Rh-Hr/inmunologíaRESUMEN
Initial Rh phenotyping of a man with hemolytic anemia, his wife, and son appeared to exclude paternity. No exclusion was found in other blood groups or in the human leukocyte antigen (HLA) system; excluding Rh, the paternity index was 98.58 percent. Samples from these three family members, and two other family members, were tested with additional Rh antisera. The results indicated that the propositus has an Rhmod phenotype with expression of c, weak e, and very weak D, E, and G antigens. To support this hypothesis, DNA analysis of the RHD and RHCE genes was performed on the five family members. Polymerase chain reaction (PCR) products from exons 2 and 5 were analyzed by denaturing gradient gel electrophoresis (DGGE). The DNA results corroborated the serologic findings and refuted the exclusion of paternity.
RESUMEN
We have investigated the arrangement of genes in the rare Rh (Rhesus) partial null condition D--. Southern blot and PCR studies under conditions which distinguish the highly homologous RH D and RH C/E genes show that in an Icelandic family with the D-- haplotype at least 85% of the RH C/E gene is deleted. This finding is in contrast to one other published case of this phenotype, where intact RH D and C/E genes were found, and also to the full amorph Rhnull phenotype, where an intact RH C/E gene was found, accompanied by the deletion of the RH D gene typical of Rh D-negative individuals.
Asunto(s)
Eliminación de Gen , Sistema del Grupo Sanguíneo Rh-Hr/genética , Secuencia de Bases , Exones , Heterocigoto , Homocigoto , Humanos , Islandia , Intrones , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Alu element-primed PCR was performed on genomic clones containing human RH blood group genes. When used as a probe, the Alu PCR product detected a restriction fragment-length polymorphism which is in complete linkage disequilibrium with the Rh C/c serological polymorphism, irrespective of the Rh D or E serological type it is coupled with. This provides the opportunity to type individuals for their RH C gene directly at the DNA level. RFLP analysis of two individuals with the amorph Rh null phenotype revealed that in one case this phenotype occurred on an RH C background, whereas in the other it was on an RH c background. Taken together these results indicate that the Rh C/c polymorphism has arisen only once, but that the amorph Rh null phenotype, although exceedingly rare, is the result of at least two independent mutations.
Asunto(s)
Sistema del Grupo Sanguíneo Rh-Hr/genética , Alelos , Análisis Mutacional de ADN , Femenino , Humanos , Desequilibrio de Ligamiento , Masculino , Linaje , Fenotipo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Eliminación de SecuenciaRESUMEN
AIMS: To determine whether myeloid proliferation was monoclonal or polyclonal in a woman with chronic neutrophilic leukaemia and myeloma. METHODS: The X-linked probe, M27 beta was used to determine the clonality of the neutrophil population by analysis of restriction fragment length polymorphisms and X inactivation pattern. RESULTS: A polyclonal pattern of X inactivation was obtained for the neutrophil population in this patient. CONCLUSION: The myeloid expansion in chronic neutrophilic leukaemia associated with myeloma represents a polyclonal reactive response to the plasma cell clone rather than a co-existent myeloproliferative disorder.
Asunto(s)
Sondas de ADN , Leucemia Neutrofílica Crónica/etiología , Mieloma Múltiple/complicaciones , Anciano , Southern Blotting , Femenino , Ligamiento Genético , Humanos , Leucemia Neutrofílica Crónica/genética , Mieloma Múltiple/genética , Polimorfismo de Longitud del Fragmento de Restricción , Cromosoma XRESUMEN
Mice with severe combined immunodeficiency (SCID) were reconstituted with peripheral blood mononuclear cells (PBMC) obtained from D-negative individuals who had been sensitized to D-positive erythrocytes. Anti-D was spontaneously secreted in mice reconstituted with PBMC obtained from donors within 14 days of re-immunization with D-positive erythrocytes, but was not detected in murine plasma when mice were reconstituted with PBMC obtained from the same donors many years after sensitization, even though anti-D was still present in the serum of these donors. In the murine plasma the anti-D titres were not related to the total human immunoglobulin concentrations. SCID mice reconstituted with PBMC from some donors immune to D-positive erythrocytes (but not immunized within 34 days of donating the sample) made a recall response to the D antigen, which in some cases was maintained for at least 84 days. Depletion of adherent cells from the reconstituting PBMC reduced the total concentration of human IgG obtained. These results show that SCID mice reconstituted with human PBMC (Hu PBMC-SCID) can make a recall response to the D antigen which cannot be attributed to non-specific polyclonal B-lymphocyte activation, and that efficient antigen processing and presentation of the integral erythrocyte membrane D polypeptide occurs in the Hu PBMC-SCID model.