RESUMEN
Serologic analysis of two families identified an HLA-DR haplotype in which DR1 and DR2 cosegregated. DNA-RFLP analysis of these families with an HLA-DRB probe revealed a pattern of hybridization suggestive of a recombination between DR1 and DR15. Following amplification, cloning, and nucleotide sequencing of HLA-DRB-gene second-exon DNA sequences, three DRB amplification products associated with the novel haplotype were identified: these corresponded to DRB1*0101, DR2 pseudogene, and DRB5*0101. Clones representing the DRB1*1501 and DR1 pseudogenes were not identified: oligonucleotide typing with DRB1*1501-specific probes confirmed the absence of this gene within the DR1/DR2 haplotype. We postulate that the DR1/DR2 haplotype represents a recombinant between those of DR1-Dw1 and DR15-Dw2, and that the crossing-over may have been between the DRB1*0101 gene and the DR2 pseudogene. This is further supported by DNA-RFLP analysis with HLA-DQB and DQA CDNA probes, which revealed conserved linkage genes between the DQB1*0501, DQA1*0101, and DRB1*0101 genes.
Asunto(s)
Antígeno HLA-DR1/genética , Antígeno HLA-DR2/genética , Haplotipos , Recombinación Genética , Secuencia de Bases , Ligamiento Genético , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Cadenas HLA-DRB1 , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Datos de Secuencia Molecular , Linaje , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
The use of DNA restriction fragment length polymorphism (DNA-RFLP) typing for HLA-DR and DQ genes was assessed in 96 patients who were HLA-A,B and DR matched by serology with one or more potential unrelated marrow donors (UD). Two hundred recipient-donor pairs from 10 transplant centres in the UK were studied. DNA-RFLP revealed serological errors in HLA-DR typing and identified additional recipient-donor mismatches. Of the 200 allegedly serologically matched pairs, 55 (28%) were mismatched for HLA-DR and/or DQ by DNA-RFLP typing. Of the 200 pairs, 68 (34%) mixed lymphocyte reactions (MLR) were negative and 132 (66%) positive. There was a significant correlation between DNA-RFLP defined mismatch and MLR positivity. However quantitative studies revealed no trend towards increasing MLR relative response index (RRI) with cumulative RFLP defined mismatch (i.e. for DR plus DQ compared with DR or DQ alone). RFLP matching for HLA-DR and DQ failed to predict a negative MLR. The RRI in the graft-versus-host direction was greater in RFLP matched pairs who carried HLA-DR4 than in matched pairs lacking DR4, suggesting that failure of RFLP to characterize DR4 subtypes was one reason why routine prediction of a negative MLR was not possible. Despite these limitations we have shown that DNA-RFLP is a reliable method for HLA-DR, DQ typing in routine UD searches in diverse clinical centres. By reducing the number of UD tested in MLR, RFLP typing can substantially reduce the work involved in donor selection.
Asunto(s)
Trasplante de Médula Ósea/inmunología , Antígenos HLA-DQ , Antígenos HLA-DR , Donantes de Tejidos , Trasplante de Médula Ósea/efectos adversos , ADN/genética , Enfermedad Injerto contra Huésped/etiología , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Prueba de Histocompatibilidad , Humanos , Prueba de Cultivo Mixto de Linfocitos , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Thirty-seven DR4-positive patient-unrelated bone marrow donor pairs previously DR/DQ restriction fragment length polymorphism (RFLP) typed and tested in mixed lymphocyte culture (MLC), have been DR4-Dw subtyped retrospectively using sequence specific oligonucleotide probes. We found that DR4-Dw subtyping substantially increased the accuracy of pre-MLC matching and could potentially accelerate donor searches by avoiding unnecessary MLC tests on Dw-mismatched donors.
Asunto(s)
Trasplante de Médula Ósea/inmunología , Antígeno HLA-DR4/genética , Prueba de Histocompatibilidad/métodos , Reacción en Cadena de la Polimerasa/métodos , Sondas de ADN , Antígeno HLA-DR4/clasificación , Humanos , Técnicas In Vitro , Prueba de Cultivo Mixto de Linfocitos , Oligodesoxirribonucleótidos/genética , Donantes de TejidosRESUMEN
A statistically significant association was observed between alleles of the HLA-DQA2 and of the DR/DQ complex in a DNA-restriction fragment length polymorphism study of 219 members from 21 multiplex families of patients with hyperthyroid Graves' disease and 773 unrelated individuals selected for homozygosity of the HLA-DQA2 alleles. This data provides evidence for linkage disequilibrium rather than for a hot spot of recombination within the HLA-DQ subregion.
Asunto(s)
Alelos , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Desequilibrio de Ligamiento , Humanos , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Serological identification of the HLA-DQw1(w5)-associated or HLA-DQw3(w7)-associated DR'Br' (DRB1*0103) allele cannot be accomplished in the presence of a second DQw1(w5)-positive or DQw3(w7)-positive haplotype, respectively. DNA-restriction fragment length polymorphism (RFLP) analysis assists in identification of DR'Br', though not in the presence of DR1. We describe an alternative or complementary method for identification of DR'Br' using two oligonucleotide probes which target HLA-DRB1 gene HV3 regions.
Asunto(s)
Sondas de ADN , Antígeno HLA-DR1/genética , Oligodesoxirribonucleótidos/genética , Alelos , Secuencia de Bases , Antígeno HLA-DR1/clasificación , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la PolimerasaRESUMEN
A detailed understanding of the structure and function of the human major histocompatibility complex (MHC) has ensued from studies by molecular biologist during the last decade. Virtually all of the HLA genes have now been cloned, and the nucleotide sequences of their different allelic forms have been determined. Typing for these HLA alleles is a fundamental prerequisite for tissue matching in allogeneic organ transplantation. Until very recently, typing procedures have been dominated by serological and cellular methods. The availability of cloned DNA from HLA genes has now permitted the technique of restriction fragment length polymorphism (RFLP) analysis to be applied, with remarkable success and advantage, to phenotyping of both HLA Class I and Class II determinants. For the HLA Class II genes DR and DQ, a simple two-stage RFLP analysis permits the accurate identification of all specificities defined by serology, and of many which are defined by cellular typing. At the present time, however, RFLP typing of HLA Class I genes is not as practicable or as informative as that for HLA Class II genes. The present clinical applications of HLA-DR and DQ RFLP typing are predominantly in phenotyping of living donors, including selection of HLA-matched volunteer bone marrow donors, in allograft survival studies, and in studies of HLA Class II-associated diseases. However, the time taken to perform RFLP analysis precludes its use for the typing of cadaveric kidney donors. Nucleotide sequence data for the alleles of HLA Class II genes have now permitted the development of allele-specific oligonucleotide (ASO) typing, a second category of DNA analysis. This has been greatly facilitated by the ability to amplify specific HLA Class II DNA 'target' sequences using the polymerase chain reaction (PCR) technique. The accuracy of DNA typing techniques should ensure that this methodology will eventually replace conventional HLA phenotyping.
Asunto(s)
ADN/análisis , Genes MHC Clase II/genética , Antígenos HLA-D/genética , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Humanos , Fenotipo , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
The association of HLA-DR3 with Graves' disease in Caucasoids is well established but its significance is unclear and its clinical value as a predictive parameter for relapse after a course of antithyroid drug therapy is controversial. We have further investigated the predictive value at the genomic level in 51 patients with Graves' disease who were treated with a 6-month course of carbimazole and followed up for 2 years. Using DNA-restriction fragment length polymorphism (DNA-RFLP) allogenotyping, (i) complete concordance of HLA-DR assignment was observed between serological and DNA-RFLP analysis of all but one of 51 patients with Graves' disease; (ii) the DR beta 17(1)-DQ alpha 2-DQ beta 2a (a DNA-RFLP allogenotype of the classical Northern European haplotype of HLA-B8 DR3) was significantly (corrected P = Pcorr less than 0.02) associated with Graves' disease particularly in patients who relapsed (Pcorr less than 0.005); (iii) HLA-DR3 was highly associated with DQA2 U allele (chi 2 = 18.53, d.f.2, P less than 0.0005); (iv) a strong correlation between the DQA2 U allele and the outcome of the disease was observed. Relapse occurred in 91% (10/11) of the patients who were homozygous for the DQA2 U allele whilst only 65% (15/23) and 41% (7/17) of patients who were hetero or homo-zygous for the DQA2 L allele (DQA2 U/L and DQA2 L/L) relapsed within the same period of follow-up (chi 2 = 7.18, d.f.2, P less than 0.05). Though the relapse rate in patients with the DQA2 U/U genotype was not significantly higher than the relapse rate in patients with the DQA2 U/L genotype, it was significantly higher than the relapse rate in patients with DQA2 L/L genotype (P less than 0.0001) with a relative risk of 14.3.
Asunto(s)
Alelos , Genes MHC Clase II/fisiología , Enfermedad de Graves/genética , Southern Blotting , Carbimazol/uso terapéutico , Femenino , Marcadores Genéticos/análisis , Enfermedad de Graves/tratamiento farmacológico , Enfermedad de Graves/inmunología , Antígeno HLA-DR3/análisis , Humanos , Masculino , Polimorfismo de Longitud del Fragmento de Restricción , RecurrenciaRESUMEN
The exon 2 nucleotide sequences of HLA-DQwl-associated and DQw3-associated HLA-DR"Br" alleles were determined from genomic DNA amplified by the Taq polymerase chain reaction technique. Both alleles reveal identical exon 2 nucleotide sequences. Comparison with other DR alleles suggests that DR"Br" may have originated from DR1 by gene conversion with DR4-Dw10, DR5, or DRw6-Dw18 third hypervariable region sequences.
Asunto(s)
Exones , Conversión Génica , Antígenos HLA-DR/genética , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN , ADN Polimerasa Dirigida por ADN , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
The HLA-DR antigens DRw8, DRw11, DRw12, DRw13, DRw14 and DRw17 are strongly associated with the supertypic specificity DRw52. This association has been used to assist in assignment of serological specificity. However, difficulties in the identification of these antigens arise since they are serologically crossreactive. This report describes the application of restriction fragment length polymorphism (RFLP) allogenotyping to assist in the positive identification of DRw8, DRw11, DRw12, DRw13, DRw14, DRw17, DRw52a and DRw52b, and in addition describes further subtypes defined by RFLP which correlate with DQ or Dw associations. We also describe a novel TaqI/DR beta RFLP in a Caucasian family which types serologically as DRw12.
Asunto(s)
Antígenos HLA-DR/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , ADN/genética , Epítopos/inmunología , Genotipo , Antígenos HLA-DQ/inmunología , Subtipos Serológicos HLA-DR , Prueba de Histocompatibilidad , Humanos , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
A single enzyme/multiple probe system of HLA-DR and DQ typing using restriction fragment-length polymorphism (RFLP) analysis is presented. TaqI-digested genomic DNAs are hybridized sequentially with short DR beta, DQ beta, and DQ alpha cDNA probes. The DR beta probe discriminates between the DR allelic specificities DR1 to DRw14, with the two exceptions of some DR3/DRw13 and some DR7/DRw9 combinations. We describe the positive identification of a DRw10-specific RFLP and demonstrate its segregation in families. The DQ beta probe defines an allelic system that identifies the alleles DQw1, DQw2, and DQw3. This permits the resolution of DR3/DRw13 and DR7/DRw9 alleles by defining the DR/DQ association caused by linkage disequilibrium. The DQ alpha probe defines another allelic series interrelated with, but independent from, the DQ beta series. Specific DQ beta/DQ alpha RFLP combinations correlate with known Dw splits of DR2, DRw6, and DR7. Combined use of the three probes permits the identification of HLA-DR, DQ, and certain Dw specificities and provides an effective and easily interpretable system for major histocompatibility complex class II allogenotyping.
Asunto(s)
ADN/genética , Antígenos HLA-D/genética , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Alelos , Genotipo , Humanos , Isoantígenos/genética , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
HLA-DR and -DQ serotyped cell lines and peripheral blood leucocytes were analysed by Southern blot allogenotyping. Using a short DQ beta cDNA probe, a DQ beta allelic series was defined by restriction fragment length polymorphism (RFLP) with the restriction endonuclease TaqI. This DQ beta allelic series correlates with, and defines splits of, the HLA-DQ serological specificities DQw1 (DQ beta 1a and DQ beta 1b RFLPs), DQw2 (DQ beta 2a and DQ beta 2b RFLPs) and DQw3 (DQ beta 3a and DQ beta 3b RFLPs). By sequential use of a short DQ alpha cDNA probe a second, DQ alpha allelic series is defined by RFLP. This series correlates to a lesser extent than DQ beta RFLPs with the HLA-DQ serological specificities. Thus, two DQ alpha RFLPs correlate with a single DQ serotype (DQ alpha 1a and DQ alpha 1c with DQw1), but three DQ alpha RFLPs correlate with more than one DQ serotype (DQ alpha 1b with DQw1 and DQw3; DQ alpha 2 with DQw2 and DQw3; DQ alpha 3 with DQw2 and DQw3). Individual DQ beta and DQ alpha RFLP subtypes appear to correlate with single, or associated HLA-DR specificities. Specific combinations of DQ beta with DQ alpha RFLPs also correlate with HLA-Dw splits of DR2 and DRw6. A system for HLA-DNA typing is described, which uses RFLP patterns generated by sequential hybridization of TaqI-digested DNAs with short DR beta, DQ beta and DQ alpha cDNA probes. The DQ beta and DQ alpha probes not only identify the DQ allele, but because of linkage disequilibrium with DR, help to assign the DR allele, which may not always be identified with a DR beta probe alone.