RESUMO
BACKGROUND: The Dombrock (Do) blood group system consists of five distinct antigens: Do(a), Do(b), Gy(a), Hy, and Jo(a). Our finding of a patient whose plasma contained a Do-related alloantibody suggested the presence of a sixth antigen. STUDY DESIGN AND METHODS: Standard hemagglutination, flow cytometry, and polymerase chain reaction (PCR)-based methods were used throughout. Protein homology modeling was used to map the amino acid change on the protein structure. RESULTS: The patient's red blood cells (RBCs) typed as Do(a-b-), Hy+(w), Jo(a+(w)), and Gy(a+(w)). The patient's plasma agglutinated RBCs with common Dombrock phenotypes. Reactivity with Hy- and Jo(a-) RBC samples was weak, and Gy(a-) RBC samples were nonreactive. DNA analysis showed the patient to be DO*793A (DO*A/DO*A), DO*323G, and DO*350C, which predicts the Do(a+b-), Hy+, and Jo(a+) phenotype, and revealed a homozygous single-nucleotide change of 547T>G in Exon 2 that is predicted to change tyrosine at Amino Acid Position 183 to aspartic acid. This missense substitution introduced a BtgZI restriction enzyme site. The sequence data were confirmed with a PCR-restriction fragment length polymorphism assay and revealed that the patient's parents and children were heterozygous DO*547T/G. Homology modeling predicted that the 183Tyr substitution by Asp altered the Cys182 environment and influenced the formation and/or stability of the Cys182-Cys231 disulfide bond. CONCLUSION: The patient's DO genes have a single-nucleotide change, which leads to the absence of the high-prevalence antigen DOYA. The absence of this antigen is associated with 183Asp and silencing of Do(a) and weakening of Gy(a), Hy, and Jo(a) antigens.
Assuntos
ADP Ribose Transferases/biossíntese , Antígenos de Grupos Sanguíneos/biossíntese , Eritrócitos/metabolismo , Regulação da Expressão Gênica , Antígeno H-Y/biossíntese , Proteínas de Membrana/biossíntese , Mutação de Sentido Incorreto , ADP Ribose Transferases/genética , ADP Ribose Transferases/imunologia , Adulto , Substituição de Aminoácidos , Antígenos de Grupos Sanguíneos/genética , Dissulfetos/imunologia , Dissulfetos/metabolismo , Feminino , Antígeno H-Y/genética , Antígeno H-Y/imunologia , Homozigoto , Humanos , Isoanticorpos/química , Isoanticorpos/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Polimorfismo de Fragmento de Restrição/genética , Polimorfismo de Fragmento de Restrição/imunologiaRESUMO
BACKGROUND: The JAL antigen (Rh48) was discovered more than 30 years ago when it caused hemolytic disease of the fetus and newborn in an African American family. A decade later it was found to cause hemolytic disease of the fetus and newborn in a Caucasian family. The presence of the same low-prevalence antigen in two different ethnic groups is rare, but additional JAL+ in both groups was subsequently identified. This study was undertaken to investigate the RH gene(s) responsible for expression of JAL and to determine the structural relationship between JAL and other Rh antigens. STUDY DESIGN AND METHODS: Samples from 17 JAL+ people were included: 2 Caucasian, 6 African American, 7 African Brazilian, 1 Caribbean, and 1 Puerto Rican. RHCE and RHD were investigated at the genomic level, and Rh cDNAs were cloned and sequenced for some samples. RESULTS: Caucasian JAL+ probands had RHCE*Ce, while JAL+ probands with African ancestry had RHCE*ce, but all had a nucleotide 340C>T change in Exon 3 of RHCE predicted to encode Arg114Trp. The JAL-encoding RHCE*ce also had 733C>G (Leu245Val) and was linked to conventional RHD or to RHD*DAU0. CONCLUSIONS: JAL+ results from a nucleotide 340C>T (Arg114Trp) on either a Ce or ce background. Homology modeling of the JAL+ RhCE protein suggests that the Arg-->Trp change eliminates a critical loop-stabilizing H-bond between the side chain of Arg114 and the e-specific amino acid Ala226. Additionally, accommodation of the bulky tryptophan would disrupt the conformation of the extracellular loops containing C/c- and e-specific amino acids, providing a structural hypothesis for the simultaneous altered expression of C/c, e, and V/VS antigens.
Assuntos
Substituição de Aminoácidos , Sistema do Grupo Sanguíneo Rh-Hr/genética , Substituição de Aminoácidos/imunologia , Anticorpos/sangue , Anticorpos/genética , Arginina/genética , Sequência de Bases , Análise Mutacional de DNA , Eritrócitos/metabolismo , Genótipo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Sistema do Grupo Sanguíneo Rh-Hr/química , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Sistema do Grupo Sanguíneo Rh-Hr/metabolismo , Homologia de Sequência de Aminoácidos , Triptofano/genéticaRESUMO
BACKGROUND: The gene polymorphisms responsible for the antigens Doa, Dob, Hy, and Joa in the Dombrock (Do) blood group system have been identified. Four different mutations have been reported to cause the Dombrock null [Gy(a-)] phenotype. These include splice mutations, an eight-nucleotide deletion, and insertion of a stop codon. Here a Dombrock null caused by a single-amino-acid substitution in the full-length protein is reported. STUDY DESIGN AND METHODS: DOA and DOB were determined by polymerase chain reaction-restriction fragment length polymorphism, and DO (ART4) exons and flanking regions were sequenced from genomic DNA. Expression analysis was performed by transfection of wild-type and mutant cDNAs into HEK 293T cells followed by flow cytometry and immunoblotting. Homology modeling was used to map the mutation on the protein structure. RESULTS: The patient's sample carried nt 793G/G, indicating a DOB/DOB background. Exon 2 sequencing showed the sample carried a new mutation, nt 185T>C, causing a Phe62Ser substitution. This variant Do was not expressed on the surface of transfected HEK 293T cells. The mutation maps to a highly conserved FDDQY motif located between the beta1-strand and alpha1-helix near the COOH terminus in the native molecule. CONCLUSIONS: The Dombrock null reported here is due to a single Phe62Ser mutation. The expression data confirmed that 62Ser is responsible for lack of cell surface Do, and protein modeling suggests the mutation disrupts important aromatic side chain interactions between Phe62 and His160. Production of an antibody to a high prevalence Dombrock antigen (anti-Gya) in this patient was consistent with complete absence of Dombrock/ART4 protein.
Assuntos
ADP Ribose Transferases/genética , Alelos , Proteínas de Membrana/genética , ADP Ribose Transferases/química , Idoso , Motivos de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular , Feminino , Humanos , Proteínas de Membrana/química , Modelos Moleculares , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNARESUMO
The kappa light chain locus of swine has been mapped to chromosome 3q12-q14 but at this time, there is not enough information comprising the variable region genes or their transcripts. Here we report the sequences of five transcripts of swine kappa light chain variable region obtained from the spleen of two adult Yorkshire pigs. The lengths of the deduced sequences of these transcripts were variable (between 107 to 112 amino acids). Comparisons of the nucleotide sequences of their variable regions with other species like human and murine VL genes shows a high degree of identity, indicating the use of at least two different families of variable light genes. The contribution of these genes to the generation of variability in swine light chain variable genes as well as their therapeutic use in humans is discussed. An interesting possibility would be the development of an antiretroviral vaccine, useful to protect against a potential risk of infections due to xenotransplantation.
Assuntos
Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Animais , Diversidade de Anticorpos/genética , Diversidade de Anticorpos/imunologia , Sequência de Bases , DNA Complementar/genética , DNA Complementar/imunologia , Humanos , Região Variável de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Dados de Sequência Molecular , Análise de Sequência de DNA , SuínosRESUMO
The kappa light chain locus of swine has been mapped to chromosome 3q12-q14 but at this time, there is not enough information comprising the variable region genes or their transcripts. Here we report the sequences of five transcripts of swine kappa light chain variable region obtained from the spleen of two adult Yorkshire pigs. The lengths of the deduced sequences of these transcripts were variable (between 107 to 112 amino acids). Comparisons of the nucleotide sequences of their variable regions with other species like human and murine VL genes shows a high degree of identity, indicating the use of at least two different families of variable light genes. The contribution of these genes to the generation of variability in swine light chain variable genes as well as their therapeutic use in humans is discussed. An interesting possibility would be the development of an antiretroviral vaccine, useful to protect against a potential risk of infections due to xenotransplantation (au)
Assuntos
Animais , Região Variável de Imunoglobulina , Diversidade de Anticorpos , DNA Complementar , Região Variável de Imunoglobulina , Análise de Sequência de DNA , SuínosRESUMO
The kappa light chain locus of swine has been mapped to chromosome 3q12-q14 but at this time, there is not enough information comprising the variable region genes or their transcripts. Here we report the sequences of five transcripts of swine kappa light chain variable region obtained from the spleen of two adult Yorkshire pigs. The lengths of the deduced sequences of these transcripts were variable (between 107 to 112 amino acids). Comparisons of the nucleotide sequences of their variable regions with other species like human and murine VL genes shows a high degree of identity, indicating the use of at least two different families of variable light genes. The contribution of these genes to the generation of variability in swine light chain variable genes as well as their therapeutic use in humans is discussed. An interesting possibility would be the development of an antiretroviral vaccine, useful to protect against a potential risk of infections due to xenotransplantation (au)
Assuntos
Animais , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Análise de Sequência de DNA , Suínos , Diversidade de Anticorpos , DNA Complementar/genética , DNA Complementar/imunologiaRESUMO
The kappa light chain locus of swine has been mapped to chromosome 3q12-q14 but at this time, there is not enough information comprising the variable region genes or their transcripts. Here we report the sequences of five transcripts of swine kappa light chain variable region obtained from the spleen of two adult Yorkshire pigs. The lengths of the deduced sequences of these transcripts were variable (between 107 to 112 amino acids). Comparisons of the nucleotide sequences of their variable regions with other species like human and murine VL genes shows a high degree of identity, indicating the use of at least two different families of variable light genes. The contribution of these genes to the generation of variability in swine light chain variable genes as well as their therapeutic use in humans is discussed. An interesting possibility would be the development of an antiretroviral vaccine, useful to protect against a potential risk of infections due to xenotransplantation.
