RESUMEN
Killer-cell immunoglobulin-like receptors (KIR) control natural killer (NK) cell functions by recognizing HLA molecules and modulating the activity of NK cells. The KIR gene cluster contains polymorphic and highly homologous genes. Diversity of the KIR region is achieved through differences in gene content, allelic polymorphism, and gene copy number, which result in unrelated individuals having different KIR genotypes and individualized immune responses that are relevant to multiple aspects of human health and disease. Therefore, KIR genotyping is increasingly used in epidemiological studies. Here, we developed multiplex polymerase chain reaction with sequence-specific primers (PCR-SSP) to compensate for the shortcomings of the conventional PCR-SSP method, which is most commonly used for KIR analysis. Multiplex PCR-SSP method involves six multiplex reactions that detect 16 KIR genes and distinguish variant types of some KIR genes by adding two reactions. The assay was evaluated in a blind survey using a panel of 40 reference DNA standards from the UCLA KIR Exchange Program. The results are 100% concordant with the genotype determined using Luminex-based reverse sequence-specific oligonucleotide typing systems. Additionally, we investigated the currently known 16 KIR genes and their common variants in 120 unrelated Korean individuals. The results were consistent with the KIR genotype previously reported by Hwang et al. This multiplex PCR-SSP is an efficient method for analyzing KIR genotypes in both small- and large-scale studies with minimal labor, reagents, and DNA. Furthermore, by providing a better definition of KIR polymorphisms it can contribute to developments in immunogenetics.
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Reacción en Cadena de la Polimerasa Multiplex , Receptores KIR , Humanos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Genotipo , Análisis Costo-Beneficio , Alelos , Receptores KIR/genética , ADN/genética , Frecuencia de los GenesRESUMEN
BACKGROUND: Middle East respiratory syndrome (MERS) is a lower respiratory tract disease caused by a beta coronavirus (CoV) called MERS-CoV, characterized by a high mortality rate. We aimed to evaluate the association between genetic variation in killer cell immunoglobulin-like receptors (KIRs) and the risk of MERS in South Koreans. METHODS: KIR genes were genotyped by multiplex polymerase chain reaction with sequence-specific primers (PCR-SSP). A case-control study was performed to identify the odds ratios (OR) of KIR genes for MERS and the association of KIR genes and their ligands, human leukocyte antigens (HLA) genes. RESULTS: KIR2DS4D and KIR3DP1F showed higher frequencies in the group of all patients infected with MERS-CoV than in the control group (p = 0.023, OR = 2.4; p = 0.039, OR = 2.7). KIR2DL1, KIR2DP1, and KIR3DP1D were significantly associated with moderate/mild (Mo/Mi) cases. KIR2DL2, KIR2DS1, and KIR3DP1F were affected in severe cases. When we investigated the association between KIR genes and their ligands in MERS patient and control groups, KIR3DL1+/Bw4(80I)+, KIR3DL1+/Bw6+, KIR3DL1+/Bw6-, KIR2DS1+/C2+, and KIR3DS+/Bw4(80I)+ were associated with MERS. KIR3DL1+/Bw6- was found in Mo/Mi cases. KIR2DS1+/C2+ and KIR2DS2+/C1+ were found in severe cases. CONCLUSION: Further investigations are needed to prove the various immune responses of MERS-CoV-infected cells according to variations in the KIR gene and ligand gene. A treatment strategy based on current research on the KIR gene and MERS-CoV will suggest potential treatment targets.
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The range of HLA typing for successful hematopoietic stem cell transplantation (HSCT) is gradually expanding with the next-generation sequencing (NGS)-based improvement in its quality. However, it is influenced by the allocation of finances and laboratory conditions. HLA-A, -B, -C, -DRB1/3/4/5, -DQA1, -DQB1, -DPA1, and -DPB1 alleles were genotyped at the 3-field level by amplicon-based NGS using MiSeqDx system and compared to our previous study employing long-range PCR and NGS using TruSight HLA v2 kit, in healthy donors from South Korea. Exon 2, exons 2/3, exons 2/3/4 or 5 of 11-loci were amplified by multiplex PCR. The sequence reads of over 53 depth counts were consistently obtained in each sample exon, depending on the target exon determined to match the reference sequence contained in the IPD-IMGT/HLA Database. HLA alleles were investigated by combinations of the determined exons. A total of 18 alleles with a frequency over 10% were found at the 11 HLA loci. Three ambiguities of HLA-A, -C, and -DRB1 were resolved. We observed a total of 26 HLA-A ~ C ~ B and 6 HLA-DRB1 ~ DQA1 ~ DQB1 ~ DPA1 ~ DPB1 haplotypes having significant linkage disequilibrium between alleles at all neighboring HLA loci. This result was compatible with the previous one, using TruSight HLA v2 kit. Advantages are simple and short progress time because one plate is used for each PCR step in one PCR machine and 11-loci HLA typing is possible even if only eight samples. These data suggested that expanded 11-loci HLA typing data by amplicon-based NGS might help perform HSCT.
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Pueblos del Este de Asia , Antígenos HLA-B , Humanos , Alelos , Haplotipos , Antígenos HLA-B/genética , Prueba de Histocompatibilidad , Reacción en Cadena de la Polimerasa Multiplex , Antígenos HLA-A/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Frecuencia de los GenesRESUMEN
Chemokines are chemotactic cytokines that can cause directed migration of leukocytes. The aim of this study was to examine differences in single nucleotide polymorphisms (SNP) of chemokine in AITD patients compared to normal controls. A total of 86 Korean pediatric patients were included in the patient group and 183 adults were included in the normal control group. To compare influences of several chemokine gene polymorphisms, 25 SNPs in 16 chemokine genes were analyzed. Genotype frequencies of CCL11(rs3744508)AA(OR = 6.9) and CCR2(rs1799864)AA(OR = 3.8) were higher in the AITD patients than in the controls, whereas CCL17(rs223828)CC was lower in the AITD patients than in the controls(OR = 0.4). In comparison between Graves' disease (GD) patients and controls, genotype frequency of CCL17(rs223828)CC(OR = 0.4) was lower in the GD group, whereas those of CCR2(rs1799864)AA(OR = 4.8) were higher in the GD group. The genotype frequency of CCL11(rs3744508)AA(OR = 11.3) was higher in Hashimoto's thyroiditis (HT) patients, whereas that of CXCL8(rs2227306)CC(OR = 0.4) was lower in HT patients. Polymorphisms of CCL11(rs3744508), CCL17(rs223828), and CCR2(rs1799864) might be associated with AITD, with CCL17(rs223828), CCR2(rs1799864) and CXCR2(rs2230054, rs1126579) affecting GD and CCL11(rs3744508) and CXCL8(rs2227306) affecting HT in Korean children.
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Enfermedad de Graves , Enfermedad de Hashimoto , Adulto , Niño , Humanos , Genotipo , Enfermedad de Graves/genética , Enfermedad de Hashimoto/genética , Polimorfismo de Nucleótido Simple , República de Corea , Pueblos del Este de Asia/genéticaRESUMEN
Latent membrane protein 2A (LMP2A), a latent Ag commonly expressed in Epstein-Barr virus (EBV)-infected host cells, is a target for adoptive T cell therapy in EBV-associated malignancies. To define whether individual human leukocyte antigen (HLA) allotypes are used preferentially in EBV-specific T lymphocyte responses, LMP2A-specific CD8+ and CD4+ T cell responses in 50 healthy donors were analyzed by ELISPOT assay using artificial Ag-presenting cells expressing a single allotype. CD8+ T cell responses were significantly higher than CD4+ T cell responses. CD8+ T cell responses were ranked from highest to lowest in the order HLA-A, HLA-B, and HLA-C loci, and CD4+ T cell responses were ranked in the order HLA-DR, HLA-DP, and HLA-DQ loci. Among the 32 HLA class I and 56 HLA class II allotypes, 6 HLA-A, 7 HLA-B, 5 HLA-C, 10 HLA-DR, 2 HLA-DQ, and 2 HLA-DP allotypes showed T cell responses higher than 50 spot-forming cells (SFCs)/5×105 CD8+ or CD4+ T cells. Twenty-nine donors (58%) showed a high T cell response to at least one allotype of HLA class I or class II, and 4 donors (8%) had a high response to both HLA class I and class II allotypes. Interestingly, we observed an inverse correlation between the proportion of LMP2A-specific T cell responses and the frequency of HLA class I and II allotypes. These data demonstrate the allele dominance of LMP2A-specific T cell responses among HLA allotypes and their intra-individual dominance in response to only a few allotypes in an individual, which may provide useful information for genetic, pathogenic, and immunotherapeutic approaches to EBV-associated diseases.
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PURPOSE: The immune responses of natural killer (NK) cells against cancer cells vary by patient. Killer Ig-like receptors (KIRs), which are some of the major receptors involved in regulating NK cell activity for killing cancer cells, have significant genetic variation. Numerous studies have suggested a potential association between the genetic variation of KIR genes and the risk of development or prognosis of various cancer types. However, an association between genetic variations of KIR genes and glioblastoma (GB) remains uncertain. We sought to evaluate the association of genetic variations of KIRs and their ligand genes with the risk of GB development in Koreans. METHODS: A case-control study was performed to identify the odds ratios (ORs) of KIR genes and Classes A, B, and, C of the human leukocyte antigen (HLA) for GB. The GB group was comprised of 77 patients with newly diagnosed IDH-wildtype GB at our institution, and the control group consisted of 200 healthy Korean volunteers. RESULTS: There was no significant difference in the frequency of KIR genes and KIR haplotypes between the GB and control groups. Genetic variations of KIR-2DL1, 3DL1, and 3DS1 with their ligand genes (HLA-C2, HLA-Bw4/6, and Bw4, respectively) had effects on the risk of GB in Korean patients. The frequency of KIR-2DL1 with HLA-C2 (OR 2.05, CI 1.19-3.52, p = 0.009), the frequency of KIR-3DL1 without HLA-Bw4 (80I) (OR 8.36, CI 4.06-17.18, p < 0.001), and the frequency of KIR-3DL1 with Bw6 (OR 4.54, CI 2.55-8.09, p < 0.001) in the GB group were higher than in the control group. In addition, the frequency of KIR-2DL1 without HLA-C2 (OR 0.44, CI 0.26-0.75, p = 0.003), the frequency of KIR-3DL1 with HLA-Bw4 (80T) (OR 0.13, CI 0.06-0.27, p < 0.001), the frequency of KIR-3DL1 without Bw6 (OR 0.27, CI 0.15-0.49, p < 0.001), and the frequency of KIR-3DS1 with Bw4 (80I) (OR 0.03, CI 0.00-0.50, p < 0.001) in the GB group were lower than in the control group. CONCLUSIONS: This study suggests that genetic variations of KIRs and their ligand genes may affect GB development in the Korean population. Further investigations are needed to demonstrate the different immune responses for GB cells according to genetic variations of KIR genes and their ligand genes.
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BACKGROUND: Autoimmune thyroid disease (AITD) manifests with a female predominance, and much attention has been directed towards the integral membrane protein 2 A (ITM2A) gene located on the X chromosome. METHODS: In a study of 166 pediatric patients with autoimmune thyroid disease (AITD), the ITM2A rs1751094 single-nucleotide polymorphism (SNP) was genotyped. The sample comprised 143 females and 23 males, with 67 patients diagnosed with Hashimoto chronic thyroiditis (HD) and 99 with Graves' disease (GD). In the 99 GD patients, 49 (49.5%) exhibited thyroid-associated ophthalmopathy (TAO). Among the 85 GD patients, 70.6% (60/85) were considered intractable GD. The results were compared to those from 198 healthy Korean individuals, including 97 females and 101 males. RESULTS: The frequency of the rs1751094 C allele and CC/AC genotype were higher in AITD, GD and HD patients compared to controls, while the frequency of the A allele and AA genotype were lower. The results were more pronounced in female AITD and GD patients compared to male patients. The association was also found in intractable GD and TAO patients. Target SNP fits Hardy-Weinberg equilibrium. CONCLUSIONS: These findings indicate that the ITM2A gene polymorphism on the X chromosome may contribute to the immunological basis of female-predominant AITD in Korean children.
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Enfermedad de Graves , Enfermedad de Hashimoto , Humanos , Masculino , Niño , Femenino , Predisposición Genética a la Enfermedad , Frecuencia de los Genes , Enfermedad de Hashimoto/genética , Enfermedad de Hashimoto/diagnóstico , Enfermedad de Graves/genética , Enfermedad de Graves/diagnóstico , Polimorfismo de Nucleótido Simple , Cromosoma X , República de Corea , Proteínas de la Membrana/genéticaRESUMEN
Marie Unna hereditary hypotrichosis (MUHH) is a rare autosomal dominant hair disorder. Through the study of a mouse model, we identified a mutation in the 5'-untranslated region of the hairless (HR) gene in patients with MUHH in a Caucasian family. The corresponding mutation, named 'hairpoor', was found in mutant mice that were generated through N-ethyl-N-nitrosourea mutagenesis. Hairpoor mouse mutants display partial hair loss at an early age and progress to near alopecia, which resembles the MUHH phenotype. This mutation conferred overexpression of HR through translational derepression and, in turn, decreased the expression of Sfrp2, an inhibitor of the Wnt signaling pathway. This study indicates that the gain in function of HR also results in alopecia, as seen with the loss of function of HR, via abnormal upregulation of the Wnt signaling pathway.
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Expresión Génica , Hipotricosis/congénito , Hipotricosis/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Proteínas Wnt/metabolismo , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Línea Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Hipotricosis/genética , Masculino , Ratones , Ratones Pelados , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutación , Linaje , Factores de Transcripción/metabolismo , Regulación hacia Arriba , Proteínas Wnt/genéticaRESUMEN
Artificial antigen-presenting cells (aAPCs) that stably express particular HLA and co-stimulatory molecules by gene transfer have been developed to effectively stimulate T cells. To investigate whether cytochalsin-B-induced membrane vesicles derived from aAPCs (AP-CIMVs) have similar antigen-presenting functions as a cell-free system, T cell responses to different types of antigen presentation were measured using Jurkat reporter cells. First, the aggregation of AP-CIMV, which affects the measurement of function, was inhibited by nuclease treatment to produce uniform AP-CIMVs. The Green fluorescent protein (GFP) expression in Jurkat reporter cells was induced in a dose-dependent manner in groups stimulated with anti-CD3 antibody-coated AP-CIMVs and aAPCs, and anti-CD3/CD28 Dynabead. When Jurkat reporter cells expressing specific T cell receptors were stimulated by AP-CIMVs and aAPCs loaded with CMV pp65 peptide, AP-CIMVs showed similar stimulatory effects to that by aAPC. However, when these Jurkat reporter cells were stimulated by aAPCs endogenously expressing CMV pp65 antigen and their AP-CIMVs, the GFP expression rate by AP-CIMVs was 8.4%, which was significantly lower than 53.2% by aAPCs. Although this study showed a limited T-cell-stimulating effect of AP-CIMVs on endogenously processed antigen presentation, these results provide useful information for the development of improved cell-free systems for T cell stimulation in the future.
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HLA-B*51:353 differs from HLA-B*51:01:01:01 in codon 95 in exon 3.
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Trasplante de Células Madre Hematopoyéticas , Donantes de Tejidos , Alelos , Secuencia de Bases , Antígenos HLA-B/genética , Prueba de Histocompatibilidad , Humanos , Análisis de Secuencia de ADN , VoluntariosRESUMEN
Advances in next-generation sequencing (NGS) have improved the resolution of T-cell receptor (TCR) repertoire analysis, and recent single-cell sequencing has made it possible to obtain information about TCR pairs. In our previous study, cytomegalovirus (CMV) pp65-specific T-cell response restricted by a single human leukocyte antigen (HLA) class I allotype was observed in an individual. Therefore, to effectively clone an antigen-specific TCR from these T cells, we developed a TCR cloning system that does not require a single cell level. First, we established the improved Jurkat reporter cell line, which was TCRαß double knock-out and expressed CD8αß molecules. Furthermore, functional TCRs were directly obtained by reverse TCR cloning using unique CDR3-specific PCR primers after bulk TCR sequencing of activation marker-positive CD8 T cells by NGS. A total of 15 TCRα and 14 TCRß strands were successfully amplified by PCR from cDNA of 4-1BB-positive CD8 T cells restricted by HLA-A*02:01, HLA-A*02:06, HLA-B*07:02, and HLA-B*40:06. The panels with combinations of TCRα and TCRß genes were investigated using Jurkat reporter cell line and artificial antigen-presenting cells (APCs). In two TCR pairs restricted by HLA-A*02:01, one TCR pair by HLA-A*02:06, four TCR pairs by HLA-B*07:02, and one TCR pair by HLA-B*40:06, their specificity and affinity were confirmed. The TCR pair of A*02:01/1-1 showed alloreactivity to HLA-A*02:06. The one TCR pair showed a higher response to the naturally processed antigen than that of the peptide pool. This reverse TCR cloning system will not only provide functional information to TCR repertoire analysis by NGS but also help in the development of TCR-T therapy.
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Infecciones por Citomegalovirus , Receptores de Antígenos de Linfocitos T , Humanos , Receptores de Antígenos de Linfocitos T/genética , Membrana Celular , Reparación del ADN , Clonación MolecularRESUMEN
Recently, long synthetic peptides or in silico-predicted epitope peptides have been used to identify T cell epitopes, but these approaches may not be suitable for investigating naturally processed epitopes. Here, mRNAs, including fragments or predicted epitope sequences of HCMV pp65 antigen, were generated by in vitro transcription following transcriptionally active PCR. Then, artificial antigen-presenting cells (aAPCs) expressing a single HLA allotype were transfected with mRNAs to identify epitopes in donors with T cell responses that recognize pp65 antigen restricted to HLA-A*02:01, -A*02:06, or -B*07:02. T cells restricted to a particular HLA allotype showed positive responses in some of the 10 fragment antigens. Among predicted epitopes within these positive fragments, three epitopes of HLA-A*02:01, -A*02:06, and -B*07:02 were confirmed. In addition, T cells expanded by anti-CD3 stimulation for two weeks could also be effectively used for the identification of these T cell epitopes, although there were individual differences. These results demonstrated that fragment antigens and epitopes can be rapidly generated using mRNA, and naturally processed antigenic regions can be detected using aAPCs without a T cell cloning procedure. This method will help to identify novel T cell epitopes for developing immunotherapy and vaccines against infectious diseases and cancer.
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HLA-DRB1*12:97 differs from HLA-DRB1*12:02:01:01 in codon 63 in exon 2.
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Trasplante de Células Madre Hematopoyéticas , Alelos , Secuencia de Bases , Cadenas HLA-DRB1/genética , Prueba de Histocompatibilidad , Humanos , Análisis de Secuencia de ADN , Donantes de Tejidos , VoluntariosRESUMEN
HLA-B*07:457 differs from HLA-B*07:02:01:01 in codon 117 in exon 3.
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Trasplante de Células Madre Hematopoyéticas , Alelos , Secuencia de Bases , Antígeno HLA-B7 , Prueba de Histocompatibilidad , Humanos , Análisis de Secuencia de ADN , Homología de Secuencia , Donantes de Tejidos , VoluntariosRESUMEN
Mycobacterium tuberculosis infection is generally asymptomatic as latent tuberculosis, but it is still known as the world's leading bacterial cause of death. The diagnosis of latent tuberculosis infection relies on the evidence of cellular immunity to mycobacterial antigens. Since the association between HLA class II and tuberculosis infection has been reported in several population groups, a detailed study on the CD4+ T cell response to major tuberculosis antigens is needed. To elucidate which HLA class II allotypes in an individual are preferentially used in tuberculosis, CD4+ T cells specific to TB10.4, Ag85b, ESAT-6, and CFP-10 of Mycobacterium tuberculosis antigens were analyzed comprehensively. A total of 33 healthy donors were analyzed by ex vivo and cultured ELISPOT using panels of artificial antigen-presenting cells expressing a single HLA class II allotype. The CD4+ T cell responses were increased by an average of 39-fold in cultured ELISPOT compared with ex vivo ELISPOT. In ex vivo and cultured ELISPOT, CD4+ T cell responses showed significantly higher by HLA-DR than those of HLA-DQ and HLA-DP locus. In cultured ELISPOT, 9 HLA-DR allotypes, 4 HLA-DQ allotypes, and 3 HLA-DP allotypes showed positive CD4+ T cell responses. Among ten donors with positive CD4+ T cell responses when tested for mixed Mycobacterium tuberculosis antigens, seven donors were positive for only a single allotype, and three were positive for two allotypes in an individual. However, only one allotype was used for a single antigen-specific response when a single tuberculosis antigen was used individually. These results on the distribution of HLA class II allotypes showing high CD4+ T-cell responses to Mycobacterium tuberculosis antigens and the intra-individual allotype dominance will provide valuable information for understanding the immunobiology and immunogenetics of tuberculosis, which can contribute to the development of more effective vaccines.
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Tuberculosis Latente , Mycobacterium tuberculosis , Tuberculosis Ganglionar , Antígenos Bacterianos , Linfocitos T CD4-Positivos , Antígenos HLA-DP , Antígenos HLA-DQ/metabolismo , Antígenos HLA-DR , HumanosRESUMEN
INTRODUCTION: Middle East Respiratory Syndrome (MERS) caused by MERS-coronavirus (CoV) is a lower respiratory tract disease characterized by a high mortality rate. MERS-CoV spread from Saudi Arabia to other countries, including South Korea. Dysfunction of the human leukocyte antigen (HLA) system has many effects due to genetic complexity and its role in the adaptive immune response. We investigated the association of HLA class I and II alleles with MERS-CoV in 32 patients with MERS. METHODS: HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 were genotyped by polymerase chain reaction sequence-based typing. RESULTS: HLA-DQB1*03:02 are significantly associated with moderate/mild cases of MERS-CoV. Other alleles are no statistical significance. CONCLUSIONS: Treatment strategies based on current research on the HLA gene and MERS-CoV will provide potential therapeutic targets.
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Genes MHC Clase II , Genes MHC Clase I , Coronavirus del Síndrome Respiratorio de Oriente Medio , Cadenas beta de HLA-DQ/genética , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , República de CoreaRESUMEN
HLA-B*13:144 differs from HLA-B*13:01:01:01 in codon 12 in exon 2.
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Trasplante de Células Madre Hematopoyéticas , Alelos , Secuencia de Bases , Antígenos HLA-B/genética , Prueba de Histocompatibilidad , Humanos , Análisis de Secuencia de ADN , Homología de Secuencia , Donantes de Tejidos , VoluntariosRESUMEN
HLA-A*24:514N differs from HLA-A*24:02:01:01 in codon 167 in exon 3.
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Trasplante de Células Madre Hematopoyéticas , Alelos , Secuencia de Bases , Antígenos HLA-A/genética , Prueba de Histocompatibilidad , Humanos , Análisis de Secuencia de ADN , Homología de Secuencia , Donantes de Tejidos , VoluntariosRESUMEN
HLA-A*02:954 differs from HLA-A*02:06:01:01 in codon 82 in exon 2.
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Trasplante de Células Madre Hematopoyéticas , Alelos , Secuencia de Bases , Antígenos HLA-A/genética , Prueba de Histocompatibilidad , Humanos , Análisis de Secuencia de ADN , Homología de Secuencia , Donantes de Tejidos , VoluntariosRESUMEN
HLA-DRB1*09:45 differs from HLA-DRB1*09:01:02:01 in codon 19 in exon 2.