RESUMO
HLA-DQB2 is a gene of limited polymorphism, with unknown function that presents at least two transcript variants: v1, which encodes the full-length beta-chain, and v2, which lacks exon 4 and could give rise to a soluble protein. We previously showed a strong correlation between high v2 expression in preimplantation biopsies (PIB) of kidneys from young (18- to 49-year olds) but not from old, deceased donors and 1-year posttransplant low (estimated glomerular filtration rate < 45 ml/min/1.73 m2 ) graft function (GF). In this study, we aimed to investigate the impact of posttransplant soluble HLA-DQB2 (sDQB2) serum levels, v1 expression in PIB, and recipient HLA-DQB2 rs7453920 A/G polymorphism on GF. sDQB2 was evaluated by enzyme-linked immunosorbent assay in sera from 114 recipients, collected at least 1 year (median 2.1 years) after transplantation. Higher sDQB2 levels were observed in recipients of kidneys from young, but not from old, donors that had a ≥30% decline in GF within 1 year after blood collection for sDQB2 determination. Among the 15 recipients of kidneys from young donors with sDQB2 ≥ 1.52 ng/ml, 40% presented a ≥30% decline in GF, whereas this occurred in none of the 43 recipients with lower sDQB2 levels (p = 0.007; OR: 36.5). Expression of HLA-DQB2 variant 1, measured by reverse transcription-polymerase chain reaction (RT-PCR) in 92 PIB from young or old donors, did not significantly differ between transplants with high or low 4-year GF. HLA-DQB2 rs7453920 single nucleotide polymorphism (SNP) frequencies did not significantly differ between recipients with low or high 4-year GF. We conclude that HLA-DQB2 variant 1 expression in PIB and recipient rs7453920 SNP polymorphism are not associated with graft outcome. On the other hand, the association, in transplants of kidneys from young donors, between high posttransplant serum sDQB2 levels and decline in GF is a very interesting finding that deserves a validation study in a larger cohort.
Assuntos
Sobrevivência de Enxerto , Transplante de Rim , Estudos de Coortes , Rejeição de Enxerto , Humanos , Rim , Transplante de Rim/efeitos adversos , Doadores de TecidosRESUMO
The plethora of RNA-seq data which have been generated in the recent years constitutes an attractive resource to investigate HLA variation and its relationship with normal and disease phenotypes, such as cancer. However, next generation sequencing (NGS) brings new challenges to HLA analysis because of the mapping bias introduced by aligning short reads originated from polymorphic genes to a single reference genome. Here we describe HLApers, a pipeline which adapts widely used tools for analysis of standard RNA-seq data to infer HLA genotypes and estimate expression. By generating reliable expression estimates for each HLA allele that an individual carries, HLApers allows a better understanding of the relationship between HLA alleles and phenotypes manifested by an individual.
Assuntos
Antígenos HLA/genética , Teste de Histocompatibilidade/métodos , Análise de Sequência de RNA/métodos , Alelos , Expressão Gênica , Frequência do Gene , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , FenótipoRESUMO
BACKGROUND: Accurate pre-transplant prediction of late graft function remains an unmet need in kidney transplantation. The aim of this study was to evaluate HLA genes expression levels in pre-implantation biopsies (PIB) of deceased donor kidneys as markers for long-term graft outcome. METHODS: HLA genes expression analysis was initially performed using microarray data of 53 PIB, previously generated by our laboratory. The validation analysis was performed by real-time PCR in 116 PIB from an independent cohort. RESULTS: The microarray data showed association between high expression levels of HLA class II genes, especially HLA-DQB1 and -DQB2, in kidneys from young (18 to 49-year-old) donors and poor (eGFRâ¯<â¯45â¯mL/min/1.73â¯m2) 1- and 5-year graft function. A subsequent study in an independent cohort, in which only HLA-DQB2 expression was evaluated, validated the association between increased HLA-DQB2 expression in PIB of kidneys from young donors and poor 1-year graft function: expression levels ≥0.0025 relative units conferred an odds ratio of 22.5, with positive and negative predictive values of 71.4% and 90.0%, respectively. CONCLUSION: Heightened expression of HLA-DQB1 and -DQB2 in PIB are promising tools for pre-transplant risk assessment of poor late graft function in transplants with kidneys from 18 to 49-year-old donors.
Assuntos
Rejeição de Enxerto/diagnóstico , Antígenos HLA-DQ/metabolismo , Cadeias beta de HLA-DQ/metabolismo , Transplante de Rim , Rim/metabolismo , Complicações Pós-Operatórias/diagnóstico , Adolescente , Adulto , Biópsia , Feminino , Rejeição de Enxerto/etiologia , Antígenos HLA-DQ/genética , Cadeias beta de HLA-DQ/genética , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Risco , Regulação para Cima , Adulto JovemRESUMO
The assignment of null alleles is clinically relevant in stem cell transplantation, in particular for donor selection. It is unclear how questionable (Q) alleles, having an unknown expression profile, should be considered in matching criteria. In this study we analyzed the RNA and protein expression profile of a questionable allele encountered in a sample of the Guadeloupe population: GD23Q, HLA-A(∗)23:19Q, 29:02:01. Full-length DNA sequencing of HLA-A(∗)23:19Q revealed a single polymorphism at position 619 (G>A) compared to HLA-A(∗)23:01:01. Serological typing showed only the presence of HLA-A29; HLA-A(∗)23:19Q was not detected on the cell surface. The absence of HLA-A(∗)23:19Q surface expression was shown by flow cytometry using a directly labeled monoclonal antibody and a panel of five indirectly labeled polyclonal antibodies all directed against HLA-A23 (HLA-A9) molecules. Allele specific amplification revealed the absence of intact full-length mRNA, but the presence of two major alternatively spliced mRNAs: sequencing identified that in one variant exon 3 is missing and in the other variant introns 2 and 3 are retained. Based upon the lack of HLA-A(∗)23:19Q surface expression and the presence of aberrant mRNA transcripts only, this study shows that HLA-A(∗)23:19Q is non-expressed.