RESUMEN
Associations between HLA genotype and disease susceptibility encompass almost all the classic HLA loci. The level of typing resolution enabling a correct identification of an HLA disease susceptibility gene depends on the disease itself and/or on the accumulated knowledge about the molecular involvement of the HLA allele(s) engaged. Therefore, the application of Next Generation Sequencing technologies to HLA disease association, which would improve typing resolution, could prove useful to better understand disease severity. In the present study, we tested a nanopore sequencing approach developed by Omixon Biocomputing Ltd, dedicated to on-demand locus typing for HLA and disease, as an alternative to the conventional widely used sequence specific oligoprobe (SSO) approach. A total of 145 DNA samples used in routine diagnosis by SSO were retrospectively analyzed with nanopore technology, for HLA-A*02 immunotherapy decision for A*29, B*27, B*51, B*57 identification in class I, and DRB1, DQA1, and DQB1 for bullous dermatosis, rheumatoid arthritis, diabetes, and celiac disease requests in class II. Each locus was typed in a separate experiment, except for DQB1 and DQA1, which were analyzed together. Concordance between typings reached 100% for all the loci tested. Ambiguities by nanopore were only found for missing exon coverage. This approach was found to be very well adapted to the routine flow imposed by the SSO technique. This study illustrates the use of the new NanoTYPE MONO kit for single locus HLA sequencing for HLA and disease association diagnosis.
Asunto(s)
Nanoporos , Humanos , Susceptibilidad a Enfermedades , Estudios Retrospectivos , Prueba de Histocompatibilidad/métodos , Alelos , Secuenciación de Nucleótidos de Alto Rendimiento , Haplotipos , Frecuencia de los GenesRESUMEN
The current practice of HLA genotyping in deceased donors poses challenges due to limited resolution within time constraints. Nevertheless, the assessment of compatibility between anti-HLA sensitized recipients and mismatched donors remains a critical medical need, particularly when dealing with allele-specific (second field genotyping level) donor-specific antibodies. In this study, we present a customized protocol based on the NanoTYPE® HLA typing kit, employing the MinION® sequencer, which enables rapid HLA typing of deceased donors within a short timeframe of 3.75 h on average at a three-field resolution with almost no residual ambiguities. Through a prospective real-time analysis of HLA typing in 18 donors, we demonstrated the efficacy and precision of our nanopore-based method in comparison to the conventional approach and without delaying organ allocation. Indeed, this duration was consistent with the deceased donor organ donation procedure leading to organ allocation via the French Biomedicine Agency. The improved resolution achieved with our protocol enhances the security of organ allocation, particularly benefiting highly sensitized recipients who often present intricate HLA antibody profiles. By overcoming technical challenges and providing comprehensive genotyping data, this approach holds the potential to significantly impact deceased donor HLA genotyping, thereby facilitating optimal organ allocation strategies.
Asunto(s)
Secuenciación de Nanoporos , Humanos , Estudios Prospectivos , Antígenos HLA/genética , Alelos , Donantes de Tejidos , Prueba de Histocompatibilidad/métodosRESUMEN
HLA-DRB1*07:143N and HLA-DRB1*07:144 differ from DRB1*07:01:01:01 by single mismatches in exons 3 and 2 respectively.
Asunto(s)
Cadenas HLA-DRB1 , Humanos , Cadenas HLA-DRB1/genética , Secuencia de Bases , Alineación de Secuencia , Alelos , Exones/genéticaRESUMEN
The novel HLA-C*05:278N allele has a premature stop codon in exon 4.
Asunto(s)
Genes MHC Clase I , Antígenos HLA-C , Humanos , Antígenos HLA-C/genética , Alelos , Exones/genética , Codón sin Sentido , Análisis de Secuencia de ADNRESUMEN
The novel HLA-DPB1*1455:01N allele differs from DPB1*05:01:01:01 by one amino acid deletion in exon 3.
Asunto(s)
Donantes de Tejidos , Humanos , Secuencia de Bases , Alelos , Cadenas beta de HLA-DP/genéticaRESUMEN
The novel HLA-C*08:258 and -C*12:374 alleles differ from HLA-C*08:02:01:01 and -C*12:03:01:01 by one nucleotide substitution.
Asunto(s)
Antígenos HLA-C , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Antígenos HLA-C/genética , Alelos , Prueba de Histocompatibilidad/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Genes MHC Clase I , Análisis de Secuencia de ADN/métodosRESUMEN
The new HLA-DRB1*15:213 allele results from one nucleotide substitution in exon 3 of HLA-DRB1*15:02:01.
Asunto(s)
Trasplante de Corazón , Humanos , Cadenas HLA-DRB1/genética , Alelos , Secuencia de Bases , ExonesRESUMEN
The novel allele B*08:304 differs from B*08:01:01:01 by one nucleotide substitution in exon 2.
Asunto(s)
Antígenos HLA-B , Nucleótidos , Humanos , Secuencia de Bases , Alelos , Exones/genética , Antígenos HLA-B/genética , Análisis de Secuencia de ADNRESUMEN
The novel HLA-C*03:618N allele has one change in exon 1 leading to a premature stop codon.
Asunto(s)
Genes MHC Clase I , Antígenos HLA-C , Humanos , Antígenos HLA-C/genética , Alelos , Exones/genética , Codón sin Sentido , Análisis de Secuencia de ADNRESUMEN
The novel HLA-A*01:420N allele has two changes in exon 4 leading to premature stop codon.