DONOR HLA GENOTYPING BY NEXT GENERATION SEQUENCING OF DNA FROM URINE-DERIVED DONOR CELLS OF RENAL ALLOGRAFT RECIPIENTS

Abstract

Introduction: Antibody-mediated rejection (AMR), dominantly induced by anti-HLA antibody, remains the most important cause of long-term graft loss after kidney transplantation. Donor-specific anti-HLA antibody (DSA) can’t often be determined at a specific allele level since historical donor DNA sample is not available and the existing donor HLA typing information is incomplete or even missing. Here, we developed a novel non-invasive technique by selectively expanding donor cells from urine of renal transplant recipients, and thus generating unlimited donor DNA to perform reliable allele-level HLA typing with next generation sequencing method for all HLA loci. Materials and Methods: This study enrolled 28 renal transplant recipients. The fresh midstream urine from each recipient was collected. After urine sample was centrifuged, the supernatant was discarded and washed twice with PBS. The resulting sediment was resuspended in the culture medium. The cells were plated and non-adherent cells were removed. DNA was extracted from the urine-derived donor cells (UDDCs). NGS sequencing is carried out to determine the HLA loci. Results: UDDCs from 26 patients were successfully isolated. High-resolution HLA results were unambiguously determined in 25 out of 26 DNA samples (25/26, 96%) and they are 100% concordant with donor HLA. We showed five HLA sequencing cases of UDDCs (Fig 1). The HLA typing of UDDCs is completely matched with HLA of donor peripheral blood. We further isolated UDDCs from a patient whose donor HLA information is missing at 10 years after kidney transplantation. Based on the UDDCs HLA typing and LSA results, DQA1*05:01 DQB1*02:01 were established as the culprit DSA loci of this patient (Fig 2). Discussion: DNA could be extracted from fresh kidney graft tissues for HLA genotyping; however, this approach is invasive and may aggravate graft injury. The amount of tissues obtained may be minimal and cannot yield sufficient quantity of DNA for HLA typing[1]. Other investigators extract DNA from paraffin embedded tissues or urinary sediments of KTx patients, and mismatched donor antigens could be deduced by subtracting the recipient’s HLA from the allograft HLA[2,3]. However, robust high-resolution HLA typing data can not be obtained by these methods. With an ex vivo expandable source of exfoliated cells, we provide a novel noninvasive urine-based molecular diagnostic method for donor HLA genotyping. Conclusion: The UDDCs-based HLA genotyping strategy successfully establishes complete donor DNA genotypes and provides a feasible and clinically relevant molecular diagnostic method that help to determine DSA loci for kidney transplant patients with historically insufficient donor HLA information.