P048 NGS performance on a transplanted liver biopsy sample: A case study

Abstract

A male pediatric patient was diagnosed with biliary atresia and was transplanted with liver from a deceased donor. The surgery went well, but years later the patient developed antibodies to Cw12, DR14, DQ7 and DQA1∗04:01 and various DP alleles. The need for high resolution typing to determine the identity of de novo DSA and the lack of any remaining pre-transplant sample on a deceased donor led to the use of NGS to determine chimeric typing. Genomic DNA was extracted from a post-transplant liver biopsy from this patient by using Gentra Puregene Tissue kit. We used the Immucor NGS FLEX HLA typing system for amplification and sequencing of HLA genes HLA -A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1 followed by analysis with MIA FORA software. The NGS methodology is a clonal sequencing approach which solves the phase ambiguity problems inherent with Sanger-based sequencing when alleles are amplified and sequenced together. The genomic DNA extracted from the transplanted liver biopsy is chimeric, with residual recipient DNA at very low but detectable levels. The presence of chimeric reads is the result of co-amplification of both donor and recipient HLA genes. The majority of sequencing reads with zero mismatches belonged to the donor and were concordant with the deceased donor HLA type provided by UNOS. Unclaimed sequencing reads appeared as grey background and derived from the recipient HLA type (previously obtained with pre-transplant samples). In this case and a few other chimeric cases examined in our laboratory, the MIA FORA NGS software detected the HLA type of donor as the called alleles and showed some unclaimed reads that did not align to the donor type but rather to the HLA type of recipient. We note that if the donor and recipient HLA genes are perfectly matched in one or both alleles then they are indistinguishable. The clonal sequencing nature of the NGS reveals PCR primer specificity by identifying the sequences of all co-amplified genomic regions within the intended target region in the same biopsy sample. This case demonstrates the clinical utility of high resolution HLA typing by NGS technology in the clinical setting to de convolute a solid organ biopsy admixture i.e. the ability to do chimeric typing.