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Use of coagulation factor XIII (F13) gene as an internal control for normalization of genomic DNA's for HLA typing.

ABSTRACT: Genomic DNA (gDNA) obtained from whole blood samples is a critical element for genomic research and clinical diagnosis. PCR efficiencies of the targeted genes like HLA-A, -B, -C, DPB1 and DRB1 using such isolated gDNAs were variable in spite of having similar amounts of gDNA taken for PCR. We addressed such PCR variabilities by normalizing the gDNA's using an internal control of human coagulation factor XIII that was found to be variable with all samples and did not correlate with the observed A₂₆₀ nm readings. The PCR and Q-PCR methodologies for the human coagulation factor XIII have been optimized, and the advantages of normalizing gDNA preparations based on F13 copy numbers have been discussed. This method will serve as a suitable choice to be used in laboratories and research centres, particularly when dealing with a large number of samples for the next-generation sequencing purposes, and in forensic labs with limited sample availability.

SUBMITTER: Neduvat AC

PROVIDER: S-EPMC6107889 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Use of coagulation factor XIII (F13) gene as an internal control for normalization of genomic DNA's for HLA typing.

Neduvat Anupama Cheleri AC Murthy Prerana Madhusudhana PM Sundarrajan Sudarson S Padmanabhan Sriram S

MethodsX 20180803

Genomic DNA (gDNA) obtained from whole blood samples is a critical element for genomic research and clinical diagnosis. PCR efficiencies of the targeted genes like HLA-A, -B, -C, DPB1 and DRB1 using such isolated gDNAs were variable in spite of having similar amounts of gDNA taken for PCR. We addressed such PCR variabilities by normalizing the gDNA's using an internal control of human coagulation factor XIII that was found to be variable with all samples and did not correlate with the observed A ...[more]

PMID: 30151348

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Project description:IntroductionAntibody mediated rejection (ABMR) is the most common cause of long-term allograft loss in kidney transplantation (KT). Therefore, a low human leukocyte antigen (HLA) mismatch (MM) load is favorable for KT outcomes. Hitherto, serological or low-resolution molecular HLA typing have been adapted in parallel. Here, we aimed to identify previously missed HLA mismatches and corresponding antibodies by high resolution HLA genotyping in a living-donor KT cohort.Methods103 donor/recipient pairs transplanted at the University of Leipzig Medical Center between 1998 and 2018 were re-typed using next generation sequencing (NGS) of the HLA loci -A, -B, -C, -DRB1, -DRB345, -DQA1, -DQB1, -DPA1, and -DPB1. Based on these data, we compiled HLA MM counts for each pair and comparatively evaluated genomic HLA-typing with pre-transplant obtained serological/low-resolution HLA (=one-field) typing results. NGS HLA typing (=two-field) data was further used for reclassification of de novo HLA antibodies as "donor-specific".ResultsBy two-field HLA re-typing, we were able to identify additional MM in 64.1% (n=66) of cases for HLA loci -A, -B, -C, -DRB1 and -DQB1 that were not observed by one-field HLA typing. In patients with biopsy proven ABMR, two-field calculated MM count was significantly higher than by one-field HLA typing. For additional typed HLA loci -DRB345, -DQA1, -DPA1, and -DPB1 we observed 2, 26, 3, and 23 MM, respectively. In total, 37.3% (69/185) of de novo donor specific antibodies (DSA) formation was directed against these loci (DRB345 ➔ n=33, DQA1 ➔ n=33, DPA1 ➔ n=1, DPB1 ➔ n=10).ConclusionOur results indicate that two-field HLA typing is feasible and provides significantly more sensitive HLA MM recognition in living-donor KT. Furthermore, accurate HLA typing plays an important role in graft management as it can improve discrimination between donor and non-donor HLA directed cellular and humoral alloreactivity in the long range. The inclusion of additional HLA loci against which antibodies can be readily detected, HLA-DRB345, -DQA1, -DQB1, -DPA1, and -DPB1, will allow a more precise virtual crossmatch and better prediction of potential DSA. Furthermore, in living KT, two-field HLA typing could contribute to the selection of the immunologically most suitable donors.

Dataset Information

Use of coagulation factor XIII (F13) gene as an internal control for normalization of genomic DNA's for HLA typing.

Publications

Use of coagulation factor XIII (F13) gene as an internal control for normalization of genomic DNA's for HLA typing.

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