Unveiling APOL1 Haplotypes: A Novel Classification Through Probe-Independent Quantitative Real-Time PCR

Dogan, M.; Watkins, C.; Ingram, H.; Moore, N. A.; Rucker, G. M.; Gower, E. G.; Eason, J. D.; Bhalla, A.; Talwar, M.; Nezakatgoo, N.; Eymard, C.; Helmick, R.; Vanatta, J. M.; Bajwa, A.; Kuscu, C.

doi:10.1101/2023.10.16.562539

bioRxiv · 10.1101/2023.10.16.562539

Unveiling APOL1 Haplotypes: A Novel Classification Through Probe-Independent Quantitative Real-Time PCR

Dogan, M.·Watkins, C.·Ingram, H.·Moore, N. A.·Rucker, G. M.·Gower, E. G.·Eason, J. D.·Bhalla, A.·Talwar, M.·Nezakatgoo, N.·Eymard, C.·Helmick, R.·Vanatta, J. M.·Bajwa, A.·Kuscu, C.

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Abstract

IntroductionApolipoprotein-L1 (APOL1) is a primate-specific protein component of high- density lipoprotein (HDL). Two variants of APOL1 (G1 and G2), provide resistance to parasitic infections in African Americans but are also implicated in kidney-related diseases and transplant outcomes in recipients. This study aims to identify these risk variants using a novel probe- independent quantitative real-time PCR method in a high African American recipient cohort. Additionally, it aims to develop a new stratification approach based on haplotype-centric model. MethodsGenomic DNA was extracted from recipient PBMCs using SDS lysis buffer and proteinase K. Quantitative PCR assay with modified forward primers and a common reverse primer enabled us to identify single nucleotide polymorphisms (SNPs) and the 6-bp deletion quantitatively. Additionally, we used sanger sequencing to verify our QPCR findings. ResultsOur novel probe-independent qPCR effectively distinguished homozygous wild-type, heterozygous SNPs/deletion, and homozygous SNPs/deletion, with at least 4-fold differences. High prevalence of APOL1 variants was observed (18% two-risk alleles, 34% one-risk allele) in our recipient cohort. Intriguingly, up to 12-month follow-up revealed no significant impact of recipient APOL1 variants on transplant outcomes. Ongoing research will encompass more time points and a larger patient cohort, allowing a comprehensive evaluation of G1/G2 variant subgroups categorized by new haplotype scores, enriching our understanding. ConclusionsOur cost-effective and rapid qPCR technique facilitates APOL1 genotyping within hours. Prospective and retrospective studies will enable comparisons with long-term allograft rejection, potentially predicting early/late-stage transplant outcomes based on haplotype evaluation in this diverse group of kidney transplant recipients.

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Dogan, M., Watkins, C., Ingram, H., Moore, N. A., Rucker, G. M., Gower, E. G., Eason, J. D., Bhalla, A., Talwar, M., Nezakatgoo, N., Eymard, C., Helmick, R., Vanatta, J. M., Bajwa, A., Kuscu, C.. 2023-10-19. Unveiling APOL1 Haplotypes: A Novel Classification Through Probe-Independent Quantitative Real-Time PCR. https://doi.org/10.1101/2023.10.16.562539

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