Unveiling APOL1 haplotypes in a predominantly African-American cohort of kidney transplant patients: a novel classification using probe-independent quantitative real-time PCR

Murat Dogan,Christine Watkins, Holly Ingram, Nicholas Moore, Grace M. Rucker, Elizabeth G. Gower, James D. Eason, Anshul Bhalla, Manish Talwar,Nosratollah Nezakatgoo,Corey Eymard,Ryan Helmick, Jason Vanatta,Amandeep Bajwa,Canan Kuscu,Cem Kuscu

FRONTIERS IN MEDICINE(2024)

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摘要
Introduction Apolipoprotein-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 a haplotype-centric model.Methods Genomic DNA was extracted from recipient PBMCs using SDS lysis buffer and proteinase K. A quantitative PCR assay with modified forward primers and a common reverse primer enabled us to quantitatively identify single nucleotide polymorphisms (SNPs) and the 6-bp deletion. Additionally, we used Sanger sequencing to verify our QPCR findings.Results Our novel probe-independent qPCR effectively distinguished homozygous wild-type, heterozygous SNPs/deletions, and homozygous SNPs/deletions, with at least 4-fold differences. A high prevalence of APOL1 variants was observed (18% two-risk alleles, 34% one-risk allele) in our recipient cohort. Intriguingly, no significant impact of recipient APOL1 variants on transplant outcomes was observed up to 12-month of follow-ups. Ongoing research will encompass more time points and a larger patient cohort, allowing for a comprehensive evaluation of G1/G2 variant subgroups categorized by new haplotype scores, enriching our understanding.Conclusion Our 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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关键词
APOL1 gene,APOL1 G0/G1/G2 gene variants,kidney tranplantation,qPCR (quantitative PCR),kidney functions
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