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

doi:10.3389/fmed.2024.1325128

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

Front Med (Lausanne). 2024 Apr 10:11:1325128. doi: 10.3389/fmed.2024.1325128. eCollection 2024.

Authors

Murat Dogan^{1

2}, Christine Watkins^{1

2}, Holly Ingram^{1

2}, Nicholas Moore¹, Grace M Rucker^{1

2}, Elizabeth G Gower³, James D Eason⁴, Anshul Bhalla^{1

2

3}, Manish Talwar^{1

2

3}, Nosratollah Nezakatgoo^{1

2

3}, Corey Eymard^{1

2

3}, Ryan Helmick^{1

2

3}, Jason Vanatta^{1

2

3}, Amandeep Bajwa^{1

2}, Canan Kuscu^{1

2}, Cem Kuscu^{1

2}

Affiliations

¹ Transplant Research Institute, Memphis, TN, United States.
² Department of Surgery, College of Medicine, University of Tennessee Health Science Center, Memphis, TN, United States.
³ Methodist Hospital, Memphis, TN, United States.
⁴ HCA Florida Largo Hospital, Largo, FL, United States.

Abstract

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.

Keywords: APOL1 G0/G1/G2 gene variants; APOL1 gene; kidney functions; kidney tranplantation; qPCR (quantitative PCR).

Abstract

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