Development of a PCR-based methodology to differentiate donor and recipient-derived cell-free DNA for its application in pediatric liver transplant patients

ARRIGONE, AGOSTINA; TREZEGUET RINATTI, GUIDO; ARROSSAGARAY, GUILLERMO; CAMACHO, MARÍA FERNANDA; GAMBA, CECILIA; IMVERTARZA, OSCAR; HALAC, ESTEBAN; BELLI, CAROLINA B; SCHAIQUEVICH, PAULA

Mar del Plata

Congreso; Reunión Conjunta LXVIII SAIC, SAB, AAFE, AACyTAL; 2023

SAIC

Cell-free DNA (cfDNA) has emerged as a promising biomarker due to its variable concentration in diverse physiological and pathological conditions. In transplant patients, cfDNA is shed to blood from both the transplanted organ and recipients tissues due to cellular turnover. Under certain clinical conditions cfDNA levels in blood can increase due to necrosis, being a promising biomarker to monitor allograft health. Genotyping both individuals is essential to differentiate alleles and quantify cfDNA from both origins. One approximation is the use of biallelic deletions and insertions polymorphisms (DIP). The aim of this study was to evaluate the capacity of different molecular techniques to distinguish 9 DIPs from two individuals in the same specimen for ultimately monitoring cfDNA levels of transplant patients. For that purpose, genomic DNA (gDNA) from healthy volunteers was used. To characterize each gDNA, two approaches were considered. First, real-time PCR (qPCR) reactions were performed using allele-specific primers (ASP) for each DIP. Then, a unique qPCR reaction using universal typing primers (UTP) followed by High-resolution melting analysis (HRMA) was performed per DIP. Initially, qPCR conditions (e.g., annealing temperature, primer concentration) were optimized. Both techniques allowed us to identifythe 9 DIPs of each of 10 healthy volunteers´gDNA. Nonetheless, the ASP approach required 18 reactions while using UTP and HRMA to obtain the complete genotype for each individual requiredonly 9 reactions. In silico calculations revealed that the developed techniques are able to discriminate DIPs among 44 of 45 possible individual pairs of gDNA out of the 10 genotyped individuals. This technique has the potential to act as a non-invasive biomarker forevaluating the transplant tissue health and drug efficacy in rejection treatment. The system could offer a method for regularly assessing post-transplant individuals after further refined validation.