Avaliação da qualidade do DNA extraído de biópsias renais preservadas em diferentes soluções e investigação de variantes de risco do gene APOL1 em pacientes com glomerulopatias

Abstract

Chronic Kidney Disease (CKD) represents a serious public health issue, often associated with progressive glomerulopathies. The identification of genetic variants, such as those in the APOL1 gene, has been fundamental in understanding susceptibility and the progression of these diseases, particularly in populations of African ancestry. This study aimed to evaluate the quality and integrity of genomic DNA extracted from kidney biopsies preserved in different media (Michel, Bouin, and formalin) and to investigate the presence of the G1 and G2 APOL1 gene variants in patients treated at the Nephrology Service of LIME-HUUFMA. Paraffin-embedded, fresh, and frozen samples were used, with DNA extraction performed using protocols specific to each preservation method. DNA quality was assessed by spectrophotometry (A260/A280), agarose gel electrophoresis, and PCR amplification tests. The results clearly demonstrated the influence of the preservation medium on the yield and purity of the extracted genetic material. Samples stored in Michel’s solution showed the best purification parameters, with concentrations ranging from 3.3 ng/μL to 200.7 ng/μL and A260/A280 ratios between 1.8 and 1.9 ideal values for pure DNA. These samples also showed a higher success rate in PCR amplification, with visible and well-defined bands. Larger fragments derived from the renal cortex significantly contributed to obtaining higher quality DNA due to the greater cellular density. On the other hand, samples fixed in Bouin's solution were found to be highly degraded, with low concentrations, absence of bands in electrophoresis, and purity ratios outside the ideal range. The presence of picric acid and the prolonged action of the fixative resulted in chemical modifications to the DNA, compromising the extraction and purification steps. The data reinforce that the choice of preservation medium and the fragment's origin area are determining factors for the recovery of viable DNA. Standardizing collection and preservation protocols, with preference for Michel-embedded samples, can optimize molecular studies especially those focused on genotyping variants such as those of the APOL1 gene with relevant diagnostic and prognostic implications.