The present report evaluated the feasibility of using buccal mucosa cells as a proxy for blood samples to perform APOL-1 genotyping evaluated.
Samples of variable origin (secretions, tissues, organs, etc.) and various methods (including PCR, restriction fragment length polymorphism-RFLP, random amplified polymorphic detection-RAPD, amplified fragment length polymorphism-AFLP, DNA sequencing, allele-specific oligonucleotide (ASO) probes and microarray analysis) can be used for genotyping, allowing for the identification of genetic variations among individuals [11, 12]. Each sample type and method presents unique advantages and disadvantages. Traditionally, blood samples are most commonly used for genetic studies. In these samples, the DNA extracted from white blood cells is generally of high quality, leading to elevated genotyping success rates. However, blood is also cumbersome to handle. As a more feasible alternative, mucosa cells were evaluated herein with respect to APOL1 genotyping potential. Compared to blood collection and handling, buccal mucosa cell sampling presents several advantages, e.g., the use of a flocked swab is rapid, minimally-invasive, and painless; moreover, the transportation and storage of mucosa cell samples is simpler and less expensive than handling blood samples.
Paired blood (positive control) and buccal mucosa cell samples from 23 patients submitted to biopsy for the diagnosis of kidney disease were used for genotyping. The latter are composed of epithelial cells and leukocytes, but may also contain oral flora . In addition, swabbing may also capture dead buccal mucosa cells . The presence of bacteria and dead cells could contribute to the partial DNA degradation seen in this type of sample, thus leading to PCR amplification failure . Indeed, assays relying on the amplification of long DNA segments (~ 10 kbp) by PCR, such as HLA genotyping, in buccal mucosa cell samples can fail due to partial DNA degradation (14). By contrast, the amplification of relatively short DNA fragments by PCR in buccal mucosa samples has been reported to be reliable .
Our results demonstrate the successful amplification of a DNA segment in the APOL1 gene in 23 buccal mucosa cell samples collected via flocked swab (Fig. 1), suggesting that the DNA obtained from this sampling technique is suitable for APOL1 genotyping. In addition, the analysis of chromatograms and nucleotide sequences following Sanger sequencing revealed all possible APOL1 alleles (G0, G1, and G2) and genotypes (G0/G0, G0/G1, G0/G2, G1/G1, and G1/G2, and G2/G2) in the 23 buccal mucosa cell samples evaluated (Supplementary Figure and Table 2). Correlation analysis between genotypes obtained from blood and buccal mucosa cell samples demonstrated almost perfect agreement (Cohen´s correlation coefficient = 1.0).
Taken together, the data presented herein indicate that the quality of DNA obtained from buccal mucosa cells collected via flocked swabs is of sufficient quality to reliably perform APOL1 genotyping. We therefore suggest that buccal mucosa cells samples represent a suitable alternative to blood samples for APOL1 genotyping purposes.
One limitation of this study is related to the small sample size used to carry out the genotyping. Although there was complete agreement between genotypes of APOL1 in paired samples from buccal mucosa cells and blood in 23 individuals, it is worth analyzing a larger number of samples from individuals in different clinical settings to confirm the reported results.
Finally, we hope that the technical approach presented herein may contribute to integrate APOL-1 screening to clinical nephrology practice.