Use of dried blood spots for the determination of genetic variation of interleukin‐10, killer immunoglobulin‐like receptor and HLA class I genes

Abstract

Optimal methods for using dried blood spots (DBS) for population genetics-based studies have not been well-established. Using DBS stored for 8 years from 21 pregnant South African women we evaluated 3 methods of gDNA extraction with and without whole genome amplification (WGA) to characterize immune-related genes: Interleukin 10 (IL-10), Killer Immunoglobulin-like Receptors (KIR) and Human Leukocyte Antigen (HLA) class I. We found that the QIAamp DNA mini kit yielded the highest gDNA quality (p<0.05; Wilcoxon Signed Rank Test) with sufficient yield for subsequent analyses. In contrast, we found that WGA was not reliable for SSP-PCR analysis of KIR2DL1, KIR2DS1, KIR2DL5, and KIR2DL3 or high-resolution HLA genotyping using a sequence-based approach. We speculate that unequal template amplification by WGA under-represents gene repertoires determined by sequencing-based approaches.

High density FTA plates serve as efficient long‐term sample storage for HLA genotyping

“…Despite the low volume of only 10 µl of blood deposited per DBS, PCR‐dependent SBT can be performed very reliably following the protocols described here. Our results thus contrast with one recent study that failed to perform high‐resolution HLA typing of DBS‐derived DNA . However, Ndlovu et al amplified DNA using WGA, which presumably increased typing error rate.…”

“…Our results thus contrast with one recent study that failed to perform high‐resolution HLA typing of DBS‐derived DNA . However, Ndlovu et al amplified DNA using WGA, which presumably increased typing error rate. The increased typing error rate may, however, also be due to HLA‐typing methods (including polymerases and PCR‐buffer mixes).…”

Development and validation of a sample sparing strategy for HLA typing utilizing next generation sequencing

“…A Whole Genome Amplification (WGA) method using a commercial product developed by Qiagen, REPLI-g, was utilized for this purpose. This product has been used in similar applications [15, 20, 21], and for HLA typing [22–24]. We validated this assay in a comparative study using DNA prepared by standard methods (Qiagen) as well as REPLI-g, and titrated the number of input cells from 3×10 4 down to 30 cells.…”

Dried blood spots can help decrease the burden on patients dually infected with multidrug-resistant tuberculosis and HIV

“…We showed that DBS can be used for different aspects such as measuring drug concentrations and genotypic resistance testing of HIV for the use in TDM [9,11,13,14]. TDM could be a tool to decrease toxicity, increase efficacy and detect resistance early, thereby preventing further resistance development in a potentially cost-effective way, by preventing hospitalisation due to toxicity or drug-drug interactions.…”

“…In addition, human leukocyte antigen genotyping through whole-genome sequencing of DBS-derived samples has already been performed. The use of whole-genome sequencing opens perspectives for future genetic studies from DBS; for instance, studies into Mycobacterium tuberculosis mutations leading to resistance to second-line anti-TB drugs resulting in XDR-TB [14].…”