Abstract
Introduction:
The leucyl-cystinyl aminopeptidase gene rs4869317 single nucleotide polymorphism (SNP) is associated with multiple conditions; for example, patients with the rs4869317 TT genotype experiencing septic shock have increased plasma clearance of vasopressin versus AA or AT genotypes. The current aim was to assess the appropriateness of a TaqMan® SNP genotyping assay as a tool to identify the rs4869317 SNP in human samples, given its association with serious medical conditions.
Methods:
Genomic DNA was amplified using sequence-specific forward/reverse primers, and allelic discrimination was achieved using VIC™- or FAM™-labeled TaqMan probes. The Applied BiosystemsTM QuantStudioTM 12 K Flex Real-Time PCR System was used for SNP genotyping, and data were analyzed by the TaqMan Genotyper software.
Results:
Intra- and interassay precisions were established, with results consistent within and between genotyping assays and between instruments and operators. Assay robustness was established across a range of input DNA (11–56 ng). The assay was 100% accurate for discriminating between alleles of the rs4869317 SNP (30 samples) and was concordant with results from independently performed Sanger sequencing. The detection of leucyl-cystinyl aminopeptidase rs4869317 SNP using TaqMan genotyping methods was shown to be an appropriate, reliable, and rapid analytical tool for generating allelic calls.
Conclusion:
This assay is a valid tool for genotyping analyses in clinical trials and potential applications in precision medicine.
Introduction
The hormone vasopressin is produced in the hypothalamus and plays an important role in osmoregulation.1 Leucyl-cystinyl aminopeptidase (LNPEP) is an enzyme responsible for the cleavage of vasopressin, oxytocin, and other peptide hormones.2 Variants of the LNPEP gene, such as the rs4869317 single nucleotide polymorphism (SNP), have been associated with premature birth, development of eating disorders (A allele), and septic shock-related mortality.1 , 3 , 4 In patients with septic shock, the TT genotype of the rs4869317 SNP was associated with an increased plasma clearance of vasopressin and increased risk of 28-day mortality compared with the AA or AT genotypes.1 Furthermore, as the application of precision (personalized) medicine continues to broaden (e.g., in sepsis),5 the identification of individuals with the TT genotype may have practical implications for improving individualized care in certain medical conditions. In contrast with slower and more expensive Sanger sequencing to detect SNPs, a limitation for a large number of clinical samples, TaqMan genotyping results in less time and allows for SNP detection in a large number of samples.6 , 7 Several comparative studies of genotyping technologies have shown the advantages and/or suitability of TaqMan.8–10 As a first step toward a high-throughput assay appropriate for clinical use, the aim of this study was to test a newly developed analytic tool to identify the LNPEP rs4869317 SNP in human samples, given its association with serious medical conditions.
Materials and Methods
Human genomic DNA, which was purified using standard procedures, was obtained from Coriell Cell Repository (Coriell Institute for Medical Research, Camden, New Jersey). Genomic DNA was quantified using the Nanodrop spectrophotometer ND-1000 or ND-2000 and diluted to a concentration of 2.5 ng/µL in TE buffer. Single-use vials were stored at 2–8° C for up to 7 days or at –70° C for longer periods.
The sequence used to identify LNPEP gene rs4869317 SNP in the genotyping assays was obtained from dbSNP (https://www.ncbi.nlm.nih.gov/snp/rs4869317; Fig. S1). Alleles of the rs4869317 SNP were detected by amplification of genomic DNA using sequence-specific forward and reverse unlabeled primers and allele-specific fluorescently labeled minor groove binding probes. Discrimination between the A and T alleles was achieved by using VIC™- or FAM™-labeled TaqMan® probes, respectively. SNP genotyping was performed using standard manufacturer protocols for Applied Biosystems TaqMan SNP Genotyping Assay (Thermo Fisher Scientific, Waltham, MA). Each TaqMan genotyping reaction was carried out in a 96-well plate, with each well containing a total volume of 25 µL of 1x TaqMan Universal Master Mix (Thermo Fisher Scientific), 1x working primer-probe mix (Thermo Fisher Scientific), and 11.2–56 ng genomic DNA. Thermal cycling conditions were as follows: polymerase activation at 95° C for 10 minutes, followed by 40 cycles of denaturation at 95° C for 15 seconds and annealing/extension at 60° C for 1 minute. The TaqMan genotyping assay was run using the Applied Biosystems QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific). The TaqMan Genotyper software version 1.3 was used to analyze the genotyping data.
Positive control genomic DNA samples were used in each genotyping assay (i.e., homozygous A/A control [NA11920], heterozygous A/T controls [NA18562], and homozygous T/T control [NA18558]). The no template control should be absent of a genotype using the TaqMan Genotyper software. Sanger sequencing was conducted at an independent laboratory to confirm genotypes.
Results
Precision
Intra-assay (within run) precision (i.e., variability of replicate results for a given sample) was examined using four samples tested in triplicate. All replicates (controls and test samples) generated the expected allele calls, with 100% reproducibility between replicates. These findings were concordant across the three replications performed and consistent with the genotype results obtained using Sanger sequencing ( Table 1). Further, SNP genotyping was repeated on the same days with the same operators using a second real-time PCR instrument, and findings were the same for the controls and test samples.
Accuracy of the rs4869317 single nucleotide polymorphism genotyping assay
Interassay (between run) precision was assessed using six independent real-time genotyping experiments with more than two operators (Table S1). All replicates generated the expected allele calls, with 100% reproducibility between replicates and operators on different days. These findings were concordant between the experiments and with the genotype findings from Sanger sequencing obtained at an independent laboratory.
Robustness
Assay robustness is demonstrated when varying amounts of input DNA are shown to be within the dynamic range of the assay and do not affect the genotyping result. Robustness was tested using three different amounts of input DNA (low [1.0 µg/mL; 11.2 ng], mid [2.5 µg/mL; 28 ng], and high [5.0 µg/mL; 56 ng]) in the same experiment, and the three replicates for each concentration of each sample had concordant genotyping results. All were in agreement with expected genotype results obtained by Sanger sequencing (Table 1), confirming that the rs4869317 SNP genotyping assay could tolerate input DNA levels from low (1.0 µg/mL; 11.2 ng) to high (5.0 µg/mL; 56 ng; Table 2).
Robustness of the rs4869317 single nucleotide polymorphism genotyping assay*
Instrument 2.
Accuracy
Accuracy was determined by the agreement of the observed results and accepted reference data. A total of 30 genomic DNA samples representing each of the three alleles equally (A/A [n = 10]; A/T [n = 10]; T/T [n = 10]) were assayed as single samples. The TaqMan genotyping results were compared with results of Sanger sequencing conducted by an independent laboratory to determine assay accuracy. The 30 samples tested had acceptable allele calls using two different real-time PCR instruments, findings which had 100% concordance with Sanger sequencing results (Table 1). The results of these experiments, in combination with the data from the precision experiments, indicated that the TaqMan genotyping assay could accurately identify LNPEP rs4869317 alleles A/A, A/T, and T/T.
Carryover
There was no evidence of specimen carryover from samples with high LNPEP SNP rs4869317 to samples with low LNPEP SNP rs4869317, as evidenced by the absence of detectable signal in the no template control samples.
Discussion
The purpose of validating a genotyping assay is to confirm that the methodology used can correctly recognize and identify the SNP of interest in a sample to be tested.11 The aim of the current study was to confirm, using validation techniques, that the TaqMan genotyping assay would be a reliable method for discriminating among alleles of the LNPEP rs4869317 SNP in clinical studies. The methods used are consistent with US Food and Drug Administration guidance regarding the development of genetic tests for heritable markers (i.e., SNP analysis), including assay specifics (e.g., DNA sequence, primer sequences, assay kits, and controls), required DNA quantity, and assay precision.12
In this study, the TaqMan genotyping of the rs4869317 SNP demonstrated intra- and interassay precision and accuracy, with consistency between results within experiments, between instruments and operators, and on different days. Genomic DNA samples were accurately called for the rs4869317 SNP using two different instruments; samples used in these experiments may be used as positive controls in future studies. Further, the genotyping assay was shown to be robust over a range of DNA levels varying from 11 to 56 ng. A limitation of the study is that the genomic DNA used was obtained from a commercial source, and it is unknown if potentially lower-quality DNA (e.g., obtained in the context of a clinical study) would impact test performance, as poor DNA quality has been shown to affect genotyping results in other situations.13 Further, PCR inhibitors may be present in samples, which can lead to undetermined results.
The detection of the rs4869317 SNP has been previously described using a PCR-restriction fragment length polymorphism method.14 However, this technique is labor-intensive, particularly for analysis of a large number of clinical samples, as the process involves analysis of PCR products by ethidium bromide–stained agarose gels.14 , 15 In contrast, TaqMan genotyping assays are less laborious due to utilization of fluorescent-labeled probes along with primers designed to amplify the sequence of interest.16 These allele-specific probes are then detected by fluorescence signal intensity after the PCR reaction.16 Further, TaqMan genotyping occurs in a single well, absent of the potential for post-PCR contamination of samples, as is possible with PCR-restriction fragment length polymorphism.17
Conclusion
The TaqMan genotyping assay for the rs4869317 SNP is appropriate as a high throughput analytical tool for use in future clinical studies, including its application in the field of precision medicine for identifying patients with specific polymorphisms associated with disease risk.18 , 19 In fact, this tool is being used to allow high-volume testing in a phase 1 clinical trial (ClinicalTrials.gov identifier NCT04093050) examining plasma clearance of vasopressin in healthy individuals.
Footnotes
Acknowledgments
Technical editorial and medical writing assistance were provided, under the direction of the authors, by Mary Beth Moncrief, PhD, and Sophie Bolick, PhD, Synchrony Medical Communications, LLC, West Chester, Pennsylvania, United States. Funding for this assistance was provided by Endo Pharmaceuticals Inc., Malvern, Pennsylvania, United States.