Cote, H. C. | Search bioRxiv

bioRxiv2021

Rare instances of non-random dropout with the monochrome multiplex qPCR assay for mitochondrial DNA copy number

Mitochondrial DNA copy number (mtDNA-CN) is a proxy for mitochondrial function and has been of increasing interest to the mitochondrial research community. There are a number of ways to measure mtDNA-CN, ranging from qPCR to whole genome sequencing [1]. A recent article in the Journal of Molecular Diagnostics [2] described a novel method for measuring mtDNA-CN that is both inexpensive and reproducible. After adapting the assay for use in our lab, we have found it to be reproducible and well-correlated with mtDNA-CN derived from whole genome sequencing. However, certain individuals show poor concordance between the two measures, particularly individuals with qPCR mtDNA-CN measurements >3 standard deviations below the sample mean, which corresponds to roughly 1% of assayed individuals (Figure 1). After examining whole genome sequencing data, this seems to be due to specific polymorphisms within the D-loop primer region, at positions MT 338, 340, 452, 457, 458, 460, 461, 466, and 467. All individuals with a variant in at least one of these positions have non-concordant mtDNA-CN measurements. Meanwhile, variants observed at other positions within the primer region do not appear to cause dropout. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/463983v1_fig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@134ca51org.highwire.dtl.DTLVardef@ce9196org.highwire.dtl.DTLVardef@1b82af6org.highwire.dtl.DTLVardef@c9701_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Discrepancy between the monochrome multiplex qPCR mtDNA-CN and the whole genome sequencing mtDNA-CN for 1,732 distinct individuals. Data are centered at 0 and scaled so that the standard deviation = 1. The dotted red line represents 3 standard deviations beneath the sample mean. Individuals in the U, L1, L4, and T haplogroups have a disproportionately higher risk of discordant measures between the two assays. C_FIG

genetics↗

bioRxiv2020

Robust Tobacco Smoking Self-Report in two Cohorts of Vulnerable Pregnant Women and Adults

BackgroundStigma associated with tobacco smoking, especially during pregnancy, may lead to underreporting and possible bias in studies relying on self-reported smoking data. Cotinine, a nicotine metabolite with a [~]20h half-life in blood, is often used as a biomarker of smoking. The objective of this study was to examine the concordance between self-reported smoking and plasma cotinine concentration among participants enrolled in two related cohorts of vulnerable individuals: human immunodeficiency virus (HIV)-positive and HIV-negative pregnant women enrolled in the CARMA-PREG cohort and HIV-positive and HIV-negative non-pregnant women and men enrolled in the CARMA-CORE cohort. MethodsFor HIV-positive (n=76) and negative (n=24) pregnant women, plasma cotinine was measured by ELISA in specimens collected during the third trimester, between 28 and 38 weeks of gestation. Plasma cotinine was also measured in HIV-positive (n=43) and negative (n=57) women and men enrolled in the CARMA-CORE cohort. ResultsSelf-reported smokers were more likely to have low income (p<0.001) in both cohorts, and to deliver preterm (p=0.007) in CARMA-PREG. In the CARMA-PREG cohort, concordance between plasma cotinine was 95% for self-reported smoking, and 89% for self-reported non-smoking. In the CARMA-CORE cohort we observed similarly high concordances of 96% and 92% for self-reported smoking and non-smoking, respectively. In this sample, the odds of discordance between self-reported smoking status and cotinine levels were not significantly different between self-reported smokers and non-smokers, nor between pregnant women and others. Taken together, the overall concordance between plasma cotinine and self-reported data was 94% with a Cohens kappa coefficient of 0.860 among all participants. ConclusionsGiven the high proportion of vulnerable people in the CARMA-PREG and CARMA-CORE cohorts, our results may not be fully generalizable to the general population. However, they demonstrate that participant surveying in a non-judgemental context can lead to accurate and robust self-report data. ImplicationsReliable self-reported smoking data is necessary to account for smoking status in subsequent studies. Our results suggest that future studies should ensure that study participants feel sale to speak candidly to non-judgemental research staff to obtain reliable self-report data.

pathology↗