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bioRxiv · 10.1101/2021.10.11.463983

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

Yang, S. Y.·Newcomb, C. E.·Battle, S. L.·Hsieh, A. Y.·Chapman, H. L.·Cote, H. C.·Arking, D. E.
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Abstract

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

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Yang, S. Y., Newcomb, C. E., Battle, S. L., Hsieh, A. Y., Chapman, H. L., Cote, H. C., Arking, D. E.. 2021-10-11. Rare instances of non-random dropout with the monochrome multiplex qPCR assay for mitochondrial DNA copy number. https://doi.org/10.1101/2021.10.11.463983

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