Digital Polymerase Chain Reaction for Assessment of Mutant Mitochondrial Carry-over after Nuclear Transfer for In Vitro Fertilization. (5th April 2021)

• Record Type: Journal Article
• Authors: Tytgat, Olivier; Tang, Mao-Xing; van Snippenberg, Willem; Boel, Annekatrien; Guggilla, Ramesh Reddy; Gansemans, Yannick; Van Herp, Michiel; Symoens, Sofie; Trypsteen, Wim; Deforce, Dieter; Heindryckx, Björn; Coucke, Paul; De Spiegelaere, Ward; Van Nieuwerburgh, Filip
• Is Part Of: Clinical chemistry. Volume 67:Number 7(2021)
• Page Start: 968
• Page End: 976
• Publication Date: 2021-04-05
• Subjects: Clinical chemistry -- Periodicals; Pharmaceutical chemistry -- Periodicals; Biochemistry -- Periodicals; Biochimie -- Périodiques; Diagnostics biologiques -- Périodiques; Biochemistry; Clinical chemistry; Pharmaceutical chemistry; Biochemistry; Laboratory Techniques and Procedures; Klinische chemie; Periodicals; 616.075605
• DOI: 10.1093/clinchem/hvab021
• Languages: English
• ISSNs: 0009-9147

Abstract

Abstract: Background: The quantification of mitochondrial DNA heteroplasmy for the diagnosis of mitochondrial disease or after mitochondrial donation, is performed mainly using next-generation sequencing strategies (NGS). Digital PCR (dPCR) has the potential to offer an accurate alternative for mutation load quantification. Methods: We assessed the mutation load of 23 low-input human samples at the m.11778 locus, which is associated with Leber's hereditary optic neuropathy (LHON) using 2 droplet digital PCR platforms (Stilla Naica and Bio-Rad QX200) and the standard NGS strategy. Assay validation was performed by analyzing a titration series with mutation loads ranging from 50% to 0.01%. Results: A good concordance in mutation rates was observed between both dPCR techniques and NGS. dPCR established a distinctly lower level of background noise compared to NGS. Minor alleles with mutation loads lower than 1% could still be detected, with standard deviations of the technical replicates varying between 0.07% and 0.44% mutation load. Although no significant systematic bias was observed when comparing dPCR and NGS, a minor proportional bias was detected. A slight overestimation of the minor allele was observed for the NGS data, most probably due to amplification and sequencing errors in the NGS workflow. Conclusion: dPCR has proven to be an accurate tool for the quantification of mitochondrial heteroplasmy, even for samples harboring a low mutation load (<1%). In addition, this alternative technique holds multiple benefits compared to NGS (e.g., less hands-on time, more straightforward data-analysis, and a lower up-front capital investment).