β-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies

PLoS One. 2018 Feb 23;13(2):e0192151. doi: 10.1371/journal.pone.0192151. eCollection 2018.

Abstract

Relationships between Fragile X Mental Retardation 1 (FMR1) mRNA levels in blood and intragenic FMR1 CGG triplet expansions support the pathogenic role of RNA gain of function toxicity in premutation (PM: 55-199 CGGs) related disorders. Real-time PCR (RT-PCR) studies reporting these findings normalised FMR1 mRNA level to a single internal control gene called β-glucuronidase (GUS). This study evaluated FMR1 mRNA-CGG correlations in 33 PM and 33 age- and IQ-matched control females using three normalisation strategies in peripheral blood mononuclear cells (PBMCs): (i) GUS as a single internal control; (ii) the mean of GUS, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA); and (iii) the mean of EIF4A2 and SDHA (with no contribution from GUS). GUS mRNA levels normalised to the mean of EIF4A2 and SDHA mRNA levels and EIF4A2/SDHA ratio were also evaluated. FMR1mRNA level normalised to the mean of EIF4A2 and SDHA mRNA levels, with no contribution from GUS, showed the most significant correlation with CGG size and the greatest difference between PM and control groups (p = 10-11). Only 15% of FMR1 mRNA PM results exceeded the maximum control value when normalised to GUS, compared with over 42% when normalised to the mean of EIF4A2 and SDHA mRNA levels. Neither GUS mRNA level normalised to the mean RNA levels of EIF4A2 and SDHA, nor to the EIF4A2/SDHA ratio were correlated with CGG size. However, greater variability in GUS mRNA levels were observed for both PM and control females across the full range of CGG repeat as compared to the EIF4A2/SDHA ratio. In conclusion, normalisation with multiple control genes, excluding GUS, can improve assessment of the biological significance of FMR1 mRNA-CGG size relationships.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Confounding Factors, Epidemiologic
  • Fragile X Mental Retardation Protein / genetics*
  • Glucuronidase / genetics*
  • Humans
  • RNA, Messenger / genetics*
  • Real-Time Polymerase Chain Reaction

Substances

  • FMR1 protein, human
  • RNA, Messenger
  • Fragile X Mental Retardation Protein
  • Glucuronidase

Grants and funding

This study was supported by an Australian Research Council (ARC) Discovery grant (DP110103346) to K.M.C, an Australian Postgraduate Award Scholarship, Monash University Faculty of Medicine, Nursing and Health Sciences Bridging Postdoctoral Fellowship and NHMRC Early Career fellowship to C.M.K. (no. 1112934); a Next Generation Clinical Researchers Program - Career Development Fellowship, funded by the Medical Research Future Fund (MRFF 1141334) to D.E.G.; and by the Victorian Government’s Operational Infrastructure Support Program, with the salaries for the molecular component supported by NHMRC project grants (no. 104299, and no. 1103389 to H.R.S. and D.E.G.) and Murdoch Children’s Research Institute, Royal Children’s Hospital Foundation (D.E.G.). The authors thank the Fragile X Association of Australia and Fragile X Alliance for supporting recruitment. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.