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

Claudine M Kraan1,2,3, Kim M Cornish2, Quang M Bui4

  • 1Cyto-molecular Diagnostic Research Laboratory, Victorian Clinical Genetics Services and Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia.

Plos One
|February 24, 2018
PubMed

Insights

Using multiple control genes improves Fragile X Mental Retardation 1 (FMR1) mRNA analysis in premutation disorders. Normalizing FMR1 mRNA levels with Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA) revealed stronger correlations with CGG size.

Area of Science:

  • Genetics
  • Molecular Biology
  • Neuroscience

Background:

  • Fragile X Mental Retardation 1 (FMR1) mRNA gain of function is implicated in premutation disorders.
  • Current studies often normalize FMR1 mRNA levels using a single control gene, β-glucuronidase (GUS).

Purpose of the Study:

  • To evaluate the impact of different normalization strategies on assessing FMR1 mRNA-CGG correlations in peripheral blood mononuclear cells (PBMCs).
  • To determine the optimal method for quantifying FMR1 mRNA levels in relation to CGG repeat size.

Main Methods:

  • Compared FMR1 mRNA-CGG correlations in 33 premutation (PM) and 33 control females using three normalization strategies: GUS alone, a mean of GUS/EIF4A2/SDHA, and a mean of EIF4A2/SDHA.
  • Assessed FMR1 mRNA levels normalized to GUS, EIF4A2, and SDHA.

Main Results:

  • Normalization using the mean of EIF4A2 and SDHA (excluding GUS) showed the strongest correlation with CGG size (p = 10-11) and the greatest difference between PM and control groups.
  • This method identified over 42% of PM results exceeding control values, compared to only 15% when using GUS alone.
  • GUS normalization exhibited greater variability in mRNA levels across CGG repeat ranges compared to the EIF4A2/SDHA ratio.

Conclusions:

  • Multiple control genes, specifically EIF4A2 and SDHA, provide a more accurate assessment of FMR1 mRNA-CGG size relationships than single-gene normalization (GUS).
  • This improved normalization strategy enhances the detection of biological significance in FMR1-associated disorders.

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