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.
Highlights
The prevalence of Fragile X Mental Retardation 1 (FMR1) Premutation (PM: CGG 55–199) alleles in the general population has been reported to be as high as 1 in 150 females and 1 in 450 males [1]
Three different methodologies for normalisation of FMR1 mRNA levels in blood were compared between PM and control groups: FMR1/GUS, FMR1/3IC and FMR1/2IC
This study demonstrates that correlation between FMR1 mRNA levels and CGG triplet repeat size is weakened when GUS is used as an internal control gene for mRNA analysis studies in peripheral blood mononuclear cells (PBMCs) of PM females
Summary
The prevalence of FMR1 Premutation (PM: CGG 55–199) alleles in the general population has been reported to be as high as 1 in 150 females and 1 in 450 males [1]. Transmitted PM alleles have the propensity to expand in future generations to full mutation (CGG !200) alleles that cause fragile X syndrome (FXS) [2]. PM alleles have been associated with adult onset Fragile X-associated disorders: Fragile X-associated tremor/ataxia syndrome (FXTAS: 40% males and 8–16% females over 50 years old) and Fragile X-associated primary ovarian insufficiency (FXPOI: ~20% females) [4, 5]. The mechanism most extensively studied in the context of Fragile X-associated disorders is FMR1 mRNA gain of function toxicity [10]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.