β-glucuronidase mRNA levels are correlated with gait and working memory in premutation females: understanding the role of FMR1 premutation alleles

Abstract

Fragile X tremor ataxia syndrome (FXTAS) is a late-onset disorder manifesting in a proportion of FMR1 premutation individuals (PM: 55-199 CGG triplet expansions). FXTAS is associated with elevated levels of FMR1 mRNA which are toxic. In this study, relationships between neurocognitive and intra-step gait variability measures with mRNA levels, measured in blood samples, were examined in 35 PM and 35 matched control females. The real-time PCR assays measured FMR1 mRNA, and previously used internal control genes: β-Glucuronidase (GUS), Succinate Dehydrogenase 1 (SDHA) and Eukaryotic Translation Initiation Factor 4A (EI4A2). Although there was significant correlation of gait variability with FMR1 mRNA levels (p = 0.004) when normalized to GUS (FMR1/GUS), this was lost when FMR1 was normalized to SDHA and EI4A2 (2IC). In contrast, GUS mRNA level normalized to 2IC showed a strong correlation with gait variability measures (p < 0.007), working memory (p = 0.001) and verbal intelligence scores (p = 0.008). PM specific changes in GUS mRNA were not mediated by FMR1 mRNA. These results raise interest in the role of GUS in PM related disorders and emphasise the importance of using appropriate internal control genes, which have no significant association with PM phenotype, to normalize FMR1 mRNA levels.

DNA Methylation at Birth Predicts Intellectual Functioning and Autism Features in Children with Fragile X Syndrome

“…Peripheral blood mononuclear cells (PBMC) were isolated from 5 millilitres of venous blood, for RNA extraction for gene expression analyses using the reverse transcription real-time PCR on a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific, Carlsbad, CA, USA). The relative standard curve method was utilised for FMR1 5′ and 3′ mRNA quantification normalised to mRNA of 2 internal control genes ( EIF4A2 and SDHA ) [ 30 ].…”

“…The differences in numbers analysed for outcomes from ( A ) to ( D ) reflect differences due to proportion of participants that: (1) did not provide blood at recruitment, but did provide consent for NBS retrieval, where NBS samples could be located and retrieved; (2) did provide blood at recruitment and consent for NBS retrieval, where NBS samples could not be located and retrieved; (3) did not complete neuropsychological assessments and/or did not obtain valid scores. FMR1 mRNA levels in PBMCs were analysed using the reverse transcription real-time PCR relative standard curve method [ 30 ]. FREE2m of NBS and DBS samples was analysed using Methylation Specific Quantitative Melt Analysis (MS-QMA) and the EpiTYPER system [ 19 ].…”

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

“…All study participants provided signed informed consent and the study procedures were consistent with the Declaration of Helsinki and approved by the Southern Health Ethics Committee (project 10147B). Participants included 35 PM females and 35 age- and IQ-matched control females recruited as part of previous studies [ 30 ]. Groups were matched on height, body mass index (BMI), age and Wechsler Abbreviated Scale of Intelligence (WASI) Full Scale IQ score (see details in [ 30 ]).…”

“…Analysis of GUS mRNA stability as compared to other genes in lymphoid malignancies or B and T cell enriched and stimulated leukocyte fractions found that GUS had less stable transcription when compared to other internal control genes [ 29 ]. More recently, the geNorm approach was used to determine the most stably expressed genes in peripheral blood mononuclear cells (PBMCs) of PM and control males [ 30 ], to determine the ‘optimal’ method for FMR1 mRNA normalisation. The EIF4A2 and SDHA mRNA average was found to be the optimal normalisation method.…”

“…The EIF4A2 and SDHA mRNA average was found to be the optimal normalisation method. This normalisation method was then applied to PM and control females, where correlations to the phenotype were also investigated [ 30 ].…”