Elsevier

Experimental Cell Research

Volume 313, Issue 2, 15 January 2007, Pages 244-253
Experimental Cell Research

Research Article
Elevated Fmr1 mRNA levels and reduced protein expression in a mouse model with an unmethylated Fragile X full mutation

https://doi.org/10.1016/j.yexcr.2006.10.002Get rights and content

Abstract

The human FMR1 gene contains a CGG repeat in its 5′ untranslated region. The repeat length in the normal population is polymorphic (5–55 CGG repeats). Lengths beyond 200 CGGs (full mutation) result in the absence of the FMR1 gene product, FMRP, through abnormal methylation and gene silencing. This causes Fragile X syndrome, the most common inherited form of mental retardation. Elderly carriers of the premutation, defined as a repeat length between 55 and 200 CGGs, can develop a progressive neurodegenerative syndrome: Fragile X-associated tremor/ataxia syndrome (FXTAS). In FXTAS, FMR1 mRNA levels are elevated and it has been hypothesised that FXTAS is caused by a pathogenic RNA gain-of-function mechanism. We have developed a knock in mouse model carrying an expanded CGG repeat (98 repeats), which shows repeat instability and displays biochemical, phenotypic and neuropathological characteristics of FXTAS. Here, we report further repeat instability, up to 230 CGGs. An expansion bias was observed, with the largest expansion being 43 CGG units and the largest contraction 80 CGG repeats. In humans, this length would be considered a full mutation and would be expected to result in gene silencing. Mice carrying long repeats (∼ 230 CGGs) display elevated mRNA levels and decreased FMRP levels, but absence of abnormal methylation, suggesting that modelling the Fragile X full mutation in mice requires additional repeats or other genetic manipulation.

Introduction

The Fragile X mental retardation gene 1 (FMR1), involved in Fragile X syndrome, contains a CGG repeat in its 5′-untranslated region. Depending on the length of this trinucleotide repeat, different clinical outcomes are possible. Repeats of normal individuals are within the range of 5 to 55 CGGs [1]. Repeat lengths greater than 200 CGGs (full mutation: FM) typically lead to methylation of both the CGG repeat and the FMR1 promoter resulting in transcriptional silencing of the gene. The consequent absence of FMRP in neurons is the cause of the mental retardation in Fragile X patients. Fragile X syndrome is the most common genetic disorder associated with mental retardation [2].

The premutation (PM), comprising ∼ 55 to ∼ 200 unmethylated CGG repeats, was long thought to be associated only with a high risk of expansion to a full mutation upon maternal transmission. However, elevated FMR1 mRNA levels and normal or slightly reduced FMR1 protein (FMRP) were reported [3], [4]. Additionally, 20% of female PM carriers are at risk of developing premature ovarian failure [5]. In 2001, the first evidence of a new neurological syndrome (Fragile X-associated tremor/ataxia syndrome: FXTAS) was published by Hagerman and colleagues, observed in five elderly male PM carriers. Patients presented with progressive intention tremor, leading to executive function deficits and generalised brain atrophy on MRI scans. Since this neurological syndrome is restricted to the PM range, reduced levels of FMRP are unlikely to be the underlying cause [6]. However, since elevation of FMR1 mRNA levels seems to be correlated to the length of the CGG repeat [4], [7], [8], and cognitive and functional impairment increases with the number of CGG repeats [9], an RNA-gain-of-function effect has been proposed, in which elevated levels of FMR1 mRNA containing an expanded CGG repeat lead to progressive neurodegeneration [6].

Little is known about when and how CGG repeat instability takes place. In order to be able to elucidate the timing and mechanism of CGG repeat instability and methylation of the FMR1 gene, a mouse model was generated by Bontekoe and colleagues [10]. In this model, the endogenous mouse CGG repeat (8 trinucleotides) of a wild type mouse was exchanged with a human CGG repeat of 98 trinucleotide units, which is in the PM range in humans. This ‘knock in’ CGG triplet mouse shows moderate repeat instability upon both maternal and paternal transmission and displays biochemical, phenotypic and neuropathological characteristics of FXTAS [10], [11].

In this paper, we report further repeat instability, up to lengths above 200 CGG units. In humans, this would implicate a full mutation, thus silencing of the gene. Mice carrying these long repeats (∼ 230 CGGs) display elevated mRNA levels and decreased FMRP levels, but absence of CpG methylation, which would mean that in mice a full mutation has not occurred as yet. However, additional mice with long CGG repeats will be necessary to fully evaluate this issue.

Section snippets

Mice

Both the knock in CGG triplet mice and wild type mice (parent of the knock in mouse, with a mouse endogenous (CGG)8 repeat) were housed in standard conditions. All experiments were carried out with permission of the local ethical committee. Repeat lengths were determined for the whole mouse colony, but only male mice were used for the experiments.

Isolation of DNA from mouse tails

DNA was extracted from mouse tails by incubating with 0.2 mg/ml Proteinase K (Roche Diagnostics) in 335 μl lysis buffer (50 mM Tris–HCl pH 7.5, 10 mM

Results

The original knock in construct introduced 98 CGG repeats into the mouse Fmr1 locus and has been used as a model for the human premutation disorder, FXTAS [10], [11], [14]. This line was initially developed to study repeat instability of long CGG repeats in the mouse Fmr1 locus and has been maintained for several years now. In the course of breeding, animals with longer repeats were born and selected for breeding in an attempt to further increase the size of the CGG array, with the hypothesis

Discussion

We have managed to obtain mice carrying full mutation length CGG repeats in the Fmr1 locus through selective breeding of a line of mice that were created by knocking in 98 CGGs. In this study, we have characterised these animals for repeat instability during transmission and for expression of the Fmr1 mRNA and protein. We find that mice carrying large CGG repeat alleles express increased amounts of mRNA, but significantly reduced levels of Fmrp. These findings are consistent with the

Acknowledgments

We are thankful to Dr. K. Usdin for providing the PCR protocol for amplification of the CGG repeat. Furthermore, we would like to thank Prof. J. Fallon for kindly providing us with the 2F5 antibody against FMRP. Furthermore, we are grateful to Elisabeth Lodder for useful discussion and to Ruud Koppenol and Tom de Vries Lentsch for support with graphics. This study was financially supported by the Prinses Beatrix Fonds (JB), NFXF (RW) and NIH ROI HD38038 (B.A.O. and D.L.N).

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    Both authors contributed equally to this work.

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