Polymerase Chain Reaction, Nuclease Digestion, and Mass Spectrometry Based Assay for the Trinucleotide Repeat Status of the Fragile X Mental Retardation 1 Gene

Department of Chemistry, University of California Davis, One Shields Avenue, Davis, California 95616, USA.
Analytical Chemistry (Impact Factor: 5.83). 07/2009; 81(13):5533-40. DOI: 10.1021/ac9008918
Source: PubMed

ABSTRACT CGG repeat expansions in the 5' noncoding region of the fragile X mental retardation 1 gene (FMR1) give rise to both neurodevelopmental and neurodegenerative human diseases depending on the length of the expansion. Expansions beyond 200 repeats (full mutation) generally result in gene silencing and fragile X syndrome (FXS), the leading heritable form of cognitive impairment and autism. Smaller expansions (55-200 CGG repeats; "premutation") give rise to the neurodegenerative disorder fragile X-associated tremor/ataxia syndrome (FXTAS) through an entirely distinct, toxic mRNA gain-of-function mechanism. A rapid means for both high-risk and newborn screening for allele size would provide a greater opportunity for early intervention and family counseling as well as furnish critical data on repeat size distribution and expanded allele frequencies. In the current work, we propose a novel mass spectrometry (MS) based method for the rapid identification of expanded CGG repeats to complement a recently described polymerase chain reaction (PCR) method for large population screening. In this combined approach, the optimized PCR method is used to amplify the relevant region of FMR1, followed by extensive nonspecific nuclease digestion. The resulting oligonucleotides are analyzed by MS in a manner that provides the relative proportion of triplet repeat oligonucleotides in seconds per sample. This assay enables swift and reproducible detection of expanded CGG alleles using a single blood spot and in principle is suitable for large scale studies and newborn screening. Moreover, this analytical scheme establishes a unique new intersection of MS with molecular biology, with potential for significant interdisciplinary impact.

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    ABSTRACT: Fragile X is the most common inherited cause of intellectual disability and is frequently associated with autism. The syndrome is due to mutations of the FMR1 gene that result in the absence of fragile X mental retardation protein (FMRP). We have developed a rapid, highly sensitive method for quantifying FMRP from dried blood spots and lymphocytes. This assay uses two new antibodies, a bacterially expressed abbreviated FMRP standard, and a Luminex platform to quantify FMRP. The assay readily distinguished between samples from males with fragile X full mutations and samples from normal males. It also differentiated mosaic from nonmosaic full-mutation male samples. This assay, because of its methodology and minimal cost, could be the basis for newborn or population screening.
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    ABSTRACT: The fragile X mental retardation (FXMR) syndrome is one of the most frequent causes of mental retardation. Affected individuals display a wide range of additional characteristic features including behavioural and physical phenotypes, and the extent to which individuals are affected is highly variable. For these reasons, elucidation of the pathophysiology of this disease has been an important challenge to the scientific community. 1991 marks the year of the discovery of both the FMR1 gene mutations involved in this disease, and of their dynamic nature. Although a mouse model for the disease has been available for 16 years and extensive research has been performed on the FMR1 protein (FMRP), we still understand little about how the disease develops, and no treatment has yet been shown to be effective. In this review, we summarise current knowledge on FXMR with an emphasis on the technical challenges of molecular diagnostics, on its prevalence and dynamics among populations, and on the potential of screening for FMR1 mutations.
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