Polymerase chain reaction, nuclease digestion, and mass spectrometry based assay for the trinucleotide repeat status of the fragile X mental retardation 1 gene

Eric D. Dodds, Flora Tassone, Paul J. Hagerman, Carlito B. Lebrilla

Research output: Contribution to journalArticle

13 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)5533-5540
Number of pages8
JournalAnalytical chemistry
Volume81
Issue number13
DOIs
StatePublished - Jul 1 2009

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Trinucleotide Repeats
Polymerase chain reaction
Mass spectrometry
Assays
Genes
Screening
Oligonucleotides
Molecular biology
Poisons
Blood
Messenger RNA

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Polymerase chain reaction, nuclease digestion, and mass spectrometry based assay for the trinucleotide repeat status of the fragile X mental retardation 1 gene. / Dodds, Eric D.; Tassone, Flora; Hagerman, Paul J.; Lebrilla, Carlito B.

In: Analytical chemistry, Vol. 81, No. 13, 01.07.2009, p. 5533-5540.

Research output: Contribution to journalArticle

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