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 10.1021/ac9008918.s001 https://acs.figshare.com/articles/journal_contribution/Polymerase_Chain_Reaction_Nuclease_Digestion_and_Mass_Spectrometry_Based_Assay_for_the_Trinucleotide_Repeat_Status_of_the_Fragile_X_Mental_Retardation_1_Gene/2846596 CGG repeat expansions in the 5′ noncoding region of the fragile X mental retardation 1 gene (<i>FMR1</i>) 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 <i>FMR1</i>, 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. 2009-07-01 00:00:00 nuclease digestion family counseling Polymerase Chain Reaction neurodegenerative disorder Trinucleotide Repeat Status Fragile X Mental Retardation 1 GeneCGG FXTAS oligonucleotide region MS Nuclease Digestion Mass Spectrometry retardation 1 gene novel mass spectrometry allele frequencies CGG alleles FMR heritable form polymerase chain reaction optimized PCR method X syndrome allele size size distribution interdisciplinary impact population screening FXS blood spot scale studies