Abstract
Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult onset neurodegenerative disorder affecting carriers of premutation expansions (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. The clinical features of FXTAS, as well as various forms of clinical involvement in carriers without FXTAS, are thought to arise through a direct toxic gain-of-function of the FMR1 mRNA containing the expanded CGG repeat. A singular difficulty in the study of cellular regulation in FXTAS, as with most other neurogenetic disorders, is background genetic variation between/among cases and controls. To circumvent this problem, a novel approach was taken whereby single allele sub-clones were derived from a single individual who was profoundly mosaic for CGG repeat size, with CGG repeats in individual alleles ranging from the normal range (<50 CGG repeats) to well into the full mutation range (>700 CGG repeats). Sub-clones with expanded CGG repeat alleles, and those with CGG repeats in the normal range, thus represent background isogenic case-control comparison groups, since all clones derive from a single individual. In order to generate clonal fibroblast cell lines from the CGG repeat size mosaic, it was necessary to first develop a protocol that would successfully isolate single cells and allow for their efficient expansion. Once the protocol was established, a PCR-based method was used to genotype with respect to CGG repeat size each of the clonal cell lines. Through this process, fifteen clonal lines have been established to date, with representatives from normal (<55 CGG repeats), premutation (55-200 repeats) and full mutation (>200 repeats) ranges. These clonal lines are currently being characterized with respect to FMR1 gene expression (FMR1 mRNA and protein levels). The current clonal library forms the basis for our effort to reprogram the fibroblast cell lines into induced Pluripotent Stem Cells (iPSCs) with the intention of further differentiating the cells into neural progenitor cells, and subsequently, functional neurons. This research project was conducted in the department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, in the laboratory of Dr. Paul J Hagerman.