Use of human-derived stem cells to create a novel, in vitro model designed to explore FMR1 CGG repeat instability amongst female premutation carriers

Stephanie L.F. Gustin, Guangwen Wang, Valerie M. Baker, Gary Latham, Vittorio Sebastiano

Research output: Contribution to journalArticlepeer-review

Abstract

Objective: Create a model, using reprogrammed cells, to provide a platform to identify the mechanisms of CGG repeat instability amongst female fragile X mental retardation 1 gene (FMR1) premutation (PM) carriers. Methods: Female PM carriers (with and without POI) and healthy controls were enrolled from June 2013 to April 2014. Patient-derived fibroblasts (FB) were reprogrammed to induced pluripotent stem cells (iPSC) using viral vectors, encoding KLF4, OCT4, SOX2, and MYC. FMR1 CGG repeat-primed PCR was used to assess the triplet repeat structure of the FMR1 gene. FMR1 promoter methylation (%) was determined using FMR1 methylation PCR (mPCR). Quantification of FMR1 transcripts by RT-qPCR was used to evaluate the effect of reprogramming on gene transcription, as well as to correlate patient phenotype with FMR1 expression. Production of FMR1 protein (FMRP) was determined using a liquid bead array-based immunoassay. Results: Upon induction to pluripotency, all control clones exhibited maintenance of progenitor cell CGG repeat number, whereas 10 of 12 clones derived from PM carriers maintained their input CGG repeat number, one of which expanded and one contracted. As compared to parent FB, iPSC clones exhibited a skewed methylation pattern; however, downstream transcription and translation appeared unaffected. Further, the PM carriers, regardless of phenotype, exhibited similar FMR1 transcription and translation to the controls. Conclusions: This is the first study to establish a stem cell model aimed to understand FMR1 CGG repeat instability amongst female PM carriers. Our preliminary data indicate that CGG repeat number, transcription, and translation are conserved upon induction to pluripotency.

Original languageEnglish (US)
Pages (from-to)1443-1455
Number of pages13
JournalJournal of Assisted Reproduction and Genetics
Volume35
Issue number8
DOIs
StatePublished - Aug 1 2018
Externally publishedYes

Keywords

  • CGG repeat
  • FMR1
  • Premutation
  • Primary ovarian insufficiency
  • Stem cell
  • iPSC

ASJC Scopus subject areas

  • Reproductive Medicine
  • Genetics
  • Obstetrics and Gynecology
  • Developmental Biology
  • Genetics(clinical)

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