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Molecular Autism

, 7:42

First Online: 06 October 2016Received: 12 May 2016Accepted: 26 September 2016


BackgroundFragile X syndrome FXS, a common cause of intellectual disability and autism, results from the expansion of a CGG-repeat tract in the 5′ untranslated region of the FMR1 gene to >200 repeats. Such expanded alleles, known as full mutation FM alleles, are epigenetically silenced in differentiated cells thus resulting in the loss of FMRP, a protein important for learning and memory. The timing of repeat expansion and FMR1 gene silencing is controversial.

MethodsWe monitored the repeat size and methylation status of FMR1 alleles with expanded CGG repeats in patient-derived induced pluripotent stem cells iPSCs and embryonic stem cells ESCs that were grown for extended period of time either as stem cells or differentiated into neurons. We used a PCR assay optimized for the amplification of large CGG repeats for sizing, and a quantitative methylation-specific PCR for the analysis of FMR1 promoter methylation. The FMR1 mRNA levels were analyzed by qRT-PCR. FMRP levels were determined by western blotting and immunofluorescence. Chromatin immunoprecipitation was used to study the association of repressive histone marks with the FMR1 gene in FXS ESCs.

ResultsWe show here that while FMR1 gene silencing can be seen in FXS embryonic stem cells ESCs, some silenced alleles contract and when the repeat number drops below ~400, DNA methylation erodes, even when the repeat number remains >200. The resultant active alleles do not show the large step-wise expansions seen in stem cells from other repeat expansion diseases. Furthermore, there may be selection against large active alleles and these alleles do not expand further or become silenced on neuronal differentiation.

ConclusionsOur data support the hypotheses that i large expansions occur prezygotically or in the very early embryo, ii large unmethylated alleles may be deleterious in stem cells, iii methylation can occur on alleles with >400 repeats very early in embryogenesis, and iv expansion and contraction may occur by different mechanisms. Our data also suggest that the threshold for stable methylation of FM alleles may be higher than previously thought. A higher threshold might explain why some carriers of FM alleles escape methylation. It may also provide a simple explanation for why silencing has not been observed in mouse models with >200 repeats.

KeywordsFragile X syndrome Repeat expansion mutation Repeat-mediated gene silencing Repeat contractions Stem cells AbbreviationsChIPChromatin immunoprecipitation

ESCsEmbryonic stem cells

FMFull mutation

FMR1Fragile X mental retardation 1

FXSFragile X syndrome

FXTASFragile X-associated tremor-ataxia syndrome

iPSCsInduced pluripotent stem cells

MS RPT-PCRMethylation-specific repeat PCR


qMS-PCRQuantitative methylation-specific PCR

qRT-PCRQuantitative reverse transcription PCR


UFMUnmethylated full mutation

Electronic supplementary materialThe online version of this article doi:10.1186-s13229-016-0105-9 contains supplementary material, which is available to authorized users.

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Autor: Yifan Zhou - Daman Kumari - Nicholas Sciascia - Karen Usdin


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