Dataset Information

FMR1 CGG repeat expansion mutation detection and linked haplotype analysis for reliable and accurate preimplantation genetic diagnosis of fragile X syndrome.

ABSTRACT: Fragile X mental retardation 1 (FMR1) full-mutation expansion causes fragile X syndrome. Trans-generational fragile X syndrome transmission can be avoided by preimplantation genetic diagnosis (PGD). We describe a robust PGD strategy that can be applied to virtually any couple at risk of transmitting fragile X syndrome. This novel strategy utilises whole-genome amplification, followed by triplet-primed polymerase chain reaction (TP-PCR) for robust detection of expanded FMR1 alleles, in parallel with linked multi-marker haplotype analysis of 13 highly polymorphic microsatellite markers located within 1 Mb of the FMR1 CGG repeat, and the AMELX/Y dimorphism for gender identification. The assay was optimised and validated on single lymphoblasts isolated from fragile X reference cell lines, and applied to a simulated PGD case and a clinical in vitro fertilisation (IVF)-PGD case. In the simulated PGD case, definitive diagnosis of the expected results was achieved for all 'embryos'. In the clinical IVF-PGD case, delivery of a healthy baby girl was achieved after transfer of an expansion-negative blastocyst. FMR1 TP-PCR reliably detects presence of expansion mutations and obviates reliance on informative normal alleles for determining expansion status in female embryos. Together with multi-marker haplotyping and gender determination, misdiagnosis and diagnostic ambiguity due to allele dropout is minimised, and couple-specific assay customisation can be avoided.

SUBMITTER: Rajan-Babu IS

PROVIDER: S-EPMC5733830 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Publications

FMR1 CGG repeat expansion mutation detection and linked haplotype analysis for reliable and accurate preimplantation genetic diagnosis of fragile X syndrome.

Rajan-Babu Indhu-Shree IS Lian Mulias M Cheah Felicia S H FSH Chen Min M Tan Arnold S C ASC Prasath Ethiraj B EB Loh Seong Feei SF Chong Samuel S SS

Expert reviews in molecular medicine 20170719

Fragile X mental retardation 1 (FMR1) full-mutation expansion causes fragile X syndrome. Trans-generational fragile X syndrome transmission can be avoided by preimplantation genetic diagnosis (PGD). We describe a robust PGD strategy that can be applied to virtually any couple at risk of transmitting fragile X syndrome. This novel strategy utilises whole-genome amplification, followed by triplet-primed polymerase chain reaction (TP-PCR) for robust detection of expanded FMR1 alleles, in parallel w ...[more]

PMID: 28720156

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Project description:The Fragile-X related disorders (FXDs) are Repeat Expansion Diseases (REDs) that result from expansion of a CGG-repeat tract located at the 5' end of the FMR1 gene. While expansion affects transmission risk and can also affect disease risk and severity, the underlying molecular mechanism responsible is unknown. Despite the fact that expanded alleles can be seen both in humans and mouse models in vivo, existing patient-derived cells do not show significant repeat expansions even after extended periods in culture. In order to develop a good tissue culture model for studying expansions we tested whether mouse embryonic stem cells (mESCs) carrying an expanded CGG repeat tract in the endogenous Fmr1 gene are permissive for expansion. We show here that these mESCs have a very high frequency of expansion that allows changes in the repeat number to be seen within a matter of days. CRISPR-Cas9 gene editing of these cells suggests that this may be due in part to the fact that non-homologous end-joining (NHEJ), which is able to protect against expansions in some cell types, is not effective in mESCs. CRISPR-Cas9 gene editing also shows that these expansions are MSH2-dependent, consistent with those seen in vivo. While comparable human Genome Wide Association (GWA) studies are not available for the FXDs, such studies have implicated MSH2 in expansion in other REDs. The shared unusual requirement for MSH2 for this type of microsatellite instability suggests that this new cell-based system is relevant for understanding the mechanism responsible for this peculiar type of mutation in humans. The high frequency of expansions and the ease of gene editing these cells should expedite the identification of factors that affect expansion risk. Additionally, we found that, as with cells from human premutation (PM) carriers, these cell lines have elevated mitochondrial copy numbers and Fmr1 hyperexpression, that we show here is O2-sensitive. Thus, this new stem cell model should facilitate studies of both repeat expansion and the consequences of expansion during early embryonic development.

Dataset Information

FMR1 CGG repeat expansion mutation detection and linked haplotype analysis for reliable and accurate preimplantation genetic diagnosis of fragile X syndrome.

Publications

FMR1 CGG repeat expansion mutation detection and linked haplotype analysis for reliable and accurate preimplantation genetic diagnosis of fragile X syndrome.

Similar Datasets

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