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.

Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer

“…Contrary to multiplex PCR, primers with similar annealing temperature were not required with multiplex LT-RPA. We also showed that LT-RPA allowed the amplification of CAG, CTG and GAA trinucleotide repeat microsatellites (Supplementary Figures S8 and S9) but not CGG triplet repeat, the latter being a CG-rich sequence that is particularly difficult to amplify by PCR (55). The expansion of these triplet repeats is implicated in several genetic disorders (48,49) and we showed that the detection of HTT CAG expanded repeats was possible by LT-RPA (Supplementary Figure S9), suggesting that this isothermal amplification approach could be used for the diagnosis of trinucleotide repeat disorders.…”

Síndrome del X frágil en fecundación in vitro. Reporte de caso

“…En el DGP el SB no puede utilizarse dado que las cantidades DNA obtenidas no son suficientes para realizar la prueba. Así es que, las pruebas moleculares para el SXF en diagnóstico preimplantación son posibles y se realizan en laboratorios especializados [16][17][18] .…”

Non-coding RNAs in Nervous System Development and Disease

“…Fragile X mental retardation 1 (FMR1) locus is characterized by the production of multiple non-coding transcripts (FMR5, FMR6, FMR4) in addition to the FMR1 mRNA. Expansion of the CGG triplet in the FMR1 gene (>200 repeats for complete penetrance) is attributed as the main cause of Fragile X syndrome (FXS), and in a pre-mutation state (55-200 repeats) is responsible for Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS) (Rajan-Babu et al, 2017). Although the pathogenic relevance of all the FMR1 associated transcripts remains to be fully defined, FMR4 is a lncRNA antisense to FMR1 that spans the repeated region and that was observed to significantly affect human cell proliferation and apoptosis in vitro (Khalil et al, 2008).…”