Direct detection of FMR1 CGG repeats causative for fragile X syndrome coupled with 24-chromosome aneuploidy screening in single cells

Abstract

Fragile X syndrome (FXS) is caused by the expansion of CGG sequences in the 5’ untranslated region of the Fragile X Mental Retardation 1 (FMR1) gene. Most current pre-implantation genetic diagnosis (PGD) protocols are based on indirect analysis of linkage markers because amplification of trinucleotide repeats is difficult. The aim of our study is to develop a dual test that can achieve direct diagnosis of FXS and screen for 24-chromosome aneuploidy from the same sample while being sensitive at the single cell level and applicable to PGD for IVF. Experimental study. Four single-cell and five-cell samples of (1) a lymphocyte cell line carrying an intermediate mutation for FXS, (2) a fibroblast cell line carrying a full mutation for the FXS, (3) a trisomic cell line for chromosome 13, (4) a cell line monosomic for chromosome 21 and (5 and 6) two normal cell lines (46 XX and 46 XY) were subjected to whole genome amplification (WGA). All cell lines were established and commercially available. After WGA, a first aliquot was processed through the AmplideX™ protocol (Asuragen) for CGG repeats assessment of the FMR1 gene. A second aliquot was used for 24-chromosome aneuploidy screening on array-CGH from BlueGnome (Illumina). Normal alleles (27 and 30), intermediate mutation (52) and full mutation of FMR1 (>200) were respectively detected in control cells (8/8), lymphocyte (8/8) and fibroblast (8/8) cell lines affected with FXS, in a very sensitive and reproducible manner. Concurrent aneuploidy screening was effectively achieved from the same WGA sample, as shown by the confirmation diagnosis of known trisomic 13, monosomic 21 or normal cell lines. This study is the first report of direct diagnosis for FXS coupled with 24-chromosome aneuploidy screening. The advantages of this new test are (1) it provides accurate sizing of FMR1 alleles up to 200 CGG repeats, allowing to define a pre-, intermediate or full Fragile X mutation, (2) it can be achieved from a unique biopsy specimen, offering the possibility to diagnose for multiple conditions on limited starting material, (3) it is reliable on a single cell, therefore applicable to pre-implantation genetic diagnosis on D3 blastomere as well as on D5/6 trophectoderm biopsy, (4) it can be completed within 24 to 48 hours, hence allowing for Day3 biopsy with Day5 fresh transfer.