Improving preimplantation genetic diagnosis for Fragile X syndrome: two new powerful single-round multiplex indirect and direct tests

Abstract

Fragile X syndrome (FraX) is caused by the expansion of an unstable CGG repeat located in the Fragile X mental retardation 1 gene (FMR1) gene. Preimplantation genetic diagnosis (PGD) can be proposed to couples at risk of transmitting the disease, that is, when the female carries a premutation or a full mutation. We describe two new single-cell, single-round multiplex PCR for indirect and direct diagnosis of FraX on biopsied embryos. These tests include five unpublished, highly heterozygous simple sequence repeats, and the co-amplification of non-expanded CGG repeats for the direct test. Heterozygosity of the new markers ranged from 69 to 81%. The mean rate of non-informative marker included in the tests was low (26% and 23% for the new indirect and direct tests, respectively). This strategy allows offering a PGD for FraX to 96% of couples requesting it in our centre. A conclusive genotype was obtained in all cells with a rate of cells presenting an allele dropout ranging from 17% for the indirect test to 26% for the direct test. The new indirect test was applied for eight PGD cycles: 32 embryos were analysed, 9 were transferred and 3 healthy babies were born. By multiplexing these highly informative markers, robustness of the diagnosis is improved and the loss of potentially healthy embryos (because they are non-diagnosed or misdiagnosed) is limited. This may increase the chances of success of couples requesting a PGD for FraX, in particular, when premature ovarian insufficiency in premutated women leads to a reduced number of embryos available for analysis.

Identification of microsatellite markers <1 Mb from the FMR1 CGG repeat and development of a single-tube tetradecaplex PCR panel of highly polymorphic markers for preimplantation genetic diagnosis of fragile X syndrome

“…18 We have now more than doubled the number of markers that can be simultaneously amplified by combining 13 closely linked and polymorphic markers with AMELX/Y into a single-tube multiplex PCR panel. This panel is expected to have at least two informative markers on either side of the FMR1 CGG repeat in a majority of at-risk couples, which will significantly reduce the need for assay customization.…”

“…9,10,[13][14][15]17,18 The larger mutant (PM and FM) FMR1 allele is known to be highly refractory to PCR amplification and detection, especially when amplified from the limiting DNA of a single cell. This difficulty is further accentuated in the female heterozygote, where the normal allele is preferentially amplified and detected, at the expense of the expanded allele that fails to be detected.…”

“…Even when a couple's normal FMR1 alleles are informative, linked marker analysis is performed in combination with FMR1 CGG repeat analysis to minimize misdiagnosis caused by ADO and/or maternal/paternal DNA contamination. [13][14][15][16][17][18] Several linkage-based assays have been published for the diagnosis of FXS. [11][12][13][14][15][16][17][18][28][29][30][31][32][33][34] Several of the reported markers are located more than 1 Mb away from the FMR1 CGG repeat, which is not ideal due to the increased probability of recombination between marker and mutation.…”

“…[14][15][16] Thus far, at least 14 different makers have been used in PGD of FXS, with up to six markers successfully coamplified from a single cell. 18 Different combinations of informative markers have been used, 11,15,16 with each new marker combination requiring some customization and optimization to ensure successful coamplification of markers.…”

“…9,10 For the remaining one-third of couples with uninformative normal FMR1 alleles, PGD of FXS relies entirely on linkage analysis of flanking microsatellite markers. [11][12][13][14][15][16][17][18] This usually involves prescreening each couple to identify informative markers 11,15,16 and PGD analysis involving multiplex PCR of a selected subset of markers, followed by nested secondary PCR of individual markers. [14][15][16] Thus far, at least 14 different makers have been used in PGD of FXS, with up to six markers successfully coamplified from a single cell.…”