Multiple displacement amplification improves PGD for fragile X syndrome

Abstract

We report an improvement in the PGD test for fragile X syndrome (FXS). Recently, multiple displacement amplification (MDA) has been reported to yield large amounts of DNA from single cells. Taking into account this technique, we developed a new PGD test for FXS, enabling combined analysis of linked polymorphic markers with the study of the non-expanded CGG repeat. Single cell amplification efficiency was first assessed on single lymphocytes. Amplification rate of the different markers ranged from 85 to 95% with an allele drop-out (ADO) rate comprised between 7 and 34%. Using this test, eight PGD cycles were carried out for six couples, and 37 embryos were analysed after preliminary MDA. Amplification rate was increased by this technique from 41 to 66% so that embryos with no results were rarer (14 versus 45% without MDA). Reliability of the test was considerably improved by combining direct with indirect genetic analysis. Furthermore, in cases of fully expanded alleles too large to be amplified by PCR, this test gives an internal amplification control. Embryonic transfers were carried out in all but one PGD cycles. One biochemical and one clinical pregnancy resulted, and a healthy child was born. This single diagnosis procedure could be suitable to most patients carrying FXS.

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

“…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.…”

“…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.…”

“…[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. 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.…”

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

“…A low rate of amplification and a high rate of ADO led to 67 to 86% of successfully diagnosed embryos during the PGD cycles. 8,9 PGD for FraX has been available in our centre since 1999. At that time, diagnosis relied only on normal CGG allele amplification and a Y chromosome marker (SRY).…”