PGD on a recombinant allele: crossover between the TSC2 gene and 'linked' markers impairs accurate diagnosis

Accounting for possible recombinations in developing an accurate preimplantation genetic diagnosis (PGD) protocol based on familial haplotypes.

Haplotypes were constructed from genomic DNA in a family where the male was affected with tuberous sclerosis complex (TSC). Embryos were biopsied at day 3, and single blastomeres were analyzed by multiplex polymerase chain reactions (PCRs) including multiple microsatellite markers and single-cell sequencing.

Informative markers used in the initial haplotype analysis, based on the genomic DNA of the parents and affected child, were analyzed in the first PGD cycle. All embryos appeared to show recombination and none were transferred. Prior to the next cycle, the parents of the affected male were included in the haplotype analysis, demonstrating that the affected child had a recombinant allele. In the second PGD cycle, two non-recombinant intragenic single nucleotide polymorphisms (SNPs) were used with the TSC2 mutation and four microsatellite markers for a second PGD cycle. In this cycle two wild-type embryos were transferred, resulting in the birth of a healthy girl carrying the wild-type TSC2 allele.

PGD analysis requires the use of more than one meiosis for constructing accurate haplotypes. Testing for multiple closely linked markers and the familial mutation are necessary to detect recombination events and enable precise diagnosis.