RESUMO
The aim of this study was to develop and perform a preimplantation genetic diagnosis (PGD) assay discriminating between wild-type and mutant alleles in two families with germline mosaicism. Family 1 had two children affected with severe myoclonic epilepsy (SCNA1A del exons 1-22). Family 2 had two children with tuberous sclerosis (TSC2 C1327T) and two healthy children. Neither mutation was detected in genomic DNA derived from the parents in either family. Informative microsatellite markers flanking SCNA1A and TSC2 along with the identified mutations were used to construct haplotypes. For tuberous sclerosis, single spermatozoa were analysed using a multiplex assay that included six informative markers and the TSC2 mutation. In family 1, deletion in the maternal allele was detected in the affected child. In family 2, both affected children and one healthy child shared the same paternal allele. To confirm mutant paternal transmission, single spermatozoa were analysed for the mutation along with six markers. Of 44 single spermatozoa, four showed the mutant T allele, allowing linkage between the mutation and the genetic markers. Both families delivered healthy children following IVF/PGD. In conclusion, germline mosaicism complicates allele assignment when constructing haplotypes for PGD. Sperm analysis is a useful tool for verifying allelic linkage.