PGD for germline mosaicism

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.We present two healthy couples with two affected children each. The first couple had one affected child with severe myoclonic epilepsy (Dravet syndrome) and another child died at age 2 years. The second couple had two affected children with tuberous sclerosis and two healthy children. The children in both couples were diagnosed with mutations in autosomal dominant genes, while the parents and two healthy siblings in the second couple did not bear these mutations in their genomic DNA. The birth of more than one affected children with the same syndrome in a couple in which both parents are healthy and do not bear the genetic mutation found in the children is indicative of germline mosaicism. Germline mosaicism (gonadal mosaicism) is a condition in which the precursor germ cells (ova or spermatozoa) are a mixture of two or more genetically different lines. Prior to a preimplantation genetic diagnosis (PGD) cycle, a haplotype is built based on an analysis of affected and non-affected family members. Since no mutation was found in genomic DNA in both couples, haplotype assignment based on genomic DNA from the parents and children could not be performed. We present strategies to be used in PGD for cases of germline mosaicism.