Single-tube tetradecaplex panel of highly polymorphic microsatellite markers < 1 Mb from F8 for simplified preimplantation genetic diagnosis of hemophilia A.

Essentials Preimplantation genetic diagnosis (PGD) of severe hemophilia A relies on linkage analysis. Simultaneous multi-marker screening can simplify selection of informative markers in a couple. We developed a single-tube tetradecaplex panel of polymorphic markers for hemophilia A PGD use. Informative markers can be used for linkage analysis alone or combined with mutation detection. SUMMARY: Background It is currently not possible to perform single-cell preimplantation genetic diagnosis (PGD) to directly detect the common inversion mutations of the factor VIII (F8) gene responsible for severe hemophilia A (HEMA). As such, PGD for such inversion carriers relies on indirect analysis of linked polymorphic markers. Objectives To simplify linkage-based PGD of HEMA, we aimed to develop a panel of highly polymorphic microsatellite markers located near the F8 gene that could be simultaneously genotyped in a multiplex-PCR reaction. Methods We assessed the polymorphism of various microsatellite markers located ≤ 1 Mb from F8 in 177 female subjects. Highly polymorphic markers were selected for co-amplification with the AMELX/Y indel dimorphism in a single-tube reaction. Results Thirteen microsatellite markers located within 0.6 Mb of F8 were successfully co-amplified with AMELX/Y in a single-tube reaction. Observed heterozygosities of component markers ranged from 0.43 to 0.84, and ∼70-80% of individuals were heterozygous for ≥ 5 markers. The tetradecaplex panel successfully identified fully informative markers in a couple interested in PGD for HEMA because of an intragenic F8 point mutation, with haplotype phasing established through a carrier daughter. In-vitro fertilization (IVF)-PGD involved single-tube co-amplification of fully informative markers with AMELX/Y and the mutation-containing F8 amplicon, followed by microsatellite analysis and amplicon mutation-site minisequencing analysis. Conclusions The single-tube multiplex-PCR format of this highly polymorphic microsatellite marker panel simplifies identification and selection of informative markers for linkage-based PGD of HEMA. Informative markers can also be easily co-amplified with mutation-containing F8 amplicons for combined mutation detection and linkage analysis.

Spinocerebellar ataxia type 2 with focal epilepsy--an unusual association.

INTRODUCTION: The spinocerebellar ataxias are a rare group of inherited neurodegenerative disorders. Epilepsy has not previously been associated with spinocerebellar ataxia type 2 (SCA2). CLINICAL PICTURE: We describe a family with 3 affected members who had typical phenotypic and MRI features of SCA2. Two had focal epilepsy with complex partial seizures and epileptiform discharges on electroencephalography. Trinucleotide expansions in the pathological range were found in the SCA2 gene, confirming SCA2. Sequencing of the expanded SCA2 gene did not reveal any new mutations that could account for epilepsy. TREATMENT AND OUTCOME: The focal epilepsy was well-controlled with carbamazepine. CONCLUSION: We hypothesise that the new feature of focal epilepsy is due to co-existence of a separate unlinked epilepsy susceptibility gene with the expanded SCA2 gene. Under this oligogenic model, both genes must be present, and co-inheritance of this susceptibility gene with the expanded SCA2 gene causes a complex interaction which triggers epilepsy.