title: Single-tube tetradecaplex panel of highly polymorphic microsatellite markers < 1 Mb from F8 for simplified preimplantation genetic diagnosis of hemophilia A.
abstract: 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.
  
  
  
  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.
authors.affiliation: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore.
authors.forename: M
authors.initials: M
authors.lastname: Zhao
authors.affiliation: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore.
authors.forename: M
authors.initials: M
authors.lastname: Chen
authors.affiliation: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore.|Preimplantation Genetic Diagnosis Center, Khoo Teck Puat - National University Children's Medical Institute, National University Health System, Singapore City, Singapore.
authors.forename: A S C
authors.initials: ASC
authors.lastname: Tan
authors.affiliation: Preimplantation Genetic Diagnosis Center, Khoo Teck Puat - National University Children's Medical Institute, National University Health System, Singapore City, Singapore.
authors.forename: F S H
authors.initials: FSH
authors.lastname: Cheah
authors.affiliation: Department of Obstetrics and Gynecology, Clinic for Human Reproduction, National University Health System, Singapore City, Singapore.
authors.forename: J
authors.initials: J
authors.lastname: Mathew
authors.affiliation: Department of Obstetrics and Gynecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore.|Department of Obstetrics and Gynecology, Clinic for Human Reproduction, National University Health System, Singapore City, Singapore.
authors.forename: P C
authors.initials: PC
authors.lastname: Wong
authors.affiliation: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore.|Preimplantation Genetic Diagnosis Center, Khoo Teck Puat - National University Children's Medical Institute, National University Health System, Singapore City, Singapore.|Department of Laboratory Medicine, National University Health System, Singapore City, Singapore.
authors.forename: S S
authors.initials: SS
authors.lastname: Chong
mesh_terms: D000483:Alleles; D004252:DNA Mutational Analysis; D019075:Electrophoresis, Capillary; D005091:Exons; D005169:Factor VIII / Q000235:genetics*; D005260:Female; D005307:Fertilization in Vitro; D008040:Genetic Linkage; D005819:Genetic Markers; D005838:Genotype; D006467:Hemophilia A / Q000097:blood / Q000175:diagnosis* / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D054643:INDEL Mutation; D008297:Male; D018895:Microsatellite Repeats; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D017354:Point Mutation; D011110:Polymorphism, Genetic; D011247:Pregnancy; D019836:Preimplantation Diagnosis; D017422:Sequence Analysis, DNA
chemical_list: D005819:Genetic Markers; C078147:F8 protein, human; D005169:Factor VIII
keywords: hemophilia A; linkage analysis; microsatellite; preimplantation genetic diagnosis; short tandem repeat