title: Development and successful clinical application of preimplantation genetic haplotyping for Herlitz junctional epidermolysis bullosa.
abstract: BACKGROUND
  Herlitz junctional epidermolysis bullosa (HJEB) is a severe, life-threatening, autosomal recessive blistering skin disease for which no cure is currently available. Prenatal diagnosis for couples at risk is feasible through fetal skin biopsy or analysis of DNA extracted from chorionic villi, but these methods can be applied only after pregnancy has been established. An alternative approach, which involves the analysis of single cells from embryos prior to establishment of pregnancy, is preimplantation genetic diagnosis (PGD). Until now, its clinical uptake has been hindered by lengthy delays in establishing mutation-specific protocols, and by the small amount of template DNA that can be obtained from a single cell. A new method that addresses these problems, preimplantation genetic haplotyping (PGH), relies on whole genome amplification followed by haplotyping of multiple polymorphic markers using standard DNA-based polymerase chain reaction (PCR) assays.
  
  
  OBJECTIVE
  To design and validate a generic PGH assay for HJEB and to transfer this into clinical practice.
  
  
  METHODS
  We established a multiplex PCR-based PGH assay involving 16 markers within and flanking the LAMB3 gene (the most frequently mutated gene in HJEB). The assay was then validated in 10 families with at least one previously affected offspring. After licensing by the Human Fertilisation and Embryology Authority (HFEA), the new test was used for PGD in a couple at risk of HJEB.
  
  
  RESULTS
  The chromosome 1 LAMB3 markers within the assay were shown to be of sufficient heterogeneity to have widespread application for preimplantation testing of HJEB. In one couple that were heterozygous carriers of nonsense mutations in LAMB3, we used the new assay to identify unaffected embryos in a series of PGD cycles. Pregnancy was established in the third PGD cycle and a healthy, unaffected child was born. DNA analysis of cord blood confirmed the predicted single-cell mutation status of wild-type LAMB3 alleles.
  
  
  CONCLUSIONS
  PGH represents a major step forward in widening the scope and availability of preimplantation testing for serious mapped single-gene disorders. We have established a generic test that is suitable for the majority of couples at risk of HJEB.
authors.affiliation: St John's Institute of Dermatology, King's College London (Guy's Campus), 9th Floor Tower Wing, Guy's Hospital, Great Maze Pond, London SE1 9RT, UK.
authors.forename: H
authors.initials: H
authors.lastname: Fassihi
authors.forename: L
authors.initials: L
authors.lastname: Liu
authors.forename: P J
authors.initials: PJ
authors.lastname: Renwick
authors.forename: P R
authors.initials: PR
authors.lastname: Braude
authors.forename: J A
authors.initials: JA
authors.lastname: McGrath
mesh_terms: D015815:Cell Adhesion Molecules / Q000235:genetics*; D002900:Chromosomes, Human, 1-3 / Q000235:genetics; D016109:Epidermolysis Bullosa, Junctional / Q000175:diagnosis* / Q000235:genetics; D005260:Female; D005819:Genetic Markers; D006239:Haplotypes; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000379:methods*; D000097794:Kalinin
chemical_list: D015815:Cell Adhesion Molecules; D005819:Genetic Markers; D000097794:Kalinin