title: Real-time reverse linkage using polar body analysis for preimplantation genetic diagnosis in female carriers of de novo mutations.
abstract: BACKGROUND
  Single cell diagnosis for preimplantation genetic diagnosis (PGD) requires simultaneous analysis of multiple linked polymorphic markers in addition to mutation analysis in order to reduce misdiagnosis. This type of analysis requires building family haplotypes spanning at least two generations. We present three childless couples in whom the female was a de novo mutation carrier in the Duchenne Muscular Dystrophy (DMD), incontinentia pigmenti (IKBKG) or Neurofibromatosis type 2 (NF2) genes, precluding linkage prior to the PGD cycle. We constructed haplotypes based on linked polymorphic markers in these families and performed concurrent diagnosis enabling embryo transfer from the first PGD cycle.
  
  
  METHODS
  Informative markers flanking the DMD, IKBKG and NF2 genes were used to construct non-linked haplotypes. Polar bodies 1 (PB1) and 2 (PB2) were biopsied and analyzed to determine allelic association between the mutation and markers in multiplex PCR reactions.
  
  
  RESULTS
  For each family, the first PGD cycle allowed the establishment of linked haplotypes based on homozygous PB1 and PB2 analysis; however, no embryos were available for transfer. Subsequent cycles, when performed, confirmed this linkage. A mutation-free child was born to the family affected with DMD and an ongoing pregnancy (32 weeks) was achieved with the carrier of the IKBKG deletion.
  
  
  CONCLUSIONS
  PB analysis for reverse linkage in real-time coupled with the PGD cycle is a powerful tool for diagnosis and linkage between markers and de novo mutations for maternal autosomal dominant or X-linked disorders. Simultaneous amplification of multiple informative markers in conjunction with the mutation allows the building of familial haplotypes and accurate PGD analysis.
authors.affiliation: Hebrew University School of Medicine, Jerusalem, Israel. gheona@szmc.org.il
authors.forename: Gheona
authors.initials: G
authors.lastname: Altarescu
authors.forename: Talia
authors.initials: T
authors.lastname: Eldar-Geva
authors.forename: Irit
authors.initials: I
authors.lastname: Varshower
authors.forename: Barry
authors.initials: B
authors.lastname: Brooks
authors.forename: Edith Zylber
authors.initials: EZ
authors.lastname: Haran
authors.forename: Ehud J
authors.initials: EJ
authors.lastname: Margalioth
authors.forename: Ephrat
authors.initials: E
authors.lastname: Levy-Lahad
authors.forename: Paul
authors.initials: P
authors.lastname: Renbaum
mesh_terms: D000328:Adult; D005260:Female; D006580:Genetic Carrier Screening / Q000379:methods*; D008040:Genetic Linkage; D005819:Genetic Markers; D006239:Haplotypes; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics; D009154:Mutation; D025581:Neurofibromin 2 / Q000235:genetics; D019836:Preimplantation Diagnosis / Q000379:methods*; D055815:Young Adult
chemical_list: D005819:Genetic Markers; C491731:IKBKG protein, human; D025581:Neurofibromin 2; D051550:I-kappa B Kinase