title: PGD on a recombinant allele: crossover between the TSC2 gene and 'linked' markers impairs accurate diagnosis.
abstract: OBJECTIVE
  Accounting for possible recombinations in developing an accurate preimplantation genetic diagnosis (PGD) protocol based on familial haplotypes.
  
  
  METHODS
  Haplotypes were constructed from genomic DNA in a family where the male was affected with tuberous sclerosis complex (TSC). Embryos were biopsied at day 3, and single blastomeres were analyzed by multiplex polymerase chain reactions (PCRs) including multiple microsatellite markers and single-cell sequencing.
  
  
  RESULTS
  Informative markers used in the initial haplotype analysis, based on the genomic DNA of the parents and affected child, were analyzed in the first PGD cycle. All embryos appeared to show recombination and none were transferred. Prior to the next cycle, the parents of the affected male were included in the haplotype analysis, demonstrating that the affected child had a recombinant allele. In the second PGD cycle, two non-recombinant intragenic single nucleotide polymorphisms (SNPs) were used with the TSC2 mutation and four microsatellite markers for a second PGD cycle. In this cycle two wild-type embryos were transferred, resulting in the birth of a healthy girl carrying the wild-type TSC2 allele.
  
  
  CONCLUSIONS
  PGD analysis requires the use of more than one meiosis for constructing accurate haplotypes. Testing for multiple closely linked markers and the familial mutation are necessary to detect recombination events and enable precise diagnosis.
authors.affiliation: Medical Genetics Unit, Zohar PGD Lab, Shaare Zedek Medical Center, Jerusalem, Israel.
authors.forename: Gheona
authors.initials: G
authors.lastname: Altarescu
authors.forename: Talia
authors.initials: T
authors.lastname: Eldar Geva
authors.forename: Barry
authors.initials: B
authors.lastname: Brooks
authors.forename: Ehud
authors.initials: E
authors.lastname: Margalioth
authors.forename: Ephrat
authors.initials: E
authors.lastname: Levy-Lahad
authors.forename: Paul
authors.initials: P
authors.lastname: Renbaum
mesh_terms: D000483:Alleles; D001757:Blastomeres; D003434:Crossing Over, Genetic; D004252:DNA Mutational Analysis; D008040:Genetic Linkage; D005819:Genetic Markers; D006239:Haplotypes; D006801:Humans; D018895:Microsatellite Repeats; D010375:Pedigree; D020641:Polymorphism, Single Nucleotide; D019836:Preimplantation Diagnosis / Q000379:methods*; D014402:Tuberous Sclerosis / Q000175:diagnosis* / Q000235:genetics; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: D005819:Genetic Markers; C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins