Journal Article

Real-time reverse linkage using polar body analysis for preimplantation genetic diagnosis in female carriers of <i>de novo</i> mutations

Gheona Altarescu, Talia Eldar-Geva, Irit Varshower, Barry Brooks, Edith Zylber Haran, Ehud J. Margalioth, Ephrat Levy-Lahad and Paul Renbaum

in Human Reproduction

Published on behalf of European Society of Human Reproduction and Embryology

Volume 24, issue 12, pages 3225-3229
Published in print December 2009 | ISSN: 0268-1161
Published online August 2009 | e-ISSN: 1460-2350 | DOI:
Real-time reverse linkage using polar body analysis for preimplantation genetic diagnosis in female carriers of de novo mutations

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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.


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.


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.


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.

Keywords: preimplantation genetic diagnosis; polar bodies; single cell mutiplex PCR; de novo mutations; reverse linkage

Journal Article.  3455 words.  Illustrated.

Subjects: Reproductive Medicine

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