Communicated by Mats Nilsson
Preimplantation Genetic Diagnosis in Genomic Regions with Duplications and Pseudogenes: Long-Range PCR in the Single-Cell Assay
Article first published online: 19 MAR 2013
© 2013 Wiley Periodicals, Inc.
Volume 34, Issue 5, pages 792–799, May 2013
How to Cite
Zeevi, D. A., Renbaum, P., Ron-El, R., Eldar-Geva, T., Raziel, A., Brooks, B., Strassburger, D., Margalioth, E. J., Levy-Lahad, E. and Altarescu, G. (2013), Preimplantation Genetic Diagnosis in Genomic Regions with Duplications and Pseudogenes: Long-Range PCR in the Single-Cell Assay. Hum. Mutat., 34: 792–799. doi: 10.1002/humu.22298
Additional Supporting Information may be found in the online version of this article.
- Issue published online: 11 APR 2013
- Article first published online: 19 MAR 2013
- Accepted manuscript online: 19 FEB 2013 07:21AM EST
- Manuscript Accepted: 11 FEB 2013
- Manuscript Received: 22 AUG 2012
- genetic diagnosis;
Long-range PCR is generally employed for the analysis of disease-causing mutations in genes with homologous pseudogene copies. However, long-range PCR is challenging when performed on single cells, as in preimplantation genetic diagnosis (PGD) of monogenic disorders. PGD on single cells requires concurrent analysis of a mutation together with multiple linked polymorphic markers from closely related family members to prevent misdiagnosis. In PGD cases involving childless de novo mutation carriers, linkage cannot be performed based on family members but rather must first be identified in single gametes. This can be an especially difficult task if the mutation to be assayed lies in a duplicated genomic region because gene-specific long-range PCR must be coupled with short-range PCR analysis of genetic markers on single cells. Here, we describe a novel method by which accurate PGD of pseudogene-homologous mutations can be achieved. Essentially, we performed whole genome amplification on single sperm or blastomeres followed by haplotype construction and long-range PCR-based mutation analysis. This original and universal strategy was used to establish allelic association for two different mutations in genes with one or more pseudogene copies (IKBKG and PKD1). The method was also sensitive enough to detect unexpected germline mosaicism in one mutation carrier.