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Preimplantation Genetic Diagnosis in Genomic Regions with Duplications and Pseudogenes: Long‐Range PCR in the Single‐Cell Assay
Author(s) -
Zeevi David A.,
Renbaum Paul,
RonEl Raphael,
EldarGeva Talia,
Raziel Arieh,
Brooks Baruch,
Strassburger Dvorah,
Margalioth Ehud J.,
LevyLahad Ephrat,
Altarescu Gheona
Publication year - 2013
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.22298
Subject(s) - pseudogene , biology , genetics , preimplantation genetic diagnosis , mutation , gene , haplotype , germline mosaicism , germline , allele , genome , pregnancy
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.

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