title: Preimplantation genetic diagnosis in genomic regions with duplications and pseudogenes: long-range PCR in the single-cell assay.
abstract: 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.
authors: Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel. zeevidavid@gmail.com
authors: David A
authors: DA
authors: Zeevi
authors: Paul
authors: P
authors: Renbaum
authors: Raphael
authors: R
authors: Ron-El
authors: Talia
authors: T
authors: Eldar-Geva
authors: Arieh
authors: A
authors: Raziel
authors: Baruch
authors: B
authors: Brooks
authors: Dvorah
authors: D
authors: Strassburger
authors: Ehud J
authors: EJ
authors: Margalioth
authors: Ephrat
authors: E
authors: Levy-Lahad
authors: Gheona
authors: G
authors: Altarescu
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D005260:Female; D020440:Gene Duplication; D006801:Humans; D008297:Male; D009154:Mutation; D010375:Pedigree; D016133:Polymerase Chain Reaction / Q000379:methods*; D019836:Preimplantation Diagnosis; D011544:Pseudogenes; D059010:Single-Cell Analysis; D050396:TRPP Cation Channels / Q000235:genetics
chemical_list: D017931:DNA Primers; D050396:TRPP Cation Channels