Preimplantation genetic diagnosis in genomic regions with duplications and pseudogenes: long-range PCR in the single-cell assay.

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.

Preimplantation genetic diagnosis (PGD) for SHOX-related haploinsufficiency in conjunction with trisomy 21 detection by molecular analysis.

PURPOSE: Development of a molecular PGD protocol for a male with an X-linked deletion in the SHOX gene region, located in the pseudoautosomal region of the X/Y chromosomes. Due to excessive recombination in this region, the deletion can be found in male offspring. METHODS: We developed a 13 marker multiplex fluorescent PCR protocol: 3 markers within the deleted SHOX region, 5 flanking markers, 3 informative markers on chromosome 21 (advanced maternal age) and 2 markers for sex determination. RESULTS: Of four embryos, two wild type males, diploid for chromosome 21 were transferred resulting in twin boys. One embryo was an affected female and another embryo was Turner. Amniocentesis confirmed the implanted embryos were males (46XY), with no recombinations. CONCLUSIONS: While many X-linked disorders can be analyzed by sexing, genes located in the pseudoautosomal regions have high XY recombination rates, requiring multiple markers to enable an accurate diagnosis.