Abstract
Preimplantation genetic diagnosis (PGD) has become a crucial approach in helping carriers of inherited disorders to give birth to healthy offspring. In this study, we review PGD methodologies and explore the use of amplification refractory mutation system quantitative polymerase chain reaction (ARMS-qPCR) and/or linkage analysis for PGD in neurodegenerative diseases that are clinically relevant with typical features, such as late onset, and which are severely debilitating. A total of 13 oocyte retrieval cycles were conducted in 10 cases with various neurodegenerative diseases. Among the 59 embryos analyzed, 49.2% (29/59) were unaffected and 50.8% (30/59) were affected. Of the 12 embryo transfer cycles, three resulted in pregnancy, and all pregnancies were delivered. The implantation rate and livebirth rate were 23.1% (3/13) per oocyte retrieval cycle and 25.0% (3/12) per embryo transfer cycle. Allele dropout (ADO) was noted in two embryos that were classified as unaffected by ARMS-qPCR but were evidenced as affected after prenatal diagnosis, rendering the false negative rate as 6.3% (2/32). Four among the 13 cycles underwent PGD by ARMS-qPCR coupled with linkage analysis, and all were correctly diagnosed. We conclude that PGD by ARMS-qPCR and/or linkage analysis is a feasible strategy, whereas ADO is a concern when ARMS-qPCR is used as the sole technology in PGD, especially in autosomal dominant diseases.
Highlights
As we focused on the application of Preimplantation genetic diagnosis (PGD) in neurodegenerative disorders, studies exclusively concerning ethical issues, diagnostic technique evolution, or those that included patients with non-neurodegenerative diseases were excluded
We devised a novel in-house patented ARMS-qPCR genotyping strategy (Figure 1) to address the need for timely and overnight diagnosis of fresh embryo transfer when a trophectoderm biopsy was performed at the 5/6-day blastocyst stage, since our protocol only includes 5/6-day blastocyst biopsies
By ARMS-qPCR is routinely performed before couples seeking the assistance of PGD are enrolled in the clinical PGD services
Summary
Polymerase chain reaction (PCR)-based technology is one of the major molecular technologies used in PGD. Direct mutation detection, such as Sanger sequencing and amplification refractory mutation system quantitative. PCR (ARMS-qPCR) complemented by linkage analysis of the co-amplification of polymorphic short tandem repeat (STR) markers, are being widely adopted [3,4,5] because both linkage analysis and direct mutation detection assays have shortcomings. In selected urgent cases, a timely overnight diagnosis is still feasible. Such timely genotyping is prone to allele dropout (ADO) and further confirmation by repeat experiments or by additional genotyping methods is impossible.
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