Abstract
BackgroundPreimplantation genetic testing (PGT) has already been applied in chromosomally balanced translocation carriers to improve the clinical outcome of assisted reproduction. However, traditional methods could not further distinguish embryos carrying a translocation from those with a normal karyotype prior to implantation.MethodsTo solve this problem, we developed a method named “Chromosomal Phasing on Base level” (BasePhasing), which based on Infinium Asian Screening Array-24 v1.0 (ASA) and a specially phasing pipeline. Firstly, by comparing the number of single nucleotide polymorphism (SNP) loci in different minor allele frequencies (MAFs) and in 2Mbp continuous windows of ASA chip and karyomap-12 chip, we verified whether ASA could be adopted for genome-wide haplotype linkage analysis. Besides, the whole gene amplification (WGA) of 3–10 cells of GM16457 cell line was used to verify whether ASA chip could be used for testing of WGA products. Finally, two balanced translocation families were utilized to carry out BasePhasing and to validate the feasibility of its clinical application.ResultsThe average number of SNP loci in each window of ASA (473.2) was twice of that of Karyomap-12 (201.2). The coincidence rate of SNP loci in genomic DNA and WGA products was about 97%. The 5.3Mbp deletion was detected positively in cell line GM16457 of both genomic DNA and WGA products, and haplotype linkage analysis was performed in genome wide successfully. In the two balanced translocation families, 18 blastocysts were analyzed, in which 8 were unbalanced and the other 10 were balanced or normal chromosomes. Two embryos were transferred back to the patients successfully, and prenatal cytogenetic analysis of amniotic fluid was performed in the second trimester. The results predicted by BasePhasing and prenatal diagnosis were totally consistent.ConclusionsInfinium ASA bead chip based BasePhasing pipeline shows good performance in balanced translocation carrier testing. With the characteristics of simple operation procedure and accurate results, we demonstrate that BasePhasing is one of the most suitable methods to distinguish between balanced and structurally normal chromosome embryos from translocation carriers in PGT at present.
Highlights
Preimplantation genetic testing (PGT) has already been applied in chromosomally balanced translocation carriers to improve the clinical outcome of assisted reproduction
Balanced translocation is one of the most frequent indications for preimplantation genetic testing (PGT), which occurs at an incidence of 1/500 to 1/625 in the general population and even up to 1/20 in the patients who has a history of repeated In vitro fertilization (IVF) failure or recurrent miscarriages [1]
BasePhasing Based on informative single nucleotide polymorphism (SNP) and chromosomal phasing principles we developed BasePhasing pipeline, which was programmed in Practical Extraction and Reporting Language (Perl), and was capable of obtaining the clear haplotypes of each family member in linkage analysis (Fig. 1)
Summary
Preimplantation genetic testing (PGT) has already been applied in chromosomally balanced translocation carriers to improve the clinical outcome of assisted reproduction. Balanced translocation is one of the most frequent indications for preimplantation genetic testing (PGT), which occurs at an incidence of 1/500 to 1/625 in the general population and even up to 1/20 in the patients who has a history of repeated IVF failure or recurrent miscarriages [1] They often have normal phenotypes, but the risk of producing unbalanced gametes is high (typically approximately 70%) due to the abnormal segregation of rearranged chromosomes during meiosis [2, 3]. With the development of technology, aCGH/ SNP array [12, 13] and whole genome sequencing [14, 15] methods have been employed for balanced translocation detection These traditional PGT methods can clearly identify embryos with chromosomally unbalanced translocation or aneuploidies, they can hardly further distinguish the balanced and structurally normal embryos. It remains unstable and inaccurate to identify the precise breakpoints in the highly repetitive and variable translocation regions
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