Performance comparison of two whole genome amplification techniques in frame of multifactor preimplantation genetic testing

Abstract

PurposeTo compare multiple displacement amplification and OmniPlex whole genome amplification technique performance during array comparative genome hybridization (aCGH), Sanger sequencing, SNaPshot and fragment size analysis downstream applications in frame of multifactor embryo preimplantation genetic testing.MethodsPreclinical workup included linked short tandem repeat (STR) marker selection and primer design for loci of interest. It was followed by a family haplotyping, after which an in vitro fertilization preimplantation genetic testing (IVF-PGT) cycle was carried out. A total of 62 embryos were retrieved from nine couples with a confirmed single gene disorder being transmitted in their family with various inheritance traits—autosomal dominant (genes—ACTA2, HTT, KRT14), autosomal recessive (genes—ALOX12B, TPP1, GLB1) and X-linked (genes—MTM1, DMD). Whole genome amplification (WGA) for the day 5 embryo trophectoderm single biopsies was carried out by multiple displacement amplification (MDA) or polymerase chain reaction (PCR)-based technology OmniPlex and was used for direct (Sanger sequencing, fragment size analysis, SNaPshot) and indirect mutation assessment (STR marker haplotyping), and embryo aneuploidy testing by array comparative genome hybridization (aCGH).ResultsFamily haplotyping revealed informative/semi-informative microsatellite markers for all clinical cases for all types of inheritance. Indirect testing gave a persuasive conclusion for all embryos assessed, which was confirmed through direct testing. The overall allele dropout (ADO) rate was higher for PCR-based WGA, and MDA shows a better genomic recovery scale. Five euploid embryos were subjected to elective single embryo transfer (eSET), which resulted in four clinical pregnancies and birth of two healthy children, which proved free of disease causative variants running in the family postnataly.ConclusionsA developed multifactor PGT protocol can be adapted and applied to virtually any genetic condition and is capable of improving single gene disorder preimplantation genetic testing in a patient-tailored manner thus increasing pregnancy rates, saving costs and increasing patient reliability.Electronic supplementary materialThe online version of this article (10.1007/s10815-018-1187-4) contains supplementary material, which is available to authorized users.

A systematic review on concurrent aneuploidy screening and preimplantation genetic testing for hereditary disorders: What is the prevalence of aneuploidy and is there a clinical effect from aneuploidy screening?

“…Title and abstract screening resulted in 73 papers. Full text screening resulted in 26 publications fulfilling the inclusion criteria . No randomized controlled trials were identified.…”

A comprehensive and universal approach for embryo testing in patients with different genetic disorders

“… 38 For monogenic diseases, sequencing of PCR products was initially used to directly detect variants in biopsied embryo cells. 12 , 13 , 14 However, owing to the limitations of contamination and ADO, 15 , 16 in recent years, family‐based linkage analysis has become a standard method using STR or SNP haplotype analysis to indirectly determine the variants. 17 , 18 , 19 , 20 Handyside et al developed the Karyomapping method, which could be applied to any single‐gene defect within the regions covered by the SNP loci and overcame the difficulty of ADO at the single‐cell level.…”

“… 12 , 13 , 14 However, every WGA methods can result in false negative or positive single‐nucleotide variants (SNVs) owing to amplification preference of the primers and/or allele dropout (ADO), leading to misdiagnosis. 15 , 16 Family‐based linkage analysis has now become a popularized method by testing short tandem repeats (STRs) or SNPs to indirectly determine variants by haplotype, 17 , 18 , 19 , 20 , 21 , 22 and some of these researches have also confirmed that gene variants and chromosome aneuploidies can be detected simultaneously. 18 , 19 , 20 , 21 , 22 Massively parallel sequencing, like MARSALA, enables direct genotyping and haplotype analysis.…”

Preimplantation Genetic Testing for HLA-matching: An Overview of Clinical Application and Utility

“…In the last few years, new technologies have also been applied in PGT for HLA typing, such as the use of whole genome amplification of the biopsied sample prior to STR testing. Furthermore, SNPs, instead of STRs, can be used to define the haplotypes across the genomic regions and such strategies are supported by the use of SNP arrays (for example the commercialized procedure known as karyomapping) or, more recently, NGS [44][45][46]. The latest technologies can assist in overcoming the limitations of the PCR-based linkage approach for HLA typing, such as the restriction in the number of STRs that can be co-amplified, the time required to characterize new STRs for a family (if the ones already available are not informative), and the need to optimize a new protocol for each couple.…”