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

Ludmila Volozonoka,Evija Jokste Pīmane,Anna Miskova,Natālija Novikova,Baiba Alkšere,Dmitry Perminov,Inga Kempa,Arita Blumberga,Linda Gailīte,Liene Korņejeva,Violeta Fodina

doi:10.1007/s10815-018-1187-4

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.

Highlights

Preimplantation genetic testing (PGT), formerly known as preimplantation genetic diagnostic (PGD) for monogenic disease testing or PGS for chromosome screening, is an alternative to prenatal testing for couples being at risk of transmitting a genetic disorder to their offspring [38]
The portion of each embryo Whole genome amplification (WGA) product was used for haplotyping informative or semi-informative markers detected by initial family linkage analysis
The overall short tandem repeat (STR) allele dropout (ADO) rate was 4.74% (Table 4) exceeding the 5% cut-off only in DMDcase 1, where the WGA product generated by OmniPlex had lower quality due to long-time storage and repeated freezethaw events. array comparative genome hybridization (aCGH) for this DMD-case 1 was performed firstly when haplotyping was unavailable

Summary

Introduction

Preimplantation genetic testing (PGT), formerly known as PGD for monogenic disease testing or PGS for chromosome screening, is an alternative to prenatal testing for couples being at risk of transmitting a genetic disorder to their offspring [38]. PGT allows exclusion of affected embryos before a clinical pregnancy has been established avoiding invasive prenatal testing and elective termination of pregnancy due to prenatally confirmed diagnosis. The material for PGT can be collected from day 3 or day 5 of developing embryo before its transfer to the uterus. The process initially requires controlled ovarian hyperstimulation, oocyte retrieval and subsequent oocyte in vitro fertilization (IVF), most commonly by intracytoplasmic sperm injection (ICSI) followed by embryo cultivation until the desired stage of development as well as a biopsy procedure [1]. PGT can be done with or without embryo vitrification for the time of testing. Embryos proved free of the disease-causing variant under consideration are subsequently transferred into the uterine cavity

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