Universal strategy for preimplantation genetic testing for cystic fibrosis based on next-generation sequencing.

Sandrine Chamayou,Annalisa Liprino,Carmen Ragolia,Elena Maglia,Maria Sicali,Carmelita Alecci,Simona Romano,Antonino Guglielmino,Giorgia Storaci,Debora Lombardo,Clementina Cardea

doi:10.1007/s10815-019-01636-1

Abstract

We developed and applied a universal strategy for preimplantation genetic testing for all cystic fibrosis gene mutations (PGT-CF) based on next-generation sequencing (NGS). A molecular protocol was designed to diagnose all CF mutations at preimplantation stage. The detection of CF mutations was performed by direct gene sequencing and linkage strategy testing 38 specific SNPs located upstream and inside the gene for PGT-CF. Seventeen couples at risk of CF transmission decided to undergo PGT-CF. Trophectoderm cell biopsies were performed on days 5-6 blastocysts. PGT for aneuploidy (PGT-A) was performed from the same samples. Tested embryos were transferred on further natural cycles. PGT was performed on 109 embryos. Fifteen CF mutations were tested. PGT-CF and PGT-A were conclusive for, respectively, 92.7% and 95.3% of the samples. A mean of 24.1 SNPs was informative per couple. After single embryo transfer on natural cycle, 81.3% of the transferred tested embryos implanted. The present protocol based on the entire CFTR gene sequencingtogether with informative SNPs outside and inside the gene can be applied to diagnose all CF mutations at preimplantation stage.

Highlights

Cystic fibrosis (CF) is the most widespread autosomal recessive disease in the Caucasian population as one Caucasian person in 25 is a carrier [1], and the incidence is 1 in 3500 live births [2]
The absence or severely reduced activity of CFTR protein due to genetic mutations can lead to excessive viscosity of the epididymal fluid and results in male infertility [11, 13] with congenital bilateral absence of vas deferens (CBAVD) [14, 15] when associated with (TG)m and Tn polymorphisms loci in intron 8 at the splice acceptor site of exon 9 of CFTR gene [16, 17]
According to the longitudinal screening for CF mutations of eastern infertile couples, it was calculated that 1 couple out of 105.2 of infertile Sicilian couples was at risk of having an affected child with CF (10/1052)

Summary

Introduction

Cystic fibrosis (CF) is the most widespread autosomal recessive disease in the Caucasian population as one Caucasian person in 25 is a carrier [1], and the incidence is 1 in 3500 live births [2]. The gene responsible for CF is named the cystic fibrosis transmembrane conductance regulator (CFTR) and was sequenced in 1989 [3, 4]. It is located in position 7q31.2 and is compound of 27 exons. The CFTR gene encodes for cyclic adenosine monophosphate-dependent chloride channel located in the apical membrane of secretary epithelial cells [5, 6].

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