Expanded carrier screening and preimplantation genetic diagnosis in a couple who delivered a baby affected with congenital factor VII deficiency

A couple consisting of a 34-year-old woman and a 40-year-old man with normal phenotypes who had a proband child with congenital FVII deficiency and terminated a second pregnancy because of the same disease (Fig. 1) was referred to our centre (Reproductive and Genetic Hospital of CITIC-Xiangya). Haematological testing of the proband showed an obviously prolonged prothrombin time (24.1 s, reference range: 9.9–12.8 s), and testing showed 2.03% FVII activity (reference range: 70%–120%). Genetic testing via Sanger sequencing confirmed that the husband is a carrier of a c.1238G > C (p.Arg353Pro) mutation in the F7 gene, while the wife is a carrier of a c.1126A > T (p.Lys316Term) mutation in the same gene. Furthermore, the husband was found to harbour another variation in the F7 gene, c.1238G > A (p.Arg353Gln), which could mildly reduce factor VII activity (Fig. 2) [12, 13]. The couple sought PGD and PGS to prevent the recurrence of the FVII deficiency, other common Mendelian disorders and chromosomal abnormalities.

Both members of the couple gave their written informed consent, and genomic DNA was extracted from their blood samples for ECS using a commercial panel from BGI-Tianjin. The panel was designed to screen 547 genes associated with 623 monogenic disorders and includes recessive and X-linked diseases with severe and highly penetrant phenotypes as well as high-prevalence monogenic diseases with moderate phenotypes [3, 11]. The samples were sequenced (PE100) in one lane of the Hiseq2000 platform. Sequence data analysis was performed using a bioinformatics pipeline developed in a previous study [3]. Briefly, the Illumina analysis pipepline (CASAVA1.8) was employed for base calling. We then separated each barcoded dataset and removed the low-quality data using in-house scripts. The sequencing reads were subsequently aligned to the reference human genome (hg19) using the Burrows-Wheeler Aligner. Single-nucleotide variants and small insertions or deletions were identified using the Genome Analysis Toolkit (Broad Institute), while deletion or duplication of exons in genes was detected using an in-house pipeline. Variants were annotated with in-house scripts [3].

The couple underwent IVF treatment and biopsy as previously reported [14]. Briefly, after oocyte retrieval, mature oocytes were subjected to ICSI. On day 5 after fertilization, blastocysts in which trophectoderm cells had herniated out of the zona pellucida were chosen for biopsy.

Whole-genome amplification (WGA) of trophectoderm cells was performed using a commercial kit (REPLI-gMidi Kit, QIAGEN, Germany) according to the manufacturer’s instructions, with some modifications. ECS revealed that both members of the couple carried a heterozygous disease-causative mutation in the CFTR gene. PGD was performed by directly evaluating the F7 and CFTR genes and via an embryo haplotyping strategy. Three pairs of primers were designed for direct mutation detection. For embryo genotyping, a set of short tandem repeat (STR) markers located on either side of the mutation was used to establish the paternal and maternal haplotypes. STR primers for one locus linked to the F7 gene (D13S261) and three loci linked to the CFTR gene (D7S633, D7S480, and CFTR-IVS17) were amplified in separate singleplex polymerase chain reaction (PCR) assays using HotStarTaq DNA Polymerase (QIAGEN, Germany), and the results were detected via capillary electrophoresis on an ABI 3130 XL genetic analyser (Applied Biosystems, Foster City, CA, USA). The primer sequences are shown in Table 1. After PGD, embryos that were free of the monogenic disorders were also tested for chromosomal aneuploidy using the Ion PGM™ sequencing platform (Life Technologies, San Francisco, CA) according to a standard protocol. Briefly, the WGA products were purified with Agencourt® AMPure®XP beads (BeckMan), connected with an Ion Xpress™ Barcode (Life Tech), and subsequently sequenced on the Ion PGM™ platform (Life Tech), according to a standard protocol (https://​ioncommunity.​thermofisher.​com/​community/​protocols-home). The raw sequencing data included approximately 0.5 M reads, which were mapped to the reference human genome (hg19) with a coverage rate of approximately 1%. Chromosomal copy number variation (CNV) analysis was performed for all samples using the Celloud cloud server (http://​www.​celloud.​org/), offered by JBRH (Beijing, China). The applied analysis pipepline was similar to that in a previous study [15].

Amniocentesis was performed at 19 weeks of gestation, and genomic DNA was extracted from cultured amniocytes using the QIAamp DNA Mini Kit (QIAGEN, Germany). Sanger sequencing was employed to confirm the absence or presence of the mutations in the F7 and CFTR genes. Additionally, the chromosome karyotype was confirmed by amniocyte G-banding using the Automatic Chromosome Karyotyping System (ZEISS, Germany) following a standard protocol.

In total, 1.5 Gb of sequence was generated to achieve a mean coverage of 200×. After upstream filtering, only one disorder, cystic fibrosis, was found to show a high risk, as both members of the couple exhibited a mutation in CFTR. A previously reported variation in the CFTR gene, c.3659C > T (p.Thr1220Ile), was identified in the husband. This mutation was also identified in a cystic fibrosis patient and was classified as a pathogenic mutation based on evaluation through in silico analyses [16]. The wife was also found to harbour a recurrent pathogenic mutation in the CFTR gene, c.3209G > A (p.Arg1070Gln) [17]. These variations were confirmed via Sanger sequencing (Fig. 2).

Following ovarian stimulation, 24 oocytes were retrieved, 20 of which were fertilized through ICSI. Of these 20, 11 developed to the blastocyst stage with apparently normal phenotypes, and trophectoderm cells were collected for WGA. Sanger sequencing and STR analysis were performed on the whole-genome amplification products. The results of simultaneous PGD for congenital FVII deficiency and CF are summarized in Table 2. Embryos 1, 2, 6, 9, and 10 were affected with congenital FVII deficiency; embryos 4 and 5 were compound heterozygotes for the two CFTR gene mutations; embryos 7 and 8 were diagnosed as carriers of congenital FVII deficiency and CF; and embryo 3 was a carrier of congenital FVII deficiency but did not harbour either of the CFTR gene mutations. Due to severe allele drop out (ADO) for the F7 gene and the linked STRs, the diagnosis of embryo 11 as either a carrier or patient was unclear.

Embryos 3, 7, and 8 were chosen for screening chromosomal abnormalities since they were unaffected by congenital FVII deficiency and CF. Comprehensive aneuploidy screening revealed that embryo 7 (a carrier of congenital FVII deficiency and CF) was euploid, whereas trisomy 16 and trisomy 13 were identified in embryos 3 and 8, respectively (Fig. 3). Based on the results, embryo 7 was transferred.

Two weeks after embryo transfer, the woman showed a positive HCG pregnancy test, and over time, a single heartbeat and ongoing pregnancy were observed. The results of mutational and cytogenetic analyses of amniocyte DNA were in agreement with the original embryo 7 genotype (euploid and a carrier of congenital FVII deficiency and CF), and a healthy 3200 g male infant was born in October 2015 (Fig. 2). Further follow-up has shown that the boy is healthy.