A 29-year-old woman came to the Department of Reproductive Heredity Center, Changhai Hospital, for genetic counseling to prevent the transmission of HS to the next-generation. She was married for half a year, had a normal sex life, was not pregnant for half a year without contraception, had regular menstruation, a large amount, no dysmenorrhea, and no intercourse pain. The shape of her spleen was enlarged and a splenectomy was done as previous treatment. After obtaining the written informed consent of the couple, we collected the genomic DNA of peripheral venous blood leukocytes and their clinical data. This study followed the principle of the “Helsinki Declaration” and was approved by the Ethics Committee of Changhai Hospital, Navy Medical University (2022-D-1173).

Genomic DNA was isolated from 2 to 5 mL of subjects’ peripheral blood samples using a commercial kit (TIANGEN, China). The targeted gene capture process was completed in MyGenostics, Beijing, China. Targeted next-generation sequencing with a panel of 208 genes (Supplementary Table S1) related to blood system diseases was adopted to detect the variants. The library was sequenced using a NextSeq500 instrument.

Based on the patient’s phenotype, genotype, and genetic pattern, primers were designed for the candidate pathogenic variant sites for confirmation, and at the same time perform Sanger sequencing on the parental candidate sites. The RNA was extracted from the blood of the patient and 1 healthy control, and reverse transcribed into cDNA by RT-PCR. Primers were designed for the target site. Using cDNA as a template, the target site was amplified by PCR and agarose gel electrophoresis, and Sanger and next-generation sequencing were carried out. Forward primer: GCT​GCT​GGA​GGT​GCT​CTC​T; Reverse primer: ACC​ACA​ACA​CAC​ACG​CAT​CC. The variants are described according to the Human Genome Variation Society (HGVS).

One to three blastocyst trophoblast cells were extracted and dissolved in 4 µL PBS preservation solution. Single Cell amplification was performed using the REPLI-g Single Cell Kit (150343, QIAGEN), following the kit instructions. Specifically, the cell lysis solution Buffer D2 (DTT, 3 µL; Buffer DLB, 33 µL) was prepared first, and 3 µL Buffer D2 was added into 4 µL PBS solution containing single-cell samples for the lysis reaction (65°C, 10 min; 4°C, hold; Hot cover, 70°C), add 3 µL stop solution to the reaction solution after the reaction; Then PCR Master Mix (H₂O, 9 µL, REPLI-g sc Reaction Buffer, 29 µL, REPLI-g sc DNA Polymerase, 2 µL) was prepared and 40 µL Master Mix was added into the sample. PCR was performed (30°C, 8 h; 65°C, 3 min; 4°C, hold; hot cap, 70°C). The 2 µL amplified product was tested by agarose gel electrophoresis. Part of the product (15 µL) was used for chip detection, and the other part (15 µL) was stored as backup. The remaining 18 µL were rehydrated to 40 µL and purified by 1.8-fold magnetic beads. The eluate volume was 50 µL for the whole gene construction library. The purified products were quantified using Qubit.

Firstly, based on the Asian Screening Array (ASA) chip data, using a waste embryo (embryo with a low rating, unsuitable for transfer) as the proband (the lowest allele drop-out rate), the parent target genes and the upstream and downstream haplotypes of a total of 1 Mb were derived. Then, the parental haplotype information combined with the embryo genotype information was used to deduce the embryo haplotype based on the Mendelian inheritance law. Among them, the mother’s haplotype was determined using SNP loci where the mother is heterozygous and the father is homozygous, combined with the genotype of the corresponding loci of the proband, and derived based on the rule of Mendelian inheritance. Similarly, paternal haplotypes require the selection of SNPs that are heterozygous in the father and homozygous in the mother, combined with the genotype of the proband, and derived based on the rule of Mendelian inheritance. Since the target variation in this family is derived from the mother, only the mother haplotype needs to be derived.

cfDNA (cell-free DNA) sampling vessel was used to collect 10 mL of the pregnant woman’s venous peripheral blood, and an EDTA sampling vessel was used to collect 5 mL of the father’s venous peripheral blood. gDNA was extracted using the magnetic bead method Blood genomic DNA small amount extraction kit (Shanghai Enlighten Biotechnology Co., LTD., LDB747). The blood sample of the pregnant woman was collected at 1600 g, and centrifuged at 4°C for 10 min, and the supernatant was collected into a new centrifuge tube and continued to centrifuge at 4°C for 1600 g for 10 min. The supernatant was collected for further use. The initial plasma sample size of the pregnant woman was 900 µl cfDNA was extracted using a cell-free DNA extraction kit (Shanghai LifeFeng Biotechnology Co., LTD., DK607), and the final elution volume was 50 µL. All experiments were performed according to the kit instructions. The library was constructed using the Hieff NGS OnePot DNA Library Prep Kit for Illumina kit (Yeasen Biotech Co., Ltd.,12203ES96). Hieff NGS MaxUp II DNA Library Prep Kit for Illumina All-purpose DNA library Construction Kit (Yeasen Biotech Co., Ltd., 12200ES96) was utilized to build the library. QSEP100 (BIOptic, China) was used for fragment analysis and Qubit 4.0 (Thermo, United States) was used for quantitative analysis. Twist Standard Hybridization and Wash Kit (Twist Bioscience, United States) was used to hybridize and capture the constructed whole genome library. The captured library was sequenced by MGISEQ-T7. Finally, the sequencing error rate and fetal cffDNA (Cell-free fetal DNA) concentration were calculated to construct family haplotype and fetal haplotype prediction.

The patient was admitted to the Reproductive Medicine Center of Changhai Hospital in July 2018. Clinical tests showed that the patient had more spherical cells beyond the normal range in the blood smear, an enlarged spleen, increased osmotic fragility of red blood cells, hyperbilirubinemia, and anemia (Table 1). Based on clinical evidence, the patient was diagnosed with hereditary spherocytosis. The activities of glucose-6-phosphate isomerase, phosphofructokinase, glucose-6-phosphate dehydrogenase, and pyruvate kinase were normal. The patient’s mother had mild anemia, and the patient’s father had a normal phenotype. For further confirmation, genetic testing was performed, and the panel detection of blood-related diseases showed that SPTB had a splicing variant (NM_001024858; chr14:65,271,654–65,271,654; exon2; c.300+2dup). This variant is novel and has not been reported in any public database. Family confirmation and segregation analyses showed that the father of the subject had no variant in this site, and the mother of the subject had no variation in this site, suggesting that this was a spontaneous variant (Figure 1).

To verify whether the variant (SPTB: NM_001024858: c.300+2dup) affects the mRNA splicing of SPTB, the peripheral blood RNA of the patient and a healthy control was extracted and inversely transcribed into cDNA by RT-PCR. PCR primers were designed to amplify the CDS region near the variant (Figure 2A). The theoretical size of the amplified fragment was 400 bp. The results of gel electrophoresis showed that the amplified fragment length of the patient was not significantly different from that of the control and theoretical amplification (Figure 2B). The cDNA amplification products of patients and healthy controls were sequenced, and the Sanger sequencing results showed that there were low signal peaks at c.279 (Figure 2C, starting after the black background base), but not in the control samples, indicating that c.300+2dup variation may affect RNA splicing. The cDNA products of the patient and healthy control were sequenced using next-generation Illumina sequencing. These sequencing results showed that 21 bases (missing GGT​GCT​CTC​TGG​AGA​GAT​GCT) were deleted at c. 279–299 (Figure 2D, red frame), while no deletion was found in the control samples, indicating that c.300+2dup variation affects RNA splicing.

During the in vitro fertilization cycle, 14 MII oocytes were obtained, sperm were injected into the eggs by ICSI (Intracytoplasmic sperm injection), and finally 5 developed into blastocysts. The trophoblast was biopsied by laser on the fifth and sixth day after fertilization. 1-3 cells were extracted from the trophectoderm (TE) of each blastocyst and prepared for WGA. All the samples were amplified and verified by agarose gel electrophoresis. CNV-seq sequencing (CNV-seq) results based on NGS are summarized as shown in Figure 2E. The results showed that only embryo 2 and embryo 5 had normal karyotypes. The chromosome abnormalities of the other three embryos were related to different degrees of chromosome chimerism, especially E03, which showed multiple chromosome chimerism. The carrying status of the SPTB variant of each embryo was first verified by Sanger sequencing following the same protocol as for the blood DNA samples. The results of Sanger sequencing showed that none of the other four embryos contained c.300+2dup variant except embryo No. 4.

Some embryos carrying the SPTB variant had been detected by Sanger sequencing, however, the result was based on whole-genome amplification products from embryonic biopsy cells. Allele dropout (ADO) is difficult to avoid, which may lead to misdiagnosis. To reduce its interference with the diagnosis results, we used SNP markers in the 1 Mb region to the side of the target gene for linkage analysis (Figure 3A). The ASA chip we used is a genome-wide SNP chip based on 9000+ East Asian genome sequencing data from Illumina. It contains 700,000 markers with good coverage for low-frequency variants in East Asia (MAF is 1%–5%). In this study, 22 effective SNPs were obtained to construct the mother haplotype. A waste embryo was used as the proband, and a total of 4 Mbp upstream and downstream of the target variant was used for haplotype analysis. According to the results of the ASA chip, we analyzed all SNP loci on parents and embryos and found that all embryos had normal haplotypes except embryo No. Four and the mother carrying the same SPTB variant (Figure 3B).

Regarding the decision to transfer the embryos, the parents had discussions with reproductive doctors, embryologists, and medical geneticists at the Center for Reproductive Medicine at Changhai hospital. According to the results of PGT-M and CNV analysis, the final decision was made to use embryo 2 for transplantation. Four weeks after transplantation, the B-ultrasound showed a fetal heartbeat, indicating a successful pregnancy. At 13 weeks of gestation, maternal peripheral blood was collected for NIPT-M detection according to the procedure shown in Figure 3C, and the results showed that the fetus did not carry the disease-causing variant. The pregnant woman underwent amniocentesis at 20 weeks of gestation for prenatal genetic diagnosis. The prenatal diagnosis (Figure 3D) was consistent with preimplantation genetic testing. The woman eventually gave birth to a healthy boy who did not carry the variant. All blood indicators were normal after birth.