Successful birth with preimplantation genetic diagnosis using single-cell allele-specific PCR and sequencing in a woman with hypochondroplasia due to FGFR3 mutation (c.1620C>A, p.N540K)
A 27-year-old HCH woman with a history of two consecutive abortions of HCH-affected fetuses visited our clinic for PGD. The woman had her diagnosis clinically and genetically confirmed as HCH by the orthopedist when she was 9 years old, and reconfirmed by another orthopedist just before the PGD. Previously, she had undergone prenatal diagnoses during two consecutive natural pregnancies, which resulted in affected fetuses after confirming the presence of the c.1620C>A, p.N540K mutation in the FGFR3 gene confirmed by chorionic villi sampling, which was same as her own mutation. Terminations of the pregnancies were carried out in both cases at the couple's request. Following adequate counseling, the couple decided to undergo PGD for the third pregnancy and signed informed consent.\nFor the GnRH agonist long protocol, 0.1 mg/day of subcutaneous triptorelin acetate (Decapeptyl, Ferring Phamaceuticals, Malmo, Sweden) was initiated on day 21 of the previous cycle. After pituitary suppression, the triptorelin dose was reduced to 0.05 mg/day and recombinant FSH (follitropin alfa, Gonal-F, Merck Serono, Geneva, Switzerland) was added. After recombinant hCG (Ovidrel, Merck Serono) triggering, 34 oocytes were retrieved and fertilized by ICSI. Twenty-two embryos with two pronuclei were cultured, and the biopsy was performed with 18 embryos that reached the six- to eight-cell stage. Partial zona dissection and biopsy were performed using a micromanipulator (Narishige, Tokyo, Japan) mounted on an inverted microscope (Nikon). The presence of a clearly visible nucleus guided the selection of the blastomere to be biopsied in G-PGD medium (Vitrolife, Goteborg, Sweden). In the end, 20 blastomeres biopsied from 18 embryos were analyzed with positive and negative controls and were loaded in a reaction tube containing 2.5 µL alkaline lysis buffer (200 mM NaOH, 50 mM dithiothreitol) [].\nA total of 20 blastomeres with each washing drop were immediately lysed by incubation at 65℃ for 10 minutes. The first round of PCR was performed in a 50 µL volume containing 2.5 µL of lysate, 5 µL of 10× PCR buffer, 10 mM tricine (pH 4.93), 200 µM dNTP, 10% dimethyl sulfoxide (DMSO), 0.4 µM of each outer primer (), and 1.25 U Taq DNA polymerase []. The first round PCR program was as follows: 5 minutes at 94℃, 25 cycles of 30 seconds at 94℃, 30 seconds at 60℃, and 1 minute at 72℃, then 5 minutes at 72℃. Two microliters of the first-round PCR products were then re-amplified in a second round of PCR. The second round was performed in a 50 µL volume containing 2 µL of the first-round PCR products, 5 µL of 10× PCR buffer, 200 µM dNTP, 10% DMSO, 0.4 µM of each inner primer (), and 1.25 U Taq DNA polymerase. The second round PCR program was as follows: 5 minutes at 94℃, 35 cycles of 30 seconds at 94℃, 30 seconds at 60℃, and 1 minute at 72℃, then 5 minutes at 72℃.\nThe second-round PCR products were analyzed by direct sequencing using an ABI Prism 3730 genetic analyzer (Applied Biosystems, Foster City, CA, USA). To confirm the result of the sequence analysis, allele-specific PCRs for the wild allele and mutant allele were performed on the first-round PCR product, separately. These processes are presented schematically in . Primers for the mutant allele were designed to generate the 122 bp amplicon and those for the wild allele were designed to generate the 155 bp amplicon ().\nThrough the allele-specific nested PCR and sequencing as described, blastomeres were classified as c.1629C, p.N540, homozygote or c.1620C>A, p.N540K, heterozygote (). Among the 20 blastomeres, amplifications of the first-round PCR product were successful in 16 (80%) blastomeres. Ten blastomeres were negative for the mutation with sequencing and allele-specific PCR. Five blastomeres were positive for mutation with both methods. A blastomere was positive for mutation in allele-specific PCR, although negative in sequencing. None of the washing drops showed amplification in first-round PCR.\nFinally, 9 embryos with the wild-type sequence (FGFR3:c.1620C) were selected and cryopreserved by vitrification in a fresh cycle due to the patient's high risk of ovarian hyperstimulation syndrome (OHSS). After a failure of the first frozen-thawed cycle, a successful singleton pregnancy was achieved through the second frozen-thawed cycle, in which the embryo transfer of 2 blastocysts had been attempted. An appropriate initial rise in β-hCG and fetal cardiac activity were noted sequentially. On 16 weeks of gestation, amniocentesis was carried out for verification. Genetic analysis of cultured amniotic fluid cells showed a normal karyotype, and the presence of the c.1620C>A, p.N540K mutation in the FGFR3 gene was excluded. On second trimester ultrasound findings, no evidence of skeletal dysplasia was identified. The pregnancy resulted in a full-term delivery without any significant complications: a 3,620-g male infant was delivered at 40 weeks by Cesarean section.