Single-cell high resolution melting analysis: A novel, generic, pre-implantation genetic diagnosis (PGD) method applied to cystic fibrosis (HRMA CF-PGD).
BACKGROUND
Institutions offering CF-PGD face the challenge of developing and optimizing single cell genotyping protocols that should cover for the extremely heterogeneous CF mutation spectrum. Here we report the development and successful clinical application of a generic CF-PGD protocol to facilitate direct detection of any CFTR nucleotide variation(s) by HRMA and simultaneous confirmation of diagnosis through haplotype analysis.


METHODS
A multiplex PCR was optimized supporting co-amplification of any CFTR exon-region, along with 6 closely linked STRs. Single cell genotypes were established through HRM analysis following melting of the 2nd round PCR products and were confirmed by STR haplotype analysis of the 1st PCR products. The protocol was validated pre-clinically, by testing 208 single lymphocytes, isolated from whole blood samples from 4 validation family trios. Fifteen PGD cycles were performed and 103 embryos were biopsied.


RESULTS
In 15 clinical PGD cycles, genotypes were achieved in 88/93 (94.6%) embryo biopsy samples, of which 57/88 (64.8%) were deemed genetically suitable for embryo transfer. Amplification failed at all loci for 10/103 blastomeres biopsied from poor quality embryos. Six clinical pregnancies were achieved (2 twin, 4 singletons). PGD genotypes were confirmed following conventional amniocentesis or chorionic villus sampling in all achieved pregnancies.


CONCLUSIONS
The single cell HRMA CF-PGD protocol described herein is a flexible, generic, low cost and robust genotyping method, which facilitates the analysis of any CFTR genotype combination. Single-cell HRMA can be beneficial to other clinical settings, for example the detection of single nucleotide variants in single cells derived from clinical tumor samples.
Department of Medical Genetics, University of Athens, St Sophia's Children's Hospital, Athens, Greece.
A
A
Destouni
Department of Medical Genetics, University of Athens, St Sophia's Children's Hospital, Athens, Greece. Electronic address: mrpoulou@med.uoa.gr.
M
M
Poulou
Department of Medical Genetics, University of Athens, St Sophia's Children's Hospital, Athens, Greece.
G
G
Kakourou
Department of Medical Genetics, University of Athens, St Sophia's Children's Hospital, Athens, Greece.
C
C
Vrettou
Department of Medical Genetics, University of Athens, St Sophia's Children's Hospital, Athens, Greece.
M
M
Tzetis
Department of Medical Genetics, University of Athens, St Sophia's Children's Hospital, Athens, Greece.
J
J
Traeger-Synodinos
Department of Medical Genetics, University of Athens, St Sophia's Children's Hospital, Athens, Greece.
S
S
Kitsiou-Tzeli
D000328:Adult; D001757:Blastomeres; D002460:Cell Line; D003550:Cystic Fibrosis / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D005260:Female; D005819:Genetic Markers; D005838:Genotype; D006239:Haplotypes; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000379:methods*; D012313:RNA / Q000235:genetics*
D005819:Genetic Markers; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D012313:RNA
Cystic fibrosis; High resolution melting analysis; Preimplantation genetic diagnosis; Single-cell genotyping