H Fassihi1, P J Renwick, C Black, J A McGrath. 1. Genetic Skin Disease Group, St John's Institute of Dermatology, The Guy's, King's College and St Thomas' School of Medicine, Lambeth Palace Road, London SE1 7EH, UK.
Abstract
BACKGROUND: Hallopeau-Siemens recessive dystrophic epidermolysis bullosa (HS-RDEB) is a severe inherited blistering skin disorder caused by mutations in the anchoring fibril type VII collagen gene, COL7A1. There is currently no effective treatment but DNA-based prenatal testing in families at risk of recurrence is possible, mostly involving chorionic villus sampling at 10-11 weeks' gestation. OBJECTIVES: An alternative method, for avoiding recurrence of HS-RDEB, is preimplantation genetic diagnosis (PGD). This involves DNA analysis of single blastomeres extracted from late cleavage stage embryos following in vitro fertilisation. METHODS: To establish PGD for HS-RDEB, we designed and optimised a sensitive single cell semi-duplex polymerase chain reaction (PCR) assay for two highly polymorphic dinucleotide repeat microsatellite markers, D3S1581 (telomeric) and D3S1289 (centromeric), close to the COL7A1 gene. RESULTS: We demonstrated high PCR efficiency, low allele drop out rates and no contamination in testing this assay on 50 single buccal cells of known heterozygous genotype and 13 research blastomeres from donated embryos. CONCLUSIONS: This semi-duplex PCR method provides robust, reproducible and informative amplification results for single cells. Moreover, this test has now been approved for clinical application by the UK Human Fertilisation and Embryology Authority (HFEA). As such, the development of PGD for HS-RDEB broadens the range of prenatal testing options and personal choice for couples at reproductive risk of this severe genetic skin disease.
BACKGROUND:Hallopeau-Siemens recessive dystrophic epidermolysis bullosa (HS-RDEB) is a severe inherited blistering skin disorder caused by mutations in the anchoring fibril type VII collagen gene, COL7A1. There is currently no effective treatment but DNA-based prenatal testing in families at risk of recurrence is possible, mostly involving chorionic villus sampling at 10-11 weeks' gestation. OBJECTIVES: An alternative method, for avoiding recurrence of HS-RDEB, is preimplantation genetic diagnosis (PGD). This involves DNA analysis of single blastomeres extracted from late cleavage stage embryos following in vitro fertilisation. METHODS: To establish PGD for HS-RDEB, we designed and optimised a sensitive single cell semi-duplex polymerase chain reaction (PCR) assay for two highly polymorphic dinucleotide repeat microsatellite markers, D3S1581 (telomeric) and D3S1289 (centromeric), close to the COL7A1 gene. RESULTS: We demonstrated high PCR efficiency, low allele drop out rates and no contamination in testing this assay on 50 single buccal cells of known heterozygous genotype and 13 research blastomeres from donated embryos. CONCLUSIONS: This semi-duplex PCR method provides robust, reproducible and informative amplification results for single cells. Moreover, this test has now been approved for clinical application by the UK Human Fertilisation and Embryology Authority (HFEA). As such, the development of PGD for HS-RDEB broadens the range of prenatal testing options and personal choice for couples at reproductive risk of this severe genetic skin disease.
Authors: Marta Molina Romero; Alberto Yoldi Chaure; Miguel Gañán Parra; Purificación Navas Bastida; José Luis Del Pico Sánchez; Ángel Vaquero Argüelles; Paloma de la Fuente Vaquero; Juan Pablo Ramírez López; José Antonio Castilla Alcalá Journal: J Assist Reprod Genet Date: 2022-01-29 Impact factor: 3.412