Κατεύθυνση Ιατρική Γενετική: Κλινική και Εργαστηριακή Κατεύθυνση
Library of the School of Health Sciences

2019-03-04

2019

Raftopoulou Maria

Ιωάννα Traeger Συνοδινού, Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Ελένη Φρυσίρα, Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Μαρία Τζέτη, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ

Ανάπτυξη πρωτοκόλλου για Προεμφυτευτική Γενετική Διάγνωση για σπάνιο μονογονιδιακό νόσημα

Greek

Development of PGT protocol for rare monogenic disease

Preimplantation genetic testing (PGΤ) is an alternative diagnostic method to classic prenatal diagnosis which tests embryo biopsies for inherited monogenic diseases or chromosomal abnormalities, by selecting only the unaffected embryos that derived from human assisted reproduction to be transferred to the mother’s uterus. Common PGT indications include monogenic diseases with known molecular basis. As such, achondroplasia is the most common form of dwarfism in humans, with a prevalence of 1:15.000-25.000. Achondroplasia is inherited as an autosomal dominant trait with complete penetrance and the vast majority of patients are heterozygous for the c.1138G>A point mutation in exon 10 of FGFR3 gene.
The aim of the present study is the development and optimization of a PGT protocol for achondroplasia, according to the European Society of Human Reproduction and Embryology (ESHRE) PGT consortium guidelines, for a couple that approached the Laboratory of Medical Genetics of University of Athens. The affected mother carries a «de-novo» c.1138G>A mutation of the FGFR3 gene while her husband is unaffected. A selective abortion due to prenatal diagnosis of achondroplasia is recorded on couple’s reproductive history. Sample of the amniotic fluid was available, allowing the development of a PGΤ protocol for achondroplasia.
The protocol is based on fluorescent multiplex-PCR for direct and indirect detection of the FGFR3 pathogenic mutation (c.1138G>A), with restriction enzyme analysis and linked polymorphic markers-microsatellite repeats (STRs), respectively. Using in silico tools, we were able to identify the STRs that are linked to FGFR3 gene as well as to choose a restriction enzyme that facilitated detection of the c.1138G>A mutation. Following primers design, multiplex-PCR amplification reactions were performed on genomic DNA samples of each family member, in order to identify the informative STR markers in the family, through linkage analysis. The multiplex-PCR protocol was tested and optimized on diluted genomic DNA and subsequently peripheral blood single lymphocytes of the affected mother. After evaluating the PCR efficiency (≥95%) and Allele-drop out (ADO) (≤8,3%) on 38 single lymphocytes of the candidate mother and a negative control, the PGT protocol for achondroplasia was considered reliable and suitable for clinical application at a single-cell DNA level.

Health Sciences

Achondroplasia, FGFR3, Development of PGT protocol, Linkage analysis, Fluorescent multiplex-PCR

Yes

3

Yes

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https://pergamos.lib.uoa.gr/uoa/dl/object/2864728