PRE-IMPLANTATION GENETIC DIAGNOSIS FOR β-THALASSEMIA, SICKLE CELL SYNDROMES AND CYSTIC FIBROSIS IN GREECE
Traeger-Synodinos J1,*, Vrettou C1, Tzetis M1, Palmer G2, Davis S3, Mastrominas M3, Kokali G4, Pandos K4, Kanavakis E1
*Corresponding Author: : Dr. Joanne Traeger-Synodinos, Medical Genetics, Athens University, Choremio Research Laboratory, St. Sophia’s Children’s Hospital, Thivon and Levadias Streets, Athens 11527, Greece; Tel.: +30-210-746-7461; Fax: +30-210-779-5553; E-mail: jtraeger@cc.uoa.gr
page: 25

PRE-IMPLANTATION GENETIC DIAGNOSIS FOR CYSTIC FIBROSIS

Cystic fibrosis is one of the most common genetic diseases targeted for prevention programs. It is caused by defects (>1,000 reported to date) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (250 kb), consisting of 27 exons and located on chromosome 7q31-32 [22]. Defects in the CFTR protein cause abnor­mal chloride concentration along the apical membrane of epithelial cells resulting in progressive lung disease, pan­creatic dysfunction and male infertility. Infertile men who are otherwise asymptomatic, have an increased chance of having CFTR mutations [most commonly those with con­genital bilateral absence of the vas deferens (CBAVD)] [21,23]. Thus, couples seeking assisted reproduction due to male infertility may have a markedly increased risk of initiating a CF-affected pregnancy; therefore, PGD is also indicated for such couples.

In Southern Europe, including Greece, CF is charac­terized by great allelic heterogeneity compared to the populations of Northern Europe [7,24-26]. Most of the approaches for PGD of CF that have been described to date either have not addressed the problem of diagnosing a wide spectrum of mutation combinations [27-31), or require the design of case-specific protocols each time [32]. Protocols based on analysis of CFTR gene linked polymorphic markers have been described that are quite widely applicable for some Northern European populations (33,34), although their usefulness has not been assessed elsewhere.

To apply PGD to CF we developed a flexible multi­plex PCR protocol, allowing analysis of sequence varia­tions in any combination amongst seven CFTR gene exons which include the majority of Greek CF mutations (exons 4, 10, 11, 13 in two parts, 14b, 17b and 21,) by nested-PCR and DGGE analysis, along with a fluorescently la­beled intragenic micro-satellite (IVS8CA) (Fig. 2). Pre-clinical experiments were carried out on 390 single lym­phocytes from three CF patients, one heterozygote and one non CF individual, all with known CFTR genotypes. Exons were amplified with a PCR efficiency ranging from 90-100%, with ADO from 0-3.8%. The micro-satellite IVS8CA was co-amplified with an overall PCR efficiency of 92.4 and 10.8% ADO. The method overcomes the need of separate assays for each CFTR gene mutation. Addi­tionally, it facilitates analysis of any informative linked polymorphic sequence variation (within the seven exons), along with micro-satellite useful for minimizing misdiag­nosis and for indirect diagnosis. The protocol is robust and flexible for diagnosing diverse CF genotype combinations in single cells, although, to date, we have not completed any clinical PGD cycles for CF [35].

 

Figure 2. The PGD strategy for analysis of CFTR gene mutations. 1) Multiplex first PCR for appropriate CFTR gene exons (according to parental mutations) and IVS8CA micro-satellite; 2) nested PCR for DGGE analy­sis of appropriate exons; 3a) DGGE analysis, and 3b) IVS8CA micro-satellite analysis.

 




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