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 β-THALASSEMIA AND SICK¬LE CELL SYNDROMES

The thalassemia syndromes and related hemoglobin­opathies are the commonest group of monogenic auto­somal recessive disorders world-wide [10]. They are caused by mutations in the β-globin gene located on chro­mosome 11. More than 170 point mutations or small inser­tions/deletions have been described which either reduce or abolish the synthesis of the β-globin chains of adult hemo­globin (Hb A) by the affected gene (http://globin.cse.psu. edu/globin).
When confronting a common and molecularly hetero­geneous monogenic disease such as the β hemoglobin­opathies, it is more practical to have a single PGD diag­nostic strategy applicable for a wide spectrum of potential affected genotypes, rather than designing and standardiz­ing case-specific protocols each time. Several methods applicable to PGD of hemoglobinopathies have been de­scribed for the detection of some of the numerous β gene mutations [11-14], but only a few have been described for a potentially wider application [15,16]. The initial proto­col we developed was based on nested PCR and denatur­ing gradient gel electrophoresis (DGGE), and exploited the observation that the majority of the common β-thal mutations in most populations worldwide tend to be clus­tered within the first 700 nucleotides of the β-globin gene [17,18]. Denaturing gradient gel electrophoresis is advan­tageous for application to PGD, since as a scanning method, it can identify any mutation within a single am­plified region, precluding the need for independent muta­tion assays, and additionally, it facilitates simultaneous analysis of more than one mutation within a single PCR fragment, invaluable when confronting compound geno­types. In addition, DGGE is an assay for the presence of normal as well as pathological alleles, and in practice, only blastomeres with definitive evidence of a normal allele on DGGE analysis are considered unaffected, pre­venting transfer of affected embryos, even if ADO has occurred.
The diagnostic strategy involved a first PCR to am­plify a several hundred base pair (bp) region, followed by nested PCR to generate amplicons suitable for DGGE analysis (Fig. 1). The pre-clinical experiments were car­ried out on 490 single cells (blastomeres from supernumer­ary human embryos, lymphocytes and amniocytes), through which the genotyping method was optimized to achieve a PCR efficiency of 85-90%, with less than 8% allele drop-out [17,18].

Between 1998 and 2002, 59 couples at risk for trans­mitting β hemoglobinopathies were counseled about PGD. Forty-one couples initiated 63 PGD cycles (one cycle in 22 couples, two cycles in 16 couples, and three cycles in three couples), of which 20 cycles with <4 IVF embryos were canceled before biopsy and genotype analysis. Amongst 43 completed cycles, 302 cleavage stage em­bryos were biopsied, 236 (78%) gave a genotype result, of which 125 clearly had at least one normal allele with DGGE analysis (unaffected). Transfer of 100 embryos (1-4/cycle) established 16 pregnancies (37% for each com­pleted cycle), including 13 singletons, two sets of twins and one set of triplets (20% implantation per embryo transferred). Six pregnancies were lost within the first trimester but 10 underwent second trimester PND. Nine pregnancies (13 babies) were confirmed unaffected, but one singleton was a PGD misdiagnosis, and was selec­tively terminated. The misdiagnosis was attributed to ex­traneous contamination or a tube switch, and not to inac­curacy of the genotyping method. The triplet pregnancy was selectively reduced to twins, and all pregnancies went to term, resulting in the birth of 12 healthy babies [19].
Although accurate, the DGGE-based protocol is tech­nically demanding, time consuming, and not as sensitive as methods involving fluorescent PCR. To simplify PGD analysis, reduce diagnosis time, improve sensitivity, whilst maintaining accuracy and monitoring of ADO, we recently established a protocol based on real-time PCR, using fluo­rescent hybridization probes for mutation detection. We designed, standardized and validated mutation detection probes for the common β-thal mutations worldwide (and Hb S), through mutation analysis in >200 carriers, and additionally, 25 prenatal diagnoses. We adapted the method to PGD using nested PCR, with the second reac­tion in LightcyclerÔ capillaries (Roche Diagnostics GmbH, Mannheim, Germany), including fluorescent de­tection probes for melting curve analysis and allele assign­ment. So far, we have applied the LightcyclerÔ (Roche Diagnostics) protocol in 10 PGD cycles. Results (avail­able within 5 hours) were obtained in 81/89 blastomeres (91%), of which 69 blastomeres were also analyzed with the DGGE-PGD protocol, and genotypes were completely concordant. Thus, PGD using mutation detection with real-time PCR is accurate, but additionally, it is more sensitive and rapid, when compared to the DGGE-based method [20,21].

Figure 1. The PGD strategy for b-globin gene muta­tions. 1) First round PCR (region A or B); 2) nested PCR for DGGE fragment 1 and/or fragment 2 and/or fragment 3; 3) run and analysis of the DGGE gel. The DGGE gel stained with ethidium bromide. Lane 1: normal; lane 2: control sample, compound heterozygote for mutations IVS-I-110 (G®A) and IVS-I-6 (T®C); lanes 3-7: single blastomeres from five embryos; lane 8: parent, hetero­zygote for IVS-I-110; lane 9: parent, heterozygote for IVS-I-6.

 




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