Preimplantation Genetic Diagnosis Protocol Research Articles

Preimplantation genetic diagnosis of β-thalassemia using real-time polymerase chain reaction with fluorescence resonance energy transfer hybridization probes

Preimplantation genetic diagnosis (PGD) is employed increasingly to allow transfer of embryos to the uterus in assisted reproduction procedures. There are three stages of biopsy: polar bodies, one or two blastomeres from the cleavage-stage embryos, and trophectoderm cells (∼5 cells) from the blastocyst-stage embryos. Validation of polymerase chain reaction (PCR)-based assays are challenging because only limited genetic material can be obtained for PGD. In the current study, we modified a valid single-cell PCR protocol for PGD using real-time PCR assay with fluorescence resonance energy transfer (FRET) hybridization probes followed by melting curve analysis. We optimized and clinically applied the protocol, permitting molecular genetic analysis to amplify a specific region on the beta-globin ( HBB) gene for a couple, carriers of two mutations: c.-78A>G and c.52A>T. Among a total of eight embryos obtained after ovarian stimulation, a single blastomere per embryo at the six- to eight-cell stage was biopsied. This PGD method showed that four embryos were unaffected, two embryos were selected for transfer, and one pregnancy was achieved. Finally, a healthy male baby was delivered at 38 weeks’ gestation. The results obtained using the new method, FRET hybridization probes, were compared with findings using an existing method, primer extension minisequencing.

Preimplantation genetic diagnosis (PGD) for nonsyndromic deafness by polar body and blastomere biopsy

Development of an efficient and reliable PGD protocol for nonsyndromic deafness, by polar body (PB) and blastomere PGD. The GJB2/GJB6 mutations along with 12 polymorphic markers were used in PGD analysis of blastomeres or polar bodies in 14 couples for 35 cycles. Marker informativity, diagnosis rates, Allele Drop Out (ADO) rates and PB1 heterozygosity rates were assessed. Six cycles were performed by PB biopsy, 27 by blastomere and two combined cycles, resulting in delivery of three unaffected children and five ongoing pregnancies. Diagnosis rates for PB and blastomeres were similar. Only 17% PB1s were heterozygote. ADO rates of 19% were observed in both groups. We have developed a single cell multiplex PGD protocol for nonsyndromic deafness with a high efficiency of diagnosis. Most PB1 are homozygous, and similar ADO rates were observed; therefore, blastomere biopsy appears to be the method of choice for this autosomal recessive disease.

Birth of a healthy boy after a double factor PGD in a couple carrying a genetic disease and at risk for aneuploidy: Case Report

Preimplantation genetic diagnosis (PGD) for monogenic diseases is widely applied, allowing the transfer to the uterus of healthy embryos. PGD is also employed for the detection of chromosome abnormalities for couples at high risk of producing aneuploid embryos, such as advanced maternal (>35 years). A significant number of patients requesting PGD for monogenic diseases are also indicated for chromosome testing. We optimized and clinically applied a PGD protocol permitting both cytogenetic and molecular genetic analysis. A couple, carriers of two cystic fibrosis (CF) mutations (c.3849 + 10 KbC > T and c.3408C > A) with a maternal age of 38 years and two previously failed IVF-PGD cycles, was enrolled in the study. After ovarian stimulation, six oocytes were obtained. To detect abnormalities for all 23 chromosomes of the oocyte, the first polar body (1PB) was biopsied from five of the oocytes and analyzed using comparative genomic hybridization (CGH). CGH analysis showed that 1PB 1 and 1PB 4 were aneuploid (22X,-9,-13,+19 and 22X,-6, respectively), while 1PB 2, 1PB 3 and 1PB 6 were euploid. Blastomere biopsy was only applicable on embryos formed from Oocyte 3 and Oocyte 6. After whole-genome amplification with multiple displacement amplification, a multiplex PCR, amplifying informative short tandem repeats (D7S1799; D7S1817) and DNA fragments encompassing the mutation sites, was performed. MiniSequencing was applied to directly detect each mutation. Genetic diagnosis showed that Embryo 6 was affected by CF and Embryo 3 carried only the c.3849 + 10 KbC > T mutation. Embryo 3 was transferred achieving pregnancy and a healthy boy was born. This strategy may lead to increased pregnancy rates by allowing preferential transfer of euploid embryos.

Preimplantation genetic diagnosis for BRCA1 exon 13 duplication mutation using linked polymorphic markers resulting in a live birth

The risk of breast cancer associated with inheriting a BRCA1 mutation is extremely high, in addition, there is a 50% chance of transmitting this familial cancer mutation to any offspring. A 31-year-old woman with a strong maternal family history of early onset of breast cancer had experienced 3 years of primary infertility. Presymptomatic testing confirmed the woman had inherited a 6 kb duplication of exon 13 (ins6KbEx13) in BRCA1 from her mother. Neither gamete donation or adoption were acceptable options for this infertile couple, and as termination of pregnancy after prenatal diagnosis following in vitro fertilization (IVF) was not ethically acceptable, preimplantation genetic diagnosis (PGD) was sought. A single-cell PCR protocol for PGD for the breast and ovarian cancer predisposing BRCA1 exon 13 duplication mutation was developed which involved amplification of three specific gene regions, including the BRCA1 mutation (ins6KbEx13), an intragenic marker (D17S855) and a flanking marker (D17S1185). In the first cycle of IVF, three embryos were analyzed and two were determined to be at low risk of having inherited the maternal BRCA1 mutation. Following the transfer of both embryos on day 5, a singleton pregnancy resulted. Declining confirmatory prenatal diagnosis, a male baby was subsequently delivered at term. Successful PGD for BRCA1 resulted in the delivery of a live-born male. PGD using linked polymorphic markers provides an alternate option for reproduction for couples with or at risk of having inherited a BRCA1 mutation.

Birth after pre‐implantation genetic diagnosis (PGD) of spinocerebellar ataxia 2 (Sca2)

Spinocerebellar ataxia 2 (SCA2) is an autosomal-dominant neurodegenerative disease caused by an extended polyglutamine sequence in the ATXN2 protein. We describe the development of a new single-cell multiplex PCR protocol for pre-implantation genetic diagnosis (PGD) of SCA2 and its successful clinical application. Three duplex tests have been developed, one, which combines the detection of the CAG repeats in addition to the D12S821 microsatellite, another, the amplification of the CAG repeats and the D12S1333 microsatellite and the last, the combination of both microsatellites D12S821 and D12S1333. PCR conditions were established using 226 single lymphoblasts or patient lymphocysts. Amplification was obtained in an average of 99.6%, a complete genotype in 86%, a conclusive result in 96% and an allelic drop-out (ADO) rate of 10.7% was observed. PGD for SCA2 was performed for a couple with a paternal risk of transmitting the pathology. Two cycles were done from which 18 embryos were biopsied, 8 were diagnosed as unaffected, 9 as affected and 1 gave no results. In both cycles 2 embryos were transferred, with no pregnancy at the first attempt, and a twin pregnancy at the second attempt. The patient delivered one girl and one boy at 36 weeks and 3 days.

Efficient strategies for preimplantation genetic diagnosis of spinal muscular atrophy

To develop and apply efficient multiplex preimplantation genetic diagnosis (PGD) protocols for spinal muscular atrophy (SMA). Two multiplex PGD protocols were developed allowing the detection of the common homozygous deletion of the telomeric spinal muscular atrophy gene (SMN1), together with two microsatellites located on each side of SMN1. The molecular genetics laboratory of the university hospital in Montpellier. A couple who had already given birth to a child affected with SMA. In vitro fertilization using intracytoplasmic sperm injection (ICSI) and blastomere biopsy. Improvement of PGD for SMA. Two different multiplex protocols were set up on 81 (multiplex A) and 64 single cells (multiplex B) from normal controls, affected patients, and individuals with homozygous SMN2 deletion. In one PGD cycle that used one of these protocols, two embryos were transferred, which resulted in the birth of a healthy baby. Analysis of microsatellite markers in addition to the SMN1 deletion allows the detection of contamination, the study of ploidy of the biopsied blastomeres, and the performance of an indirect genetic diagnosis, thereby increasing the reliability of the results. This PGD assay may be applied to all families with the common deletion of SMN1 and also to couples in whom one of the partners carries a small intragenic mutation in SMN1, identified in about 6% of affected individuals who do not lack both copies of SMN1.

Preimplantation genetic diagnosis of X-linked retinoschisis using multiple displacement amplification (MDA)

Preimplantation genetic diagnosis (PGD) represents an alternative to prenatal diagnosis and allows selection of unaffected IVF embryos to establish pregnancies in couples at risk of transmitting a genetic disorder. X-linked juvenile retinoschisis (RS) is a recessively inherited vitreo-retinal degeneration characterized by macular pathology and intraretinal splitting of the retina. The underlying defect in X-linked juvenile retinoschisis is due to a defective XLRS1 gene on chromosome X. We investigate the use of MDA in the RS–PGD program. We investigate the use of MDA from single cells as a universal first step for PGD of single defects using MDA to amplify single blastomere DNA, which was then used in the RS–PGD program. Two couples which the wife carries a causative mutation 599 G > A in the exon 6 of the XLRS1 gene that causes X-linked RS. The affected wife from family 1 and 2 were heterozygous at TAAA2 and CAG locus. To identify the sex chromosomes X22 informativity test were performed. MDA was used to amplify the whole-genome directly from a single cell. The MDA product was used for polymerase chain reaction (PCR) analysis of informative markers, used in the RS–PGD program. Segregation studies of the both families were performed and showed that the markers were informative. Three cycles were carried out for the two couples. The development of a RS–PGD protocol allowed for the diagnosis of 12 embryos none of which remained without diagnosis. Five embryos were shown to be affected males and 2 were carried female, while 3 were unaffected males and 2 non-carrier females of which 5 were transferred. One singleton ongoing pregnancy ensued. 12 cells were analysed, none of which showed no amplification. Only one cell showed ADO. We describe the first MDA–PGD for X-linked RS. The MDA technique is useful for overcoming the problem of insufficient genomic DNA in PGD, since enough DNA from a single cell to allow for multiple PCR analyses. At the same time, high amplification efficiency and low ADO rates are seen compared with nested multiplex PCR. The use of MDA as an initial step in PGD allows for DNA amplification in quantities sufficient for the diagnosis of a wide spectrum of single-gene defects using standard methods.

Pregnancy following preimplantation genetic diagnosis of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)

Presentation of a novel case, involving the design and implementation of preimplantation genetic diagnosis (PGD) for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). The disease-causing mutation, c.459C>T (R153C) in exon 4 of the Notch3 gene, was previously identified in the affected father. The family already had a pregnancy termination following prenatal diagnosis and chose to undergo PGD. A PGD protocol was designed to include informative, linked short tandem repeat (STR) markers and an intragenic single nucleotide polymorphism (SNP), coupled to mutation identification. Biopsy was performed at day 3 and blastocysts were transferred on day 5 after fertilization. Standard prenatal diagnosis procedures were employed to confirm the PGD results. One blastomere was removed at day 3 from each of eight embryos. Detection of the c.459C>TNotch3 mutation, coupled to informative polymorphic markers, unambiguously identified three unaffected embryos. Blastocyst transfer resulted in a singleton pregnancy and subsequent prenatal diagnosis confirmed that the fetus was disease-free. Given the dominant, highly penetrant and potentially serious effects of Notch3 mutations, PGD for CADASIL may be considered and implemented as a reproductive option, following proper genetic counseling.

Preimplantation genetic diagnosis of Von Hippel-Lindau disease cancer syndrome by combined mutation and segregation analysis

Von Hippel-Lindau (VHL) disease is an autosomal dominant cancer syndrome, associated with the development of tumors and cysts in multiple organ systems, whose expression and age of onset are highly variable. The VHL disease tumor suppressor gene (VHL) maps to 3p25-p26 and mutations ranging from a single base change to large deletions have been detected in patients with VHL disease. We developed a single cell PCR protocol for preimplantation genetic diagnosis (PGD) of VHL disease to select unaffected embryos on the basis of the detection of the specific mutation and segregation analysis of polymorphic linked markers. Multiplex-nested PCR using single buccal cells of an affected individual were performed in order to test the accuracy and reliability of this single-cell protocol. For each locus tested, amplification efficiency was 83% to 87% and allelic drop-out rates ranged from 12% to 8%. Three VHL disease PGD cycles were performed on cells from a couple with paternal transmission of a 436delC mutation in exon 2 of the VHL gene, leading to the identification of three unaffected embryos. Independent of the mutation present, this general PGD protocol for the diagnosis of VHL disease can be used in families informative for either the D3S1038 or D3S1317 microsatellite markers.

Preimplantation genetic diagnosis for Marfan syndrome

To develop and apply efficient and reliable protocols for preimplantation genetic diagnosis (PGD) for Marfan syndrome. Two mutation-specific protocols were developed, and the markers D15S1028, D15S992, D15S196, D15S576, D15S123, and D15S143 were used to set up four multiplex polymerase chain reactions (PCRs). Research Center Reproduction and Genetics. Ten couples carrying mutations in the FBN1 gene. Six PGD protocols were developed for 10 couples, and 7 of them underwent a total of 16 clinical cycles. Amplification, allele drop-out (ADO), and contamination rates during the preclinical assays. DNA analyses of blastomeres from embryos biopsied during PGD cycles. Six different protocols were set up, with the main objective being to to use one protocol for several couples. A total of 16 PGD cycles were performed, which resulted in the delivery of an unaffected boy and three ongoing pregnancies. The development of single-cell multiplex PCRs for linked markers and its use in PGD reduce the workload of the genetic diagnostic laboratory as well as the average waiting time for patients. This approach also allows for the simultaneous and accurate detection of recombination, contamination, and ADO, thereby increasing the reliability of the diagnosis.

Successful preimplantation genetic diagnosis for alpha- and beta-thalassemia in China

In southern China, the average carrier rates of alpha-thalassemia and beta-thalassemia in the population are as high as 10.3% and 2.8%, respectively. Because of the high rates, they are known as 'social diseases' in some regions. In this study, the fluorescent gap PCR, which can detect the alpha-thalassemia Southeast Asia deletion (SEA deletion), was applied in four clinical applications of preimplantation genetic diagnosis (PGD) on four couples, among whom both partners were alpha-thalassemia carriers. Two patients became pregnant and two healthy babies were born, which confirmed the PGD results. The single cell multiplex nested PCR followed by reverse dot blot (RDB), which can simultaneously detect the 16 beta-thalassemia mutations in the Chinese population, was applied in four clinical PGD cycles on four couples among whom both partners were beta-thalassemia carriers. One pregnancy was achieved and it resulted in a live healthy birth, which confirmed the results of PGD. The amplification efficiencies of the two protocols described above were 89.5% and 93.9%, respectively. The allele drop-out (ADO) rates of these two protocols were 5.9% and 10.9%, respectively. These studies represent the successful applications of PGD protocols that can detect more than 95% of alpha- and beta -thalassemia mutations in the Chinese population.

Improved single-cell protocol for preimplantation genetic diagnosis of spinal muscular atrophy

To develop and validate a simple and reliable single-cell analysis protocol for the preimplantation genetic diagnosis (PGD) of spinal muscular atrophy (SMA). Molecular tests based on specific enzymatic digestion have already been described for SMA diagnosis. We modified the amplified DNA fragments so as to introduce a novel restriction site that provides an internal control for the completeness of the digestion. The genetics and reproduction departments of two teaching hospitals. Six informed couples at risk of transmitting SMA. All patients underwent standard procedures associated with intracytoplasmic sperm injection. Improvement of SMA diagnostic efficiency and accuracy on single cell. One hundred fifty lymphocytes were analyzed with our protocol. One hundred percent diagnostic accuracy was achieved from both homozygous normal and SMN1-deleted leukocytes. Successful molecular analysis was achieved for 36 of 42 biopsied embryos (86%). Twenty-five normal embryos were transferred, but no pregnancy was achieved. We developed an improved protocol for PGD of SMA that is simple, robust, and accurate; unfortunately, no pregnancies were achieved for any of the six patients who have undergone PGD in the program thus far.

Clinical application of multiple displacement amplification in preimplantation genetic diagnosis

Multiple displacement amplification (MDA) is a technique used in the amplification of very small amounts of DNA. MDA is reported to yield large quantities of high-quality DNA. The applicability of MDA to single cells was recently demonstrated as a potential technique for preimplantation genetic diagnosis (PGD). This paper shows the first clinical application of MDA in PGD. Two cycles of PGD were performed in two diseases, resulting in two pregnancies. All the diagnoses given on blastomeres were confirmed on the non-transferred whole embryos. The blastomere diagnosis was coupled with short tandem repeat (STR) analysis (16 loci) in all cycles. Allelic drop-out (ADO) assessment and amplification efficiency were evaluated on 40 single lymphocytes derived from parents of each disease. ADO and amplification failure were 10.3 and 2.2% for β-thalassaemia and 17.9 and 2.2% for cystic fibrosis respectively. HLA matching for A, B and DR was performed successfully on single cell for the β-thalassaemia family using similar methods to genomic DNA. The PGD protocol used in all diseases consists of MDA amplification, followed by a standard polymerase chain reaction protocol. Although HLA matching was not applied to embryos, its feasibility was shown on single cell DNA amplified by MDA. Altogether, these data show the simplicity and reliability of performing PGD in combination with HLA matching and STR analysis using MDA.

Preimplantation genetic diagnosis for β‐thalassaemia: the Sardinian experience

AbstractObjectivesTo report the experiences on preimplantation genetic diagnosis (PGD) in couples at risk for ß‐thalassaemia in Sardinia.Methods23 couples at risk for ß‐thalassaemia were included in the PGD programme with a total of 42 cycles performed. Among these, 11 couples were fertile, while the remaining 12 had associated fertility problems. In vitro Fertilization (IVF), PGD and prenatal genetic molecular confirmation protocols and results are reported.ResultsAll the patients followed the protocol of ovarian stimulation, oocyte retrieval, intracytoplasmic sperm injection (ICSI), embryo biopsy and genetic analysis. A total of 272 oocytes were fertilized in the regular way, and embryo biopsy was performed on 202 embryos. Out of these 202 embryos, 192 (95%) were successful. The genetic diagnosis was performed on 150 embryos (78.1%). Ninety‐eight were identified as unaffected and 75 were transferred in 31 cycles. In the infertile patient group, two biochemical pregnancies (11.1% per transfer), in the fertile patient group, four clinical pregnancies, two twin and two singleton pregnancies (30.8% per transfer), were obtained. The genetic molecular results were confirmed in all pregnancies by first‐trimester chorionic villus sampling (CVS).ConclusionOur study shows that PGD for ß‐thalassaemia is an available procedure for couples who wish to avoid termination of pregnancy, except in cases where the IVF cycle efficiency is very poor. Copyright © 2004 John Wiley & Sons, Ltd.

Cryo-damaged blastomeres may be used efficiently for preimplantation genetic diagnosis (PGD) for the purpose of aneuploidy screening by FISH

Application of preimplantation genetic diagnosis (PGD) is rapidly growing and provides important advantages compared to the alternative method of the prenatal diagnosis. Most of the cases performed in the world have been for the indication of aneuploidy both for diagnostic and for treatment purposes. Today the standard method of PGD requires the removal of a cell from a day 3, 6–8 cell cleavage stage IVF embryo, followed by fixation of the cell and FISH analysis. Although there are some minor variations in the different PGD protocols, all of them require the use/biopsy of an intact and living cell.

Cryo-damaged single blastomeres may be used efficiently for preimplantation genetic diagnosis (PGD) for the purpose of screening for single gene defect

Application of preimplantation genetic diagnosis (PGD) is rapidly growing and provides important advantages compared to the alternative method of the prenatal diagnosis. The cases performed in the world have been for the indication either of aneuploidy or for preventing the inheritance of commonly occurring single gene defects (such as cystic fibrosis). Today the standard method of PGD requires the removal of a cell from a day 3, 6–8 cell cleavage stage IVF embryo, followed by transferring the cell into a PCR tube, performing the PCR reaction and analyzing the results. Although there are some minor variations in the different PGD protocols, all of them require the use/biopsy of an intact and living cell. The objective of the present study was to investigate if it was possible to apply the PGD protocol in a way that it can be used for PCR analyses using lysed embryonic cells instead of intact cells. Experimental research protocol. A total of 6 embryos donated to research were involved in this study. A standard slow freezing and rapid thaw protocol was applied. Both intact and degenerated cells were biopsied from thawed, disaggregated and transferred to a PCR tube. A total of 24 blastomeres were removed from the embryos; and they were separated according to intactness. PCR tubes were marked correspondingly if they contained a single living or a single degenerated/lysed blastomere. Both live and degenerated cells were submitted to fluorescent polymerase chain reaction (PCR) and subsequent restriction enzyme digestion and fragment analysis on an automated sequencer. The test was designed to analyze the region corresponding to the most frequently occurring mutation represented by deletion of 3 basepairs, at the amino acid position 508 of the putative product of the CF gene. A total of 24 blastomeres were separated from the frozen-thawed embryos. Twelve of them were completely intact and therefore considered to be alive while the other 12 blastomeres were displaying all the signs of cryodamage therefore were considered as degenerated. All the 12 intact blastomeres provided clear signals (100%) on the gene region that was amplified corresponding to a non-mutated sequence, as expected. Equally, all the 12 degenerated blastomeres provided clear signals (100%) to the same region corresponding to a non-mutated sequence. The results of the present study demonstrate that it is possible to use degenerated/lysed blastomeres for the purpose of preimplantation genetic diagnosis to detect alterations in the gene sequences. It has also been shown that amplification of genetic material obtained from a single cell is similarly efficient when performed on degenerated cells compared to live/intact cells using a non-altered protocol. As the results demonstrated, fragmentation of nuclear DNA in the degenerated cells may not present a high risk (that would interfere with amplification efficiency) depending perhaps on the timing of blastomere separation relative to the thaw procedure. This new approach is especially important for cryo-ET cycles, because it does not require the removal of live blastomere(s) from the embryo which in return would assist to preserve a higher viability and developmental potential of the embryo. Extension of the present study to test different gene sequences is warranted in order to demonstrate that this new approach is well applicable for screening various types of single gene defects.

Detailed investigation of factors influencing amplification efficiency and allele drop-out in single cell PCR: implications for preimplantation genetic diagnosis.

Preimplantation genetic diagnosis (PGD) of single gene disorders relies on PCR-based tests performed on single cells (polar bodies or blastomeres). Despite the use of increasingly robust protocols, allele drop-out (ADO; the failure to amplify one of the two alleles in a heterozygous cell) remains a significant problem for diagnosis using single cell PCR. In extreme cases ADO can affect >40% of amplifications and has already caused several PGD misdiagnoses. We suggest that an improved understanding of the origins of ADO will allow development of more reliable PCR assays. In this study we carefully varied reaction conditions in >3000 single cell amplifications, allowing factors influencing ADO rates to be identified. ADO was found to be affected by amplicon size, amount of DNA degradation, freezing and thawing, the PCR programme, and the number of cells simultaneously amplified. Factors found to have little or no affect on ADO were local DNA sequence, denaturing temperature (94 or 96 degrees C) and cell type. Consideration of the causal factors identified during this study should permit the design of PGD protocols that experience little ADO, thus improving the accuracy of PGD for single gene disorders.

First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy

Objective: To develop a preimplantation genetic diagnosis (PGD) protocol that allows any form of chromosome imbalance to be detected. Design: Case report employing a method based on whole-genome amplification and comparative genomic hybridization (CGH). Setting: Clinical IVF laboratory. Patient(s): A 40-year-old IVF patient. Intervention(s): Polar body and blastomere biopsy. Main Outcome Measure(s): Detection of aneuploidy. Result(s): Chromosome imbalance was detected in 9 of 10 polar bodies. A variety of chromosomes were aneuploid, but chromosomal size was found to be an important predisposing factor. In three cases, the resulting embryos could be tested using fluorescence in situ hybridization, and in each case the CGH diagnosis was confirmed. A single embryo could be recommended for transfer on the basis of the CGH data, but no pregnancy ensued. Conclusion(s): Evidence suggests that preferential transfer of chromosomally normal embryos can improve IVF outcomes. However, current PGD protocols do not allow analysis of every chromosome, and therefore a proportion of abnormal embryos remains undetected. We describe a method that allows every chromosome to be assessed in polar bodies and oocytes. The technique was accurate and allowed identification of aneuploid embryos that would have been diagnosed as normal by standard PGD techniques. As well as comprehensive cytogenetic analysis, this protocol permits simultaneous testing for multiple single-gene disorders.

Preimplantation genetic diagnostic protocols for alpha- and beta-thalassaemias using multiplex fluorescent PCR.

Haemoglobinopathies including alpha- and beta-thalassaemia are the world's most common class of single gene disorder. Prenatal diagnosis (PND) for beta-thalassaemia has been proven to be an effective strategy for controlling the incidence of new cases and is widely used in several countries where the disease is common. Successful preimplantation genetic diagnosis (PGD) protocols for beta-thalassaemia have been introduced using restriction fragment length polymorphism (RFLP), single-stranded conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). However, contamination and allele dropout (ADO) remain an important concern for all of these strategies. In the present study two PGD protocols for detecting beta-thalassaemia mutations (codon 41-42 and IVSI-110) and one for alpha-thalassaemia (SEA mutation) have been designed and tested. These methods contain failsafe mechanisms to reduce the risk of misdiagnosis due to ADO or contamination and utilise multiplex fluorescent PCR (F-PCR). Interestingly, amplification efficiency and ADO were significantly affected by the choice of DNA polymerase and the freshness of the single cells used. The close similarity between the DNA sequences of beta-globin and delta-globin was also found to be an important issue that necessitated careful design of primers for the beta-globin gene.

Preimplantation genetic diagnosis for sickle-cell anemia and for ?-thalassemia

We developed single-cell polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples carrying mutations in the beta-globin gene. With PGD the genetic status of an embryo obtained after intracytoplasmic sperm injection (ICSI) is determined by PCR analysis in single blastomeres, allowing only healthy embryos to be transferred to the uterus. We carried out nine PGD cycles using fluorescent PCR for two couples in whom the partners carried sickle-cell trait. Both couples achieved pregnancies, one of which was spontaneously aborted. We have developed two beta-thalassemia PGD protocols: one for the analysis of the 25-26delAA and the IVS2+1G>A mutation, and the other for the simultaneous detection of the IVS1+6T>C and the IVS1+110G>A mutations. For the second protocol, both non-labelled PCR and later fluorescent PCR were used. Both protocols were applied in clinical cycles (two non-labelled PCR cycles and one fluorescent PCR cycle) for two couples. The patient with the fluorescent PCR-PGD cycle became pregnant. Overall, the three fluorescent PCR assays were accurate and reliable with amplification efficiencies of minimum 93% and allele dropout (ADO) rates between 0 and 12%.