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7.004 Preimplantation genetic diagnosis for non-syndromic deafness by polar body and blastomere biopsy
Abstracts - PGDIS: 8th International Symposium on PGD
on our experience for consecutive PGD cycles for single gene disorders from February 2003 through to December 2007, describing clinical outcome of 36 cycles in 20 couples for 12 different disorders: Duchenne muscular dystrophy (DMD), epidermolysis bullosa (EB), ornithine transcarbamylase (OTC) deficiency, osteogenesis imperfecta (OI), pyruvate dehydrogenase (PDH) deficiency, spinal muscular atrophy (SMA), Huntington’s disease (HD), spinocerebellar ataxia 3 (SCA3), long-chain 3-hydroacyl CoA dehydrogenase (LCHAD) deficiency, fragile X syndrome (FXS), phenylketonuria (PKU) and Charcot–Marie–Tooth (CMT) disease. In 36 PGD cycles for single gene disorders, a total of 363 cleavage stage embryos were biopsied. Successful diagnosis rate of biopsied blastomeres was 93.9% (340/363) and 190 embryos (55.9%) were diagnosed as unaffected. Among them, 95 embryos were transferred in 35 cycles except one cycle for PKU, resulting in 12 clinical pregnancies (34.3% per embryo transfer). There was no clinical abortion in the cases of pregnancies. All 12 clinical pregnancies were confirmed to be unaffected, and 13 healthy babies were delivered without any complications in five of DMD and each of EB, OTC deficiency, OI, SCA3 and SMA, respectively. Two pregnancies in each case of PKU and OI are still ongoing and the PGD results were confirmed by amniocentesis. There was no misdiagnosis in all pregnancies after PGD for single gene disorders. Our data show that PGD is a realistic option for couples at risk to avoid the birth of affected children with single gene disorders and to have a healthy unaffected baby of their own. 7.004 Preimplantation genetic diagnosis for nonsyndromic deafness by polar body and blastomere biopsy Altarescu G, Eldar-Geva T, Brooks B, Haran E, Margalioth EJ, Levy-Lahad E, Renbaum P Medical Genetics Unit and IVF Clinic, Zohar PGD Lab, Shaare Zedek Medical Center, Jerusalem, Israel Objective: (i) To develop an efficient and reliable universal protocol for PGD of non-syndromic deafness; and (ii) assess heterozygosity rates of polar body 1 (PB1), marker informativity and allele drop out (ADO) rates for polar body and blastomere preimplantation genetic diagnosis (PGD). Materials/Methods: Polar bodies 1 and 2 (PB1 and PB2) and blastomeres were biopsied by mechanical drilling.Three mutations, GJB2 35delG, 167delT and GJB6 delD13S1830 and the markers D13S141, D13S175, D13S633, D13S1275, D13S250, D13S232, GJB2-AT1, GJB2-AT2, GJB2-TG1, GJB2TG2, GJB2-AC1 were used to set up a universal multiplex PCR protocol to be used for PGD in non-syndromic deafness. Results were only assigned for samples with at least three informative flanking markers (including the familial mutation). Results: Eight couples underwent 19 PGD cycles resulting in a pregnancy rate of 21% and delivery of three unaffected children. Six cycles (32%) were performed by polar body biopsy, six by blastomere biopsy, six by frozen blastomere biopsy and in one cycle by both PB and blastomere biopsies. In 17 cycles (90%), at least two embryos were transferred and in one cycle one embryo was transferred. The rate of successful diagnosis for blastomere PGD was 91% and for PB PGD, 87%. Only 11 of 68 PB1 (17%) were heterozygotic. Similar ADO rates (19%) were observed for both heterozygotic PB1 and blastomeres. While all families had at least two informative markers on each side of the gene for PB PGD, none had four fully informative markers for blastomere PGD. Conclusion: We have developed a universal single-cell
S-46 Reproductive BioMedicine Online, Vol. 16, Suppl. 3, April 2008
multiplex protocol for non-syndromic deafness with a high efficiency of diagnosis for PGD. Although PB PGD allows more informative marker assessment, most PB1 are homozygous and ADO rates were seen to be similar. Therefore, blastomere biopsy appears to be the method of choice for this autosomal recessive disease. 7.005 A de-novo factor VIII (F8) mutation creates a challenge for PGD Laurie AD1, Hill A2, Smith MP 1, George PM2 1Canterbury Health Laboratories, Christchurch, New Zealand; 2The Fertility Centre, Christchurch. New Zealand Gonadal mosaicism presents a challenge in preimplantation genetic diagnosis (PGD) because detection of linked markers does not imply presence of a disease mutation. In a young boy with haemophilia A, we detected the splicing variant c.6901– 2A>G in the factor VIII gene (F8), but we did not detect this mutation in his mother. This suggests two possibilities: either this mutation arose de novo early in embryonic development of the boy, or that his mother is a gonadal mosaic for F8 c.6901–2A>G. Since our panel of F8-linked microsatellite markers would not be fully informative for PGD for this woman, we developed a mini-sequencing assay to genotype F8 c.6901–2, which we used in addition to the linked markers. We performed cleavage-stage PGD on two embryos from a single IVF cycle, one of which had the F8 microsatellite haplotype associated with the mutation but did not have the c.6901–2A>G variant. No pregnancy resulted from this cycle. Later, this woman conceived naturally and prenatal analysis of the fetus detected the same haplotype as the boy, without c.6901–2A>G. This data has not resolved where this mutation first arose, but demonstrates the complexities associated with de-novo mutations and possible gonadal mosaicism. 7.006 Application of PGD for single gene disorders Ismailoglu B1, Ozgon G1, Fiorentino F2, Demir S1, Karagozoglu H1, Unal S, Karlikaya G1, Kahraman S1 1Istanbul Memorial Hospital ART and Reproductive Genetics Unit. Istanbul, Turkey; 2Genoma, Italy Introduction: Preimplantation genetic diagnosis (PGD) is an essential alternative to prenatal diagnosis for couples at risk of having an affected child because of single gene disorder. When prenatal diagnosis was made, if the result were an affected fetus, termination of pregnancy would be requested. So, it could cause ethical problems and high stress for the couples. Selection of unaffected embryos generated by IVF is only way to avoid these problems. This study consists of PGD applications for 29 different single gene disorders and their molecular tools. Materials/Methods: A total of 84 couples (111 cycles) came to our genetic centre for PGD of single gene diseases between 2002 and 2007. in all, 1603 oocytes were collected, 1255 of those were fertilized and 887 embryos were selected for biopsy. Nested PCR was used as a molecular technique. Multiplex PCR was performed in first step on the blastomeres biopsied from day-3 embryos. Desired regions were amplified in a second step. According to mutation of single gene, minisequencing method, fragment analysis or linkage analysis were used to determine mutations or mutation-containing alleles. Results: A total of 753 (85%) embryos were diagnosed for 29 different single gene diseases (beta-thalassaemia, cystic fibrosis, Neiman pick, fragile X, CMT, SMA, NF, FMF, etc.).
on our experience for consecutive PGD cycles for single gene disorders from February 2003 through to December 2007, describing clinical outcome of 36 cycles in 20 couples for 12 different disorders: Duchenne muscular dystrophy (DMD), epidermolysis bullosa (EB), ornithine transcarbamylase (OTC) deficiency, osteogenesis imperfecta (OI), pyruvate dehydrogenase (PDH) deficiency, spinal muscular atrophy (SMA), Huntington’s disease (HD), spinocerebellar ataxia 3 (SCA3), long-chain 3-hydroacyl CoA dehydrogenase (LCHAD) deficiency, fragile X syndrome (FXS), phenylketonuria (PKU) and Charcot–Marie–Tooth (CMT) disease. In 36 PGD cycles for single gene disorders, a total of 363 cleavage stage embryos were biopsied. Successful diagnosis rate of biopsied blastomeres was 93.9% (340/363) and 190 embryos (55.9%) were diagnosed as unaffected. Among them, 95 embryos were transferred in 35 cycles except one cycle for PKU, resulting in 12 clinical pregnancies (34.3% per embryo transfer). There was no clinical abortion in the cases of pregnancies. All 12 clinical pregnancies were confirmed to be unaffected, and 13 healthy babies were delivered without any complications in five of DMD and each of EB, OTC deficiency, OI, SCA3 and SMA, respectively. Two pregnancies in each case of PKU and OI are still ongoing and the PGD results were confirmed by amniocentesis. There was no misdiagnosis in all pregnancies after PGD for single gene disorders. Our data show that PGD is a realistic option for couples at risk to avoid the birth of affected children with single gene disorders and to have a healthy unaffected baby of their own. 7.004 Preimplantation genetic diagnosis for nonsyndromic deafness by polar body and blastomere biopsy Altarescu G, Eldar-Geva T, Brooks B, Haran E, Margalioth EJ, Levy-Lahad E, Renbaum P Medical Genetics Unit and IVF Clinic, Zohar PGD Lab, Shaare Zedek Medical Center, Jerusalem, Israel Objective: (i) To develop an efficient and reliable universal protocol for PGD of non-syndromic deafness; and (ii) assess heterozygosity rates of polar body 1 (PB1), marker informativity and allele drop out (ADO) rates for polar body and blastomere preimplantation genetic diagnosis (PGD). Materials/Methods: Polar bodies 1 and 2 (PB1 and PB2) and blastomeres were biopsied by mechanical drilling.Three mutations, GJB2 35delG, 167delT and GJB6 delD13S1830 and the markers D13S141, D13S175, D13S633, D13S1275, D13S250, D13S232, GJB2-AT1, GJB2-AT2, GJB2-TG1, GJB2TG2, GJB2-AC1 were used to set up a universal multiplex PCR protocol to be used for PGD in non-syndromic deafness. Results were only assigned for samples with at least three informative flanking markers (including the familial mutation). Results: Eight couples underwent 19 PGD cycles resulting in a pregnancy rate of 21% and delivery of three unaffected children. Six cycles (32%) were performed by polar body biopsy, six by blastomere biopsy, six by frozen blastomere biopsy and in one cycle by both PB and blastomere biopsies. In 17 cycles (90%), at least two embryos were transferred and in one cycle one embryo was transferred. The rate of successful diagnosis for blastomere PGD was 91% and for PB PGD, 87%. Only 11 of 68 PB1 (17%) were heterozygotic. Similar ADO rates (19%) were observed for both heterozygotic PB1 and blastomeres. While all families had at least two informative markers on each side of the gene for PB PGD, none had four fully informative markers for blastomere PGD. Conclusion: We have developed a universal single-cell
S-46 Reproductive BioMedicine Online, Vol. 16, Suppl. 3, April 2008
multiplex protocol for non-syndromic deafness with a high efficiency of diagnosis for PGD. Although PB PGD allows more informative marker assessment, most PB1 are homozygous and ADO rates were seen to be similar. Therefore, blastomere biopsy appears to be the method of choice for this autosomal recessive disease. 7.005 A de-novo factor VIII (F8) mutation creates a challenge for PGD Laurie AD1, Hill A2, Smith MP 1, George PM2 1Canterbury Health Laboratories, Christchurch, New Zealand; 2The Fertility Centre, Christchurch. New Zealand Gonadal mosaicism presents a challenge in preimplantation genetic diagnosis (PGD) because detection of linked markers does not imply presence of a disease mutation. In a young boy with haemophilia A, we detected the splicing variant c.6901– 2A>G in the factor VIII gene (F8), but we did not detect this mutation in his mother. This suggests two possibilities: either this mutation arose de novo early in embryonic development of the boy, or that his mother is a gonadal mosaic for F8 c.6901–2A>G. Since our panel of F8-linked microsatellite markers would not be fully informative for PGD for this woman, we developed a mini-sequencing assay to genotype F8 c.6901–2, which we used in addition to the linked markers. We performed cleavage-stage PGD on two embryos from a single IVF cycle, one of which had the F8 microsatellite haplotype associated with the mutation but did not have the c.6901–2A>G variant. No pregnancy resulted from this cycle. Later, this woman conceived naturally and prenatal analysis of the fetus detected the same haplotype as the boy, without c.6901–2A>G. This data has not resolved where this mutation first arose, but demonstrates the complexities associated with de-novo mutations and possible gonadal mosaicism. 7.006 Application of PGD for single gene disorders Ismailoglu B1, Ozgon G1, Fiorentino F2, Demir S1, Karagozoglu H1, Unal S, Karlikaya G1, Kahraman S1 1Istanbul Memorial Hospital ART and Reproductive Genetics Unit. Istanbul, Turkey; 2Genoma, Italy Introduction: Preimplantation genetic diagnosis (PGD) is an essential alternative to prenatal diagnosis for couples at risk of having an affected child because of single gene disorder. When prenatal diagnosis was made, if the result were an affected fetus, termination of pregnancy would be requested. So, it could cause ethical problems and high stress for the couples. Selection of unaffected embryos generated by IVF is only way to avoid these problems. This study consists of PGD applications for 29 different single gene disorders and their molecular tools. Materials/Methods: A total of 84 couples (111 cycles) came to our genetic centre for PGD of single gene diseases between 2002 and 2007. in all, 1603 oocytes were collected, 1255 of those were fertilized and 887 embryos were selected for biopsy. Nested PCR was used as a molecular technique. Multiplex PCR was performed in first step on the blastomeres biopsied from day-3 embryos. Desired regions were amplified in a second step. According to mutation of single gene, minisequencing method, fragment analysis or linkage analysis were used to determine mutations or mutation-containing alleles. Results: A total of 753 (85%) embryos were diagnosed for 29 different single gene diseases (beta-thalassaemia, cystic fibrosis, Neiman pick, fragile X, CMT, SMA, NF, FMF, etc.).