P-38 Preimplantation genetic diagnosis for nemaline myopathy

POSTER PRESENTATIONS, PGD & Genetic Diseases designed all protocols and primers for our cases. Mutation testing combined with indirect analysis by polymorphic markers (STR mainly) was used. PGDs were performed at single blastomeres of day 3 embryos. Results: Ten to fifteen markers were tested for each family, and on the average they were informative in 80%. All our protocols were successfully tested on single lymphocytes or sperms. We tried to achieve a good result for mutation detection in single cells in each case. A big deletion of GALC gene was perfect at DNA but failed at single cells. So, only linkage analysis was used in this PGD case. IVS8 5/7/9-polyT polymorphism of CFTR gene was difficult to detect even at DNA but in our nested PCR it was good at single cells. In the case of Bechterew syndrome we developed nested PCR with Real-time PCR for second PCR of HLA-B27 combined with sex detection. As for this syndrome we had a consilium about the necessity for PGD. Only PGD setup was done for this genetic condition because the couple had a spontaneous pregnancy. Each PGD setup took about two months. In four PGD-IVF-ICSI cycles 20 embryos were tested and 17 were genotyped successfully. From 1 to 5 embryos were recommended for transfer and 1 or 2 were transferred on the 5th day. Three patients (26 34 years old) were pregnant, two of them with twins. Four healthy children were born. One pregnancy is proceeding. A patient aged 40 was negative. Conclusion: Carefully prepared PGD cases in combination with good reproductive potential give good results in preventing genetic diseases. Keywords: PGD, Single Gene Disorders. P-36 Preimplantation genetic diagnosis for mucopolysaccharidose type I: Analysis of a novel indel mutation O. Ayvaz1 , E. Unsal1 , A. Baltaci2 , T. Duman3 , L. Ozer4 , F. Akyigit2 , V. Baltaci1 . 1 Istanbul Bilim University School of Medicine, Istanbul, Turkey, 2 Genart Woman Health and Reproductive Biotechnology Center, Ankara, Turkey, 3 Ankara University School of Medicine, Ankara, Turkey, 4 Mikrogen Genetik Tani Laboratuvari, Ankara, Turkey Objective: Mucopolysaccharidosis type I (MPSI) is an autosomal recessive disorder caused by a deficiency in alpha-L iduronidase (IDUA), which leads to lysosomal accumulation of large sugar molecules called glycosaminoglycans (GAGs) dermatan and heparan sulfate. Severe MPS I occurs in approximately 1 in 100,000 newborns. Attenuated MPS I is less common and occurs in about 1 in 500,000 newborns. The use of in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) may help couples at risk to avoid pregnancies with known genetic diseases; in this case to achieve pregnancy without MPS I. A novel indel mutation (c.956_972+9delinsTA) that was firstly identified in a Turkish family was analysed. Materials and Methods: The deletion of 26 nucleotides and insertion of a dinucleotide in IDUA gene was firstly monitorized via sequence analysis in family members. Multiplex nested PCR tecnique was performed to detect the mutation in embryos. By means of fragment analysis, six informative STR markers were used to confirm that there were no allelic drop out (ADO the random non amplification of one of the alleles). Results: PGD cycle resulted in 10 embryos of which three were heterozygous and four were mutant. Two of three normal embryos were transferred resulting in a healthy baby born at term. Conclusion: The related indel mutation was only released by Bertoli (Hum. Mut. 2011) in a Turkish family. Analysis of novel mutations using PGD contributes to successful clinical applications in assisted reproductive technology especially in populations with high consanguineousy. The experience provided

S41 by this study encourages the development of standardized molecular PGD protocols for many rare diseases. Keywords: PGD, IVF, Mucopolysaccharidose Type I. P-37 Preimplantation genetic diagnosis for hypoparathyroidism deafness renal dysplasia syndrome H. Karadayi1,2 , E. Atli1 , A. Arslan1 , J. Caferler1 , Y.H. Ozon1 , H. Berkil1 , M. Bahce3 . 1 GENETIKS Genetic Diagnosis and Research Center, Istanbul, Turkey, 2 Heliks DNA Technologies, Istanbul, Turkey, 3 MBGENLAB Genetic Diagnosis Center, Ankara, Turkey Objectives: The Hypoparathyroidism Deafness Renal dysplasia (HDR) syndrome is an autosomal dominant disorder caused by mutations of the dual zinc finger transcription factor, GATA3 gene located at chromosome 10p15. GATA3 is a member of the GATA family of transcription factors. This gene is involved in vertebrate embryonic development. Material and Method: A PGD workup was performed initially on the parents DNA and then tested on single cells. The female patient then underwent a cycle of IVF. Five embryos were suitable for PGD and the single blastomeres were obtained by biopsy. Multiplex nested PCR analysis was performed for GATA3 gene c.829C>T (p.R277X) mutation, simultaneously with the linked polymorphic markers called D10S1728, D10S1712, D10S189, and D10S1751, all representing short tandem repeats (STR) associated with GATA3 gene. Results: The results show that there were 3 homozygous affected and 2 normal genotype embryos. Normal embryos were transferred back to the patient and a singleton pregnancy was obtained. Amniocentesis confirmed the presence of the normal fetus. Conclusion: These results show the feasibility of PGD for HDR syndrome, GATA3 gene. This technique could be applicable to families suffering from HDR syndrome GATA3 gene mutation carriers as well since they share similar mutation and clinical manifestations. Key words: HDR Syndrome, GATA3 gene, PGD P-38 Preimplantation genetic diagnosis for nemaline myopathy E. Atli1 , H. Karadayi1,2 , A. Arslan1 , H. Berkil1 , J. Caferler1 , C. Ozen3 , Y.H. Ozon1 . 1 GENETIKS Genetic Diagnosis and Research Center, Istanbul, Turkey, 2 Heliks DNA Technologies, Istanbul, Turkey, 3 Gayrettepe Florence Nightingale Hospital, Istanbul, Turkey Objectives: Nemaline Myopathy (NM) constitutes a heterogeneous group of congenital myopathies characterized by the presence of distinct rod-like inclusions, “nemaline bodies”, in the sarcoplasm of skeletal muscle fibers. Mutations in seven genes have been associated with NM, but the most commonly mutated gene is nebulin (NEB). Mutations in the NEB gene are the main cause of recessively inherited NM. NEB gene is one of the largest genes in human genome. Material and Method: A PGD workup was performed initially on the parents DNA and then tested on single cells. The female patient then underwent a cycle of IVF. Eighteen embryos were suitable for PGD and the single blastomeres were obtained by biopsy. Multiplex nested PCR analysis was performed for NEB gene c.9832-1G>A mutation, simultaneously with the linked polymorphic markers called D2S2277, D2S2275, D2S2299, D2S2241, and D2S2324, all representing short tandem repeats (STR) associated with NEB gene, known to be located at 2nd chromosome. Results: The results show that there were 10 heterozygous, 6 homozygous affected and 2 normal genotype embryos. Normal

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12th International Conference on Preimplantation Genetic Diagnosis

embryos were transferred to the patient and a singleton pregnancy was obtained. Our patient gave birth and we confirmed the healthy birth. Conclusion: These results show the feasibility of PGD for NM, NEB gene. This technique could be applicable to families suffering from NM disease NEB gene mutation carriers as well since they share similar mutation and clinical manifestations. Key words: Nemaline Myopathy, NEB gene, PGD P-39 Preimplantation genetic diagnosis for epidermolysis bullosa H. Karadayi1,2 , E. Atli1 , C. Aktas1 , J. Caferler1 , H. Berkil1 , M. Bahce3 , Y.H. Ozon1 . 1 GENETIKS Genetic Diagnosis and Research Center, Istanbul, Turkey, 2 Heliks DNA Technologies, Istanbul, Turkey, 3 MBGENLAB Genetic Diagnosis Center, Ankara, Turkey Objectives: Epidermolysis Bullosa (EB) comprises a group of disorders characterized by congenital skin fragility. EB has been classified into EB simplex, junctional epidermolysis bullosa (JEB), dystrophic EB and Kindler syndrome. Mutations in the gene encoding b4 integrin subunit (ITGB4) are responsible for JEB. JEB is characterized by generalized blistering and occlusion of the pylorus at birth, which usually leads to early demise. We performed PGD for an at-risk couple with a history of neonatal death due to EB. Materials and Methods: A PGD workup was performed initially on the parents’ DNA and then tested on single cells. The female patient then underwent a cycle of IVF. Five embryos were suitable for PGD and the single blastomeres were obtained by biopsy. Multiplex nested PCR analysis was performed for ITGB4 gene Intron 16, IVS16-2 A>G mutation, simultaneously with the linked polymorphic markers called D17S535, D17S650, D17S1301, D17S1839, and D17S1817, all representing short tandem repeats (STR) associated with ITGB4 gene, known to be located at 17th chromosome. Results: The results show that there were 1 heterozygous, 3 homozygous affected and 1 normal genotype embryos. One heterozygous and 1 normal embryo were transferred back to the patient and a singleton pregnancy was obtained. Amniocentesis confirmed the presence of the normal fetus. Conclusion: These results show the feasibility of PGD for EB, ITGB4 gene. This technique could be applicable to families suffering from EB disease ITGB4 gene mutation carriers as well similar clinical manifestations. Keywords: Epidermolysis Bullosa, ITGB4 gene, PGD P-40 Combined molecular and chromosomal analysis in embryos: Our experience T.M. Alberola, R. Bautista-Ll´ acer, M. Pardo, C. S´ anchezMatamoros, E. García-Mengual, X. Vendrell. Reproductive Genetics Unit. Sistemas Genomicos, Paterna (Valencia), Spain Objective: To offer an alternative to couples who have reproductive problems plus are at risk of transmitting monogenic diseases to offspring. Materials and Methods: Genomic amplification of each blastomere and its corresponding blank with Sureplex Amplification System (BlueGnome® ) with modifications. Specific amplification by fluorescent heminested PCR of the mutation-bearing region and linked short tandem repeat microsatellite markers. Capillary electrophoresis to detect the fluorescent fragments. Minisequencing (for point mutations) of the corresponding position using the SNaPshot kit (Applied Biosystems, USA). Bioinformatic analysis using the GeneMapper v3.7 to determine allele size and nucleotide sequence. Labeling and hybridization of DNA amplified by Sureplex with 24Sure or 24Sure+ microarrays, following the manufacturer’s protocol. Scanning of microarrays

with Agilent’s G2565CA scanner and analysis with the BlueFuse Multi software of BlueGnome® . Results: Our laboratory has set up this combined analysis for several monogenic diseases, using the amplified DNA to interrogate by direct methods the disease-causing mutation and, furthermore, to give a chromosomal diagnosis. Previous to PGD it is necessary to perform preclinical studies to ensure the viability of the diagnosis. The conditions are tested in parents’ lymphocytes before issuing a favorable report. We have analyzed 18 embryos for 3 monogenic diseases to date. Only 3 of them (16.6%), where disease-free and chromosomally normal. The results will be presented as updated. Disease

Mutation

No. of cycles

Arrhythmogenic right ventricular dysplasia Charcot Marie Tooth 1B

c.3115dupG DSP gene c.131C>T MPZ gene c.211A>G BRCA1 gene

1

6

2

NO

1

5

0

–

1

7

1

No data for the moment

Breast ovarian cancer

No. of embryos analyzed

No. of transferable embryos

Pregnancy?

Conclusions: Vitrification and accumulation of cycles are necessary to achieve a certain number of transferable embryos and increase the probability of pregnancy. Keywords: Combines PGD, monogenic disease, chromosomal analysis. P-41 Preimplantation genetic diagnosis for lamellar ichthyosis type 2 E. Atli1 , H. Karadayi1,2 , C. Aktas1 , H. Berkil1 , M. Ulug3 , A.A. Ulug3 , J. Caferler1 , Y.H. Ozon1 . 1 GENETIKS Genetic Diagnosis and Research Center, Istanbul, Turkey, 2 Heliks DNA Technologies, Istanbul, Turkey, 3 Jinemed Hospital, Istanbul, Turkey Objectives: Lamellar Ichthyosis Type 2 (LI2) is clinically characterized by the scales, which are large, adherent, dark and pigmented, and by the absence of erythema. LI2 is a rare autosomal recessive skin disorder for which a gene has been localized on chromosome 2q33 35. It has been vital to elucidate the biological importance of the protein ABCA12 in skin-barrier permeability, following the discovery that ABCA12 gene mutations can result in this rare disease. Materials and Methods: A PGD workup was performed initially on the parents’ DNA and then tested on single cells. The female patient then underwent a cycle of IVF. Seven embryos were suitable for PGD and the single blastomers were obtained by biopsy. Multiplex nested PCR analysis was performed for ABCA12 gene c.2122C>G p.Y634X mutation, simultaneously with the linked polymorphic markers called D2S128, D2S137, D2S1345, and D2S2382, all representing short tandem repeats (STR) associated with ABCA12 gene located in 2th chromosome. Results: The results show that there were 5 homozygous affected, 2 heterozygous genotype embryos. Two heterozygous embryos were transferred back to the patient and a singleton pregnancy was obtained. Amniocentesis confirmed the presence of the normal fetus. Our patient gave birth and we confirmed the healthy birth. Conclusion: These results show the feasibility of PGD for LI2 disease, ABCA12 gene. This technique could be applicable to families suffering from LI2, ABCA12 gene mutation carriers as well since they share similar mutation and clinical manifestations. Keywords: Lamellar Ichthyosis, ABCA12 gene, PGD