Article Pre-embryonic diagnosis for Sandhoff disease

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1 RBMOnline - Vol 12. No Reproductive BioMedicine Online; on web 9 January 2006 Article Pre-embryonic diagnosis for Sandhoff disease Dr Anver Kuliev received his PhD in Clinical Cytogenetics from Moscow Research Institute of Human Morphology in In 1979 he took the responsibility for the World Health Organization (WHO) s Hereditary Diseases Program in Geneva, where he developed community-based programmes for prevention of genetic disorders and early approaches for prenatal diagnosis. He moved to the Reproductive Genetics Institute in 1990, where he heads the WHO Collaborating Center for Prevention of Genetic Disorders, and scientific research in prenatal and preimplantation genetics. He is an author on 144 papers and 11 books in the above areas, including five books in the field of preimplantation genetics. Dr Anver Kuliev Anver Kuliev, Svetlana Rechitsky, Katya Laziuk, Oleg Verlinsky, Ilan Tur-Kaspa, Yury Verlinsky Reproductive Genetics Institute, Chicago, IL, USA Correspondence: anverkuliev@hotmail.com Abstract Embryos found to be abnormal during preimplantation genetic diagnosis (PGD) are discarded or analysed to confirm the diagnosis. To overcome this limitation, which is unacceptable in some communities and ethnic groups, pre-embryonic genetic diagnosis has been introduced, involving sequential first and second polar body analysis followed by transfer of embryos deriving from the mutation-free oocytes, while removing from culture and freezing the mutant oocytes at the pronuclear stage. The technique is applied here to PGD of Sandhoff disease caused by 16-kb deletion of the hexosaminidase B gene for a couple with a religious objection to discarding embryos irrespective of embryo genotype. Of 16 oocytes tested in a standard IVF protocol for 16-kb deletion, simultaneously with five linked polymorphic markers, eight were predicted mutant and frozen prior to syngamy, with the remaining eight, found to be free of mutation, further cultured and confirmed unaffected using blastomere biopsy. The transfer of two of these embryos resulted in birth of an unaffected child, demonstrating feasibility of pre-embryonic diagnosis to avoid embryo discard. Keywords: PGD, polar body PCR analysis, pre-embryonic genetic diagnosis, pronuclear stage freezing, Sandhoff disease 328 Introduction Pre-embryonic genetic diagnosis (PEGD) was first introduced for sickle cell disease, to predict the potential embryo genotype prior to pronuclei fusion, following intracytoplasmic sperm injection (ICSI) (Kuliev et al., 2001). This technique is of special interest in those ethnic groups that cannot benefit from preimplantation genetic diagnosis (PGD) because of their negative attitude to micromanipulation and the discarding of embryos even if they are affected. Although PEGD can be performed by first polar body (PB1) testing only (Verlinsky et al., 1990, 1995; Munné et al., 1998), it may not be sufficient for the accurate prediction of the embryo genotype without the second PB (PB2) analysis, which allows identification of the mutation-free zygotes deriving from the heterozygous metaphase II oocytes in PGD for single gene disorders (Verlinsky et al. 1997), and the exclusion from transfer of embryos originating from the oocytes with meiosis II errors in PGD for chromosomal disorders (Kuliev et al., 2003). Accordingly, only euploid oocytes, or those predicted to have a normal maternal allele, could then be allowed to progress to pronuclei fusion (syngamy), embryo development and transfer either in the same, or a subsequent menstrual cycle, avoiding the formation and possible discarding of any affected embryo. This is a principally new type of genetic diagnosis, which moves the predictive genotyping to an even earlier stage than a traditional PGD, making the diagnosis more ethically acceptable, as it overcomes the negative reaction to the embryo micromanipulation and discard. In the abovementioned case, PEGD was not performed on request, but necessitated by an incidental hyperstimulation, thus the embryo transfer had to be postponed to the next cycle anyway. Pronuclear stage freezing of the preselected mutation-free oocytes was therefore the only realistic option to resume their culture and transfer in a subsequent cycle, which resulted in an unaffected pregnancy and birth of a healthy child (Kuliev et al., 2001). The present paper describes PEGD for Sandhoff disease (SD) performed on patient request, resulting in the same cycle embryo transfer and resulting in birth of an unaffected child, thus avoiding any establishment, micromanipulation and discard of the affected embryos.

2 Materials and methods A 32-year-old women and her spouse at risk for producing a child with SD requested PGD to be performed without any possible discard of embryos, even if affected. As seen from the pedigree shown in Figure 1, the couple had one affected son with classical features of SD, who died at the age of 1 year and 3 months despite bone marrow transplantation. SD results from a defect in the beta chain of hexaminidase B gene (HEXB) on chromosome 5, which consists of 14 exons distributed over 40 kb of DNA (MIM ; ). Mutation in this gene causes beta-hexasaminidase deficiency, resulting in the lysosomal storage disease GM2- gangliosidosis. The same condition is also caused by Tay Sachs disease, resulting from the defect of hexaminidase A gene (HEXA). The child inherited two different mutations from his parents: the paternally derived I207V mutation in exon 5 of HEXB gene, resulting from ATT to GTT substitution, and a large maternal 16-kb deletion (16 kb Del), involving as many as five exons, from exon 1 to exon 5 (Figure 2). The paternal mutation was identified by the HinfI restriction digestion, which cuts the normal allele into two fragments of 32 and 25 bp, leaving the mutant allele uncut, and the maternal 16 kb Del was detected by a fragment size analysis (Figure 2). Five closely linked polymorphic markers, D5S1982, D5S1988, D5S2003, D5S349 and D5S1404, were tested simultaneously with the HEXB gene in a multiplex heminested PCR system. The maternal and paternal haplotypes, established by family studies and PB analysis, are presented in Figure 1, while primer sequences are listed in Table 1. A single PGD cycle was initiated, which was performed according to the following modification of the timetable of the applied procedures of sequential PB1 and PB2 analysis, described elsewhere (Verlinsky and Kuliev, 2005). PB1 was removed as usual 3.5 h after aspiration, followed by ICSI. PB2 was removed soon after it was extruded, approximately within 6.5 h after ICSI, to allow sufficient time for completion of the DNA analysis before pronuclei fusion. DNA analysis is currently performed in less than 9 h overall, making it realistic to freeze the oocytes predicted to contain the deleted HEXB allele before syngamy (within 24 h after aspiration or 12 h after PB2 removal), and culture the HEXB deletion-free oocytes to blastocyst and transfer at day 5, following confirmation of the maternal mutation free status of the embryos by the day 3 blastomere biopsy. Following informed consent, approved by the Institutional Review Board, the embryos deriving from the mutation-free oocytes were preselected for transfer back to patient, while the pronuclear stage mutant oocytes and the spare normal embryos were frozen, to be available for the couple in future cycles. Results Of 18 oocytes available for testing in a single PGD cycle, 16 were with conclusive PB1 and PB2 results, of which eight contained the maternal 16 kb Del and were frozen at the pronuclear stage (Figure 1). Four of these oocytes contained heterozygous PB1 and normal PB2 (oocytes 3, 9, 11 and 14), and four contained homozygous normal PB1 and mutant PB2 (Figure 2B). The remaining eight oocytes were free of the deletion, two originating from the oocytes with heterozygous PB1 and mutant PB2 (oocytes 1 and 5), and the others from the oocytes with homozygous mutant PB1 and normal PB2. As the predicted genotypes in these oocytes may erroneously appear opposite, due to a possible undetected allele dropout of one of the alleles in the actually heterozygous PB1, similar to the four mutant oocytes predicted on the basis of homozygous normal PB1 and mutant PB2, the testing for five closely linked polymorphic markers was essential, confirming all the predicted oocyte genotypes mentioned. A follow-up blastomere analysis of the embryos deriving from the oocytes predicted to be free of maternal deletion showed complete correspondence to the PB diagnosis. Six of these embryos also appeared to contain a normal paternal allele (embryos 1, 4, 5, 6, 8 and 10), while only two (embryos 16 and 18) inherited the paternally derived mutant allele, confirmed by all five linked polymorphic markers tested (Figures 1 and 2). Two of these embryos (embryos 1 and 10), with both maternal and paternal normal alleles, were transferred, resulting in a singleton pregnancy and birth of an unaffected child. The remaining six unaffected embryos were frozen to be available for the couple in the future, while eight mutant oocytes were frozen at the pronuclear stage. Discussion The presented results show that PEGD is a realistic option for those couples who cannot accept traditional PGD, because of their objection to any micromanipulation and potential discarding of the tested embryos. In contrast to the previous PEGD attempt mentioned, involving the freezing of all the tested oocytes at the pronuclear stage immediately after ICSI and extrusion of PB2 (Kuliev et al., 2001), the presented result was achieved without freezing of the mutation-free oocytes, which were detected well before the pronuclei fusion, after which the discarding of embryos could not be avoided. Although all the oocytes could have been frozen irrespective of the DNA diagnosis, as described in the previously reported case mentioned, not all frozen pronuclear stage oocytes could potentially be successfully recovered, which may incidentally also include a few preselected unaffected embryos for transfer and could have affected the PEGD outcome. The realization of PEGD in the same clinical cycle is clearly an important practical step, which has become realistic because of DNA analysis being completed within less than 9 h. This approach may currently be applied to autosomal recessive, X-linked, and maternally derived dominant and chromosomal mutations, detectable by sequential PB1 and PB2 analysis. To perform PEGD for paternally derived dominant and chromosomal mutations, a technique for sperm duplication prior to genetic analysis may be required, recently shown to be feasible through sperm nuclear 329

3 330 Figure 1. Preimplantation genetic diagnosis (PGD) for Sandhoff disease (SD). (A) Family pedigree with mutation and haplotype analysis based on parental (1.1 and 1.2) and affected child s (2.1) genomic DNA testing. The marker order is presented on the upper left for father and upper right for mother. Maternal and paternal mutations and the linked markers are shown in non-bold, while normal alleles and their linked markers are shown in bold. (B) Results of sequential first and second polar body analysis of 16 oocytes, showing eight normal (bold) and eight mutant oocytes (non-bold) that were frozen prior to syngamy. (C) Blastomere analysis of embryos, resulting from the mutation-free oocytes, which confirms the polar body diagnosis. Two of these embryos, nos 1 and 10, free of both maternal and paternal mutations, were transferred, resulting in birth of an unaffected child. The remaining six embryos, two of which were heterozygous, were frozen (Fr) for future use by the couple. ET = embryo transfer; N = normal; M = maternal.

4 Figure 2. Map of human hexaminidase B (HEXB) gene and results of maternal and paternal mutation testing in polar bodies and blastomeres. (A) Schematic presentation of maternal and paternal mutations and linked polymorphic markers. (B) Polar body analysis of the maternal 16kb deletion (N = normal; D = deletion). (C) Restriction map: HinfI enzyme created two fragments in normal gene, leaving the paternal mutation I207V uncut. (D) Blastomere analysis for maternal deletion and paternal mutation, confirming the polar body diagnosis. L = ladder; M = mother; F = father; C = control. 331

5 Table 1. Primers for the detection of the 16-kb deletion and I207V mutation in the hexaminidase B gene (HEXB) causing Sandhoff disease, and the linked markers in heminested polymerase chain reaction analysis. NA = not applicable. Gene/ Accession Hetero- No. Upper Lower Annealing polymorphism no. zygosity alleles primer primer temperature index (ºC) 16-kb deletion ENST0000 NA NA Outside: 5 ACCTCTTTATGGC 5 AATTATGGGATGA (heminested); TGGCTCC 3 CTGCCTATT 3 amplifies only (Ensembl) Inside: 5 AGACACGGCAAG 5 AATTATGGGATGA 55 deleted sequence ATTAGAGTAATAT 3 CTGCCTATT 3 Exon 4 exon 5; ENST0000 NA NA Outside: 5 TAGAGACCTTTAG 5 GCTAAGACAAATA amplifies only CCAGTTAGTTTA 3 TCTGGGGAAA normal sequence (Ensembl) Inside: 5 TAGAGACCTTTAG 5 CTAAACAGGTTAC 55 CCAGTTAGTTTA 3 ATTTTTTTCTAT 3 I207V ENST0000 NA NA Outside: 5 AATAGATTTAGTCT 5 ATTACTTACCAGA heminested (Hinf TCATTGAGTTC 3 GTTTTAAGAATA 3 I cuts normal (Ensembl) Inside: 5 AATAGATTTAGTCT 5 GCAGATAATGT 55 sequence) TCATTGAGTTC 3 CTGGATGTATGA 3 D5S1982 Z Outside: 5 AGAGTTTGGGCAA 5 GGAAAGACATTT (heminested) GGCGTA 3 AACCCTTTCTCT 3 Inside: 5 GATGAGAATGAAG 5 GGAAAGACATTT 55 GTTAAAAAGTCC 3 AACCCTTTCTCT 3 D5S1988 Z Outside: 5 AGCTTACTTCACT 5 AAGAAATGGA (heminested) TGGCATAA 3 AGCAACCTAAG 3 Inside: 5 AGCTTACTTCACT 5 GTCCACCGATGG 55 TGGCATAA 3 ATGAATG 3 D5S2003 Z Outside: 5 AGCCTAAGTGACA 5 CTCACAGAGGGT (heminested) AAGTGAGACA 3 GTGTTATAATAGA 3 Inside: 5 AGCCTAAGTGACA 5 TAGAGTCCTTTTC 55 AAGTGAGACA 3 ATTGCCAA 3 D5S349 M Outside: 5 ATATTTGGTTTCCA 5 CCACCAGATTAA (heminested) TAGAATCTGAG 3 GCGTGAATC 3 Inside: 5 ATATTTGGTTTCCA 5 CCTCTAGAAAA 55 TAGAATCTGAG 3 TGGTAGTTGGG 3 D5S1404 L Outside: 5 GCCAATTTCTTGT 5 TAATTTACCCACT (heminested) CTATTCCTTAG 3 GTATCAGTCAGG 3 Inside: 5 GCCAATTTCTTGT 5 GGTTCCATGAGA 55 CTATTCCTTAG 3 AGTAAGAGATCTA transfer into anucleated metaphase II oocytes (Willadsen et al., 2004). The technique will allow genetic analysis of one of the sperm duplicates, using the other one for fertilization and transfer of the resulting embryos, if the corresponding duplicate shows normal genotype. In this way, the establishment and discarding of any embryo containing paternal mutation may be avoided. However, more data might be necessary to work out special conditions supporting the faithful replication of human sperm genome, to ensure that the haploid cell pairs obtained from sperm duplication are identical (Verlinsky and Kuliev, 2005). As mentioned, PEGD may be applied for aneuploidy testing, as the majority of chromosomal disorders deriving from the female meiosis can be tested by PB analysis. Available experience is presently limited to translocation or aneuploidy testing by PB1 analysis, which, as mentioned, leaves meiosis II errors undetected (Verlinsky et al., 1995; Munné et al., 1998; Montag et al., 2004). As seen from the presented results, the detection of the second meiosis errors is currently feasible within the time available prior to pronuclei fusion, so PEGD for chromosomal disorders may in future also be applied in those countries where PGD is still not acceptable because of the potential discarding of the affected embryos with the currently used methods. It should also be mentioned that the presented case is the only attempt of PGD for SD in the overall experience of more than 2000 PGD cycles for single gene disorders, with the previous experience involving PGD for Tay Sachs disease, the other severe lysosomal storage disease with lethal outcome (ESHRE 2002; Kuliev and Verlinsky, 2004). With addition of the PEGD approach, the presently available techniques make it possible to offer a great variety of methods for predicting and avoiding not only the birth, but also conception or implantation of the affected embryos. This provides at-risk couples with all possible options for avoiding offspring with genetic and chromosomal disorders, independent of their attitudes to oocyte or embryo micromanipulation, testing and discard, so they may achieve their goal of having healthy unaffected children of their own.

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