Abstract. Introduction. RBMOnline - Vol 9. No Reproductive BioMedicine Online; on web 23 June 2004

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1 RBMOnline - Vol 9. No Reproductive BioMedicine Online; on web 23 June 2004 Article Preimplantation genetic diagnosis with HLA matching Dr Svetlana Rechitsky is a graduate of Kharkov University's Genetics Faculty, and received her PhD in Experimental Molecular Embryology from the Second Moscow Medical Institute in She moved to the Reproductive Genetics Institute in 1989 to head the DNA laboratory, which has performed the largest preimplantation genetic diagnosis (PGD) series for single gene disorders, with PGD design for the majority of these disorders developed for the first time. She has published more than 30 papers in the field of PGD, including a key contribution to the recently published Atlas of Preimplantation Genetic Diagnosis. Dr Svetlana Rechitsky Svetlana Rechitsky, Anver Kuliev 1, Illan Tur-Kaspa, Randy Morris, Yury Verlinsky Reproductive Genetics Institute, Chicago, IL, USA 1 Correspondence: 2825 North Halsted Street, Chicago, IL, USA; anverkuliev@hotmail.com or rgi@flash.net Abstract Preimplantation genetic diagnosis (PGD) has recently been offered in combination with HLA typing, which allowed a successful haematopoietic reconstitution in affected siblings with Fanconi anaemia by transplantation of stem cells obtained from the HLA-matched offspring resulting from PGD. This study presents the results of the first PGD practical experience performed in a group of couples at risk for producing children with genetic disorders. These parents also requested preimplantation HLA typing for treating the affected children in the family, who required HLA-matched stem cell transplantation. Using a standard IVF procedure, oocytes or embryos were tested for causative gene mutations simultaneously with HLA alleles, selecting and transferring only those unaffected embryos, which were HLA matched to the affected siblings. The procedure was performed for patients with children affected by Fanconi anaemia (FANC) A and C, different thalassaemia mutations, Wiscott Aldrich syndrome, X-linked adrenoleukodystrophy, X-linked hyperimmunoglobulin M syndrome and X-linked hypohidrotic ectodermal displasia with immune deficiency. Overall, 46 PGD cycles were performed for 26 couples, resulting in selection and transfer of 50 unaffected HLA-matched embryos in 33 cycles, yielding six HLA-matched clinical pregnancies and the birth of five unaffected HLA-matched children. Despite the controversy of PGD use for HLA typing, the data demonstrate the usefulness of this approach for at-risk couples, not only to avoid the birth of affected children with an inherited disease, but also for having unaffected children who may also be potential HLA-matched donors of stem cells for treatment of affected siblings. Keywords: Fanconi anaemia, preimplantation genetic diagnosis, preimplantation HLA typing, thalassaemia, Wiscott Aldrich syndrome, X-linked diseases 210 Introduction It has previously been demonstrated that preimplantation genetic diagnosis (PGD) may be offered in combination with HLA typing, allowing successful haematopoietic reconstitution in affected siblings by transplantation of stem cells obtained from HLA-matched offspring resulting from PGD (Verlinsky et al., 2001). Since then, preimplantation HLA matching has become one of the indications for PGD (Kuliev and Verlinsky, 2004; Van de Velde et al., 2004), which has recently been performed even without testing for the causative gene, also resulting in haematopoietic reconstitution in the affected sibling (Verlinsky et al., 2004). Despite extensive discussions and concerns about the ethical and legal issues involved in preimplantation HLA typing (Damewood, 2001; Edwards, 2003; Fost, 2004), an increasing number of patients regard the procedure as an attractive option for establishing a mutation-free pregnancy, which will also provide an HLA match for treatment of the affected sibling. In addition to Fanconi anaemia complementation group C (FA-C) described earlier (Verlinsky et al., 2001), combined PGD and HLA testing has now been undertaken for FA complementation group A (FA-A), different thalassaemia mutations, Wiscott Aldrich syndrome (WAS), X-linked adrenoleukodystrophy (X-ALD), X-linked hyper-igm syndrome (HIGM) and X-linked hypohidrotic ectodermal dysplasia with immune deficiency (HED-ID) (OMIM, 2001). Among these conditions the most prevalent ones are thalassaemias, which are the commonest autosomal recessive

2 diseases in the Mediterranean region, Middle East and South East Asia, with heterozygous frequency of thalassaemia mutations reaching 14% in Greece and Cyprus. The prevalence of the other conditions is much lower, 1 in 17,000 for X-ALD, 1 in 100,000 for FA, and even rarer for WA, HIGM and HED-ID. All these conditions may be effectively treated only by HLA-matched bone marrow transplantation, which makes PGD the method of choice for those who cannot find an HLA match among their relatives. This paper describes the first practical experience of PGD with HLA typing, involving the above conditions, which resulted in establishing unaffected clinical pregnancies and the birth of healthy HLA-matched children to siblings in need of stem cell transplantation. Materials and methods Twenty-six couples overall presented for PGD combined with HLA typing, for whom 46 clinical cycles were performed, including 11 from five couples with FA-A and FA-C, 30 from 17 couples with thalassaemia, two from a couple with HIGM, and one each for couples with WAS, X-ALD and HED-ID affected siblings in a family (Table 1). As mentioned, one of the cases performed for FA-C has been reported previously (Verlinsky et al., 2001). PGD cycles were performed using a standard IVF protocol and intracytoplasmic sperm injection (ICSI), coupled with micromanipulation procedures as described elsewhere (Verlinsky and Kuliev, 2000). For testing of maternal mutations and maternal HLA match, the first and second polar bodies (PB1 and PB2) were removed sequentially following maturation and fertilization of oocytes, while single blastomeres were removed from the 8-cell embryos for testing of both the paternal and maternal mutations and the paternal and maternal HLA match. HLA genes were tested using only closely linked polymorphic short tandem repeat (STR) markers located throughout the HLA region. For each family, heterozygous markers not shared by the parents were selected. Such markers provide information about the origin of chromosome 6. A haplotype analysis for father, mother and the affected child was performed for each family prior to preimplantation HLA typing. This allowed detecting and avoiding misdiagnosis due to preferential amplification and allele drop-out (ADO), exceeding 10% in polymerase chain reaction (PCR) of single blastomeres, potential recombination within the HLA region, and a possible aneuploidy or uniparental disomy of chromosome 6, which may also affect the diagnostic accuracy of HLA typing of the embryo. A multiplex hemi-nested PCR was used, with the first round of PCR requiring similar annealing temperature for the outside primers. Thirty cycles of PCR were performed with a denaturation step at 95 C for 20 s, annealing at C for 1 min and elongation at 72 C for 30 s. Twenty minutes of incubation at 96 C were performed before starting cycling. After cycling, 10 min of elongation at 72 C took place. The annealing temperature for the second round was programmed at 55 C (details of the method have been published earlier; Verlinsky and Kuliev, 2000; Verlinsky et al., 2001) The strategy for PGD of two of five couples at risk for producing progeny with FA-C has been reported previously (Verlinsky et al., 2001). The remaining three couples were carriers of FA-A gene mutations, including one with different maternal and paternal mutations, the maternal one involving ATG AAG substitution in exon 1, and the paternal mutation a14 bp deletion in exon 2, representing a frameshift mutation. The paternal mutation was detected as the size difference in capillary electrophoresis of the PCR product, while the maternal mutation was detected by NlaIII restriction digestion, which cuts the normal sequence, leaving the mutant sequence uncut. In the other couple, only the paternal mutation was known; it was T1131A mutation, due to ACT GCT substitution in exon 34, which creates a restriction site for Fsp4HI. In addition, another restriction enzyme, TspRI, was used, which cuts the normal sequence. No mutation was identified in the third couple, leaving the only possibility of choosing unaffected embryos by linkage analysis, using five closely linked polymorphic markers. A couple at risk for producing a progeny with X-ALD had an affected son with the G343D mutation, caused by a single (G to A) sequence change in the nucleotide 1414 (G1414A) of Table 1. Results and outcome of PGD with preimplantation HLA typing. Disease Maternal Patient No. No. No. No. Pregnancy/ age /cycle embryos, normal abnormal transfers/ birth (years) total/ embryos embryos no. amplified Non-match Match Non-match Match embryos Thalassaemia 36.1 a 17/30 307/ /36 2/1 FANCA + C 34.2 a 5/11 73/ /9 2/2 b WAS /1 2/ /1 0 XALD /1 4/ Hyper IgM /2 24/ /2 1/1 HED + ID /1 16/ /2 1/1 Total 26/46 426/ /50 6/5 a Mean maternal age. b One couple decided to transfer partially matched embryo carrier of the paternal mutation in FANCA gene. 211

3 212 ABCD gene. PGD was based on FokI restriction digestion, which creates two fragments in the PCR product of normal gene, leaving the mutant one uncut. The other couple at risk for producing a progeny with WAS had two affected sons carrying the missence mutation in exon 1 of the WAS gene, due to a single nucleotide (CTT CCT) substitution at position 150. The mutation testing was done using ScrFI restriction digestion, cutting the mutant and leaving the normal gene product intact. Seventeen couples had thalassaemia mutations, including 619-bp deletion, 28, codon 39, IVS I-5, R30T, IVS I-110, IVSI-1, IVS 2 1, codon 41/42, codon 8/9 (+G) and cap site 1. The maternal mutations were analysed by PB1 and PB2, as described elsewhere (Kuliev et al., 1999; Verlinsky and Kuliev, 2000), followed by paternal mutation testing and HLA typing in blastomeres, particularly when parents were carriers of different mutations, as shown in Figure 1. A single couple with an HIGM child had mutation in exon 5, eliminating restriction site for Cac8I, which creates two fragments in the PCR product from the normal gene, leaving the mutant gene product uncut. For greater accuracy, another restriction enzyme (MnlI) was applied, which creates three fragments in the mutant PCR product, compared with two fragments in the normal gene (Figure 2). Finally, in one couple, the mother was a carrier of the mutation, resulting from T to G change (CTG CGG) in exon 4 of the NEMO (IKBKG) gene. Because of the presence of a closely linked pseudogene with a normal sequence at position of the mutation, which is co-amplified with the transcribed gene, a special design was developed to avoid misdiagnosis (Figure 3). Approaches for PGD and combined HLA typing have been described previously (Verlinsky and Kuliev, 2000; Verlinsky et al., 2001, 2004). In brief, DNA testing was performed by multiplex nested PCR analysis, amplifying mutations simultaneously with linked markers, HLA alleles and short tandem repeats in the HLA region. Linkage analysis was performed for each couple to avoid a possible misdiagnosis, which requires special care because of the phenomena of ADO and preferential amplification, known to be frequent in a single-cell DNA analysis. For example, six informative linked markers are available for testing, together with mutation analysis for thalassaemias (three of them are shown in Figure 1). In couples with the FA mutation, six markers were used, representing dinucleotide repeats essential in confirming the presence of the normal gene in the embryos selected for transfer. In the couple with HIGM, four closely linked dinucleotide markers were applied, together with CYS218STOP mutation in exon 5 and HLA alleles, improving the accuracy of PGD (Figure 2). Six closely linked STR were used in couple with HED-ID (Figure 3). Because patients referring for combined PGD and HLA typing are usually of advanced reproductive age, the copy number of the chromosome, in which causative gene tested was localized was analysed using corresponding satellite markers, as well as the copy number of chromosome 6, where HLA genes are localized. A set of STR throughout HLA region and chromosome specific satellite makers, allowed identification of monosomy, trisomy or uniparental disomy of chromosome 6, as well as the possibility of recombination, which strongly affect the accuracy of HLA matching (Foissac et al., 2000; Verlinsky et al., 2004). Primers for multiplex PCR analysis for thalassaemias and FANC were described earlier (Kuliev et al., 1999; Verlinsky and Kuliev, 2000; Verlinsky et al., 2001) and others, including FANCA, X-ALD, HIGM, WAS and HED-ID, as well as marker analysis, are listed in Table 2. As usual, the patients gave consent, which was approved by Institutional Review Board, that, based on the multiplex mutation and marker analysis, unaffected HLA embryos would be preselected for transferring back to the patients, unaffected but non-hla matched would be frozen for future possible use by the couple, while those predicted as mutant would be exposed to confirmatory analysis using the genomic DNA from these embryos to evaluate the accuracy of the single cell based PGD. Results Overall, of 426 oocytes or embryos tested in 46 PGD cycles performed for 26 couples carrying the above mentioned mutations (approximately 9.3 per cycle), 396 produced results (8.6 per cycle), from which 278 unaffected embryos (six per cycle) were selected. Although the overall number of HLA matched embryos was 71 (17.8%), only 54 were also unaffected (13.6%), of which 50 developed appropriately to be acceptable for transfer in 33 of 46 PGD cycles (1.5 embryos per transfer on average). This yielded six unaffected HLA matched pregnancies (18.2% per transfer), and the birth of five healthy children HLA matched to affected siblings (Table 1), including two free from FA, one free from thalassaemia, one free from HIGM, and one free from HED-ID, following confirmation of PGD by amniocentesis. The largest group of PGD cycles (30 cycles for 17 couples) was performed for thalassaemias. Of 307 oocytes and embryos tested, 285 showed conclusive results, of which 84 were predicted to be homozygous mutant and 201 unaffected carriers or normal. The HLA typing revealed 50 (17.5%) embryos to be matched to the affected siblings, but only 40 were also unaffected (14%), of which 36 developed appropriately to be acceptable for transfer in 21 of 30 PGD cycles, resulting in two unaffected HLA-matched pregnancies. One of these pregnancies resulted in a stillbirth and the other in the birth of an unaffected HLA-matched child (Figures 1 and 4). As the parents were carriers of different mutations (IVSI-5 and Cd8+G), both PB and blastomere analysis were performed so that mutation-free oocytes could be selected (oocytes 1, 2, 3, 6, 8 and 9; the latter two oocytes are not shown) prior to blastomere analysis, used mainly for selection of paternal mutation-free embryos (embryos 1, 3, 5 and 9) and HLA matching, simultaneously with linked marker analysis (Figure 4). In addition, HLA typing was also done in PB1 and PB2 to identify oocytes with maternal HLA match, which is useful for interpretation of HLA matching results in blastomeres. Only embryos 2 and 6 appeared to be HLAmatched to the affected sibling; these were therefore transferred, resulting in the birth of a healthy child, who was

4 Figure 1. Preimplantation HLA typing combined with PGD for betaglobin mutation. Upper panel: position of two different mutations and informative linked polymorphic markers used in PGD. Coloured arrows represent hemi-nested primers designed for mutation analysis. Application of outside primers for testing both maternal and paternal mutation improves the allele drop-out (ADO) detection rate. Two different artificially created restriction site primers (ACRS) were used in the second round of heminested PCR to detect the maternal and paternal mutations. Middle panel: restriction maps for the maternal (left) and paternal (right) mutation. The mentioned ACRS introduces BsaI restriction site to the mutant paternal sequence. Two different ACRS primers were designed to detect maternal mutation; one introduced the NheI restriction site to the mutant sequence, and the other introduced the RsaI restriction site to the normal betaglobin sequence, both increasing the reliability of the mutation analysis to overcome potential problems due to incomplete digestion of the PCR product. Bottom left: PB1 and PB2 analysis for the maternal mutation IVS I-5. Polyacrylamide gel electrophoresis of PCR product digested with RsaI (upper panel) and NheI (lower panel) restriction enzymes. Based on this analysis, oocytes 4 and 5 are predicted to contain the affected allele and the remaining oocytes (1, 2, 3 and 6) are mutation free. Bottom right: blastomere analysis of paternal and maternal mutations. Polyacrylamide gel electrophoresis of PCR product from blastomere amplification digested with BsaI restriction enzyme for the paternal (cd 8+G) mutation (top) and RsaI (bottom) confirming the presence of maternal mutation (IVS I-5). (haplotypes are shown on Figure 4). Two embryos (numbers 2 and 6) predicted to be carriers of the paternal mutation and representing full HLA match to the affected baby, were transferred, resulting in clinical pregnancy and the birth of a healthy child (see Figure 4). Std = size standard; A = affected; N = normal; M = maternal genomic DNA; P = paternal genomic DNA; PB1 = first polar body; PB2 = second polar body; Und = undigested PCR product; * = transferred embryos; ET = embryo transfer. 213

5 Figure 2. Preimplantation HLA typing combined with PGD for X-linked hyper-igm syndrome. Upper panel: position of the C218X mutation in exon 5 of CD40 ligand gene (Xq26.3) and tightly linked dinucleotide polymorphic markers inside the gene (exon 5) and outside the gene (DXS1187, DXS8094, DXS1062). Horizontal arrows represent primer positions. Vertical arrows indicate the location of MnlI and Cac8I restriction sites, and the positions of the dinucleotide polymorphic markers. Middle panel: restriction map of Cac8I restriction digestion, and polyacrylamide gel analysis of PCR product from 11 blastomeres digested with Cac8I restriction enzyme. All but two embryos (4 and 5) were predicted to be normal, based on the presence of two bands of 113 bp and 82 bp. Mutant 195-bp fragments were detected in blastomeres 4 and 5. Bottom panel: restriction map for MnlI digestion and polyacrylamide gel analysis of PCR product from 10 blastomeres digested with MnlI restriction enzyme. Embryos 1, 2, 3, 6, 7, 9 and 10 were predicted to be normal based on the presence of 82-bp bands. A mutant 60-bp fragment was detected along with the normal 82-bp band in embryos 4 and 5. By the presence of both normal and affected sequences and two polymorphic markers DXS1187, DXS 8094 and DXS 1062, these embryos were predicted to be carriers (smaller 13-bp and 24-bp bands are not shown, while the invariant 99-bp fragment created by the presence of the constant restriction site is seen in all samples). L = size standard; bp = base pairs; N = normal control DNA; Un = undigested PCR product. 214

6 Figure 3. PGD for X-linked hypohidrotic ectodermal dysplasia with immune deficiency (HED-ID). Upper panel: position of the L153R mutation in exon 5 of CD40 ligand gene (Xq26.3) and tightly linked dinucleotide polymorphic markers. Middle panel: restriction maps for AciI restriction enzyme. Bottom left: capillary electrophoregrams of fluorescently labelled PCR products of some of the blastomeres from embryos 12 and 26 for four polymorphic markers shown in upper panel. Bottom right: polyacrylamide gel electrophoresis of PCR product digested with AciI restriction enzyme. Based on this analysis, embryos 12 and 26 are predicted to be free from the affected maternal allele and, as will be seen in Figure 6, also HLA matched, so transferred. 215

7 Table 2. List of mutations and primers used for PGD of FAA, WAS, XALD, HYGM and HED-ID. Gene/ Upper primer Lower primer Annealing polymorphism temp 216 WAS (WASP gene) 5 TCAGCAGAACATACCCTCC 3 5 AGAGAGAGAAGGAGGAGAGG C Leu39Pro 5 TCAGCAGAACATACCCTCC 3 5 GAAGAAACGGTGGGGAC 3 53 C Hemi-nested PCR ScrFI cuts mutant allele WASP DXS TCAAATTGTGGGACGTACAC 3 5 GGTCCCCGTTCTTTTTG C Hemi-nested FL-PCR 5 FAM 5 GGTCCCCGTTCTTTTTG 3 53 C CAACAAACAGACCAGGGAC 3 HED-ID (NEMO- 5 GCTGACAGGAAGTGGCTTTTTA 3 5 CTCATTGCTTTTGGAAACCCT C IKBKG gene) Leu153Arg (T G) 5 GCTGACAGGAAGTGGCTTTTTA 3 5 CGACTCACCGACCCTCCA 3 60 C AciI cuts mutant allele Hemi-nested PCR HED-ID DXYS154 5 ACTCTCACCTATCCTATTCAACTTA 3 5 AAGTGATCCTCCCGCTTC C Hemi-nested FL-PCR 5 HEX 5 AAGTGATCCTCCCGCTTC 3 50 C ACATGATATTATATGTAGAAAATCC 3 HED-ID DXS AGCACCCAGTAAGAGACTG 3 5 TGAATCAATCTATCCATCTCTC C Nested FL-PCR 5 FAM CAGGCCACTACCACTTATG 3 5 TACTGTTTTCCACTCTAATGC 3 55 C HED-ID DXS GTGAAGCCAAGGTGGGAGGAT 3 5 GCCCTGGGGTACACAAGCC C Hemi-nested FL-PCR 5 HEX 5 GCCCTGGGGTACACAAGCC 3 55 C CACAGGCGTTCAAAACCAGC 3 HED-ID DXS TGAGGCAGGGCGCACTTG 3 5 CAGGAGGCCGTGTGAGAGC C Hemi-nested FL-PCR 5 TGAGGCAGGGCGCACTTG 3 5 FAM GGCTGCGCCAGTGAACAA 3 55 C HED-ID DXS GTCCATGAGGTATCCAAACAGG 3 5 GTCACAGTGACTCCATCCCAT C Hemi-nested FL-PCR 5 GTCCATGAGGTATCCAAACAGG 3 5 HEX GCTTGTAATCTCCAACCCCTA 3 55 C HED-ID DXS AGAGGGAGGCCAGCCATC 3 5 CCTTGGTGACATCGCTGTA C Nested FL-PCR 5 CACAGGCCTCCTGCATGATG 3 5 HEX TTCCAGGCTGGGGCTGCAC 3 55 C X-ALD (ABCD1 5 TTCTGGAACGCCTGTGGT 3 5 CTGGGTCTCACCTGACTCTG C gene) G343D (G A) 5 TTCTGGAACGCCTGTGGT 3 5 TGGCAGTGATGATGGGGA 3 55 C Hemi-nested PCR HaeIII cuts normal allele ABCD1 DXS GAAACTTAGAGGGTTGGCTT 3 5 CCCCAAAGAATGCCCT C Hemi-nested PCR 5 ACACTGCTCCCCTTGCC 3 5 HEX CCGAGTTATTACAAAGAAGCAC 55 C HIGM (CD40 gene) 5 TTCGAGTCAAGCTCCATTTATAG 3 5 AAAGGACGTGAAGCCAGTG C Cys218Stop (C A) 5 AGCCTCTGCCTAAAGTCCC 3 5 AAAGGACGTGAAGCCAGTG 3 63 C Hemi-nested PCR Cac8I cuts Normal allele MnlI cuts mutant allele CD40 DXS AATGTCACCATTAACAGTTTGG 3 5 GTAGCAACCCCATCAAACTAAA C Hemi-nested FL-PCR 5 AATGTCACCATTAACAGTTTGG 3 5 HEX 55 C CTTGTCCAGGACAGTTACATGA 3 CD40 DXS1187 Hemi-nested FL-PCR 5 TTAGAGCAGAGGTTTCTAGTCTTTC 3 5 GAGAAAGTCACTGAACAGAGGAGTT C 5 HEX TCCTTTCTACCCACTTTTTTCAA 3 5 GAGAAAGTCACTGAACAGAGGAGTT 3 55 C CD40 DXS CCCACCTAATAGGATTTTATGAA 3 5 CTAGTATTTCAAGAGCCAATGATTA C Hemi-nested FL-PCR 5 CCCACCTAATAGGATTTTATGAA 3 5 FAM CCTCAGTCAGTTGCCTGTTAAG 3 55 C 3 CD40 (CA)n 5 TTCCAATCTCTCTTTCTCAATCTCT 3 5 AACTGACTAGCAACGGCCTG C Hemi-nested FL-PCR 5 FAM TGTCAGTCTCTTCCCTCCCC 3 5 AACTGACTAGCAACGGCCTG 3 63 C FAA (FANCA gene) 5 AGCCAACGGGTGTGCG 3 5 GCTCTGGCGGGAAGGGA C Met1Lys (T A) 5 CAATAGGAAGGCAGCGCG 3 5 GAGTTCGGGACCCACGAG 3 47 C Nla III cuts normal allele Exon1 Nested PCR FAA (FANCA gene) 5 GATGGTGGGTTTCTCCGC 3 5 GGTCCTGATGGCTTCGCA C PCR Continued

8 Table 2. (continued). Exon 2 14-bp 5 GGGACCCCGTGTGTGAAT 3 5 GGTCCTGATGGCTTCGCA 3 55 C deletion Hemi-nested FAA (FANCA gene) 5 CTGCCTTGAACTCTTTTGC 3 5 GGAGGTCAGCGGTTTGTG C Thr1131Ala (A G) 5 CTGCCTTGAACTCTTTTGC 3 5 TCAGCGGTTTGTGAGGAC 3 55 C Exon 34 Fnu4HI cuts mutant allele TspRI cuts normal allele Hemi-nested PCR FAA D16S520 5 CGGTATTAGCAATCAGGG 3 5 CTTACCAACCTCCACAGC C Hemi-nested FL-PCR 5 FAM TTTCCAGAGAAAAAGAACAC 3 5 CTTACCAACCTCCACAGC 3 55 C FAA D16S TATTCCCCACAGTGCTAAG 3 5 AGCTTTTATTTCCCAGGG C Hemi-nested FL-PCR 5 HEX TGCCACATCTGGTCACTTA 3 5 AGCTTTTATTTCCCAGGG 3 55 C FAA D16S CTCCCTGAGCAACAAACA 3 5 CGCCTGATTTAGGCTTT C Hemi-nested FL-PCR 5 FAM CGTGGCTTCACTAAAACA 3 5 CGCCTGATTTAGGCTTT 3 55 C FAA D16S TCAGCCACACAGATAAACC 3 5 TTGTTTCCAATGTGTCTCC C Hemi-nested FL-PCR 5 HEX TGAGTTTGTTTTTTTCTGCT 5 TTGTTTCCAATGTGTCTCC 3 55 C FAA D16S408 5 TCTTATCCCAGGAACCC 3 5 CATCTGAGCAACCACCA C Hemi-nested FL-PCR 5 HEX CATGGTGATTGGGTCAAG 3 5 CATCTGAGCAACCACCA 3 55 C Figure 4. Preimplantation HLA matching combined with PGD for thalassemia. Upper panel: family pedigree with HLA haplotype analysis based on parental (1.1 and 1.2) and affected child s (2.1) genomic DNA testing. HLA marker s order is presented on the bottom left. Boldfaced numbers represent the affected maternal and paternal chromosomes 11. Paternal and maternal STR markers used for HLA testing, inherited by the affected child (2.1) are shown in boldface. Bottom panel: mutation and HLA testing in nine embryos, the upper row showing one affected embryo (embryo 4), five carriers and two normal embryos, and the lower row HLA typing, showing only two full matches (embryos 2 and 6), which were transferred, resulting in an HLA-matched unaffected child (testing of the baby showed that it originates from embryo 2). Paternally derived markers for each embryo are shown on the left and maternally derived markers on the right. Paternally and maternally derived matched markers are shown in boldface. 217

9 218 confirmed to be thalassaemia-free as well as HLA-matched to the affected sibling with thalassaemia (Figure 4). The diagnosis of unaffected HLA-matched status was also confirmed in the stillbirth, resulting from the other clinical pregnancy mentioned (the data are not shown). Of 73 embryos tested in 11 cycles for FA, 68 embryos were with conclusive data, of which 20 were predicted to contain two copies of the mutant gene and 48 were either homozygous normal or heterozygous; nine of the latter (13.2%) were also HLA-matched to the affected siblings in the family. These embryos, plus one with only maternal HLA match, were transferred in eight of 13 cycles, resulting in the birth of one full HLA-matched, and one maternal matched child free of FA-C in one and of FA-A in the other. The successful cord blood transplantation in one of the cases has been reported earlier, and the report of the other is still in preparation. Of the remaining five PGD cycles, performed for couples at risk for WAS, X-ALD, HIGM and HED-ID, the latter two resulted in the birth of unaffected HLA-matched children (Figures 5 and 6). In one of them performed for HIGM, of 15 oocytes tested by PB1 and PB2, five of 11 oocytes with conclusive results appeared to be free of maternal mutation, but only one was a maternal HLA match (see resulting embryo 2 in Figure 5). In addition, three of five oocytes with the maternal mutation were HLA matches (see embryos 11, 13 and 15 in Figure 5). However, embryos 13 and 15 were affected and a non-paternal match, while only maternal mutant chromosome was detected in embryo 11. Only one embryo (embryo 2), predicted to be mutation-free and maternal match by PB analysis, appeared to be a normal female with also a paternal match. The transfer of this single embryo resulted in a singleton pregnancy, confirmed to be unaffected and an HLA match by amniocentesis, resulting in the birth of a healthy HLA-matched baby girl. In a single cycle performed for HED-ID, 16 embryos yielded conclusive results, of which six were free of maternal mutation, based on PB1 and PB2 testing, but none of these was a maternal HLA match. As seen from Figures 3 and 6, of 16 resulting embryos for which blastomere biopsy results were available both for mutation analysis and HLA typing, three were affected males (embryos 17, 20 and 21; only the latter being HLA-matched), four were female carriers, two of which were non-matched (embryos 3 and 4), one was HLA recombinant (embryo 13) and one was HLA-matched (embryo 12). The remaining seven embryos were unaffected, including two male non-matched embryos (embryos 16 and 24), the former containing an extra maternal X-chromosome, and five normal female embryos, of which only one (embryo 26) was HLA-matched. This embryo, together with embryo 12, which was a normal female carrier, were transferred, resulting in a singleton pregnancy and the birth of an unaffected child which was confirmed to be HLA-matched to affected sibling. The other normal embryos, which were not HLA-matched to the affected sibling, were frozen for future use by the couple. Discussion The data presented here demonstrate the feasibility of practical application of PGD with HLA typing, despite the expected low probability of selecting HLA-matched unaffected embryos. As predicted in the first case report on PGD with HLA typing for FA (Verlinsky et al., 2001), the approach appeared extremely attractive for couples who had a child affected with a lethal congenital disease, for whom HLA-matched stem cell transplantation is the only hope (Kuliev and Verlinsky, 2004; Van develde et al., 2004). Overall, six unaffected HLAmatched pregnancies have been established, representing the first practical experience in the field, and complementing the most recent experience on preimplantation HLA typing without testing for the causative gene (Verlinsky et al., 2004). Because embryo selection in the latter series was based solely on HLA matching, 23% of the embryos were transferred, in agreement with the predicted probability, in contrast to the present data, which involved searching for HLA-matched embryos only among those that were also unaffected. Although the sample size is too small for comparison, only 13.6% of the HLA-matched embryos were also unaffected, which is lower than the expected 18.7%, and of course much lower than that in a sole preimplantation HLA typing (Verlinsky et al., 2004). This relatively low number of unaffected HLA-matched embryos detected in the present study may be influenced by the advanced reproductive age of the women involved (approximately 35 years), which is known to affect significantly the number of available embryos for testing and also the success rate of assisted reproduction. The mean number of embryos available from these women per cycle was under 10, which clearly limits the chances of finding a sufficient number of unaffected embryos that are also HLAmatched to a sibling. Assuming that one in four embryos is expected to be HLA-matched and three of four unaffected, the overall probability of HLA-matched embryos to be also unaffected could not be expected to be higher than one in 5.3 embryos. Therefore, with the availability of only 8.8 embryos on average with conclusive results in the current material, only 1.6 HLA-matched unaffected embryos are likely to have been detected. Assuming also that not all embryos develop to a level acceptable for transfer, approximately one embryo per cycle would have been available for transfer, which is of course below the optimal number of embryos to be replaced to ensure a clinical pregnancy and successful birth. Overall, 50 unaffected HLA identical embryos were transferred in 33 of 46 cycles performed (1.5 on the average), which resulted in an 18.2% pregnancy rate, representing the range expected in woman of advanced reproductive age. The other limitation associated with advanced reproductive age is a higher prevalence of aneuploidies, which should be tested at least for those chromosomes in which the causative genes are located, and for chromosome 6, where HLA genes are located, as this may affect the accuracy of PGD and HLA typing. Testing for chromosome 6 and for those chromosomes in which the causative genes for the above conditions are localized revealed 39 (9.8%) embryos with aneuploidies, including a total of 14 trisomies, 24 monosomies and one uniparental disomy, which may have affected the accuracy of diagnosis if not detected. Currently, information has been collected on testing aneuploidy rate for chromosome 6 in 397 embryos overall; 26 (6.6%) showed aneuploidy, and nine (2.3%) of these had trisomy 6 while 17 (4.3%) had monosomy 6 (unpublished data). This suggests that aneuploidies for

10 Figure 5. Preimplantation HLA matching combined with PGD for hyper-igm. Upper panel: family pedigree with HLA haplotype analysis based on parental (1.1 and 1.2) and affected child s (2.1) genomic DNA testing. HLA markers order is presented on the bottom left. Boldfaced numbers represent the affected maternal chromosome X. Paternal and maternal STR markers used for HLA testing, inherited by the affected child (2.1) are shown in boldface. Bottom panel: mutation and HLA testing in 12 embryos, the upper row showing three affected embryos (embryos 11, 13, 15, the former being monosomy X), two carriers and seven normal embryos, and the lower row HLA typing, showing only two full matches (embryos 2 and 11), of which only embryo 2 was also unaffected and transferred, resulting in an HLA-matched unaffected child. Paternally derived markers for each embryo are shown on the left and maternally derived markers on the right. Paternally and maternally derived matched markers are shown in boldface. 219

11 Figure 6. Preimplantation HLA matching combined with PGD for HED-ID. Upper panel: family pedigree with HLA haplotype analysis based on parental (1.1 and 1.2) and affected child s (2.1) genomic DNA testing. HLA marker s order is presented on the bottom left. Boldfaced numbers represent the affected maternal chromosome X. Paternal and maternal STR markers used for HLA testing, inherited by the affected child (2.1) are shown in boldface. Bottom panel: mutation and HLA testing in 16 embryos, the upper row showing three affected embryos (embryos 17, 20 and 21), four carriers and nine normal embryos, and the lower row HLA typing, showing only two full matches (embryos 12 and 26), both being also unaffected and transferred, resulting in an HLAmatched unaffected child. Paternally derived markers for each embryo are shown on the left and maternally derived markers on the right. Paternally and maternally derived matched markers are shown in boldface. 220

12 chromosome 6 and any other chromosome in which the causative gene is localized should be routinely tested simultaneously with HLA typing and mutation testing in the same multiplex PCR system. This allows detection of the copy number of chromosomes, as well as uniparental disomies of chromosome 6, which may result in misdiagnosis of the HLA matching. Finally, one of the most important phenomena affecting the accuracy of preimplantation HLA typing is possible recombination in the HLA region, which may lead to misdiagnosis if not detected. As demonstrated in this study, the microsatellite markers linked to the HLA alleles may be tested simultaneously in the multiplex PCR system, allowing detecting recombination in the HLA region, observed in 4.3% of cases in the present data (not shown). Because preimplantation HLA typing cannot identify matches for recombinant siblings, haplotype analysis prior to initiation of the actual cycle is required, to inform the couples about their options. For example, in one of the cases performed for thalassaemia, the fact that the child was recombinant became obvious only during PB1 analysis, without which maternal haplotypes cannot be established. While paternal haplotypes may be identified through sperm typing, testing for maternal haplotypes requires maternal somatic cell haploidization, which may be performed by somatic cell nuclei transfer and fusion with matured oocytes (Tesarik and Mendoza, 2004). Despite the need for further improvement of the technique as mentioned, the presented results show that couples with affected children requiring HLA compatible stem cell transplantation have a realistic option to undergo IVF and PGD with combined preimplantation HLA typing, so as to have an unaffected HLA-matched child as a potential donor of compatible stem cells for sibling. Together with the previously reported application of PGD for FA-C, and the most recent report on preimplantation HLA typing not involving testing for the causative gene, at least 10 HLA-matched children have already been born, and confirmed to be HLA matches for affected siblings. Although not all these sibling have yet been treated, preliminary results provide encouraging results. At the present time, umbilical cord blood stem cells collected from these children have already been transplanted in siblings with FA-C (Verlinsky et al., 2001) and Diamond Blackfan anaemia (Verlinsky et al., 2004), resulting in successful hsematopoietic reconstitution in both, which suggests that it is an acceptable approach for the couples at risk, despite being highly controversial. On the other hand, it also overcomes an important controversy in therapeutic cloning, as it avoids the use of surplus human embryos to obtain embryonic stem cells. In addition, no embryo is discarded based on the results of preimplantation HLA typing, as all unaffected embryos may be frozen for future use by the couple. Therefore, couples at risk of having children with congenital bone marrow disorders have to be informed about currently available options not only for avoiding the birth of affected child, but also for selecting a suitable stem cell donor for affected siblings, which may presently be the only hope for treating of siblings with congenital bone marrow failure. Acknowledgements We are indebted to our colleagues in DNA and embryology laboratories, T Sharapova, S Ozen, K Lazyuk, G Wolf, Y Illkevitch, V Galat and V Kuznetsov, and also to our genetic counsellors, C Lavin, R Beck, R Genoveze and D Pauling, for their participation in acquisition of the data and technical assistance. References Damewood MD 2001 Ethical implications of a new application of preimplantation diagnosis. Journal of the American Medical Association 285, Edwards RG 2003 Social and ethical issues of PGD, cloning and gene therapy. Reproductive BioMedicine Online 6, Foissac A, Salhi M, Cambon-Thomsen A 2000 Microsatellite in the HLA region: 1999 update. Tissue Antigens 55, Fost NC 2004 Conception for donation. Journal of the American Medical Association 291, Kuliev A, Verlinsky Y 2004 Thirteen years experience of preimplantation diagnosis: report of the Fifth International Symposium on Preimplantation Genetics. Reproductive BioMedicine Online 8, Kuliev A, Rechitsky S, Verlinsky O et al Birth of healthy children after reimplantation diagnosis of thalassemias. Journal of Assisted Reproduction and Genetics 16, Online Mendelian Inheritance in Man (OMIM) 2001 Johns Hopkins University [accessed 21 June 2004]. Tesarik J, Mendoza C 2003 Somatic cell haploidization: an update. Reproductive BioMedicine Online 6, Van de Velde H, Georgiou I, De Rycke M et al Novel universal approach for preimplantation genetic diagnosis of β-thalassemia in combination with HLA matching of embryos. Human Reproduction 19, Verlinsky Y, Kuliev A 2000 Atlas of Preimplantation Genetic Diagnosis. Parthenon, New York, London. Verlinsky Y, Rechitsky S, Schoolcraft W et al Preimplantation diagnosis for Fanconi anemia combined with HLA matching. Journal of the American Medical Association 285, Verlinsky Y, Rechitsky S, Sharapova T et al Preimplantation HLA typing. Journal of the American Medical Association 291, Received 21 May 2004; refereed 10 June 2004; accepted 16 June