PGD for inherited cardiac diseases

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1 Reproductive Bioedicine Online (2012) 24, ARTICLE for inherited cardiac diseases Anver Kuliev *, Ekaterina Pomerantseva, Dana Polling, Oleg Verlinsky, Svetlana Rechitsky Reproductive Genetics Institute, 2825 Halsted Street, Chicago, IL 60657, USA * Corresponding author. address: anverkuliev@hotmail.com (A Kuliev). Dr Anver Kuliev received his PhD in clinical cytogenetics in In 1979, he took the responsibility for the World Health Organization s Hereditary Diseases Programme in Geneva, where he developed the communitybased programmes for prevention of genetic disorders and early approaches for prenatal diagnosis. He moved to the Reproductive Genetics Institute in 1990, where he heads scientific research in prenatal and preimplantation genetics. He is an author on 185 papers, including 11 books in the above areas, five of which in the field of preimplantation genetics. Abstract Preimplantation genetic diagnosis () has been applied for more than 200 different inherited conditions, with expanding application to common disorders with genetic predisposition. One of the recent indications for has been inherited cardiac disease, for which no preclinical diagnosis and preventive management may exist and which may lead to premature or sudden death. This paper presents the first, as far as is known, cumulative experience of for inherited cardiac diseases, including familial hypertrophic and dilated cardiomyopathy, cardioencephalomyopathy and Emery Dreifuss muscular dystrophy. A total of 18 cycles were performed, resulting in transfer in 15 of them, which yielded nine unaffected pregnancies and the births of seven disease- or disease predisposition-free children. The data open the prospect of for inherited cardiac diseases, allowing couples carrying cardiac disease predisposing genes to reproduce without much fear of having offspring with these genes, which are at risk for premature or sudden death. RBOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: cardioencephalomyopathy, Emery Dreifuss muscular dystrophy, familial hypertrophic and dilated cardiomyopathy, inherited cardiac disease,, premature or sudden death Introduction Preimplantation genetic diagnosis () is currently an established clinical procedure in assisted reproduction and genetic practices (ESHRE, 2011; IS, 2008), which has been applied for more than 230 different inherited conditions with extremely high accuracy (Kuliev and Rechitsky, 2011; Liebaers et al., 2010; IS (2010); Rechitsky et al., 2011; Verlinsky and Kuliev, 2006). Its application has been expanding beyond traditional indications of prenatal diagnosis and currently includes common disorders with genetic predisposition, such as inherited forms of cancer (Rechitsky et al., 2002; Verlinsky and Kuliev, 2006). This applies also to the diseases with no current prospect of treatment, which may manifest despite presymptomatic diagnosis and follow up, when may provide the only relief for the at-risk couples to reproduce. The available experience already includes for dozens of couples at risk, who have had success in producing healthy children free from predisposition to common diseases (Rechitsky and Kuliev, 2010; Rechitsky et al., 2002) /$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi: /j.rbmo

2 444 A Kuliev et al. The first case of for inherited cardiac disease was described for a couple at risk for producing offspring with Holt Oram syndrome, which is an autosomal dominant condition determined by mutation in TBX5 (He et al., 2004). Holt Oram syndrome is characterized by atrial septal defect and cardiac conduction disease, together with upper extremity malformations, although these clinical manifestations may be extremely variable. They rarely present at birth or only present with a sinus bradycardia, as the only clinical sign, which might also be left unnoticed. As in for other common disorders, the fact that inherited cardiac disorders may not be realized even during a lifetime, makes the application of controversial, perhaps explaining the limited application of for inherited cardiac diseases at the present time. The majority of inherited cardiac disorders are dominant, for which no cure may be administered, because their first and only clinical occurrence may be a premature or sudden death. One of such conditions is the familial hypertrophic cardiomyopathy (HC), which clinically manifests at different ages, with no symptoms observed for years until provoked by different factors, such as excessive exercise. Different conditions leading to HC have been reported, two of which, HC4 and HC7, will be described in this paper. HC4 is caused by mutation in the gene YBPC3 located on chromosome 11 (11p11.2), encoding the cardiac isoform of myosin-binding protein C, exclusively in heart muscle (I ). HC7 is caused by a mutation in TI3 located on chromosome 19 (19q13.4), leading to an asymmetric ventricular hypertrophy and defect in interventricular septum, with high risk of cardiac failure and sudden death (I ). Hypertrophic cardiomyopathy is also one of the clinical manifestations of fatal infantile cytochrome C oxidase deficiency (I ), for which is strongly indicated, as described below. In contrast to above conditions, this is an autosomal recessive cardiac disease, presenting within the first month after birth and characterized by a generalized congenital muscular dystrophy, similar to spinal muscular atrophy, but with significant reduction or lack of cytochrome C oxidase in muscles (Papadopoulou et al., 1999). This devastating disease is caused by the defect in SCO2 located on chromosome 22 (22q13), although the same condition may also be determined by mutations in at least 10 other genes involved in Cox activity. The other condition, for which is strongly indicated, is dilated cardiomyopathy (CD), which is an autosomal dominant disease, caused by different mutations in LA located on chromosome 1 (1q21.2; I 100). This cardiac disease is characterized by ventricular dilation and impaired systolic function, resulting in a heart failure and arrhythmia, which causes premature or sudden death. While the large phenotypic variability of patients may be determined by different mutations in LA, differences from one family to another may be observed within the same mutation, with possible involvement of skeletal muscles that leads to the muscles weakness, similar to that in Emery Dreifuss muscular dystrophy (ED), which is an X-linked disease, also characterized by cardiomyopathy, although presenting within the first year after birth (I ). This paper presents the first cumulative experience of application in 18 cycles of inherited cardiac disorders. aterials and methods A total of 18 cycles for nine couples at risk for producing an affected progeny with the above conditions were performed, including nine cycles for CD, three for CH4, one for CH7, three for cardioencephalomyopathy and two for ED (Table 1). One of the four couples at risk for producing a progeny with CD (Figure 1) requested prospectively, with no previous pregnancies attempted, because the male partner was the carrier of the LA mutation predisposing to CD. He first experienced cardiac symptoms, such as palpitations, at the age of 22 and then was diagnosed to have a ventricular tachycardia in a 48-h Halter monitoring at the age of 26. To prevent the risk for the development of cardiomyopathy and arrhythmias, which can lead to sudden death, a cardioverter defibrillator was implanted. As seen from Figure 1, the patient s father passed away from sudden death at age 32, after experiencing heart failure due to cardiomyopathy. Also his father s sister was diagnosed with cardiomyopathy at age 49 and his grandfather and great aunt and her son died at age from cardiovascular complications. The patient had a dominant mutation in LA as a result of C to T change in codon 1033 (c.1033c>t), leading to amino acid change from Arg to Trp in position 335 of the proteins lamin A and lamin B, involved in the heart muscle work. This mutation was detected by spi digestion, which creates two fragments of 90 and 95 bp in the PCR product of the normal LA allele, leaving the mutant one uncut. As seen from Table 2, four polymorphic markers were also tested simultaneously with the mutation analysis, including D1S2714, D1S2777, D1S2624 and D1S506, to avoid misdiagnosis due to preferential amplification or allele drop out (ADO) of the genes tested. Four cycles were performed for three patients with CH4 and CH7, determined by mutation in YBPC3 and TI3, respectively. one of these couples had previous progeny, but had a family history of premature or sudden death. As seen from Figure 2A, CH4 in one of the families was due to frameshift mutation D1076fs in YBPC3, while CH7 in the other family was caused by the AV mutation in TI3 (Figure 2B). The D1076fs mutation in YBPC3 was detected by RsaI and BsaHI digestion, the first cutting the mutant gene into two fragments of 72 and 60 bp, and the second cutting the normal one into two fragments of the same size. In addition, five polymorphic markers were also used to exclude the possibility of ADO, including D11S1978, D11S4, D11S4117, D11S1350 and D11S4147. The AV mutation in TI3 was detected by the use of the two enzymes, HaeII, cutting the normal gene, and BspI, cutting the mutant gene into two fragments, as presented in Table 2. Three cycles were performed for cardioencephalomyopathy in a couple with a child affected with left ventricular hypertrophic cardiomyopathy, whose first symptoms were manifested as early as 1.5 months, with a severe respiratory attack. A maternal mutation, EK of SCO2, in this case was detected by HindIII and BsrBI digestion, the first cutting the mutant and the second cutting the normal gene

3 Table 1 Reproductive outcome of for cardiac diseases. Disease and gene (mutation) Patients (cycles) Embryos Transfers Embryos Total received ormal or Abnormal a Inconclusive b transferred (amplified) carrier Clinical pregnancies Cardioencephalomyopathy (AR) SCO2 (R262del(CA); EK) 1 (3) 33 (32) Cardiomyopathy dilated; CD1 (AD) LA (K270K) 1 (4) 51 (47) c LA (R335T) 1 (1) 11 (11) LA (R1P) 1 (1) 2 (2) LA (T528K) 1 (3) 44 (34) Cardiomyopathy familial, hypertrophic 4; CH4 (AD) YBPC3 (D1076fs) 1 (1) 7 (6) YBPC3 (IVS11 10C A) 1 (2) 10 (8) Cardiomyopathy familial, hypertrophic 7; CH7 (AD) TI3 (AV) 1 (1) 11 (10) Emery Dreifuss muscular dystrophy 1, X-linked ED 1 (2) 31 (31) Total 9 (18) 200 (181) a Including aneuploidies. b Shared markers in parents, making impossible to exclude allele drop out. c Including one pair of twins. Births for inherited cardiac diseases 445

4 446 A Kuliev et al. A Pt. DOB 08/04/80 B Embryo # arkers: D1S2714 D1S2777 LA-R335T D1S2624 D1S R335T R335T FA 156 R335T FR FR FR Figure 1 for dilated cardiomyopathy (CD), determined by the dominant mutation R335T in LA. (A) Family pedigree of a couple with affected husband carrying the R335T mutation in LA. Paternal linked polymorphic markers are shown on the left and maternal on the right, and the order of the markers and mutation in LA are shown on the far left. (B) Blastomere results revealed two embryos carrying the R335T mutation in LA (embryos 9 and 12), while nine embryos were free of the mutation. Two of these embryos (1 and 8) were transferred, resulting in a singleton pregnancy and the birth of a healthy child without the predisposing gene to CD (as indicated in the family pedigree by ). = embryo transfer; FA = failed amplification; FR = frozen. (Table 2). The paternal mutation R262del(CA) was tested by sequencing, which resulted in detection of a 139-bp fragment in the normal gene and a -bp fragment in the mutant gene. Five polymorphic markers, D22S1153, D22S1160, D22S1161, D22S922 and SP laiii, were also tested simultaneously, to avoid misdiagnosis due to ADO (Table 2). Finally, two cycles were performed for a couple at risk for producing offspring with ED, through testing for maternal mutation IVS2+1G T, using BpmI digestion, which cuts the normal gene into two fragments of 115 and 6 bp, with the mutant one left uncut. In addition, five polymorphic markers, DXS8103, DX1684, DXS8087, DXS1073 and DYS154, were tested to exclude the presence of ADO (Table 2). All cycles were performed using a standard IVF protocol coupled with micromanipulation procedures for sequential first and second polar body (PB) sampling and/or embryo biopsy, described elsewhere (Verlinsky and Kuliev, 2005). The biopsied PB and blastomeres were tested by the multiplex nested PCR analysis, involving the above-mentioned mutations and linked marker analysis in a multiplex hemi-nested system (Verlinsky and Kuliev, 2006). Except for the case of ED, for which PB biopsy procedure was performed, all others were tested by embryo biopsy at the cleavage stage. In cases of advanced reproductive age, aneuploidy testing by fluorescent in-situ hybridization, described previously (Verlinsky and Kuliev, 2006), or by microarray technique for 24 chromosomes, using array comparative genomic hybridization (24sure; BlueGnome, UK), was performed, the latter requiring a blastocyst biopsy and embryo freezing (Verlinsky and Kuliev, 2005), with their transfer in a subsequent cycle. Pregnancy outcome was defined as the presence of a gestational sac with fetal cardiac activity. As per the informed consent, approved by the Institutional Review Board (RGI IRB/ ), the embryos derived from the oocytes free of genetic predisposition to cardiac disease, based on mutation and polymorphic marker information, were preselected for transfer back to patients, while the affected ones were tested to confirm the diagnosis. Results As seen from Table 1, of 18 cycles performed for nine at-risk couples, 30 cardiac disease predisposition-free embryos were preselected for transfer in 15 transfer cycles (two embryos per transfer cycle, on average), resulting in nine pregnancies (60% pregnancy rate per transfer) and the births of seven disease- or disease predisposition-free children. In nine cycles performed for four patients with CD, a total of 108 embryos were tested, of which 37 of 94 with

5 Table 2 Primers and reaction conditions for of cardiac diseases. utation or polymorphism Cardiomyopathy, dilated, CD1A, LA R335T Outside: R335T-1: GTCTCCTACACCGACCCACGT Inside: R335T-3: GCTCACCAAACCCTCCCAC D1S2714 hemi-nested Outside: : TGTGGGGGCTGAGATGAAT Inside: : Hex CCCAGGATTTTAAGACCAGC D1S2777 hemi-nested Outside: : CACCACGGAACTCCAGTAT Inside: : CACCACGGAACTCCAGTAT D1S2624 hemi-nested Outside: : GAGGCAGAGGCAGACACAGATG Inside: : Hex ATGGGGCTGACACTCTATGAGG D1S506 hemi-nested Outside: 506-1: CTGGACTCAGCCTGAGAAGAATATG Inside: 506-3: Fam AGAAAGGGAGGGATCGTTCAG Cardiomyopathy, familial hypertrophic, CH4, YBPC3 D1076fs Outside: D1076fs-1: CTGGTTGGCAGGGTGG Inside: D1076fs-3: AGGCGTGGTGACCCAACTG D11S1978 hemi-nested Outside: : TGCACTCCACAAATACACACAATT Inside: : Hex CAGAATGTTAGTATAAGTGTGCATGTG D11S4 hemi-nested Outside: 4-1: GCCTCCTGTTCTGTTATTTCACTTA Inside: 4-3: Fam TGACTTTAGCCTTGTGCTGAACTG D11S4117 hemi-nested Outside: : TTGTCTTCTTTCTAATCTTCCTTCCA Inside: : TTGTCTTCTTTCTAATCTTCCTTCCA Primers ( ) Product sizes (bp) Annealing temperature ( C) Forward Reverse R335T-2: CGTGGATCTCCATGTCCAGG spi: mutant 185, normal R335T-4: GTCCAGAAGCTCCTGGTACTCGT : AGACTCTGGAGTAGCAGGGACTA : AGACTCTGGAGTAGCAGGGACTA : CAAGTAATCCTCCTGCCTCAG : Hex TGTTGGGATTACAGGTGTGAG : GACTCAGCGTCCTGCACAGAGT : GACTCAGCGTCCTGCACAGAGT 506-2: GCTATGCTGGGGCAAGGG : GCTATGCTGGGGCAAGGG D1076fs-2: TCTTCTTGTCGGCTTTCTGCA D1076fs-4: TCCGTGTTGCCGACATCCT : ACTTAGATGTCCATCGACAGATGAA : ACTTAGATGTCCATCGACAGATGAA 4-2: CAGCGCCTGGCTTGTACATAT 4-2: CAGCGCCTGGCTTGTACATAT : GTGAGCAAGAGATCACGCCAC : Fam TGACAGAGCGAGACTCCATCTAAAA RsaI: mutant , normal ; BsaHI: mutant, normal (continued on next page) for inherited cardiac diseases 447

6 Table 2 (continued) utation or polymorphism Primers ( ) Product sizes (bp) Annealing temperature Forward Reverse ( C) D11S1350 hemi-nested Outside: : : CAAATTAAATCATTCTGGGGTCTTT AAACTACCAGCAGTAGAGCACACCT Inside: : Fam : AAACACCTGCTCTCCAAGAATATC AAACTACCAGCAGTAGAGCACACCT D11S4147 hemi-nested Outside: : : AGCTTTTCCCTTGTGGGTGTT GCCAGCCTATCTAAACTGTATAATT Inside: : Fam AAGGGGAAGACGGACATAAAAC : GCCAGCCTATCTAAACTGTATAATT Cardiomyopathy, familial hypertrophic, CH7, TI3 AV Outside: AV-1: AAAAAGGAGTGTAGGATGGAGGAGT Inside: AV-3: GGTGTGCGGGAAATGGAAG D19S867 Outside: 867-1: CAATGAAAATGCTTTGTAAAACTCTT Inside: 867-1: CAATGAAAATGCTTTGTAAAACTCTT D19S904 Outside: 904-1: AATCACACCATTGTACTCCAGCC Inside: 904-3: HEX AGGGCAAGACTCCGTTTCAA D19S246 Outside: 246-1: GTGAGCCAAGACTACGCCACT Inside: 246-3: Fam AGAGTGAGATTCCACCTTTCAAAAA D19S Outside: -1: TTTTCCTATTTTATCTGGCGGG Inside: -3: FA AAGTGAAAGCCGAAGTCTTTTCA D19S571 Outside: 571-1: TGAACTCCAGCCTGGGTGAG Inside: 571-1: TGAACTCCAGCCTGGGTGAG Cardioencephalomyopathy, SCO2 EK Outside: -1: AGCAGCAAAAGCGAACAGAAG Inside: -1 AV-2: TTCCCCTCAGCATCCTCTTTC HaeII: mutant 226, normal + 26; BspI; mutant , normal AV-4: TTCTCGGTGTCCTCCTTCTTCA 867-2: TTGGTTTCCTTCTGTCATGTCATC 867-3: Fam TCAGAGGTGACCAGTTCTTTCATAC 904-2: TCGGAGATGTTAAAATGTGAAAAAC 904-2: TCGGAGATGTTAAAATGTGAAAAAC 246-2: CCAGAAACACATCATTTACCCACTT 246-2: CCAGAAACACATCATTTACCCACTT -2: TCATCAAGTCTGTTCCAGCCAA : TCATCAAGTCTGTTCCAGCCAA 571-2: TTGACAGCATGTATTTGAAATATGG 571-3: Hex AGTTACACGTATACATGAAATGACTGA : TCGGGGTCCACAGTGATGAAG HindIII: mutant , normal mismatch: CACCTGCACCAGCTTCTCAA -7 mismatch: ACCTGCACCAGCTTCTCCCG BsrBI: mutant 185, normal (continued on next page) 448 A Kuliev et al.

7 Table 2 utation or polymorphism (continued) Primers ( ) Product sizes (bp) Annealing temperature Forward Reverse ( C) R262del(CA) Outside: 262-3: CAAGGATGAGGACCAGGACTACA 262-2: CCAGACTGCAGTGGCTCAAGA utant, normal 139 Inside: 262-4: Fam TGCCATCTACCTGCTCAACCC 262-2: CCAGACTGCAGTGGCTCAAGA D22S1153 Outside: : GTAGAGGTTGCAGTGAGCCATGA : AACCCTGCTCCTAGCCTTCCT Inside: : AACCCTGCTCCTAGCCTTCCT D22S1160 Outside: : TTTGGGGAAGCAGTGAGTCACTA : TCTCAGGGATGCTTTCCCATG Inside: : TTTGGGGAAGCAGTGAGTCACTA : Hex ATTTGCAGATGACGAACATGTATCA D22S1161 Outside: : ACAAGGTGGACCCGAATCAGA : CGAGTTTGTGGTGGTTTGTTACAG Inside: : ACAAGGTGGACCCGAATCAGA : Fam TAGCAGGCCAAGCCGAAGA D22S922 Outside: 922-1: CGATGGGATGTCTGTGGGG 922-2: GGTTTCCTCAGTTTTACCTGTGCT Inside: 922-3: FA 922-2: GGTTTCCTCAGTTTTACCTGTGCT GGGTTGGAACTGTTAGGTATCTTG SP laiii Outside: laiii-1: TGCCAAGAACATAGTGGGTGA laiii-2: CAGCCTCTGAGCCACCGA +/ Inside: laiii-1: TGCCAAGAACATAGTGGGTGA laiii-3: CCACACCTCTAAGTCACAAAGC Emery Dreifuss muscular dystrophy X-linked, ED IVS2+1G T Outside: IVS2-1: CAACTCGTAGGCTTTACGAGAA IVS2-2: CTTTCTCCAGTGCCGCTCT BpmI: mutant, normal Inside: IVS2-1: CAACTCGTAGGCTTTACGAGAA IVS2-3 mismatch: CCACAGGCGAGGCTCTCT DXS8103 Outside: GTGAAGCCAAGGTGGGAGGAT GCCCTGGGGTACACAAGCC Inside: Hex CACAGGCGTTCAAAACCAGC GCCCTGGGGTACACAAGCC DXS1684 Outside: AGCACCCAGTAAGAGACTG TGAATCAATCTATCCATCTCTC Inside: Fam CAGGCCACTACCACTTATG TACTGTTTTCCACTCTAATGC DXS8087 Outside: TGAGGCAGGGCGCACTTG CAGGAGGCCGTGTGAGAGC Inside: TGAGGCAGGGCGCACTTG Fam GGCTGCGCCAGTGAACAA DXS1073 Outside: GAAACTTAGAGGGTTGGCTT CCCCAAAGAATGCCCT Inside: ACACTGCTCCCCTTGCC Hex CCGAGTTATTACAAAGAAGCAC DYS154 Outside: ACTCTCACCTATCCTATTCAACTTA AAGTGATCCTCCCGCTTC Hex ACATGATATTATATGTAGAAAATCC AAGTGATCCTCCCGCTTC for inherited cardiac diseases 449

8 450 A Kuliev et al. A C ARKERS: D11S4 D11S4 YBPC3-D1076fs D11S4117 D11S1350 D11S D1-76fs D19S902 D19S867 D19S904 D19S246 TI 3-AV D19S D19S AV B D EBRYO # F 1 FA D1-76fs F D1-76fs D1-76fs AV AV AV AV AV 123 FA AV AV 123 F* FR* FR* * ormal for 24 chromosome by microarray Figure 2 for hypertrophic cardiomyopathy (CH). (A, B) for CH4. (A) Family pedigree of a couple with affected mother carrying the frameshift mutation D1076fs in YBPC3. Paternal linked polymorphic markers are shown on the left and maternal on the right, and the order of the markers and frameshift mutation in YBPC3 are shown on the far left. (B) Blastomere results revealed three embryos (embryos 7, 9 and 10) carrying the frameshift mutation D1076fs in YBPC3, three unaffected embryos and one embryo that did not amplify. Two of the normal embryos were transferred (embryos 3 and 8), following cryopreservation (frozen embryo transfer, F), resulting in an unaffected pregnancy (as indicated in the family pedigree by ). (C, D) for CH7. (A) Family pedigree of a couple with affected father carrying the AV mutation in TI3. Paternal linked polymorphic markers are shown on the left and maternal on the right, and the order of the markers and mutation in TI3 are shown on the far left. (B) Blastomere results revealed three mutation-free embryos, based on the testing of the mutation and six polymorphic markers (embryos 4, 5 and 11), seven mutant embryos and one embryo that did not amplify. Unaffected embryos were tested for 24 chromosome aneuploidy at the blastocyst stage, of which one (embryo 4) was euploid and transferred in the subsequent cycle. FA = failed amplification; F = frozen embryo transfer; FR = frozen. results were free of mutant genes tested, 48 were mutant and nine had with inconclusive results, due to shared markers in parents, making it impossible to exclude the possibility of ADO. Fifteen of the mutation-free embryos were transferred in eight cycles, yielding the births of four healthy children, including one pair of twins, free from predisposition to sudden death. One of the cases of for CD, determined by dominant mutation in LA, is demonstrated in Figure 1, showing that of 10 of 11 embryos tested for mutation and four linked polymorphic markers, two were found to carry the R335T mutation in LA and eight were free of the R335T mutation. Two of these embryos were transferred, resulting in a singleton pregnancy and the birth of a healthy child without the gene predisposing to CD. Of four cycles performed for three couples at risk for producing offspring with CH, a total of 28 embryos were tested, of which four did not amplify, 16 with results were mutant, seven were free of mutation and one had inconclusive results due to the same reason as above. Three of the mutation-free embryos were transferred in two cycles, yielding a singleton pregnancy, presented in Figure 2A. As seen from this figure, of the seven embryos tested, three (embryos 7, 9 and 10) were carriers of the frameshift mutation D1076fs in YBPC3, three were unaffected and one did not amplify. Two of the normal embryos were transferred following freezing, resulting in an unaffected pregnancy. The results of the cycle for the patient at risk for producing offspring with CH7 are presented in Figure 2B. Of 11 tested embryos, 10 amplified, of which three (embryos 4, 5 and 11) were unaffected, based on the testing of the mutation and six polymorphic markers. Because these embryos were also tested for 24 chromosome aneuploidy by array comparative genomic hybridization at the blastocyst stage, the embryos were frozen and one of them (embryo 4), which was also aneuploidy free, was transferred in the subsequent cycle. Of three cycles performed for cardioencephalopathy, 32 of 33 embryos tested amplified, of which 16 were unaffected. Seven of these embryos were transferred, resulting in two unaffected pregnancies and the birth of a healthy child free from cardioencephalopathy. The results of one of these cycles are presented in Figure 3, showing that of nine embryos tested, two embryos (embryos 1 and 2) were

9 for inherited cardiac diseases 451 A ARKERS: D22S1153 D22S1160 D22S1161 D22S922 SCO2 SP-laII R262delCA EK R262delCA EK + +, RECOBIAT B EBRYO # R262EK delca R262EK delca R262 delca R262 delca R262 delca + CARRIER CARRIER OOSOY22 Figure 3 for cardioencephalomyopathy. (A) Family pedigree of a couple with a previous affected child, who was double heterozygous for EK and R262del(CA) in SCO2. Paternal polymorphic markers are shown on the left and maternal on the right, with the order of the markers and mutation shown on the far left. (B) Blastomere results revealed two homozygous affected embryos (embryos 1 and 2), two carriers of the paternal mutation (embryos 4 and 7), four mutation-free embryos (embryos 3, 5, 6 and 8) and one embryo monosomic for chromosome 22 (embryo 9), based on the testing of the mutation and six polymorphic markers. Two mutation-free embryos (embryos 3 and 5) were transferred, resulting in a singleton pregnancy and the birth of an unaffected child (as indicated in the family pedigree by ). = embryo transfer. homozygous affected, two (embryos 4 and 7) were carriers of the mutant gene, one (embryo 9) was monosomic for chromosome 22 and four (embryos 3, 5, 6 and 8) were free of the mutation. Two of these embryos (embryos 3 and 5) were transferred, resulting in a singleton pregnancy and the birth of an unaffected child. In two cycles performed for ED, all 31 embryos amplified, of which 17 disease-free embryos were detected. Five of these embryos were transferred, yielding an unaffected pregnancy in each cycle and the births of two ED-free children. One of these cycles is presented in Figure 4, demonstrating the results of sequential PB1 and PB2 analysis for IVS2+1G T mutation, followed by mutation and aneuploidy testing at the cleavage stage. Three embryos (embryos 4, 5 and 9) originating from mutant oocytes were male and embryo 1 originating from the oocyte with unknown genotype due to failed amplification could not be preselected for transfer. The other embryo (embryo 10), also originating from a mutant oocyte, was male and also monosomic for the X chromosome. The remaining two embryos (embryos 6 and 11) were free of mutation and aneuploidy and were transferred, resulting in the birth of an unaffected child. Discussion Presented results show that may be a realistic option for couples at risk for producing offspring with cardiac disease, determined by inherited predisposition. Inheritance of such susceptibility factors place the individual at risk of serious cardiac disease clinically manifested either in early childhood, such as in cardioencephalopathy, or later in adult life, with the only clinical realization manifesting in premature or sudden death, as in CD and CH. Conditions in the family history of the couples at risk that may indicate a possible need for may be a heart attack and sudden death at young ages, family members with pacemakers or internal cardiac defibrillators, arrhythmia and heart surgery. The chances that the offspring of these patients will develop the same heart disease will differ depending on the mode of inheritance, but their penetrance is difficult to predict, because many inherited cardiac conditions are difficult to diagnose and will develop with age and may be induced by certain medications or activities, such as excessive exercise, which may lead to cardiac arrest or sudden death, justifying the parents requests for. In

10 452 A Kuliev et al. A DXS8103 DXS1684 DXS8061 DXS8087 ED-IVS2+1G-T DXS1073 F8 IT1 F8IT 13 DXY154 I. II. III /167 Y 1 Y ED Y B OOCYTES C PCR EBRYO FA FA PCR/FISH 1 Y XY XY XX XY Y0 1 Y Figure 4 for Emery Dreifus muscular dystrophy (ED). (A) Family pedigree of a couple with mother (II-2) carrying X-linked ED IVS2+1G T mutation, inherited from her father (I-1). Paternal polymorphic markers are shown on the left and maternal on the right, with the order of the markers and mutation shown on the far left. The haplotypes for the patient s father (I-1) are also shown on the left. (B) Sequential first and second polar body analysis resulted in eight mutation-free oocytes, five mutant oocytes (2, 4, 5, 9 and 10) and two oocytes that did not amplify (1 and 13). (C) Blastomere results of seven resulting embryos for gender determination by fluorescent in-situ hybridization and PCR showed that embryos resulting from mutant oocytes 4, 5 and 9 were males and therefore affected, so only embryos 6 and 11 originating from mutation-free oocytes, regardless of X,Y genotype, were transferred, resulting in a singleton pregnancy and the birth of an unaffected child (as indicated in the family pedigree by ). = embryo transfer; FA = failed amplification. fact, in some cases a common, apparently milder, disease susceptibility gene may contribute to premature death, major disability or hardship in a family. However, only the personal experience may alter a family s perception of severity of the condition, as the basis for their decision to undertake. any couples already going through IVF for fertility treatment may have questions about the implications of genetic susceptibility factors for offspring, the option to test embryos and the appropriateness of using in testing for susceptibility to inherited cardiac disease. Because the symptoms of inherited cardiac disease may be easily overlooked, as seen from description of the cases above, the family history may be the only reason to test for the presence of predisposing gene mutations and consideration about the need for, which may appear to be the life-saving procedure for individuals at risk. So with the future identification of the genes predisposing to inherited cardiac disease, might appear a useful tool for couples at risk to avoid the chance of producing offspring with inherited cardiac diseases with high probability of premature or sudden death within their lifespan.

11 for inherited cardiac diseases 453 So appeared to be acceptable to the couples at risk, despite important ethical implications as some of the above conditions do not present at birth and may not be realized even during the lifetime. However, the patients at risk of having children with a strong genetic predisposition to late-onset disorders should be informed about the availability of, without which some of these couples may remain childless because of their fear to opt for prenatal diagnosis and possible pregnancy termination. This seems to be ethically more acceptable than a denial of the information on the availability of. The available experience in offering for the above cardiac diseases and other conditions with inherited predisposition to common disorders showed that the availability of allows couples to reproduce, which otherwise would never be attempted. This especially applies to the diseases with no current prospect for treatment, arising despite presymptomatic diagnosis and follow up, as in the described predisposition to inherited cardiac diseases, when may be offered as a relief for at-risk couples. References ESHRE Preimplantation Genetic Diagnosis () Consortium, Best practice guidelines for polar body and embryo biopsy for preimplantation genetic diagnosis/screening (/PGS). Hum. Reprod. 26, He, J., cdermont, D.A., Song, Y., Gilbert, F., Kligman, I., Basson, C., Preimplantation genetic diagnosis of human congenital heart disease and Holt Oram syndrome. AJG A, 93. Kuliev, A., Rechitsky, S., Polar body based preimplantation genetic diagnosis for endelian disorders. ol. Hum. Reprod. 17, Liebaers, I., Desmyttere, S., Verpoest, W., De Rycke,., Staessen, C., Sermon, K., Devroey, P., Haentjens, P., Bonduelle,., Report on a consecutive series of 581 children born after blastomere biopsy for preimplantation genetic diagnosis. Hum. Reprod. 25, Papadopoulou, L.C., Sue, C.., Davidson,.., Tanji, K., ishino, I., Sadlock, J.E., Krishna, S., Walker, W., Selby, J., Glerum, D.., Coster, R.V., Lyon, G., Scalais, E., Lebel, R., Kaplan, P., Shanske, S., De Vivo, D.C., Bonilla, E., Hirano,., Diauro, S., Schon, E.A., Fatal infantile cardioencephalomyopathy with COX deficiency and mutations in SCO2, a COX assembly gene. ature Genet 23, Preimplantation Genetic Diagnosis International Society (IS), Guidelines for good practice in : program requirements and laboratory quality assurance. Reprod. Biomed. Online 16, 147. Preimplantation Genetic Diagnosis International Society (IS), th International Congress on Preimplantation Genetic Diagnosis. Reprod. Biomed. Online 20, S1 S42. Rechitsky, S., Kuliev, A., ovel indications for preimplantation genetic diagnosis. Reprod. Biomed. Online 20, S1 S2. Rechitsky, S., Verlinsky, O., Chistokhina, A., Sharapova, T., Ozen, S., asciangelo, C., Kuliev, A., Verlinsky, Y., Preimplantation Genetic Diagnosis for Cancer Predisposition. Reprod. Biomed. 4, Rechitsky, S., Pomerantseva, K., Pakhalchuk, T., Polling, D., Verlinsky, O., Kuliev, A., First systematic experience of preimplantation genetic diagnosis for de novo mutations. Reprod. Biomed. Online 22, Verlinsky, Y., Kuliev, A., Atlas of Preimplantation Genetic Diagnosis, second ed. Taylor and Francis, London and ew York, p Verlinsky, Y., Kuliev, A., Practical Preimplantation Genetic Diagnosis. Springer, Berlin, Y, 204p. Declaration: The authors report no financial or commercial conflicts of interest. Received 16 October 2011; refereed 18 December 2011; accepted 21 December 2011.