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PGD for inherited cardiac diseases
Reproductive BioMedicine Online (2012) 24, 443– 453
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ARTICLE
PGD for inherited cardiac diseases Anver Kuliev *, Ekaterina Pomerantseva, Dana Polling, Oleg Verlinsky, Svetlana Rechitsky Reproductive Genetics Institute, 2825 N Halsted Street, Chicago, IL 60657, USA * Corresponding author. E-mail address: [email protected] (A Kuliev). Dr Anver Kuliev received his PhD in clinical cytogenetics in 1969. 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 (PGD) has been applied for more than 200 different inherited conditions, with expand-
ing application to common disorders with genetic predisposition. One of the recent indications for PGD 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 PGD for inherited cardiac diseases, including familial hypertrophic and dilated cardiomyopathy, cardioencephalomyopathy and Emery–Dreifuss muscular dystrophy. A total of 18 PGD 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 PGD 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. RBMOnline 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, PGD, premature or sudden death
Introduction Preimplantation genetic diagnosis (PGD) is currently an established clinical procedure in assisted reproduction and genetic practices (ESHRE, 2011; PGDIS, 2008), which has been applied for more than 230 different inherited conditions with extremely high accuracy (Kuliev and Rechitsky, 2011; Liebaers et al., 2010; PGDIS (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 PGD may provide the only relief for the at-risk couples to reproduce. The available experience already includes PGD 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).
1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2011.12.009
444 The first case of PGD 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 PGD for other common disorders, the fact that inherited cardiac disorders may not be realized even during a lifetime, makes the application of PGD controversial, perhaps explaining the limited application of PGD 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 (HCM), 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 HCM have been reported, two of which, HCM4 and HCM7, will be described in this paper. HCM4 is caused by mutation in the gene MYBPC3 located on chromosome 11 (11p11.2), encoding the cardiac isoform of myosin-binding protein C, exclusively in heart muscle (MIM 115197). HCM7 is caused by a mutation in TNNI3 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 (MIM 613690). Hypertrophic cardiomyopathy is also one of the clinical manifestations of fatal infantile cytochrome C oxidase deficiency (MIM 604377), for which PGD 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 PGD is strongly indicated, is dilated cardiomyopathy (CMD), which is an autosomal dominant disease, caused by different mutations in LMNA located on chromosome 1 (1q21.2; MIM 115200). 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 LMNA, 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 (EMD), which is an X-linked disease, also characterized by cardiomyopathy, although presenting within the first year after birth (MIM 310300). This paper presents the first cumulative experience of PGD application in 18 cycles of inherited cardiac disorders.
A Kuliev et al.
Materials and methods A total of 18 PGD cycles for nine couples at risk for producing an affected progeny with the above conditions were performed, including nine cycles for CMD, three for CMH4, one for CMH7, three for cardioencephalomyopathy and two for EMD (Table 1). One of the four couples at risk for producing a progeny with CMD (Figure 1) requested PGD prospectively, with no previous pregnancies attempted, because the male partner was the carrier of the LMNA mutation predisposing to CMD. 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 49–50 from cardiovascular complications. The patient had a dominant mutation in LMNA 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 MspI digestion, which creates two fragments of 90 and 95 bp in the PCR product of the normal LMNA 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 CMH4 and CMH7, determined by mutation in MYBPC3 and TNNI3, respectively. None of these couples had previous progeny, but had a family history of premature or sudden death. As seen from Figure 2A, CMH4 in one of the families was due to frameshift mutation D1076fs in MYBPC3, while CMH7 in the other family was caused by the A157V mutation in TNNI3 (Figure 2B). The D1076fs mutation in MYBPC3 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, D11S1344, D11S4117, D11S1350 and D11S4147. The A157V mutation in TNNI3 was detected by the use of the two enzymes, HaeII, cutting the normal gene, and BspMI, 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, E140K of SCO2, in this case was detected by HindIII and BsrBI digestion, the first cutting the mutant and the second cutting the normal gene
Reproductive outcome of PGD for cardiac diseases.
Disease and gene (mutation)
Cardioencephalomyopathy (AR) SCO2 (R262del(CA); E140K) Cardiomyopathy dilated; CMD1 (AD) LMNA (K270K) LMNA (R335T) LMNA (R189P) LMNA (T528K) Cardiomyopathy familial, hypertrophic 4; CMH4 (AD) MYBPC3 (D1076fs) MYBPC3 (IVS11–10C–A) Cardiomyopathy familial, hypertrophic 7; CMH7 (AD) TNNI3 (A157V) Emery–Dreifuss muscular dystrophy 1, X-linked EMD Total
Patients (cycles)
Embryos Total received (amplified)
Normal or carrier
Abnormal
1 (3)
33 (32)
16
13
3
3
7
2
1
1 1 1 1
51 (47) 11 (11) 2 (2) 44 (34)
20 7 1 9
26 2 1 19
1 2 0 6
4 1 1 2
9 2 1 3
2 1 0 1
3c 1 0 0
1 (1) 1 (2)
7 (6) 10 (8)
3 1
3 6
0 1
1 0
2 0
1 0
0 0
1 (1)
11 (10)
3
7
0
1
1
0
0
1 (2) 9 (18)
31 (31) 200 (181)
17 77
14 91
0 13
2 15
5 30
2 9
2 7
(4) (1) (1) (3)
Transfers a
Inconclusive
b
Embryos transferred
Clinical pregnancies
Births
PGD for inherited cardiac diseases
Table 1
a
Including aneuploidies. Shared markers in parents, making impossible to exclude allele drop out. c Including one pair of twins. b
445
446
A Kuliev et al.
A
Pt. DOB 08/04/80 Markers: D1S2714 D1S2777 LMNA-R335T D1S2624 D1S506
140 156 R335T 132 95
143 150 N 126 89
PGD PGD 143 150 N 126 89
PGD
B
Embryo # 1
143 150 N 126 89
143 152 N 128 97
2
143 150 N 126 89
140 152 N 128 95
N
N
ET
FR
4
143 150 N 126 89
143 152 N 128 97
N
5
143 150 N 126 89
143 152 N 128 97
7
6
143 150 N 126 89
143 152 N 128 97
N
N
FR
FR
143 150 N 126 89
143 152 N 128 97
N
140 152 N 128 95
8
143 143 150 152 N N 126 128 89 97
N
143 152 N 128 97
M
N
143 152 N 128 97
9
140 156 R335T 132 95
143 152 N 128 97
10
143 150 N 126 89
140 152 N 128 95
N
11
FA
12
140 156 R335T 132 95
143 152 N 128 97
M
ET
Figure 1 PGD for dilated cardiomyopathy (CMD), determined by the dominant mutation R335T in LMNA. (A) Family pedigree of a couple with affected husband carrying the R335T mutation in LMNA. Paternal linked polymorphic markers are shown on the left and maternal on the right, and the order of the markers and mutation in LMNA are shown on the far left. (B) Blastomere results revealed two embryos carrying the R335T mutation in LMNA (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 CMD (as indicated in the family pedigree by PGD). ET = 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 137-bp fragment in the mutant gene. Five polymorphic markers, D22S1153, D22S1160, D22S1161, D22S922 and SNP NlaIII, 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 EMD, 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 PGD 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 EMD, 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/2.29.2002), 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 CMD, a total of 108 embryos were tested, of which 37 of 94 with
Primers and reaction conditions for PGD of cardiac diseases.
Mutation or polymorphism
Cardiomyopathy, dilated, CMD1A, LMNA R335T
D1S2714 hemi-nested
D1S2777 hemi-nested
D1S2624 hemi-nested
D1S506 hemi-nested
Cardiomyopathy, familial hypertrophic, CMH4, MYBPC3 D1076fs
D11S1978 hemi-nested
D11S1344 hemi-nested
D11S4117 hemi-nested
Primers (50 –30 ) Forward
Reverse
Outside: R335T-1: GTCTCCTACACCGACCCACGT Inside: R335T-3: GCTCACCAAACCCTCCCAC Outside: 2714-1: TGTGGGGGCTGAGATGAAT Inside: 2714-3: Hex CCCAGGATTTTAAGACCAGC Outside: 2777-1: CACCACGGAACTCCAGTAT Inside: 2777-1: CACCACGGAACTCCAGTAT Outside: 2624-1: GAGGCAGAGGCAGACACAGATG Inside: 2624-3: Hex ATGGGGCTGACACTCTATGAGG Outside: 506-1: CTGGACTCAGCCTGAGAAGAATATG Inside: 506-3: Fam AGAAAGGGAGGGATCGTTCAG
R335T-2: CGTGGATCTCCATGTCCAGG R335T-4: GTCCAGAAGCTCCTGGTACTCGT 2714-2: AGACTCTGGAGTAGCAGGGACTA 2714-2: AGACTCTGGAGTAGCAGGGACTA 2777-2: CAAGTAATCCTCCTGCCTCAG 2777-3: Hex TGTTGGGATTACAGGTGTGAG 2624-2: GACTCAGCGTCCTGCACAGAGT 2624-2: GACTCAGCGTCCTGCACAGAGT 506-2: GCTATGCTGGGGCAAGGG
Outside: D1076fs-1: CTGGTTGGCAGGGTGG Inside: D1076fs-3: AGGCGTGGTGACCCAACTG Outside: 1978-1: TGCACTCCACAAATACACACAATT Inside: 1978-3: Hex CAGAATGTTAGTATAAGTGTGCATGTG Outside: 1344-1: GCCTCCTGTTCTGTTATTTCACTTA Inside: 1344-3: Fam TGACTTTAGCCTTGTGCTGAACTG Outside: 4117-1: TTGTCTTCTTTCTAATCTTCCTTCCA Inside: 4117-1: TTGTCTTCTTTCTAATCTTCCTTCCA
D1076fs-2: TCTTCTTGTCGGCTTTCTGCA D1076fs-4: TCCGTGTTGCCGACATCCT 1978-2: ACTTAGATGTCCATCGACAGATGAA 1978-2: ACTTAGATGTCCATCGACAGATGAA 1344-2: CAGCGCCTGGCTTGTACATAT 1344-2: CAGCGCCTGGCTTGTACATAT 4117-2: GTGAGCAAGAGATCACGCCAC 4117-3: Fam TGACAGAGCGAGACTCCATCTAAAA
Product sizes (bp)
Annealing temperature (C)
MspI: mutant 185, normal 90 + 95
62–45
140–150
62–45
PGD for inherited cardiac diseases
Table 2
55 140–150
62–45 55
120–130
62–45 55
80–100
62–45
506-2: GCTATGCTGGGGCAAGGG
55
RsaI: mutant 72 + 60, normal 132; BsaHI: mutant 132, normal 72 + 60
62–45 55
160–190
62–45 55
130–180
62–45 55
100–120
62–45 55
447
(continued on next page)
(continued)
Mutation or polymorphism
D11S1350 hemi-nested
D11S4147 hemi-nested
Cardiomyopathy, familial hypertrophic, CMH7, TNNI3 A157V
D19S867
D19S904
D19S246
D19S206
D19S571
Cardioencephalomyopathy, SCO2 E140K
448
Table 2
Primers (50 –30 )
Product sizes (bp)
Annealing temperature (C) 62–45
Forward
Reverse
Outside: 1350-1: CAAATTAAATCATTCTGGGGTCTTT Inside: 1350-3: Fam AAACACCTGCTCTCCAAGAATATC Outside: 4147-1: AGCTTTTCCCTTGTGGGTGTT Inside: 4147-3: Fam AAGGGGAAGACGGACATAAAAC
1350-2: AAACTACCAGCAGTAGAGCACACCT 1350-2: AAACTACCAGCAGTAGAGCACACCT 4147-2: GCCAGCCTATCTAAACTGTATAATT 4147-2: GCCAGCCTATCTAAACTGTATAATT
180–200
Outside: A157V-1: AAAAAGGAGTGTAGGATGGAGGAGT Inside: A157V-3: GGTGTGCGGGAAATGGAAG Outside: 867-1: CAATGAAAATGCTTTGTAAAACTCTT Inside: 867-1: CAATGAAAATGCTTTGTAAAACTCTT Outside: 904-1: AATCACACCATTGTACTCCAGCC Inside: 904-3: HEX AGGGCAAGACTCCGTTTCAA Outside: 246-1: GTGAGCCAAGACTACGCCACT Inside: 246-3: Fam AGAGTGAGATTCCACCTTTCAAAAA Outside: 206-1: TTTTCCTATTTTATCTGGCGGG Inside: 206-3: FAM AAGTGAAAGCCGAAGTCTTTTCA Outside: 571-1: TGAACTCCAGCCTGGGTGAG Inside: 571-1: TGAACTCCAGCCTGGGTGAG
A157V-2: TTCCCCTCAGCATCCTCTTTC A157V-4: TTCTCGGTGTCCTCCTTCTTCA 867-2: TTGGTTTCCTTCTGTCATGTCATC 867-3: Fam TCAGAGGTGACCAGTTCTTTCATAC 904-2: TCGGAGATGTTAAAATGTGAAAAAC 904-2: TCGGAGATGTTAAAATGTGAAAAAC 246-2: CCAGAAACACATCATTTACCCACTT 246-2: CCAGAAACACATCATTTACCCACTT 206-2: TCATCAAGTCTGTTCCAGCCAA
HaeII: mutant 226, normal 150 + 26; BspMI; mutant 190 + 35 + 1, normal 134 + 56 + 35 + 1
571-2: TTGACAGCATGTATTTGAAATATGG 571-3: Hex AGTTACACGTATACATGAAATGACTGA
100–130
Outside: 140-1: AGCAGCAAAAGCGAACAGAAG Inside: 140-1
140-2: TCGGGGTCCACAGTGATGAAG
HindIII: mutant 166 + 19, normal 185
55 130–150
55
62–45 55
110–130
62–45 55
115–130
62–45 55
200–230
62–45 55
120–140
206-2: TCATCAAGTCTGTTCCAGCCAA
62–45 55 62–45 55
62–65 55
BsrBI: mutant 185, normal 168 + 17 (continued on next page)
A Kuliev et al.
140-4 mismatch: CACCTGCACCAGCTTCTCAA 140-7 mismatch: ACCTGCACCAGCTTCTCCCG
62–45
(continued)
Mutation or polymorphism R262del(CA) D22S1153 D22S1160
D22S1161 D22S922
SNP NlaIII Emery–Dreifuss muscular dystrophy X-linked, EMD IVS2+1G–T
DXS8103 DXS1684 DXS8087 DXS1073 DYS154
Primers (50 –30 )
Product sizes (bp)
Forward
Reverse
Outside: 262-3: CAAGGATGAGGACCAGGACTACA Inside: 262-4: Fam TGCCATCTACCTGCTCAACCC Outside: 1151-1: GTAGAGGTTGCAGTGAGCCATGA Inside: 1153-3 Outside: 1160-1: TTTGGGGAAGCAGTGAGTCACTA Inside: 1160-1: TTTGGGGAAGCAGTGAGTCACTA
262-2: CCAGACTGCAGTGGCTCAAGA 262-2: CCAGACTGCAGTGGCTCAAGA 1153-2: AACCCTGCTCCTAGCCTTCCT 1153-2: AACCCTGCTCCTAGCCTTCCT 1160-1: TCTCAGGGATGCTTTCCCATG 1160-3: Hex ATTTGCAGATGACGAACATGTATCA 1161-2: CGAGTTTGTGGTGGTTTGTTACAG 1161-3: Fam TAGCAGGCCAAGCCGAAGA 922-2: GGTTTCCTCAGTTTTACCTGTGCT 922-2: GGTTTCCTCAGTTTTACCTGTGCT
Annealing temperature (C)
Mutant 137, normal 139 140–160 130–170
150–160
62–45 55 62–45 55
Outside: 1161-1: ACAAGGTGGACCCGAATCAGA Inside: 1161-1: ACAAGGTGGACCCGAATCAGA Outside: 922-1: CGATGGGATGTCTGTGGGG Inside: 922-3: FAM GGGTTGGAACTGTTAGGTATCTTG Outside: NlaIII-1: TGCCAAGAACATAGTGGGTGA Inside: NlaIII-1: TGCCAAGAACATAGTGGGTGA
NlaIII-2: CAGCCTCTGAGCCACCGA NlaIII-3: CCACACCTCTAAGTCACAAAGC
+/
62–45 55
Outside: IVS2-1: CAACTCGTAGGCTTTACGAGAA
IVS2-2: CTTTCTCCAGTGCCGCTCT
BpmI: mutant 121, normal 115 + 6
62–45
Inside: IVS2-1: CAACTCGTAGGCTTTACGAGAA Outside: GTGAAGCCAAGGTGGGAGGAT Inside: Hex CACAGGCGTTCAAAACCAGC Outside: AGCACCCAGTAAGAGACTG Inside: Fam CAGGCCACTACCACTTATG Outside: TGAGGCAGGGCGCACTTG Inside: TGAGGCAGGGCGCACTTG Outside: GAAACTTAGAGGGTTGGCTT Inside: ACACTGCTCCCCTTGCC Outside: ACTCTCACCTATCCTATTCAACTTA Hex ACATGATATTATATGTAGAAAATCC
IVS2-3 mismatch: CCACAGGCGAGGCTCTCT GCCCTGGGGTACACAAGCC GCCCTGGGGTACACAAGCC TGAATCAATCTATCCATCTCTC TACTGTTTTCCACTCTAATGC CAGGAGGCCGTGTGAGAGC Fam GGCTGCGCCAGTGAACAA CCCCAAAGAATGCCCT Hex CCGAGTTATTACAAAGAAGCAC AAGTGATCCTCCCGCTTC AAGTGATCCTCCCGCTTC
130–140
130–150 130–140 150–160 190–220 160–180
62–45 55 62–45 55
PGD for inherited cardiac diseases
Table 2
55 62–45 55 62–45 55 62–45 55 62–45 55 62–45 55
449
450
A Kuliev et al.
A
MARKERS: D11S4174 D11S1344 MYBPC3-D1076fs D11S4117 D11S1350 D11S1978
C
144 172 N 116 194 186
148 174 N 114 187 167
PGD
148 174 N 114 187 167
3
137 159 N 116 191 186
N FET 1
137 159 N 116 191 186
PGD
B EMBRYO #
144 164 D1-76fs 112 179 182
D19S902 D19S867 D19S904 D19S246 TNNI 3-A157V D19S206 D19S571
5
FA
7
144 144 172 164 N D1-76fs 116 112 194 179 186 182
M
137 159 N 116 191 186
N FET 2
9
144 144 172 164 N D1-76fs 116 112 194 179 186 182
M
10
144 144 172 164 N D1-76fs 116 112 194 179 186 182
M
1
12
148 174 N 114 187 167
137 159 N 116 191 186
N
149 117 119 219 A157V 126 123
M
134 126 119 222 N 121 127
134 132 127 206 N 121 100
134 126 119 222 N 121 127
PGD
D
8
148 174 N 114 187 167
149 117 119 219 A157V 126 123
145 126 127 183 N 136 104
2
149 117 119 219 A157V 126 123
3
134 132 127 206 N 121 100
M
145 126 127 183 A157V 126 123
4
134 132 127 206 N 121 100
M
145 126 127 183 N 136 104
134 126 119 222 N 121 127
5
145 126 127 183 N 136 104
134 132 127 206 N 121 100
N N FET* FR*
6
145 117 119 219 A157V 126 123
7
134 132 127 206 N 121 100
M
8
149 117 119 219 A157V 126 123
134 126 119 222 N 121 127
M
FA
9
149 117 119 219 A157V 126 123
10
134 132 127 206 N 121 100
M
149 117 119 219 A157V 126 123
134 126 119 222 N 121 127
M
11
145 126 127 183 N 136 104
134 132 127 206 N 121 100
N FR*
* Normal for 24 chromosome by microarray
Figure 2 PGD for hypertrophic cardiomyopathy (CMH). (A, B) PGD for CMH4. (A) Family pedigree of a couple with affected mother carrying the frameshift mutation D1076fs in MYBPC3. Paternal linked polymorphic markers are shown on the left and maternal on the right, and the order of the markers and frameshift mutation in MYBPC3 are shown on the far left. (B) Blastomere results revealed three embryos (embryos 7, 9 and 10) carrying the frameshift mutation D1076fs in MYBPC3, 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, FET), resulting in an unaffected pregnancy (as indicated in the family pedigree by PGD). (C, D) PGD for CMH7. (A) Family pedigree of a couple with affected father carrying the A157V mutation in TNNI3. Paternal linked polymorphic markers are shown on the left and maternal on the right, and the order of the markers and mutation in TNNI3 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; FET = 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 PGD for CMD, determined by dominant mutation in LMNA, 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 LMNA 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 CMD. Of four cycles performed for three couples at risk for producing offspring with CMH, 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 MYBPC3, 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 PGD cycle for the patient at risk for producing offspring with CMH7 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
PGD for inherited cardiac diseases A
451
MARKERS:
D22S1153 D22S1160 D22S1161 D22S922 SCO2 SNP-NlaII
150 162 157 138 R262delCA +
148 150 133 162 155 157 138 136 E140K N +
158 133 155 138 N -
PGD 150 150 162 162 157 157 138 136 R262delCA E140K + +
158 133 155 138 N -
M, RECOMBINANT B
EMBRYO #
1
2
150 148 162 133 157 155 138 138 R262 E140K delCA + +
150 148 162 133 157 155 138 138 R262 E140K delCA + +
M
M
3
158 133 155 138 N -
N
4
150 150 150 162 162 162 157 157 157 136 138 136 R262 N N delCA + -
N CARRIER ET
150 162 157 136 N -
5
158 133 155 138 N -
150 162 157 136 N -
N
6
158 133 155 138 N -
7
150 150 150 162 162 162 157 157 157 136 138 136 N R262 N delCA + -
N
CARRIER
8
158 133 155 138 N -
9
150 162 157 136 N -
150 162 157 138 R262 delCA +
N MONOSOMY22
ET
Figure 3 PGD for cardioencephalomyopathy. (A) Family pedigree of a couple with a previous affected child, who was double heterozygous for E140K 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 PGD). ET = 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 PGD cycles performed for EMD, 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 EMD-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 PGD 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 CMD and CMH. Conditions in the family history of the couples at risk that may indicate a possible need for PGD 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 PGD. In
452
A Kuliev et al. DXS8103 DXS1684 DXS8061 DXS8087 EMD-IVS2+1G-T DXS1073 F8 INT1 F8INT 13 DXY154
A
143 140 157 198 112 98 174
I.
Y
1
145 140 161 200 114 100 165/167
II.
137 134 155 212 114 104 165
Y
2
143 140 157 198 112 98 174
2
1 PGD
1 137 134 155 212 114 104 165
OOCYTES
1
2
FA
M
PCR
C
143 140 157 198 112 98 174
1
3
137 134 155 212 114 104 165
EMD
III.
B
139 134 157 202 112 98 165
4
5
6
N
M
M
137 134 155 212 114 104 165
143 140 157 198 112 98 174
143 140 157 198 112 98 174
N
4
5
6
M XY
M XY
N XX
137 134 155 212 114 104 165
2 137 134 155 212 114 104 165
Y
7
9
10
N
M
M
137 134 155 212 114 104 165
143 140 157 198 112 98 174
143 140 157 198 112 98 174
9
10
139 134 157 202 112 98 165
11
12
13
N
N
FA
137 134 155 212 114 104 165
137 134 155 212 114 104 165
14
15
16
N
N
N
137 134 155 212 114 104 165
137 134 155 212 114 104 165
137 134 155 212 114 104 165
11
EMBRYO
PCR/FISH 143 140 157 198 112 98 174
Y ET
M XY
Y0
137 134 155 212 114 104 165
Y
ET
Figure 4 PGD for Emery–Dreifus muscular dystrophy (EMD). (A) Family pedigree of a couple with mother (II-2) carrying X-linked EMD–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 PGD). ET = 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 PGD. Many 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 PGD 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 PGD, 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, PGD 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.
PGD for inherited cardiac diseases So PGD 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 PGD, 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 PGD. The available experience in offering PGD for the above cardiac diseases and other conditions with inherited predisposition to common disorders showed that the availability of PGD 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 PGD may be offered as a relief for at-risk couples.
References ESHRE Preimplantation Genetic Diagnosis (PGD) Consortium, 2011. Best practice guidelines for polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS). Hum. Reprod. 26, 14–46. He, J., McDermont, D.A., Song, Y., Gilbert, F., Kligman, I., Basson, C., 2004. Preimplantation genetic diagnosis of human congenital heart disease and Holt–Oram syndrome. AJMG 126A, 93–98. Kuliev, A., Rechitsky, S., 2011. Polar body based preimplantation genetic diagnosis for Mendelian disorders. Mol. Hum. Reprod. 17, 275–285. Liebaers, I., Desmyttere, S., Verpoest, W., De Rycke, M., Staessen, C., Sermon, K., Devroey, P., Haentjens, P., Bonduelle, M., 2010. Report on a consecutive series of 581 children born after
453 blastomere biopsy for preimplantation genetic diagnosis. Hum. Reprod. 25, 275–282. Papadopoulou, L.C., Sue, C.M., Davidson, M.M., Tanji, K., Nishino, I., Sadlock, J.E., Krishna, S., Walker, W., Selby, J., Glerum, D.M., Coster, R.V., Lyon, G., Scalais, E., Lebel, R., Kaplan, P., Shanske, S., De Vivo, D.C., Bonilla, E., Hirano, M., DiMauro, S., Schon, E.A., 1999. Fatal infantile cardioencephalomyopathy with COX deficiency and mutations in SCO2, a COX assembly gene. Nature Genet 23, 333–337. Preimplantation Genetic Diagnosis International Society (PGDIS), 2008. Guidelines for good practice in PGD: program requirements and laboratory quality assurance. Reprod. Biomed. Online 16, 134–147. Preimplantation Genetic Diagnosis International Society (PGDIS), 2010. 10th International Congress on Preimplantation Genetic Diagnosis. Reprod. Biomed. Online 20, S1–S42. Rechitsky, S., Kuliev, A., 2010. Novel indications for preimplantation genetic diagnosis. Reprod. Biomed. Online 20, S1–S2. Rechitsky, S., Verlinsky, O., Chistokhina, A., Sharapova, T., Ozen, S., Masciangelo, C., Kuliev, A., Verlinsky, Y., 2002. Preimplantation Genetic Diagnosis for Cancer Predisposition. Reprod. Biomed. 4, 148–155. Rechitsky, S., Pomerantseva, K., Pakhalchuk, T., Polling, D., Verlinsky, O., Kuliev, A., 2011. First systematic experience of preimplantation genetic diagnosis for de novo mutations. Reprod. Biomed. Online 22, 350–361. Verlinsky, Y., Kuliev, A., 2005. Atlas of Preimplantation Genetic Diagnosis, second ed. Taylor and Francis, London and New York, p. 288. Verlinsky, Y., Kuliev, A., 2006. Practical Preimplantation Genetic Diagnosis. Springer, Berlin, NY, 204p. Declaration: The authors report no financial or commercial conflicts of interest. Received 16 October 2011; refereed 18 December 2011; accepted 21 December 2011.
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ARTICLE
PGD for inherited cardiac diseases Anver Kuliev *, Ekaterina Pomerantseva, Dana Polling, Oleg Verlinsky, Svetlana Rechitsky Reproductive Genetics Institute, 2825 N Halsted Street, Chicago, IL 60657, USA * Corresponding author. E-mail address: [email protected] (A Kuliev). Dr Anver Kuliev received his PhD in clinical cytogenetics in 1969. 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 (PGD) has been applied for more than 200 different inherited conditions, with expand-
ing application to common disorders with genetic predisposition. One of the recent indications for PGD 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 PGD for inherited cardiac diseases, including familial hypertrophic and dilated cardiomyopathy, cardioencephalomyopathy and Emery–Dreifuss muscular dystrophy. A total of 18 PGD 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 PGD 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. RBMOnline 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, PGD, premature or sudden death
Introduction Preimplantation genetic diagnosis (PGD) is currently an established clinical procedure in assisted reproduction and genetic practices (ESHRE, 2011; PGDIS, 2008), which has been applied for more than 230 different inherited conditions with extremely high accuracy (Kuliev and Rechitsky, 2011; Liebaers et al., 2010; PGDIS (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 PGD may provide the only relief for the at-risk couples to reproduce. The available experience already includes PGD 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).
1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2011.12.009
444 The first case of PGD 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 PGD for other common disorders, the fact that inherited cardiac disorders may not be realized even during a lifetime, makes the application of PGD controversial, perhaps explaining the limited application of PGD 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 (HCM), 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 HCM have been reported, two of which, HCM4 and HCM7, will be described in this paper. HCM4 is caused by mutation in the gene MYBPC3 located on chromosome 11 (11p11.2), encoding the cardiac isoform of myosin-binding protein C, exclusively in heart muscle (MIM 115197). HCM7 is caused by a mutation in TNNI3 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 (MIM 613690). Hypertrophic cardiomyopathy is also one of the clinical manifestations of fatal infantile cytochrome C oxidase deficiency (MIM 604377), for which PGD 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 PGD is strongly indicated, is dilated cardiomyopathy (CMD), which is an autosomal dominant disease, caused by different mutations in LMNA located on chromosome 1 (1q21.2; MIM 115200). 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 LMNA, 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 (EMD), which is an X-linked disease, also characterized by cardiomyopathy, although presenting within the first year after birth (MIM 310300). This paper presents the first cumulative experience of PGD application in 18 cycles of inherited cardiac disorders.
A Kuliev et al.
Materials and methods A total of 18 PGD cycles for nine couples at risk for producing an affected progeny with the above conditions were performed, including nine cycles for CMD, three for CMH4, one for CMH7, three for cardioencephalomyopathy and two for EMD (Table 1). One of the four couples at risk for producing a progeny with CMD (Figure 1) requested PGD prospectively, with no previous pregnancies attempted, because the male partner was the carrier of the LMNA mutation predisposing to CMD. 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 49–50 from cardiovascular complications. The patient had a dominant mutation in LMNA 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 MspI digestion, which creates two fragments of 90 and 95 bp in the PCR product of the normal LMNA 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 CMH4 and CMH7, determined by mutation in MYBPC3 and TNNI3, respectively. None of these couples had previous progeny, but had a family history of premature or sudden death. As seen from Figure 2A, CMH4 in one of the families was due to frameshift mutation D1076fs in MYBPC3, while CMH7 in the other family was caused by the A157V mutation in TNNI3 (Figure 2B). The D1076fs mutation in MYBPC3 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, D11S1344, D11S4117, D11S1350 and D11S4147. The A157V mutation in TNNI3 was detected by the use of the two enzymes, HaeII, cutting the normal gene, and BspMI, 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, E140K of SCO2, in this case was detected by HindIII and BsrBI digestion, the first cutting the mutant and the second cutting the normal gene
Reproductive outcome of PGD for cardiac diseases.
Disease and gene (mutation)
Cardioencephalomyopathy (AR) SCO2 (R262del(CA); E140K) Cardiomyopathy dilated; CMD1 (AD) LMNA (K270K) LMNA (R335T) LMNA (R189P) LMNA (T528K) Cardiomyopathy familial, hypertrophic 4; CMH4 (AD) MYBPC3 (D1076fs) MYBPC3 (IVS11–10C–A) Cardiomyopathy familial, hypertrophic 7; CMH7 (AD) TNNI3 (A157V) Emery–Dreifuss muscular dystrophy 1, X-linked EMD Total
Patients (cycles)
Embryos Total received (amplified)
Normal or carrier
Abnormal
1 (3)
33 (32)
16
13
3
3
7
2
1
1 1 1 1
51 (47) 11 (11) 2 (2) 44 (34)
20 7 1 9
26 2 1 19
1 2 0 6
4 1 1 2
9 2 1 3
2 1 0 1
3c 1 0 0
1 (1) 1 (2)
7 (6) 10 (8)
3 1
3 6
0 1
1 0
2 0
1 0
0 0
1 (1)
11 (10)
3
7
0
1
1
0
0
1 (2) 9 (18)
31 (31) 200 (181)
17 77
14 91
0 13
2 15
5 30
2 9
2 7
(4) (1) (1) (3)
Transfers a
Inconclusive
b
Embryos transferred
Clinical pregnancies
Births
PGD for inherited cardiac diseases
Table 1
a
Including aneuploidies. Shared markers in parents, making impossible to exclude allele drop out. c Including one pair of twins. b
445
446
A Kuliev et al.
A
Pt. DOB 08/04/80 Markers: D1S2714 D1S2777 LMNA-R335T D1S2624 D1S506
140 156 R335T 132 95
143 150 N 126 89
PGD PGD 143 150 N 126 89
PGD
B
Embryo # 1
143 150 N 126 89
143 152 N 128 97
2
143 150 N 126 89
140 152 N 128 95
N
N
ET
FR
4
143 150 N 126 89
143 152 N 128 97
N
5
143 150 N 126 89
143 152 N 128 97
7
6
143 150 N 126 89
143 152 N 128 97
N
N
FR
FR
143 150 N 126 89
143 152 N 128 97
N
140 152 N 128 95
8
143 143 150 152 N N 126 128 89 97
N
143 152 N 128 97
M
N
143 152 N 128 97
9
140 156 R335T 132 95
143 152 N 128 97
10
143 150 N 126 89
140 152 N 128 95
N
11
FA
12
140 156 R335T 132 95
143 152 N 128 97
M
ET
Figure 1 PGD for dilated cardiomyopathy (CMD), determined by the dominant mutation R335T in LMNA. (A) Family pedigree of a couple with affected husband carrying the R335T mutation in LMNA. Paternal linked polymorphic markers are shown on the left and maternal on the right, and the order of the markers and mutation in LMNA are shown on the far left. (B) Blastomere results revealed two embryos carrying the R335T mutation in LMNA (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 CMD (as indicated in the family pedigree by PGD). ET = 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 137-bp fragment in the mutant gene. Five polymorphic markers, D22S1153, D22S1160, D22S1161, D22S922 and SNP NlaIII, 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 EMD, 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 PGD 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 EMD, 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/2.29.2002), 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 CMD, a total of 108 embryos were tested, of which 37 of 94 with
Primers and reaction conditions for PGD of cardiac diseases.
Mutation or polymorphism
Cardiomyopathy, dilated, CMD1A, LMNA R335T
D1S2714 hemi-nested
D1S2777 hemi-nested
D1S2624 hemi-nested
D1S506 hemi-nested
Cardiomyopathy, familial hypertrophic, CMH4, MYBPC3 D1076fs
D11S1978 hemi-nested
D11S1344 hemi-nested
D11S4117 hemi-nested
Primers (50 –30 ) Forward
Reverse
Outside: R335T-1: GTCTCCTACACCGACCCACGT Inside: R335T-3: GCTCACCAAACCCTCCCAC Outside: 2714-1: TGTGGGGGCTGAGATGAAT Inside: 2714-3: Hex CCCAGGATTTTAAGACCAGC Outside: 2777-1: CACCACGGAACTCCAGTAT Inside: 2777-1: CACCACGGAACTCCAGTAT Outside: 2624-1: GAGGCAGAGGCAGACACAGATG Inside: 2624-3: Hex ATGGGGCTGACACTCTATGAGG Outside: 506-1: CTGGACTCAGCCTGAGAAGAATATG Inside: 506-3: Fam AGAAAGGGAGGGATCGTTCAG
R335T-2: CGTGGATCTCCATGTCCAGG R335T-4: GTCCAGAAGCTCCTGGTACTCGT 2714-2: AGACTCTGGAGTAGCAGGGACTA 2714-2: AGACTCTGGAGTAGCAGGGACTA 2777-2: CAAGTAATCCTCCTGCCTCAG 2777-3: Hex TGTTGGGATTACAGGTGTGAG 2624-2: GACTCAGCGTCCTGCACAGAGT 2624-2: GACTCAGCGTCCTGCACAGAGT 506-2: GCTATGCTGGGGCAAGGG
Outside: D1076fs-1: CTGGTTGGCAGGGTGG Inside: D1076fs-3: AGGCGTGGTGACCCAACTG Outside: 1978-1: TGCACTCCACAAATACACACAATT Inside: 1978-3: Hex CAGAATGTTAGTATAAGTGTGCATGTG Outside: 1344-1: GCCTCCTGTTCTGTTATTTCACTTA Inside: 1344-3: Fam TGACTTTAGCCTTGTGCTGAACTG Outside: 4117-1: TTGTCTTCTTTCTAATCTTCCTTCCA Inside: 4117-1: TTGTCTTCTTTCTAATCTTCCTTCCA
D1076fs-2: TCTTCTTGTCGGCTTTCTGCA D1076fs-4: TCCGTGTTGCCGACATCCT 1978-2: ACTTAGATGTCCATCGACAGATGAA 1978-2: ACTTAGATGTCCATCGACAGATGAA 1344-2: CAGCGCCTGGCTTGTACATAT 1344-2: CAGCGCCTGGCTTGTACATAT 4117-2: GTGAGCAAGAGATCACGCCAC 4117-3: Fam TGACAGAGCGAGACTCCATCTAAAA
Product sizes (bp)
Annealing temperature (C)
MspI: mutant 185, normal 90 + 95
62–45
140–150
62–45
PGD for inherited cardiac diseases
Table 2
55 140–150
62–45 55
120–130
62–45 55
80–100
62–45
506-2: GCTATGCTGGGGCAAGGG
55
RsaI: mutant 72 + 60, normal 132; BsaHI: mutant 132, normal 72 + 60
62–45 55
160–190
62–45 55
130–180
62–45 55
100–120
62–45 55
447
(continued on next page)
(continued)
Mutation or polymorphism
D11S1350 hemi-nested
D11S4147 hemi-nested
Cardiomyopathy, familial hypertrophic, CMH7, TNNI3 A157V
D19S867
D19S904
D19S246
D19S206
D19S571
Cardioencephalomyopathy, SCO2 E140K
448
Table 2
Primers (50 –30 )
Product sizes (bp)
Annealing temperature (C) 62–45
Forward
Reverse
Outside: 1350-1: CAAATTAAATCATTCTGGGGTCTTT Inside: 1350-3: Fam AAACACCTGCTCTCCAAGAATATC Outside: 4147-1: AGCTTTTCCCTTGTGGGTGTT Inside: 4147-3: Fam AAGGGGAAGACGGACATAAAAC
1350-2: AAACTACCAGCAGTAGAGCACACCT 1350-2: AAACTACCAGCAGTAGAGCACACCT 4147-2: GCCAGCCTATCTAAACTGTATAATT 4147-2: GCCAGCCTATCTAAACTGTATAATT
180–200
Outside: A157V-1: AAAAAGGAGTGTAGGATGGAGGAGT Inside: A157V-3: GGTGTGCGGGAAATGGAAG Outside: 867-1: CAATGAAAATGCTTTGTAAAACTCTT Inside: 867-1: CAATGAAAATGCTTTGTAAAACTCTT Outside: 904-1: AATCACACCATTGTACTCCAGCC Inside: 904-3: HEX AGGGCAAGACTCCGTTTCAA Outside: 246-1: GTGAGCCAAGACTACGCCACT Inside: 246-3: Fam AGAGTGAGATTCCACCTTTCAAAAA Outside: 206-1: TTTTCCTATTTTATCTGGCGGG Inside: 206-3: FAM AAGTGAAAGCCGAAGTCTTTTCA Outside: 571-1: TGAACTCCAGCCTGGGTGAG Inside: 571-1: TGAACTCCAGCCTGGGTGAG
A157V-2: TTCCCCTCAGCATCCTCTTTC A157V-4: TTCTCGGTGTCCTCCTTCTTCA 867-2: TTGGTTTCCTTCTGTCATGTCATC 867-3: Fam TCAGAGGTGACCAGTTCTTTCATAC 904-2: TCGGAGATGTTAAAATGTGAAAAAC 904-2: TCGGAGATGTTAAAATGTGAAAAAC 246-2: CCAGAAACACATCATTTACCCACTT 246-2: CCAGAAACACATCATTTACCCACTT 206-2: TCATCAAGTCTGTTCCAGCCAA
HaeII: mutant 226, normal 150 + 26; BspMI; mutant 190 + 35 + 1, normal 134 + 56 + 35 + 1
571-2: TTGACAGCATGTATTTGAAATATGG 571-3: Hex AGTTACACGTATACATGAAATGACTGA
100–130
Outside: 140-1: AGCAGCAAAAGCGAACAGAAG Inside: 140-1
140-2: TCGGGGTCCACAGTGATGAAG
HindIII: mutant 166 + 19, normal 185
55 130–150
55
62–45 55
110–130
62–45 55
115–130
62–45 55
200–230
62–45 55
120–140
206-2: TCATCAAGTCTGTTCCAGCCAA
62–45 55 62–45 55
62–65 55
BsrBI: mutant 185, normal 168 + 17 (continued on next page)
A Kuliev et al.
140-4 mismatch: CACCTGCACCAGCTTCTCAA 140-7 mismatch: ACCTGCACCAGCTTCTCCCG
62–45
(continued)
Mutation or polymorphism R262del(CA) D22S1153 D22S1160
D22S1161 D22S922
SNP NlaIII Emery–Dreifuss muscular dystrophy X-linked, EMD IVS2+1G–T
DXS8103 DXS1684 DXS8087 DXS1073 DYS154
Primers (50 –30 )
Product sizes (bp)
Forward
Reverse
Outside: 262-3: CAAGGATGAGGACCAGGACTACA Inside: 262-4: Fam TGCCATCTACCTGCTCAACCC Outside: 1151-1: GTAGAGGTTGCAGTGAGCCATGA Inside: 1153-3 Outside: 1160-1: TTTGGGGAAGCAGTGAGTCACTA Inside: 1160-1: TTTGGGGAAGCAGTGAGTCACTA
262-2: CCAGACTGCAGTGGCTCAAGA 262-2: CCAGACTGCAGTGGCTCAAGA 1153-2: AACCCTGCTCCTAGCCTTCCT 1153-2: AACCCTGCTCCTAGCCTTCCT 1160-1: TCTCAGGGATGCTTTCCCATG 1160-3: Hex ATTTGCAGATGACGAACATGTATCA 1161-2: CGAGTTTGTGGTGGTTTGTTACAG 1161-3: Fam TAGCAGGCCAAGCCGAAGA 922-2: GGTTTCCTCAGTTTTACCTGTGCT 922-2: GGTTTCCTCAGTTTTACCTGTGCT
Annealing temperature (C)
Mutant 137, normal 139 140–160 130–170
150–160
62–45 55 62–45 55
Outside: 1161-1: ACAAGGTGGACCCGAATCAGA Inside: 1161-1: ACAAGGTGGACCCGAATCAGA Outside: 922-1: CGATGGGATGTCTGTGGGG Inside: 922-3: FAM GGGTTGGAACTGTTAGGTATCTTG Outside: NlaIII-1: TGCCAAGAACATAGTGGGTGA Inside: NlaIII-1: TGCCAAGAACATAGTGGGTGA
NlaIII-2: CAGCCTCTGAGCCACCGA NlaIII-3: CCACACCTCTAAGTCACAAAGC
+/
62–45 55
Outside: IVS2-1: CAACTCGTAGGCTTTACGAGAA
IVS2-2: CTTTCTCCAGTGCCGCTCT
BpmI: mutant 121, normal 115 + 6
62–45
Inside: IVS2-1: CAACTCGTAGGCTTTACGAGAA Outside: GTGAAGCCAAGGTGGGAGGAT Inside: Hex CACAGGCGTTCAAAACCAGC Outside: AGCACCCAGTAAGAGACTG Inside: Fam CAGGCCACTACCACTTATG Outside: TGAGGCAGGGCGCACTTG Inside: TGAGGCAGGGCGCACTTG Outside: GAAACTTAGAGGGTTGGCTT Inside: ACACTGCTCCCCTTGCC Outside: ACTCTCACCTATCCTATTCAACTTA Hex ACATGATATTATATGTAGAAAATCC
IVS2-3 mismatch: CCACAGGCGAGGCTCTCT GCCCTGGGGTACACAAGCC GCCCTGGGGTACACAAGCC TGAATCAATCTATCCATCTCTC TACTGTTTTCCACTCTAATGC CAGGAGGCCGTGTGAGAGC Fam GGCTGCGCCAGTGAACAA CCCCAAAGAATGCCCT Hex CCGAGTTATTACAAAGAAGCAC AAGTGATCCTCCCGCTTC AAGTGATCCTCCCGCTTC
130–140
130–150 130–140 150–160 190–220 160–180
62–45 55 62–45 55
PGD for inherited cardiac diseases
Table 2
55 62–45 55 62–45 55 62–45 55 62–45 55 62–45 55
449
450
A Kuliev et al.
A
MARKERS: D11S4174 D11S1344 MYBPC3-D1076fs D11S4117 D11S1350 D11S1978
C
144 172 N 116 194 186
148 174 N 114 187 167
PGD
148 174 N 114 187 167
3
137 159 N 116 191 186
N FET 1
137 159 N 116 191 186
PGD
B EMBRYO #
144 164 D1-76fs 112 179 182
D19S902 D19S867 D19S904 D19S246 TNNI 3-A157V D19S206 D19S571
5
FA
7
144 144 172 164 N D1-76fs 116 112 194 179 186 182
M
137 159 N 116 191 186
N FET 2
9
144 144 172 164 N D1-76fs 116 112 194 179 186 182
M
10
144 144 172 164 N D1-76fs 116 112 194 179 186 182
M
1
12
148 174 N 114 187 167
137 159 N 116 191 186
N
149 117 119 219 A157V 126 123
M
134 126 119 222 N 121 127
134 132 127 206 N 121 100
134 126 119 222 N 121 127
PGD
D
8
148 174 N 114 187 167
149 117 119 219 A157V 126 123
145 126 127 183 N 136 104
2
149 117 119 219 A157V 126 123
3
134 132 127 206 N 121 100
M
145 126 127 183 A157V 126 123
4
134 132 127 206 N 121 100
M
145 126 127 183 N 136 104
134 126 119 222 N 121 127
5
145 126 127 183 N 136 104
134 132 127 206 N 121 100
N N FET* FR*
6
145 117 119 219 A157V 126 123
7
134 132 127 206 N 121 100
M
8
149 117 119 219 A157V 126 123
134 126 119 222 N 121 127
M
FA
9
149 117 119 219 A157V 126 123
10
134 132 127 206 N 121 100
M
149 117 119 219 A157V 126 123
134 126 119 222 N 121 127
M
11
145 126 127 183 N 136 104
134 132 127 206 N 121 100
N FR*
* Normal for 24 chromosome by microarray
Figure 2 PGD for hypertrophic cardiomyopathy (CMH). (A, B) PGD for CMH4. (A) Family pedigree of a couple with affected mother carrying the frameshift mutation D1076fs in MYBPC3. Paternal linked polymorphic markers are shown on the left and maternal on the right, and the order of the markers and frameshift mutation in MYBPC3 are shown on the far left. (B) Blastomere results revealed three embryos (embryos 7, 9 and 10) carrying the frameshift mutation D1076fs in MYBPC3, 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, FET), resulting in an unaffected pregnancy (as indicated in the family pedigree by PGD). (C, D) PGD for CMH7. (A) Family pedigree of a couple with affected father carrying the A157V mutation in TNNI3. Paternal linked polymorphic markers are shown on the left and maternal on the right, and the order of the markers and mutation in TNNI3 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; FET = 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 PGD for CMD, determined by dominant mutation in LMNA, 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 LMNA 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 CMD. Of four cycles performed for three couples at risk for producing offspring with CMH, 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 MYBPC3, 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 PGD cycle for the patient at risk for producing offspring with CMH7 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
PGD for inherited cardiac diseases A
451
MARKERS:
D22S1153 D22S1160 D22S1161 D22S922 SCO2 SNP-NlaII
150 162 157 138 R262delCA +
148 150 133 162 155 157 138 136 E140K N +
158 133 155 138 N -
PGD 150 150 162 162 157 157 138 136 R262delCA E140K + +
158 133 155 138 N -
M, RECOMBINANT B
EMBRYO #
1
2
150 148 162 133 157 155 138 138 R262 E140K delCA + +
150 148 162 133 157 155 138 138 R262 E140K delCA + +
M
M
3
158 133 155 138 N -
N
4
150 150 150 162 162 162 157 157 157 136 138 136 R262 N N delCA + -
N CARRIER ET
150 162 157 136 N -
5
158 133 155 138 N -
150 162 157 136 N -
N
6
158 133 155 138 N -
7
150 150 150 162 162 162 157 157 157 136 138 136 N R262 N delCA + -
N
CARRIER
8
158 133 155 138 N -
9
150 162 157 136 N -
150 162 157 138 R262 delCA +
N MONOSOMY22
ET
Figure 3 PGD for cardioencephalomyopathy. (A) Family pedigree of a couple with a previous affected child, who was double heterozygous for E140K 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 PGD). ET = 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 PGD cycles performed for EMD, 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 EMD-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 PGD 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 CMD and CMH. Conditions in the family history of the couples at risk that may indicate a possible need for PGD 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 PGD. In
452
A Kuliev et al. DXS8103 DXS1684 DXS8061 DXS8087 EMD-IVS2+1G-T DXS1073 F8 INT1 F8INT 13 DXY154
A
143 140 157 198 112 98 174
I.
Y
1
145 140 161 200 114 100 165/167
II.
137 134 155 212 114 104 165
Y
2
143 140 157 198 112 98 174
2
1 PGD
1 137 134 155 212 114 104 165
OOCYTES
1
2
FA
M
PCR
C
143 140 157 198 112 98 174
1
3
137 134 155 212 114 104 165
EMD
III.
B
139 134 157 202 112 98 165
4
5
6
N
M
M
137 134 155 212 114 104 165
143 140 157 198 112 98 174
143 140 157 198 112 98 174
N
4
5
6
M XY
M XY
N XX
137 134 155 212 114 104 165
2 137 134 155 212 114 104 165
Y
7
9
10
N
M
M
137 134 155 212 114 104 165
143 140 157 198 112 98 174
143 140 157 198 112 98 174
9
10
139 134 157 202 112 98 165
11
12
13
N
N
FA
137 134 155 212 114 104 165
137 134 155 212 114 104 165
14
15
16
N
N
N
137 134 155 212 114 104 165
137 134 155 212 114 104 165
137 134 155 212 114 104 165
11
EMBRYO
PCR/FISH 143 140 157 198 112 98 174
Y ET
M XY
Y0
137 134 155 212 114 104 165
Y
ET
Figure 4 PGD for Emery–Dreifus muscular dystrophy (EMD). (A) Family pedigree of a couple with mother (II-2) carrying X-linked EMD–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 PGD). ET = 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 PGD. Many 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 PGD 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 PGD, 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, PGD 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.
PGD for inherited cardiac diseases So PGD 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 PGD, 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 PGD. The available experience in offering PGD for the above cardiac diseases and other conditions with inherited predisposition to common disorders showed that the availability of PGD 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 PGD may be offered as a relief for at-risk couples.
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453 blastomere biopsy for preimplantation genetic diagnosis. Hum. Reprod. 25, 275–282. Papadopoulou, L.C., Sue, C.M., Davidson, M.M., Tanji, K., Nishino, I., Sadlock, J.E., Krishna, S., Walker, W., Selby, J., Glerum, D.M., Coster, R.V., Lyon, G., Scalais, E., Lebel, R., Kaplan, P., Shanske, S., De Vivo, D.C., Bonilla, E., Hirano, M., DiMauro, S., Schon, E.A., 1999. Fatal infantile cardioencephalomyopathy with COX deficiency and mutations in SCO2, a COX assembly gene. Nature Genet 23, 333–337. Preimplantation Genetic Diagnosis International Society (PGDIS), 2008. Guidelines for good practice in PGD: program requirements and laboratory quality assurance. Reprod. Biomed. Online 16, 134–147. Preimplantation Genetic Diagnosis International Society (PGDIS), 2010. 10th International Congress on Preimplantation Genetic Diagnosis. Reprod. Biomed. Online 20, S1–S42. Rechitsky, S., Kuliev, A., 2010. Novel indications for preimplantation genetic diagnosis. Reprod. Biomed. Online 20, S1–S2. Rechitsky, S., Verlinsky, O., Chistokhina, A., Sharapova, T., Ozen, S., Masciangelo, C., Kuliev, A., Verlinsky, Y., 2002. Preimplantation Genetic Diagnosis for Cancer Predisposition. Reprod. Biomed. 4, 148–155. Rechitsky, S., Pomerantseva, K., Pakhalchuk, T., Polling, D., Verlinsky, O., Kuliev, A., 2011. First systematic experience of preimplantation genetic diagnosis for de novo mutations. Reprod. Biomed. Online 22, 350–361. Verlinsky, Y., Kuliev, A., 2005. Atlas of Preimplantation Genetic Diagnosis, second ed. Taylor and Francis, London and New York, p. 288. Verlinsky, Y., Kuliev, A., 2006. Practical Preimplantation Genetic Diagnosis. Springer, Berlin, NY, 204p. Declaration: The authors report no financial or commercial conflicts of interest. Received 16 October 2011; refereed 18 December 2011; accepted 21 December 2011.