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Preimplantation genetic diagnosis (PGD) 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 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.
Preimplantation genetic diagnosis (PGD) is currently an established clinical procedure in assisted reproduction and genetic practices. 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. 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. 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. We present here our first 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 for these disorders was performed, resulting in transfer in 15 of them, which yielded nine unaffected pregnancies and birth 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 at risk for premature or sudden death.
). 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 (
). 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 (
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 (
). 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 (
). 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.
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).
Table 1Reproductive outcome of PGD for cardiac diseases.
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.
Figure 1PGD 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.
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.
Figure 2PGD 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.
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 (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 (
). 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 (
), 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 (
), 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 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 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.
Figure 3PGD 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.
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.
Figure 4PGD 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.
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 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.
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.
Declaration: The authors report no financial or commercial conflicts of interest.
Footnotes
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 community-based programmes for prevention of genetic disorders and early approaches for prenatal diagnosis. He moved to the Reproductive Genetics Institute in 1990, where he heads 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.
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 community-based programmes for prevention of genetic disorders and early approaches for prenatal diagnosis. He moved to the Reproductive Genetics Institute in 1990, where he heads 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.
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