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Review The Molecular Genetic Revolution in Congenital Heart Disease Raymond W. Sze 1,2 and Katherine E. Yutzey 3
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xtraordinary advances have been made in understanding the molecular basis of cardiovascular disease. In adult cardiology, gene therapy to trigger myocardial neoangiogenesis for ischemic heart disease may replace current therapies [1]. However, it is in congenital heart disease that fundamental changes in our approach to understanding and ultimately to treating cardiac disease are occurring. Our colleagues in cardiology and cardiothoracic surgery have recognized the bench-to-bedside importance of molecular genetic research, and one needs to only glance through their journals to see the prominence with which these insights are featured in their research and clinical articles. The four overarching organizations of the radiology community have recently agreed to reemphasize cardiac imaging in training, practice, and research. Courses and workshops in cardiac imaging have featured prominently in recent national conferences. What have been typically and appropriately emphasized are basic cardiac anatomy and physiology and MR imaging techniques. What has been neglected thus far is any mention of the molecular genetic approach to cardiac disease. If the radiology community wishes to be an active player
in the diagnosis and treatment of congenital heart disease, then clearly the language and research that form the foundation and concepts of its future must be understood. In this review, several fundamental concepts in molecular developmental biology will be introduced by contrasting the classic, descriptive embryologic approach to cardiac looping with the molecular genetic approach. Clinical applications of molecular genetic research will be described using the abnormalities that result from deletions in chromosome 22q11 and familial hypertrophic cardiomyopathy syndromes as examples. Finally, opportunities for radiologists and the radiology community to contribute to developmental research will be described. As will be emphasized throughout this article, modifications of traditional imaging techniques and the advancing science of molecular imaging will have a critical role in assembling the seemingly separate genetic pieces of the developmental puzzle into a unified and coherent whole.
Molecular Foundations of Congenital Heart Disease
Classic descriptive embryology teaches us that the primitive heart tube is formed by fusion
of the lateral endocardial tubes [2]. Bulges and constrictions of the primitive heart tube mark future chambers, and as the heart tube elongates, it loops to the right. Cardiac looping marks the first break in left–right symmetry of the developing embryo. However, the mechanisms that control looping have been poorly understood. Of importance is that excised hearts from experimental animals have an intrinsic ability to loop; looping occurs even when the heart tube is isolated from the pericardium, the cardiac jelly, and the hemodynamic effects of blood flow. During the past 3–5 years, molecular developmental biologists have made exciting progress in elucidating the mechanisms that control cardiac looping and development [3]. Asymmetric gene expression in the primitive heart tube provides important clues and markers for normal and abnormal looping (Fig. 1). Abnormal cardiac looping likely contributes to many developmental cardiac anomalies. Mutations in genes with a variety of functions in early heart development have been associated with human congenital heart disease [4]. The broad spectrum of genetic mutations that can lead to congenital disease underscores the importance of accurate early diagnosis of these developmental anomalies.
Received June 30, 2000; accepted after revision September 6, 2000. Presented at the annual meeting of the American Roentgen Ray Society, Washington, DC, May 2000. R. W. Sze is a 2000–2001 American Roentgen Ray Society Scholar. K. E. Yutzey is supported by grants from the National Institutes of Health and the American Heart Society, Ohio Valley affiliate. 1
Department of Radiology, Children’s Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039.
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Present address: Department of Radiology, Children’s Hospital & Regional Medical Center, 4800 Sand Point Way N.E., CH-69, Seattle, WA 98105. Address correspondence to R. W. Sze.
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Department of Pediatrics, Division of Molecular Cardiovascular Biology, Children’s Hospital Research Foundation, Cincinnati, OH 45229-3039.
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Sze and Yutzey which the genetic cause of cardiovascular disease has been identified, there is variable penetrance and the genotype is not predictive of the phenotype. Therefore, the development of improved imaging techniques is critical for the treatment of individuals with identified mutations in genes that can cause cardiac disease. Classic embryology deals with “what”; molecular developmental biology attempts to explain “how” and “why.” However, the reductionist strategy of dissecting the individual genes associated with heart development must then be redirected toward understanding the precise effects of these genes in isolation and in concert. Just because a gene is present during a developmental event does not prove its importance, nor does it explain its function. Techniques to visualize the developing organ will be critical to establishing causal relationships between gene and gene products and their actual contribution to embryogenesis. Fig. 1.—Drawing shows asymmetric gene expression in cardiac looping. Paracrine signaling molecules (A) are secreted by cells into their surroundings, affecting neighboring cells. Transcription factors bind to DNA (B) and regulate transcription of DNA sequences (genes) into messenger RNA; note helix–loop–helix motif. Extracellular matrix proteins (C) are macromolecules that serve as support structure of cells.
Paracrine Signaling Molecules
Extracellular Matrix Proteins
Paracrine signaling molecules are secreted by cells into their surroundings, with effects on target cells in close proximity (Fig. 1). This is in contrast to endocrine signaling, in which molecules are secreted into the blood stream with effects on distant target cells. An example of paracrine signaling in a nondevelopmental context is the transmission of nerve impulses via neurotransmitters across synapses. BMP4 (bone morphogenetic protein 4) is a paracrine signaling molecule expressed in the primitive heart tube of the zebrafish. The shift in expression from radially symmetric to left-predominant marks the first break in left–right symmetry of the developing heart [5]. In mutant animals with asymmetric or right-sided expression of BMP4, cardiac looping is randomized.
Extracellular matrix proteins are macromolecules secreted by cells that serve as scaffolding for developing cells (Fig. 1). These proteins may separate developing groups of cells, direct cell migration, and even induce differentiation in adjacent cells. The extracellular matrix protein JB3 is asymmetrically expressed on the right side of the primitive heart tube, and flectin and hLAMP1 are asymmetrically expressed on the left. When extracellular matrix protein expression is experimentally disrupted, such as in vitamin A–deficient quail hearts, looping is arrested [7]. Mutations in human genes encoding for paracrine signaling molecules, transcription factors, and extracellular matrix proteins have been associated with a variety of syndromes with congenital cardiovascular defects. An example of a paracrine signaling mechanism linked to congenital heart disease in humans is Alagille syndrome [8]. Deletions in the jagged1 gene, which encodes a signaling molecule in the Notch pathway, have been identified in patients with this syndrome. Mutations in the transcription factor genes NKX2.5 and TBX5 (Fig. 1) have been associated with a variety of congenital cardiac malformations in humans [9–11]. Marfan syndrome, which includes cardiovascular defects, is the result of mutations in the gene encoding the extracellular matrix protein fibrillin [12]. In each case in
Transcription Factors
Transcription factors are proteins that bind to DNA and regulate the transcription of DNA to messenger RNA (Fig. 1). In embryonic chicks and mice, two muscle-specific transcription factors, dHAND and eHAND, are asymmetrically expressed in the right and left ventricles, respectively. Loss of dHAND expression leads to arrest of cardiac development at the looping stage [6]. Based on these studies, dHAND has been hypothesized to have a critical role in heart chamber development. 576
Clinical Applications CATCH-22
CATCH-22 is the acronym for Cardiac defects, Abnormal facies, Thymic hypoplasia, Cleft palate, and Hypocalcemia from deletions in chromosome 22 [4] (Fig. 2). The cardiac defects involve abnormalities of conotruncal development, such as persistent truncus arteriosus, tetralogy of Fallot, and interrupted aortic arch. The association of conotruncal cardiac lesions and abnormalities of third and fourth pharyngeal pouch derivatives was first recognized in the DiGeorge syndrome [13]. Two other syndromes, the velocardiofacial [14] and conotruncal anomaly face syndromes [15], were recognized as similar but were considered distinct because of different patterns of facial dysmorphism and distribution of cardiac anomalies. Recent cytogenetic and molecular studies, however, revealed microdeletions in the same specific region of chromosome 22 (22q11) (Fig. 2) in 80–95% of patients. Furthermore, genotyping of nonsyndromic patients with isolated conotruncal cardiac lesions revealed that between 20% and 30% of these patients had deletions in the identical chromosomal region. Likely, 10% of patient’s requiring surgery for congenital heart disease have abnormalities in this specific genetic neighborhood [4]. DiGeorge [16] was first to propose perturbation of the third and fourth pharyngeal pouches as an underlying mechanism for the syndrome that he had first described. CATCH-22 is currently understood as a defect of neural crest migration and development. The neural crest is the pleuripotential cell population, sometimes reAJR:176, March 2001
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The Molecular Genetic Revolution in Congenital Heart Disease
Fig. 2.—Drawing shows cardiac defects, abnormal facies, thymic hypoplasia, cleft palate, and hypocalcemia from deletions in chromosome 22 (CATCH-22). 22q11 locus (A), DiGeorge-critical segment, is located on long arm of chromosome 22. Neural crest migration (B) is shown by stippled arrows, which depict migrational routes of neural crest cells to various primordia affected in CATCH-22. Clinical manifestations of CATCH-22 (C), including facial dysmorphism, hypocalcemia, immunodeficiency resulting from absence of parathyroid and thymic development, and congenital heart and aortic arch diseases, are illustrated.
Fig. 3.—Drawing illustrates sacromeric protein mutations, which cause hypertrophic cardiomyopathy. Sarcomere (A) is basic unit of myocardial contraction. Magnified view of region shown in inset (B) depicts individual protein components of thick and thin filaments of sarcomere. Morphology and prognosis (C) of various hypertrophic cardiomyopathies due to mutations of specific sarcomeric contractile proteins are shown. LV = left ventricle.
ferred to as the fourth germ cell layer, that arises from the lateral margins of the neural plate (Fig. 2). These cells detach from the neural plate durAJR:176, March 2001
ing neurulation, migrate throughout the body, and differentiate into structures as diverse as the dermal bones of the skull, adrenal medulla, dor-
sal root and chain ganglia, and melanocytes of the skin. In the developing cardiovasculature, neural crest contributes to the truncoconal septum and the aortic arch (Fig. 2); ablation of the neural crest in experimental chicken embryos re-creates the phenotype of DiGeorge syndrome. Current research is focused on determining which gene or genes within the 22q11 region are responsible for perturbing neural crest development and causing the CATCH-22 syndrome. Researchers hope to create a mouse model of the 22q11 deletion, but a major obstacle will be recognizing and understanding the development of this structural heart disease in experimental embryos and animals. High-resolution imaging techniques to study the morphology of the developing heart in transgenic animals, most commonly the mouse, could be critical to the success of these efforts. In the future, identification of early morphologic indicators of aortic arch anomalies could translate into the clinic with more accurate prenatal diagnoses in fetuses at genetic risk for cardiovascular disease. Familial Hypertrophic Cardiomyopathy
Familial hypertrophic cardiomyopathy is defined as ventricular wall thickening without a cause for increased cardiac mass [17]. The estimated prevalence is 0.2%, but it may be higher (patients who meet sonographic diagnostic criteria can be asymptomatic). Familial hypertrophic cardiomyopathy is the most common cause of sudden death in young adults [18]. The severity and anatomic manifestations of disease are variable. Recent molecular genetic research reveals that familial hypertrophic cardiomyopathy is not one disease with multiple manifestations but, instead, is multiple diseases of the contractile proteins of the myocardium or sarcomere (Fig. 3). To date, mutations of eight distinct proteins have been implicated in familial hypertrophic cardiomyopathy [19]. Clinical studies of patients with familial hypertrophic cardiomyopathy and experimental studies of transgenic mouse models of familial hypertrophic cardiomyopathy have provided insight into this disease. Mutations of the myosin light chain are associated with the mid-cavitary form of hypertophic cardiomyopathy. Mutations of the ß-myosin heavy chain cause a high risk of sudden death (Fig. 3). Beyond gene-specific morphology and prognosis is the hope for gene-specific treatment. Investigators recognized that certain sarcomeric mutations lead to increased intracellular calcium with the subsequent activation of the enzyme calcineurin. Calcineurin triggers a molecular cascade ultimately leading 577
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Sze and Yutzey to abnormal transcription of cardiac proteins. In a dramatic experiment, mice with certain sarcomeric mutations were treated with cyclosporin, a calcineurin inhibitor [20]. Treated mice had normal hearts. Genetically identical but untreated littermates developed severe hypertrophic cardiomyopathy, myofibrillar disarray histologically, and early development of congestive failure (Fig. 4). The clinical syndromes of CATCH-22 and familial hypertrophic cardiomyopathy exemplify fundamental changes in clinical understanding and eventual management of congenital heart disease. In CATCH-22, multiple syndromes based on historical description are unified by a common chromosomal defect; in familial hypertrophic cardiomyopathy, a single disease with an inexplicable variety of clinical manifestations is found to be multiple distinct abnormalities of the fundamental contractile unit of the heart. The molecular etiologic classification will eventually replace classifications based on historical descriptions, and further research will lead to gene-specific diagnosis, prognosis, and treatment of congenital heart disease. Imaging Applications and Opportunities Traditional Imaging
The molecular revolution will provide important opportunities for the radiology com-
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munity to contribute to basic science and clinical research. As has been emphasized throughout this article, the success of the reductionist approach in dissecting out the genes associated with development must be counterbalanced by the realization that identification of the molecular pieces (genes) of a puzzle does not solve the puzzle. Even if a gene or gene product is temporally and spatially related to a developmental event, it is not certain that it plays an important role in that event. The power of current genotyping techniques must be complemented by tools to study the dynamic anatomy and function of the developing organism. It is in the determination of the outward physical manifestation of a gene or gene product, or phenotype, that the radiologic sciences stand to make tremendous contributions to the understanding of developmental biology. Molecular cardiovascular biology presents several challenges. The mouse is the most common experimental animal because of its well-characterized genome, comparability with the human heart, and relatively low breeding and maintenance costs. However, its small size and rapid heart rate (basal heart rate of 550–620 beats per minute) make anatomic and physiologic characterization extremely difficult [21, 22]. MR microscopy (MR imaging on super high-field magnets with resolution on the 10-
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Fig. 4.—Prevention of cardiac hypertrophy in transgenic mice by calcineurin inhibition. Heart specimens were taken from genetically identical mice (tropomodulin-overexpressing transgenic). Images were digitized with slide scanner and then enlarged using software (Photoshop; Adobe Systems, Mountain View, CA). rv = right ventricle, lv = left ventricle. (Reprinted with permission from [20]) A, Histologic specimen from untreated mouse shows characteristic cardiomyopathy that develops by 24 days after birth. (H and E) B, Histologic specimen from mouse treated with cyclosporin reveals normal heart. (H and E)
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to 100-µ scale) has been applied to studying the vasculature in fixed embryos [23] and embryos in utero [24]. Our laboratory has reported a high-resolution three-dimensional MR microscopic technique for morphometric analysis of a transgenic mouse model of dilated cardiomyopathy. The technique allowed calculation of atrial and ventricular chamber volumes, and the isotropic voxel acquisition allowed volumetric postprocessing and display [25] (Fig. 5). Gated MR images of transgenic mice have also been obtained, with good correlation of cardiac output and stroke volume calculations with an invasive thermodilution technique [26]. Continued refinement of coils, contrast agents, and pulse sequences may ultimately provide developmental biologists with a much-needed assay: a high-resolution, noninvasive, in utero tool that can be used repeatedly to study the precise sequence of events involved in normal and abnormal development. Molecular Imaging
Molecular imaging seeks specific molecules as the source of imaging contrast, as opposed to traditional imaging, which targets nonspecific physical and physiologic properties [27]. Challenges include the creation of specific molecular probes, systems to deliver the probe to the area of interest, strategies to amplify the typically pico- to nanomolar concentration signal, and high-resolution and high-sensitivity imaging systems. Although most current molecular imaging research is focused on tumor imaging, techniques developed should be equally applicable to answering developmental questions. Of particular interest is the recent report of MR imaging visualization of gene expression in living Xenopus laevis tadpoles [28]. Investigators designed a gadolinium enhancement technique that capitalizes on the enzyme ß-galactosidase, the workhorse of in vitro staining for gene expression in developmental biology. The gadolinium contrast is shielded from water until it encounters ß-galactosidase, at which time the protective cage is opened. The unpaired electrons then interact with the protons in water to cause signal enhancement (Fig. 6). The technique should be applicable to studying developmental questions such as the anatomic location and timing of genes believed to be involved in developmental processes. Apoptosis, or programmed cell death, is a critical process in cardiac development. It is thought to be involved in the fusion of the endocardial tubes, remodeling of the AJR:176, March 2001
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The Molecular Genetic Revolution in Congenital Heart Disease
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Fig. 5.—Three-dimensional T1-weighted gradient-echo MR images (TR/TE, 50/5; flip angle 45°; isotropic; 150-mm voxels) of heart; chambers were perfused with gadolinium gelatin solution before scanning. (Reprinted with permission from [25]) A and B, Normal mouse heart showing direct-acquisition horizontal long-axis section (A) and postprocessed volumetric rendering of isolated atria and ventricles (B). C and D, Heart of transgenic mouse model (overexpression of myosin light chain) of dilated cardiomyopathy showing direct acquisition horizontal long-axis section (C) and postprocessed volumetric rendering of isolated atria and ventricles (D). Note attenuated ventricular walls and enlarged chamber volumes in cardiomyopathy model.
C muscular septum, and sculpting of the cardiac valves, among other things. Investigators have recently described an in vivo nuclear medicine technique to study apoptosis in transplant rejection. The technique involves radiolabeling annexin V, a protein that binds to phosphatidylserine, a membrane protein externalized during the early execution phase of apoptosis [29]. Although these investigators have concentrated on noninvasive cardiac and liver transplant rejection detection (Fig. 7), the availability of an in vivo method to study apoptosis in the developing fetus has potential promise in clarifying the precise role of this developmental event in normal and abnormal organogenesis. AJR:176, March 2001
Conclusion
The concerted effort by the radiology community to reemphasize cardiac imaging in both training and practice will advance cardiovascular diagnosis and treatment and will ultimately benefit patients with congenital and acquired diseases of the heart and great vessels. It is critical that radiologists who plan to be involved in the care of patients with congenital heart disease realize that molecular genetic research is fundamentally changing our approach to and understanding of these malformations. Insights obtained through chromosomal studies of patients and through genetic manipulation of experimental animals already have clinical applications. The recently de-
D scribed CATCH-22 syndrome unifies conotruncal cardiac lesions with and without other anomalies of neural crest development under deletions of a single chromosomal locus, and familial hypertrophic cardiomyopathy is now understood to be multiple distinct diseases of the sarcomere. Insights gained from basic science research may eventually allow gene-specific diagnosis, prognosis, and treatment. The sequencing of the human genome undoubtedly will result in the identification of additional genes associated with developmental defects in the cardiovascular system [11]. The cardiology community is rapidly adopting the molecular approach to congenital heart disease, and radiologists who intend 579
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Sze and Yutzey to be a part of the future of congenital heart disease must be able to understand and speak its language. The molecular revolution offers important opportunities for the radiology community to participate in basic science and clinical research. As basic science researchers discover more and more genes associated with cardiac development, it becomes increasingly clear that the specific role of most of these genes in isolation and in concert is largely unknown. A fundamental problem is the lack of techniques to determine the precise morphologic consequences of a genetic perturbation. The molecular developmental reductionists have made rapid strides in identifying the pieces, or genes, of the cardiac developmental puzzle. The radiology community now has the opportunity, through modification of traditional imaging methods and through pioneering molecular imaging techniques, to help put the pieces together.
Fig. 6.—MR imaging detection of β-galactosidase messenger RNA expression in living Xenopus laevis tadpoles. Gadolinium is initially enclosed in chemical cage. When cage encounters enzyme β-galactosidase, protective gate is cleaved open, exposing gadolinium (purple) to water and causing signal enhancement. β-galactosidase messenger RNA expression is seen in eye (e), cement gland (c), brachial arches (b), and somites (s). (Reprinted with permission from [27])
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Acknowledgments
We thank Glenn Miñano for preparation of the illustrations and Nikki Turner for secretarial assistance.
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Fig. 7.—Noninvasive monitoring of acute cardiac transplant rejection and response to immunosuppression using radiolabeled annexin V in 12-week-old male SpragueDawley rats. Cardiac allograft was anastomosed to abdominal aorta and inferior vena cava. Immunosuppression was initiated on postoperative day 5. (Courtesy of Blankenberg F, Palo Alto, CA) A, Prone anterior nuclear imaging scan shows no evidence of rejection on postoperative day 1. B, Serial anterior acquisition obtained on postoperative day 4 shows apoptotic acute rejection and increased radiolabeled annexin V uptake (arrow ). Immunosuppression was initiated on postoperative day 5. C, Prone anterior nuclear imaging scan shows evidence of mild rejection on postoperative day 10. D, Prone anterior nuclear imaging scan shows no evidence of rejection on postoperative day 18; diminished radionuclide activity represents response to therapy.
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