Marfan syndrome: from molecular pathogenesis to clinical treatment Francesco Ramirez and Harry C Dietz

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1 Marfan syndrome: from molecular pathogenesis to clinical treatment Francesco Ramirez and Harry C Dietz Marfan syndrome is a connective tissue disorder with ocular, musculoskeletal and cardiovascular manifestations that are caused by mutations in fibrillin-1, the major constituent of extracellular microfibrils. Mouse models of Marfan syndrome have revealed that fibrillin-1 mutations perturb local TGFb signaling, in addition to impairing tissue integrity. This discovery has led to the identification of a new syndrome with overlapping Marfan syndrome-like manifestations that is caused by mutations in TGFb receptor types I and II. It has also prompted the idea that TGFb antagonism will be a productive treatment strategy in Marfan syndrome and perhaps in other related disorders. More generally, these studies have established that Marfan syndrome is part of a group of developmental disorders with broad and complex effects on morphogenesis, homeostasis and organ function. Addresses Institute of Genetic Medicine, Howard Hughes Medical Institute, Johns Hopkins University, School of Medicine, BRB 539, 733 North Broadway, Baltimore, MD 21205, USA Corresponding author: Dietz, Harry C (hdietz@jhmi.edu) This review comes from a themed issue on Genetics of disease Edited by Robert Nussbaum and Leena Peltonen Available online 27th April X/$ see front matter # 2007 Elsevier Ltd. All rights reserved. DOI /j.gde Introduction Marfan syndrome (Online Mendelian Inheritance in Man [OMIM] ) is a systemic disorder caused by mutations in the extracellular matrix protein fibrillin-1. First described by Antoine-Bernard Marfan in an 1896 case report of a young girl with unusual musculoskeletal features [1], Marfan syndrome occupies a special place in the history of medicine and science owing to the number of seminal discoveries and conceptual breakthroughs that have been associated with this disorder. A 50-year-long analysis of the clinical and genetic features of Marfan syndrome ultimately led Victor McKusick to delineate it as the founding member of a larger group of congenital conditions that he defined as the heritable disorders of the connective tissue, and which he predicted to be the result of structural or metabolic dysfunctions of extracellular matrix proteins [2]. The demonstration in 1991 that mutations in the fibrillin-1 gene (FBN1) cause Marfan syndrome confirmed McKusick s prediction, and in addition represented an early successful example of the discovery of a disease-causing gene based on the convergence of genetic linkage studies and the candidate gene approach [3]. Fifteen years later, the unexpected finding that increased transforming growth factor beta (TGFb) signaling is part of the molecular pathogenesis of Fbn1-deficient mice has paved the way to a new drugbased strategy against the life-threatening manifestations of Marfan syndrome [4,5 ]. This review highlights the most exciting developments in the past two years of Marfan syndrome research and discusses their impact on the clinical management of this and related conditions, including more common and non-syndromic presentations of Marfan syndrome. Clinical manifestations and differential diagnosis The phenotype of Marfan syndrome typically involves manifestations in the cardiovascular, skeletal and ocular systems; additionally, the skin, integument, lung, muscle, adipose tissue and dura can also be affected (Table 1) [6 ]. Inherited as an autosomal dominant trait, Marfan syndrome has an estimated incidence of 2 3 per individuals. Approximately 25% of cases are caused by de novo mutations. The disease has no ethnic or gender predilection and shows high penetrance but marked inter- and intra-familial variability. The most striking and immediately evident manifestation in Marfan syndrome patients often involves a disproportionate increase in linear bone growth that causes overt malformations of the digits, limbs and anterior chest wall. Craniofacial abnormalities, scoliosis and joint hypermobility are common skeletal findings as well. Early and severe myopia and dislocation of one or both lenses of the eye occur in the majority of Marfan syndrome patients. Cardiovascular manifestations include progressive aortic root enlargement and abnormally thick and elongated valve leaflets. Ascending aortic aneurysm can precipitate life-threatening complications such as aortic regurgitation, dissection or rupture. Treatment of vascular disease in Marfan syndrome includes regular imaging to monitor aneurysm progression, b-adrenergic blockade to slow aortic growth, and prophylactic surgery to prevent aortic complications. A major problem with the clinical management of cardiovascular complications in Marfan syndrome is the difficulty to diagnose the disorder, particularly in young children, because of extensive phenotypic variability, age-dependent onset of informative manifestations, high degree of

2 Marfan syndrome: from molecular pathogenesis to clinical treatment Ramirez and Dietz 253 Table 1 Clinical features of the Marfan and Loeys-Dietz syndromes. Marfan syndrome Loeys-Dietz syndrome Ocular system Early and severe myopia +++ Ectopia lentis +++ Glaucoma + Cataract + Retinal detachment + + Blue sclerae ++ Skeletal system Pectus deformity Scoliosis Dolichostenomelia +++ +/ Arachnodactyly Joint laxity Pes planus Cervical spine instability ++ Osteoporosis +/ ++ Club foot deformity ++ Camptodactyly + ++ Cardiovascular system Aortic root aneurysm Aortic root dissection Other primary aneurysms +/ +++ Other primary dissections +/ +++ Arterial tortuosity +++ Mitral valve prolapse Patent ductus arteriosus ++ Atrial septal defect ++ Other septal defects + Bicuspid aortic valve + Craniofacial Long and narrow face +++ +/ Down-slant of palpebral fissures Enophthalmos ++ + Malar hypoplasia Micrognatia or retrognathia High-arched palate Craniosynostosis ++(1) Hypertelorism +++(1) Cleft palate ++(1) Bifid uvula +++(2) Hydrocephalus + Neurocognitive Developmental delay + Cutaneous Striae distensae Dystrophic scars ++ Translucent skin +++ Easy bruising +++ Velvety skin texture +++ Integument Hernia Dural ectasia Visceral Splenic rupture + Bowel perforation + Uterine rupture or hemorrhage ++ Inflammatory bowel symptoms +, not associated; +/, rare and/or subtle; +, occasionally observed; ++, commonly observed; +++, generally observed; (1), by definition, not observed in Loeys-Dietz syndrome type II; (2), rare and/or subtle in Loeys-Dietz syndrome type II. spontaneous mutations, or clinical overlap with several other conditions. As our understanding of the genetic and clinical features of Marfan syndrome has evolved, so have the clinical criteria used to identify individuals at risk. The current diagnostic criteria, known as the Ghent Nosology, are the product of this evolving process and provide more stringent guidelines than before to differentiate classic Marfan syndrome from a number of connective tissue disorders that share some Marfan syndrome-like manifestations but differ in their genetic cause, repertoire of manifestations, natural history, and response to treatment [7]. Molecular genetics and pathophysiology Fibrillin-1 and the closely related fibrillin-2 protein (mutated in congenital contractural arachnodactyly [CCA]; OMIM ] are large glycoproteins that are primarily made of multiple repeated domains homologous to the calcium-binding epidermal growth factor (cbegf) module, and a distinct 8-cysteine motif (TB/8-cys) found uniquely in the latent TGFb binding proteins (LTBPs) (Figure 1) [8 ]. Fibrillin monomers polymerize into microfibrils that incorporate or are decorated by additional proteins, in addition to associating with elastin in the elastic fibers (Figure 1). Microfibrils and elastic fibers give rise to morphologically discrete architectural assemblies that fulfill the mechanical demands of individual organ systems, such as the elastic fibers that together with the interposed layers of vascular muscle cells impart elasticity to the aortic wall. Although experimental evidence and biosynthetic considerations had suggested that loss of tissue integrity is the underlying pathophysiology in Marfan syndrome [9], clinical observations pointed to a larger role of fibrillinrich microfibrils in organ physiology. First, the high degree of clinical variability in the absence of informative genotype phenotype correlations implied that modifier genes modulate phenotypic severity in Marfan syndrome. Second, selected manifestations of Marfan syndrome, such as bone overgrowth, craniofacial features, valve and lung abnormalities, and muscle and fat hypoplasia, argued for altered cell behavior during morphogenesis. These considerations were indirectly supported by biochemical evidence that fibrillin-1 can interact with, and presumably influence, a large variety of cell surface and extracellular proteins, including LTBPs, molecules that target latent TGFb to the matrix [10,11]. Binding to the extracellular matrix provides functional context to growth factors by regulating the spatial and temporal release, as well as the duration and intensity of productive signaling events [11]. In this view, FBN1 mutations in Marfan syndrome might also promote promiscuous activation of TGFb with adverse consequences to diverse cellular activities. Fibrillin-1 regulates TGFb bioactivity Mice homozygous for a hypomorphic in-frame deletion (mgd) in Fbn1 that replicate the neonatal lethal form of

3 254 Genetics of disease Figure 1 Schematic representation of the fibrillin-1 protein with its main structural motifs (a) as well as the steps leading to elastic fiber and microfibril assembly (b). Fibrillin-1 monomers contain multiple repetitive motifs including epidermal growth factor-like (EGF) motifs, some of which adhere to the consensus for calcium binding (cbegf); an 8-cysteine motif with homology to that found in the latent transforming growth factor-b binding proteins (TB/8-cys) and a hybrid motif containing features of both EGF and TB/8-cys motifs (hybrid). Upon secretion, fibrillin-1 monomers aggregate to form complex beaded structures that in turn form macro-aggregates called microfibrils. Microfibrils can occur independently of elastin or can surround and become embedded within elastic fibers during embryogenesis. Marfan syndrome have provided the first in vivo evidence supporting a role for fibrillin-1 in TGFb modulation [12]. Many individuals with Marfan syndrome show chronic obstructive lung disease and a predisposition for pneumothorax, a process that had been equated with destructive emphysema due to impaired tissue integrity [6 ]. However, homozygous mgd mice display widening of the distal pre-alveolar saccules at birth, without signs of inflammation or tissue destruction [4]. Importantly, the mouse phenotype was correlated with elevated TGFb activity in lung tissue, in addition to being rescued by systemic administration of TGFb-neutralizing antibodies. These early findings raised the possibility that a similar mechanism underlies other manifestations of Marfan syndrome, a prediction that has been validated recently in different strains of Marfan syndrome-like mice. Ng et al. [13] have associated architectural alterations in the mitral valves of mice heterozygous for a structural mutation in fibrillin-1 (substitution of an obligatory cysteine in a cbegf module; C1039G) with increased cell proliferation, decreased apoptosis and abnormally high TGFb activity. They have also been able to prevent the mitral valve phenotype by TGFb antagonism. Other reports have documented excess TGFb activation and signaling in the dura of mgr/mgr mice, which produce less than the normal amount of wild type fibrillin-1, and in the aortic wall and skeletal muscles of C1039G/+ mice [14,15 ].

4 Marfan syndrome: from molecular pathogenesis to clinical treatment Ramirez and Dietz 255 Early analyses of mgr/mgr mice had indicated that aneurysm progression in this adult lethal model of Marfan syndrome is largely driven by secondary cellular events, which are consistent with an unproductive effort by resident vascular muscle cells to remodel an intrinsically faulty but morphologically normal elastic matrix [16,17]. In temporal succession, these events include inappropriate production of matrix proteins and metalloproteinases, elastic fiber calcification, vascular wall inflammation, intimal hyperplasia, and structural collapse of the vessel wall. Addition of synthetic fibrillin-1 peptides or aortic extracts from mgr/mgr mice to cell culture systems has more recently suggested that proteolysis of fibrillin-rich microfibrils also contributes to aneurysm progression by stimulating the expression of metalloproteinases and macrophage chemotaxis [18,19 ]. Judge et al. [20] have shown that the aortic wall of C1039G/+ mice recapitulates the same histopathology in the absence of a clinical end point. Owing to their survival, C1039G/+ mice have been employed to document that TGFb antagonism can effectively prevent histological signs of aneurysm progression in this Marfan syndrome model [5 ]. These results demonstrate that fibrillin-1 microfibrils serve an essential role in aortic matrix homeostasis during extra-uterine life, and that structural deficits in microfibrils promote abnormal activation of TGFb with deleterious consequences on vascular muscle cell performance and tissue remodeling (Figure 2). Analysis of vascular disease in mgn/mgn mice (which do not produce fibrillin-1) has recently shown that dissecting aortic aneurysm in this neonatal lethal model of Marfan syndrome is accounted for by impaired maturation of the vessel wall, even in the presence of normally cross-linked elastin [21]. Clinical spectrum of TGFb signaling disorders of the connective tissue The discovery that perturbed TGFb signaling contributes to Marfan syndrome pathogenesis predicted that conditions that display Marfan syndrome-like manifestations might be caused by mutations in different components of the TGFb signaling network (regulators or transducers). Loeys-Dietz syndrome (OMIM ) is an illustrative example of this prediction [22 ]. Loeys- Dietz syndrome is an autosomal dominant disorder with both unique and Marfan syndrome-like manifestations, such as aortic root aneurysm, aneurysms and dissections throughout the arterial tree, and generalized arterial tortuosity (Table 1). Loeys-Dietz syndrome is caused by heterozygous substitution of evolutionarily conserved obligatory residues in the kinase domains of the type I or II TGFb receptor (TbR-I or TbR-II), which in theory should attenuate TGFb signaling. Contrary to this Figure 2 Model of normal regulation of TGFb by microfibrils and perturbations associated with microfibrillar deficiency in Marfan syndrome. Extracellular microfibrils normally bind the large latent complex of TGFb, composed of the mature cytokine (TGFb), latency-associated peptide (LAP) and one of three latent transforming growth factor-b binding proteins (LTBPs). This interaction is proposed to suppress the release of free and active TGFb (TGFb activation). In the absence of a sufficient quotient of microfibrils (e.g. Marfan syndrome), failure of matrix sequestration of the large latent complex leads to promiscuous activation of TGFb (lightening bolt). Free and active TGFb (star burst) interacts with its cell surface receptor, culminating in phosphorylation (P) of the receptor-activated smad proteins (R-Smads 2 and 3), which then bind to Smad4 and translocate from the cytoplasm to the nucleus, where, in combination with transcription factors (TFs), they mediate TGFb-induced transcriptional responses. Genes downstream of TGFb induce phenotypic consequences in Marfan syndrome including developmental emphysema, myxomatous changes of the mitral valve and mitral valve prolapse, aortic aneurysm formation and skeletal muscle myopathy. Losartan is believed to rescue these phenotypes by decreasing the expression of TGFb, by decreasing expression of TGFb receptor or by decreasing the expression of activators of TGFb such as thrombospondin-1.

5 256 Genetics of disease prediction and an earlier report [23], the vessel wall of Loeys-Dietz syndrome patients has been found to exhibit increased TGFb signaling, and cells from these patients have been shown to maintain TGFb responsiveness [22 ]. The paradoxical findings in Loeys-Dietz syndrome suggest that heterozygous TGFBR mutations either trigger unproductive compensatory events or have themselves gain-of-function properties. It also seems possible that some phenotypic manifestations of Loeys- Dietz syndrome reflect blunted TGFb responsiveness. This might vary in a context-specified manner. For example, one could imagine that transient but high-level bursts of TGFb, as might be expected during temporally constrained developmental events, might exhaust the capacity to propagate signal in patients with a half-normal quotient of functional receptors. By contrast, this quotient might be capable of handling the more subtle level of TGFb associated with tissue homeostasis, but the chronicity of such near-threshold signaling might predispose to altered activity of signal transducers or regulators, culminating in paradoxically enhanced signaling. Fibrillin-1 deficiency might also have dual effects on TGFb activity, because microfibrils appear to act as both positive regulators by optimizing cytokine concentration at sites of intended function and negative regulators by sequestering and suppressing activation of the latent TGFb complex. This mode of regulation might extend to other TGF-b superfamily members, such as the BMPs. Indeed, syndactyly in Fbn2 null mice has been linked genetically with a deficiency in Bmp7 signaling, and biochemical interaction between fibrillin-1 and the pro-domain of Bmp-7 has recently been demonstrated [24,25 ]. It is therefore plausible that complex connective tissue disorders, such as Marfan syndrome and Loeys-Dietz syndrome, integrate both an excess and a deficiency of signaling by multiple cytokines. A subset of patients with features reminiscent of the vascular form of Ehlers-Danlos syndrome (OMIM ), a condition typically caused by mutations in type III collagen, harbor heterozygous mutations in TGFRB1 or TGFRB2 [26 ]. Such patients (designated Loeys-Dietz syndrome type II) do not have the typical craniofacial features previously associated with Loeys-Dietz syndrome (now termed type I) but do show arterial tortuosity and similarly aggressive vascular disease. Patients with TGFBR mutations have also been described as having typical Marfan syndrome or isolated thoracic aortic aneurysm [27,28]. Both groups showed atypically widespread and/or severe vascular manifestations for these conditions. Given that identical mutations have been observed in individuals with Loeys-Dietz syndrome, further clinical evaluations are needed to assess whether these mutations cause additional features of Loeys-Dietz syndrome in these patients; if not, such individuals might prove a valuable resource in the identification of genetic modifiers that restrict phenotypic expression of TGFBR mutations. Arterial tortuosity syndrome (OMIM ) is another disorder associated with elevated TGFb activity and characterized by vascular and skeletal manifestations that overlap with Loeys-Dietz syndrome [29]. The underlying defect in arterial tortuosity syndrome is loss of function of the glucose transporter GLUT10, a defect that impairs glucose-dependent expression of decorin, a potent extracellular inhibitor of TGFb. Finally, there are other disorders that do not fulfill the Ghent nosology but which are occasionally caused by FBN1 mutations, such as familial ectopia lentis, Shprintzen-Goldberg syndrome and Weill- Marchesani syndrome [30 32]. It is yet to be determined whether the majority of these patients harbor mutations in discrete components of the TGFb signaling network. Therapeutic applications The demonstration that TGFb antagonism can rescue aortic aneurysm in C1039G/+ mice prompted the idea to test the efficacy of losartan, a widely used angiotensin II type 1 receptor (AT1) antagonist, because of its antihypertensive properties and ability to counteract TGFb in animal models of chronic renal disease and cardiomyopathy [33,34]. Drug administration to 2-month-old C1039G/+ mice and 14-day-old C1039G/+ embryos for 6 and 10 months, respectively, blocked the development of histological signs of aortic aneurysm in both cases [5 ]. The treatment also had a beneficial effect on alveolar septation and muscle hypoplasia. Although the precise mechanism whereby losartan exerts systemic TGFb blockage remains to be elucidated, these experiments have provided proof-of-principle that TGFb antagonism is a general strategy against aneurysm progression in Marfan syndrome and other disorders of the TGFb signaling network. Similar considerations apply for the use of losartan in the clinical management of congenital and acquired myopathies. As in Marfan syndrome, muscle hypoplasia is also observed in Fbn1 mutant mouse strains and is associated with impaired muscle regeneration in response to injury or physiologic signals for hypertrophy. Once again, the phenotype has been shown to be accounted for by increased TGFb signaling and to be rescued by TGFb antagonism with either neutralizing antibody or losartan [15 ]. A far-reaching finding of these studies is that the same TGFb-dependent failure of muscle regeneration and therapeutic response to losartan was also seen in a dystrophin-deficient mouse model of Duchenne muscular dystrophy, with significant improvement of muscle regeneration, architecture and function [15 ]. Conclusions Marfan syndrome research continues to yield novel insights into the genetic etiology and pathophysiology of a wide variety of human disorders. The new paradigm

6 Marfan syndrome: from molecular pathogenesis to clinical treatment Ramirez and Dietz 257 that matrix sequestration crucially regulates the local activation of latent TGFb has already had several important benefits. First, the TGFb signaling pathway is now considered an attractive target to counteract aneurysm progression in Marfan syndrome using traditional pharmacological means of therapy. Second, TGFb involvement in Marfan syndrome pathogenesis helps to conceptualize the origin of clinical variability by providing a number of candidate modifiers that are part of the TGFb signaling network. Third, the genes encoding regulators and effectors of TGFb signaling have emerged as attractive candidates for the sites of mutations causing phenotypes that overlap with Marfan syndrome or Loeys-Dietz syndrome. Lastly, the definition of Marfan syndrome has changed from a structural disorder of the connective tissue to a developmental abnormality with broad and complex effects on the morphogenesis and function of multiple organ systems. The expanding concept of the extracellular matrix as both a structural and instructional entity will probably have relevance to many other disorders with extreme implications for therapeutic interventions. Acknowledgements We thank Ms Karen Johnson for preparing the manuscript, and members of our laboratories for their enthusiastic involvement in the work described in this review. These studies were supported by grants from the National Institutes of Health (AR-42044, AR , AR41135), as well as by the Howard Hughes Medical Institute, Smilow Center for Marfan Syndrome Research, Broccoli Foundation and the National Marfan Foundation. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Marfan AB: Un cas de déformation congénitale des quatre membres plus prononcée aux extrémites charactérisée par l allongement des os avec uncertain degré d amincissement. Bull Mem Soc Med Hop 1896, 13: McKusick VA: Heritable disorders of connective tissue. III. The Marfan syndrome. J Chronic Dis 1955, 2: Dietz HC, Cutting GR, Pyeritz RE, Maslen CL, Sakai LY, Corson GM, Puffenberger EG, Hamosh A, Nanthakumar EJ, Curristin SM et al.: Marfan syndrome caused by a recurrent de novo missense mutation in the fibrillin gene. Nature 1991, 352: Neptune ER, Frischmeyer PA, Arking E, Myers L, Bunton TE, Gayraud B, Ramirez F, Sakai LY, Dietz HC: Dysregulation of TGF-b activation contributes to pathogenesis in Marfan syndrome. 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