RESEARCH REVIEW

Genetic Basis of Potential Therapeutic Strategies for Craniosynostosis

Heather Melville,1 Yingli Wang,1 Peter J. Taub,2,3 and Ethylin Wang Jabs1,3,4*
1 2 3 4

Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York Department of Surgery, Mount Sinai School of Medicine, New York, New York Department of Pediatrics, Mount Sinai School of Medicine, New York, New York Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, New York

Received 23 June 2010; Accepted 17 August 2010

Craniosynostosis, the premature fusion of one or more cranial sutures, is a common malformation of the skull that can result in facial deformity and increased intracranial pressure. Syndromic craniosynostosis is present in 15% of craniosynostosis patients and often is clinically diagnosed by neurocranial phenotype as well as various other skeletal abnormalities. The most common genetic mutations identied in syndromic craniosynostosis involve the broblast growth factor receptor (FGFR) family with other mutations occurring in genes for transcription factors TWIST, MSX2, and GLI3, and other proteins EFNB1, RAB23, RECQL4, and POR, presumed to be involved either upstream or downstream of the FGFR signaling pathway. Both syndromic and nonsyndromic craniosynostosis patients require early diagnosis and intervention. The premature suture fusion can impose pressure on the growing brain and cause continued abnormal postnatal craniofacial development. Currently, treatment options for craniosynostosis are almost exclusively surgical. Serious complications can occur in infants requiring either open or endoscopic repair and therefore the development of nonsurgical techniques is highly desirable although arguably difcult to design and implement. Genetic studies of aberrant signaling caused by mutations underlying craniosynostosis in in vitro calvarial culture and in vivo animal model systems have provided promising targets in designing genetic and pharmacologic strategies for systemic or adjuvant nonsurgical treatment. Here we will review the current literature and provide insights to future possibilities and limitations of therapeutic applications.
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How to Cite this Article:

Melville H, Wang Y, Taub PJ, Jabs EW. 2010. Genetic basis of potential therapeutic strategies for craniosynostosis. Am J Med Genet Part A 152A:30073015.

however, the metopic and lambdoid sutures may also be affected. Depending on the specic sutural synostosis, different types of altered skull morphology and facial deformity may occur and confer functional abnormalities in patients. Cranial sutures are brous joints that act as growth sites and provide a malleable quality to the head, thus allowing the neonate to pass through the birth canal. This separation of the at bones of the skull also allows for brain growth during early development. Premature fusion of one or more of these sutures can signicantly affect cranial and brain development while also increasing risk for elevated intracranial pressure [reviewed in Cohen and Persing, 1998], impaired cerebral blood ow, airway obstruction, and various adverse neurologic and physiological side effects such as deafness, blindness, and seizures [reviewed in Cohen, 2006]. Craniosynostosis events can increase risk of developmental delay, learning disability, or both [Lekovic et al., 2004; reviewed in Speltz et al., 2004; Kapp-Simon et al., 2007]. Fifteen percent of individuals with craniosynostosis present with syndromic conditions and are more severely affected than those with single suture synostosis (Table I). Muenke, Crouzon, Pfeiffer,
Grant sponsor: NIH/NIDCR; Grant Numbers: 5 R01 DE018500, 5 R01 DE018500-02S1, 5 R01 DE016887; Grant sponsor: CDC; Grant Number: 5 R01 DD000350. Heather Melville and Yingli Wang contributed equally to this work. *Correspondence to: Ethylin Wang Jabs, M.D., Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1497, New York, New York 10029. E-mail: ethylin.jabs@mssm.edu Published online 15 November 2010 in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/ajmg.a.33703

Key words: craniosynostosis; Apert syndrome; surgery; treatment; mutation; broblast growth factor receptor; inhibitor

INTRODUCTION
Craniosynostosis, the premature fusion of cranial sutures, is a common congenital craniofacial abnormality observed in 1:2,500 live births worldwide [Lajeunie et al., 1995; reviewed in Cohen and Maclean, 2000; Boulet et al., 2008]. Particular sutures affected most commonly include the sagittal or coronal sutures;

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TABLE I. Craniosynostosis Syndromes in Humans Characterized by Brachycephaly and Related Mouse Models Syndrome Crouzon JacksonWeiss Pfeiffer Apert Clinical features Ocular proptosis, hypertelorism, midface hypoplasia Bony syndactyly of toes, broad great toe Broad thumbs and great toes, syndactyly of ngers and toes Syndactyly of hands and feet, broad distal phalanx of thumb and big toe, visceral organ abnormalities Abnormalities of the hands or feet including carpal and tarsal fusions, hearing loss Acanthosis nigricans, choanal atresia, hydrocephalus requiring shunt Cutis gyrata; acanthosis nigricans; anogenital anomalies; prominent umbilical stump Choanal atresia, radiohumeral synostosis, arachnodactyly AntleyBixler syndrome-like bone-malformations with ambiguous genitalia Facial asymmetry, ptosis of the eyelid, prominent ear crus, syndactyly of 2nd and 3rd ngers or toes Ocular hypertelorism, broad nasal root, bid nasal tip, typically more severe in affected females Acrocephaly, preaxial polydactyly and syndactyly of feet, short stature, lateral displacement of inner canthi Mutant gene/protein FGFR2 FGFR2 FGFR1, -2 FGFR2 Mouse model Fgfr2C342Y/ Fgfr1P250R/; Fgfr2-IIIc/D Fgfr2S252W/; Fgfr2S252W/; Fgfr2P253R/; Fgfr2P253R/ Fgfr3P244R/ Prx1-Cre; PORlox/lox a Twist1/ Refs. (mouse model) Eswarakumar et al. [2004] Zhou et al. [2000] and Hajihosseini et al. [2001] Chen et al. [2003], Wang et al. [2005, 2010], and Yin et al. [2008] Twigg et al. [2009] Schmidt et al. [2009] El Ghouzzi et al. [1997], Bourgeois et al. [1998], Carver et al. [2002], and Merrill et al. [2006] Compagni et al. [2003] and Davy et al. [2004] G unther et al. [1994], Kasarskis et al. [1998], and Eggenschwiler et al. [2001] Mann et al. [2005]

FGFR3-associated Coronal synostosis syndrome (Muenke) Crouzonodermoskeletal BeareStevenson AntleyBixler POR deciency Saethre-Chotzen

FGFR3 FGFR3 FGFR2 FGFR2 POR TWIST1

Craniofrontonasal dysplasia Carpenter

EFNB1 RAB23

BallerGerold

Radial aplasia, absent or hypoplastic thumbs, curved ulna, short stature, imperforate anus, poikiloderma

RECQL4

Boston type

Frontal orbital retrusion, clover-leaf skull shape, myopia or hyperopia Greig Preaxial and postaxial polydactyly, cephalopolysyndactyly syndactyly, frontal bossing
a

MSX2 GLI3

EphrinB1KO/, ephrin B1lox, ephrin B1null Open brain mouse (opb, recessive, K39X and R80X mutations), opb1, opb2, RAB23 nulla Recql4/, resembles RothmundThomson syndromea TIMP1-Msx2Pro7His Extra toes (Xt dominant)

Liu et al. [1995, 1999] Hui and Joyner [1993]

Mutant mice do not resemble the human craniosynostosis condition.

and Saethre-Chotzen syndromes represent the most common of the syndromic craniosynostosis conditions [reviewed in Hehr and Muenke, 1999]. Besides calvarial synostosis, these syndromes can be associated with other phenotypic manifestations, such as syndactyly of the hands and feet in Apert syndrome, abnormally broad thumbs and toes in Pfeiffer syndrome, or acanthosis nigricans in Crouzonodermoskeletal and BeareStevenson cutis gyrata syndromes.

MOLECULAR ETIOLOGY OF CRANIOSYNOSTOSIS

These craniosynostosis syndromes may be caused by activating dominant mutations in the broblast growth factor receptor (FGFR), particularly FGFR1, -2, and -3 [Wilkie et al., 2007], or inactivating mutations in the transcription factor TWIST1 [El Ghouzzi et al., 1997; Howard et al., 1997]. Other less frequent mutations are found in MSX2, EFNB1, RAB23, POR, GLI3, and

MELVILLE ET AL. RECQL4 for rare syndromes such as Boston-type craniosynostosis, craniofrontonasal, Carpenter, POR (AntleyBixler-like phenotype with disordered steroidogenesis), Greig cephalopolysyndactyly, and BallerGerold, respectively [Vortkamp et al., 1992; Jabs et al., ck et al., 2004; Twigg et al., 2004; Wieland et al., 2005; Van 1993; Flu Maldergem et al., 2006; Jenkins et al., 2007]. The discovery of these mutant genes is helping to elucidate their role in the developmental pathways of skull development, especially FGF/FGFR signaling (Table I). Genetic studies in both humans and mice have provided further evidence that FGFR signaling is involved in modulating osteoblast activity in both intramembranous and endochondral bone ossication [reviewed in Marie et al., 2005]. During intramembranous ossication throughout human fetal development, FGFR1, -2, and -3 are expressed in the cranial sutures [Delezoide et al., 1998], supporting the ndings that mutations in FGFR1, -2, and -3 genes were associated with syndromic and nonsyndromic craniosynostosis [reviewed in McIntosh et al., 2000; Wilkie, 2005]. FGFRs contain three major immunoglobulin-like regions, IgI, -II, and -III; these extracellular domains express variable binding afnities for the 22 available FGF ligands [reviewed in Hajihosseini, 2008; Katoh and Katoh, 2009]. The FGFR pathway is stimulated by FGF ligands, and this receptorligandheparan sulfate proteoglycan complex facilitates homodimerization of FGFRs and promotes tyrosine kinase phosphorylation, thus triggering downstream signaling via the docking protein FRS2 and primarily the MAPK/ERK and PI3K/ AKT signaling cascades [reviewed in Hajihosseini, 2008; Katoh and Katoh, 2009; Turner and Grose, 2010]. In some craniosynostosis syndromes, such as Crouzon with the FGFR2 C342Y mutation, the receptor is identied in Crouzon syndrome, the receptor is constitutively activated, triggering abnormal signaling pathways without presence of ligand [Neilson and Friesel, 1995]; whereas in the Apert syndrome, the receptor and downstream pathways are activated in ligand-dependent manner [Anderson et al., 1998; Yu et al., 2000]. Protein blot analysis of various in vivo tissues in the Apert mouse model [Shukla et al., 2007; Holmes et al., 2009], and in vitro calvarial organ and osteoblast cultures stimulated by FGFs [Chaudhary and Avioli, 1997; Chikazu et al., 2000; Spector et al., 2005] indicated that MAPK signaling is also an important pathway accompanying aberrant FGFR activation.

3009 allow for brain growth, as well as create a more natural head shape. Age and correct timing are critical parameters in successful correction of craniosynostosis given the infants ability to ossify small cranial defects, thereby minimizing the need for bone grafting; however, risking the possibility of resynostosing the extirbated suture [Marchac et al., 1994; reviewed in McCarthy and Cutting, 1990; Persing et al., 2009]. Therefore, surgical intervention is undertaken between 3 and 12 months of life to maximize therapeutic outcomes at an early stage of development. The surgical procedure carries risk for infection, bleeding, venous air emboli, and seizures, despite advances in anesthesia that have reduced morbidity and mortality associated with these invasive cranioplasty procedures [Whitaker et al., 1979; Jones et al., 1992; reviewed in Cohen et al., 2008; Persing et al., 2009]. Recent literature indicates that an estimated 80100% of patients who undergo surgical correction associated with craniosynostosis, specically frontoorbital advancement procedures, require blood transfusions [Meara et al., 2005]. Although complications may arise, in a retrospective analysis of 104 patients presenting with pansynostosis and craniofacial synostosis, 87.5% of patients were considered to have at least a satisfactory cranial form (categories III) with the most critical parameter of success being early surgical intervention [McCarthy et al., 1995]. Complications are; however, worsened by the need for multiple surgeries for correction of secondary defects, such as midface hypoplasia, particularly in patients presenting with syndromic craniosynostosis conditions like Apert and Crouzon. Thomas et al. [2005] conducted a retrospective study of 76 patients with isolated coronal synostosis that were operated at a single unit and found that those patients with the FGFR3 P250R mutation were signicantly associated with transcranial reoperation and 20.7% underwent reoperation because of increased intracranial pressure. They also observed that cases with the mutation had early intervention with a posterior release to prevent excessive frontal bulging at around 6 months of age. Although mortality rates are reportedly very low at 2.6% in a 14-year analysis of 120 craniosynostosis patients [Ferreira et al., 2006], the need to develop minimally invasive therapies via biologically based methods or pharmacologic intervention would be benecial for both the patient and clinician.

SURGICALLY RELATED TREATMENTS FOR CRANIOSYNOSTOSIS Surgical Intervention

Strategies currently in use to treat craniosynostosis are almost exclusively surgical. These surgical modalities for correcting the premature fusion closures focus on removal of the fused suture and various patterns of osteotomies to reconstruct the skull. Recently, techniques utilizing distraction osteogenesis have been applied to the calvarial vault for cases of craniosynostosis. This technique involves creation of an osteotomy and application of external or internal devices to gradually separate the components [reviewed in Cohen et al., 2008]. Ultimately, these invasive procedures are designed to increase the intracranial volume so as to decrease intracranial pressure and

Cranial Orthotic Molding Therapy

Cranial orthotic devices or skull molding caps have demonstrated variable success across decades of use in reshaping infant skull structure [Persing et al., 1986]. These devices inuence growth and skull shape by exerting external pressure on key locations of the skull to oppose the internal pressure forces of the growing brain. A renewed interest in external cranial vault molding, or cranial helmets, has improved cranial remodeling after endoscopic surgical intervention for patients presenting with craniosynostosis. Although there are limited clinical studies regarding the efcacy of molding, some retrospective analyses of patients after endoscopic craniosynostosis surgery with or without postoperative banding indicate that molding helmet therapy can promote more normal cranial growth and operative correction [Seymour-Dempsey et al., 2002; Tellado and Lema, 2009].

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AMERICAN JOURNAL OF MEDICAL GENETICS PART A normal at birth, but had inconsistent postnatal stability of phenotypic response [Shukla et al., 2007]. This variable response to treatment, including lethality in some cases, suggests that delicate manipulation of dosage and delivery in vivo is necessary for ultimately translating these agents to therapeutic application in the future.

NONSURGICAL THERAPEUTIC STRATEGIES FOR CRANIOSYNOSTOSIS

In recent years, the understanding of biochemical and molecular signaling specically of the FGFR pathway has revealed that similar mutations underlie both craniosynostosis and various cancers. FGFRs have been implicated in pathogenesis of cancer via aberrant splicing, various activating mutations, and FGF-driven tumorigenesis [Pollock et al., 2007; reviewed in Wilkie, 2007; Katoh and Katoh, 2009; Turner and Grose, 2010]. Interestingly, the majority of the somatic mutations identied in a screen of endometrial tumors were identical to the activating germ line mutations in FGFR2 that cause Apert and BeareStevenson syndromes [Pollock et al., 2007]. Small molecule inhibitors designed to target the aberrant FGFR pathway have been tested in preclinical and clinical trials for treatment of cancer and are now under investigation for the treatment of craniosynostosis given the commonality of underlying mutations. Studies undertaken to investigate potential pharmacologic, genetic, and other methods of nonsurgical modulation of aberrant signaling in craniosynostosis are summarized in Table II.

Genetic and Other Strategies

Genetic approaches to treat craniosynostosis caused by gain-offunction mutations of Fgfrs has been conducted in animal models and have continued to provide informative data regarding therapeutic targets in nonsurgical treatment. Substitution of two amino acids, L424A and R426A (two substitutions designated as LR) in the juxtamembrane domain of an activated Fgfr2c carrying a Crouzonlike mutation C342Y (designated as Fgfr2-CLR) prevents the recruitment and tyrosine phosphorylation of docking protein Frs2a, resulting in normal skull development [Eswarakumar et al., 2006]. Further studies have employed alternative approaches, particularly designing strategies to modulate protein levels involved in aberrant FGFR signaling. In rats, to prevent fusion of the posterior frontal suture, which normally demonstrates postnatal suture fusion, infection with a dominant-negative FGFR1 construct abrogated overactive FGFR signaling in vivo [Greenwald et al., 2001]. Others utilized glycosaminoglycans (GAGs) proteins, such as heparan sulfate, that are involved in facilitation of FGF-FGFR ligand binding and osteoblastic differentiation. Manipulating levels of GAGs and FGF ligands has illustrated variable cooperative binding activity and promoted inhibition of mutant FGFR signaling in Apert syndrome [McDowell et al., 2006]. Recombinant proteins, human antibodies, and small interfering RNAs (siRNAs) are other technologies that have been used as therapeutics in animal models of craniosynostosis. Study of the mouse skull showed that NOGGIN, an antagonist of bone morphogenetic proteins, is expressed postnatally in the patent cranial sutures and the expression is suppressed by overactive FGF/FGFR signaling [Warren et al., 2003]. In a rat model transplanted with mutant FGFR2 osteoblasts and exhibiting craniosynostosis, Shen et al. [2009] found downregulation of Noggin in abnormal fusing sutures; the topical application of recombinant human NOGGIN protein prevented craniosynostosis. Local application of cells expressing exogenous NOGGIN was found to be an effective inhibitor of cranial resynostosis in mice after extirbation of the cranial suture [Cooper et al., 2009]. Examination of treated sutures indicated that the NOGGIN treatment inhibited bone formation, providing compelling evidence that adjuvant NOGGIN therapy may provide benet to traditional surgical repair of craniosynostosis [Cooper et al., 2009]. Human and animal studies have shown that the synostosis of cranial sutures may be related to the overexpression of transforming growth factor beta2 (TGFb2) [Opperman et al., 1997; Roth et al., 1997a,b]. By studying an in vivo New Zealand white rabbit model with bilateral coronal suture synostosis [Mooney et al., 1994a,b], the overexpression of TGFb2 was abrogated with neutralizing antibodies in the synostotic sutures after surgical excision. Local application of anti-TGFb2 antibody infused in a collagen matrix inhibited postsurgical resynostosis and improved intracranial

Pharmacologic Strategies
FGFR signaling plays a critical role in osteogenesis, particularly with respect to craniosynostosis. Investigations of treatment on osteoblast-like cells or osteoblast cultures obtained from coronal sutures of Apert syndrome with small molecule inhibitors have elucidated the possible role of the various signaling pathways in abnormal osteoblast proliferation, differentiation, and apoptosis [Lemonnier et al., 2000, 2001a,b; Lomri et al., 2001; Kim et al., 2003; Miraoui et al., 2009, 2010]. However, these cellular assays represent relatively nonphysiological systems as compared to in vitro models of craniosynostosis using calvarial organ culture derived from animal models; the latter provides preliminary and more direct evidence for the effect of small molecule inhibitors in treatment of craniosynostosis (Table II). Mouse calvaria obtained from the Crouzon-like Fgfr2C342Y/ mouse model cultured in the presence of FGFR tyrosine kinase inhibitor PD173074 exhibited coronal suture patency, as opposed to the premature suture fusion seen in untreated mutants [Perlyn et al., 2006]. An alternative FGFR inhibitor, PLX052, was also used in calvarial organ culture of the Crouzon-like Fgfr2cC342Y/ mouse and prevented premature coronal suture fusion in mutant skulls without affecting normal development in wild-type explants [Eswarakumar et al., 2006]. An in vitro calvarial culture model of the Apert mouse harboring the FGFR2 P253R mutation showed that treatment with MEK1 inhibitor PD98059 partially alleviated coronal suture fusion [Yin et al., 2008]. These studies suggest that the application of small molecule inhibitors, such as PD173074 and U0126, to decrease aberrant FGFR tyrosine kinase activity and/or downstream activation of ERK1/2 and other pathways may give insights into pharmacological strategies to treat craniosynostosis. Initial efforts have been performed to explore the potential of these inhibitors in vivo. Shukla and colleagues intraperitoneally injected MEK1/2 inhibitor U0126 into female mice during pregnancy and early postnatal stages to treat their Apert Fgfr2/S252W offspring. They noted that mutant pups appeared phenotypically

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TABLE II. Review of Nonsurgical Therapeutic Strategies for Craniosynostosis Culture or model system In vitro Mouse cranial suture culture from wild-type CD1 strain; in utero rat SpragueDawley calvarial sutures In vitro Mouse calvarial organ culture from Crouzon syndrome, Fgfr2 C342Y/ murine model In vitro Mouse calvarial culture from Crouzon syndrome, Fgfr2 C342Y/ murine model In vitro Mouse organ cultures of calvaria and long bones from Apert Fgfr2 P253R/ murine model In vivo Mouse Fgfr2c CLR/ with Crouzon syndrome, Fgfr2 C342Y/ mutation and additional juxtamembrane mutations Target/pathway Fgfr1 Treatment Dominant negative Fgfr1 Effect Inhibited posterior frontal suture fusion Refs. Greenwald et al. [2001]

Fgfr2

FGFR tyrosine kinase inhibitor PLX052 FGFR tyrosine kinase inhibitor PD173074 MEK1 inhibitor PD98059

Prevented coronal suture fusion Retained patency of coronal suture

Eswarakumar et al. [2006] Perlyn et al. [2006]

Fgfr2

ERK1/2

Partially alleviated Yin et al. coronal suture fusion [2008] and growth retardation of femur Prevented premature fusion of the coronal suture Eswarakumar et al. [2006]

Frs2a docking Additional L424A and protein-dependent R426A mutations in Fgfr2c Crouzon mouse prevent recruitment and tyrosine phosphorylation of Frs2a Intraperitoneal injection of MEK1/2 inhibitor U0126 Heterozygous U6-Fgfr2 S252W shRNA transgenic mouse mated with Apert mouse

In vivo Mouse with knock-in Apert ERK1/2 syndrome, Fgfr2 S252W/ mutation Mouse with knock-in Apert Fgfr2 syndrome, Fgfr2 S252W/ mutation In vivo Chimeric human/nude (athymic) rat NOGGIN xenotransplant model of craniosynostosis containing Crouzon and Apert FGFR2 mutant human osteoblasts In vivo Mouse model with NOGGIN postoperative resynostosis treated with suturectomy In vivo Rabbit model with bilateral TGFb2 coronal suture synostosis received suturectomy

Repressed or rescued craniosynostosis phenotype in Apert mouse Repressed or rescued craniosynostosis phenotype in Apert mouse

Shukla et al. [2007] Shukla et al. [2007]

Recombinant human NOGGIN Inhibited suture fusion

Shen et al. [2009]

Cells expressing NOGGIN

Inhibited bone formation Cooper et al. [2009] Mooney et al. [2007a,b] and Frazier et al. [2008]

Neutralizing TGFb2 antibody Inhibited postoperative resynostosis and improved intracranial volume and cranial vault growth

3012 volume and craniofacial growth [Mooney et al., 2007a,b; Frazier et al., 2008]. Recent studies have explored the application of RNA interference, both in vitro [Gosain et al., 2009] and in vivo [Shukla et al., 2007]. Gosain and colleagues used endogenous anti-TGFb1 small interfering RNA to target and knockdown TGFb1 mRNA transcripts, which are expressed during cranial suture formation [Opperman et al., 1997] and may affect Fgfr signaling, ultimately showing a decrease in mRNA levels of Fgf2 and Fgfr1 as well as a successful knockdown of TGFb1. Their results indicate that TGFb1 siRNA has the potential to change signaling in the mouse dura, which is responsible for suture fusion in vitro [Gosain et al., 2009]. In vivo studies were also performed by using small hairpin RNA to target the mutant Fgfr2 S252W transcripts in the Apert syndrome mouse model and achieved restoration of wild-type phenotype [Shukla et al., 2007].

AMERICAN JOURNAL OF MEDICAL GENETICS PART A Yoon et al., 2009]. Since this provides little reason to presuppose that a fetus has a point mutation such as those that underlie syndromic craniosynostosis, the chance of early detection of the skeletal abnormality and treatment in utero is difcult. Another problem is that many of the discussed pharmacological agents are tyrosine kinase inhibitors, which activate multiple cytoplasmic signaling pathways and play an important role in diverse normal cellular regulatory processes. Negative side effects from systemic delivery of these inhibitors have been documented in clinical cancer research therefore it may be difcult to insure a normal overall outcome for infants treated for craniosynostosis. Postsurgical local delivery of these drugs may help to alleviate complications associated with systemic treatment by limiting the application to the site of suture excision. Genetic study in the eld of craniosynostosis will continue to reveal new targets for nonsurgical treatment and drive the exploration of new technologies. The development of nonsurgical treatment modalities for syndromic craniosynostosis may ultimately provide a complement, or possibly an alternative, to the invasive surgical interventions currently in use. The systematic analysis of the pathogenic mechanisms involved in craniosynostosis, particularly the syndromic conditions, will be undoubtedly helpful in designing and implementing these new treatment strategies.

INSIGHTS AND FUTURE DIRECTIONS IN NONSURGICAL TREATMENT STRATEGIES

The genetic study of craniosynostosis syndromes and their underlying etiology has clearly provided targets for nonsurgical treatment for craniosynostosis. Although Shukla et al. [2007] obtained a partial rescue of the phenotype in the Apert mutant mouse treated with MEK1/2 inhibitor, this study and other in vitro organ culture studies, indicate that many variables, such as inhibitor targets, drug delivery, drug dosage, and treatment intervals are critical to the efcacy of a particular treatment. Due to the various signaling pathways that are involved in the mechanisms of craniosynostosis, complex treatment strategies may be required, such as applying these drugs in combination. Although the progress in abrogating the cranial suture synostosis in animal models has been promising, there are obvious limitations and practical difculties related to systemic application of these pharmacological inhibitor-based therapies without surgical intervention. For one, according to mouse studies, early detection and treatment in utero appears critical for successful therapeutic outcomes. Extensive characterization of the Apert mouse models [Chen et al., 2003; Wang et al., 2005, 2010; Holmes et al., 2009] indicates coronal suture fusion in an early embryonic event, occurring by embryonic days 13.515.5, a gestational period in mice that corresponds with 1012 weeks in humans. Since the Apert syndrome mice treated with MEK1/2 inhibitor U0126 appeared normal at birth, but developed skull abnormalities by postnatal day 12 [Shukla et al., 2007], it is likely that continued inhibition of ERK1/2 activation with daily postnatal administration of U0126 is required to prevent development of cranial abnormalities in this model. These data suggest that both in utero and postnatal treatment with small molecule inhibitors would be optimal to signicantly reduce the effects of craniosynostosis and prevent premature suture fusion in humans, but there are constraints to implementing such treatment. Currently, no early screening procedure is in place for identication of de novo syndromic craniosynostosis mutations. The autosomal dominant point mutations that underlie Apert syndrome occur at an average rate of 105 per male gamete, the likelihood of which increases with paternal age [Tolarova et al., 1997; Crow, 2000; Goriely et al., 2003;

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