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REVIEW A Genetic-Pathophysiological Framework for Craniosynostosis Stephen R.F. Twigg 1 and Andrew O.M. Wilkie 1,2, * Craniosynostosis, the premature fusion of one or more cranial sutures of the skull, provides a paradigm for investigating the interplay of genetic and environmental factors leading to malformation. Over the past 20 years molecular genetic techniques have provided a new approach to dissect the underlying causes; success has mostly come from investigation of clinical samples, and recent advances in high- throughput DNA sequencing have dramatically enhanced the study of the human as the preferred ‘‘model organism.’’ In parallel, how- ever, we need a pathogenetic classification to describe the pathways and processes that lead to cranial suture fusion. Given the prenatal onset of most craniosynostosis, investigation of mechanisms requires more conventional model organisms; principally the mouse, because of similarities in cranial suture development. We present a framework for classifying genetic causes of craniosynostosis based on current understanding of cranial suture biology and molecular and developmental pathogenesis. Of note, few pathologies result from complete loss of gene function. Instead, biochemical mechanisms involving haploinsufficiency, dominant gain-of-function and recessive hypomorphic mutations, and an unusual X-linked cellular interference process have all been implicated. Although few of the genes involved could have been predicted based on expression patterns alone (because the genes play much wider roles in embryonic development or cellular homeostasis), we argue that they fit into a limited number of functional modules active at different stages of cranial suture development. This provides a useful approach both when defining the potential role of new candidate genes in cranio- synostosis and, potentially, for devising pharmacological approaches to therapy. Introduction Cranial sutures, superficially simple fibrocellular structures separating the rigid plates of the skull bones, are wonder- fully subtle in executing their roles. Mature sutures are bridged by fibers 1 that unite the bone fronts and resist deformation in both tension and compression. Their prin- cipal function is to enable the growth of the skull in coor- dination with expansion of the developing brain, 2 which occurs particularly rapidly in humans and during fetal and infant life; the intracranial pressure produces quasi- static tensile strains, which could act either directly on the suture or indirectly through mechanotransduction by the dura mater, the tough membrane that adheres to the inner surface of the calvaria (skull vault) and separates it from the brain. 3 In addition, sutures permit deformation of the skull during birth, absorb cyclic loading during mastication and locomotion, and act as shock absorbers against external impacts. 4 Although cranial sutures start off as simple lines of demarcation between developing bones, they become increasingly interdigitated with age, a feature that is more marked on the external (ectocranial) surface. 5 These meandering patterns can be described in terms of fractal geometry, with the fractal dimension increasing with age. Mathematical attempts to account for this behavior have employed reaction-diffusion models incorporating diffusible factors, positive and negative feedback loops, mechanical strain, and time-dependent processes. 1,5 The explanatory success of these theoretical studies cap- tures the underlying idea that growth at sutures is likely to involve nested sets of cellular signaling pathways controlled by secretion of paracrine factors and responsive to mechanical strain. Failure of the mechanisms that maintain suture patency leads to craniosynostosis, the premature fusion of one or more of the cranial sutures. This occurs in about 1 in 2,250 children 6,7 and usually becomes apparent between the last third of pregnancy and the end of the first year of life. The fusion of a suture abolishes further growth of the abutting bones in a direction perpendicular to the suture. As a consequence, continued enlargement of the brain promotes compensatory overgrowth at other sutures, leading to progressive distortion in the skull shape. 8 Multi- ple complications can arise because of raised intracranial pressure, facial deformities affecting vision, breathing, and dentition, and other features such as hearing loss or in- tellectual disability that might be caused by the underlying gene defect or alternatively might occur secondary to cra- niosynostosis. Surgery to remodel the skull and create extra volume for the brain, with the secondary aim to improve psychosocial adjustment, is currently the mainstay of management. 9–11 No pharmacological interventions are currently validated for prevention of suture fusion. Broadly speaking, three interacting factors predispose to the abnormal fusion of a suture (Figure 1A). Least well un- derstood is the effect of mechanical force (strain) trans- mitted by the growing brain to maintain suture patency. The relationship between brain growth and failure of suture function is complex, because although micro- cephaly is a recognized risk factor for craniosynostosis (see GeneReviews in Web Resources), most individuals with microcephaly do not develop this complication. 1 Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK; 2 Craniofacial Unit, Department of Plastic and Reconstructive Surgery, Oxford University Hospitals NHS Trust, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ajhg.2015.07.006. Ó2015 by The American Society of Human Genetics. All rights reserved. The American Journal of Human Genetics 97, 359–377, September 3, 2015 359

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Page 1: A Genetic-Pathophysiological Framework for Craniosynostosis · A Genetic-Pathophysiological Framework for Craniosynostosis Stephen R.F. Twigg 1and Andrew O.M. Wilkie ,2 * Craniosynostosis,the

REVIEW

A Genetic-PathophysiologicalFramework for Craniosynostosis

Stephen R.F. Twigg1 and Andrew O.M. Wilkie1,2,*

Craniosynostosis, the premature fusion of one or more cranial sutures of the skull, provides a paradigm for investigating the interplay of

genetic and environmental factors leading to malformation. Over the past 20 years molecular genetic techniques have provided a new

approach to dissect the underlying causes; success has mostly come from investigation of clinical samples, and recent advances in high-

throughput DNA sequencing have dramatically enhanced the study of the human as the preferred ‘‘model organism.’’ In parallel, how-

ever, we need a pathogenetic classification to describe the pathways and processes that lead to cranial suture fusion. Given the prenatal

onset of most craniosynostosis, investigation of mechanisms requires more conventional model organisms; principally the mouse,

because of similarities in cranial suture development. We present a framework for classifying genetic causes of craniosynostosis based

on current understanding of cranial suture biology and molecular and developmental pathogenesis. Of note, few pathologies result

from complete loss of gene function. Instead, biochemical mechanisms involving haploinsufficiency, dominant gain-of-function and

recessive hypomorphic mutations, and an unusual X-linked cellular interference process have all been implicated. Although few of

the genes involved could have been predicted based on expression patterns alone (because the genes playmuchwider roles in embryonic

development or cellular homeostasis), we argue that they fit into a limited number of functional modules active at different stages of

cranial suture development. This provides a useful approach both when defining the potential role of new candidate genes in cranio-

synostosis and, potentially, for devising pharmacological approaches to therapy.

Introduction

Cranial sutures, superficially simple fibrocellular structures

separating the rigid plates of the skull bones, are wonder-

fully subtle in executing their roles. Mature sutures are

bridged by fibers1 that unite the bone fronts and resist

deformation in both tension and compression. Their prin-

cipal function is to enable the growth of the skull in coor-

dination with expansion of the developing brain,2 which

occurs particularly rapidly in humans and during fetal

and infant life; the intracranial pressure produces quasi-

static tensile strains, which could act either directly on

the suture or indirectly through mechanotransduction by

the dura mater, the tough membrane that adheres to the

inner surface of the calvaria (skull vault) and separates it

from the brain.3 In addition, sutures permit deformation

of the skull during birth, absorb cyclic loading during

mastication and locomotion, and act as shock absorbers

against external impacts.4

Although cranial sutures start off as simple lines of

demarcation between developing bones, they become

increasingly interdigitated with age, a feature that is

more marked on the external (ectocranial) surface.5 These

meandering patterns can be described in terms of fractal

geometry, with the fractal dimension increasing with

age. Mathematical attempts to account for this behavior

have employed reaction-diffusion models incorporating

diffusible factors, positive and negative feedback loops,

mechanical strain, and time-dependent processes.1,5 The

explanatory success of these theoretical studies cap-

tures the underlying idea that growth at sutures is likely

to involve nested sets of cellular signaling pathways

1Weatherall Institute of Molecular Medicine, University of Oxford, John R

Department of Plastic and Reconstructive Surgery, Oxford University Hospital

*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.ajhg.2015.07.006. �2015 by The American Societ

The American

controlled by secretion of paracrine factors and responsive

to mechanical strain.

Failure of the mechanisms that maintain suture patency

leads to craniosynostosis, the premature fusion of one or

more of the cranial sutures. This occurs in about 1 in

2,250 children6,7 and usually becomes apparent between

the last third of pregnancy and the end of the first year

of life. The fusion of a suture abolishes further growth of

the abutting bones in a direction perpendicular to the

suture. As a consequence, continued enlargement of the

brain promotes compensatory overgrowth at other sutures,

leading to progressive distortion in the skull shape.8 Multi-

ple complications can arise because of raised intracranial

pressure, facial deformities affecting vision, breathing,

and dentition, and other features such as hearing loss or in-

tellectual disability that might be caused by the underlying

gene defect or alternatively might occur secondary to cra-

niosynostosis. Surgery to remodel the skull and create extra

volume for the brain, with the secondary aim to improve

psychosocial adjustment, is currently the mainstay of

management.9–11 No pharmacological interventions are

currently validated for prevention of suture fusion.

Broadly speaking, three interacting factors predispose to

the abnormal fusion of a suture (Figure 1A). Least well un-

derstood is the effect of mechanical force (strain) trans-

mitted by the growing brain to maintain suture patency.

The relationship between brain growth and failure of

suture function is complex, because although micro-

cephaly is a recognized risk factor for craniosynostosis

(see GeneReviews in Web Resources), most individuals

with microcephaly do not develop this complication.

adcliffe Hospital, Headington, Oxford OX3 9DS, UK; 2Craniofacial Unit,

s NHS Trust, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK

y of Human Genetics. All rights reserved.

Journal of Human Genetics 97, 359–377, September 3, 2015 359

Page 2: A Genetic-Pathophysiological Framework for Craniosynostosis · A Genetic-Pathophysiological Framework for Craniosynostosis Stephen R.F. Twigg 1and Andrew O.M. Wilkie ,2 * Craniosynostosis,the

Figure 1. Overview of Clinical Causes and Classification of Craniosynostosis(A) Cross-section of coronal suture showing the developing parietal bone (p) overlying the frontal bone (f). Internally, the dura mater (d)separates the calvaria from the brain; skin (s) is external. Left, normal growth of the skull vault is regulated by a delicate balance of pro-liferation and differentiation occurring within the suture (green shading) co-ordinated with enlargement of the underlying brain (blackarrows). Right, in craniosynostosis this balance has been disturbed by excessive external force on the skull, usually during pregnancy(unfilled arrows), inefficient transduction of stretch from the growing brain (gray arrows), or intrinsic abnormality of signaling withinthe suture itself (red shading).(B) Approaches to clinical classification. Left: view of skull from above (front at top). Normal skull with major vault sutures identified iscentral. To either side, the examples show skull shapes resulting from sagittal synostosis (left) and bilateral coronal synostosis (right).Note, themetopic suture closes physiologically at the age of 3–9months.12 Center: the clinical history can reveal possible environmentalpredispositions such as teratogen exposure (most commonly maternal treatment with the anticonvulsant sodium valproate13) or twin-ning (intrauterine constraint);14 affected relatives with craniosynostosis (filled symbols in pedigree) suggest a likely genetic cause, whichcan be confirmed by diagnostic genetic testing. Right: clinical examination can reveal facial dysmorphic features such as hypertelorism(wide spaced eyes) and grooved nasal tip, suggesting craniofrontonasal syndrome (left), or other physical features such as syndactylycharacteristic of Apert syndrome (right). Clinical photographs reproduced, with permission, from Johnson and Wilkie9 andTwigg et al.15

Along similar lines, craniosynostosis has been docu-

mented in association with many chromosomal abnor-

malities,16 but usually only a minority of those with

similar chromosome imbalances actually develop the con-

dition, suggesting non-specific mechanisms through poor

brain growth and/or transduction of strain.17 The second

factor, the intrinsic property of the suture itself, is the

focus of this review. Finally, extrinsic forces acting on

the skull, especially during fetal life, might frequently pre-

cipitate craniosynostosis, especially in the uncomplicated

non-syndromic, single-suture-fusion cases. Epidemiolog-

ical data consistent with a contribution from fetal head

constraint include positive associations of craniosynosto-

sis with primiparity, multiple pregnancy, prematurity,

and high birth weight.7,18 Compressive strain has been

shown experimentally to increase osteogenesis at the

suture (reviewed by Herring3). In addition, there is some

in vivo support for the role of head constraint in

craniosynostosis from experimental manipulations per-

formed in mice.19

360 The American Journal of Human Genetics 97, 359–377, Septemb

As might be anticipated given this plethora of potential

causes, craniosynostosis is extremely heterogeneous in its

presentation. Three main axes of clinical classification

exist (Figure 1B), which enumerate (1) the pattern of suture

fusion and consequent skull shape, (2) the occurrence of

environmental or genetic predispositions, and (3) the pres-

ence of additional clinical features (such as facial dysmor-

phism, limb anomalies, or learning disability) suggestive

of a syndrome.9,10,20 Fusion of multiple sutures, positive

family history, and additional syndromic features all sug-

gest an underlying genetic predisposition; in some cases,

accompanying dysmorphic features are caused by fusion

of sutures separating the facial bones. Over the past two

decades our understanding of the molecular processes in

craniosynostosis, and hence underlying normal suture

development, has been transformed by human genetics

studies. To date, mutations in 57 genes have been identi-

fied as recurrently causing craniosynostosis, and the

number of genes is growing rapidly as high-throughput

sequencing of exomes (and, increasingly, whole genomes)

er 3, 2015

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is applied to the problem. Although further genes, muta-

tions of which are highly penetrant for craniosynostosis,

no doubt await discovery, and despite the lag period before

animalmodeling can be accomplished, distinct modules of

pathogenesis are emerging. It is therefore timely to pro-

pose a genetic-pathophysiological framework for classi-

fying craniosynostosis, based on integrating knowledge

of clinical genetics, suture biology, biochemical studies of

mutant molecules, and cellular and developmental obser-

vations of abnormal suture formation in mouse disease

models.

Monogenic Causes of Craniosynostosis

Starting with the identification in 1993 of an MSX2 (MIM:

123101) point mutation encoding a specific missense

amino acid substitution segregating in a 3-generation

family with craniosynostosis (MIM: 604757),21 there are

presently 57 human genes for which there is reasonable

evidence (based on at least two affected individuals with

congruent phenotypes) that mutations are causally related

to craniosynostosis. Inspection of the complete list (Table

S1) suggests that these genes fall into two broad groups.

First, those for which mutations of a particular molecular

type (based on the encoded variant protein) are frequently

(>50% of independent mutations) associated with cranio-

synostosis, which might therefore be considered a core

characteristic of the particular gene/mutation combina-

tion; second, those for which the occurrence of craniosy-

nostosis, although probably causally associated, arises in

only a minority of cases with the mutation. The latter

grouping includes many (such as filaminopathy, hypo-

phosphatasia, mucopolysaccharidoses, osteosclerosis, and

pycnodysostosis) that primarily represent perturbations

in osteogenesis (for example affecting the balance of oste-

oblast and osteoclast activity) rather than in the biology of

cranial suture development per se. These general osteo-

genic genes are not considered further. This review focuses

on trying to place the first category, comprising the 20 core

genes listed in Table 1, into a pathophysiological context,

quoting examples from the wider gene set (Table S1) only

where they provide additional support for a salient point.

Processes in Suture Formation

The cranial vault bones are relatively unusual in arising by

intramembranous ossification, without a cartilaginous

intermediate,4 and the cranial sutures are key to under-

standing their development. To formulate our pathophys-

iological framework, we distinguish five processes in suture

formation. These are stem cell specification andmigration,

lineage commitment, boundary formation and integrity,

osteogenic proliferation/differentiation, and resorption/

homeostasis. We can use this classification to distinguish

events both on a temporal basis (for example, early versus

late events) and on an anatomical basis (for example,

events primarily affecting undifferentiated cells located

centrally in the suture versus more mature peripheral oste-

ogenic cells). These processes are exquisitely coordinated

The American

and overlapping in both time and space, so that the dis-

tinctions are to some extent artificial, but we think a useful

perspective emerges from this analysis. This overview

focuses primarily on the coronal suture, which has the

most complex biogenesis and is the suture most

commonly fused in genetic forms of craniosynostosis.17

The other major vault sutures (metopic, sagittal, lambdoid;

Figure 1B) are anatomically more simple, being formed

when initially distant bone fronts approximate each other

later in embryonic development.4,53

Stem Cell Specification and Migration

It has long been presumed that sutures contain a popula-

tion of undifferentiated osteogenic cells with stem cell-

like properties,54,55 and this has recently received further

experimental support with the demonstration that expres-

sion ofGli1 (a classical marker of hedgehog [HH] signaling)

probably defines sutural stem cells at postnatal stages; cells

emanating from the suture populate the periosteum on

both surfaces of the growing bones, and genetic ablation

of Gli1-expressing cells in 1-month-old mice caused coro-

nal synostosis within a further month.56 An elegant study

(which also exploited Gli1 as the key marker of cell iden-

tity) pinpoints that the precursors of these cells originate

from cephalic paraxial mesoderm, in the region of the

rostral mesencephalon/caudal diencephalon and located

immediately adjacent to the neural tube, at embryonic

day (E)7.5 in response to sonic hedgehog (SHH) accumula-

tion in the adjacent notochord (Figure 2A).57 Lineage

tracing using genetically marked cells shows that this

population migrates laterally during E8.5–E9.5 to locate

above the developing eye.57 Of note, expression of Gli1 is

transient because calvarial structures do not label after

E8, indicating that the influence of SHH on these cells is

rapidly lost.

Lineage Commitment

During a critical period from E9.5 to E11.5, the cells above

the developing eye pattern the future coronal suture. Deck-

elbaum et al.57 have proposed the term ‘‘supraorbital regu-

latory center’’ for this region, in recognition of its role as an

organizing center. Put simply, groups of cells with osteo-

genic potential, originating from neural crest (future

frontal bone) and mesoderm (future parietal bone), are

separated by the undifferentiated stem cell population

originating from a localized section of paraxial mesoderm

(Figure 2B). During E11.5–E13.5, and co-ordinated with

growth of the underlying brain, cells from the supraorbital

region extend apically, centered above the diencephalic-

telencephalic boundary, to overlay the surface of the

rapidly growing brain.57,62,63 Descendants of these cells

can still be identified in the mid-sutural mesenchyme of

the definitive coronal suture at birth (P0), demonstrating

that they make a permanent contribution to the popula-

tion of undifferentiated cells.57 A separate population of

descendants becomes integrated into the parietal bone

mesenchyme and adopts an osteogenic fate; the future

Journal of Human Genetics 97, 359–377, September 3, 2015 361

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Table 1. Core Genes for which Specific Types of Mutation Are Associated with Craniosynostosis in More than Half of Affected Individuals

Gene (MIM#)aInheritancePattern Clinical Disorder (MIM#) Prevalence (%)b

Typical SutureFusion Major Phenotypic Features Ref(s)

ASXL1 (612990) AD (n) Bohring-Opitz syndrome (605039) – metopic forehead nevus flammeus, ulnar deviation and flexion of wristsand metacarpalophalangeal joints, severe intellectual disability

22

CDC45 (603465) AR – – coronal thin eyebrows, small ears, variable short stature 23

COLEC11 (612502) AR 3MC syndrome 2 (265050) – metopic hypertelorism, blepharoptosis, arched eyebrows, cleft lip/palate, hearing loss, radio-ulnar synostosis, genital andvesicorenal anomalies

24

EFNB1 (300035) XLD (malesparing)

craniofrontonasal syndrome (304110) 0.8 coronal hypertelorism, notched nasal tip, chest anomalies, longitudinalsplitting of nails; heterozygous females more severely affectedthan hemizygous males

15,25

ERF (611888) AD ERF-related craniosynostosis (600775) 1.1 multisuture exorbitism, midface hypoplasia, Chiari type I malformation,postnatal onset of craniosynostosis

26

FGFR1 (136350) AD Pfeiffer syndrome (101600) – coronal mild craniofacial features, broad medially deviated thumbs andhalluces, cutaneous syndactyly, specific amino acid substitutionp.Pro252Arg

27

FGFR1 AD (n) osteoglophonic dysplasia (166250) – multisuture prominent brow ridges, depressed nasal bridge, rhizomelicdwarfism, localized lytic lesions of metaphyses

28

FGFR2 (176943) AD (n) Apert syndrome (101200) 3.6 coronal, multisuture midface hypoplasia, dilated cerebral ventricles, complexsyndactyly of the hands and feet

29

FGFR2 AD (n) Beare-Stevenson syndrome (123790) * multisuture choanal atresia, prominent umbilical stump, furrowed scalp/neck skin, acanthosis nigricans in survivors

30

FGFR2 AD Crouzon syndrome (123500) 2.4 multisuture, coronal,sagittal

exorbitism, midface hypoplasia, beaked nose (‘‘crouzonoid’’facies), clinically normal hands and feet

31,32

FGFR2 AD (n) Pfeiffer syndrome (101600) 0.8 multisuture broad thumbs and halluces; in severe cases, cloverleaf skull,brain anomalies, tracheal sleeve, fused elbows

33–35

FGFR2 AD (n) bent bone dysplasia (614592) * coronal osteopenia, reduced mineralization of the calvaria, bentlong bones; perinatal lethal

36

FGFR3 (134934) AD Muenke syndrome (602849) 4.0 coronal defined by specific amino acid substitution p.Pro250Arg; mayinclude sensorineural hearing loss, mild brachydactyly,cone-shaped epiphyses

37

AD (n) Crouzon/acanthosis nigricans (612247) 0.4 multisuture crouzonoid facies, choanal stenosis, hydrocephalus, acanthosisnigricans; specific amino acid substitution p.Ala391Glu

38

(Continued on next page)

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Table 1. Continued

Gene (MIM#)aInheritancePattern Clinical Disorder (MIM#) Prevalence (%)b

Typical SutureFusion Major Phenotypic Features Ref(s)

AD (n) thanatophoric dysplasia II (187601) * multisuture lethal skeletal dysplasia, micromelic limb shortening,straight femora; specific amino acid substitution p.Lys650Glu

39

IHH (600726) AD Philadelphia craniosynostosis (185900) – sagittal cutaneous and osseous syndactyly 40

IL11RA (600939) AR craniosynostosis and dental anomalies(614188)

– multisuture maxillary hypoplasia, delayed tooth eruption, supernumeraryteeth, minor digit abnormalities, conductive hearing loss

41

MEGF8 (604267) AR Carpenter syndrome 2 (614796) – metopic hypertelorism, arched eyebrows, lateralization defects,brachydactyly, syndactyly, preaxial polydactyly

42

MSX2 (123101) AD Boston craniosynostosis (604757) – sagittal, coronal,multisuture

none diagnostic; syndrome defined by specific amino acidsubstitutions p.Pro148His, p.Pro148Leu

21

POR (124015) AR Antley-Bixler syndrome (201750) – bicoronal, multisuture choanal stenosis, radio-humeral synostosis, bowed femora,multiple joint contractures, genital abnormalities; abnormalsteroidogenesis

43

RAB23 (606144) AR Carpenter syndrome 1 (201000) – multisuture obesity, cardiac defects, polysyndactyly, brachydactyly, genuvalgum, hypogenitalism, umbilical hernia, learning disability

44

RUNX2 (600211) AD (n) – – multisuture none diagnostic; syndrome defined by specific gene duplication 45,46

SKI (164780) AD (n) Shprintzen-Goldberg syndrome (182212) – sagittal, multisuture hypertelorism, micrognathia, high arched palate, arachnodactyly,joint contractures, pectus deformity, aortic root aneurysm, mitralvalve prolapse, learning disability

47

TCF12 (600480) AD TCF12-related craniosynostosis (615314) 1.3 coronal resembles mild Saethre-Chotzen syndrome; diagnosis defined bypresence of mutations in the gene, ~50% non-penetrance

48

TWIST1 (601622) AD Saethre-Chotzen syndrome (101400) 3.6 coronal low frontal hairline, hypertelorism, eyelid ptosis, downslantingpalpebral fissures, blocked tear ducts, small ears with prominentcrus helicis

49,50

WDR35 (613602) AR cranioectodermal dysplasia 2 (613610) – sagittal facial dysmorphism, narrow thorax, short long bones,brachydactyly, sparse hair, hypoplastic teeth, cystic kidneys,hepatic fibrosis

51

ZIC1 (600470) AD (n) ZIC1- craniosynostosis 0.2 coronal severe learning disability 52

Abbreviations are as follows: AD, autosomal dominant; AR, autosomal recessive; XLD, X-linked dominant; (n), usually arises by new mutation; * usually lethal at birth.aInformation has been extracted from Table S1, which contains further details on mutational category and phenotype and additional references.bThe prevalence figures are for percent total craniosynostosis cases with specified mutation, from the cohort attending the Craniofacial Unit, Oxford, born between 1998 and 2008 (n¼ 531), and surgically treated before endof 2013 (updated from Wilkie et al.17).

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Figure 2. Key Embryological Processes inCoronal Suture Formation(A) At E7.5, sonic hedgehog secreted by thenotochord (orange shading) induces Gli1expression in adjacent cells of cephalicparaxial mesoderm (red dots). Over thenext 48 hr, these cells migrate laterally(curved arrow) to a position above thedeveloping eye.(B) Supraorbital regulatory center at E10.5,showing cells of mesodermal and neuralcrest origin (pink/red and blue, respec-tively). These cells migrate to populatethe future coronal suture (red), parietalbone (mesodermal cells, pink), and frontalbone (neural crest, with small contributionfrommesoderm). Dashed line indicates thediencephalic-telencephalic boundary. BA1,first branchial arch.(C) Top: cross-section of coronal suture atE17.5, b-galactosidase staining of Wnt1-Cre/R26R mice to demonstrate neural-crest-derived tissues (dark blue). Note thatthe frontal bone (f) and underlying duramater (d) are of neural crest origin, whereasthe parietal bone (p; dashed outline) andsutural gap (s) show no blue staining, indi-

cating a mesodermal origin. Bottom: dual origin of the skull bones from neural crest (blue) and cephalic mesoderm (red).(D) Simplified view of an established coronal suture (E14 onward). For continued patency, a population of undifferentiated stem cells(red dots) must be maintained in the mid-sutural mesenchyme. The proliferation-differentiation balance between these cells and thosein the growing margins of the bones is maintained by a hierarchy of paracrine signaling feedback loops, such as those provided by IHHand FGF receptor signaling.Figure redrawn from original data presented elsewhere.56–61 Part C adapted from Jiang et al.58 with permission.

frontal bone originates mostly from cells of neural crest

origin (next section).

Key transcription factors involved in orchestrating this

early lineage commitment are EN1 (engrailed 1), MSX2,

and TWIST1, all of which are present in the supraorbital

regulatory center at E11.57 Recently, individuals with

severe bilateral coronal synostosis were described with

heterozygous truncations or missense substitution of

another transcription factor, ZIC1.23,52 Constructs con-

taining these ZIC1 mutations caused altered or enhanced

expression of a target gene, engrailed-2, in a Xenopus em-

bryo assay, consistent with a gain of function.52 Given

the epistatic relationship of ZIC homologs (odd-paired in

Drosophila) upstream of engrailed in both Drosophila64 and

Xenopus,65,66 and the expression of murine Zic1 in the ter-

ritory corresponding to supraorbital regulatory center,52

ZIC1 (MIM: 600470) mutations are likely to disrupt early

lineage commitment in the coronal suture.

A complex and incompletely understood set of positive

andnegative feedback loops links these transcription factors

to the major signaling pathways required to commit cells

to an osteogenic fate, including bone morphogenetic pro-

teins (BMPs), wingless-related family members (WNTs),

and fibroblast growth factors (FGFs); there is evidence for

activity of each of these pathways at this time (reviewed

in Mishina and Snider67). However, expression studies do

not enable disentanglement of the exact sequence of events

or signaling hierarchies involved; neither do classical

knockout approaches answer the question, because the

364 The American Journal of Human Genetics 97, 359–377, Septemb

multiple roles of these pathways in organogenesis at earlier

stages of embryonic development tend to lead to lethality

before the cranial sutures are established.67 The availability

of suitable drivers for suture expression, together with tem-

poral cell labeling and sorting, should enable considerable

progress in teasing apart these processes over the coming

decade. Within the next 24–48 hr of development (E12.0–

E13.5), the expression of master regulators of osteogenic

differentiation Runx2 and Sp7 is initiated.

Boundary Formation and Integrity

Coordinated with the processes described above, guidance

cues are required to ensure that cells migrate along the cor-

rect path. Of particular significance for the coronal suture

is that it lies at the boundary between tissues with distinct

embryological origins: trigeminal neural crest (frontal

bone) and cephalic mesoderm (parietal bone) (reviewed

in Morriss-Kay and Wilkie53). Lineage tracing using the

Wnt1-Cre/R26R system to label cells of neural crest origin

and their progeny shows that the coronal suture itself

arises from mesoderm, and this was confirmed using a

reciprocal mesodermal driver, Mesp1-Cre.58,62 The neural

crest/mesoderm boundary can be identified from E9.5,

and so includes the critical period during which the defin-

itive coronal sutures are forming. The characteristic over-

lapping of the frontal bone by the parietal bone, forming

the oblique cross-section of the coronal suture (Figure 2C),

can be understood from the expansion of the underlying

cerebral hemispheres toward the hindbrain, taking the

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neural crest underneath the mesoderm.58 The dura mater

underlying both bones is of neural crest origin (Figure 2C).

Interestingly, the other major sutures have different tissue

origins and relationships: the metopic suture uniquely

forms within neural crest and the sagittal and lambdoid su-

tures, like the coronal, have dual neural crest/mesodermal

origins, but unlike the coronal, this is not the case along

their entire length (Figure 2C).53 For this reason, in the

subset of cases of metopic synostosis that are associated

with neurocognitive abnormalities and/or dysmorphic fea-

tures, a disturbance of neural crest development is the

likely mechanism.68

Although it was originally thought that the coronal su-

ture boundary did not permit migration of sutural cells

either into or from the neural crest, evidence has emerged

that the barrier is in fact unidirectional, in that cells of neu-

ral crest origin cannot normally cross into the suture, but

the reverse does not apply.57 This latter conclusion is

consistent with the evidence from Zhao et al.56 that the

coronal suture contains stem cells; the progeny of these

cells can be expected to fuel the growth of both the adja-

cent bones, parietal and frontal (Figures 2B and 2D). Mol-

ecules thought to be important in the maintenance of

the suture boundary include the transcription factors

EN1, TWIST1, and MSX2 (previous section) and members

of the EPHRIN/EPH receptor and JAGGED/NOTCH fam-

ilies; all these molecules have been implicated in tissue

boundary formation in multiple other contexts.63,69,70

Osteogenic Proliferation and Differentiation

The importance of SHH in the earliest stages of coronal su-

ture development was described above. Gli1, a general

marker of HH signaling, is also expressed in the undifferen-

tiated mesenchyme of established postnatal sutures;56

however, in this context the paralogous molecule Indian

hedgehog (IHH) is the instructive ligand. Mice lacking

IHH (Ihh�/�) have reduced sizes of the developing frontal

and parietal bones, reduced Bmp2/Bmp4 expression, and

correspondingly widened midline sutures at E15.5; this

phenotype is thought to result from a differentiation

defect, because proliferation was found to be unaf-

fected.56,71 Secretion of IHH by differentiating cells is pro-

posed to maintain the recruitment of undifferentiated

osteoprogenitors from mid-sutural mesenchyme56 (Fig-

ure 2D). For calvarial expansion to occur, this differentia-

tion process must be exquisitely tuned to ongoing cell

proliferation.55 Notably, the observation that craniosynos-

tosis is frequently associated with defects in certain key cell

division genes (discussed later) points to the need for rapid

mitotic turnover in the cranial sutures. Twist1, expressed in

mid-sutural mesenchyme in the established coronal suture

(E16),59 is directly antagonistic to RUNX2, potentially

providing an important mechanism to prevent the mid-

sutural cells from undergoing osteogenesis.72 TWIST1

also has inhibitory activity on levels of bone sialoprotein

and osteocalcin, two of the downstream targets of

RUNX2: this requires heterodimerization with a type I

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basic-helix-loop-helix partner.73 In vivo, the strong

epistatic genetic interaction between Twist1 and Tcf12 in

the murine coronal suture shows that TCF12/HEB is the

key partner protein.48

Apart from the importance of maintaining the undiffer-

entiated fate of mid-sutural stem cells, a second key

element of cranial vault growth is provided by positive

differentiation signals emanating from osteoid, the collag-

enous unmineralized matrix produced by osteoblasts

along the expanding osteogenic fronts (reviewed in

Morriss-Kay and Wilkie53). Redundant signaling by

several fibroblast growth factors (there is evidence to

implicate particularly FGF2, FGF9, FGF10, and FGF18) is

crucial to orchestrating this process.60,74–80 The receptor

Fgfr2 is expressed in the rapidly proliferating osteoproge-

nitor cells, whereas Fgfr1 is associated with a more differ-

entiated state (Figure 2D); increased FGF-signaling flux

drives a switch from Fgfr2 to Fgfr1 expression, associated

with the onset of osteogenic differentiation.60,61 The

dura mater underlying the sutures also provides a source

of growth factors including FGF2, BMP4, and TGFb15 (re-

viewed in Levi et al.81).

As is the case for cartilaginous bones, the transcription

factor Runx2 represents a well-established positive regu-

lator of commitment to terminal osteogenic differentia-

tion. However, for reasons that remain poorly understood,

bones undergoing intramembraneous ossification (such as

the skull vault) are dependent on a higher RUNX2 dosage

than those undergoing endochondral ossification, so that

Runx2/RUNX2 haploinsufficiency is associated with patho-

logically widened cranial sutures in both mice and hu-

mans.82 Conversely, craniosynostosis occurs in most

individuals with RUNX2 duplication.45,46

Genetic studies have illuminated the role of another

molecule in osteoblast differentiation, retinoic acid. Reces-

sive mutations in POR (MIM: 124015), the flavoprotein

that donates electrons to all microsomal P450 enzymes,

cause Antley-Bixler syndrome (MIM: 201750), in which

craniosynostosis is associated with abnormal steroidogen-

esis.43 A genotype-phenotype correlation is apparent,

such that individuals with skeletal malformations are usu-

ally compound heterozygous for variants encoding a

combination of a missense and a null allele; moreover,

no subjects harboring homozygous null mutations have

been encountered, supporting the conclusion from mouse

studies that complete loss of function is lethal.83,84 Resid-

ual POR activity could differentially affect the function of

the many P450 enzymes, raising the question as to which

aspect of steroidogenesis is related to craniosynostosis. Ho-

mozygous mutations of CYP26B1 (MIM: 605207), which

encodes one of the P450 enzymes required for degradation

of retinoic acid, lead to mineralization defects of the

skull and/or craniosynostosis (MIM: 614416), related to

increased differentiation of osteoblasts to terminal osteo-

cytes.85 This evidence, together with the observation of

elevated retinoic acid levels in blood or tissues of POR

mutants in both mice86,87 and humans,88 suggests that

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disturbed retinoic acid metabolism is a key contributor to

the pathology.89Whether this manifests with craniosynos-

tosis ormineralization defects might depend on the precise

extent of osteoblast/osteocyte imbalance.85

How mechanical forces are integrated into the prolifera-

tion/differentiation response is uncertain, but primary

cilia are likely to play a key role.90 In calvarial cultures,

stretched sutures released FGF2 and demonstrated an im-

mediate increase in permeability to Ca2þ ions.91 Poten-

tially linked to these observations, mice with a neural

crest-driven knockout of Pkd2, encoding a calcium channel

that is a key component of the ciliary mechanotransduc-

tion system, exhibited postnatal fusions of sutures in the

facial bones (causing a bent snout) and features such as

fractured molar roots that were suggestive of increased

trauma, attributed to failure of normal feedback linking tis-

sue strength to mechanical loading.92 Furthermore, mice

with a mutation in Fuz, a key regulator of ciliogenesis,

have coronal synostosis.93 Cilia are also involved in inte-

grating HH and WNT signaling, both of which are impli-

cated in suture biogenesis, and mice with a Wnt1-driven

neural crest conditional mutation of Kif3a have metopic

synostosis.68,94 Given the proposed role of the IHH-Gli1

feedback loop in suture maintenance (Figure 2D), it is

tempting to speculate that mechanotransduction in the

suture could feed directly into this pathway; evidence is

lacking at present but this would be a fruitful field for

future investigation.

Resorption/Homeostasis

The steady state in the mature suture represents a balance

between osteogenesis and resorption, the latter of which is

mediated by osteoclasts of hematopoietic origin. Many of

the genes associated with generalized skeletal dysplasias

but with only low frequency of craniosynostosis (Table

S1) are likely to affect this balance; for example, it is not

surprising that osteopetroses, characterized by excessively

dense bones and deficient osteoclast function, sometimes

feature craniosynostosis in the clinical presentation. A

more suture-specific mechanism is likely in the case of

interleukin 11 (IL11) signaling, because loss-of-function

mutations in the co-receptor IL11RA are predictably associ-

ated with craniosynostosis (MIM: 614188) in humans (and

fusion of facial sutures in the case of mouse Il11ra�/� mu-

tants).41 However, interpretation is complicated because of

evidence that both osteoblast and osteoclast function are

compromised. Although IL11 has a stimulatory role in os-

teoblasts,95 predicting that deficient signaling would be

associated with osteopenia, the opposite turns out to be

the case; trabecular bone volume was increased in

knockout mice and osteoclast precursors showed a cell-

autonomous defect in differentiation.96 This suggests

that the osteoclast defect might predominate in the

context of loss of IL11 function. Circumstantial support

is provided by the observation that individuals homozy-

gous for IL11RA (MIM: 600939) mutations often have

delayed eruption of the secondary dentition, because local-

366 The American Journal of Human Genetics 97, 359–377, Septemb

ized resorption of the jaw bone by osteoclasts is a prerequi-

site for tooth eruption.41

The role and importance of cartilage intermediates in

craniosynostosis is unclear. Although the skull bones are

classically described as forming by intramembranous ossi-

fication, transient cartilage intermediates are well known

to occur.60 Behr et al.97,98 have identified cartilage both

in the normally fusing posterior frontal suture of wild-

type mice (equivalent to the human metopic suture) and

in the fusing coronal sutures of Twist1þ/� mice. Of note,

Twist1 inhibits chondrogenesis and is regulated by b-cate-

nin (canonical WNT signaling).99 Consistent with this, the

appearance of cartilage is accompanied by downregulation

of canonical WNT signaling (revealed, for example, by loss

of AXIN2 protein). However, these events were described

during postnatal stages, long after the initiating processes

leading to craniosynostosis, consistent with the possibility

that these represent secondary, downstream consequences

of altered signaling arising from earlier events.

Genetic and Biochemical Mechanisms in

Craniosynostosis

Moving on from defining the developmental role of genes,

another key element toward constructing a pathophysio-

logical framework is to delineate the activity of mutations

at a genetic and biochemical level. Here it is important to

appreciate that the constellation of gene mutations that

cause craniosynostosis is highly idiosyncratic. Unlike the

situation, for example, with the BBSome,100 there are few

cases where craniosynostosis results from simple loss of

gene function (RAB23 [MIM: 606144] and IL11RA muta-

tions [Table 2] are exceptions). One explanation could be

that skull growth and cranial suture development occur

relatively late during mammalian embryogenesis; loss of

function of many key genes in suture development would

probably already cause lethality owing to a separate and

earlier requirement in organogenesis. Rather, craniosynos-

tosis often seems to result as an ‘‘accidental’’ phenotypic

consequence of particular genotypes, which emerges

when mutations evade multiple earlier developmental

sieves. A striking example is provided by the seemingly

cytotoxic gain-of-function mechanisms associated with

FGF receptor mutations, which—given the near-universal

importance of FGF-mediated signaling in development—

might be expected to have widespread adverse effects on

many other aspects of organogenesis. Presumably in

most other tissues and time points, the toxic effects of

increased signal (Table 2) and associated defects in cellular

processing of mutant protein117–119 are efficiently miti-

gated by tight feedback regulation. At present, however,

this is speculation, because systems biology description

of the cellular processes is too crude to provide explanatory

power.

Emphasizing the unpredictability of these phenotypes,

species differences between human and mouse are

frequent for identical mutations in orthologous genes.

For example, mice with relevant mutations in Fgfr3 and

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Table 2. Biochemical Mechanisms in Craniosynostosis and Relationship to Pattern of Mendelian Inheritance

Biochemical Mechanism(and Pattern of MendelianInheritance) Example Genes Example Alleles Comments References

Complete loss of function(recessive)

RAB23, IL11RA homozygous nonsense orframeshifting mutations; alsoaa substitutions

consanguinity leading to autozygosityfrequent

41,44

Partial loss of function(recessive; hypomorphicmutation)

POR, MEGF8,CDC45

missense, weak splice sitemutations (outside invariantag/ and /gt sequences)

consanguinity leading to autozygosityoccasional. Leaky mutations in essentialgenes; combinations of compoundheterozygous alleles give moreopportunity for combined output to fallwithin narrow window of activity forphenotypic effect

23,42,83,84,89

Partial loss of function(dominant; haploinsufficiency)

TWIST1, TCF12,ERF

complete and partial deletions;nonsense, frameshift; some aasubstitutions

pathogenic aa substitutions localized tokey DNA binding/dimerization regions.Biochemical and genetic evidence ofinteraction between Twist1 and Tcf12

26,48,73

Partial loss of function(dominant negative)

STAT3 aa substitutions aa substitutions cluster in SH2 or DNAbinding domains

101

Gain of function: increasedligand affinity/broadenedspecificity (dominant)

FGFR1, FGFR2,FGFR3

equivalent Pro>Arg substitutionsin IgII-IgIII linker of FGFR1, -2,and -3

substitution to bulky arginine residueintroduces additional contacts to FGFs,causing increased binding affinity andillegitimate binding by FGF10

102–106

MSX2 Pro>His and Pro>Leu substitutionsat 7th position of MSX2homeodomain

contacts minor groove of DNA,associated with altered DNA-bindingspecificity

107

Gain of function: constitutiveactivation (dominant)

FGFR2, FGFR3 aa substitutions to or fromcysteine

results in covalent cross-linking ofreceptor monomers

108,109

FGFR2, FGFR3 aa substitutions in kinase domain abrogates requirement for dimerization 110

FGFR3 aa substitutions near membrane-spanning region

enhances transient interactions betweenmonomers

111

ZIC1 loss of inhibitory motif enhanced transcriptional activation 52,65,66

Gain of function: increaseddosage (dominant)

MSX2, IHH,RUNX2

complete gene or regulatoryelement duplications

opposite phenotype (reduced ossification)associated with deletions in MSX2 andRUNX2 further illustrating dosagesensitivity

40,46,112

Gain of function: ectopicisoform expression (dominant)

FGFR2 deletions or insertions involvingexon encoding FGFR2c spliceform

drives illegitimate expression of FGFR2bin usually non-expressing tissues

74,113,114

Cellular interference (X-linkeddominant with paradoxical malesparing)

EFNB1 partial and complete heterozygousdeletions and missense substitutionsassociated with similar phenotypesin females

occurs in females owing to randomX-inactivation of X-linked EFNB1;secondary abnormality of receptorlevels (EPHB2, EPHA4) demonstratedin mice

115,116

Abbreviation is as follows: aa, amino acid.

Efnb1 lack the coronal synostosis associated with the

equivalent human mutations,120,121 and mice with exact

mutationsmimicking Apert syndrome usually lack syndac-

tyly.122–124 There are many examples of phenomena that

rely on particular levels of wild-type or mutant protein

selectively affecting suture biology, including the partic-

ular sensitivity of the sutures to haploinsufficiency

(TWIST1, TCF12, ERF) and increased dosage states (IHH,

MSX2, RUNX2), the role of X-inactivation interacting

with heterozygous EFNB1 (MIM: 300035) mutations to

disrupt tissue boundaries in craniofrontonasal syndrome,

and the particular sensitivity of the cranial sutures to hypo-

morphic mutations in essential genes involved in cell divi-

sion (CDC45 [MIM: 603465], ESCO2 [MIM: 609353],

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RECQL4 [MIM: 603780]). Selected examples are summa-

rized, with references, in Table 2.

One observation of particular note is that equivalent

amino acid substitutions in the extracellular IgII-IgIII

linker of three FGFR paralogs (p.Pro252Arg in FGFR1,

p.Pro253Arg in FGFR2, and p.Pro250Arg in FGFR3) each

causes a distinct craniosynostosis syndrome (Pfeiffer

[MIM: 101600], Apert [MIM: 101200], and Muenke

[MIM: 602849] syndromes, respectively).27,29,37 Moreover,

each of these syndromes is characteristically associated

with coronal synostosis, whereas allelic mutations in

each gene more typically present with multisuture fusion

(Table 1). The likely common factor is that all three mutant

receptors illegitimately bind FGF10,102–104 creating an

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Figure 3. Early Pathological Processes in Coronal Sutures ofMutant Mice(A) Apert-Fgfr2Ser252Trp/þ mutant. Mid-sutural region containingundifferentiated stem cells becomes populated by osteogenic cells(ovals; evidence supports both increased proliferation and failureof mechanisms preventing differentiation) at E12.5–E13.5. Abbre-viations are as follows: f, frontal bone; p, parietal bone.(B) Neural crest cell migration defect in Twist1þ/� mutant. Nor-mally, the neural crest cells (blue) are restricted to the future fron-tal bone territory. In mutant mice, cells do not observe the normalboundary and additionally populate the coronal suture and devel-oping parietal bone.(C) Primary failure to maintain a population of undifferentiatedsutural stem cells. This is reflected in abnormal NOTCH1 proteinlevels (gray shading) at E12.5–E13.5.Figure based on original data presented elsewhere.63,69,70,131

abnormal paracrine signaling loop (see next section).

Despite being caused by such very specific mutations,

Muenke and Apert syndromes are two of the most com-

mon genetic diagnoses in craniosynostosis (Table 1);

remarkably, new mutations at these particular nucleotides

arise in themale germline at a frequency 500- to 1,000-fold

higher than the background rate, because of a selective

advantage of mutant cells in the testis (termed selfish sper-

matogonial selection).125

Pathophysiology

Given the specific temporal sequence of developmental

processes taking place in the cranial sutures, the mouse

provides the most accurate animal model for investigating

the details of pathophysiology in craniosynostosis. How-

ever, as noted above, even accurate genetic models do not

always have true craniosynostosis involving the cranial

vault, although synostosis of facial bones is frequently

identified in these mutants.41,120,121 Currently, the models

that most accurately phenocopy the onset of vault

craniosynostosis include several different gain-of-function

368 The American Journal of Human Genetics 97, 359–377, Septemb

mutations of Fgfr2 (Apert-Fgfr2Ser252Trp/þ,123 Apert-

Fgfr2Pro253Arg/þ,124 Apert splicing Fgfr2DIIIc/þ,113 Crouzon-

Fgfr2Trp290Arg/þ,126 Crouzon-Fgfr2Cys342Tyr/þ,127 Beare-

Stevenson-Fgfr2Tyr394Cys/þ,128), Pfeiffer- Fgfr1Pro250Arg/þ,129

Twist1þ/� haploinsufficiency,49,130 and ErfloxP/� dosage

reduction.26 Additional mouse models are well reviewed

in Holmes.122 In documenting pathogenesis in these

models, it is particularly important to devote efforts to

identifying the earliest developmental abnormality, ideally

before any anatomical change is evident; once craniosynos-

tosis has become manifest, it becomes extremely chal-

lenging to distinguish primary effects from secondary

consequences of earlier abnormal processes in develop-

ment. In this section we focus on studies that fulfil this

requirement to illuminate the underlying pathogenic

processes.

Apert-Fgfr2Ser252Trp/þ Mutant and Encroachment by Osteogenic

Fronts

An exemplar for a carefully conducted disease model study

is that on the Apert-Fgfr2Ser252Trp/þ mutant.131 Although

no abnormality could be detected in mutant embryos at

E12.5, by E13.5 multiple changes in expression of osteo-

genic genes were apparent, associated with narrowing of

the nascent gap at the base of the coronal suture. Although

more apical parts of the coronal suture remained patent for

several further embryonic days, effectively its fate was

already sealed because the bones could not grow separately

at their bases. Having identified this critical E13.5 time

point, studies of sutures from 1 day earlier (E12.5) were

conducted in an attempt to identify even earlier markers

for these events. A small but consistent increase in prolifer-

ation (assessed by bromodeoxyuridine incorporation and

detection of Ki67 antigen) was found, demonstrating a bio-

logical abnormality at E12.5, corresponding to the later

stages in organization of the supraorbital regulatory center.

By further analysis of osteoblast cultures (postnatal day 1),

which showed both accelerated proliferation and differen-

tiation, it was proposed that the primary defect involved

failure of the osteogenic fronts to halt their progress across

the undifferentiatedmesenchyme at the base of the cranial

suture. In other words, the delicate balance that maintains

the existence of a population of undifferentiated cells in

the mid-sutural mesenchyme in the face of rapid prolifera-

tion and differentiation to each side is disturbed, and the

stem cells are overwhelmed (Figure 3A).

How do these findings relate to knowledge of craniosy-

nostosis in humans? Embryonic day 13.0 in the mouse is

equivalent to 6 weeks post-conception in terms of human

skull development (8 weeks’ gestational age).4 At this stage

it would be technically challenging to identify a causative

mutation and, if a decision was made to terminate an

affected pregnancy, study the embryonic material. The

earliest available human pathological fetal material is

from around 19 weeks,132 illustrating the critical role of

the mouse studies in understanding the earliest stages of

pathogenesis of craniosynostosis. However, Mathijssen

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et al.,133 by calibration against the separation of the frontal

and parietal bone ossification centers in a series of human

fetal skulls, found that the distance between ossification

centers in two skulls of neonates who had Apert syndrome

corresponded to 15 weeks’ gestation, not long after coronal

sutures are first apparent. This corroborates the mouse

study described above,131 that in Apert syndrome there is

a primary failure in the function of the coronal suture

around the time of its formation. Unfortunately, most

functional studies of clinical material from craniosynosto-

sis cases utilize cells obtained months or years after disease

onset, and from poorly controlled tissue sources; hence,

their pathogenic interpretation should be treated with

particular caution.

Further analysis of the Apert-Fgfr2Ser252Trp/þ mutant was

undertaken using tissue-specific drivers to enable selective

expression of the mutation in cells of mesodermal or neu-

ral crest origin.134 This demonstrated that craniosynostosis

still occurs in themesoderm-driven Fgfr2Ser252Trp/þmutant,

typically with bony bridging from the frontal bone edge to

a point behind the parietal bone edge. This suggests that

Fgfr2Ser252Trp/þ expressed in early mesodermal osteoproge-

nitors is sufficiently sensitive to allow paracrine osteogenic

induction by FGFs secreted from neural crest-derived tis-

sue.134 By contrast, the reciprocal neural-crest-driven

Fgfr2Ser252Trp/þ mutant does not have craniosynostosis,

eliminating a deterministic role in pathology for either

the nascent frontal bone or the underlying dura mater.

However, the onset of the earliest abnormalities in the

mesoderm-conditional mutant was 2–3 days later (E15.5)

than in the germline mutant, indicating that the presence

of mutant cells of neural crest origin does have a synergis-

tic effect.

The mechanism outlined above falls into the general

category of altered proliferation/differentiation balance,

which at this early time point severely compromises

coronal suture function. As indicated in Table 2, Apert sub-

stitutions result in both increased ligand affinity and broad-

ened specificity of mutant FGFR2 receptors. Illegitimate

binding to FGF10 is strongly implicated in the coronal

(and facial) suture pathology, because reduction in Fgf10

dosage in the Apert-Fgfr2DIIIc/þ mouse model prevents the

occurrence of craniosynostosis.74 The selective early coro-

nal closure, combined with rapid expansion of the brain,

might act to keep open the other major sutures even in a

pro-osteogenic environment, leading to the widely open

metopic and sagittal sutures seen in most individuals with

Apert syndrome.135 A similar FGF10-mediated mechanism

is likely to explain the selective coronal synostosis that oc-

curs in Muenke syndrome (FGFR3-p.Pro250Arg heterozy-

gote). Although the equivalent Fgfr3Pro244Arg/þ mouse

model doesnotdevelop craniosynostosis, impeding adirect

test of this idea, characteristic hearing loss in this model is

rescued by reduced Fgf10 expression.105

Unlike in Apert syndrome, the activation mechanisms

occurring for the FGFR2 mutations associated with Crou-

zon (MIM: 123500) and Pfeiffer syndromes are typically

The American

constitutive rather than ligand dependent (Table 2).

Perhaps reflecting this distinct biochemical abnormality,

these syndromes show a different pattern of craniosynos-

tosis with less marked predilection for the coronal suture;

instead, sagittal or multisuture synostoses are also com-

mon presentations. In severe Pfeiffer syndrome, all sutures

either fail to form or fuse before birth, so the skull can grow

only by remodelling; this leads to cloverleaf skull, a very

severe disorder.136,137 A milder manifestation occurs in

some Crouzon syndrome and also ERF-related craniosy-

nostosis, whereby delayed closure of all sutures is associ-

ated with a relatively normal skull shape but high risk of

raised intracranial pressure.26,138,139 Interestingly, some

of the mouse mutants initially show paradoxically delayed

markers of early osteogenesis, followed by later craniosy-

nostosis,26,126 or differences in ossification potential be-

tween bones of differing origins,140 illustrating a shift of

the proliferation-differentiation balance occurring over

time. Neben et al.141 propose that this shift might reflect

differential utilization of non-canonical nucleolar FGF re-

ceptor signaling.

The key effector pathway perturbed by FGF receptor mu-

tations is the RAS-ERK pathway, as demonstrated by the

marked reduction in abnormal phenotypes associated

with genetic inhibition of FRS2-mediated signal transduc-

tion,142 in vivo inhibition of MEK1/2,143 and the craniosy-

nostosis associated with Erf mutations.26 However, the

complex relationship of RAS-ERK activation with craniosy-

nostosis is highlighted by the fact that only a minority of

individuals harboring RASopathy mutations develop cra-

niosynostosis.144

Twist1þ/� Mutant, Neural Crest Migration, and Boundary

Integrity

The second process that has been subject to the most

rigorous investigation in mouse mutants has been that of

boundary formation at the coronal suture. The starting

point for this work was the observation that in Saethre-

Chotzen syndrome, caused by TWIST1 halpoinsufficiency,

the coronal sutures are typically fused. By use ofWnt1-Cre/

R26R labeling of cells of neural crest origin, Merrill et al.70

showed that in Twist1þ/� mouse mutants, abnormal

migration of cells derived from neural crest could be de-

tected within the coronal suture and extending into the

parietal bone territory, first observed at E14.5 (Figure 3B).

Importantly, this cell-mixing process was shown to corre-

late with craniosynostosis. Given the frequent role of

EPHRIN-EPH interactions in tissue boundary formation,

protein levels of family members were examined and this

revealed disturbed distribution of EPHRINs-A2 and -A4

and their receptor EPHA4 at E14.5.70 Consistent with

this, EphA4�/� mutant mice were found to have craniosy-

nostosis and Twist1þ/�EphA4þ/� embryos exhibited

abnormal partitioning of DiI-labeled osteogenic cells,

which occupied the coronal suture territory.63 Postnatally,

the number of Gli1-expressing, presumptive stem cells was

reduced in the Twist1þ/� mutants.56

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Mutations in another ligand, the X-chromosome-en-

coded EPHRIN-B1, cause craniofrontonasal syndrome

(CFNS), which is characteristically associated with coronal

synostosis; orthologous Efnb1 is expressed in neural crest

but not cephalic mesoderm in mice.15 Based on studies

of mouse limb and craniofacial development,115,116 there

is strong evidence to implicate the female-restricted pro-

cess of X inactivation in disrupting the normal coronal su-

ture boundary, although this could not be directly proved

because Efnb1þ/� mice do not develop craniosynostosis.121

This unusual mechanism has been termed cellular interfer-

ence (Table 2).25

Twist1þ/� Mutant and Mesoderm Specification

NOTCH signaling also contributes to boundary formation,

although no genes from this pathway have been identified

as being frequentlymutated in clinical craniosynostosis. In

mice, the earliest abnormality identified in the sutures of

Twist1þ/� mutants, observed at E12.5 (the identical time

point as for the Fgfr2Ser252Trp/þ mutant reviewed above,

but 48 hr prior to observation of abnormal cell mixing),

was spreading of NOTCH2 protein to include the mid-su-

tural mesenchyme (Figure 3C).69 Also of note, crossing of

a conditional transgenic allele of Spry1 (encoding an inhib-

itor of FGF receptor signaling) with Twist1þ/� mutant mice

largely prevented the craniosynostosis from occurring.145

Together, these observations show that TWIST1 lies primar-

ily upstream of FGF receptor signaling (as in nematode

worms and fruit flies)146,147 and implicate TWIST1 inmain-

taining the identity of sutural cells, whichmight be impor-

tant to prevent later cell mixing. Importantly, however, the

conditional Spry1 experiment demonstrates that these

abnormalities can be overridden in an environment that

reduces theoverall osteogenic drive.Hence the relative con-

tributions that loss of boundary integrity versus increased

osteogenic differentiation make to the craniosynostosis

associated with TWIST1 mutation are difficult to separate.

Synthesis: A Genetic-Pathophysiological Framework

By combining information on (1) human gene mutations

and their genetic/biochemical characteristics (Tables 1

and 2), (2) processes in suture formation and gene expres-

sion patterns (previous sections and Figure 2), and (3)

detailed studies of pathophysiology (section above and

Figure 3), we propose a disease framework for craniosynos-

tosis built around the key processes of tissue boundary for-

mation andproliferation/differentiation balance (Figure 4).

This model includes earlier developmental stages (stem cell

specification and migration, lineage commitment, and or-

ganization of supraorbital regulatory center) for which the

link between genes and pathogenetic mechanisms is less

certain, either because the mouse model does not pheno-

copy the human disorder (for example, murine Rab23

loss of function causes a severe neural tube defect)148,149

or because relevant studies have not yet been performed.

The emerging importance of signaling through the

IL11RA-STAT3 pathway, which probably affects remodel-

370 The American Journal of Human Genetics 97, 359–377, Septemb

ling, is also highlighted and provides an unexpected link

between craniosynostosis and immunity. Although there

is experimental evidence for TGFb signaling providing an

instructive role for the dura mater in suture homeostasis

(reviewed in Levi et al.81), to our knowledge no genetic mu-

tants have been shown to act primarily by disturbing dura

mater-suture interactions. Analysis of Ski mutants targeted

to contain the localized mutations found in Shprintzen-

Goldberg syndrome would be interesting, because among

TGFb or BMP-related disorders, this is the one most consis-

tently associated with craniosynostosis.47

Therapeutic Approaches?

Currently the mainstay of treatment of craniosynostosis is

surgery, allied to support from specialties such as speech

therapy and clinical psychology. Although it is attractive

to envisage the surgeons losing business to novel medical

therapies, serious challenges will need to be overcome.

The majority of severe craniosynostosis is unanticipated

at birth (many arise by new mutation), yet the associated

suture fusion(s) have already initiated prenatally. More-

over, the idea that medical therapy could provide an

adjunct to surgery (for example, by maintaining the

patency of sutures post-operatively) is weakened by the

counterintuitive fact that surgery frequently involves

the destruction of patent sutures; rather, surgery aims to

create an expanded but rigid skull vault (the separate bones

being wired together),10,11 with further increase in brain

growth accommodated within the remaining space, plus

remodelling of the inner surface of the calvaria. The crea-

tion of artificial sutures as simple gaps between the bones

could potentially lead to progressive skull deformity, as

illustrated by occurrence of vertex bulge arising from strip

craniectomy,150 unless the mechanical properties of

normal sutures were accurately replicated.

Even with these caveats, the experiment of Shukla

et al.,143 in which they virtually cured progeny mice with

the Apert Fgfr2Ser252Trp/þmutation by intraperitoneal injec-

tion of the pregnant mother with U0126, an inhibitor of

MEK1/2, was undoubtedly spectacular.151 This treatment

was able to reverse the coronal synostosis, runting, and

infertility in some heterozygous mutant mice. However,

the independent replication of these findings has not yet

been reported and subsequent progress has been relatively

slow, with themost notable development being the discov-

ery that an inhibitor of p38 MAP kinase ameliorated the

associated skin disorder (but not craniosynostosis) in the

murine Beare-Stevenson-Fgfr2Y394C/þ model;128 the acces-

sibility of skin makes this a therapeutically tractable target.

Clearly the intelligent design of therapies requires detailed

knowledge of the mechanisms of craniosynostosis and

pathways affected, as surveyed above; recent reviews81,152

provide further details. In parallel with the development

of potential therapeutics, careful natural history studies

need to be conducted so that potential benefits and draw-

backs can be assessed realistically in relation to outcomes

based on current care.

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Figure 4. A Genetic-Pathophysiological Framework for CraniosynostosisProcesses in suture formation described in the text are displayed on the left (gray boxes). Other panels show relative timing of events inmouse, genes mutated (blue boxes; black type for core genes and gray type for additional genes), and pathways proposed to be affected(green boxes). Patterning of the supraorbital regulatory center and boundary formation are events particular to coronal suture develop-ment; correspondingly, mutations disrupting these processes lead predominantly to coronal craniosynostosis. Later developmental pro-cesses (proliferation-differentiation balance, homeostasis) apply to all sutures and correspondingly, pathological suture involvementtends to be more generalized. Mutations involving BMP signaling (MSX2, SKI) are not placed in this framework, pending further infor-mation from specific mouse models. Note that the origin of the metopic suture within the neural crest suggests that abnormal matura-tion of this tissue (through disturbed dynamics of cell identity or migration) might be a common factor predisposing to metopicsynostosis, which is not reviewed here (yellow boxes, bottom).

Conclusions

The study of craniosynostosis has been transformed over

the past two decades by the identification of pathogenic

mutations in the most common classical syndromes. This

has had many direct benefits, both for affected individuals

and their families (improved diagnostic testing, genetic

counselling, risk estimation, reproductive advice, and prog-

nostic guidance) and for identifying key molecules and

pathways in cranial suture development. Since 2010, the

availability of whole exome and genome approaches to

molecular genetics research has accelerated the rate of

discovery of genes enriched for mutations in subjects with

craniosynostosis.23,26,42,48,51 Here we have illustrated how

these human genetics discoveries can be understood by

integrating the information with multiple complementary

approaches from biochemistry, structural studies, cell and

developmental biology, and mouse genetics. Maintenance

of cranial sutures requires an ongoing, exquisitely delicate

balance to sustain a contiguous population of stem cells

correctly located in the mid-sutural mesenchyme, while

allowing for the rapid proliferation and differentiation

of closely adjacent cells to enable skull growth. As further

genes are discovered and reported, we anticipate that the

pathophysiological framework described here (Figure 4)

The American

will prove usefulwhen trying to incorporate newmolecular

components into the picture. In this review we have indi-

cated areas needing further research; we think the analysis

of mouse models using conditional drivers of expression

combined with tracing of genetically marked cells, and

understanding how genetic and biomechanical signals

integrate through primary cilia (and other mechanisms)

to effect coordinated brain/suture growth, are particularly

exciting topics for the next decade. Given the importance

of environmental factors in craniosynostosis and observa-

tion of clinical variability, including frequent asymmetry

in suture fusion and non-penetrance, the contribution of

genetic and epigenetic influences on allelic expression,

somatic mosaicism, and mutation in regulatory motifs are

all lines of inquiry that should be explored further and are

likely to yield diagnostic answers in individual cases.

An alternative methodology in human genetics, more

suited to identifying common susceptibility alleles confer-

ring modest relative risk, is to use genome wide association

studies (GWASs). The first GWAS for sagittal synostosis, re-

ported in 2012, identified two significantly associated loci;

one located 120 kb downstream of BMP2 (encoding a

ligand in BMP signaling) and the other within an intron

of BBS9 (component of the BBSome, involved in moving

Journal of Human Genetics 97, 359–377, September 3, 2015 371

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cargo molecules in and out of cilia).153 Although it should

not be assumed that genetic susceptibility factors will

occupy the same pathophysiological landscape as mono-

genic ones, both these components affect processes that

overlap with those disrupted by previously identified sin-

gle gene mutations.

Supplemental Data

Supplemental Data include one table and can be found with this

article online at http://dx.doi.org/10.1016/j.ajhg.2015.07.006.

Acknowledgments

We thankAimee Fenwick, AnneGoriely,DeborahLloyd, and anon-

ymous referees for helpful comments on the manuscript, Simeon

Boyd and Wanda Lattanzi for suggesting genes to include in Table

S1, and Ryan McAllister (http://www.handjazz.com) for artwork.

We are grateful to Steven Wall and David Johnson, lead surgeons

at the Oxford Craniofacial Unit, for facilitating genetic research

in their patients, and to the Wellcome Trust for sustained funding

over a period of more than 20 years, which has made this work

possible. The laboratory is currently supported by the Wellcome

Trust (Senior Investigator Award to A.O.M.W., ref. 102731), the

NIHR Oxford Biomedical Research Centre, and the NIH.

Web Resources

The URLs for data presented herein are as follows:

GeneReviews, Verloes, A., Drunat, S., Gressens, P., et al. (2013).

Primary autosomal recessive microcephalies and Seckel syn-

drome spectrum disorders, http://www.ncbi.nlm.nih.gov/

books/NBK9587/

OMIM, http://www.omim.org/

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