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
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
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
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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
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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97,359–377,September3,2015
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
er 3, 2015
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
Journal of Human Genetics 97, 359–377, September 3, 2015 365
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
er 3, 2015
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],
The American
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
Journal of Human Genetics 97, 359–377, September 3, 2015 367
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
er 3, 2015
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
Journal of Human Genetics 97, 359–377, September 3, 2015 369
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.
er 3, 2015
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
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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