Craniosynostosis, Fibroblast Growth Factor Receptors and Gastrointestinal Malformations – A Possible Link

by

Christine Elizabeth Hibberd

A thesis submitted in conformity with the requirements for the degree of Masters of Science

Graduate Department of Dentistry University of Toronto

© Copyright by Christine E Hibberd 2014

ii

Craniosynostosis, Fibroblast Growth Factor Receptors and

Gastrointestinal Malformations – A Possible Link

Christine Elizabeth Hibberd

Masters of Science

Graduate Department of Dentistry University of Toronto

2014

Abstract

Syndromic craniosynostosis is most commonly associated with mutations in Fibroblast Growth

Factor Receptor genes (FGFR)-1, 2 and 3. Clinical and animal reports suggest a link between

FGFR-associated craniosynostosis and defects in the gastrointestinal tract (GIT).

Objective: to determine whether GIT malformations occur more frequently in the

craniosynostosis population with a known FGFR mutation when compared to the general

population.

Methods: A retrospective chart review of patients diagnosed with Crouzon, Pfeiffer or Apert

syndromes between 1990 and 2011 was performed at the Hospital for Sick Children in Toronto.

Thirty-two charts meeting inclusion criteria were analyzed for any history of GIT abnormalities.

Results: Three out of 32 patients had documented intestinal/bowel malrotations while 7 had

gastroesophageal reflux disease. All patients had documented FGFR2 mutations, a finding in line

with previous studies and published case reports.

Conclusions: Results suggest an association between FGFR-associated craniosynostosis and

GIT malformations.

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Acknowledgments

There are many people who offered their endless support throughout this process, whom I am

incredibly grateful to.

To Dr. Sarah Bowdin, thank you for your mentorship, encouragement, guidance and

patience over the past three years. I appreciate your vast knowledge in the field of genetics and

craniofacial anomalies that inspires me to become involved in treating patients with craniofacial

differences. It was an absolute pleasure to work with you.

To Dr. Siew-Ging Gong, thank you for your all of your advice and guidance throughout

this project. You have taught me to truly appreciate the importance of basic science in the

context of a very clinical world.

To Dr. Bryan Tompson, thank you for all of your support as the Head of the Department

of Orthodontics and member of my research advisory committee. I will always be grateful for all

you have taught me over the past three years

To Dr. Christopher Forrest, thank you for taking the time out of your busy schedule to

serve on my committee. Your expertise is invaluable and very much appreciated.

To my classmates and friends, Dr. Sean Chung, Dr. Heather Ford and Dr. Marc

Yarascavitch, my sincerest thanks for all of your support, suggestions, advice and

encouragement. The past three years would not have been the same without you.

A very special thank you to my family. To my parents, Graeme and Elizabeth and my

sister Jennifer, thank you for all your support and the endless hours you spent watching Jack and

recently, Henry, in order to make it possible for me to complete my orthodontic program and

thesis. To my adorable sons Jack and Henry, you brighten everyday of my life with love and

excitement. Thank you for being the sweetest boys and making me the happiest mommy in the

world. I love you both so much. And finally to husband and best friend, Andrei, thank you for

your love, continual support, patience and constant encouragement that made it possible for me

to realize and fulfill my dreams. Words cannot describe how much you mean to me and how

much I appreciate all you do. I am very lucky to have you. I love you. I dedicate this thesis to my

loving and supportive family.

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TABLE OF CONTENTS

Abstract ii

Acknowledgements iii

List of Tables vii

List of Figures viii

List of Abbreviations ix

I. INTRODUCTION AND LITERATURE REVIEW 1

1. CRANIOSYNOSTOSIS 1

1.1. Cranial sutures – development and fusion: 1

1.2. Craniosynostosis terminology 3

Simple/complex and primary/secondary synostosis 4

Syndromic craniosynostosis 4

1.3 Incidence 6

1.4 Management 6

1.5 Causes 7

1.6 Genetic overview of craniosynostosis syndromes 7

2. FIBROBLAST GROWTH FACTOR (FGF) RECEPTORS 9

2.1 Structure 9

2.2 Evolution 9

2.3 Biological roles 10

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2.4 Mutations of FGFRs in craniosynostosis syndromes 10

2.5 Genotype-phenotype correlations 11

3. GASTROINTESTINAL TRACT (GIT) 16

3.1 Gastrointestinal development and FGF signalling 16

3.2 GI malformations 17

EUROCAT classification system 17

Intestinal malrotation 19

Gastroesophageal Reflux Disease (GERD) 19

3.3 Craniosynostosis and structural/functional anomalies of the GI system 20

II. HYPOTHESIS AND OBJECTIVES 22

III. MATERIALS AND METHODS 23

IV. RESULTS 25

1. Patient sample: 25

2. Craniosynostosis syndromes and mutations: 25

3. GIT anomalies 28

4. Summary 30

V. DISCUSSION 31

1. Genetic findings in sample population 31

2. Intestinal malrotation in sample population 32

3. Proposed mechanism 34

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4. Study limitations 35

VI. FUTURE DIRECTIONS 37

VII. CONCLUSION 38

VIII. REFERENCES 39

IX. APPENDICES

Appendix A: The Hospital for Sick Children Research Ethics Board Approval 52

Appendix B: The Hospital for Sick Children Research Ethics Board Reapproval 54

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List of Tables

Table 1: FGFR mutations and associated disorders.

Table 2: EUROCAT category and description of gastrointestinal anomaly.

Table 3: Number and type of mutations identified in a sample of 32 FGFR-associated

craniosynostosis syndromic patients at SickKids (Toronto, Ontario) between the years of 1990-

2011.

Table 4: Observed Fibroblast Growth Factor Receptor mutations and associated findings.

Table 5: Frequency of Intestinal/Bowel Malrotation and GERD in the sample population of CS,

PS and AS patients.

viii

List of Figures

Figure 1: Major bones and sutures of the adult human cranium.

Figure 2: Distribution of germline mutations in FGFR1, FGFR2 and FGFR3 causing missense

substitutions or probable splicing abnormalities.

Figure 3: Distribution and types of mutations observed in the sample population of CS, PS and

AS patients (pictured above FGFR2 protein).

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List of Abbreviations

Apert syndrome AS

Arginine Arg

Base pairs bp

Central nervous system CNS

Cleft palate CP

Crouzon syndrome CS

Crouzon syndrome with acanthosis nigricans CS-AN

Cysteine Cys

Extracellular signal-regulated kinases ERKs

European Surveillance of Congenital Anomalies EUROCAT

Fibroblast Growth Factor FGF

Fibroblast Growth Factor Receptor FGFR

Gastroesophogeal reflux disease GERD

Gastrointestinal GI

Gastrointestinal tract GIT

Heparan sulfate proteoglycan HSPG

Hospital for Sick Children SickKids

Immunoglobulin Ig

Left-right L/R

Mitogen-activated protein MAP

Pfeiffer syndrome PS

Phenylalanine Phe

x

Proline Pro

Protein kinase C PKC

Transforming growth factor TGF-β

Tyrosine Tyr

Tyrosine kinase TK

Serine Ser

1

I. INTRODUCTION AND LITERATURE REVIEW

Craniosynostosis is a condition characterized by the premature fusion of one or more

cranial sutures. Craniosynostosis may be isolated or as part of multisystem syndromes. When a

child is suspected to have a craniosynostosis syndrome they may require further diagnostic and

genetic tests as these individuals often have a higher risk of other physical and developmental

conditions. The genetic cause of syndromic craniosynostosis is heterogeneous; however, the

most commonly mutated genes are the Fibroblast Growth Factor Receptor (FGFR) genes,

specifically FGFR1, FGFR2 and FGFR3.

Several published reports present gastrointestinal (GI) malformations, in particular

intestinal malrotations, as comorbidities associated with syndromic craniosynostosis. These

reports, along with evidence from mouse models, suggest an association. Expression of the

FGFRs in the GI tract (GIT) further strengthens a possible genetic link between FGFR-

associated craniosynostosis syndromes and the presence of GI defects in these patients. This

study is focused on analyzing the incidence of specific GI defects in craniosynostosis patients

with known mutations in the FGFR genes. This section will begin with a brief review of the

formation of cranial sutures, followed by the incidence, types, causes, and management of

craniosynostosis and a discussion of the associated syndromes. The genetic basis of FGFR-

associated craniosynostosis and the related GIT changes will be discussed in the following

sections.

1. CRANIOSYNOSTOSIS

1.1 Cranial sutures – development and fusion:

The neurocranium is composed of flat calvarial bones and a cranial base (Fig.1).

Intramembranous bone formation is responsible for generating the flat calvarial bones that give

rise to the roof and sides of the skull whereas the cranial base develops by endochondral bone

formation. Needle-like bone spicules radiate peripherally at the margins of the frontal, parietal

and occipital bones to produce the fontanelles and sutures (Cohen, 2002). Fontanelles are soft

membranous gaps found between incompletely formed cranial bones on an infant’s skull.

2

Sutures develop at the periphery of the extending bone fields, due to a wedge-shaped

proliferation of cells, referred to as osteogenic fronts (Decker & Hall, 1985). It is these

osteogenic fronts that seem to direct the morphogenetic determination of sutural patterning. The

osteogenic fronts can approach each other in one of two ways, yielding either an edge-to-edge

suture or a beveled suture (Johansen & Hall, 1982). End-to-end sutures form in the midline of the

skull with both the sagittal and metopic sutures being formed this way (Kokich, 1976). Coronal,

lambdoidal and frontozygomatic sutures are formed away from the midline and form a beveled

suture (Cohen, 2002). It has been proposed that the end-to-end suture is the result of

biomechanical forces equal in magnitude acting on each opposing side of the midline and

initiating suture formation while maintaining the flat calvarial bones in the same plane of space

(Cohen, 2002). In contrast, sutures formed away from the midline are likely to be affected by

unequal biomechanical forces, thus moving the bones into different planes of space yielding an

overlapping, beveled suture (Cohen, 2002). Fusion of the sutures located along the cranial

midline, such as the sagittal and metopic sutures, seem to produce more prominent ridging

compared to the ridging observed with synostosis of the sutures occurring away from the midline

such as the coronal and lambdoidal sutures (Cohen, 2002).

The timing of fusion of these sutures follows a strict temporal pattern, with most of the

sutures normally fusing after birth. In infancy, flexible sutures and open fontanelles allow for

deformation of the calvarium by enabling the bones of the skull to overlap as an infant passes

through the birth canal during delivery without damaging the infant’s brain (Levi et al., 2012).

During childhood, the brain grows and expands rapidly. Patent sutures allow the brain to grow

quickly without being constricted, while still providing a protective shell for the brain. The

fontanelles are the first to close during infancy and early childhood, followed by sutural fusion in

early adulthood when growth is complete (Cohen, 2002). The closure of the posterior fontanelle

generally occurs by the third month of life, while the anterior fontanelle typically closes between

the ages of 7-19 months (Aisenson, 1950). The metopic suture is the only suture that fuses early

on in life, undergoing fusion between six to eight months of age (Weinzweig et al., 2003). All

other cranial sutures usually begin to close when an individual enters his/her early twenties,

commencing most often with the sagittal suture. Fusion of the coronal suture closure occurs in

the mid-twenties and lastly the lambdoid suture in the late twenties (Morriss-Kay & Wilkie,

3

2005). Thus, a failure in this sequencing resulting in the premature fusion of one or more of

these sutures causes a birth defect known as craniosynostosis.

Figure 1: Major bones and sutures of the adult human cranium. Lateral (left) and top (right)

view demonstrating the bones (red line) and sutures (blue) of the calvarium. The metopic suture

separating the right and left halves of the frontal bone generally closes by the second year of

life.

Reprinted with permission from Levi et al., 2012

1.2 Craniosynostosis terminology

Craniosynostosis is recognized clinically and radiographically as the premature fusion of one or

more cranial sutures. It is the most common congenital defect of the human skull (Cohen, 1988;

Robin et al., 2011). To aid in diagnosis and treatment, different classification systems are

employed to describe the specific type of craniosynostosis. Classification is based upon the

number of sutures involved, their anatomical locations, shape of the head, etiology, and whether

the synostosis is an isolated physical difference or part of a syndrome.

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Simple/complex and primary/secondary synostosis

Simple craniosynostosis is a term used to describe the premature fusion when only one

suture fuses prematurely. When multiple sutures are involved, this is termed complex or

compound craniosynostosis (Kimonis et al., 2007).

Primary synostosis results from a defect of ossification and may involve a single or

multiple sutures, for example coronal synostosis only or coronal and sagittal synostosis (Cohen,

2002). It is believed that this premature fusion is the direct result of a developmental error during

embryogenesis (Nagaraja et al., 2013). Secondary craniosynostosis is caused as a result of

another medical condition or environmental issue, which subsequently impedes normal suture

formation (Cohen, 2002; Kimonis et al., 2007). Numerous conditions are associated with

secondary craniosynostosis, including metabolic disorders, hematological disorders and

teratogens (Cohen & MacLean, 2000).

Syndromic craniosynostosis

When craniosynostosis is accompanied by other physical or developmental anomalies it

is designated as syndromic craniosynostosis (Kimonis et al., 2007). All craniosynostosis

syndromes have defects in common with each other as a result of the premature fusion of the

cranial sutures. The shape of the skull is distorted due to the inability of growth to occur

perpendicularly to the prematurely fused suture and the resultant supplementary overgrowth at

the non-fused sutures of the cranium. In addition to craniosynostosis, several defects occur in the

midfacial region such as midfacial hypoplasia, exophthalmos, dental problems, and orofacial

clefting.

More than one hundred syndromes have been described in which craniosynostosis is a

documented feature (Cohen, 2002; Cornejo-Roldan et al., 1999; Robin et al., 2011). Examples

of craniosynostosis syndromes include Crouzon, Pfeiffer and Apert syndrome. These syndromes

share characteristics such as midface hypoplasia, hypertelorism, exophthalmos, a hypoplastic

maxilla and relative mandibular prognathism. Although similar in many respects, each syndrome

differs, in particular in the extent of involvement of the limbs and the associated abnormalities

that are observed in both Pfeiffer and Apert syndrome. Crouzon syndrome (CS) is the most

common syndrome of the three and is generally the most mild in presentation with individuals

5

often having normal or near normal intelligence (Forrest & Hopper, 2013). CS is characterized

by the absence of limb defects, in contrast to Pfeiffer and Apert syndromes (Johnson & Wilkie,

2011)

Pfeiffer syndrome (PS) is characterized by craniosynostosis of variable degrees, with

distinct phenotypic features that include midface hypoplasia, severe exophthalmos, ocular

hypertelorism, psittichorhina, broad radially deviated thumbs and toes, and in some cases partial

cutaneous syndactyly of the hands and feet (Johnson & Wilkie, 2011; Lajeunie et al., 2006).

Recognized central nervous system (CNS) malformations in PS include hydrocephalus and

congenital or acquired Chiari malformation (Kabbani & Raghuveer, 2004; Ranger et al., 2010).

Other recognized associations are radio-ulnar synostosis, and fusion of the cartilaginous tracheal

rings (Akai et al., 2006; C.-P. Chen et al., 2008; Gonzales et al., 2005; Hockstein et al., 2004;

Lajeunie et al., 2006; Oliveira et al., 2006; Ranger et al., 2010; Stevens & Roeder, 2006; Zackai

et al., 2003). These severely affected patients frequently have a high mortality rate in infancy or

early childhood (Akai et al., 2006; Lajeunie et al., 2006). PS is subdivided into three clinical

types, 1, 2 and 3. Type 1 is a milder form of PS whereas both type 2 and 3 exhibit severe

phenotypic expression. PS type 1 characteristics include craniosynostosis, broad thumbs and

toes, and partial syndactyly of the hands and feet to varying degrees (Barone et al., 1993;

Johnson & Wilkie, 2011; Koga et al., 2012). These individuals have normal or near normal

intelligence (Barone et al., 1993). PS type 2 characteristics include craniosynostosis manifesting

as a cloverleaf skull, broad thumbs and toes, severe ocular proptosis and central nervous system

malformations. These individuals can also be affected with elbow ankylosis or synostosis and

other anomalies (Barone et al., 1993). PS type 3 is very similar to type 2, but is differentiated by

the lack of the cloverleaf skull. Further characteristics include shallow orbits, and a

foreshortened anterior cranial base (Barone et al., 1993). Limb defects found in PS involves the

broadening of the thumbs and/or big toes and may exhibit either unilateral or bilateral

involvement. Some cases may also involve cutaneous syndactyly (Lajeunie et al., 2006). The

severity and extent of involvement of the limbs is a useful diagnostic aid to distinguish between

PS and Apert syndromes.

Apert syndrome (AS) is characterized by bicoronal craniosynostosis, midface hypoplasia

and severe bilateral and symmetrical syndactyly of the hands and feet (Johnson & Wilkie, 2011;

Kaplan, 1991; Wilkie et al., 1995). Another finding frequently found associated with AS is a

6

cleft palate (Johnson & Wilkie, 2011). Individuals with AS also have a high risk of learning

impairments and disability (Johnson & Wilkie, 2011). The extent of the involvement and

symmetry in the malformations of the hands and feet in AS distinguishes this syndrome from PS.

1.3 Incidence

The overall incidence of craniosynostosis has been estimated to range between 1:2100 to

1:3000 live births (Lajeunie et al., 1995). It has been estimated that approximately 85% of

documented cases are isolated, involving only a single suture, with the remaining 15% of cases

being syndromic (Chumas et al., 1997). The incidence of syndromic craniosynostosis is reported

to be between one in 25,000 and one in 100,000 infants (Forrest et al., 2013).

The location of the synostosis, ranked from most to least commonly observed are the

sagittal, coronal (either unilaterally or bilaterally), metopic, and then lambdoidal sutures (Cohen

& MacLean, 2000; Johnson & Wilkie, 2011). Sagittal synostosis accounts for approximately 40-

55% of non-syndromic synostotic cases, with coronal synostosis accounting for 20-25%, metopic

synostosis accounting for 10-15% and finally lambdoidal synostosis occurring rarely up to 5%

(Slater et al., 2008). Recent reports suggest that the involvement of the metopic suture may be

more prevalent than previously reported (Forrest & Hopper, 2013). These studies indicate that

metopic synostosis is found to in one in 10,000 live births compared to coronal synostosis

occurring in one in 15,000 live births (Selber et al., 2008; van der Meulen et al., 2009). In

general, there is a male predominance of the symmetric craniosynostoses (sagittal and metopic)

and a female predominance of the asymmetric subtypes (Forrest & Hopper, 2013).

1.4 Management

The management of individuals with craniosynostosis can range from minimal

involvement to requiring extensive care. Specifically, patients with syndromic craniosynostoses

require complex care which is best approached by a multidisciplinary team to effectively address

the vast array of their needs (Cunningham et al., 2007; Derderian & Seaward, 2012). Patients

often require multiple staged procedures which are carried out throughout childhood and

7

adolescence at age-appropriate intervals (Forrest & Hopper, 2013). As an example, procedures

may involve cranial vault remodeling, cleft repair, tracheostomies or limb reconstruction,

depending upon the presenting problems. A multidisciplinary team should ideally include

genetics, plastic surgery, neurosurgery, neuroradiology, dentistry, orthodontics, oral and

maxillofacial surgery, ophthalmology, otolaryngology, anesthesiology, psychiatry, social work,

occupational therapy, speech and language pathology and nursing in a tertiary care facility with

expertise in this area (Forrest & Hopper, 2013). Expertise is established based upon volumes of

patients and interest in the area (Cunningham et al., 2007).

1.5 Causes

The premature fusion of a suture or sutures may be the result of mechanical pressures,

metabolic, or genetic defects or teratogenic exposure (Nagaraja et al., 2013). Intrauterine

compression of the skull against the maternal pelvis could result in a mechanical cause of

synostosis (Johnson & Wilkie, 2011; Kimonis et al., 2007; Levi et al., 2012; Oliveira et al.,

2006). Hyperthyroidism is an example of a metabolic condition that can induce premature fusion

(Kabbani & Raghuveer, 2004; Nagaraja et al., 2013). Numerous genetic factors have been

implicated in the cause of synostosis. These genes encode proteins that are known to be

involved in the control of intramembranous ossification of the skull (Coussens et al., 2007).

1.6 Genetic overview of craniosynostosis syndromes

The identification of specific genetic mutations, in both syndromic and non-syndromic

cases of craniosynostosis, has led to a greater understanding of the etiology, development and

presentation of these disorders.

In general, evidence supports an autosomal dominant mode of inheritance and it is

believed that approximately 8% of all individuals with craniosynostosis have a dominant family

history of this condition (Cohen, 2002). However, the phenotype is not always consistent among

affected relatives of the same family. While some pedigrees do display fusion of a specific suture

such as coronal, sagittal or metopic, others will exhibit synostosis of different sutures (Cohen,

8

2002). Intrafamilial phenotypic variation can be extensive, leading to significant differences in

management and morbidity.

Over 60 different mutations have been identified to be causal in syndromic forms of

craniosynostosis. The majority of these mutations occur in the FGFR2 gene. As part of a 10-

year prospective cohort study testing 326 affected children, Wilkie and colleagues (2010)

identified FGFR2 as the most frequent mutation (32% of all genetic cases) followed by FGFR3

(25%), TWIST1 (19%) and EFNB1 (7%). Other well established, but considerably rarer,

associated genes include FGFR1 (mild PS), POR (Antley-Bixler syndrome) and RAB23

(Carpenter syndrome). Furthermore, other single-gene defects have been reported and include

mutations in EFNA4 (non-syndromic coronal synostosis), ESCO2 (Roberts syndrome), GLI3

(Greig syndrome), JAG1 (Alagille syndrome), KRAS (Noonan syndrome), RECQL4 (Baller

Gerold syndrome), TGFBR1 or TGFBR2 (Loeys-Dietz syndrome) and MSX2 (Boston type)

(Florisson et al., 2013; Jabs et al., 1993; Johnson & Wilkie, 2011; Merrill et al., 2006; Wilkie et

al., 2010).

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2. FIBROBLAST GROWTH FACTOR (FGF) RECEPTORS

2.1 Structure

The activities of fibroblast growth factors (FGF) are mediated by four cell surface

tyrosine kinase (TK) receptors, FGFR 1-4 (Ornitz & Marie, 2002). The receptors consist of an

extracellular region, a single hydrophobic membrane-spanning segment and a cytoplasmic TK

domain (Johnson et al, 1991). The extracellular area serves as the ligand recognition and binding

site and it is composed of three immunoglobulin (Ig)-like domains, designated IgI–IgIII, a stretch

of seven to eight acidic residues in the linker connecting IgI and IgII, designated the “acid box”

and a conserved positively charged region in IgII that serves as a binding site for heparin

(Schlessinger et al., 2000). Two highly conserved cysteine residues form an intramolecular

disulfide bridge stabilizing the loop structure of the three Ig-like domains. The cytoplasmic

domain contains the catalytic protein TK core which serves to recruit and activate docking and

signaling proteins (Eswarakumar et al., 2005). Ligand binding to the FGFR in conjunction with

a heparan sulfate proteoglycan (HSPG) causes receptor dimerization, transphosphorylation and

activation of an intracellular TK domain (Jaye, Schlessinger, & Dionne, 1992). The dimeric

FGFR is stabilized by both FGF-mediated interactions, direct receptor–receptor interactions as

well as by binding of the HSPG to the heparin binding domains as well as the FGF molecules

(Schlessinger et al., 2000). Receptor activation leads to the docking of adapter proteins followed

by the activation of a signal transduction cascade that regulates genetic expression causing

diverse biological responses (Szebenyi & Fallon, 1999). Receptors differ from one another

mainly in their ligand affinities and tissue distribution (Burke et al., 1998).

2.2 Evolution

The genes coding for FGFs and their receptors have been identified in multicellular

organisms ranging from the nematode, Caenorhabditis elegans, to the mouse, Mus musculus, and

the human, Homo sapiens. However, they have not been identified in unicellular organisms such

as Escherichia coli and Saccharomyces cerevisiae. Evidence exists indicating that both the FGF

and FGFR gene families have greatly expanded during the evolution of primitive metazoa to

vertebrates. The co-evolution of FGF and FGFR genes has permitted increased ligand-receptor

10

specificity, enabling the formation of preferred ligand-receptor interactions offering this

signaling system a great functional diversity and therefore an almost ubiquitous involvement in

developmental and physiologic processes (Itoh & Ornitz, 2004).

2.3 Biological roles

In recent years, the analysis of FGFR mutations involved in human skeletal disorders and

advances in mouse genetics have revealed essential molecular mechanisms by which FGF/FGFR

signaling controls tissue formation. Reported roles of FGFRs have included regulation of cell

division, cell growth and maturation, formation of blood vessels, wound healing and bone

growth during embryonic development (Hughes, 1997). FGFRs have been intensely studied as a

possible anticancer target for their role in tumour cell proliferation, angiogenesis and migration

(Kumar, Narasu, Gundla, Dayam, & J A R P, 2013) and for their involvement in bone formation

and metabolism (Du et al., 2012).

In the skeleton, FGF/FGFR signaling is an important regulator of prenatal and postnatal

bone development (Marie, 2003; Ornitz & Marie, 2002). During embryonic development, the

FGF/FGFR interaction has been shown to control the switch between adipocyte and osteoblast

differentiation in mesenchymal bone marrow stromal cells (Xiao et al., 2010). Genetic studies in

mice and humans have highlighted the important role of FGFR1/2 signaling in the control of

osteoblast gene expression and bone formation (Wilkie, 2005). FGF2 signaling activates many

internal signaling pathways in osteoblasts, including mitogen-activated protein (MAP),

extracellular signal-regulated kinases (ERKs), p38 MAP kinase and protein kinase C (PKC).

These pathways were known to mediate osteoblast proliferation, expression of specific osteoblast

genes and survival (Marie, 2003)

2.4 Mutations of FGFRs in craniosynostosis syndromes

Mutations of FGFR1-3 have been associated with a variety of phenotypic abnormalities

and often significant phenotypic variability is observed even among familial cases (Passos-

Bueno et al., 1999). Few phenotype-genotype correlations are absolute suggesting that

11

phenotypic features may also be determined by epigenetic factors or unlinked modifier genes

(Robin et al., 2011).

What is known is that the majority of FGFR mutations act in an autosomal dominant

way, are frequently de novo and result in increased activation of the mutant receptor, sometimes

independently of the ligand (Passos-Bueno et al., 1999). This is termed a “gain-of-function”

mutation.

Missense or splice-site mutations account for the majority of all observed nucleotide

changes with the occasional occurrence of small inframe insertions and deletions being

documented in FGFR-associated craniosynostosis syndromes (Cohen, 2002). FGFR mutations

may affect the extracellular or intracellular domains (Cohen & MacLean, 2000). When FGFR

mutations create or destroy cysteine residues, an unpaired cysteine remains that can generate

intermolecular disulfide bonding resulting in constitutive receptor activation (Neilson & Friesel,

1996). These gain of functions mutations cause alterations that can cause varying degrees of

ligand-independent signaling. An FGFR mutation can allow early human development to

proceed normally but may exert its interference with later bone development (Cohen, 2002).

2.5 Genotype-phenotype correlations

Missense mutations in FGFR1-3 have been associated with craniosynostosis and human

chondrodysplasia (Table 1). Typically, these mutations account for eight predominant

syndromes, comprising the FGFR-related craniosynostosis spectrum (Robin et al., 2011). These

include: AS, PS, CS, CS with acanthosis nigricans (CS-AN), Beare-Stevenson syndrome,

FGFR2-relatated isolated coronal synostosis, Jackson-Weiss syndrome and Muenke syndrome.

All of these mutations are autosomal dominant and often arise spontaneously (Ornitz & Marie,

2002).

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Table 1: FGFR mutations and associated disorders. Gene Characteristic mutation (%

responsible) Disorder

FGFR1 Widespread (haploinsufficiency) P252R (100%) N330I, Y374C, C381R

Kallman syndrome Pfeiffer syndrome (PS) Osteoglophonic dysplasia

FGFR2 S252W (66%), P253R (32%) IgIIIc (71%), IgIIIa (23%), TK, IgI Y375C (>85%), S372C

Apert syndrome (AS) Crouzon(CS)/Pfeiffer(PS)/ Jackson-Weiss/Antley-Bixler syndromes Beare-Stevenson syndrome

FGFR3 P250R (>99%) A391E (100%) G380R (99%) K650M (100%) N540K (42%) R248C (56%), Y373C (22%), stop codon (12%) K650E (100%)

Meunke syndrome Crouzon syndrome with acanthosis nigricans(CS-AN) Achondroplasia Severe achondroplasia with acanthosis nigricans Hypochondroplasia Thanatophoric dysplasia I (TDI) Thanatophoric dysplasia II (TDII)

Adapted with permission from Wilkie, 2005

Three distinct craniosynostosis syndromes (CS, PS and AS) are usually caused by

heterozygous mutations of FGFR2 (Johnson & Wilkie, 2011). The mutations can be widely

distributed across the FGFR2 gene, yet the majority are found in the IgIIIa/IIIc domain. Several

critical mutational “hot spots” have been described in this domain for CS and PS patients in

codons 278, 290 and 342 (Kress et al., 2000; Schaefer et al., 1998). Mutations in these areas can

result in unpaired cysteine residues, ultimately disrupting the protein structure by forming

intermolecular dimerization that leads to constitutive activation of the receptor (Neilson &

Friesel, 1996).

Interestingly, mutations occurring on the same codon can exhibit very different

phenotypes as a result of which nucleotide substitution occurs. For example, two FGFR2

mutations creating cysteine residues (p.Trp290Cys and p.Tyr340Cys) cause severe forms of PS

whereas conversion of the same residues into another amino acid (p.Trp290Gly, p.Trp290Arg,

p.Tyr340His) results exclusively in the CS phenotype (Lajeunie et al., 2006). This suggests that

alteration of the protein may cause a different functionality of the gene itself.

13

Figure 2: Distribution of germline mutations in FGFR1, FGFR2 and FGFR3 causing missense

substitutions (above protein) or probable splicing abnormalities (below protein). The mutation

prevalence is indicated on a logarithmic scale and different phenotypes are color-coded. Where

multiple phenotypes occur with mutation at a single position, the column colors are partitioned

proportionately using a linear scale. Substitutions recorded in more than 20 independent subjects

14

are identified individually. For clarity, insertions, deletions and nonsense mutations (none of

which has occurred as a recurrent mutation) are omitted; ‘‘Antley– Bixler’’ and ‘‘Jackson–

Weiss’’ phenotypes are lumped with Pfeiffer syndrome. Data for FGFR1 and FGFR2 are from

Wilkie’s unpublished database; data for FGFR3 are from Passos-Bueno et al. (1999),

supplemented with additional mutations. In each case the alternatively spliced IIIc spliceform is

shown; no mutations have been described in any of the IIIb exons.

Reprinted with permission from Wilkie, 2005

More than 50 mutations have been described in CS with approximately 95% of them

being located in just two exons of the gene encoding the extracellular IgIII domain, i.e., the IIIa

and IIIc domains (Meyers et al., 1996). Many of these mutations are believed to alter

downstream receptor signaling (Anderson et al., 1998; Ibrahimi et al., 2001; Mangasarian et al.,

1997; Mansukhani et al., 2000; Plotnikov et al., 2000). Mutations causing AS have been shown

to result in the loss of ligand binding specificity and thus can be activated by inappropriate

ligands, whereas CS and PS mutations in FGFR2 result in ligand independent activation and

dramatic decreased ligand binding (Ibrahimi et al., 2001; Mangasarian et al., 1997; Marie et al.,

2012; Plotnikov et al., 2000).

The mutations associated with PS overlap considerably with that of CS. PS type 1 can be

caused by a mutation in FGFR1 or FGFR2 (Koga et al., 2012). PS type 2 and 3 manifest as more

severe forms of the disorder, both occurring as de novo mutations. A small subset of

substitutions in the FGFR2 gene encoding p.Trp290Cys, p.Try340Cys, p.Cys342Arg or

p.Ser351Cys account for a large majority of the severely affected cases (Johnson & Wilkie,

2011).

Over 98% of patients diagnosed with AS have been found to have a missense mutation of

FGFR2 in the linker between the IgII and IgIII domains at codon p.Ser252Trp (66%) or

p.Pro253Arg (32%) (Johnson & Wilkie, 2011). In these cases some phenotypic correlations can

be drawn depending upon the specific mutation. Individuals with the FGFR2 mutation

p.Ser252Trp tend to present more often with cleft palates, severe ocular problems and

nasolacrimal stenosis (Akai et al., 2006). Intellectual disability and syndactyly appears to be

15

more severe in individuals with the with the p.Pro253Arg mutation in the FGFR2 gene (Akai et

al., 2006; Jadico et al., 2006; Johnson & Wilkie, 2011; Slaney et al., 1996).

Mutations in FGFR3 are involved in both craniosynostosis and human

chondrodysplasias. A correlation has been observed between a mutation in FGFR3

(p.Ala391Glu) and the development of acanthosis nigricans (AN) in patients with CS; however,

patients with CS without AN are very unlikely to display this specific mutation (Robin et al.,

2011). Achondroplasia, the most common form of short-limb dwarfism, has been linked to

specific mutations within the transmembrane region of FGFR3 with G380R accounting for

nearly all cases (Rousseau et al., 1996; Shiang et al., 1994; Wilkie, 2005)

16

3. GASTROINTESTINAL TRACT (GIT)

3.1 GI development and FGF signaling

The molecular basis for the development of the GIT is not completely understood.

Morphogens, substances that originate from a localized source and form a concentration gradient

throughout a tissue, are involved in the building of complex organs with intricate patterns of

cellular specialization (Wolpert, 1996). A few highly conserved morphogenetic signaling

pathways have now been identified in the development of the GIT. The four most important

morphogenetic signaling pathways that have been shown to play a role in patterning of the early

gut tube are FGF, Hedgehog, Wnt and Transforming Growth Factor (TGF)-β. These pathways

are conserved among organisms from the fruit fly to the human (Dab et al., 2013; van den Brink,

2007).

At a molecular level, studies have demonstrated that the FGFRs and the FGF ligands are

expressed along the entire GIT and are known to play a critical role in the development of GI

structures and multiple other organ systems. High levels of expression of FGFR2 have been

found throughout the GIT in adult human tissues (Hughes, 1997), specifically throughout the

gastric epithelium and submucosal macro- and microvasculature, with marked expression in the

muscularis mucosae and muscularis. FGFR2 expression is widespread in the entire mucosal

epithelium and muscularis in the entire ileum and appendix, with minimal expression observed in

hepatocytes and bile duct (Hughes, 1997).

Mouse models have been instrumental in uncovering many of the potential abnormalities

of the GIT associated with Fgfr2 mutations. Fairbanks et al., (2004) reported that duodenal

atresia was present with a 35% penetrance in Fgfr2b mutant mice embryos. A CS mouse model

with a specific Fgfr2 mutation (in codon 290 of Fgfr2), a mutation also found in humans with

CS, has been characterized to present with numerous GI defects (Dab et al., 2013; Mai et al.,

2010). The Fgfr2W290R mutants had numerous defective organ formation such as bones, teeth,

glands, and lungs (Mai et al., 2010). Additionally, they also presented with specific histological,

structural and functional defects in the esophagus (Dab et al., 2013). The GIT were much

shorter with a relatively empty peritoneal cavity. The mutant mice exhibited a decreased

thickness in the both the muscle layers and epithelium of the esophagus with a reduction in cell

proliferation noted. The esophageal layers also contained reduced collagen contention in

17

conjunction with a distinct patterning change in the orientation of muscle fibers of these mutant

mice and demonstrated reduced contractile activity compared to wildtype mice (Dab et al.,

2013). The findings in the Fgfr2W290R mutant mice therefore suggest a strong correlation between

the Fgfr mutation and GI malformations. The correlation therefore lends a plausible scientific

explanation for the anecdotal and observed findings of GI problems in Fgfr2 related

craniosynostosis patients, such as CS.

3.2 GI malformations

The GIT system is complex both developmentally and structurally. During embryonic

development, abnormalities of development of the GIT may involve one or multiple areas with

or without involvement of the other systems in the body. Some anomalies are readily detected,

yet others may not initially be apparent.

Common congenital GI malformations found in patients treated at a tertiary/quaternary

care academic hospital, the Hospital for Sick Children (SickKids) in Toronto, Ontario, Canada,

include esophageal atresia with or without tracheo-esophageal fistula, intestinal atresia,

malrotation with volvulus, Hirschsprung's disease, ano-rectal malformations and abdominal wall

defects. These anomalies are often classified using the European Surveillance of Congenital

Anomalies (EUROCAT) system.

EUROCAT classification system

EUROCAT is a network of population-based registries for the epidemiologic surveillance

of congenital anomalies. This database is useful for providing incidence data for rare congenital

anomalies, including those of the digestive system and abdominal wall defects. This registry has

documented 1.7 million births in 21 countries of Europe. (http://www.eurocat-network.eu/).

Categories classified under the EUROCAT heading of Digestive System include:

oesophageal atresia with or without tracheo-oesophageal fistula, duodenal atresia or stenosis,

atresia or stenosis of other parts of small intestine, ano-rectal atresia and stenosis, Hirschsprung’s

disease, atresia of bile ducts, annular pancreas, diaphragmatic hernia. Additional categories to be

reviewed included intestinal malrotation and gastroesophogeal reflux disease (GERD).

18

Categories classified under the EUROCAT heading of abdominal wall defects include

gastroschisis and omphalocele. A description of each anomaly is provided in the Table 2 below.

Table 2: EUROCAT category and description of gastrointestinal anomaly. Digestive system

CATEGORY DESCRIPTION Oesophageal atresia with or without tracheo-oesophageal fistula

Occlusion or narrowing of the oesophagus with or without tracheo-oesophageal fistula

Duodenal atresia or stenosis Occlusion or narrowing of duodenum

Atresia or stenosis of other parts of small intestine

Occlusion or narrowing of other parts of the small intestine

Ano-rectal atresia and stenosis Imperforate anus or absence or narrowing of the communication canal between the rectum and anus with or without fistula to neighbouring organs

Hirschsprung’s disease Absence of the parasympathetic ganglion nerve cells (aganglionosis) of the wall of the colon or rectum. May result in cong. megacolon.

Atresia of bile ducts Congenital absence of the lumen of the extrahepatic bile ducts

Annular pancreas Pancreas surrounds the duodenum causing stenosis

Diaphragmatic hernia Defect in the diaphragm with protrusion of abdominal content into the thoracic cavity. Various degree of lung hypoplasia on the affected side

Abdominal wall defects

CATEGORY DESCRIPTION Gastroschisis Protrusion of abdominal contents through an

abdominal wall defect lateral to an intact umbilical cord and not covered by a membrane

Omphalocele Herniation of abdominal content through the umbilical ring, the contents being covered by a membrane sometimes at the time of delivery.

Source: European Surveillance of Congenital Anomalies (http://www.eurocat-network.eu/)

19

Intestinal malrotation

Intestinal malrotation is a serious and potentially fatal developmental anomaly. Both the

small and large intestines can be affected during development in fetal life involving both the

position and peritoneal attachments leading to malrotation (Cassart et al., 2006). In majority of

cases, diagnosis is made during the first year of life (Pickhardt & Bhalla, 2002). Diagnostic

symptoms include abdominal pain, bile stained vomit secondary to bowel obstruction, bowel

dilatation, ascites or meconium peritonitis (Nehra & Goldstein, 2011). Nevertheless, the true

incidence of intestinal malrotation remains unknown (Nehra & Goldstein, 2011). The literature

cites incidences ranging from 1:200 to 1:6000, with an incidence of 1:500 being commonly

referenced (Cassart et al., 2006; Nehra & Goldstein, 2011; Pickhardt & Bhalla, 2002).

Gastroesophageal Reflux Disease (GERD)

GERD is a very common disorder of the GIT (Bredenoord, Pandolfino, & Smout, 2013).

It has been defined as a chronic condition developing due to significant quantities of reflux of

gastric contents into the esophagus causing symptoms with or without mucosal erosions and/or

associated complications (Chen & Hsu, 2013).

The true incidence of GERD in the general population is difficult to discern, but can be

classified in terms of its prevalence. In the adult population, GERD can be defined by at least

weekly heartburn and/or acid regurgitation, with prevalence estimates ranging from 10 to 20% in

western countries and less than 5% in Asian countries (Dent et al., 2005; El-Serag et al., 2009;

Moayyedi & Talley, 2006). The estimated prevalence of GERD in infants is considered to have a

peak incidence of approximately 50% at 4 months of age and then to decline affecting only 5%

to 10% of infants at 12 months of age (Lightdale & Gremse, 2013). Vomiting will settle in

majority of infants by 13 or 14 months of age (A. Martin et al., 2002). While this is merely an

estimate, the reported prevalence of GERD in patients of all ages worldwide is increasing

(Sherman et al., 2009). Diagnosis of GERD in infants and children can be difficult and is usually

made based upon presenting symptoms. In infants, common symptoms include regurgitation or

vomiting associated with irritability, anorexia or feeding refusal, poor weight gain, dysphagia,

presumably painful swallowing, and arching of the back during feedings. In young children, aged

1 to 5 years old, common symptoms of GERD include regurgitation, vomiting, abdominal pain,

anorexia, and feeding refusal (Gupta et al., 2006). Older children and adolescents are likely to

20

resemble a similar clinical presentation to adults with GERD including symptoms such as

heartburn, epigastric pain, chest pain, nocturnal pain, dysphagia, and unpleasant tasting burps

(Lightdale & Gremse, 2013). The two typical symptoms of GERD are recognized as pyrosis,

commonly referred to as heartburn and regurgitation, with some patients also noting extra-

oesophageal symptoms such as hoarseness, cough, and asthma (Bredenoord et al., 2013). There

is no gold standard test for objectively diagnosing GERD, and thus diagnoses have relied on a

combination of disease characteristics (Bredenoord et al., 2013).

3.3 Craniosynostosis and structural/functional anomalies of the GI system

Children with craniosynostosis syndromes may have other associated health problems

including GI malformations (Cohen & Kreiborg, 1993). Interestingly, mutations in FGFR2, the

most common gene associated with craniosynostosis syndromes have also been linked to case

reports of GI disorders. The literature on this topic is surprisingly sparse; however, there are few

studies and several of case reports on the subject.

Cohen and Kreiborg (1993) examined 135 patients with AS and concluded that only

1.5% presented with GI problems compared to 10% affected with cardiovascular anomalies,

9.6% with genitourinary anomalies 9.6%, and 1.5% with respiratory anomalies. Notably, when

autopsies were performed on 12 of these 135 patients, visceral anomalies including congenital

heart defects, anomalies of the respiratory system, GIT anomalies and genitourinary

abnormalities, were found in 9 of 12. This suggests that the percentage of anomalies from

clinical examination and history should be considered the minimum estimate because of the

possibility of clinically silent visceral anomalies, minor internal anomalies and anatomic

variations.

A recent study by Koga et al. (2012) investigated the clinical expression of PS types 2

and 3 in a Japanese population. Records of a total of 23 patients with confirmed PS diagnosis,

treated in one Japanese hospital between 1980-2011,were examined. Striking findings included

the detection of GI malformations in 22% of these patients. The detected problems included an

imperforate anus, intestinal malrotation and intestinal atresia in the affected individuals (Koga et

al., 2012).

21

Only a handful of case reports are cited in the literature ranging from 1975 to the present

documenting craniosynostosis patients who also presented with GI problems. In 1975, Eaton and

colleagues identified a patient with PS with features suggestive of type 2, who was found to have

intestinal malrotation. Barone et al, (1993) examined two patients with PS type 3, who developed

bowel obstruction and were also found to have intestinal malrotation. Park et al., (1995)

identified a mother and son both with CS and both diagnosed with epithelial- derived anal

atresia. More recently, Zarate and colleagues (2010) reported a case of a 16 month old male with

craniosynostosis who underwent cranial vault remodeling, placement of VP shunts for

hydrocephalus, gastrostomy tube for suspected GERD, tracheostomy due to bilateral choanal

stenosis, craniofacial reconstruction and posterior fossa decompression. The diagnosis of GI

malformation was very delayed resulting in significant morbidity including numerous infections

and respiratory problems. In another recent unpublished case report (2010) from the Hospital for

Sick Children (SickKids) in Toronto, an infant presented with severe bicoronal synostosis and a

diagnosis of PS, who subsequently presented with symptoms of a small bowel obstruction,

secondary to volvulus and a duodenal web.

These documented case reports have indicated the possibility that GI malformations

could be a direct effect of the FGFR gene mutation associated with craniosynostosis, a

possibility further supported by research in animal models. Specifically, all the discussed case

reports have a common finding of intestinal rotation. We therefore propose that a correlation

between FGFR-associated craniosynostosis and GIT malformations exists.

22

II. HYPOTHESIS AND OBJECTIVES

Hypothesis

We hypothesized that FGFR-related craniosynostosis is associated with GI

malformations at a greater incidence than the general population.

Objective

The aim of this study was to determine whether GI malformations occur more frequently

in the craniosynostosis population with a known FGFR mutation compared to the general

population.

23

III. MATERIALS AND METHODS

A retrospective chart review of patients diagnosed with CS, PS or AS at the Hospital for

Sick Children (SickKids) between 1990 and 2011 was performed. Approval was obtained from

the Research Ethics Board at SickKids (Appendix A & B),

Inclusion criteria of charts to be included for review included the following:

a) Record of genetic counseling, testing or discussion.

b) Diagnosis of a craniosynostosis syndrome (CS, PS or AS)

c) Identification of a mutation in FGFR1, 2 or 3 genes

d) Documentation of a GI malformation according to the EUROCAT classification system

and common congenital GI malformations seen at SickKids

Exclusion criteria included:

a) Incomplete medical records

b) Lack of documented evidence of mutations in FGFR1, 2 or 3.

Three databases were searched for patients meeting the study inclusion criterion:

i) Division of Clinical and Metabolic Genetics clinical database (SHIRE systems)

ii) Department of Paediatric Laboratory Medicine molecular genetics database (SHIRE

systems), and

iii) Division of Plastic Surgery craniofacial database.

Medical records of the selected patients treated at SickKids included review of molecular

genetic test reports, general clinic letters, consultations, diagnostic imaging reports, clinic notes

for plastic surgery, genetics and GI visits and emergency records. These charts were reviewed

using a systematic approach that included a step-by-step analysis of all the records contained in

each patient folder to assess for a definitive diagnosis of craniosynostosis, the presence of

mutations in FGFR1, 2 or 3 genes, and any record of gastrointestinal abnormalities. If GIT

abnormalities were noted, they were classified using the EUROCAT classification system

(http://www.eurocat-network.eu/) for the following GIT anomalies:

i) oeshophageal atresia with or without tracheo-oesophageal fistula  

ii) intestinal malrotation, stenosis or atresia,

24

iii) GERD

iv) ano-rectal atresia or stenosis

v) Hirschsprung's disease,

vi) atresia of the bile ducts

vii) annular pancreas,

viii) diaphragmatic hernia,

ix) gastroschisis,

x) omphalocele

In addition, the investigation was further expanded to include findings of clefts of the

palate and/or lip. Findings were documented in a coded spreadsheet to preserve patient

anonymity.

Statistical analysis:

Statistical analysis was carried out by an independent statistician. A power calculation

and a binomial test was conducted to establish the statistical significance of the deviation in the

proportion of patients with GIT malformations in this sample compared to that of the general

population, at the 0.05 significance level.

25

IV. RESULTS

1. Patient sample:

A total of 32 patient charts were identified that met all the inclusion criteria of the study.

An initial review of the three databases of patients treated at SickKids between 1990 and 2011

identified a total of 50 charts of patients with clinical diagnoses of CS, PS and AS. As SickKids

is a hospital for children, all patients were 18 years of age or younger. Out of the 50 charts

identified initially, 18 patients were excluded from the study: 5 were due to inadequate health

records, 8 to a lack of documentation of genetic testing for FGFR1, 2 or 3, and 5 due to the

documented absence of a detectable FGFR mutation.

The 32 patient charts were reviewed for the congenital GIT malformations as described

in the EUROCAT classification system and/or frequently encountered at SickKids.

2. Craniosynostosis syndromes and mutations:

Of the 32 patients that met the inclusion criteria, 10 were diagnosed with CS, 7 with PS

and 15 with AS (Tables 3 and 4). All 32 patients were found to have documented FGFR2

mutations. Information on the specific mutations was only available for 25 patients. In total, 16

different mutations were identified in the 25 patients (Table 4 and Figure 3).

Of the 10 patients diagnosed with CS, all mutations were missense mutations in the IgIII

domain of FGFR2. Three out of the 10 mutations were in codon 342, with the change of the

cysteine (Cys) residue to tyrosine (Tyr), phenylalanine (Phe) and serine (Ser). The other

mutations included changes in codons 290, 328, and 344.

PS patients in the sample contained missense mutations in the IgIII domain, and in the

linker region between IgII and IgIII (S252) of FGFR2. In two of these patients, there was a

change in codon 342 from Cys to arginine (Arg). A splice-site mutation was identified in the

IgIII domain of FGFR2.

26

Of the 15 patients diagnosed with AS, 4 lacked documentation regarding the specific

FGFR2 mutation. One patient was found to have a large intronic deletion of 1372 base pairs (bp)

between FGFR2 exons IIIb and IIIc. The remaining 10 patients had missense mutations in the

linker region between IgII and IgIII in either Ser252 or proline (Pro)253. Seven of these 10

patients presented with a mutation at codon 252 from Ser to tryptophan (Trp), while three

patients presented with a mutation at codon 253 from Pro to Arg.

Table 3: Number and type of mutations identified in a sample of 32 FGFR-associated

craniosynostosis syndromic patients at the Hospital for Sick Children (Toronto, Ontario) between

the years of 1990-2011.

Crouzon Syndrome (CS)

Pfeiffer Syndrome (PS)

Apert Syndrome (AS)

No. of patients 10 7 15 Summary of type and Location of Mutations

-­‐ Missense mutations in IgIII domain of FGFR2

-­‐ Missense mutations in IgIII domain of FGFR2

-­‐ Missense mutations in Ser252 of FGFR2

-­‐ Missense mutations in Ser252 and Ppo253 of FGFR2

-­‐ Deletion mutation in FGFR2

27

Table 4: Observed Fibroblast Growth Factor Receptor mutations and associated findings. IM -

intestinal (bowel) malrotation; GERD - gastroesophageal reflux disease; CP- cleft palate; CL -

cleft lip; * likely reporting error in mutation nomenclature.

28

Figure 3: Distribution and types of mutations observed in the sample population of CS, PS and

AS patients (pictured above FGFR2 protein).

3. GIT anomalies

The review of the charts for GIT abnormalities according to the EUROCAT classification

system revealed a relative absence of documentation of most GI problems. The most common

abnormalities observed were intestinal/bowel malrotation (IM) and GERD.

Three out of 32 patients were found to have IM, and also exhibited GERD (Table 4). Of

the three patients with IM, two of them were diagnosed with PS, with the mutation cysteine to

arginine in codon 342. The third patient was diagnosed with AS with a mutation in codon 252

with a change from serine to tryptophan.

29

Table 5: Frequency of Intestinal/Bowel Malrotation and GERD in the sample population of CS,

PS and AS patients.

GERD Malrotation

Sample Size Frequency (% of total) Frequency (% of total)

Apert 15 3 9.375 1 3.13 Pfeiffer 7 2 6.25 2 6.25 Crouzon 10 2 6.25 0 0 Total 32 7 21.875 3 9.38

The incidence in the general population of IM is 1 in 500 (pgenral=0.002) (Cassart et al.,

2006; Nehra & Goldstein, 2011; Pickhardt & Bhalla, 2002). Statistical analysis comparing the

incidence of IM in this sample population revealed a significantly higher incidence of this GI

abnormality compared to the general population (p < 0.05), thus we conclude that the proportion

with intestinal malrotation in our sample is greater than that of the general population.

Seven out of 32 patients were found to have GERD (Table 5). Three were AS patients,

two were PS and one was a CS patient. No apparent pattern of genetic mutations is evident. The

true incidence of GERD in the general population is very difficult to discern and estimated at 10-

20% in western countries (Dent et al., 2005; El-Serag et al., 2009; Moayyedi & Talley, 2006).

The incidence of 21.75% (7/32) in the sample population appeared to be similar to the incidence

found in the general population in western countries.

Cleft palate (CP) was identified in a total of 12 cases. Nine cases were found in patients

with AS, 1 in a patient with PS and two in patients with CS. A total of seven cases with CP

displayed a mutation in codon 252 with a change from serine to tryptophan, while one case of CP

was found in a patient with a mutational change from cysteine to arginine in codon 342. The

remaining four cases identified with CP did not have documentation available of specific

mutation involved.

30

4. Summary

In summary, the major findings of our study are:

1. The sample of 32 patient charts reveals 16 different genetic mutations in the FGFR2 gene.

2. Mutations in CS patients were found in the IgIII domains, mutations in PS patients occurred in

both the IgIII domain, and in the linker region between IgII and IgIII (Ser252) of FGFR2, and

mutations in AS patients were found in the linker region between IgII and IgIII in either Ser252

or Pro253.

3. The incidence of IM was significantly higher in the sample population compared to the

incidence reported in the general population.

4. The incidence of CP was found almost exclusively in AS involving the p.Ser252Trp mutation.

31

V. DISCUSSION

This study showed a statistically significant association between FGFR-associated

craniosynostosis and specific GI defects. To our knowledge, no studies to date have examined

this population to look for this specific morbidity involving the GIT. Our study addresses this by

reviewing the hospital records of a group of FGFR-associated craniosynostosis syndromic

patients for the occurrence of GIT malformations.

1. Genetic findings in sample population

The current study found a total of 16 different genetic mutations in the 32 patient charts

reviewed. Generally, the mutations observed in our cohort are similar to those previously

documented in the literature. There has been quite extensive documentation of mutations in

FGFR2 at codon 342, as well as 290 and 278 in both CS and PS patients and are regarded as

mutational hotspots (Schaefer et al., 1998; Kress et al., 2001) The literature shows that

mutations at codon 342, such as p.Cys342Arg and p.Cys342Trp tend to occur most frequently on

the FGFR2 gene (Kress et al., 2001). Other commonly found mutations in individuals with CS

and PS include p.Trp290Gly, p.Trp290Arg, p.Tyr340His and p.Cys278Phe mutation (Lajeunie et

al, 2006; Kress et al, 2001, Park et al , 1995). Indeed, this was also the observation made in our

study. Codon 342 appeared to be the most common codon affected in the sample population.

Mutational changes of the cysteine residue to either Tyr, Phe or Ser were found in three CS

patients and to Cys or Arg in two PS patients. Further, a mutation at codon 290 from Trp to Gly

was observed in a CS patient. In addition to the missense mutations noted, one PS patient in our

cohort sample exhibited a splice site mutation at 952-1G>A. The mutation of this patient has

been previously described by Teebi and colleagues (2002).

In our study, two patients exhibiting intestinal malrotation were found to have the

p.Cys342Arg mutation in the FGFR2 gene. This particular mutation, along with p.Ser351Cys,

p.Trp290Cys has been detected in the more severe phenotypic displays of PS (Johnson & Wilkie,

2011).

32

Previous studies have reported that 98% of AS cases can be accounted for by two

mutations 1) p.Ser252Trp and 2) p.Pro253Arg (Ferreira et al., 1999; Johnson & Wilkie, 2011).

Literature suggests that the p.Ser252Trp mutation accounts for approximately two-thirds of the

AS cases and the p.Pro253Arg mutation for the other third (Ferreira et al., 1999; Johnson &

Wilkie, 2011). Similar proportions were observed in our study, with the p.Ser252Trp mutation

occurring more often than the p.Pro253Arg mutation. Phenotype-genotype correlations for these

specific mutations have been previously documented. Individuals with an FGFR2 mutation of

p.Ser252Trp tend to present more often with cleft palates, severe ocular problems and

nasolacrimal stenosis (Akai et al., 2006). Patients with p.Pro253Arg in the FGFR2 gene often

display more intellectual disability and greater degrees of syndactyly (Akai et al., 2006; Jadico et

al., 2006; Johnson & Wilkie, 2011; Slaney et al., 1996). The sample population in our study also

supports these previously documented findings with six out of seven individuals with

p.Ser252Trp mutation displaying cleft palates. The AS patient exhibiting a large intronic deletion

was found to harbour a heterozygous 1372 bp deletion between FGFR2 exons IIIb and IIIc, This

apparently originated from recombination between 13 bp of identical DNA sequence present in

both exons and was not found in the unaffected parents (Fenwick et al., 2011).

2. Intestinal malrotation in sample population

Our findings suggest an association between FGFR-related craniosynostosis and GI

malformations, in particular bowel malrotation. The results show a statistically significant

difference in the number of FGFR-associated craniosynostosis patients that present with an

intestinal (bowel) malrotation when compared to the normal population. Patients in our cohort

are approximately 47 times more likely to have bowel malrotation. These results support our

hypothesis that FGFR-related craniosynostosis is associated with GI malformations. These

findings are not surprising given what is already known about FGFR involvement in normal

growth and development. They are further supported by multiple mouse model experiments, case

reports and clinical studies, revealing GIT anomalies in the syndromic craniosynostosis

populations (Barone et al., 1993; Cohen & Kreiborg, 1993; Eaton et al., 1975; Fairbanks et al.,

2004; Gong, 2012; Johnson & Wilkie, 2011; Lajeunie et al., 1999; Mai et al., 2010; Oldridge et

33

al., 1997; Park et al., 1995; Passos-Bueno et al., 1999; Wilkie, 2001; Wilkie et al., 1995; Zarate

et al., 2010).

Several studies examining phenotypic expression in the human FGFR-associated

craniosynostosis populations similarly point towards such correlations. A retrospective study by

Koga and colleagues (2012), carried out in Japan, has cited repeated malformations in the GIT as

part of their findings. In their sample population, 22% of the PS patients exhibited GIT

malformations including an imperforate anus, and intestinal malrotation (Koga et al., 2012). Our

study showed similar results with 25% of PS patients presenting with some type of GIT

malformation. Cohen and Kreiborg's, (1993) study thoroughly examined 136 AS cases and

concluded that 1.5% of living patients presented with clinically identifiable GIT problems.

Furthermore, a large majority of deceased patients (9 out of 12) possessed visceral anomalies.

This suggests that the percentage of anomalies identified during clinical examination and history

taking should be considered the minimum estimate. They suggest that clinically silent visceral

anomalies, minor internal anomalies and other anatomic variations in syndromic

craniosynostosis cases may be more prevalent than initial presentation suggests.

Our study also proposes that GI anomalies in the FGFR-associated craniosynostosis

population may not be an infrequent finding. Over the past three and a half decades, six case

reports have documented the finding of a GIT anomaly in FGFR-associated craniosynostosis

patients. Some of these case reports illustrate the importance of early recognition of GIT

malrotation as a possible comorbidity associated with FGFR-related craniosynostosis syndromes.

Our study identified two PS patients and an AS patient with intestinal malrotation. One of the

PS patients exhibited findings consistent with intestinal malrotation that was diagnosed during

her second year of life. This patient had a jejunum that lay to the right of the midline, her cecum

was abnormally located superomedially and the ascending colon was on a horizontal plane with

the cecum directed medially. Another one of our identified PS was an infant who presented with

PS and subsequently presented with a small bowel obstruction secondary to volvulus and a

duodenal web. Fortunately, prompt diagnosis was able to avoid further morbidity in this case.

Lastly, the AS patient with intestinal malrotation identified in this study was discovered later in

life as a result of a history of recurrent vomiting and abdominal pain. These previous case

reports and the three cases identified in this study highlight the importance of recognizing GIT

malformation as a possible comorbidity to FGFR-related craniosynostosis syndromes.

34

3. Proposed mechanism

The anatomical development of the GIT is a complex process occurring in three distinct

phases: i) an early phase of umbilical cord herniation, lasting from weeks 5-10 weeks of

embryogenesis followed by ii) a stage of reduction of the midgut loop back into the abdomen

occurring at weeks 10-11 and finally iii) a period of fixation from the 12th week of development

lasting until after birth (Frazer & Robbins, 1915; Mall, 1898; Strouse, 2004). In the early

embryo, the GIT is a straight tube supplied by the superior mesenteric artery (SMA). The midgut

lengthens disproportionally to the embryo, forming a U-shaped loop that herniates into the base

of the umbilical cord at approximately 6 weeks of gestation. Coincident with this growth, the

small intestine rotates a total of 270 degrees in an anti-clockwise direction being completed by

the 10th week when the intestine transitions back to the abdomen (Martin & Shaw-Smith, 2010).

Malrotation of the intestine is a broad term used to collectively describe any failure of rotation

that alters the normal developmental process of any part of the intestinal tract. It is not a single

distinct entity, but rather a continuum of abnormalities occurring during development of the

midgut. Martin and Shaw-Smith (2010) suggest that four etiologies may be attributed for

intestinal malrotations. These include: i) abnormalities of left and right patterning, ii)

abnormalities of the dorsal mesentery, iii) abnormalities of the bowel and iv) incorrect placement

of the intestine or abdominal organs.

For our purposes, we can consider the proposed roles of FGF signaling during

embryogenesis to suggest a link between FGFR-associated craniosynostosis and GIT

malrotation. It has been shown that FGF signaling is crucial in many aspects of early

embryogenesis, including the coordination of morphogenetic movements including dorsoventral,

anterioposterior and left-right (L/R) axis determination (Dorey & Amaya, 2010). In the sea

urchin both the fgfA ligand, and the fgfr2 receptor are necessary for the migration of the primary

mesenchyme cells (Röttinger et al., 2008). Similarly in Drosophila, a mutation in the fgfr2 gene

results in the failure of mesodermal cells to migrate away from the midline during gastrulation

(Beiman et al., 1996; Gisselbrecht, et al., 1996). The L/R axis is the third axis to be established in

the embryo and is responsible for the asymmetric placement of the thoracic and abdominal

organs, including what is considered the normal positioning of the bowel (Dorey & Amaya,

2010).

35

Studies in mouse, chick, rabbit and zebrafish have demonstrated a direct role in FGF

signaling in L/R axis determination (Albertson & Yelick, 2005; Boettger et al., 1999; Fischer et

al., 2002; Meyers & Martin, 1999). In all vertebrates, the L/R axis appears to originate from a

fluid flow in the node, a closed depression on the ventral surface of the embryo (Essner et al.,

2002; Nonaka et al., 1998). This extraembryonic fluid undergoes a leftward flow that is set into

motion by polarized monocilia and is thought to be responsible for normal situs (Tanaka et al.,

2005). FGF signaling seems to play several important roles during this early step of L/R axis

determination and may be responsible for the formation of cilia, cilia length (Neugebauer et al.,

2009), and for the release of vesicular nodal parcels (VNPs) containing morphogens–Sonic

hedgehog (SHH) and retinoic acid (RA)–into the node of the mouse (Tanaka et al., 2005). Any

genetic mutation that could interfere with nodal fluid flow altering L/R patterning of the dorsal

mesentery, the intestine and other abdominal contents may lead to intestinal malrotation.

Further support for this plausible proposed mechanism can be extrapolated from the

Cohen and Kreiborg (1993) study of AS patients. These authors report many different visceral

anomalies afflicting these patients including examples of L/R patterning problems such as

pulmonary stenosis, dextocardia, mesocardia and dextrorotation in the cardiovascular system and

partial biliary atresia with agenesis of the gallbladder in the GI system (Cohen & Kreiborg, 1993;

Lindsay, Black, & Jr., 1975; Ofodile & Adeloye, 1982).

4. Study limitations

This study was retrospective in nature. Inherent limitations of retrospective study designs

include the reliance on the accuracy of written record, which at times may be sparse or

inaccessible. Although SickKids maintains a thorough record of all patients treated, while

reviewing hospital records, inconsistencies in nomenclature and reporting errors were identified.

We observed a probable documentation error in the charts of three CS patients who likely had a

mutation recorded incorrectly. In these cases, the documented amino acid change did not match

the genomic sequence; thus, we conclude that a typographical error must have occurred.

Furthermore, potential biases could exist in our sample population. It is possible that

since SickKids is a leading paediatric hospital in North America, with a craniofacial program,

36

parents of children with craniosynostosis may tend to seek out a multidisciplinary center such as

SickKids for all of their care. This could potentially increase the number of patients with

intestinal malrotation seen at SickKids. Alternatively, and more likely, many patients travel great

distances to seek care in the craniofacial program at SickKids, but when a GIT issue arises, these

individuals may be more likely to see their local physician or hospital for relatively mild

symptoms or because they may not associated the two problems of craniofacial and GIT

anomalies together. Thus, it is quite possible that our sample may represent an

underascertainment of the true incidence of rotation in this craniosynostosis population.

37

VI. FUTURE DIRECTIONS

The findings of this study suggest a correlation between FGFR-related craniosynostosis

syndromes and GIT malformations. However, a direct cause and effect relationship cannot be

ascertained. Further research will need to be conducted to confirm this relationship. While the

findings of retrospective studies can be very useful in generating thought provoking data, they

are only hypothesis generating, and thus are most useful to suggest future directions for research.

We proposed that an FGFR gene mutation is involved in the malformation of the gut

solely based upon the evidence presented in current literature. Future directions for research

could involve using a multi-center approach to look at a greater quantity of these FGFR-

associated syndromic cases. Conducting a study to examine patients with craniosynostosis and a

known FGFR mutation compared to patients with craniosynostosis but no FGFR mutation to

discern any differences that are noted in the GIT may shed further light on the potential

widespread impact an FGFR mutation can exert.

38

VII. CONCLUSION

Our study supports the theory that intestinal/bowel malrotations in an FGFR-associated

craniosynostoses population are more likely to occur compared to the general population.

According to the current management protocol, screening of GIT disorders is not a part of the

investigative procedures in the care of CS patients. This study provides a sound scientific

rationale for the possible implementation of changes, e.g., additional testing and screening

procedures for GIT problems, in the clinical management of patients with FGFR-associated

craniosynostosis.

The findings of our study support a plausible explanation for the discovery of GIT

anomalies, specifically intestinal malrotation in craniosynostosis syndromes. The involvement of

FGFR in the normal growth and development of the GIT provides a sound scientific rationale for

the anomalies occurring in cases where mutations involving this gene occur. In addition,

research in animal models, the few studies and case reports demonstrating GIT further supports

the plausible correlation between FGFR-associated craniosynostosis and GIT malformations.

This, we speculate that GIT anomalies are a direct result of mutations occurring in the FGFR

altering the normal pathway of development and growth yielding malformations.

39

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IX. Appendices

Appendix A – Hospital for Sick Children Research Ethics Board Approval

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Appendix B - The Hospital for Sick Children Research Ethics Board reapproval letter

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