American Journal of Medical Genetics Part C (Seminars in Medical Genetics) 163C:213–217 (2013)

I N T R O D U C T I O N

Perspectives and Challenges in AdvancingResearch Into Craniofacial AnomaliesTIMOTHY C. COX,* DANIELA V. LUQUETTI, AND MICHAEL L. CUNNINGHAM

Timothy CoPediatrics (DiviInstitute. His re

Daniela LuqCraniofacial Mbirth defects.

Michael CunMedical Directcraniofacial co

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Development of the craniofacial region is a remarkably complex and tightly orchestrated process. It is thereforenot surprising that genetic and environmental insults frequently result in craniofacial anomalies. Nonetheless, ourknowledge of their etiology and pathogenesis is still scarce, limiting our efforts at prevention. Furthermore, fewstandardized protocols have been developed to guide clinical and surgical interventions. In this Issue of theSeminars, reviews on the most recent research advances on craniofacial conditions, from genomics andepigenetics to ontology and medical care are discussed with emphasis on the most common anomalies of thecraniofacial region: orofacial clefts, craniosynostosis, craniofacial microsomia, facial dysostosis, Robin sequence,jaw and dentition anomalies, and anterior neural tube defects. Phenotypic variability and the importance ofdetailed characterization using standardized terminology to better distinguish between phenotypes, newtechnologies (and their limitations) for genetic diagnosis, and the use of mouse models to study these conditionsin both their complex phenotypic and genetic aspects are highlighted. © 2013 Wiley Periodicals, Inc.

How to cite this article: Cox TC, Luquetti DV, Cunningham ML. 2013. Perspectives and challenges inadvancing research into craniofacial anomalies. Am J Med Genet Part C Semin Med Genet 163C:213–217.

INTRODUCTION

Why Craniofacial?

Craniofacial form is inextricably linkedto our societal structure, essentiallyplaying a major role in defining whowe are. Not only does it provide most ofthe major senses that determine ourability to efficiently interact with oursurroundings and the natural route bywhich we obtain nutrition to sustain life,but it also serves as the principal basis ofour recognition of self and family (i.e.,familiar and heritable features). To servethese roles, the overall structure of thecraniofacial region is necessarily compli-cated—from the exquisite morphoge-netic events that coordinate its assembly

x, Ph.D. is Professor and Laurel Endsion of Craniofacial Medicine) andsearch employs animal models to unuetti, M.D., Ph.D. is a medical genetedicine, Department of Pediatrics, Un

ningham,M.D., Ph.D. is the Jean Reor of the Seattle Children's Craniofanditions as well as basic and transladence to: Timothy C. Cox, Ph.D., c/oC9S‐5, 1900 9th Avenue, Seattle,

2/ajmg.c.31383ublished online in Wiley Online Lib

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to the complexity of tissues and cell typesthat enable all its functions. It is notsurprising then that it is also the singlemost impacted structure during devel-opment, whether as a result of genetic orenvironmental insult or a combinationof the two.

The unfortunate downside to beinga centerpiece of societal structure is thedifferent treatment of, or inability toaccept into “society,” those with atypicalcraniofacial form. While much of ourhealthcare is devoted to fixing thefunctional deficits of such birth defects,there is also a need to “normalize” anindividual’s appearance so they canmore easily integrate into society. How-ever, our goal should not only be todo a better job at diagnosing or correct-

owed Chair in Pediatric Craniofacial Research in tthe Center for Developmental Biology and Regenederstand the genetic and epigenetic contributions toicist and epidemiologist in the Craniofacial Center ativersity of Washington. Her research includes studyi

nny Professor of Craniofacial Medicine in the Universicial Center. For over 20 years Dr. Cunningham's cational research to identify causes and treatments.Center for Developmental Biology & Regenerative MWA 98101. E‐mail: tccox@u.washington.edu

rary (wileyonlinelibrary.com): 18 October 2013

ing craniofacial dysmorphologies, butshould rather also be focused on mitiga-tion and/or prevention. The latter goal isespecially true if we are to makesignificant inroads into alleviating theglobal burden of these conditions notjust in Western societies, which alreadyhave access to quality healthcare, but tothose societies that are less fortunate.

As health care professionals, there isa responsibility to keep abreast of thelatest research advances and continuallyreview practices to ensure the mostoptimal outcomes for patients. Fromexperiences in our own CraniofacialCenter, a coordinated multidisciplinaryapproach is essential for optimal care ofthe patient with a craniofacial anomaly.However, regular multidisciplinary

he University of Washington's Department ofrative Medicine at Seattle Children's Researchcraniofacial development and dysmorphology.Seattle Children's Hospital and the Division ofng potential genetic and non‐genetic causes of

ty ofWashington Department of Pediatrics andreer has focused on the care of children with

edicine, Seattle Children's Research Institute,

214 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) INTRODUCTION

discourse involving all health care spe-cialties as well as researchers (epidemi-ologists, geneticists, developmentalbiologists, and epigeneticists to name afew) is required if significant advances aregoing to be made in understanding notonly the causes of craniofacial anomaliesbut also the most effective evidence‐based interventional and treatmentstrategies.

A Vision for the Future

“Why understand the genetic basis ofcraniofacial birth defects? What are youhoping to achieve with such an under-standing? It is not as if you’ll ever be ableto correct the genes in the developinghuman embryo. Plus, can’t all craniofa-cial malformations be surgically cor-rected anyway?” These are the types ofquestions and comments many of usused to hear frequently 20, and even 10,years ago both from grant reviewers andlay people who ventured unwittinglyinto conversation about what we did fora living. It is pleasing, not just as grantwriters, that we no longer hear thesecomments. So what has changed?

First and foremost have been thetremendous advances in genetic tech-nologies over the past decade andspecifically in the way we analyzegenomes—from arrayCGH, SNP arraysand, more recently, massively parallel (ordeep) sequencing methods such asexome sequencing. These methodolo-gies have proven exceptionally usefuland powerful for family studies wherethere is a clear heritable phenotype andin studies with parent‐case trios ininstances where there is a robust andreproducible phenotype. Consequently,these technologies have raised so muchhope for patients and families, in asmuch as a genetic diagnosis can bringwith it some degree of certainty, closure,and understanding whilst also ascribingtrue recurrence risks. But even thoughthese approaches have quickly becomestandard weapons in the geneticist’sarsenal, there are still numerous limi-tations, both technical and practical, asoutlined in the review by Kriti Khan-delwal and colleagues. These limitationsare particularly evident when dealing

with common craniofacial disorderssuch as orofacial clefts (see review byElizabeth Leslie and Mary Marazita) andcraniofacial microsomia (review by Car-rie Heike et al.) where phenotypicdiversity, that is: variable expressivityand incomplete penetrance, is a majorconfounding factor.

The other major advance that hasinfluenced perception is arguably in thefield of epigenetics, and most signifi-cantly that involving the maternal–fetalinterface, that is, hormonal, teratogenic,and nutritional impact on fetal diseasesusceptibility and long‐term health andwell‐being. While gene–environmentinteractions have long been acknowl-edged as important, our ability toquantify specific effects both at thegenome level and now at the phenotypiclevel has unveiled enormous possibilities.In this Issue, the extensive review byClaudia Kappen on anterior neural tubedefects elegantly highlights the power ofboth epidemiological studies and partic-ularly animal model investigations inteasing apart the important interrela-tionship between diet and genotype. Asimilar convergence of epidemiologicaland biochemical evidence is also raisinghope for interventional and/or preven-tative therapeutic possibilities in othercraniofacial conditions, such as thecraniosynostosis and midface deficien-cies, although much work is still to bedone. In all cases, inroads can only bemade with more detailed and quantita-tive phenotyping, an understanding ofthe functional impact of genetic variants,and a much better appreciation of thefull spectrum of clinical variability in allcraniofacial conditions. With this inmind, the face and the craniofacialskeleton have considerable advantagesover other organ systems in that manytools are already available for quantita-tive 2D and 3D imaging, includingcomputed tomography (CT), magneticresonance imaging (MRI) and 3Dphotogrammetry, and in at least thelarger craniofacial centers are a routinepart of treatment planning and clinicalcare. The key to rapid progress in thisarea however will be the standardizationof protocols used by clinicians in differ-ent centers. The review by Carrie Heike

and colleagues expounds the benefits ofsuch an approach for both understandingand managing craniofacial microsomia,providing a framework on which we canmeasure the effects of ever changingtreatment strategies and serving as anexcellent model for other conditions.

DEALING WITH THECHALLENGES POSED BYPHENOTYPIC VARIABILITY

As many of the articles in this Issueillustrate, even with the extraordinarypower to detect causative mutations thatis afforded by different genetic technol-ogies, we still cannot predict who willpresent with a particular craniofacialcondition even when carrying an iden-tified causative mutation. And for manyconditions, there is even variability insurgical outcomes for children with thesame clinical diagnosis that is not relatedto the surgeons expertise. Understand-ing the major factors that contribute tothese issues endure as some of the biggestchallenges in craniofacial medicine.

Phenotypic variability can take theform of variable expressivity (the varia-tion in the degree to which tissues areaffected) and incomplete penetrance(the absolute variation of severity from“full blown” to the apparent absenceof clinical signs). So what factorscontribute to this phenotypic variability?In the past decade or so, emphasis was on‘other’ genetic variation within anindividual’s genome, largely becausethe technology to address this issuewas much further advanced than forother areas. However, in more recenttimes there has been a significant shiftto focus on the role of epigenetics—in

there has been a significantshift to focus on therole of epigenetics

the broadest sense, being defined asfactors not determined per se by anindividual’s DNA sequence but thatnevertheless influence gene expressionand activity. In humans, the discordanceof phenotypes in identical twins is the

INTRODUCTION AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 215

classic indicator of the relative contribu-tion of genetic variation and epigeneticfactors.

Animal models, particularly mousemodels, also provide many spectacularexamples of phenotypic variation due tothe influence of epigenetic factors.Because most investigators generateandmaintain their models of craniofacialdysmorphology on inbred (i.e., pure)genetic backgrounds, any inter‐individ-ual variation in phenotype can beconsidered a result of epigenetic influ-ences. Just as striking can be the differ-ences in phenotypic presentation(including variability) when specificmutations are introduced or maintainedon different inbred genetic backgroundsin this species. Classic examples of thisare the Tcof1 mutations (which inhumans cause Treacher Collins syn-drome) and the epistatic clf (clf1 andclf2) alleles that confer cleft susceptibility.In fact, it is expected that most mutationswould show phenotypic diversity ifplaced on different inbred or evenoutbred backgrounds, thus extendingthe power of these mouse models to alsomap genetic modifiers and dissect gene–environment interactions. However, aswith clinical studies, the power of suchapproaches is currently limited by theability to quantify changes in geneexpression and phenotypes with suffi-cient resolution and sensitivity (and ofcourse funding!). For gene expression,microarrays still offer a relatively cheapmeans of detecting more robust changes,whereas deep sequencing‐based ap-proaches such as RNAseq and evenChIPseq (and variations thereof) arepromising to provide unprecedentedinsight into changes in expression,tissue‐specific patterns of mRNA splic-ing and epigenetic mechanisms of generegulation. And for phenotypes, partic-ularly in animal models, significantadvances are being made using technol-ogies such as micro‐CT, micro‐MRI,and various new laser‐based microscopictechniques.

Phenotyping and Subphenotyping

In this Issue of the Seminars, we not onlywanted to provide updates on the state‐

of‐the‐field for the most common, andarguably the more significant, craniofa-cial conditions that present in clinic (seereviews by Elizabeth Leslie and MaryMarazita, Carrie Heike et al., TonyRoscioli et al., and Claudia Kappen),but also to highlight the advantages ofcombining animal model investigationswith clinical studies. To this end, anumber of reviews in this Issue are co‐authored by developmental biologistsand clinicians who are either alreadyworking together or have come togetherfor this Issue to provide a uniqueperspective on specific craniofacial con-ditions (see reviews by David Clouthieret al., Paul Trainor and Brian Andrews,Peter Farlie et al., and Ophir Kleinet al.).

It is noteworthy that a number ofthese articles cross into topics covered inother reviews in the Issue because ofphenotypic similarity between thesegroups of craniofacial disorders. In factin some cases, these other disorders wereoriginally considered as differential di-agnoses. For example, in the review oncraniofacial microsomia, both the facialdysostoses, auriculocondylar syndrome,and Pierre Robin sequence are dis-cussed, and vice versa for the reviews oneach of these three conditions. Whilethese examples highlight the benefits ofcareful phenotypic descriptions andrecognition of facial gestalts, they alsoextol the virtues of obtaining a definitivegenetic diagnosis. Using the auriculo-condylar syndrome as a case in point,retrospective assessment of individualsharboring either PLCB4 or GNAI3mutations suggests there may be featuresdistinguishing each of the genetic sub-types (see review by David Clouthieret al.). The same applies to the facialdysostoses such as Mandibulofacial Dys-ostosis with Microcephaly (caused bymutations in EFTUD2) and Nagersyndrome (caused by mutations inSF3B4) (see review by Paul Trainor &Brian Andrews). While these and theother facial dysostoses show manysimilarities because the causative genesare believed to function in the samebiological processes, the individual dis-orders can be distinguished inmost cases.However, this ability relies on an

appreciation of the full phenotypicspectrum associated with each gene,and in some cases this may require ashift to quantitative phenotyping, whichby its nature requires normative data forcomparison.

While the notion of detailed phe-notyping is not new, there is nowcertainly a new appreciation for thelevel of precision that is required todistinguish closely related phenotypes,and in many cases this may only bepossible through quantitative measures.Such detailed assessment is relativelysimple if conducted retrospectively withgenetic data in hand, but can, as a result,unwittingly lead to bias. Therefore,careful subphenotyping ideally shouldbe performed first to provide therecommendation for targeted geneanalysis rather than the other wayaround. This does not only make sensefrom a research perspective but also froma cost perspective. The review by TonyRoscioli and colleagues on their expe-riences in Australia with a nationaldiagnostic service for the craniosynos-toses provides a simple example of a cost‐effective service to gather genetic dataon a large volume of phenotypicallywell‐defined patients that would other-wise not be possible through individualclinical activities. There are many otheropportunities where detailed and evenquantitative phenotyping could easilyhave an impact. For example, it issomewhat surprising that today thevast majority of dysmorphic patientsare still screened for “del22q11.2” evenif the typical facial gestalt and cardiacdefects of this well‐characterized syn-drome are absent. Does this reflectinexperience of the referring cliniciansor a “catch all” in case it is an atypicalpresentation? There are two things toconsider here: (1) cost and (2) interpret-ability. If the result came back positivefor the typical 22q11.2 deletion, can itreally be interpreted as causing thevariant phenotype or is it a simple caseof non‐penetrance, with some otherlesion being responsible? If it really isn’tinterpretable, then the cost is notjustifiable. Now, with the costs of thenewer genome technologies, suchas exome sequencing, become more

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affordable, such approaches will nodoubt be added to the list of potentiallyun‐interpretable exploratory tests “per-formed” on poorly phenotyped patients.Yet it is hoped that in the future thesenew technologies can be paired withboth highly detailed phenotypingprotocols as well as “intelligent” dataand bioinformatic search algorithms toinstead generate testable research hy-potheses (i.e., using knowledge of geneexpression in model organisms, geneticpathways, or cell and developmentalprocesses either for targeted locus se-quencing or for prioritizing analyses ofgenomic scale data). This topic isaddressed in the review by Jim Brinkleyand colleagues.

For more genetically complex cra-niofacial conditions, such as orofacialclefting, the need for careful, detailedsubphenotyping is already well recog-nized and nicely outlined by Elizabeth

the need for careful, detailedsubphenotyping is already

well recognized

Leslie andMaryMarazita in their review.As part of this, and also emphasized byOphir Klein and colleagues, is theimportance of noting associated features,especially those derived from embryo-logically related tissues or processes. Forexample, although noted decades ago, arecent flurry of articles has seen the re‐investigation of the co‐presentation oftooth defects in individuals with orofa-cial clefts. In each of these studies, asignificantly increased frequency of anarray of dental anomalies, includingmissing and supernumery teeth, mal-formed teeth, as well as defects inmineralization, has been found, particu-larly in those with cleft lip/palate. Thesedata support the notion of a commonetiology. Consistent with this is the factthat most major cleft lip/palate genes areexpressed in the embryonic oral epithe-lia (not the neural crest derived mesen-chyme, as commonly assumed), whichalso plays a major role in both inductionand mineralization of teeth. Animal

models will now be critical for iden-tifying the common molecular anddevelopmental mechanisms, which may

Animal models will now becritical for identifying thecommon molecular and

developmental mechanisms

ultimately provide new clues to bettermanage the dental issues faced by thisgroup of patients.

ANIMAL MODELS—APPRECIATING THEBENEFITS ANDLIMITATIONS

As emphasized above and elegantlydiscussed in many of the reviews inthis Issue of Seminars, animal models—particularly the mouse—continue toplay a major role in helping us under-stand the molecular and developmentalbasis of craniofacial disorders. In fact,with the massive international expansionin mutant mouse resources, throughnumerous federally and internationallyfunded gene knockout, chemical muta-genesis and spontaneous mutant surveil-lance programs, this contribution isexpected to exponentially grow. How-ever, other model systems, such as thezebrafish (as outlined by Ophir Kleinet al.) and the chick, are also beingincreasingly used in craniofacial researchas genetic resources and techniques forgenetic manipulation in these species arerapidly being developed. Although thebroader utility and relevance of thesealternate model systems may seemquestionable on the surface, they eachhave distinct advantages and limitationsthat must be considered when contem-plating them as a research model forcraniofacial phenotypes. For example, asKlein et al. point out, zebrafish offermany advantages for dissecting geneticpathways that are typically conserved invertebrate craniofacial development.They can justifiably be used as modelsfor early mandibular development and

branchial arch patterning as well as evenstudies on calvarial ossification andsuture biology. However, their suitabilityfor studies of midfacial development isuncertain because of the questionablehomology of many of the zebrafishmidfacial cartilages to the mammalianfacial skeleton. Although the samemightonce have been considered for teeth, asKlein et al. explain, zebrafish still haveancestral palatal teeth that appear toform and mineralize using many of thesame molecular pathways as in mamma-lian dentition.

THE IMPORTANCE OFTERMINOLOGY

Aside from the difficulty of obtainingfunding, two simple things provide themost source of frustration from aresearch perspective: a lack of detail inphenotypic descriptions (as discussedabove), and the poor or inappropriateuse of terminology. In fact, these issuespertain equally to clinical studies andanimal model investigations. In partthey can reflect an availability of neces-sary resources (e.g., insufficient tools forassessment, or lack of time or expertiseof the investigator). However, they alsocan result from a lack of appreciationfor the need of detail or accuracy, oralternatively the acceptance and propa-gation in the literature of ill‐definedor misused terms. An example of thelatter that remarkably still occasionallyappears in the current literature is theinappropriate use of the term “cleft lipwith or without cleft palate” (CL/P) todescribe individuals with only a second-ary palatal cleft. Many clinical investi-gators have previously acknowledgedor recognized similar issues, which ledto the coordinated effort and publica-tion, in this Journal, of the Elements ofMorphology [Allanson et al., 2009],a large set of well‐defined (andagreed upon) clinical terms to describenon‐canonical or atypical craniofacialmorphology [Carey et al., 2012]. Thiswas an important step in the rightdirection.

In this Issue we therefore also felt itprudent to include an update on recentefforts, facilitated by the National

INTRODUCTION AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 217

Institute of Dental and CraniofacialResearch’s FaceBase Initiative, to for-malize and extend an ontological re-source that underpins research intocraniofacial development and dysmor-phology (see review by Jim Brinkley andcolleagues). This large project, TheOntology of Craniofacial Developmentand Dysmorphology (OCDM), is builtaround the Foundational Model ofAnatomy (FMA), which is the mostwidely utilized biomedical ontology.While the goals of the OCDM are notto force researchers to use specificterminology, its purpose is to defineterminology (in humans and mice) andprovide their anatomical relationshipsboth to other terminology, to related orderivative anatomical structures (evenacross species), as well as to originatingembryologic anatomy—both in thecontext of canonical anatomy and

dysmorphology. With the need formore detailed phenotyping and thetechnology capable of better discerningor distinguishing similar phenotypes, wefeel it is important that all craniofacialresearchers are aware of these efforts andthe resources they will ultimatelyprovide.

CONCLUDING REMARKS

The ability to understand the causes andimprove the treatment of craniofacialdisorders does not just come with agenetic diagnosis but requires consider-ably more detailed information onpathways and developmental processesthat can only come from research onappropriate animal models. But in theclinic, there also remain many chal-lenges. To improve diagnoses and, as aconsequence, prognoses we need to

utilize more precise and detailed meth-ods for describing phenotypes, especiallywhere marked variability is presumedor anticipated. Understanding both thegenetic and epigenetic contributions tophenotypic variability will not only helpinterpret how these factors influenceclinical outcomes and treatment practi-ces, but ultimately will also provideinsight into the exciting realm ofinterventional or preventative therapeu-tic strategies.

REFERENCESAllanson JE, Cunniff C, HoymeHE,McGaughran

J, Muenke M, Neri G. 2009. Elements ofmorphology: Standard terminology for thehead and face. Am J Med Genet A 149A:6–28.

Carey JC, Allanson JE, Hennekam RC, BieseckerLG. 2012. Standard terminology for pheno-typic variations: The elements of morpho-logy project, its current progress, and futuredirections. Hum Mutat 33:781–786.