The Society of Craniofacial Genetics and Developmental Biology 34th Annual Meeting


  • Dr. Dwight R. Cordero M.D.

    Corresponding author
    1. Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts
    • Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA.

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This article is an introduction to active areas of research in craniofacial genetics and developmental biology as highlighted by Dr. Brian Hall's accompanying article in this issue of AJMG (Hall BK 2012. AJMG XX) and the publication in this issue of the abstracts which were presented as either talks or posters at the 34th annual meeting of the Society of Craniofacial Genetics and Developmental Biology (SCGDB). The meeting hosted by the faculty of Dentistry at McGill University convened in October 2011, in Montreal, Quebec, Canada. The Society met in conjunction with the International Congress of Human Genetics-American Society of Human Genetics meeting. Clinical Craniofacial Dysmorphology was the theme of the scientific sessions given by invited speakers.

The SCGDB is an international organization established in 1975 as the Society of Craniofacial Genetics. In 2011, the name of the society was expanded to include developmental biology to reflect the tight yet hierarchical integration between genetics and development/embryogenesis. The objectives of the Society are to promote understanding, research, and interdisciplinary communication concerning craniofacial genetics and developmental biology, and to apply the results of basic and clinical research to the care and management of individuals with craniofacial problems. ( for detailed objectives).

The society is enormously active in pursuit of its objectives. To bring to attention the work of those who have and are contributing to the advancement of the Society two awards were established in 2011 for graduate students demonstrating research excellence. The first was to recognize the significant contributions to craniofacial genetics, developmental biology and the Society by Dr. Geoffrey Sperber. As befits Geoff's status in the field and role in the Society, the “Geoffrey Sperber Award for Excellence in Craniofacial Research” was presented at the Montreal meeting. Zohreh Khavandgar of the Faculty of Dentistry at McGill University received this award for her research on mechanisms of bone mineralization. A second award, the genesis Award for Excellence in Craniofacial Research, generously funded by the journal genesis was won by Christopher Percival for his research on bone mineral density in a mouse model for Beare–Stevenson Cutis Gyrata Syndrome.

The 2012 annual meeting of the SCFDB is scheduled to convene on November 5th in San Francisco in conjunction with the annual meeting of the American Society of Human Genetics (ASHG) to be held November 6–10 ( Mark your calendar and monitor the SCGDB and ASHG web sites for details.

Abstracts of the 34th Annual Meeting of the Society of Craniofacial Genetics and Developmental Biology (SCGDB) in Montreal, Quebec on 11 October 2011

Inherited Retinal Disorders: Hope for Treatment

Roderick R. McInnes1

1Lady Davis Institute, Jewish General Hospital, Department of Human Genetics, McGill University, Montreal, Quebec, Canada

Inherited retinal diseases can be broadly classified into developmental defects and retinal degenerations. I will give a brief overview of these conditions, and focus in particular on our current understanding of the mechanisms that underlie photoreceptor death in the inherited degenerations. In our work on these diseases, we are addressing two fundamental questions: First, why do the neurons die? And second, how is it that they can function perfectly normally for decades, yet still be at risk of death? Are they sick? What are the biochemical changes that result from the mutation, and that eventually kill the cells? Some insight into these difficult questions has been obtained by many groups over the past decade (reviewed in Bramall et al. [2010]). I will also review exciting progress in the treatment of inherited retinal disease, both cell replacement therapy and gene therapy. In Phase 1 clinical trials, gene therapy appears to have been effective in partially correcting the blindness of at least one retinal degeneration, and cell replacement therapy in mouse models of retinal degeneration is very promising. Finally, I will report new findings on the role of one transcription factor gene, Prdm8, in retinal development. Loss of function of this gene leads to a virtual absence of bipolar cells, the major interneurons in the retina, and also to fascinating neuromuscular abnormalities [Ross et al., 2011].

Supported in part by grants from the Canadian Institutes of Health Research and the Macula Vision Research Foundation.


Bramall AN, Wright AF, Jacobson SG, McInnes RR. 2010. The genomic, biochemical, and cellular responses of the retina in inherited photoreceptor degenerations and prospects for the treatment of these disorders. Annu Rev Neurosci 33:441–472.

Ross SE, McCord AE, Jung C, Atan D, Mok SI, Hemberg M, Kim TK, Salogiannis J, Hu L, Cohen S, Lin Y, Harrar D, McInnes RR, Greenberg ME. 2012. Bhlhb5 and prdm8 form a repressor complex involved in neuronal circuit assembly. Neuron. Jan 26;73(2):292–303.

What Can One Learn From Fetal Facies: Is it a Clue to Diagnosis?

Deborah Krakow, MD1

1Department of Orthopaedic Surgery, Human Genetics and Obstetrics and Gynecology, David Geffen School of Medicine, UCLA, Los Angeles, California

Improvements in prenatal fetal ultrasound based on technological advancements have yielded superior images of many organ systems throughout gestational ages. This is especially true of the fetal facies. Ultrasound can evaluate both the bony structures as well as the soft tissue contours. Absolute measurements of the orbital diameters, philtrum, and mandible can give definite evidence for hypo- and hypertelorism, abnormal philtrums, and micrognathia. The recognition of well-described craniofacial disorders can be translated into the fetal period, as soon as the early second trimester. Abnormal craniofacial finding can be readily appreciated in the skeletal dysplasia group of disorders, craniosynostosis group of disorders, and cleft/lip palate syndromes. Determining the constellation of abnormal facial findings can help direct the prenatal geneticists toward differential diagnoses, including recognition of novel disorders. These diagnoses can then be refined based on abnormalities in other organ systems and well as using molecular diagnostics to help identify the causative mechanisms.

Clinical Approach to the Child With Cleft or Craniofacial Anomalies

Marilyn C. Jones1

1Division of Genetics, Department of Pediatrics, University of California, San Diego and Rady Children's Hospital, San Diego, California

Facial clefts and other craniofacial anomalies (CFA) constitute a group of birth defects that are both pathogenically and etiologically heterogeneous. With respect to pathogenesis, CFA's can be classified as malformations, deformations, disruptions, or dysplasias. Malformations do not change over time. Most are due to multifactorial inheritance and the treatment is typically surgical. Deformations have the potential to improve with time and postural intervention. Disruptions do not recur, but the treatment is surgical. The altered growth potential in dysplasias raises concerns about progression and the development of neoplasia over time.

With respect to etiology, CFAs are the result of genomic changes (dosage imbalance with gain or loss of groups of genes), genetic changes (at the level of the DNA itself), environmental factors, or the interaction of environmental factors with a specific genetic background that renders an individual at risk for a specific CFA. The clinical approach starts with defining the pathogenesis of the specific craniofacial anomaly (such as failure of formation of the frontonasal process leading to a midline cleft lip) and using the history and physical examination to elucidate etiology (such as parent with a single central incisor might suggest a mutation in SHH where as a history of poorly controlled diabetes and the presence of sacral agenesis might suggest diabetic embryopathy).

Genetic Regulation of Bone Extracellular Matrix Mineralization

Monzur Murshed1

1Department of Medicine and Faculty of Dentistry, McGill University, Montreal, Quebec, Canada

Mineralization of vertebrate bone extracellular matrix (ECM) is a physiologic process. In contrast, soft tissue mineralization is a pathologic condition. Initiation of ECM mineralization requires a scaffold of fibrillar proteins such as collagen or elastin within which critically sized nuclei of salts of calcium and inorganic phosphate precipitate and become stable. These precipitates later grow and mature into hydroxyapatite crystals. Interestingly, although suitable scaffolding proteins are present in many soft tissues, normally, ECMs in these tissues do not mineralize. There are two possibilities that may explain this phenomenon. Firstly, it is possible that an activator of mineralization is missing in these soft tissues, and secondly, that soft tissue mineralization is actively prevented by the presence of mineralization inhibitors. Several key studies now identify the latter as the most likely explanation. In fact, absence or removal of mineralization inhibitors is a prerequisite for the initiation of mineralization in the bone microenvironment. We provided genetic evidence suggesting that bone mineralization can be explained, at least in part, by the matrix composition and by the enzymatic removal of pyrophosphate, a ubiquitously present small-molecule mineralization inhibitor, from the bone ECM. More recently, we demonstrated a local role for Sphingomyelin phosphodiesterase 3 (SMPD3), a lipid metabolizing enzyme, in bone mineralization. A deletion mutation in Smpd3 leads to severe skeletal dysplasia in mice. Our in vivo genetic experiments suggest that SMPD3 enzymatic activity is necessary for normal bone mineralization and skeletal development.

Mammalian Mandibular Modules: 20 Years Since the “Atchley–Hall” Model

Brian K. Hall1

1Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada

It is 20 years since Atchley and Hall [1991] published a model for the development and evolution of complex morphological structures using the mammalian mandible (dentary) as the exemplar anatomical structure. Included in the model was the development of the concept of modularity of morphology, modules consisting of aggregations (condensations) of cells that are the primary resource for the development of individual bones or cartilages. In this first of a series of presentations/papers to review the model, I examine our current understanding of cellular modules of the murine dentary. The 1991 model postulated that the dentary arose from six cell condensations of neural-crest-derived cells. Four were skeletogenic forming the ramus and the three processes of the dentary. Two were odontogenic, forming the incisor and molar teeth and associated alveolar bone. Subsequent studies reveal that a single skeletogenic unit forms the bone of the ramus and the angular, condylar, and coronoid processes. In mice the distal cartilages on these processes are secondary, arising from the periosteum. In rats and humans, these cartilages are sesamoids arising in separate condensations outside the dentary. Thus, the single osteo-chondrogenic condensation in mice is represented in rats and humans by four cell populations; one osteogenic and three sesamoid (chondrogenic) condensations. The significance of these differences for our understanding of the cellular and molecular mechanism underlying mandibular development and for the application of studies from other mammalian species to human craniofacial development will be documented and discussed.

Supported by NSERC of Canada (A5056).


Atchley WR, Hall BK. 1991. A model for development and evolution of complex morphological structures. Biol Rev Camb Philos Soc 66:101–157.

GWAS Follow-Up Mutation Screen and Expression Analysis Implicate ARHGAP29 as a Novel Candidate Gene for Nonsyndromic Cleft Lip/Palate

Elizabeth J. Leslie1, M. Adela Mansilla1, Leah C. Biggs1, Kristi Schuette1, Steve Bullard2, Tian-Xiao Zhang3, Margaret Cooper4, Martine Dunnwald1, Andrew C. Lidral2, Mary L. Marazita4, Terri H. Beaty3, Jeffrey C. Murray1

1Department of Pediatrics, University of Iowa, Iowa City, Iowa

2Department of Orthodontics, University of Iowa, Iowa City, Iowa

3Department of Epidemiology, School of Public Health, Johns Hopkins University, Baltimore, Maryland

4Center for Craniofacial and Dental Genetics, Department of Oral Biology, School of Dental Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania

Nonsyndromic cleft lip and/or palate (NSCL/P) is a common birth defect with complex etiology. Genome-wide association studies have successfully identified novel loci associated with NSCL/P including one near the ABCA4 gene, mutations in which cause several retinal disorders. Neither expression analysis nor mutation screening support a role for ABCA4 in the etiology of NSCL/P, so we investigated the adjacent gene, ARHGAP29, encoding Rho GTPase activating protein 29. ARHGAP29 has preferential activity toward RhoA, which has many functions related to cellular shape, movement, and proliferation, all critical for craniofacial development. Expression analysis using a mouse demonstrated that Arhgap29 is present in the epithelium and mesenchyme of the medial and lateral nasal processes and the mandibular processes at E10.5, and the oral and medial edge epithelia and palatal mesenchyme at E14.5. Sequencing of ARHGAP29 in 962 individuals with NSCL/P and 972 unrelated controls from the Philippines and the US revealed one nonsense, one frameshift, and 14 missense variants, which are overrepresented in cases (P = 0.03). We tested the most associated SNP (rs560426) near ABCA4 and ARHGAP29 for genetic interaction with other candidate genes, identifying a possible interaction with IRF6 (rs2235371; P = 0.04). This interaction is supported by reduced expression of Arhgap29 in the oral epithelium of an Irf6-null mouse, suggesting a novel pathway for clefting involving the transcription factor IRF6 interacting with the Rho pathway via ARHGAP29. The combination of genome-wide association, rare coding sequence variants, craniofacial expression, and interactions with a known clefting gene support a role for ARHGAP29 in NSCL/P.

Supported by NIH DE08559 and DE020057.

Autosomal Dominant Multiple Natal Teeth With Selective Tooth Agenesis

John M. Graham Jr1, Nancy Kramer1, Vincent Funari1, Ophir Klein2, Kerstin Seidel2, Piranit Kantaputra3, Kent D. Taylor1

1Medical Genetics Institute, Cedars Sinai Medical Center, Los Angeles, California

2Department Orofacial Sciences, University of California, San Francisco, California

3Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand

We report a 5-generation family with autosomal dominant multiple natal teeth followed by selective tooth agenesis, which is not associated with any nondental features. Natal teeth are usually a sporadic isolated finding in an otherwise normal infant. Familial occurrence is rare but has been reported to be autosomal dominant. Autosomal dominant agenesis of teeth can be caused by mutations in the homeobox gene MSX1. Other families with autosomal dominant oligodontia have mutations in PAX9, and selective agenesis of only the permanent teeth has been linked to 10q11.2-q21. A family with isolated X-linked selective tooth agenesis resulted from mutations EDA. Mutations in WNT10A have been associated with isolated hypodontia. The genetic basis for isolated natal teeth is unknown. DNA from 28 family members was analyzed on the Illumina OMNI-express chip using 733,120 SNPs and mapped to an approximately 2 Mb segment on chromosome 1q36.11 with LOD score 2.97 at 23.8–25.8 Mb (GRCh37/hg19; MERLIN). By dividing the pedigree into three 3-generation families, a region of association was found located between LOC284632 and GRHL3 (parenTDT, P = 0.005 for rs11249039, rs11249045, or rs7526505). GRHL3 is a gene expressed exclusively in surface ectoderm in drosophila, where it plays an essential role in cuticle formation. Expression of the murine Grhl3 gene is found in ectodermally derived tissues including the oral epithelium. Sequencing of the region of association is underway, and experimental models are being developed to test the hypothesis that variation in the regulation of this gene might play a role in this phenotype.

Is the Craniofacial Phenotype Sufficient to Characterize FGFR-Related Craniosynostosis Syndromes?

Yann Heuzé1, Neus Martínez-Abadías1, Jennifer M. Stella1, Federico Di Rocco2, Corinne Collet3, Gemma García Fructuoso4, Mariana Alamar4, Lun-Jou Lo5, Simeon A. Boyadjiev6, Joan T. Richtsmeier1

1Department of Anthropology, Pennsylvania State University, University Park, Pennsylvania

2Craniofacial Surgery Unit, Department of Pediatric Neurosurgery, Hôpital Necker–Enfants Malades,University Paris V, Paris, France

3Laboratoire de Biochimie et de Biologie Moléculaire, INSERM U606, Paris, France

4Servei de Neurocirurgia, Hospital Sant Joan de Déu, Barcelona, Spain

5Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan

6Section of Genetics, Department of Pediatrics, University of California Davis, Sacramento, California

More than 180 craniosynostosis syndromes (CS) have been described, the more common CS being associated with mutations in fibroblast growth factor receptors (FGFRs). These “FGFR-related CS” show the characteristic premature fusion of one or several cranial sutures along with additional craniofacial, neural, limb, heart, lung, and/or skin anomalies. Despite the potentially high number of causative mutations for some of these FGFR-related CS, clinical diagnoses are quite reliable. However, our morphometric analysis based on landmarks collected from skull CT images of patients with Apert (n = 19), Crouzon (n = 9), Pfeiffer (n = 5), and Muenke (n = 4) syndromes along with those of unaffected children (n = 20) shows that these diagnostic categories are more difficult to establish when skull shape is the only trait considered. Indeed, we observe substantial overlap between craniofacial phenotypes, particularly of Apert, Pfeiffer and Muenke syndromes. The craniofacial phenotype as characterized by CT data is not sufficient to characterize FGFR-related CS. The cases that are most different from the unaffected individuals are the syndromic cases with bicoronal craniosynostosis. When syndromic cases displaying bicoronal craniosynostosis (n = 17) are compared with unaffected individuals (n = 20) and with children presenting with nonsyndromic bicoronal craniosynostosis (n = 14), our data provide a clear separation between craniofacial phenotypes of syndromic and nonsyndromic cases. The syndromic cases with bicoronal craniosynostosis display frontal bossing and severe midfacial hypoplasia. These last results indicate that in the case of bicoronal craniosynostosis the FGFR-related causative mutation and/or the molecular pathway affected by this mutation generates additional cranial dysmorphologies.

Supported in part by NIH/NIDCR R01DE018500, 3R01 DE018500-02S1, R01DE016886, and CDC 5R01 DD000350.

Understanding Phenotypic Variability in Neurodevelopmental Disorders

Santhosh Girirajan1, Jill A. Rosenfeld2, Blake C. Ballif2, Lisa G. Shaffer2, Evan E. Eichler1

1Department of Genome Sciences, University of Washington, Seattle, Washington

2Signature Genomics Laboratory, Spokane, Washington

We recently proposed a two-hit model to explain the phenotypic variability associated with a 520-kbp microdeletion on chromosome 16p12.1, wherein, the microdeletion both predisposes to neuropsychiatric phenotypes as a single event and exacerbates neurodevelopmental phenotypes in association with other large (>500 kbp) copy number variants (CNVs). We extended our model to include 72 genomic disorders and examined CNV data from 32,587 cases with intellectual disability and congenital malformation for the presence of two large CNVs compared to 8,635 controls. Of the 2,312 cases with a known genomic disorder, 233 (10.2%) cases carried another CNV >500 kbp and 373 carried another CNV >150 kbp elsewhere in the genome. For 45/233 (19%) of these two-hit carriers, the second CNV was also associated with a genomic disorder. While the frequency of second hits was higher in CNVs associated with variable expressivity such as del15q13.3, del16p11.2, dup16p13.11, del16p12.1, and del and dup1q21.1, we found a positive correlation (Spearman correlation, r = 0.64, P < 0.001) between the proportion of inherited cases and the prevalence of the second hit. Analysis of parental DNA shows a combination of inherited and de novo events contributing to the occurrence of two hits in the probands. Pathway analysis of genes within the second hit CNVs shows disruption of genes involved in cellular signaling, neurological, and developmental functions. Our data provide strong support for the two-hit model to explain variable expressivity in genomic disorders and, overall, presents an oligogenic basis for the study of complex diseases.

Supported by NIH HD065285 to EEE.

Sphingomyelin Phosphodiesterase 3, a Novel Regulator of Skeletal Development and Mineralization

Zohreh Khavandgar1, Robert Scott Kiss2, Jingjing Li3, Monzur Murshed1,3

1Faculty of Dentistry, McGill University, Montreal, Quebec, Canada

2Division of Cardiology, McGill University Health Center, Montreal, Quebec, Canada

3Department of Medicine, McGill University, Montreal, Quebec, Canada

Mineralization of vertebrate bone and tooth extracellular matrix is a genetically regulated process. One of the latest additions to the growing list of mineralization regulators is Sphingomyelin phosphodiesterase 3 (Smpd3). Smpd3 encodes a neutral sphingomyelinase that cleaves sphingomyelin to generate bioactive lipid metabolites. A deletion mutation called fragilitas ossium (fro) in the murine Smpd3 gene leads to severe skeletal dysplasia and perinatal death. In a recent study, it has been suggested that SMPD3 activity in the brain regulates skeletal development through endocrine factors. To further understand the role of SMPD3 in skeletal development, we examined endochondral ossification during early skeletogenesis in fro/fro mice. We observed an impaired apoptosis of the hypertrophic chondrocytes and severely under-mineralized cortical bones in E15.5 fro/fro embryos. To investigate whether SMPD3 plays a cell-autonomous role in these tissues, we generated fro/fro;Col1a1-Smpd3 mice, in which osteoblast-specific expression of Smpd3 corrected the fro/fro skeletal abnormalities and prevented perinatal deaths. Although the bone mineralization defects were fully corrected in fro/fro;Col1a1-Smpd3 embryos, their cartilage phenotype was largely unaffected. In the current study, we demonstrate a critical role for SMPD3 metabolites during in vitro mineral deposition by MC3T3-E1 pre-osteoblasts. Our data identify SMPD3 as a novel regulator of skeletal development and mineralization.

Supported by Canadian Institute of Health Research and Osteogenesis Imperfecta Foundation, USA.

A Rare DNA Variant in a cis-Overlapping Motif (COM) in an IRF6 Enhancer Element is Associated With Van der Woude Syndrome

Walid D. Fakhouri1, Fedik Rahimov2, Huiqing Zhou3, Tianli Du1, Evelyn N. Kouwenhoven3, Hans van Bokhoven3,4, Jeffrey C. Murray2, Brian C. Schutte1,5

1Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan

2Department of Pediatrics, The University of Iowa, Iowa City, Iowa

3Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands

4Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition, and Behavior, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

5Department of Pediatrics and Human Development, Michigan State University, East Lansing, Michigan

Cleft lip and palate (CLP) is one of the most common birth defects in humans. Mutations in interferon regulatory factor 6 (IRF6) cause Van der Woude syndrome (VWS), an autosomal dominant form of CLP, and contribute risk for isolated CLP, including a common DNA variant rs642961. Rs642961 is located in MCS9.7, a multi-species conserved sequence that is near IRF6. MCS9.7 element was shown to possess enhancer activity that mimicked the expression of endogenous Irf6. In order to identify possible etiologic DNA variants, we sequenced MCS9.7 in DNA samples obtained from individuals with VWS. We screened 48 DNA samples for which no disease-causing mutation was detected in IRF6 exons. We observed one new DNA variant that is an A insertion and is predicted to disrupt the DNA binding for both p63 and for bHLH transcription factors. We focused on four members of bHLH family whose expression pattern appeared to overlap with Irf6. Using a DNA binding assay, we observed that this DNA variant abrogated binding by p63 and reduced the binding affinity for the bHLH trans factors. In a transient transactivation assay, we observed strong enhancer activity by the MCS9.7 element. This activation was highly dependent on p63, and the activation was abrogated by the A insertion mutation. In conclusion, these data are consistent with the hypothesis that the rare DNA variant at the cis-overlapping motif in MCS9.7 is etiologic for VWS, and supports the rationale for additional mutation screening of the MCS9.7 enhancer element in patients with CLP.

Supported in part by NIH-DE13513.

A Systems Biology Approach to Cleft Lip and Palate

Evelyn J. Bowers1

1Department of Anthropology, Ball State University, Muncie, Indiana

The etiology of clefts may follow a multifactorial-threshold model. That model, however, fits poorly. Some clefts are teratogenic; others, genetic, occasionally following a Mendelian pattern. Although human sibships are usually too small for testing, the recurrence frequency may approximate what would be expected for a double recessive in a two factor cross, 1/16 or 6.25%. About 25% of CLP is attributable to known genetic pathways. It appears that CLPs result from alleles or teratogens which slow neural crest cell migration as the known pathways may. My group (Bowers) have found that CLPs are systematic disorders, not alterations of the head and face alone. Affected children sometimes have reduced heights and delayed maturation, and frequently have reduced elbow breadths, with normal triceps skinfolds and arm circumferences. We found significantly negative standard deviation scores (Zs) for elbow breadth in a sample of 209 children, ages 2–18:11, divided by sex, age group, and whether the cleft was unilateral or bilateral. Average Zs ranged from −0.40 (P < 0.05) to −1.27 (P < 0.001). Only boys above age 7:7 with bilateral CLP had non-significant average Zs, and these too were negative. This suggests that one of the molecules contributing to the formation of both membranous and endochondral bone, such as the transcription factor RUNX2, may be involved. Here, I start to trace the regulatory circuitry which may link Runx2 to the pathways with known involvement.


Bowers EJ, Mayro RF, Whitaker LA, Pasquariello PS, LaRossa D, Randall P. 1987. General body growth in children with clefts of the lip, palate and craniofacial structure. Scand J Plastic Reconstr Surg 21:7–14.

Bowers EJ, Mayro RF, Whitaker LA, Pasquariello PS, LaRossa D, Randall P. 1988. General body growth in children with cleft palate and related disorders: Age differences. Am J Phys Anth 75:503–515.

Bowers EJ. 2011. Growth in children with clefts: Serial hand-wrist X-ray evidence. Cleft Palate Craniofacial J 48(6):762–772.

Alterations in Postnatal Craniofacial Bone Mineral Density and Volume in the Fgfr2Y394C/+ Beare–Stevenson Cutis Gyrata Syndrome Mouse Model

Christopher Percival1, Yingli Wang2, Xueyan Zhou2, Ethylin Jabs2, Joan Richtsmeier1

1Department of Anthropology, Pennsylvania State University, University Park, Pennsylvania

2Department of Genetics and Genomic Sciences, Mt. Sinai School of Medicine, New York, New York

A novel technique is used to quantify individual cranial bone volume and relative bone mineral density across the murine skull from micro-computed tomography images and, in doing so, highlights the association between low bone density, low bone volume, and craniosynostosis in the Fgfr2Y394C/+ mouse model of Beare–Stevenson cutis gyrata syndrome at P0 and at P8. While landmark based morphometric analysis indicates that the severity of dysmorphology in craniofacial form varies across the skull, the influence of the Fgfr2 Y394C mutation on rates of bone volume increase appear standard for all bones measured. These results suggest that this mutation influences bone cell activity across the skull, even at sites quite distant from the prematurely fused sutures that define craniosynostosis syndromes. The net volume reduction of high-density material in some mutant bones suggests that osteoclast activity, in addition to that of osteoblast, is affected during this early postnatal period. This novel study provides important information on the effect of the Fgfr2 Y394C mutation on endochondral and intramembranous bone development across the skull, complementing the results of morphometric analyses, and providing the basis for hypotheses that can be tested with more in depth histological, molecular, and cellular studies.

Supported in part by NIH/NIDCR R01-DE018500 (JTR), 3R01 DE018500-02S1 (JTR and EWJ), and NSF BCS-0725227.

Assessing the Oral Microbiota of Healthy and Alcohol-Treated Rats Using Whole-Genome DNA Probes From Human Bacteria

Zaher Jabbour1, Cássio do Nascimento2, Michel El-Hakim3, Janet E. Henderson4, Rubens Albuquerque1

1Faculty of Dentistry, Division of Restorative Dentistry, McGill University, Montreal, Quebec, Canada

2Faculty of Dentistry of Ribeirao Preto, Department of Dental Materials and Prosthodontics, University of Sao Paulo, Sao Paulo, Brazil

3Division of Oral and Maxillofacial Surgery, Faculty of Dentistry, McGill University, Montreal, Quebec, Canada

4Division of Orthopedic Surgery, Faculty of Medicine, McGill University, Montreal, Quebec, Canada

Molecular methods for bacterial identification and quantification have been considered faster and more reliable than conventional methods. This study aimed to evaluate the capacity of whole-genome DNA probes prepared from human oral bacteria to detect and quantify oral bacterial species of rats, and to assess the influence of alcohol ingestion on the oral biofilm. Twenty-four mature Wistar rats were equally divided in two groups. One group (control) was fed balanced diet of rat pellets and water. The alcohol-treated group (AT) received the same diet and 20% ethanol solution. Upon euthanasia after 30 days, bacterial samples from the oral biofilm covering the animals' teeth were collected using microbrushes. Bacteria identification and quantification were based on the intensity of chemiluminescent signals released by DNA–DNA checkerboard hybridization with 33 probes prepared from human oral bacteria. Bacteria levels were compared using a Mann–Whitney U-test with a significance level < 0.05. All targeted strains, except Streptococcus mutans and Streptococcus mitis, were detected in the control group. Escherichia coli, Psuedomonas aeruginosa, Porphyromonas endodontalis, and Veillonella parvula were the only species detected in the AT group. Significantly higher bacteria levels were found in the control group compared to the AT group (P = 0.001). The percentage of E. coli was highest in both groups. Whole-genome DNA probes prepared from human oral bacteria can cross-react with rats' oral bacterial strains. Alcohol consumption is associated with lower bacterial diversity and numbers in the oral cavity of rats.

Supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP).

Nonsyndromic Sagittal Craniosynostosis Associated With Novel Variants in FGFR1, TWIST1, and RAB23 Genes

Xiaoqian Ye1,2, Audrey Guilmatre1, Ethylin Wang Jabs1, Yann Heuzé3, Joan Richtsmeier3, Deborah J. Fox4,5, Charlotte M. Druschel4,5, Rhinda J. Goedken6, Paul A. Romitti6

1Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York

2The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST), Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China

3Department of Anthropology, Pennsylvania State University, University Park, Pennsylvania

4Department of Epidemiology and Biostatistics, School of Public Health, University at Albany State University of New York, Albany, New York

5Congenital Malformations Registry, New York State Department of Health, Troy, New York, New York

6Department of Epidemiology, The University of Iowa College of Public Health, Iowa City, Iowa

Craniosynostosis, the premature fusion of one or more cranial sutures, occurs in approximately one in 2,500 live births. Among various forms, midline sagittal nonsyndromic craniosynostosis represents the most prevalent type. In syndromic craniosynostosis, causative mutations are known on fibroblast growth factor receptors (FGFRs), TWIST1, RAB23, and other genes. However, the etiology of nonsyndromic craniosynostosis is largely unknown. We used data from an ongoing, population-based case-control study in Iowa and New York State to identify novel candidate genes for nonsyndromic sagittal craniosynostosis. In this study, we evaluated 96 nonsyndromic sagittal craniosynostosis cases and performed extensive candidate genes analysis by direct sequencing. Two isoforms (intron 7 of isoform 1, exon 6 of isoform 5-6) of FGFR1, intron 9 of FGFR2, TWIST1, and RAB23 were selected based on previous publications, their expression patterns, animal models, and/or roles in known human craniosynostosis syndromes. Novel nucleotide variants were found in FGFR1 (isoform 5 and 6, n = 2), TWIST1 (n = 1), and RAB23 (n = 1). These variants detected in our study were unique and did not occur in 316 alleles from healthy controls or in the NCBI dbSNP, Human Gene Mutation Database and CHIP Bioinformatics databases. Our aggregate data suggest that mutations in these candidate genes are likely to contribute to nonsyndromic sagittal craniosynostosis, although they would account for only a small proportion of the total cases. These findings add to the perception of nonsyndromic sagittal craniosynostosis as a complex developmental anomaly under potential polygenic control.

Supported in part by grant from CDC 5R01 DD000350.

Differential Gene Expression in the Face of CL/Fr Mouse Embryos at E11.5 Based on Microarray Analysis

Brennan Takagi1, T.J. Hynd1, S. Jack Somponpun1, Kazuaki Nonaka2, Scott Lozanoff1

1Department of Anatomy, Biochemistry and Physiology, University of Hawaii School of Medicine, Honolulu, Hawaii

2Faculty of Dentistry, Section of Pediatric Dentistry, Kyushu University, Fukuoka, Japan

The CL/Fr mouse demonstrates heritable bilateral and unilateral cleft lip and palate (CLP) at a rate of approximately 35%, generally above the background “A” strain mouse. Using classical mouse breeding strategies, it has been suggested that at least two disease loci, clf1 and clf2, are involved in the defect and candidate genes have been identified. Additionally, gene-targeting analyses strongly suggest that Wnt9b contributes to CLP in the “A” strain mice. The aim of this study was to test the expression of clf1 and clf2 candidate genes in the facial prominences of CL/Fr embryos, utilizing microarray analysis. Medial nasal, lateral nasal, and maxillary prominences of phenotypically normal as well as cleft E11.5 CL/Fr mice were dissected and RNA was extracted using standard techniques for Agilent-microarray protocol. Results indicate that expression of the clf1 candidate genes, Wnt9b and Wnt3, and the clf2 candidate genes, Adcy2 and Ube2ql1, are significantly reduced (−3.11, −1.50, −2.22, and −1.83-fold, respectively) in the CL/Fr cleft tissues, suggesting that all four genes may be involved in the CLP mutation in CL/Fr mice. Future gene expression studies through quantitative RT-PCR and regional expression analyses through immunohistochemistry will be performed to further test the expression of these clf1 and clf2 candidate genes.

Supported in part by NIH/NCRR 5P20RR024206 (SJS) and R01-DK-064752 (SL).

Differential Gene Expression in Mice With Misexpression of six2 Associated With Frontonasal Dysplasia

Thomas Hynd1, Ben Fogelgren1, S. Jack Somponpun1, Sheri F.T. Fong1, Scott Lozanoff1

1Department of Anatomy, Biochemistry, and Physiology, University of Hawaii School of Medicine, Honolulu, Hawaii

We have previously described the Br mutant mouse displaying heritable frontonasal dysplasia. Linkage analysis mapped the mutation near the homeobox transcription factor six2, normally expressed in the facial mesenchyme during embryonic development. The purpose of this study is to determine expression patterns of six2, as well as possible upstream and downstream targets of six2, in the developing midface. The three sets of paired facial prominences (medial, lateral, and maxillary) of E11.5 embryos were dissected and RNA extracted for qPCR assays and Agilent microarray analysis. Medial nasal prominences (MNP) were also taken for cell culture. qPCR results indicated six2 expression is highest in the MNP and demonstrated haploinsufficient down-regulation in each of the three facial prominence sets in the Br mouse. Microarray results suggested the misregulation of several genes involved in a wide variety of genetic pathways, including the transcription factor six3. Further validation will be required to corroborate these microarray results, including qPCR, immunohistochemistry and RNA interference. Preliminary results using an in vitro knockdown of six2, performed on an MNP cell culture system utilizing siRNA, demonstrated a 65–70% knockdown of six2. These results may enable further in vitro work in order to elucidate a pathway in the developing midface involving six2.

This work was supported, in part, by NIH/NCRR R01DK064752 (SL) & 5P20RR024206 (SJS).

Genome-Wide Meta-Analysis of Nonsyndromic Cleft Lip With or Without Cleft Palate (NSCL/P) Identifies Multiple New Loci

Kerstin U. Ludwig1,2, Stefan Herms1,2, Michael Knapp3, Markus M. Nöthen1,2, Elisabeth Mangold1

1Institute of Human Genetics, University of Bonn, Bonn, Germany

2Department of Genomics, Life and Brain Center, Institute of Human Genetics, University of Bonn, Bonn, Germany

3Institute of Medical Biometry, Informatics, and Epidemiology, University of Bonn, Bonn, Germany

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is amongst the most common birth defects. The etiology of this malformation, which involves environmental and genetic factors, has recently been enlightened by the discovery of six genetic susceptibility loci in genome-wide association studies (GWAS). To identify additional loci we conducted a meta-analysis of the two largest GWAS on NSCL/P, that is, a case–control study of Central Europeans [Mangold et al., 2010] and a family-based study involving European and Asian trios [Beaty et al., 2010, data retrieved from dbGaP]. Our analysis confirms all previously identified loci and identifies six new susceptibility regions for the European population (1p36, 2p21, 3p11.1, 8q21.3 13q31.1, and 15q22). Population-specific analysis revealed that five of them also play a role in the Asian population, suggesting that we have identified common genetic risk factors for NSCL/P. Candidate genes within these regions include SPRY2, THADA, PAX7, and EPHA3, opening new starting points for subsequent in-depth genetic and functional studies.

Supported by DFG grants (MA 2546/3-1, KR 1912/7-1, NO 246/6-1, WI 1555/5-1), dbGaP-data were obtained at through dbGaP accession number phs000094.v1.p1.


Mangold E, Ludwig KU, Birnbaum S, Baluardo C, Ferrian M, Herms S, Reutter H, de Assis NA, Chawa TA, Mattheisen M, Steffens M, Barth S, Kluck N, Paul A, Becker J, Lauster C, Schmidt G, Braumann B, Scheer M, Reich RH, Hemprich A, Pötzsch S, Blaumeiser B, Moebus S, Krawczak M, Schreiber S, Meitinger T, Wichmann HE, Steegers-Theunissen RP, Kramer FJ, Cichon S, Propping P, Wienker TF, Knapp M, Rubini M, Mossey PA, Hoffmann P, Nöthen MM. 2010. Genome-wide association study identifies two susceptibility loci for nonsyndromic cleft lip with or without cleft palate. Nat Genet 42:24–26.

Beaty TH, Murray JC, Marazita ML, Munger RG, Ruczinski I, Hetmanski JB, Liang KY, Wu T, Murray T, Fallin MD, Redett RA, Raymond G, Schwender H, Jin SC, Cooper ME, Dunnwald M, Mansilla MA, Leslie E, Bullard S, Lidral AC, Moreno LM, Menezes R, Vieira AR, Petrin A, Wilcox AJ, Lie RT, Jabs EW, Wu-Chou YH, Chen PK, Wang H, Ye X, Huang S, Yeow V, Chong SS, Jee SH, Shi B, Christensen K, Melbye M, Doheny KF, Pugh EW, Ling H, Castilla EE, Czeizel AE, Ma L, Field LL, Brody L, Pangilinan F, Mills JL, Molloy AM, Kirke PN, Scott JM, Arcos-Burgos M, Scott AF. 2010. A genome-wide association study of cleft lip with and without cleft palate identifies risk variants near MAFB and ABCA4. Nat Genet 42:525–529.

Growth of the Skull and Brain in a Mouse Model for Apert Syndrome

Cheryl A. Hill1, Jordan R. Austin1, Joan T. Richtsmeier2, Susan Motch2, Neus Martínez-Abadías2, Yingli Wang3, Ethylin Wang Jabs3, Kristina Aldridge1

1Department of Pathology and Anatomical Sciences, University of Missouri-Columbia, Columbia, Missouri

2Department of Anthropology, Pennsylvania State University, University Park, Pennsylvania

3Department of Genetics and Genomic Sciences, Mt. Sinai School of Medicine, New York, New York

Craniofacial and neural tissues develop in concert throughout pre- and postnatal growth. Craniosynostosis syndromes, such as Apert syndrome (AS), are associated with specific phenotypes involving both the skull and the brain. Analysis of a mouse model for AS, the Fgfr2+/P253Rmouse, allows for study of the effects of this specific mutation on these two tissues simultaneously over the course of development. Previous work on this mouse model has demonstrated specific and localized differences in the brain and skull. The purpose of this study is to compare theFgfr2+/P253Rmouse and their wild-type littermates at two developmental time points, to determine whether growth patterns differ in brain and skull. Both three-dimensional micro-magnetic resonance images and computed tomography scans were acquired from mice with the Fgfr2+/P253R mutation and their wild-type littermates. The sample consisted of newborn (P0) mice (N = 28) and 2-day-old (P2) mice (N = 23). Coordinate data for 15 brain and 24 skull landmarks were collected using Amira© and Analyze 10.0© software and statistically compared using Euclidean Distance Matrix Analysis. Results demonstrate that the Fgfr2+/P253R mice show reduced growth in the cerebrum and the face, while the height and width of the neurocranium and posterior regions of the brain show increased growth as compared to wild-type mice. This localized correspondence of differential growth patterns in skull and brain point to their continued interaction through development, while also demonstrating that both tissues display divergent postnatal growth patterns as compared to their wild-type littermates.

Supported in part by NIH/NIDCR R01 DE018500 (JTR), R01DE18500-02S1 (JTR and EWJ).

Phenotypic Continuum in FGFR Syndromic Craniosynostosis? Evidence From Human Patients and Mouse Models

Neus Martínez-Abadías1, Yann Heuzé1, Yingli Wang2, Susan Motch1, Talia Pankratz1, Jennifer M. Stella1, Gemma García Fructuoso3, Mariana Alamar3, Federico Di Rocco4, Corinne Collet5, Lun-Jou Lo6, Simeon A. Boyadjiev7, Ethylin Wang Jabs2, Joan T. Richtsmeier1

1Department of Anthropology, Pennsylvania State University, University Park, Pennsylvania

2Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York

3Servei de Neurocirurgia, Hospital Sant Joan de Déu, Barcelona, Spain

4Craniofacial Surgery Unit, Department of Pediatric Neurosurgery, Hôpital Necker–Enfants Malades, University Paris V, Paris, France

5Laboratoire de Biochimie et de Biologie Moléculaire, INSERM U606, Paris, France

6Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan

7Section of Genetics, Department of Pediatrics, University of California Davis, Sacramento, California

Of the more than 180 craniosynostosis syndromes (CS) with a prevalence of 1/10,000 live births, many are caused by mutations in fibroblast growth factor receptors (FGFRs). The FGFR-related CS involve premature fusion of cranial sutures associated with craniofacial, neural, limb, and visceral malformations. The precise definition and classification of FGFR-related CS can be difficult due to phenotypic variation within diagnostic categories, such that genetic testing is often required. However, there is no one-to-one correspondence between genetic mutations and phenotypes: a single mutation can result in different phenotypes, and mutations in different genes may produce similar phenotypes. We focus on a subset of CS caused by mutations in FGFR1, 2, and 3 (Apert, Crouzon, Pfeiffer and Muenke syndromes) to precisely quantify the phenotypic spectrum of these disorders based on geometric morphometric analysis of 3D landmark coordinates collected from CT reconstructions of the skull of human patients (N = 37), unaffected individuals (N = 20) and mouse models for CS (N = 96 mutant; 109 non-mutant littermates). Our analyses suggest that there is correspondence between human and mouse data and that within both organisms the cranial morphologies associated with CS are distributed over a phenotypic continuum that ranges from no dysmorphology to various degrees of mild and severe dysmorphology. Individuals with Apert syndrome are the most severely affected, whereas individuals with Crouzon, Pfeiffer, and Muenke syndromes display mild to severe dysmorphologies. Along this phenotypic spectrum fairly well-defined diagnostic groups overlap due to high degrees of within-group variation, suggesting that common genetic and phenotypic variation underlie FGFR-related CS.

Supported in part by NIH/NIDCR (R01 DE018500, 3R01 DE018500-02S1, R01 DE016886), CDC (5R01DD000350), and Beca Postdoctoral Beatriu de Pinós, AGAUR, Generalitat de Catalunya.

Proteins Regulation of Enamel Crystallographic Ultrastructure

Hazem Eimar1, Benedetto Marelli1, Showan Nazhat1, Samer Abinader1, Wala Amin2, Jesus Torres3, Rubens Albuquerque1, Faleh Tamimi1

1Faculty of Dentistry McGill University, Montreal, Quebec, Canada

2Faculty of Dentistry, Jordan University, Amman, Jordan

3Department of Health Science III, Universidad Rey Juan Carlos, Alcorcon, Madrid, Spain

Enamel is a composite material that comprises an inorganic matrix composed of hierarchically organized carbonated-hydroxyapatite (HA) crystals, and an organic matrix mainly composed of the protein amelogenin. Understanding the relation between tooth enamel chemical components is of special interest for the interpretation of the variations in tooth development among humans. Accordingly, this study was designed to investigate how variations in the enamel proteins may affect its ultrastructure. One-hundred extracted sound teeth were collected from adult patients attending McGill-Undergraduate Dental Clinic. FTIR and XRD were used to assess enamel chemical composition (protein content and degree of HA carbonization) and crystallography (crystal size, lattice parameters: a-axis and c-axis). The data obtained were analyzed for correlation, and statistical significance was set at P < 0.05. Tooth enamel protein content and crystallographic structure varied dramatically within the studied population. Enamel protein content was inversely correlated with its HA crystal size (R = −0.352, B = −19.4). Further analysis revealed that this correlation was not purely linear. Instead, it followed a curve; in which at specific enamel protein content, tooth enamel samples had the maximum HA crystal size. However, below or above that specific enamel protein content, teeth expressed smaller HA crystals [(R = 0.32, B = 271.4) and (R = −0.36, B = −15.1), respectively]. Moreover, the amount of tooth enamel protein was positively correlated with the carbonization degree of HA crystals (R = 0.474, B = 0.410). From the present study, we conclude that tooth enamel proteins exhibited a dual behavioral effect on the size of HA crystals. Moreover, the degree of HA carbonization was also regulated by enamel proteins.

Supported in part by Fondation de l'Ordre des dentistes du Québec (FODQ).

Reading Between the Lines: The Development of Negative Spaces in a Crouzon/Pfeiffer Syndrome Mouse Model at Birth

Susan M. Motch1, Neus Martínez-Abadías1, Talia L. Pankratz1, Yingli Wang2, Kristina Aldridge3, Ethylin W. Jabs2, Thomas Neuberger4, Timothy M. Ryan1,5, Joan T. Richtsmeier1

1Department of Anthropology, Pennsylvania State University, University Park, Pennsylvania

2Department of Genetics and Genomic Sciences, Mt. Sinai School of Medicine, New York, New York

3Department of Pathology and Anatomical Sciences, University of Missouri-School of Medicine, Columbia, Missouri

4High Field MRI Facility, Pennsylvania State University, University Park, Pennsylvania

5Center for Quantitative Imaging, Pennsylvania State University, University Park, Pennsylvania

Crouzon syndrome is associated with nearly 50 known FGFR2 mutations, one of which, the FGFR2 C342Y mutation, is causative for Crouzon and Pfeiffer craniosynostosis syndromes. Individuals with Crouzon and Pfeiffer syndromes show marked phenotypic variation but usually display premature closure of cranial suture(s), additional craniofacial malformations, as well as defects involving other systems including respiratory disorders and auditory impairments. We used µCT and µMR images of newborn littermates of the Fgfr2cC342Y/+ mouse model for Crouzon/Pfeiffer syndromes, to investigate the global and regional impact of this mutation on the developing skull and negative spaces of the head at P0. Negative spaces were defined as the air-filled space of the nasopharynx that develop within the intramembranous facial skeleton and fluid filled structures of the cochlea and vestibular canals that develop within the otic skeleton, which is still cartilaginous at birth. Global and regional differences in skull morphology were observed using configurations of 3D landmark coordinates measured on µCT isosurfaces in Fgfr2cC342Y/+ mice (n = 28) relative to non-mutant littermates (n = 31). Results revealed dysmorphology of the facial skeleton, cranial base and cranial vault in Fgfr2cC342Y/+ mice. Volumetric measurement using µMR images indicated restriction of the nasopharynx of Fgfr2cC342Y/+ mice (n = 8) compared to non-mutant littermates (n = 11), but no difference in cochlear and semicircular canal volume. Future work aims to determine whether differences in the effect of the FGFR2 C342Y mutation on these negative spaces are due to differential effects of the mutation on endochondral and intramembranous forming bone.

Supported in part by NIH/NIDCR R01 DE018500 (JTR); 3R01 DE018500-02S1 (JTR and EWJ).

Reduced Bone Mass in Mice Lacking the Men1 Gene in Osteoblasts

Ippei Kanazawa1, Geetanjali Nayak1, Lucie Canaff1, Monzur Murshed1, Geoffrey N. Hendy1

1Department of Medicine, McGill University Health Centre, Montreal, Quebec, Canada

Homozygous inactivation of the multiple endocrine neoplasia type 1 (Men1) gene encoding menin in mice is embryonic lethal and fetuses exhibit clear defects in cranial and facial development. We have shown previously that menin has an important role in osteoblastogenesis and osteoblast differentiation by in vitro studies. To further understand the physiological role of menin in bone development in vivo we are generating mouse models in which the expression of the Men1 gene is altered only in osteoblasts. Mice lacking Men1 exons 3–8 in osteoblasts driven by Osteocalcin-Cre (Men1 KO mice) displayed no differences in growth rate compared to wild-type (WT) littermates. In 9-month-old female mice, micro-CT revealed that trabecular bone volume and cortical bone thickness were significantly reduced in the Men1 KO mice. Histomorphometric analysis showed that bone volume/total volume, numbers of osteoblasts and osteoclasts, as well as mineral apposition rate were all significantly reduced in the Men1 KO mice. In mice overexpressing human menin in osteoblasts from a human menin cDNA driven by a Col1a1 promoter (Men1 TG mice), at 6 months of age, the Men1 TG mice were not different from WT littermates in growth rate and bone mineral density by DXA. Taken together, depletion of menin in the osteoblast leads to decreased osteoblast and osteoclast numbers as well as impaired bone remodeling, resulting in a reduction in trabecular and cortical bone whereas overexpression has no effect at least in younger mice. Therefore, maintenance of menin expression and function in bone is important to avoid decreased bone mass.

Supported in part by grants from the Canadian Institutes of Health Research (CIHR).

Replication of GWAS Candidate Genes in Four Independent Populations Confirm the Role of Common Variants and Identifies Rare Variants in PAX7 and VAX1 Contributing to the Etiology of Non-Syndromic CL(P)

A. Butali1, S. Suzuki1,2, M.A. Mansilla1, A.L. Petrin1, E. Leslie, J. L'Heureux1, M.E. Cooper4, N. Natsume2, T.H. Beaty3, M.L. Marazita4, J.C. Murray1

1Department of Pediatrics, University of Iowa, Iowa City, Iowa

2School of Dentistry, Aichi-Gakuin University, Japan

3Johns Hopkins University, School of Public Health, Baltimore, Maryland

4Center for Craniofacial and Dental Genetics, Department of Oral Biology, School of Dental Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania

GWAS of CL(P) have identified several significant and near-significant genetic associations for non-syndromic CL(P) [Beaty et al., 2010]. To replicate two of the near-significant GWAS signals, the present study investigated the role of both common and rare variants in the PAX7 and VAX1 genes. Direct sequencing in and around the PAX7 and VAX1 genes in individuals of European and Asian ancestry was done. TaqMan genotyping was carried out for SNPs in VAX1 and PAX7 and Transmission Disequilibrium Test (TDT) was performed to investigate family based association in each population. Nineteen variants were found in PAX7. Eleven unreported variants were found in VAX1. TDT analysis showed strong associations with markers in VAX1 (rs7078160, P= 2.7E-06 and rs475202, P= 0.0002) in a combined Mongolian and Japanese CL (P) case–parent triads. Further analysis of parent-of-origin effects showed a significant maternal to child transmission (P = 6.7E-05, OR = 2.02) and paternal to child transmission (P = 0.009, OR = 1.62) for VAX1 marker rs7078160 in Mongolian and Japanese combined CL(P). CL(P) males were mostly responsible for the parental effects in the combined Japanese and Mongolian populations (rs7078160, P = 1.5E-05, OR = 2.95 for maternal transmission, and P = 0.05, OR = 1.65 for paternal transmission). The rs6659735 trinucleotide marker in PAX7 was significantly associated with all the Iowan cleft groups combined [P = 0.007 in all clefts and P = 0.01 in CL(P)]. Our study replicated previous GWAS findings for markers in VAX1 across three independent Asian populations, and identified rare variants in PAX7 that may contribute to the etiology of CL(P). Elucidating the role of these rare variants warrants further investigation.

Supported by NIH DE-08559, DE016148, KAKENHI, Grant-in-Aid for Young Scientist (B) No. 20791560 (Aichi-Gakuin), U01 DE-20057 and U01-DE-018993.


Beaty TH, Murray JC, Marazita ML, Munger RG, Ruczinski I, Hetmanski JB, Liang KY, Wu T, Murray T, Fallin MD, Redett RA, Raymond G, Schwender H, Jin SC, Cooper ME, Dunnwald M, Mansilla MA, Leslie E, Bullard S, Lidral AC, Moreno LM, Menezes R, Vieira AR, Petrin A, Wilcox AJ, Lie RT, Jabs EW, Wu-Chou YH, Chen PK, Wang H, Ye X, Huang S, Yeow V, Chong SS, Jee SH, Shi B, Christensen K, Melbye M, Doheny KF, Pugh EW, Ling H, Castilla EE, Czeizel AE, Ma L, Field LL, Brody L, Pangilinan F, Mills JL, Molloy AM, Kirke PN, Scott JM, Arcos-Burgos M, Scott AF. 2010. A genome-wide association study of cleft lip with and without cleft palate identifies risk variants near MAFB and ABCA4 Nat Genet 42:525–529.