Michael L. Cunningham

Parameters of Care for Craniosynostosis

Background A multidisciplinary meeting was held from March 4 to 6, 2010, in Atlanta, Georgia, entitled “Craniosynostosis: Developing Parameters for Diagnosis, Treatment, and Management.” The goal of this meeting was to create parameters of care for individuals with craniosynostosis. Methods Fifty-two conference attendees represented a broad range of expertise, including anesthesiology, craniofacial surgery, dentistry, genetics, hand surgery, neurosurgery, nursing, ophthalmology, oral and maxillofacial surgery, orthodontics, otolaryngology, pediatrics, psychology, public health, radiology, and speech-language pathology. These attendees also represented 16 professional societies and peer-reviewed journals. The current state of knowledge related to each discipline was reviewed. Based on areas of expertise, four breakout groups were created to reach a consensus and draft specialty-specific parameters of care based on the literature or, in the absence of literature, broad clinical experience. In an iterative manner, the specialty-specific draft recommendations were presented to all conference attendees. Participants discussed the recommendations in multidisciplinary groups to facilitate exchange and consensus across disciplines. After the conference, a pediatric intensivist and social worker reviewed the recommendations. Results Consensus was reached among the 52 conference attendees and two post hoc reviewers. Longitudinal parameters of care were developed for the diagnosis, treatment, and management of craniosynostosis in each of the 18 specialty areas of care from prenatal evaluation to adulthood. Conclusions To our knowledge, this is the first multidisciplinary effort to develop parameters of care for craniosynostosis. These parameters were designed to help facilitate the development of educational programs for the patient, families, and health-care professionals; stimulate the creation of a national database and registry to promote research, especially in the area of outcome studies; improve credentialing of interdisciplinary craniofacial clinical teams; and improve the availability of health insurance coverage for all individuals with craniosynostosis.

A case-control study of infant, maternal and perinatal characteristics associated with deformational plagiocephaly.

Deformational plagiocephaly, an abnormal asymmetric flattening of infants heads, is diagnosed in approximately 10% of infants. The prevalence of plagiocephaly has increased dramatically since 1992 when it was first recommended that infants be placed to sleep in a non-prone position to reduce the risk of sudden infant death syndrome. The authors conducted a case-control study to evaluate associations between plagiocephaly and perinatal characteristics. The authors assessed whether risk factors for plagiocephaly have changed since 1992. Cases were born 1987-2002 in Washington State and diagnosed with plagiocephaly at the Craniofacial Center at Seattle Childrens Hospital. Risk factor information was abstracted from birth certificate and hospital discharge data and unconditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI). Cases (n = 2764) were more likely than controls (n = 13 817) to have been injured at birth (OR 1.4; 95% CI 1.2, 1.7) or diagnosed with a congenital anomaly (OR 2.0; 95% CI 1.8, 2.3). Cases were more likely to have been male, a twin, or small-for-gestational-age. This first large-scale, case-control study of risk factors for plagiocephaly in a U.S. population provides new evidence that birth injuries and congenital anomalies are associated with plagiocephaly risk.

Heterogeneity of mutational mechanisms and modes of inheritance in auriculocondylar syndrome

Background Auriculocondylar syndrome (ACS) is a rare craniofacial disorder consisting of micrognathia, mandibular condyle hypoplasia and a specific malformation of the ear at the junction between the lobe and helix. Missense heterozygous mutations in the phospholipase C, β 4 (PLCB4) and guanine nucleotide binding protein (G protein), α inhibiting activity polypeptide 3 (GNAI3) genes have recently been identified in ACS patients by exome sequencing. These genes are predicted to function within the G protein-coupled endothelin receptor pathway during craniofacial development. Results We report eight additional cases ascribed to PLCB4 or GNAI3 gene lesions, comprising six heterozygous PLCB4 missense mutations, one heterozygous GNAI3 missense mutation and one homozygous PLCB4 intragenic deletion. Certain residues represent mutational hotspots; of the total of 11 ACS PLCB4 missense mutations now described, five disrupt Arg621 and two disrupt Asp360. The narrow distribution of mutations within protein space suggests that the mutations may result in dominantly interfering proteins, rather than haploinsufficiency. The consanguineous parents of the patient with a homozygous PLCB4 deletion each harboured the heterozygous deletion, but did not present the ACS phenotype, further suggesting that ACS is not caused by PLCB4 haploinsufficiency. In addition to ACS, the patient harbouring a homozygous deletion presented with central apnoea, a phenotype that has not been previously reported in ACS patients. Conclusions These findings indicate that ACS is not only genetically heterogeneous but also an autosomal dominant or recessive condition according to the nature of the PLCB4 gene lesion.

Three-Dimensional Head Shape Quantification for Infants With and Without Deformational Plagiocephaly

Objective The authors developed and tested three-dimensional (3D) indices for quantifying the severity of deformational plagiocephaly (DP). Design The authors evaluated the extent to which infants with and without DP (as determined by clinic referral and two experts’ ratings) could be correctly classified. Participants Infants aged 4 to 11 months, including 154 with diagnosed DP and 100 infants without a history of DP or other craniofacial condition. After excluding participants with discrepant expert ratings, data from 90 infants with DP and 50 infants without DP were retained. Measurements Two-dimensional (2D) histograms of surface normal vector angles were extracted from 3D mesh data and used to compute the severity scores. Outcome Measures Left posterior flattening score (LPFS), right posterior flattening score (RPFS), asymmetry score (AS), absolute asymmetry score (AAS), and an approximation of a previously described 2D measure, the oblique cranial length ratio (aOCLR). Two-dimensional histograms localized the posterior flatness for each participant. Analysis The authors fit receiver operating characteristic curves and calculated the area under the curves (AUC) to evaluate the relative accuracy of DP classification using the above measures. Results The AUC statistics were AAS = 91 %, LPFS = 97%, RPFS = 91 %, AS = 99%, and aOCLR = 79%. Conclusion Novel 3D-based plagiocephaly posterior severity scores provided better sensitivity and specificity in the discrimination of plagiocephalic and typical head shapes than the 2D measurements provided by a close approximation of OCLR. These indices will allow for more precise quantification of the DP phenotype in future studies on the prevalence of this condition, which may lead to improved clinical care.

Little evidence of association between severity of trigonocephaly and cognitive development in infants with single-suture metopic synostosis

OBJECTIVESTo measure severity of trigonocephaly among infants with single-suture metopic craniosynostosis by using a novel shape descriptor, the trigonocephaly severity index (TSI), and to evaluate whether degree of trigonocephaly correlates with their neurodevelopmental test scores. METHODSWe conducted a multicenter cross-sectional and longitudinal study, identifying and recruiting 65 infants with metopic synostosis before their corrective surgery. We obtained computed tomography images for 49 infants and measured the presurgical TSI, a 3-dimensional outline-based cranial shape descriptor. We evaluated neurodevelopment by administering the Bayley Scales of Infant Development, Second Edition, and the Preschool Language Scale, Third Edition, before surgery and at 18 and 36 months of age. We fit linear regression models to estimate associations between test scores and TSI values adjusted for age at testing and race/ethnicity. We fit logistic regression models to estimate whether the odds of developmental delay were increased among children with more severe trigonocephaly. RESULTSWe observed little adjusted association between neurodevelopmental test scores and TSI values, and no associations that persisted at 3 years. Trigonocephaly was less severe among children referred at older ages. CONCLUSIONWe observed little evidence of an association between the severity of trigonocephaly among metopic synostosis patients and their neurodevelopmental test scores. Detecting such a relationship with precision may require larger sample sizes or alternative phenotypic quantifiers. Until studies are conducted to explore these possibilities, it appears that although associated with the presence of metopic synostosis, the risk of developmental delays in young children is unrelated to further variation in trigonocephalic shape.

Further characterization of atypical features in auriculocondylar syndrome caused by recessive PLCB4 mutations

Auriculocondylar syndrome (ACS) is a branchial arch syndrome typically inherited in an autosomal dominant fashion. Patients with ACS display the following core symptoms with varying severity: a specific malformation of the external ear, known as a “question mark ear,” micrognathia and mandibular condyle hypoplasia. Recently, phospholipase C, β 4 (PLCB4) mutations were identified as the major cause of autosomal dominant ACS, with mutations of the PLCB4 catalytic domain predicted to have a dominant negative effect. In addition, one ACS patient born to related parents harbored a homozygous partial deletion of PLCB4, and presented with ACS plus central apnea and macropenis; these features had not been previously reported in association with ACS. His parents, each with a heterozygous partial PLCB4 deletion, were phenotypically normal, suggesting autosomal recessive inheritance of ACS, with complete loss of function of PLCB4 predicted in the patient. We herein describe two brothers with ACS caused by compound heterozygous splice site mutations in PLCB4. The patients were born to the same unrelated and healthy parents, with each parent harboring one of the mutations, indicating autosomal recessive ACS. Both patients reported here had mixed apneas, gastrointestinal transit defects and macropenis, in addition to typical craniofacial features of ACS. This is the first example of ACS caused by compound heterozygous splice site mutations in PLCB4, the second autosomal recessive case of ACS confirmed by molecular analysis, and strengthens the link between complete loss of function of PLCB4 and extra‐craniofacial features.

Osteoblast differentiation profiles define sex specific gene expression patterns in craniosynostosis

Single suture craniosynostosis (SSC) is the premature fusion of one calvarial suture and occurs in 1-1700-2500 live births. Congenital fusion of either the sagittal, metopic, or coronal sutures represents 95% of all cases of SSC. Sagittal and metopic synostosis have a male preponderance (3:1) while premature fusion of the coronal suture has a female preponderance (2:1). Although environmental and genetic factors contribute to SSC, the etiology of the majority of SSC cases remains unclear. In this study, 227 primary calvarial osteoblast cell lines from patients with coronal, metopic, or sagittal synostosis and unaffected controls were established and assayed for ALP activity and BrdU incorporation (n = 226) as respective measures of early stage osteoblast differentiation and proliferation. Primary osteoblast cell lines from individuals with sagittal synostosis demonstrated higher levels of ALP activity and reduced proliferation when compared to control lines. In order to address the sex differences in SSC types, the data was further stratified by sex. Osteoblasts from males and females with sagittal synostosis as well as males with metopic synostosis demonstrated higher levels of ALP activity when compared to sex matched controls, and males with sagittal or metopic synostosis demonstrated reduced levels of proliferation. In order to elucidate genes and pathways involved in these observed phenotypes, correlation analyses comparing ALP activity and proliferation to global gene expression was performed. Transcripts related to osteoblast differentiation were identified both differentially up and downregulated, correlated with ALP activity when compared to controls, and demonstrated a striking sex specific gene expression pattern. These data support that the dysregulation of osteoblast differentiation plays a role in the development of SSC and that genetic factors contribute to the observed sex related differences.

Transcriptional analysis of human cranial compartments with different embryonic origins

OBJECTIVEnPrevious investigations suggest that the embryonic origins of the calvarial tissues (neural crest or mesoderm) may account for the molecular mechanisms underlying sutural development. The aim of this study was to evaluate the differences in the gene expression of human cranial tissues and assess the presence of an expression signature reflecting their embryonic origins.nnnMETHODSnUsing microarray technology, we investigated global gene expression of cells from the frontal and parietal bones and the metopic and sagittal intrasutural mesenchyme (ISM) of four human foetal calvaria. qRT-PCR of a selected group of genes was done to validate the microarray analysis. Paired comparison and correlation analyses were performed on microarray results.nnnRESULTSnOf six paired comparisons, frontal and parietal compartments (distinct tissue types of calvaria, either bone or intrasutural mesenchyme) had the most different gene expression profiles despite being composed of the same tissue type (bone). Correlation analysis revealed two distinct gene expression profiles that separate frontal and metopic compartments from parietal and sagittal compartments. TFAP2A, TFAP2B, ICAM1, SULF1, TNC and FOXF2 were among differentially expressed genes.nnnCONCLUSIONnTranscriptional profiles of two groups of tissues, frontal and metopic compartments vs. parietal and sagittal compartments, suggest differences in proliferation, differentiation and extracellular matrix production. Our data suggest that in the second trimester of human foetal development, a gene expression signature of neural crest origin still exists in frontal and metopic compartments while gene expression of parietal and sagittal compartments is more similar to mesoderm.

Single nucleotide polymorphism discovery in TBX1 in individuals with and without 22q11.2 deletion syndrome

BACKGROUNDnChildren with 22q11.2 deletion syndrome (22q11.2DS) have a wide range of clinical features. TBX1 has been proposed as a candidate gene for some of the features in this condition. Polymorphisms in the nondeleted TBX1, which may affect the function of the sole TBX1 gene in individuals with the 22q11.2DS, may be a key to understanding the phenotypic variability among individuals with a shared deletion. Comprehensive single nucleotide polymorphism (SNP) discovery by resequencing candidate genes can identify genetic variants that influence a given phenotype. The purpose of this study was to further characterize the sequence variability in TBX1 by identifying all common SNPs in this gene.nnnMETHODSnWe resequenced TBX1 in 29 children with a documented 22q11.2 deletion and 95 nondeleted, healthy individuals. We estimated allele frequencies, performed tagSNP selection, and inferred haplotypes. We also compared SNP frequencies between 22q11.2DS and control samples.nnnRESULTSnWe identified 355 biallelic markers among the 190 chromosomes resequenced in the control panel. The vast majority of the markers identified were SNPs (n = 331), and the remainder indels (n = 24). We did not identify SNPs or indels in the cis- regulatory element (FOX-binding site) upstream of TBX1. In children with 22q11.2DS we detected 187 biallelic markers, six of which were indels. Four of the seven coding SNPs identified in the controls were identified in children with 22q11.2DS.nnnCONCLUSIONSnThis comprehensive SNP discovery data can be used to select SNPs to genotype for future association studies assessing the role of TBX1 and phenotypic variability in individuals with 22q11.2DS.

A genotype-specific surgical approach for patients with Pfeiffer syndrome due to W290C pathogenic variant in FGFR2 is associated with improved developmental outcomes and reduced mortality

PurposeAmong children with FGFR2-associated Pfeiffer syndrome, those with the W290C pathogenic variant (PV) are reported to have the worst clinical outcomes. Mortality is high, and severe neurocognitive impairment has been reported in all surviving patients. However, it is unclear whether these poor outcomes are an unavoidable consequence of the PV itself, or could be improved with a genotype-specific treatment approach. The purpose of this report is to describe the more intensive surgical approach used for each of the three patients with W290C PV in FGFR2 at our center, all of whom survived and have normal neurocognitive functioning.MethodsRetrospective chart review.ResultsIn contrast to other patients with Pfeiffer syndrome at our center, all three patients who were subsequently found to have a W290C PV required a similar and more aggressive approach based on early cephalocranial disproportion. In contrast to previously reported W290C cases, each of our three patients survived and demonstrate normal neurocognitive functioning.ConclusionWhile previously reported outcomes in W290C-associated Pfeiffer syndrome have been extremely poor, we present three patients who underwent an intensive surgical approach and have normal development. This suggests that a personalized and aggressive surgical approach for children with W290C PV may dramatically improve clinical outcome.