Objective. The aim of this study was to assess the utility of ultrasound (US) imaging for diagnosis of abnormal tracheal morphology, such as tracheal cartilaginous sleeves (TCS), in patients with syndromic craniosynostosis (SC). Study Design. Age-matched cohort study. Setting. Tertiary pediatric hospital. Methods. Two age-matched cohorts were identified: patients with SC and known TCS based upon airway endoscopy and normal controls without tracheal pathology. Enrolled patients underwent awake US of the neck which were randomized and reviewed by blinded pediatric radiologists and rated on presence or absence of normal tracheal cartilage morphology and visualization or nonvisualization of a tracheostomy tube. Fisher's exact test was used to assess pooled data. Fleiss' Kappa (kappa) was calculated to assess inter-rater reliability. Results. Ten patients were included in each cohort. Control patients were gender and age-matched to TCS patients with a mean difference of 3.7 months (+/- 3.9 months). Across all raters, cartilage type was correctly identified in 93% (95% confidence interval [CI]: 84%-98%) and tracheostomy visualization in 97% (95% CI: 89%-99%). The sensitivity and specificity for detection of abnormal cartilage pathology was 87% and 100%, respectively. Inter-rater reliability for cartilage assessment was kappa = 0.88 (95% CI: 0.67-1.00, P < .05) and 0.83 (95% CI: 0.58-1.00, P < .05) for tracheostomy presence. Conclusion. This study demonstrated that tracheal US is a feasible, accurate screening tool for TCS, and can be successfully performed non-sedated in patients up to 18 years of age, both with and without tracheostomy tubes in place.

OBJECTIVE Occurring once in every 2000 live births, craniosynostosis (CS) is the most frequent cranial birth defect. Although the genetic etiologies of syndromic CS cases are well defined, the genetic cause of most nonsyndromic cases remains unknown. METHODS The authors analyzed exome or RNA sequencing data from 876 children with nonsyndromic CS, including 291 case-parent trios and 585 additional probands. The authors also utilized the GeneMatcher platform and the Gabriella Miller Kids First genome sequencing project to identify additional CS patients with AXIN1 mutations. RESULTS The authors describe 11 patients with nonsyndromic CS harboring rare, damaging mutations in AXIN1, an inhibitor of Wnt signaling. AXIN1 regulates signaling upstream of key mediators of osteoblast differentiation. Three of the 6 mutations identified in trios occurred de novo in the proband, while 3 were transmitted from unaffected parents. Patients with nonsyndromic CS were highly enriched for mutations in AXIN1 compared to both expectation (p = 0.0008) and exome sequencing data from > 76,000 healthy controls (p = 2.3 × 10−6), surpassing the thresholds for genome-wide significance. CONCLUSIONS These findings describe the first phenotype associated with mutations in AXIN1, with mutations identified in approximately 1% of nonsyndromic CS cases. The results strengthen the existing link between Wnt signaling and maintenance of cranial suture patency and have implications for genetic testing in families with CS.

Purpose: Craniofacial microsomia (CFM) represents a spectrum of craniofacial malformations, ranging from isolated microtia with or without aural atresia to underdevelopment of the mandible, maxilla, orbit, facial soft tissue, and/or facial nerve. The genetic causes of CFM remain largely unknown.Methods: We performed genome sequencing and linkage analysis in patients and families with microtia and CFM of unknown genetic etiology. The functional consequences of damaging missense variants were evaluated through expression of wild-type and mutant proteins in vitro.Results: We studied a 5-generation kindred with microtia, identifying a missense variant in FOXI3 (p.Arg236Trp) as the cause of disease (logarithm of the odds = 3.33). We subsequently identified 6 individuals from 3 additional kindreds with microtia-CFM spectrum phenotypes harboring damaging variants in FOXI3, a regulator of ectodermal and neural crest development. Missense variants in the nuclear localization sequence were identified in cases with isolated microtia with aural atresia and found to affect subcellular localization of FOXI3. Loss of function variants were found in patients with microtia and mandibular hypoplasia (CFM), suggesting dosage sensitivity of FOXI3.Conclusion: Damaging variants in FOXI3 are the second most frequent genetic cause of CFM, causing 1% of all cases, including 13% of familial cases in our cohort.(c) 2022 The Authors. Published by Elsevier Inc. on behalf of American College of Medical Genetics and Genomics. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PURPOSE: Craniofacial microsomia (CFM) represents a spectrum of craniofacial malformation, ranging from isolated microtia with or without aural atresia to underdevelop-ment of the mandible, maxilla, orbit, facial soft tissue, and/ or facial nerve. The genetic causes of CFM remain largely unknown. METHODS: We performed whole exome or whole genome sequencing of 415 kindreds with microtia/CFM spectrum phenotypes. RESULTS: We studied a five generation kindred with microtia, identifying a missense mutation in FOXI3 (p.R236W) as the cause of disease (LOD=3.33). We subse-quently identified 6 individuals from 3 additional kindreds with microtia-CFM spectrum phenotypes harboring mutations in FOXI3, a regulator of ectodermal and neural crest development. Missense mutations in the nuclear localization sequence were identified in cases with isolated microtia with aural atresia, and loss of function mutations were found in CFM patients, suggesting dosage sensitivity. Mutations were found to affect subcellular localization of FOXI3. CONCLUSIONS: The results

Craniosynostosis, the premature fusion of the calvarial bones, has numerous etiologies. Among them, several involve mutations in genes related to the TGFb signaling pathway, a critical molecular mediator of human development. These TGFb pathway-associated craniosynostosis syndromes include Loeys–Dietz syndrome (LDS) and Shprintzen–Goldberg syndrome (SGS). LDS and SGS have many similarities common to fibrillinopathies, specifically Marfan syndrome (MFS), which is caused by mutations in FBN1. Historically discriminating features of MFS from LDS and SGS are (1) the presence of ectopia lentis (the subluxation/dislocation of the ocular lens) and (2) the absence of craniosynostosis. Curiously, several instances of a seemingly novel syndrome involving only craniosynostosis and ectopia lentis have recently been reported to be caused by recessive mutations in ADAMTSL4, a poorly characterized gene as of yet. Here, we report on two new cases of craniosynostosis with ectopia lentis, each harboring recessive mutations in ADAMTSL4. We also discuss a proposed mechanism for the relationship between ADAMTSL4, FBN1, and TGFb pathway-related syndromes.

Auriculocondylar syndrome 2 (ARCND2) is a rare autosomal dominant craniofacial malformation syndrome linked to multiple genetic variants in the coding sequence of phospholipase C β4 (PLCB4). PLCB4 is a direct signaling effector of the endothelin receptor type A (EDNRA)-Gq/11 pathway, which establishes the identity of neural crest cells (NCCs) that form lower jaw and middle ear structures. However, the functional consequences of PLCB4 variants on EDNRA signaling is not known. Here, we show, using multiple signaling reporter assays, that known PLCB4 variants resulting from missense mutations exert a dominant-negative interference over EDNRA signaling. In addition, using CRISPR/Cas9, we find that F0 mouse embryos modeling one PLCB4 variant have facial defects recapitulating those observed in hypomorphic Ednra mouse models, including a bone that we identify as an atavistic change in the posterior palate/oral cavity. Remarkably, we have identified a similar osseous phenotype in a child with ARCND2. Our results identify the disease mechanism of ARCND2, demonstrate that the PLCB4 variants cause craniofacial differences and illustrate how minor changes in signaling within NCCs may have driven evolutionary changes in jaw structure and function. This article has an associated First Person interview with the first author of the paper.

Crouzon syndrome is an autosomal dominant condition characterized by craniosynostosis and distinctive facial features. Pathogenic variants in the gene encoding fibroblast growth factor receptor type 2 (FGFR2), localized in two exons (termed IIIa and IIIc) account for the majority of classic cases. Here we describe a case with a pathogenic variant affecting the tyrosine kinase domain of the FGFR2 presenting with atypical clinical features. A 26-year-old male presented for genetic evaluation given his medical history of multiple anomalies at birth. He was born at term and was on prolonged mechanical ventilation for almost > 1 year after birth. His past medical history was significant for congenital diaphragmatic hernia and tracheoesophageal fistula, both of which were repaired. Other significant medical history included left lung hypoplasia, pectus excavatum, and bilaterally undescended testes at birth. He was on G-tube feeds for the first 8 years of his life. He received speech therapy and occupational therapy growing up. He had normal somatic growth and weight gain and was an average student and finished college. Now he is completely functional, independent, and has a job. No family history of any congenital conditions. His physical examination was significant for macrocephaly (Head circumference – 62.75 cm, > 98th percentile), creases and furrows on the forehead, proptosis, hypertelorism, esotropia, high arched palate, mid-face hypoplasia, and pectus excavatum. He did not have any hand or feet abnormalities. The only head imaging that could be tracked down from his childhood was a cranial ultrasound done at three months of age suggestive of an increase in mild symmetric dilatation of the lateral ventricles. Genetic testing was done, which showed pathogenic variant in FGFR2, p. Arg678Gly suggestive of autosomal dominant FGFR related craniosynostosis syndrome which affects the tyrosine kinase 2 domain of the FGFR2 receptor. The variant has been observed in an individual with Crouzon syndrome and functional studies have demonstrated a damaging gain of function effect of the variant. Congenital diaphragmatic hernia (CDH) has never been reported to be associated with Crouzon syndrome though it has been described in association with Apert syndrome. The definitive mechanism of its association is not known but is thought to affect the fibroblast growth factor pathway which is an important pathway for limb and diaphragm development. Through this, we would like to emphasize that Crouzon syndrome should not be excluded from the differential in the presence of CDH. Our patient also had macrocephaly which is not a typical feature of Crouzon syndrome as it is usually associated with craniosynostosis. Hence, we would consider working him up to rule out an underlying undetected hydrocephalus.

Craniosynostosis (CS) is a major birth defect in which one or more skull sutures fuse prematurely. We previously performed a genome-wide association study (GWAS) for sagittal non-syndromic CS (sNCS), identifying associations downstream from BMP2 on 20p12.3 and intronic to BBS9 on 7p14.3; analyses of imputed variants in DLG1 on 3q29 were also genome-wide significant. We followed this work with a GWAS for metopic non-syndromic NCS (mNCS), discovering a significant association intronic to BMP7 on 20q13.31. In the current study, we sequenced the associated regions on 3q29, 7p14.3, and 20p12.3, including two candidate genes (BMP2 and BMPER) near some of these regions in 83 sNCS child-parent trios, and sequenced regions on 7p14.3 and 20q13.2-q13.32 in 80 mNCS child-parent trios. These child-parent trios were selected from the original GWAS cohorts if the probands carried at least one copy of the top associated GWAS variant (rs1884302 C allele for sNCS; rs6127972 T allele for mNCS). Many of the variants sequenced in these targeted regions are strongly predicted to be within binding sites for transcription factors involved in craniofacial development or bone morphogenesis. Variants enriched in more than one trio and predicted to be damaging to gene function are prioritized for functional studies.

Craniosynostosis is the premature fusion of one or more of the cranial sutures. The etiology of craniosynostosis is complex, with both environmental and genetic risk factors. With input from multiple stakeholders, including clinical geneticists and craniofacial pediatricians, our laboratory recently launched a comprehensive genetic testing panel for craniosynostosis using targeted next generation sequencing and copy number analysis via exon-level microarray. Testing options include single gene analysis or a focused panel, which includes seven genes (EFNB1, ERF, FGFR1, FGFR2, FGFR3, TCF12, TWIST1). Pathogenic variants in these seven genes account for approximately 75% of individuals with syndromic craniosynostosis. An expanded panel is also available that includes 42 additional genes that have been linked to craniosynostosis in at least two published reports. To date, ten clinical samples have been tested in our lab, with variants reported in seven (four pathogenic, one likely pathogenic, and two variants of uncertain significance). A pathogenic TCF12 nonsense variant was detected in a 14 year old female with bicoronal craniosynostosis and a shortened right forefoot. A ∼1 kb deletion encompassing exon 4 within GLI3 was detected in an 8 month old male with sagittal craniosynostosis and preaxial polydactyly of the right foot, consistent with a diagnosis of Greig cephalopolysyndactyly syndrome. Sequencing was performed for a 4 month old female with coronal craniosynostosis and polysyndactyly of the left foot and a known diagnosis of 22q11.2 deletion syndrome. A rare missense variant was detected in CDC45, which lies within the 22q11.2 critical region. Involving our institution's clinicians in test development enabled development of panels most appropriate for our patient population, with high detection rates. Future goals for the panel include streamlining the laboratory workflow by detection of copy number variants via NGS data and regular updates to panel content to include genes recently implicated in craniosynostosis.

Our previous genome-wide association study (GWAS) for sagittal nonsyndromic craniosynostosis (sNCS) provided important insights into the genetics of midline CS. In this study, we performed a GWAS for a second midline NCS, metopic NCS (mNCS), using 215 non-Hispanic white case-parent triads. We identified six variants with genome-wide significance (P ≤ 5 × 10–8): rs781716 (P = 4.71 × 10–9; odds ratio [OR] = 2.44) intronic to SPRY3; rs6127972 (P = 4.41 × 10–8; OR = 2.17) intronic to BMP7; rs62590971 (P = 6.22 × 10–9; OR = 0.34), located ~ 155 kb upstream from TGIF2LX; and rs2522623, rs2573826, and rs2754857, all intronic to PCDH11X (P = 1.76 × 10–8, OR = 0.45; P = 3.31 × 10–8, OR = 0.45; P = 1.09 × 10–8, OR = 0.44, respectively). We performed a replication study of these variants using an independent non-Hispanic white sample of 194 unrelated mNCS cases and 333 unaffected controls; only the association for rs6127972 (P = 0.004, OR = 1.45; meta-analysis P = 1.27 × 10–8, OR = 1.74) was replicated. Our meta-analysis examining single nucleotide polymorphisms common to both our mNCS and sNCS studies showed the strongest association for rs6127972 (P = 1.16 × 10–6). Our imputation analysis identified a linkage disequilibrium block encompassing rs6127972, which contained an enhancer overlapping a CTCF transcription factor binding site (chr20:55,798,821–55,798,917) that was significantly hypomethylated in mesenchymal stem cells derived from fused metopic compared to open sutures from the same probands. This study provides additional insights into genetic factors in midline CS.

Purpose Enrichment of heterozygous missense and truncating SMAD6 variants was previously reported in nonsyndromic sagittal and metopic synostosis, and interaction of SMAD6 variants with a common polymorphism near BMP2 (rs1884302) was proposed to contribute to inconsistent penetrance. We determined the occurrence of SMAD6 variants in all types of craniosynostosis, evaluated the impact of different missense variants on SMAD6 function, and tested independently whether rs1884302 genotype significantly modifies the phenotype. Methods We performed resequencing of SMAD6 in 795 unsolved patients with any type of craniosynostosis and genotyped rs1884302 in SMAD6 -positive individuals and relatives. We examined the inhibitory activity and stability of SMAD6 missense variants. Results We found 18 (2.3%) different rare damaging SMAD6 variants, with the highest prevalence in metopic synostosis (5.8%) and an 18.3-fold enrichment of loss-of-function variants comparedwith gnomAD data ( P < 10 −7 ). Combined with eight additional variants, ≥20/26 were transmitted from an unaffected parent but rs1884302 genotype did not predict phenotype. Conclusion Pathogenic SMAD6 variants substantially increase the risk of both nonsyndromic and syndromic presentations of craniosynostosis, especially metopic synostosis. Functional analysis is important to evaluate missense variants. Genotyping of rs1884302 is not clinically useful. Mechanisms to explain the remarkable diversity of phenotypes associated with SMAD6 variants remain obscure.

Objective The World Health Organization recommends that infants unable to feed directly at the breast in low resource settings be cup fed with hand expressed breastmilk. No standard feeding cup exists. The aim of this study was to evaluate the design of the Nifty cup, a newly designed feeding cup, as compared to the paladai and to assess acceptability among mothers and health care providers. Methods This study was conducted at Sri Ramachandra Medical Center and Research Institute in Chennai. Eligible caregivers were primary caregivers of infants who were less than 12 mo old, born prematurely or with an oral cleft, and who were fed by cup. Health care providers who prescribed cup feeding for infants at least 4 times in the past year were also eligible. Caregivers and health care providers fed each infant with a paladai and a Nifty cup. They completed an interviewer-administered survey. The design and acceptability parameters of the Nifty cup were compared to those of paladai using a Wilcoxon signed rank test. Results Forty three caregivers and 28 health care providers were enroled. Among caregivers, the Nifty cup as compared to the paladai was less problematic on most parameters including spillage, regurgitation, difficulty in use, and duration of feeding (all p -values <0.01). Findings were similar for health care providers. Conclusions The Nifty cup is a promising feeding cup for feeding infants with breastfeeding difficulties to support growth and nutrition.

The authors have no financial conflicts to disclose.

Craniosynostosis is the premature fusion of the sutures of the calvaria and is principally designated as being either syndromic (demonstrating characteristic extracranial malformations) or non-syndromic. While many forms of syndromic craniosynostosis are known to be caused by specific mutations, the genetic etiology of non-syndromic, single-suture craniosynostosis (SSC) is poorly understood. Based on the low recurrence rate (4-7%) and the fact that recurrent mutations have not been identified for most cases of SSC, we propose that some cases of isolated, single suture craniosynostosis may be polygenic. Previous work in our lab identified a disproportionately high number of rare and novel gain-of-function IGF1R variants in patients with SSC as compared to controls. Building upon this result, we used expression array data from calvarial osteoblasts isolated from infants with and without SSC to ascertain correlations between high IGF1 expression and expression of other osteogenic genes of interest. We identified a positive correlation between increased expression of IGF1 and RUNX2, a gene known to cause SSC with increased gene dosage. Subsequent phosphorylation assays revealed that osteoblast cell lines from cases with high IGF1 expression demonstrated inhibition of GSK3β, a serine/threonine kinase known to inhibit RUNX2, thus activating osteogenesis through the IRS1-mediated Akt pathway. With these findings, we have utilized established mouse strains to examine a novel model of polygenic inheritance (a phenotype influenced by more than one gene) of SSC. Compound heterozygous mice with selective disinhibition of RUNX2 and either overexpression of IGF1 or loss of function of GSK3β demonstrated an increase in the frequency and severity of synostosis as compared to mice with the RUNX2 disinhibition alone. These polygenic mouse models reinforce, in-vivo, that the combination of activation of the IGF1 pathway and disinhibition of the RUNX2 pathway leads to an increased risk of developing craniosynostosis and serves as a model of human SSC.

Introduction: Early fusion of the sagittal suture is the most common form of craniosynostosis. A number of techniques have been developed for the management of this condition while the “optimum” approach is yet to be known. Computational models have great potential in optimisation of the calvarial reconstruction. The aim of this study was to develop a patient-specific computational model of the calvarial growth to predict the morphology of the calvaria following the surgery based on the pre-operative computed-tomography (CT) images of the same patient. Methods: CT images of a sagittal sysnotosis patient were obtained pre and post-operatively and in a follow up visit at the ages of 6,9 and 24 months respectively. A 3D finite element model of the skull based on the pre-operative images was developed. The model included the brain, bones and sutures. The treatment approach i.e. here full calvarial remodelling, was virtually performed on the model. Input parameters to the model were estimated based on our previous studies. Several sensitivity analyses to the input parameters were performed and outputs were compared in terms of overall shape to follow up calvarial morphology. Results: As expected, sensitivity analyses highlighted that model predictions were sensitive to the choice of input parameters. The most important parameters were the way that the bone-brain interface was modelled and modelling bone formation during the development. It was also demonstrated that the calvarial morphology at the 24 month of age could be predicted based on the model at the 6month of the age. Conclusion: The model developed in this study is the first patient-specific validated model of the calvarial growth. The close match between the predicted shape of the calvarial and the follow up CT build confidence in the modelling approach. However, further studies are required to compare the biomechanics of different reconstruction approaches.

INTRODUCTION:Preterm infants make up the majority of the 9 million babies born in Africa and South Asia requiring supplemental feedings as they transition to exclusive breastfeeding. The World Health Organization recommends the use of a cup to feed newborns with breastfeeding difficulties in low-resource settings. We set out to evaluate the Nifty cup, a new feeding cup designed specifically for infants with breastfeeding difficulties.MATERIALS AND METHODS:We conducted a randomized clinical trial in Ghana. We hypothesized infants would prefer the Nifty cup and that it would have less spillage as compared to a medicine cup. We enrolled mothers and preterm infants with breastfeeding difficulties indicated to cup feed at Komfo Anokye Teaching Hospital. Each mother-infant pair used the Nifty cup and a standard medicine cup; and two feeding assessments with each cup were conducted. We employed an intent-to-treat analysis comparing cup preference using a Wilcoxon signed rank test and spillage using generalized estimating equations.RESULTS:We enrolled 200 mothers and 237 infants. Many infants were very low birth weight (62%), less than two weeks old (62%), and multiple birth (29%). In response to separate questions about each cup, more mothers reported liking the Nifty cup a lot as compared to the medicine cup (85% versus 57%, p<0.001). When asked to choose between the two cups, more than 75% preferred the Nifty cup (p < 0.001). There was slightly less spillage with the Nifty cup (8.9%) versus the medicine cup (9.3%), which was not statistically significant (p = 0.35). Mothers reported greater confidence and ease of using the Nifty cup and greater use one-month post-discharge compared to the medicine cup (p-values <0.001). Nearly all mothers were breastfeeding and cup feeding their infants at study initiation and at one-month post-discharge.DISCUSSION:This is the first randomized clinical trial of cup feeding in sub-Saharan Africa. Mothers prefer the Nifty cup to a medicine cup for supplemental feeds to their preterm infant. The Nifty cup was used with greater ease and confidence. The Nifty cup can offer an improved feeding experience for the mother-infant pair.

DOI: 10.1002/ajmg.a.38540 After publishing this manuscript two data errors were identified. Neither error affects the interpretation of the data. Table 3 lists three patients with TCF12 variants. Proband 95628 is listed as a female with a c.1907A>G; p.(Lys636Arg) variant. This proband is male, not female. This proband is correctly listed as a male in Supplemental Table S1a. Table 4 and Supplemental Table S1b list four probands with SCARF2 variants. Proband 95585 is inaccurately listed as having c.2593dupG: p.Ala865Serfs*184. The correct variant designation is c.2596dupG: p.Ala866Glyfs*191

Blepharocheilodontic syndrome (BCDS) consists of lagophthalmia, ectropion of the lower eyelids, distichiasis, euryblepharon, cleft lip/palate and dental anomalies and has autosomal dominant inheritance with variable expression. We identified heterozygous variants in two genes of the cadherin–catenin complex, CDH1, encoding E-cadherin, and CTNND1, encoding p120 catenin delta1 in 15 of 17 BCDS index patients, as was recently described in a different publication. CDH1 plays an essential role in epithelial cell adherence; CTNND1 binds to CDH1 and controls the stability of the complex. Functional experiments in zebrafish and human cells showed that the CDH1 variants impair the cell adhesion function of the cadherin–catenin complex in a dominant-negative manner. Variants in CDH1 have been linked to familial hereditary diffuse gastric cancer and invasive lobular breast cancer; however, no cases of gastric or breast cancer have been reported in our BCDS cases. Functional experiments reported here indicated the BCDS variants comprise a distinct class of CDH1 variants. Altogether, we identified the genetic cause of BCDS enabling DNA diagnostics and counseling, in addition we describe a novel class of dominant negative CDH1 variants.