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Craniosynostosis, the premature ossification of cranial sutures, is a developmental disorder of the skull vault, occurring in approximately 1 in 2250 births. The causes are heterogeneous, with a monogenic basis identified in ~25% of patients. Using whole-genome sequencing, we identified a novel, de novo variant in BCL11B, c.7C>A, encoding an R3S substitution (p.R3S), in a male patient with coronal suture synostosis. BCL11B is a transcription factor that interacts directly with the nucleosome remodelling and deacetylation complex (NuRD) and polycomb-related complex 2 (PRC2) through the invariant proteins RBBP4 and RBBP7. The p.R3S substitution occurs within a conserved amino-terminal motif (RRKQxxP) of BCL11B and reduces interaction with both transcriptional complexes. Equilibrium binding studies and molecular dynamics simulations show that the p.R3S substitution disrupts ionic coordination between BCL11B and the RBBP4–MTA1 complex, a subassembly of the NuRD complex, and increases the conformational flexibility of Arg-4, Lys-5 and Gln-6 of BCL11B. These alterations collectively reduce the affinity of BCL11B p.R3S for the RBBP4–MTA1 complex by nearly an order of magnitude. We generated a mouse model of the BCL11B p.R3S substitution using a CRISPR-Cas9-based approach, and we report herein that these mice exhibit craniosynostosis of the coronal suture, as well as other cranial sutures. This finding provides strong evidence that the BCL11B p.R3S substitution is causally associated with craniosynostosis and confirms an important role for BCL11B in the maintenance of cranial suture patency.

Mutations in the ERF gene, coding for ETS2 repressor factor, a member of the ETS family of transcription factors cause a recently recognized syndromic form of craniosynostosis (CRS4) with facial dysmorphism, Chiari‐1 malformation, speech and language delay, and learning difficulties and/or behavioral problems. The overall prevalence of ERF mutations in patients with syndromic craniosynostosis is around 2%, and 0.7% in clinically nonsyndromic craniosynostosis. Here, we present findings from 16 unrelated probands with ERF‐related craniosynostosis, with additional data from 20 family members sharing the mutations. Most of the probands exhibited multisutural (including pan‐) synostosis but a pattern involving the sagittal and lambdoid sutures (Mercedes‐Benz pattern) predominated. Importantly the craniosynostosis was often postnatal in onset, insidious and progressive with subtle effects on head morphology resulting in a median age at presentation of 42 months among the probands and, in some instances, permanent visual impairment due to unsuspected raised intracranial pressure (ICP). Facial dysmorphism (exhibited by all of the probands and many of the affected relatives) took the form of orbital hypertelorism, mild exorbitism and malar hypoplasia resembling Crouzon syndrome but, importantly, a Class I occlusal relationship. Speech delay, poor gross and/or fine motor control, hyperactivity and poor concentration were common. Cranial vault surgery for raised ICP and/or Chiari‐1 malformation was expected when multisutural synostosis was observed. Variable expressivity and nonpenetrance among genetically affected relatives was encountered. These observations form the most complete phenotypic and developmental profile of this recently identified craniosynostosis syndrome yet described and have important implications for surgical intervention and follow‐up.

Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or inherited heterozygous mutations in Tousled-like kinase 2 (TLK2) in 38 unrelated individuals and two affected mothers, using whole-exome and whole-genome sequencing technologies, matchmaker databases, and international collaborations. Affected individuals had a consistent phenotype, characterized by mild-borderline neurodevelopmental delay (86%), behavioral disorders (68%), severe gastro-intestinal problems (63%), and facial dysmorphism including blepharophimosis (82%), telecanthus (74%), prominent nasal bridge (68%), broad nasal tip (66%), thin vermilion of the upper lip (62%), and upslanting palpebral fissures (55%). Analysis of cell lines from three affected individuals showed that mutations act through a loss-of-function mechanism in at least two case subjects. Genotype-phenotype analysis and comparison of computationally modeled faces showed that phenotypes of these and other individuals with loss-of-function variants significantly overlapped with phenotypes of individuals with other variant types (missense and C-terminal truncating). This suggests that haploinsufficiency of TLK2 is the most likely underlying disease mechanism, leading to a consistent neurodevelopmental phenotype. This work illustrates the power of international data sharing, by the identification of 40 individuals from 26 different centers in 7 different countries, allowing the identification, clinical delineation, and genotype-phenotype evaluation of a distinct NDD caused by mutations in TLK2.

INTRODUCTION: Recent evidence has emerged linking mutations in CDK13 to syndromic congenital heart disease. We present here genetic and phenotypic data pertaining to 16 individuals with CDK13 mutations.METHODS: Patients were investigated by exome sequencing, having presented with developmental delay and additional features suggestive of a syndromic cause.RESULTS: Our cohort comprised 16 individuals aged four to 16 years. All had developmental delay, including six with autism spectrum disorder. Common findings included feeding difficulties (15/16), structural cardiac anomalies (9/16), seizures (4/16) and abnormalities of the corpus callosum (4/11 patients who had undergone MRI). All had craniofacial dysmorphism, with common features including short, upslanting palpebral fissures, hypertelorism or telecanthus, medial epicanthic folds, low-set, posteriorly rotated ears and a small mouth with thin upper lip vermilion. Fifteen patients had predicted missense mutations, including five identical p.(Asn842Ser) substitutions and two p.(Gly717Arg) substitutions. One patient had a canonical splice acceptor site variant (c.2898-1G>A). All mutations were located within the protein kinase domain of CDK13. The affected amino acids are highly conserved, and in silico analyses including comparative protein modelling predict that they will interfere with protein function. The location of the missense mutations in a key catalytic domain suggest that they are likely to cause loss of catalytic activity but retention of cyclin K binding, resulting in a dominant negative mode of action. Although the splice site mutation was predicted to produce a stable internally deleted protein, this was not supported by expression studies in lymphoblastoid cells. A loss of function contribution to the underlying pathological mechanism therefore cannot be excluded, and the clinical significance of this variant remains uncertain.CONCLUSIONS These patients demonstrate that heterozygous, likely dominant negative mutations affecting the protein kinase domain of the CDK13 gene result in a recognisable, syndromic form of intellectual disability, with or without congenital heart disease.

Burn-McKeown syndrome (BMKS) is a rare syndrome characterized by choanal atresia, prominent ears, abnormalities of the outer third of the lower eyelids, cardiac abnormalities, hearing loss, and unilateral cleft lip. Recently, compound heterozygous mutations were identified in TXNL4A. We analyzed a subject with clinical features of BMKS and her parents by whole genome sequencing and also identified compound heterozygous mutations in TXNL4 (a novel splice site mutation (c.258-2A>G, p.?) and a 34 bp type 1Δ promoter deletion (c.-222_-189del34, p.?) in the proband). Subsequently, we tested a cohort of 16 subjects with clinical features of BMKS and 15 subjects with isolated choanal atresia for mutations in TXNL4A by dideoxy-sequence analysis. In one individual with BMKS unrelated to the first family, we identified the identical compound heterozygous mutations. In an individual with isolated choanal atresia, we found homozygosity for the same type 1Δ promoter deletion, whilst in two cousins from a family with choanal atresia and other minor anomalies we found homozygosity for a different deletion (type 2Δ) within the promoter. Hence, we identified recessive mutations in TXNL4A in 2 subjects with BMKS, but also in 3 patients (2 families) with isolated choanal atresia.

Curry-Jones syndrome (CJS) is a pattern of malformation that includes craniosynostosis, pre-axial polysyndactyly, agenesis of the corpus callosum, cutaneous and gastrointestinal abnormalities. A recurrent, mosaic mutation of SMO (c.1234 C>T; p.Leu412Phe) causes CJS. This report describes the gastrointestinal and surgical findings in a baby with CJS who presented with abdominal obstruction and reviews the spectrum of gastrointestinal malformations in this rare disorder. A 41 week, 4165 gram, female presented with craniosynostosis, pre-axial polysyndactyly, and cutaneous findings consistent with a clinical diagnosis of CJS. The infant developed abdominal distension beginning on the second day of life. Surgical exploration revealed an intestinal malrotation for which she underwent a Ladd procedure. Multiple small nodules were found on the surface of the small and large bowel in addition to an apparent intestinal duplication that seemed to originate posterior to the pancreas. Histopathology of serosal nodules revealed bundles of smooth muscle with associated ganglion cells. Molecular analysis demonstrated the SMO c.1234 C>T mutation in varying amounts in affected skin (up to 35%) and intestinal hamartoma (26%). Gastrointestinal features including structural malformations, motility disorders, and upper GI bleeding are major causes of morbidity in CJS. Smooth muscle hamartomas are a recognized feature of children with CJS typically presenting with abdominal obstruction requiring surgical intervention. A somatic mutation in SMO likely accounts for the structural malformations and predisposition to form bowel hamartomas and myofibromas. The mutation burden in the involved tissues likely accounts for the variable manifestations.

We report the use of the Novalung® interventional Lung Assist extracorporeal carbon dioxide removal device, (Novalung GmbH, Lotzenacker 3, D-72379 Hechingen, Germany) to treat a 46-year-old female with life-threatening bronchospasm secondary to influenza infection. Despite maximal treatment she developed severe hypercapnia and acidosis. The necessity for high inflation pressures led to the development of gross surgical emphysema. Use of the interventional Lung Assist enabled a rapid correction of hypercapnoea and acidosis, allowing a reduction in airway pressures, reducing further barotrauma. Subsequent resolution of the inflammatory process allowed removal of the interventional Lung Assist after 11 days. She was successfully weaned from mechanical ventilation and made a full recovery.

We present a case report of a patient who developed acute swelling of the tongue during anaesthesia using the laryngeal mask airway. The swelling was thought to be due to obstruction of the venous drainage of the tongue. This was associated with isolated cyanosis of the tongue and paraesthesia. The swelling and cyanosis of the tongue resolved rapidly after removal of the laryngeal mask airway. The patient suffered paraesthesia of the tip of the tongue that lasted for two weeks.

The RET proto-oncogene encodes a receptor tyrosine kinase activated by the binding of factors from the glial cell line-derived neurotrophic factor (GDNF) family to receptor-alpha components such as GDNF family receptor alpha-1 (GFR alpha-1). Mutations within the sequence of the RET proto-oncogene are associated with multiple endocrine neoplasia type 2 (MEN 2), an inherited tumor syndrome characterized by the development of medullary thyroid carcinoma (MTC) and other neuroendocrine tumors. Despite Northern analysis showing that RET is expressed in the majority of MTCs, the factors regulating this expression are poorly understood. To address this issue we examined RET expression in response to glucocorticoids in the TT cell line, derived from a metastatic MTC. The synthetic glucocorticoid dexamethasone was found to reduce RET expression at both mRNA and protein levels. This effect was dose responsive and maximal at 24 h. The reduction in RET mRNA was shown to be specific to glucocorticoids and was also seen in a primary MTC culture. Nuclear run-on studies revealed the reduction in steady-state RNA to be due to a decrease in RET mRNA transcription and the effect was shown to be independent of new protein synthesis or RNA stability. Dexamethasone was also found to exert an inhibitory effect upon cell growth, suggesting a potential use for glucocorticoids in the treatment of medullary carcinoma and MEN 2.

Craniosynostosis, the premature fusion of one or more cranial sutures, is one of the most common craniofacial abnormalities. There is considerable genetic heterogeneity and the underlying pathophysiological mechanisms are diverse, occurring at multiple stages of cranial suture biogenesis. A genetic diagnosis, currently made in ~25% of cases, is an important aspect of the multidisciplinary approach to clinical management and genetic counselling. The aim of this thesis is to identify novel genetic causes of craniosynostosis in unsolved familial cases. Four families were selected and investigated using a combination of next generation sequencing and copy number analysis. Candidate changes were followed up through segregation and functional analysis to determine if they were causal. The most convincing finding was a homozygous deletion identified in the 5′ UTR (untranslated region) of dehydrogenase/reductase 3 (DHRS3) in a consanguineous family with syndromic coronal synostosis. This deletion was shown to lead to loss of DHRS3 activity and an increase of all-trans-retinoic acid (ATRA), a known teratogen that had previously been deduced to contribute to craniosynostosis. These findings are consistent with a novel embryopathy caused by excess ATRA, leading to skeletal and cardiac defects. In a second large family with syndromic metopic synostosis, a nonsynonymous variant in HIST1H2AD, encoding one of the canonical histone H2A components of the nucleosome, was identified. Although a link between histone mutation and craniosynostosis is unknown, we speculated that this variant could perturb gene expression during development of the metopic suture. To gain support for causality, two approaches were undertaken; 1. Search for additional patients with a variant in H2A genes including resequencing in a large cohort of patients with craniosynostosis. 2. Creation of Hist1h2ad mutant Embryonic Stem cells using CRISPR/CAS9; Generation of the mouse model is ongoing. Chromosomal rearrangements can be pathogenic through altering the topological associated domain (TAD) structure. A de novo tandem duplication on chromosome 4 including FGF5 and C4orf22 was identified in a third family (affected mother and daughter) with multiple suture synostosis. The duplication overlaps TAD boundaries, leading to the hypothesis that this may induce mis-expression of genes in or near the duplication. Gene expression analysis was undertaken. To investigate this further, work using Capture-C, a technique used to discover local chromatin structure and interactions, is ongoing. In summary, I have identified a new craniosynostosis disease gene, DHRS3, suggesting an additional aetiology in osteogenesis of the cranial suture caused by disruption of retinoic metabolism. In addition, two loci, HIST1H2AD and FGF5/C4orf22 duplication, are plausible candidates for craniosynostosis with possible roles in disturbing neural crest development and RAS/MAPK signalling, respectively. Finally, this work illustrates a combined genomic technology approach to resolve difficult cases that may be applicable apply to other diseases in which a genetic cause is suspected.