Andrew T. Timberlake

Two locus inheritance of non-syndromic midline craniosynostosis via rare SMAD6 and common BMP2 alleles

Premature fusion of the cranial sutures (craniosynostosis), affecting 1 in 2000 newborns, is treated surgically in infancy to prevent adverse neurologic outcomes. To identify mutations contributing to common non-syndromic midline (sagittal and metopic) craniosynostosis, we performed exome sequencing of 132 parent-offspring trios and 59 additional probands. Thirteen probands (7%) had damaging de novo or rare transmitted mutations in SMAD6, an inhibitor of BMP – induced osteoblast differentiation (p<10−20). SMAD6 mutations nonetheless showed striking incomplete penetrance (<60%). Genotypes of a common variant near BMP2 that is strongly associated with midline craniosynostosis explained nearly all the phenotypic variation in these kindreds, with highly significant evidence of genetic interaction between these loci via both association and analysis of linkage. This epistatic interaction of rare and common variants defines the most frequent cause of midline craniosynostosis and has implications for the genetic basis of other diseases. DOI: http://dx.doi.org/10.7554/eLife.20125.001

The role of mechanical forces and adenosine in the regulation of intestinal enterochromaffin cell serotonin secretion

Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin (5-HT) with activation of gut motility, secretion, and pain. These cells express adenosine (ADORA) receptors and are considered to function as mechanosensors. Physiological pathways mediating mechanosensitivity and adenosine responsiveness remain to be fully elucidated, as do their roles in inflammatory bowel disease (IBD) and neoplasia. Pure (98-99%) FACS-sorted normal and IBD human EC cells and neoplastic EC cells (KRJ-I) were studied. IBD-EC cells and KRJ-I overexpressed ADORA2B. NECA, a general ADORA receptor agonist, stimulated, whereas the A2B receptor antagonist MRS1754 inhibited, 5-HT release (EC50 = 1.8 × 10-6 M; IC50 = 3.7 × 10-8 M), which was associated with corresponding alterations in intracellular cAMP levels and pCREB (Ser133). Mechanical stimulation using a rhythmic flex model induced transcription and activation of Tph1 (tryptophan hydroxylase) and VMAT₁ (vesicular monoamine transporter 1) and the release of 5-HT, which could be inhibited by MRS1754 and amplified by NECA. Secretion was also inhibited by H-89 (PKA inhibitor) while Tph1 and VMAT₁ transcription was regulated by PKA/MAPK and PI₃K-mediated signaling. Normal and IBD-EC cells also responded to NECA and mechanical stimulation with PKA activation, cAMP production, and 5-HT release, effects reversible by MRS1754. EC cells express stimulatory ADORA2B, and rhythmic stretch induces A2B activation, PKA/MAPK/IP3-dependent transcription, and PKA-dependent secretion of 5-HT synthesis and secretion. Receptor expression is amplified in IBD and neoplasia, and 5-HT release is increased. Determination of factors that regulate EC cell function are necessary for understanding its role as a mechanosensory cell and to facilitate the development of agents that can selectively target cell function in EC cell-associated disease.

Serotonin and the 5-HT7 receptor: The link between hepatocytes, IGF-1 and small intestinal neuroendocrine tumors

Platelet‐derived serotonin (5‐HT) is involved in liver regeneration. The liver is also the metastatic site for malignant enterochromaffin (EC) cell “carcinoid” (neuroendocrine) neoplasms, the principal cellular source of 5‐HT. We hypothesized that 5‐HT produced by metastatic EC cells played a role in the hepatic tumor‐microenvironment principally via 5‐HT7 receptor‐mediated activation of hepatocyte IGF‐1 synthesis and secretion. Using isolated rat hepatocytes, we evaluated 5‐HT7 receptor expression (using PCR, sequencing and western blot). ELISA, cell transfection and western blots delineated 5‐HT‐mediated signaling pathways (pCREB, AKT and ERK). IGF‐1 synthesis/secretion was evaluated using QPCR and ELISA. IGF‐1 was tested on small intestinal neuroendocrine neoplasm proliferation, while IGF‐1 production and 5‐HT7 expression were examined in an in vivo SCID metastasis model. Our results demonstrated evidence for a functional 5‐HT7 receptor. 5‐HT activated cAMP/PKA activity, pCREB (130–205%, P < 0.05) and pERK/pAKT (1.2–1.75, P < 0.05). Signaling was reversed by the 5‐HT7 receptor antagonist SB269970. IGF‐1 significantly stimulated proliferation of two small intestinal neuroendocrine neoplasm cell lines (EC50: 7–70 pg/mL) and could be reversed by the small molecule inhibitor BMS‐754807. IGF‐1 and 5‐HT were elevated (40–300×) in peri‐tumoral hepatic tissue in nude mice, while 5‐HT7 was increased fourfold compared to sham‐operated animals. We conclude that hepatocytes express a cAMP‐coupled 5‐HT7 receptor, which, at elevated 5‐HT concentrations that occur in liver metastases, signals via CREB/AKT and is linked to IGF‐1 synthesis and secretion. Because IGF‐1 regulates NEN proliferation, identification of a role for 5‐HT7 in the hepatic metastatic tumor microenvironment suggests the potential for novel therapeutic strategies for amine‐producing mid‐gut tumors.

De novo mutations in inhibitors of Wnt, BMP, and Ras/ERK signaling pathways in non-syndromic midline craniosynostosis

Significance Craniosynostosis is a common congenital malformation resulting from premature fusion of the bones that comprise the cranial vault, requiring surgery in infancy to prevent adverse neurologic outcomes. Eighty-five percent of cases are non-syndromic and of unknown cause. By exome sequencing of families with non-syndromic midline craniosynostosis, we show that 5% of cases have de novo damaging mutations in negative regulators of the Wnt, bone morphogenetic protein (BMP), and Ras/ERK signaling pathways, developmental cascades that converge on common nuclear targets to promote bone formation. Another 5% have transmitted mutations in these pathways. Common variants near BMP2 show genetic interaction with these rare mutations. The results provide insight into pathophysiology and have immediate implications for the diagnosis and genetic counseling of families with craniosynostosis. Non-syndromic craniosynostosis (NSC) is a frequent congenital malformation in which one or more cranial sutures fuse prematurely. Mutations causing rare syndromic craniosynostoses in humans and engineered mouse models commonly increase signaling of the Wnt, bone morphogenetic protein (BMP), or Ras/ERK pathways, converging on shared nuclear targets that promote bone formation. In contrast, the genetics of NSC is largely unexplored. More than 95% of NSC is sporadic, suggesting a role for de novo mutations. Exome sequencing of 291 parent–offspring trios with midline NSC revealed 15 probands with heterozygous damaging de novo mutations in 12 negative regulators of Wnt, BMP, and Ras/ERK signaling (10.9-fold enrichment, P = 2.4 × 10−11). SMAD6 had 4 de novo and 14 transmitted mutations; no other gene had more than 1. Four familial NSC kindreds had mutations in genes previously implicated in syndromic disease. Collectively, these mutations contribute to 10% of probands. Mutations are predominantly loss-of-function, implicating haploinsufficiency as a frequent mechanism. A common risk variant near BMP2 increased the penetrance of SMAD6 mutations and was overtransmitted to patients with de novo mutations in other genes in these pathways, supporting a frequent two-locus pathogenesis. These findings implicate new genes in NSC and demonstrate related pathophysiology of common non-syndromic and rare syndromic craniosynostoses. These findings have implications for diagnosis, risk of recurrence, and risk of adverse neurodevelopmental outcomes. Finally, the use of pathways identified in rare syndromic disease to find genes accounting for non-syndromic cases may prove broadly relevant to understanding other congenital disorders featuring high locus heterogeneity.

Co-occurrence of frameshift mutations in SMAD6 and TCF12 in a child with complex craniosynostosis

Non-syndromic craniosynostosis (CS) affects 1 in 2350 live births. Recent studies have shown that a significant fraction of cases are caused by de novo or rare transmitted mutations that promote premature osteoblast differentiation in cranial sutures. Rare heterozygous loss-of-function (LOF) mutations in SMAD6 and TCF12 are highly enriched in patients with non-syndromic sagittal and coronal CS, respectively. Interestingly, both mutations show striking incomplete penetrance, suggesting a role for modifying alleles; in the case of SMAD6, a common variant near BMP2 drastically increases penetrance of sagittal CS. Here, we report a proband presenting with both sagittal and coronal craniosynostosis with the highly unusual recurrence of CS within two months of initial surgery, requiring a second operation to re-establish suture patency at six months of age. Exome sequencing revealed a rare transmitted frameshift mutation in SMAD6 (p. 152 fs*27) inherited from an unaffected parent, absence of the common BMP2 risk variant, and a de novo frameshift mutation in TCF12 (p.E548fs*14). SMAD6 and TCF12 independently inhibit transcriptional targets of BMP signaling. The findings are consistent with epistasis of these mutations, increasing penetrance and severity of CS in this proband. They also add to the list of composite phenotypes resulting from two Mendelian mutations, and support the utility of exome sequencing in atypical CS cases.

The Etiology of Neuronal Development in Craniosynostosis: A Working Hypothesis

Craniosynostosis is one of the most common craniofacial conditions treated by neurologic and plastic surgeons. In addition to disfigurement, children with craniosynostosis experience significant cognitive dysfunction later in life. Surgery is performed in infancy to correct skull deformity; however, the field is at a crossroads regarding the best approach for correction. Since the cause of brain dysfunction in these patients has remained uncertain, the role and type of surgery might have in attenuating the later-observed cognitive deficits through impact on the brain has been unclear. Recently, however, advances in imaging such as event-related potentials, diffusion tensor imaging, and functional MRI, in conjunction with more robust clinical studies, are providing important insight into the potential etiologies of brain dysfunction in syndromic and nonsyndromic craniosynostosis patients. This review aims to outline the cause(s) of such brain dysfunction including the role extrinsic vault constriction might have on brain development and the current evidence for an intrinsic modular developmental error in brain development. Illuminating the cause of brain dysfunction will identify the role of surgery can play in improving observed functional deficits and thus direct optimal primary and adjuvant treatment.

9p24 triplication in syndromic hydrocephalus with diffuse villous hyperplasia of the choroid plexus

Hydrocephalus, a disorder of impaired cerebrospinal fluid (CSF) homeostasis, often results from an imbalance between CSF production and reabsorption. Rarely, hydrocephalus is the consequence of CSF hypersecretion in the context of diffuse villous hyperplasia of the choroid plexus (DVHCP). The limited genetic information in previously reported cases suggests a high prevalence of gains of Chromosome 9p in this disease, although the critical genes involved in DVHCP pathogenesis have not been identified. Here, we report a patient with syndromic hydrocephalus with DVHCP associated with a novel 9p24.3-11.2 triplication and 15q13.2-q13.3 microdeletion. We review the clinical, radiological, and pathological features of DVHCP, as well as its surgical management. A better understanding of the genetic basis of DVHCP could spur the development of rational, targeted nonsurgical hydrocephalus treatments.

Three-Dimensional Imaging in Rhinoplasty: A Comparison of the Simulated versus Actual Result

PurposeComputer imaging has become increasingly popular for rhinoplasty. Three-dimensional (3D) analysis permits a more comprehensive view from multiple vantage points. However, the predictability and concordance between the simulated and actual result have not been morphometrically studied. The purpose of this study was to aesthetically and quantitatively compare the simulated to actual rhinoplasty result.MethodsA retrospective review of 3D images (VECTRA, Canfield) for rhinoplasty patients was performed. Images (preop, simulated, and actual) were randomized. A blinded panel of physicians rated the images (1 = poor, 5 = excellent). The image series considered “best” was also recorded. A quantitative assessment of nasolabial angle and tip projection was compared. Paired and two-sample t tests were performed for statistical analysis (P < 0.05 as significant).ResultsForty patients were included. 67.5% of preoperative images were rated as poor (mean = 1.7). The simulation received a mean score of 2.9 (good in 60% of cases). 82.5% of actual cases were rated good to excellent (mean 3.4) (P < 0.001). Overall, the panel significantly preferred the actual postoperative result in 77.5% of cases compared to the simulation in 22.5% of cases (P < 0.001). The actual nasal tip was more projected compared to the simulations for both males and females. There was no significant difference in nasal tip rotation between simulated and postoperative groups.Conclusion3D simulation is a powerful communication and planning tool in rhinoplasty. In this study, the actual result was deemed more aesthetic than the simulated image. Surgeon experience is important to translate the plan and achieve favorable postoperative results.Level of Evidence IVThis journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Abstract 24: Two Locus Inheritance of Non-Syndromic Midline Craniosynostosis Via Rare SMAD6 and Common BMP2 Alleles

PURPOSE: More than 30,000 people receive organ transplants every year in the US. Vascularized composite allotransplantation (VCA) is the newest realm of solid organ transplantation. The skin component of VCA is highly antigenic and mandates high doses of systemic immunosuppressive drugs. Oral dosing of immunosuppressive drugs such as tacrolimus (TAC), rapamycin (Rapa), and mycophenolic acid (MPA) leads to fluctuating, erratic, or unpredictable blood levels risking toxicity or lack of efficacy. We propose a drug delivery platform that can not only provide sustained drug release but also on-cue triggered drug release upon ultrasound stimulation (USS) in graft tissues with stable, low blood levels, minimizing overall drug exposure and facilitating long-term VCA survival with no systemic complications.

Xp22.2 Chromosomal Duplication in Familial Intracranial Arachnoid Cyst

Xp22.2 Chromosomal Duplication in Familial Intracranial Arachnoid Cyst Arachnoid cysts (ACs) are congenital fluid-filled malformations that account for approximately 1% of all intracranial, space-occupying lesions in the central nervous system.1 Despite an estimated prevalence of 1.4%, little is known about the pathogenesis of these presumed developmental anomalies of the arachnoid.2 The coincidence of ACs in known mendelian cystic disorders, such as autosomal dominant polycystic kidney disease,3 along with rare clinical reports of familial AC occurrence,4,5 suggests a genetic basis for the disorder. However, no gene or chromosomal abnormalities have been detected in familial intracranial AC. We present a familial form of isolated intracranial AC showing an X-linked dominant inheritance pattern and characterized by the presence of large, bilateral, and symmetric middle fossa ACs (Figure 1) in 4 family members of a nuclear kindred (Figure 2).