Bop1 is required to establish precursor domains of craniofacial tissues.

Bop1 can promote cell proliferation and is a component of the Pes1-Bop1-WDR12 (PeBoW) complex that regulates ribosomal RNA processing and biogenesis. In embryos, however, bop1 mRNA is highly enriched in the neural plate, cranial neural crest and placodes, and potentially may interact with Six1, which also is expressed in these tissues. Recent work demonstrated that during development, Bop1 is required for establishing the size of the tadpole brain, retina and cranial cartilages, as well as controlling neural tissue gene expression levels. Herein, we extend this work by assessing the effects of Bop1 knockdown at neural plate and larval stages. Loss of Bop1 expanded neural plate gene expression domains (sox2, sox11, irx1) and reduced neural crest (foxd3, sox9), placode (six1, sox11, irx1, sox9) and epidermal (dlx5) expression domains. At larval stages, Bop1 knockdown reduced the expression of several otic vesicle genes (six1, pax2, irx1, sox9, dlx5, otx2, tbx1) and branchial arch genes that are required for chondrogenesis (sox9, tbx1, dlx5). The latter was not the result of impaired neural crest migration. Together these observations indicate that Bop1 is a multifunctional protein that in addition to its well-known role in ribosomal biogenesis functions during early development to establish the craniofacial precursor domains.

The sulfotransferase XB5850668.L is required to apportion embryonic ectodermal domains.

BACKGROUND: Members of the sulfotransferase superfamily (SULT) influence the activity of a wide range of hormones, neurotransmitters, metabolites and xenobiotics. However, their roles in developmental processes are not well characterized even though they are expressed during embryogenesis. We previously found in a microarray screen that Six1 up-regulates LOC100037047, which encodes XB5850668.L, an uncharacterized sulfotransferase. RESULTS: Since Six1 is required for patterning the embryonic ectoderm into its neural plate, neural crest, preplacodal and epidermal domains, we used loss- and gain-of function assays to characterize the role of XB5850668.L during this process. Knockdown of endogenous XB5850668.L resulted in the reduction of epidermal, neural crest, cranial placode and otic vesicle gene expression domains, concomitant with neural plate expansion. Increased levels had minimal effects, but infrequently expanded neural plate and neural crest gene domains, and infrequently reduced cranial placode and otic vesicle gene domains. Mutation of two key amino acids in the sulfotransferase catalytic domain required for PAPS binding and enzymatic activity tended to reduce the effects of overexpressing the wild-type protein. CONCLUSIONS: Our analyses indicates that XB5850668.L is a member of the SULT2 family that plays important roles in patterning the embryonic ectoderm. Some aspects of its influence likely depend on sulfotransferase activity.

Overgrowth syndromes, diagnosis and management.

PURPOSE OF REVIEW: This review will focus on the current knowledge of the diagnosis and management of overgrowth syndromes with specific focus on mosaic conditions and treatment strategies. RECENT FINDINGS: With the implementation of massively parallel sequencing, the genetic etiology of many classically described overgrowth syndromes have been identified. More recently, the role of mosaic genetic changes has been well described in numerous syndromes. Furthermore, the role of imprinting and methylation, especially of the 11p15 region, has been shown to be instrumental for growth. Perhaps most importantly, many overgrowth syndromes carry an increased risk of neoplasm formation especially in the first 10 years of life and possibly beyond. The systematic approach to the child with overgrowth will aide in timely diagnosis and efficiently align them with appropriate screening strategies. In some cases, precision medical interventions are available to target the perturbed growth signaling pathways. SUMMARY: The systematic approach to the child with overgrowth aids in the standardization of the diagnostic pathway for these young patients, thereby expediting the diagnostic timeline, enabling rigorous monitoring, and delivering tailored therapeutic interventions.

Changes in Memory, Sedation, and Receptor Kinetics Imparted by the ß2-N265M and ß3-N265M GABAA Receptor Point Mutations.

Point mutations in the ß2 (N265S) and ß3 (N265M) subunits of γ-amino butyric acid type A receptors (GABAARs) that render them insensitive to the general anesthetics etomidate and propofol have been used to link modulation of ß2-GABAARs to sedation and ß3-GABAARs to surgical immobility. These mutations also alter GABA sensitivity, and mice carrying the ß3-N265M mutation have been reported to have impaired baseline memory. Here, we tested the effects of the ß2-N265M and ß3-N265M mutations on memory, movement, hotplate sensitivity, anxiety, etomidate-induced sedation, and intrinsic kinetics. We found that both ß2-N265M and ß3-N265M mice exhibited baseline deficits in the Context Preexposure Facilitation Effect learning paradigm. Exploratory activity was slightly greater in ß2-N265M mice, but there were no changes in either genotype in anxiety or hotplate sensitivity. ß2-N265M mice were highly resistant to etomidate-induced sedation, and heterozygous mice were partially resistant. In rapid solution exchange experiments, both mutations accelerated deactivation two- to three-fold compared to wild type receptors and prevented modulation by etomidate. This degree of change in the receptor deactivation rate is comparable to that produced by an amnestic dose of etomidate but in the opposite direction, indicating that intrinsic characteristics of GABAARs are optimally tuned under baseline conditions to support mnemonic function.

Natural size variation among embryos leads to the corresponding scaling in gene expression.

Xenopus laevis frogs from laboratory stocks normally lay eggs exhibiting extensive size variability. We find that these initial size differences subsequently affect the size of the embryos prior to the onset of growth, and the size of tadpoles during the growth period. Even though these tadpoles differ in size, their tissues, organs, and structures always seem to be properly proportioned, i.e. they display static allometry. Initial axial patterning events in Xenopus occur in a spherical embryo, allowing easy documentation of their size-dependent features. We examined the size distribution of early Xenopus laevis embryos and measured diameters that differed by about 38% with a median of about 1.43 â€‹mm. This range of embryo sizes corresponds to about a 1.9-fold difference in surface area and a 2.6-fold difference in volume. We examined the relationship between embryo size and gene expression and observed a significant correlation between diameter and RNA content during gastrula stages. In addition, we investigated the expression levels of genes that pattern the mesoderm, induce the nervous system and mediate the progression of ectodermal cells to neural precursors in large and small embryos. We found that most of these factors were expressed at levels that scaled with the different embryo sizes and total embryo RNA content. In agreement with the changes in transcript levels, the expression domains in larger embryos increased proportionally with the increase in surface area, maintaining their relative expression domain size in relation to the total size of the embryo. Thus, our study identified a mechanism for adapting gene expression domains to embryo size by adjusting the transcript levels of the genes regulating mesoderm induction and patterning. In the neural plate, besides the scaling of the expression domains, we observed similar cell sizes and cell densities in small and large embryos suggesting that additional cell divisions took place in large embryos to compensate for the increased size. Our results show in detail the size variability among Xenopus laevis embryos and the transcriptional adaptation to scale gene expression with size. The observations further support the involvement of BMP/ADMP signaling in the scaling process.

Identifying surgeon and institutional drivers of cost in total shoulder arthroplasty: a multicenter study.

BACKGROUND: Despite rapid increases in the demand for total shoulder arthroplasty, data describing cost trends are scarce. We aim to (1) describe variation in the cost of shoulder arthroplasty performed by different surgeons at multiple hospitals and (2) determine the driving factors of such variation. METHODS: A standardized, highly accurate cost accounting method, time-driven activity-based costing, was used to determine the cost of 1571 shoulder arthroplasties performed by 12 surgeons at 4 high-volume institutions between 2016 and 2018. Costs were broken down into supply costs (including implant price and consumables) and personnel costs, including physician fees. Cost parameters were compared with total cost for surgical episodes and case volume. RESULTS: Across 4 institutions and 12 surgeons, surgeon volume and hospital volume did not correlate with episode-of-care cost. Average cost per case of each institution varied by factors of 1.6 (P = .47) and 1.7 (P = .06) for anatomic total shoulder arthroplasty (TSA) and reverse total shoulder arthroplasty (RSA), respectively. Implant (56% and 62%, respectively) and personnel costs from check-in through the operating room (21% and 17%, respectively) represented the highest percentages of cost and highly correlated with the cost of the episode of care for TSA and RSA. CONCLUSIONS: Variation in episode-of-care total costs for both TSA and RSA had no association with hospital or surgeon case volume at 4 high-volume institutions but was driven primarily by variation in implant and personnel costs through the operating room. This analysis does not address medium- or long-term costs.

Mutations in the sonic hedgehog pathway cause macrocephaly-associated conditions due to crosstalk to the PI3K/AKT/mTOR pathway.

The hedgehog (Hh) pathway is highly conserved and required for embryonic patterning and determination. Mutations in the Hh pathway are observed in sporadic tumors as well as under syndromic conditions. Common to these syndromes are the findings of polydactyly/syndactyly and brain overgrowth. The latter is also a finding most commonly observed in the cases of mutations in the PI3K/AKT/mTOR pathway. We have identified novel Hh pathway mutations and structural copy number variations in individuals with somatic overgrowth, macrocephaly, dysmorphic facial features, and developmental delay, which phenotypically closely resemble patients with phosphatase and tensin homolog (PTEN) mutations. We hypothesized that brain overgrowth and phenotypic overlap with syndromic overgrowth syndromes in these cases may be due to crosstalk between the Hh and PI3K/AKT/mTOR pathways. To test this, we modeled disease-associated variants by generating PTCH1 and Suppressor of Fused (SUFU) heterozygote cell lines using the CRISPR/Cas9 system. These cells demonstrate activation of PI3K signaling and increased phosphorylation of its downstream target p4EBP1 as well as a distinct cellular phenotype. To further investigate the mechanism underlying this crosstalk, we treated human neural stem cells with sonic hedgehog (SHH) ligand and performed transcriptional analysis of components of the mTOR pathway. These studies identified decreased expression of a set of mTOR negative regulators, leading to its activation. We conclude that there is a significant crosstalk between the SHH and PI3K/AKT/mTOR. We propose that this crosstalk is responsible for why mutations in PTCH1 and SUFU lead to macrocephaly phenotypes similar to those observed in PTEN hamartoma and other overgrowth syndromes associated with mutations in PI3K/AKT/mTOR pathway genes.

The Role of Self-reports and Behavioral Measures of Interpretation Biases in Children with Varying Levels of Anxiety.

We investigated the role of self-reports and behavioral measures of interpretation biases and their content-specificity in children with varying levels of spider fear and/or social anxiety. In total, 141 selected children from a community sample completed an interpretation bias task with scenarios that were related to either spider threat or social threat. Specific interpretation biases were found; only spider-related interpretation bias and self-reported spider fear predicted unique variance in avoidance behavior on the Behavior Avoidance Task for spiders. Likewise, only social-threat related interpretation bias and self-reported social anxiety predicted anxiety during the Social Speech Task. These findings support the hypothesis that fearful children display cognitive biases that are specific to particular fear-relevant stimuli. Clinically, this insight might be used to improve treatments for anxious children by targeting content-specific interpretation biases related to individual disorders.

Truncating mutations in APP cause a distinct neurological phenotype.

Dominant missense mutations in the amyloid ß (Aß) precursor protein (APP) gene have been implicated in early onset Alzheimer disease. These mutations alter protein structure to favor the pathologic production of Aß. We report that homozygous nonsense mutations in APP are associated with decreased somatic growth, microcephaly, hypotonia, developmental delay, thinning of the corpus callosum, and seizures. We compare the phenotype of this case to those reported in mouse models and demonstrate multiple similarities, strengthening the role of amyloid precursor protein in normal brain function and development. Ann Neurol 2016;80:456-460.

De novo loss-of-function variants in STAG2 are associated with developmental delay, microcephaly, and congenital anomalies.

The cohesin complex is an evolutionarily conserved multi-subunit protein complex which regulates sister chromatid cohesion during mitosis and meiosis. Additionally, the cohesin complex regulates DNA replication, DNA repair, and transcription. The core of the complex consists of four subunits: SMC1A, SMC3, RAD21, and STAG1/2. Loss-of-function mutations in many of these proteins have been implicated in human developmental disorders collectively termed "cohesinopathies." Through clinical exome sequencing (CES) of an 8-year-old girl with a clinical history of global developmental delay, microcephaly, microtia with hearing loss, language delay, ADHD, and dysmorphic features, we describe a heterozygous de novo variant (c.205C>T; p.(Arg69*)) in the integral cohesin structural protein, STAG2. This variant is associated with decreased STAG2 protein expression. The analyses of metaphase spreads did not exhibit premature sister chromatid separation; however, delayed sister chromatid cohesion was observed. To further support the pathogenicity of STAG2 variants, we identified two additional female cases from the DECIPHER research database with mutations in STAG2 and phenotypes similar to our patient. Interestingly, the clinical features of these three cases are remarkably similar to those observed in other well-established cohesinopathies. Herein, we suggest that STAG2 is a dosage-sensitive gene and that heterozygous loss-of-function variants lead to a cohesinopathy.

Early neural ectodermal genes are activated by Siamois and Twin during blastula stages.

BMP signaling distinguishes between neural and non-neural fates by activating epidermis-specific transcription and repressing neural-specific transcription. The neural ectoderm forms after the Organizer secrets antagonists that prevent these BMP-mediated activities. However, it is not known whether neural genes also are transcriptionally activated. Therefore, we tested the ability of nine Organizer transcription factors to ectopically induce the expression of four neural ectodermal genes in epidermal precursors. We found evidence for two pathways: Foxd4 and Sox11 were only induced by Sia and Twn, whereas Gmnn and Zic2 were induced by Sia, Twn, as well as seven other Organizer transcription factors. The induction of Foxd4, Gmnn and Zic2 by Sia/Twn was both non-cell autonomous (requiring an intermediate protein) and cell autonomous (direct), whereas the induction of Sox11 required Foxd4 activity. Because direct induction by Sia/Twn could occur endogenously in the dorsal-equatorial blastula cells that give rise to both the Organizer mesoderm and the neural ectoderm, we knocked down Sia/Twn in those cells. This prevented the blastula expression of Foxd4 and Sox11, demonstrating that Sia/Twn directly activate some neural genes before the separation of the Organizer mesoderm and neural ectoderm lineages.

Expanding the phenotype of mutations in DICER1: mosaic missense mutations in the RNase IIIb domain of DICER1 cause GLOW syndrome.

BACKGROUND: Constitutional DICER1 mutations have been associated with pleuropulmonary blastoma, cystic nephroma, Sertoli-Leydig tumours and multinodular goitres, while somatic DICER1 mutations have been reported in additional tumour types. Here we report a novel syndrome termed GLOW, an acronym for its core phenotypic findings, which include Global developmental delay, Lung cysts, Overgrowth and Wilms tumour caused by mutations in the RNase IIIb domain of DICER1. METHODS AND RESULTS: We performed whole exome sequencing on peripheral mononuclear blood cells of an affected proband and identified a de novo missense mutation in the RNase IIIb domain of DICER1. We confirmed an additional de novo missense mutation in the same domain of an unrelated case by Sanger sequencing. These missense mutations in the RNase IIIb domain of DICER1 are suspected to affect one of four metal binding sites located within this domain. Pyrosequencing was used to determine the relative abundance of mutant alleles in various tissue types. The relative mutation abundance is highest in Wilms tumour and unaffected kidney samples when compared with blood, confirming that the mutation is mosaic. Finally, we performed bioinformatic analysis of microRNAs expressed in murine cells carrying specific Dicer1 RNase IIIb domain metal binding site-associated mutations. We have identified a subset of 3p microRNAs that are overexpressed whose target genes are over-represented in mTOR, MAPK and TGF-ß signalling pathways. CONCLUSIONS: We propose that mutations affecting the metal binding sites of the DICER1 RNase IIIb domain alter the balance of 3p and 5p microRNAs leading to deregulation of these growth signalling pathways, causing a novel human overgrowth syndrome.

Is there an indication for digital subtraction angiography in the assessment of irradiation-induced vascular damage before free flap surgery by the means of the internal mammary vessels?

Secondary breast reconstruction is increasingly performed after postmastectomy radiotherapy. Damage to blood vessel walls is one of the adverse effects of irradiation therapy, which may jeopardize reconstructive free flap surgery. It would be of great importance to be informed about the quality of the recipient vessel before reconstructive surgery. The aim of this study was to prospectively assess the value of preoperative angiography in the assessment of radiation-induced arterial damage and to relate the findings to the degree of vascular damage found during the operation and with histology. This study included women who had been treated with thoracic radiotherapy and required free flap breast reconstruction. Preoperative angiographic, intraoperative quality and histological findings of vessels were scored and compared together with the occurrence of postoperative complications. In 34 patients a total of 40 free flaps breast reconstruction were performed. Total 21 internal mammary arteries had been within the field of irradiation. In only two out of six patients with aberrant angiographies the internal mammary artery has been within the field of irradiation. This study concludes that damage to the internal mammary vessels cannot always be detected preoperatively by angiography, or even by intraoperative examination.

Can glucose facilitate fear exposure? Randomized, placebo-controlled trials on the effects of glucose administration on fear extinction processes.

Previous studies showed that glucose has beneficial effects on memory function and can enhance contextual fear learning. To derive potential therapeutic interventions, further research is needed regarding the effects of glucose on fear extinction. In two experimental studies with healthy participants (Study 1: N = 68, 39 females; Study 2: N = 89, 67 females), we investigated the effects of glucose on fear extinction learning and its consolidation. Participants completed a differential fear conditioning paradigm consisting of acquisition, extinction, and return of fear tests: reinstatement, and extinction recall. US-expectancy ratings, skin conductance response (SCR), and fear potentiated startle (FPS) were collected. Participants were pseudorandomized and double-blinded to one of two groups: They received either a drink containing glucose or saccharine 20 min before (Study 1) or immediately after extinction (Study 2). The glucose group showed a significantly stronger decrease in differential FPS during extinction (Study 1) and extinction recall (Study 2). Additionally, the glucose group showed a significantly lower contextual anxiety at test of reinstatement (Study 2). Our findings provide first evidence that glucose supports the process of fear extinction, and in particular the consolidation of fear extinction memory, and thus has potential as a beneficial adjuvant to extinction-based treatments. Registered through the German Clinical Trials Registry (https://www.bfarm.de/EN/BfArM/Tasks/German-Clinical-Trials-Register/_node.html; Study 1: DRKS00010550; Study 2: DRKS00018933).

Specific domains of FoxD4/5 activate and repress neural transcription factor genes to control the progression of immature neural ectoderm to differentiating neural plate.

FoxD4/5, a forkhead transcription factor, plays a critical role in establishing and maintaining the embryonic neural ectoderm. It both up-regulates genes that maintain a proliferative, immature neural ectoderm and down-regulates genes that promote the transition to a differentiating neural plate. We constructed deletion and mutant versions of FoxD4/5 to determine which domains are functionally responsible for these opposite activities, which regulate the critical developmental transition of neural precursors to neural progenitors to differentiating neural plate cells. Our results show that up-regulation of genes that maintain immature neural precursors (gem, zic2) requires the Acidic blob (AB) region in the N-terminal portion of the protein, indicating that the AB is the transactivating domain. Additionally, down-regulation of those genes that promote the transition to neural progenitors (sox) and those that lead to neural differentiation (zic, irx) involves: 1) an interaction with the Groucho co-repressor at the Eh-1 motif in the C-terminus; and 2) sequence downstream of this motif. Finally, the ability of FoxD4/5 to induce the ectopic expression of neural precursor genes in the ventral ectoderm also involves both the AB region and the Eh-1 motif; FoxD4/5 accomplishes ectopic neural induction by both activating neural precursor genes and repressing BMP signaling and epidermal genes. This study identifies the specific, conserved domains of the FoxD4/5 protein that allow this single transcription factor to regulate a network of genes that controls the transition of a proliferative neural ectodermal population to a committed neural plate population poised to begin differentiation.

Disruption of Model Cell Membranes by Carbon Nanotubes.

Carbon nanotubes (CNTs) have one of the highest production volumes among carbonaceous engineered nanoparticles (ENPs) worldwide and are have potential uses in applications including biomedicine, nanocomposites, and energy conversion. However, CNTs possible widespread usage and associated likelihood for biological exposures have driven concerns regarding their nanotoxicity and ecological impact. In this work, we probe the responses of planar suspended lipid bilayer membranes, used as model cell membranes, to functionalized multi-walled carbon nanotubes (MWCNT), CdSe/ZnS quantum dots, and a control organic compound, melittin, using an electrophysiological measurement platform. The electrophysiological measurements show that MWCNTs in a concentration range of 1.6 to 12 ppm disrupt lipid membranes by inducing significant transmembrane current fluxes, which suggest that MWCNTs insert and traverse the lipid bilayer membrane, forming transmembrane carbon nanotubes channels that allow the transport of ions. This paper demonstrates a direct measurement of ion migration across lipid bilayers induced by CNTs. Electrophysiological measurements can provide unique insights into the lipid bilayer-ENPs interactions and have the potential to serve as a preliminary screening tool for nanotoxicity.

Evaluation of the lower limb vasculature before free fibula flap transfer. A prospective blinded comparison between magnetic resonance angiography and digital subtraction angiography.

Introduction The aim of this study was to compare magnetic resonance angiography (MRA) with digital subtraction angiography (DSA) in the preoperative assessment of crural arteries and their skin perforators prior to free fibular transfer. Patients and methods Fifteen consecutive patients, scheduled for free vascularized fibular flap transfer, were subjected to DSA as well as MRA of the crural arteries of both legs (n = 30). All DSA and MRA images were assessed randomly, blindly, and independently by two radiologists. Each of the assessors scored the degree of stenosis of various segments on a 5 point scale from 0 (occlusive) to 4 (no stenosis). The Cohen's Kappa coefficient was used to assess the agreement between DSA and MRA scores. In addition, the number of cutaneous perforators were scored and the assessors were asked if they would advise against fibula harvest and transplantation based on the images. Results A Cohen's Kappa of 0.64, indicating "substantial agreement of stenosis severity scores" was found between the two imaging techniques. The sensitivity of MRA to detect a stenosis compared with DSA was 79% (CI 95%:60-91), and a specificity of 98% (CI 95%: 97-99). In 53 out of 60 assessments, advice on suitability for transfer were equal between DSA and MRA. The median number of cutaneous perforators that perfuse the skin overlying the fibula per leg was one for DSA as well as MRA (P = 0.142).Conclusions A substantial agreement in the assessment of stenosis severity was found between DSA and MRA. The results suggest that MRA is a good alternative to DSA in the preoperative planning of free fibula flap transplantation.

Occurrence of Hepatoblastomas in Patients with Beckwith-Wiedemann Spectrum (BWSp).

Patients with Beckwith-Wiedemann syndrome (BWS), an epigenetic imprinting disorder involving alterations in genes at the 11p15 chromosomal location, are predisposed to develop hepatoblastomas (HBs), which are rare embryonal liver tumors. Tumors can develop after a BWS diagnosis or, conversely, can be the presenting feature leading to a subsequent diagnosis. While HBs are the cardinal tumors of BWS, not all patients with the BWS spectrum will develop HBs. This observation has led to many hypotheses, including genotype-associated risk, tissue mosaicism, and tumor-specific second hits. To explore these hypotheses, we present the largest cohort of patients with BWS and HBs to date. Our cohort comprised 16 cases, and we broadened our sample size by searching the literature for all cases of BWS with HBs. From these isolated case studies, we amassed another 34 cases, bringing the total number to 50 cases of BWS-HB. We observed that paternal uniparental isodisomy (upd(11)pat) was the most common genotype, representing 38% of cases. The next most common genotype was IC2 LOM, representing 14% of cases. Five patients had clinical BWS without a molecular diagnosis. To investigate the potential mechanism of HBs in BWS, we analyzed normal liver and HB samples from eight cases and isolated tumor samples from another two cases. These samples underwent methylation testing, and 90% of our tumor samples underwent targeted cancer next-generation sequencing (NGS) panels. These matched samples provided novel insights into the oncogenesis of HBs in BWS. We found that 100% of the HBs that underwent NGS panel testing had variants in the CTNNB1 gene. We further identified three distinct groups of BWS-HB patients based on epigenotype. We also demonstrated epigenotype mosaicism, where 11p15 alterations can differ between the blood, HB, and normal liver. In light of this epigenotype mosaicism, tumor risk assessment based on blood profiling may not be accurate. Therefore, universal screening is recommended for all patients with BWS.

Phenotypic progression of skeletal anomalies in CLOVES syndrome.

Overgrowth syndromes, defined as genetic disorders in which there is disproportionate somatic growth, are challenging to diagnose due to their heterogeneous presentations and possible differing genetic etiologies. CLOVES syndrome is characterized by congenital lipomatous overgrowth, vascular malformations, epidermal nevi, and skeletal abnormalities (Scoliosis). We describe a developmental follow up of the skeletal changes in CLOVES syndrome and a detailed account of its management. We demonstrate the asymmetric growth rate of toes responsible for the macrodactyly observed, and present additional phenotypic findings, including postnatal onset of abdominal symmetry and hepatomegaly. While the etiology of CLOVES is still a mystery, its similarity to Klippel-Trenaunay syndrome suggests a shared defect in a common signaling pathway, and its asymmetric bone overgrowth supports a mosaic genetic defect as its etiology.