TNF Promoter Hypomethylation Is Associated With Mucosal Inflammation in IBD and Anti-TNF Response.

Background and Aims: Inflammatory bowel diseases (IBDs) are chronic inflammatory conditions influenced heavily by environmental factors. DNA methylation is a form of epigenetic regulation linking environmental stimuli to gene expression changes and inflammation. Here, we investigated how DNA methylation of the tumor necrosis factor (TNF) promoter differs between inflamed and uninflamed mucosa of IBD patients, including anti-TNF responders and nonresponders. Methods: We obtained mucosal biopsies from 200 participants (133 IBDs and 67 controls) and analyzed TNF promoter methylation using bisulfite sequencing, comparing inflamed with uninflamed segments, in addition to paired inflamed/uninflamed samples from individual patients. We conducted similar analyses on purified intestinal epithelial cells from bowel resections. We also compared TNF methylation levels of inflamed and uninflamed mucosa from a separate cohort of 15 anti-TNF responders and 17 nonresponders. Finally, we sequenced DNA methyltransferase genes to identify rare variants in IBD patients and functionally tested them using rescue experiments in a zebrafish genetic model of DNA methylation deficiency. Results: TNF promoter methylation levels were decreased in inflamed mucosa of IBD patients and correlated with disease severity. Isolated intestinal epithelial cells from inflamed tissue showed proportional decreases in TNF methylation. Anti-TNF nonresponders showed lower levels of TNF methylation than responders in uninflamed mucosa. Our sequencing analysis revealed 2 missense variants in DNA methyltransferase 1, 1 of which had reduced function in vivo. Conclusion: Our study reveals an association of TNF promoter hypomethylation with mucosal inflammation, suggesting that IBD patients may be particularly sensitive to inflammatory environmental insults affecting DNA methylation. Together, our analyses indicate that TNF promoter methylation analysis may aid in the characterization of IBD status and evaluation of anti-TNF therapy response.

Identification of Environmental Compounds That May Trigger Early Female Puberty by Activating Human GnRHR and KISS1R.

There has been an alarming trend toward earlier puberty in girls, suggesting the influence of an environmental factor(s). As the reactivation of the reproductive axis during puberty is thought to be mediated by the hypothalamic neuropeptides kisspeptin and gonadotropin-releasing hormone (GnRH), we asked whether an environmental compound might activate the kisspeptin (KISS1R) or GnRH receptor (GnRHR). We used GnRHR or KISS1R-expressing HEK293 cells to screen the Tox21 10K compound library, a compendium of pharmaceuticals and environmental compounds, for GnRHR and KISS1R activation. Agonists were identified using Ca2+ flux and phosphorylated extracellularly regulated kinase (p-ERK) detection assays. Follow-up studies included measurement of genes known to be upregulated upon receptor activation using relevant murine or human cell lines and molecular docking simulation. Musk ambrette was identified as a KISS1R agonist, and treatment with musk ambrette led to increased expression of Gnrh1 in murine and human hypothalamic cells and expansion of GnRH neuronal area in developing zebrafish larvae. Molecular docking demonstrated that musk ambrette interacts with the His309, Gln122, and Gln123 residues of the KISS1R. A group of cholinergic agonists with structures similar to methacholine was identified as GnRHR agonists. When applied to murine gonadotrope cells, these agonists upregulated Fos, Jun, and/or Egr1. Molecular docking revealed a potential interaction between GnRHR and 5 agonists, with Asn305 constituting the most conservative GnRHR binding site. In summary, using a Tox21 10K compound library screen combined with cellular, molecular, and structural biology techniques, we have identified novel environmental agents that may activate the human KISS1R or GnRHR.

Heart failure symptom burden, dietary intake, and inflammation: An integrative review of the literature.

Heart failure (HF) is characterized by high symptom burden including, but not limited to fatigue, dyspnea, and edema. Up to 21.5% of HF patients experience significant depressive symptoms, much higher than 7.1% in adults without HF. Diet, metabolites, and other inflammatory mechanisms have gained notable attention in recent studies for contributions to symptoms in HF. Symptoms for black adults (B/As) with HF are often influenced by lifestyle factors, which may influence their higher mortality rates; few studies address these factors. Distinguishing the links between key elements with diet, inflammation, and symptoms may bring clarity for new dietary strategies in HF clinical care. The purpose of this integrative review is to examine the existing literature regarding relationships among physiologic pathways in HF along with physical and emotional symptoms in the context of inflammation, dietary intake, tumor necrosis factor-alpha (TNF-α), a biomarker of inflammation, and trimethylamine-N-Oxide (TMAO). Based on available evidence, inflammation may be a key link between physical symptoms, diet, depression, TMAO, and TNF-α in persons with HF and warrants further examination to clarify pathological links to solidify evidence for better guidance with dietary modifications. The literature reviewed in this study demonstrates that more work is needed to examine dietary planning, social support, and differences between men and women in the B/A community. Results of this literature review call attention to the essential, personalized care needs related to symptom monitoring and dietary planning which is expected to decrease symptom burden in the HF population.

Association of deep phenotyping with diagnostic yield of prenatal exome sequencing for fetal brain abnormalities.

PURPOSE: To evaluate whether deep prenatal phenotyping of fetal brain abnormalities (FBAs) increases diagnostic yield of trio-exome sequencing (ES) compared with standard phenotyping. METHODS: Retrospective exploratory analysis of a multicenter prenatal ES study. Participants were eligible if an FBA was diagnosed and subsequently found to have a normal microarray. Deep phenotyping was defined as phenotype based on targeted ultrasound plus prenatal/postnatal magnetic resonance imaging, autopsy, and/or known phenotypes of other affected family members. Standard phenotyping was based on targeted ultrasound alone. FBAs were categorized by major brain findings on prenatal ultrasound. Cases with positive ES results were compared with those that have negative results by available phenotyping, as well as diagnosed FBAs. RESULTS: A total of 76 trios with FBAs were identified, of which 25 (33%) cases had positive ES results and 51 (67%) had negative results. Individual modalities of deep phenotyping were not associated with diagnostic ES results. The most common FBAs identified were posterior fossa anomalies and midline defects. Neural tube defects were significantly associated with receipt of a negative ES result (0% vs 22%, P = .01). CONCLUSION: Deep phenotyping was not associated with increased diagnostic yield of ES for FBA in this small cohort. Neural tube defects were associated with negative ES results.

The p190 RhoGAPs, ARHGAP35, and ARHGAP5 are implicated in GnRH neuronal development: Evidence from patients with idiopathic hypogonadotropic hypogonadism, zebrafish, and in vitro GAP activity assay.

PURPOSE: The study aimed to identify novel genes for idiopathic hypogonadotropic hypogonadism (IHH). METHODS: A cohort of 1387 probands with IHH underwent exome sequencing and de novo, familial, and cohort-wide investigations. Functional studies were performed on 2 p190 Rho GTPase-activating proteins (p190 RhoGAP), ARHGAP35 and ARHGAP5, which involved in vivo modeling in larval zebrafish and an in vitro p190A-GAP activity assay. RESULTS: Rare protein-truncating variants (PTVs; n = 5) and missense variants in the RhoGAP domain (n = 7) in ARHGAP35 were identified in IHH cases (rare variant enrichment: PTV [unadjusted P = 3.1E-06] and missense [adjusted P = 4.9E-03] vs controls). Zebrafish modeling using gnrh3:egfp phenotype assessment showed that mutant larvae with deficient arhgap35a, the predominant ARHGAP35 paralog in the zebrafish brain, display decreased GnRH3-GFP+ neuronal area, a readout for IHH. In vitro GAP activity studies showed that 1 rare missense variant [ARHGAP35 p.(Arg1284Trp)] had decreased GAP activity. Rare PTVs (n = 2) also were discovered in ARHGAP5, a paralog of ARHGAP35; however, arhgap5 zebrafish mutants did not display significant GnRH3-GFP+ abnormalities. CONCLUSION: This study identified ARHGAP35 as a new autosomal dominant genetic driver for IHH and ARHGAP5 as a candidate gene for IHH. These observations suggest a novel role for the p190 RhoGAP proteins in GnRH neuronal development and integrity.

Testing for rare genetic causes of obesity: findings and experiences from a pediatric weight management program.

BACKGROUND: Genetic screening for youth with obesity in the absence of syndromic findings has not been part of obesity management. For children with early onset obesity, genetic screening is recommended for those having clinical features of genetic obesity syndromes (including hyperphagia). OBJECTIVES: The overarching goal of this work is to report the findings and experiences from one pediatric weight management program that implemented targeted sequencing analysis for genes known to cause rare genetic disorders of obesity. SUBJECTS/METHODS: This exploratory study evaluated youth tested over an 18-month period using a panel of 40-genes in the melanocortin 4 receptor pathway. Medical records were reviewed for demographic and visit information, including body mass index (BMI) percent of 95th percentile (%BMIp95) and two eating behaviors. RESULTS: Of 117 subjects: 51.3% were male; 53.8% Hispanic; mean age 10.2 years (SD 3.8); mean %BMIp95 157% (SD 29%). Most subjects were self- or caregiver-reported to have overeating to excess or binge eating (80.3%) and sneaking food or eating in secret (59.0%). Among analyzed genes, 72 subjects (61.5%) had at least one variant reported; 50 (42.7%) had a single variant reported; 22 (18.8%) had 2-4 variants reported; most variants were rare (<0.05% minor allele frequency [MAF]), and of uncertain significance; all variants were heterozygous. Nine subjects (7.7%) had a variant reported as PSCK1 "risk" or MC4R "likely pathogenic"; 39 (33.3%) had a Bardet-Biedl Syndrome (BBS) gene variant (4 with "pathogenic" or "likely pathogenic" variants). Therefore, 9 youth (7.7%) had gene variants previously identified as increasing risk for obesity and 4 youth (3.4%) had BBS carrier status. CONCLUSIONS: Panel testing identified rare variants of uncertain significance in most youth tested, and infrequently identified variants previously reported to increase the risk for obesity. Further research in larger cohorts is needed to understand how genetic variants influence the expression of non-syndromic obesity.

Impact on hospital-wide antipsychotic prescribing practices through physician peer comparison letters.

Peer comparison is a behavioral strategy that provides feedback to individuals on how they compare with others. It is used to improve health care quality, reduce inappropriate prescribing, and improve physician performance. There is very little data on peer comparison and the impact on system-wide prescribing practices, particularly with antipsychotics. To that end, the Maryland statewide pharmacy and therapeutics committee reviews hospital-level antipsychotic data for 5 facilities on a quarterly basis, including high doses and polypharmacy. One facility, Springfield Hospital Center, consistently stood out in 2016 as having higher rates of high doses of haloperidol, olanzapine, and quetiapine as well as patients receiving 3 or more antipsychotics. The pharmacist began to send out individual letters to the psychiatrists detailing their prescribing habits in these areas compared with other psychiatrists and the other state facilities. Over the course of 4 years, the percentage of patients on high doses of 3 antipsychotics substantially decreased. The percentage of patients on polypharmacy in the facility decreased, but not at the same rate as the other hospitals, leaving the facility even higher than the state average at the end of the 4-year period. Pharmacist-initiated physician peer comparison letters were associated with a considerable decrease in the prevalence of high-dose olanzapine, haloperidol, and quetiapine but did not appear to impact antipsychotic polypharmacy. This type of communication may be beneficial for stimulating system-wide changes in prescribing practices for high doses of antipsychotics; however, more individualized interventions are likely needed to reduce antipsychotic polypharmacy.

Probable haloperidol decanoate-induced fever in an African American with benign ethnic neutropenia: A case report.

We present a case in which a patient developed fever and leukocytosis subsequent to each monthly haloperidol decanoate injection, an adverse reaction that does not meet the diagnostic criteria of neuroleptic malignant syndrome (NMS) or any previously reported adverse reaction for this medication. A patient being treated with haloperidol decanoate for psychosis experienced a fever within 3 days of injection and leukocytosis along with swelling, pain, and a "knot" feeling at the injection site. This recurred after each injection for several months. Muscle rigidity or changes in vital signs other than temperature were not noted. Temperature and injection site reactions resolved with administration of acetaminophen and ibuprofen. The elevation in temperature was discovered as a result of universal twice daily temperature monitoring implemented due to the COVID-19 pandemic. Reports of fever with antipsychotics are typically associated with NMS or heat stroke; the details of this case do not meet the clinical criteria for either. Similar reactions are reported for other antipsychotics, such as clozapine and olanzapine, but not for haloperidol. The recommendation was to discontinue use of the medication due to an unclear mechanism of the reaction.

CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module.

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

Loss of CBY1 results in a ciliopathy characterized by features of Joubert syndrome.

Ciliopathies are clinically and genetically heterogeneous diseases. We studied three patients from two independent families presenting with features of Joubert syndrome: abnormal breathing pattern during infancy, developmental delay/intellectual disability, cerebellar ataxia, molar tooth sign on magnetic resonance imaging scans, and polydactyly. We identified biallelic loss-of-function (LOF) variants in CBY1, segregating with the clinical features of Joubert syndrome in the families. CBY1 localizes to the distal end of the mother centriole, contributing to the formation and function of cilia. In accordance with the clinical and mutational findings in the affected individuals, we demonstrated that depletion of Cby1 in zebrafish causes ciliopathy-related phenotypes. Levels of CBY1 transcript were found reduced in the patients compared with controls, suggesting degradation of the mutated transcript through nonsense-mediated messenger RNA decay. Accordingly, we could detect CBY1 protein in fibroblasts from controls, but not from patients by immunofluorescence. Furthermore, we observed reduced ability to ciliate, increased ciliary length, and reduced levels of the ciliary proteins AHI1 and ARL13B in patient fibroblasts. Our data show that CBY1 LOF-variants cause a ciliopathy with features of Joubert syndrome.

TCF12 haploinsufficiency causes autosomal dominant Kallmann syndrome and reveals network-level interactions between causal loci.

Dysfunction of the gonadotropin-releasing hormone (GnRH) axis causes a range of reproductive phenotypes resulting from defects in the specification, migration and/or function of GnRH neurons. To identify additional molecular components of this system, we initiated a systematic genetic interrogation of families with isolated GnRH deficiency (IGD). Here, we report 13 families (12 autosomal dominant and one autosomal recessive) with an anosmic form of IGD (Kallmann syndrome) with loss-of-function mutations in TCF12, a locus also known to cause syndromic and non-syndromic craniosynostosis. We show that loss of tcf12 in zebrafish larvae perturbs GnRH neuronal patterning with concomitant attenuation of the orthologous expression of tcf3a/b, encoding a binding partner of TCF12, and stub1, a gene that is both mutated in other syndromic forms of IGD and maps to a TCF12 affinity network. Finally, we report that restored STUB1 mRNA rescues loss of tcf12 in vivo. Our data extend the mutational landscape of IGD, highlight the genetic links between craniofacial patterning and GnRH dysfunction and begin to assemble the functional network that regulates the development of the GnRH axis.

CSGALNACT1-congenital disorder of glycosylation: A mild skeletal dysplasia with advanced bone age.

Congenital disorders of glycosylation (CDGs) comprise a large number of inherited metabolic defects that affect the biosynthesis and attachment of glycans. CDGs manifest as a broad spectrum of disease, most often including neurodevelopmental and skeletal abnormalities and skin laxity. Two patients with biallelic CSGALNACT1 variants and a mild skeletal dysplasia have been described previously. We investigated two unrelated patients presenting with short stature with advanced bone age, facial dysmorphism, and mild language delay, in whom trio-exome sequencing identified novel biallelic CSGALNACT1 variants: compound heterozygosity for c.1294G>T (p.Asp432Tyr) and the deletion of exon 4 that includes the start codon in one patient, and homozygosity for c.791A>G (p.Asn264Ser) in the other patient. CSGALNACT1 encodes CSGalNAcT-1, a key enzyme in the biosynthesis of sulfated glycosaminoglycans chondroitin and dermatan sulfate. Biochemical studies demonstrated significantly reduced CSGalNAcT-1 activity of the novel missense variants, as reported previously for the p.Pro384Arg variant. Altered levels of chondroitin, dermatan, and heparan sulfate moieties were observed in patients' fibroblasts compared to controls. Our data indicate that biallelic loss-of-function mutations in CSGALNACT1 disturb glycosaminoglycan synthesis and cause a mild skeletal dysplasia with advanced bone age, CSGALNACT1-CDG.

Randomized Controlled Trial of a Natural Food-Based Fiber Solution to Prevent Constipation in Postoperative Spine Fusion Patients.

BACKGROUND: Constipation after orthopaedic surgery occurs frequently, likely due to a combination of high levels of opioid medications for severe pain management and mobility limitations after surgery. It can result in serious complications, increased cost, and patient discomfort. PURPOSE: This study evaluated a natural food-based fiber solution to prevent constipation in postoperative orthopaedic patients. METHODS: A posttest control group-randomized study design was used. Dependent variables were presence of postoperative constipation, time to first bowel movement (BM), and total number of postoperative BMs. Descriptive statistics, Student's t tests, and Mann-Whitney nonparametric 2-group tests with chi-square analysis were used. Level of significance for all tests was p < .05. Forty-six participants were evaluated. RESULTS: Ages were similar for both the intervention and control groups. Bowel Function Index (BFI) scores were not significantly different (p = .448). No significant group differences were present for the individual BFI item scores (p > .05). The number of patients with a BM during the first 3 days was not significantly different (p = .489). There were no significant differences found between the 2 groups regarding laxative administration (p > .05 for all laxatives). CONCLUSION: Further studies are indicated that address natural fibers and pharmaceutical methods for the prevention of constipation after spinal surgery.

Impact of a Preset Daily Cell Phone Alarm on Medication Adherence for Aspirin as Antithrombotic Therapy.

The ability of patients to adhere to medication regimens is considered critical to achieving optimal results. Many patients are discharged with aspirin (ASA) as an anticoagulant for venous thrombosis embolism prophylaxis after joint replacement surgery. In studies where ASA was prescribed as an antithrombotic after selected orthopaedic surgeries, both lack of understanding and missing doses were identified as factors that affected adherence rates (D. , ). The purpose of this study was to explore the preliminary impact of a preset telephone alarm on medication adherence in adults prescribed ASA for 35 days after knee or hip arthroplasty. This was a randomized controlled trial (n = 79). Adherence was measured with a four-question self-reporting tool. Average age was 61 years. The majority were female (59.5%) and Caucasian (62.0%) with college or graduate degree-level education (78.5%). When comparing the groups, there were no significant differences between the two groups for the demographics of age, gender, and race. The alarm group had significantly better adherence rates, with fewer people who forgot to take their ASA; only 29.7% of the alarm group ever forgot to take their medication compared with 59.5% of the no-alarm group (p = .008). It seems that simple cell phone alarms can serve as effective reminders to patients to take selected medications as prescribed.

Germline-Activating RRAS2 Mutations Cause Noonan Syndrome.

Noonan syndrome (NS) is characterized by distinctive craniofacial appearance, short stature, and congenital heart disease. Approximately 80% of individuals with NS harbor mutations in genes whose products are involved in the RAS/mitogen-activating protein kinase (MAPK) pathway. However, the underlying genetic causes in nearly 20% of individuals with NS phenotype remain unexplained. Here, we report four de novo RRAS2 variants in three individuals with NS. RRAS2 is a member of the RAS subfamily and is ubiquitously expressed. Three variants, c.70_78dup (p.Gly24_Gly26dup), c.216A>T (p.Gln72His), and c.215A>T (p.Gln72Leu), have been found in cancers; our functional analyses showed that these three changes induced elevated association of RAF1 and that they activated ERK1/2 and ELK1. Notably, prominent activation of ERK1/2 and ELK1 by p.Gln72Leu associates with the severe phenotype of the individual harboring this change. To examine variant pathogenicity in vivo, we generated zebrafish models. Larvae overexpressing c.70_78dup (p.Gly24_Gly26dup) or c.216A>T (p.Gln72His) variants, but not wild-type RRAS2 RNAs, showed craniofacial defects and macrocephaly. The same dose injection of mRNA encoding c.215A>T (p.Gln72Leu) caused severe developmental impairments and low dose overexpression of this variant induced craniofacial defects. In contrast, the RRAS2 c.224T>G (p.Phe75Cys) change, located on the same allele with p.Gln72His in an individual with NS, resulted in no aberrant in vitro or in vivo phenotypes by itself. Together, our findings suggest that activating RRAS2 mutations can cause NS and expand the involvement of RRAS2 proto-oncogene to rare germline disorders.

Bi-allelic Variants in DYNC1I2 Cause Syndromic Microcephaly with Intellectual Disability, Cerebral Malformations, and Dysmorphic Facial Features.

Cargo transport along the cytoplasmic microtubular network is essential for neuronal function, and cytoplasmic dynein-1 is an established molecular motor that is critical for neurogenesis and homeostasis. We performed whole-exome sequencing, homozygosity mapping, and chromosomal microarray studies in five individuals from three independent pedigrees and identified likely-pathogenic variants in DYNC1I2 (Dynein Cytoplasmic 1 Intermediate Chain 2), encoding a component of the cytoplasmic dynein 1 complex. In a consanguineous Pakistani family with three affected individuals presenting with microcephaly, severe intellectual disability, simplification of cerebral gyration, corpus callosum hypoplasia, and dysmorphic facial features, we identified a homozygous splice donor site variant (GenBank: NM_001378.2:c.607+1G>A). We report two additional individuals who have similar neurodevelopmental deficits and craniofacial features and harbor deleterious variants; one individual bears a c.740A>G (p.Tyr247Cys) change in trans with a 374 kb deletion encompassing DYNC1I2, and an unrelated individual harbors the compound-heterozygous variants c.868C>T (p.Gln290∗) and c.740A>G (p.Tyr247Cys). Zebrafish larvae subjected to CRISPR-Cas9 gene disruption or transient suppression of dync1i2a displayed significantly altered craniofacial patterning with concomitant reduction in head size. We monitored cell death and cell cycle progression in dync1i2a zebrafish models and observed significantly increased apoptosis, likely due to prolonged mitosis caused by abnormal spindle morphology, and this finding offers initial insights into the cellular basis of microcephaly. Additionally, complementation studies in zebrafish demonstrate that p.Tyr247Cys attenuates gene function, consistent with protein structural analysis. Our genetic and functional data indicate that DYNC1I2 dysfunction probably causes an autosomal-recessive microcephaly syndrome and highlight further the critical roles of the dynein-1 complex in neurodevelopment.

Adherence to the WHO surgical safety checklist: an observational study in a Swiss academic center.

BACKGROUND: The World Health Organization (WHO) Surgical Safety Checklist is used globally to ensure patient safety during surgery. Two years after its implementation in the University Hospital Basel's operating rooms, adherence to the protocol was evaluated. METHODS: This mixed method observational study took place in the surgical department of the University Hospital of Basel, Switzerland from April to August 2017. Data collection was via individual structured interviews with selected OR team members regarding checklist adherence and on-site non-participant observations of Team Time Out and Team Sign Out sequences in the OR. Data were subjected to thematic analysis and descriptive statistics compiled. RESULTS: Comprehensive local expert interviews indicated that individual, procedural and contextual variables influenced the application of the checklist. Facilitating factors included well-informed specialists who advocated the use of the Checklist, as well as teams focused on the checklist's intended process and on its content. In contrast, factors such as staff insecurity, a generally negative attitude towards the checklist, a lack of teamwork, and hesitance to complete the checklist, hindered its implementation.The checklist's application was evaluated in 104 on-site observations comprising of 72 Team Time Out (TTO) and 32 Team Sign Out (TSO) sections. Adherence to the protocol ranged between 96 and 100% in TTO and 22% in TSO respectively. Lack of implementation of the TSO was mainly due to the absence of one of the key OR team members, who were busy with other tasks or no longer present in the operating room. CONCLUSION: The study illustrates factors, which foster and hinder consistent application of the WHO surgical safety checklist namely individual, procedural and contextual. It also demonstrates that the TTO was consistently and correctly applied, while the unavailability of key OR team members at sign-out time was the most common reason for omission or incomplete use of the TSO.

Mutations in NCAPG2 Cause a Severe Neurodevelopmental Syndrome that Expands the Phenotypic Spectrum of Condensinopathies.

The use of whole-exome and whole-genome sequencing has been a catalyst for a genotype-first approach to diagnostics. Under this paradigm, we have implemented systematic sequencing of neonates and young children with a suspected genetic disorder. Here, we report on two families with recessive mutations in NCAPG2 and overlapping clinical phenotypes that include severe neurodevelopmental defects, failure to thrive, ocular abnormalities, and defects in urogenital and limb morphogenesis. NCAPG2 encodes a member of the condensin II complex, necessary for the condensation of chromosomes prior to cell division. Consistent with a causal role for NCAPG2, we found abnormal chromosome condensation, augmented anaphase chromatin-bridge formation, and micronuclei in daughter cells of proband skin fibroblasts. To test the functional relevance of the discovered variants, we generated an ncapg2 zebrafish model. Morphants displayed clinically relevant phenotypes, such as renal anomalies, microcephaly, and concomitant increases in apoptosis and altered mitotic progression. These could be rescued by wild-type but not mutant human NCAPG2 mRNA and were recapitulated in CRISPR-Cas9 F0 mutants. Finally, we noted that the individual with a complex urogenital defect also harbored a heterozygous NPHP1 deletion, a common contributor to nephronophthisis. To test whether sensitization at the NPHP1 locus might contribute to a more severe renal phenotype, we co-suppressed nphp1 and ncapg2, which resulted in significantly more dysplastic renal tubules in zebrafish larvae. Together, our data suggest that impaired function of NCAPG2 results in a severe condensinopathy, and they highlight the potential utility of examining candidate pathogenic lesions beyond the primary disease locus.

Small molecule inhibition of RAS/MAPK signaling ameliorates developmental pathologies of Kabuki Syndrome.

Kabuki Syndrome (KS) is a rare disorder characterized by distinctive facial features, short stature, skeletal abnormalities, and neurodevelopmental deficits. Previously, we showed that loss of function of RAP1A, a RAF1 regulator, can activate the RAS/MAPK pathway and cause KS, an observation recapitulated in other genetic models of the disorder. These data suggested that suppression of this signaling cascade might be of therapeutic benefit for some features of KS. To pursue this possibility, we performed a focused small molecule screen of a series of RAS/MAPK pathway inhibitors, where we tested their ability to rescue disease-relevant phenotypes in a zebrafish model of the most common KS locus, kmt2d. Consistent with a pathway-driven screening paradigm, two of 27 compounds showed reproducible rescue of early developmental pathologies. Further analyses showed that one compound, desmethyl-Dabrafenib (dmDf), induced no overt pathologies in zebrafish embryos but could rescue MEK hyperactivation in vivo and, concomitantly, structural KS-relevant phenotypes in all KS zebrafish models (kmt2d, kmd6a and rap1). Mass spectrometry quantitation suggested that a 100 nM dose resulted in sub-nanomolar exposure of this inhibitor and was sufficient to rescue both mandibular and neurodevelopmental defects. Crucially, germline kmt2d mutants recapitulated the gastrulation movement defects, micrognathia and neurogenesis phenotypes of transient models; treatment with dmDf ameliorated all of them significantly. Taken together, our data reinforce a causal link between MEK hyperactivation and KS and suggest that chemical suppression of BRAF might be of potential clinical utility for some features of this disorder.

The Human FSGS-Causing ANLN R431C Mutation Induces Dysregulated PI3K/AKT/mTOR/Rac1 Signaling in Podocytes.

BACKGROUND: We previously reported that mutations in the anillin (ANLN) gene cause familial forms of FSGS. ANLN is an F-actin binding protein that modulates podocyte cell motility and interacts with the phosphoinositide 3-kinase (PI3K) pathway through the slit diaphragm adaptor protein CD2-associated protein (CD2AP). However, it is unclear how the ANLN mutations cause the FSGS phenotype. We hypothesized that the R431C mutation exerts its pathogenic effects by uncoupling ANLN from CD2AP. METHODS: We conducted in vivo complementation assays in zebrafish to determine the effect of the previously identified missense ANLN variants, ANLNR431C and ANLNG618C during development. We also performed in vitro functional assays using human podocyte cell lines stably expressing wild-type ANLN (ANLNWT ) or ANLNR431C . RESULTS: Experiments in anln-deficient zebrafish embryos showed a loss-of-function effect for each ANLN variant. In human podocyte lines, expression of ANLNR431C increased cell migration, proliferation, and apoptosis. Biochemical characterization of ANLNR431C -expressing podocytes revealed hyperactivation of the PI3K/AKT/mTOR/p70S6K/Rac1 signaling axis and activation of mTOR-driven endoplasmic reticulum stress in ANLNR431C -expressing podocytes. Inhibition of mTOR, GSK-3ß, Rac1, or calcineurin ameliorated the effects of ANLNR431C . Additionally, inhibition of the calcineurin/NFAT pathway reduced the expression of endogenous ANLN and mTOR. CONCLUSIONS: The ANLNR431C mutation causes multiple derangements in podocyte function through hyperactivation of PI3K/AKT/mTOR/p70S6K/Rac1 signaling. Our findings suggest that the benefits of calcineurin inhibition in FSGS may be due, in part, to the suppression of ANLN and mTOR. Moreover, these studies illustrate that rational therapeutic targets for familial FSGS can be identified through biochemical characterization of dysregulated podocyte phenotypes.