RNA variant assessment using transactivation and transdifferentiation.

Understanding the impact of splicing and nonsense variants on RNA is crucial for the resolution of variant classification as well as their suitability for precision medicine interventions. This is primarily enabled through RNA studies involving transcriptomics followed by targeted assays using RNA isolated from clinically accessible tissues (CATs) such as blood or skin of affected individuals. Insufficient disease gene expression in CATs does however pose a major barrier to RNA based investigations, which we show is relevant to 1,436 Mendelian disease genes. We term these "silent" Mendelian genes (SMGs), the largest portion (36%) of which are associated with neurological disorders. We developed two approaches to induce SMG expression in human dermal fibroblasts (HDFs) to overcome this limitation, including CRISPR-activation-based gene transactivation and fibroblast-to-neuron transdifferentiation. Initial transactivation screens involving 40 SMGs stimulated our development of a highly multiplexed transactivation system culminating in the 6- to 90,000-fold induction of expression of 20/20 (100%) SMGs tested in HDFs. Transdifferentiation of HDFs directly to neurons led to expression of 193/516 (37.4%) of SMGs implicated in neurological disease. The magnitude and isoform diversity of SMG expression following either transactivation or transdifferentiation was comparable to clinically relevant tissues. We apply transdifferentiation and/or gene transactivation combined with short- and long-read RNA sequencing to investigate the impact that variants in USH2A, SCN1A, DMD, and PAK3 have on RNA using HDFs derived from affected individuals. Transactivation and transdifferentiation represent rapid, scalable functional genomic solutions to investigate variants impacting SMGs in the patient cell and genomic context.

Proteomic analysis of the developing mammalian brain links PCDH19 to the Wnt/ß-catenin signalling pathway.

Clustering Epilepsy (CE) is a neurological disorder caused by pathogenic variants of the Protocadherin 19 (PCDH19) gene. PCDH19 encodes a protein involved in cell adhesion and Estrogen Receptor α mediated-gene regulation. To gain further insights into the molecular role of PCDH19 in the brain, we investigated the PCDH19 interactome in the developing mouse hippocampus and cortex. Combined with a meta-analysis of all reported PCDH19 interacting proteins, our results show that PCDH19 interacts with proteins involved in actin, microtubule, and gene regulation. We report CAPZA1, αN-catenin and, importantly, ß-catenin as novel PCDH19 interacting proteins. Furthermore, we show that PCDH19 is a regulator of ß-catenin transcriptional activity, and that this pathway is disrupted in CE individuals. Overall, our results support the involvement of PCDH19 in the cytoskeletal network and point to signalling pathways where PCDH19 plays critical roles.

Application of multiple mosaic callers improves post-zygotic mutation detection from exome sequencing data.

PURPOSE: The gold standard for identification of post-zygotic variants (PZVs) is droplet digital polymerase chain reaction or high-depth sequencing across multiple tissues types. These approaches are yet to be systematically implemented for monogenic disorders. We developed PZV detection pipelines for correct classification of de novo variants. METHOD: Our pipelines detect PZV in parents (gonosomal mosaicism [pGoM]) and children (somatic mosaicism, "M3"). We applied them to research exome sequencing (ES) data from the Australian Cerebral Palsy Biobank (n = 145 trios) and Simons Simplex Collection (n = 405 families). Candidate mosaic variants were validated using deep amplicon sequencing or droplet digital polymerase chain reaction. RESULTS: 69.2% (M3trio), 63.9% (M3single), and 92.7% (pGoM) of detected variants were validated, with 48.6%, 56.7%, and 26.2% of variants, respectively, meeting strict criteria for mosaicism. In the Australian Cerebral Palsy Biobank, 16.6% of probands and 20.7% of parents had at least 1 true-positive somatic or pGoM variant, respectively. A large proportion of PZVs detected in Simons Simplex Collection parents (79.8%) and child (94.5%) were not previously reported. We reclassified 3.7% to 8.0% of germline de novo variants as mosaic. CONCLUSION: Many PZVs were incorrectly classified as germline variants or missed by previous approaches. Systematic application of our pipelines could increase genetic diagnostic rate, improve estimates of recurrence risk in families, and benefit novel disease gene identification.

Cognitive impairment and exploitation: connecting fragments of a bigger picture through data.

BACKGROUND: Exploitation poses a significant public health concern. This paper highlights 'jigsaw pieces' of statistical evidence, indicating cognitive impairment as a pre- or co-existing factor in exploitation. METHODS: We reviewed English Safeguarding Adults Collection (SAC) data and Safeguarding Adults Reviews (SARs) from 2017 to 22. Data relevant to exploitation and cognitive impairment were analysed using summary statistics and 'analysis of variance'. RESULTS: Despite estimates suggesting cognitive impairments may be prevalent among people experiencing exploitation in England, national datasets miss opportunities to illuminate this issue. Although SAC data include statistics on support needs and various forms of abuse and exploitation, they lack intersectional data. Significant regional variations in recorded safeguarding investigations and potential conflation between abuse and exploitation also suggest data inconsistencies. Increased safeguarding investigations for people who were not previously in contact with services indicate that adults may be 'slipping through the net'. SARs, although representing serious cases, provide stronger evidence linking cognitive impairment with risks of exploitation. CONCLUSIONS: This study identifies opportunities to collect detailed information on cognitive impairment and exploitation. The extremely limited quantitative evidence-base could be enhanced using existing data channels to build a more robust picture, as well as improve prevention, identification and response efforts for 'at-risk' adults.

Ex vivo biomechanical evaluation of tissue construct strength in an equine colopexy model.

OBJECTIVE: To compare strength of left paramedian colopexies using various techniques in equine ex vivo models. STUDY DESIGN: Experimental study. SAMPLE POPULATION: Equine cadavers euthanized for nongastrointestinal pathology (36 specimens derived from 9 horses). METHODS: Colopexies were performed after euthanasia. Suture pattern (horizontal mattress vs. cruciate) and incorporation of dorsal sheath of the rectus abdominis (partial-thickness) versus incorporation of dorsal and ventral sheath of the rectus abdominis (full-thickness) were evaluated. Single cycle load to failure, work to peak load, stiffness, and mode of failure of colopexy tissue constructs were assessed. RESULTS: Mean load to failure of all constructs ranged from 102.26 to 166.38 N. Partial-thickness bites demonstrated a mean load to failure and standard deviation (SD) of 111.91 (35.88) N and 102.26 (30.06) N (p < .05) which was significantly lower than the mean and SD of full-thickness bites (166.3 [72.42] N and 163.21 [51.40 N]), respectively. All full-thickness bites regardless of suture pattern and over half of partial-thickness bites failed at the colonic wall. There was no significant difference in load to failure compared to mode of failure. CONCLUSION: A stronger colopexy was achieved with a full-thickness bite regardless of the suture pattern. The most common mode of failure was the colon wall. CLINICAL SIGNIFICANCE: Incorporating ventral and dorsal fascia of the rectus abdominus provided a stronger colopexy structure, which may necessitate a second incision or subcutaneous palpation of the needle when performing a colopexy. The lateral band of the colon failed in most constructs (77%) regardless of technique, which could weaken the colonic wall and risk colonic rupture.

Using Virtual Emergency Medicine Clinicians as a Health System Entry Point (Virtual First): Cross-Sectional Survey Study.

BACKGROUND: The COVID-19 pandemic accelerated the use and acceptance of telemedicine. Simultaneously, emergency departments (EDs) have experienced increased ED boarding. With this acceptance of telemedicine and the weighty increase in patient boarding, we proposed the innovative Virtual First (VF) program to leverage emergency medicine clinicians' (EMCs) ability to triage patients. VF seeks to reduce unnecessary ED visits by connecting patients with EMCs prior to seeking in-person care rather than using traditional ED referral systems. OBJECTIVE: The goal of this study is to investigate how patients' access to EMCs from home via the establishment of VF changed how patients sought care for acute care needs. METHODS: VF is a synchronous virtual video visit at a tertiary care academic hospital. VF was staffed by EMCs and enabled full management of patient complaints or, if necessary, referral to the appropriate level of care. Patients self-selected this service as an alternative to seeking in-person care at a primary care provider, urgent care center, or ED. A postvisit convenience sample survey was collected through a phone SMS text message or email to VF users. This is a cross-sectional survey study. The primary outcome measure is based on responses to the question "How would you have sought care if a VF visit was not available to you?" Secondary outcome measures describe valued aspects and criticisms. Results were analyzed using descriptive statistics. RESULTS: There were 3097 patients seen via VF from July 2021 through May 2022. A total of 176 (5.7%) patients completed the survey. Of these, 87 (49.4%) would have sought care at urgent care centers if VF had not been available. There were 28 (15.9%) patients, 26 (14.8%) patients, and 1 (0.6%) patient that would have sought care at primary care providers, EDs, or other locations, respectively. Interestingly, 34 (19.3%) patients would not have sought care. The most valued aspect of VF was receiving care in the comfort of the home (n=137, 77.8%). For suggested improvements, 58 (33%) patients most commonly included "Nothing" as free text. CONCLUSIONS: VF has the potential to restructure how patients seek medical care by connecting EMCs with patients prior to ED arrival. Without the option of VF, 64.2% (113/177) of patients would have sought care at an acute care facility. VF's innovative employment of EMCs allows for acute care needs to be treated virtually if feasible. If not, EMCs understand the local resources to better direct patients to the appropriate site. This has the potential to substantially decrease patient costs because patients are given the appropriate destination for in-person care, reducing the likelihood of the need for transfer and multiple ED visits.

Assessment and comparison of microcirculation and macrocirculation in horses undergoing emergency exploratory celiotomy versus elective surgical procedures.

OBJECTIVE: To assess oral buccal microcirculation by hand-held videomicroscopy in horses during colic surgery, comparing microcirculation values with macrocirculatory parameters and with those of healthy elective surgical horses. STUDY DESIGN: Clinical prospective study. ANIMALS: Client-owned horses (nine in the colic group; 11 in the elective group). METHODS: In the colic group, buccal mucosal side stream dark-field microscopy (DFM) videos, cardiac output (CO), mean arterial pressure (MAP), and lactate were obtained at three timepoints under general anesthesia (30, 90, and 150 min after induction). Video analysis was used to determine total vessel density, proportion of perfused vessels, perfused vessel density, and heterogeneity index. Dark-field microscopy videos, MAP, and lactate were obtained at a single timepoint under general anesthesia (45 min after induction) in the elective group. RESULTS: There were no differences in microcirculatory parameters between colic and elective horses, nor was there a difference across timepoints in the colic group. There was a weak negative correlation between microvascular parameters and CO (rho = -0.23). CONCLUSION: The colic group did not have decreased microcirculation in comparison with the healthy elective group. Dark-field microscopy did not correlate well with macrocirculatory parameters in the colic group. IMPACT: Dark-field microscopy may not be a sensitive enough indicator to detect differences in microcirculation between colic and elective groups. The lack of difference in microcirculation may be due to sample size, probe location, or variation in disease severity.

Computed Tomography Scans in Children With Hemophilia After Minor Head Trauma.

OBJECTIVES: Children with hemophilia have the usual childhood risk of falls and head trauma. Head computed tomographies (HCTs) are fast, detailed, and readily available, but increased radiation exposure in the pediatric population is now recognized as causing increased brain malignancy. By examining the incidence of intracranial cerebral hemorrhage in this population, we will be able to weigh risks and benefits of HCT use more accurately. METHODS: Using a retrospective chart review, we examined past medical records of pediatric patients, aged 0 to 15 years, with hemophilia presenting to 1 academic medical center. Primary outcomes included number of head CTs ordered, total and per patient over the years studied, and the incidence of positive findings, as defined by presence of blood products as documented by radiologist final read/interpretation. RESULTS: The mean number of head CTs per child was 2.5 (range, 1-10). None of the HCT scans were read as intracranial cerebral hemorrhage, and none of the patients had findings that lead to neurosurgical intervention. In a sensitivity analysis, applying Pediatric Emergency Care Applied Research Network head injury criteria, 11 HCT scans would be ordered for a reduction of 80 HCTs, or a decrease of 2 HCT scans per child. No incidence of intracranial cerebral hemorrhage would have been missed. CONCLUSIONS: Our findings suggest that in the child with hemophilia and a history of minor head trauma, exposure to the radiation of a HCT based on the diagnosis of hemophilia alone may not be necessary but that imaging decisions need to be made in conjunction with clinical examination findings and neurologic status.

A Goat Cadaver as a Cost-effective Resource for Teaching Emergency Medicine Procedures in Kijabe, Kenya.

BACKGROUND: A pediatric emergency medicine and critical care fellowship was recently developed in Kenya through the University of Nairobi/Kenyatta National Hospital and AIC Kijabe Hospital. As part of this training, a week-long trauma and emergency medicine course was developed with emphasis on trauma and emergency medicine procedures. Given limited resources, we developed a course with simulation of procedures centered around utilization of a goat cadaver. OBJECTIVE: The aim of the study was to describe fellow and faculty experiences and perspectives when using a goat cadaver to teach emergency medicine procedures by simulation in Kijabe, Kenya. METHODS: A 5-day course was given to 2 fellows with a variety of didactics and simulations after which fellows completed a questionnaire to rate their satisfaction with the content and teaching effectiveness. RESULTS: The course was rated very highly, with an average content satisfaction score of 4.5 5 and average teaching effectiveness score of 4.4 of 5. Qualitative faculty feedback was positive, with specific learnings allowing ongoing adaptation of this model. CONCLUSIONS: A goat cadaver is a cost-effective resource not often considered that can be adequately used to teach several emergency medicine skills by simulation.

Different types of disease-causing noncoding variants revealed by genomic and gene expression analyses in families with X-linked intellectual disability.

The pioneering discovery research of X-linked intellectual disability (XLID) genes has benefitted thousands of individuals worldwide; however, approximately 30% of XLID families still remain unresolved. We postulated that noncoding variants that affect gene regulation or splicing may account for the lack of a genetic diagnosis in some cases. Detecting pathogenic, gene-regulatory variants with the same sensitivity and specificity as structural and coding variants is a major challenge for Mendelian disorders. Here, we describe three pedigrees with suggestive XLID where distinctive phenotypes associated with known genes guided the identification of three different noncoding variants. We used comprehensive structural, single-nucleotide, and repeat expansion analyses of genome sequencing. RNA-Seq from patient-derived cell lines, reverse-transcription polymerase chain reactions, Western blots, and reporter gene assays were used to confirm the functional effect of three fundamentally different classes of pathogenic noncoding variants: a retrotransposon insertion, a novel intronic splice donor, and a canonical splice variant of an untranslated exon. In one family, we excluded a rare coding variant in ARX, a known XLID gene, in favor of a regulatory noncoding variant in OFD1 that correlated with the clinical phenotype. Our results underscore the value of genomic research on unresolved XLID families to aid novel, pathogenic noncoding variant discovery.

Integrated in silico and experimental assessment of disease relevance of PCDH19 missense variants.

PCDH19 is a nonclustered protocadherin molecule involved in axon bundling, synapse function, and transcriptional coregulation. Pathogenic variants in PCDH19 cause infantile-onset epilepsy known as PCDH19-clustering epilepsy or PCDH19-CE. Recent advances in DNA-sequencing technologies have led to a significant increase in the number of reported PCDH19-CE variants, many of uncertain significance. We aimed to determine the best approaches for assessing the disease relevance of missense variants in PCDH19. The application of the American College of Medical Genetics and Association for Molecular Pathology (ACMG-AMP) guidelines was only 50% accurate. Using a training set of 322 known benign or pathogenic missense variants, we identified MutPred2, MutationAssessor, and GPP as the best performing in silico tools. We generated a protein structural model of the extracellular domain and assessed 24 missense variants. We also assessed 24 variants using an in vitro reporter assay. A combination of these tools was 93% accurate in assessing known pathogenic and benign PCDH19 variants. We increased the accuracy of the ACMG-AMP classification of 45 PCDH19 variants from 50% to 94%, using these tools. In summary, we have developed a robust toolbox for the assessment of PCDH19 variant pathogenicity to improve the accuracy of PCDH19-CE variant classification.

Truncating Variants in NAA15 Are Associated with Variable Levels of Intellectual Disability, Autism Spectrum Disorder, and Congenital Anomalies.

N-alpha-acetylation is a common co-translational protein modification that is essential for normal cell function in humans. We previously identified the genetic basis of an X-linked infantile lethal Mendelian disorder involving a c.109T>C (p.Ser37Pro) missense variant in NAA10, which encodes the catalytic subunit of the N-terminal acetyltransferase A (NatA) complex. The auxiliary subunit of the NatA complex, NAA15, is the dimeric binding partner for NAA10. Through a genotype-first approach with whole-exome or genome sequencing (WES/WGS) and targeted sequencing analysis, we identified and phenotypically characterized 38 individuals from 33 unrelated families with 25 different de novo or inherited, dominantly acting likely gene disrupting (LGD) variants in NAA15. Clinical features of affected individuals with LGD variants in NAA15 include variable levels of intellectual disability, delayed speech and motor milestones, and autism spectrum disorder. Additionally, mild craniofacial dysmorphology, congenital cardiac anomalies, and seizures are present in some subjects. RNA analysis in cell lines from two individuals showed degradation of the transcripts with LGD variants, probably as a result of nonsense-mediated decay. Functional assays in yeast confirmed a deleterious effect for two of the LGD variants in NAA15. Further supporting a mechanism of haploinsufficiency, individuals with copy-number variant (CNV) deletions involving NAA15 and surrounding genes can present with mild intellectual disability, mild dysmorphic features, motor delays, and decreased growth. We propose that defects in NatA-mediated N-terminal acetylation (NTA) lead to variable levels of neurodevelopmental disorders in humans, supporting the importance of the NatA complex in normal human development.

Constraint and conservation of paired-type homeodomains predicts the clinical outcome of missense variants of uncertain significance.

The need to interpret the pathogenicity of novel missense variants of unknown significance identified in the homeodomain of X-chromosome aristaless-related homeobox (ARX) gene prompted us to assess the utility of conservation and constraint across these domains in multiple genes compared to conventional in vitro functional analysis. Pathogenic missense variants clustered in the homeodomain of ARX contribute to intellectual disability (ID) and epilepsy, with and without brain malformation in affected males. Here we report novel c.1112G>A, p.Arg371Gln and c.1150C>T, p.Arg384Cys variants in male patients with ID and severe seizures. The third case of a male patient with a c.1109C>T, p.Ala370Val variant is perhaps the first example of ID and autism spectrum disorder (ASD), without seizures or brain malformation. We compiled data sets of pathogenic variants from ClinVar and presumed benign variation from gnomAD and demonstrated that the high levels of sequence conservation and constraint of benign variation within the homeodomain impacts upon the ability of publicly available in silico prediction tools to accurately discern likely benign from likely pathogenic variants in these data sets. Despite this, considering the inheritance patterns of the genes and disease variants with the conservation and constraint of disease variants affecting the homeodomain in conjunction with current clinical assessments may assist in predicting the pathogenicity of missense variants, particularly for genes with autosomal recessive and X-linked patterns of disease inheritance, such as ARX. In vitro functional analysis demonstrates that the transcriptional activity of all three variants was diminished compared to ARX-Wt. We review the associated phenotypes of the published cases of patients with ARX homeodomain variants and propose expansion of the ARX-related phenotype to include severe ID and ASD without brain malformations or seizures. We propose that the use of the constraint and conservation data in conjunction with consideration of the patient phenotype and inheritance pattern may negate the need for the experimental functional validation currently required to achieve a diagnosis.

Burnout Rates in Pediatric Emergency Medicine Physicians.

OBJECTIVE: Burnout is a syndrome in which a reduced sense of personal accomplishment, depersonalization, and emotional exhaustion develop in response to prolonged stress. It is well known that physicians suffer high rates of burnout; emergency medicine physicians experience significantly increased rates of burnout, whereas physicians in other specialties, like pediatrics, may be spared. Pediatric emergency medicine physicians are on the frontline of care for the critically ill child, which could put them at high risk for burnout. This study evaluates the rate of burnout in pediatric emergency medicine physicians. METHODS: We conducted a survey assessing burnout using a sample of pediatric emergency medicine physicians who subscribe to an open Listserv server maintained by Brown University. Burnout was measured using a validated instrument, the Maslach Burnout Inventory-Human Services Survey, which was distributed by e-mail to the study group. RESULTS: Respondents averaged a score of 9 (95% confidence interval [CI], 8-10), 23 (95% CI, 21-25), and 39 (95% CI, 38-40) in the subscales of depersonalization, emotional exhaustion, and personal accomplishment, respectively. This placed our cohort into the average range for all subscales. The percentage of respondents who scored in the high levels of burnout (moderate to high scores in both depersonalization and emotional exhaustion and low to moderate scores in personal accomplishment) was 25% (95% CI, 18-32). CONCLUSIONS: Unlike previous literature showing burnout prevalence in excess of 60% in emergency medicine physicians and 38% in pediatricians, our pediatric emergency medicine physicians fared better with only 25% (95% CI, 18-32), showing elevated levels of burnout.

Not All Stridor Is Croup.

BACKGROUND: Emergency providers often attribute stridor to croup in pediatric patients. However, even in children who are having other symptoms of a viral etiology, several other causes need to be considered. CASE: A 6-month-old term male without significant past medical history presented to the emergency department with stridor with likely underlying laryngospasm. He was initially ascribed the diagnosis of croup and was discharged home after receiving steroids and racemic epinephrine. However, he returned hours later after a seizure event at home. A thorough evaluation revealed an ionized calcium of 0.49 mmol/L, and further history revealed the patient was being fed a coconut water-based homemade solution for several months. He was subsequently found to have rickets and delay in milestone achievement. Awareness of hypocalcemia as a possible cause of laryngospasm is important because of the potential life-threatening effects of critically low calcium. Hypocalcemia should be included in the differential diagnosis of any child who presents with stridor, especially if lacking other symptoms of a viral illness.

Intrasinus bolstering of traumatic maxillary sinus fractures by using Foley catheter balloons in two foals.

OBJECTIVE: To describe the repair of unstable facial fractures by using Foley catheter balloons as intrasinus bolsters. STUDY DESIGN: Case report ANIMALS: Two weanling foals with unilateral fractures of the sinus and orbit secondary to kick injuries. Preoperative imaging that included positive contrast dacrocystorhinography and computed tomography confirmed severe comminution of facial fractures and nasolacrimal duct disruption in both foals. METHODS: Small bone fragments were surgically removed, and large fragments were retained even when denuded of periosteum. Repair procedures included nasolacrimal canaliculosinusotomy and suturing fracture fragments together with polydioxanone sutures. After fixation, the fracture fragments could be depressed into the sinus with manual pressure, so two intrasinus Foley catheters were placed to bolster the sinus wall, with the tubing exiting through a frontal sinus trephine. The skin was completely closed over the fractures. Catheters and nasolacrimal stenting were maintained in place during fracture healing. RESULTS: One foal prematurely dislodged catheters and nasolacrimal stent 11 days after fixation. The catheters and stenting were removed as planned 4 weeks after surgery in the second foal. Wound, fracture healing, and overall cosmesis was good in both foals, and epiphora resolved. Surgical site infection, sinusitis, and sequestration did not occur. Both foals became high-level performance horses with acceptable cosmetic outcome and good bilateral nasal airflow. CONCLUSION: Foley catheter balloons supported sinus fracture repair and maintained stability of the surgical reconstruction during convalescence. CLINICAL SIGNIFICANCE: Suture repair of comminuted sinus fractures can be supported by using Foley catheters, which are readily available.

Severe neurocognitive and growth disorders due to variation in THOC2, an essential component of nuclear mRNA export machinery.

Highly conserved TREX-mediated mRNA export is emerging as a key pathway in neuronal development and differentiation. TREX subunit variants cause neurodevelopmental disorders (NDDs) by interfering with mRNA export from the cell nucleus to the cytoplasm. Previously we implicated four missense variants in the X-linked THOC2 gene in intellectual disability (ID). We now report an additional six affected individuals from five unrelated families with two de novo and three maternally inherited pathogenic or likely pathogenic variants in THOC2 extending the genotypic and phenotypic spectrum. These comprise three rare missense THOC2 variants that affect evolutionarily conserved amino acid residues and reduce protein stability and two with canonical splice-site THOC2 variants that result in C-terminally truncated THOC2 proteins. We present detailed clinical assessment and functional studies on a de novo variant in a female with an epileptic encephalopathy and discuss an additional four families with rare variants in THOC2 with supportive evidence for pathogenicity. Severe neurocognitive features, including movement and seizure disorders, were observed in this cohort. Taken together our data show that even subtle alterations to the canonical molecular pathways such as mRNA export, otherwise essential for cellular life, can be compatible with life, but lead to NDDs in humans.

THOC2 Mutations Implicate mRNA-Export Pathway in X-Linked Intellectual Disability.

Export of mRNA from the cell nucleus to the cytoplasm is essential for protein synthesis, a process vital to all living eukaryotic cells. mRNA export is highly conserved and ubiquitous. Mutations affecting mRNA and mRNA processing or export factors, which cause aberrant retention of mRNAs in the nucleus, are thus emerging as contributors to an important class of human genetic disorders. Here, we report that variants in THOC2, which encodes a subunit of the highly conserved TREX mRNA-export complex, cause syndromic intellectual disability (ID). Affected individuals presented with variable degrees of ID and commonly observed features included speech delay, elevated BMI, short stature, seizure disorders, gait disturbance, and tremors. X chromosome exome sequencing revealed four missense variants in THOC2 in four families, including family MRX12, first ascertained in 1971. We show that two variants lead to decreased stability of THOC2 and its TREX-complex partners in cells derived from the affected individuals. Protein structural modeling showed that the altered amino acids are located in the RNA-binding domains of two complex THOC2 structures, potentially representing two different intermediate RNA-binding states of THOC2 during RNA transport. Our results show that disturbance of the canonical molecular pathway of mRNA export is compatible with life but results in altered neuronal development with other comorbidities.

Molecular Basis of Altered hERG1 Channel Gating Induced by Ginsenoside Rg3.

Outward current conducted by human ether-à-go-go-related gene type 1 (hERG1) channels is a major determinant of action potential repolarization in the human ventricle. Ginsenoside 20(S)-Rg3 [Rg3; (2S,3R,4S,5S,6R)-2-[(2R,3R,4S,5S,6R)-4,5-dihydroxy-2-[[(3S,5R,8R,9R,10R,12R,13R,14R,17S)-12-hydroxy-17-[(2S)-2-hydroxy-6-methylhept-5-en-2-yl]-4,4,8,10,14-pentamethyl-2,3,5,6,7,9,11,12,13,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl]oxy]-6-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol], an alkaloid isolated from the root of Panax ginseng, slows the rate of hERG1 deactivation, induces channels to open at more negative potentials than normal, and increases current magnitude. The onset of Rg3 action is extremely fast, suggesting that it binds to an extracellular accessible site on the channel to alter its gating. Here we used a scanning mutagenesis approach to identify residues in the extracellular loops and transmembrane segments of hERG1 that might interact with Rg3. Single or multiple residues of hERG1 were mutated to Ala or Cys and the resulting mutant channels were heterologously expressed in Xenopus oocytes. The effects of Rg3 on the voltage dependence of activation and the deactivation rate of mutant channel currents were characterized using the two-microelectrode voltage clamp technique. Mutation to Ala of specific residues in the S1 (Tyr420), S2 (Leu452, Phe463), and S4 (Ile521, Lys525) segments partially inhibited the effects of Rg3 on hERG1. The double mutant Y420A/L452A nearly eliminated the effects of Rg3 on voltage-dependent channel gating but did not prevent the increase in current magnitude. These findings together with molecular modeling suggest that Rg3 alters the gating of hERG1 channels by interacting with and stabilizing the voltage sensor domain in an activated state.

Molecular mechanisms of Slo2 K+ channel closure.

KEY POINTS: Intracellular Na+ -activated Slo2 potassium channels are in a closed state under normal physiological conditions, although their mechanisms of ion permeation gating are not well understood. A cryo-electron microscopy structure of Slo2.2 suggests that the ion permeation pathway of these channels is closed by a single constriction of the inner pore formed by the criss-crossing of the cytoplasmic ends of the S6 segments (the S6 bundle crossing) at a conserved Met residue. Functional characterization of mutant Slo2 channels suggests that hydrophobic interactions between Leu residues in the upper region of the S6 segments contribute to stabilizing the inner pore in a non-conducting state. Mutation of the conserved Met residues in the S6 segments to the negatively-charged Glu did not induce constitutive opening of Slo2.1 or Slo2.2, suggesting that ion permeation of Slo2 channels is not predominantly gated by the S6 bundle crossing. ABSTRACT: Large conductance K+ -selective Slo2 channels are in a closed state unless activated by elevated [Na+ ]i . Our previous studies suggested that the pore helix/selectivity filter serves as the activation gate in Slo2 channels. In the present study, we evaluated two other potential mechanisms for stabilization of Slo2 channels in a closed state: (1) dewetting and collapse of the inner pore (hydrophobic gating) and (2) constriction of the inner pore by tight criss-crossing of the cytoplasmic ends of the S6 α-helical segments. Slo2 channels contain two conserved Leu residues in each of the four S6 segments that line the inner pore region nearest the bottom of the selectivity filter. To evaluate the potential role of these residues in hydrophobic gating, Leu267 and Leu270 in human Slo2.1 were each replaced by 15 different residues. The relative conductance of mutant channels was highly dependent on hydrophilicity and volume of the amino acid substituted for Leu267 and was maximal with L267H. Consistent with their combined role in hydrophobic gating, replacement of both Leu residues with the isosteric but polar residue Asn (L267N/L270N) stabilized channels in a fully open state. In a recent cryo-electron microscopy structure of chicken Slo2.2, the ion permeation pathway of the channel is closed by a constriction of the inner pore formed by criss-crossing of the S6 segments at a conserved Met. Inconsistent with the S6 segment crossing forming the activation gate, replacement of the homologous Met residues in human Slo2.1 or Slo2.2 with the negatively-charged Glu did not induce constitutive channel opening.