A novel SMARCC1 BAFopathy implicates neural progenitor epigenetic dysregulation in human hydrocephalus.

Hydrocephalus, characterized by cerebral ventriculomegaly, is the most common disorder requiring brain surgery in children. Recent studies have implicated SMARCC1, a component of the BRG1-associated factor (BAF) chromatin remodelling complex, as a candidate congenital hydrocephalus gene. However, SMARCC1 variants have not been systematically examined in a large patient cohort or conclusively linked with a human syndrome. Moreover, congenital hydrocephalus-associated SMARCC1 variants have not been functionally validated or mechanistically studied in vivo. Here, we aimed to assess the prevalence of SMARCC1 variants in an expanded patient cohort, describe associated clinical and radiographic phenotypes, and assess the impact of Smarcc1 depletion in a novel Xenopus tropicalis model of congenital hydrocephalus. To do this, we performed a genetic association study using whole-exome sequencing from a cohort consisting of 2697 total ventriculomegalic trios, including patients with neurosurgically-treated congenital hydrocephalus, that total 8091 exomes collected over 7 years (2016-23). A comparison control cohort consisted of 1798 exomes from unaffected siblings of patients with autism spectrum disorder and their unaffected parents were sourced from the Simons Simplex Collection. Enrichment and impact on protein structure were assessed in identified variants. Effects on the human fetal brain transcriptome were examined with RNA-sequencing and Smarcc1 knockdowns were generated in Xenopus and studied using optical coherence tomography imaging, in situ hybridization and immunofluorescence. SMARCC1 surpassed genome-wide significance thresholds, yielding six rare, protein-altering de novo variants localized to highly conserved residues in key functional domains. Patients exhibited hydrocephalus with aqueductal stenosis; corpus callosum abnormalities, developmental delay, and cardiac defects were also common. Xenopus knockdowns recapitulated both aqueductal stenosis and cardiac defects and were rescued by wild-type but not patient-specific variant SMARCC1. Hydrocephalic SMARCC1-variant human fetal brain and Smarcc1-variant Xenopus brain exhibited a similarly altered expression of key genes linked to midgestational neurogenesis, including the transcription factors NEUROD2 and MAB21L2. These results suggest de novo variants in SMARCC1 cause a novel human BAFopathy we term 'SMARCC1-associated developmental dysgenesis syndrome', characterized by variable presence of cerebral ventriculomegaly, aqueductal stenosis, developmental delay and a variety of structural brain or cardiac defects. These data underscore the importance of SMARCC1 and the BAF chromatin remodelling complex for human brain morphogenesis and provide evidence for a 'neural stem cell' paradigm of congenital hydrocephalus pathogenesis. These results highlight utility of trio-based whole-exome sequencing for identifying pathogenic variants in sporadic congenital structural brain disorders and suggest whole-exome sequencing may be a valuable adjunct in clinical management of congenital hydrocephalus patients.

Pleiotropic role of TRAF7 in skull-base meningiomas and congenital heart disease.

While somatic variants of TRAF7 (Tumor necrosis factor receptor-associated factor 7) underlie anterior skull-base meningiomas, here we report the inherited mutations of TRAF7 that cause congenital heart defects. We show that TRAF7 mutants operate in a dominant manner, inhibiting protein function via heterodimerization with wild-type protein. Further, the shared genetics of the two disparate pathologies can be traced to the common origin of forebrain meninges and cardiac outflow tract from the TRAF7-expressing neural crest. Somatic and inherited mutations disrupt TRAF7-IFT57 interactions leading to cilia degradation. TRAF7-mutant meningioma primary cultures lack cilia, and TRAF7 knockdown causes cardiac, craniofacial, and ciliary defects in Xenopus and zebrafish, suggesting a mechanistic convergence for TRAF7-driven meningiomas and developmental heart defects.

A novel SMARCC1 -mutant BAFopathy implicates epigenetic dysregulation of neural progenitors in hydrocephalus.

Importance: Hydrocephalus, characterized by cerebral ventriculomegaly, is the most common disorder requiring brain surgery. A few familial forms of congenital hydrocephalus (CH) have been identified, but the cause of most sporadic cases of CH remains elusive. Recent studies have implicated SMARCC1 , a component of the B RG1- a ssociated factor (BAF) chromatin remodeling complex, as a candidate CH gene. However, SMARCC1 variants have not been systematically examined in a large patient cohort or conclusively linked with a human syndrome. Moreover, CH-associated SMARCC1 variants have not been functionally validated or mechanistically studied in vivo . Objectives: The aims of this study are to (i) assess the extent to which rare, damaging de novo mutations (DNMs) in SMARCC1 are associated with cerebral ventriculomegaly; (ii) describe the clinical and radiographic phenotypes of SMARCC1 -mutated patients; and (iii) assess the pathogenicity and mechanisms of CH-associated SMARCC1 mutations in vivo . Design setting and participants: A genetic association study was conducted using whole-exome sequencing from a cohort consisting of 2,697 ventriculomegalic trios, including patients with neurosurgically-treated CH, totaling 8,091 exomes collected over 5 years (2016-2021). Data were analyzed in 2023. A comparison control cohort consisted of 1,798 exomes from unaffected siblings of patients with autism spectrum disorder and their unaffected parents sourced from the Simons simplex consortium. Main outcomes and measures: Gene variants were identified and filtered using stringent, validated criteria. Enrichment tests assessed gene-level variant burden. In silico biophysical modeling estimated the likelihood and extent of the variant impact on protein structure. The effect of a CH-associated SMARCC1 mutation on the human fetal brain transcriptome was assessed by analyzing RNA-sequencing data. Smarcc1 knockdowns and a patient-specific Smarcc1 variant were tested in Xenopus and studied using optical coherence tomography imaging, in situ hybridization, and immunofluorescence microscopy. Results: SMARCC1 surpassed genome-wide significance thresholds in DNM enrichment tests. Six rare protein-altering DNMs, including four loss-of-function mutations and one recurrent canonical splice site mutation (c.1571+1G>A) were detected in unrelated patients. DNMs localized to the highly conserved DNA-interacting SWIRM, Myb-DNA binding, Glu-rich, and Chromo domains of SMARCC1 . Patients exhibited developmental delay (DD), aqueductal stenosis, and other structural brain and heart defects. G0 and G1 Smarcc1 Xenopus mutants exhibited aqueductal stenosis and cardiac defects and were rescued by human wild-type SMARCC1 but not a patient-specific SMARCC1 mutant. Hydrocephalic SMARCC1 -mutant human fetal brain and Smarcc1 -mutant Xenopus brain exhibited a similarly altered expression of key genes linked to midgestational neurogenesis, including the transcription factors NEUROD2 and MAB21L2 . Conclusions: SMARCC1 is a bona fide CH risk gene. DNMs in SMARCC1 cause a novel human BAFopathy we term " S MARCC1- a ssociated D evelopmental D ysgenesis S yndrome (SaDDS)", characterized by cerebral ventriculomegaly, aqueductal stenosis, DD, and a variety of structural brain or cardiac defects. These data underscore the importance of SMARCC1 and the BAF chromatin remodeling complex for human brain morphogenesis and provide evidence for a "neural stem cell" paradigm of human CH pathogenesis. These results highlight the utility of trio-based WES for identifying risk genes for congenital structural brain disorders and suggest WES may be a valuable adjunct in the clinical management of CH patients. KEY POINTS: Question: What is the role of SMARCC1 , a core component of the B RG1- a ssociated factor (BAF) chromatin remodeling complex, in brain morphogenesis and congenital hydrocephalus (CH)? Findings: SMARCC1 harbored an exome-wide significant burden of rare, protein-damaging de novo mutations (DNMs) (p = 5.83 × 10 -9 ) in the largest ascertained cohort to date of patients with cerebral ventriculomegaly, including treated CH (2,697 parent-proband trios). SMARCC1 contained four loss-of-function DNMs and two identical canonical splice site DNMs in a total of six unrelated patients. Patients exhibited developmental delay, aqueductal stenosis, and other structural brain and cardiac defects. Xenopus Smarcc1 mutants recapitulated core human phenotypes and were rescued by the expression of human wild-type but not patient-mutant SMARCC1 . Hydrocephalic SMARCC1 -mutant human brain and Smarcc1 -mutant Xenopus brain exhibited similar alterationsin the expression of key transcription factors that regulate neural progenitor cell proliferation. Meaning: SMARCC1 is essential for human brain morphogenesis and is a bona fide CH risk gene. SMARCC1 mutations cause a novel human BAFopathy we term " S MARCC1- a ssociated D evelopmental D ysgenesis S yndrome (SaDDS)". These data implicate epigenetic dysregulation of fetal neural progenitors in the pathogenesis of hydrocephalus, with diagnostic and prognostic implications for patients and caregivers.

A retrospective cohort analysis of the Yale pediatric genomics discovery program.

The Pediatric Genomics Discovery Program (PGDP) at Yale uses next-generation sequencing (NGS) and translational research to evaluate complex patients with a wide range of phenotypes suspected to have rare genetic diseases. We conducted a retrospective cohort analysis of 356 PGDP probands evaluated between June 2015 and July 2020, querying our database for participant demographics, clinical characteristics, NGS results, and diagnostic and research findings. The three most common phenotypes among the entire studied cohort (n = 356) were immune system abnormalities (n = 105, 29%), syndromic or multisystem disease (n = 103, 29%), and cardiovascular system abnormalities (n = 62, 17%). Of 216 patients with final classifications, 77 (36%) received new diagnoses and 139 (64%) were undiagnosed; the remaining 140 patients were still actively being investigated. Monogenetic diagnoses were found in 67 (89%); the largest group had variants in known disease genes but with new contributions such as novel variants (n = 31, 40%) or expanded phenotypes (n = 14, 18%). Finally, five PGDP diagnoses (8%) were suggestive of novel gene-to-phenotype relationships. A broad range of patients can benefit from single subject studies combining NGS and functional molecular analyses. All pediatric providers should consider further genetics evaluations for patients lacking precise molecular diagnoses.

Xenopus Tadpole Craniocardiac Imaging Using Optical Coherence Tomography.

Optical coherence tomography (OCT) imaging can be used to visualize craniocardiac structures in the Xenopus model system. OCT is analogous to ultrasound, utilizing light instead of sound to create a gray-scale image from the echo time delay of infrared light reflected from the specimen. OCT is a high-speed, cross-sectional, label-free imaging modality, which can outline dynamic in vivo morphology at resolutions approaching histological detail. OCT imaging can acquire 2D and 3D data in real time to assess cardiac and facial structures. Additionally, during cardiac imaging, Doppler imaging can be used to assess the blood flow pattern in relation to the intracardiac structures. Importantly, OCT can reproducibly and efficiently provide comprehensive, nondestructive in vivo cardiac and facial phenotyping. Tadpoles do not require preprocessing and thus can be further raised or analyzed after brief immobilization during imaging. The rapid development of the Xenopus model combined with a rapid OCT imaging protocol allows the identification of specific gene/teratogen phenotype relationships in a short period of time. Loss- or gain-of-function experiments can be evaluated in 4-5 d, and OCT imaging only requires ∼5 min per tadpole. Thus, we find this pairing an efficient workflow for screening numerous candidate genes derived from human genomic studies to in-depth mechanistic studies.

PPIL4 is essential for brain angiogenesis and implicated in intracranial aneurysms in humans.

Intracranial aneurysm (IA) rupture leads to subarachnoid hemorrhage, a sudden-onset disease that often causes death or severe disability. Although genome-wide association studies have identified common genetic variants that increase IA risk moderately, the contribution of variants with large effect remains poorly defined. Using whole-exome sequencing, we identified significant enrichment of rare, deleterious mutations in PPIL4, encoding peptidyl-prolyl cis-trans isomerase-like 4, in both familial and index IA cases. Ppil4 depletion in vertebrate models causes intracerebral hemorrhage, defects in cerebrovascular morphology and impaired Wnt signaling. Wild-type, but not IA-mutant, PPIL4 potentiates Wnt signaling by binding JMJD6, a known angiogenesis regulator and Wnt activator. These findings identify a novel PPIL4-dependent Wnt signaling mechanism involved in brain-specific angiogenesis and maintenance of cerebrovascular integrity and implicate PPIL4 gene mutations in the pathogenesis of IA.

DLG5 variants are associated with multiple congenital anomalies including ciliopathy phenotypes.

BACKGROUND: Cilia are dynamic cellular extensions that generate and sense signals to orchestrate proper development and tissue homeostasis. They rely on the underlying polarisation of cells to participate in signalling. Cilia dysfunction is a well-known cause of several diseases that affect multiple organ systems including the kidneys, brain, heart, respiratory tract, skeleton and retina. METHODS: Among individuals from four unrelated families, we identified variants in discs large 5 (DLG5) that manifested in a variety of pathologies. In our proband, we also examined patient tissues. We depleted dlg5 in Xenopus tropicalis frog embryos to generate a loss-of-function model. Finally, we tested the pathogenicity of DLG5 patient variants through rescue experiments in the frog model. RESULTS: Patients with variants of DLG5 were found to have a variety of phenotypes including cystic kidneys, nephrotic syndrome, hydrocephalus, limb abnormalities, congenital heart disease and craniofacial malformations. We also observed a loss of cilia in cystic kidney tissue of our proband. Knockdown of dlg5 in Xenopus embryos recapitulated many of these phenotypes and resulted in a loss of cilia in multiple tissues. Unlike introduction of wildtype DLG5 in frog embryos depleted of dlg5, introduction of DLG5 patient variants was largely ineffective in restoring proper ciliation and tissue morphology in the kidney and brain suggesting that the variants were indeed detrimental to function. CONCLUSION: These findings in both patient tissues and Xenopus shed light on how mutations in DLG5 may lead to tissue-specific manifestations of disease. DLG5 is essential for cilia and many of the patient phenotypes are in the ciliopathy spectrum.

Paired Box 9 (PAX9), the RNA polymerase II transcription factor, regulates human ribosome biogenesis and craniofacial development.

Dysregulation of ribosome production can lead to a number of developmental disorders called ribosomopathies. Despite the ubiquitous requirement for these cellular machines used in protein synthesis, ribosomopathies manifest in a tissue-specific manner, with many affecting the development of the face. Here we reveal yet another connection between craniofacial development and making ribosomes through the protein Paired Box 9 (PAX9). PAX9 functions as an RNA Polymerase II transcription factor to regulate the expression of proteins required for craniofacial and tooth development in humans. We now expand this function of PAX9 by demonstrating that PAX9 acts outside of the cell nucleolus to regulate the levels of proteins critical for building the small subunit of the ribosome. This function of PAX9 is conserved to the organism Xenopus tropicalis, an established model for human ribosomopathies. Depletion of pax9 leads to craniofacial defects due to abnormalities in neural crest development, a result consistent with that found for depletion of other ribosome biogenesis factors. This work highlights an unexpected layer of how the making of ribosomes is regulated in human cells and during embryonic development.

Author Correction: Visualizing flow in an intact CSF network using optical coherence tomography: implications for human congenital hydrocephalus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Projection of new thresholds for hypertension to outpatient clinic patients and impact of risk factors: a cross-sectional study.

BACKGROUND: The 2017 American College of Cardiology (ACC)/American Heart Association (AHA) guidelines on hypertension management recommend new stage 1 hypertension thresholds (130-139/80-89 mmHg) for starting antihypertensive treatment. OBJECTIVE: To analyze the impact of the 2017 ACC/AHA guidelines on patients' diagnoses within daily practice, in comparison with management using the 2018 European hypertension guidelines, regarding the new thresholds. DESIGN AND SETTING: Cross-sectional study conducted in a hypertension outpatient clinic at a tertiary-level public hospital. METHODS: The diagnosis of hypertension was defined separately using each guideline. The participants were patients who were attending the hypertension clinic, who were evaluated using the thresholds of two guidelines, based on cardiovascular risk factors, including age, gender, smoking status, diabetes mellitus, dyslipidemia, obesity, osteoporosis, chronic renal failure and family history of hypertension. RESULTS: After adapting the guidelines to the blood pressure values of our sample, 74.5% (n = 277) of the patients were diagnosed as hypertensive according to the blood pressure classification of the European Society of Cardiology (ESC) guidelines published in 2018, while 91.1% (n = 339) of the patients were hypertensive according to the new 2017 ACC/AHA guidelines. Multivariate regression analysis revealed that the significant demographic and cardiovascular risk factors associated with hypertension, based on the 2018 European Society of Hypertension (ESH)/ESC guidelines, were age (odds ratio, OR: 1.027; 95% confidence interval, CI: 1.001-1.054; P = 0.042), obesity (OR: 4.534; 95% CI: 1.830-11.237; P = 0.001) and family history of hypertension (OR: 2.199; 95% CI: 1.252-3.862; P = 0.006). CONCLUSIONS: The factors associated with the definition of hypertension may vary through changing the threshold values.

Quantitative Phenotyping of Xenopus Embryonic Heart Pathophysiology Using Hemoglobin Contrast Subtraction Angiography to Screen Human Cardiomyopathies.

Congenital heart disease (CHD) is a significant cause of mortality in infants and adults. Currently human genomic analysis has identified a number of candidate genes in these patients. These genes span diverse categories of gene function suggesting that despite the similarity in cardiac lesion, the underlying pathophysiology may be different. In fact, patients with similar CHDs can have drastically different outcomes, including a dramatic decrease in myocardial function. To test these human candidate genes for their impact on myocardial function, we need efficient animals models of disease. For this purpose, we paired Xenopus tropicalis with our microangiography technique, hemoglobin contrast subtraction angiography (HCSA). To demonstrate the gene-teratogen-physiology relationship, we modeled human cardiomyopathy in tadpoles. First we depleted the sarcomeric protein myosin heavy chain 6 (myh6) expression using morpholino oligos. Next, we exposed developing embryos to the teratogen ethanol and in both conditions showed varying degrees of cardiac dysfunction. Our results demonstrate that HCSA can distinguish biomechanical phenotypes in the context of gene dysfunction or teratogen. This approach can be used to screen numerous candidate CHD genes or suspected teratogens for their effect on cardiac function.

Gaussian process post-processing for particle tracking velocimetry.

Particle tracking velocimetry (PTV) gives quantitative estimates of fluid flow velocities from images. But particle tracking is a complicated problem, and it often produces results that need substantial post-processing. We propose a novel Gaussian process regression-based post-processing step for PTV: The method smooths ("denoises") and densely interpolates velocity estimates while rejecting track irregularities. The method works under a large range of particle densities and fluid velocities. In addition, the method calculates standard deviances (error bars) for the velocity estimates, opening the possibility of propagating the standard deviances through subsequent processing and analysis. The accuracy of the method is experimentally evaluated using Optical Coherence Tomography images of particles in laminar flow in a pipe phantom. Following this, the method is used to quantify cilia-driven fluid flow and vorticity patterns in a developing Xenopus embryo.

Projection of new thresholds for hypertension to outpatient clinic patients and impact of risk factors: a cross-sectional study

ABSTRACT BACKGROUND: The 2017 American College of Cardiology (ACC)/American Heart Association (AHA) guidelines on hypertension management recommend new stage 1 hypertension thresholds (130-139/80-89 mmHg) for starting antihypertensive treatment. OBJECTIVE: To analyze the impact of the 2017 ACC/AHA guidelines on patients' diagnoses within daily practice, in comparison with management using the 2018 European hypertension guidelines, regarding the new thresholds. DESIGN AND SETTING: Cross-sectional study conducted in a hypertension outpatient clinic at a tertiary-level public hospital. METHODS: The diagnosis of hypertension was defined separately using each guideline. The participants were patients who were attending the hypertension clinic, who were evaluated using the thresholds of two guidelines, based on cardiovascular risk factors, including age, gender, smoking status, diabetes mellitus, dyslipidemia, obesity, osteoporosis, chronic renal failure and family history of hypertension. RESULTS: After adapting the guidelines to the blood pressure values of our sample, 74.5% (n = 277) of the patients were diagnosed as hypertensive according to the blood pressure classification of the European Society of Cardiology (ESC) guidelines published in 2018, while 91.1% (n = 339) of the patients were hypertensive according to the new 2017 ACC/AHA guidelines. Multivariate regression analysis revealed that the significant demographic and cardiovascular risk factors associated with hypertension, based on the 2018 European Society of Hypertension (ESH)/ESC guidelines, were age (odds ratio, OR: 1.027; 95% confidence interval, CI: 1.001-1.054; P = 0.042), obesity (OR: 4.534; 95% CI: 1.830-11.237; P = 0.001) and family history of hypertension (OR: 2.199; 95% CI: 1.252-3.862; P = 0.006). CONCLUSIONS: The factors associated with the definition of hypertension may vary through changing the threshold values.

Effects of General Anaesthesia on the Middle Ear Pressure.

OBJECTIVE: Inhalation agents can have different effects on the middle ear pressure (MEP). We aimed to investigate the effect of sevoflurane and desflurane, the agents used in patients who do not have any ear pathology and who undergo surgery under general anaesthesia, on MEP. METHODS: Fifty adult patients who were scheduled to undergo inguinal hernia and lower extremity surgery were included in our study. All patients were aged between 20 and 60 years and belonged to the American Society of Anesthesiologists (ASA) I-III class. Patients were divided into two groups, according to the inhalation agent administered for the surgery: sevofluran, Group S (n=25); and desfluran, Group D (n=25). Anaesthetic agents, intraoperative end tidal carbon dioxide and airway pressures were recorded. The MEP was measured for both ears preoperatively, at the intraoperative 5th, 10th, 15th minutes, and at the postoperative 10th and 30th minutes. RESULTS: The MEP at the intraoperative 10th minute was significantly higher in Group D compared to Group S. In Group D, the MEP increased significantly at the intraoperative 10th and 15th minutes, and postoperative 30th minute, compared to preoperative values. In Group S, the MEP increased significantly at the postoperative 10th minute, compared to preoperative values. CONCLUSION: We found that desflurane increases the MEP during the intraoperative and postoperative period, compared to sevoflurane.

Visualizing flow in an intact CSF network using optical coherence tomography: implications for human congenital hydrocephalus.

Cerebrospinal fluid (CSF) flow in the brain ventricles is critical for brain development. Altered CSF flow dynamics have been implicated in congenital hydrocephalus (CH) characterized by the potentially lethal expansion of cerebral ventricles if not treated. CH is the most common neurosurgical indication in children effecting 1 per 1000 infants. Current treatment modalities are limited to antiquated brain surgery techniques, mostly because of our poor understanding of the CH pathophysiology. We lack model systems where the interplay between ependymal cilia, embryonic CSF flow dynamics and brain development can be analyzed in depth. This is in part due to the poor accessibility of the vertebrate ventricular system to in vivo investigation. Here, we show that the genetically tractable frog Xenopus tropicalis, paired with optical coherence tomography imaging, provides new insights into CSF flow dynamics and role of ciliary dysfunction in hydrocephalus pathogenesis. We can visualize CSF flow within the multi-chambered ventricular system and detect multiple distinct polarized CSF flow fields. Using CRISPR/Cas9 gene editing, we modeled human L1CAM and CRB2 mediated aqueductal stenosis. We propose that our high-throughput platform can prove invaluable for testing candidate human CH genes to understand CH pathophysiology.

Familial Dilated Cardiomyopathy Associated With a Novel Combination of Compound Heterozygous TNNC1 Variants.

Familial dilated cardiomyopathy (DCM), clinically characterized by enlargement and dysfunction of one or both ventricles of the heart, can be caused by variants in sarcomeric genes including TNNC1 (encoding cardiac troponin C, cTnC). Here, we report the case of two siblings with severe, early onset DCM who were found to have compound heterozygous variants in TNNC1: p.Asp145Glu (D145E) and p.Asp132Asn (D132N), which were inherited from the parents. We began our investigation with CRISPR/Cas9 knockout of TNNC1 in Xenopus tropicalis, which resulted in a cardiac phenotype in tadpoles consistent with DCM. Despite multiple maneuvers, we were unable to rescue the tadpole hearts with either human cTnC wild-type or patient variants to investigate the cardiomyopathy phenotype in vivo. We therefore utilized porcine permeabilized cardiac muscle preparations (CMPs) reconstituted with either wild-type or patient variant forms of cTnC to examine effects of the patient variants on contractile function. Incorporation of 50% WT/50% D145E into CMPs increased Ca2+ sensitivity of isometric force, consistent with prior studies. In contrast, incorporation of 50% WT/50% D132N, which had not been previously reported, decreased Ca2+ sensitivity of isometric force. CMPs reconstituted 50-50% with both variants mirrored WT in regard to myofilament Ca2+ responsiveness. Sinusoidal stiffness (SS) (0.2% peak-to-peak) and the kinetics of tension redevelopment (k TR) at saturating Ca2+ were similar to WT for all preparations. Modeling of Ca2+-dependence of k TR support the observation from Ca2+ responsiveness of steady-state isometric force, that the effects on each mutant (50% WT/50% mutant) were greater than the combination of the two mutants (50% D132N/50% D145E). Further studies are needed to ascertain the mechanism(s) of these variants.

RPSA, a candidate gene for isolated congenital asplenia, is required for pre-rRNA processing and spleen formation in Xenopus.

A growing number of tissue-specific inherited disorders are associated with impaired ribosome production, despite the universal requirement for ribosome function. Recently, mutations in RPSA, a protein component of the small ribosomal subunit, were discovered to underlie approximately half of all isolated congenital asplenia cases. However, the mechanisms by which mutations in this ribosome biogenesis factor lead specifically to spleen agenesis remain unknown, in part due to the lack of a suitable animal model for study. Here we reveal that RPSA is required for normal spleen development in the frog, Xenopus tropicalis Depletion of Rpsa in early embryonic development disrupts pre-rRNA processing and ribosome biogenesis, and impairs expression of the key spleen patterning genes nkx2-5, bapx1 and pod1 in the spleen anlage. Importantly, we also show that whereas injection of human RPSA mRNA can rescue both pre-rRNA processing and spleen patterning, injection of human mRNA bearing a common disease-associated mutation cannot. Together, we present the first animal model of RPSA-mediated asplenia and reveal a crucial requirement for RPSA in pre-rRNA processing and molecular patterning during early Xenopus development.

CRISPR/Cas9 F0 Screening of Congenital Heart Disease Genes in Xenopus tropicalis.

In the US and Europe, birth defects are the leading cause of infant mortality. Among birth defects, Congenital Heart Disease (CHD) occurs in approximately 8 out of 1000 live births, affects 1.3 million newborns per year worldwide, and has the highest mortality rate. While there is evidence to indicate that CHD does have a genetic basis, most of the CHD burden remains unexplained genetically. Fortunately, new genomics technologies are enabling genetic analyses of CHD patients. Whole exome sequencing of trios as well as copy number variations assayed by high-density SNP arrays can now be obtained at high efficiency and relatively low cost. These efforts are identifying a number of sequence variations in patients with CHD, but only a small percentage have second unrelated alleles to validate them as disease causing. Importantly, most of these candidate genes do not have an identified molecular mechanism implicating them in cardiac development. Therefore, there is a pressing need to develop rapid functional assays to evaluate candidate genes for a role in cardiac development, and then to investigate the underlying developmental mechanisms. Most recently, the advent of CRISPR/Cas9 genome editing technology has greatly enhanced the ability to manipulate and observe the function of the genome in model systems and cell culture. Incorporating the power of a developmental system such as Xenopus tropicalis with the CRISPR/Cas9 system and the microscale imaging modality optical coherence tomography (OCT), the analysis of thousands of different genes in cardiac development becomes possible.

A case with CMTX1 disease showing transient ischemic-attack-like episodes.

Charcot-Marie-Tooth (CMT) disease is a hereditary neurologic disease which affects the sensorial and motor fibers of the peripheral nerves. CMTX1 is an X-linked dominantly inherited subtype of CMT and is caused by mutations in gap junction beta 1 gene (GJB1). A small proportion of GJB1 mutations are associated with recurrent central nervous system findings. We describe a 15-year-old male patient with CMTX1 who had stroke-like findings along with foot deformities and peripheral neuropathy. Strokes and stroke-like attacks are rarely seen in children and adolescents. Herein, neurological signs, MRI findings and genetic results of a CMTX1 case are presented and discussed.