Rare EIF4A2 variants are associated with a neurodevelopmental disorder characterized by intellectual disability, hypotonia, and epilepsy.

Eukaryotic initiation factor-4A2 (EIF4A2) is an ATP-dependent RNA helicase and a member of the DEAD-box protein family that recognizes the 5' cap structure of mRNAs, allows mRNA to bind to the ribosome, and plays an important role in microRNA-regulated gene repression. Here, we report on 15 individuals from 14 families presenting with global developmental delay, intellectual disability, hypotonia, epilepsy, and structural brain anomalies, all of whom have extremely rare de novo mono-allelic or inherited bi-allelic variants in EIF4A2. Neurodegeneration was predominantly reported in individuals with bi-allelic variants. Molecular modeling predicts these variants would perturb structural interactions in key protein domains. To determine the pathogenicity of the EIF4A2 variants in vivo, we examined the mono-allelic variants in Drosophila melanogaster (fruit fly) and identified variant-specific behavioral and developmental defects. The fruit fly homolog of EIF4A2 is eIF4A, a negative regulator of decapentaplegic (dpp) signaling that regulates embryo patterning, eye and wing morphogenesis, and stem cell identity determination. Our loss-of-function (LOF) rescue assay demonstrated a pupal lethality phenotype induced by loss of eIF4A, which was fully rescued with human EIF4A2 wild-type (WT) cDNA expression. In comparison, the EIF4A2 variant cDNAs failed or incompletely rescued the lethality. Overall, our findings reveal that EIF4A2 variants cause a genetic neurodevelopmental syndrome with both LOF and gain of function as underlying mechanisms.

Unique variants in CLCN3, encoding an endosomal anion/proton exchanger, underlie a spectrum of neurodevelopmental disorders.

The genetic causes of global developmental delay (GDD) and intellectual disability (ID) are diverse and include variants in numerous ion channels and transporters. Loss-of-function variants in all five endosomal/lysosomal members of the CLC family of Cl- channels and Cl-/H+ exchangers lead to pathology in mice, humans, or both. We have identified nine variants in CLCN3, the gene encoding CIC-3, in 11 individuals with GDD/ID and neurodevelopmental disorders of varying severity. In addition to a homozygous frameshift variant in two siblings, we identified eight different heterozygous de novo missense variants. All have GDD/ID, mood or behavioral disorders, and dysmorphic features; 9/11 have structural brain abnormalities; and 6/11 have seizures. The homozygous variants are predicted to cause loss of ClC-3 function, resulting in severe neurological disease similar to the phenotype observed in Clcn3-/- mice. Their MRIs show possible neurodegeneration with thin corpora callosa and decreased white matter volumes. Individuals with heterozygous variants had a range of neurodevelopmental anomalies including agenesis of the corpus callosum, pons hypoplasia, and increased gyral folding. To characterize the altered function of the exchanger, electrophysiological analyses were performed in Xenopus oocytes and mammalian cells. Two variants, p.Ile607Thr and p.Thr570Ile, had increased currents at negative cytoplasmic voltages and loss of inhibition by luminal acidic pH. In contrast, two other variants showed no significant difference in the current properties. Overall, our work establishes a role for CLCN3 in human neurodevelopment and shows that both homozygous loss of ClC-3 and heterozygous variants can lead to GDD/ID and neuroanatomical abnormalities.

Heterozygous Variants in KDM4B Lead to Global Developmental Delay and Neuroanatomical Defects.

KDM4B is a lysine-specific demethylase with a preferential activity on H3K9 tri/di-methylation (H3K9me3/2)-modified histones. H3K9 tri/di-demethylation is an important epigenetic mechanism responsible for silencing of gene expression in animal development and cancer. However, the role of KDM4B on human development is still poorly characterized. Through international data sharing, we gathered a cohort of nine individuals with mono-allelic de novo or inherited variants in KDM4B. All individuals presented with dysmorphic features and global developmental delay (GDD) with language and motor skills most affected. Three individuals had a history of seizures, and four had anomalies on brain imaging ranging from agenesis of the corpus callosum with hydrocephalus to cystic formations, abnormal hippocampi, and polymicrogyria. In mice, lysine demethylase 4B is expressed during brain development with high levels in the hippocampus, a region important for learning and memory. To understand how KDM4B variants can lead to GDD in humans, we assessed the effect of KDM4B disruption on brain anatomy and behavior through an in vivo heterozygous mouse model (Kdm4b+/-), focusing on neuroanatomical changes. In mutant mice, the total brain volume was significantly reduced with decreased size of the hippocampal dentate gyrus, partial agenesis of the corpus callosum, and ventriculomegaly. This report demonstrates that variants in KDM4B are associated with GDD/ intellectual disability and neuroanatomical defects. Our findings suggest that KDM4B variation leads to a chromatinopathy, broadening the spectrum of this group of Mendelian disorders caused by alterations in epigenetic machinery.

De novo variants in MPP5 cause global developmental delay and behavioral changes.

Membrane Protein Palmitoylated 5 (MPP5) is a highly conserved apical complex protein essential for cell polarity, fate and survival. Defects in cell polarity are associated with neurologic disorders including autism and microcephaly. MPP5 is essential for neurogenesis in animal models, but human variants leading to neurologic impairment have not been described. We identified three patients with heterozygous MPP5 de novo variants (DNV) and global developmental delay (GDD) and compared their phenotypes and magnetic resonance imaging (MRI) to ascertain how MPP5 DNV leads to GDD. All three patients with MPP5 DNV experienced GDD with language delay/regression and behavioral changes. MRI ranged from normal to decreased gyral folding and microcephaly. The effects of MPP5 depletion on the developing brain were assessed by creating a heterozygous conditional knock out (het CKO) murine model with central nervous system (CNS)-specific Nestin-Cre drivers. In the het CKO model, Mpp5 depletion led to microcephaly, decreased cerebellar volume and cortical thickness. Het CKO mice had decreased ependymal cells and Mpp5 at the apical surface of cortical ventricular zone compared with wild type. Het CKO mice also failed to maintain progenitor pools essential for neurogenesis. The proportion of cortical cells undergoing apoptotic cell death increased, suggesting that cell death reduces progenitor population and neuron number. Het CKO mice also showed behavioral changes, similar to our patients. To our knowledge, this is the first report to show that variants in MPP5 are associated with GDD, behavioral abnormalities and language regression/delay. Murine modeling shows that neurogenesis is likely altered in these individuals, with cell death and skewed cellular composition playing significant roles.

Alkylglycerol monooxygenase, a heterotaxy candidate gene, regulates left-right patterning via Wnt signaling.

Congenital heart disease (CHD) is a major cause of morbidity in the pediatric population yet its genetic and molecular causes remain poorly defined. Previously, we identified AGMO as a candidate heterotaxy disease gene, a disorder of left-right (LR) patterning that can have a profound effect on cardiac function. AGMO is the only known alkylglycerol monooxygenase, an orphan tetrahydrobiopterin dependent enzyme that cleaves the ether linkage in alkylglycerols. However, whether AGMO plays a role in LR patterning was unexplored. Here we reveal that Agmo is required for correct development of the embryonic LR axis in Xenopus embryos recapitulating the patient's heterotaxy phenotype. Mechanistically, we demonstrate that Agmo is a regulator of canonical Wnt signaling, required during gastrulation for normal formation of the left - right organizer. Mutational analysis demonstrates that this function is dependent on Agmo's alkylglycerol monooxygenase activity. Together, our findings identify Agmo as a regulator of canonical Wnt signaling, demonstrate a role for Agmo in embryonic axis formation, and provide insight into the poorly understood developmental requirements for ether lipid cleavage.

Xenopus as a model organism for birth defects-Congenital heart disease and heterotaxy.

Congenital heart disease is the leading cause of birth defects, affecting 9 out of 1000 newborns each year. A particularly severe form of congenital heart disease is heterotaxy, a disorder of left-right development. Despite aggressive surgical management, patients with heterotaxy have poor survival rates and severe morbidity due to their complex congenital heart disease. Recent genetic analysis of affected patients has found novel candidate genes for heterotaxy although their underlying mechanisms remain unknown. In this review, we discuss the importance and challenges of birth defects research including high locus heterogeneity and few second alleles that make defining disease causality difficult. A powerful strategy moving forward is to analyze these candidate genes in a high-throughput human disease model. Xenopus is ideal for these studies. We present multiple examples demonstrating the power of Xenopus in discovering new biology from the analysis of candidate heterotaxy genes such as GALNT11, NEK2 and BCOR. These genes have diverse roles in embryos and have led to a greater understanding of complex signaling pathways and basic developmental biology. It is our hope that the mechanistic analysis of these candidate genes in Xenopus enabled by next generation sequencing of patients will provide clinicians with a greater understanding of patient pathophysiology allowing more precise and personalized medicine, to help patients more effectively in the future.

Homozygous nonsense mutation in SGCA is a common cause of limb-girdle muscular dystrophy in Assiut, Egypt.

INTRODUCTION: The genetic causes of limb-girdle muscular dystrophy (LGMD) have been studied in numerous countries, but such investigations have been limited in Egypt. METHODS: A cohort of 30 families with suspected LGMD from Assiut, Egypt, was studied using immunohistochemistry, homozygosity mapping, Sanger sequencing, and whole exome sequencing. RESULTS: Six families were confirmed to have pathogenic mutations, 4 in SGCA and 2 in DMD. Of these, 3 families harbored a single nonsense mutation in SGCA, suggesting that this may be a common mutation in Assiut, Egypt, originating from a founder effect. CONCLUSIONS: The Assiut region in Egypt appears to share at least several of the common LGMD genes found in other parts of the world. It is notable that 4 of the 6 mutations were ascertained by means of whole exome sequencing, even though it was the last approach adopted. This illustrates the power of this technique for identifying causative mutations for muscular dystrophies. Muscle Nerve 54: 690-695, 2016.

Mutations in the satellite cell gene MEGF10 cause a recessive congenital myopathy with minicores.

We ascertained a nuclear family in which three of four siblings were affected with an unclassified autosomal recessive myopathy characterized by severe weakness, respiratory impairment, scoliosis, joint contractures, and an unusual combination of dystrophic and myopathic features on muscle biopsy. Whole genome sequence from one affected subject was filtered using linkage data and variant databases. A single gene, MEGF10, contained nonsynonymous mutations that co-segregated with the phenotype. Affected subjects were compound heterozygous for missense mutations c.976T > C (p.C326R) and c.2320T > C (p.C774R). Screening the MEGF10 open reading frame in 190 patients with genetically unexplained myopathies revealed a heterozygous mutation, c.211C > T (p.R71W), in one additional subject with a similar clinical and histological presentation as the discovery family. All three mutations were absent from at least 645 genotyped unaffected control subjects. MEGF10 contains 17 atypical epidermal growth factor-like domains, each of which contains eight cysteine residues that likely form disulfide bonds. Both the p.C326R and p.C774R mutations alter one of these residues, which are completely conserved in vertebrates. Previous work showed that murine Megf10 is required for preserving the undifferentiated, proliferative potential of satellite cells, myogenic precursors that regenerate skeletal muscle in response to injury or disease. Here, knockdown of megf10 in zebrafish by four different morpholinos resulted in abnormal phenotypes including unhatched eggs, curved tails, impaired motility, and disorganized muscle tissue, corroborating the pathogenicity of the human mutations. Our data establish the importance of MEGF10 in human skeletal muscle and suggest satellite cell dysfunction as a novel myopathic mechanism.

Molecular diagnosis of hereditary inclusion body myopathy by linkage analysis and identification of a novel splice site mutation in GNE.

BACKGROUND: Many myopathies share clinical features in common, and diagnosis often requires genetic testing. We ascertained a family in which five siblings presented with distal muscle weakness of unknown etiology. METHODS: We performed high-density genomewide linkage analysis and mutation screening of candidate genes to identify the genetic defect in the family. Preserved clinical biopsy material was reviewed to confirm the diagnosis, and reverse transcriptase PCR was used to determine the molecular effect of a splice site mutation. RESULTS: The linkage scan excluded the majority of known myopathy genes, but one linkage peak included the gene GNE, in which mutations cause autosomal recessive hereditary inclusion body myopathy type 2 (HIBM2). Muscle biopsy tissue from a patient showed myopathic features, including small basophilic fibers with vacuoles. Sequence analysis of GNE revealed affected individuals were compound heterozygous for a novel mutation in the 5' splice donor site of intron 10 (c.1816+5G>A) and a previously reported missense mutation (c.2086G>A, p.V696M), confirming the diagnosis as HIBM2. The splice site mutation correlated with exclusion of exon 10 from the transcript, which is predicted to produce an in-frame deletion (p.G545_D605del) of 61 amino acids in the kinase domain of the GNE protein. The father of the proband was heterozygous for the splice site mutation and exhibited mild distal weakness late in life. CONCLUSIONS: Our study expands on the extensive allelic heterogeneity of HIBM2 and demonstrates the value of linkage analysis in resolving ambiguous clinical findings to achieve a molecular diagnosis.

COVID-19: neonatal-perinatal perspectives.

The coronavirus disease 2019 (COVID-19) pandemic, resulting from infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused severe and widespread illness in adults, including pregnant women, while rarely infecting neonates. An incomplete understanding of disease pathogenesis and viral spread has resulted in evolving guidelines to reduce transmission from infected mothers to neonates. Fortunately, the risk of neonatal infection via perinatal/postnatal transmission is low when recommended precautions are followed. However, the psychosocial implications of these practices and racial/ethnic disparities highlighted by this pandemic must also be addressed when caring for mothers and their newborns. This review provides a comprehensive overview of neonatal-perinatal perspectives of COVID-19, ranging from the basic science of infection and recommendations for care of pregnant women and neonates to important psychosocial, ethical, and racial/ethnic topics emerging as a result of both the pandemic and the response of the healthcare community to the care of infected individuals.

Efficient identification of novel mutations in patients with limb girdle muscular dystrophy.

Limb girdle muscular dystrophy type 2 (LGMD2) is a genetically heterogeneous autosomal recessive disorder caused by mutations in 15 known genes. DNA sequencing of all candidate genes can be expensive and laborious, whereas a selective sequencing approach often fails to provide a molecular diagnosis. We aimed to efficiently identify pathogenic mutations via homozygosity mapping in a population in which the genetics of LGMD2 has not been well characterized. Thirteen consanguineous families containing a proband with LGMD2 were recruited from Saudi Arabia, and for 11 of these families, selected individuals were genotyped at 10,204 single nucleotide polymorphisms. Linkage analysis excluded all but one or two known genes in ten of 11 genotyped families, and haplotype comparisons between families allowed further reduction in the number of candidate genes that were screened. Mutations were identified by DNA sequencing in all 13 families, including five novel mutations in four genes, by sequencing at most two genes per family. One family was reclassified as having a different myopathy based on genetic and clinical data after linkage analysis excluded all known LGMD2 genes. LGMD2 subtypes A and B were notably absent from our sample of patients, indicating that the distribution of LGMD2 mutations in Saudi Arabian families may be different than in other populations. Our data demonstrate that homozygosity mapping in consanguineous pedigrees offers a more efficient means of discovering mutations that cause heterogeneous disorders than comprehensive sequencing of known candidate genes.

RAPGEF5 Regulates Nuclear Translocation of ß-Catenin.

Canonical Wnt signaling coordinates many critical aspects of embryonic development, while dysregulated Wnt signaling contributes to common diseases, including congenital malformations and cancer. The nuclear localization of ß-catenin is the defining step in pathway activation. However, despite intensive investigation, the mechanisms regulating ß-catenin nuclear transport remain undefined. In a patient with congenital heart disease and heterotaxy, a disorder of left-right patterning, we previously identified the guanine nucleotide exchange factor, RAPGEF5. Here, we demonstrate that RAPGEF5 regulates left-right patterning via Wnt signaling. In particular, RAPGEF5 regulates the nuclear translocation of ß-catenin independently of both ß-catenin cytoplasmic stabilization and the importin ß1/Ran-mediated transport system. We propose a model whereby RAPGEF5 activates the nuclear GTPases, Rap1a/b, to facilitate the nuclear transport of ß-catenin, defining a parallel nuclear transport pathway to Ran. Our results suggest new targets for modulating Wnt signaling in disease states.

Ontogeny and the effects of exogenous and endogenous glucocorticoids on tight junction protein expression in ovine cerebral cortices.

Maternal glucocorticoid treatment reduces blood-brain permeability early, but not late in fetal development, and pretreatment with glucocorticoids does not affect barrier permeability in newborn lambs. In addition, endogenous increases in plasma cortisol levels are associated with decreases in blood-brain barrier permeability during normal fetal development. Therefore, we tested the hypotheses that development as well as endogenous and exogenous glucocorticoids alters the expression of tight junction proteins in the cerebral cortex of sheep. Cerebral cortices from fetuses at 60%, 70%, and 90% of gestation, newborn and adult sheep were snap frozen after four 6-mg dexamethasone or placebo injections were given over 48-h to the ewes and adult sheep. Lambs were treated similarly with 0.25 mg/kg-dexamethasone or placebo. Tight junction protein expression was measured by Western immunoblot. Claudin-1 was higher (P<0.05) in fetuses at 60% of gestation than in newborn and adult sheep. Claudin-5 was higher at 60% than 70% of gestation, and than in newborn and adult sheep. ZO-1 was higher in newborn than adult sheep. ZO-2 was higher at 90% gestation, in newborn and adult sheep than 60% gestation. Claudin-5 was higher in dexamethasone than placebo-treated lambs, and ZO-2 was higher in fetuses of dexamethasone than placebo-treated ewes at 90% gestation. ZO-2 expression demonstrated a direct correlation with increases in plasma cortisol during fetal development. We conclude that claudin-1, claudin-5, ZO-1, and ZO-2 expression exhibit differential developmental regulation, exogenous glucocorticoids regulate claudin-5 and ZO-2 in vivo at some, but not all ages, and increases in endogenous fetal glucocorticoids are associated with increases in ZO-2 expression, but not with occludin, claudin-1, claudin-5 or ZO-1 expression in ovine cerebral cortices.