Disruption of NEUROD2 causes a neurodevelopmental syndrome with autistic features via cell-autonomous defects in forebrain glutamatergic neurons.

While the transcription factor NEUROD2 has recently been associated with epilepsy, its precise role during nervous system development remains unclear. Using a multi-scale approach, we set out to understand how Neurod2 deletion affects the development of the cerebral cortex in mice. In Neurod2 KO embryos, cortical projection neurons over-migrated, thereby altering the final size and position of layers. In juvenile and adults, spine density and turnover were dysregulated in apical but not basal compartments in layer 5 neurons. Patch-clamp recordings in layer 5 neurons of juvenile mice revealed increased intrinsic excitability. Bulk RNA sequencing showed dysregulated expression of many genes associated with neuronal excitability and synaptic function, whose human orthologs were strongly associated with autism spectrum disorders (ASD). At the behavior level, Neurod2 KO mice displayed social interaction deficits, stereotypies, hyperactivity, and occasionally spontaneous seizures. Mice heterozygous for Neurod2 had similar defects, indicating that Neurod2 is haploinsufficient. Finally, specific deletion of Neurod2 in forebrain excitatory neurons recapitulated cellular and behavioral phenotypes found in constitutive KO mice, revealing the region-specific contribution of dysfunctional Neurod2 in symptoms. Informed by these neurobehavioral features in mouse mutants, we identified eleven patients from eight families with a neurodevelopmental disorder including intellectual disability and ASD associated with NEUROD2 pathogenic mutations. Our findings demonstrate crucial roles for Neurod2 in neocortical development, whose alterations can cause neurodevelopmental disorders including intellectual disability and ASD.

De Novo Variants in CNOT1, a Central Component of the CCR4-NOT Complex Involved in Gene Expression and RNA and Protein Stability, Cause Neurodevelopmental Delay.

CNOT1 is a member of the CCR4-NOT complex, which is a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. We report on 39 individuals with heterozygous de novo CNOT1 variants, including missense, splice site, and nonsense variants, who present with a clinical spectrum of intellectual disability, motor delay, speech delay, seizures, hypotonia, and behavioral problems. To link CNOT1 dysfunction to the neurodevelopmental phenotype observed, we generated variant-specific Drosophila models, which showed learning and memory defects upon CNOT1 knockdown. Introduction of human wild-type CNOT1 was able to rescue this phenotype, whereas mutants could not or only partially, supporting our hypothesis that CNOT1 impairment results in neurodevelopmental delay. Furthermore, the genetic interaction with autism-spectrum genes, such as ASH1L, DYRK1A, MED13, and SHANK3, was impaired in our Drosophila models. Molecular characterization of CNOT1 variants revealed normal CNOT1 expression levels, with both mutant and wild-type alleles expressed at similar levels. Analysis of protein-protein interactions with other members indicated that the CCR4-NOT complex remained intact. An integrated omics approach of patient-derived genomics and transcriptomics data suggested only minimal effects on endonucleolytic nonsense-mediated mRNA decay components, suggesting that de novo CNOT1 variants are likely haploinsufficient hypomorph or neomorph, rather than dominant negative. In summary, we provide strong evidence that de novo CNOT1 variants cause neurodevelopmental delay with a wide range of additional co-morbidities. Whereas the underlying pathophysiological mechanism warrants further analysis, our data demonstrate an essential and central role of the CCR4-NOT complex in human brain development.

De Novo Frameshift Variants in the Neuronal Splicing Factor NOVA2 Result in a Common C-Terminal Extension and Cause a Severe Form of Neurodevelopmental Disorder.

The neuro-oncological ventral antigen 2 (NOVA2) protein is a major factor regulating neuron-specific alternative splicing (AS), previously associated with an acquired neurologic condition, the paraneoplastic opsoclonus-myoclonus ataxia (POMA). We report here six individuals with de novo frameshift variants in NOVA2 affected with a severe neurodevelopmental disorder characterized by intellectual disability (ID), motor and speech delay, autistic features, hypotonia, feeding difficulties, spasticity or ataxic gait, and abnormal brain MRI. The six variants lead to the same reading frame, adding a common proline rich C-terminal part instead of the last KH RNA binding domain. We detected 41 genes differentially spliced after NOVA2 downregulation in human neural cells. The NOVA2 variant protein shows decreased ability to bind target RNA sequences and to regulate target AS events. It also fails to complement the effect on neurite outgrowth induced by NOVA2 downregulation in vitro and to rescue alterations of retinotectal axonal pathfinding induced by loss of NOVA2 ortholog in zebrafish. Our results suggest a partial loss-of-function mechanism rather than a full heterozygous loss-of-function, although a specific contribution of the novel C-terminal extension cannot be excluded.

De Novo Variants in MAPK8IP3 Cause Intellectual Disability with Variable Brain Anomalies.

Using exome sequencing, we have identified de novo variants in MAPK8IP3 in 13 unrelated individuals presenting with an overlapping phenotype of mild to severe intellectual disability. The de novo variants comprise six missense variants, three of which are recurrent, and three truncating variants. Brain anomalies such as perisylvian polymicrogyria, cerebral or cerebellar atrophy, and hypoplasia of the corpus callosum were consistent among individuals harboring recurrent de novo missense variants. MAPK8IP3 has been shown to be involved in the retrograde axonal-transport machinery, but many of its specific functions are yet to be elucidated. Using the CRISPR-Cas9 system to target six conserved amino acid positions in Caenorhabditis elegans, we found that two of the six investigated human alterations led to a significantly elevated density of axonal lysosomes, and five variants were associated with adverse locomotion. Reverse-engineering normalized the observed adverse effects back to wild-type levels. Combining genetic, phenotypic, and functional findings, as well as the significant enrichment of de novo variants in MAPK8IP3 within our total cohort of 27,232 individuals who underwent exome sequencing, we implicate de novo variants in MAPK8IP3 as a cause of a neurodevelopmental disorder with intellectual disability and variable brain anomalies.

Multicenter study of mortality in achondroplasia.

Sudden death and higher mortality are recognized in achondroplasia, with acute brainstem compression, a common cause of mortality in children <4 years and cardiovascular deaths being more prevalent in adults. Although, changes in clinical management have improved survival, mortality is still higher than in the general population. The aim of this multicenter clinic-based study was to assess the rate and causes of mortality in patients seen in clinic since 1986. Information was ascertained for achondroplasia patients clinically assessed in four skeletal dysplasia clinics. Data was sent to the National Death Index to identify vital status and cause of death. Standardized mortality rates (SMR) were calculated based on U.S. populations from 1975, 1995, and 2000. Eight hundred fifty-five patients were identified, contributing 12,117 person-years and a total of 12 deaths. One case died in infancy. In the 1-4 year age group, which had the highest age-adjusted SMR, three out of five deaths were because of cerebrovascular/cardiovascular events. Half the deaths in ages 5 through 24 were because of accidental events, including motor vehicle accidents. Decreased mortality in children with achondroplasia was noted, particularly in younger age groups. This improvement in childhood survival is outpaced by improved survival in the general population. Causes of death in these patients have shifted over the last 30 years, with fewer sudden death and deaths because of pneumonia or hydrocephalus countered by more cardiovascular or cerebrovascular and accidental deaths. Clinicians should be aware of the apparent increased risk of vehicular accidents and counsel patients accordingly.

Novel homozygous likely-pathogenic intronic variant in INS causing permanent neonatal diabetes in siblings.

Permanent neonatal diabetes (PNDM) is a rare genetic condition characterized by hyperglycemia, insulinopenia, and failure to thrive beginning in the first 6 months of life. Recessive mutations in INS lead to decreased production of insulin via a variety of mechanisms. We present a case of two brothers, born to consanguineous parents, with a novel homozygous intronic variant in the INS gene. Each patient presented with intrauterine growth restriction (IUGR) and significant hyperglycemia within the first 24 h of life. All the grandparents have a diagnosis of diabetes, one of them requiring insulin treatment and the parents currently deny personal histories of diabetes. Although this mutation has not previously been described, given the segregation of the mutation, absence of heterozygosity (AOH) in the genomic region encompassing the INS locus, documented insulinopenia, and high neonatal insulin requirements, we suspect that this variant is pathogenic. Possible implications for personalized treatment of the underlying molecular etiology for an individual's diabetes are discussed.

Pseudoachondroplasia and painful sequelae.

Pseudoachondroplasia (PSACH) is a well-described autosomal dominant short limb dwarfing condition caused by mutations in the cartilage oligomeric matrix protein gene (COMP). The most debilitating complication of the disorder is joint pain starting in childhood, the extent and severity of which is poorly defined. The aim of this study was to fully assess the pain and identify additional clinical complications affecting those with PSACH. An online survey was distributed to individuals with PSACH. Of the 77 surveys analyzed, 83% reported chronic pain starting as early as the newborn period. Pain was most frequently reported in weight bearing joints including the knees, hips, and back, and significantly interfered with their overall quality of life. For pain relief, patients with PSACH used a wide variety of treatments. However, patients reported only a 60% resolution of pain with their current treatments. An increase in other comorbidities was not found, specifically osteoporosis was not increased. This study documents for the first time that pain is the most common presenting symptom in PSACH and is often overlooked until short stature becomes obvious. The recognition of chronic pain as one of the earliest manifestations of PSACH is important to allow for prompt diagnosis.

Mutations in COL27A1 cause Steel syndrome and suggest a founder mutation effect in the Puerto Rican population.

Osteochondrodysplasias represent a large group of developmental structural disorders that can be caused by mutations in a variety of genes responsible for chondrocyte development, differentiation, mineralization and early ossification. The application of whole-exome sequencing to disorders apparently segregating as Mendelian traits has proven to be an effective approach to disease gene identification for conditions with unknown molecular etiology. We identified a homozygous missense variant p.(Gly697Arg) in COL27A1, in a family with Steel syndrome and no consanguinity. Interestingly, the identified variant seems to have arisen as a founder mutation in the Puerto Rican population.