Mono and biallelic variants in HCN2 cause severe neurodevelopmental disorders.

Hyperpolarization activated Cyclic Nucleotide (HCN) gated channels are crucial for various neurophysiological functions, including learning and sensory functions, and their dysfunction are responsible for brain disorders, such as epilepsy. To date, HCN2 variants have only been associated with mild epilepsy and recently, one monoallelic missense variant has been linked to developmental and epileptic encephalopathy. Here, we expand the phenotypic spectrum of HCN2- related disorders by describing twenty-one additional individuals from fifteen unrelated families carrying HCN2 variants. Seventeen individuals had developmental delay/intellectual disability (DD/ID), two had borderline DD/ID, and one had borderline DD. Ten individuals had epilepsy with DD/ID, with median age of onset of 10 months, and one had epilepsy with normal development. Molecular diagnosis identified thirteen different pathogenic HCN2 variants, including eleven missense variants affecting highly conserved amino acids, one frameshift variant, and one in-frame deletion. Seven variants were monoallelic of which five occurred de novo, one was not maternally inherited, one was inherited from a father with mild learning disabilities, and one was of unknown inheritance. The remaining six variants were biallelic, with four homozygous and two compound heterozygous variants. Functional studies using two-electrode voltage-clamp recordings in Xenopus laevis oocytes were performed on three monoallelic variants, p.(Arg324His), p.(Ala363Val), and p.(Met374Leu), and three biallelic variants, p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp). The p.(Arg324His) variant induced a strong increase of HCN2 conductance, while p.(Ala363Val) and p.(Met374Leu) displayed dominant negative effects, leading to a partial loss of HCN2 channel function. By confocal imaging, we found that the p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp) pathogenic variants impaired membrane trafficking, resulting in a complete loss of HCN2 elicited currents in Xenopus oocytes. Structural 3D-analysis in depolarized and hyperpolarized states of HCN2 channels, revealed that the pathogenic variants p.(His205Gln), p.(Ser409Leu), p.(Arg324Cys), p.(Asn369Ser) and p.(Gly460Asp) modify molecular interactions altering HCN2 function. Taken together, our data broadens the clinical spectrum associated with HCN2 variants, and disclose that HCN2 is involved in developmental encephalopathy with or without epilepsy.

An autosomal-dominant childhood-onset disorder associated with pathogenic variants in VCP.

Valosin-containing protein (VCP) is an AAA+ ATPase that plays critical roles in multiple ubiquitin-dependent cellular processes. Dominant pathogenic variants in VCP are associated with adult-onset multisystem proteinopathy (MSP), which manifests as myopathy, bone disease, dementia, and/or motor neuron disease. Through GeneMatcher, we identified 13 unrelated individuals who harbor heterozygous VCP variants (12 de novo and 1 inherited) associated with a childhood-onset disorder characterized by developmental delay, intellectual disability, hypotonia, and macrocephaly. Trio exome sequencing or a multigene panel identified nine missense variants, two in-frame deletions, one frameshift, and one splicing variant. We performed in vitro functional studies and in silico modeling to investigate the impact of these variants on protein function. In contrast to MSP variants, most missense variants had decreased ATPase activity, and one caused hyperactivation. Other variants were predicted to cause haploinsufficiency, suggesting a loss-of-function mechanism. This cohort expands the spectrum of VCP-related disease to include neurodevelopmental disease presenting in childhood.

A mutational hotspot in AMOTL1 defines a new syndrome of orofacial clefting, cardiac anomalies, and tall stature.

AMOTL1 encodes angiomotin-like protein 1, an actin-binding protein that regulates cell polarity, adhesion, and migration. The role of AMOTL1 in human disease is equivocal. We report a large cohort of individuals harboring heterozygous AMOTL1 variants and define a core phenotype of orofacial clefting, congenital heart disease, tall stature, auricular anomalies, and gastrointestinal manifestations in individuals with variants in AMOTL1 affecting amino acids 157-161, a functionally undefined but highly conserved region. Three individuals with AMOTL1 variants outside this region are also described who had variable presentations with orofacial clefting and multi-organ disease. Our case cohort suggests that heterozygous missense variants in AMOTL1, most commonly affecting amino acid residues 157-161, define a new orofacial clefting syndrome, and indicates an important functional role for this undefined region.

N-methyl-d-aspartate (NMDA) receptor genetics: The power of paralog homology and protein dynamics in defining dominant genetic variants.

Predicting genotype-to-phenotype correlations from genomic variants has been challenging, particularly for genes that have a complex balance of dominant and recessive inheritance for phenotypes. Variants in NMDA receptor components GRIN1, GRIN2A, and GRIN2B cause a myriad of dominant disease phenotypes, with the most common being epilepsy and autism spectrum disorder. Starting from the analysis of a variant of uncertain significance (VUS, GRIN2A G760S), we realized the need for tools to map dominant variants for the components of the NMDA receptor. Some variants within GRIN1, GRIN2A, and GRIN2B exert dominant epilepsy and developmental delay, yet other amino acid variants are conserved and predicted to alter protein function but do not have dominant phenotypes. Common variant annotation tools are not powered to determine pathogenic dominant outcomes. To address this gap, we integrated sequence and structural analyses for GRIN1, GRIN2A, and GRIN2B. Using this approach, we determined that paralog homology mapping and topology can segregate dominant variants, with an elevation of intermolecular contacts between the subunits. Furthermore, demonstrating the general utility of our methodology, we show that 25 VUS within ClinVar also reach a dominant variant annotation, including the GRIN2A G760S variant. Our work suggests paralog homology and protein topology as a powerful strategy within the receptor complex to resolve dominant genetic variants relative to variants that would fit a recessive inheritance, requiring two damaging variants. These strategies should be tested in additional dominant genetic disorders to determine the broader utility.

PTPN4 germline variants result in aberrant neurodevelopment and growth.

Protein-tyrosine phosphatases (PTPs) are pleomorphic regulators of eukaryotic cellular responses to extracellular signals that function by modulating the phosphotyrosine of specific proteins. A handful of PTPs have been implicated in germline and somatic human disease. Using exome sequencing, we identified missense and truncating variants in PTPN4 in six unrelated individuals with varying degrees of intellectual disability or developmental delay. The variants occurred de novo in all five subjects in whom segregation analysis was possible. Recurring features include postnatal growth deficiency or excess, seizures, and, less commonly, structural CNS, heart, or skeletal anomalies. PTPN4 is a widely expressed protein tyrosine phosphatase that regulates neuronal cell homeostasis by protecting neurons against apoptosis. We suggest that pathogenic variants in PTPN4 confer risk for growth and cognitive abnormalities in humans.

Perinatal Manifestations of DARS2-Associated Leukoencephalopathy With Brainstem and Spinal Cord Involvement and Lactate Elevation (LBSL).

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a progressive disorder associated with deficiency of mitochondrial aspartyl-tRNA synthetase, a homodimer encoded by the gene DARS2. There is a wide range in age of onset of symptoms, typically from childhood to adulthood, with very few cases of infantile onset disease reported. We report a child at age 10 years with perinatal onset of symptoms evidenced by congenital microcephaly with progression to severe but non-lethal epileptic encephalopathy and spastic quadriplegia. A comprehensive epilepsy focused gene panel performed as a trio with parents detected a novel homozygous DARS2 variant. This variant is located at the dimer interface in a critical catalytic domain and is expected to result in markedly reduced enzyme activity which likely explains the severe and early onset symptoms in this case.

A dyadic approach to the delineation of diagnostic entities in clinical genomics.

The delineation of disease entities is complex, yet recent advances in the molecular characterization of diseases provide opportunities to designate diseases in a biologically valid manner. Here, we have formalized an approach to the delineation of Mendelian genetic disorders that encompasses two distinct but inter-related concepts: (1) the gene that is mutated and (2) the phenotypic descriptor, preferably a recognizably distinct phenotype. We assert that only by a combinatorial or dyadic approach taking both of these attributes into account can a unitary, distinct genetic disorder be designated. We propose that all Mendelian disorders should be designated as "GENE-related phenotype descriptor" (e.g., "CFTR-related cystic fibrosis"). This approach to delineating and naming disorders reconciles the complexity of gene-to-phenotype relationships in a simple and clear manner yet communicates the complexity and nuance of these relationships.

Expanding the molecular spectrum and the neurological phenotype related to CAMTA1 variants.

The CAMTA1-associated phenotype was initially defined in patients with intragenic deletions and duplications who showed nonprogressive congenital ataxia, with or without intellectual disability. Here, we describe 10 individuals with CAMTA1 variants: nine previously unreported (likely) pathogenic variants comprising one missense, four frameshift and four nonsense variants, and one missense variant of unknown significance. Six patients were diagnosed following whole exome sequencing and four individuals with exome-based targeted panel analysis. Most of them present with developmental delay, manifesting in speech and motor delay. Other frequent findings are hypotonia, cognitive impairment, cerebellar dysfunction, oculomotor abnormalities, and behavioral problems. Feeding problems occur more frequently than previously observed. In addition, we present a systematic review of 19 previously published individuals with causal variants, including copy number, truncating, and missense variants. We note a tendency of more severe cognitive impairment and recurrent dysmorphic features in individuals with a copy number variant. Pathogenic variants are predominantly observed in and near the N- and C- terminal functional domains. Clinical heterogeneity is observed, but 3'-terminal variants seem to associate with less pronounced cerebellar dysfunction.

Virus-induced genetics revealed by multidimensional precision medicine transcriptional workflow applicable to COVID-19.

Precision medicine requires the translation of basic biological understanding to medical insights, mainly applied to characterization of each unique patient. In many clinical settings, this requires tools that can be broadly used to identify pathology and risks. Patients often present to the intensive care unit with broad phenotypes, including multiple organ dysfunction syndrome (MODS) resulting from infection, trauma, or other disease processes. Etiology and outcomes are unique to individuals, making it difficult to cohort patients with MODS, but presenting a prime target for testing/developing tools for precision medicine. Using multitime point whole blood (cellular/acellular) total transcriptomics in 27 patients, we highlight the promise of simultaneously mapping viral/bacterial load, cell composition, tissue damage biomarkers, balance between syndromic biology versus environmental response, and unique biological insights in each patient using a single platform measurement. Integration of a transcriptome workflow yielded unexpected insights into the complex interplay between host genetics and viral/bacterial specific mechanisms, highlighted by a unique case of virally induced genetics (VIG) within one of these 27 patients. The power of RNA-Seq to study unique patient biology while investigating environmental contributions can be a critical tool moving forward for translational sciences applied to precision medicine.

Perinatal distress in 1p36 deletion syndrome can mimic hypoxic ischemic encephalopathy.

1p36 deletion syndrome is a well-described condition with a recognizable phenotype, including cognitive impairment, seizures, and structural brain anomalies such as periventricular leukomalacia (PVL). In a large series of these individuals by Battaglia et al., "birth history was notable in 50% of the cases for varying degrees of perinatal distress." Given the potential for perinatal distress, seizures and PVL, we questioned if this disorder has clinical overlap with hypoxic ischemic encephalopathy (HIE). We reviewed the medical records of 69 individuals with 1p36 deletion to clarify the perinatal phenotype of this disorder and determine if there is evidence of perinatal distress and/or hypoxic injury. Our data provides evidence that these babies have signs of perinatal distress. The majority (59% term; 75% preterm) needed resuscitation and approximately 18% had cardiac arrest. Most had abnormal brain imaging (84% term; 73% preterm) with abnormal white matter findings in over half of patients. PVL or suggestion of "hypoxic insult" was present in 18% of term and 45% of preterm patients. In conclusion, individuals with 1p36 deletion have evidence of perinatal distress, white matter changes, and seizures, which can mimic HIE but are likely related to their underlying chromosome disorder.

Correction to: De novo and inherited TCF20 pathogenic variants are associated with intellectual disability, dysmorphic features, hypotonia, and neurological impairments with similarities to Smith-Magenis syndrome.

It was highlighted that the original article [1] contained a typographical error in the Results section. Subject 17 was incorrectly cited as Subject 1. This Correction article shows the revised statement. The original article has been updated.

De novo and inherited TCF20 pathogenic variants are associated with intellectual disability, dysmorphic features, hypotonia, and neurological impairments with similarities to Smith-Magenis syndrome.

BACKGROUND: Neurodevelopmental disorders are genetically and phenotypically heterogeneous encompassing developmental delay (DD), intellectual disability (ID), autism spectrum disorders (ASDs), structural brain abnormalities, and neurological manifestations with variants in a large number of genes (hundreds) associated. To date, a few de novo mutations potentially disrupting TCF20 function in patients with ID, ASD, and hypotonia have been reported. TCF20 encodes a transcriptional co-regulator structurally related to RAI1, the dosage-sensitive gene responsible for Smith-Magenis syndrome (deletion/haploinsufficiency) and Potocki-Lupski syndrome (duplication/triplosensitivity). METHODS: Genome-wide analyses by exome sequencing (ES) and chromosomal microarray analysis (CMA) identified individuals with heterozygous, likely damaging, loss-of-function alleles in TCF20. We implemented further molecular and clinical analyses to determine the inheritance of the pathogenic variant alleles and studied the spectrum of phenotypes. RESULTS: We report 25 unique inactivating single nucleotide variants/indels (1 missense, 1 canonical splice-site variant, 18 frameshift, and 5 nonsense) and 4 deletions of TCF20. The pathogenic variants were detected in 32 patients and 4 affected parents from 31 unrelated families. Among cases with available parental samples, the variants were de novo in 20 instances and inherited from 4 symptomatic parents in 5, including in one set of monozygotic twins. Two pathogenic loss-of-function variants were recurrent in unrelated families. Patients presented with a phenotype characterized by developmental delay, intellectual disability, hypotonia, variable dysmorphic features, movement disorders, and sleep disturbances. CONCLUSIONS: TCF20 pathogenic variants are associated with a novel syndrome manifesting clinical characteristics similar to those observed in Smith-Magenis syndrome. Together with previously described cases, the clinical entity of TCF20-associated neurodevelopmental disorders (TAND) emerges from a genotype-driven perspective.

Evolution of the phenotype of craniosynostosis with dental anomalies syndrome and report of IL11RA variant population frequencies in a Crouzon-like autosomal recessive syndrome.

In 2011, biallelic loss-of-function variants in the interleukin receptor 11 alpha gene IL11RA were found to be associated with a Crouzon-like craniosynostosis syndrome with associated dental anomalies (CRSDA). Since then, a total of 41 similar patients have been reported with IL11RA variants. We report two adult brothers diagnosed with Crouzon syndrome as children, in which the clinical diagnosis of CRSDA was made on reevaluation. Laboratory testing detected biallelic IL11RA variants, c.916_924dup (p.Thr306_Ser308dup) and c.781C > T (p.Arg261Cys), both of which have now been reported in other families. Protein modeling and conservation analysis show that these two mutation sites cluster together near a WSXWS motif that likely plays a significant role in regulating IL11RA protein function. Population analysis from gnomAD shows that Non-Finnish Europeans (similar to ethnicity of this family), have an allele frequency for p.Thr306_Ser308dup of 0.014% and p.Arg261Cys of 0.008%. We found other ethnicities have functional IL11RA missense variants at higher frequencies. With this report, we provide a summary of the clinical findings including details of middle ear anomalies associated with conductive hearing loss. We also provide data supporting the populations at risk for this condition to increase recognition and diagnosis of this rare autosomal recessive craniosynostosis syndrome.

Lethal NARS2-Related Disorder Associated With Rapidly Progressive Intractable Epilepsy and Global Brain Atrophy.

BACKGROUND: Infantile epileptic encephalopathy is a heterogeneous condition that has been associated with variants in more than 200 genes. The variability in findings and prognosis creates challenges to making the correct diagnosis and initiating the appropriate therapy. Biallelic variants in NARS2, a mitochondrial aminoacyl-tRNA synthetase gene, were recently associated with neurodegenerative disorders that include epilepsy. METHODS: We describe two infant brothers who presented with focal status epilepticus that progressed to lethal epileptic encephalopathy. We compared the cost of diagnostic laboratory evaluation for each child. Detailed NARS2 protein analysis was performed using a sequence-to-structure-to-function workflow, merging multiple homologous structures, to suggest biologic impact of the NARS2 variants. RESULTS: Brain magnetic resonance imaging showed rapid progression to generalized atrophy. Extensive metabolic, infectious, chromosomal and genetic testing of the first infant failed to reach a specific diagnosis. The younger brother presented similarly. Rapid whole exome sequencing was performed revealing novel biallelic variants in NARS2. The variants c.167A>G (p.Gln56Arg) and c.631T>A (p.Phe211Ile) were confirmed in a reserved sample from the older brother. Management was then redirected toward palliative care and the child died at age nine months. CONCLUSIONS: NARS2-related disorder should be considered in infants presenting with refractory seizures and rapid brain atrophy. Metabolic screening tests may be normal or yield nonspecific findings. Rapid whole exome sequencing in children with fulminant onset intractable epilepsy may minimize extensive diagnostic evaluation and aid in prognosis and medical management.

ßIV Spectrinopathies Cause Profound Intellectual Disability, Congenital Hypotonia, and Motor Axonal Neuropathy.

ßIV spectrin links ankyrinG (AnkG) and clustered ion channels at axon initial segments (AISs) and nodes of Ranvier to the axonal cytoskeleton. Here, we report bi-allelic pathogenic SPTBN4 variants (three homozygous and two compound heterozygous) that cause a severe neurological syndrome that includes congenital hypotonia, intellectual disability, and motor axonal and auditory neuropathy. We introduced these variants into ßIV spectrin, expressed these in neurons, and found that 5/7 were loss-of-function variants disrupting AIS localization or abolishing phosphoinositide binding. Nerve biopsies from an individual with a loss-of-function variant had reduced nodal Na+ channels and no nodal KCNQ2 K+ channels. Modeling the disease in mice revealed that although ankyrinR (AnkR) and ßI spectrin can cluster Na+ channels and partially compensate for the loss of AnkG and ßIV spectrin at nodes of Ranvier, AnkR and ßI spectrin cannot cluster KCNQ2- and KCNQ3-subunit-containing K+ channels. Our findings define a class of spectrinopathies and reveal the molecular pathologies causing nervous-system dysfunction.