MSL2 variants lead to a neurodevelopmental syndrome with lack of coordination, epilepsy, specific dysmorphisms, and a distinct episignature.

Epigenetic dysregulation has emerged as an important etiological mechanism of neurodevelopmental disorders (NDDs). Pathogenic variation in epigenetic regulators can impair deposition of histone post-translational modifications leading to aberrant spatiotemporal gene expression during neurodevelopment. The male-specific lethal (MSL) complex is a prominent multi-subunit epigenetic regulator of gene expression and is responsible for histone 4 lysine 16 acetylation (H4K16ac). Using exome sequencing, here we identify a cohort of 25 individuals with heterozygous de novo variants in MSL complex member MSL2. MSL2 variants were associated with NDD phenotypes including global developmental delay, intellectual disability, hypotonia, and motor issues such as coordination problems, feeding difficulties, and gait disturbance. Dysmorphisms and behavioral and/or psychiatric conditions, including autism spectrum disorder, and to a lesser extent, seizures, connective tissue disease signs, sleep disturbance, vision problems, and other organ anomalies, were observed in affected individuals. As a molecular biomarker, a sensitive and specific DNA methylation episignature has been established. Induced pluripotent stem cells (iPSCs) derived from three members of our cohort exhibited reduced MSL2 levels. Remarkably, while NDD-associated variants in two other members of the MSL complex (MOF and MSL3) result in reduced H4K16ac, global H4K16ac levels are unchanged in iPSCs with MSL2 variants. Regardless, MSL2 variants altered the expression of MSL2 targets in iPSCs and upon their differentiation to early germ layers. Our study defines an MSL2-related disorder as an NDD with distinguishable clinical features, a specific blood DNA episignature, and a distinct, MSL2-specific molecular etiology compared to other MSL complex-related disorders.

Substrate specificity controlled by the exit site of human P4-ATPases, revealed by de novo point mutations in neurological disorders.

The maintenance of lipid asymmetry on the plasma membrane is regulated by flippases, such as ATP8A2, ATP11A, and ATP11C, which translocate phosphatidylserine and phosphatidylethanolamine from the outer leaflet to the inner leaflet. We previously identified a patient-derived point mutation (Q84E) in ATP11A at the phospholipid entry site, which acquired the ability to flip phosphatidylcholine (PtdCho). This mutation led to elevated levels of sphingomyelin (SM) in the outer leaflet of the plasma membrane. We herein present two de novo ATP11A dominant mutations (E114G and S399L) in heterozygous patients exhibiting neurological and developmental disorders. These mutations, situated near the predicted phospholipid exit site, similarly confer the ability for ATP11A to recognize PtdCho as a substrate, resulting in its internalization into cells. Cells expressing these mutants had increased SM levels on their surface, attributed to the up-regulated expression of the sphingomyelin synthase-1 gene, rendering them more susceptible to SM phosphodiesterase-mediated cell lysis. Corresponding mutations in ATP11C and ATP8A2, paralogs of ATP11A, exerted similar effects on PtdCho-flipping activity and increased SM levels on the cell surface. Molecular dynamics simulations, based on the ATP11C structure, suggest that the E114G and S399L mutations enhance ATP11C's affinity toward PtdCho. These findings underscore the importance of the well-conserved exit and entry sites in determining phospholipid substrate specificity and indicate that aberrant flipping of PtdCho contributes to neurological disorders.

Bi-allelic TTI1 variants cause an autosomal-recessive neurodevelopmental disorder with microcephaly.

Telomere maintenance 2 (TELO2), Tel2 interacting protein 2 (TTI2), and Tel2 interacting protein 1 (TTI1) are the three components of the conserved Triple T (TTT) complex that modulates activity of phosphatidylinositol 3-kinase-related protein kinases (PIKKs), including mTOR, ATM, and ATR, by regulating the assembly of mTOR complex 1 (mTORC1). The TTT complex is essential for the expression, maturation, and stability of ATM and ATR in response to DNA damage. TELO2- and TTI2-related bi-allelic autosomal-recessive (AR) encephalopathies have been described in individuals with moderate to severe intellectual disability (ID), short stature, postnatal microcephaly, and a movement disorder (in the case of variants within TELO2). We present clinical, genomic, and functional data from 11 individuals in 9 unrelated families with bi-allelic variants in TTI1. All present with ID, and most with microcephaly, short stature, and a movement disorder. Functional studies performed in HEK293T cell lines and fibroblasts and lymphoblastoid cells derived from 4 unrelated individuals showed impairment of the TTT complex and of mTOR pathway activity which is improved by treatment with Rapamycin. Our data delineate a TTI1-related neurodevelopmental disorder and expand the group of disorders related to the TTT complex.

Exome/Genome Sequencing in Undiagnosed Syndromes.

Exome sequencing (ES) and genome sequencing (GS) have radically transformed the diagnostic approach to undiagnosed rare/ultrarare Mendelian diseases. Next-generation sequencing (NGS), the technology integral for ES, GS, and most large (100+) gene panels, has enabled previously unimaginable diagnoses, changes in medical management, new treatments, and accurate reproductive risk assessments for patients, as well as new disease gene discoveries. Yet, challenges remain, as most individuals remain undiagnosed with current NGS. Improved NGS technology has resulted in long-read sequencing, which may resolve diagnoses in some patients who do not obtain a diagnosis with current short-read ES and GS, but its effectiveness is unclear, and it is expensive. Other challenges that persist include the resolution of variants of uncertain significance, the urgent need for patients with ultrarare disorders to have access to therapeutics, the need for equity in patient access to NGS-based testing, and the study of ethical concerns. However, the outlook for undiagnosed disease resolution is bright, due to continual advancements in the field.

Bi-allelic variants in neuronal cell adhesion molecule cause a neurodevelopmental disorder characterized by developmental delay, hypotonia, neuropathy/spasticity.

Cell adhesion molecules are membrane-bound proteins predominantly expressed in the central nervous system along principal axonal pathways with key roles in nervous system development, neural cell differentiation and migration, axonal growth and guidance, myelination, and synapse formation. Here, we describe ten affected individuals with bi-allelic variants in the neuronal cell adhesion molecule NRCAM that lead to a neurodevelopmental syndrome of varying severity; the individuals are from eight families. This syndrome is characterized by developmental delay/intellectual disability, hypotonia, peripheral neuropathy, and/or spasticity. Computational analyses of NRCAM variants, many of which cluster in the third fibronectin type III (Fn-III) domain, strongly suggest a deleterious effect on NRCAM structure and function, including possible disruption of its interactions with other proteins. These findings are corroborated by previous in vitro studies of murine Nrcam-deficient cells, revealing abnormal neurite outgrowth, synaptogenesis, and formation of nodes of Ranvier on myelinated axons. Our studies on zebrafish nrcamaΔ mutants lacking the third Fn-III domain revealed that mutant larvae displayed significantly altered swimming behavior compared to wild-type larvae (p < 0.03). Moreover, nrcamaΔ mutants displayed a trend toward increased amounts of α-tubulin fibers in the dorsal telencephalon, demonstrating an alteration in white matter tracts and projections. Taken together, our study provides evidence that NRCAM disruption causes a variable form of a neurodevelopmental disorder and broadens the knowledge on the growing role of the cell adhesion molecule family in the nervous system.

The best of both worlds: Blending cutting-edge research with clinical processes for a productive exome clinic.

Genomic medicine has been transformed by next-generation sequencing (NGS), inclusive of exome sequencing (ES) and genome sequencing (GS). Currently, ES is offered widely in clinical settings, with a less prevalent alternative model consisting of hybrid programs that incorporate research ES along with clinical patient workflows. We were among the earliest to implement a hybrid ES clinic, have provided diagnoses to 45% of probands, and have identified several novel candidate genes. Our program is enabled by a cost-effective investment by the health system and is unique in encompassing all the processes that have been variably included in other hybrid/clinical programs. These include careful patient selection, utilization of a phenotype-agnostic bioinformatics pipeline followed by manual curation of variants and phenotype integration by clinicians, close collaborations between the clinicians and the bioinformatician, pursuit of interesting variants, communication of results to patients in categories that are predicated upon the certainty of a diagnosis, and tracking changes in results over time and the underlying mechanisms for such changes. Due to its effectiveness, scalability to GS and its resource efficiency, specific elements of our paradigm can be incorporated into existing clinical settings, or the entire hybrid model can be implemented within health systems that have genomic medicine programs, to provide NGS in a scientifically rigorous, yet pragmatic setting.

The book is just being written: The enduring journey of parents of children with emerging- ultrarare disorders.

Ultra rare disorders are being diagnosed at an unprecedented rate, due to genomic sequencing. These diagnoses are often a new gene association, for which little is known, and few share the diagnosis. For these diagnoses, we use the term emerging-ultrarare disorder (E-URD), defined as <100 diagnosed individuals. We contacted 20 parents of children diagnosed with an E-URD through the Duke University Research Sequencing Clinic. Seventeen completed semi-structured interviews exploring parental perspectives (7/17 had children in publications describing the phenotype; 4/17 had children in the first publication establishing a new disorder). Data were analyzed using a directed content approach informed by an empowerment framework. Parents reported a range of responses, including benefits of a diagnosis and challenges of facing the unknown, some described feeling lost and confused, while others expressed empowerment. Empowerment characteristics were hope for the future, positive emotions, engagement, and confidence/self-efficacy to connect with similar others, partner with healthcare providers, and seek new knowledge. We identified a subset of parents who proactively engaged researchers, supported research and publications, and created patient advocacy and support organizations to connect with and bolster similarly diagnosed families. Other parents reported challenges of low social support, low tolerance for uncertainty, limited knowledge about their child's disorder, as well as difficulty partnering with HCPs and connecting to an E-URD community. An overarching classification was developed to describe parental actions taken after an E-URD diagnosis: adjusting, managing, and pioneering. These classifications may help genetic counselors identify and facilitate positive steps with parents of a child with an E-URD.

Unraveling non-participation in genomic research: A complex interplay of barriers, facilitators, and sociocultural factors.

Although genomic research offering next-generation sequencing (NGS) has increased the diagnoses of rare/ultra-rare disorders, populations experiencing health disparities infrequently participate in these studies. The factors underlying non-participation would most reliably be ascertained from individuals who have had the opportunity to participate, but decline. We thus enrolled parents of children and adult probands with undiagnosed disorders who had declined genomic research offering NGS with return of results with undiagnosed disorders (Decliners, n = 21) and compared their data to those who participated (Participants, n = 31). We assessed: (1) practical barriers and facilitators, (2) sociocultural factors-genomic knowledge and distrust, and (3) the value placed upon a diagnosis by those who declined participation. The primary findings were that residence in rural and medically underserved areas (MUA) and higher number of barriers were significantly associated with declining participation in the study. Exploratory analyses revealed multiple co-occurring practical barriers, greater emotional exhaustion and research hesitancy in the parents in the Decliner group compared to the Participants, with both groups identifying a similar number of facilitators. The parents in the Decliner group also had lower genomic knowledge, but distrust of clinical research was not different between the groups. Importantly, despite their non-participation, those in the Decliner group indicated an interest in obtaining a diagnosis and expressed confidence in being able to emotionally manage the ensuing results. Study findings support the concept that some families who decline participation in diagnostic genomic research may be experiencing pile-up with exhaustion of family resources - making participation in the genomic research difficult. This study highlights the complexity of the factors that underlie non-participation in clinically relevant NGS research. Thus, approaches to mitigating barriers to NGS research participation by populations experiencing health disparities need to be multi-pronged and tailored so that they can benefit from state-of -the art genomic technologies.

Expanding the Spectrum of BAF-Related Disorders: De Novo Variants in SMARCC2 Cause a Syndrome with Intellectual Disability and Developmental Delay.

SMARCC2 (BAF170) is one of the invariable core subunits of the ATP-dependent chromatin remodeling BAF (BRG1-associated factor) complex and plays a crucial role in embryogenesis and corticogenesis. Pathogenic variants in genes encoding other components of the BAF complex have been associated with intellectual disability syndromes. Despite its significant biological role, variants in SMARCC2 have not been directly associated with human disease previously. Using whole-exome sequencing and a web-based gene-matching program, we identified 15 individuals with variable degrees of neurodevelopmental delay and growth retardation harboring one of 13 heterozygous variants in SMARCC2, most of them novel and proven de novo. The clinical presentation overlaps with intellectual disability syndromes associated with other BAF subunits, such as Coffin-Siris and Nicolaides-Baraitser syndromes and includes prominent speech impairment, hypotonia, feeding difficulties, behavioral abnormalities, and dysmorphic features such as hypertrichosis, thick eyebrows, thin upper lip vermilion, and upturned nose. Nine out of the fifteen individuals harbor variants in the highly conserved SMARCC2 DNA-interacting domains (SANT and SWIRM) and present with a more severe phenotype. Two of these individuals present cardiac abnormalities. Transcriptomic analysis of fibroblasts from affected individuals highlights a group of differentially expressed genes with possible roles in regulation of neuronal development and function, namely H19, SCRG1, RELN, and CACNB4. Our findings suggest a novel SMARCC2-related syndrome that overlaps with neurodevelopmental disorders associated with variants in BAF-complex subunits.

Expanding the phenotypic spectrum of ARCN1-related syndrome.

PURPOSE: This study aimed to describe the phenotypic and molecular characteristics of ARCN1-related syndrome. METHODS: Patients with ARCN1 variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. RESULTS: In total, we identified 14 cases of ARCN1-related syndrome, (9 pediatrics, and 5 fetal cases from 3 families). The clinical features these newly identified cases were compared to 6 previously reported cases for a total of 20 cases. Intrauterine growth restriction, micrognathia, and short stature were present in all patients. Other common features included prematurity (11/15, 73.3%), developmental delay (10/14, 71.4%), genitourinary malformations in males (6/8, 75%), and microcephaly (12/15, 80%). Novel features of ARCN1-related syndrome included transient liver dysfunction and specific glycosylation abnormalities during illness, giant cell hepatitis, hepatoblastoma, cataracts, and lethal skeletal manifestations. Developmental delay was seen in 73% of patients, but only 3 patients had intellectual disability, which is less common than previously reported. CONCLUSION: ARCN1-related syndrome presents with a wide clinical spectrum ranging from a severe embryonic lethal syndrome to a mild syndrome with intrauterine growth restriction, micrognathia, and short stature without intellectual disability. Patients with ARCN1-related syndrome should be monitored for liver dysfunction during illness, cataracts, and hepatoblastoma. Additional research to further define the phenotypic spectrum and possible genotype-phenotype correlations are required.

The broad phenotypic spectrum of PPP2R1A-related neurodevelopmental disorders correlates with the degree of biochemical dysfunction.

PURPOSE: Neurodevelopmental disorders (NDD) caused by protein phosphatase 2A (PP2A) dysfunction have mainly been associated with de novo variants in PPP2R5D and PPP2CA, and more rarely in PPP2R1A. Here, we aimed to better understand the latter by characterizing 30 individuals with de novo and often recurrent variants in this PP2A scaffolding Aα subunit. METHODS: Most cases were identified through routine clinical diagnostics. Variants were biochemically characterized for phosphatase activity and interaction with other PP2A subunits. RESULTS: We describe 30 individuals with 16 different variants in PPP2R1A, 21 of whom had variants not previously reported. The severity of developmental delay ranged from mild learning problems to severe intellectual disability (ID) with or without epilepsy. Common features were language delay, hypotonia, and hypermobile joints. Macrocephaly was only seen in individuals without B55α subunit-binding deficit, and these patients had less severe ID and no seizures. Biochemically more disruptive variants with impaired B55α but increased striatin binding were associated with profound ID, epilepsy, corpus callosum hypoplasia, and sometimes microcephaly. CONCLUSION: We significantly expand the phenotypic spectrum of PPP2R1A-related NDD, revealing a broader clinical presentation of the patients and that the functional consequences of the variants are more diverse than previously reported.

Defining the genotypic and phenotypic spectrum of X-linked MSL3-related disorder.

PURPOSE: We sought to delineate the genotypic and phenotypic spectrum of female and male individuals with X-linked, MSL3-related disorder (Basilicata-Akhtar syndrome). METHODS: Twenty-five individuals (15 males, 10 females) with causative variants in MSL3 were ascertained through exome or genome sequencing at ten different sequencing centers. RESULTS: We identified multiple variant types in MSL3 (ten nonsense, six frameshift, four splice site, three missense, one in-frame-deletion, one multi-exon deletion), most proven to be de novo, and clustering in the terminal eight exons suggesting that truncating variants in the first five exons might be compensated by an alternative MSL3 transcript. Three-dimensional modeling of missense and splice variants indicated that these have a deleterious effect. The main clinical findings comprised developmental delay and intellectual disability ranging from mild to severe. Autism spectrum disorder, muscle tone abnormalities, and macrocephaly were common as well as hearing impairment and gastrointestinal problems. Hypoplasia of the cerebellar vermis emerged as a consistent magnetic resonance image (MRI) finding. Females and males were equally affected. Using facial analysis technology, a recognizable facial gestalt was determined. CONCLUSION: Our aggregated data illustrate the genotypic and phenotypic spectrum of X-linked, MSL3-related disorder (Basilicata-Akhtar syndrome). Our cohort improves the understanding of disease related morbidity and allows us to propose detailed surveillance guidelines for affected individuals.

De novo variants in SNAP25 cause an early-onset developmental and epileptic encephalopathy.

PURPOSE: This study aims to provide a comprehensive description of the phenotypic and genotypic spectrum of SNAP25 developmental and epileptic encephalopathy (SNAP25-DEE) by reviewing newly identified and previously reported individuals. METHODS: Individuals harboring heterozygous missense or loss-of-function variants in SNAP25 were assembled through collaboration with international colleagues, matchmaking platforms, and literature review. For each individual, detailed phenotyping, classification, and structural modeling of the identified variant were performed. RESULTS: The cohort comprises 23 individuals with pathogenic or likely pathogenic de novo variants in SNAP25. Intellectual disability and early-onset epilepsy were identified as the core symptoms of SNAP25-DEE, with recurrent findings of movement disorders, cerebral visual impairment, and brain atrophy. Structural modeling for all variants predicted possible functional defects concerning SNAP25 or impaired interaction with other components of the SNARE complex. CONCLUSION: We provide a comprehensive description of SNAP25-DEE with intellectual disability and early-onset epilepsy mostly occurring before the age of two years. These core symptoms and additional recurrent phenotypes show an overlap to genes encoding other components or associated proteins of the SNARE complex such as STX1B, STXBP1, or VAMP2. Thus, these findings advance the concept of a group of neurodevelopmental disorders that may be termed "SNAREopathies."

De novo variants in TCF7L2 are associated with a syndromic neurodevelopmental disorder.

TCF7L2 encodes transcription factor 7-like 2 (OMIM 602228), a key mediator of the evolutionary conserved canonical Wnt signaling pathway. Although several large-scale sequencing studies have implicated TCF7L2 in intellectual disability and autism, both the genetic mechanism and clinical phenotype have remained incompletely characterized. We present here a comprehensive genetic and phenotypic description of 11 individuals who have been identified to carry de novo variants in TCF7L2, both truncating and missense. Missense variation is clustered in or near a high mobility group box domain, involving this region in these variants' pathogenicity. All affected individuals present with developmental delays in childhood, but most ultimately achieved normal intelligence or had only mild intellectual disability. Myopia was present in approximately half of the individuals, and some individuals also possessed dysmorphic craniofacial features, orthopedic abnormalities, or neuropsychiatric comorbidities including autism and attention-deficit/hyperactivity disorder (ADHD). We thus present an initial clinical and genotypic spectrum associated with variation in TCF7L2, which will be important in informing both medical management and future research.

Autism and developmental disability caused by KCNQ3 gain-of-function variants.

OBJECTIVE: Recent reports have described single individuals with neurodevelopmental disability (NDD) harboring heterozygous KCNQ3 de novo variants (DNVs). We sought to assess whether pathogenic variants in KCNQ3 cause NDD and to elucidate the associated phenotype and molecular mechanisms. METHODS: Patients with NDD and KCNQ3 DNVs were identified through an international collaboration. Phenotypes were characterized by clinical assessment, review of charts, electroencephalographic (EEG) recordings, and parental interview. Functional consequences of variants were analyzed in vitro by patch-clamp recording. RESULTS: Eleven patients were assessed. They had recurrent heterozygous DNVs in KCNQ3 affecting residues R230 (R230C, R230H, R230S) and R227 (R227Q). All patients exhibited global developmental delay within the first 2 years of life. Most (8/11, 73%) were nonverbal or had a few words only. All patients had autistic features, and autism spectrum disorder (ASD) was diagnosed in 5 of 11 (45%). EEGs performed before 10 years of age revealed frequent sleep-activated multifocal epileptiform discharges in 8 of 11 (73%). For 6 of 9 (67%) recorded between 1.5 and 6 years of age, spikes became near-continuous during sleep. Interestingly, most patients (9/11, 82%) did not have seizures, and no patient had seizures in the neonatal period. Voltage-clamp recordings of the mutant KCNQ3 channels revealed gain-of-function (GoF) effects. INTERPRETATION: Specific GoF variants in KCNQ3 cause NDD, ASD, and abundant sleep-activated spikes. This new phenotype contrasts both with self-limited neonatal epilepsy due to KCNQ3 partial loss of function, and with the neonatal or infantile onset epileptic encephalopathies due to KCNQ2 GoF. ANN NEUROL 2019;86:181-192.

A pathogenic variant in the SETBP1 hotspot results in a forme-fruste Schinzel-Giedion syndrome.

Schinzel-Giedion syndrome (SGS; OMIM 269150) is an ultra-rare genetic disorder associated with a distinctive facial gestalt, congenital malformations, severe intellectual disability, and a progressive neurological course. The prognosis for SGS is poor, with survival beyond the first decade rare. Germline, de novo heterozygous variants in the SETBP1 gene cause SGS with the pathogenic variants associated with the SGS phenotype missense and confined to exon 4 of the gene, clustered in a four amino acid (12 bp) hotspot in the SKI homologous region of the SETBP1 protein. We report a patient with a de novo I871S variant within the SKI homologous region, which has been associated with the severe phenotype previously; but our patient has fewer features of SGS and a milder course. This is the first report of a forme-fruste phenotype in a patient with a pathogenic variant within the SGS hotspot on the SETBP1 gene and it highlights the importance of considering atypical clinical presentations in the context of severe ultra-rare genetic disorders.

Rationally designed AAV2 and AAVrh8R capsids provide improved transduction in the retina and brain.

The successful application of adeno-associated virus (AAV) gene delivery vectors as a therapeutic paradigm will require efficient gene delivery to the appropriate cells in affected organs. In this study, we utilized a rational design approach to introduce modifications to the AAV2 and AAVrh8R capsids and the resulting variants were evaluated for transduction activity in the retina and brain. The modifications disrupted either capsid/receptor binding or altered capsid surface charge. Specifically, we mutated AAV2 amino acids R585A and R588A, which are required for binding to its receptor, heparan sulfate proteoglycans, to generate a variant referred to as AAV2-HBKO. In contrast to parental AAV2, the AAV2-HBKO vector displayed low-transduction activity following intravitreal delivery to the mouse eye; however, following its subretinal delivery, AAV2-HBKO resulted in significantly greater photoreceptor transduction. Intrastriatal delivery of AAV2-HBKO to mice facilitated widespread striatal and cortical expression, in contrast to the restricted transduction pattern of the parental AAV2 vector. Furthermore, we found that altering the surface charge on the AAVrh8R capsid by modifying the number of arginine residues on the capsid surface had a profound impact on subretinal transduction. The data further validate the potential of capsid engineering to improve AAV gene therapy vectors for clinical applications.

Looking beyond the exome: a phenotype-first approach to molecular diagnostic resolution in rare and undiagnosed diseases.

PurposeTo describe examples of missed pathogenic variants on whole-exome sequencing (WES) and the importance of deep phenotyping for further diagnostic testing.MethodsGuided by phenotypic information, three children with negative WES underwent targeted single-gene testing.ResultsIndividual 1 had a clinical diagnosis consistent with infantile systemic hyalinosis, although WES and a next-generation sequencing (NGS)-based ANTXR2 test were negative. Sanger sequencing of ANTXR2 revealed a homozygous single base pair insertion, previously missed by the WES variant caller software. Individual 2 had neurodevelopmental regression and cerebellar atrophy, with no diagnosis on WES. New clinical findings prompted Sanger sequencing and copy number testing of PLA2G6. A novel homozygous deletion of the noncoding exon 1 (not included in the WES capture kit) was detected, with extension into the promoter, confirming the clinical suspicion of infantile neuroaxonal dystrophy. Individual 3 had progressive ataxia, spasticity, and magnetic resonance image changes of vanishing white matter leukoencephalopathy. An NGS leukodystrophy gene panel and WES showed a heterozygous pathogenic variant in EIF2B5; no deletions/duplications were detected. Sanger sequencing of EIF2B5 showed a frameshift indel, probably missed owing to failure of alignment.ConclusionThese cases illustrate potential pitfalls of WES/NGS testing and the importance of phenotype-guided molecular testing in yielding diagnoses.

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.