Neurodevelopmental and synaptic defects in DNAJC6 parkinsonism, amenable to gene therapy.

DNAJC6 encodes auxilin, a co-chaperone protein involved in clathrin-mediated endocytosis (CME) at the presynaptic terminal. Biallelic mutations in DNAJC6 cause a complex, early-onset neurodegenerative disorder characterized by rapidly progressive parkinsonism-dystonia in childhood. The disease is commonly associated with additional neurodevelopmental, neurological and neuropsychiatric features. Currently, there are no disease-modifying treatments for this condition, resulting in significant morbidity and risk of premature mortality. To investigate the underlying disease mechanisms in childhood-onset DNAJC6 parkinsonism, we generated induced pluripotent stem cells (iPSC) from three patients harboring pathogenic loss-of-function DNAJC6 mutations and subsequently developed a midbrain dopaminergic (mDA) neuronal model of disease. When compared to age-matched and CRISPR-corrected isogenic controls, the neuronal cell model revealed disease-specific auxilin deficiency as well as disturbance of synaptic vesicle (SV) recycling and homeostasis. We also observed neurodevelopmental dysregulation affecting ventral midbrain patterning and neuronal maturation. In order to explore the feasibility of a viral vector-mediated gene therapy approach, iPSC-derived neuronal cultures were treated with lentiviral DNAJC6 gene transfer, which restored auxilin expression and rescued CME. Our patient-derived neuronal model provides deeper insights into the molecular mechanisms of auxilin deficiency as well as a robust platform for the development of targeted precision therapy approaches.

Unbiased phenotype and genotype matching maximizes gene discovery and diagnostic yield.

PURPOSE: Widespread application of next-generation sequencing, combined with data exchange platforms, has provided molecular diagnoses for countless families. To maximize diagnostic yield, we implemented an unbiased semi-automated genematching algorithm based on genotype and phenotype matching. METHODS: Rare homozygous variants identified in 2 or more affected individuals, but not in healthy individuals, were extracted from our local database of ∼12,000 exomes. Phenotype similarity scores (PSS), based on human phenotype ontology terms, were assigned to each pair of individuals matched at the genotype level using HPOsim. RESULTS: 33,792 genotype-matched pairs were discovered, representing variants in 7567 unique genes. There was an enrichment of PSS ≥0.1 among pathogenic/likely pathogenic variant-level pairs (94.3% in pathogenic/likely pathogenic variant-level matches vs 34.75% in all matches). We highlighted founder or region-specific variants as an internal positive control and proceeded to identify candidate disease genes. Variant-level matches were particularly helpful in cases involving inframe indels and splice region variants beyond the canonical splice sites, which may otherwise have been disregarded, allowing for detection of candidate disease genes, such as KAT2A, RPAIN, and LAMP3. CONCLUSION: Semi-automated genotype matching combined with PSS is a powerful tool to resolve variants of uncertain significance and to identify candidate disease genes.

A loss-of-function mutation in human Oxidation Resistance 1 disrupts the spatial-temporal regulation of histone arginine methylation in neurodevelopment.

BACKGROUND: Oxidation Resistance 1 (OXR1) gene is a highly conserved gene of the TLDc domain-containing family. OXR1 is involved in fundamental biological and cellular processes, including DNA damage response, antioxidant pathways, cell cycle, neuronal protection, and arginine methylation. In 2019, five patients from three families carrying four biallelic loss-of-function variants in OXR1 were reported to be associated with cerebellar atrophy. However, the impact of OXR1 on cellular functions and molecular mechanisms in the human brain is largely unknown. Notably, no human disease models are available to explore the pathological impact of OXR1 deficiency. RESULTS: We report a novel loss-of-function mutation in the TLDc domain of the human OXR1 gene, resulting in early-onset epilepsy, developmental delay, cognitive disabilities, and cerebellar atrophy. Patient lymphoblasts show impaired cell survival, proliferation, and hypersensitivity to oxidative stress. These phenotypes are rescued by TLDc domain replacement. We generate patient-derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identify that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1-dependent mechanisms regulating gene expression during neurodevelopment. We model the function of OXR1 in early human brain development using patient-derived brain organoids revealing that OXR1 contributes to the spatial-temporal regulation of histone arginine methylation in specific brain regions. CONCLUSIONS: This study provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency in patients.

WiTNNess: An international natural history study of infantile-onset TNNT1 myopathy.

OBJECTIVE: We created WiTNNess as a hybrid prospective/cross-sectional observational study to simulate a clinical trial for infantile-onset TNNT1 myopathy. Our aims were to identify populations for future trial enrollment, rehearse outcome assessments, specify endpoints, and refine trial logistics. METHODS: Eligible participants had biallelic pathogenic variants of TNNT1 and infantile-onset proximal weakness without confounding conditions. The primary endpoint was ventilator-free survival. "Thriving" was a secondary endpoint defined as the ability to swallow and grow normally without non-oral feeding support. Endpoints of gross motor function included independent sitting and standing as defined by the Word Health Organization, a novel TNNT1 abbreviated motor score, and video mapping of limb movement. We recorded adverse events, concomitant medications, and indices of organ function to serve as comparators of safety in future trials. RESULTS: Sixteen children were enrolled in the aggregate cohort (6 prospective, 10 cross-sectional; median census age 2.3 years, range 0.5-13.8). Median ventilator-free survival was 20.2 months and probability of death or permanent mechanical ventilation was 100% by age 60 months. All six children (100%) in the prospective arm failed to thrive by age 12 months. Only 2 of 16 (13%) children in the aggregate cohort sat independently and none stood alone. Novel exploratory motor assessments also proved informative. Laboratory and imaging data suggest that primary manifestations of TNNT1 deficiency are restricted to skeletal muscle. INTERPRETATION: WiTNNess allowed us to streamline and economize the collection of historical control data without compromising scientific rigor, and thereby establish a sound operational framework for future clinical trials.

Neurodevelopmental disorder mutations in the purine biosynthetic enzyme IMPDH2 disrupt its allosteric regulation.

Inosine 5' monophosphate dehydrogenase (IMPDH) is a critical regulatory enzyme in purine nucleotide biosynthesis that is inhibited by the downstream product GTP. Multiple point mutations in the human isoform IMPDH2 have recently been associated with dystonia and other neurodevelopmental disorders, but the effect of the mutations on enzyme function has not been described. Here, we report the identification of two additional missense variants in IMPDH2 from affected individuals and show that all of the disease-associated mutations disrupt GTP regulation. Cryo-EM structures of one IMPDH2 mutant suggest this regulatory defect arises from a shift in the conformational equilibrium toward a more active state. This structural and functional analysis provides insight into IMPDH2-associated disease mechanisms that point to potential therapeutic approaches and raises new questions about fundamental aspects of IMPDH regulation.

Point mutations in IMPDH2 which cause early-onset neurodevelopmental disorders disrupt enzyme regulation and filament structure.

Inosine 5' monophosphate dehydrogenase (IMPDH) is a critical regulatory enzyme in purine nucleotide biosynthesis that is inhibited by the downstream product GTP. Multiple point mutations in the human isoform IMPDH2 have recently been associated with dystonia and other neurodevelopmental disorders, but the effect of the mutations on enzyme function has not been described. Here, we report identification of two additional affected individuals with missense variants in IMPDH2 and show that all of the disease-associated mutations disrupt GTP regulation. Cryo-EM structures of one IMPDH2 mutant suggest this regulatory defect arises from a shift in the conformational equilibrium toward a more active state. This structural and functional analysis provides insight into IMPDH2-associated disease mechanisms that point to potential therapeutic approaches and raises new questions about fundamental aspects of IMPDH regulation.

SPTSSA variants alter sphingolipid synthesis and cause a complex hereditary spastic paraplegia.

Sphingolipids are a diverse family of lipids with critical structural and signalling functions in the mammalian nervous system, where they are abundant in myelin membranes. Serine palmitoyltransferase, the enzyme that catalyses the rate-limiting reaction of sphingolipid synthesis, is composed of multiple subunits including an activating subunit, SPTSSA. Sphingolipids are both essential and cytotoxic and their synthesis must therefore be tightly regulated. Key to the homeostatic regulation are the ORMDL proteins that are bound to serine palmitoyltransferase and mediate feedback inhibition of enzymatic activity when sphingolipid levels become excessive. Exome sequencing identified potential disease-causing variants in SPTSSA in three children presenting with a complex form of hereditary spastic paraplegia. The effect of these variants on the catalytic activity and homeostatic regulation of serine palmitoyltransferase was investigated in human embryonic kidney cells, patient fibroblasts and Drosophila. Our results showed that two different pathogenic variants in SPTSSA caused a hereditary spastic paraplegia resulting in progressive motor disturbance with variable sensorineural hearing loss and language/cognitive dysfunction in three individuals. The variants in SPTSSA impaired the negative regulation of serine palmitoyltransferase by ORMDLs leading to excessive sphingolipid synthesis based on biochemical studies and in vivo studies in Drosophila. These findings support the pathogenicity of the SPTSSA variants and point to excessive sphingolipid synthesis due to impaired homeostatic regulation of serine palmitoyltransferase as responsible for defects in early brain development and function.

Nociception and pain in humans lacking a functional TRPV1 channel.

BACKGROUNDChronic pain is a debilitating illness with currently limited therapy, in part due to difficulties in translating treatments derived from animal models to patients. The transient receptor potential vanilloid 1 (TRPV1) channel is associated with noxious heat detection and inflammatory pain, and reports of adverse effects in human trials have hindered extensive efforts in the clinical development of TRPV1 antagonists as novel pain relievers.METHODSWe examined 2 affected individuals (A1 and A2) carrying a homozygous missense mutation in TRPV1, rendering the channel nonfunctional. Biochemical and functional assays were used to analyze the mutant channel. To identify possible phenotypes of the affected individuals, we performed psychophysical and medical examinations.RESULTSWe demonstrated that diverse TRPV1 activators, acting at different sites of the channel protein, were unable to open the cloned mutant channel. This finding was not a consequence of impairment in the expression, cellular trafficking, or assembly of protein subunits. The affected individuals were insensitive to application of capsaicin to the mouth and skin and did not demonstrate aversive behavior toward capsaicin. Furthermore, quantitative sensory testing of A1 revealed an elevated heat-pain threshold but also, surprisingly, an elevated cold-pain threshold and extensive neurogenic inflammatory, flare, and pain responses following application of the TRPA1 channel activator mustard oil.CONCLUSIONOur study provides direct evidence in humans for pain-related functional changes linked to TRPV1, which is a prime target in the development of pain relievers.FUNDINGSupported by the Israel Science Foundation (368/19); Teva's National Network of Excellence in Neuroscience grant (no. 0394886) and Teva's National Network of Excellence in Neuroscience postdoctoral fellowship.

Infantile SOD1 deficiency syndrome caused by a homozygous SOD1 variant with absence of enzyme activity.

Pathogenic variants in SOD1, encoding superoxide dismutase 1, are responsible for about 20% of all familial amyotrophic lateral sclerosis cases, through a gain-of-function mechanism. Recently, two reports showed that a specific homozygous SOD1 loss-of-function variant is associated with an infantile progressive motor-neurological syndrome. Exome sequencing followed by molecular studies, including cDNA analysis, SOD1 protein levels and enzymatic activity, and plasma neurofilament light chain levels, were undertaken in an infant with severe global developmental delay, axial hypotonia and limb spasticity. We identified a homozygous 3-bp in-frame deletion in SOD1. cDNA analysis predicted the loss of a single valine residue from a tandem pair (p.Val119/Val120) in the wild-type protein, yet expression levels and splicing were preserved. Analysis of SOD1 activity and protein levels in erythrocyte lysates showed essentially no enzymatic activity and undetectable SOD1 protein in the child, whereas the parents had ∼50% protein expression and activity relative to controls. Neurofilament light chain levels in plasma were elevated, implying ongoing axonal injury and neurodegeneration. Thus, we provide confirmatory evidence of a second biallelic variant in an infant with a severe neurological syndrome and suggest that the in-frame deletion causes instability and subsequent degeneration of SOD1. We highlight the importance of the valine residues at positions V119-120, and suggest possible implications for future therapeutics research.

Delineation of the phenotype of MED17-related disease in Caucasus-Jewish families.

BACKGROUND: and Purpose: Postnatal progressive microcephaly, with seizures and brain atrophy (OMIM # 613668) is a rare disorder caused by a homozygous founder missense mutation c.1112T>C (p.L371P) in the MED17 gene on chromosome 11 that was identified in 2010 in Caucasus Jewish families. The present study aimed to delineate the phenotype and developmental outcomes in patients diagnosed with this mutation to date. METHODS: We conducted a medical charts review to collect the clinical, laboratory and neuroimaging findings in patients from several unrelated families of Caucasus-Jewish origin, who were diagnosed with the same homozygous c.1112T>C MED17 mutation. RESULTS: The study cohort, including the previously reported patients, comprised 10 males and 5 females from 11 families. All subjects had at birth a normal head circumference, which steeply declined to -6SD within a few months. None of the patients achieved developmental milestones. All patients had progressive spasticity and were wheelchair bound due to spastic quadriplegia. All of them eventually developed profound intellectual disability. Epilepsy of varied severity was present in all patients. Most patients required enteral feeding due to aspirations. Eight patients died before puberty (age range 2-13 years). Brain MRI showed marked cerebral atrophy and early prominent cerebellar atrophy (vermian > hemispheres) accompanied by pontine ventral flattening. CONCLUSIONS: The founder c.1112T>C mutation in MED17 gene is expressed by a unique and homogeneous clinical phenotype with distinctive MRI findings. This mutation should be considered in patients of Caucasus-Jewish ancestry presenting with clinical features and a MRI pattern of progressive cerebral and cerebellar atrophy.

Biallelic mutations in neurofascin cause neurodevelopmental impairment and peripheral demyelination.

Axon pathfinding and synapse formation are essential processes for nervous system development and function. The assembly of myelinated fibres and nodes of Ranvier is mediated by a number of cell adhesion molecules of the immunoglobulin superfamily including neurofascin, encoded by the NFASC gene, and its alternative isoforms Nfasc186 and Nfasc140 (located in the axonal membrane at the node of Ranvier) and Nfasc155 (a glial component of the paranodal axoglial junction). We identified 10 individuals from six unrelated families, exhibiting a neurodevelopmental disorder characterized with a spectrum of central (intellectual disability, developmental delay, motor impairment, speech difficulties) and peripheral (early onset demyelinating neuropathy) neurological involvement, who were found by exome or genome sequencing to carry one frameshift and four different homozygous non-synonymous variants in NFASC. Expression studies using immunostaining-based techniques identified absent expression of the Nfasc155 isoform as a consequence of the frameshift variant and a significant reduction of expression was also observed in association with two non-synonymous variants affecting the fibronectin type III domain. Cell aggregation studies revealed a severely impaired Nfasc155-CNTN1/CASPR1 complex interaction as a result of the identified variants. Immunofluorescence staining of myelinated fibres from two affected individuals showed a severe loss of myelinated fibres and abnormalities in the paranodal junction morphology. Our results establish that recessive variants affecting the Nfasc155 isoform can affect the formation of paranodal axoglial junctions at the nodes of Ranvier. The genetic disease caused by biallelic NFASC variants includes neurodevelopmental impairment and a spectrum of central and peripheral demyelination as part of its core clinical phenotype. Our findings support possible overlapping molecular mechanisms of paranodal damage at peripheral nerves in both the immune-mediated and the genetic disease, but the observation of prominent central neurological involvement in NFASC biallelic variant carriers highlights the importance of this gene in human brain development and function.

Homozygous frameshift variant in NTNG2, encoding a synaptic cell adhesion molecule, in individuals with developmental delay, hypotonia, and autistic features.

Regulation of neuronal connectivity and synaptic communication are key to proper functioning of the brain. The Netrin-G subfamily and their cognate receptors are vertebrate-specific synaptic cell adhesion molecules with a role in synapse establishment and function, which seem to have co-evolved to contribute to higher brain functions. We identified a homozygous frameshift variant in NTNG2 (NM_032536.3: c.376dup), encoding Netrin-G2, in eight individuals from four families with global developmental delay, hypotonia, secondary microcephaly, and autistic features. Comparison of haplotypes established this as a founder variant. Previous studies showed that Ntng2-knockout mice have impaired visual, auditory, and motor coordination abilities required for demanding tasks, as well as possible spatial learning and memory deficits. Knockout of Ntng2 in a cellular model resulted in short neurites, and knockout of its trans-synaptic partner Ngl2/Lrrc4 in mice revealed autistic-like behavior and reduced NMDAR synaptic plasticity. The Ngl2/Lrrc4-knockout mouse phenotype was rescued by NMDAR activation, suggesting a mechanistic link to autism spectrum disorder. We thus propose NTNG2 as a candidate disease gene and provide further support for the involvement of Netrin-G2 in neuropsychiatric phenotypes.

Pathogenic Variants in NUP214 Cause "Plugged" Nuclear Pore Channels and Acute Febrile Encephalopathy.

We report biallelic missense and frameshift pathogenic variants in the gene encoding human nucleoporin NUP214 causing acute febrile encephalopathy. Clinical symptoms include neurodevelopmental regression, seizures, myoclonic jerks, progressive microcephaly, and cerebellar atrophy. NUP214 and NUP88 protein levels were reduced in primary skin fibroblasts derived from affected individuals, while the total number and density of nuclear pore complexes remained normal. Nuclear transport assays exhibited defects in the classical protein import and mRNA export pathways in affected cells. Direct surface imaging of fibroblast nuclei by scanning electron microscopy revealed a large increase in the presence of central particles (known as "plugs") in the nuclear pore channels of affected cells. This observation suggests that large transport cargoes may be delayed in passage through the nuclear pore channel, affecting its selective barrier function. Exposure of fibroblasts from affected individuals to heat shock resulted in a marked delay in their stress response, followed by a surge in apoptotic cell death. This suggests a mechanistic link between decreased cell survival in cell culture and severe fever-induced brain damage in affected individuals. Our study provides evidence by direct imaging at the single nuclear pore level of functional changes linked to a human disease.

Heterozygous RNF13 Gain-of-Function Variants Are Associated with Congenital Microcephaly, Epileptic Encephalopathy, Blindness, and Failure to Thrive.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) initiates a stress response mechanism to clear out the unfolded proteins by either facilitating their re-folding or inducing their degradation. When this fails, an apoptotic cascade is initiated so that the affected cell is eliminated. IRE1α is a critical sensor of the unfolded-protein response, essential for initiating the apoptotic signaling. Here, we report an infantile neurodegenerative disorder associated with enhanced activation of IRE1α and increased apoptosis. Three unrelated affected individuals with congenital microcephaly, infantile epileptic encephalopathy, and profound developmental delay were found to carry heterozygous variants (c.932T>C [p.Leu311Ser] or c.935T>C [p.Leu312Pro]) in RNF13, which codes for an IRE1α-interacting protein. Structural modeling predicted that the variants, located on the surface of the protein, would not alter overall protein folding. Accordingly, the abundance of RNF13 and IRE1α was not altered in affected individuals' cells. However, both IRE1α-mediated stress signaling and stress-induced apoptosis were increased in affected individuals' cells. These results indicate that the RNF13 variants confer gain of function to the encoded protein and thereby lead to altered signaling of the ER stress response associated with severe neurodegeneration in infancy.

Further delineation of the clinical spectrum of de novo TRIM8 truncating mutations.

De novo mutations of the TRIM8 gene, which codes for a tripartite motif protein, have been identified using whole exome sequencing (WES) in two patients with epileptic encephalopathy (EE), but these reports were not sufficient to conclude that TRIM8 was a novel gene responsible for EE. Here we report four additional patients presenting with EE and de novo truncating mutations of TRIM8 detected by WES, and give further details of the patient previously reported by the Epi4K consortium. Epilepsy of variable severity was diagnosed in children aged 2 months to 3.5 years of age. All patients had developmental delay of variable severity with no or very limited language, often associated with behavioral anomalies and unspecific facial features or MRI brain abnormalities. The phenotypic variability observed in these patients appeared related to the severity of the epilepsy. One patient presented pharmacoresistant EE with regression, recurrent infections and nephrotic syndrome, compatible with the brain and kidney expression of TRIM8. Interestingly, all mutations were located at the highly conserved C-terminus section of TRIM8. This collaborative study confirms that TRIM8 is a novel gene responsible for EE, possibly associated with nephrotic syndrome. This report brings new evidence on the pathogenicity of TRIM8 mutations and highlights the value of data-sharing to delineate the phenotypic characteristics and biological basis of extremely rare disorders.

A patient-specific induced pluripotent stem cell model for West syndrome caused by ST3GAL3 deficiency.

ST3GAL3 encodes the Golgi enzyme beta-galactoside-alpha-2,3-sialyltransferase-III that in humans forms, among others, the sialyl Lewis a (sLea) epitope on proteins. Functionally deleterious variants in this gene were previously identified in patients with either non-syndromic or syndromic intellectual disability such as West syndrome, an age-dependent epileptic encephalopathic syndrome associated with developmental arrest or regression. The aim of this study was to further elucidate the molecular and cellular mechanisms causing West syndrome by lack of ST3GAL3 function. For this purpose we generated induced pluripotent stem cell (iPSC) lines from fibroblasts obtained from a patient with West syndrome, carrying a variant in exon 12 (c.958G>C, p.(Ala320Pro)) of ST3GAL3, and a healthy sibling, using lentiviral reprogramming. iPSCs and cortical neurons derived thereof were analysed by lectin blots, mRNA sequencing, adherence assays, and FACS. While no significant difference was observed at stem cell or fibroblast level between patient and control cells, patient-derived cortical neurons displayed an altered lectin blot staining pattern, enhanced adherence to a poly-L-ornithine/laminin-coated surface and decreased levels of neurons expressing T-box transcription factor brain 1. Our results suggest that changes in the sialylation pattern on the surface of specific neuronal cell types affect adhesive interactions during development, which in turn may cause subtle changes in tissue composition that could result in the occurrence of epilepsy and might impair neural development to an extent that is detrimental to the development and maintenance of normal cognitive functions.

[EXOME ANALYSIS - A GAME CHANGER IN PEDIATRICS].

INTRODUCTION: Thirteen years after the completion of the human genome project, the determination of the genomic sequence of the coding parts of the DNA (the exones, hence the exome), has turned into a primary diagnostic tool in daily use in clinical practice. The Department of Genetics at Hadassah was the first in Israel to introduce exome analysis as a robust diagnostic tool into the pediatric departments. Till now 2600 exomes were analyzed at Hadassah, 850 of them in 2016 alone. Exome analysis is cheap and fast, enabling precise and non-invasive diagnosis for a vast array of genetic disorders and congenital malformations. The unique composition of the population which the hospital serves (marked by a high rate of consanguinity) enabled reaching diagnosis in 65% of the cases, twice the rate in medical centers worldwide. The results of this analysis enable genetic counseling to patients' families and prevention of serious disorders. Moreover, the results contribute to the understanding of the biological basis of newly identified disorders and in certain cases assist in the management of the patients. The major limitation of exome analysis is the multitude of identified variants which exist in any individual and which challenge our ability to pick the disease-causing variant. In the case of a disease-causing variant in a new gene, experimental proof is required to validate the causality of the variant; occasionally, an incidental finding with possible clinical significance is identified, raising serious ethical concerns. In this article, we will review the use of this technology through the experience of three pediatric departments at Hadassah.

Clinical and experimental evidence suggest a link between KIF7 and C5orf42-related ciliopathies through Sonic Hedgehog signaling.

Acrocallosal syndrome (ACLS) is an autosomal recessive neurodevelopmental disorder caused by KIF7 defects and belongs to the heterogeneous group of ciliopathies related to Joubert syndrome (JBTS). While ACLS is characterized by macrocephaly, prominent forehead, depressed nasal bridge, and hypertelorism, facial dysmorphism has not been emphasized in JBTS cohorts with molecular diagnosis. To evaluate the specificity and etiology of ACLS craniofacial features, we performed whole exome or targeted Sanger sequencing in patients with the aforementioned overlapping craniofacial appearance but variable additional ciliopathy features followed by functional studies. We found (likely) pathogenic variants of KIF7 in 5 out of 9 families, including the original ACLS patients, and delineated 1000 to 4000-year-old Swiss founder alleles. Three of the remaining families had (likely) pathogenic variants in the JBTS gene C5orf42, and one patient had a novel de novo frameshift variant in SHH known to cause autosomal dominant holoprosencephaly. In accordance with the patients' craniofacial anomalies, we showed facial midline widening after silencing of C5orf42 in chicken embryos. We further supported the link between KIF7, SHH, and C5orf42 by demonstrating abnormal primary cilia and diminished response to a SHH agonist in fibroblasts of C5orf42-mutated patients, as well as axonal pathfinding errors in C5orf42-silenced chicken embryos similar to those observed after perturbation of Shh signaling. Our findings, therefore, suggest that beside the neurodevelopmental features, macrocephaly and facial widening are likely more general signs of disturbed SHH signaling. Nevertheless, long-term follow-up revealed that C5orf42-mutated patients showed catch-up development and fainting of facial features contrary to KIF7-mutated patients.

A homozygous deleterious CDK10 mutation in a patient with agenesis of corpus callosum, retinopathy, and deafness.

The primary cilium is a key organelle in numerous physiological and developmental processes. Genetic defects in the formation of this non-motile structure, in its maintenance and function, underlie a wide array of ciliopathies in human, including craniofacial, brain and heart malformations, and retinal and hearing defects. We used exome sequencing to study the molecular basis of disease in an 11-year-old female patient who suffered from growth retardation, global developmental delay with absent speech acquisition, agenesis of corpus callosum and paucity of white matter, sensorineural deafness, retinitis pigmentosa, vertebral anomalies, patent ductus arteriosus, and facial dysmorphism reminiscent of STAR syndrome, a suspected ciliopathy. A homozygous variant, c.870_871del, was identified in the CDK10 gene, predicted to cause a frameshift, p.Trp291Alafs*18, in the cyclin-dependent kinase 10 protein. CDK10 mRNAs were detected in patient cells and do not seem to undergo non-sense mediated decay. CDK10 is the binding partner of Cyclin M (CycM) and CDK10/CycM protein kinase regulates ciliogenesis and primary cilium elongation. Notably, CycM gene is mutated in patients with STAR syndrome. Following incubation, the patient cells appeared less elongated and more densely populated than the control cells suggesting that the CDK10 mutation affects the cytoskeleton. Upon starvation and staining with acetylated-tubulin, γ-tubulin, and Arl13b, the patient cells exhibited fewer and shorter cilia than control cells. These findings underscore the importance of CDK10 for the regulation of ciliogenesis. CDK10 defect is likely associated with a new form of ciliopathy phenotype; additional patients may further validate this association.

Heterozygous De Novo UBTF Gain-of-Function Variant Is Associated with Neurodegeneration in Childhood.

Ribosomal RNA (rRNA) is transcribed from rDNA by RNA polymerase I (Pol I) to produce the 45S precursor of the 28S, 5.8S, and 18S rRNA components of the ribosome. Two transcription factors have been defined for Pol I in mammals, the selectivity factor SL1, and the upstream binding transcription factor (UBF), which interacts with the upstream control element to facilitate the assembly of the transcription initiation complex including SL1 and Pol I. In seven unrelated affected individuals, all suffering from developmental regression starting at 2.5-7 years, we identified a heterozygous variant, c.628G>A in UBTF, encoding p.Glu210Lys in UBF, which occurred de novo in all cases. While the levels of UBF, Ser388 phosphorylated UBF, and other Pol I-related components (POLR1E, TAF1A, and TAF1C) remained unchanged in cells of an affected individual, the variant conferred gain of function to UBF, manifesting by markedly increased UBF binding to the rDNA promoter and to the 5'- external transcribed spacer. This was associated with significantly increased 18S expression, and enlarged nucleoli which were reduced in number per cell. The data link neurodegeneration in childhood with altered rDNA chromatin status and rRNA metabolism.