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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 inIMPDH2and 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.Significance StatementPoint mutations in the human enzyme IMPDH2, a critical regulator of nucleotide biosynthesis, are linked to neurodevelopmental disorders, such as dystonia. Here, we report two additional IMPDH2 point mutants associated with similar disorders. We investigate the effects of each mutation on IMPDH2 structure and functionin vitroand find that all mutations are gain of function, preventing allosteric regulation of IMPDH2 activity. We report high resolution structures of one variant and present a structure-based hypothesis for its dysregulation. This work provides a biochemical basis for understanding diseases caused byIMPDH2mutation and lays a foundation for future therapeutic development.

The ionotropic glutamate delta receptor GluD1, encoded by the GRID1 gene, is involved in synapse formation, function, and plasticity. GluD1 does not bind glutamate, but instead cerebellin and D-serine, which allow the formation of trans-synaptic bridges, and trigger transmembrane signaling. Despite wide expression in the nervous system, pathogenic GRID1 variants have not been characterized in humans so far. We report homozygous missense GRID1 variants in five individuals from two unrelated consanguineous families presenting with intellectual disability and spastic paraplegia, without (p.Thr752Met) or with (p.Arg161His) diagnosis of glaucoma, a threefold phenotypic association whose genetic bases had not been elucidated previously. Molecular modeling indicated that Arg161His and Thr752Met mutations alter the hinge between GluD1 cerebellin and D-serine binding domains and the stiffness of this latter domain, respectively. Expression, trafficking, physical interaction with metabotropic glutamate receptor mGlu1, and cerebellin binding of GluD1 mutants were not conspicuously altered. Conversely, we found that both GluD1 mutants hampered signaling of metabotropic glutamate receptor mGlu1/5 via the ERK pathway in neurons of primary cortical culture. Moreover, both mutants impaired dendrite morphology and excitatory synapse density in neurons of primary hippocampal culture. These results show that the clinical phenotypes are distinct entities segregating in the families as an autosomal recessive trait, and caused by pathophysiological effects of GluD1 mutants involving metabotropic glutamate receptor signaling and neuronal connectivity. Our findings unravel the importance of the GluD1 receptor signaling in sensory, cognitive and motor functions of the human nervous system.

Mutations in the KCNA1 gene, encoding the voltage-gated potassium channel Kv1.1, have been associated with a spectrum of neurological phenotypes, including episodic ataxia type 1 and developmental and epileptic encephalopathy. We have recently identified a de novo variant in KCNA1 in the highly conserved Pro-Val-Pro motif within the pore of the Kv1.1 channel in a girl affected by early onset epilepsy, ataxia and developmental delay. Other mutations causing severe epilepsy are located in Kv1.1 pore domain. The patient was initially treated with a combination of antiepileptic drugs with limited benefit. Finally, seizures and ataxia control were achieved with lacosamide and acetazolamide. The aim of this study was to functionally characterize Kv1.1 mutant channel to provide a genotype–phenotype correlation and discuss therapeutic options for KCNA1-related epilepsy. To this aim, we transfected HEK 293 cells with Kv1.1 or P403A cDNAs and recorded potassium currents through whole-cell patch-clamp. P403A channels showed smaller potassium currents, voltage-dependent activation shifted by +30 mV towards positive potentials and slower kinetics of activation compared with Kv1.1 wild-type. Heteromeric Kv1.1+P403A channels, resembling the condition of the heterozygous patient, confirmed a loss-of-function biophysical phenotype. Overall, the functional characterization of P403A channels correlates with the clinical symptoms of the patient and supports the observation that mutations associated with severe epileptic phenotype cluster in a highly conserved stretch of residues in Kv1.1 pore domain. This study also strengthens the beneficial effect of acetazolamide and sodium channel blockers in KCNA1 channelopathies.

MSTO1 is a cytoplasmic protein that modulates mitochondrial dynamics by promoting mitochondrial fusion. Mutations in the MSTO1 gene are responsible for an extremely rare condition characterized by early-onset myopathy and cerebellar ataxia. We report here two siblings from a large Ashkenazi Jewish family, presenting with a progressive neuromuscular disease characterized by ataxia and myopathy. By whole exome sequencing, we found a novel homozygous missense mutation (c.1403T>A, p.Leu468Gln) in MSTO1. Studies performed on fibroblasts from the index patient demonstrated the pathogenic role of the identified variant; we found that MSTO1 protein level was reduced and that mitochondrial network was fragmented or formed enlarged structures. Moreover, patient's cells showed reduced mitochondrial DNA amount. Our report confirms that MSTO1 mutations are typically recessive, and associated with clinical phenotypes characterized by early-onset muscle impairment and ataxia, often with upper motor neuron signs and varied cognitive impairment.

Isolated defects in the mitochondrial respiratory chain complex II (CII; succinate-ubiquinone oxidoreductase) are extremely rare and mainly result from bi-allelic mutations in one of the nuclear encoded subunits: SDHA, SDHB and SDHD, which comprise CII and the assembly CII factor SDHAF1. We report an adolescent female who presented with global developmental delay, intellectual disability and childhood onset progressive bilateral optic atrophy. Whole exome sequencing of the patient and her unaffected parents identified the novel heterozygous de novo variant c.1984C > T [NM_004168.4] in the SDHA gene. Biochemical assessment of CII in the patient’s derived fibroblasts and lymphocytes displayed considerably decreased CII residual activity compared with normal controls, when normalized to the integral mitochondrial enzyme citrate synthase. Protein modeling of the consequent p.Arg662Cys variant [NP-004159.2] suggested that this substitution will compromise the structural integrity of the FAD-binding protein at the C-terminus that will ultimately impair the FAD binding to SDHA, thus decreasing the entire CII activity. Our study emphasizes the role of certain heterozygous SDHA mutations in a distinct clinical phenotype dominated by optic atrophy and neurological impairment. This is the second mutation that has been reported to cause this phenotype. Furthermore, it adds developmental delay and cognitive disability to the expanding spectrum of the disorder. We propose to add SDHA to next generation sequencing gene panels of optic atrophy.

Calpainopathies constitute a heterogeneous group of disorders resulting from deficiencies in calpains, calcium-specific proteases that modulate substrates by limited proteolysis. Clinical manifestations depend on tissue-specific expression of the defective calpain and substrate specificity. CAPN15, encoding the Drosophila small optic lobes (sol) homolog, was recently found to cause various eye defects in individuals carrying bi-allelic missense variants. Here we report on two siblings with manifestations reminiscent of Johanson-Blizzard syndrome including failure to thrive, microcephaly, global developmental delay, dysmorphic features, endocrine abnormalities and congenital malformations, in addition to eye abnormalities. Exome sequencing identified a homozygous 47 base-pair deletion in a minimal intron of CAPN15, including the splice donor site. Sequencing of cDNA revealed single exon skipping, resulting in an out-of-frame deletion with a predicted premature termination codon. These findings expand the phenotypic spectrum associated with CAPN15 variants, and suggest that complete loss-of-function is associated with a recognizable syndrome of congenital malformations and developmental delay, overlapping Johanson-Blizzard syndrome and the recently observed brain defects in Capn15 knockout (KO) mice. Moreover, the data highlight the unique opportunity for indel detection in minimal introns.

The GTPase of immunity-associated proteins (GIMAPs) are a family of genes believed to contribute to lymphocyte development, signaling, and apoptosis, thus playing an important role in immune system homeostasis. While models of gene derangement have been described in both mice and immortalized cell lines, human examples of these diseases remain exceptionally rare. In this manuscript we describe the first documented human cases of a homozygous deleterious GIMAP6 variant in the GIMAP6 gene and their subsequent clinical and immunological phenotype. In order to interrogate the patients' immune defect, we performed whole-exome sequencing, western blot, flow cytometry analysis, lymphocyte activation and proliferation studies, cytokine release assays, and apoptosis studies. We found two siblings with a predicted deleterious homozygous variant in the GIMAP6 gene with no expression of GIMAP6 protein on western blot. Patients demonstrated accelerated apoptosis, but largely normal lymphocyte subpopulations, activation and proliferation and cytokine release. There appears to be a spectrum of clinical features associated with deficiency of GIMAP6 protein, with one patient suffering lymphopenia and recurrent sinopulmonary infections, and the other clinically asymptomatic. Biallelic variants in the GIMAP6 gene have now been shown to demonstrate disease in humans. The absence of GIMAP6 protein is associated with a spectrum of clinical manifestations and much remains to be learnt about the pathogenic mechanisms underlying this disease. We suggest that biallelic variants in the gene for GIMAP6 should be considered in children with lymphopenia and recurrent sinopulmonary infections.

We report a loss-of-function mutation in the TLDc domain of human Oxidation Resistance 1 (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 generated patient derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identified that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1 dependent mechanisms regulating gene expression during neurodevelopment. We modeled 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. Our work provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency, highlighting the therapeutic potential of OXR1 in numerous neurodegenerative and neurodevelopmental disorders.

BackgroundMAPK-activated protein kinase 5 (MAPKAPK5) is an essential enzyme for diverse cellular processes. Dysregulation of the pathways regulated by MAPKAPK enzymes can lead to the development of variable diseases. Recently, homozygous loss-of-function variants inMAPKAPK5were reported in four patients from three families presenting with a recognisable neurodevelopmental disorder, so-called ‘neurocardiofaciodigital’ syndrome.Objective and methodsIn order to improve characterisation of the clinical features associated with biallelicMAPKAPK5variants, we employed a genotype-first approach combined with reverse deep-phenotyping of three affected individuals.ResultsIn the present study, we identified biallelic loss-of-function and missenseMAPKAPK5variants in three unrelated individuals from consanguineous families. All affected individuals exhibited a syndromic neurodevelopmental disorder characterised by severe global developmental delay, intellectual disability, characteristic facial morphology, brachycephaly, digital anomalies, hair and nail defects and neuroradiological findings, including cerebellar hypoplasia and hypomyelination, as well as variable vision and hearing impairment. Additional features include failure to thrive, hypotonia, microcephaly and genitourinary anomalies without any reported congenital heart disease.ConclusionIn this study, we consolidate the causality of loss of MAPKAPK5 function and further delineate the molecular and phenotypic spectrum associated with this new ultra-rare neurodevelopmental syndrome.

The circadian clock, which drives a wide range of bodily rhythms in synchrony with the day–night cycle, is based on a molecular oscillator that ticks with a period of approximately 24 h. Timed proteasomal degradation of clock components is central to the fine-tuning of the oscillator’s period. FBXL3 is a protein that functions as a substrate-recognition factor in the E3 ubiquitin ligase complex, and was originally shown in mice to mediate degradation of CRY proteins and thus contribute to the mammalian circadian clock mechanism. By exome sequencing, we have identified a FBXL3 mutation in patients with syndromic developmental delay accompanied by morphological abnormalities and intellectual disability, albeit with a normal sleep pattern. We have investigated the function of FBXL3 in the zebrafish, an excellent model to study both vertebrate development and circadian clock function and, like humans, a diurnal species. Loss of fbxl3a function in zebrafish led to disruption of circadian rhythms of promoter activity and mRNA expression as well as locomotor activity and sleep–wake cycles. However, unlike humans, no morphological effects were evident. These findings point to an evolutionary conserved role for FBXL3 in the circadian clock system across vertebrates and to the acquisition of developmental roles in humans.

Mutations in FBXO7 have been discovered associated with an atypical parkinsonism. We report here a new homozygous missense mutation in a paediatric patient that causes an L250P substitution in the dimerization domain of Fbxo7. This alteration selectively ablates the Fbxo7-PI31 interaction and causes a significant reduction in Fbxo7 and PI31 levels in patient cells. Consistent with their association with proteasomes, L250P patient fibroblasts have reduced proteasome activity and proteasome subunits. We also show PI31 interacts directly with the MiD49/51 fission adaptor proteins, and unexpectedly, PI31 acts as an adaptor enabling SCFFbxo7 ligase to ubiquitinate MiD49. Thus, the L250P mutation changes the function of Fbxo7 by altering its substrate repertoire. Although MiD49/51 expression was reduced in L250P patient cells, there was no effect on the mitochondrial network. However, patient cells had higher levels of ROS and reduced viability under stress. Our study shows that Fbxo7 and PI31 affect each other’s functions in regulating both proteasomal and mitochondrial function and demonstrate a new function for PI31, as an adaptor for the SCFFbxo7 E3 ubiquitin ligase.

Malignant infantile osteopetrosis (MIOP) is the autosomal recessive, severe form of osteopetrosis. This rare genetic syndrome usually presents soon after birth and is often fatal if left untreated. Early diagnosis is key for proper management but clinical presentation is diverse, and oftentimes diagnosis may be challenging. In this study, we retrospectively collected data of genetic mutations and phenotypic characteristics at the initial presentation of 81 MIOP patients and analyzed genotype-phenotype correlations. The most common genetic mutation was in the TCIRG1 gene (n = 46, 56.8%), followed by SNX10 (n = 20, 25%). Other genetic mutations included RANK (n = 7, 8.7%), CLCN7 (n = 5, 6.2%) and CA2 (n = 3, 3.7%). More than half of the patients presented with growth retardation (n = 46, 56.8%). Twenty-one of the patients were blind (26%) and thirty-seven patients had other neurological deficits (45.7%) at the time of initial presentation. Most patients presented with hematological signs of bone marrow failure including anemia (n = 69, 85.2%) and thrombocytopenia (n = 33, 40.7%). Thrombocytopenia at initial presentation was significantly more prevalent in patients with mutations in the TCIRG1 gene (p = 0.036). Other phenotypic presenting features were not found to be significantly correlated to specific gene mutations. In conclusion, the initial presentation of MIOP is variable, but some features are common such as growth retardation, visual impairment, and cytopenias. High awareness of MIOP presenting signs is essential for prompt diagnosis of this challenging disease.

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.

The ATP/GTP-Binding Protein 1 (AGTPBP1) gene (OMIM *606830) catalyzes deglutamylation of polyglutamylated proteins, and its deficiency manifests by cerebellar ataxia and peripheral neuropathy in mice and lower motor neuron-like disease in sheep. In the mutant mice, cerebellar atrophy due to Purkinje cell degeneration is observed, likely due to increased tubulin polyglutamylation in affected brain areas. We report two unrelated individuals who presented with early onset cerebellar atrophy, developmental arrest with progressive muscle weakness, and feeding and respiratory difficulties, accompanied by severe motor neuronopathy. Whole exome sequencing followed by segregation analysis in the families and cDNA studies revealed deleterious biallelic variants in the AGTPBP1 gene. We conclude that complete loss-of-function of AGTPBP1 in humans, just like in mice and sheep, is associated with cerebellar and motor neuron disease, reminiscent of Pontocerebellar Hypoplasia Type 1 (PCH1).

Nuclear envelopathies comprise a heterogeneous group of diseases caused by mutations in genes encoding nuclear envelope proteins. Mutations affecting lamina-associated polypeptide 1 (LAP1) result in two discrete phenotypes of muscular dystrophy and progressive dystonia with cerebellar atrophy. We report 7 patients presenting at birth with severe progressive neurological impairment, bilateral cataract, growth retardation and early lethality. All the patients are homozygous for a nonsense mutation in the TOR1AIP1 gene resulting in the loss of both protein isoforms LAP1B and LAP1C. Patient-derived fibroblasts exhibit changes in nuclear envelope morphology and large nuclear-spanning channels containing trapped cytoplasmic organelles. Decreased and inefficient cellular motility is also observed in these fibroblasts. Our study describes the complete absence of both major human LAP1 isoforms, underscoring their crucial role in early development and organogenesis. LAP1-associated defects may thus comprise a broad clinical spectrum depending on the availability of both isoforms in the nuclear envelope throughout life., Nuclear envelopathies are a group of diseases caused by genetic mutations in essential nuclear envelope genes. Here, the authors report a nuclear envelopathy with a homozygous nonsense variant in TOR1AIP1 which leads to changes in the nuclear morphology including large nuclear-spanning channels in patients’ fibroblasts.

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.

RBL2/p130 is one of three highly conserved members of the retinoblastoma (RB) protein family. It is strongly upregulated during neuronal differentiation and brain development, and is critical for survival of post-mitotic neurons. Similar to RB1, it has been implicated as a tumor suppressor gene and has been shown to be dysregulated in various types of cancer. Recent publications describe biallelic, germline loss of function variants in RBL2 in individuals with profound developmental delay. We report a child with profound developmental delay, microcephaly, and hypotonia, who developed fulminant exophthalmos at age 6 years. Brain MRI followed by a biopsy of an intra-orbital mass revealed a mesenchymal tumor. Post-surgical histopathologic examination of the resected tumor was compatible with diagnosis of nodular fasciitis. Exome sequencing from peripheral blood identified a biallelic frameshift variant (c.901dupT) in RBL2. Notably, no malignancies were reported in previous cases with RBL2 variants. This case provides a possible association between RBL2 and orbital tumors.

We report a patient who presented with congenital hypotonia, hypoventilation, and cerebellar histopathological alterations. Exome analysis revealed a homozygous mutation in the initiation codon of the NME3 gene, which encodes an NDP kinase. The initiation-codon mutation leads to deficiency in NME3 protein expression. NME3 is a mitochondrial outer-membrane protein capable of interacting with MFN1/2, and its depletion causes dysfunction in mitochondrial dynamics. Consistently, the patient’s fibroblasts were characterized by a slow rate of mitochondrial dynamics, which was reversed by expression of wild-type or catalytic-dead NME3. Moreover, glucose starvation caused mitochondrial fragmentation and cell death in the patient’s cells. The expression of wild-type and catalytic-dead but not oligomerization-attenuated NME3 restored mitochondrial elongation. However, only wild-type NME3 sustained ATP production and viability. Thus, the separate functions of NME3 in mitochondrial fusion and NDP kinase cooperate in metabolic adaptation for cell survival in response to glucose starvation. Given the critical role of mitochondrial dynamics and energy requirements in neuronal development, the homozygous mutation in NME3 is linked to a fatal mitochondrial neurodegenerative disorder.

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 nrcama(Delta) 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, nrcama(Delta) mutants displayed a trend toward increased amounts of alpha-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.

Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.1668F is a founder variant among Ashkenazi Jews (allele frequency of -.2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function.

8 pages, 4 figures, 1 table. The online version of this article (https://doi.org/10.1038/s41436-020-0833-2) contains supplementary material, which is available to authorized users., Purpose: Pathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the nonspecific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy. Methods: Using CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. Results: We identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. Conclusions: We designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD-deficient individuals who could benefit from uridine therapy., This work was supported by The Rocket Fund, by R01DK99551 to H.H.F., and by grants BFU2016–80570-R and RTI2018–098084- B-100 from the Spanish Ministry of Science and Innovation (AEI/ FEDER, UE) and with partial support from U54 NS115198. The University of Washington Center for Mendelian Genomics for exome sequencing and analysis of CDG-0117

RNA polymerase (Pol) III has a noncanonical role of viral DNA sensing in the innate immune system. This polymerase transcribes viral genomes to produce RNAs that lead to induction of type I interferons (IFNs). However, the genetic and functional links of Pol III to innate immunity in humans remain largely unknown. Here, we describe a rare homozygous mutation (D40H) in the POLR3E gene, coding for a protein subunit of Pol III, in a child with recurrent and systemic viral infections and Langerhans cell histiocytosis. Fibroblasts derived from the patient exhibit impaired induction of type I IFN and increased susceptibility to human cytomegalovirus (HCMV) infection. Cultured cell lines infected with HCMV show induction of POLR3E expression. However, induction is not restricted to DNA virus, as sindbis virus, an RNA virus, enhances the expression of this protein. Likewise, foreign nonviral DNA elevates the steady-state level of POLR3E and elicits promoter-dependent and -independent transcription by Pol III. Remarkably, the molecular mechanism underlying the D40H mutation of POLR3E involves the assembly of defective initiation complexes of Pol III. Our study links mutated POLR3E and Pol III to an innate immune deficiency state in humans.

Consanguinity, commonplace in many regions around the globe, is associated with an increased risk of autosomal recessive (AR) genetic disorders. Consequently, consanguineous couples undergoing preimplantation genetic diagnosis (PGD) for one Mendelian disorder may be at increased risk for a child with a second, unrelated AR genetic disorder. We examined the yield of exome analysis for carrier screening of additional AR disorders, beyond the primary diagnosis, amongst consanguineous vs. non-consanguineous populations. Parental samples from trio exomes of 102 consanguineous families and 105 non-consanguineous controls were evaluated for shared carrier status, after disregarding the primary molecular diagnosis. Results were sub-classified according to disease severity. Secondary shared carrier status for pathogenic and likely pathogenic variants leading to AR disorders of moderate to profound severity was identified in 10/102 (9.8%) consanguineous couples, as compared to 1/105 (0.95%) non-consanguineous couples (χ(2) = 8.0565, p value

Rab proteins coordinate inter-organellar vesicle-mediated transport, facilitating intracellular communication, protein recycling, and signaling processes. Dysfunction of Rab proteins or their direct interactors leads to a wide range of diseases with diverse manifestations. We describe seven individuals from four consanguineous Arab Muslim families with an infantile-lethal syndrome, including failure to thrive (FTT), chronic diarrhea, neonatal respiratory distress, variable pituitary dysfunction, and distal arthrogryposis. Exome sequencing analysis in the independent families, followed by an internal gene-matching process using a local exome database, identified a homozygous splice-site variant in MADD (c.2816 + 1 G > A) on a common haplotype. The variant segregated with the disease in all available family members. Determination of cDNA sequence verified single exon skipping, resulting in an out-of-frame deletion. MADD encodes a Rab guanine nucleotide exchange factor (GEF), which activates RAB3 and RAB27A/27B and is thus a crucial regulator of neuromuscular junctions and endocrine secretory granule release. Moreover, MADD protects cells from caspase-mediated TNF-α-induced apoptosis. The combined roles of MADD and its downstream effectors correlate with the phenotypic spectrum of disease, and call for additional studies to confirm the pathogenic mechanism and to investigate possible therapeutic avenues through modulation of TNF-α signaling.

Isolated defects in the mitochondrial respiratory chain complex II (CII; succinate-ubiquinone oxidoreductase) are extremely rare and mainly result from bi-allelic mutations in one of the nuclear encoded subunits: SDHA, SDHB and SDHD, which comprise CII and the assembly CII factor SDHAF1. We report an adolescent female who presented with global developmental delay, intellectual disability and childhood onset progressive bilateral optic atrophy. Whole exome sequencing of the patient and her unaffected parents identified the novel heterozygous de novo variant c.1984C T [NM_004168.4] in the SDHA gene. Biochemical assessment of CII in the patient's derived fibroblasts and lymphocytes displayed considerably decreased CII residual activity compared with normal controls, when normalized to the integral mitochondrial enzyme citrate synthase. Protein modeling of the consequent p.Arg662Cys variant [NP-004159.2] suggested that this substitution will compromise the structural integrity of the FAD-binding protein at the C-terminus that will ultimately impair the FAD binding to SDHA, thus decreasing the entire CII activity. Our study emphasizes the role of certain heterozygous SDHA mutations in a distinct clinical phenotype dominated by optic atrophy and neurological impairment. This is the second mutation that has been reported to cause this phenotype. Furthermore, it adds developmental delay and cognitive disability to the expanding spectrum of the disorder. We propose to add SDHA to next generation sequencing gene panels of optic atrophy.

Despite the wide use of genomics to investigate the molecular basis of rare congenital malformations, a significant fraction of patients remains bereft of diagnosis. As part of our continuous effort to recruit and perform genomic and functional studies on such cohorts, we investigated the genetic and mechanistic cause of disease in two independent consanguineous families affected by overlapping craniofacial, cardiac, laterality and neurodevelopmental anomalies. Using whole exome sequencing, we identified homozygous frameshift CCDC32 variants in three affected individuals. Functional analysis in a zebrafish model revealed that ccdc32 depletion recapitulates the human phenotypes. Because some of the patient phenotypes overlap defects common to ciliopathies, we asked if loss of CCDC32 might contribute to the dysfunction of this organelle. Consistent with this hypothesis, we show that ccdc32 is required for normal cilia formation in zebrafish embryos and mammalian cell culture, arguing that ciliary defects are at least partially involved in the pathomechanism of this disorder.