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Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here we identify the non-coding RNA RNU4-2as a syndromic NDD gene. RNU4-2encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 base pair region of RNU4-2mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 115 individuals with NDD. Most individuals (77.4%) have the same highly recurrent single base insertion (n.64_65insT). In 54 individuals in whom it could be determined, the de novo variants were all on the maternal allele. We demonstrate that RNU4-2is highly expressed in the developing human brain, in contrast to RNU4-1and other U4 homologues. Using RNA sequencing, we show how 5′ splice-site use is systematically disrupted in individuals with RNU4-2variants, consistent with the known role of this region during spliceosome activation. Finally, we estimate that variants in this 18 base pair region explain 0.4% of individuals with NDD. This work underscores the importance of non-coding genes in rare disorders and will provide a diagnosis to thousands of individuals with NDD worldwide.

Tandem repeat DNA sequences constitute a significant proportion of the human genome. While previously considered to be functionally inert, these sequences are now broadly accepted as important contributors to genetic diversity. However, the polymorphic nature of these sequences can lead to expansion beyond a gene-specific threshold, causing disease. More than 50 pathogenic repeat expansions have been identified to date, many of which have been discovered in the last decade as a result of advances in sequencing technologies and associated bioinformatic tools. Commonly utilised diagnostic platforms including Sanger sequencing, capillary array electrophoresis, and Southern blot are generally low throughput and are often unable to accurately determine repeat size, composition, and epigenetic signature, which are important when characterising repeat expansions. The rapid advances in bioinformatic tools designed specifically to interrogate short-read sequencing and the development of long-read single molecule sequencing is enabling a new generation of high throughput testing for repeat expansion disorders. In this review, we discuss some of the challenges surrounding the identification and characterisation of disease-causing repeat expansions and the technological advances that are poised to translate the promise of genomic medicine to individuals and families affected by these disorders.

We report a child with a history of temporal‐parietal‐occipital disconnection for epilepsy secondary to posterior quadrantic dysplasia who developed recurrent and prolonged bouts of distress and autonomic disturbance associated with EEG and PET evidence of status epilepticus confined to his disconnected cortex. These bouts were refractory to antiseizure medications but resolved following resection of the disconnected cortex. In the absence of synaptic connections, we hypothesise that his seizure‐related symptoms were mediated either by neurochemical transmission in preserved vascular and lymphatic channels or by ephaptic transmission to trigeminal nerve fibres in overlying dura, producing symptoms akin to migraine. The case highlights potential means by which seizures may manifest clinically, without synaptic connections, and adds to the differential for symptoms post‐disconnection surgery.

Several neurological disorders, such as myotonic dystrophy are caused by expansions of short tandem repeats (STRs) which can be difficult to detect by molecular tools. Methodological advances have made repeat expansion (RE) detection with whole genome sequencing (WGS) feasible. We recruited a multi-generational family (family A) ascertained for genetic studies of autism spectrum disorder. WGS was performed on seven children from four nuclear families from family A and analyzed for REs of STRs known to cause neurological disorders. We detected an expansion of a heterozygous intronic CCTG STR in CNBPin two siblings. This STR causes myotonic dystrophy type 2 (DM2). The expansion did not segregate with the ASD phenotype. Repeat-primed PCR showed that the DM2 CCTG motif was expanded above the pathogenic threshold in both children and their mother. On subsequent examination, the mother had mild features of DM2. We show that screening of STRs in WGS datasets has diagnostic utility, both in the clinical and research domain, with potential management and genetic counseling implications.

Coffin–Siris syndrome (CSS, MIM#135900) is a congenital disorder characterized by coarse facial features, intellectual disability, and hypoplasia of the fifth digit and nails. Pathogenic variants for CSS have been found in genes encoding proteins in the BAF (BRG1-associated factor) chromatin-remodeling complex. To date, more than 150 CSS patients with pathogenic variants in nine BAF-related genes have been reported. We previously reported 71 patients of whom 39 had pathogenic variants. Since then, we have recruited an additional 182 CSS-suspected patients. We performed comprehensive genetic analysis on these 182 patients and on the previously unresolved 32 patients, targeting pathogenic single nucleotide variants, short insertions/deletions and copy number variations (CNVs). We confirmed 78 pathogenic variations in 78 patients. Pathogenic variations in ARID1B, SMARCB1, SMARCA4, ARID1A, SOX11, SMARCE1, and PHF6were identified in 48, 8, 7, 6, 4, 1, and 1 patients, respectively. In addition, we found three CNVs including SMARCA2. Of particular note, we found a partial deletion of SMARCB1in one CSS patient and we thoroughly investigated the resulting abnormal transcripts.

Avoidance of environmental dangers depends on nociceptive topognosis, or the ability to localize painful stimuli. This is proposed to rely on somatotopic maps arising from topographically organized point-to-point connections between the body surface and the CNS. To determine the role of topographic organization of spinal ascending projections in nociceptive topognosis, we generated a conditional knockout mouse lacking expression of the netrin1 receptor DCC in the spinal cord. These mice have an increased number of ipsilateral spinothalamic connections and exhibit aberrant activation of the somatosensory cortex in response to unilateral stimulation. Furthermore, spinal cord-specific Dccknockout animals displayed mislocalized licking responses to formalin injection, indicating impaired topognosis. Similarly, humans with DCCmutations experience bilateral sensation evoked by unilateral somatosensory stimulation. Collectively, our results constitute functional evidence of the importance of topographic organization of spinofugal connections for nociceptive topognosis.

Gene-disruptive mutations contribute to the biology of neurodevelopmental disorders (NDDs), but most of the related pathogenic genes are not known. We sequenced 208 candidate genes from >11,730 cases and >2,867 controls. We identified 91 genes, including 38 new NDD genes, with an excess of de novo mutations or private disruptive mutations in 5.7% of cases. Drosophila functional assays revealed a subset with increased involvement in NDDs. We identified 25 genes showing a bias for autism versus intellectual disability and highlighted a network associated with high-functioning autism (full-scale IQ >100). Clinical follow-up for NAA15, KMT5B, and ASH1L highlighted new syndromic and nonsyndromic forms of disease.

Brain malformations involving the corpus callosum are common in children with developmental disabilities. We identified DCC mutations in four families and five sporadic individuals with isolated agenesis of the corpus callosum (ACC) without intellectual disability. DCC mutations result in variable dominant phenotypes with decreased penetrance, including mirror movements and ACC associated with a favorable developmental prognosis. Possible phenotypic modifiers include the type and location of mutation and the sex of the individual.

To define causative somatic mutations in resected brain tissue from an infant with intractable epilepsy secondary to hemispheric cortical dysplasia.

Spinocerebellar ataxia, type 2 (SCA2), results from an expansion of a stretch of polyglutamine repeats within the coding sequence of the ataxin-2 gene (ATX2), localized to chromosome 12q23-24. Recent studies have widened the clinical phenotype, notably for individuals with repeats of intermediate size, from 32 to 35 glutamine residues. This narrow range necessitates precise determination of repeat size. Diagnostic laboratories most often perform direct genotyping of ATX2 from polymerase chain-amplified patient DNA with subsequent sizing utilizing slab gel polyacrylamide gel electrophoresis (PAGE) or capillary electrophoresis. Using cloning and sequencing methods, we have constructed a ladder of ATX2 alleles of known size and sequence composition. This freely available size ladder will facilitate future quantification of expansions of the ATX2 locus.

A set of low copy number plasmid vectors for mammalian gene expression has been constructed. These vectors are derived from the previously described bacterial low copy number expression vectors, pWSK29 and pWKS30, which are present at six to eight copies per cell. The new plasmids also have the following useful properties: (1) they contain antibiotic resistance markers for the selection of stable mammalian cell lines; (2) they have either constitutive or inducible promoters; (3) a chimeric intron, for enhancing gene expression, is present; (4) they contain unique cloning sites; (5) they have an SV40 polyadenylation signal, and a subset of the vectors have an SV40 origin of replication for episomal replication and transient gene expression. A cDNA encoding the Menkes disease protein was cloned into two of these vectors, and transient expression studies in COS-7 cells showed that both constitutive and inducible expression was possible. This set of expression vectors will provide a useful tool for the manipulation, inEscherichia coli,of mammalian genes or cDNAs that are unstable in the high copy number vectors that are currently available.

Menkes' disease is a fatal, X-linked, copper deficiency disorder that results from defective copper efflux from intestinal cells and inadequate copper delivery to other tissues, leading to deficiencies of critical copper-dependent enzymes. Wilson's disease is an autosomally inherited, copper toxicosis disorder resulting from defective biliary excretion of copper, which leads to copper accumulation in the liver. TheATP7Aand ATP7Bgenes that are defective in patients with Menkes' and Wilson's diseases, respectively, encode transmembrane, P-type ATPase proteins (ATP7A or MNK and ATP7B or WND, respectively) that function to translocate copper across cellular membranes. In this study, the cDNAs derived from a normal humanATP7Agene and the murine ATP7Bhomologue,Atp7b, were separately transfected into an immortalized fibroblast cell line obtained from a Menkes' disease patient. Both MNK and WND expressed from plasmid constructs were able to correct the copper accumulation and copper retention phenotype of these cells. However, the two proteins responded differently to elevated extracellular copper levels. Although MNK showed copper-induced trafficking from the trans-Golgi network to the plasma membrane, in the same cell line the intracellular location of WND did not appear to be affected by elevated copper.

Hypertrophic cardiomyopathy (HCM) is a disease characterised by hypertrophy of the left ventricle of the heart. HCM has a prevalence of ~1 in 500 individuals and is the leading cause of sudden cardiac death among young individuals. Over 20 HCM genes have been identified but at least 20% of cases remain of unknown aetiology.

The reactive changes in different types of astrocytes were analyzed in parkinsonian syndromes in order to identify common reactions and their relationship to disease severity. Immunohistochemistry was used on formalin-fixed, paraffin-embedded sections from the putamen, pons, and substantia nigra from 13 Parkinson disease (PD), 29 multiple-system atrophy (MSA), 34 progressive supranuclear palsy (PSP), 10 corticobasal degeneration(CBD), and 13 control cases. Classic reactive astrocytes were observed in MSA, PSP, and CBD, but not PD cases; the extent of reactivity correlated with indices of neurodegeneration and disease stage. Approximately 40% to 45% of subcortical astrocytes in PD and PSP accumulated α-synuclein and phospho-tau, respectively; subcortical astrocytes in MSA and CBD cases did not accumulate these proteins. Protoplasmic astrocytes were identified from fibrous astrocytes by their expression of parkin coregulated gene and apolipoprotein D, and accumulated abnormal proteins in PD, PSP, and CBD, but not MSA. The increased reactivity of parkin coregulated gene-immunoreactive protoplasmic astrocytes correlated with parkin expression in PSP and CBD. Nonreactive protoplasmic astrocytes were observed in PD and MSA cases; in PD, they accumulated α-synuclein, suggesting that the attenuated response might be due to an increase in the level of α-synuclein. These heterogenous astroglial responses in PD, MSA, PSP, and CBD indicate distinct underlying pathogenic mechanisms in each disorder.