De novo variants in the non-coding spliceosomal snRNA gene RNU4-2 are a frequent cause of syndromic neurodevelopmental disorders.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Increasingly, large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a novel syndromic NDD gene. RNU4-2 encodes 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 bp region of RNU4-2 mapping 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 119 individuals with NDD. The vast majority of individuals (77.3%) have the same highly recurrent single base-pair insertion (n.64_65insT). We estimate that variants in this region explain 0.41% of individuals with NDD. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to its contiguous counterpart RNU4-1 and other U4 homologs, supporting RNU4-2's role as the primary U4 transcript in the brain. Overall, this work underscores the importance of non-coding genes in rare disorders. It will provide a diagnosis to thousands of individuals with NDD worldwide and pave the way for the development of effective treatments for these individuals.

Narrowing the diagnostic gap: Genomes, episignatures, long-read sequencing, and health economic analyses in an exome-negative intellectual disability cohort.

PURPOSE: Genome sequencing (GS)-specific diagnostic rates in prospective tightly ascertained exome sequencing (ES)-negative intellectual disability (ID) cohorts have not been reported extensively. METHODS: ES, GS, epigenetic signatures, and long-read sequencing diagnoses were assessed in 74 trios with at least moderate ID. RESULTS: The ES diagnostic yield was 42 of 74 (57%). GS diagnoses were made in 9 of 32 (28%) ES-unresolved families. Repeated ES with a contemporary pipeline on the GS-diagnosed families identified 8 of 9 single-nucleotide variations/copy-number variations undetected in older ES, confirming a GS-unique diagnostic rate of 1 in 32 (3%). Episignatures contributed diagnostic information in 9% with GS corroboration in 1 of 32 (3%) and diagnostic clues in 2 of 32 (6%). A genetic etiology for ID was detected in 51 of 74 (69%) families. Twelve candidate disease genes were identified. Contemporary ES followed by GS cost US$4976 (95% CI: $3704; $6969) per diagnosis and first-line GS at a cost of $7062 (95% CI: $6210; $8475) per diagnosis. CONCLUSION: Performing GS only in ID trios would be cost equivalent to ES if GS were available at $2435, about a 60% reduction from current prices. This study demonstrates that first-line GS achieves higher diagnostic rate than contemporary ES but at a higher cost.

Integration of EpiSign, facial phenotyping, and likelihood ratio interpretation of clinical abnormalities in the re-classification of an ARID1B missense variant.

Heterozygous ARID1B variants result in Coffin-Siris syndrome. Features may include hypoplastic nails, slow growth, characteristic facial features, hypotonia, hypertrichosis, and sparse scalp hair. Most reported cases are due to ARID1B loss of function variants. We report a boy with developmental delay, feeding difficulties, aspiration, recurrent respiratory infections, slow growth, and hypotonia without a clinical diagnosis, where a previously unreported ARID1B missense variant was classified as a variant of uncertain significance. The pathogenicity of this variant was refined through combined methodologies including genome-wide methylation signature analysis (EpiSign), Machine Learning (ML) facial phenotyping, and LIRICAL. Trio exome sequencing and EpiSign were performed. ML facial phenotyping compared facial images using FaceMatch and GestaltMatcher to syndrome-specific libraries to prioritize the trio exome bioinformatic pipeline gene list output. Phenotype-driven variant prioritization was performed with LIRICAL. A de novo heterozygous missense variant, ARID1B p.(Tyr1268His), was reported as a variant of uncertain significance. The ACMG classification was refined to likely pathogenic by a supportive methylation signature, ML facial phenotyping, and prioritization through LIRICAL. The ARID1B genotype-phenotype has been expanded through an extended analysis of missense variation through genome-wide methylation signatures, ML facial phenotyping, and likelihood-ratio gene prioritization.

Dominant-negative variants in CBX1 cause a neurodevelopmental disorder.

PURPOSE: This study aimed to establish variants in CBX1, encoding heterochromatin protein 1ß (HP1ß), as a cause of a novel syndromic neurodevelopmental disorder. METHODS: Patients with CBX1 variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. To investigate the pathogenicity of identified variants, we performed in vitro cellular assays and neurobehavioral and cytological analyses of neuronal cells obtained from newly generated Cbx1 mutant mouse lines. RESULTS: In 3 unrelated individuals with developmental delay, hypotonia, and autistic features, we identified heterozygous de novo variants in CBX1. The identified variants were in the chromodomain, the functional domain of HP1ß, which mediates interactions with chromatin. Cbx1 chromodomain mutant mice displayed increased latency-to-peak response, suggesting the possibility of synaptic delay or myelination deficits. Cytological and chromatin immunoprecipitation experiments confirmed the reduction of mutant HP1ß binding to heterochromatin, whereas HP1ß interactome analysis demonstrated that the majority of HP1ß-interacting proteins remained unchanged between the wild-type and mutant HP1ß. CONCLUSION: These collective findings confirm the role of CBX1 in developmental disabilities through the disruption of HP1ß chromatin binding during neurocognitive development. Because HP1ß forms homodimers and heterodimers, mutant HP1ß likely sequesters wild-type HP1ß and other HP1 proteins, exerting dominant-negative effects.

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.

Whole exome and genome sequencing in mendelian disorders: a diagnostic and health economic analysis.

Whole genome sequencing (WGS) improves Mendelian disorder diagnosis over whole exome sequencing (WES); however, additional diagnostic yields and costs remain undefined. We investigated differences between diagnostic and cost outcomes of WGS and WES in a cohort with suspected Mendelian disorders. WGS was performed in 38 WES-negative families derived from a 64 family Mendelian cohort that previously underwent WES. For new WGS diagnoses, contemporary WES reanalysis determined whether variants were diagnosable by original WES or unique to WGS. Diagnostic rates were estimated for WES and WGS to simulate outcomes if both had been applied to the 64 families. Diagnostic costs were calculated for various genomic testing scenarios. WGS diagnosed 34% (13/38) of WES-negative families. However, contemporary WES reanalysis on average 2 years later would have diagnosed 18% (7/38 families) resulting in a WGS-specific diagnostic yield of 19% (6/31 remaining families). In WES-negative families, the incremental cost per additional diagnosis using WGS following WES reanalysis was AU$36,710 (£19,407;US$23,727) and WGS alone was AU$41,916 (£22,159;US$27,093) compared to WES-reanalysis. When we simulated the use of WGS alone as an initial genomic test, the incremental cost for each additional diagnosis was AU$29,708 (£15,705;US$19,201) whereas contemporary WES followed by WGS was AU$36,710 (£19,407;US$23,727) compared to contemporary WES. Our findings confirm that WGS is the optimal genomic test choice for maximal diagnosis in Mendelian disorders. However, accepting a small reduction in diagnostic yield, WES with subsequent reanalysis confers the lowest costs. Whether WES or WGS is utilised will depend on clinical scenario and local resourcing and availability.

De Novo ZMYND8 variants result in an autosomal dominant neurodevelopmental disorder with cardiac malformations.

PURPOSE: ZMYND8 encodes a multidomain protein that serves as a central interactive hub for coordinating critical roles in transcription regulation, chromatin remodeling, regulation of super-enhancers, DNA damage response and tumor suppression. We delineate a novel neurocognitive disorder caused by variants in the ZMYND8 gene. METHODS: An international collaboration, exome sequencing, molecular modeling, yeast two-hybrid assays, analysis of available transcriptomic data and a knockdown Drosophila model were used to characterize the ZMYND8 variants. RESULTS: ZMYND8 variants were identified in 11 unrelated individuals; 10 occurred de novo and one suspected de novo; 2 were truncating, 9 were missense, of which one was recurrent. The disorder is characterized by intellectual disability with variable cardiovascular, ophthalmologic and minor skeletal anomalies. Missense variants in the PWWP domain of ZMYND8 abolish the interaction with Drebrin and missense variants in the MYND domain disrupt the interaction with GATAD2A. ZMYND8 is broadly expressed across cell types in all brain regions and shows highest expression in the early stages of brain development. Neuronal knockdown of the DrosophilaZMYND8 ortholog results in decreased habituation learning, consistent with a role in cognitive function. CONCLUSION: We present genomic and functional evidence for disruption of ZMYND8 as a novel etiology of syndromic intellectual disability.

Germline variants in tumor suppressor FBXW7 lead to impaired ubiquitination and a neurodevelopmental syndrome.

Neurodevelopmental disorders are highly heterogenous conditions resulting from abnormalities of brain architecture and/or function. FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. We used a genotype-first approach and global data-sharing platforms to identify 35 individuals harboring de novo and inherited FBXW7 germline monoallelic chromosomal deletions and nonsense, frameshift, splice-site, and missense variants associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. Collectively, the data presented herein provide compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.

Clinically Responsive Genomic Analysis Pipelines: Elements to Improve Detection Rate and Efficiency.

Massively parallel sequencing has markedly improved mendelian diagnostic rates. This study assessed the effects of custom alterations to a diagnostic genomic bioinformatic pipeline in response to clinical need and derived practice recommendations relative to diagnostic rates and efficiency. The Genomic Annotation and Interpretation Application (GAIA) bioinformatics pipeline was designed to detect panel, exome, and genome sample integrity and prioritize gene variants in mendelian disorders. Reanalysis of selected negative cases was performed after improvements to the pipeline. GAIA improvements and their effect on sensitivity are described, including addition of a PubMed search for gene-disease associations not in the Online Mendelian Inheritance of Man database, inclusion of a process for calling low-quality variants (known as QPatch), and gene symbol nomenclature consistency checking. The new pipeline increased the diagnostic rate and reduced staff costs, resulting in a saving of US$844.34 per additional diagnosis. Recommendations for genomic analysis pipeline requirements are summarized. Clinically responsive bioinformatics pipeline improvements increase diagnostic sensitivity and increase cost-effectiveness.

Germline AGO2 mutations impair RNA interference and human neurological development.

ARGONAUTE-2 and associated miRNAs form the RNA-induced silencing complex (RISC), which targets mRNAs for translational silencing and degradation as part of the RNA interference pathway. Despite the essential nature of this process for cellular function, there is little information on the role of RISC components in human development and organ function. We identify 13 heterozygous mutations in AGO2 in 21 patients affected by disturbances in neurological development. Each of the identified single amino acid mutations result in impaired shRNA-mediated silencing. We observe either impaired RISC formation or increased binding of AGO2 to mRNA targets as mutation specific functional consequences. The latter is supported by decreased phosphorylation of a C-terminal serine cluster involved in mRNA target release, increased formation of dendritic P-bodies in neurons and global transcriptome alterations in patient-derived primary fibroblasts. Our data emphasize the importance of gene expression regulation through the dynamic AGO2-RNA association for human neuronal development.

Impacts of genomics on the health and social costs of intellectual disability.

BACKGROUND: This study provides an integrated assessment of the economic and social impacts of genomic sequencing for the detection of monogenic disorders resulting in intellectual disability (ID). METHODS: Multiple knowledge bases were cross-referenced and analysed to compile a reference list of monogenic disorders associated with ID. Multiple literature searches were used to quantify the health and social costs for the care of people with ID. Health and social expenditures and the current cost of whole-exome sequencing and whole-genome sequencing were quantified in relation to the more common causes of ID and their impact on lifespan. RESULTS: On average, individuals with ID incur annual costs in terms of health costs, disability support, lost income and other social costs of US$172 000, accumulating to many millions of dollars over a lifetime. CONCLUSION: The diagnosis of monogenic disorders through genomic testing provides the opportunity to improve the diagnosis and management, and to reduce the costs of ID through informed reproductive decisions, reductions in unproductive diagnostic tests and increasingly targeted therapies.

Fetal diagnosis of Mowat-Wilson syndrome by whole exome sequencing.

Mowat-Wilson syndrome (MWS) is a complex genetic disorder associated with heterozygous variation in ZEB2. It is mainly characterized by moderate-to-severe intellectual disability, facial dysmorphism, epilepsy, and various malformations including Hirschsprung disease, corpus callosum anomalies, and congenital heart defects. It is rarely diagnosed prenatally and there is limited information available on the prenatal phenotype associated with MWS. Here we report the detection of a heterozygous de novo nonsense variant in ZEB2 by whole exome sequencing in a fetus with microphthalmia in addition to cardiac defects and typical MWS facial dysmorphism. As the prenatal phenotypic spectrum of MWS expands, the routine addition of fetal genomic testing particularly in the presence of multiple malformations will increase both the sensitivity and specificity of prenatal diagnostics.