De novo variants in the RNU4-2 snRNA cause a frequent neurodevelopmental syndrome.

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 RNU4-2 as a 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 115 individuals with NDD. Most individuals (77.4%) have the same highly recurrent single base insertion (n.64_65insT). In 54 individuals where it could be determined, the de novo variants were all on the maternal allele. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to RNU4-1 and other U4 homologs. Using RNA-sequencing, we show how 5' splice site usage is systematically disrupted in individuals with RNU4-2 variants, consistent with the known role of this region during spliceosome activation. Finally, we estimate that variants in this 18 bp 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.

AI-MARRVEL - A Knowledge-Driven AI System for Diagnosing Mendelian Disorders.

BACKGROUND: Diagnosing genetic disorders requires extensive manual curation and interpretation of candidate variants, a labor-intensive task even for trained geneticists. Although artificial intelligence (AI) shows promise in aiding these diagnoses, existing AI tools have only achieved moderate success for primary diagnosis. METHODS: AI-MARRVEL (AIM) uses a random-forest machine-learning classifier trained on over 3.5 million variants from thousands of diagnosed cases. AIM additionally incorporates expert-engineered features into training to recapitulate the intricate decision-making processes in molecular diagnosis. The online version of AIM is available at https://ai.marrvel.org. To evaluate AIM, we benchmarked it with diagnosed patients from three independent cohorts. RESULTS: AIM improved the rate of accurate genetic diagnosis, doubling the number of solved cases as compared with benchmarked methods, across three distinct real-world cohorts. To better identify diagnosable cases from the unsolved pools accumulated over time, we designed a confidence metric on which AIM achieved a precision rate of 98% and identified 57% of diagnosable cases out of a collection of 871 cases. Furthermore, AIM's performance improved after being fine-tuned for targeted settings including recessive disorders and trio analysis. Finally, AIM demonstrated potential for novel disease gene discovery by correctly predicting two newly reported disease genes from the Undiagnosed Diseases Network. CONCLUSIONS: AIM achieved superior accuracy compared with existing methods for genetic diagnosis. We anticipate that this tool may aid in primary diagnosis, reanalysis of unsolved cases, and the discovery of novel disease genes. (Funded by the NIH Common Fund and others.).

Exploring the complexity of systemic sclerosis etiology by trio whole genome sequencing.

Systemic sclerosis (SSc) is a heterogeneous rare autoimmune fibrosing disorder affecting connective tissue. The etiology of systemic sclerosis is largely unknown and many genes have been suggested as susceptibility loci of modest impact by genome-wide association study (GWAS). Multiple factors can contribute to the pathological process of the disease, which makes it more difficult to identify possible disease-causing genetic alterations. In this study, we have applied whole genome sequencing (WGS) in 101 indexed family trios, supplemented with transcriptome sequencing on cultured fibroblast cells of four patients and five family controls where available. Single nucleotide variants (SNVs) and copy number variants (CNVs) were examined, with emphasis on de novo variants. We also performed enrichment test for rare variants in candidate genes previously proposed in association with systemic sclerosis. We identified 42 exonic and 34 ncRNA de novo SNV changes in 101 trios, from a total of over 6000 de novo variants genome wide. We observed higher than expected de novo variants in PRKXP1 gene. We also observed such phenomenon along with increased expression in patient group in NEK7 gene. Additionally, we also observed significant enrichment of rare variants in candidate genes in the patient cohort, further supporting the complexity/multi-factorial etiology of systemic sclerosis. Our findings identify new candidate genes including PRKXP1 and NEK7 for future studies in SSc. We observed rare variant enrichment in candidate genes previously proposed in association with SSc, which suggest more efforts should be pursued to further investigate possible pathogenetic mechanisms associated with those candidate genes.

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.

The clinical utility and diagnostic implementation of human subject cell transdifferentiation followed by RNA sequencing.

RNA sequencing (RNA-seq) has recently been used in translational research settings to facilitate diagnoses of Mendelian disorders. A significant obstacle for clinical laboratories in adopting RNA-seq is the low or absent expression of a significant number of disease-associated genes/transcripts in clinically accessible samples. As this is especially problematic in neurological diseases, we developed a clinical diagnostic approach that enhanced the detection and evaluation of tissue-specific genes/transcripts through fibroblast-to-neuron cell transdifferentiation. The approach is designed specifically to suit clinical implementation, emphasizing simplicity, cost effectiveness, turnaround time, and reproducibility. For clinical validation, we generated induced neurons (iNeurons) from 71 individuals with primary neurological phenotypes recruited to the Undiagnosed Diseases Network. The overall diagnostic yield was 25.4%. Over a quarter of the diagnostic findings benefited from transdifferentiation and could not be achieved by fibroblast RNA-seq alone. This iNeuron transcriptomic approach can be effectively integrated into diagnostic whole-transcriptome evaluation of individuals with genetic disorders.

Heterozygous MAP3K20 variants cause ectodermal dysplasia, craniosynostosis, sensorineural hearing loss, and limb anomalies.

Biallelic pathogenic variants in MAP3K20, which encodes a mitogen-activated protein kinase, are a rare cause of split-hand foot malformation (SHFM), hearing loss, and nail abnormalities or congenital myopathy. However, heterozygous variants in this gene have not been definitively associated with a phenotype. Here, we describe the phenotypic spectrum associated with heterozygous de novo variants in the linker region between the kinase domain and leucine zipper domain of MAP3K20. We report five individuals with diverse clinical features, including craniosynostosis, limb anomalies, sensorineural hearing loss, and ectodermal dysplasia-like phenotypes who have heterozygous de novo variants in this specific region of the gene. These individuals exhibit both shared and unique clinical manifestations, highlighting the complexity and variability of the disorder. We propose that the involvement of MAP3K20 in endothelial-mesenchymal transition provides a plausible etiology of these features. Together, these findings characterize a disorder that both expands the phenotypic spectrum associated with MAP3K20 and highlights the need for further studies on its role in early human development.

De novo variants in PLCG1 are associated with hearing impairment, ocular pathology, and cardiac defects.

Phospholipase C isozymes (PLCs) hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol, important signaling molecules involved in many cellular processes. PLCG1 encodes the PLCγ1 isozyme that is broadly expressed. Hyperactive somatic mutations of PLCG1 are observed in multiple cancers, but only one germline variant has been reported. Here we describe three unrelated individuals with de novo heterozygous missense variants in PLCG1 (p.Asp1019Gly, p.His380Arg, and p.Asp1165Gly) who exhibit variable phenotypes including hearing loss, ocular pathology and cardiac septal defects. To model these variants in vivo, we generated the analogous variants in the Drosophila ortholog, small wing (sl). We created a null allele slT2A and assessed the expression pattern. sl is broadly expressed, including in wing discs, eye discs, and a subset of neurons and glia. Loss of sl causes wing size reductions, ectopic wing veins and supernumerary photoreceptors. We document that mutant flies exhibit a reduced lifespan and age-dependent locomotor defects. Expressing wild-type sl in slT2A mutant rescues the loss-of-function phenotypes whereas expressing the variants causes lethality. Ubiquitous overexpression of the variants also reduces viability, suggesting that the variants are toxic. Ectopic expression of an established hyperactive PLCG1 variant (p.Asp1165His) in the wing pouch causes severe wing phenotypes, resembling those observed with overexpression of the p.Asp1019Gly or p.Asp1165Gly variants, further arguing that these two are gain-of-function variants. However, the wing phenotypes associated with p.His380Arg overexpression are mild. Our data suggest that the PLCG1 de novo heterozygous missense variants are pathogenic and contribute to the features observed in the probands.

De novo missense variants in ZBTB47 are associated with developmental delays, hypotonia, seizures, gait abnormalities, and variable movement abnormalities.

The collection of known genetic etiologies of neurodevelopmental disorders continues to increase, including several syndromes associated with defects in zinc finger protein transcription factors (ZNFs) that vary in clinical severity from mild learning disabilities and developmental delay to refractory seizures and severe autism spectrum disorder. Here we describe a new neurodevelopmental disorder associated with variants in ZBTB47 (also known as ZNF651), which encodes zinc finger and BTB domain-containing protein 47. Exome sequencing (ES) was performed for five unrelated patients with neurodevelopmental disorders. All five patients are heterozygous for a de novo missense variant in ZBTB47, with p.(Glu680Gly) (c.2039A>G) detected in one patient and p.(Glu477Lys) (c.1429G>A) identified in the other four patients. Both variants impact conserved amino acid residues. Bioinformatic analysis of each variant is consistent with pathogenicity. We present five unrelated patients with de novo missense variants in ZBTB47 and a phenotype characterized by developmental delay with intellectual disability, seizures, hypotonia, gait abnormalities, and variable movement abnormalities. We propose that these variants in ZBTB47 are the basis of a new neurodevelopmental disorder.

Gain-of-function and loss-of-function variants in GRIA3 lead to distinct neurodevelopmental phenotypes.

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs) mediate fast excitatory neurotransmission in the brain. AMPARs form by homo- or heteromeric assembly of subunits encoded by the GRIA1-GRIA4 genes, of which only GRIA3 is X-chromosomal. Increasing numbers of GRIA3 missense variants are reported in patients with neurodevelopmental disorders (NDD), but only a few have been examined functionally. Here, we evaluated the impact on AMPAR function of one frameshift and 43 rare missense GRIA3 variants identified in patients with NDD by electrophysiological assays. Thirty-one variants alter receptor function and show loss-of-function (LoF) or gain-of-function (GoF) properties, whereas 13 appeared neutral. We collected detailed clinical data from 25 patients (from 23 families) harbouring 17 of these variants. All patients had global developmental impairment, mostly moderate (9/25) or severe (12/25). Twelve patients had seizures, including focal motor (6/12), unknown onset motor (4/12), focal impaired awareness (1/12), (atypical) absence (2/12), myoclonic (5/12), and generalized tonic-clonic (1/12) or atonic (1/12) seizures. The epilepsy syndrome was classified as developmental and epileptic encephalopathy in eight patients, developmental encephalopathy without seizures in 13 patients, and intellectual disability with epilepsy in four patients. Limb muscular hypotonia was reported in 13/25, and hypertonia in 10/25. Movement disorders were reported in 14/25, with hyperekplexia or non-epileptic erratic myoclonus being the most prevalent feature (8/25). Correlating receptor functional phenotype with clinical features revealed clinical features for GRIA3-associated NDDs and distinct NDD phenotypes for LoF and GoF variants. GoF variants were associated with more severe outcomes: patients were younger at the time of seizure onset (median age one month), hypertonic, and more often had movement disorders, including hyperekplexia. Patients with LoF variants were older at the time of seizure onset (median age 16 months), hypotonic, and had sleeping disturbances. LoF and GoF variants were disease-causing in both sexes but affected males often carried de novo or hemizygous LoF variants inherited from healthy mothers, whereas all but one affected females had de novo heterozygous GoF variants.

Dominant negative variants in KIF5B cause osteogenesis imperfecta via down regulation of mTOR signaling.

BACKGROUND: Kinesin motor proteins transport intracellular cargo, including mRNA, proteins, and organelles. Pathogenic variants in kinesin-related genes have been implicated in neurodevelopmental disorders and skeletal dysplasias. We identified de novo, heterozygous variants in KIF5B, encoding a kinesin-1 subunit, in four individuals with osteogenesis imperfecta. The variants cluster within the highly conserved kinesin motor domain and are predicted to interfere with nucleotide binding, although the mechanistic consequences on cell signaling and function are unknown. METHODS: To understand the in vivo genetic mechanism of KIF5B variants, we modeled the p.Thr87Ile variant that was found in two patients in the C. elegans ortholog, unc-116, at the corresponding position (Thr90Ile) by CRISPR/Cas9 editing and performed functional analysis. Next, we studied the cellular and molecular consequences of the recurrent p.Thr87Ile variant by microscopy, RNA and protein analysis in NIH3T3 cells, primary human fibroblasts and bone biopsy. RESULTS: C. elegans heterozygous for the unc-116 Thr90Ile variant displayed abnormal body length and motility phenotypes that were suppressed by additional copies of the wild type allele, consistent with a dominant negative mechanism. Time-lapse imaging of GFP-tagged mitochondria showed defective mitochondria transport in unc-116 Thr90Ile neurons providing strong evidence for disrupted kinesin motor function. Microscopy studies in human cells showed dilated endoplasmic reticulum, multiple intracellular vacuoles, and abnormal distribution of the Golgi complex, supporting an intracellular trafficking defect. RNA sequencing, proteomic analysis, and bone immunohistochemistry demonstrated down regulation of the mTOR signaling pathway that was partially rescued with leucine supplementation in patient cells. CONCLUSION: We report dominant negative variants in the KIF5B kinesin motor domain in individuals with osteogenesis imperfecta. This study expands the spectrum of kinesin-related disorders and identifies dysregulated signaling targets for KIF5B in skeletal development.

Health-related quality of life in a systematically assessed cohort of children and adults with urea cycle disorders.

PURPOSE: Individuals with urea cycle disorders (UCDs) may develop recurrent hyperammonemia, episodic encephalopathy, and neurological sequelae which can impact Health-related Quality of Life (HRQoL). To date, there have been no systematic studies of HRQoL in people with UCDs. METHODS: We reviewed HRQoL and clinical data for 190 children and 203 adults enrolled in a multicenter UCD natural history study. Physical and psychosocial HRQoL in people with UCDs were compared to HRQoL in healthy people and people with phenylketonuria (PKU) and diabetes mellitus. We assessed relationships between HRQoL, UCD diagnosis, and disease severity. Finally, we calculated sample sizes required to detect changes in these HRQoL measures. RESULTS: Individuals with UCDs demonstrated worse physical and psychosocial HRQoL than their healthy peers and peers with PKU and diabetes. In children, HRQoL scores did not differ by diagnosis or severity. In adults, individuals with decreased severity had worse psychosocial HRQoL. Finally, we show that a large number of individuals would be required in clinical trials to detect differences in HRQoL in UCDs. CONCLUSION: Individuals with UCDs have worse HRQoL compared to healthy individuals and those with PKU and diabetes. Future work should focus on the impact of liver transplantation and other clinical variables on HRQoL in UCDs.

Monitoring the treatment of urea cycle disorders using phenylbutyrate metabolite analyses: Still many lessons to learn.

Medications that elicit an alternate pathway for nitrogen excretion such as oral sodium phenylbutyrate (NaPBA) and glycerol phenylbutyrate (GPB) and intravenous sodium phenylacetate (NaPAA) are important for the management of urea cycle disorders (UCDs). Plasma concentrations of their primary metabolite, phenylacetate (PAA), as well as the ratio of PAA to phenylacetylglutamine (PAGN) are useful for guiding dosing and detecting toxicity. However, the frequency of toxic elevations of metabolites and associated clinical covariates is relatively unknown. A retrospective analysis was conducted on 1255 plasma phenylbutyrate metabolite measurements from 387 individuals. An additional analysis was also conducted on a subset of 68 individuals in whom detailed clinical information was available. In the course of these analyses, abnormally elevated plasma PAA and PAA:PAGN were identified in 39 individuals (4.15% of samples) and 42 individuals (4.30% of samples), respectively. Abnormally elevated PAA and PAA:PAGN values were more likely to occur in younger individuals and associate positively with dose of NAPBA and negatively with plasma glutamine and glycine levels. These results demonstrate that during routine clinical management, the majority of patients have PAA levels that are deemed safe. As age is negatively associated with PAA levels however, children undergoing treatment with NaPBA may need close monitoring of their phenylbutyrate metabolite levels.

Delayed skeletal development and IGF-1 deficiency in a mouse model of lysinuric protein intolerance.

SLC7A7 deficiency, or lysinuric protein intolerance (LPI), causes loss of function of the y+LAT1 transporter critical for efflux of arginine, lysine and ornithine in certain cells. LPI is characterized by urea cycle dysfunction, renal disease, immune dysregulation, growth failure, delayed bone age and osteoporosis. We previously reported that Slc7a7 knockout mice (C57BL/6×129/SvEv F2) recapitulate LPI phenotypes, including growth failure. Our main objective in this study was to characterize the skeletal phenotype in these mice. Compared to wild-type littermates, juvenile Slc7a7 knockout mice demonstrated 70% lower body weights, 87% lower plasma IGF-1 concentrations and delayed skeletal development. Because poor survival prevents evaluation of mature knockout mice, we generated a conditional Slc7a7 deletion in mature osteoblasts or mesenchymal cells of the osteo-chondroprogenitor lineage, but no differences in bone architecture were observed. Overall, global Slc7a7 deficiency caused growth failure with low plasma IGF-1 concentrations and delayed skeletal development, but Slc7a7 deficiency in the osteoblastic lineage was not a major contributor to these phenotypes. Future studies utilizing additional tissue-specific Slc7a7 knockout models may help dissect cell-autonomous and non-cell-autonomous mechanisms underlying phenotypes in LPI.

Argininosuccinate lyase deficiency causes blood-brain barrier disruption via nitric oxide-mediated dysregulation of claudin expression.

Nitric oxide (NO) is a critical signaling molecule that has been implicated in the pathogenesis of neurocognitive diseases. Both excessive and insufficient NO production have been linked to pathology. Previously, we have shown that argininosuccinate lyase deficiency (ASLD) is a novel model system to investigate cell-autonomous, nitric oxide synthase-dependent NO deficiency. Humans with ASLD are at increased risk for developing hyperammonemia due to a block in ureagenesis. However, natural history studies have shown that individuals with ASLD have multisystem disease including neurocognitive deficits that can be independent of ammonia. Here, using ASLD as a model of NO deficiency, we investigated the effects of NO on brain endothelial cells in vitro and the blood-brain barrier (BBB) in vivo. Knockdown of ASL in human brain microvascular endothelial cells (HBMECs) led to decreased transendothelial electrical resistance, indicative of increased cell permeability. Mechanistically, treatment with an NO donor or inhibition of Claudin-1 improved barrier integrity in ASL-deficient HBMECs. Furthermore, in vivo assessment of a hypomorphic mouse model of ASLD showed increased BBB leakage, which was partially rescued by NO supplementation. Our results suggest that ASL-mediated NO synthesis is required for proper maintenance of brain microvascular endothelial cell functions as well as BBB integrity.

TMEM161B regulates cerebral cortical gyration, Sonic Hedgehog signaling, and ciliary structure in the developing central nervous system.

Sonic hedgehog signaling regulates processes of embryonic development across multiple tissues, yet factors regulating context-specific Shh signaling remain poorly understood. Exome sequencing of families with polymicrogyria (disordered cortical folding) revealed multiple individuals with biallelic deleterious variants in TMEM161B, which encodes a multi-pass transmembrane protein of unknown function. Tmem161b null mice demonstrated holoprosencephaly, craniofacial midline defects, eye defects, and spinal cord patterning changes consistent with impaired Shh signaling, but were without limb defects, suggesting a CNS-specific role of Tmem161b. Tmem161b depletion impaired the response to Smoothened activation in vitro and disrupted cortical histogenesis in vivo in both mouse and ferret models, including leading to abnormal gyration in the ferret model. Tmem161b localizes non-exclusively to the primary cilium, and scanning electron microscopy revealed shortened, dysmorphic, and ballooned ventricular zone cilia in the Tmem161b null mouse, suggesting that the Shh-related phenotypes may reflect ciliary dysfunction. Our data identify TMEM161B as a regulator of cerebral cortical gyration, as involved in primary ciliary structure, as a regulator of Shh signaling, and further implicate Shh signaling in human gyral development.

Loss-of-function variants in MYCBP2 cause neurobehavioural phenotypes and corpus callosum defects.

The corpus callosum is a bundle of axon fibres that connects the two hemispheres of the brain. Neurodevelopmental disorders that feature dysgenesis of the corpus callosum as a core phenotype offer a valuable window into pathology derived from abnormal axon development. Here, we describe a cohort of eight patients with a neurodevelopmental disorder characterized by a range of deficits including corpus callosum abnormalities, developmental delay, intellectual disability, epilepsy and autistic features. Each patient harboured a distinct de novo variant in MYCBP2, a gene encoding an atypical really interesting new gene (RING) ubiquitin ligase and signalling hub with evolutionarily conserved functions in axon development. We used CRISPR/Cas9 gene editing to introduce disease-associated variants into conserved residues in the Caenorhabditis elegans MYCBP2 orthologue, RPM-1, and evaluated functional outcomes in vivo. Consistent with variable phenotypes in patients with MYCBP2 variants, C. elegans carrying the corresponding human mutations in rpm-1 displayed axonal and behavioural abnormalities including altered habituation. Furthermore, abnormal axonal accumulation of the autophagy marker LGG-1/LC3 occurred in variants that affect RPM-1 ubiquitin ligase activity. Functional genetic outcomes from anatomical, cell biological and behavioural readouts indicate that MYCBP2 variants are likely to result in loss of function. Collectively, our results from multiple human patients and CRISPR gene editing with an in vivo animal model support a direct link between MYCBP2 and a human neurodevelopmental spectrum disorder that we term, MYCBP2-related developmental delay with corpus callosum defects (MDCD).

RNA Sequencing as a Diagnostic Tool.

This JAMA Insights discusses RNA sequencing, which allows detection of qualitative and quantitative changes in RNA expression across the genome in clinical samples, and how it may improve molecular diagnostic rates achieved by diagnostic exome sequencing and whole-genome sequencing alone.

A recurrent single-exon deletion in TBCK might be under-recognized in patients with infantile hypotonia and psychomotor delay.

Advanced bioinformatics algorithms allow detection of multiple-exon copy-number variations (CNVs) from exome sequencing (ES) data, while detection of single-exon CNVs remains challenging. A retrospective review of Baylor Genetics' clinical ES patient cohort identified four individuals with homozygous single-exon deletions of TBCK (exon 23, NM_001163435.2), a gene associated with an autosomal recessive neurodevelopmental phenotype. To evaluate the prevalence of this deletion and its contribution to disease, we retrospectively analyzed single nucleotide polymorphism (SNP) array data for 8194 individuals undergoing ES, followed by PCR confirmation and RT-PCR on individuals carrying homozygous or heterozygous exon 23 TBCK deletions. A fifth individual was diagnosed with the TBCK-related disorder due to a heterozygous exon 23 deletion in trans with a c.1860+1G>A (NM_001163435.2) pathogenic variant, and three additional heterozygous carriers were identified. Affected individuals and carriers were from diverse ethnicities including European Caucasian, South Asian, Middle Eastern, Hispanic American and African American, with only one family reporting consanguinity. RT-PCR revealed two out-of-frame transcripts related to the exon 23 deletion. Our results highlight the importance of identifying single-exon deletions in clinical ES, especially for genes carrying recurrent deletions. For patients with early-onset hypotonia and psychomotor delay, this single-exon TBCK deletion might be under-recognized due to technical limitations of ES.