A Mild PUM1 Mutation Is Associated with Adult-Onset Ataxia, whereas Haploinsufficiency Causes Developmental Delay and Seizures.

Certain mutations can cause proteins to accumulate in neurons, leading to neurodegeneration. We recently showed, however, that upregulation of a wild-type protein, Ataxin1, caused by haploinsufficiency of its repressor, the RNA-binding protein Pumilio1 (PUM1), also causes neurodegeneration in mice. We therefore searched for human patients with PUM1 mutations. We identified eleven individuals with either PUM1 deletions or de novo missense variants who suffer a developmental syndrome (Pumilio1-associated developmental disability, ataxia, and seizure; PADDAS). We also identified a milder missense mutation in a family with adult-onset ataxia with incomplete penetrance (Pumilio1-related cerebellar ataxia, PRCA). Studies in patient-derived cells revealed that the missense mutations reduced PUM1 protein levels by ∼25% in the adult-onset cases and by ∼50% in the infantile-onset cases; levels of known PUM1 targets increased accordingly. Changes in protein levels thus track with phenotypic severity, and identifying posttranscriptional modulators of protein expression should identify new candidate disease genes.

Stretch-activated ion channel TMEM63B associates with developmental and epileptic encephalopathies and progressive neurodegeneration.

By converting physical forces into electrical signals or triggering intracellular cascades, stretch-activated ion channels allow the cell to respond to osmotic and mechanical stress. Knowledge of the pathophysiological mechanisms underlying associations of stretch-activated ion channels with human disease is limited. Here, we describe 17 unrelated individuals with severe early-onset developmental and epileptic encephalopathy (DEE), intellectual disability, and severe motor and cortical visual impairment associated with progressive neurodegenerative brain changes carrying ten distinct heterozygous variants of TMEM63B, encoding for a highly conserved stretch-activated ion channel. The variants occurred de novo in 16/17 individuals for whom parental DNA was available and either missense, including the recurrent p.Val44Met in 7/17 individuals, or in-frame, all affecting conserved residues located in transmembrane regions of the protein. In 12 individuals, hematological abnormalities co-occurred, such as macrocytosis and hemolysis, requiring blood transfusions in some. We modeled six variants (p.Val44Met, p.Arg433His, p.Thr481Asn, p.Gly580Ser, p.Arg660Thr, and p.Phe697Leu), each affecting a distinct transmembrane domain of the channel, in transfected Neuro2a cells and demonstrated inward leak cation currents across the mutated channel even in isotonic conditions, while the response to hypo-osmotic challenge was impaired, as were the Ca2+ transients generated under hypo-osmotic stimulation. Ectopic expression of the p.Val44Met and p.Gly580Cys variants in Drosophila resulted in early death. TMEM63B-associated DEE represents a recognizable clinicopathological entity in which altered cation conductivity results in a severe neurological phenotype with progressive brain damage and early-onset epilepsy associated with hematological abnormalities in most individuals.

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.

Australian Genomics: Outcomes of a 5-year national program to accelerate the integration of genomics in healthcare.

Australian Genomics is a national collaborative partnership of more than 100 organizations piloting a whole-of-system approach to integrating genomics into healthcare, based on federation principles. In the first five years of operation, Australian Genomics has evaluated the outcomes of genomic testing in more than 5,200 individuals across 19 rare disease and cancer flagship studies. Comprehensive analyses of the health economic, policy, ethical, legal, implementation and workforce implications of incorporating genomics in the Australian context have informed evidence-based change in policy and practice, resulting in national government funding and equity of access for a range of genomic tests. Simultaneously, Australian Genomics has built national skills, infrastructure, policy, and data resources to enable effective data sharing to drive discovery research and support improvements in clinical genomic delivery.

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.

Critically unwell infants and children with mitochondrial disorders diagnosed by ultra-rapid genomic sequencing.

PURPOSE: To characterize the diagnostic and clinical outcomes of a cohort of critically ill infants and children with suspected mitochondrial disorders (MD) undergoing ultra-rapid genomic testing as part of a national program. METHODS: Ultra-rapid genomic sequencing was performed in 454 families (genome sequencing: n=290, exome sequencing +/- mitochondrial DNA sequencing: n=164). In 91 individuals, MD was considered, prompting analysis using an MD virtual gene panel. These individuals were reviewed retrospectively and scored according to modified Nijmegen Mitochondrial Disease Criteria. RESULTS: A diagnosis was achieved in 47% (43/91) of individuals, 40% (17/43) of whom had an MD. Seven additional individuals in whom an MD was not suspected were diagnosed with an MD following broader analysis. Gene-agnostic analysis led to the discovery of two novel disease genes, with pathogenicity validated through targeted functional studies (CRLS1 and MRPL39). Functional studies enabled diagnosis in another four individuals. Of the 24 individuals ultimately diagnosed with an MD, 79% had a change in management, which included 53% whose care was redirected to palliation. CONCLUSION: Ultra-rapid genetic diagnosis of MD in acutely unwell infants and children is critical for guiding decisions about the need for additional investigations and clinical management.

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.

De novo MCM6 variants in neurodevelopmental disorders: a recognizable phenotype related to zinc binding residues.

The minichromosome maintenance (MCM) complex acts as a DNA helicase during DNA replication, and thereby regulates cell cycle progression and proliferation. In addition, MCM-complex components localize to centrosomes and play an independent role in ciliogenesis. Pathogenic variants in genes coding for MCM components and other DNA replication factors have been linked to growth and developmental disorders as Meier-Gorlin syndrome and Seckel syndrome. Trio exome/genome sequencing identified the same de novo MCM6 missense variant p.(Cys158Tyr) in two unrelated individuals that presented with overlapping phenotypes consisting of intra-uterine growth retardation, short stature, congenital microcephaly, endocrine features, developmental delay and urogenital anomalies. The identified variant affects a zinc binding cysteine in the MCM6 zinc finger signature. This domain, and specifically cysteine residues, are essential for MCM-complex dimerization and the induction of helicase activity, suggesting a deleterious effect of this variant on DNA replication. Fibroblasts derived from the two affected individuals showed defects both in ciliogenesis and cell proliferation. We additionally traced three unrelated individuals with de novo MCM6 variants in the oligonucleotide binding (OB)-fold domain, presenting with variable (neuro)developmental features including autism spectrum disorder, developmental delay, and epilepsy. Taken together, our findings implicate de novo MCM6 variants in neurodevelopmental disorders. The clinical features and functional defects related to the zinc binding residue resemble those observed in syndromes related to other MCM components and DNA replication factors, while de novo OB-fold domain missense variants may be associated with more variable neurodevelopmental phenotypes. These data encourage consideration of MCM6 variants in the diagnostic arsenal of NDD.

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.

Heterozygous NOTCH1 Variants Cause CNS Immune Activation and Microangiopathy.

NOTCH1 belongs to the NOTCH family of proteins that regulate cell fate and inflammatory responses. Somatic and germline NOTCH1 variants have been implicated in cancer, Adams-Oliver syndrome, and cardiovascular defects. We describe 7 unrelated patients grouped by the presence of leukoencephalopathy with calcifications and heterozygous de novo gain-of-function variants in NOTCH1. Immunologic profiling showed upregulated CSF IP-10, a cytokine secreted downstream of NOTCH1 signaling. Autopsy revealed extensive leukoencephalopathy and microangiopathy with vascular calcifications. This evidence implicates that heterozygous gain-of-function variants in NOTCH1 lead to a chronic central nervous system (CNS) inflammatory response resulting in a calcifying microangiopathy with leukoencephalopathy. ANN NEUROL 2022;92:895-901.

Biallelic variants in TUBGCP6 result in microcephaly and chorioretinopathy 1: Report of four cases and a literature review.

Autosomal recessive microcephaly and chorioretinopathy-1 (MCCRP1) is a rare Mendelian disorder resulting from biallelic loss of function variants in Tubulin-Gamma Complex Associated Protein 6 (TUBGCP6, MIM#610053). Clinical features of this disorder include microcephaly, cognitive impairment, dysmorphic features, and variable ophthalmological anomalies including chorioretinopathy. Microcephaly can be recognized prenatally and visual impairment becomes evident during the first year of life. The clinical presentation resembles the findings in some acquired conditions such as congenital toxoplasmosis and cytomegalovirus infections; thus, it is important to recognize and diagnose this syndrome in view of its impact on patient health management and familial reproductive plans. To date, only seven molecularly confirmed patients from five unrelated families have been reported. We report an additional four unrelated patients with TUBGCP6 variants including one prenatal diagnosis and review the clinical phenotypes and genotypes of all the known cases. This report expands the molecular and phenotypic spectrum of TUBGCP6 and includes additional prenatal findings associated with MCCRP1.

A mutational hotspot in AMOTL1 defines a new syndrome of orofacial clefting, cardiac anomalies, and tall stature.

AMOTL1 encodes angiomotin-like protein 1, an actin-binding protein that regulates cell polarity, adhesion, and migration. The role of AMOTL1 in human disease is equivocal. We report a large cohort of individuals harboring heterozygous AMOTL1 variants and define a core phenotype of orofacial clefting, congenital heart disease, tall stature, auricular anomalies, and gastrointestinal manifestations in individuals with variants in AMOTL1 affecting amino acids 157-161, a functionally undefined but highly conserved region. Three individuals with AMOTL1 variants outside this region are also described who had variable presentations with orofacial clefting and multi-organ disease. Our case cohort suggests that heterozygous missense variants in AMOTL1, most commonly affecting amino acid residues 157-161, define a new orofacial clefting syndrome, and indicates an important functional role for this undefined region.

Biallellic variants in CACNA1S cause fetal akinesia sequence, progressive hydrops and stillbirth.

Fetal arthrogryposis is a well-recognised ultrasonographic phenotype, caused by both genetic, maternal and extrinsic factors. When present with fetal growth restriction, pulmonary hypoplasia and multiple joint contractures, it is often referred to as fetal akinesia deformation sequence (FADS). Historically, elucidating genetic causes of arthryogryposis/FADS has been challenging; there are now more than 150 genes known to cause arthrogryposis through myopathic, neuromuscular and metabolic pathways affecting fetal movement. FADS is associated with over 400 medical conditions making prenatal diagnosis challenging. Here we present a case of FADS diagnosed at 19 weeks gestation with progression to severe fetal hydrops and stillbirth at 26-weeks gestation. Initial investigations including combined first trimester screening, TORCH (infection) screen and chromosomal microarray were normal. Trio whole exome sequencing (WES) detected compound heterozygous likely pathogenic CACNA1S gene variants associated with autosomal dominant (AD) and autosomal recessive (AR) congenital myopathy and FADS. To our knowledge, this is the first prenatal diagnosis of this condition.

Identification of Regulatory Molecular "Hot Spots" for LH/PLOD Collagen Glycosyltransferase Activity.

Hydroxylysine glycosylations are post-translational modifications (PTMs) essential for the maturation and homeostasis of fibrillar and non-fibrillar collagen molecules. The multifunctional collagen lysyl hydroxylase 3 (LH3/PLOD3) and the collagen galactosyltransferase GLT25D1 are the human enzymes that have been identified as being responsible for the glycosylation of collagen lysines, although a precise description of the contribution of each enzyme to these essential PTMs has not yet been provided in the literature. LH3/PLOD3 is thought to be capable of performing two chemically distinct collagen glycosyltransferase reactions using the same catalytic site: an inverting beta-1,O-galactosylation of hydroxylysines (Gal-T) and a retaining alpha-1,2-glucosylation of galactosyl hydroxylysines (Glc-T). In this work, we have combined indirect luminescence-based assays with direct mass spectrometry-based assays and molecular structure studies to demonstrate that LH3/PLOD3 only has Glc-T activity and that GLT25D1 only has Gal-T activity. Structure-guided mutagenesis confirmed that the Glc-T activity is defined by key residues in the first-shell environment of the glycosyltransferase catalytic site as well as by long-range contributions from residues within the same glycosyltransferase (GT) domain. By solving the molecular structures and characterizing the interactions and solving the molecular structures of human LH3/PLOD3 in complex with different UDP-sugar analogs, we show how these studies could provide insights for LH3/PLOD3 glycosyltransferase inhibitor development. Collectively, our data provide new tools for the direct investigation of collagen hydroxylysine PTMs and a comprehensive overview of the complex network of shapes, charges, and interactions that enable LH3/PLOD3 glycosyltransferase activities, expanding the molecular framework and facilitating an improved understanding and manipulation of glycosyltransferase functions in biomedical applications.

De novo putative loss-of-function variants in TAF4 are associated with a neuro-developmental disorder.

TATA-binding protein associated factor 4 (TAF4) is a subunit of the Transcription Factor IID (TFIID) complex, a central player in transcription initiation. Other members of this multimeric complex have been implicated previously as monogenic disease genes in human developmental disorders. TAF4 has not been described to date as a monogenic disease gene. We here present a cohort of eight individuals, each carrying de novo putative loss-of-function (pLoF) variants in TAF4 and expressing phenotypes consistent with a neuro-developmental disorder (NDD). Common features include intellectual disability, abnormal behavior, and facial dysmorphisms. We propose TAF4 as a novel dominant disease gene for NDD, and coin this novel disorder "TAF4-related NDD" (T4NDD). We place T4NDD in the context of other disorders related to TFIID subunits, revealing shared features of T4NDD with other TAF-opathies.

Prenatal phenotyping: A community effort to enhance the Human Phenotype Ontology.

Technological advances in both genome sequencing and prenatal imaging are increasing our ability to accurately recognize and diagnose Mendelian conditions prenatally. Phenotype-driven early genetic diagnosis of fetal genetic disease can help to strategize treatment options and clinical preventive measures during the perinatal period, to plan in utero therapies, and to inform parental decision-making. Fetal phenotypes of genetic diseases are often unique and at present are not well understood; more comprehensive knowledge about prenatal phenotypes and computational resources have an enormous potential to improve diagnostics and translational research. The Human Phenotype Ontology (HPO) has been widely used to support diagnostics and translational research in human genetics. To better support prenatal usage, the HPO consortium conducted a series of workshops with a group of domain experts in a variety of medical specialties, diagnostic techniques, as well as diseases and phenotypes related to prenatal medicine, including perinatal pathology, musculoskeletal anomalies, neurology, medical genetics, hydrops fetalis, craniofacial malformations, cardiology, neonatal-perinatal medicine, fetal medicine, placental pathology, prenatal imaging, and bioinformatics. We expanded the representation of prenatal phenotypes in HPO by adding 95 new phenotype terms under the Abnormality of prenatal development or birth (HP:0001197) grouping term, and revised definitions, synonyms, and disease annotations for most of the 152 terms that existed before the beginning of this effort. The expansion of prenatal phenotypes in HPO will support phenotype-driven prenatal exome and genome sequencing for precision genetic diagnostics of rare diseases to support prenatal care.

De Novo Variants Disrupting the HX Repeat Motif of ATN1 Cause a Recognizable Non-Progressive Neurocognitive Syndrome.

Polyglutamine expansions in the transcriptional co-repressor Atrophin-1, encoded by ATN1, cause the neurodegenerative condition dentatorubral-pallidoluysian atrophy (DRPLA) via a proposed novel toxic gain of function. We present detailed phenotypic information on eight unrelated individuals who have de novo missense and insertion variants within a conserved 16-amino-acid "HX repeat" motif of ATN1. Each of the affected individuals has severe cognitive impairment and hypotonia, a recognizable facial gestalt, and variable congenital anomalies. However, they lack the progressive symptoms typical of DRPLA neurodegeneration. To distinguish this subset of affected individuals from the DRPLA diagnosis, we suggest using the term CHEDDA (congenital hypotonia, epilepsy, developmental delay, digit abnormalities) to classify the condition. CHEDDA-related variants alter the particular structural features of the HX repeat motif, suggesting that CHEDDA results from perturbation of the structural and functional integrity of the HX repeat. We found several non-homologous human genes containing similar motifs of eight to 10 HX repeat sequences, including RERE, where disruptive variants in this motif have also been linked to a separate condition that causes neurocognitive and congenital anomalies. These findings suggest that perturbation of the HX motif might explain other Mendelian human conditions.

Expanding the Spectrum of BAF-Related Disorders: De Novo Variants in SMARCC2 Cause a Syndrome with Intellectual Disability and Developmental Delay.

SMARCC2 (BAF170) is one of the invariable core subunits of the ATP-dependent chromatin remodeling BAF (BRG1-associated factor) complex and plays a crucial role in embryogenesis and corticogenesis. Pathogenic variants in genes encoding other components of the BAF complex have been associated with intellectual disability syndromes. Despite its significant biological role, variants in SMARCC2 have not been directly associated with human disease previously. Using whole-exome sequencing and a web-based gene-matching program, we identified 15 individuals with variable degrees of neurodevelopmental delay and growth retardation harboring one of 13 heterozygous variants in SMARCC2, most of them novel and proven de novo. The clinical presentation overlaps with intellectual disability syndromes associated with other BAF subunits, such as Coffin-Siris and Nicolaides-Baraitser syndromes and includes prominent speech impairment, hypotonia, feeding difficulties, behavioral abnormalities, and dysmorphic features such as hypertrichosis, thick eyebrows, thin upper lip vermilion, and upturned nose. Nine out of the fifteen individuals harbor variants in the highly conserved SMARCC2 DNA-interacting domains (SANT and SWIRM) and present with a more severe phenotype. Two of these individuals present cardiac abnormalities. Transcriptomic analysis of fibroblasts from affected individuals highlights a group of differentially expressed genes with possible roles in regulation of neuronal development and function, namely H19, SCRG1, RELN, and CACNB4. Our findings suggest a novel SMARCC2-related syndrome that overlaps with neurodevelopmental disorders associated with variants in BAF-complex subunits.

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.

Different types of disease-causing noncoding variants revealed by genomic and gene expression analyses in families with X-linked intellectual disability.

The pioneering discovery research of X-linked intellectual disability (XLID) genes has benefitted thousands of individuals worldwide; however, approximately 30% of XLID families still remain unresolved. We postulated that noncoding variants that affect gene regulation or splicing may account for the lack of a genetic diagnosis in some cases. Detecting pathogenic, gene-regulatory variants with the same sensitivity and specificity as structural and coding variants is a major challenge for Mendelian disorders. Here, we describe three pedigrees with suggestive XLID where distinctive phenotypes associated with known genes guided the identification of three different noncoding variants. We used comprehensive structural, single-nucleotide, and repeat expansion analyses of genome sequencing. RNA-Seq from patient-derived cell lines, reverse-transcription polymerase chain reactions, Western blots, and reporter gene assays were used to confirm the functional effect of three fundamentally different classes of pathogenic noncoding variants: a retrotransposon insertion, a novel intronic splice donor, and a canonical splice variant of an untranslated exon. In one family, we excluded a rare coding variant in ARX, a known XLID gene, in favor of a regulatory noncoding variant in OFD1 that correlated with the clinical phenotype. Our results underscore the value of genomic research on unresolved XLID families to aid novel, pathogenic noncoding variant discovery.