Tandem mass spectrometry is currently used by the Ohio Department of Health to screen newborn infants for 36 medically actionable inborn errors of metabolism. As a complementary test for infants with abnormal biochemical screens, whole genome sequencing (WGS) theoretically could reduce false-positive results, facilitate timely case resolution and, in some instances, indicate a more specific diagnosis than obtained initially. Menkes disease is a X-linked recessive disorder of human copper metabolism with a predicted minimum birth prevalence of 1 in 34,810 live male births. Recent progress in treatment options for Menkes heighten the importance of newborn screening (NBS) to identify this illness within the window of therapeutic opportunity, the first 4 to 6 weeks of life. We developed a DNA extraction protocol from dried blood spots that yielded high quality DNA for whole genome sequencing and studied 24 subjects with known ATP7A variants. Analysis was confined to genes (n = 55) for neurometabolic conditions currently screened for in Ohio (n = 36) plus ATP7A (Menkes disease). WGS detected all ATP7A variants including 7 missense, 5 splice site, 4 copy number variants, 4 nonsense, 3 indels, and 1 missense/splice site. WGS also detected 5 heterozygous pathogenic variants in 5 genes that encode conditions for which Ohio screens. Our results confirm the feasibility and reliability of this approach for early Menkes disease detection and to complement tandem mass spectroscopy for detection of other actionable inherited disorders.

Syndromic cardiac malformations can result in morbidity, yet their genetic etiology is only understood for a subset of individuals. Genome sequencing efforts in congenital anomaly cohorts may identify disease-associated variants in previously unrecognized genes. Through international matchmaking efforts, we identified eighteen individuals in total with de novo or loss-of-function variants in EIF3A (n = 4) or EIF3B (n = 14). The clinical phenotype varied but predominantly included cardiac defects, craniofacial dysmorphisms, mild developmental delays, and behavioral abnormalities. These genes encode core subunits of the eukaryotic initiation factor 3 (eIF3) complex, which plays a critical role in binding mRNA transcripts to the 40S ribosomal subunit during translation initiation. Both genes are highly constrained against loss of function, and animal models have demonstrated that disruptions in the eIF3 complex result in a range of developmental defects, including cardiovascular malformations. Additionally, EIF3B is located within the minimally overlapping region implicated in cardiac anomalies associated with 7p22.3 microdeletions. We sought to further study the role of these genes in syndromic congenital heart disease. To explore their functional impact, we generated zebrafish models with mutations in the orthologous eif3s10 and eif3ba genes, which resulted in developmental abnormalities, including thin heart tubes, lack of craniofacial cartilage, and embryonic lethality. We propose that pathogenic variants in EIF3A, as well as pathogenic variants or microdeletions involving EIF3B, cause a distinct autosomal-dominant neurodevelopmental syndrome characterized by cardiovascular and craniofacial manifestations.

Hippocampal sclerosis is a frequent finding in pediatric epilepsy surgery and has traditionally been regarded as an acquired lesion. It commonly co-occurs with focal cortical dysplasia (FCD IIIa), yet whether hippocampal injury is secondary to seizures or reflects a shared underlying etiology remains unresolved. Here we identified somatic variants activating the RAS-MAPK pathway in 40% of patients with hippocampal sclerosis, but in none with non-sclerotic hippocampus. Gain-of-function variants in PTPN11 were the most common finding, with mutations present in both cortex and hippocampus and enriched in hippocampal neurons, consistent with a shared developmental origin. In mice, Ptpn11 D61Y mutants developed profound hippocampal degeneration and gliosis following subthreshold kainic acid exposure, whereas wild-type controls were unaffected. p38-dependent stress pathways were upregulated in patients and mice, suggesting a mechanism through which ERK-p38 crosstalk lowers the threshold for seizure-induced injury. These results provide a genetic explanation for FCD IIIa, elucidate the role of somatic mutations within the RAS-MAPK pathway in driving hippocampal sclerosis, and provide a target for pathway-specific interventions for intractable seizures.

Somatic variants are a prominent cause of epilepsy-associated cortical malformations, but about half of patients undergoing genetic testing have no finding due partly to limitations in variant detection. Most studies have focused on single-nucleotide variants or small indels that are accessible to short-read sequencing technologies, but somatic structural variants are also emerging as important contributors despite their unique detection challenges. Optical genome mapping (OGM) is a promising methodology for the detection of structural variants, but requires high quality, high molecular weight DNA from clinical specimens. Here we successfully optimize a protocol for OGM of surgically-resected patient brain tissue which yields ~450x effective coverage - suitable for detecting somatic variants at low allele fractions. We apply this approach to brain specimens from four patients with epilepsy. OGM identifies large and complex mosaic structural variants ranging from 7-40% variant allele fraction, most of which are not captured by short-read exome sequencing of the same specimen. In one patient with a known germline DEPDC5 variant, OGM reveals a somatic variant - a 13.2kb deletion in DEPDC5 at approximately 20% VAF - consistent with the established two-hit model in DEPDC5-associated lesional epilepsies. By resolving the breakpoints in PacBio HiFi sequencing data, we identify a mechanism for this somatic deletion, mediated by recombination of two Alu elements flanking the region. Our findings demonstrate that OGM is a robust and complementary tool for detecting somatic structural variation in human brain tissue, with potential to improve diagnostic yield and refine genotype-phenotype correlations in neurological disorders.

Alagille syndrome (ALGS) is a rare, typically multisystem genetic disorder that impacts the liver, heart, eyes, vertebrae, and other areas of development. A clinical diagnosis can be established through defined clinical diagnostic criteria, while a molecular diagnosis requires the presence of a heterozygous pathogenic variant in JAG1 or NOTCH2 detected through molecular genetic testing. Disruption of either gene product results in disrupted Notch signaling pathways, resulting in the broad spectrum of clinical manifestations. The estimated incidence of ALGS is approximately 1 in 30,000-70,000 newborns. Here, we present a case report of a female neonate that presented at birth to the cardiothoracic intensive care unit with critical congenital heart disease (CHD), specifically, Tetralogy of Fallot (ToF). The patient and her parents were enrolled in a translational genomics protocol, COURAGE for Kids, aimed at performing research genome sequencing (GS) on neonates with critical CHD, in a phenotype-agnostic manner, to characterize genetic associations with disease and longitudinal outcomes. This family's GS uncovered a paternally inherited heterozygous missense variant in JAG1 (NM_000214.3:c.806C>T:p.Pro269Leu), which is associated with ALGS type 1 (MIM #118450). Segregation analysis revealed that the proband's paternal grandfather, who also had ToF as a child, carries the same familial variant. A subsequent dysmorphology assessment revealed typical ALGS facial features in the proband, as well as her father, who does not have a history of CHD (prominent forehead, deeply set eyes, wide nasal bridge, bulbous nose, and pointed chin). Thus, this variant is likely causing incomplete ALGS in the proband, father, and paternal grandfather. This variant may have also contributed to vascular events that have occurred within this family, an important example of how an uncovered molecular diagnosis can guide care even for isolated disease. We report a familial JAG1 variant (p.Pro269Leu) in a family with incomplete ALGS presenting as isolated ToF. Given the proband's clinical features alone were not suspicious of a molecular diagnosis of ALGS or other multisystem genetic disorder, this case underscores the potential importance of phenotype-agnostic genetic workup for critical CHD in the neonatal setting.

Rare germline and somatic variants in SLC35A2 cause a spectrum of severe glycosylation disorders that commonly present with epilepsy. SLC35A2 encodes the Golgi transporter for UDP-galactose, but how its deficiency leads to severe neurodevelopmental disorders is unknown. Using a mouse model deficient for Slc35a2 in the forebrain, we identified a specific defect in O-GalNAc glycan synthesis, while other galactose-containing glycoconjugates remained intact. O-GalNAc glycans were absent from their normal location within neuronal tracts of the corpus callosum, and truncated precursors accumulated in the cortex on critical extracellular matrix molecules. Cultured primary neurons lacking Slc35a2 showed impaired development, hyperexcitability, and impaired O-GalNAc glycosylation. Finally, human brain tissue from cases of SLC35A2-associated intractable epilepsy displayed a strong correlation between variant burden and truncated O-GalNAc glycans. These findings provide a mechanistic link between genetic causes of SLC35A2-associated epilepsy and protein O-glycosylation that can be targeted for biomarker and therapeutic development.

Chromatin regulation is critical for neurodevelopment, and its disruption has emerged as a key pathogenic mechanism in neurodevelopmental disease, including autism spectrum disorder (ASD), a condition known for genetic and phenotypic heterogeneity. We previously identified an ASD gene, KDM5A, encoding a histone H3 lysine 4 demethylase, and reported de novo and inherited variants in nine individuals with severe ASD and other neurodevelopmental phenotypes. Here, we expand the genetic and phenotypic spectrum of KDM5A-related neurodevelopmental disorders and investigate the functional impact of identified variants. Through international collaborations, we assembled a cohort of 24 additional individuals from 21 families with rare, protein-altering KDM5A variants. All individuals presented with severe speech impairment and intellectual disability, often alongside ASD and other neurodevelopmental features. The variants include missense, nonsense, frameshift, and splice site, distributed across nearly all functional domains of the protein. Structural modeling revealed localized conformational disruptions, particularly at conserved residues in enzymatic or chromatin-interacting domains. For a subset of variants, we demonstrated reduced KDM5A protein levels in cell lines derived from affected individuals. Transcriptomic profiling revealed variant-specific gene expression changes, most pronounced in variants affecting the PLU1 chromatin binding motif and the Jumonji C domain of the enzymatic core. American College of Medical Genetics and Genomics-guided reclassification supported pathogenicity for the majority of variants, including multiple upgrades from uncertain significance to pathogenic or likely pathogenic. Together, these findings implicate diverse KDM5A alleles in a rare but recurrent form of ASD, and establish KDM5A as a key regulator of neurodevelopment and chromatin-mediated ASD pathogenesis.

Biallelic hypomorphic variants in PRORP cause the rare autosomal recessive disorder combined oxidative phosphorylation deficiency type 54 (COXPD54). COXPD54 encompasses a clinical spectrum of sensorineural hearing loss and ovarian insufficiency (Perrault syndrome) to leukodystrophy with developmental delay and epilepsy. Here, we report two new affected individuals with biallelic PRORP variants with clinical features consistent with COXPD54. One individual was homozygous for c.1505G > A p.Arg502Gln, whereas the other was compound heterozygous for c.1510C > T, p.His504Tyr and c.893C > A, p.Ser298Ter (NM_014672.4). In vitro tRNA processing assays revealed decreased mitochondrial 5′ tRNA leader cleavage by human RNase P complex with the two novel missense PRORP metallonuclease domain variants. These data provide further evidence that biallelic PRORP variants disrupt 5’ tRNA leader cleavage and are associated with a pleiotropic phenotype of COXPD54.

The establishment of neuronal polarity, whereby somatodendritic and axonal cellular compartments are defined, is a critical determinant for the development of neuronal networks and patterning during neurogenesis. The axon initial segment (AIS), a key structure in the establishment of this polarity, is formed through interactions between the microtubule and actin cytoskeleton, Ankyrin G, TRIM46 and multiple transmembrane and perimembranous proteins. Here we implicate a component of the septin cytoskeleton, Septin-2, in the maintenance and function of the AIS through the study of mutations found in five unrelated human individuals and one mother-daughter duo with a majority presenting with cognitive impairment. Septins form octameric rods that assemble into higher order filamentous scaffolds driven by Septin-2 homodimerization. Mutant Septin-2 is predicted to impart a dominant negative blockade on septin octamers forming these structures by precluding Septin-2 homodimerization. Expression of mutant Septin-2 constructs in neurons leads to the disappearance of canonical hallmarks of the AIS. This includes loss of Ankyrin G in the AIS, aberrant localization of MAP2 within the distal axon, axonal shortening and electrophysiological hypoexcitability. We further show that Septin-2 binds to a neuron-specific domain of Ankyrin G, an interaction that is largely ablated by these mutations. These data establish a role for Septin-2 in the maintenance and function of the AIS and implicate cytoskeletal structures composed of septin oligomers in the establishment of higher cognitive functions in humans.

Biallelic disease-causing variants in IGHMBP2 cause spinal muscular atrophy with respiratory distress type I (SMARD1) and Charcot-Marie-Tooth type 2S (CMT2S). We present 12 unrelated patients with clinically suspected IGHMBP2-related-disease, each carrying a variant deep in intron 8 of IGHMBP2 (c.1235+1076G>A (n=6), c.1235+450G>A (n=5), and c.1235+894C>A (n=1)), along with a known deleterious variant in trans. To assess aberrant pathogenic splicing induced by these deep intronic variants in a relevant model, patient-derived induced pluripotent stem cells were differentiated into motor neurons (iMNs). Long-read RNA sequencing revealed introduction of different pseudoexons by each variant: c.1235+450G>A (626bp), c.1235+1076G>A (112bp and 77bp) and c.1235+894C>A (182bp). Although each variant utilizes a unique splice acceptor site, they all activate the same cryptic donor site, enabling a therapeutic approach to redirect aberrant splicing for all the variants using a single shared antisense oligonucleotide (ASO). Treatment of iMNs with this single ASO restored full-length IGHMBP2 protein in c.1235+894G>A and c.1235+1076G>A by decreasing the use of the novel acceptor site. In contrast, ASO treatment did not correct the splicing in c.1235+450G>A, suggesting that additional splice correction will be needed for this specific variant. A CRISPR interference screen of IGHMBP2 loss-of-function in iMNs identified ribonucleoprotein complex biogenesis (RNP), and rRNA and tRNA processing as top pathways implicated in motor neuron vulnerability. Proteomics and transcriptomics analysis of successfully treated patient iMNs revealed correction of RNP biogenesis and rRNA processing defects. This study highlights the importance of characterizing deep intronic variants in disease-relevant cells to assist the diagnostic process and inform therapeutics development.

Pathogenic variants in GNAS can cause a wide range of diseases including pseudohypoparathyroidism, pseudopseudohypoparathyroidism, McCune-Albright syndrome, among others. The specific phenotypic features that may be seen are influenced by the variant type and location in the gene, whether it causes loss or gain of function, and whether it is germline or somatic in nature. The GNAS locus is imprinted, which also results in a parent-of-origin effect. Typically, germline loss of function variants on the maternal allele are associated with variable hormonal resistances, obesity, intrauterine growth restriction, and cognitive impairment. Here, we describe a mother and daughter with a unique splicing variant near exon 5 of the GNAS gene (NM_000516.5:c.432 + 5G>A), shown to cause alternative splicing through RNA sequencing (RNA-seq), likely resulting in a loss-of-function effect. Segregation testing revealed that the variant arose de novo in the mother, and phasing showed it was on her paternal allele. The resultant phenotype includes a SHOX deficiency-like disorder with Madelung deformity in the mother, and significant growth restriction with brachydactyly in the daughter, further expanding the phenotypic spectrum of GNAS inactivation disorders.

The nucleosome remodeling and deacetylation (NuRD) complex is a major chromatin regulator and plays a critical role in regulating gene transcription, genome integrity, and cell cycle progression. Heterozygous variants in GATAD2B, a core NuRD component, have been reported to cause GATAD2B-Associated Neurodevelopmental Disorder (GAND), an autosomal dominant neurodevelopmental disorder characterized by intellectual disability, developmental delay, hypotonia, and distinctive craniofacial features. The vast majority of disease-causing variants in GATAD2B reported to date are loss-of-function (nonsense, frameshift, or splice site) variants. Here, we report a 6-year-old male patient with profound global developmental delay and dysmorphic features, who was found to have a de novo ~97 kbp partial duplication of the GATAD2B gene. Using long-read transcriptome and genome sequencing on the Pacific BioSciences (PacBio) platform, we show that the duplication is a tandem event whose breakpoint in the 3' UTR of the gene causes skipping of the last exon and transcriptional read-through. The resulting transcript contains two incomplete copies of GATAD2B, one with exons 1-10 and the other with exons 2-7, likely representing a loss-of-function allele. Follow-up clinical evaluations confirmed the patient's diagnosis of GAND, ending a years-long diagnostic odyssey for the family and highlighting an unusual mechanism of gene disruption in GATAD2B.

Germline ZFX variants are associated with an X-linked neurodevelopmental disorder, with 14 males and 16 females reported to date. We describe a 20-year-old female with a heterozygous ZFX frameshift variant, p.(Met666Valfs*2), identified by genome sequencing, previously reported in an affected male. She exhibited motor and speech delays with hypotonia in early childhood, and was later diagnosed with congenital heart defects, autism spectrum disorder, mild intellectual disability, and absence seizures. She further developed sensorineural hearing loss, skin hyperpigmentation, and ophthalmoplegia. Novel phenotypic features included inferior cerebellar vermian hypoplasia, hypoplastic right vertebral artery, aberrant subclavian artery, long palpebral fissures, ophthalmoplegia, skin hyperpigmentation, and a short uvula, expanding the known clinical spectrum. Female carriers of pathogenic ZFX variants demonstrate highly variable expressivity, ranging from apparently unaffected individuals to syndromic presentations. Individuals with heterozygous missense variants often exhibit hyperparathyroidism, suggesting a genotype-phenotype correlation. Reanalysis of published RNA-sequencing data identified 15 ZFX target genes involved in neurodevelopment, suggesting a role for these genes in disease pathogenesis. These findings confirm the pathogenicity of the p.(Met666Valfs*2) variant in the proband and highlight the phenotypic heterogeneity of the disorder in females. Clinical care should include cardiac and endocrine monitoring, with endocrine testing offered to unaffected females carrying missense variants.

A recurrent de novo germline variant in the MAX gene, p.(Arg60Gln), has recently been associated with polydactyly-macrocephaly syndrome in six unrelated individuals. Affected individuals presented with progressive macrocephaly, post-axial polydactyly, developmental delay, autistic features and a series of craniofacial, brain, cardiac, ocular, and renal anomalies. Here, we describe two unrelated female probands with the known recurrent MAX variant, c.179G>A p.(Arg60Gln), who presented with the emerging phenotypes of the MAX-associated syndrome. We also propose that genitourinary abnormalities, including Mayer-Rokitanski-Kuster-Hauser syndrome in one individual, may constitute an expansion of the known phenotype. These findings contribute to the current knowledge regarding the phenotypic spectrum of MAX-associated polydactyly-macrocephaly syndrome.

De novo variants in CSNK2A1 cause autosomal dominant Okur-Chung neurodevelopmental syndrome (OCNDS). OCNDS has an evolving clinical phenotype predominantly characterized by intellectual disability, global delays, dysmorphic features, and immunological manifestations. Microcephaly, defined as a small head circumference, is not widely recognized as a classical clinical presentation. Here, we describe four individuals from three unrelated families who shared several clinical features characteristic of an underlying syndromic neurodevelopmental condition. Trio clinical exome and research genome sequencing revealed that all affected individuals had heterozygous pathogenic missense variants in CSNK2A1. Two variants (c.468T>A p.Asp156Glu and c.149A>G p.Tyr50Cys) were de novo and previously reported, but the third variant (c.137G>T p.Gly46Val) is novel and segregated in two affected individuals in a family. This adds to growing evidence of inherited disease-causing variants in CSNK2A1, an observation reported only twice previously. A detailed phenotypic analysis of our cohort together with those individuals reported in the literature revealed that OCNDS individuals, on average, have a smaller head circumference with one-third presenting with microcephaly. We also show that the incidence of microcephaly is significantly correlated with the location of the variant in the encoded protein. Our findings suggest that small head circumference is a common but under-recognized feature of OCNDS, which may not be apparent at birth.

DDX17 is an RNA helicase shown to be involved in critical processes during the early phases of neuronal differentiation. Globally, we compiled a case series of 11 patients with neurodevelopmental phenotypes harbouring de novo monoallelic variants in DDX17. All 11 patients in our case series had a neurodevelopmental phenotype, whereby intellectual disability, delayed speech and language, and motor delay predominated. We performed in utero cortical electroporation in the brain of developing mice, assessing axon complexity and outgrowth of electroporated neurons, comparing wild-type and Ddx17 knockdown. We then undertook ex vivo cortical electroporation on neuronal progenitors to quantitatively assess axonal development at a single cell resolution. Mosaic ddx17 crispants and heterozygous knockouts in Xenopus tropicalis were generated for assessment of morphology, behavioural assays and neuronal outgrowth measurements. We further undertook transcriptomic analysis of neuroblastoma SH-SY5Y cells, to identify differentially expressed genes in DDX17-KD cells compared to controls. Knockdown of Ddx17 in electroporated mouse neurons in vivo showed delayed neuronal migration as well as decreased cortical axon complexity. Mouse primary cortical neurons revealed reduced axon outgrowth upon knockdown of Ddx17 in vitro. The axon outgrowth phenotype was replicated in crispant ddx17 tadpoles and in heterozygotes. Heterozygous tadpoles had clear neurodevelopmental defects and showed an impaired neurobehavioral phenotype. Transcriptomic analysis identified a statistically significant number of differentially expressed genes involved in neurodevelopmental processes in DDX17-KD cells compared to control cells. We have identified potential neurodevelopment disease-causing variants in a gene not previously associated with genetic disease, DDX17. We provide evidence for the role of the gene in neurodevelopment in both mammalian and non-mammalian species and in controlling the expression of key neurodevelopment genes.

INTRODUCTION:Perlman syndrome is a rare autosomal recessive overgrowth disorder with a predisposition to Wilms tumor, caused by biallelic variants in DIS3L2. The majority of patients die in infancy due to respiratory and/or renal failure, limiting the reports of patients surviving into childhood. METHODS:Exome sequencing was performed in the proband and her older brother. A younger sibling subsequently underwent targeted variant analysis. RNA sequencing was utilized to investigate the functional impact of the missense variant. RESULTS:Three siblings presented at birth with fetal macrosomia, dysmorphic facial features, and facial hypotonia. The proband had early speech delay and was diagnosed with Wilms tumor at 3 years old. Her brothers both had developmental delay presenting within the first year of life. Genetic testing identified compound heterozygous variants in DIS3L2 (NM_152383.5): c.127C>T (p.Arg43Ter) (paternal)/c.2381G>A (p.Arg794His) (maternal). CONCLUSION:Our findings expand the genetic and clinical spectrums associated with Perlman syndrome and increase the understanding of the phenotype observed in childhood. They also support consideration of genetic testing for Perlman syndrome in individuals and sibships with macrosomia, developmental delay, and characteristic facial dysmorphisms, with or without the presence of Wilms tumor.

The primary cilium is a small organelle that plays key roles in cellular signaling. Defects in primary cilia formation, morphology, and function cause a heterogeneous group of developmental syndromes termed ciliopathies. The inturned planar cell polarity protein (INTU) gene acts in the CPLANE complex to facilitate ciliogenesis and support cilia signaling. Bi-allelic genetic variants in INTU have previously been reported in seven patients with pleiotropic disorders, but a core set of phenotypes from these patients has not been codified and functional studies into these variants have failed to fully demonstrate mechanistic perturbations caused by INTU dysfunction. Here, we report on a person with cardiac abnormalities, distinctive craniofacial features, developmental delays, tongue hamartomas, bilateral clinodactyly, and polydactyly of the left great toe. Trio whole-exome sequencing identified compound heterozygous variants in the INTU gene. Functional studies provide evidence that these INTU variants confer human disease through altered ciliogenesis and/or cilia signaling. Furthermore, we suggest that this study along with previous reports sufficiently establishes an association between a pleiotropic disorder and variants in the INTU gene to enhance clinical interpretation of INTU variants in future studies.