PSMD11 loss-of-function variants correlate with a neurobehavioral phenotype, obesity, and increased interferon response.

Primary proteasomopathies have recently emerged as a new class of rare early-onset neurodevelopmental disorders (NDDs) caused by pathogenic variants in the PSMB1, PSMC1, PSMC3, or PSMD12 proteasome genes. Proteasomes are large multi-subunit protein complexes that maintain cellular protein homeostasis by clearing ubiquitin-tagged damaged, misfolded, or unnecessary proteins. In this study, we have identified PSMD11 as an additional proteasome gene in which pathogenic variation is associated with an NDD-causing proteasomopathy. PSMD11 loss-of-function variants caused early-onset syndromic intellectual disability and neurodevelopmental delay with recurrent obesity in 10 unrelated children. Our findings demonstrate that the cognitive impairment observed in these individuals could be recapitulated in Drosophila melanogaster with depletion of the PMSD11 ortholog Rpn6, which compromised reversal learning. Our investigations in subject samples further revealed that PSMD11 loss of function resulted in impaired 26S proteasome assembly and the acquisition of a persistent type I interferon (IFN) gene signature, mediated by the integrated stress response (ISR) protein kinase R (PKR). In summary, these data identify PSMD11 as an additional member of the growing family of genes associated with neurodevelopmental proteasomopathies and provide insights into proteasomal biology in human health.

Biallelic variants in GTF3C5, a regulator of RNA polymerase III-mediated transcription, cause a multisystem developmental disorder.

General transcription factor IIIC subunit 5 (GTF3C5) encodes transcription factor IIIC63 (TFIIIC63). It binds to DNA to recruit another transcription factor, TFIIIB, and RNA polymerase III (Pol III) to mediate the transcription of small noncoding RNAs, such as tRNAs. Here, we report four individuals from three families presenting with a multisystem developmental disorder phenotype with biallelic variants in GTF3C5. The overlapping features include growth retardation, developmental delay, intellectual disability, dental anomalies, cerebellar malformations, delayed bone age, skeletal anomalies, and facial dysmorphism. Using lymphoblastoid cell lines (LCLs) from two affected individuals, we observed a reduction in TFIIIC63 protein levels compared to control LCLs. Genome binding of TFIIIC63 protein is also reduced in LCL from one of the affected individuals. Additionally, approximately 40% of Pol III binding regions exhibited reduction in the level of Pol III occupancy in the mutant genome relative to the control, while approximately 54% of target regions showed comparable levels of Pol III occupancy between the two, indicating partial impairment of Pol III occupancy in the mutant genome. Yeasts with subject-specific variants showed temperature sensitivity and impaired growth, supporting the notion that the identified variants have deleterious effects. gtf3c5 mutant zebrafish showed developmental defects, including a smaller body, head, and eyes. Taken together, our data show that GTF3C5 plays an important role in embryonic development, and that biallelic variants in this gene cause a multisystem developmental disorder. Our study adds GTF3C5-related disorder to the growing list of genetic disorders associated with Pol III transcription machinery.

Diagnostic Yield of Exome Sequencing in Pediatric Cardiomyopathy.

OBJECTIVE: To assess the diagnostic yield of exome sequencing (ES) in pediatric cardiomyopathy. STUDY DESIGN: A single-institution, retrospective chart review of 91 patients with pediatric cardiomyopathy was performed. While pediatric cardiomyopathy is often genetic in nature, no genetic test is recommended as standard of care. All our patients were diagnosed with cardiomyopathy and evaluated by a medical geneticist between January 2010 through September 2022. Demographic information and clinical data were abstracted. RESULTS: Of 91 patients with pediatric cardiomyopathy, 36 (39.6%) received a diagnosis by ES. Twenty-two (61.1%) of these diagnoses would have been missed on cardiac multigene panel testing. The diagnostic yield for cardiomyopathy presenting under 1 year of age was 38.3%, while the yield for patients over 1 year of age was 41.9%. CONCLUSIONS: ES has a high diagnostic yield in pediatric cardiomyopathy compared with a gene panel. Over 60% of patients with diagnosis by ES would not have received their molecular genetic diagnosis if only multigene panel testing was sent. Diagnostic yield did not vary significantly between the subtypes of cardiomyopathy and patient age groups, highlighting the likely clinical utility of ES for all pediatric cardiomyopathy patients.

Expansion of the prenatal phenotype of Baraitser-Winter syndrome: Presentation of two cases of multiple congenital anomaly syndrome.

Baraitser-Winter cerebrofrontofacial syndrome (BWCFF) is a variable multiple congenital anomaly condition, typically presenting postnatally with neurocognitive delays, distinctive facial features, cortical brain malformations, and in some, a variety of additional congenital malformations. However, only a few cases have reported the prenatal presentation of this syndrome. Here, we report two cases of BWCFF and their associated prenatal findings. One case presented with non-immune hydrops fetalis and a horseshoe kidney and was found to have a de novo heterozygous variant in ACTB (c.158A>G). The second case presented with gastroschisis, bilateral cleft lip and palate, and oligohydramnios, and was found to harbor a different de novo variant in ACTB (c.826G>A). Limited reports exist describing prenatally identified anomalies that include fetal growth restriction, increased nuchal fold, bilateral hydronephrosis, rocker bottom foot, talipes, cystic hygroma, omphalocele, and hydrops fetalis. In addition, only three of these cases have included detailed prenatal imaging findings. The two prenatal cases presented here demonstrate an expansion of the prenatal phenotype of BWCFF to include gastroschisis, lymphatic involvement, and oligohydramnios, which should each warrant consideration of this diagnosis in the setting of additional anomalies.

Expanding the clinical phenotype and variant spectrum associated with RFX7.

RFX7 encodes a transcription factor that is ubiquitously expressed and important for neural development. Haploinsufficiency of RFX7 is associated with intellectual disability, developmental delay, and diverse malformations of brain structures. Currently, there are only 16 clinically described individuals who have variants in RFX7. A recognizable pattern of malformation associated with mutation in RFX7 has not yet been uncovered. Here we describe the phenotypic presentation of two additional individuals who have novel de novo variants in RFX7. One of the individuals we describe is from an under-represented Afro-Caribbean population.

Utility of genome sequencing in exome-negative pediatric patients with neurodevelopmental phenotypes.

Exome sequencing (ES) has emerged as an essential tool in the evaluation of neurodevelopmental disorders (NDD) of unknown etiology. Genome sequencing (GS) offers advantages over ES due to improved detection of structural, copy number, repeat number and non-coding variants. However, GS is less commonly utilized due to higher cost and more intense analysis. Here, we present nine cases of pediatric NDD that were molecularly diagnosed with GS between 2017 and 2022, following non-diagnostic ES. All individuals presented with global developmental delay or regression. Other features present in our cohort included epilepsy, white matter abnormalities, brain malformation and dysmorphic features. Two cases were diagnosed on GS due to newly described gene-disease relationship or variant reclassification (MAPK8IP3, CHD3). Additional features missed on ES that were later detected on GS were: intermediate-size deletions in three cases who underwent ES that were not validated for CNV detection, pathogenic variants within the non-protein coding genes SNORD118 and RNU7-1, pathogenic variant within the promoter region of GJB1, and a coding pathogenic variant within BCAP31 which was not sufficiently covered on ES. GS following non-diagnostic ES led to the identification of pathogenic variants in this cohort of nine cases, four of which would not have been identified by reanalysis alone.

Hospital-level variation in genetic testing in children's hospitals' neonatal intensive care units from 2016 to 2021.

PURPOSE: This study aimed to examine variation in genetic testing between neonatal intensive care units (NICUs) across hospitals over time. METHODS: We performed a multicenter large-scale retrospective cohort study using NICU discharge data from the Pediatric Hospital Information System database between 2016 and 2021. We analyzed the variation in the percentage of NICU patients who had any genetic testing across hospitals and over time. We used a multivariable multilevel logistic regression model to investigate the potential association between patient characteristics and genetic testing. RESULTS: The final analysis included 207,228 neonates from 38 hospitals. Overall, 13% of patients had at least 1 genetic test sent, although this varied from 4% to 50% across hospitals. Over the study period, the proportion of patients tested increased, with the increase disproportionately borne by hospitals already testing high proportions of patients. On average, patients who received genetic testing had higher illness severity. Controlling for severity, however, only minimally reduced the degree of hospital-level variation in genetic testing. CONCLUSION: The percentage of NICU patients who undergo genetic testing varies among hospitals and increasingly so over time. Variation is largely unexplained by differences in severity between hospitals. The degree of variation suggests that clearer guidelines for NICU genetic testing are warranted.

Monoallelic loss-of-function BMP2 variants result in BMP2-related skeletal dysplasia spectrum.

PURPOSE: Bone morphogenic proteins (BMPs) regulate gene expression that is related to many critical developmental processes, including osteogenesis for which they are named. In addition, BMP2 is widely expressed in cells of mesenchymal origin, including bone, cartilage, skeletal and cardiac muscle, and adipose tissue. It also participates in neurodevelopment by inducing differentiation of neural stem cells. In humans, BMP2 variants result in a multiple congenital anomaly syndrome through a haploinsufficiency mechanism. We sought to expand the phenotypic spectrum and highlight phenotypes of patients harboring monoallelic missense variants in BMP2. METHODS: We used retrospective chart review to examine phenotypes from an international cohort of 18 individuals and compared these with published cases. Patient-derived missense variants were modeled in zebrafish to examine their effect on the ability of bmp2b to promote embryonic ventralization. RESULTS: The presented cases recapitulated existing descriptions of BMP2-related disorders, including craniofacial, cardiac, and skeletal anomalies and exhibit a wide phenotypic spectrum. We also identified patients with neural tube defects, structural brain anomalies, and endocrinopathies. Missense variants modeled in zebrafish resulted in loss of protein function. CONCLUSION: We use this expansion of reported phenotypes to suggest multidisciplinary medical monitoring and management of patients with BMP2-related skeletal dysplasia spectrum.

Retrospective identification of patients with SRRM2-related neurodevelopmental disorder in a single tertiary children's hospital.

SRRM2-related neurodevelopmental disorder is a recently described genetic diagnosis caused by loss-of-function variants in SRRM2. In order to understand the clinical spectrum of SRRM2-related neurodevelopmental disorder, we performed a retrospective exome data and clinical chart review at a single tertiary children's hospital, Children's Hospital of Philadelphia (CHOP). Among approximately 3100 clinical exome sequencing cases performed at CHOP, we identified three patients with SRRM2 loss-of-function pathogenic variants, in addition to one patient previously described in the literature. Common clinical features include developmental delay, attention deficit hyperactivity disorder, macrocephaly, hypotonia, gastroesophageal reflux, overweight/obesity, and autism. While developmental disabilities are commonly seen in all individuals with SRRM2 variants, the degree of developmental delay and intellectual disability is variable. Our data suggest that SRRM2-related neurodevelopmental disorder can be identified in 0.3% of individuals with developmental disabilities receiving exome sequencing.

Expanding the reproductive organ phenotype of CHD7-spectrum disorder.

CHD7 disorder is a multiple congenital anomaly syndrome with a highly variable phenotypic spectrum, and includes CHARGE syndrome. Internal and external genital phenotypes frequently seen in CHD7 disorder include cryptorchidism and micropenis in males, and vaginal hypoplasia in females, both thought to be secondary to hypogonadotropic hypogonadism. Here, we report 14 deeply phenotyped individuals with known CHD7 variants (9 pathogenic/likely pathogenic and 5 VOUS) and a range of reproductive and endocrine phenotypes. Reproductive organ anomalies were observed in 8 of 14 individuals and were more commonly noted in males (7/7), most of whom presented with micropenis and/or cryptorchidism. Kallmann syndrome was commonly observed among adolescents and adults with CHD7 variants. Remarkably, one 46,XY individual presented with ambiguous genitalia, cryptorchidism with Müllerian structures including uterus, vagina and fallopian tubes, and one 46,XX female patient presented with absent vagina, uterus and ovaries. These cases expand the genital and reproductive phenotype of CHD7 disorder to include two individuals with genital/gonadal atypia (ambiguous genitalia), and one with Müllerian aplasia.

Missense Variants in the Histone Acetyltransferase Complex Component Gene TRRAP Cause Autism and Syndromic Intellectual Disability.

Acetylation of the lysine residues in histones and other DNA-binding proteins plays a major role in regulation of eukaryotic gene expression. This process is controlled by histone acetyltransferases (HATs/KATs) found in multiprotein complexes that are recruited to chromatin by the scaffolding subunit transformation/transcription domain-associated protein (TRRAP). TRRAP is evolutionarily conserved and is among the top five genes intolerant to missense variation. Through an international collaboration, 17 distinct de novo or apparently de novo variants were identified in TRRAP in 24 individuals. A strong genotype-phenotype correlation was observed with two distinct clinical spectra. The first is a complex, multi-systemic syndrome associated with various malformations of the brain, heart, kidneys, and genitourinary system and characterized by a wide range of intellectual functioning; a number of affected individuals have intellectual disability (ID) and markedly impaired basic life functions. Individuals with this phenotype had missense variants clustering around the c.3127G>A p.(Ala1043Thr) variant identified in five individuals. The second spectrum manifested with autism spectrum disorder (ASD) and/or ID and epilepsy. Facial dysmorphism was seen in both groups and included upslanted palpebral fissures, epicanthus, telecanthus, a wide nasal bridge and ridge, a broad and smooth philtrum, and a thin upper lip. RNA sequencing analysis of skin fibroblasts derived from affected individuals skin fibroblasts showed significant changes in the expression of several genes implicated in neuronal function and ion transport. Thus, we describe here the clinical spectrum associated with TRRAP pathogenic missense variants, and we suggest a genotype-phenotype correlation useful for clinical evaluation of the pathogenicity of the variants.

Expanding the phenotypic spectrum of ARCN1-related syndrome.

PURPOSE: This study aimed to describe the phenotypic and molecular characteristics of ARCN1-related syndrome. METHODS: Patients with ARCN1 variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. RESULTS: In total, we identified 14 cases of ARCN1-related syndrome, (9 pediatrics, and 5 fetal cases from 3 families). The clinical features these newly identified cases were compared to 6 previously reported cases for a total of 20 cases. Intrauterine growth restriction, micrognathia, and short stature were present in all patients. Other common features included prematurity (11/15, 73.3%), developmental delay (10/14, 71.4%), genitourinary malformations in males (6/8, 75%), and microcephaly (12/15, 80%). Novel features of ARCN1-related syndrome included transient liver dysfunction and specific glycosylation abnormalities during illness, giant cell hepatitis, hepatoblastoma, cataracts, and lethal skeletal manifestations. Developmental delay was seen in 73% of patients, but only 3 patients had intellectual disability, which is less common than previously reported. CONCLUSION: ARCN1-related syndrome presents with a wide clinical spectrum ranging from a severe embryonic lethal syndrome to a mild syndrome with intrauterine growth restriction, micrognathia, and short stature without intellectual disability. Patients with ARCN1-related syndrome should be monitored for liver dysfunction during illness, cataracts, and hepatoblastoma. Additional research to further define the phenotypic spectrum and possible genotype-phenotype correlations are required.

A Recurrent De Novo PACS2 Heterozygous Missense Variant Causes Neonatal-Onset Developmental Epileptic Encephalopathy, Facial Dysmorphism, and Cerebellar Dysgenesis.

Developmental and epileptic encephalopathies (DEEs) represent a large clinical and genetic heterogeneous group of neurodevelopmental diseases. The identification of pathogenic genetic variants in DEEs remains crucial for deciphering this complex group and for accurately caring for affected individuals (clinical diagnosis, genetic counseling, impacting medical, precision therapy, clinical trials, etc.). Whole-exome sequencing and intensive data sharing identified a recurrent de novo PACS2 heterozygous missense variant in 14 unrelated individuals. Their phenotype was characterized by epilepsy, global developmental delay with or without autism, common cerebellar dysgenesis, and facial dysmorphism. Mixed focal and generalized epilepsy occurred in the neonatal period, controlled with difficulty in the first year, but many improved in early childhood. PACS2 is an important PACS1 paralog and encodes a multifunctional sorting protein involved in nuclear gene expression and pathway traffic regulation. Both proteins harbor cargo(furin)-binding regions (FBRs) that bind cargo proteins, sorting adaptors, and cellular kinase. Compared to the defined PACS1 recurrent variant series, individuals with PACS2 variant have more consistently neonatal/early-infantile-onset epilepsy that can be challenging to control. Cerebellar abnormalities may be similar but PACS2 individuals exhibit a pattern of clear dysgenesis ranging from mild to severe. Functional studies demonstrated that the PACS2 recurrent variant reduces the ability of the predicted autoregulatory domain to modulate the interaction between the PACS2 FBR and client proteins, which may disturb cellular function. These findings support the causality of this recurrent de novo PACS2 heterozygous missense in DEEs with facial dysmorphim and cerebellar dysgenesis.

Phenotypic expansion of CACNA1C-associated disorders to include isolated neurological manifestations.

PURPOSE: CACNA1C encodes the alpha-1-subunit of a voltage-dependent L-type calcium channel expressed in human heart and brain. Heterozygous variants in CACNA1C have previously been reported in association with Timothy syndrome and long QT syndrome. Several case reports have suggested that CACNA1C variation may also be associated with a primarily neurological phenotype. METHODS: We describe 25 individuals from 22 families with heterozygous variants in CACNA1C, who present with predominantly neurological manifestations. RESULTS: Fourteen individuals have de novo, nontruncating variants and present variably with developmental delays, intellectual disability, autism, hypotonia, ataxia, and epilepsy. Functional studies of a subgroup of missense variants via patch clamp experiments demonstrated differential effects on channel function in vitro, including loss of function (p.Leu1408Val), neutral effect (p.Leu614Arg), and gain of function (p.Leu657Phe, p.Leu614Pro). The remaining 11 individuals from eight families have truncating variants in CACNA1C. The majority of these individuals have expressive language deficits, and half have autism. CONCLUSION: We expand the phenotype associated with CACNA1C variants to include neurodevelopmental abnormalities and epilepsy, in the absence of classic features of Timothy syndrome or long QT syndrome.

Disruption of RFX family transcription factors causes autism, attention-deficit/hyperactivity disorder, intellectual disability, and dysregulated behavior.

PURPOSE: We describe a novel neurobehavioral phenotype of autism spectrum disorder (ASD), intellectual disability, and/or attention-deficit/hyperactivity disorder (ADHD) associated with de novo or inherited deleterious variants in members of the RFX family of genes. RFX genes are evolutionarily conserved transcription factors that act as master regulators of central nervous system development and ciliogenesis. METHODS: We assembled a cohort of 38 individuals (from 33 unrelated families) with de novo variants in RFX3, RFX4, and RFX7. We describe their common clinical phenotypes and present bioinformatic analyses of expression patterns and downstream targets of these genes as they relate to other neurodevelopmental risk genes. RESULTS: These individuals share neurobehavioral features including ASD, intellectual disability, and/or ADHD; other frequent features include hypersensitivity to sensory stimuli and sleep problems. RFX3, RFX4, and RFX7 are strongly expressed in developing and adult human brain, and X-box binding motifs as well as RFX ChIP-seq peaks are enriched in the cis-regulatory regions of known ASD risk genes. CONCLUSION: These results establish a likely role of deleterious variation in RFX3, RFX4, and RFX7 in cases of monogenic intellectual disability, ADHD and ASD, and position these genes as potentially critical transcriptional regulators of neurobiological pathways associated with neurodevelopmental disease pathogenesis.

Ciliopathies: Coloring outside of the lines.

Ciliopathy syndromes are a diverse spectrum of disease characterized by a combination of cystic kidney disease, hepatobiliary disease, retinopathy, skeletal dysplasia, developmental delay, and brain malformations. Though generally divided into distinct disease categories based on the pattern of system involvement, ciliopathy syndromes are known to display certain phenotypic overlap. We performed next-generation sequencing panel testing, clinical exome sequencing, and research-based exome sequencing reanalysis on patients with suspected ciliopathy syndromes with additional features. We identified biallelic pathogenic variants in BBS1 in a child with features of cranioectodermal dysplasia, and biallelic variants in BBS12 in a child with the clinical stigmata of Bardet-Biedl syndrome, but also with anal atresia. We additionally identified biallelic pathogenic variants in WDR35 and DYNC2H1 in children with predominant liver disease and ductal plate malformation without skeletal dysplasia. Our study highlights the phenotypic and genetic diversity of ciliopathy syndromes, the importance of considering ciliopathy syndromes as a disease-spectrum and screening for all associated complications in all patients, and describes exclusive extra-skeletal manifestations in two classical skeletal dysplasia syndromes.

Expanding the phenotypic spectrum of Mendelian connective tissue disorders to include prominent kidney phenotypes.

Heritable connective tissue disorders are a group of diseases, each rare, characterized by various combinations of skin, joint, musculoskeletal, organ, and vascular involvement. Although kidney abnormalities have been reported in some connective tissue disorders, they are rarely a presenting feature. Here we present three patients with prominent kidney phenotypes who were found by whole exome sequencing to have variants in established connective tissue genes associated with Loeys-Dietz syndrome and congenital contractural arachnodactyly. These cases highlight the importance of considering connective tissue disease in children presenting with structural kidney disease and also serves to expand the phenotype of Loeys-Dietz syndrome and possibly congenital contractural arachnodactyly to include cystic kidney disease and cystic kidney dysplasia, respectively.

Exon skip-inducing variants in FLNA in an attenuated form of frontometaphyseal dysplasia.

Pathogenic variation in the X-linked gene FLNA causes a wide range of human developmental phenotypes. Loss-of-function is usually male embryonic-lethal, and most commonly results in a neuronal migration disorder in affected females. Gain-of-function variants cause a spectrum of skeletal dysplasias that present with variable additional, often distinctive, soft-tissue anomalies in males and females. Here we present two, unrelated, male individuals with novel, intronic variants in FLNA that are predicted to be pathogenic. Their phenotypes are reminiscent of the gain-of-function spectrum without the skeletal manifestations. Most strikingly, they manifest urethral anomalies, cardiac malformations, and keloid scarring, all commonly encountered features of frontometaphyseal dysplasia. Both variants prevent inclusion of exon 40 into the FLNA transcript, predicting the in-frame deletion of 42 amino acids, however the abundance of FLNA protein was equivalent to that observed in healthy individuals. Loss of these 42 amino acids removes sites that mediate key FLNA functions, including binding of some ligands and phosphorylation. This phenotype further expands the spectrum of the FLNA filaminopathies.

Nonlethal presentations of CYP26B1-related skeletal anomalies and multiple synostoses syndrome.

Retinoic acid exposures as well as defects in the retinoic acid-degrading enzyme CYP26B1 have teratogenic effects on both limb and craniofacial skeleton. An initial report of four individuals described a syndrome of fetal and infantile lethality with craniosynostosis and skeletal anomalies caused by homozygous pathogenic missense variants in CYP26B1. In contrast, a 22-year-old female was reported with a homozygous missense pathogenic variant in CYP26B1 with complex multisuture craniosynostosis and intellectual disability, suggesting that in some cases, biallelic pathogenic variants of CYP26B1 may be compatible with life. Here we describe four additional living individuals from two families with compound heterozygous pathogenic missense variants in CYP26B1. Structural assessment of these additional missense variants places them further from the catalytic site and supports a model consistent with milder nonlethal disease. In addition to previously reported findings of multisuture craniosynostosis, conductive hearing loss, joint contractures, long slender fingers, camptodactly, broad fingertips, and developmental delay/intellectual disability, skeletal imaging in our cases also revealed gracile long bones, gracile ribs, radioulnar synostosis, and carpal and/or tarsal fusions. These individuals broaden the phenotypic range of biallelic pathogenic variants in CYPB26B1 and most significantly clarify that mortality can range from perinatal lethality to survival into adulthood.

A Recurrent De Novo Variant in NACC1 Causes a Syndrome Characterized by Infantile Epilepsy, Cataracts, and Profound Developmental Delay.

Whole-exome sequencing (WES) has increasingly enabled new pathogenic gene variant identification for undiagnosed neurodevelopmental disorders and provided insights into both gene function and disease biology. Here, we describe seven children with a neurodevelopmental disorder characterized by microcephaly, profound developmental delays and/or intellectual disability, cataracts, severe epilepsy including infantile spasms, irritability, failure to thrive, and stereotypic hand movements. Brain imaging in these individuals reveals delay in myelination and cerebral atrophy. We observe an identical recurrent de novo heterozygous c.892C>T (p.Arg298Trp) variant in the nucleus accumbens associated 1 (NACC1) gene in seven affected individuals. One of the seven individuals is mosaic for this variant. NACC1 encodes a transcriptional repressor implicated in gene expression and has not previously been associated with germline disorders. The probability of finding the same missense NACC1 variant by chance in 7 out of 17,228 individuals who underwent WES for diagnoses of neurodevelopmental phenotypes is extremely small and achieves genome-wide significance (p = 1.25 × 10-14). Selective constraint against missense variants in NACC1 makes this excess of an identical missense variant in all seven individuals more remarkable. Our findings are consistent with a germline recurrent mutational hotspot associated with an allele-specific neurodevelopmental phenotype in NACC1.