Loss-of-function of KMT5B leads to neurodevelopmental disorder and impairs neuronal development and neurogenesis.

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders that cause severe social, communication, and behavioral problems. Recent studies show that the variants of a histone methyltransferase gene KMT5B cause neurodevelopmental disorders (NDDs), including ASD, and the knockout of Kmt5b in mice is embryonic lethal. However, the detailed genotype-phenotype correlations and functional effects of KMT5B in neurodevelopment are unclear. By targeted sequencing of a large Chinese ASD cohort, analyzing published genome-wide sequencing data, and mining literature, we curated 39 KMT5B variants identified from NDD individuals. A genotype-phenotype correlation analysis for 10 individuals with KMT5B pathogenic variants reveals common symptoms, including ASD, intellectual disability, languages problem, and macrocephaly. In vitro knockdown of the expression of Kmt5b in cultured mouse primary cortical neurons leads to a decrease in neuronal dendritic complexity and an increase in dendritic spine density, which can be rescued by expression of human KMT5B but not that of pathogenic de novo missense mutants. In vivo knockdown of the Kmt5b expression in the mouse embryonic cerebral cortex by in utero electroporation results in decreased proliferation and accelerated migration of neural progenitor cells. Our findings reveal essential roles of histone methyltransferase KMT5B in neuronal development, prenatal neurogenesis, and neuronal migration.

SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis.

High myopia is one of the leading causes of visual impairment worldwide with high heritability. We have previously identified the genetic contribution of SLC39A5 to nonsyndromic high myopia and demonstrated that disease-related mutations of SLC39A5 dysregulate the TGF-ß pathway. In this study, the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia are determined. We observed the morphogenesis and migration abnormalities of the SLC39A5 knockout (KO) human embryonic kidney cells (HEK293) and found a significant injury of ECM constituents. RNA-seq and qRT-PCR revealed the transcription decrease in COL1A1, COL2A1, COL4A1, FN1 and LAMA1 in the KO cells. Further, we demonstrated that TGF-ß signalling, the regulator of ECM, was inhibited in SLC39A5 depletion situation, wherein the activation of receptor Smads (R-Smads) via phosphorylation was greatly blocked. SLC39A5 re-expression reversed the phenotype of TGF-ß signalling and ECM synthesis in the KO cells. The fact that TGF-ß signalling was zinc-regulated and that SLC39A5 was identified as a zinc transporter urged us to check the involvement of intracellular zinc in TGF-ß signalling impairment. Finally, we determined that insufficient zinc chelation destabilized Smad proteins, which naturally inhibited TGF-ß signalling. Overall, the SLC39A5 depletion-induced zinc deficiency destabilized Smad proteins, which inhibited the TGF-ß signalling and downstream ECM synthesis, thus contributing to the pathogenesis of high myopia. This discovery provides a deep insight into myopic development.

Mutation pattern and genotype-phenotype correlations of SETD2 in neurodevelopmental disorders.

SETD2 encodes an important protein for epigenetic modification of histones which plays an essential role in early development. Variants in SETD2 have been reported in neurodevelopmental disorders including autism spectrum disorder (ASD). However, most de novo SETD2 variants were reported in different large-cohort sequencing studies, mutation pattern and comprehensive genotype-phenotype correlations for SETD2 are still lacking. We have applied target sequencing to identify rare, clinical-relevant SETD2 variants and detected two novel de novo SETD2 variants, including a de novo splicing variant (NM_014159: c.4715+1G>A) and a de novo missense variant (c.3185C>T: p.P1062L) in two individuals with a diagnosis of ASD. To analyze the correlations between SETD2 mutations and corresponding phenotypes, we systematically review the reported individuals with de novo SETD2 variants, classify the pathogenicity, and analyze the detailed phenotypes. We subsequently manually curate 17 SETD2 de novo variants in 17 individuals from published literature. Individuals with de novo SETD2 variants present common phenotypes including speech and motor delay, intellectual disability, macrocephaly, ASD, overgrowth and recurrent otitis media. Our study reveals new SETD2 mutations and provided a relatively homozygous phenotype spectrum of SETD2-related neurodevelopmental disorders which will be beneficial for disease classification and diagnosis in clinical practice.

Biallelic loss-of-function variants in NEMF cause central nervous system impairment and axonal polyneuropathy.

We aimed to detect the causative gene in five unrelated families with recessive inheritance pattern neurological disorders involving the central nervous system, and the potential function of the NEMF gene in the central nervous system. Exome sequencing (ES) was applied to all families and linkage analysis was performed on family 1. A minigene assay was used to validate the splicing effect of the relevant discovered variants. Immunofluorescence (IF) experiment was performed to investigate the role of the causative gene in neuron development. The large consanguineous family confirms the phenotype-causative relationship with homozygous frameshift variant (NM_004713.6:c.2618del) as revealed by ES. Linkage analysis of the family showed a significant single-point LOD of 4.5 locus. Through collaboration in GeneMatcher, four additional unrelated families' likely pathogenic NEMF variants for a spectrum of central neurological disorders, two homozygous splice-site variants (NM_004713.6:c.574+1G>T and NM_004713.6:c.807-2A>C) and a homozygous frameshift variant (NM_004713.6: c.1234_1235insC) were subsequently identified and segregated with all affected individuals. We further revealed that knockdown (KD) of Nemf leads to impairment of axonal outgrowth and synapse development in cultured mouse primary cortical neurons. Our study demonstrates that disease-causing biallelic NEMF variants result in central nervous system impairment and other variable features. NEMF is an important player in mammalian neuron development.

NCKAP1 Disruptive Variants Lead to a Neurodevelopmental Disorder with Core Features of Autism.

NCKAP1/NAP1 regulates neuronal cytoskeletal dynamics and is essential for neuronal differentiation in the developing brain. Deleterious variants in NCKAP1 have been identified in individuals with autism spectrum disorder (ASD) and intellectual disability; however, its clinical significance remains unclear. To determine its significance, we assemble genotype and phenotype data for 21 affected individuals from 20 unrelated families with predicted deleterious variants in NCKAP1. This includes 16 individuals with de novo (n = 8), transmitted (n = 6), or inheritance unknown (n = 2) truncating variants, two individuals with structural variants, and three with potentially disruptive de novo missense variants. We report a de novo and ultra-rare deleterious variant burden of NCKAP1 in individuals with neurodevelopmental disorders which needs further replication. ASD or autistic features, language and motor delay, and variable expression of intellectual or learning disability are common clinical features. Among inherited cases, there is evidence of deleterious variants segregating with neuropsychiatric disorders. Based on available human brain transcriptomic data, we show that NCKAP1 is broadly and highly expressed in both prenatal and postnatal periods and demostrate enriched expression in excitatory neurons and radial glias but depleted expression in inhibitory neurons. Mouse in utero electroporation experiments reveal that Nckap1 loss of function promotes neuronal migration during early cortical development. Combined, these data support a role for disruptive NCKAP1 variants in neurodevelopmental delay/autism, possibly by interfering with neuronal migration early in cortical development.

Author Correction: Large-scale targeted sequencing identifies risk genes for neurodevelopmental disorders.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Large-scale targeted sequencing identifies risk genes for neurodevelopmental disorders.

Most genes associated with neurodevelopmental disorders (NDDs) were identified with an excess of de novo mutations (DNMs) but the significance in case-control mutation burden analysis is unestablished. Here, we sequence 63 genes in 16,294 NDD cases and an additional 62 genes in 6,211 NDD cases. By combining these with published data, we assess a total of 125 genes in over 16,000 NDD cases and compare the mutation burden to nonpsychiatric controls from ExAC. We identify 48 genes (25 newly reported) showing significant burden of ultra-rare (MAF < 0.01%) gene-disruptive mutations (FDR 5%), six of which reach family-wise error rate (FWER) significance (p < 1.25E-06). Among these 125 targeted genes, we also reevaluate DNM excess in 17,426 NDD trios with 6,499 new autism trios. We identify 90 genes enriched for DNMs (FDR 5%; e.g., GABRG2 and UIMC1); of which, 61 reach FWER significance (p < 3.64E-07; e.g., CASZ1). In addition to doubling the number of patients for many NDD risk genes, we present phenotype-genotype correlations for seven risk genes (CTCF, HNRNPU, KCNQ3, ZBTB18, TCF12, SPEN, and LEO1) based on this large-scale targeted sequencing effort.

Excess of RALGAPB de novo variants in neurodevelopmental disorders.

Autism spectrum disorder is a neurodevelopmental disorder (NDD) with complex genetic architecture marked primarily by social and communication impairments along with deficits in restrictive and repetitive behaviors. Due to the complex nature and genetic heterogeneity of the disease, genotype and phenotype correlation remains challenging. Prior studies have implicated RALGAPB as a candidate gene for ASD, but stringent analysis is required to determine the pathogenicity. By targeted sequencing, we identified a new de novo RALGAPB missense variant (c.1238C> T; p.T413M) in an ASD family. By leveraging published large-scale genome sequencing studies, we curated five de novo likely gene-disruptive (LGD) variants and 5 de novo missense variants in ASD and related NDDs and revealed a genome-wide significant excess of RALGAPB de novo LGD variants (P_adjust = 0.0053). Quantitative reverse transcription PCR revealed that the frameshift variant c.1927dupA; p.N643fs*3 reduced mRNA expression levels confirming the loss-of-function effect. Co-expression analysis using human brain transcriptome data provide the potential functional link of RALGAPB and 38 ASD and/or NDD genes. Our study suggests RALGAPB as a new NDD risk gene which should be considered in clinical diagnosis of ASD and related NDDs.

Genotype and Phenotype Correlations for TBL1XR1 in Neurodevelopmental Disorders.

TBL1XR1 is a member of the WD40 repeat-containing gene family. Mutations of TBL1XR1 have been reported in neurodevelopmental disorders (NDDs). Although the phenotypes of some patients have been described in single studies, few studies have reviewed the genotype and phenotype relationships using a relatively large cohort of patients with TBL1XR1 mutations. Herein, we report a new de novo frameshift mutation in TBL1XR1 (NM_024665.4, c.388_389delAC, p.T130Sfs*14) in a patient with autism spectrum disorder (ASD). To explore the correlations between genotypes and phenotypes for TBL1XR1 in NDDs, we manually curated and analyzed 38 variants and the associated phenotypes from 50 individuals with NDDs. TBL1XR1 mutations lead to a wide range of phenotypic defects. We conclude that the most common phenotypes associated with TBL1XR1 mutations were language and motor developmental delay, intellectual disabilities, facial deformity, hypotonia, and microcephaly. Our study provides a comprehensive spectrum of neurodevelopmental phenotypes caused by TBL1XR1 mutations, which is important for genetic diagnosis and precision clinical management.

Phenotype-to-genotype approach reveals head-circumference-associated genes in an autism spectrum disorder cohort.

The genotype-first approach has been successfully applied and has elucidated several subtypes of autism spectrum disorder (ASD). However, it requires very large cohorts because of the extensive genetic heterogeneity. We investigate the alternate possibility of whether phenotype-specific genes can be identified from a small group of patients with specific phenotype(s). To identify novel genes associated with ASD and abnormal head circumference using a phenotype-to-genotype approach, we performed whole-exome sequencing on 67 families with ASD and abnormal head circumference. Clinically relevant pathogenic or likely pathogenic variants account for 23.9% of patients with microcephaly or macrocephaly, and 81.25% of those variants or genes are head-size associated. Significantly, recurrent pathogenic mutations were identified in two macrocephaly genes (PTEN, CHD8) in this small cohort. De novo mutations in several candidate genes (UBN2, BIRC6, SYNE1, and KCNMA1) were detected, as well as one new candidate gene (TNPO3) implicated in ASD and related neurodevelopmental disorders. We identify genotype-phenotype correlations for head-size-associated ASD genes and novel candidate genes for further investigation. Our results also suggest a phenotype-to-genotype strategy would accelerate the elucidation of genotype-phenotype relationships for ASD by using phenotype-restricted cohorts.

Disruptive variants of CSDE1 associate with autism and interfere with neuronal development and synaptic transmission.

RNA binding proteins are key players in posttranscriptional regulation and have been implicated in neurodevelopmental and neuropsychiatric disorders. Here, we report a significant burden of heterozygous, likely gene-disrupting variants in CSDE1 (encoding a highly constrained RNA binding protein) among patients with autism and related neurodevelopmental disabilities. Analysis of 17 patients identifies common phenotypes including autism, intellectual disability, language and motor delay, seizures, macrocephaly, and variable ocular abnormalities. HITS-CLIP revealed that Csde1-binding targets are enriched in autism-associated gene sets, especially FMRP targets, and in neuronal development and synaptic plasticity-related pathways. Csde1 knockdown in primary mouse cortical neurons leads to an overgrowth of the neurites and abnormal dendritic spine morphology/synapse formation and impaired synaptic transmission, whereas mutant and knockdown experiments in Drosophila result in defects in synapse growth and synaptic transmission. Our study defines a new autism-related syndrome and highlights the functional role of CSDE1 in synapse development and synaptic transmission.

Disruptive mutations in TANC2 define a neurodevelopmental syndrome associated with psychiatric disorders.

Postsynaptic density (PSD) proteins have been implicated in the pathophysiology of neurodevelopmental and psychiatric disorders. Here, we present detailed clinical and genetic data for 20 patients with likely gene-disrupting mutations in TANC2-whose protein product interacts with multiple PSD proteins. Pediatric patients with disruptive mutations present with autism, intellectual disability, and delayed language and motor development. In addition to a variable degree of epilepsy and facial dysmorphism, we observe a pattern of more complex psychiatric dysfunction or behavioral problems in adult probands or carrier parents. Although this observation requires replication to establish statistical significance, it also suggests that mutations in this gene are associated with a variety of neuropsychiatric disorders consistent with its postsynaptic function. We find that TANC2 is expressed broadly in the human developing brain, especially in excitatory neurons and glial cells, but shows a more restricted pattern in Drosophila glial cells where its disruption affects behavioral outcomes.

AMPA receptor GluA2 subunit defects are a cause of neurodevelopmental disorders.

AMPA receptors (AMPARs) are tetrameric ligand-gated channels made up of combinations of GluA1-4 subunits encoded by GRIA1-4 genes. GluA2 has an especially important role because, following post-transcriptional editing at the Q607 site, it renders heteromultimeric AMPARs Ca2+-impermeable, with a linear relationship between current and trans-membrane voltage. Here, we report heterozygous de novo GRIA2 mutations in 28 unrelated patients with intellectual disability (ID) and neurodevelopmental abnormalities including autism spectrum disorder (ASD), Rett syndrome-like features, and seizures or developmental epileptic encephalopathy (DEE). In functional expression studies, mutations lead to a decrease in agonist-evoked current mediated by mutant subunits compared to wild-type channels. When GluA2 subunits are co-expressed with GluA1, most GRIA2 mutations cause a decreased current amplitude and some also affect voltage rectification. Our results show that de-novo variants in GRIA2 can cause neurodevelopmental disorders, complementing evidence that other genetic causes of ID, ASD and DEE also disrupt glutamatergic synaptic transmission.

POGZ de novo missense variants in neuropsychiatric disorders.

BACKGROUND: De novo likely gene-disrupting variants of POGZ cause autism spectrum disorder (ASD) and intellectual disability. However, de novo missense variants of this gene were not well explored in neuropsychiatric disorders. METHODS: The single-molecule molecular inversion probes-based targeted sequencing method was performed on the proband. Variant was validated using Sanger sequencing in both proband and parents. Immunoblot analysis was performed to examine the expression of POGZ in patient-derived peripheral blood lymphocytes. Published POGZ de novo missense variants in neuropsychiatric disorders were reviewed. RESULTS: We detected a novel de novo missense variant in POGZ (c.1534C>A, p.H512N, NM_015100.4) in an individual with ASD. Immunoblot analysis revealed a dramatic reduction in POGZ protein in patient-derived peripheral blood lymphocytes suggesting a loss-of-function mechanism of this de novo missense variant. In addition, we collected and annotated additional eight POGZ de novo missense variants identified in neuropsychiatric disorders from literatures. CONCLUSION: Our findings will be beneficial to the functional analysis of POGZ in ASD pathogenesis, and for genetic counseling and clinical diagnosis of patients with POGZ de novo missense variants.

Rare inherited missense variants of POGZ associate with autism risk and disrupt neuronal development.

Excess de novo likely gene-disruptive and missense variants within dozens of genes have been identified in autism spectrum disorder (ASD) and other neurodevelopmental disorders. However, many rare inherited missense variants of these high-risk genes have not been thoroughly evaluated. In this study, we analyzed the rare missense variant burden of POGZ in a large cohort of ASD patients from the Autism Clinical and Genetic Resources in China (ACGC) and further dissected the functional effect of disease-associated missense variants on neuronal development. Our results showed a significant burden of rare missense variants in ASD patients compared to the control population (P = 4.6 × 10-5, OR = 3.96), and missense variants in ASD patients showed more severe predicted functional outcomes than those in controls. Furthermore, by leveraging published large-scale sequencing data of neurodevelopmental disorders (NDDs) and sporadic case reports, we identified 8 de novo missense variants of POGZ in NDD patients. Functional analysis revealed that two inherited, but not de novo, missense variants influenced the cellular localization of POGZ and failed to rescue the defects in neurite and dendritic spine development caused by Pogz knockdown in cultured mouse primary cortical neurons. Significantly, L1CAM, an autism candidate risk gene, is differentially expressed in POGZ deficient cell lines. Reduced expression of L1cam was able to partially rescue the neurite length defects caused by Pogz knockdown. Our study showed the important roles of rare inherited missense variants of POGZ in ASD risk and neuronal development and identified the potential downstream targets of POGZ, which are important for further molecular mechanism studies.

Pathogenic missense mutation pattern of forkhead box genes in neurodevelopmental disorders.

BACKGROUND: Forkhead box (FOX) proteins are a family of transcription factors. Mutations of three FOX genes, including FOXP1, FOXP2, and FOXG1, have been reported in neurodevelopmental disorders (NDDs). However, due to the lack of site-specific statistical significance, the pathogenicity of missense mutations of these genes is difficult to determine. METHODS: DNA and RNA were extracted from peripheral blood lymphocytes. The mutation was detected by single-molecule molecular inversion probe-based targeted sequencing, and the variant was validated by Sanger sequencing. Real-time quantitative PCR and western blot were performed to assay the expression of the mRNA and protein. To assess the pattern of disorder-related missense mutations of NDD-related FOX genes, we manually curated de novo and inherited missense or inframeshift variants within FOXP1, FOXP2, and FOXG1 that co-segregated with phenotypes in NDDs. All variants were annotated by ANNOVAR. RESULTS: We detected a novel de novo missense mutation (NM_001244815: c.G1444A, p.E482K) of FOXP1 in a patient with intellectual disability and severe speech delay. Real-time PCR and western blot revealed a dramatic reduction of mRNA and protein expression in patient-derived lymphocytes, indicating a loss-of-function mechanism. We observed that the majority of the de novo or transmitted missense variants were located in the FOX domains, and 95% were classified as pathogenic mutations. However, 10 variants were located outside of the FOX domain and were classified as likely pathogenic or variants of uncertain significance. CONCLUSION: Our study shows the pathogenicity of missense and inframeshift variants of NDD-related FOX genes, which is important for clinical diagnosis and genetic counseling. Functional analysis is needed to determine the pathogenicity of the variants with uncertain clinical significance.

Inherited and multiple de novo mutations in autism/developmental delay risk genes suggest a multifactorial model.

Background: We previously performed targeted sequencing of autism risk genes in probands from the Autism Clinical and Genetic Resources in China (ACGC) (phase I). Here, we expand this analysis to a larger cohort of patients (ACGC phase II) to better understand the prevalence, inheritance, and genotype-phenotype correlations of likely gene-disrupting (LGD) mutations for autism candidate genes originally identified in cohorts of European descent. Methods: We sequenced 187 autism candidate genes in an additional 784 probands and 85 genes in 599 probands using single-molecule molecular inversion probes. We tested the inheritance of potentially pathogenic mutations, performed a meta-analysis of phase I and phase II data and combined our results with existing exome sequence data to investigate the phenotypes of carrier parents and patients with multiple hits in different autism risk genes. Results: We validated recurrent, LGD, de novo mutations (DNMs) in 13 genes. We identified a potential novel risk gene (ZNF292), one novel gene with recurrent LGD DNMs (RALGAPB), as well as genes associated with macrocephaly (GIGYF2 and WDFY3). We identified the transmission of private LGD mutations in genes predominantly associated with DNMs and showed that parental carriers tended to share milder autism-related phenotypes. Patients that carried DNMs in two or more candidate genes show more severe phenotypes. Conclusions: We identify new risk genes and transmission of deleterious mutations in genes primarily associated with DNMs. The fact that parental carriers show milder phenotypes and patients with multiple hits are more severe supports a multifactorial model of risk.

Genotype and phenotype correlations for SHANK3 de novo mutations in neurodevelopmental disorders.

SHANK3 has been identified as the causative gene of 22q13.3 microdeletion syndrome phenotype. De novo mutations (DNMs) of SHANK3 were subsequently identified in patients with several neurodevelopmental disorders, including autism spectrum disorders (ASDs), schizophrenia (SCZ), a Rett syndrome-like phenotype, and intellectual disability (ID). Although broad developmental phenotypes of these patients have been described in single studies, few studies have reviewed the genotype and phenotype relationships using a relatively large cohort of patients with SHANK3 DNMs. In this study, we identified a de novo splice mutation (NM_033517.1: c.2265+1G>A) that functionally impairs mRNA splicing, produces multiple splice variants, and results in the reduction of the amounts of mRNA. To analyze the genotype and phenotype correlations for SHANK3 DNMs, we reviewed 37 previously published patients with 28 SHANK3 DNMs. Our results revealed that haploinsufficiency of SHANK3 causes a broad spectrum of neurodevelopmental phenotypes with impaired social interaction, repetitive behavior, speech impairment, ID, and regression as the most common observations. Seizures, hypotonia, global development delay, dysmorphic features, and several other features also occurred recurrently. Specific phenotypes are also observed in certain genotypes. Our study provides the frequency of the heterogeneous co-occurring conditions caused by SHANK3 DNMs, which will be beneficial for diagnosis and clinical management.

De novo genic mutations among a Chinese autism spectrum disorder cohort.

Recurrent de novo (DN) and likely gene-disruptive (LGD) mutations contribute significantly to autism spectrum disorders (ASDs) but have been primarily investigated in European cohorts. Here, we sequence 189 risk genes in 1,543 Chinese ASD probands (1,045 from trios). We report an 11-fold increase in the odds of DN LGD mutations compared with expectation under an exome-wide neutral model of mutation. In aggregate, ∼4% of ASD patients carry a DN mutation in one of just 29 autism risk genes. The most prevalent gene for recurrent DN mutations is SCN2A (1.1% of patients) followed by CHD8, DSCAM, MECP2, POGZ, WDFY3 and ASH1L. We identify novel DN LGD recurrences (GIGYF2, MYT1L, CUL3, DOCK8 and ZNF292) and DN mutations in previous ASD candidates (ARHGAP32, NCOR1, PHIP, STXBP1, CDKL5 and SHANK1). Phenotypic follow-up confirms potential subtypes and highlights how large global cohorts might be leveraged to prove the pathogenic significance of individually rare mutations.