Kun Xia

Targeted sequencing identifies 91 neurodevelopmental-disorder risk genes with autism and developmental-disability biases

Gene-disruptive mutations contribute to the biology of neurodevelopmental disorders (NDDs), but most of the related pathogenic genes are not known. We sequenced 208 candidate genes from >11,730 cases and >2,867 controls. We identified 91 genes, including 38 new NDD genes, with an excess of de novo mutations or private disruptive mutations in 5.7% of cases. Drosophila functional assays revealed a subset with increased involvement in NDDs. We identified 25 genes showing a bias for autism versus intellectual disability and highlighted a network associated with high-functioning autism (full-scale IQ >100). Clinical follow-up for NAA15, KMT5B, and ASH1L highlighted new syndromic and nonsyndromic forms of disease.

Genes with de novo mutations are shared by four neuropsychiatric disorders discovered from NPdenovo database

Currently, many studies on neuropsychiatric disorders have utilized massive trio-based whole-exome sequencing (WES) and whole-genome sequencing (WGS) to identify numerous de novo mutations (DNMs). Here, we retrieved 17 104 DNMs from 3555 trios across four neuropsychiatric disorders: autism spectrum disorder, epileptic encephalopathy, intellectual disability and schizophrenia, in addition to unaffected siblings (control), from 36 studies by WES/WGS. After eliminating non-exonic variants, we focused on 3334 exonic DNMs for evaluation of their association with these diseases. Our results revealed a higher prevalence of DNMs in the probands of all four disorders compared with the one in the controls (P<1.3 × 10−7). The elevated DNM frequency is dominated by loss-of-function/deleterious single-nucleotide variants and frameshift indels (that is, extreme mutations, P<4.5 × 10−5). With extensive annotation of these ‘extreme’ mutations, we prioritized 764 candidate genes in these four disorders. A combined analysis of Gene Ontology, microRNA targets and transcription factor targets revealed shared biological process and non-coding regulatory elements of candidate genes in the pathology of neuropsychiatric disorders. In addition, weighted gene co-expression network analysis of human laminar-specific neocortical expression data showed that candidate genes are convergent on eight shared modules with specific layer enrichment and biological process features. Furthermore, we identified that 53 candidate genes are associated with more than one disorder (P<0.000001), suggesting a possibly shared genetic etiology underlying these disorders. Particularly, DNMs of the SCN2A gene are frequently occurred across all four disorders. Finally, we constructed a freely available NPdenovo database, which provides a comprehensive catalog of the DNMs identified in neuropsychiatric disorders.

Exome sequencing identifies MVK mutations in disseminated superficial actinic porokeratosis

Disseminated superficial actinic porokeratosis (DSAP) is an autosomal dominantly inherited epidermal keratinization disorder whose etiology remains unclear. We performed exome sequencing in one unaffected and two affected individuals from a DSAP family. The mevalonate kinase gene (MVK) emerged as the only candidate gene located in previously defined linkage regions after filtering against existing SNP databases, eight HapMap exomes and 1000 Genomes Project data and taking into consideration the functional implications of the mutations. Sanger sequencing in 57 individuals with familial DSAP and 25 individuals with sporadic DSAP identified MVK mutations in 33% and 16% of these individuals (cases), respectively. All 14 MVK mutations identified in our study were absent in 676 individuals without DSAP. Our functional studies in cultured primary keratinocytes suggest that MVK has a role in regulating calcium-induced keratinocyte differentiation and could protect keratinocytes from apoptosis induced by type A ultraviolet radiation. Our results should help advance the understanding of DSAP pathogenesis.

SNP Cutter: a comprehensive tool for SNP PCR–RFLP assay design

The Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) is a relatively simple and inexpensive method for genotyping single nucleotide polymorphisms (SNPs). It requires minimal investment in instrumentation. Here, we describe a web application, ‘SNP Cutter,’ which designs PCR–RFLP assays on a batch of SNPs from the human genome. NCBI dbSNP rs IDs or formatted SNPs are submitted into the SNP Cutter which then uses restriction enzymes from a pre-selected list to perform enzyme selection. The program is capable of designing primers for either natural PCR–RFLP or mismatch PCR–RFLP, depending on the SNP sequence data. SNP Cutter generates the information needed to evaluate and perform genotyping experiments, including a PCR primers list, sizes of original amplicons and different allelic fragment after enzyme digestion. Some output data is tab-delimited, therefore suitable for database archiving. The SNP Cut-ter is available at .

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.

Significant linkage and association between a functional (GT)n polymorphism in promoter of the N-methyl-D-aspartate receptor subunit gene (GRIN2A) and schizophrenia.

Dysfunction of the N-methyl-d-aspartate (NMDA) type glutamate receptor has been proposed as a mechanism in the etiology of schizophrenia, based on the observation that non-competitive antagonists of the NMDA receptor, such as phencyclidine, induce schizophrenia-like symptoms. Previous study identified a variable (GT)n polymorphism in the promoter region of the N-methyl-d-aspartate (NMDA) subunit gene (GRIN2A), and showed its association with schizophrenia in a case-control study, together with a correlation between the length of the repeat and severity of chronic outcome. Our present study was aimed at confirming the association of the (GT)n polymorphism of GRIN2A promoter with schizophrenia using 122 Han Chinese sib-pair families. Non-parametric linkage analysis and transmission/disequilibrium test (TDT) were undertaken using the GENEHUNTER, v2.1. In non-parametric linkage analysis, suggestive linkage was found for the (GT)n polymorphism (NPL=2.77, P=0.002902). The TDT was significant for (GT)n polymorphism and that the (GT)23 was preferentially transmitted to schizophrenia-affected children (T/NT: 123:72, chi(2)=13.34, P=0.000260). Our results indicate that the (GT)n polymorphism in the promoter of GRIN2A gene may play a significant role in the etiology of schizophrenia among our samples.

Disruption of POGZ Is Associated with Intellectual Disability and Autism Spectrum Disorders

Intellectual disability (ID) and autism spectrum disorders (ASD) are genetically heterogeneous, and a significant number of genes have been associated with both conditions. A few mutations in POGZ have been reported in recent exome studies; however, these studies do not provide detailed clinical information. We collected the clinical and molecular data of 25 individuals with disruptive mutations in POGZ by diagnostic whole-exome, whole-genome, or targeted sequencing of 5,223 individuals with neurodevelopmental disorders (ID primarily) or by targeted resequencing of this locus in 12,041 individuals with ASD and/or ID. The rarity of disruptive mutations among unaffected individuals (2/49,401) highlights the significance (p = 4.19 × 10(-13); odds ratio = 35.8) and penetrance (65.9%) of this genetic subtype with respect to ASD and ID. By studying the entire cohort, we defined common phenotypic features of POGZ individuals, including variable levels of developmental delay (DD) and more severe speech and language delay in comparison to the severity of motor delay and coordination issues. We also identified significant associations with vision problems, microcephaly, hyperactivity, a tendency to obesity, and feeding difficulties. Some features might be explained by the high expression of POGZ, particularly in the cerebellum and pituitary, early in fetal brain development. We conducted parallel studies in Drosophila by inducing conditional knockdown of the POGZ ortholog row, further confirming that dosage of POGZ, specifically in neurons, is essential for normal learning in a habituation paradigm. Combined, the data underscore the pathogenicity of loss-of-function mutations in POGZ and define a POGZ-related phenotype enriched in specific features.

Hotspots of missense mutation identify neurodevelopmental disorder genes and functional domains

Although de novo missense mutations have been predicted to account for more cases of autism than gene-truncating mutations, most research has focused on the latter. We identified the properties of de novo missense mutations in patients with neurodevelopmental disorders (NDDs) and highlight 35 genes with excess missense mutations. Additionally, 40 amino acid sites were recurrently mutated in 36 genes, and targeted sequencing of 20 sites in 17,688 patients with NDD identified 21 new patients with identical missense mutations. One recurrent site substitution (p.A636T) occurs in a glutamate receptor subunit, GRIA1. This same amino acid substitution in the homologous but distinct mouse glutamate receptor subunit Grid2 is associated with Lurcher ataxia. Phenotypic follow-up in five individuals with GRIA1 mutations shows evidence of specific learning disabilities and autism. Overall, we find significant clustering of de novo mutations in 200 genes, highlighting specific functional domains and synaptic candidate genes important in NDD pathology.

mirTrios: an integrated pipeline for detection of de novo and rare inherited mutations from trios-based next-generation sequencing

Objectives Recently, several studies documented that de novo mutations (DNMs) play important roles in the aetiology of sporadic diseases. Next-generation sequencing (NGS) enables variant calling at single-base resolution on a genome-wide scale. However, accurate identification of DNMs from NGS data still remains a major challenge. We developed mirTrios, a web server, to accurately detect DNMs and rare inherited mutations from NGS data in sporadic diseases. Methods The expectation-maximisation (EM) model was adopted to accurately identify DNMs from variant call files of a trio generated by GATK (Genome Analysis Toolkit). The GATK results, which contain certain basic properties (such as PL, PRT and PART), are iteratively integrated into the EM model to strike a threshold for DNMs detection. Training sets of true and false positive DNMs in the EM model were built from whole genome sequencing data of 64 trios. Results With our in-house whole exome sequencing datasets from 20 trios, mirTrios totally identified 27 DNMs in the coding region, 25 of which (92.6%) are validated as true positives. In addition, to facilitate the interpretation of diverse mutations, mirTrios can also be employed in the identification of rare inherited mutations. Embedded with abundant annotation of DNMs and rare inherited mutations, mirTrios also supports known diagnostic variants and causative gene identification, as well as the prioritisation of novel and promising candidate genes. Conclusions mirTrios provides an intuitive interface for the general geneticist and clinician, and can be widely used for detection of DNMs and rare inherited mutations, and annotation in sporadic diseases. mirTrios is freely available at http://centre.bioinformatics.zj.cn/mirTrios/.

Targeted sequencing and functional analysis reveal brain-size-related genes and their networks in autism spectrum disorders

Autism spectrum disorder (ASD) represents a set of complex neurodevelopmental disorders with large degrees of heritability and heterogeneity. We sequenced 136 microcephaly or macrocephaly (Mic–Mac)-related genes and 158 possible ASD-risk genes in 536 Chinese ASD probands and detected 22 damaging de novo mutations (DNMs) in 20 genes, including CHD8 and SCN2A, with recurrent events. Nine of the 20 genes were previously reported to harbor DNMs in ASD patients from other populations, while 11 of them were first identified in present study. We combined genetic variations of the 294 sequenced genes from publicly available whole-exome or whole-genome sequencing studies (4167 probands plus 1786 controls) with our Chinese population (536 cases plus 1457 controls) to optimize the power of candidate-gene prioritization. As a result, we prioritized 67 ASD-candidate genes that exhibited significantly higher probabilities of haploinsufficiency and genic intolerance, and significantly interacted and co-expressed with each another, as well as other known ASD-risk genes. Probands with DNMs or rare inherited mutations in the 67 candidate genes exhibited significantly lower intelligence quotients, supporting their strong functional impact. In addition, we prioritized 39 ASD-related Mic–Mac-risk genes, and showed their interaction and co-expression in a functional network that converged on chromatin remodeling, synapse transmission and cell cycle progression. Genes within the three functional subnetworks exhibited distinct and recognizable spatiotemporal-expression patterns in human brains and laminar-expression profiles in the developing neocortex, highlighting their important roles in brain development. Our results indicate some of Mic–Mac-risk genes are involved in ASD.