Association of PLXND1 with a novel subtype of anomalous pulmonary venous return.

Anomalous pulmonary venous return (APVR) is a potentially lethal congenital heart disease. Elucidating the genetic etiology is crucial for understanding its pathogenesis and improving clinical practice, whereas its genetic basis remains largely unknown because of complex genetic etiology. We thus performed whole-exome sequencing for 144 APVR patients and 1636 healthy controls and report a comprehensive atlas of APVR-related rare genetic variants. Novel singleton, loss-of-function and deleterious missense variants (DVars) were enriched in patients, particularly for genes highly expressed in the developing human heart at the critical time point for pulmonary veins draining into the left atrium. Notably, PLXND1, encoding a receptor for semaphorins, represents a strong candidate gene of APVR (adjusted P = 1.1e-03, odds ratio: 10.9-69.3), accounting for 4.17% of APVR. We further validated this finding in an independent cohort consisting of 82 case-control pairs. In these two cohorts, eight DVars were identified in different patients, which convergently disrupt the GTPase-activating protein-related domain of PLXND1. All variant carriers displayed strikingly similar clinical features, in that all anomalous drainage of pulmonary vein(s) occurred on the right side and incorrectly connected to the right atrium, which may represent a novel subtype of APVR for molecular diagnosis. Studies in Plxnd1 knockout mice further revealed the effects of PLXND1 deficiency on severe heart and lung defects and cellular abnormalities related to APVR such as abnormal migration and vascular formation of vascular endothelial cells. These findings indicate the important role of PLXND1 in APVR pathogenesis, providing novel insights into the genetic etiology and molecular subtyping for APVR.

Polyadenylation-related isoform switching in human evolution revealed by full-length transcript structure.

Rhesus macaque is a unique nonhuman primate model for human evolutionary and translational study, but the error-prone gene models critically limit its applications. Here, we de novo defined full-length macaque gene models based on single molecule, long-read transcriptome sequencing in four macaque tissues (frontal cortex, cerebellum, heart and testis). Overall, 8 588 227 poly(A)-bearing complementary DNA reads with a mean length of 14 106 nt were generated to compile the backbone of macaque transcripts, with the fine-scale structures further refined by RNA sequencing and cap analysis gene expression sequencing data. In total, 51 605 macaque gene models were accurately defined, covering 89.7% of macaque or 75.7% of human orthologous genes. Based on the full-length gene models, we performed a human-macaque comparative analysis on polyadenylation (PA) regulation. Using macaque and mouse as outgroup species, we identified 79 distal PA events newly originated in humans and found that the strengthening of the distal PA sites, rather than the weakening of the proximal sites, predominantly contributes to the origination of these human-specific isoforms. Notably, these isoforms are selectively constrained in general and contribute to the temporospatially specific reduction of gene expression, through the tinkering of previously existed mechanisms of nuclear retention and microRNA (miRNA) regulation. Overall, the protocol and resource highlight the application of bioinformatics in integrating multilayer genomics data to provide an intact reference for model animal studies, and the isoform switching detected may constitute a hitherto underestimated regulatory layer in shaping the human-specific transcriptome and phenotypic changes.

HTAADVar: Aggregation and fully automated clinical interpretation of genetic variants in heritable thoracic aortic aneurysm and dissection.

PURPOSE: Early detection and pathogenicity interpretation of disease-associated variants are crucial but challenging in molecular diagnosis, especially for insidious and life-threatening diseases, such as heritable thoracic aortic aneurysm and dissection (HTAAD). In this study, we developed HTAADVar, an unbiased and fully automated system for the molecular diagnosis of HTAAD. METHODS: We developed HTAADVar (http://htaadvar.fwgenetics.org) under the American College of Medical Genetics and Genomics/Association for Molecular Pathology framework, with optimizations based on disease- and gene-specific knowledge, expert panel recommendations, and variant observations. HTAADVar provides variant interpretation with a self-built database through the web server and the stand-alone programs. RESULTS: We constructed an expert-reviewed database by integrating 4373 variants in HTAAD genes, with comprehensive metadata curated from 697 publications and an in-house study of 790 patients. We further developed an interpretation system to assess variants automatically. Notably, HTAADVar showed a multifold increase in performance compared with public tools, reaching a sensitivity of 92.64% and specificity of 70.83%. The molecular diagnostic yield of HTAADVar among 790 patients (42.03%) also matched the clinical data, independently demonstrating its good performance in clinical application. CONCLUSION: HTAADVar represents the first fully automated system for accurate variant interpretation for HTAAD. The framework of HTAADVar could also be generalized for the molecular diagnosis of other genetic diseases.

Targeted resequencing of 358 candidate genes for autism spectrum disorder in a Chinese cohort reveals diagnostic potential and genotype-phenotype correlations.

Autism spectrum disorder (ASD) is a childhood neuropsychiatric disorder with a complex genetic architecture. The diagnostic potential of a targeted panel of ASD genes has only been evaluated in small cohorts to date and is especially understudied in the Chinese population. Here, we designed a capture panel with 358 genes (111 syndromic and 247 nonsyndromic) for ASD and sequenced a Chinese cohort of 539 cases evaluated with the Autism Diagnostic Interview-Revised (ADI-R) and the Autism Diagnostic Observation Schedule (ADOS) as well as 512 controls. ASD cases were found to carry significantly more ultra-rare functional variants than controls. A subset of 78 syndromic and 54 nonsyndromic genes was the most significantly associated and should be given high priority in the future screening of ASD patients. Pathogenic and likely pathogenic variants were detected in 9.5% of cases. Variants in SHANK3 and SHANK2 were the most frequent, especially in females, and occurred in 1.2% of cases. Duplications of 15q11-13 were detected in 0.8% of cases. Variants in CNTNAP2 and MEF2C were correlated with epilepsy/tics in cases. Our findings reveal the diagnostic potential of ASD genetic panel testing and new insights regarding the variant spectrum. Genotype-phenotype correlations may facilitate the diagnosis and management of ASD.

Emergence, Retention and Selection: A Trilogy of Origination for Functional De Novo Proteins from Ancestral LncRNAs in Primates.

While some human-specific protein-coding genes have been proposed to originate from ancestral lncRNAs, the transition process remains poorly understood. Here we identified 64 hominoid-specific de novo genes and report a mechanism for the origination of functional de novo proteins from ancestral lncRNAs with precise splicing structures and specific tissue expression profiles. Whole-genome sequencing of dozens of rhesus macaque animals revealed that these lncRNAs are generally not more selectively constrained than other lncRNA loci. The existence of these newly-originated de novo proteins is also not beyond anticipation under neutral expectation, as they generally have longer theoretical lifespan than their current age, due to their GC-rich sequence property enabling stable ORFs with lower chance of non-sense mutations. Interestingly, although the emergence and retention of these de novo genes are likely driven by neutral forces, population genetics study in 67 human individuals and 82 macaque animals revealed signatures of purifying selection on these genes specifically in human population, indicating a proportion of these newly-originated proteins are already functional in human. We thus propose a mechanism for creation of functional de novo proteins from ancestral lncRNAs during the primate evolution, which may contribute to human-specific genetic novelties by taking advantage of existed genomic contexts.

RhesusBase: a knowledgebase for the monkey research community.

Although the rhesus macaque is a unique model for the translational study of human diseases, currently its use in biomedical research is still in its infant stage due to error-prone gene structures and limited annotations. Here, we present RhesusBase for the monkey research community (http://www.rhesusbase.org). We performed strand-specific RNA-Seq studies in 10 macaque tissues and generated 1.2 billion 90-bp paired-end reads, covering >97.4% of the putative exon in macaque transcripts annotated by Ensembl. We found that at least 28.7% of the macaque transcripts were previously mis-annotated, mainly due to incorrect exon-intron boundaries, incomplete untranslated regions (UTRs) and missed exons. Compared with the previous gene models, the revised transcripts show clearer sequence motifs near splicing junctions and the end of UTRs, as well as cleaner patterns of exon-intron distribution for expression tags and cross-species conservation scores. Strikingly, 1292 exon-intron boundary revisions between coding exons corrected the previously mis-annotated open reading frames. The revised gene models were experimentally verified in randomly selected cases. We further integrated functional genomics annotations from >60 categories of public and in-house resources and developed an online accessible database. User-friendly interfaces were developed to update, retrieve, visualize and download the RhesusBase meta-data, providing a 'one-stop' resource for the monkey research community.

Hominoid-specific de novo protein-coding genes originating from long non-coding RNAs.

Tinkering with pre-existing genes has long been known as a major way to create new genes. Recently, however, motherless protein-coding genes have been found to have emerged de novo from ancestral non-coding DNAs. How these genes originated is not well addressed to date. Here we identified 24 hominoid-specific de novo protein-coding genes with precise origination timing in vertebrate phylogeny. Strand-specific RNA-Seq analyses were performed in five rhesus macaque tissues (liver, prefrontal cortex, skeletal muscle, adipose, and testis), which were then integrated with public transcriptome data from human, chimpanzee, and rhesus macaque. On the basis of comparing the RNA expression profiles in the three species, we found that most of the hominoid-specific de novo protein-coding genes encoded polyadenylated non-coding RNAs in rhesus macaque or chimpanzee with a similar transcript structure and correlated tissue expression profile. According to the rule of parsimony, the majority of these hominoid-specific de novo protein-coding genes appear to have acquired a regulated transcript structure and expression profile before acquiring coding potential. Interestingly, although the expression profile was largely correlated, the coding genes in human often showed higher transcriptional abundance than their non-coding counterparts in rhesus macaque. The major findings we report in this manuscript are robust and insensitive to the parameters used in the identification and analysis of de novo genes. Our results suggest that at least a portion of long non-coding RNAs, especially those with active and regulated transcription, may serve as a birth pool for protein-coding genes, which are then further optimized at the transcriptional level.

Adaptive functions of structural variants in human brain development.

Quantifying the structural variants (SVs) in nonhuman primates could provide a niche to clarify the genetic backgrounds underlying human-specific traits, but such resource is largely lacking. Here, we report an accurate SV map in a population of 562 rhesus macaques, verified by in-house benchmarks of eight macaque genomes with long-read sequencing and another one with genome assembly. This map indicates stronger selective constrains on inversions at regulatory regions, suggesting a strategy for prioritizing them with the most important functions. Accordingly, we identified 75 human-specific inversions and prioritized them. The top-ranked inversions have substantially shaped the human transcriptome, through their dual effects of reconfiguring the ancestral genomic architecture and introducing regional mutation hotspots at the inverted regions. As a proof of concept, we linked APCDD1, located on one of these inversions and down-regulated specifically in humans, to neuronal maturation and cognitive ability. We thus highlight inversions in shaping the human uniqueness in brain development.

CHDbase: A Comprehensive Knowledgebase for Congenital Heart Disease-related Genes and Clinical Manifestations.

Congenital heart disease (CHD) is one of themost common causes of major birth defects, with a prevalence of 1%. Although an increasing number of studies have reported the etiology of CHD, the findings scattered throughout the literature are difficult to retrieve and utilize in research and clinical practice. We therefore developed CHDbase, an evidence-based knowledgebase of CHD-related genes and clinical manifestations manually curated from 1114 publications, linking 1124susceptibility genes and 3591 variations to more than 300 CHD types and related syndromes. Metadata such as the information of each publication and the selected population and samples, the strategy of studies, and the major findings of studies were integrated with each item of the research record. We also integrated functional annotations through parsing ∼ 50 databases/tools to facilitate the interpretation of these genes and variations in disease pathogenicity. We further prioritized the significance of these CHD-related genes with a gene interaction network approach and extracted a core CHD sub-network with 163 genes. The clear genetic landscape of CHD enables the phenotype classification based on the shared genetic origin. Overall, CHDbase provides a comprehensive and freely available resource to study CHD susceptibilities, supporting a wide range of users in the scientific and medical communities. CHDbase is accessible at http://chddb.fwgenetics.org.

6mA-Sniper: Quantifying 6mA sites in eukaryotes at single-nucleotide resolution.

While N6-methyldeoxyadenine (6mA) modification is a fundamental regulation in prokaryotes, its prevalence and functions in eukaryotes are controversial. Here, we report 6mA-Sniper to quantify 6mA sites in eukaryotes at single-nucleotide resolution, and delineate a 6mA profile in Caenorhabditis elegans with 2034 sites. Twenty-six of 39 events with Mnl I restriction endonuclease sites were verified, demonstrating the feasibility of this method. The levels of 6mA sites pinpointed by 6mA-Sniper are generally increased after Pseudomonas aeruginosa infection, but decreased in strains with the removal of METL-9, the dominant 6mA methyltransferase. The enrichment of these sites on specific motif of [GC]GAG, the selective constrains on them, and their coordinated changes with METL-9 levels thus support an active shaping of the 6mA profile by methyltransferase. Moreover, for regions marked by 6mA sites that emerged after infection, an enrichment of up-regulated genes was detected, possibly mediated through a mutual exclusive cross-talk between 6mA and H3K27me3 modification. We thus highlight 6mA regulation as a previously neglected regulator in eukaryotes.

De novo genes with an lncRNA origin encode unique human brain developmental functionality.

Human de novo genes can originate from neutral long non-coding RNA (lncRNA) loci and are evolutionarily significant in general, yet how and why this all-or-nothing transition to functionality happens remains unclear. Here, in 74 human/hominoid-specific de novo genes, we identified distinctive U1 elements and RNA splice-related sequences accounting for RNA nuclear export, differentiating mRNAs from lncRNAs, and driving the origin of de novo genes from lncRNA loci. The polymorphic sites facilitating the lncRNA-mRNA conversion through regulating nuclear export are selectively constrained, maintaining a boundary that differentiates mRNAs from lncRNAs. The functional new genes actively passing through it thus showed a mode of pre-adaptive origin, in that they acquire functions along with the achievement of their coding potential. As a proof of concept, we verified the regulations of splicing and U1 recognition on the nuclear export efficiency of one of these genes, the ENSG00000205704, in human neural progenitor cells. Notably, knock-out or over-expression of this gene in human embryonic stem cells accelerates or delays the neuronal maturation of cortical organoids, respectively. The transgenic mice with ectopically expressed ENSG00000205704 showed enlarged brains with cortical expansion. We thus demonstrate the key roles of nuclear export in de novo gene origin. These newly originated genes should reflect the novel uniqueness of human brain development.

Meta-analysis and genome-wide interpretation of genetic susceptibility to drug addiction.

BACKGROUND: Classical genetic studies provide strong evidence for heritable contributions to susceptibility to developing dependence on addictive substances. Candidate gene and genome-wide association studies (GWAS) have sought genes, chromosomal regions and allelic variants likely to contribute to susceptibility to drug addiction. RESULTS: Here, we performed a meta-analysis of addiction candidate gene association studies and GWAS to investigate possible functional mechanisms associated with addiction susceptibility. From meta-data retrieved from 212 publications on candidate gene association studies and 5 GWAS reports, we linked a total of 843 haplotypes to addiction susceptibility. We mapped the SNPs in these haplotypes to functional and regulatory elements in the genome and estimated the magnitude of the contributions of different molecular mechanisms to their effects on addiction susceptibility. In addition to SNPs in coding regions, these data suggest that haplotypes in gene regulatory regions may also contribute to addiction susceptibility. When we compared the lists of genes identified by association studies and those identified by molecular biological studies of drug-regulated genes, we observed significantly higher participation in the same gene interaction networks than expected by chance, despite little overlap between the two gene lists. CONCLUSIONS: These results appear to offer new insights into the genetic factors underlying drug addiction.

AutismKB 2.0: a knowledgebase for the genetic evidence of autism spectrum disorder.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with strong genetic contributions. To provide a comprehensive resource for the genetic evidence of ASD, we have updated the Autism KnowledgeBase (AutismKB) to version 2.0. AutismKB 2.0 integrates multiscale genetic data on 1379 genes, 5420 copy number variations and structural variations, 11 669 single-nucleotide variations or small insertions/deletions (SNVs/indels) and 172 linkage regions. In particular, AutismKB 2.0 highlights 5669 de novo SNVs/indels due to their significant contribution to ASD genetics and includes 789 mosaic variants due to their recently discovered contributions to ASD pathogenesis. The genes and variants are annotated extensively with genetic evidence and clinical evidence. To help users fully understand the functional consequences of SNVs and small indels, we provided comprehensive predictions of pathogenicity with iFish, SIFT, Polyphen etc. To improve user experiences, the new version incorporates multiple query methods, including simple query, advanced query and batch query. It also functionally integrates two analytical tools to help users perform downstream analyses, including a gene ranking tool and an enrichment analysis tool, KOBAS. AutismKB 2.0 is freely available and can be a valuable resource for researchers.

Statistical analysis of twenty years (1993 to 2012) of data from mainland China's first intervention center for children with autism spectrum disorder.

BACKGROUND: Autism spectrum disorder (ASD) is characterized by persistent deficits in social communication and interaction, and restrictive and repetitive patterns of behavior, interests or activities. This study aimed to analyze trends in ASD diagnosis and intervention in 20 years of data from the Beijing Stars and Rain Education Institute for Autism (SR), the first autism intervention center in mainland China, and from a recent survey of members of the Heart Alliance, an industry association of autism intervention centers in China. METHODS: We analyzed the registration data at the SR from 1993 to 2012 for a total of 2,222 children who had a parent-reported diagnosis of ASD and 612 of 'autistic tendencies'. Most of the children who were the primary focus of our analyses were age six and under. We also analyzed results of a survey we conducted in 2013 of 100 member centers of the Heart Alliance. Generalized Estimating Equations, multiple linear regression and the Mann-Whitney test were used for data analysis. Statistically significant findings are reported here. RESULTS: The number of hospitals where SR children received their diagnosis increased from several in the early 1990s to 276 at present. The proportion of 'autistic tendencies' diagnosis increased 2.04-fold from 1998 to 2012 and was higher for children diagnosed at a younger age. The mean age at first diagnosis of ASD or 'autistic tendencies' decreased by 0.27 years every decade. A higher level of parental education was statistically significantly associated with an earlier diagnosis of the child. The mean parental age at childbirth increased by about 1.48 years per decade, and the mean maternal age was 1.40 and 2.10 years higher than that in the national population censuses of 2000 and 2010, respectively. At the time of the survey 3,957 children with ASD were being trained at the 100 autism intervention centers. Ninety-seven of these centers opened after the year 2000. Economically underdeveloped regions are still underserved. CONCLUSIONS: This study revealed encouraging trends and remaining challenges in ASD diagnosis and intervention among children at the SR over the past 20 years and the 100 autism intervention centers in China at present.

Prophylactic effect of glyceryl trinitrate on post-endoscopic retrograde cholangiopancreatography pancreatitis: a randomized placebo-controlled trial.

AIM: To examine the prophylactic effect of glyceryl trinitrate on post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis and hyperamylasemia. METHODS: Patients scheduled for ERCP were randomly divided into study group and placebo group. Patients in study group and placebo group were treated with 5 mg glyceryl trinitrate and 100 mg vitamin C, respectively, 5 min before endoscopic maneuvers. RESULTS: A total of 74 patients were enrolled in the final analysis. Post-ERCP pancreatitis occurred in 3 patients (7.9%) of the study group and 9 patients (25%) in the placebo group (P = 0.012). Hyperamylasemia occurred in 8 patients of the study group (21.1%) and 13 patients (36.1%) of the placebo group (P = 0.037). CONCLUSION: Glyceryl trinitrate before ERCP can effectively prevent post-ERCP and hyperamylasemia.