Synaptic, transcriptional and chromatin genes disrupted in autism.

The genetic architecture of autism spectrum disorder involves the interplay of common and rare variants and their impact on hundreds of genes. Using exome sequencing, here we show that analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, plus a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways. These include voltage-gated ion channels regulating the propagation of action potentials, pacemaking and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodellers-most prominently those that mediate post-translational lysine methylation/demethylation modifications of histones.

Whole-Genome Sequencing and Integrative Genomic Analysis Approach on Two 22q11.2 Deletion Syndrome Family Trios for Genotype to Phenotype Correlations.

The 22q11.2 deletion syndrome (22q11DS) affects 1:4,000 live births and presents with highly variable phenotype expressivity. In this study, we developed an analytical approach utilizing whole-genome sequencing (WGS) and integrative analysis to discover genetic modifiers. Our pipeline combined available tools in order to prioritize rare, predicted deleterious, coding and noncoding single-nucleotide variants (SNVs), and insertion/deletions from WGS. We sequenced two unrelated probands with 22q11DS, with contrasting clinical findings, and their unaffected parents. Proband P1 had cognitive impairment, psychotic episodes, anxiety, and tetralogy of Fallot (TOF), whereas proband P2 had juvenile rheumatoid arthritis but no other major clinical findings. In P1, we identified common variants in COMT and PRODH on 22q11.2 as well as rare potentially deleterious DNA variants in other behavioral/neurocognitive genes. We also identified a de novo SNV in ADNP2 (NM_014913.3:c.2243G>C), encoding a neuroprotective protein that may be involved in behavioral disorders. In P2, we identified a novel nonsynonymous SNV in ZFPM2 (NM_012082.3:c.1576C>T), a known causative gene for TOF, which may act as a protective variant downstream of TBX1, haploinsufficiency of which is responsible for congenital heart disease in individuals with 22q11DS.

The disruption of Celf6, a gene identified by translational profiling of serotonergic neurons, results in autism-related behaviors.

The immense molecular diversity of neurons challenges our ability to understand the genetic and cellular etiology of neuropsychiatric disorders. Leveraging knowledge from neurobiology may help parse the genetic complexity: identifying genes important for a circuit that mediates a particular symptom of a disease may help identify polymorphisms that contribute to risk for the disease as a whole. The serotonergic system has long been suspected in disorders that have symptoms of repetitive behaviors and resistance to change, including autism. We generated a bacTRAP mouse line to permit translational profiling of serotonergic neurons. From this, we identified several thousand serotonergic-cell expressed transcripts, of which 174 were highly enriched, including all known markers of these cells. Analysis of common variants near the corresponding genes in the AGRE collection implicated the RNA binding protein CELF6 in autism risk. Screening for rare variants in CELF6 identified an inherited premature stop codon in one of the probands. Subsequent disruption of Celf6 in mice resulted in animals exhibiting resistance to change and decreased ultrasonic vocalization as well as abnormal levels of serotonin in the brain. This work provides a reproducible and accurate method to profile serotonergic neurons under a variety of conditions and suggests a novel paradigm for gaining information on the etiology of psychiatric disorders.

Spectrum of elastin sequence variants and cardiovascular phenotypes in 49 patients with Williams-Beuren syndrome.

Haploinsufficiency of the elastin gene (ELN) on 7q11.23 is responsible for supravalvular aortic stenosis (SVAS) and other arteriopathies in patients with Williams-Beuren syndrome (WBS). These defects occur with variable penetrance and expressivity, but the basis of this is unknown. To determine whether DNA variations in ELN could serve as genetic modifiers, we sequenced the 33 exons and immediately surrounding sequence of the ELN gene (9,455 bp of sequence) in 49 DNAs from patients with WBS and compared cardiovascular phenotypes. Four missense, and four novel intronic variants were identified from a total of 24 mostly intronic single nucleotide variations and one indel. Two missense changes were present in one patient each, one published, p.Gly610Ser in exon 27 (MAF, 0.003) and one novel, p.Cys714Tyr, in exon 33 (MAF, 0.001), were rare in the general population. To identify a statistical association between the variants identified here and cardiovascular phenotypes a larger cohort would be needed.

The effect of graphitized carbon on the adsorption and photocatalytic degradation of methylene blue over TiO2/C composites.

The TiO2/C composites with approximately 40 wt% of carbon were prepared by calcination of precursors, formed from a one-pot liquid phase reaction between Ti(SO4)2 and flour. All TiO2/C composites displayed mesoporous structures with high BET surface areas (117-138 m2 g-1) and small crystal sizes of TiO2 (8-27 nm). The contents of graphitic carbon and rutile TiO2 increased, while the surface area and TiO2 crystal size decreased for the TiO2/C composite on increasing the calcination temperature from 650 to 800 °C; when calcinated at 800 °C, the anatase TiO2 completely changed into rutile TiO2 in the TiO2/C composite. The TiO2/C composite calcinated at higher temperatures exhibited better adsorptive and photocatalytic degradation performance in the removal of methylene blue (MB). For the entire rutile TiO2/C-800 composite, the adsorption process of MB can be well described by the pseudo-second-order kinetic model and is governed by chemical adsorption with the maximum adsorption capacity value equal to about 15 mg g-1. Under continuous illumination with a 254 nm UV lamp (15 W) for 3 h, the percentage of MB (14 mg l-1) photocatalytic degradation on 50 mg of TiO2/C-800 was 25.1% higher than that of the maximum adsorption removal. These results suggest that the graphitized carbon has a significant effect on the adsorptivity and photocatalytic activity of the TiO2/C composite.

Whole-Genome Sequencing and iPLEX MassARRAY Genotyping Map an EMS-Induced Mutation Affecting Cell Competition in Drosophila melanogaster.

Cell competition, the conditional loss of viable genotypes only when surrounded by other cells, is a phenomenon observed in certain genetic mosaic conditions. We conducted a chemical mutagenesis and screen to recover new mutations that affect cell competition between wild-type and RpS3 heterozygous cells. Mutations were identified by whole-genome sequencing, making use of software tools that greatly facilitate the distinction between newly induced mutations and other sources of apparent sequence polymorphism, thereby reducing false-positive and false-negative identification rates. In addition, we utilized iPLEX MassARRAY for genotyping recombinant chromosomes. These approaches permitted the mapping of a new mutation affecting cell competition when only a single allele existed, with a phenotype assessed only in genetic mosaics, without the benefit of complementation with existing mutations, deletions, or duplications. These techniques expand the utility of chemical mutagenesis and whole-genome sequencing for mutant identification. We discuss mutations in the Atm and Xrp1 genes identified in this screen.

Reciprocal Relationship between Head Size, an Autism Endophenotype, and Gene Dosage at 19p13.12 Points to AKAP8 and AKAP8L.

Microcephaly and macrocephaly are overrepresented in individuals with autism and are thought to be disease-related risk factors or endophenotypes. Analysis of DNA microarray results from a family with a low functioning autistic child determined that the proband and two additional unaffected family members who carry a rare inherited 760 kb duplication of unknown clinical significance at 19p13.12 are macrocephalic. Consideration alongside overlapping deletion and duplication events in the literature provides support for a strong relationship between gene dosage at this locus and head size, with losses and gains associated with microcephaly (p=1.11x10(-11)) and macrocephaly (p=2.47x10(-11)), respectively. Data support A kinase anchor protein 8 and 8-like (AKAP8 and AKAP8L) as candidate genes involved in regulation of head growth, an interesting finding given previous work implicating the AKAP gene family in autism. Towards determination of which of AKAP8 and AKAP8L may be involved in the modulation of head size and risk for disease, we analyzed exome sequencing data for 693 autism families (2591 individuals) where head circumference data were available. No predicted loss of function variants were observed, precluding insights into relationship to head size, but highlighting strong evolutionary conservation. Taken together, findings support the idea that gene dosage at 19p13.12, and AKAP8 and/or AKAP8L in particular, play an important role in modulation of head size and may contribute to autism risk. Exome sequencing of the family also identified a rare inherited variant predicted to disrupt splicing of TPTE / PTEN2, a PTEN homologue, which may likewise contribute to both macrocephaly and autism risk.

A Novel C-Terminal CIB2 (Calcium and Integrin Binding Protein 2) Mutation Associated with Non-Syndromic Hearing Loss in a Hispanic Family.

Hearing loss is a complex disorder caused by both genetic and environmental factors. Previously, mutations in CIB2 have been identified as a common cause of genetic hearing loss in Pakistani and Turkish populations. Here we report a novel (c.556C>T; p.(Arg186Trp)) transition mutation in the CIB2 gene identified through whole exome sequencing (WES) in a Caribbean Hispanic family with non-syndromic hearing loss. CIB2 belongs to the family of calcium-and integrin-binding (CIB) proteins. The carboxy-termini of CIB proteins are associated with calcium binding and intracellular signaling. The p.(Arg186Trp) mutation is localized within predicted type II PDZ binding ligand at the carboxy terminus. Our ex vivo studies revealed that the mutation did not alter the interactions of CIB2 with Whirlin, nor its targeting to the tips of hair cell stereocilia. However, we found that the mutation disrupts inhibition of ATP-induced Ca2+ responses by CIB2 in a heterologous expression system. Our findings support p.(Arg186Trp) mutation as a cause for hearing loss in this Hispanic family. In addition, it further highlights the necessity of the calcium binding property of CIB2 for normal hearing.

Coex-Rank: An approach incorporating co-expression information for combined analysis of microarray data.

Microarrays have been widely used to study differential gene expression at the genomic level. They can also provide genome-wide co-expression information. Biologically related datasets from independent studies are publicly available, which requires robust combined approaches for integration and validation. Previously, meta-analysis has been adopted to solve this problem. As an alternative to meta-analysis, for microarray data with high similarity in biological experimental design, a more direct combined approach is possible. Gene-level normalization across datasets is motivated by the different scale and distribution of data due to separate origins. However, there has been limited discussion about this point in the past. Here we describe a combined approach for microarray analysis, including gene-level normalization and Coex-Rank approach. After normalization, a linear modeling process is used to identify lists of differentially expressed genes. The Coex-Rank approach incorporates co-expression information into a rank-aggregation procedure. We applied this computational approach to our data, which illustrated an improvement in statistical power and a complementary advantage of the Coex-Rank approach from a biological perspective. Our combined approach for microarray data analysis (Coex-rank) is based on normalization, which is naturally driven. The Coex-rank process not only takes advantage of merging the power of multiple methods regarding normalization but also assists in the discovery of functional clusters of genes.