Analysis of CHRNA7 rare variants in autism spectrum disorder susceptibility.

Chromosome 15q13.3 recurrent microdeletions are causally associated with a wide range of phenotypes, including autism spectrum disorder (ASD), seizures, intellectual disability, and other psychiatric conditions. Whether the reciprocal microduplication is pathogenic is less certain. CHRNA7, encoding for the alpha7 subunit of the neuronal nicotinic acetylcholine receptor, is considered the likely culprit gene in mediating neurological phenotypes in 15q13.3 deletion cases. To assess if CHRNA7 rare variants confer risk to ASD, we performed copy number variant analysis and Sanger sequencing of the CHRNA7 coding sequence in a sample of 135 ASD cases. Sequence variation in this gene remains largely unexplored, given the existence of a fusion gene, CHRFAM7A, which includes a nearly identical partial duplication of CHRNA7. Hence, attempts to sequence coding exons must distinguish between CHRNA7 and CHRFAM7A, making next-generation sequencing approaches unreliable for this purpose. A CHRNA7 microduplication was detected in a patient with autism and moderate cognitive impairment; while no rare damaging variants were identified in the coding region, we detected rare variants in the promoter region, previously described to functionally reduce transcription. This study represents the first sequence variant analysis of CHRNA7 in a sample of idiopathic autism.

Convergence of genes and cellular pathways dysregulated in autism spectrum disorders.

Rare copy-number variation (CNV) is an important source of risk for autism spectrum disorders (ASDs). We analyzed 2,446 ASD-affected families and confirmed an excess of genic deletions and duplications in affected versus control groups (1.41-fold, p = 1.0 × 10(-5)) and an increase in affected subjects carrying exonic pathogenic CNVs overlapping known loci associated with dominant or X-linked ASD and intellectual disability (odds ratio = 12.62, p = 2.7 × 10(-15), ∼3% of ASD subjects). Pathogenic CNVs, often showing variable expressivity, included rare de novo and inherited events at 36 loci, implicating ASD-associated genes (CHD2, HDAC4, and GDI1) previously linked to other neurodevelopmental disorders, as well as other genes such as SETD5, MIR137, and HDAC9. Consistent with hypothesized gender-specific modulators, females with ASD were more likely to have highly penetrant CNVs (p = 0.017) and were also overrepresented among subjects with fragile X syndrome protein targets (p = 0.02). Genes affected by de novo CNVs and/or loss-of-function single-nucleotide variants converged on networks related to neuronal signaling and development, synapse function, and chromatin regulation.

Use of array CGH to detect exonic copy number variants throughout the genome in autism families detects a novel deletion in TMLHE.

Autism is a neurodevelopmental disorder with increasing evidence of heterogeneous genetic etiology including de novo and inherited copy number variants (CNVs). We performed array comparative genomic hybridization using a custom Agilent 1 M oligonucleotide array intended to cover 197 332 unique exons in RefSeq genes; 98% were covered by at least one probe and 95% were covered by three or more probes with the focus on detecting relatively small CNVs that would implicate a single protein-coding gene. The study group included 99 trios from the Simons Simplex Collection. The analysis identified and validated 55 potentially pathogenic CNVs, categorized as de novo autosomal heterozygous, inherited homozygous autosomal, complex autosomal and hemizygous deletions on the X chromosome of probands. Twenty percent (11 of 55) of these CNV calls were rare when compared with the Database of Genomic Variants. Thirty-six percent (20 of 55) of the CNVs were also detected in the same samples in an independent analysis using the 1 M Illumina single-nucleotide polymorphism array. Findings of note included a common and sometimes homozygous 61 bp exonic deletion in SLC38A10, three CNVs found in lymphoblast-derived DNA but not present in whole-blood derived DNA and, most importantly, in a male proband, an exonic deletion of the TMLHE (trimethyllysine hydroxylase epsilon) that encodes the first enzyme in the biosynthesis of carnitine. Data for CNVs present in lymphoblasts but absent in fresh blood DNA suggest that these represent clonal outgrowth of individual B cells with pre-existing somatic mutations rather than artifacts arising in cell culture. GEO accession number GSE23765 (http://www.ncbi.nlm.nih.gov/geo/, date last accessed on 30 August 2011). Genboree accession: http://genboree.org/java-bin/gbrowser.jsp?refSeqId=1868&entryPointId=chr17&from=53496072&to=53694382&isPublic=yes, date last accessed on 30 August 2011.

Multiple recurrent de novo CNVs, including duplications of the 7q11.23 Williams syndrome region, are strongly associated with autism.

We have undertaken a genome-wide analysis of rare copy-number variation (CNV) in 1124 autism spectrum disorder (ASD) families, each comprised of a single proband, unaffected parents, and, in most kindreds, an unaffected sibling. We find significant association of ASD with de novo duplications of 7q11.23, where the reciprocal deletion causes Williams-Beuren syndrome, characterized by a highly social personality. We identify rare recurrent de novo CNVs at five additional regions, including 16p13.2 (encompassing genes USP7 and C16orf72) and Cadherin 13, and implement a rigorous approach to evaluating the statistical significance of these observations. Overall, large de novo CNVs, particularly those encompassing multiple genes, confer substantial risks (OR = 5.6; CI = 2.6-12.0, p = 2.4 × 10(-7)). We estimate there are 130-234 ASD-related CNV regions in the human genome and present compelling evidence, based on cumulative data, for association of rare de novo events at 7q11.23, 15q11.2-13.1, 16p11.2, and Neurexin 1.

Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna).

The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids (Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.

Annotation, phylogenetics, and expression of the nuclear receptors in Daphnia pulex.

BACKGROUND: The nuclear receptor superfamily currently consists of seven gene subfamilies that encompass over 80 distinct receptor proteins. These transcription factors typically share a common five-domain structure with a highly conserved DNA-binding domain. Some nuclear receptors are ubiquitous among the metazoans, while others are unique to specific phylogenetic groups. Crustaceans represent the second largest group of arthropods with insects being the largest. However, relative to insects, little is known about the nuclear receptors of crustaceans. The aim of this study was to identify putative nuclear receptors from the first assembled genome of a crustacean Daphnia pulex http://wFleaBase.org. Nuclear receptor expression was evaluated and receptors were subjected to phylogenetic analyses to gain insight into evolution and function. RESULTS: Twenty-five putative nuclear receptors were identified in D. pulex based on the presence of a conserved DNA-binding domain. All of the nuclear receptor protein sequences contain a highly homologous DNA-binding domain and a less conserved ligand-binding domain with the exception of the NR0A group. These receptors lack a ligand-binding domain. Phylogenetic analysis revealed the presence of all seven receptor subfamilies. The D. pulex genome contains several nuclear receptors that have vertebrate orthologs. However, several nuclear receptor members that are represented in vertebrates are absent from D. pulex. Notable absences include receptors of the 1C group (peroxisome proliferators-activated receptors), the 3A group (estrogen receptor), and the 3C group (androgen, progestogen, mineralcorticoid, and glucocorticoid receptors). The D. pulex genome also contains nuclear receptor orthologs that are present in insects and nematodes but not vertebrates, including putative nuclear receptors within the NR0A group. A novel group of receptors, designated HR97, was identified in D. pulex that groups with the HR96/CeNHR8/48/DAF12 clade, but forms its own sub-clade. Gene products were detected in adult female D. pulex for 21 of the 25 receptors. CONCLUSION: Nuclear receptors are ancient proteins with highly conserved DNA-binding domains. The DNA-binding domains of the nuclear receptors of D. pulex contain the same degree of conservation that is typically found within nuclear receptors of other species. Most of the receptors identified in D. pulex have orthologs within the vertebrate and invertebrate lineages examined with the exception of the novel HR97 group and the Dappu-HR10 and potentially the Dappu-HR11 receptors found in D. pulex. These groups of receptors may harbour functions that are intrinsic to crustacean physiology.

An orthotopic xenograft model of intraneural NF1 MPNST suggests a potential association between steroid hormones and tumor cell proliferation.

Malignant peripheral nerve sheath tumors (MPNST) are the most aggressive cancers associated with neurofibromatosis type 1 (NF1). Here we report a practical and reproducible model of intraneural NF1 MPNST, by orthotopic xenograft of an immortal human NF1 tumor-derived Schwann cell line into the sciatic nerves of female scid mice. Intraneural injection of the cell line sNF96.2 consistently produced MPNST-like tumors that were highly cellular and showed extensive intraneural growth. These xenografts had a high proliferative index, were angiogenic, had significant mast cell infiltration and rapidly dominated the host nerve. The histopathology of engrafted intraneural tumors was consistent with that of human NF1 MPNST. Xenograft tumors were readily examined by magnetic resonance imaging, which also was used to assess tumor vascularity. In addition, the intraneural proliferation of sNF96.2 cell tumors was decreased in ovariectomized mice, while replacement of estrogen or progesterone restored tumor cell proliferation. This suggests a potential role for steroid hormones in supporting tumor cell growth of this MPNST cell line in vivo. The controlled orthotopic implantation of sNF96.2 cells provides for the precise initiation of intraneural MPNST-like tumors in a model system suitable for therapeutic interventions, including inhibitors of angiogenesis and further study of steroid hormone effects on tumor cell growth.

Plexiform-like neurofibromas develop in the mouse by intraneural xenograft of an NF1 tumor-derived Schwann cell line.

Plexiform neurofibromas are peripheral nerve sheath tumors that arise frequently in neurofibromatosis type 1 (NF1) and have a risk of malignant progression. Past efforts to establish xenograft models for neurofibroma involved the implantation of tumor fragments or heterogeneous primary cultures, which rarely achieved significant tumor growth. We report a practical and reproducible animal model of plexiform-like neurofibroma by xenograft of an immortal human NF1 tumor-derived Schwann cell line into the peripheral nerve of scid mice. The S100 and p75 positive sNF94.3 cell line was shown to possess a normal karyotype and have apparent full-length neurofibromin by Western blot. These cells were shown to have a constitutional NF1 microdeletion and elevated Ras-GTP activity, however, suggesting loss of normal neurofibromin function. Localized intraneural injection of the cell line sNF94.3 produced consistent and slow growing tumors that infiltrated and disrupted the host nerve. The xenograft tumors resembled plexiform neurofibromas with a low rate of proliferation, abundant extracellular matrix (hypocellularity), basal laminae, high vascularity, and mast cell infiltration. The histologic features of the developed tumors were particularly consistent with those of human plexiform neurofibroma as well. Intraneural xenograft of sNF94.3 cells enables the precise initiation of intraneural, plexiform-like tumors and provides a highly reproducible model for the study of plexiform neurofibroma tumorigenesis. This model facilitates testing of potential therapeutic interventions, including angiogenesis inhibitors, in a relevant cellular environment.

Comparison of regional gene expression differences in the brains of the domestic dog and human.

Comparison of the expression profiles of 2,721 genes in the cerebellum, cortex and pituitary gland of three American Staffordshire terriers, one beagle and one fox hound revealed regional expression differences in the brain but failed to reveal marked differences among breeds, or even individual dogs. Approximately 85 per cent (42 of 49 orthologue comparisons) of the regional differences in the dog are similar to those that differentiate the analogous human brain regions. A smaller percentage of human differences were replicated in the dog, particularly in the cortex, which may generally be evolving more rapidly than other brain regions in mammals. This study lays the foundation for detailed analysis of the population structure of transcriptional variation as it relates to cognitive and neurological phenotypes in the domestic dog.

Formation of Hirano bodies induced by expression of an actin cross-linking protein with a gain-of-function mutation.

Hirano bodies are paracrystalline actin filament-containing structures reported to be associated with a variety of neurodegenerative diseases. However, the biological function of Hirano bodies remains poorly understood, since nearly all prior studies of these structures were done with postmortem samples of tissue. In the present study, we generated a full-length form of a Dictyostelium 34-kDa actin cross-linking protein with point mutations in the first putative EF hand, termed 34-kDa DeltaEF1. The 34-kDa DeltaEF1 protein binds calcium normally but has activated actin binding that is unregulated by calcium. The expression of the 34-kDa DeltaEF1 protein in Dictyostelium induces the formation of Hirano bodies, as assessed by both fluorescence microscopy and transmission electron microscopy. Dictyostelium cells bearing Hirano bodies grow normally, indicating that Hirano bodies are not associated with cell death and are not deleterious to cell growth. Moreover, the expression of the 34-kDa DeltaEF1 protein rescues the phenotypes of cells lacking the 34-kDa protein and cells lacking both the 34-kDa protein and alpha-actinin. Finally, the expression of the 34-kDa DeltaEF1 protein also initiates the formation of Hirano bodies in cultured mouse fibroblasts. These results show that the failure to regulate the activity and/or affinity of an actin cross-linking protein can provide a signal for the formation of Hirano bodies. More generally, the formation of Hirano bodies is a cellular response to or a consequence of aberrant function of the actin cytoskeleton.

Calcium regulation of actin crosslinking is important for function of the actin cytoskeleton in Dictyostelium.

The actin cytoskeleton is sensitive to changes in calcium, which affect contractility, actin-severing proteins, actin-crosslinking proteins and calmodulin-regulated enzymes. To dissect the role of calcium control on the activity of individual proteins from effects of calcium on other processes, calcium-insensitive forms of these proteins were prepared and introduced into living cells to replace a calcium-sensitive form of the same protein. Crosslinking and bundling of actin filaments by the Dictyostelium 34 kDa protein is inhibited in the presence of micromolar free calcium. A modified form of the 34 kDa protein with mutations in the calcium binding EF hand (34 kDa deltaEF2) was prepared using site-directed mutagenesis and expressed in E. coli. Equilibrium dialysis using [(45)Ca]CaCl(2) revealed that the wild-type protein is able to bind one calcium ion with a Kd of 2.4 microM. This calcium binding is absent in the 34 kDa deltaEF2 protein. The actin-binding activity of the 34 kDa deltaEF2 protein was equivalent to wildtype but calcium insensitive in vitro. The wild-type and 34 kDa deltaEF2 proteins were expressed in 34-kDa-null and 34 kDa/alpha-actinin double null mutant Dictyostelium strains to test the hypothesis that calcium regulation of actin crosslinking is important in vivo. The 34 kDa deltaEF2 failed to supply function of the 34 kDa protein important for control of cell size and for normal growth to either of these 34-kDa-null strains. Furthermore, the distribution of the 34 kDa protein and actin were abnormal in cells expressing 34 kDa deltaEF2. Thus, calcium regulation of the formation and/or dissolution of crosslinked actin structures is required for dynamic behavior of the actin cytoskeleton important for cell structure and growth.

NF1 mutations and molecular testing.

Neurofibromatosis 1 is a progressive autosomal dominant condition caused by mutations in the NF1 gene on chromosome 17. The condition shows clinical variable expressivity, with varying features even between family members who share the same mutation. Furthermore, it is impossible to precisely predict the severity and course of the condition, a source of frustration for families and physicians. Neurofibromatosis 1 is also heterogeneous at the mutation level, with more than 300 independent mutations having been reported in this gene. The mutation data have accumulated slowly owing to the variability of the mutation types and the size and complexity of the gene. This is also reflected in the lack of a simple, inexpensive, highly accurate DNA-based test for neurofibromatosis 1 at present. This article reviews current NF1 mutation spectrum and testing, discussing and illustrating mutation mechanisms and pathogenetic effects, as well as factors affecting DNA testing and interpretation/diagnosis.

RT-PCR splicing analysis of the NF1 open reading frame.

Neurofibromatosis 1 (NF1) is an autosomal dominant condition whose molecular diagnosis is challenging because of the large size of the gene and the vast number of unique NF1 gene mutations. Some splicing and nonsense mutations have been shown to cause exon skipping. Recently, temperature-induced abnormal splicing has been found in NF1 in ex-vivo tissues. This prompted us to investigate the entire NF1 transcript for such aberrant splicing. We found several novel exon skips that appeared de novo or were present initially and increased in aged/cooled blood: exon 20, exons 20 and 21 combined, exon 33, exon 34, exon 37, exon 40, exon 45, exons 43 and 45 combined, part of exon 43, and the first codon of exon 12b. Some aberrant splice forms were undetectable when blood was drawn into Qiagen PAXgene tubes, rather than EDTA vacutainers, and we demonstrate how these aberrant splicing events are a potential pitfall for RNA-based NF1 mutation characterization. The same reverse transcription/polymerase chain reaction strategy was used to screen for novel NF1 alternative splicing in Schwann cells and seven other tissues. Even though no Schwann-specific alternative exons were identified, we found minor novel splicing isoforms differentially expressed such as skips of exon 37 and exon 40. Skipping of exon 43, part of exon 43, and the first codon of exon 12b were found in all tissues analyzed. These forms suggest greater tissue-based variability in the NF1 message than was previously thought and may indicate minor amounts of heterogeneity at the protein level.