ASD restricted and repetitive behaviors associated at 17q21.33: genes prioritized by expression in fetal brains.

Autism spectrum disorder (ASD) is a behaviorally defined condition that manifests in infancy or early childhood as deficits in communication skills and social interactions. Often, restricted and repetitive behaviors (RRBs) accompany this disorder. ASD is polygenic and genetically complex, so we hypothesized that focusing analyses on intermediate core component phenotypes, such as RRBs, can reduce genetic heterogeneity and improve statistical power. Applying this approach, we mined Caucasian genome-wide association studies (GWAS) data from two of the largest ASD family cohorts, the Autism Genetics Resource Exchange and Autism Genome Project (AGP). Of the 12 RRBs measured by the Autism Diagnostic Interview-Revised, seven were found to be significantly familial and substantially variable, and hence, were tested for genome-wide association in 3104 ASD-affected children from 2045 families. Using a stringent significance threshold (P<7.1 × 10-9), GWAS in the AGP revealed an association between 'the degree of the repetitive use of objects or interest in parts of objects' and rs2898883 (P<6.8 × 10-9), which resides within the sixth intron of PHB. To identify the candidate target genes of the associated single-nucleotide polymorphisms at that locus, we applied chromosome conformation studies in developing human brains and implicated three additional genes: SLC35B1, CALCOCO2 and DLX3. Gene expression, brain imaging and fetal brain expression quantitative trait locus studies prioritize SLC35B1 and PHB. These analyses indicate that GWAS of single heritable features of genetically complex disorders followed by chromosome conformation studies in relevant tissues can be successful in revealing novel risk genes for single core features of ASD.

Additive effects of oxytocin receptor gene polymorphisms on reward circuitry in youth with autism.

Several common alleles in the oxytocin receptor gene (OXTR) are associated with altered brain function in reward circuitry in neurotypical adults and may increase risk for autism spectrum disorders (ASD). Yet, it is currently unknown how variation in the OXTR relates to brain functioning in individuals with ASD, and, critically, whether neural endophenotypes vary as a function of aggregate genetic risk. Here, for we believe the first time, we use a multi-locus approach to examine how genetic variation across several OXTR single-nucleotide polymorphisms (SNPs) affect functional connectivity of the brain's reward network. Using data from 41 children with ASD and 41 neurotypical children, we examined functional connectivity of the nucleus accumbens (NAcc) - a hub of the reward network - focusing on how connectivity varies with OXTR risk-allele dosage. Youth with ASD showed reduced NAcc connectivity with other areas in the reward circuit as a function of increased OXTR risk-allele dosage, as well as a positive association between risk-allele dosage and symptom severity, whereas neurotypical youth showed increased NAcc connectivity with frontal brain regions involved in mentalizing. In addition, we found that increased NAcc-frontal cortex connectivity in typically developing youth was related to better scores on a standardized measure of social functioning. Our results indicate that cumulative genetic variation on the OXTR impacts reward system connectivity in both youth with ASD and neurotypical controls. By showing differential genetic effects on neuroendophenotypes, these pathways elucidate mechanisms of vulnerability versus resilience in carriers of disease-associated risk alleles.

The cost of feeding bred dairy heifers on native warm-season grasses and harvested feedstuffs.

Heifer rearing is one of the largest production expenses for dairy cattle operations, which is one reason milking operations outsource heifer rearing to custom developers. The cost of harvested feedstuffs is a major expense in heifer rearing. A possible way to lower feed costs is to graze dairy heifers, but little research exists on this topic in the mid-south United States. The objectives of this research were to determine the cost of feeding bred dairy heifers grazing native warm-season grasses (NWSG), with and without legumes, and compare the cost of grazing with the cost of rearing heifers using 3 traditional rations. The 3 rations were corn silage with soybean meal, corn silage with dry distillers grain, and a wet distillers grain-based ration. Bred Holstein heifers between 15- and 20-mo-old continuously grazed switchgrass (SG), SG with red clover (SG+RC), a big bluestem and Indiangrass mixture (BBIG), and BBIG with red clover (BBIG+RC) in Tennessee during the summer months. Total grazing days were calculated for each NWSG to determine the average cost/animal per grazing day. The average daily gain (ADG) was calculated for each NWSG to develop 3 harvested feed rations that would result in the same ADG over the same number of grazing day as each NWSG treatment. The average cost/animal per grazing day was lowest for SG ($0.48/animal/grazing d) and highest for BBIG+RC ($1.10/animal/grazing d). For both BBIG and SG, legumes increased the average cost/animal per grazing day because grazing days did not increase enough to account for the additional cost of the legumes. No difference was observed in ADG for heifers grazing BBIG (0.85 kg/d) and BBIG+RC (0.94 kg/d), and no difference was observed in ADG for heifers grazing SG (0.71 kg/d) and SG+RC (0.70 kg/d). However, the ADG for heifers grazing SG and SG+RC was lower than the ADG for heifers grazing either BBIG or BBIG+RC. The average cost/animal per grazing day was lower for all NWSG treatments than the average cost/animal per day for all comparable feed rations at a low, average, and high yardage fee. Results of this study suggest that SG was the most cost-effective NWSG alternative to harvested feeds for bred dairy heifer rearing.

Using large clinical data sets to infer pathogenicity for rare copy number variants in autism cohorts.

Copy number variants (CNVs) have a major role in the etiology of autism spectrum disorders (ASD), and several of these have reached statistical significance in case-control analyses. Nevertheless, current ASD cohorts are not large enough to detect very rare CNVs that may be causative or contributory (that is, risk alleles). Here, we use a tiered approach, in which clinically significant CNVs are first identified in large clinical cohorts of neurodevelopmental disorders (including but not specific to ASD), after which these CNVs are then systematically identified within well-characterized ASD cohorts. We focused our initial analysis on 48 recurrent CNVs (segmental duplication-mediated 'hotspots') from 24 loci in 31 516 published clinical cases with neurodevelopmental disorders and 13 696 published controls, which yielded a total of 19 deletion CNVs and 11 duplication CNVs that reached statistical significance. We then investigated the overlap of these 30 CNVs in a combined sample of 3955 well-characterized ASD cases from three published studies. We identified 73 deleterious recurrent CNVs, including 36 deletions from 11 loci and 37 duplications from seven loci, for a frequency of 1 in 54; had we considered the ASD cohorts alone, only 58 CNVs from eight loci (24 deletions from three loci and 34 duplications from five loci) would have reached statistical significance. In conclusion, until there are sufficiently large ASD research cohorts with enough power to detect very rare causative or contributory CNVs, data from larger clinical cohorts can be used to infer the likely clinical significance of CNVs in ASD.

Gestational immune activation and Tsc2 haploinsufficiency cooperate to disrupt fetal survival and may perturb social behavior in adult mice.

Approximately 40-50% of individuals affected by tuberous sclerosis (TSC) develop autism spectrum disorders (ASDs). One possible explanation for this partial penetrance is an interaction between TSC gene mutations and other risk factors such as gestational immune activation. In this study, we report the interactive effects of these two ASD risk factors in a mouse model of TSC. Combined, but not single, exposure had adverse effects on intrauterine survival. Additionally, provisional results suggest that these factors synergize to disrupt social approach behavior in adult mice. Moreover, studies in human populations are consistent with an interaction between high seasonal flu activity in late gestation and TSC mutations in ASD. Taken together, our studies raise the possibility of a gene × environment interaction between heterozygous TSC gene mutations and gestational immune activation in the pathogenesis of TSC-related ASD.

Modeling the functional genomics of autism using human neurons.

Human neural progenitors from a variety of sources present new opportunities to model aspects of human neuropsychiatric disease in vitro. Such in vitro models provide the advantages of a human genetic background combined with rapid and easy manipulation, making them highly useful adjuncts to animal models. Here, we examined whether a human neuronal culture system could be utilized to assess the transcriptional program involved in human neural differentiation and to model some of the molecular features of a neurodevelopmental disorder, such as autism. Primary normal human neuronal progenitors (NHNPs) were differentiated into a post-mitotic neuronal state through addition of specific growth factors and whole-genome gene expression was examined throughout a time course of neuronal differentiation. After 4 weeks of differentiation, a significant number of genes associated with autism spectrum disorders (ASDs) are either induced or repressed. This includes the ASD susceptibility gene neurexin 1, which showed a distinct pattern from neurexin 3 in vitro, and which we validated in vivo in fetal human brain. Using weighted gene co-expression network analysis, we visualized the network structure of transcriptional regulation, demonstrating via this unbiased analysis that a significant number of ASD candidate genes are coordinately regulated during the differentiation process. As NHNPs are genetically tractable and manipulable, they can be used to study both the effects of mutations in multiple ASD candidate genes on neuronal differentiation and gene expression in combination with the effects of potential therapeutic molecules. These data also provide a step towards better understanding of the signaling pathways disrupted in ASD.

Low-pass genome-wide sequencing and variant inference using identity-by-descent in an isolated human population.

Whole-genome sequencing in an isolated population with few founders directly ascertains variants from the population bottleneck that may be rare elsewhere. In such populations, shared haplotypes allow imputation of variants in unsequenced samples without resorting to complex statistical methods as in studies of outbred cohorts. We focus on an isolated population cohort from the Pacific Island of Kosrae, Micronesia, where we previously collected SNP array and rich phenotype data for the majority of the population. We report identification of long regions with haplotypes co-inherited between pairs of individuals and methodology to leverage such shared genetic content for imputation. Our estimates show that sequencing as few as 40 personal genomes allows for inference in up to 60% of the 3000-person cohort at the average locus. We ascertained a pilot data set of whole-genome sequences from seven Kosraean individuals, with average 5× coverage. This assay identified 5,735,306 unique sites of which 1,212,831 were previously unknown. Additionally, these variants are unusually enriched for alleles that are rare in other populations when compared to geographic neighbors (published Korean genome SJK). We used the presence of shared haplotypes between the seven Kosraen individuals to estimate expected imputation accuracy of known and novel homozygous variants at 99.6% and 97.3%, respectively. This study presents whole-genome analysis of a homogenous isolate population with emphasis on optimal rare variant inference.

Comparison of MultiMap and TSP/CONCORDE for constructing radiation hybrid maps.

Radiation hybrid (RH) map construction allows investigators to locate both type I and type II markers on a given genome map. The process is composed of two steps. The first consists of determining the pattern distribution of a set of markers within the different cell lines of an RH panel. This is mainly done by polymerase chain reaction (PCR) amplification and gel electrophoresis, and results in a series of numbers indicating the presence or the absence of each marker in each cell line. The second step consists of a comparison of these numbers, using various algorithms, to group and then order markers. Because different algorithms may provide (slightly) different orders, we have compared the merits of the MultiMap and TSP/CONCORDE packages using a data set of information currently under analysis for construction of the canine genome RH map.

DNA sequence and physical mapping of the canine transglutaminase 1 gene.

The transglutaminase 1 gene (TGM1) encodes an enzyme necessary for cross-linking the structural proteins that form the cornified envelope, an essential component of the outermost layer of the skin, the stratum corneum. Reported here is the complete coding region of canine TGM1, its chromosome localization, and its map position in the integrated canine linkage-radiation hybrid map. Canine TGM1 consists of 2,448 nucleotides distributed over 15 exons. The nucleotide sequence has 90% identity to human TGM1. The deduced canine TGM1 protein is 816 amino acids long and is 92% identical to human TGM1. Using fluorescence in situ hybridization, we localized canine TGM1 to dog (Canis familiaris) chromosome 8 (CFA 8q). Canine TGM1 localized to CFA 8 on the integrated linkage-radiation hybrid map in the interval FH2149-MYH7. Characterizing the coding region of canine TGM1 is a first step in examining the role of this enzyme in normal and defective cornification in the dog.

Physical and radiation hybrid mapping of canine chromosome 12, in a region corresponding to human chromosome 6p12-q12.

The positional cloning of the hypocretin receptor 2, the gene for autosomal recessive canine narcolepsy, has led to the development of a physical map spanning a large portion of canine chromosome 12 (CFA12), in a region corresponding to human chromosome 6p12-q13. More than 40 expressed sequence tags (ESTs) were used in homology search experiments, together with chromosome walking, to build both physical and radiation hybrid maps of the CFA12 13-21 region. The resulting map of bacterial artificial chromosome ends, ESTs, and microsatellite markers represents the longest continuous high-density map of the dog genome reported to date. These data further establish the dog as a system for studying disease genes of interest to human populations and highlight feasible approaches for positional cloning of disease genes in organisms where genomic resources are limited.

Cloning, sequence analysis and radiation hybrid mapping of a mammalian KRT2p gene.

We report here on the cloning, characterization and radiation hybrid mapping of the canine basic keratin gene KRT2p. The gene spans 8.3 kb, consists of nine exons and eight introns, and is characterized by the typical features of both basic keratins and keratins in general, including glycine-rich head and tail domains, which flank an alpha-helical rod domain of approximately 310 amino acids. Comparisons of sequence and structure reveal that canine KRT2p is strikingly similar to human KRT2p. Alignment of the predicted amino acid sequences for human and dog reveals greater than 80% identity. In the rod domain, the amino acid identity exceeds 90%. We note, however, that canine KRT2p encodes a protein 21 residues longer than human K2p due to the insertion of a glycine repeat motif, GG(G)X, in the head and tail domains of the canine gene. This is the first report of the nearly complete genome sequence for KRT2p of any organism. Radiation hybrid mapping of canine KRT2p to chromosome 27 of the dog is also reported.

Reversible polymers formed from self-complementary monomers using quadruple hydrogen bonding.

Units of 2-ureido-4-pyrimidone that dimerize strongly in a self-complementary array of four cooperative hydrogen bonds were used as the associating end group in reversible self-assembling polymer systems. The unidirectional design of the binding sites prevents uncontrolled multidirectional association or gelation. Linear polymers and reversible networks were formed from monomers with two and three binding sites, respectively. The thermal and environmental control over lifetime and bond strength makes many properties, such as viscosity, chain length, and composition, tunable in a way not accessible to traditional polymers. Hence, polymer networks with thermodynamically controlled architectures can be formed, for use in, for example, coatings and hot melts, where a reversible, strongly temperature-dependent rheology is highly advantageous.

Partial deficiency of hypoxanthine-guanine phosphoribosyltransferase with reduced affinity for PP-ribose-P in four related males with gout.

A family is described in which four affected males, spanning two generations, have hyperuricemia and gout accompanied by hematuria but are without severe neurologic involvement. The affected males were found to have markedly reduced levels of erythrocytic hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity; these were 5-12% with hypoxanthine and 0.5-3% with guanine as compared to controls. Erythrocytic adenine phosphoribosyltransferase (APRT) was approximately three-fold elevated in the affected individuals. The residual HGPRT activity in affected males enabled characterization of some of the properties of this mutation. The apparent Michaelis constants (km) for both hypoxanthine and guanine were essentially unchanged, whereas the km for PP-ribose-P was approximately 10-20-fold elevated for all four affected males. The enzyme was more sensitive to product inhibition by IMP and GMP than controls, and exhibited greater thermal lability at 65 degrees C than found with control lysates.

Deoxyadenosine metabolism and toxicity in cultured L5178Y cells.

The growth of cultured L5178Y cells is inhibited by relatively low concentrations fo deoxyadenosine in the presence of deoxycoformycin, an inhibitor of adenosine deaminase. Cell viability is reduced, presumably as a consequence of the induced state of unbalanced growth which is characterized by inhibition in DNA synthesis, accumulation of cells in G1 or early S phase, a continuation in RNA synthesis, and increasing cell volume. The intracellular concentrations of purine and pyrimidine ribonucleoside phosphates remain essentially unchanged. The significant changes in the intracellular deoxynucleoside triphosphate pools are an increase in deoxyadenosine triphosphate and a decrease in deoxycytidine triphosphate.

Biological effects of inhibition of guanine nucleotide synthesis by mycophenolic acid in cultured neuroblastoma cells.

Mycophenolic acid, an inhibitor of inosinate dehydrogenase, had cytostatic and cytotoxic effects on cultured neuroblastoma cells. Proliferation was inhibited by 50% when cells were incubated with 0.07 micrometerM mycophenolic acid, and cell viability was reduced by 83% when cells were treated with 10 micrometerM mycophenolic acid for 24 hr. Treatment of monolayer cultures with mycophenolic acid reduced intracellular concentrations of guanosine triphosphate by 70% within 3 hr, whereas cytidine triphosphate and uridine triphosphate concentrations were significantly elevated, and adenosine triphosphate concentrations were increased only slightly. Reduction of cellular guanine nucleotides had differential effects on rates of macromolecular synthesis: incorporation of radioactive thymidine into acid-insoluble material was inhibited by mycophenolic acid to a much greater extent than was that of adenosine and leucine. Although proliferation of neuroblastoma cells was inhibited, differentiation, as judged by formation of neuronlike processes in serum-free medium, was unaffected by decreased intracellular concentrations of guanosine triphosphate.

Consequences of inhibition of guanine nucleotide synthesis by mycophenolic acid and virazole.

Mycophenolic acid and virazole are inhibitors of inosinate dehydrogenase and produce growth inhibition and loss of viability in cultured murine lymphoma L5178Y cells. Treatment with 1 muM mycophenolic acid produced the following changes in concentrations of acid-soluble nucleotides: (a) guanosine triphosphate decreased to less than 10% of control within 2 hr; (b) uridine triphosphate and cytidine triphosphate concentrations increased markedly; (c) adenosine triphosphate did not change; (d) deoxyguanosine triphosphate decreased; and (e) thymidine triphosphate increased. DNA synthesis was inhibited by 90% within 2 hr, whereas the incorporation of adenosine into RNA and of leucine into protein were much less affected. Virazole (100 muM) produces similar effects. These biochemical effects of mycophenolic acid, as well as its effects on cell growth, can be prevented by addition of guanylate to the medium. Mycophenolic acid treatment also appears to cause breakdown of high-molecular-weight DNA.

Purine and pyrimidine metabolism: pathways, pitfalls and perturbations.

The conceptual framework which underlies many studies of purine and pyrimidine metabolism in intact cells has been critically evaluated. The model that is implicit in many such studies is the single, partially purified enzyme. This paper gives examples both of instances in which the extrapolation of results of enzymes studies to intact cells has been successful and of instances in which enzymes behave differently in the intact cell than in cell extracts. Pitfalls in the extrapolation of results of enzyme studies to intact cells concern (a) metabolic pathways, (b) intracellular enzyme activities, (c) enzyme regulation, and (d) intracellular metabolite concentrations. Examples are also given of situations in which perturbations in one aspect of purine or pyrimidine metabolism lead to changes in other aspects, often distant in the network of reactions.