Empirical Evaluation of Oligonucleotide Probe Selection for DNA Microarrays

DNA-based microarrays are increasingly central to biomedical research. Selecting oligonucleotide sequences that will behave consistently across experiments is essential to the design, production and performance of DNA microarrays. Here our aim was to improve on probe design parameters by empirically and systematically evaluating probe performance in a multivariate context. We used experimental data from 19 array CGH hybridizations to assess the probe performance of 385,474 probes tiled in the Duchenne muscular dystrophy (DMD) region of the X chromosome. Our results demonstrate that probe melting temperature, single nucleotide polymorphisms (SNPs), and homocytosine motifs all have a strong effect on probe behavior. These findings, when incorporated into future microarray probe selection algorithms, may improve microarray performance for a wide variety of applications

High level of complexity and global diversity of the 3q29 locus revealed by optical mapping and long-read sequencing.

BACKGROUND: High sequence identity between segmental duplications (SDs) can facilitate copy number variants (CNVs) via non-allelic homologous recombination (NAHR). These CNVs are one of the fundamental causes of genomic disorders such as the 3q29 deletion syndrome (del3q29S). There are 21 protein-coding genes lost or gained as a result of such recurrent 1.6-Mbp deletions or duplications, respectively, in the 3q29 locus. While NAHR plays a role in CNV occurrence, the factors that increase the risk of NAHR at this particular locus are not well understood. METHODS: We employed an optical genome mapping technique to characterize the 3q29 locus in 161 unaffected individuals, 16 probands with del3q29S and their parents, and 2 probands with the 3q29 duplication syndrome (dup3q29S). Long-read sequencing-based haplotype resolved de novo assemblies from 44 unaffected individuals, and 1 trio was used for orthogonal validation of haplotypes and deletion breakpoints. RESULTS: In total, we discovered 34 haplotypes, of which 19 were novel haplotypes. Among these 19 novel haplotypes, 18 were detected in unaffected individuals, while 1 novel haplotype was detected on the parent-of-origin chromosome of a proband with the del3q29S. Phased assemblies from 44 unaffected individuals enabled the orthogonal validation of 20 haplotypes. In 89% (16/18) of the probands, breakpoints were confined to paralogous copies of a 20-kbp segment within the 3q29 SDs. In one del3q29S proband, the breakpoint was confined to a 374-bp region using long-read sequencing. Furthermore, we categorized del3q29S cases into three classes and dup3q29S cases into two classes based on breakpoints. Finally, we found no evidence of inversions in parent-of-origin chromosomes. CONCLUSIONS: We have generated the most comprehensive haplotype map for the 3q29 locus using unaffected individuals, probands with del3q29S or dup3q29S, and available parents, and also determined the deletion breakpoint to be within a 374-bp region in one proband with del3q29S. These results should provide a better understanding of the underlying genetic architecture that contributes to the etiology of del3q29S and dup3q29S

Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3

Seizure protein 6 (SEZ6) is required for the development and maintenance of the nervous system, is a major substrate of the protease BACE1 and is linked to Alzheimer's disease (AD) and psychiatric disorders, but its molecular functions are not well understood. Here, we demonstrate that SEZ6 controls glycosylation and cell surface localization of kainate receptors composed of GluK2/3 subunits. Loss of SEZ6 reduced surface levels of GluK2/3 in primary neurons and reduced kainate-evoked currents in CA1 pyramidal neurons in acute hippocampal slices. Mechanistically, loss of SEZ6 in vitro and in vivo prevented modification of GluK2/3 with the human natural killer-1 (HNK-1) glycan, a modulator of GluK2/3 function. SEZ6 interacted with GluK2 through its ectodomain and promoted post-endoplasmic reticulum transport of GluK2 in the secretory pathway in heterologous cells and primary neurons. Taken together, SEZ6 acts as a new trafficking factor for GluK2/3. This novel function may help to better understand the role of SEZ6 in neurologic and psychiatric diseases

Parkin regulates kainate receptors by interacting with the GluK2 subunit

Although loss-of-function mutations in the PARK2 gene, the gene that encodes the protein parkin, cause autosomal recessive juvenile parkinsonism, the responsible molecular mechanisms remain unclear. Evidence suggests that a loss of parkin dysregulates excitatory synapses. Here we show that parkin interacts with the kainate receptor (KAR) GluK2 subunit and regulates KAR function. Loss of parkin function in primary cultured neurons causes GluK2 protein to accumulate in the plasma membrane, potentiates KAR currents and increases KAR-dependent excitotoxicity. Expression in the mouse brain of a parkin mutant causing autosomal recessive juvenile parkinsonism results in GluK2 protein accumulation and excitotoxicity. These findings show that parkin regulates KAR function in vitro and in vivo, and suggest that KAR upregulation may have a pathogenetic role in parkin-related autosomal recessive juvenile parkinsonism

The Cost of Autism Spectrum Disorders

Objective: A diagnosis of an autism spectrum disorders is usually associated with substantial lifetime costs to an individual, their family and the community. However, there remains an elusive factor in any cost-benefit analysis of ASD diagnosis, namely the cost of not obtaining a diagnosis. Given the infeasibility of estimating the costs of a population that, by its nature, is inaccessible, the current study compares expenses between families whose children received a formal ASD diagnosis immediately upon suspecting developmental atypicality and seeking advice, with families that experienced a delay between first suspicion and formal diagnosis. Design: A register based questionnaire study covering all families with a child with ASD in Western Australia. Participants: Families with one or more children diagnosed with an ASD, totalling 521 children diagnosed with an ASD; 317 records were able to be included in the final analysis.Results: The median family cost of ASD was estimated to be AUD $34,900 per annum with almost 90$29,200) due to loss of income from employment. For each additional symptom reported, approximately $1,400 cost for the family per annum was added. While there was little direct influence on costs associated with a delay in the diagnosis, the delay was associated with a modest increase in the number of ASD symptoms, indirectly impacting the cost of ASD. Conclusions: A delay in diagnosis was associated with an indirect increased financial burden to families. Early and appropriate access to early intervention is known to improve a child's long-term outcomes and reduce lifetime costs to the individual, family and society. Consequently, a per symptom dollar value may assist in allocation of individualised funding amounts for interventions rather than a nominal amount allocated to all children below a certain age, regardless of symptom presentation, as is the case in Western Australia

Molecular and functional interactions between tumor necrosis factor-alpha receptors and the glutamatergic system in the mouse hippocampus : implications for seizure susceptibility

Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine acting on two distinct receptor subtypes, namely p55 and p75 receptors. TNF-alpha p55 and p75 receptor knockout mice were previously shown to display a decreased or enhanced susceptibility to seizures, respectively, suggesting intrinsic modifications in neuronal excitability. We investigated whether alterations in glutamate system function occur in these naive knockout mice with perturbed cytokine signaling that could explain their different propensity to develop seizures. Using Western blot analysis of hippocampal homogenates, we found that p55(-/-) mice have decreased levels of membrane GluR3 and NR1 glutamate receptor subunits while GluR1, GluR2, GluR6/7 and NR2A/B were unchanged as compared to wild-type mice. In p75(-/-) mice, GluR2, GluR3, GluR6/7 and NR2A/B glutamate receptor subunits were increased in the hippocampus while GluR1 and NR1 did not change. Extracellular single-cell recordings of the electrical activity of hippocampal neurons were carried out in anesthetized mice by standard electrophysiological techniques. Microiontophoretic application of glutamate increased the basal firing rate of hippocampal neurons in p75(-/-) mice versus wild-type mice, and this effect was blocked by 2-amino-5-phosphopentanoic acid and 6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione denoting the involvement of N-methyl-D-aspartic acid and AMPA receptors. In p55(-/-) mice, hippocampal neurons responses to glutamate were similar to wild-type mice. Spontaneous glutamate release measured by in vivo hippocampal microdialysis was significantly decreased only in p55(-/-) mice. No changes were observed in KCl-induced glutamate release in both receptor knockout mice strains versus wild-type mice. These findings highlight specific molecular and functional interactions between p55 and p75 receptor-mediated signaling and the glutamate system. These interactions may be relevant for controlling neuronal excitability in physiological and pathological conditions.peer-reviewe

Regulation of Kainate Receptor Subunit mRNA by Stress and Corticosteroids in the Rat Hippocampus

Kainate receptors are a class of ionotropic glutamate receptors that have a role in the modulation of glutamate release and synaptic plasticity in the hippocampal formation. Previous studies have implicated corticosteroids in the regulation of these receptors and recent clinical work has shown that polymorphisms in kainate receptor subunit genes are associated with susceptibility to major depression and response to anti-depressant treatment. In the present study we sought to examine the effects of chronic stress and corticosteroid treatments upon the expression of the mRNA of kainate receptor subunits GluR5-7 and KA1-2. Our results show that, after 7 days, adrenalectomy results in increased expression of hippocampal KA1, GluR6 and GluR7 mRNAs, an effect which is reversed by treatment with corticosterone in the case of KA1 and GluR7 and by aldosterone treatment in the case of GluR6. 21 days of chronic restraint stress (CRS) elevated the expression of the KA1 subunit, but had no effect on the expression of the other subunits. Similarly, 21 days of treatment with a moderate dose of corticosterone also increased KA1 mRNA in the dentate gyrus, whereas a high corticosterone dose has no effect. Our results suggest an interaction between hippocampal kainate receptor composition and the hypothalamic-pituitary-adrenal (HPA) axis and show a selective chronic stress induced modulation of the KA1 subunit in the dentate gyrus and CA3 that has implications for stress-induced adaptive structural plasticity

Conformational changes in α7 acetylcholine receptors underlying allosteric modulation by divalent cations

Allosteric modulation of membrane receptors is a widespread mechanism by which endogenous and exogenous agents regulate receptor function. For example, several members of the nicotinic receptor family are modulated by physiological concentrations of extracellular calcium ions. In this paper, we examined conformational changes underlying this modulation and compare these with changes evoked by ACh. Two sets of residues in the α7 acetylcholine receptor extracellular domain were mutated to cysteine and analyzed by measuring the rates of modification by the thiol-specific reagent 2-aminoethylmethane thiosulfonate. Using Ba2+ as a surrogate for Ca2+, we found a divalent-dependent decrease the modification rates of cysteine substitutions at M37 and M40, residues at which rates were also slowed by ACh. In contrast, Ba2+ had no significant effect at N52C, a residue where ACh increased the rate of modification. Thus divalent modulators cause some but not all of the conformational effects elicited by agonist. Cysteine substitution of either of two glutamates (E44 or E172), thought to participate in the divalent cation binding site, caused a loss of allosteric modulation, yet Ba2+ still had a significant effect on modification rates of these residues. In addition, the effect of Ba2+ at these residues did not appear to be due to direct occlusion. Our data demonstrate that modulation by divalent cations involves substantial conformational changes in the receptor extracellular domain. Our evidence also suggests the modulation occurs via a binding site distinct from one which includes either (or both) of the conserved glutamates at E44 or E172

High frequency of known copy number abnormalities and maternal duplication 15q11-q13 in patients with combined schizophrenia and epilepsy

<p>Abstract</p> <p>Background</p> <p>Many copy number variants (CNVs) are documented to be associated with neuropsychiatric disorders, including intellectual disability, autism, epilepsy, schizophrenia, and bipolar disorder. Chromosomal deletions of 1q21.1, 3q29, 15q13.3, 22q11.2, and <it>NRXN1 </it>and duplications of 15q11-q13 (maternal), 16p11, and 16p13.3 have the strongest association with schizophrenia. We hypothesized that cases with both schizophrenia and epilepsy would have a higher frequency of disease-associated CNVs and would represent an enriched sample for detection of other mutations associated with schizophrenia.</p> <p>Methods</p> <p>We used array comparative genomic hybridization (CGH) to analyze 235 individuals with both schizophrenia and epilepsy, 80 with bipolar disorder and epilepsy, and 191 controls.</p> <p>Results</p> <p>We detected 10 schizophrenia plus epilepsy cases in 235 (4.3%) with the above mentioned CNVs compared to 0 in 191 controls (p = 0.003). Other likely pathological findings in schizophrenia plus epilepsy cases included 1 deletion 16p13 and 1 duplication 7q11.23 for a total of 12/235 (5.1%) while a possibly pathogenic duplication of 22q11.2 was found in one control for a total of 1 in 191 (0.5%) controls (p = 0.008). The rate of abnormality in the schizophrenia plus epilepsy of 10/235 for the more definite CNVs compares to a rate of 75/7336 for these same CNVs in a series of unselected schizophrenia cases (p = 0.0004).</p> <p>Conclusion</p> <p>We found a statistically significant increase in the frequency of CNVs known or likely to be associated with schizophrenia in individuals with both schizophrenia and epilepsy compared to controls. We found an overall 5.1% detection rate of likely pathological findings which is the highest frequency of such findings in a series of schizophrenia patients to date. This evidence suggests that the frequency of disease-associated CNVs in patients with both schizophrenia and epilepsy is significantly higher than for unselected schizophrenia.</p

PAK1 Protein Expression in the Auditory Cortex of Schizophrenia Subjects

Deficits in auditory processing are among the best documented endophenotypes in schizophrenia, possibly due to loss of excitatory synaptic connections. Dendritic spines, the principal post-synaptic target of excitatory projections, are reduced in schizophrenia. p21-activated kinase 1 (PAK1) regulates both the actin cytoskeleton and dendritic spine density, and is a downstream effector of both kalirin and CDC42, both of which have altered expression in schizophrenia. This study sought to determine if there is decreased auditory cortex PAK1 protein expression in schizophrenia through the use of quantitative western blots of 25 schizophrenia subjects and matched controls. There was no significant change in PAK1 level detected in the schizophrenia subjects in our cohort. PAK1 protein levels within subject pairs correlated positively with prior measures of total kalirin protein in the same pairs. PAK1 level also correlated with levels of a marker of dendritic spines, spinophilin. These latter two findings suggest that the lack of change in PAK1 level in schizophrenia is not due to limited sensitivity of our assay to detect meaningful differences in PAK1 protein expression. Future studies are needed to evaluate whether alterations in PAK1 phosphorylation states, or alterations in protein expression of other members of the PAK family, are present in schizophrenia