A Population Genetic Approach to Mapping Neurological Disorder Genes Using Deep Resequencing

Deep resequencing of functional regions in human genomes is key to identifying potentially causal rare variants for complex disorders. Here, we present the results from a large-sample resequencing (n = 285 patients) study of candidate genes coupled with population genetics and statistical methods to identify rare variants associated with Autism Spectrum Disorder and Schizophrenia. Three genes, MAP1A, GRIN2B, and CACNA1F, were consistently identified by different methods as having significant excess of rare missense mutations in either one or both disease cohorts. In a broader context, we also found that the overall site frequency spectrum of variation in these cases is best explained by population models of both selection and complex demography rather than neutral models or models accounting for complex demography alone. Mutations in the three disease-associated genes explained much of the difference in the overall site frequency spectrum among the cases versus controls. This study demonstrates that genes associated with complex disorders can be mapped using resequencing and analytical methods with sample sizes far smaller than those required by genome-wide association studies. Additionally, our findings support the hypothesis that rare mutations account for a proportion of the phenotypic variance of these complex disorders

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

A dominant-negative mutation in the TRESK potassium channel is linked to familial migraine with aura. Nature Medicine, 2010(advance online publication

Migraine with aura is a common, debilitating, recurrent headache disorder associated with transient and reversible focal neurological symptoms 1 . A role has been suggested for the two-pore domain (K2P) potassium channel, TWIK-related spinal cord potassium channel (TRESK, encoded by KCNK18), in pain pathways and general anaesthesia 2 . We therefore examined whether TRESK is involved in migraine by screening the KCNK18 gene in subjects diagnosed with migraine. Here we report a frameshift mutation, F139WfsX24, which segregates perfectly with typical migraine with aura in a large pedigree. We also identified prominent TRESK expression in migrainesalient areas such as the trigeminal ganglion. Functional characterization of this mutation demonstrates that it causes a complete loss of TRESK function and that the mutant subunit suppresses wild-type channel function through a dominantnegative effect, thus explaining the dominant penetrance of this allele. These results therefore support a role for TRESK in the pathogenesis of typical migraine with aura and further support the role of this channel as a potential therapeutic target. Migraine is a common recurrent headache disorder, with an annual prevalence estimated at 18.2% in females and 6.5% in males 1 . One third of attacks, which can last from 4 to 72 h, are preceded by transient neurological disturbances known as aura. These are commonly visual, taking the form of scintillating shapes, hallucinations or black spots. Cortical spreading depression (CSD) underlies the aura, and, although its precise relationship to headache is unclear, there is evidence in rats that CSD can activate trigeminal nociceptors 3,4 . During a migraine headache, trigeminal ganglion nerve afferents innervating the meningeal nociceptors secrete pro-inflammatory peptides (such as CGRP and substance P) that cause local inflammation, intensify activatio

WNK1/HSN2 Mutation in Human Peripheral Neuropathy Deregulates <em>KCC2</em> Expression and Posterior Lateral Line Development in Zebrafish (<em>Danio rerio</em>)

<div><p>Hereditary sensory and autonomic neuropathy type 2 (HSNAII) is a rare pathology characterized by an early onset of severe sensory loss (all modalities) in the distal limbs. It is due to autosomal recessive mutations confined to exon “HSN2” of the WNK1 (with-no-lysine protein kinase 1) serine-threonine kinase. While this kinase is well studied in the kidneys, little is known about its role in the nervous system. We hypothesized that the truncating mutations present in the neural-specific HSN2 exon lead to a loss-of-function of the WNK1 kinase, impairing development of the peripheral sensory system. To investigate the mechanisms by which the loss of WNK1/HSN2 isoform function causes HSANII, we used the embryonic zebrafish model and observed strong expression of WNK1/HSN2 in neuromasts of the peripheral lateral line (PLL) system by immunohistochemistry. Knocking down wnk1/hsn2 in embryos using antisense morpholino oligonucleotides led to improper PLL development. We then investigated the reported interaction between the WNK1 kinase and neuronal potassium chloride cotransporter KCC2, as this transporter is a target of WNK1 phosphorylation. <em>In situ</em> hybridization revealed <em>kcc2</em> expression in mature neuromasts of the PLL and semi-quantitative RT–PCR of wnk1/hsn2 knockdown embryos showed an increased expression of <em>kcc2</em> mRNA. Furthermore, overexpression of human KCC2 mRNA in embryos replicated the wnk1/hsn2 knockdown phenotype. We validated these results by obtaining double knockdown embryos, both for wnk1/hsn2 and kcc2, which alleviated the PLL defects. Interestingly, overexpression of inactive mutant KCC2-C568A, which does not extrude ions, allowed a phenocopy of the PLL defects. These results suggest a pathway in which WNK1/HSN2 interacts with KCC2, producing a novel regulation of its transcription independent of KCC2's activation, where a loss-of-function mutation in WNK1 induces an overexpression of KCC2 and hinders proper peripheral sensory nerve development, a hallmark of HSANII.</p> </div

KCC2 is overexpressed in WNK1/HSN2 knockdown embryos.

<p>A) RT-PCR against <i>slc12a5</i> (coding for kcc2) shows higher levels of RNA in WNK1/HSN2 knockdown embryos when compared with WT at 72 hpf as well as at 24 hpf. We overexpressed human KCC2 mRNA in WT embryos (morphology presented in (B)) to validate this result and were able to replicate the WNK1/HSN2 knockdown phenotype as assayed by (C) the number of structural and functional hair cells in PLL neuromasts and by (D) the 4-di-2-ASP score. We also validated this result by obtaining a partial rescue of the WNK1/HSN2 knockdown phenotype by knocking down kcc2 using MO1-slc12a5 in WNK1/HSN2 embryos (double knockdown-KD experiments). The embryos lacking kcc2 have morphological defects and a lower 4-di-2-ASP score (D) due to their smaller length (B), but double knockdown embryos, which are also morphologically abnormal and smaller in size have a significantly higher 4-di-2-ASP score (D) indicative of a partial rescue. For (C) The number of neuromasts counted per condition is indicated in the boxes and the total number of embryos obtained per condition is indicated in parenthesis at the bottom of the box plots. For (D), the total number of embryos is indicated in the boxes. (E) Overexpression of inactive mutant KCC2-C568A mimics hKCC2 overexpression and WNK1/HSN2 knockdown phenotype by producing PLL defects as assayed by 4-di-2-ASP vital dye staining. This indicates that WNK1/HSN2 interacts with KCC2 and regulates its transcription independent of the cotransporter's activation. Neuromast scores were tabulated as previously done and presented as a box plot. The number of neuromasts counted per condition is indicated in the boxes. Scale bar: 100 µm.</p

KCC2 is found in the embryonic zebrafish PLL.

<p>A) Presence of kcc2 in the zebrafish embryo was assessed by <i>in situ</i> hybridization against slc12a5 and reveals staining in the hindbrain (hb) and spinal cord (sc) at 72 hpf as well as staining in hair cells of a PLL neuromast (nm) at 4dpf. B) Neuromasts of 4 dpf transgenic Tg(<i>NBT:MAPT-GFP</i>) embryos expressing GFP (green) under neuron-specific beta-tubulin promoter (left upper and middle images) and primordium of a 2dpf transgenic Tg(<i>-8.0cldnb:lynEGFP</i>) embryos expressing GFP under the claudin-b promoter in the membranes of cells composing the primordium (left lower image) were labeled with the Ca²⁺ indicator Rhod 2-AM (deep red) to show that ionophoresis of glycine failed to evoke Ca²⁺ transients in 3–4 dpf neuromasts (right black upper traces) but did so in primordium cells (right red bottom traces). In contrast, neuromasts of embryos of equivalent stage respond to glutamate (right, green middle trace). The dashed line illustrates the primordium or neuromast region and the heavy lines illustrates the position of the pipet. C)Transgenic embryos expressing GFP under the claudin-b promoter, which labels the membranes of cells composing the primordium, were used to observe the size of this migrating group of PLL neuromast progenitors for both non-injected and WNK1/HSN2 knockdown embryos. Close-up image of the primordium cells shows no difference in organization between non-injected and WNK1/HSN2 knockdown embryos. D) The primordium area was measured on one side of the embryos and data was tabulated in a scatter plot which shows that WNK1/HSN2 embryos, as well as embryos overexpressing human KCC2, have a significantly smaller primordium area than non-injected embryos. The number of primordial measured is indicated in parenthesis at the bottom of the graph. The knockdown embryo presented in (C) is a representative results at 22 hpf obtained from MO-wnk1-ATG injection. Scale bars: (A) 100 µm and 20 µm for neuromast image, (B)20 µm, (C) 80 µm for full primordium and 20 µm for cell close-up.</p

WNK1/HSN2 knockdown leads to abnormal neuromast development.

<p>A) The number of structural hair cells was assessed using transgenic embryos expressing GFP under the beta-tubulin promoter, revealing only the neuronal hair cells within PLL neuromasts. C) The number of functional hair cells was assessed by using transgenic embryos expressing GFP under the claudin-b promoter, rendering the neuromast fluorescent. The functional hair cells were revealed by incubation in the styryl dye FM-464FX, shown in red. B, D) Hair cells were counted for each PLL neuromast and totals were tabulated in box plots showing that WNK1/HSN2 knockdown embryos have a significantly lower number of structural and functional hair cells within their neuromasts when compared with non-injected embryos. The knockdown embryos presented in (A) and (C) are representative results at 72 hpf obtained from MO-hsn2-SB3′ injection. The number of neuromasts counted per condition is indicated in the boxes and the total number of embryos obtained per condition is indicated in parenthesis at the bottom of the box plots. Scale bar: 20 µm.</p

Expression of the WNK1 kinase in zebrafish embryos.

<p>A) Structure of the zebrafish WNK1 ortholog which conserved the HSN2 exon, <i>wnk1b</i>. The split exon 10 and fused exons 11–13 have been indicated on the sequence, respectively in pale blue and dark blue, as well as antisense morpholino oligonucleotide targets (red lines). B) Both copies of the zebrafish WNK1 ortholog, <i>wnk1a</i> and <i>wnk1b</i> are expressed early at the 16 cells stage and persist at 18, 24 and 72 hours post-fertilization (hpf) and until 7 days post-fertilization (dpf). The RT-PCR was done using primers set in the HSN2 exon for <i>wnk1b</i>, targeting a sequence spanning exons 1–8 for <i>wnk1a</i> and using as control the housekeeping gene <i>GAPDH</i>. C) Zebrafish WNK1/HSN2 from the <i>wnk1b</i> gene was detected in the neuromasts of the posterior lateral line by whole-mount immunohistochemistry using an anti-HSN2 antibody. The inset shows a closer view of a stained neuromast. Scale bar: 100 µm.</p

WNK1/HSN2 knockdown in zebrafish using antisense morpholino oligonucleotides (AMO).

<p>A) Knockdown embryos show no morphological phenotype, but reveal posterior lateral line defects (PLL) as observed under fluorescence with the 4-di-2-ASP vital dye when compared with non-injected WT embryos at 72 hours post-fertilization. The knockdown embryo presented is a representative result obtained from MO-hsn2-SB5′ injection. B) Each neuromast of the PLL observed with 4-di-2-ASP is assigned a score and totals for each fish is tabulated by condition and presented as a box plot, showing significantly lower scores for knockdown embryos when compared with WT. C) Two human sequence constructs of WNK1/HSN2 were designed for the rescue of the knockdown phenotype: a partial sequence containing exons 1-HSN2 and a complete construct containing exons 1–28 (but missing exons 11 and 12). D) 4-di-2-ASP score was assessed for embryos injected with MO-hsn2-SB3′ and concentrations of 30, 50 or 75 ng/µl of either partial or complete human WNK1/HSN2 constructs, revealing a partial rescue of the knockdown phenotype for embryos injected with 50 and 75 ng/µl of the complete construct. Scale bar: 100 µm.</p