Overview of the investigated WTCCC data sets.

<p>The SNPs were genotyped with an Affymetrix GeneChip 500K. The 1958 British Birth cohort with 1,504 samples was used as control.</p

Consistently significant pathways.

<p>To avoid that a pathway is only significant due to a small number of significant SNPs, we performed the multistage integrative pathway (MIP) analysis pipeline four times with different constraints. In the first MIP run, all SNPs are included. In the next three runs, only those having a <i>p</i>-value smaller than a threshold of 10⁻³, 10⁻⁴, and 10⁻⁵, respectively, are included. This table lists all pathways that had a <i>p</i>-value smaller than 0.05 during all four MIP runs. The complete results of each disease are shown in supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078577#pone.0078577.s003" target="_blank">Tables S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078577#pone.0078577.s004" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078577#pone.0078577.s005" target="_blank">S4</a>, and a more detailed overview of all four MIP runs which also includes a comparison to the literature is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078577#pone.0078577.s006" target="_blank">Table S5</a>.</p

Pathway set creation example.

<p>This example shows how the different pathway sets are built for a given pathway <i>x</i>. The pathway <i>x</i> is depicted as blue rectangle and the genes 1 to 5 as orange rectangles. For the pathway <i>x</i>, six different pathway sets are created. First, the <b>pathway set</b> containing the SNPs of all genes occurring in pathway <i>x</i>. Second, the <b>characteristic pathway set</b> that only contains the SNPs of those genes occurring exclusively in pathway <i>x</i>, i.e., genes 2, 3, 4 and 5. Since gene 1 also shows up in pathways <i>y</i> and <i>z</i>, the SNPs of these genes are not considered in the characteristic pathway interaction set. Third, two <b>pathway interaction sets</b> are created: first, the ultra-set for the SNPs of the genes assigned to the EV interaction class and second, the high-set with SNPs of genes assigned to the HC and EV class. Finally, the <b>characteristic interaction pathway sets</b> are generated. These sets are built similar to the interaction pathway sets but they contain only those SNPs of genes occurring exclusively in pathway <i>x</i>. In contrast to the ultra interaction set, the ultra characteristic interaction set does not include the SNPs 1, 2 and 3 because the corresponding gene also occurs in pathways <i>y</i> and <i>z</i>.</p

Definition of the interaction pathway sets.

<p>We built four interaction pathway sets: ultra (yellow), high (red), medium (blue) and low (green) depending on the interaction classes of the genes. These interaction classes are either EV (experimentally validated), HC (high interaction confidence), MC (medium interaction confidence) and LC (low interaction confidence). The low interaction set is the superset of all interaction sets because it includes genes of all interaction classes. In contrast, the smallest ultra set only contains the genes of the EV class.</p

Established analysis pipeline for a multistage integrative pathway analysis.

<p>The analysis consists of three steps: 1) Construction of the SNP data structure, 2) Creation of the pathway sets, and 3) evaluation of these sets and determination of the best ones. The data structure is built by mapping all SNPs to their corresponding gene. Depending on the domain-interactions of the gene's proteins, each gene is assigned to a gene interaction class, which describes the number of interactions and the interaction confidence of the encoded proteins. For this purpose, information from UniProt, Pfam, DOMINE, and KEGG is used. Additionally, the KEGG pathways of the genes are determined. In the second step, four different pathway sets are built (for more details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078577#pone-0078577-g003" target="_blank">Figure 3</a>). In the final step, these sets are statistically evaluated with a variation of the Fisher's test statistic. Since there are several pathway sets built for one pathway, a best list is determined containing exclusively one set per pathway which has a <i>p</i>-value smaller or equal 0.05.</p

Consistency of pathway sets generated with the proposed analysis methods.

<p>The procedure described in this manuscript includes multiple analysis methods to identify significant pathways that are related to the phenotype of a given GWAS. This diagram shows with which analysis methods the consistent pathway sets that are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078577#pone-0078577-t002" target="_blank">Table 2</a> have been determined. In average, 61% of the sets are determined with the interaction methods. In contrast, the characteristic interaction methods only identified less significant pathway sets. Concluding, it is more important to focus on the interaction based methods for the identification of important SNP sets.</p

Video_8_Case Report: Deep brain stimulation improves tremor in FGF-14 associated spinocerebellar ataxia.MP4

ObjectivesSpinocerebellar ataxia 27 (SCA 27) is a rare heredodegenerative disorder caused by mutations in the fibroblast growth factor 14 (FGF14) and characterized by early-onset tremor and progressive ataxia later during the disease course. We investigated the effect of deep brain stimulation (DBS) of the ventralis intermedius nucleus of the thalamus (VIM) and subthalamic projections on tremor and ataxia.MethodsAt baseline, we studied the effects of high-frequency VIM stimulation and low-frequency stimulation of subthalamic projections on tremor and ataxia. The patient then adopted the best individual high-frequency stimulation programme at daytime and either 30 Hz-stimulation of the subthalamic contacts or StimOFF at night during two separate 5-weeks follow-up intervals. Both patient and rater were blinded to the stimulation settings.ResultsHigh-frequency stimulation of the VIM effectively attenuated tremor. At follow-up, intermittent 30 Hz-stimulation at night resulted in a superior tremor response compared to StimOFF at night. Ataxia was not affected.DiscussionStimulation of the VIM and adjacent subthalamic projections effectively attenuated tremor in a patient with confirmed SCA 27. Cycling between daytime high-frequency and night-time low-frequency stimulation led to a more sustained tremor response. This suggests to study in future if low-frequency stimulation of the subthalamic projection fibers may help overcome tolerance of tremor that is observed as a long-term limitation of VIM-DBS.</p

Establishing conditions that maintain CD surface marker labeling with detection of intracellular antigens.

<p>CD24 surface antigen detection on SH-SY5Y cells remained largely stable with 4% PFA fixation after staining (<b>A</b>, <b>B</b>), yet was greatly reduced with permeabilization using Triton X-100(C, D). Lowering PFA concentration combined with Tween-20 for permeabilization restored CD24 surface staining to levels approximating those seen on live cells (<b>E</b>, <b>F</b>; see <b>A</b>). Optimizing fixation and permeabilization (<b>H</b> to <b>K</b>) enabled simultaneous detection of intracellular antigens, here TUJ1 (β-III-tubulin) while preserving cell surface staining (see <b>F</b>, <b>K</b>). Panel (<b>G</b>) provides a quantitative overview of the different conditions (i-vi indicating the conditions as labeled in <b>A</b> to <b>F</b>). Incubation of live or solely fixed cells with antibodies targeting intracellular epitopes without permeabilization results in no detection. Representative scatter plots for ≥ three independent experimental repeats are shown.</p

Video_1_Case Report: Deep brain stimulation improves tremor in FGF-14 associated spinocerebellar ataxia.MP4

ObjectivesSpinocerebellar ataxia 27 (SCA 27) is a rare heredodegenerative disorder caused by mutations in the fibroblast growth factor 14 (FGF14) and characterized by early-onset tremor and progressive ataxia later during the disease course. We investigated the effect of deep brain stimulation (DBS) of the ventralis intermedius nucleus of the thalamus (VIM) and subthalamic projections on tremor and ataxia.MethodsAt baseline, we studied the effects of high-frequency VIM stimulation and low-frequency stimulation of subthalamic projections on tremor and ataxia. The patient then adopted the best individual high-frequency stimulation programme at daytime and either 30 Hz-stimulation of the subthalamic contacts or StimOFF at night during two separate 5-weeks follow-up intervals. Both patient and rater were blinded to the stimulation settings.ResultsHigh-frequency stimulation of the VIM effectively attenuated tremor. At follow-up, intermittent 30 Hz-stimulation at night resulted in a superior tremor response compared to StimOFF at night. Ataxia was not affected.DiscussionStimulation of the VIM and adjacent subthalamic projections effectively attenuated tremor in a patient with confirmed SCA 27. Cycling between daytime high-frequency and night-time low-frequency stimulation led to a more sustained tremor response. This suggests to study in future if low-frequency stimulation of the subthalamic projection fibers may help overcome tolerance of tremor that is observed as a long-term limitation of VIM-DBS.</p

Experimental outline.

<p>Schematic illustrating the research strategy of identifying novel surface marker combinations on a target population in neural and other stem cell differentiation systems for which intracellular, standard immunocytochemical markers are well established. Following harvesting, the resulting single cell suspension is subject to surface antigen candidate staining, followed by gentle fixation, permeabilization and subsequent co-staining with known intracellular markers. CD markers co-labeling the target population serve as positive markers, those absent on the target population serve as negative markers. In a separate, subsequent step, a combination of the identified positive and/or negative CD markers enables the flow cytometric enrichment of the viable population of interest from a heterogeneous cell suspension for further study and biomedical applications.</p