Genetic interactions between <i>alfa-1(ok3062)</i>, <i>TDP-43</i>, and FUS.

<div><p>(A) <i>TDP-43</i>^<i>A315T</i>; <i>alfa-1(ok3062)</i> worms had a higher rate of paralysis that either <i>TDP-43</i>^<i>A315T</i> or <i>alfa-1(ok3062)</i> worms alone (P<0.005).</p> <p>(B) <i>FUS</i>^S57∆ worms, <i>alfa-1(ok3062)</i> worms, and <i>FUS</i>^S57∆; <i>alfa-1(ok3062)</i> worms showed similar rates of paralysis.</p></div

Deletion of C9ORF72 Results in Motor Neuron Degeneration and Stress Sensitivity in <i>C. elegans</i>

<div><p>An expansion of the hexanucleotide GGGGCC repeat in the first intron of <i>C9ORF72</i> gene was recently linked to amyotrophic lateral sclerosis. It is not known if the mutation results in a gain of function, a loss of function or if, perhaps both mechanisms are linked to pathogenesis. We generated a genetic model of ALS to explore the biological consequences of a null mutation of the <i>Caenorhabditis elegans C9ORF72</i> orthologue, <i>F18A1.6</i>, also called <i>alfa-1</i>. <i>alfa-1</i> mutants displayed age-dependent motility defects leading to paralysis and the specific degeneration of GABAergic motor neurons. <i>alfa-1</i> mutants showed differential susceptibility to environmental stress where osmotic stress provoked neurodegeneration. Finally, we observed that the motor defects caused by loss of <i>alfa-1</i> were additive with the toxicity caused by mutant TDP-43 proteins, but not by the mutant FUS proteins. These data suggest that a loss of <i>alfa-1/C9ORF72</i> expression may contribute to motor neuron degeneration in a pathway associated with other known ALS genes.</p> </div

<i>alfa-1(ok3062)</i> mutants are sensitive to osmotic stress.

<div><p>(A) In thermal stress resistance assays, <i>alfa-1(ok3062)</i> mutants were indistinguishable from wild type N2 worms, and <i>daf-2</i>(e1370) were not statistically different <i>alfa-1(ok3062)</i>;<i>daf-2</i>(e1370) mutants.</p> <p>(B) In oxidative stress resistance assays, <i>alfa-1(ok3062)</i> mutants were indistinguishable from wild type N2 worms, and <i>daf-2</i>(e1370) were not statistically different <i>alfa-1(ok3062)</i>;<i>daf-2</i>(e1370) mutants.</p> <p>(C) <i>alfa-1(ok3062)</i> mutants were more sensitive to osmotic stress than N2 worms (P<0.005), while <i>alfa-1(ok3062)</i>;<i>daf-2</i>(e1370) worms are slightly more sensitive when compared to <i>daf-2</i>(e1370) worms alone.</p> <p>(D) The difference in sensitivity between <i>alfa-1(ok3062)</i>;<i>daf-2</i>(e1370) and <i>daf-2</i>(e1370) increases at 500 mM NaCl (P<0.005). At 600 mM NaCl, the effect of NaCl is too drastic to see a difference.</p> <p>(E) During thermal stress, <i>alfa-1(ok3062)</i> worms are not more sensitive to neurodegeneration than the <i>unc-47p::GFP</i> worms.</p> <p>(F) When exposed to 400 mM NaCl, <i>alfa-1(ok3062)</i> worms had a higher rate of neurodegeneration than <i>unc-47p::GFP</i> worms (*P<0.05).</p></div

ALFA-1 is the orthologue of C9ORF72 in <i>C. elegans</i>.

<div><p>(A) Protein sequence alignment using Clustal W and BoxShade of C9ORF72 isoform 1 and ALFA-1 isoform 1. Overall, these sequences share 26% identify and 59 % similarity. </p> <p>(B) ALFA-1 has two predicted transcripts and the <i>ok3062</i> deletion mutation spans exons 3 and 4 for both transcripts.</p> <p>(B) RT-PCR confirming the complete loss of expression of the <i>alfa-1</i> transcripts. <i>act-3</i> was used as a control.</p></div

Age-dependent motility defects and neurodegeneration in <i>alfa-1(ok3062)</i> mutants.

<div><p>(A) <i>alfa-1(ok3062)</i> mutants showed motility defects leading to paralysis of 60% of the population by day 12 of adulthood compared to 20% for N2 worms (P<0.0001).</p> <p>(B) <i>alfa-1(ok3062)</i> worms are more sensitive to aldicarb-induced paralysis than N2 worms (P<0.0001).</p> <p>(C) Percentage of wild type N2 or <i>alfa-1(ok3062)</i> worms displaying a swimming-induced paralysis phenotype after 8 hours in liquid culture (** P<0.001).</p> <p>(D) Example of gap (indicated by arrow) along a neuronal process in animals expressing the <i>unc-47p::GFP</i> reporter.</p> <p>(E) Quantification of neurodegeneration at day 9 of adulthood associated with <i>alfa-1(ok3062)</i> in different neuronal populations including cholinergic neurons marked by <i>unc-17p::GFP</i>, dopaminergic neurons visualized with <i>dat-1p::GFP</i>, or GABAergic neurons revealed by <i>unc-47p::GFP</i>. Significant neurodegeneration was observed in the GABAergic neurons of <i>alfa-1(ok3062)</i> mutants (**P<0.001).</p></div

Lithium response in lymphoblastoid cell line samples.

<p>ANCOVA analysis was performed to compare the three groups (Controls, Responders, and Non-responders to lithium treatment) separately for Syn2a and Syn2b expression. The variables “Age at sampling” and “LCL frozen storage” were used as covariates.</p

Cell lines expression for SYN2a.

<p>Expression in (A) HEK293 embryonic kidney cells, (B) SK-N-AS neuroblastoma cells, and (C) and U-118 MG glioblastoma/astrocytoma cells for the Synapsin IIa variant compared to GAPDH. P-values depicting the mean differences between 3 independent experiments for each cell line at each of the 3 treatment concentration of either lithium or vehicle (0.5 mM, 1.0 mM, and 2.0 mM).</p

Cell lines expression for SYN2b.

<p>Expression in (A) HEK293 embryonic kidney cells, (B) SK-N-AS neuroblastoma cells, and (C) and U-118 MG glioblastoma/astrocytoma cells for the Synapsin IIb variant compared to GAPDH. P-values depicting the mean differences between 3 independent experiments for each cell line at each of the 3 treatment concentration of either lithium or vehicle (0.5 mM, 1.0 mM, and 2.0 mM).</p

The Impact of Phenotypic and Genetic Heterogeneity on Results of Genome Wide Association Studies of Complex Diseases

<div><p>Phenotypic misclassification (between cases) has been shown to reduce the power to detect association in genetic studies. However, it is conceivable that complex traits are heterogeneous with respect to individual genetic susceptibility and disease pathophysiology, and that the effect of heterogeneity has a larger magnitude than the effect of phenotyping errors. Although an intuitively clear concept, the effect of heterogeneity on genetic studies of common diseases has received little attention. Here we investigate the impact of phenotypic and genetic heterogeneity on the statistical power of genome wide association studies (GWAS). We first performed a study of simulated genotypic and phenotypic data. Next, we analyzed the Wellcome Trust Case-Control Consortium (WTCCC) data for diabetes mellitus (DM) type 1 (T1D) and type 2 (T2D), using varying proportions of each type of diabetes in order to examine the impact of heterogeneity on the strength and statistical significance of association previously found in the WTCCC data. In both simulated and real data, heterogeneity (presence of “non-cases”) reduced the statistical power to detect genetic association and greatly decreased the estimates of risk attributed to genetic variation. This finding was also supported by the analysis of loci validated in subsequent large-scale meta-analyses. For example, heterogeneity of 50% increases the required sample size by approximately three times. These results suggest that accurate phenotype delineation may be more important for detecting true genetic associations than increase in sample size.</p></div

Genome-wide analysis of the Wellcome Trust Case-Control Consortium (WTCCC) data for diabetes type 1 (T1D) and type 2 (T2D) under heterogeneity: twenty most significant associations [−log(p-values)].

<p>SNP = single nucleotide polymorphism; β = admixture.</p