<i>MEK1</i> sequence variations identified in ovarian cancer cell lines.

*<p><a href="http://www.ncbi.nlm.nih.gov/SNP/" target="_blank">www.ncbi.nlm.nih.gov/SNP/</a></p>**<p>blocks.fhcrc.org/sift/SIFT.html</p

<i>MEK1</i> and <i>MEK2</i> Sequencing Primers.

<p><i>MEK1</i> and <i>MEK2</i> Sequencing Primers.</p

Electropherograms of <i>BRAF</i> and <i>MEK1</i> mutations compared to normal controls.

<p>Four <i>BRAF</i> mutations were identified in four individual cell lines. A) OVCAR 10 contained a nt 603 G→T transversion causing a heterozygous missense substitution p.Q201H in exon 4. B) OV90 contained a novel heterozygous deletion starting at nt 1457 (arrow) resulting in a 5 amino acid deletion, p.N486-P490del, in exon 12. C) Hey contained a nt 1391 G→A transition resulting in loss of heterozygosity. D) ES-2 contained an exon 15, T→A transversion at nt 1799, substituting glutamic acid for valine at position 600 (p.V600E). E) A nt 199 G→A transition in <i>MEK1,</i> exon 2 resulted in a heterozygous missense substitution, p.D67N.</p

<i>BRAF</i> Sequencing Primers.

<p><i>BRAF</i> Sequencing Primers.</p

Functional characterization of the MEK1 p.D67N mutant identified in ES-2.

<p>Human embryonic kidney 293T cells were transiently transfected with empty vector, wild-type MEK1, MEK1 p.Y130C (positive control mutant which has known high activity level<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001279#pone.0001279-RodriguezViciana1" target="_blank">[18]</a>) and the MEK1 p.D67N mutant. ERK (p44 ERK1 and p42 ERK2) phosphorylation was assayed by Western blotting using phospho-specific antibodies. The p.D67N MEK1 mutant had increased ERK phosphorylation compared to the level induced by empty vector and wildtype MEK1. The level of ERK phosphorylation induced by p.D67N MEK1 is slightly less than the CFC MEK1 p.Y130C mutant which is known to have increased activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001279#pone.0001279-RodriguezViciana1" target="_blank">[18]</a>. Myc-tagged MEK1 is shown for transfection efficiency and total ERK is shown as a loading control.</p

<i>MEK2</i> synonymous SNPs identified in ovarian cancer cell lines.

*<p><a href="http://www.ncbi.nlm.nih.gov/SNP/" target="_blank">www.ncbi.nlm.nih.gov/SNP/</a></p>**<p>blocks.fhcrc.org/sift/SIFT.html</p

Reverse Pathway Genetic Approach Identifies Epistasis in Autism Spectrum Disorders

<div><p>Although gene-gene interaction, or epistasis, plays a large role in complex traits in model organisms, genome-wide by genome-wide searches for two-way interaction have limited power in human studies. We thus used knowledge of a biological pathway in order to identify a contribution of epistasis to autism spectrum disorders (ASDs) in humans, a reverse-pathway genetic approach. Based on previous observation of increased ASD symptoms in Mendelian disorders of the Ras/MAPK pathway (RASopathies), we showed that common SNPs in RASopathy genes show enrichment for association signal in GWAS (<i>P</i> = 0.02). We then screened genome-wide for interactors with RASopathy gene SNPs and showed strong enrichment in ASD-affected individuals (<i>P</i> < 2.2 x 10⁻¹⁶), with a number of pairwise interactions meeting genome-wide criteria for significance. Finally, we utilized quantitative measures of ASD symptoms in RASopathy-affected individuals to perform modifier mapping via GWAS. One top region overlapped between these independent approaches, and we showed dysregulation of a gene in this region, <i>GPR141</i>, in a RASopathy neural cell line. We thus used orthogonal approaches to provide strong evidence for a contribution of epistasis to ASDs, confirm a role for the Ras/MAPK pathway in idiopathic ASDs, and to identify a convergent candidate gene that may interact with the Ras/MAPK pathway.</p></div

Ras/MAPK ASD epistasis top results.

<p>The unique epistatic SNP pairs with <i>P</i> < 2.9x10^-9 are listed in the table. For each SNP, the following is listed in order of columns: rsID (Epistatic SNP), chromosome (CHR), position (BP, reference version hg19), minor allele frequency in the ASD dataset (MAF), nearest gene to the epistatic SNP, Ras/MAPK gene associated with the interacting SNP, and <i>P</i>-value for epistasis in cases (Epistasis ASD <i>P</i>) and pseudo-controls (Epistasis Control <i>P</i>). Locus pairs meeting genome-wide significance criteria (<i>P</i> < 7.6 x 10⁻¹⁰) are bolded. Main effects for epistatic and Ras/MAPK SNPs listed here are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006516#pgen.1006516.s004" target="_blank">S4 Table</a>, with no SNPs showing <i>P</i> < 0.01.</p

Comparison of number of Ras/MAPK gene epistasis results in ASD cases versus pseudo-controls.

<p>The graph displays number of epistasis tests (y-axis) in the ASD cases (dark gray, circle) and ASD pseudo-controls (light gray, triangle) with <i>P</i>-value thresholds (x-axis, left to right): <i>P</i> < 2.9x10^-9, < 1.0x10^-8, <i>P</i> < 1.0x10^-7, <i>P</i> < 1.0x10^-6, <i>P</i> < 1.0x10^-5, and <i>P</i> < 1.0x10^-4. The 2x2 chi-square test <i>P</i>-value and odds ratio (OR) are included for the epistasis results meeting nominal significance (<i>P</i> < 10⁻⁶).</p

Social responsiveness association in RASopathy top results.

<p>The independent SNPs with social responsiveness score (SRS) association in RASopathy (random effects meta-analysis <i>P</i> < 1.0x10^-4) are listed. The data underlying the top six candidate modifiers are graphically illustrated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006516#pgen.1006516.s014" target="_blank">S6 Fig</a>. For each SNP, the following is listed in order of columns: SNP rsID, chromosome (CHR), position (BP, reference version hg19), minor allele frequency in the dataset (MAF), groups contributing to the RASopathy association (group with the most significant association <i>P</i>-value is listed first and groups with similar direction of effect are in parentheses), Cochran’s Q <i>P</i>-value for all four RASopathy groups, RASopathy (CFC, CS, NF1, and NS) SRS association (random effects meta-analysis) <i>P</i>-value, control sibling SRS <i>P</i>-value (linear regression), gene(s) containing or flanking SNP.</p