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

Linkage disequilibrium (LD) map of the region chromosome 7: 37.7Mb– 38.1 Mb.

<p>The graph displays LD between the SNPs rs114490548 (<i>P</i> = 7.8 x10^-11, Ras/MAPK ASD epistasis analysis) and rs62621010 (<i>P</i> = 5.6x10^-7, RASopathy QTL analysis). LD (D′) values for each pairwise comparison of SNPs were calculated based on LD and recombination rate data in 1000 Genomes May 2013 release and plotted using HAPLOVIEW(126) (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006516#sec027" target="_blank">web resources</a>) default settings and standard color theme. The red color corresponds to D’ = 1 and log of odds (LOD) ≥ 2, white corresponds to D’<1 and LOD <2, and blue to D’ = 1 and LOD<2.</p

Enrichment of Ras/MAPK SNPs in ASD association results.

<p>The histogram displays the distribution of results with percent of SNPs meeting q = 0.2 for 100 randomly permuted gene sets compared to the Ras/MAPK SNP set (dashed line, 9.05%) in an ASD transmission disequilibrium test (TDT) for association. The y-axis displays the proportion of permutation results; the x-axis displays the percent SNPs meeting q = 0.2.</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

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

Gene expression in neural cell lines.

<p>For the genes <i>ELMO1</i>, <i>GPR141</i>, <i>SFRP4</i>, <i>EPDR1</i>, <i>and STARD3NL</i>, the graph displays the normalized mRNA expression relative to controls measured by qPCR for two independent experiments. <i>NME8</i> had undetermined quantities in the first experiment (1) and extreme variance in the second experiment (2) due to low expression level, and therefore was excluded from the graph (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006516#pgen.1006516.s003" target="_blank">S3 Table</a>). The error bars denote the standard error of the sample measurements, and the asterisk denotes a t-test <i>P</i>-value < 0.05 between CFC (dark gray) and control (light gray).</p

Novel Molecular Pathways Elicited by Mutant FGFR2 May Account for Brain Abnormalities in Apert Syndrome

<div><p>Apert syndrome (AS), the most severe form craniosynostosis, is characterized by premature fusion of coronal sutures. Approximately 70% of AS patients carry S252W gain-of-function mutation in <i>FGFR2</i>. Besides the cranial phenotype, brain dysmorphologies are present and are not seen in other <i>FGFR2</i>-asociated craniosynostosis, such as Crouzon syndrome (CS). Here, we hypothesized that S252W mutation leads not only to overstimulation of FGFR2 downstream pathway, but likewise induces novel pathological signaling. First, we profiled global gene expression of wild-type and S252W periosteal fibroblasts stimulated with FGF2 to activate FGFR2. The great majority (92%) of the differentially expressed genes (DEGs) were divergent between each group of cell populations and they were regulated by different transcription factors. We than compared gene expression profiles between AS and CS cell populations and did not observe correlations. Therefore, we show for the first time that S252W mutation in FGFR2 causes a unique cell response to FGF2 stimulation. Since our gene expression results suggested that novel signaling elicited by mutant FGFR2 might be associated with central nervous system (CNS) development and maintenance, we next investigated if DEGs found in AS cells were also altered in the CNS of an AS mouse model. Strikingly, we validated <i>Strc</i> (stereocilin) in newborn Fgfr2^S252W/+ mouse brain. Moreover, immunostaining experiments suggest a role for endothelial cells and cerebral vasculature in the establishment of characteristic CNS dysmorphologies in AS that has not been proposed by previous literature. Our approach thus led to the identification of new target genes directly or indirectly associated with FGFR2 which are contributing to the pathophysiology of AS.</p> </div

Human periosteal fibroblast experiments.

<p>(A) Validation of differentially expressed genes showing the correlation between fold-changes obtained from the Affymetrix microarray experiment and the fold-change values for each gene in each cell line. The correlations between the values of microarray and qRT-PCR fold-changes were calculated through Spearman correlation test. (B) Immunofluorescence staining of TCF19 (green) in two lineages of S252W fibroblasts not included in microarray experiment after 24 h treatment with PBS (control) or FGF2. Blue staining refers to nuclei (DAPI), magnification: 10×; scale bar = 500 µm. (C) Fold-change of the mRNA levels of <i>BAT3</i>, <i>BDP1</i>, <i>CYP51A1</i>, <i>RFC3</i> and <i>TCF19</i> in FGF2 treated C342Y human fibroblasts and S252W human fibroblasts. Note that there was no <i>TCF19</i> expression detected in C342Y human fibroblasts.</p