Characteristics of Patients with Sporadic CAA.

<p>Characteristics of Patients with Sporadic CAA.</p

Boston criteria for CAA diagnosis.

<p>*As defined in reference 20.</p>‡<p>Other causes of intracerebral hemorrhage include:</p><p>• excessive warfarin dosing (INR >3.0)</p><p>• antecedent head trauma or ischemic stroke</p><p>• CNS tumor</p><p>• vascular malformation</p><p>• CNS vasculitis</p><p>• blood dyscrasia</p><p>• coagulopathy.</p><p>Note: INR.3.0 or other nonspecific laboratory abnormalities permitted for diagnosis of possible CAA.</p

EGFR Missense Mutations Are Transforming and Tumorigenic

<div><p>(A) Anchorage-independent growth of NIH-3T3 cells expressing various <i>EGFR</i> alleles as mean number of colonies ± standard deviation (bar graph, above). The lanes (below) show EGFR and actin immunoblots of whole cell lysates from NIH-3T3 subclones plated in soft agar. EGF (10 ng/ml) was added to the top agar where indicated.</p> <p>(B) Tumorigenicity of NIH-3T3 cells stably expressing the indicated <i>EGFR</i> alleles in nude mice. Mean tumor size ± standard deviation was determined 3–4 wk after subcutaneous inoculation into nude mice (<i>n</i> = 6 per cell line).</p></div

EGFR Missense Mutations in Glioblastoma Cluster in the Extracellular Domain and Are Associated with Increased <i>EGFR</i> Gene Dose

<div><p>(A) Location of missense mutations within the EGFR protein in a panel of 151 gliomas (132 glioblastomas, 11 WHO grade III gliomas, and eight glioblastoma cell lines). Each diamond represents one sample harboring the indicated mutation. Amino acid (AA) numbers are based on the new convention for EGFR numbering, which starts at the initiator methionine of pro-EGFR. Ligand-binding domains (I and III), cysteine-rich domains (II and IV), kinase domain (kinase), and the extracellular deletion mutant EGFRvIII [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030485#pmed-0030485-b045" target="_blank">45</a>] are indicated as reference.</p> <p>(B) Increased <i>EGFR</i> gene dose in tumors harboring <i>EGFR</i> missense mutations. The array (left) shows a high-resolution view of Affymetrix 100K SNP array at the <i>EGFR</i> gene locus for ten glioblastoma tumors and three normal controls (sample numbers are indicated above each column). <i>EGFR</i> mutation and log₂ ratio (see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030485#st2" target="_blank">Methods</a>) are indicated below each column. The plot (left) shows a comparison of <i>EGFR</i> gene copy number determination by SNP array (y-axis, EGFR log₂ ratios) and FISH (x-axis). AMP, amplified; NON-AMP, non amplified.</p> <p>(C) RT-PCR for <i>EGFRvIII</i> and full-length <i>EGFR</i> in 14 fresh-frozen glioblastoma tumors (see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030485#st2" target="_blank">Methods</a>). The upper band represents full-length <i>EGFR</i> (1,044 bp), the lower band <i>EGFRvIII</i> (243 bp), and the inset shows glyceraldehyde-3-phosphate dehydrogenase <i>(GAPDH)</i> RT-PCR results.</p></div

<i>EGFR</i> Missense Mutations Sensitize Cells to EGFR Kinase Inhibitors

<div><p>(A) Effect of increasing concentrations of the EGFR inhibitor erlotinib (0–10 μM) on the viability of IL-3 independent Ba/F3 subclones expressing EGFR ectodomain mutants (R108K, T263P, A289V, G598V, and EGFRvIII), the EGFR kinase domain mutants (L858R and L861Q), or the erlotinib-resistant EGFR double mutant L858R-T790M (LTM). Parental Ba/F3 cells and Ba/F3 cells expressing wild-type EGFR are not IL-3 independent and were included as controls. Viability (a mean percent of control ± standard deviation) was determined after exposure to erlotinib for 48 h.</p> <p>(B) Oncogenic EGFR ectodomain mutations map to interdomain interfaces. Shown are ribbon and surface diagrams of the EGFR [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030485#pmed-0030485-b046" target="_blank">46</a>] with sites of amino acid substitutions highlighted. Blue, domain I; green, domain II; red, domain III; and yellow, domain IV. Sites of the most prevalent amino acid substitutions are shown in red. Images were created with PyMOL (<a href="http://pymol.sourceforge.net/" target="_blank">http://pymol.sourceforge.net/</a>). P596 is not visible in this view.</p></div

<i>G6PC2</i> gene-based association with FG levels using SKAT and BURDEN test

<p><i>G6PC2</i> gene-based association with FG levels using SKAT and BURDEN test</p

Functional characterization of wild type and variant G6PC2 proteins.

<p>(A) Expression levels in HEK293 and (B) INS-1E cells were determined by western blot and densitometry analysis. The multiple bands on the western blot are likely to represent glycosylated G6PC2 protein products. Data are presented as mean ± standard error of the mean for at least three independent experiments. Significant differences are indicated as ** <i>P</i><0.01; *** <i>P</i><0.001; **** <i>P</i><0.0001. EV, empty vector; WT, wild type. (C) Expression levels in HEK293 and INS-1E cells in the presence of proteasomal inhibitor MG-132 or lysosomal inhibitor chloroquine were determined by western blot. (D) Cellular localization in HEK293 cells was assessed by immunofluorescence microscopy. Cells were double immunostained for FLAG tag (green) and calnexin (red), and merged images with a DNA stain (blue) are shown. Images were taken with laser settings that were optimized separately for each sample. Scale bar, 10µm.</p

Haplotypes of the lead non-coding GWAS SNP rs560887 and the three coding variants.

<p>rs138726309 (p.His177Tyr), rs2232323 (p.Tyr207Ser), and rs492594 (p.Val219Leu), obtained from 4,442 unrelated individuals from the Oxford Biobank. (A) Percentage minor allele frequency (MAF) and effect size estimates (<math><mi>β</mi><mo>^</mo></math>) of the four variants reported for the minor allele in mmol/L of FG after adjustment for age, sex, and BMI. (B) Haplotypes of the four associated variants in G6PC2 revealed that the glucose-lowering Leu219 allele was carried exclusively in cis with the glucose-raising allele at the GWAS SNP. Wild-type, glucose-raising alleles are circled in blue and the mutant, glucose-lowering alleles are circled in red. Diameter of the circle is proportional to the effect size estimates. Haplotype association was performed with FG derived residuals (after adjustment for age, sex, and BMI) using the most frequent haplotype as baseline.</p