Validation array coverage.

<p>Validation array coverage.</p

Comparison of hematopoietic potential of iPSCs to mouse ESCs.

<p>Fractions of Lin⁻ cells (A), KLS cells (B), Kit+Lin-Sca-1- Progenitors (C), GMPs (D), CMPs (E), and MEPs (F) from iPSCs relative to mouse ESCs after 7 days of OP9 coculture (unsorted).</p

Sequencing of iPSC clones.

<p><b>A.</b> Number of variants identified per iPSC clone. <b>B.</b> Variant allele frequencies of all validated mutations for each clone. Samples are ranked by the number of variants in decreasing order. No mutations were identified in Ax2-11, thus it is not listed in panel B.</p

Generation of iPSC clones from a single mouse C57BL/6 male mouse.

<p><b>A.</b> Schematic of experimental approach (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120585#sec002" target="_blank">Material and Methods</a> for full protocol). <b>B.</b> Representative bright-field images (left) and alkaline-phosphastase stains (right) of B6/BLU ESCs (top) and a representative iPSC (bottom, Ax1-10). All images at 100x magnification. <b>C.</b> Images from the teratoma derived from Ax1-10 demonstrating ectoderm (neural tissue), mesoderm (cartilage) and endoderm (ciliated respiratory epithelium).</p

Hematopoietic differentiation potential of the 24 iPSC clones.

<p>100,000 cells from OP9 cocultured mESCs (B6/BLU) or iPSCs were plated in methylcellulose media containing hematopoietic cytokines (SCF, IL-3, IL-6, and Epo). <b>A.</b> CFUs were counted after 7 additional days of culture. The relative number of CFUs per 100,000 cells plated from Day7 iPSC-derived progenitors vs. Day7 ESC (B6/BLU)-derived progenitors are shown. iPSC clones are ranked from the highest to the lowest average of two independent experiments. Error bars represent the means +/− one standard deviation. <b>B.</b> Morphology of day 7 OP9 cocultured ESC-derived cells after 7–8 days of additional culture in MethoCult media containing hematopoietic cytokines (SCF, IL-3, IL-6, and Epo). A scale bar of 20 μm is shown. <b>(C-E)</b>. Fractions of CD11b⁺ (<b>C</b>), CD34⁺Kit⁺ (<b>D</b>), and Ter119⁺ (<b>E</b>) cells obtained after 7 days of methylcellulose culture containing hematopoietic cytokines (SCF, IL-3, IL-6, and Epo), comparing iPSC-derived progenitors relative to ESC-derived progenitors, in the same order as panel A. <b>F.</b> Lack of correlation between the number of mutations and the hematopoietic differentiation potential of the iPSC clones (r² = 0.0006065).</p

Hematopoietic differentiation from murine ESCs.

<p><b>A.</b> Morphology of wild type ESC-derived cells after 7 days of OP9 coculture (unsorted) by Wright-Giemsa staining. A scale bar of 20 μm is shown. (B-D). Immunophenotyping of hematopoietic progenitor cells from wild-type mouse bone marrow cells (panel B), murine ESCs after 7 days of OP9 coculture (panel C), and iPSC clone Ax1-14 after 7 days of OP9 coculture (panel D). Lineage⁻ (Lin⁻), KLS (Lin⁻Kit⁺Sca-1⁺), progenitors (Lin⁻Kit⁺Sca-1⁻), CMPs (Lin⁻Kit⁺Sca-1⁻CD34⁺FCγ⁻), GMPs (Lin⁻Kit⁺Sca-1⁻CD34⁺FCγ⁺), and MEPs (Lin⁻Kit⁺Sca-1⁻CD34⁻FCγ-).</p

Whole exome sequencing coverage.

<p>Whole exome sequencing coverage.</p

<i>TYK2</i> protein-coding variants identified by exon-sequencing of RA cases and controls.

<p>Using dense genotyping, we demonstrate that three <i>TYK2</i> protein-coding variants predicted to be damaging, P1104A, A928V, and I684S, protect against RA (highlighted in red). By exon-sequencing in 1,118 RA cases and 1,118 controls, we identified 23 additional missense variants predicted to be damaging (PolyPhen-2 and SIFT), with no strong evidence of association to RA in gene-based association tests. The <i>TYK2</i> coding exons, the protein domains, and the minor allele count (MAC) of the rare variants (MAC<5) in cases and controls are shown.</p

Results from stepwise conditional analysis of the <i>TYK2</i> locus.

<p>We fine-mapped the <i>TYK2</i> locus using Immunochip data available for 7,222 ACPA+ RA cases and 15,870 controls (MAF>0). (A) In the meta-analysis, the best signal of association was at the <i>TYK2</i> missense variant P1104A (rs34536443).(B) Conditional on P1104A, the best signal of association was at the <i>TYK2</i> missense variant A928V (rs35018800). (C) Conditional on P1104A and A928V variants, the best signal of association is at the <i>TYK2</i> missense variant I684S (rs12720356). (D) Conditional on the 3 RA-protective variants in <i>TYK2</i>, we observed no additional signal of association at the locus (best signal is rs3176768, P = 0.01). P-values from meta-analyses of logistic regressions results from 6 Immunochip collections are shown. The three <i>TYK2</i> missense variants predicted to be damaging and independently associated with RA risk are highlighted in green.</p