FEV₁ survival adjusted CF specific percentiles (KNoRMA) distribution in CF subjects according to <i>AGER</i>

<p>-<b>429 T/C genotype.</b> Distribution of the lung function according to the FEV₁ adjusted on the age, the height and the mortality; expressed in KNoRMA in <i>AGER</i> -429 CC and CT carriers (light-grey bars, n = 258) and <i>AGER</i> -429 TT carriers (dark-grey bars, n = 709).</p

<i>In vitro</i> influence of <i>AGER</i>

<p>-<b>429T/C polymorphism on the promoter activity.</b> Constructs containing either <i>AGER</i>-<i>429C</i> or <i>AGER</i>-<i>429T</i> and a luciferase reporter gene were transfected into BEAS-2B (A) and A549 (B) cells. Luciferase activity assays were performed in triplicate in six independent experiments. Relative luciferase activity is represented as a ratio against the luciferase activity in cells transfected with the AGER-429T plasmid. Luciferase activity, reflecting activity of the <i>AGER</i> gene promoter, was significantly higher in cells containing <i>AGER</i>-<i>429C</i> plasmid compared to the cells containing <i>AGER</i>-<i>429T</i> plasmid (p = 0.016 and 0.031 respectively in BEAS-2B (A) and A549 (B) cells).</p

Clinical characteristics of the CF subjects from the French CF Gene Modifier Study overall and according to AGER -429T/C distribution.

<p>BMI: body mass index, FEV₁: forced expired volume in 1 second.</p

Lung development in late gestation IGF-1R^−/− mice.

<p><b>A–L</b>, Lungs prepared from IGF-1R^+/+ and IGF-1R^−/− embryos at developmental stages E14.5, E17.5 and E19.5. <b>A–F</b>, Ventral view of whole lungs. <b>G–L</b>, Rim of lung lobe. Abbreviations: AL, apical lobe; AzL, azygous lobe; CL, cardiac lobe; DL, diaphragmatic lobes; LL, left lobe. <b>M–X</b>, Lung histology of IGF-1R^+/+ versus IGF-1R^−/− embryos. H&E stained lung sections at developmental stages E14.5 (<b>M</b>–<b>P</b>), E17.5 (<b>Q</b>–<b>T</b>) and E19.5 (<b>U</b>–<b>X</b>), showing that saccular walls are thicker and acinar buds smaller in IGF-1R^−/− embryos as compared with controls of the same stage. Note that histomorphological appearance is similar when comparing E19.5 IGF-1R^−/− (<b>V</b>, <b>X</b>) with two days younger E17.5 IGF-1R^+/+ lungs (<b>Q</b>, <b>S</b>).</p

Lung histomorphology and cell turnover in the absence of IGF-1R.

<p><b>A</b>, Saccular airspace and <b>B</b>, saccular wall thickness (mean ± SEM) at developmental stages E17.5 in IGF-1R^+/+ (n = 4) and IGF-1R^−/− embryos (n = 4). Wilcoxon Mann-Whitney U test. <b>C–F,</b> Extended gestation period and lung histology. H&E stain of lung tissue from embryos at 19.5 (<b>C</b>, <b>D</b>) and 21.5 days (<b>E</b>, <b>F</b>). To extend gestation period up to 21.5 days, pregnant mothers were treated with progesterone from E17.5 onwards. Note the presence of red blood cell extravasation in E21.5 lung samples from IGF-1R^+/+ and IGF-1R^−/− mice. <b>G–O,</b> Cell turnover in IGF-1R^−/− embryonic lung at E17.5. Lung histology from IGF-1R^+/+ embryos (G, J and M) and IGF-1R^−/− embryos (H, K and N) at E17.5. Bar graphs (I, L and O) show quantification (mean ± SEM; n = 3–7 individuals per group; Student’s <i>t</i>-test). <b>G–I</b>, Cells were counted using DAPI staining (blue signal). <b>J–L</b>, Cell proliferation was measured using phospho–histone H3 immunohistochemistry (brown staining). <b>M–O</b>, Apoptosis was detected using cleaved caspase-3 immunohistochemistry (brown staining). Cleaved caspase-3 (P-U) and phospho-histone H3 labeling (V-AA) at high magnification showing examples for IHC-positive epithelial (red arrows), vascular endothelial (blue) and mesenchymal cells (green), as identified by their anatomical location. Note that many of the proliferating cells are located in areas that are composed of mostly mesenchymal cells.</p

Development of diaphragm and chest in the absence of IGF-1R.

<p><b>A</b>, Hematoxylin-eosin stained transversal section of thoracic wall and diaphragm in control (left) and IGF-1R^−/− embryos (right) at E17.5. Bar graphs compare <b>B</b>, diaphragm thickness, <b>C</b>, rib diameter, and <b>D</b>, diaphragm-to-rib ratio (mean ± SEM) in IGF-1R^+/+ embryos (n = 4) and IGF-1R^−/− embryos (n = 4). R, Rib; D, diaphragm. Wilcoxon Mann-Whitney U test.</p

Lung, heart, liver and kidney weight relative to body weight, in IGF-1R^+/+ and IGF-1R^−/− embryos.

<p>Values are mean ± SEM. * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001 compared with IGF-1R^+/+ mice. Student’s <i>t</i>-test.</p>a<p>n = 5; BW, body weight; ND, not determined.</p

IGF-1R protein levels, lung histology and respiratory function in adult IGF-1R^neo/− mice. A

<p>, Western immunoblot of IGF-1R in lung from mice with distinct combinations of mutant IGF-1R alleles. Total proteins were extracted from lung tissue from IGF-1R^neo/−, IGF-1R^+/−, IGF-1R^+/neo and IGF-1R^+/+ mice (n = 3 for each genotype), and IGF-1R^−/− embryo (negative control), and were probed with anti-IGF-1Rβ (upper panel) or anti-β-actin antibodies (lower panel). IGF-1R^neo/− mice have 22% of receptor levels present in IGF-1R^+/+ mice (quantified in B), IGF-1R^+/− have 50%, IGF-1R^+/neo mice are between 70 and 80%, and IGF-1R^−/− mice lack IGF-1R completely. <b>B</b>, IGF-1R abundance determined in lung tissue. Bar graph shows IGF-1R levels relative to β-actin from 5 IGF-1R^+/+ and 6 IGF-1R^neo/− individuals (Error bars SEM; Student’s <i>t</i>-test). Image shows 4 representative lanes from western immunoblot. <b>C</b>, Hematoxylin-eosin stained lung sections from IGF-1R^+/+ and IGF-1R^neo/− males. <b>D</b>, Alveolar airspace, <b>E</b>, alveolar boundary length density, <b>F</b>, alveolar wall thickness, in IGF-1R^+/+ (n = 4) and IGF-1R^neo/− mice (n = 4). Error bars indicate SEM; Wilcoxon Mann-Whitney U test. <b>G-I</b>, Respiratory function in adult IGF-1R^neo/− mice. Mice were challenged with 6% and 8% CO₂. <b>G</b>, Minute ventilation (V_E), <b>H</b>, tidal volume (V_T), and <b>I</b>, respiratory frequency (BR) were measured in 6 individuals per group. Differences between room air and hypercapnia were significant, but no significant differences were found between genotypes. Values labeled <i>b</i> were different from <i>a</i> (<i>P</i><0.005); Error bars indicate SEM; Wilcoxon Mann-Whitney U test.</p

Immunohistochemistry of lung differentiation markers.

<p><b>A</b> and <b>B,</b> Representative tissue sections from IGF-1R^+/+ and IGF-1R^−/− embryos at stage E17.5 showing CD31-immunoreactivity specific for capillary endothelia. <b>C</b>, Morphometric comparison of CD31 signal between genotypes (n = 5 per group; two-tailed <i>t</i>-test). <b>D–F</b>, Capillary complexity was estimated calculating the density of capillary junctions from CD31 IHC. <b>G–J</b>, Sections from IGF-1R^+/+ and IGF-1R^−/− embryos at E17.5 and E19.5 show IHC of blood vessel-specific von Willebrand protein. Arrows (I, J) point to small blood vessels developing in saccular walls. Large blood vessels were similarly marked in all specimen. <b>K–M</b>, Representative lung histology from IGF-1R^+/+ and IGF-1R^−/− embryos at E17.5. NKX2-1 distal-to-proximal IHC signal ratio was measured in 6 IGF-1R^+/+ and 5 IGF-1R^−/− embryos. NS, not significant; Wilcoxon Mann-Whitney U test. <b>N-Q</b>, Epithelial cell-specific NKX2-1 transcription factor was detected in IGF-1R^+/+ and IGF-1R^−/− embryos at E17.5 and E19.5. <b>R–Y</b>, IHC of type 2-specific pro-SP-C at low (R-U) and high magnification (V-Y). Interestingly, for NKX2-1 and pro-SP-C, the IHC pattern of IGF-1R^−/− lungs at E19.5 resembles controls at E17.5 (panel N <i>versus</i> Q, R <i>versus</i> U, and V <i>versus</i> Y), suggesting an approximately 2-day developmental delay in IGF-1R^−/− end-gestational lungs.</p

Additional file 1: of Whole exome sequencing in three families segregating a pediatric case of sarcoidosis

Table S1. Recessive variants found in at least two affected children of different trios. Possibly pathogenic recessive variants (polymorphisms) found by whole-exome -sequencing in at least two affected children of the trios (T). Chr., chromosome; SNP, single nucleotide polymorphism; QUAL., a quality parameter measuring the probability p that the observation of the variant is due to chance (for ex: QUAL = n, p = 1/n). As detailed in the text, Alamut® Visual integrates missense variant pathogenicity prediction tools and in silico study of variants’ effect on RNA splicing, allowing the assessment of their potential impact on splice junctions and splicing regulatory sequences. Alamut® Visual helped us also to exclude well known mutations identified in recessive diseases for those genes which have been related to known genetic diseases (as shown in Table 3). (DOCX 23 kb