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

Table_1_SLC26A9 Gene Is Associated With Lung Function Response to Ivacaftor in Patients With Cystic Fibrosis.DOC

<p>Ivacaftor is a drug used to treat cystic fibrosis (CF) patients carrying specific gating CFTR mutations. Interpatient variability in the lung response has been shown to be partly explained by rs7512462 in the Solute Carrier Family 26 Member 9 (SLC26A9) gene. In an independent and larger cohort, we aimed to evaluate whether SLC26A9 variants contribute to the variability of the lung phenotype and if they influence the lung response to ivacaftor. We genotyped the French CF Gene Modifier Study cohort (n = 4,840) to investigate whether SLC26A9 variants were involved in the lung phenotype heterogeneity. Their influence in the response to ivacaftor was tested in the 30 treated patients who met the inclusion criteria: older than 6 years of age, percent-predicted forced expiratory volume measured in 1 s (FEV_1pp) in the 3 months before treatment initiation ranging between 40 and 90%. Response to treatment was determined by the change in FEV_1pp from baseline, averaged in 15–75 days, and the 1st-year post-treatment. We observed that SLC26A9 variants were not associated with lung function variability in untreated patients and that gain of lung function in patients treated with ivacaftor was similar to clinical trials. We confirmed that rs7512462 was associated with variability in ivacaftor-lung response, with a significant reduction in lung function improvement for patients with the C allele. Other SLC26A9 SNPs also contributed to the ivacaftor-response. Interindividual variability in lung response to ivacaftor is associated with SLC26A9 variants in French CF patients. Pharmacogenomics and personalized medicine will soon be part of CF patient care.</p

Table_2_SLC26A9 Gene Is Associated With Lung Function Response to Ivacaftor in Patients With Cystic Fibrosis.docx

<p>Ivacaftor is a drug used to treat cystic fibrosis (CF) patients carrying specific gating CFTR mutations. Interpatient variability in the lung response has been shown to be partly explained by rs7512462 in the Solute Carrier Family 26 Member 9 (SLC26A9) gene. In an independent and larger cohort, we aimed to evaluate whether SLC26A9 variants contribute to the variability of the lung phenotype and if they influence the lung response to ivacaftor. We genotyped the French CF Gene Modifier Study cohort (n = 4,840) to investigate whether SLC26A9 variants were involved in the lung phenotype heterogeneity. Their influence in the response to ivacaftor was tested in the 30 treated patients who met the inclusion criteria: older than 6 years of age, percent-predicted forced expiratory volume measured in 1 s (FEV_1pp) in the 3 months before treatment initiation ranging between 40 and 90%. Response to treatment was determined by the change in FEV_1pp from baseline, averaged in 15–75 days, and the 1st-year post-treatment. We observed that SLC26A9 variants were not associated with lung function variability in untreated patients and that gain of lung function in patients treated with ivacaftor was similar to clinical trials. We confirmed that rs7512462 was associated with variability in ivacaftor-lung response, with a significant reduction in lung function improvement for patients with the C allele. Other SLC26A9 SNPs also contributed to the ivacaftor-response. Interindividual variability in lung response to ivacaftor is associated with SLC26A9 variants in French CF patients. Pharmacogenomics and personalized medicine will soon be part of CF patient care.</p

Table_3_SLC26A9 Gene Is Associated With Lung Function Response to Ivacaftor in Patients With Cystic Fibrosis.doc

<p>Ivacaftor is a drug used to treat cystic fibrosis (CF) patients carrying specific gating CFTR mutations. Interpatient variability in the lung response has been shown to be partly explained by rs7512462 in the Solute Carrier Family 26 Member 9 (SLC26A9) gene. In an independent and larger cohort, we aimed to evaluate whether SLC26A9 variants contribute to the variability of the lung phenotype and if they influence the lung response to ivacaftor. We genotyped the French CF Gene Modifier Study cohort (n = 4,840) to investigate whether SLC26A9 variants were involved in the lung phenotype heterogeneity. Their influence in the response to ivacaftor was tested in the 30 treated patients who met the inclusion criteria: older than 6 years of age, percent-predicted forced expiratory volume measured in 1 s (FEV_1pp) in the 3 months before treatment initiation ranging between 40 and 90%. Response to treatment was determined by the change in FEV_1pp from baseline, averaged in 15–75 days, and the 1st-year post-treatment. We observed that SLC26A9 variants were not associated with lung function variability in untreated patients and that gain of lung function in patients treated with ivacaftor was similar to clinical trials. We confirmed that rs7512462 was associated with variability in ivacaftor-lung response, with a significant reduction in lung function improvement for patients with the C allele. Other SLC26A9 SNPs also contributed to the ivacaftor-response. Interindividual variability in lung response to ivacaftor is associated with SLC26A9 variants in French CF patients. Pharmacogenomics and personalized medicine will soon be part of CF patient care.</p

Overview of gene expression profiles.

<p>A- The heat-map of the mean expression levels of all the genes in each condition revealed two distinct clusters that separated cystic-fibrosis (CF) cells from CTRL cells. B and C- Venn diagram of differentially expressed genes between cystic-fibrosis and CTRL cells upon <i>P</i>. <i>aeruginosa</i> infection. Circles: Differentially expressed genes upregulated (B) or downregulated (C) at a single postinfection time point or at two or three consecutive postinfection time points (thus, 0h was not considered). We selected the genes whose fold-change in expression level was ≥2 in the event of upregulation and ≤0.5 in the event of downregulation. Squares: Differentially expressed genes upregulated (B) or downregulated (C) at two, three, or four consecutive time points (thus, 0h was considered). We selected the genes for which the ratio of FC in infected cells over FC in noninfected cells was ≥1.5 in the event of upregulation or ≤0.6 in the event of downregulation.</p

List of genes in the protein-binding gene-ontology category that were most upregulated (FC ≥ 3) in CTRL cells 6 hours after <i>P</i>. <i>aeruginosa</i> infection.

<p>Genes were classified according their molecular function, using the PANTHER Classification System (<a href="http://www.pantherdb.org/" target="_blank">http://www.pantherdb.org/</a>). 0 h, nonstimulated; 6 h, 6 hours postinfection; FC, fold change; adjpBH, adjusted <i>P</i> value to which the α cutoff was applied.</p><p>List of genes in the protein-binding gene-ontology category that were most upregulated (FC ≥ 3) in CTRL cells 6 hours after <i>P</i>. <i>aeruginosa</i> infection.</p

Genes in the catalytic-activity (A) and in the protein-binding (B) gene-ontology category: five downregulated genes with the greatest difference in expression level between cystic-fibrosis and CTRL cells after infection.

<p>FC, fold change; h, hours; adjpBH, adjusted <i>P</i> value to which the α cutoff was applied.</p><p>Genes in the catalytic-activity (A) and in the protein-binding (B) gene-ontology category: five downregulated genes with the greatest difference in expression level between cystic-fibrosis and CTRL cells after infection.</p

Gene-ontology molecular-function categories upregulated in infected cystic-fibrosis cells compared to CTRL cells.

<p>Gene-ontology molecular-function categories upregulated in infected cystic-fibrosis cells compared to CTRL cells.</p