Identification of single nucleotide variants in SLC26A9 gene in patients with cystic fibrosis (p.Phe508del homozygous) and its association to Orkambi® (Lumacaftor and Ivacaftor) response in vitro.

BACKGROUND: Since patients with cystic fibrosis with different Cystic Fibrosis Transmembrane Regulator (CFTR) genotypes present a wide response variability for modulator drugs such as Orkambi®, it is important to screen variants in candidate genes with an impact on precision and personalized medicine, such as Solute Carrier Family 26, member 9 (SLC26A9) gene. METHODS: Sanger sequencing for the exons and intron-exon boundary junctions of the SLC26A9 gene was employed in nine individuals with p.Phe508del homozygous genotype for the CFTR gene who were not under CFTR modulators therapy. The sequencing variants were evaluated by in silico prediction tools. The CFTR function was measured by cAMP-stimulated current (ΔIsc-eq-FSK) in polarized CFTR of human nasal epithelial cells cultured in micro-Ussing chambers with Orkambi®. RESULTS: We found 24 intronic variants, three in the coding region (missense variants - rs74146719 and rs16856462 and synonymous - rs33943971), and three in the three prime untranslated region (3' UTR) region in the SLC26A9 gene. Twenty variants were considered benign according to American College of Medical Genetics and Genomics guidelines, and ten were classified as uncertain significance. Although some variants had deleterious predictions or possible alterations in splicing, the majority of predictions were benign or neutral. When we analyzed the ΔIsc-eq-FSK response to Orkambi®, there were no significant differences within the genotypes and alleles for all 30 variants in the SLC26A9 gene. CONCLUSIONS: Among the nine individuals with p.Phe508del homozygous genotype for the CFTR gene, no pathogenic SLC26A9 variants were found, and we did not detect associations from the 30 SLC26A9 variants and the response to the Orkambi® in vitro.

Novel, rare and common pathogenic variants in the CFTR gene screened by high-throughput sequencing technology and predicted by in silico tools.

Cystic fibrosis (CF) is caused by ~300 pathogenic CFTR variants. The heterogeneity of which, challenges molecular diagnosis and precision medicine approaches in CF. Our objective was to identify CFTR variants through high-throughput sequencing (HTS) and to predict the pathogenicity of novel variants through in 8 silico tools. Two guidelines were followed to deduce the pathogenicity. A total of 169 CF patients had genomic DNA submitted to a Targeted Gene Sequencing and we identified 63 variants (three patients had three variants). The most frequent alleles were: F508del (n = 192), G542* (n = 26), N1303K (n = 11), R1162* and R334W (n = 9). The screened variants were classified as follows: 41 - pathogenic variants [classified as (I) n = 23, (II) n = 6, (III) n = 1, (IV) n = 6, (IV/V) n = 1 and (VI) n = 4]; 14 - variants of uncertain significance; and seven novel variants. To the novel variants we suggested the classification of 6b-16 exon duplication, G646* and 3557delA as Class I. There was concordance among the predictors as likely pathogenic for L935Q, cDNA.5808T>A and I1427I. Also, Y325F presented two discordant results among the predictors. HTS and in silico analysis can identify pathogenic CFTR variants and will open the door to integration of precision medicine into routine clinical practice in the near future.

Extent of rescue of F508del-CFTR function by VX-809 and VX-770 in human nasal epithelial cells correlates with SNP rs7512462 in SLC26A9 gene in F508del/F508del Cystic Fibrosis patients.

BACKGROUND: We analyzed the CFTR response to VX-809/VX-770 drugs in conditionally reprogrammed cells (CRC) of human nasal epithelium (HNE) from F508del/F508del patients based on SNP rs7512462 in the Solute Carrier Family 26, Member 9 (SLC26A9; MIM: 608481) gene. METHODS: The Isc-eq measurements of primary nasal epithelial cells from F508del/F508del patients (n = 12) for CFTR function were performed in micro Ussing chambers and compared with non-CF controls (n = 2). Data were analyzed according to the rs7512462 genotype which were determined by real-time PCR. RESULTS: The CRC-HNE cells from F508del/F508del patients evidenced high variability in the basal levels of CFTR function. Also, the rs7512462*C allele showed an increased basal CFTR function and higher responses to VX-809 + VX-770. The rs7512462*CC + CT genotypes together evidenced CFTR function levels of 14.89% relatively to wt/wt (rs7512462*CT alone-15.29%) i.e., almost double of rs7512462*TT (7.13%). Furthermore, sweat [Cl-] and body mass index of patients also evidenced an association with the rs7512462 genotype. CONCLUSION: The CFTR function can be performed in F508del/F508del patient-derived CRC-HNEs and its function and responses to VX-809 + VX-770 combination as well as clinical data, are all associated with the rs7512462 variant, which partially sheds light on the generally inter-individual phenotypic variability and in personalized responses to CFTR modulator drugs.

Association of clinical severity of cystic fibrosis with variants in the SLC gene family (SLC6A14, SLC26A9, SLC11A1 and SLC9A3).

INTRODUCTION: Cystic fibrosis (CF) manifests with clinical and histopathological variability depending on environmental and genetic factors. Moreover, the genes encoding ion channels[rs3788766(SLC6A14), rs7512462(SLC26A9), rs17235416(SLC11A1) and rs17563161(SLC9A3)] have been insufficiently studied as modifier genes. Then, our objective was associate the variants in the genes of SLC family with 43 CF severity markers. METHODS: The variants were identified by real-time-PCR in 188 CF patients considering the CFTR genotype. Statistical analyses were performed by parametric and nonparametric tests. The correction by multiple testing was performed by the False Rate Discovery test, alpha=0.05. RESULTS: Depending on the CFTR mutations, we found association of: (i) rs3788766*CC with mucoid Pseudomonas aeruginosa (OR=0.171; 95%CI=0.029-0.696), non-mucoid P. aeruginosa (OR=0.283; 95%CI=0.094-0.853) and Staphyloccocus aureus (OR=4.443; 95%CI=1.019-40.64), largest FEFmax(p=0.041) and best response to bronchodilator for FEF50%(p=0.033) and FEV1/FVC(p=0.044); (ii) rs3788766*CT with early start of pulmonary symptom (OR=3.524; 95%CI=1.229-10.1) and osteoporosis (OR=0.203; 95%CI=0.022-0.883); (iii) rs3788766*TT with lowest body mass index (OR=4.242; 95%CI=1.505-11.95), presence of mucoid P. aeruginosa (OR=3.176; 95%CI=1.29-7.819) and S. aureus (OR=0.116; 95%CI=0.004-0.881), highest Bhalla score (p=0.047) and lowest FEFmax(p=0.028) and FEF25%(p=0.031) values; (iv) rs7512462*CC with highest Shwachman-Kulczycki score (p=0.019), FVC(p=0.043), FEV1(p=0.047), FEV1/FVC(p=0.022), FEF50%(p=0.038) and FEF25-75%(p=0.016); (v) rs7512462*CT with lowest values of FVC(p=0.034), FEV1(p=0.047), FEV1/FVC(p=0.022), FEF25%(p=0.012), FEF50%(p=0.038), FEF75%(p=0.008), FEF25-75%(p=0.016) and ERV(p=0.023); (vi) rs7512462*TT with best response to the inhaled bronchodilator for FEV1(p=0.011), FEF50%(p=0.019), FEF75%(p=0.036) and FEF25-75%(p=0.008); (vii) rs17234516*Normal allele with lowest value of SaO2 (p=0.010) and S. aureus (OR=3.333; 95%CI=1.085-10.24); (viii) rs17563161*GG with lowest age for onset of digestive symptoms (OR=2.564; 95%CI=1.234-5.33). CONCLUSIONS: The clinical and laboratory variability of CF were associated with the variants in the genes of SLC family in our sample.