Increased expression of anion transporter SLC26A9 delays diabetes onset in cystic fibrosis
Anh-Thu N. Lam, … , Scott M. Blackman, Garry R. Cutting
Anh-Thu N. Lam, … , Scott M. Blackman, Garry R. Cutting
Published October 3, 2019
Citation Information: J Clin Invest. 2020;130(1):272-286. https://doi.org/10.1172/JCI129833.
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Research Article Endocrinology Genetics

Increased expression of anion transporter SLC26A9 delays diabetes onset in cystic fibrosis

Abstract

Diabetes is a common complication of cystic fibrosis (CF) that affects approximately 20% of adolescents and 40%–50% of adults with CF. The age at onset of CF-related diabetes (CFRD) (marked by clinical diagnosis and treatment initiation) is an important measure of the disease process. DNA variants associated with age at onset of CFRD reside in and near SLC26A9. Deep sequencing of the SLC26A9 gene in 762 individuals with CF revealed that 2 common DNA haplotypes formed by the risk variants account for the association with diabetes. Single-cell RNA sequencing (scRNA-Seq) indicated that SLC26A9 is predominantly expressed in pancreatic ductal cells and frequently coexpressed with CF transmembrane conductance regulator (CFTR) along with transcription factors that have binding sites 5′ of SLC26A9. These findings were replicated upon reanalysis of scRNA-Seq data from 4 independent studies. DNA fragments derived from the 5′ region of SLC26A9-bearing variants from the low-risk haplotype generated 12%–20% higher levels of expression in PANC-1 and CFPAC-1 cells compared with the high- risk haplotype. Taken together, our findings indicate that an increase in SLC26A9 expression in ductal cells of the pancreas delays the age at onset of diabetes, suggesting a CFTR-agnostic treatment for a major complication of CF.

Authors

Anh-Thu N. Lam, Melis A. Aksit, Briana Vecchio-Pagan, Celeste A. Shelton, Derek L. Osorio, Arianna F. Anzmann, Loyal A. Goff, David C. Whitcomb, Scott M. Blackman, Garry R. Cutting

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Figure 6

Reporter gene expression driven by DNA fragments derived from the 5′ region of SLC26A9.

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Reporter gene expression driven by DNA fragments derived from the 5′ reg...
(A) Diagram depicting the location and length of the regions studied relative to SLC26A9. (B) Luciferase expression levels obtained from PANC-1 cells transfected with various SLC26A9 DNA fragments bearing either LR or HR variants for CFRD. The 1.172 kb region generated robust expression of luciferase consistent with a promoter. Levels do not differ between the LR and HR bearing fragments. The 1.173 kb region generated little to no activity, similar to negative controls. The 2.3 kb region composed of the 1.172 kb and 1.173 kb regions generated a combined expression of luciferase that was 12% higher for LR compared with HR haplotype (P = 5.15 × 10–09). The 4.8 kb region generated a combined 19% higher expression level compared with HR (P = 6.28 × 10–07). (C) Transfections in CFPAC-1 cells resulted in the same trend being observed. The 2.3 kb region drove a combined expression of luciferase that was 20% higher for LR compared with HR haplotype (P = 2.00 × 10–03). For plots in B and C, results are shown for 2 to 3 separate transfections of PANC-1 and CFPAC-1 cells with 2 to 4 independent plasmid constructs (A–D), each containing alleles corresponding to the LR (blue) or HR (red) haplotypes in their native orientation. For each transfection, the data points to the left of the vertical line show results from each independent clone. On the right, data points from all clones are combined. *P ≤ 0.05; ***P ≤ 0.001. Negative controls (pGL4.10 empty vector and renilla) are shown in gray. Total data points (n) are listed below each construct. Significance was assessed using Student’s t test. Error bars show SEM.