Defining Molecular and Environmental Stressors that Impact Severity of Cystic Fibrosis-Causing Variants Restricted; Files Only

Abstract

Variants in the cystic fibrosis gene (CFTR) result in defective or absent CFTR protein, leading to life threatening dysfunction to respiratory, endocrine, and digestive systems. Approximately ~13% are linked to premature termination codons (PTCs), or “nonsense” variants, with G542X being the most common. Patients harboring CFTR PTCs typically exhibit severe disease and are ineligible for clinically approved CFTR “modulators” (i.e. small molecules). Notably, PTCs are disproportionately represented in minoritized populations, including those from Latin American ancestries. These groups exhibit a strong monotonic association between reduced median household income and high mortality rate along with lower pulmonary function.

 

Based on gene-gene interaction networks, we identified several genetic modifiers of PTC synthesis that rescue processing of CFTR nonsense alleles. Prominent “hits” were comprised of ribosomal proteins such as RPL12 and RPL8. Partially suppressing these targets resulted in reduced speed and/or fidelity of translation, conferring read-through of CFTR PTCs. Together with these gene knockdowns and small molecule read-through agents, negative social determinants of health were examined to determine whether they exacerbated the in vivo effects of PTCs and contribute to racial and ethnic inequities in CF health outcomes. Variables such as dysglycemia, poor nutritional quality, and exposure to chemical pollutants were tested.

 

In experimental treatments with modulators, only G418 significantly increased G542X-CFTR surface localization, while Escin showed modest and variable effects, consistent with its nonspecific mechanism of action. ETI, effective in folding and gating mutations, showed insignificant effects on G542X PTC variant. Other agents such as PTC-124 and ELX-02 failed to improve trafficking as well. However, combining siRNA-mediated knockdowns with modulators produced synergistic effects. SiRPL12+ETI and siMAEA+ETI combinations enhanced G542X-CFTR localization, showing the potential of targeting read-through, correction, and potentiation mechanisms simultaneously.

 

Molecular and environmental stressors were tested to determine their effects on therapeutic efficacy. Under dysglycemic conditions, increased glucose concentrations reduced baseline trafficking of G542X-CFTR, but had insignificant effects in G418, VTD, or ETI. Supplementation with omega-3 or omega-6 polyunsaturated fatty acid (PUFA) reduced CFTR trafficking across treatments with the ETI+ARA condition showing elevated trafficking along with reduced cell viability. These findings emphasize the need to manipulate dietary PUFA ratios. Lastly, exposure to polyfluorooctane sulfonic acid, a common PFAS pollutant, led to decreased CFTR surface expression with VTD treatment, suggesting environmental toxicants may undermine CFTR modulator efficacy by disrupting protein stability.

 

Together, these findings support the hypothesis that gene-gene networks regulating ABC protein synthesis are conserved and can be targeted to enhance readthrough of CFTR nonsense mutations. Furthermore, it highlights the importance of assessing both genetic and background factors in CF treatment and care.

Table of Contents

Section 1: Introduction………………………………………………………………………………….1

Background………………………………………………………………………………………………...1

Preliminary Data………………………………………………………………………………………….4

Figure 1. YOR1 alleles used for plasmid-based mutagenesis reveal

new gene targets that augment W1282X-CFTR cell surface localization…………………....5

Table 1. Gene deletions that restore functional expression of the yor1

nonsense variant, G704X…………………………………………………………………………........6

Figure 2: Effects of hyperglycemia and/or CFTR modulators on

immortalized human CF bronchial epithelia (CFBE)……………………………………………..7

Hypotheses………………………………………………………………………………………………...7

Section 2: Methods……………………………………………………………………………………....9

Experimental Design…………………………………………………………………………………….9

Figure 3. 96-well plate layout for G542X-CFTR treatment screening…………………………9

Figure 4. 96-well plate layout for G542X-CFTR combination of

well-performing” drugs +/- gene knockdown (set1)………………………………………………10

Figure 5. 96-well plate layout for G542X-CFTR combination of

“well-performing” drugs +/- gene knockdown (set 2)…………………………………………….10

Figure 6. 96-well plate layout for assessing environmental stressors

on G542X-CFTR processing…………………………………………………………………………....11

Experimental Precision………………………………………………………………………………....12

Section 3: Results……………………………………………………………………………………......12

Single Drug Treatments………………………………………………………………………………...12

Figure 7. G542X cell surface localization in response to single drug treatments………..…13

Single siRNA Treatments……………………………………………………………………………….14

Figure 8. G542X plasma membrane (PM) density following

single gene knockdown treatments……………………………………………………………….....14

Combination Treatments………………………………………………………………………….......14

Figure 9. G542X trafficking consequent to combined

gene knockdown(s) and drug treatments………………………………………………………......15

Dysregulated Glucose Metabolism………………………………………………………………......16

Figure 10. G542X cell surface localization under normal

and dysglycemic conditions…....................................................................................17

Nutritional Quality……………………………………………………………………………………...18

Figure 11. G542X plasma membrane density in response to

fatty acid supplementation.......................................................................................19

Chemical Pollutants……………………………………………………………………………….......19

Figure 12. G542X trafficking in the presence of the “forever chemical,” PFOS…………....20

Section 4: Discussion…………………………………………………………………………………...21

Section 5: Conclusion……………………………………………………………………………….....24

Section 6: References………………………………………………………………………………......26

 

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Genetics Biology, General Biology, Molecular
Keyword	Environmental Cystic Fibrosis Genetic
Committee Chair / Thesis Advisor	Kathryn E. Oliver, Ph.D., Emory University
Committee Members	Nitya P. Jacob, Ph.D., Emory University Natalia J. Bayona Vasquez, Ph.D., Emory University

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