Discovery of New Gene Interaction Networks that Govern ABC Protein Biogenesis
- Our work focuses on elucidating mechanistic and therapeutic impact of genetic modifiers that influence synthesis of CFTR variants, with particular attention to rare mutations such as premature termination codons (PTCs).
- Through collaboration with Dr. John L. Hartman IV (University of Alabama at Birmingham), we employed genome-wide yeast phenomics to ascertain gene-gene interactions responsible for functional restoration of PTCs and other defects expressed in the CFTR homologue, YOR1.
- Mutations corresponding to CF-causing variants were introduced into YOR1, and “hits” that emerged from subsequent yeast deletion library screens included evolutionarily conserved translation initiation factors, tRNA processing enzymes, and ribosomal proteins (among others).
- Translational relevance of molecular targets has been confirmed in a number of CF relevant models – e.g. Fischer Rat Thyroid (FRT) cells, CF bronchial epithelia (CFBE41o-), and primary human bronchial or nasal epithelia (HBE, HNE).
- These studies illustrate the utility of yeast phenomics as a method for deducing principles of ABC protein biogenesis.
Investigating Ribosomal Speed as a Novel Modulator of CF Molecular Phenotype
- Approximately 50% knockdown of RPL12 (uL11) or RPL8 (uL2) enhances maturation, plasma membrane density, open channel probability, and/or transepithelial ion transport of certain CFTR variants such as ΔF508 and W1282X.
- RPL12 silencing reduces translation initiation and elongation rates, which allows the ribosome and associated chaperones more time to achieve a native-like CFTR protein conformation.
- RPL12 suppression is well tolerated in vivo, as Rpl12+/- mice are haplosufficient and without a detectable phenotype (collaboration with Dr. Craig Hodges, Case Western).
- RPL8 depletion induces structural rearrangements to the 60S and 40S subunits, facilitating alterations to translational fidelity.
- Synonymous single nucleotide polymorphisms (sSNPs) also exert significant consequences on CFTR biogenesis through altered rates of translation (collaboration with Dr. Zoya Ignatova, University of Hamburg).
- The c.2562T>G sSNP decreases ribosome velocity by ~50% at p.Thr854 in CFTR, thereby altering folding trajectory.
- For the rare D579G-CFTR variant, c.2562T>G amplifies protein expression, channel activity, and pharmacologic response.
Antisense Oligonucleotide Therapeutics
- Our previous findings indicate RPL12 and RPL8 should be considered as therapeutic targets for overcoming PTCs and other specific CFTR variants.
- Through collaboration with Ionis Pharmaceuticals, we aim to determine whether ASOs (single-stranded deoxyribonucleotides) directed against these ribosomal proteins may rescue mutant CFTR functional expression.
- Two human ASOs decreased RPL12 protein, as well as augmented ΔF508-CFTR mRNA levels, band C production, and channel function (unpublished).
- This work is being extended to other genetic conditions with etiology similar to CF (e.g. primary ciliary dyskinesia).
Addressing Racial/Ethnic Inequities in CF NBS
- In collaboration with Dr. Rachel Linnemann and other Emory-Children’s team members, our goal is to improve CF newborn screening (NBS) in Georgia by diminishing false negatives and delayed diagnoses, thereby enhancing health quality and equity for all people living with the disease.
- The state performs CFTR DNA analysis using a 39-variant panel that exhibits lower detection rates of pathogenic variants among infants of color.
- In Georgia, the population of Black or African American individuals is ~2.5-fold higher than the national average.
- Approximately 900 people with CF are followed at CF Care Centers in Atlanta (Emory-Children’s) and Augusta. Our pediatric programs serve 2-3 times more Black or African American individuals than national trends.
- We are conducting a multi-center quality improvement project to accelerate changes to the Georgia CF NBS algorithm.