Downstream Alternate Start Site Allows N-Terminal Nonsense Variants to Escape NMD and Results in Functional Recovery by Readthrough and Modulator Combination

Abstract

Genetic variants that introduce premature termination codons (PTCs) have remained difficult to therapeutically target due to lack of protein product. Nonsense mediated mRNA decay (NMD) targets PTC-bearing transcripts to reduce the potentially damaging effects of truncated proteins. Readthrough compounds have been tested on PTC-generating variants in attempt to permit translation through a premature stop. However, readthrough compounds have not proved efficacious in a clinical setting due to lack of stable mRNA. Here, we investigate N-terminal variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which have been shown to escape NMD, potentially through a mechanism of alternative translation initiation at downstream AUG codons. We hypothesized that N-terminal variants in CFTR that evade NMD will produce stable transcript, allowing CFTR function to be restored by a combination of readthrough and protein modulator therapy. We investigate this using two cell line models expressing CFTR-expression minigenes (EMG; HEK293s and CFBEs) and primary human nasal epithelial (NE) cells, and we test readthrough compounds G418 and ELX-02 in combination with CFTR protein modulators. HEK293 cells expressing the variants E60X and L88X generate CFTR-specific core glycosylated products that are consistent with downstream translation initiation. Mutation of downstream methionines at codons 150 and 152 does not result in changes in CFTR protein processing in cells expressing L88X-CFTR-EMG. However, mutation of methionine at 265 results in loss of detectable CFTR protein in cells expressing E60X, L88X, and Y122X CFTR-EMGs, indicating that downstream translation initiation is occurring at the AUG codon at position M265. In HEK293 stable cells harboring L88X, treatment with readthrough compounds alone allows for formation of full-length, but misfolded CFTR protein. Upon addition of protein modulators in combination with readthrough, we observe formation of mature, complex-glycosylated CFTR. In CFBE and NE cells, addition of readthrough ELX-02 and modulator therapy results in substantial recovery of CFTR function. Our work indicates that N-terminal variants generate stable CFTR transcript due to translation initiation at a downstream AUG codon. Thus, individuals with CF bearing 5′ nonsense variants that evade NMD are ideal candidates for treatment with clinically safe readthrough compounds and modulator therapy.

Quantitative determination of SLC2A1 variant functional effects in GLUT1 deficiency syndrome

“…Prior work on DDX3X 15 variants, studied using the same assay, suggested that mildly damaging variants may be slowly depleting and required modifications of the growth assay. Our functional data for SLC2A1 p.V87I, which is a VUS reported in three affected and one unaffected individual in a small family, 27 suggest that this variant is benign. The clinical phenotype in this family, while consistent with mild Glut1DS, is not specific for this syndrome given the many causes of mild cognitive impairment and migraine.…”

“…Interestingly, we found a positive functional score suggesting that it is a benign variant for one missense variant (p.V87I) that had previously been identified in a child with a mild phenotype, including focal epilepsy with onset at 3.5 years old and mild cognitive impairment. 27 The variant was also identified in the child's mother who had migraine, paroxysmal hemiparesis, and late onset PED, and one of a pair of twin siblings who had mild absence epilepsy, eyelid myoclonia, mild developmental delay and cognitive impairment. However, the other unaffected twin also had the variant, making this VUS difficult to interpret and less likely to be pathogenic.…”

“…In addition, we cannot fully explain why the functional score for p.Y28X was consistently less negative than the other two nonsense variants we tested, though they were statistically not significantly different. We note that p.Y28X is the most proximal of the three nonsense variants, leading us to be consider the possibility that in an alternative downstream start site may yield a protein with a possible partial function 28–31 …”

“…We note that p.Y28X is the most proximal of the three nonsense variants, leading us to be consider the possibility that in an alternative downstream start site may yield a protein with a possible partial function. 28 , 29 , 30 , 31 …”

Emerging Personalized Opportunities for Enhancing Translational Readthrough in Rare Genetic Diseases and Beyond

“…The production of full length CFTR was efficiently induced in primary human nasal epithelial cells by treatment with G418 (at concentrations of 100 µM and 400 µM) or ELX-02 (at concentrations of 100 µM and 200 µM). Importantly, combinatorial treatment with CFTR modulators was obligatory for achieving functional CFTR reconstitution, since treatment with readthrough inducers alone produced misfolded CFTR [ 131 ]. Another recent evaluation of ELX-02 in rare CFTR nonsense mutations revealed a favorable “U UGA C” sequence context for CFTR mRNA stabilization and readthrough [ 14 ], while the co-treatment of patient-derived human nasal epithelial cells with ELX-02 (1 mM) and an NMD inhibitor (SMG1i, 5 µM) increased CFTR mRNA levels compared to ELX-02 treatment alone, it did not result in significant increases in CFTR activity [ 14 ].…”