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Published on: March 16, 2022
Rescue of melanocortin 4 receptor (MC4R) nonsense mutations by aminoglycoside-mediated read-through
Harald Brumm1, Jessica Mühlhaus, Florian Bolze
1Institute of Experimental Pediatric Endocrinology, Charité Universitätsmedizin Berlin, Berlin, Germany.
Abstract:
Aminoglycoside-mediated read-through of stop codons was recently demonstrated for a variety of diseases in vitro and in vivo. About 30 percent of human genetic diseases are the consequence of nonsense mutations. Nonsense mutations in obesity-associated genes like the melanocortin 4 receptor (MC4R), expressed in the hypothalamus, show the impact of premature stop codons on energy homeostasis. Therefore, the MC4R could be a potential pharmaceutical target for obesity treatment and targeting MC4R stop mutations could serve as proof of principle for nonsense mutations in genes expressed in the brain. We investigated four naturally occurring nonsense mutations in the MC4R (W16X, Y35X, E61X, Q307X) located at different positions in the receptor for aminoglycoside-mediated functional rescue in vitro. We determined localization and amount of full-length protein before and after aminoglycoside treatment by fluorescence microscopy, cell surface and total enzyme linked immunosorbent assay (ELISA). Signal transduction properties were analyzed by cyclic adenosine monophosphate (cAMP) assays after transient transfection of MC4R wild type and mutant receptors into COS-7 cells. Functional rescue of stop mutations in the MC4R is dependent on: (i) triplet sequence of the stop codon, (ii) surrounding sequence, (iii) location within the receptor, (iv) applied aminoglycoside and ligand. Functional rescue was possible for W16X, Y35X (N-terminus), less successful for Q307X (C-terminus) and barely feasible for E61X (first transmembrane domain). Restoration of full-length proteins by PTC124 could not be confirmed. Future pharmaceutical applications must consider the potency of aminoglycosides to restore receptor function as well as the ability to pass the blood-brain barrier.
Insights
Aminoglycosides can restore function of the melanocortin 4 receptor (MC4R) with nonsense mutations, offering a potential treatment for obesity. Functional rescue depends on mutation location and drug specifics.
Area of Science:
- Genetics
- Pharmacology
- Molecular Biology
Background:
- Approximately 30% of human genetic diseases stem from nonsense mutations, which introduce premature stop codons.
- Nonsense mutations in obesity-associated genes, such as the melanocortin 4 receptor (MC4R), disrupt energy homeostasis.
- MC4R is a potential pharmaceutical target for obesity, and targeting its stop mutations could validate this approach for brain-expressed genes.
Purpose of the Study:
- To investigate aminoglycoside-mediated functional rescue of four naturally occurring nonsense mutations in the MC4R.
- To assess the impact of mutation location, codon context, and drug choice on functional restoration.
- To evaluate MC4R as a proof-of-principle target for treating genetic diseases caused by nonsense mutations.
Main Methods:
- Investigated four MC4R nonsense mutations (W16X, Y35X, E61X, Q307X) for aminoglycoside-mediated rescue in vitro.
- Quantified full-length protein levels using fluorescence microscopy and ELISA.
- Analyzed signal transduction via cyclic adenosine monophosphate (cAMP) assays in transfected COS-7 cells.
Main Results:
- Functional rescue varied significantly based on mutation position: N-terminal mutations (W16X, Y35X) showed rescue, C-terminal (Q307X) showed less, and within the transmembrane domain (E61X) showed minimal feasibility.
- Rescue efficiency depended on the stop codon's triplet sequence, surrounding nucleotides, mutation location, specific aminoglycoside, and ligand.
- PTC124 failed to restore full-length MC4R protein production.
Conclusions:
- Aminoglycoside-mediated functional rescue of MC4R nonsense mutations is feasible but highly dependent on specific factors.
- MC4R represents a viable target for exploring therapeutic strategies for obesity linked to nonsense mutations.
- Future drug development must consider aminoglycoside potency, receptor-specific rescue parameters, and blood-brain barrier penetration for neurological targets.
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