Readthrough Activators and Nonsense-Mediated mRNA Decay Inhibitor Molecules: Real Potential in Many Genetic Diseases Harboring Premature Termination Codons.

Nonsense mutations that generate a premature termination codon (PTC) can induce both the accelerated degradation of mutated mRNA compared with the wild type version of the mRNA or the production of a truncated protein. One of the considered therapeutic strategies to bypass PTCs is their "readthrough" based on small-molecule drugs. These molecules promote the incorporation of a near-cognate tRNA at the PTC position through the native polypeptide chain. In this review, we detailed the various existing strategies organized according to pharmacological molecule types through their different mechanisms. The positive results that followed readthrough molecule testing in multiple neuromuscular disorder models indicate the potential of this approach in peripheral neuropathies.

Amlexanox: Readthrough Induction and Nonsense-Mediated mRNA Decay Inhibition in a Charcot-Marie-Tooth Model of hiPSCs-Derived Neuronal Cells Harboring a Nonsense Mutation in GDAP1 Gene.

Nonsense mutations are involved in multiple peripheral neuropathies. These mutations induce the presence of a premature termination codon (PTC) at the mRNA level. As a result, a dysfunctional or truncated protein is synthesized, or even absent linked to nonsense-mediated mRNA degradation (NMD) system activation. Readthrough molecules or NMD inhibitors could be innovative therapies in these hereditary neuropathies, particularly molecules harboring the dual activity as amlexanox. Charcot-Marie-Tooth (CMT) is the most common inherited pathology of the peripheral nervous system, affecting 1 in 2500 people worldwide. Nonsense mutations in the GDAP1 gene have been associated with a severe form of CMT, prompting us to investigate the effect of readthrough and NMD inhibitor molecules. Although not clearly defined, GDAP1 could be involved in mitochondrial functions, such as mitophagy. We focused on the homozygous c.581C>G (p.Ser194*) mutation inducing CMT2H using patient human induced pluripotent stem cell (hiPSC)-derived neuronal cells. Treatment during 20 h with 100 µM of amlexanox on this cell model stabilized GDAP1 mRNAs carrying UGA-PTC and induced a restoration of the mitochondrial morphology. These results highlight the potential of readthrough molecules associated to NMD inhibitors for the treatment of genetic alterations in CMT, opening the way for future investigations and a potential therapy.

From Negative to Positive Diagnosis: Structural Variation Could Be the Second Mutation You Are Looking for in a Recessive Autosomal Gene.

Next-generation sequencing (NGS) allows the detection of plentiful mutations increasing the rate of patients getting a positive diagnosis. However, while single-nucleotide variants (SNVs) or small indels can be easily detected, structural variations (SVs) such as copy number variants (CNVs) are often not researched. In Charcot-Marie-Tooth disease (CMT), the most common hereditary peripheral neuropathy, the PMP22-duplication was the first variation detected. Since then, more than 90 other genes have been associated with CMT, with point mutations or small indels mostly described. Herein, we present a personalized approach we performed to obtain a positive diagnosis of a patient suffering from demyelinating CMT. His NGS data were aligned to the human reference sequence but also studied using the CovCopCan software, designed to detect large CNVs. This approach allowed the detection of only one mutation in SH3TC2, the frequent p.Arg954*, while SH3TC2 is known to be responsible for autosomal recessive demyelinating CMT forms. Interestingly, by modifying the standard CovCopCan use, we detected the second mutation of this patient corresponding to a 922 bp deletion in SH3TC2 (Chr5:148,390,609-Chr5:148,389,687), including only one exon (exon 14). This highlights that SVs, different from PMP22 duplication, can be responsible for peripheral neuropathy and should be searched systematically. This approach could also be employed to improve the diagnosis of all inherited diseases.

New structural variations responsible for Charcot-Marie-Tooth disease: The first two large KIF5A deletions detected by CovCopCan software.

Next-generation sequencing (NGS) allows the detection of mutations in inherited genetic diseases, like the Charcot-Marie-Tooth disease (CMT) which is the most common hereditary peripheral neuropathy. The majority of mutations detected by NGS are single nucleotide variants (SNVs) or small indels, while structural variants (SVs) are often underdiagnosed. PMP22 was the first gene described as being involved in CMT via a SV of duplication type. To date, more than 90 genes are known to be involved in CMT, with mainly SNVs and short indels described. Herein targeted NGS and the CovCopCan bioinformatic tool were used in two unrelated families, both presenting with typical CMT symptoms with pyramidal involvement. We have discovered two large SVs in KIF5A, a gene known to cause axonal forms of CMT (CMT2) in which no SVs have yet been described. In the first family, the patient presented with a large deletion of 12 kb in KIF5A from Chr12:57,956,278 to Chr12:57,968,335 including exons 2-15, that could lead to mutation c.(130-943_c.1717-533del), p.(Gly44_Leu572del). In the second family, two cases presented with a large deletion of 3 kb in KIF5A from Chr12:57,974,133 to Chr12:57,977,210 including exons 24-28, that could lead to mutation c.(2539-605_*36 + 211del), p.(Leu847_Ser1032delins33). In addition, bioinformatic sequence analysis revealed that a NAHR (Non-Allelic-Homologous-Recombination) mechanism, such as those in the PMP22 duplication, could be responsible for one of the KIF5A SVs and could potentially be present in a number of other patients. This study reveals that large KIF5A deletions can cause CMT2 and highlights the importance of analyzing not only the SNVs but also the SVs during diagnosis of neuropathies.

GDAP1 Involvement in Mitochondrial Function and Oxidative Stress, Investigated in a Charcot-Marie-Tooth Model of hiPSCs-Derived Motor Neurons.

Mutations in the ganglioside-induced differentiation associated protein 1 (GDAP1) gene have been associated with demyelinating and axonal forms of Charcot-Marie-Tooth (CMT) disease, the most frequent hereditary peripheral neuropathy in humans. Previous studies reported the prevalent GDAP1 expression in neural tissues and cells, from animal models. Here, we described the first GDAP1 functional study on human induced-pluripotent stem cells (hiPSCs)-derived motor neurons, obtained from normal subjects and from a CMT2H patient, carrying the GDAP1 homozygous c.581C>G (p.Ser194*) mutation. At mRNA level, we observed that, in normal subjects, GDAP1 is mainly expressed in motor neurons, while it is drastically reduced in the patient's cells containing a premature termination codon (PTC), probably degraded by the nonsense-mediated mRNA decay (NMD) system. Morphological and functional investigations revealed in the CMT patient's motor neurons a decrease of cell viability associated to lipid dysfunction and oxidative stress development. Mitochondrion is a key organelle in oxidative stress generation, but it is also mainly involved in energetic metabolism. Thus, in the CMT patient's motor neurons, mitochondrial cristae defects were observed, even if no deficit in ATP production emerged. This cellular model of hiPSCs-derived motor neurons underlines the role of mitochondrion and oxidative stress in CMT disease and paves the way for new treatment evaluation.