NanoLuc reporters identify COL4A5 nonsense mutations susceptible to drug-induced stop codon readthrough

Omachi, Kohei; Kai, Hirofumi; Roberge, Michel; Miner, Jeffrey H.

doi:10.1016/j.isci.2022.103891

Cited by 9 publications

(11 citation statements)

References 56 publications

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“…The split NanoLuc-based protein-protein interaction assay system is a method to detect close protein interactions by luminescence associated with the proximity of the split NanoLuc fragments LgBiT and SmBiT (18). Using this technology, we previously developed a method to evaluate the formation of proper collagen α 3 α 4 α 5(IV) heterotrimers quantitatively (Figure 3A) (16, 19, 20). This split NanoLuc complementation-based α 3 α 4 α 5(IV) assay has detected loss of function and presumed pathogenicity of many variants reported in patients with Alport syndrome.…”

Section: Resultsmentioning

confidence: 99%

A COL4A4-G394S Variant and Impaired Collagen IV Trimerization in a Patient with Mild Alport Syndrome

et al. 2022

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Background: Missense variants in COL4A genes are often found in patients with an Alport syndrome-like presentation, but their pathogenicity is not always clear. We encountered a female patient with microscopic hematuria and proteinuria at 33 years of age with a diagnosis of thin basement membrane disease who was approaching end stage kidney disease at 58 years of age. We hypothesized that this patient's kidney disease was within the spectrum of Alport syndrome. Methods: We used histologic, genetic, and biochemical approaches to investigate the mechanisms of kidney disease. By immunofluorescence, we investigated collagen IV chain composition of the glomerular basement membrane (GBM). We employed targeted sequencing to search for pathogenic variants in COL4A and other relevant genes. We utilized N- and C-terminal split NanoLuciferase assays to determine the impact of a novel COL4A4 variant of uncertain significance (VUS) on collagen IV heterotrimer formation in vitro. We transfected COL4A4 expression constructs with split NanoLuciferase fragment-fused COL4A3 and COL4A5 constructs into human embryonic kidney 293T cells. To assay for α3α4α5(IV) heterotrimer formation and secretion, we measured luminescence in cell lysates and culture supernatants from transfected cells. Results: Immunostaining suggested that the collagen α3α4α5(IV) network was present throughout the patient's GBMs. DNA sequencing revealed a novel homozygous VUS, COL4A4 c.1180G>A (p.Gly394Ser). In the C-terminal split luciferase-based α3α4α5(IV) heterotrimer formation assays, luminescence levels for G394S were comparable to WT, but in the N-terminal tag assays, the extracellular luminescence levels for G394S were decreased by ~50% vs. WT. Conclusions: Our cell-based assay provides a platform to test COL4 VUS and shows that G394S impairs assembly of the α3α4α5(IV) N-terminus and subsequent trimer secretion. These data suggest that the COL4A4 G394S variant is pathogenic and causes an atypical mild form of autosomal recessive Alport syndrome.

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Section: Resultsmentioning

confidence: 99%

A COL4A4-G394S Variant and Impaired Collagen IV Trimerization in a Patient with Mild Alport Syndrome

et al. 2022

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“…Of note, one interesting study found that caffeine can attenuate the NMD activity and combine with PTC124 to enhance the readthrough and enrich the generation of full-length proteins [ 49 ]. Many small compounds have been screened and identified to have PTC readthrough activity through cell-based luciferase assays with a premature nonsense mutation in a firefly luciferase gene [ 2 , 47 , 50 ]. However, PTC124 can stabilize firefly luciferase directly [ 38 ], so this may restrict the identification of small compound(s) to enhance the PTC124 PTC readthrough.…”

Section: Discussionmentioning

confidence: 99%

PTC124 Rescues Nonsense Mutation of Two Tumor Suppressor Genes NOTCH1 and FAT1 to Repress HNSCC Cell Proliferation

et al. 2022

Biomedicines

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(1) Background: PTC124 (Ataluren) is an investigational drug for the treatment of nonsense mutation-mediated genetic diseases. With the exception of the TP53 tumor suppressor gene, there has been little research on cancers with nonsense mutation. By conducting a database search, we found that another two tumor suppressor genes, NOTCH1 and FAT1, have a high nonsense mutation rate in head and neck squamous cell carcinoma (HNSCC). PTC124 may re-express the functional NOTCH1 or FAT1 in nonsense mutation NOTCH1 or FAT1 in HSNCC (2) Methods: DOK (with NOTCH1 Y550X) or HO-1-u-1 (with FAT1 E378X) HNSCC cells were treated with PTC124, and the NOTCH1 or FAT1 expression, cell viability, and NOTCH1- or FAT1-related downstream gene profiles were assayed. (3) Results: PTC124 was able to induce NOTCH1 or FAT1 expression in DOK and HO-1-u-1 cells. PTC124 was able to upregulate NOTCH downstream genes HES5, AJUBA, and ADAM10 in DOK cells. PTC124 enhanced DDIT4, which is under the control of the FAT1–YAP1 pathway, in HO-1-u-1 cells. FLI-06 (a NOTCH signaling inhibitor) reversed PTC124-mediated cell growth inhibition in DOK cells. PTC124 could reverse TT-10 (a YAP signaling activator)-mediated HO-1-u-1 cell proliferation. (4) Conclusions: PTC124 can rescue nonsense mutation of NOTCH1 and FAT1 to repress HNSCC cell proliferation.

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“…A comprehensive NanoLuc-based investigation of known readthrough inducers in the context of a larger number of nonsense mutations in COL4A5 —the gene involved in Alport syndrome—suggested the applicability of PTC readthrough therapy for a subset of mutations (at least 11 out of 49 nonsense mutations in COL4A5 ) [ 134 ]. A NanoLuc-based translational reporter system was used to evaluate different readthrough inducers on 49 different nonsense mutations at different concentrations and in four different cell types.…”

Section: Nonsense Suppression In the Context Of Rare Genetic Diseases...mentioning

confidence: 99%

“…On the contrary, treatment with ataluren (20–50 µM) or RTC14 (25–100 µM) resulted in no detectable induction. Furthermore, combinatorial treatment with the antimalarial drug mefloquine appeared to enhance the G418- and gentamicin-induced readthrough of a nonsense mutation (R1563X) [ 134 ].…”

Section: Nonsense Suppression In the Context Of Rare Genetic Diseases...mentioning

confidence: 99%

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

Wagner

Wießner

Friedrich

et al. 2023

IJMS

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Nonsense mutations trigger premature translation termination and often give rise to prevalent and rare genetic diseases. Consequently, the pharmacological suppression of an unscheduled stop codon represents an attractive treatment option and is of high clinical relevance. At the molecular level, the ability of the ribosome to continue translation past a stop codon is designated stop codon readthrough (SCR). SCR of disease-causing premature termination codons (PTCs) is minimal but small molecule interventions, such as treatment with aminoglycoside antibiotics, can enhance its frequency. In this review, we summarize the current understanding of translation termination (both at PTCs and at cognate stop codons) and highlight recently discovered pathways that influence its fidelity. We describe the mechanisms involved in the recognition and readthrough of PTCs and report on SCR-inducing compounds currently explored in preclinical research and clinical trials. We conclude by reviewing the ongoing attempts of personalized nonsense suppression therapy in different disease contexts, including the genetic skin condition epidermolysis bullosa.

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NanoLuc reporters identify COL4A5 nonsense mutations susceptible to drug-induced stop codon readthrough

Cited by 9 publications

References 56 publications

A COL4A4-G394S Variant and Impaired Collagen IV Trimerization in a Patient with Mild Alport Syndrome

A COL4A4-G394S Variant and Impaired Collagen IV Trimerization in a Patient with Mild Alport Syndrome

PTC124 Rescues Nonsense Mutation of Two Tumor Suppressor Genes NOTCH1 and FAT1 to Repress HNSCC Cell Proliferation

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

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