Meiosis-Based Laboratory Evolution of the Thermal Tolerance in Kluyveromyces marxianus

Kluyveromyces marxianus is the fastest-growing eukaryote and a promising host for producing bioethanol and heterologous proteins. To perform a laboratory evolution of thermal tolerance in K. marxianus, diploid, triploid and tetraploid strains were constructed, respectively. Considering the genetic diversity caused by genetic recombination in meiosis, we established an iterative cycle of “diploid/polyploid - meiosis - selection of spores at high temperature” to screen thermotolerant strains. Results showed that the evolution of thermal tolerance in diploid strain was more efficient than that in triploid and tetraploid strains. The thermal tolerance of the progenies of diploid and triploid strains after a two-round screen was significantly improved than that after a one-round screen, while the thermal tolerance of the progenies after the one-round screen was better than that of the initial strain. After a two-round screen, the maximum tolerable temperature of Dip2-8, a progeny of diploid strain, was 3°C higher than that of the original strain. Whole-genome sequencing revealed nonsense mutations of PSR1 and PDE2 in the thermotolerant progenies. Deletion of either PSR1 or PDE2 in the original strain improved thermotolerance and two deletions displayed additive effects, suggesting PSR1 and PDE2 negatively regulated the thermotolerance of K. marxianus in parallel pathways. Therefore, the iterative cycle of “meiosis - spore screening” developed in this study provides an efficient way to perform the laboratory evolution of heat resistance in yeast.

Novel ratio-metric features enable the identification of new driver genes across cancer types

An emergent area of cancer genomics is the identification of driver genes. Driver genes confer a selective growth advantage to the cell. While several driver genes have been discovered, many remain undiscovered, especially those mutated at a low frequency across samples. This study defines new features and builds a pan-cancer model, cTaG, to identify new driver genes. The features capture the functional impact of the mutations as well as their recurrence across samples, which helps build a model unbiased to genes with low frequency. The model classifies genes into the functional categories of driver genes, tumour suppressor genes (TSGs) and oncogenes (OGs), having distinct mutation type profiles. We overcome overfitting and show that certain mutation types, such as nonsense mutations, are more important for classification. Further, cTaG was employed to identify tissue-specific driver genes. Some known cancer driver genes predicted by cTaG as TSGs with high probability are ARID1A, TP53, and RB1. In addition to these known genes, potential driver genes predicted are CD36, ZNF750 and ARHGAP35 as TSGs and TAB3 as an oncogene. Overall, our approach surmounts the issue of low recall and bias towards genes with high mutation rates and predicts potential new driver genes for further experimental screening. cTaG is available at https://github.com/RamanLab/cTaG.

Gene Amplification as a Mechanism of Yeast Adaptation to Nonsense Mutations in Release Factor Genes

Protein synthesis (translation) is one of the fundamental processes occurring in the cells of living organisms. Translation can be divided into three key steps: initiation, elongation, and termination. In the yeast Saccharomyces cerevisiae, there are two translation termination factors, eRF1 and eRF3. These factors are encoded by the SUP45 and SUP35 genes, which are essential; deletion of any of them leads to the death of yeast cells. However, viable strains with nonsense mutations in both the SUP35 and SUP45 genes were previously obtained in several groups. The survival of such mutants clearly involves feedback control of premature stop codon readthrough; however, the exact molecular basis of such feedback control remain unclear. To investigate the genetic factors supporting the viability of these SUP35 and SUP45 nonsense mutants, we performed whole-genome sequencing of strains carrying mutant sup35-n and sup45-n alleles; while no common SNPs or indels were found in these genomes, we discovered a systematic increase in the copy number of the plasmids carrying mutant sup35-n and sup45-n alleles. We used the qPCR method which confirmed the differences in the relative number of SUP35 and SUP45 gene copies between strains carrying wild-type or mutant alleles of SUP35 and SUP45 genes. Moreover, we compare the number of copies of the SUP35 and SUP45 genes in strains carrying different nonsense mutant variants of these genes as a single chromosomal copy. qPCR results indicate that the number of mutant gene copies is increased compared to the wild-type control. In case of several sup45-n alleles, this was due to a disomy of the entire chromosome II, while for the sup35-218 mutation we observed a local duplication of a segment of chromosome IV containing the SUP35 gene. Taken together, our results indicate that gene amplification is a common mechanism of adaptation to nonsense mutations in release factor genes in yeast.

Translational readthrough as a potential therapeutic for AIPL1-associated Leber Congenital Amaurosis in a patient-derived iPSC-retinal organoid model

Leber Congenital Amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterised by severe sight impairment in infancy and rapidly progressive degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cGMP phosphodiesterase (PDE6) compared to control or CRISPR corrected organoids. Moreover, increased levels of cGMP were detected. The translational readthrough inducing drug (TRID) PTC124 was investigated as a potential therapeutic. LCA4 retinal organoids exhibited rescue of AIPL1 and PDE6; however, the level of full-length, functional AIPL1 induced through PTC124 treatment was insufficient to reduce cGMP levels and fully rescue the LCA4 phenotype. LCA4 retinal organoids are a valuable platform for the in vitro investigation of the molecular mechanisms that drive photoreceptor loss and for the evaluation of novel therapeutics.

New Mutations in HFE2 and TFR2 Genes Causing Non HFE-Related Hereditary Hemochromatosis

Hereditary hemochromatosis (HH) is an iron metabolism disease clinically characterized by excessive iron deposition in parenchymal organs such as liver, heart, pancreas, and joints. It is caused by mutations in at least five different genes. HFE hemochromatosis is the most common type of hemochromatosis, while non-HFE related hemochromatosis are rare cases. Here, we describe six new patients of non-HFE related HH from five different families. Two families (Family 1 and 2) have novel nonsense mutations in the HFE2 gene have novel nonsense mutations (p.Arg63Ter and Asp36ThrfsTer96). Three families have mutations in the TFR2 gene, one case has one previously unreported mutation (Family A—p.Asp680Tyr) and two cases have known pathogenic mutations (Family B and D—p.Trp781Ter and p.Gln672Ter respectively). Clinical, biochemical, and genetic data are discussed in all these cases. These rare cases of non-HFE related hereditary hemochromatosis highlight the importance of an earlier molecular diagnosis in a specialized center to prevent serious clinical complications.

Coding-Complete Genome Sequences of Alpha and Delta SARS-CoV-2 Variants from Kamphaeng Phet Province, Thailand, from May to July 2021

We report coding-complete genome sequences of 44 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains of the alpha and delta variants identified from patients in Kamphaeng Phet, Thailand. Two nonsense mutations in open reading frame 3a (ORF3a) (G254*) and ORF8 (K68*) were found in the alpha variant sequences. Two lineages of the delta variant, B.1.617.2 and AY.30, were found.

The Role of POPDC Proteins in Cardiac Pacemaking and Conduction

The Popeye domain-containing (POPDC) gene family, consisting of Popdc1 (also known as Bves), Popdc2, and Popdc3, encodes transmembrane proteins abundantly expressed in striated muscle. POPDC proteins have recently been identified as cAMP effector proteins and have been proposed to be part of the protein network involved in cAMP signaling. However, their exact biochemical activity is presently poorly understood. Loss-of-function mutations in animal models causes abnormalities in skeletal muscle regeneration, conduction, and heart rate adaptation after stress. Likewise, patients carrying missense or nonsense mutations in POPDC genes have been associated with cardiac arrhythmias and limb-girdle muscular dystrophy. In this review, we introduce the POPDC protein family, and describe their structure function, and role in cAMP signaling. Furthermore, the pathological phenotypes observed in zebrafish and mouse models and the clinical and molecular pathologies in patients carrying POPDC mutations are described.

2-Guanidino-quinazoline promotes the readthrough of nonsense mutations underlying human genetic diseases

Premature termination codons (PTCs) account for 10% to 20% of genetic diseases in humans. The gene inactivation resulting from PTC can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a new reporter cell line and performed high-throughput screening (HTS) to identify potential new readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy. We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of natural stop codons.

Comprehensive Analysis of Combinatorial Pharmacological Treatments to Correct Nonsense Mutations in the CFTR Gene

Cystic fibrosis (CF) is caused by loss of function of the CFTR chloride channel. A substantial number of CF patients carry nonsense mutations in the CFTR gene. These patients cannot directly benefit from pharmacological correctors and potentiators that have been developed for other types of CFTR mutations. We evaluated the efficacy of combinations of drugs targeting at various levels the effects of nonsense mutations: SMG1i to protect CFTR mRNA from nonsense-mediated decay (NMD), G418 and ELX-02 for readthrough, VX-809 and VX-445 to promote protein maturation and function, PTI-428 to enhance CFTR protein synthesis. We found that the extent of rescue and sensitivity to the various agents is largely dependent on the type of mutation, with W1282X and R553X being the mutations most and least sensitive to pharmacological treatments, respectively. In particular, W1282X-CFTR was highly responsive to NMD suppression by SMG1i but also required treatment with VX-445 corrector to show function. In contrast, G542X-CFTR required treatment with readthrough agents and VX-809. Importantly, we never found cooperativity between the NMD inhibitor and readthrough compounds. Our results indicate that treatment of CF patients with nonsense mutations requires a precision medicine approach with the design of specific drug combinations for each mutation.

Molecular Approaches Fighting Nonsense

Nonsense mutations are the result of single nucleotide substitutions in the DNA that change a sense codon (coding for an amino acid) to a nonsense or premature termination codon (PTC) within the coding region of the mRNA [...]