Stop Mutation Research Articles

Dedicated developmental programing for group-supporting behaviors in eusocial honeybees.

The evolutionary changes from solitary to eusocial living in vertebrates and invertebrates are associated with the diversification of social interactions and the development of queen and worker castes. Despite strong innate patterns, our understanding of the mechanisms manifesting these sophisticated behaviors is still rudimentary. Here, we show that doublesex (dsx) manifests group-supporting behaviors in the honeybee (Apis mellifera) worker caste. Computer-based individual behavioral tracking of worker bees with biallelic stop mutations in colonies revealed that the dsx gene is required for the rate and duration of group-supporting behavior that scales the relationship between bees and their work. General sensorimotor functions remained unaffected. Unexpectedly, unlike in other insects, the dsx gene is required for the neuronal wiring of the mushroom body in which the gene is spatially restricted expressed. Together, our study establishes dedicated programming for group-supporting behaviors and provides insight into the connection between development in the neuronal circuitry and behaviors regulating the formation of a eusocial society.

Screening and Validation of Leaf Width-Related Genes in Inbred Maize Lines.

Leaf width is a key determinant of planting density and photosynthetic efficiency. In an effort to determine which genes regulate maize plant leaf width, we performed a genome-wide association study (GWAS) of 1.49 × 106 single nucleotide polymorphisms (SNPs) in 80 sequenced backbone inbred maize lines in Jilin Province, China, based upon phenotypic leaf width data from two years. In total, 14 SNPs were identified as being significantly related to leaf width (p < 0.000001), with these SNPs being located on chromosomes 1, 2, 3, 5, 6, 7, 8, and 9. A total of five candidate genes were identified within a mean linkage disequilibrium (LD) distance of 9.7 kb, with a significant SNP being identified within the Zm00001d044327 candidate gene. RNA was then isolated from 12 different inbred maize lines from this GWAS study cohort and was used to conduct qPCR analyses which revealed significant differences in Zm00001d044327 expression among strains exhibiting significant differences in leaf width. Based on an assessment of EMS mutant lines harboring a conserved amino acid stop mutation and two non-synonymous mutations in Zm00001d044327 that exhibited a narrow leaf width, these data suggested that Zm00001d044327 is a key regulator of maize leaf width.

R2R3-MYB transcription factor CsMYB60 controls mature fruit skin color by regulating flavonoid accumulation in cucumber.

Skin color is an important trait that determines the cosmetic appearance and quality of fruits. In cucumber, the skin color ranges from white to brown in mature fruits. However, the genetic basis for this important trait remains unclear. We conducted a genome-wide association study of natural cucumber populations, along with map-based cloning techniques, on an F2 population resulting from a cross between Pepino (with yellow-brown fruit skin) and Zaoer-N (with creamy fruit skin). We identified CsMYB60 as a candidate gene responsible for skin coloration in mature cucumber fruits. In cucumber accessions with white to pale yellow skin color, a premature stop mutation (C to T) was found in the second exon region of CsMYB60, whereas light yellow cucumber accessions exhibited splicing premature termination caused by an intronic mutator-like element insertion in CsMYB60. Transgenic CsMYB60c cucumber plants displayed a yellow-brown skin color by promoting accumulation of flavonoids, especially hyperoside, a yellow-colored flavonol. CsMYB60c encodes a nuclear protein that primarily acts as a transcriptional activator through its C-terminal activation motif. RNA sequencing and DNA affinity purification sequencing assays revealed that CsMYB60c promotes skin coloration by directly binding to the YYTACCTAMYT motif in the promoter regions of flavonoid biosynthetic genes, including CsF3'H, which encodes flavonoid 3'-hydroxylase. The findings of our study not only offer insight into the function of CsMYB60 as dominantly controlling fruit coloration, but also highlight that intronic DNA mutations can have a similar phenotypic impact as exonic mutations, which may be valuable in future cucumber breeding programs.

Positive experience of nmDMD patient treatment

Progressive Duchenne muscular dystrophy (DMD) (OMIM # 310200) is an inherited X-linked neuromuscular disease caused by a mutation in the DMD gene encoding the dystrophin protein, resulting in absent or deficient dystrophin function. It usually affects boys during childhood. Among mutations, large deletions are found in ~65% of cases; ~10% of mutations are represented by duplications, and the remaining cases are represented by point and small mutations, of which 10–15% are represented by nonsense mutations (stop mutation). Currently, pathogenetic therapy is available for a number of deletions and point nonsense mutations in this gene. Translarna® (ataluren) is the only drug for pathogenetic therapy of patients with DMD caused by nonsense mutations registered in the Russian Federation. We present a clinical case of the earliest and long- lasting effective treatment with Translarna® (ataluren) in a boy born in 2016 in St. Petersburg.

Comprehensive genomic analysis of the SARS-CoV-2 Omicron variant BA.2.76 in Jining City, China, 2022

ObjectiveThis study aims to analyze the molecular characteristics of the novel coronavirus (SARS-CoV-2) Omicron variant BA.2.76 in Jining City, China.MethodsWhole-genome sequencing was performed on 87 cases of SARS-CoV-2 infection. Evolutionary trees were constructed using bioinformatics software to analyze sequence homology, variant sites, N-glycosylation sites, and phosphorylation sites.ResultsAll 87 SARS-CoV-2 whole-genome sequences were classified under the evolutionary branch of the Omicron variant BA.2.76. Their similarity to the reference strain Wuhan-Hu-1 ranged from 99.72 to 99.74%. In comparison to the reference strain Wuhan-Hu-1, the 87 sequences exhibited 77–84 nucleotide differences and 27 nucleotide deletions. A total of 69 amino acid variant sites, 9 amino acid deletions, and 1 stop codon mutation were identified across 18 proteins. Among them, the spike (S) protein exhibited the highest number of variant sites, and the ORF8 protein showed a Q27 stop mutation. Multiple proteins displayed variations in glycosylation and phosphorylation sites.ConclusionSARS-CoV-2 continues to evolve, giving rise to new strains with enhanced transmission, stronger immune evasion capabilities, and reduced pathogenicity. The application of high-throughput sequencing technologies in the epidemic prevention and control of COVID-19 provides crucial insights into the evolutionary and variant characteristics of the virus at the genomic level, thereby holding significant implications for the prevention and control of the COVID-19 pandemic.

Identification of a DLG3 stop mutation in the MRX20 family.

Here, we identified the causal mutation in the MRX20 family, one of the larger X-linked pedigrees that have been described in which no gene had been identified up till now. In 1995, the putative disease gene had been mapped to the pericentromeric region on the X chromosome, but no follow-up studies were performed. Here, whole exome sequencing (WES) on two affected and one unaffected family member revealed the c.195del/p.(Thr66ProfsTer55) mutation in the DLG3 gene (NM_021120.4) that segregated with the affected individuals in the family. DLG3 mutations have been consequently associated with intellectual disability and are a plausible explanation for the clinical abnormalities observed in this family. In addition, we identified two other variants co-segregating with the phenotype: a stop gain mutation in SSX1 (c.358G>T/p.(Glu120Ter)) (NM_001278691.2) and a nonsynonymous SNV in USP27X (c.56 A>G/p.(Gln19Arg)) (NM_001145073.3). RNA sequencing revealed 14 differentially expressed genes (p value < 0.1) in 7 affected males compared to 4 unaffected males of the family, including four genes known to be associated with neurological disorders. Thus, in this paper we identified the c.195del/p.(Thr66ProfsTer55) mutation in the DLG3 gene (NM_021120.4) as likely responsible for the phenotype observed in the MRX20 family.

Dosage response to reduced height‐1 (Rht‐1) loss‐of‐function mutations and characterization of slender phenotype in hexaploid wheat

AbstractThe reduced height (Rht‐1) genes in wheat (Triticum aestivum L.) are integral in controlling plant height. Previous studies in other plant species have demonstrated that loss‐of‐function mutations in their orthologous Rht‐1 genes results in plants with a slender phenotype illustrated by increased plant heights, sterility, and a constitutive gibberellic acid (GA3) response; however, this phenotype has not been described in wheat. In this study, nonsense alleles occurring in the GRAS domain of Rht‐A1, B1, and D1 were combined to create single, double, and triple Rht‐1 mutants. Homozygous lines possessing none, one, two, or three Rht‐1 stop mutations were grown in replicated field trials in three environments to assess agronomic traits. Germination tests to measure GA3 responsiveness and gene expression analysis via RNA‐seq were also performed. Rht‐1 triple mutants exhibited a slender phenotype characterized by rapid growth, elongated coleoptiles and internodes, elongated spikes, decreased tiller and spikelet number, and sterile heads. The presence of a single functional Rht‐1 gene resulted in a normal phenotype. Differences in plant height among the Rht‐1 double mutants, Rht‐1 single mutants, and Rht‐1 all wild‐type dosages trended toward increased plant height with increased Rht‐1 stop mutation dosage. Differences in Rht‐1 homeolog gene expression did not equate to differences in plant height between the different Rht‐1 stop mutations.

Salmonella Derby adaptation to swine and simultaneous attenuation for humans go through decay of Salmonella Pathogenicity Island I.

We report the identification, within the global population of Salmonella Derby, of a lineage highly diffused and expanding in swine while being significantly under-represented in humans which carries stop mutations in the Salmonella Pathogenicity Island 1 (SPI-1) genes sipA and hilC. Single-cell analysis of invasion, vacuolar load, and cytosolic hyper-replication show that these mutations attenuate S. Derby virulence in human intestinal cells. Identification of this lineage follows the previous detection of another lineage over-represented in swine compared with humans which carries a loss-of-function mutation in hilD, the major SPI-1 regulator, responsible for reduced infection of human cells. Notably, this hilD-mutated lineage represents a sub-population of the wider sipA and hilC-mutated lineages identified in this study, indicating that SPI-1 is subjected to genetic decay in this part of the Salmonella Derby population adapted to swine. Virulence assessment on swine intestinal cells showed drastically lower levels for both wild-type and mutant S. Derby compared with human cells. Still, S. Derby with impaired sipA and hilC showed further attenuation also in swine cells compared with the wild type. These data suggest that invasion of swine intestinal epithelium is not needed by S. Derby to circulate in pigs, leading to the decay of SPI-1, which in turn determines attenuation for humans. Accordingly, a further S. Derby lineage under-represented in humans compared with pigs was found carrying truncated or missing SPI-1 genes, indicating that the S. Derby population is undergoing converging evolution in its adaptation to swine while attenuating for humans through loss of SPI-1 function.IMPORTANCEThis study integrated population data with in vitro assessment of virulence phenotypes to unveil that a considerable part of the global population of Salmonella Derby is evolving to enhance its host adaptation to the swine host and that this evolution is simultaneously increasing its attenuation for humans. The study shows that the fixation of deleterious mutations in SPI-1 has a role in this process. This evidence indicates that SPI-1 has a key role for S. Derby virulence in humans but not for its circulation in swine. The results show that genes generally considered essential for Salmonella pathogenesis do not play the same key role for all Salmonella serovars or lineages and/or all hosts. The study helps in understanding the molecular mechanisms underlying the ecology and host adaptation of Salmonella showing that the adaptation process can vary for different types of Salmonella and hosts.

P24 Recurrent skin ulcerations as an isolated manifestation of ISG15 deficiency

Abstract Type I interferonopathies are characterized by a very broad phenotypic spectrum; however, some overlapping clinical features are seen across the type I interferonopathies, most particularly brain and skin involvement. We report a female patient presenting at 7 months of age with ulcerations in her groin and axilla. The lesions in her axilla healed; however, she continued having recurrent ulcerations in both sides of her groin and she developed lipoatrophy of the lower abdomen. Skin biopsy revealed deep-seated inflammation with abnormal adipose tissue; no bacteria were isolated. Extensive immunology blood work-up did not reveal any abnormalities, and the inflammatory markers were normal. Because of the extent of the clinical findings and pending results of genetic screening with targeted next-generation sequencing, we commenced methotrexate at 1 year of age (15 mg m–2). Following the initiation of methotrexate, there was marked improvement in the clinical features, with resolution of active skin ulceration. Genetic screening revealed a homozygous stop mutation in ISG15 p.Q16X. A CT scan of her head did not show any calcifications. Methotrexate was stopped at age 3 years. She remains well with no further skin ulcerations, and her development continues to be age appropriate. ISG15 deficiency is a type I interferonopathy, previously described in association with intracranial calcifications and increased susceptibility to mycobacteria infections. However, more recent reports have described skin ulcerations as a main finding in addition to neurological and immunological manifestations. Our patient is not showing any signs of neurological or other immunological manifestations but remains under careful monitoring.

Abstract P1023: Nonsense Mutations In Atrial Myofilament Regulatory Protein MyBP-HL Are Subject To Proteolytic Degradation And Loss Of Functional Protein

Background: Myosin binding protein-H like (MyBP-HL) is a recently identified atrial myofilament protein encoded by the gene MYBPHL. The Arg255Stop mutant has been linked to atrial enlargement, atrial fibrillation, and dilated cardiomyopathy. Deletion of MYBPHL in mice recapitulates this human phenotype. MyBP-HL is composed of two Immunoglobulin domains (Ig) and one Fibronectin domain (FnIII), like the structure and sequence of the last three C-terminal domains (C8-C9-C10) of cardiac myosin binding protein-C (cMyBP-C). cMyBP-C nonsense mutations fail in myofilament incorporation and result in a rapid degradation of the truncated protein and are a major cause of hypertrophic cardiomyopathy. There are 9 premature stop mutations in MYBPHL identified in humans (Gln29, Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250, Arg255, and Tyr307). Hypothesis: We hypothesize that MYBPHL nonsense mutations will share a common mechanism that leads to MyBP-HL failing to integrate into the sarcomere, resulting in truncated MyBP-HL degradation and loss myofilament incorporation. Results: We demonstrate heterologous expression of MyBP-HL in neonatal rat cardiomyocytes can incorporate in a similar sarcomere pattern as cMyBP-C. Expression of the nine nonsense mutations do not localize into the sarcomere. Neonatal rat cardiomyocytes transfected with MyBP-HL constructs were treated with proteasome and/or calpain inhibitors. We observed that wild-type MyBP-HL and MyBP-HL truncating mutations are degraded mainly by the proteasome, although we also identified a significant calpain involvement in MyBP-HL degradation. Shorter MyBP-HL truncated peptides are degraded as a poisoning peptides, but longer peptides are not degraded. Conclusion: Based on these data, we demonstrated that MYBPHL nonsense mutations prevent MyBP-HL incorporation into the thick filament as are subject to degradation. The differential expression of short vs. long truncating MyBP-HL mutants suggest a differential pathogenic mechanism for subsets of these mutations.

Site-Specific RNA Editing of Stop Mutations in the CFTR mRNA of Human Bronchial Cultured Cells.

It is reported that about 10% of cystic fibrosis (CF) patients worldwide have nonsense (stop) mutations in the CFTR gene, which cause the premature termination of CFTR protein synthesis, leading to a truncated and non-functional protein. To address this issue, we investigated the possibility of rescuing the CFTR nonsense mutation (UGA) by sequence-specific RNA editing in CFTR mutant CFF-16HBEge, W1282X, and G542X human bronchial cells. We used two different base editor tools that take advantage of ADAR enzymes (adenosine deaminase acting on RNA) to edit adenosine to inosine (A-to-I) within the mRNA: the REPAIRv2 (RNA Editing for Programmable A to I Replacement, version 2) and the minixABE (A to I Base Editor). Immunofluorescence experiments show that both approaches were able to recover the CFTR protein in the CFTR mutant cells. In addition, RT-qPCR confirmed the rescue of the CFTR full transcript. These findings suggest that site-specific RNA editing may efficiently correct the UGA premature stop codon in the CFTR transcript in CFF-16HBEge, W1282X, and G542X cells. Thus, this approach, which is safer than acting directly on the mutated DNA, opens up new therapeutic possibilities for CF patients with nonsense mutations.

Suppressing ASPARTIC PROTEASE 1 prolongs photosynthesis and increases wheat grain weight.

The elongation of photosynthesis, or functional staygreen, represents a feasible strategy to propel metabolite flux towards cereal kernels. However, achieving this goal remains a challenge in food crops. Here we report the cloning of wheat CO2 assimilation and kernel enhanced 2 (cake2), the mechanism underlying the photosynthesis advantages and natural alleles amenable to breeding elite varieties. A premature stop mutation in the A-genome copy of the ASPARTIC PROTEASE 1 (APP-A1) gene increased the photosynthesis rate and yield. APP1 bound and degraded PsbO, the protective extrinsic member of photosystem II critical for increasing photosynthesis and yield. Furthermore, a natural polymorphism of the APP-A1 gene in common wheat reduced APP-A1's activity and promoted photosynthesis and grain size and weight. This work demonstrates that the modification of APP1 increases photosynthesis, grain size and yield potentials. The genetic resources could propel photosynthesis and high-yield potentials in elite varieties of tetraploid and hexaploid wheat.

WS09.01 Extending the success of Trikafta to rare mutations - insights from CFTR structure and modulator binding

ETI offers therapeutic opportunities for most CF patients but not for many with rare mutations. We established patient-derived organoids with the Q1100P mutation, not reported in CFTR2. We obtained biopsies from 3 patients heterozygous for Q1100P and a stop mutation (C225X, likely nonresponsive to ETI) or a missense (K163E). We investigated CFTR function after treatment with modulators.

Applying patient personalized organoid model system to understand complex GI manifestations in cystic fibrosis disease

Background: Stem cell-derived organotypic culture has become a revolutionary tool in transforming care for patients with multiple GI-disorders. Cystic fibrosis is a deadly genetic disorder with multiple GI complications, such as Distal intestinal obstruction syndrome (DIOS), chronic constipation, Gastroesophageal reflux disease (GERD), and in some cases pancreatitis, caused by loss of function mutations in the gene that encodes for the cystic fibrosis transmembrane conductance regulator (CFTR) chloride/bicarbonate ion channel. Due to genetic and environmental influences, CF patients exhibit large phenotypic variation. These factors make clinical trials difficult and largely impractical due to limited and heterogenous patient pools, and additionally, the benefit of approved small-molecule CF therapy in a large number of rare mutation patients is not known. Moreover, critical questions of whether CFTR contributes to the development of intestine and how it contributes to GI-related issues remain unanswered. Results: In this study, we performed a comprehensive bench-side study using in vitro patient enteroids and in vivo mice implanted Human Intestinal Organoids (HIOs) to test highly effective CFTR modulators -Ivacaftor response for a rare CF mutation patient. Based on the positive Ivacaftor response in the enteroids, the patient was enrolled in N=1 clinical trial and showed improved GI outcomes upon Ivacaftor treatment. HIO implantation model allowed in vivo modulator dosing and provided an elegant human organ-based demonstration of bench-to-bedside testing of modulator effects. Additionally, using the CF HIO model the role of CFTR function in the maturation of human intestine was reported for the first time. In HIOs derived from a stop mutation CF patient and CFTR-corrected HIOs as control, we performed single cell RNAseq analysis to generate a molecular map of GI-cell type specific transcriptomic alterations and identify key targetable pathways relevant to stop mutations in CFTR for which currently there is no small molecule therapy. Conclusion: In all, we demonstrate that these models effectively serve to translate data from the lab to the clinic and back so that patient specific therapies could be validated, or new therapies be devised. Additionally, single-cell RNA sequencing in patient HIOs will provide useful insights into cell population specific and transcriptomic level alterations that are associated with abnormalities in GI function in CF. Nothing to disclose This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Decreased or Absent B Cells in patients with Severe Reduction in All Serum Immunoglobulin Isotypes, a registry based study

BackgroundSevere Reduction in All Serum Immunoglobulin Isotypes with Profoundly Decreased or Absent B Cells is a subgroup of inborn error of immunities (IEI). 80 to 90% carry mutations in X-chromosomal Bruton tyrosine kinase (BTK) and around 5–10% of the cases are autosomal recessive Agammaglobulinemia (ARA). Very few of Hypogammaglobulinemia with no B cells classified in other subgroups. All of these patients benefit from a genetic evaluation, especially in highly consanguine populations. Materials and Methods27 patients from 11 families with hypogamma-globinemia and no B-cells registered in Immunodeficiency Diseases Research Center registry, Isfahan University of Medical Sciences were phenotypically and genetically evaluated. ResultsThe most prevalent genetic cause was mutations in BTK. The novel mutations in BTK gene includes c.176 G>A (p. Glu89Lys); c.115T>C (p. Tyr39His); c.685-686insTTAC (p. Asn229llefs5), in ARA includes a homozygous stop mutation in Immunoglobulin Heavy Constant Mu Chain (IGHM) gene, a frameshift mutation of B-Cell Antigen Receptor Complex-Associated Protein (CD79A), a bi-allelic stop-gain mutation in Transcription Factor 3 (TCF3), and in PIK3CD also in others IEI includes :novel homozygous silent mutations in Phosphoinositide-3-Kinase Regulatory Subunit (RASGRP1), homozygote SAMD9 mutation. DiscussionWe did not observe any phenotypically significant differences in XLA patients.The main clinical manifestation of ARA cases is, recurrent infections. However, patient with IGHM deficiency had earlier onset of the disease and more severe complications. In CD79A deficient case with 5 affected members and four deaths in her family, the symptoms started very early but managed properly according to the family history. Due to the late diagnosis of PIK3CD cases, more severe complications like bronchiectasis, neurologic sequels after poliomyelitis, and SLE nephropathy has been developed. Our novel SAMD9 had quite different phenotype and the novel RASPGR2 case with no B cells unfortunately managed as ARA before genetic diagnosis and has died of lymphoma as her 3 siblings. However, we observed that in families with more than one affected family member, the disease was better controlled in the second patient. ConclusionOur study broadens the genetic spectrum of hypogammaglobulinemia with no B cells in Iran. This helps differentiate phenotypes and genotypes and manage patients appropriately.

Suppression of ZEAXANTHIN EPOXIDASE 1 restricts stripe rust growth in wheat

Reducing losses caused by pathogens is an effective strategy for stabilizing crop yields. Daunting challenges remain in cloning and characterizing genes that inhibit stripe rust, a devastating disease of wheat (Triticum aestivum) caused by Puccinia striiformis f. sp. tritici (Pst). We found that suppression of wheat zeaxanthin epoxidase 1 (ZEP1) increased wheat defense against Pst. We isolated the yellow rust slower 1 (yrs1) mutant of tetraploid wheat in which a premature stop mutation in ZEP1-B underpins the phenotype. Genetic analyses revealed increased H2O2 accumulation in zep1 mutants and demonstrated a correlation between ZEP1 dysfunction and slower Pst growth in wheat. Moreover, wheat kinase START 1.1 (WKS1.1, Yr36) bound, phosphorylated, and suppressed the biochemical activity of ZEP1. A rare natural allele in the hexaploid wheat ZEP1-B promoter reduced its transcription and Pst growth. Our study thus identified a novel suppressor of Pst, characterized its mechanism of action, and revealed beneficial variants for wheat disease control. This work opens the door to stacking wheat ZEP1 variants with other known Pst resistance genes in future breeding programs to enhance wheat tolerance to pathogens.

Variants of Flavin-Containing Monooxygenase 3 Found in Subjects in an Updated Database of Genome Resources.

Single-nucleotide substitutions of human flavin-containing monooxygenase 3 (FMO3) identified in the whole-genome sequences of the updated Japanese population reference panel (now containing 38,000 subjects) were investigated. In this study, two stop codon mutations, two frameshifts, and 43 amino-acid-substituted FMO3 variants were identified. Among these 47 variants, one stop codon mutation, one frameshift, and 24 substituted variants were already recorded in the National Center for Biotechnology Information database. Functionally impaired FMO3 variants are known to be associated with the metabolic disorder trimethylaminuria; consequently, the enzymatic activities of the 43 substituted FMO3 variants were investigated. Twenty-seven recombinant FMO3 variants expressed in bacterial membranes had similar activities toward trimethylamine N-oxygenation (∼75%-125%) to that of wild-type FMO3 (98 minutes-1). However, six recombinant FMO3 variants (Arg51Gly, Val283Ala, Asp286His, Val382Ala, Arg387His, and Phe451Leu) had moderately decreased (∼50%) activities toward trimethylamine N-oxygenation, and 10 recombinant FMO3 variants (Gly11Asp, Gly39Val, Met66Lys, Asn80Lys, Val151Glu, Gly193Arg, Arg387Cys, Thr453Pro, Leu457Trp, and Met497Arg) showed severely decreased FMO3 catalytic activity (<10%). Because of the known deleterious effects of FMO3 C-terminal stop codons, the four truncated FMO3 variants (Val187SerfsTer25, Arg238Ter, Lys416SerfsTer72, and Gln427Ter) were suspected to be inactive with respect to trimethylamine N-oxygenation. The FMO3 p.Gly11Asp and p.Gly193Arg variants were located within the conserved sequences of flavin adenine dinucleotide (positions 9-14) and NADPH (positions 191-196) binding sites, which are important for FMO3 catalytic function. Whole-genome sequence data and kinetic analyses revealed that 20 of the 47 nonsense or missense FMO3 variants had moderately or severely impaired activity toward N-oxygenation of trimethylaminuria. SIGNIFICANCE STATEMENT: The number of single-nucleotide substitutions in human flavin-containing monooxygenase 3 (FMO3) recorded in the expanded Japanese population reference panel database was updated. One stop mutation, FMO3 p.Gln427Ter; one frameshift (p.Lys416SerfsTer72); and 19 novel amino-acid-substituted FMO3 variants were identified, along with p.Arg238Ter, p.Val187SerfsTer25, and 24 amino-acid-substituted variants already recorded with reference SNP (rs) numbers. Recombinant FMO3 Gly11Asp, Gly39Val, Met66Lys, Asn80Lys, Val151Glu, Gly193Arg, Arg387Cys, Thr453Pro, Leu457Trp, and Met497Arg variants showed severely decreased FMO3 catalytic activity, possibly associated with the trimethylaminuria.

Attenuation of Avian Flavivirus by Rewiring the Leucine and Serine Codons of Its E-NS1 Protein toward Stop Mutation To Redirect Virus Evolution.

Recently, a new strategy for attenuating RNA viruses by redirecting their evolution in sequence space was confirmed for Enterovirus and Influenza viruses. Using avian flavivirus as a model, the 69 serine and 53 leucine codons on the E-NS1 genes were modified to change evolutionary direction of the viral sequence space. This means that all codons encoding serine or leucine residues were substituted with codons that are only one base different from the three stop codons, resulting in the initial position of the virus genome in sequence space being closer to the detrimental areas to achieve attenuation by reducing viral adaptability. The growth curve and plaque size of CQW1-one-to-stop (CQW1-OTS) were similar to those of CQW1-wild type (CQW1-WT) in vitro, but attenuated proliferation was detected when treated with a mutagenic reagent (ribavirin). However, comparably high CQW1-OTS and CQW1-WT lethality rates were detected in 9-day-old duck embryos and 5-day-old ducklings, suggesting that this strategy works but with limitations. With that in mind, homologous hosts in nonsensitive age (25-day-old ducks) and heterologous hosts (3-week-old Kunming mice) were employed to investigate if CQW1-OTS was attenuated under host selection pressure. Minimal attenuation of CQW1-OTS in elder ducks and apparent attenuation in mice were reported, providing reduced viral titers, mild clinical signs, and lower specific infectivity. Collectively, we experimentally demonstrate that the attenuation strategy of redirecting virus evolution in sequence space works for flavivirus. Redirection of the virus is attenuated only under some outside pressure, such as heterologous hosts or antiviral drugs treatment, limiting its usage in flaviviruses. IMPORTANCE Flaviviruses are medically important arboviruses that threaten public health, but no approved treatments are currently available. Vaccines prevent flavivirus infection. We employed duck Tembusu virus (TMUV), a mosquito-borne flavivirus, to evaluate virus redirection. TMUV is native to birds and could infect mice by intracerebral injection, making it an experimental animal model to study flavivirus characteristics in vivo. The 69 serine and 53 leucine codons on the E-NS1 proteins of CQW1 were synonymously substituted to change evolutionary direction of the virus in sequence space. In vitro mutagen reagent treatment suppressed CQW1-OTS viral multiplication, but in vivo attenuation depended on host selective pressure. CQW1-OTS viral attenuation was observed in older ducks but not sensitive ducklings; considerable attenuation was also observed in heterogenous host (mice), which provides more selective pressure on viruses. Collectively, these data indicated that there are very important preconditions for application of evaluating whether this strategy shows application prospects in novel flavivirus vaccine development.

Evaluation of Pharmacological Rescue of Melanocortin-4 Receptor Nonsense Mutations by Aminoglycoside.

The melanocortin-4 receptor (MC4R) is critical for central satiety regulation, therefore presenting a potent target for pharmacological obesity treatment. Melanocortin-4 receptor mutations prevalently cause monogenetic obesity. A possibility of overcoming stop mutations is aminoglycoside-mediated translational readthrough. Promising results were achieved in COS-7 cells, but data for human cell systems are still missing, so uncertainty surrounds this potential treatment. In transfected HEK-293 cells, we tested whether translational readthrough by aminoglycoside Geneticin combined with high-affinity ligand setmelanotide, which is effective in proopiomelanocortin or leptin receptor deficiency patients, is a treatment option for affected patients. Five MC4R nonsense mutants (W16X, Y35X_D37V, E61X, W258X, Q307X) were investigated. Confocal microscopy and cell surface expression assays revealed the importance of the mutations' position within the MC4R. N-terminal mutants were marginally expressed independent of Geneticin treatment, whereas mutants with nonsense mutations in transmembrane helix 6 or helix 8 showed wild-type-like expression. For functional analysis, Gs and Gq/11 signaling were measured. N-terminal mutants (W16X, Y35X_D37V) showed no cAMP formation after challenge with alpha-MSH or setmelanotide, irrespective of Geneticin treatment. Similarly, Gs activation was almost impossible in W258X and Q307X with wild-type-like cell surface expression. Results for Gq/11 signaling were comparable. Based on our data, this approach improbably represents a therapeutic option.

Nonsense codons suppression. An acute toxicity study of three optimized TRIDs in murine model, safety and tolerability evaluation

Stop mutations cause 11% of the genetic diseases, due to the introduction of a premature termination codon (PTC) in the mRNA, followed by the production of a truncated protein. A promising therapeutic approach is the suppression therapy by Translational Readthrough Inducing Drugs (TRIDs), restoring the expression of the protein. Recently, three new TRIDs (NV848, NV914, NV930) have been proposed, and validated by several in vitro assays, for the rescue of the CFTR protein, involved in Cystic Fibrosis disease. In this work, an acute toxicological study for the three TRIDs was conducted in vivo on mice, according to the OECD No.420 guidelines. Animals were divided into groups and treated with a single dose of TRIDs molecules or Ataluren, an FDA-approved TRID molecule, as control. Mice were observed continuously for the first day post-drugs administration and the behavioral changes were recorded. On the 15th day, animals were sacrificed for histological examinations. The results showed that acute administration of 2000 mg/kg of NV914 and Ataluren and 300 mg/kg of NV848 or NV930, did not induce any mortality within 14 days. Moreover, histopathological analysis of treated mice showed no differences when compared to the experimental controls. In summary, our results suggest a good tolerability for the three molecules, and include NV848 and NV930 in a category 4 and NV914 in a category 5 of the Globally Harmonized System (GHS) of Classification and Labeling of Chemicals, classifying these compounds in a low-risk scale for health.