Stop Codon In mRNA Research Articles

HERV-W ENV transcription in B cells predicting symptomatic COVID-19 and risk for long COVID can express a full-length protein despite stop codon in mRNA from chromosome X via a ribosome readthrough.

The human genome comprises 8% of endogenous retroviruses (HERVs). Though HERVS contribute to physiological functions, copies retained pathogenic potential. The HERV-W ENV protein was shown expressed in patients with worse COVID-19 symptoms and post-COVID syndrome. A significant detection of the mRNA encoding HERV-W ENV from patients with COVID-19 in B cells from RNAseq reads obtained from peripheral blond mononuclear cells. This data stratified with increased COVID-19 symptoms or with post-acute sequelae of COVID-19 (long COVID) after 3 months. The HERV-W ENV-U3R RNA was confirmed to display the best alignment with chromosome X ERVWE2 locus. However, a stop codon precluding its translation was re-addressed after recent understandings of ribosome readthrough mechanisms. Experimental results evidenced that this HERV gene can effectively express a full-length protein in the presence of molecules allowing translation via a readthrough mechanism at the ribosome level. Results not only confirm HERV-W ENV RNA origin in these patients but show for the first time how a defective HERV copy can be translated into a complete protein when specific factors make it possible at the ribosome level. The present proof of concept now requires further studies to identify the factors involved in this newly understood mechanism, following SARS-CoV-2 exposure.

Chronic intermittent ethanol exposure-induced m6A modifications around mRNA stop codons of opioid receptor genes

ABSTRACT Chronic alcohol consumption may alter mRNA methylation and expression levels of genes related to addiction and reward in the brain, potentially contributing to alcohol tolerance and dependence. Neuron-like (SH-SY5Y) and non-neuronal (SW620) cells were utilized as models to examine chronic intermittent ethanol (CIE) exposure-induced global m6A RNA methylation changes, as well as m6A mRNA methylation changes around the stop codon of three opioid receptor genes (OPRM1, OPRD1, and OPRK1), which are known to regulate pain, reward, and addiction behaviours. CIE exposure for three weeks significantly increased global RNA methylation levels in both SH-SY5Y (t = 3.98, P = 0.007) and SW620 (t = 2.24, P = 0.067) cells. However, a 3-week CIE exposure resulted in hypomethylation around mRNA stop codon regions of OPRM1 and OPRD1 in both cell lines [OPRM1 (SH-SY5Y): t = -5.05, P = 0.0005; OPRM1 (SW620): t = -3.19, P = 0.013; OPRD1 (SH-SY5Y): t = -13.43, P < 0.00001; OPRD1 (SW620): t = -4.00, P = 0.003]. Additionally, mRNA expression levels of OPRM1, OPRD1, and OPRK1 were downregulated (corresponding to mRNA hypomethylation) in both SH-SY5Y and SW620 cells after a 3-week CIE exposure. The present study demonstrated that chronic ethanol exposure altered global RNA methylation levels, as well as mRNA methylation and expression levels of opioid receptor genes in both neuron-like and non-neuronal cells. Our findings suggest a potential epitranscriptomic mechanism by which chronic alcohol consumption remodels the expression of reward-related and alcohol responsive genes in the brain, thus increasing the risk of alcohol use disorder development. Abbreviations: OPRM1: the μ-opioid receptor; OPRD1: the δ-opioid receptor; OPRK1: the κ-opioid receptor; CIE: chronic intermittent ethanol exposure; CIE+WD: chronic intermittent ethanol exposure followed by a 24-hr withdrawal; SH-SY5Y: human neuroblastoma cell Line; SW620: human colon carcinoma cell line; RT-qPCR: reverse transcription followed by quantitative polymerase reaction; MazF-RT-qPCR: MazF digestion followed by RT-qPCR

A common intronic single nucleotide variant modifies PKD1 expression level.

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in PKD1 and PKD2 (PKD1/2), has unexplained phenotypic variability likely affected by environmental and other genetic factors. Approximately 10% of individuals with ADPKD phenotype have no causal mutation detected, possibly due to unrecognized risk variants of PKD1/2. This study was designed to identify risk variants of PKD genes through population genetic analyses. We used Wright's F-statistics (Fst) to evaluate common single nucleotide variants (SNVs) potentially favored by positive natural selection in PKD1 from 1000 Genomes Project (1KG) and genotyped 388 subjects from the Rogosin Institute ADPKD Data Repository. The variants with >90th percentile Fst scores underwent further investigation by in silico analysis and molecular genetics analyses. We identified a deep intronic SNV, rs3874648G> A, located in a conserved binding site of the splicing regulator Tra2-β in PKD1 intron 30. Reverse-transcription PCR (RT-PCR) of peripheral blood leukocytes (PBL) from an ADPKD patient homozygous for rs3874648-A identified an atypical PKD1 splice form. Functional analyses demonstrated that rs3874648-A allele increased Tra2-β binding affinity and activated a cryptic acceptor splice-site, causing a frameshift that introduced a premature stop codon in mRNA, thereby decreasing PKD1 full-length transcript level. PKD1 transcript levels were lower in PBL from rs3874648-G/A carriers than in rs3874648-G/G homozygotes in a small cohort of normal individuals and patients with PKD2 inactivating mutations. Our findings indicate that rs3874648G > A is a PKD1 expression modifier attenuating PKD1 expression through Tra2-β, while the derived G allele advantageously maintains PKD1 expression and is predominant in all subpopulations.

Extensive ribosome and RF2 rearrangements during translation termination.

Protein synthesis ends when a ribosome reaches an mRNA stop codon. Release factors (RFs) decode the stop codon, hydrolyze peptidyl-tRNA to release the nascent protein, and then dissociate to allow ribosome recycling. To visualize termination by RF2, we resolved a cryo-EM ensemble of E. coli 70S•RF2 structures at up to 3.3 Å in a single sample. Five structures suggest a highly dynamic termination pathway. Upon peptidyl-tRNA hydrolysis, the CCA end of deacyl-tRNA departs from the peptidyl transferase center. The catalytic GGQ loop of RF2 is rearranged into a long β-hairpin that plugs the peptide tunnel, biasing a nascent protein toward the ribosome exit. Ribosomal intersubunit rotation destabilizes the catalytic RF2 domain on the 50S subunit and disassembles the central intersubunit bridge B2a, resulting in RF2 departure. Our structures visualize how local rearrangements and spontaneous inter-subunit rotation poise the newly-made protein and RF2 to dissociate in preparation for ribosome recycling.

Forward Genetics Approach Reveals a Mutation in bHLH Transcription Factor-Encoding Gene as the Best Candidate for the Root Hairless Phenotype in Barley.

Root hairs are the part of root architecture contributing significantly to the root surface area. Their role is particularly substantial in maintaining plant growth under stress conditions, however, knowledge on mechanism of root hair differentiation is still limited for majority of crop species, including barley. Here, we report the results of a map-based identification of a candidate gene responsible for the lack of root epidermal cell differentiation, which results in the lack of root hairs in barley. The analysis was based on the root hairless barley mutant rhl1.b, obtained after chemical mutagenesis of spring cultivar ‘Karat’. The rhl1 gene was located in chromosome 7HS in our previous studies. Fine mapping allowed to narrow the interval encompassing rhl1 gene to 3.7 cM, which on physical barley map spans a region of 577 kb. Five high confidence genes are located within this region and their sequencing resulted in the identification of A>T mutation in one candidate, HORVU7Hr1G030250 (MLOC_38567), differing the mutant from its parent variety. The mutation, located in the 3′ splice-junction site, caused the retention of the last intron, 98 bp long, in mRNA of rhl1.b allele. This resulted in the frameshift, the synthesis of 71 abnormal amino acids and introduction of premature STOP codon in mRNA. The mutation was present in the recombinants from the mapping population (F2 rhl1.b × ‘Morex’) that lacked root hairs. The candidate gene encodes a bHLH transcription factor with LRL domain and may be involved in early stages of root hair cell development. We discuss the possible involvement of HORVU7Hr1G030250 in this process, as the best candidate responsible for early stages of rhizodermis differentiation in barley.

Origin of the omnipotence of eukaryotic release factor 1

Termination of protein synthesis on the ribosome requires that mRNA stop codons are recognized with high fidelity. This is achieved by specific release factor proteins that are very different in bacteria and eukaryotes. Hence, while there are two release factors with overlapping specificity in bacteria, the single omnipotent eRF1 release factor in eukaryotes is able to read all three stop codons. This is particularly remarkable as it is able to select three out of four combinations of purine bases in the last two codon positions. With recently determined 3D structures of eukaryotic termination complexes, it has become possible to explore the origin of eRF1 specificity by computer simulations. Here, we report molecular dynamics free energy calculations on these termination complexes, where relative eRF1 binding free energies to different cognate and near-cognate codons are evaluated. The simulations show a high and uniform discrimination against the near-cognate codons, that differ from the cognate ones by a single nucleotide, and reveal the structural mechanisms behind the precise decoding by eRF1.

Structure-Based Energetics of Stop Codon Recognition by Eukaryotic Release Factor.

In translation termination, the eukaryotic release factor (eRF1) recognizes mRNA stop codons (UAA, UAG, or UGA) in a ribosomal A site and triggers release of the nascent polypeptide chain from P-site tRNA. eRF1 is highly selective for U in the first position and a combination of purines (except two consecutive guanines, i.e., GG) in the second and third positions. Eukaryotes decode all three stop codons with a single release factor eRF1, instead of two (RF1 and RF2), in bacteria. Furthermore, unlike bacterial RF1/RF2, eRF1 stabilizes the compact U-turn mRNA configuration in the ribosomal A site by accommodating four nucleotides instead of three. Despite the available cryo-EM structures (resolution ∼3.5-3.8 Å), the energetic principle for eRF1 selectivity toward a stop codon remains a fundamentally unsolved problem. Using cryo-EM structures of eukaryotic translation termination complexes as templates, we carried out molecular dynamics free energy simulations of cognate and near-cognate complexes to quantitatively address the energetics of stop codon recognition by eRF1. Our results suggest that eRF1 has a higher discriminatory power against sense codons, compared to that reported earlier for RF1/RF2. The compact mRNA formed specific intra-mRNA interactions, which itself contributed to stop codon specificity. Furthermore, the specificity is enhanced by the loss of protein-mRNA interactions and, most importantly, by desolvation of the incorrect codons in the near-cognate complexes. Our work provides a clue to how eRF1 discriminates between cognate and near-cognate codons during protein synthesis.

The kinetic mechanism of bacterial ribosome recycling.

Bacterial ribosome recycling requires breakdown of the post-termination complex (PoTC), comprising a messenger RNA (mRNA) and an uncharged transfer RNA (tRNA) cognate to the terminal mRNA codon bound to the 70S ribosome. The translation factors, elongation factor G and ribosome recycling factor, are known to be required for recycling, but there is controversy concerning whether these factors act primarily to effect the release of mRNA and tRNA from the ribosome, with the splitting of the ribosome into subunits being somewhat dispensable, or whether their main function is to catalyze the splitting reaction, which necessarily precedes mRNA and tRNA release. Here, we utilize three assays directly measuring the rates of mRNA and tRNA release and of ribosome splitting in several model PoTCs. Our results largely reconcile these previously held views. We demonstrate that, in the absence of an upstream Shine–Dalgarno (SD) sequence, PoTC breakdown proceeds in the order: mRNA release followed by tRNA release and then by 70S splitting. By contrast, in the presence of an SD sequence all three processes proceed with identical apparent rates, with the splitting step likely being rate-determining. Our results are consistent with ribosome profiling results demonstrating the influence of upstream SD-like sequences on ribosome occupancy at or just before the mRNA stop codon.

Regulatory Role of N6 -methyladenosine (m6 A) Methylation in RNA Processing and Human Diseases.

N6 -methyladenosine (m6 A) modification is an abundant and conservative RNA modification in bacterial and eukaryotic cells. m6 A modification mainly occurs in the 3' untranslated regions (UTRs) and near the stop codons of mRNA. Diverse strategies have been developed for identifying m6 A sites in single nucleotide resolution. Dynamic regulation of m6 A is found in metabolism, embryogenesis, and developmental processes, indicating a possible epigenetic regulation role along RNA processing and exerting biological functions. It has been known that m6 A editing involves in nuclear RNA export, mRNA degradation, protein translation, and RNA splicing. Deficiency of m6 A modification will lead to kinds of diseases, such as obesity, cancer, type 2 diabetes mellitus (T2DM), infertility, and developmental arrest. Some specific inhibitors against methyltransferase and demethylase have been developed to selectively regulate m6 A modification, which may be advantageous for treatment of m6 A related diseases. J. Cell. Biochem. 118: 2534-2543, 2017. © 2017 Wiley Periodicals, Inc.

Chemical-Induced Read-Through at Premature Termination Codons Determined by a Rapid Dual-Fluorescence System Based on S. cerevisiae.

Nonsense mutations generate in-frame stop codons in mRNA leading to a premature arrest of translation. Functional consequences of premature termination codons (PTCs) include the synthesis of truncated proteins with loss of protein function causing severe inherited or acquired diseases. A therapeutic approach has been recently developed that is based on the use of chemical agents with the ability to suppress PTCs (read-through) restoring the synthesis of a functional full-length protein. Research interest for compounds able to induce read-through requires an efficient high throughput large scale screening system. We present a rapid, sensitive and quantitative method based on a dual-fluorescence reporter expressed in the yeast Saccharomyces cerevisiae to monitor and quantitate read-through at PTCs. We have shown that our novel system works equally well in detecting read-through at all three PTCs UGA, UAG and UAA.

Chemical Ligation Synthesis of Isotope‐Labeling Compatible Selenoprotein S

Selenoproteins contain the 21st amino acid selenocysteine (Sec) and play vital roles in human health, such as antioxidant defense. Although selenoproteins are expressed naturally in the human body, procuring sufficient quantities for biochemical and biophysical characterization by heterologous expression is challenging. Low yield in genetic incorporation is due to the Sec codon being identical to the UGA stop codon in mRNA. An alternative approach for their preparation is chemical ligation, a semi‐synthetic method that relies upon an amide bond formation to generate peptides and proteins from their complementary fragments. Here, we describe a new method for chemical ligation of selenoprotein S (SelS) which is compatible with simple and low cost isotopic labeling and does not rely on complete chemical synthesis of a selenoprotein. SelS is a member of the endoplasmic reticulum associated protein degradation (ERAD) pathway, a protein machinery devoted to relocating misfolded proteins from the ER to a cytoplasmic protein degradation complex. The active site lies close to the C‐terminal where a sole Sec is paired with a Cysteine 13 residues away. Previously we have characterized the activity of SelS prepared by heterologous expression, however, structural characterization with the Sec in place was prohibited by the low yield. In our approach a Sec‐containing fragment is expressed in E. coli and used as a source of the selenopeptide during the chemical ligation. This has the advantage of a high yield, with simple and efficient ligation while being compatible with isotopic‐labeling of selenoproteins. Useful applications of this method include incorporation of Selenium‐77 and Carbon‐13 for structural and biochemical characterization by NMR spectroscopy.Support or Funding InformationThis work was funded by the National Institute of General Medical Science

Design of a Confirmatory Phase 3, Multicenter, Randomized, Double Blind, Placebo-Controlled Study (ACT DMD) of Ataluren in Patients with Nonsense Mutation Duchenne Muscular Dystrophy

Aim: Approximately 13% of Duchenne muscular dystrophy (DMD) is caused by nonsense mutation (nm) in the dystrophin gene. Nonsense mutations in DNA correspond to premature stop codons in mRNA. Ataluren an oral drug designed to promote ribosomal read through of premature stop codons, enabling production of full length, and functional protein. Currently ongoing is a Phase 3, randomized, double-blind, placebo-controlled study (ACT DMD) designed to confirm the efficacy and safety of ataluren 40 mg/kg/d in boys with nmDMD, initially demonstrated in a Phase 2b study. The design of ACT DMD reflects lessons learned from previous study and evolving understanding of the natural history of DMD. The study design targets an enriched study population of patients in the ambulatory decline phase of DMD to efficiently demonstrate disease stabilization over 48 weeks.

G.P.106: Internal consistency and robustness of clinical efficacy data from the phase 2b study of ataluren (PTC124®) in nonsense mutation Duchenne muscular dystrophy

Ataluren is an oral, investigational drug designed to promote ribosomal readthrough of premature stop codons in mRNA, leading to production of full-length, functional protein. In a Phase 2b, 48-week study in nonsense mutation Duchenne muscular dystrophy (nmDMD), ataluren 40 mg/kg/day was associated with slowing of ambulatory decline (measured by 6-min walk distance [6MWD]) vs placebo. Positive trends were also seen in timed function tests of stair-climbing, stair-descending, and walking/running 10 m. Treatment differences vs placebo were larger in an ambulatory decline phase subgroup (7–16 yrs old, on corticosteroids, baseline 6MWD ¡Ý150 m but %-predicted 6MWD ¡Ü80%). Internal consistency and robustness of physical function outcomes were evaluated by statistical methods. Endpoint analyses were repeated 1000 times (Monte Carlo simulation), each time with 10% of patients in each arm excluded at random. Separately, endpoint analyses were performed after excluding one or more extreme results (best/worst exclusion) from each arm. These methods were applied to the overall study population and ambulatory decline phase subgroup. Monte Carlo simulation demonstrated that treatment differences in model-estimated mean change in 6MWD of ∼ 30 m in the overall study population and ∼45 m in the ambulatory decline phase subgroup, favoring ataluren vs placebo, were not driven by a small number of patients. Best/worst exclusion analysis confirmed these treatment differences were not distorted by outliers. Timed function test outcomes were consistent with 6MWD findings. Ambulatory decline in patients with nmDMD could potentially be delayed or stabilized with disease-modifying treatment. These analyses document the internal consistency and give evidence of robustness of the Phase 2b efficacy data for ataluren in nmDMD, confirming observations of relative stabilization of ambulatory measures of disease progression over 48 weeks with ataluren. Ataluren is an oral, investigational drug designed to promote ribosomal readthrough of premature stop codons in mRNA, leading to production of full-length, functional protein. In a Phase 2b, 48-week study in nonsense mutation Duchenne muscular dystrophy (nmDMD), ataluren 40 mg/kg/day was associated with slowing of ambulatory decline (measured by 6-min walk distance [6MWD]) vs placebo. Positive trends were also seen in timed function tests of stair-climbing, stair-descending, and walking/running 10 m. Treatment differences vs placebo were larger in an ambulatory decline phase subgroup (7–16 yrs old, on corticosteroids, baseline 6MWD ¡Ý150 m but %-predicted 6MWD ¡Ü80%). Internal consistency and robustness of physical function outcomes were evaluated by statistical methods. Endpoint analyses were repeated 1000 times (Monte Carlo simulation), each time with 10% of patients in each arm excluded at random. Separately, endpoint analyses were performed after excluding one or more extreme results (best/worst exclusion) from each arm. These methods were applied to the overall study population and ambulatory decline phase subgroup. Monte Carlo simulation demonstrated that treatment differences in model-estimated mean change in 6MWD of ∼ 30 m in the overall study population and ∼45 m in the ambulatory decline phase subgroup, favoring ataluren vs placebo, were not driven by a small number of patients. Best/worst exclusion analysis confirmed these treatment differences were not distorted by outliers. Timed function test outcomes were consistent with 6MWD findings. Ambulatory decline in patients with nmDMD could potentially be delayed or stabilized with disease-modifying treatment. These analyses document the internal consistency and give evidence of robustness of the Phase 2b efficacy data for ataluren in nmDMD, confirming observations of relative stabilization of ambulatory measures of disease progression over 48 weeks with ataluren.

Ataluren: first global approval.

Nonsense mutations are implicated in 5-70% of individual cases of most inherited diseases, including Duchenne muscular dystrophy (DMD) and cystic fibrosis. Ataluren (Translarna™) is an orally available, small molecule compound that targets nonsense mutations, and is the first drug in its class. Ataluren appears to allow cellular machinery to read through premature stop codons in mRNA, enabling the translation process to produce full-length, functional proteins. This article summarizes the milestones in the development of ataluren leading to its conditional first approval for nonsense mutation DMD.

S1 Development of a confirmatory phase 3, multicentre, randomized, double-blind, placebo-controlled study of ataluren in patients with nonsense mutation Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a rare, X-linked disorder that causes severe, progressive muscle loss and early death. In ∼13% of patients, the disease is caused by a nonsense mutation (nm) in the gene for dystrophin. Ataluren is an orally delivered, investigational drug designed to promote ribosomal readthrough of premature stop codons in mRNA, leading to production of full-length, functional protein. A confirmatory Phase 3, multicenter, randomized, double-blind, placebo-controlled study has been designed to assess the efficacy and safety of ataluren 10, 10, 20 mg/kg tid in patients with nmDMD. The study design reflects lessons learned from prior studies and targets a study population to best demonstrate the treatment effect over 48 weeks. Key study entry criteria require that patients are male with a nonsense mutation in the dystrophin gene, between the ages of 7 and 16 years, receiving a stable dose of corticosteroids, able to walk ⩾150 m during the screening 6-min walk test (6MWT), and have a screening 6MWT below the protocol-specified threshold for %-predicted 6MWD. These criteria were selected based on the results of a retrospective subgroup analysis of patients in the Phase 2b trial of ataluren in nmDMD meeting these criteria, in which the difference between the 10, 10, 20 mg/kg dose of ataluren ( n = 30) vs placebo ( n = 31) in mean change in 6MWD over 48 weeks was ∼50 m. In the planned study, 220 patients will be randomized in a 1:1 ratio to either placebo or ataluren. The primary outcome measure is the 6MWT. Secondary measures include: timed function tests, quality of life, North Star Ambulatory Assessment, patient/parent-reported activities of daily living, safety, compliance to study drug, and ataluren exposure of ataluren in blood. This study will be the largest clinical trial of an investigational drug in DMD and is designed to confirm the treatment effect of ataluren seen in the Phase 2b ataluren trial.

Backbone 1H, 13C and 15N resonance assignments of the human eukaryotic release factor eRF1

Eukaryotic translation termination is mediated by two interacting release factors, eukaryotic class 1 release factor (eRF1) and eukaryotic class 3 release factor (eRF3), which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. eRF1 consisting of three well-defined functional domains recognizes all three mRNA stop codons located in the A site of the small ribosomal subunit and triggers hydrolysis of the ester bond of peptidyl-tRNA in the peptidyl transfer center of the large ribosomal subunit. Nevertheless, various aspects of molecular mechanism of translation termination in eukaryotes remain unclear. Elucidation of the structure and dynamics of eRF1 in solution is essential for understanding molecular mechanism of its function in translation termination. To approach this problem, here we report NMR backbone signal assignments of the human eRF1 (437 a.a., 50kDa).

P.11.19 Phase 2b, dose-ranging study of ataluren (PTC124®) innonsense mutation Duchenne muscular dystrophy – results of a post hoc analysis of change in %-predicted 6-min walk distance

Duchenne muscular dystrophy (DMD) is a rare, X-linked disorder that causes severe, progressive muscle loss and early death. In ∼13% of patients, the disease is caused by a nonsense mutation in the gene for dystrophin. Ataluren is an orally delivered, investigational drug designed to promote ribosomal readthrough of premature stop codons in mRNA, leading to production of full-length, functional protein. An international, multicenter, randomized, double-blind, placebo-controlled trial (Study 007) enrolled ambulatory males ⩾5 years with nonsense mutation DMD. Patients received tid 20, 20, 40 mg/kg ataluren; 10, 10, 20 mg/kg ataluren; or placebo orally for 48 weeks. The primary endpoint was absolute change in 6-min walk distance (6MWD) over 48 weeks. The study enrolled 174 subjects (median [range] age = 8 [5–20] years, median [range] height = 123 [99–173] cm) at 37 sites in 11 countries. A post hoc analysis was performed to convert absolute 6MWD data to %-predicted 6MWD using a previously published age and height-based equation to adjust for maturational differences. Median [range] baseline %-predicted 6MWD = 63[12–96]%. Mean decline in %-predicted 6MWD at Week 48 of 7.6% was observed for placebo, vs 2.7% for ataluren 10, 10, 20 mg/kg, resulting in a difference of 4.9% (nominal p -value = 0.0273). Mean decline for ataluren 20, 20, 40 mg/kg was no different from placebo (7.7%), for a difference of 0.1% from placebo. Conversion of absolute 6MWD data to %-predicted 6MWD assists in the interpretation of disease-related progressive loss of function against the background of normal growth and development. Analysis of Study 007 using %-predicted 6MWD data adjusts for maturational influences and supports the treatment difference between ataluren 10, 10, 20 mg/kg and placebo seen in the primary analysis of 6MWD in meters.

Abstract 2029: Wild type BRCA2 mRNA splice variants in normal and breast tumor cells

Abstract BRCA2 is one of the two principal tumor suppressor genes responsible for Hereditary Breast and Ovarian Cancer (HBOC) Syndrome. Most deleterious mutations in BRCA2 are nonsense, frame-shifting insertions/deletions, or splicing mutations resulting in premature mRNA stop codons. However, numerous sequence variants of uncertain significance (VUS) are routinely seen during clinical testing. Some of these could potentially affect splicing fidelity by altering splice site choices or affecting the ratios of normally occurring splice variants, which have yet to be defined for BRCA2. To characterize the normally occurring splice variants and possible alternate protein products of BRCA2, we have performed a complete cDNA exon scan of BRCA2 cDNA in MCF7 (a breast cancer cell line), 184A1 (a non-cancerous breast cell line), and two lymphoblastoid cell lines from unaffected wild type. In all cell lines we identified three BRCA2 splice variants: delta exon 3, delta exon 12, and delta exons 6&amp;7+2bp. While the first two variants retain the BRCA2 translational reading frame and could theoretically result in alternate BRCA2 protein products, the third is an out-of-frame variant predicted to result in a premature stop codon. We have further developed a set of splice variant-specific detection assays that revealed all three splice variants were present in 14 breast tumor/adjacent normal tissue pairs. Surprisingly, all three BRCA2 splice variants were comparatively more abundant in the tumor cells than their normal adjacent tissues. To characterize the regulators of BRCA2 splicing in tumor cells, we repeated the exon scan in the presence of cyclohexamide or puromycin to inhibit nonsense-mediated mRNA decay (NMD). We saw no evidence of accumulated mis-spliced BRCA2 mRNA products in the presence of the inhibitors, indicating NMD does not play a major role in regulating the accumulation of normally occurring splice variants. To determine whether nonsense-associated alternate splicing (NAS) promotes skipping of exons containing frame shifting or nonsense mutations, we repeated the BRCA2 exon scan in four lymphoblastoid cell lines containing BRCA2 mutations 5578delAA, 852delC, 6174delT and S1955X. In all cases, the splicing patterns were the same as breast and wild type lymphoblastoid cell line patterns, indicating NAS does not generally promote alternate BRCA2 protein products in response to truncating mutations. We conclude that, in addition to full-length BRCA2 mRNA, there are three additional splice variants that accumulate to comparatively higher levels in tumors than in adjacent normal tissue. This suggests breast tumor tissues have altered splicing regulation, altered regulation of selective mRNA degradation, or possibly both, and could reflect a level of epigenetic misregulation of BRCA2 and possibly other genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2029. doi:10.1158/1538-7445.AM2011-2029

Chronic ataluren (PTC124) treatment of nonsense mutation cystic fibrosis

In a subset of patients with cystic fibrosis (CF), nonsense mutations (premature stop codons) disrupt production of full-length, functional CF transmembrane conductance regulator (CFTR). Ataluren (PTC124) allows ribosomal readthrough of premature stop codons in mRNA. We evaluated drug activity and safety in patients with nonsense mutation CF who took ataluren three times daily (morning, midday and evening) for 12 weeks at either a lower dose (4, 4 and 8 mg·kg(-1)) or higher dose (10, 10 and 20 mg·kg(-1)). The study enrolled 19 patients (10 males and nine females aged 19-57 yrs; dose: lower 12, higher seven) with a classic CF phenotype, at least one CFTR nonsense mutation allele, and an abnormal nasal total chloride transport. Both ataluren doses were similarly active, improving total chloride transport with a combined mean change of -5.4 mV (p<0.001), and on-treatment responses (at least -5 mV improvement) and hyperpolarisations (values more electrically negative than -5 mV) in 61% (p<0.001) and 56% (p = 0.002) of patients. CFTR function was greater with time and was accompanied by trends toward improvements in pulmonary function and CF-related coughing. Adverse clinical and laboratory findings were uncommon and usually mild. Chronic ataluren administration produced time-dependent improvements in CFTR activity and clinical parameters with generally good tolerability.

P3.51 Results of a Phase 2b, dose-ranging study of ataluren (PTC124®) in nonsense mutation Duchenne/Becker muscular dystrophy (nmDBMD)

Introduction: DBMD is an X-linked disorder causing severe, progressive muscle loss in children. Patients (∼13%) have DBMD due to a nonsense (premature stop codon) mutation in the gene for dystrophin, a protein needed for muscle stability. Ataluren is an investigational drug designed to promote ribosomal readthrough of premature stop codons in mRNA, leading to production of full-length, functional protein. Methods: This Phase 2b, randomized, double-blind, placebo-controlled, dose-ranging study assessed the efficacy and safety of ataluren in males ⩾5year with nmDBMD documented by dystrophin gene sequencing.