Are Nonsense Alleles of Drosophila melanogaster Genes under Any Selection?

AbstractA gene which carries a bona fide loss-of-function mutation effectively becomes a functionless pseudogene, free from selective constraint. However, there is a number of molecular mechanisms that may lead to at least a partial preservation of the function of genes carrying even drastic alleles. We performed a direct measurement of the strength of negative selection acting on nonsense alleles of protein-coding genes in the Zambian population of Drosophila melanogaster. Within those exons that carry nonsense mutations, negative selection, assayed by the ratio of missense over synonymous nucleotide diversity levels, appears to be absent, consistent with total loss of function. In other exons of nonsense alleles, negative selection was deeply relaxed but likely not completely absent, and the per site number of missense alleles declined significantly with the distance from the premature stop codon. This pattern may be due to alternative splicing which preserves function of some isoforms of nonsense alleles of genes.

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Efficient analysis of mouse genome sequences reveal many nonsense variants

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1605076113 ◽

2016 ◽

Vol 113 (20) ◽

pp. 5670-5675 ◽

Cited By ~ 4

Author(s):

Sophie Steeland ◽

Steven Timmermans ◽

Sara Van Ryckeghem ◽

Paco Hulpiau ◽

Yvan Saeys ◽

...

Keyword(s):

Stop Codon ◽

Wellcome Trust Sanger Institute ◽

Inbred Strains ◽

Sequence Difference ◽

Loss Of Function ◽

Mus Spretus ◽

Web Tool ◽

Nonsense Mutations ◽

Protein Coding ◽

The Many

Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as research models. They have been shown to influence research results, explain phenotypical differences between inbred strains, and increase the amount of interesting gene variants present in the many available inbred lines. SPRET/Ei is an inbred strain derived from Mus spretus that has ∼1% sequence difference with the C57BL/6J reference genome. We obtained a listing of all SNPs and insertions/deletions (indels) present in SPRET/Ei from the Mouse Genomes Project (Wellcome Trust Sanger Institute) and processed these data to obtain an overview of all transcripts having nonsynonymous coding sequence variants. We identified 8,883 unique variants affecting 10,096 different transcripts from 6,328 protein-coding genes, which is about 28% of all coding genes. Because only a subset of these variants results in drastic changes in proteins, we focused on variations that are nonsense mutations that ultimately resulted in a gain of a stop codon. These genes were identified by in silico changing the C57BL/6J coding sequences to the SPRET/Ei sequences, converting them to amino acid (AA) sequences, and comparing the AA sequences. All variants and transcripts affected were also stored in a database, which can be browsed using a SPRET/Ei M. spretus variants web tool (www.spretus.org), including a manual. We validated the tool by demonstrating the loss of function of three proteins predicted to be severely truncated, namely Fas, IRAK2, and IFNγR1.

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Ectopic Transcript Analysis Indicates that Allelic Exclusion is an Important Cause of Type I Protein C Deficiency in Patients with Nonsense and Frameshift Mutations in the PROC Gene

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650386 ◽

1996 ◽

Vol 75 (06) ◽

pp. 870-876 ◽

Cited By ~ 4

Author(s):

José Manuel Soria ◽

Lutz-Peter Berg ◽

Jordi Fontcuberta ◽

Vijay V Kakkar ◽

Xavier Estivill ◽

...

Keyword(s):

Splice Site ◽

Protein C ◽

Stop Codon ◽

Premature Stop Codon ◽

Allelic Exclusion ◽

Polymorphism Analysis ◽

Type I ◽

Protein C Deficiency ◽

Nonsense Mutations ◽

Stop Codons

SummaryNonsense mutations, deletions and splice site mutations are a common cause of type I protein C deficiency. Either directly or indirectly by altering the reading frame, these' lesions generate or may generate premature stop codons and could therefore be expected to result in premature termination of translation. In this study, the possibility that such mutations could instead exert their pathological effects at an earlier stage in the expression pathway, through “allelic exclusion” at the RNA level, was investigated. Protein C (PROC) mRNA was analysed in seven Spanish type I protein C deficient patients heterozygous for two nonsense mutations, a 7bp deletion, a 2bp insertion and three splice site mutations. Ectopic RNA transcripts from patient and control lymphocytes were analysed by RT-PCR and direct sequencing of amplified PROC cDNA fragments. The nonsense mutations and the deletion were absent from the cDNAs indicating that only mRNA derived from the normal allele had been expressed. Similarly for the splice site mutations, only normal PROC cDNAs were obtained. In one case, exclusion of the mutated allele could be confirmed by polymorphism analysis. In contrast to these six mutations, the 2 bp insertion was not associated with loss of mRNA from the mutated allele. In this case, cDNA analysis revealed the absence of 19 bases from the PROC mRNA consistent with the generation and utilization of a cryptic splice site 3’ to the site of mutation, which would result in a frameshift and a premature stop codon. It is concluded that allelic exclusion is a common causative mechanism in those cases of type I protein C deficiency which result from mutations that introduce premature stop codons

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Cis-Segregation of c.1171C>T Stop Codon (p.R391*) in SERPINC1 Gene and c.1691G>A Transition (p.R506Q) in F5 Gene and Selected GWAS Multilocus Approach in Inherited Thrombophilia

Genes ◽

10.3390/genes12060934 ◽

2021 ◽

Vol 12 (6) ◽

pp. 934

Author(s):

Donato Gemmati ◽

Giovanna Longo ◽

Eugenia Franchini ◽

Juliana Araujo Silva ◽

Ines Gallo ◽

...

Keyword(s):

At Risk ◽

Stop Codon ◽

Association Studies ◽

Premature Stop Codon ◽

Large Family ◽

Genome Wide Association Studies ◽

Loss Of Function ◽

Gene Markers ◽

Inherited Thrombophilia ◽

Fv Leiden

Inherited thrombophilia (e.g., venous thromboembolism, VTE) is due to rare loss-of-function mutations in anticoagulant factors genes (i.e., SERPINC1, PROC, PROS1), common gain-of-function mutations in procoagulant factors genes (i.e., F5, F2), and acquired risk conditions. Genome Wide Association Studies (GWAS) recently recognized several genes associated with VTE though gene defects may unpredictably remain asymptomatic, so calculating the individual genetic predisposition is a challenging task. We investigated a large family with severe, recurrent, early-onset VTE in which two sisters experienced VTE during pregnancies characterized by a perinatal in-utero thrombosis in the newborn and a life-saving pregnancy-interruption because of massive VTE, respectively. A nonsense mutation (CGA > TGA) generating a premature stop-codon (c.1171C>T; p.R391*) in the exon 6 of SERPINC1 gene (1q25.1) causing Antithrombin (AT) deficiency and the common missense mutation (c.1691G>A; p.R506Q) in the exon 10 of F5 gene (1q24.2) (i.e., FV Leiden; rs6025) were coinherited in all the symptomatic members investigated suspecting a cis-segregation further confirmed by STR-linkage-analyses [i.e., SERPINC1 IVS5 (ATT)5–18, F5 IVS2 (AT)6–33 and F5 IVS11 (GT)12–16] and SERPINC1 intragenic variants (i.e., rs5878 and rs677). A multilocus investigation of blood-coagulation balance genes detected the coexistence of FV Leiden (rs6025) in trans with FV HR2-haplotype (p.H1299R; rs1800595) in the aborted fetus, and F11 rs2289252, F12 rs1801020, F13A1 rs5985, and KNG1 rs710446 in the newborn and other members. Common selected gene variants may strongly synergize with less common mutations tuning potential life-threatening conditions when combined with rare severest mutations. Merging classic and newly GWAS-identified gene markers in at risk families is mandatory for VTE risk estimation in the clinical practice, avoiding partial risk score evaluation in unrecognized at risk patients.

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Loss-of-function in IRF2BPL is associated with neurological phenotypes

10.1101/322495 ◽

2018 ◽

Cited By ~ 1

Author(s):

Paul C. Marcogliese ◽

Vandana Shashi ◽

Rebecca C. Spillmann ◽

Nicholas Stong ◽

Jill A. Rosenfeld ◽

...

Keyword(s):

Nervous System ◽

Population Genomics ◽

Stop Codon ◽

Ectopic Expression ◽

Premature Stop Codon ◽

Model Organism ◽

Global Developmental Delay ◽

Mendelian Disease ◽

Loss Of Function ◽

Missense Variants

AbstractThe Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) gene encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals affected with neurological symptoms who carry damaging heterozygous variants in IRF2BPL. Five cases carrying nonsense variants in IRF2BPL resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The bioinformatics signature for IRF2BPL based on population genomics is consistent with a gene that is intolerant to variation. We show that the IRF2BPL ortholog in the fruit fly, called pits (protein interacting with Ttk69 and Sin3A), is broadly expressed including the nervous system. Complete loss of pits is lethal early in development, whereas partial knock-down with RNA interference in neurons leads to neurodegeneration, revealing requirement for this gene in proper neuronal function and maintenance. The nonsense variants in IRF2BPL identified in patients behave as severe loss-of-function alleles in this model organism, while ectopic expression of the missense variants leads to a range of phenotypes. Taken together, IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.

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Synergistic Activation of Defense Responses in Arabidopsis by Simultaneous Loss of the GSL5 Callose Synthase and the EDR1 Protein Kinase

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-23-5-0578 ◽

2010 ◽

Vol 23 (5) ◽

pp. 578-584 ◽

Cited By ~ 19

Author(s):

Anna Wawrzynska ◽

Natalie L. Rodibaugh ◽

Roger W. Innes

Keyword(s):

Powdery Mildew ◽

Positional Cloning ◽

Stop Codon ◽

Premature Stop Codon ◽

Defense Responses ◽

Loss Of Function ◽

Callose Synthase ◽

Synergistic Activation ◽

Golovinomyces Cichoracearum ◽

Inducible Gene

Loss-of-function mutations in the EDR1 gene of Arabidopsis confer enhanced resistance to Golovinomyces cichoracearum (powdery mildew). Disease resistance mediated by the edr1 mutation is dependent on an intact salicylic acid (SA) signaling pathway, but edr1 mutant plants do not constitutively express the SA-inducible gene PR-1 and are not dwarfed. To identify other components of the EDR1 signaling network, we screened for mutations that enhanced the edr1 mutant phenotype. Here, we describe an enhancer of edr1 mutant, eed3, which forms spontaneous lesions in the absence of pathogen infection, constitutively expresses both SA- and methyl jasmonate (JA)–inducible defense genes, and is dwarfed. Positional cloning of eed3 revealed that the mutation causes a premature stop codon in GLUCAN SYNTHASE-LIKE 5 (GSL5, also known as POWDERY MILDEW RESISTANT 4), which encodes a callose synthase required for pathogen-induced callose production. Significantly, gsl5 single mutants do not constitutively express PR-1 or AtERF1 (a JA-inducible gene) and are not dwarfed. Thus, loss of both EDR1 and GSL5 function has a synergistic effect. Our data suggest that EDR1 and GSL5 negatively regulate SA and JA production or signaling by independent mechanisms and that negative regulation of defense signaling by GSL5 may be independent of callose production.

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Molecular genetic study in two patients with congenital hypoaldosteronism (types I and II) in relation to previously published hormonal studies

Acta Endocrinologica ◽

10.1530/eje.0.1390096 ◽

1998 ◽

pp. 96-100 ◽

Cited By ~ 14

Author(s):

M Peter ◽

K Bunger ◽

SL Drop ◽

WG Sippell

Keyword(s):

Genetic Study ◽

Stop Codon ◽

Molecular Genetic ◽

Premature Stop Codon ◽

Type I ◽

Type Ii ◽

Loss Of Function ◽

Molecular Genetic Study ◽

Base Exchange ◽

Deficiency Type

We performed a molecular genetic study in two patients with congenital hypoaldosteronism. An original study of these patients was published in this Journal in 1982. Both index cases, a girl (patient 1) and a boy (patient 2). presented with salt-wasting and failure to thrive in the neonatal period. Parents of patient 1 were not related, whereas the parents of patient 2 were cousins. Endocrine studies had shown a defect in 18-oxidation of 18-OH-corticosterone in patient 1 and a defect in the 18-hydroxylation of corticosterone in patient 2. Plasma aldosterone was decreased in both patients, whereas 18-OH-corticosterone was elevated in patient 1 and decreased in patient 2. Plasma corticosterone and 11-deoxycorticosterone were elevated in both patients, whereas cortisol and its precursors were in the normal range. According to the nomenclature proposed by Ulick, the defects are termed corticosterone methyl oxidase (CMO) deficiency type II in patient 1, and type I in patient 2 respectively. Genetic defects in the gene CYP11B2 encoding aldosterone synthase have been described in a few cases. In patient 1, we identified only one heterozygous amino acid substitution (V386A) in exon 7, which has no deleterious effect on the enzyme activity. In patient 2 and his older brother, we identified a homozygous single base exchange (G to T) in codon 255 (GAG), causing a premature stop codon E255X (TAG). The mutant enzyme has lost the five terminal exons containing the haem binding site, and is thus a loss of function enzyme. This is only the second report of a patient with CMO deficiency type II without a mutation in the exons and exon-intron boundaries, whereas the biochemical phenotype of the two brothers with CMO deficiency type I can be explained by the patient's genotype.

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Interferon lambda 4 expression is suppressed by the host during viral infection

Journal of Experimental Medicine ◽

10.1084/jem.20160437 ◽

2016 ◽

Vol 213 (12) ◽

pp. 2539-2552 ◽

Cited By ~ 28

Author(s):

MeeAe Hong ◽

Johannes Schwerk ◽

Chrissie Lim ◽

Alison Kell ◽

Abigail Jarret ◽

...

Keyword(s):

Biochemical Characterization ◽

Splice Variants ◽

Stop Codon ◽

Association Studies ◽

Premature Stop Codon ◽

Genetic Association Studies ◽

Protein Isoforms ◽

Translation Efficiency ◽

Regulation Of Expression ◽

Protein Coding

Interferon (IFN) lambdas are critical antiviral effectors in hepatic and mucosal infections. Although IFNλ1, IFNλ2, and IFNλ3 act antiviral, genetic association studies have shown that expression of the recently discovered IFNL4 is detrimental to hepatitis C virus (HCV) infection through a yet unknown mechanism. Intriguingly, human IFNL4 harbors a genetic variant that introduces a premature stop codon. We performed a molecular and biochemical characterization of IFNλ4 to determine its role and regulation of expression. We found that IFNλ4 exhibits similar antiviral activity to IFNλ3 without negatively affecting antiviral IFN activity or cell survival. We show that humans deploy several mechanisms to limit expression of functional IFNλ4 through noncoding splice variants and nonfunctional protein isoforms. Furthermore, protein-coding IFNL4 mRNA are not loaded onto polyribosomes and lack a strong polyadenylation signal, resulting in poor translation efficiency. This study provides mechanistic evidence that humans suppress IFNλ4 expression, suggesting that immune function is dependent on other IFNL family members.

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Long antiparallel open reading frames are unlikely to be encoding essential proteins in prokaryotic genomes

10.1101/724807 ◽

2019 ◽

Author(s):

Denis Moshensky ◽

Andrei Alexeevski

Keyword(s):

Negative Selection ◽

Stop Codon ◽

Biological Significance ◽

Open Reading Frames ◽

Overlapping Genes ◽

Base Pairs ◽

Protein Coding ◽

Essential Proteins ◽

Prokaryotic Genomes ◽

Reading Frames

AbstractThe origin and evolution of genes that have common base pairs (overlapping genes) are of particular interest due to their influencing each other. Especially intriguing are gene pairs with long overlaps. In prokaryotes, co-directional overlaps longer than 60 bp were shown to be nonexistent except for some instances. A few antiparallel prokaryotic genes with long overlaps were described in the literature. We have analyzed putative long antiparallel overlapping genes to determine whether open reading frames (ORFs) located opposite to genes (antiparallel ORFs) can be protein-coding genes.We have confirmed that long antiparallel ORFs (AORFs) are observed reliably to be more frequent than expected. There are 10 472 000 AORFs in 929 analyzed genomes with overlap length more than 180 bp. Stop codons on the opposite to the coding strand are avoided in 2 898 cases with Benjamini-Hochberg threshold 0.01.Using Ka/Ks ratio calculations, we have revealed that long AORFs do not affect the type of selection acting on genes in a vast majority of cases. This observation indicates that long AORFs translations commonly are not under negative selection.The demonstrative example is 282 longer than 1 800 bp AORFs found opposite to extremely conserved dnaK genes. Translations of these AORFs were annotated “glutamate dehydrogenases” and were included into Pfam database as third protein family of glutamate dehydrogenases, PF10712. Ka/Ks analysis has demonstrated that if these translations correspond to proteins, they are not subjected by negative selection while dnaK genes are under strong stabilizing selection. Moreover, we have found other arguments against the hypothesis that these AORFs encode essential proteins, proteins indispensable for cellular machinery.However, some AORFs, in particular, dnaK related, have been found slightly resisting to synonymous changes in genes. It indicates the possibility of their translation. We speculate that translations of certain AORFs might have a functional role other than encoding essential proteins.Essential genes are unlikely to be encoded by AORFs in prokaryotic genomes. Nevertheless, some AORFs might have biological significance associated with their translations.Author summaryGenes that have common base pairs are called overlapping genes. We have examined the most intriguing case: if gene pairs encoded on opposite DNA strands exist in prokaryotes. An intersection length threshold 180 bp has been used. A few such pairs of genes were experimentally confirmed.We have detected all long antiparallel ORFs in 929 prokaryotic genomes and have found that the number of open reading frames, located opposite to annotated genes, is much more than expected according to statistical model. We have developed a measure of stop codon avoidance on the opposite strand. The lengths of found antiparallel ORFs with stop codon avoidance are typical for prokaryotic genes.Comparative genomics analysis shows that long antiparallel ORFs (AORFs) are unlikely to be essential protein-coding genes. We have analyzed distributions of features typical for essential proteins among formal translations of all long AORFs: prevalence of negative selection, non-uniformity of a conserved positions distribution in a multiple alignment of homologous proteins, the character of homologs distribution in phylogenetic tree of prokaryotes. All of them have not been observed for the majority of long AORFs. Particularly, the same results have been obtained for some experimentally confirmed AOGs.Thus, pairs of antiparallel overlapping essential genes are unlikely to exist. On the other hand, some antiparallel ORFs affect the evolution of genes opposite that they are located. Consequently, translations of some antiparallel ORFs might have yet unknown biological significance.

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Therapeutic base and prime editing of COL7A1 mutations in recessive dystrophic epidermolysis bullosa

10.1101/2021.07.12.452037 ◽

2021 ◽

Author(s):

Sung-ah Hong ◽

Song-Ee Kim ◽

A-young Lee ◽

Gue-ho Hwang ◽

Jong Hoon Kim ◽

...

Keyword(s):

Epidermolysis Bullosa ◽

Ex Vivo ◽

Stop Codon ◽

Point Mutations ◽

Premature Stop Codon ◽

Loss Of Function ◽

Dystrophic Epidermolysis Bullosa ◽

Immunodeficient Mice ◽

Type Vii Collagen ◽

Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (C7), a protein that functions in skin adherence. From 36 Korean RDEB patients, we identified a total of 69 pathogenic mutations (40 variants without recurrence), including point mutations (72.5%) and insertion/deletion mutations (27.5%). We used base and prime editing to correct mutations in fibroblasts from two patients (Pat1, who carried a c.3631C>T mutation in one allele, and Pat2, who carried a c.2005C>T mutation in one allele). We applied adenine base editors (ABEs) to correct the pathogenic mutation or to bypass a premature stop codon in Pat1-derived primary fibroblasts. To expand the targeting scope, we also utilized prime editors (PEs) to correct the mutations in Pat1- and Pat2-derived fibroblasts. Ultimately, we found that both ABE- and PE-mediated correction of COL7A1 mutations restored full-length C7 expression, reversed the impaired adhesion and proliferation exhibited by the patient-derived fibroblasts, and, following transfer of edited patient-derived fibroblasts into the skin of immunodeficient mice, led to C7 deposition within the dermal-epidermal junction. These results suggest that base and prime editing could be feasible strategies for ex vivo gene editing to treat RDEB.

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Hereditary Multiple Exostoses: Clinical, Molecular and Radiologic Survey in 9 Families

Prague Medical Report ◽

10.14712/23362936.2017.8 ◽

2017 ◽

Vol 118 (2-3) ◽

pp. 87-94

Author(s):

Karel Medek ◽

Jiří Zeman ◽

Tomáš Honzík ◽

Hana Hansíková ◽

Štěpánka Švecová ◽

...

Keyword(s):

Malignant Transformation ◽

De Novo ◽

Stop Codon ◽

Premature Stop Codon ◽

Gene Mutations ◽

Nonsense Mutations ◽

Hereditary Multiple Exostoses ◽

Multiple Exostoses ◽

The Mean

Hereditary multiple exostoses (HME) represents a heterogeneous group of diseases often associated with progressive skeletal deformities. Most frequently, mutations inEXT1andEXT2genes with autosomal dominant inheritance are responsible for HME. In our group of 9 families with HME we evaluated the clinical course of the disease and analysed molecular background using Sanger sequencing and MLPA inEXT1andEXT2genes. The mean age in our group of patients, when the first exostosis was recognised was 4.5 years (range 2–10 years) and the number of exostoses per one patient documented on X-ray ranged from 2 to 54. Most of the exostoses developed before the growth was completed and they were dominantly localised in the distal femurs, proximal tibia, proximal humerus and distal radius. In all patients, at least one to 8 surgeries were necessary due to complaints and local complications, but neither patient developed malignant transformation. In half of the patients, the disease resulted in short stature. DNA analyses were positive in 7 families. In five probands, differentEXT1gene mutations resulting in premature stop-codon (p.Gly124Argfs*65, p.Leu191*, p.Trp364Lysfs*11, p.Val371Glyfs*10, p.Leu490Profs*31) were found. In two probands, nonsense mutations were found inEXT2gene (p.Val187Profs*115, p.Cys319fs*46). Five mutations have been novel and two mutations have occurredde novoin probands. Although the risk for malignant transformation is usually low, especially in patients with low number of exostoses, early diagnostics and longitudinal follow up of patients is of a big importance, because early surgery can prevent progression of secondary bone deformities.

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