A Premature Stop Codon in RAF1 Is the Priority Candidate Causative Mutation of the Inherited Chicken Wingless-2 Developmental Syndrome

The chicken wingless-2 (wg-2) mutation is inherited in an autosomal recessive fashion, and the resulting phenotype in mutant (wg-2/wg-2) individuals is a developmental syndrome characterized by absent wings, truncated legs, craniofacial as well as skin and feather defects, and kidney malformations. Mapping and genotyping established that the mutation resides within 227 kilobases (kb) of chromosome 12 in a wg-2 congenic inbred line. A capture array was designed to target and sequence the candidate region along with flanking DNA in 24 birds from the line. Many point mutations and insertions or deletions were identified, and analysis of the linked variants indicated a point mutation predicted to cause a premature stop codon in the RAF1 gene. Expression studies were conducted inclusive of all genes in the candidate region. Interestingly, RAF1 transcription was elevated, yet the protein was absent in the mutants relative to normal individuals. RAF1 encodes a protein integral to the Ras/Raf/MAPK signaling pathway controlling cellular proliferation, and notably, human RASopathies are developmental syndromes caused by germline mutations in genes of this pathway. Our work indicates RAF1 as the priority candidate causative gene for wg-2 and provides a new animal model to study an important signaling pathway implicated in limb development, as well as RASopathies.

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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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Detection of Splicing Abnormalities and Genotype-Phenotype Correlation in X-linked Alport Syndrome

Journal of the American Society of Nephrology ◽

10.1681/asn.2018030228 ◽

2018 ◽

Vol 29 (8) ◽

pp. 2244-2254 ◽

Cited By ~ 12

Author(s):

Tomoko Horinouchi ◽

Kandai Nozu ◽

Tomohiko Yamamura ◽

Shogo Minamikawa ◽

Takashi Omori ◽

...

Keyword(s):

Confidence Interval ◽

Alport Syndrome ◽

Stop Codon ◽

Consensus Sequence ◽

Point Mutations ◽

Premature Stop Codon ◽

Transcript Level ◽

Phenotype Correlation ◽

Genotype Phenotype Correlation ◽

Splicing Patterns

BackgroundX-linked Alport syndrome (XLAS) is a progressive hereditary nephropathy caused by mutations in the COL4A5 gene. Genotype-phenotype correlation in male XLAS is relatively well established; relative to truncating mutations, nontruncating mutations exhibit milder phenotypes. However, transcript comparison between XLAS cases with splicing abnormalities that result in a premature stop codon and those with nontruncating splicing abnormalities has not been reported, mainly because transcript analysis is not routinely conducted in patients with XLAS.MethodsWe examined transcript expression for all patients with suspected splicing abnormalities who were treated at one hospital between January of 2006 and July of 2017. Additionally, we recruited 46 males from 29 families with splicing abnormalities to examine genotype-phenotype correlation in patients with truncating (n=21, from 14 families) and nontruncating (n=25, from 15 families) mutations at the transcript level.ResultsWe detected 41 XLAS families with abnormal splicing patterns and described novel XLAS atypical splicing patterns (n=14) other than exon skipping caused by point mutations in the splice consensus sequence. The median age for developing ESRD was 20 years (95% confidence interval, 14 to 23 years) among patients with truncating mutations and 29 years (95% confidence interval, 25 to 40 years) among patients with nontruncating mutations (P=0.001).ConclusionsWe report unpredictable atypical splicing in the COL4A5 gene in male patients with XLAS and reveal that renal prognosis differs significantly for patients with truncating versus nontruncating splicing abnormalities. Our results suggest that splicing modulation should be explored as a therapy for XLAS with truncating mutations.

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Deficiency in the A-subunit of coagulation factor XIII: two novel point mutations demonstrate different effects on transcript levels

Blood ◽

10.1182/blood.v84.2.517.517 ◽

1994 ◽

Vol 84 (2) ◽

pp. 517-525 ◽

Cited By ~ 116

Author(s):

H Mikkola ◽

M Syrjala ◽

V Rasi ◽

E Vahtera ◽

E Hamalainen ◽

...

Keyword(s):

Factor Xiii ◽

Solid Phase ◽

Stop Codon ◽

Point Mutations ◽

Premature Stop Codon ◽

Coagulation Factor ◽

Mrna Levels ◽

Coagulation Factor Xiii ◽

Stop Mutation ◽

A Subunit

Abstract Congenital deficiency in coagulation factor XIII is a rare autosomal recessive bleeding disorder. Although the defect was characterized over 30 years ago, little is known about the molecular basis of the disorder. Here, we show two novel point mutations in the gene of the A- subunit of factor XIII in the genetically isolated population of Finland. All eight factor XIII-deficient families identified in Finland were studied. The exons of the gene of A-subunit were amplified individually by polymerase chain reaction and subsequently screened by single-strand conformation polymorphism. Sequence analysis of the abnormally migrating fragments showed two point mutations resulting in an amino acid alteration. A C-to-T transition at Arg-661 in exon XIV created a premature stop codon. This mutation was detected in six of the eight families, thus being the major alteration causing FXIII deficiency in Finland. In two of the six families, the patients were compound heterozygotes with the Arg-661-Stop mutation in one allele and either a T-to-C point mutation in exon VI or a thus far uncharacterized mutation in the other allele. The T-to-C transition in exon VI resulted in a substitution of threonine for methionine 242. The transition was found in one family only, where it was in the heterozygote form combined with the Arg-661-Stop mutation. To evaluate the consequences of these mutations, steady-state FXIII mRNA levels were quantitated by solid-phase minisequencing. In addition to the termination of translation 70 amino acids before the initial stop codon, the Arg-661- Stop mutation causes a 10- to 30-fold reduction in FXIII mRNA levels. This is also likely to result in a low translation level in the truncated polypeptide. In contrast, Met-242-Thr mutation does not seem to affect the level of mRNA. Here, the absence of a functional and immunodetectable protein is probably caused by an altered conformation of the mutant polypeptide, resulting in early degradation of the defective protein.

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Mutation detection and prenatal diagnosis of XLHED pedigree

PeerJ ◽

10.7717/peerj.3691 ◽

2017 ◽

Vol 5 ◽

pp. e3691 ◽

Cited By ~ 4

Author(s):

Yao Lin ◽

Wei Yin ◽

Zhuan Bian

Keyword(s):

Prenatal Diagnosis ◽

Structural Changes ◽

Frameshift Mutation ◽

Stop Codon ◽

Direct Sequencing ◽

Premature Stop Codon ◽

Deciduous Teeth ◽

Chinese Family ◽

Female Carrier ◽

Causative Gene

Background The phenotypic characters of X -linked Hypohidrotic Ectodermal Dysplasia (XLHED) are the dysplasia of epithelial- and mesenchymal-derived organs. Ectodysplasin (EDA) is the causative gene of XLHED. Methods The current study reported a large Chinese XLHED pedigree. The genomic DNA of adult and fetus was extracted from peripheral blood and shed chorion cell respectively. The nucleotide variation in EDA gene was screened through direct sequencing the coding sequence. The methylation state of EDA gene’s promoter was evaluated by pyrosequencing. Results This Chinese XLHED family had two male patients and three carriers. All of them were with a novel EDA frameshift mutation. The mutation, c.172-173insGG, which leads to an immediate premature stop codon in exon one caused severe structural changes of EDA. Prenatal diagnosis suggested that the fetus was a female carrier. The follow-up observation of this child indicated that she had mild hypodontia of deciduous teeth at age six. The methylation level of EDA gene’s promoter was not related to carriers’ phenotype changes in this family. Discussion We reported a new frameshift mutation of EDA gene in a Chinese family. Prenatal diagnosis can help to predict the disease status of the fetus.

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In vivoRNA targeting of point mutations via suppressor tRNAs and adenosine deaminases

10.1101/210278 ◽

2017 ◽

Cited By ~ 2

Author(s):

Dhruva Katrekar ◽

Prashant Mali

Keyword(s):

Mouse Model ◽

Rna Editing ◽

Unnatural Amino Acids ◽

Stop Codon ◽

Genetic Diseases ◽

Point Mutations ◽

Premature Stop Codon ◽

Dystrophin Gene ◽

Multiple Challenges

ABSTRACTPoint mutations underlie many genetic diseases. In this regard, while programmable DNA nucleases have been used to repair mutations, their use for gene therapy poses multiple challenges: one, efficiency of homologous recombination is typically low in cells; two, an active nuclease presents a risk of introducing permanent off-target mutations; and three, prevalent programmable nucleases typically comprise elements of non-human origin raising the potential ofin vivoimmunogenicity. In light of these, approaches to instead directly target RNA, and use of molecular machinery native to the host would be highly desirable. Towards this, we engineered and optimized two complementary approaches, referred together hereon astRiAD, based on the use oftRNAsin codon suppression andadenosinedeaminases in RNA editing. Specifically, by delivering modified endogenous tRNAs and/or the RNA editing enzyme ADAR2 and an associated guiding RNA (adRNA) via adeno-associated viruses, we enabled premature stop codon read-through and correction in themdxmouse model of muscular dystrophy that harbors a nonsense mutation in the dystrophin gene. We further demonstrated inducible restoration of dystrophin expression by pyrolysyl-tRNA mediated incorporation of unnatural amino acids (UAAs) at the stop codon. Additionally, we also engineered ADAR2 mediated correction of a point mutation in liver RNA of thespfashmouse model of ornithine transcarbamylase (OTC) deficiency. Taken together, our results establish the use of suppressor tRNAs and ADAR2 forin vivoRNA targeting, and this integrated tRiAD approach is robust, genomically scarless, and potentially non-immunogenic as it utilizes effector RNAs and human proteins.

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Biological Characteristics and Molecular Mechanisms of Fludioxonil Resistance in Fusarium graminearum in China

Plant Disease ◽

10.1094/pdis-01-20-0079-re ◽

2020 ◽

Vol 104 (9) ◽

pp. 2426-2433

Author(s):

F. Zhou ◽

D. X. Li ◽

H. Y. Hu ◽

Y. L. Song ◽

Y. C. Fan ◽

...

Keyword(s):

Fusarium Graminearum ◽

Molecular Mechanisms ◽

Target Genes ◽

Stop Codon ◽

Point Mutations ◽

Premature Stop Codon ◽

Cross Resistance ◽

Alternative Mechanism ◽

Head Blight ◽

Resistant Mutants

Fusarium graminearum is the primary causal agent of Fusarium head blight (FHB) of wheat. The phenylpyrrole fungicide fludioxonil is not currently registered for the management of FHB in China. The current study assessed the fludioxonil sensitivity of a total of 53 F. graminearum isolates collected from the six most important wheat-growing provinces of China during 2018 and 2019. The baseline fludioxonil sensitivity distribution indicated that all of the isolates were sensitive, exhibiting a unimodal cure with a mean effective concentration for 50% inhibition value of 0.13 ± 0.12 μg/ml (standard deviation). Five fludioxonil-resistant mutants were subsequently induced by exposure to fludioxonil under laboratory conditions. Ten successive rounds of subculture in the absence of the selection pressure indicated that the mutation was stably inherited. However, the fludioxonil-resistant mutants were found to have reduced pathogenicity, higher glycerol accumulation, and higher osmotic sensitivity than the parental wild-type isolates, indicating that there was a fitness cost associated with fludioxonil resistance. In addition, the study also found a positive cross resistance between fludioxonil, procymidone, and iprodione, but not with other fungicides such as boscalid, carbendazim, tebuconazole, and fluazinam. Sequence analysis of four candidate target genes (FgOs1, FgOs2, FgOs4, and FgOs5) revealed that the HBXT2R mutant contained two point mutations that resulted in amino acid changes at K223T and K415R in its FgOs1 protein, and one point mutation at residue 520 of its FgOs5 protein that resulted in a premature stop codon. Similarly, the three other mutants contained point mutations that resulted in changes at the K192R, K293R, and K411R residues of the FgOs5 protein but none in the FgOs2 and FgOs4 genes. However, it is important to point out that the FgOs2 and FgOs4 expression of all the fludioxonil-resistant mutants was significantly (P < 0.05) downregulated compared with the sensitive isolates (except for the SQ1-2 isolate). It was also found that one of the resistant mutants did not have changes in any of the sequenced target genes, indicating that an alternative mechanism could also lead to fludioxonil resistance.

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Mutation in the RPE65 gene causing hereditary retinal dystrophy in the Briard dogs: application of a new detection method

Czech Journal of Animal Science ◽

10.17221/372-cjas ◽

2008 ◽

Vol 53 (No. 4) ◽

pp. 176-179

Author(s):

R. Bechyňová ◽

J. Dostál ◽

A. Stratil ◽

F. Jílek ◽

P. Horák

Keyword(s):

Autosomal Recessive ◽

Clinical Symptoms ◽

Stop Codon ◽

Retinal Dystrophy ◽

Premature Stop Codon ◽

Causative Mutation ◽

Functional Protein ◽

Hereditary Retinal Dystrophy ◽

Briard Dogs ◽

Autosomal Recessive Manner

Inherited eye diseases are widespread in most of the pure dog breeds and they show a severe impact on canine health, welfare and working ability. Congenital stationary night blindness (CSNB) was originally described in Briards. CSNB is slow progressive retinal degeneration with very early onset of clinical symptoms and is inherited in an autosomal recessive manner. The causative mutation (Y16567.1:c.487_490delAAGA) for CSNB was identified in exon 5 of the RPE65 gene. This deletion results in a frameshift and leads to a premature stop codon and expression of a non-functional protein. To date, only expensive, laborious or unpractical methods have been used for detection of the mutation in the canine RPE65 gene. The main goals of this study were to develop a new method for routine genotyping of the causative mutation and to assess its occurrence in the Czech population of Briards. The method of electrophoresis in the gel Spraedex EL600 can be widely used for genotyping of the RPE65 gene as a basis of proper genetic counselling and an improvement of genetic health in the Briard populations. In the studied population, the following frequencies of alleles + (wild) and – (mutant) were observed – 0.939 and 0.061, respectively.

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A Stop-Gain Mutation within MLPH Is Responsible for the Lilac Dilution Observed in Jacob Sheep

Genes ◽

10.3390/genes11060618 ◽

2020 ◽

Vol 11 (6) ◽

pp. 618 ◽

Cited By ~ 1

Author(s):

Christian J. Posbergh ◽

Elizabeth A. Staiger ◽

Heather J. Huson

Keyword(s):

Autosomal Recessive ◽

Coat Color ◽

Color Change ◽

Stop Codon ◽

Premature Stop Codon ◽

Autosomal Recessive Inheritance ◽

Pedigree Analysis ◽

Causative Mutation ◽

Nonsynonymous Mutation ◽

Original Case

A coat color dilution, called lilac, was observed within the Jacob sheep breed. This dilution results in sheep appearing gray, where black would normally occur. Pedigree analysis suggested an autosomal recessive inheritance. Whole-genome sequencing of a dilute case, a known carrier, and sixteen non-dilute sheep was used to identify the molecular variant responsible for the coat color change. Through investigation of the genes MLPH, MYO5A, and RAB27A, we discovered a nonsynonymous mutation within MLPH, which appeared to match the reported autosomal recessive nature of the lilac dilution. This mutation (NC_019458.2:g.3451931C>A) results in a premature stop codon being introduced early in the protein (NP_001139743.1:p.Glu14*), likely losing its function. Validation testing of additional lilac Jacob sheep and known carriers, unrelated to the original case, showed a complete concordance between the mutation and the dilution. This stop-gain mutation is likely the causative mutation for dilution within Jacob sheep.

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Specific point mutations in Lactobacillus casei ATCC 27139 cause a phenotype switch from Lac− to Lac+

Microbiology ◽

10.1099/mic.0.021907-0 ◽

2009 ◽

Vol 155 (3) ◽

pp. 751-760 ◽

Cited By ~ 1

Author(s):

Yu-Kuo Tsai ◽

Hung-Wen Chen ◽

Ta-Chun Lo ◽

Thy-Hou Lin

Keyword(s):

Gene Cluster ◽

Point Mutation ◽

Lactobacillus Casei ◽

Stop Codon ◽

Single Point ◽

Point Mutations ◽

Premature Stop Codon ◽

Gene Clusters ◽

Single Point Mutation ◽

Phenotype Switch

Lactose metabolism is a changeable phenotype in strains of Lactobacillus casei. In this study, we found that L. casei ATCC 27139 was unable to utilize lactose. However, when exposed to lactose as the sole carbon source, spontaneous Lac+ clones could be obtained. A gene cluster (lacTEGF–galKETRM) involved in the metabolism of lactose and galactose in L. casei ATCC 27139 (Lac−) and its Lac+ revertant (designated strain R1) was sequenced and characterized. We found that only one nucleotide, located in the lacTEGF promoter (lacTp), of the two lac–gal gene clusters was different. The protein sequence identity between the lac–gal gene cluster and those reported previously for some L. casei (Lac+) strains was high; namely, 96–100 % identity was found and no premature stop codon was identified. A single point mutation located within the lacTp promoter region was also detected for each of the 41 other independently isolated Lac+ revertants of L. casei ATCC 27139. The revertants could be divided into six classes based on the positions of the point mutations detected. Primer extension experiments conducted on transcription from lacTp revealed that the lacTp promoter of these six classes of Lac+ revertants was functional, while that of L. casei ATCC 27139 was not. Northern blotting experiments further confirmed that the lacTEGF operon of strain R1 was induced by lactose but suppressed by glucose, whereas no blotting signal was ever detected for L. casei ATCC 27139. These results suggest that a single point mutation in the lacTp promoter was able to restore the transcription of a fully functional lacTEGF operon and cause a phenotype switch from Lac− to Lac+ for L. casei ATCC 27139.

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Dissolution of Xylose Metabolism inLactococcus lactis

Applied and Environmental Microbiology ◽

10.1128/aem.66.9.3974-3980.2000 ◽

2000 ◽

Vol 66 (9) ◽

pp. 3974-3980 ◽

Cited By ~ 34

Author(s):

Karn A. Erlandson ◽

Joo-Heon Park ◽

Wissam ◽

El Khal ◽

Hsin-Hsin Kao ◽

...

Keyword(s):

Stop Codon ◽

Starter Culture ◽

Point Mutations ◽

Premature Stop Codon ◽

Xylose Isomerase ◽

Environmental Isolates ◽

Xylose Metabolism ◽

Sequence Comparisons ◽

Catalytic Sites ◽

Dairy Starter

ABSTRACT Xylose metabolism, a variable phenotype in strains ofLactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyloperon. The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L. lactis subsp. lactis B-4449 (formerlyLactobacillus xylosus), L. lactis subsp.lactis IO-1, and L. lactis subsp.lactis 210, respectively. The two environmental isolates,L. lactis B-4449 and L. lactis IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose.L. lactis 210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl−. Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl+ strains. Sequence comparisons revealed a number of point mutations in thexylA, xylB, and xylR genes inL. lactis 210, IO-1, and B-4449. None of these mutations, with the exception of a premature stop codon in xylB, are obviously lethal, since they lie outside of regions recognized as critical for activity. Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of L. lactis unable to metabolize xylose.

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