Evolution of CCL11: genetic characterization in lagomorphs and evidence of positive and purifying selection in mammals.

PubMed

Neves, Fabiana; Abrantes, Joana; Esteves, Pedro J

2016-07-01

The interactions between chemokines and their receptors are crucial for differentiation and activation of inflammatory cells. CC chemokine ligand 11 (CCL11) binds to CCR3 and to CCR5 that in leporids underwent gene conversion with CCR2. Here, we genetically characterized CCL11 in lagomorphs (leporids and pikas). All lagomorphs have a potentially functional CCL11, and the Pygmy rabbit has a mutation in the stop codon that leads to a longer protein. Other mammals also have mutations at the stop codon that result in proteins with different lengths. By employing maximum likelihood methods, we observed that, in mammals, CCL11 exhibits both signatures of purifying and positive selection. Signatures of purifying selection were detected in sites important for receptor binding and activation. Of the three sites detected as under positive selection, two were located close to the stop codon. Our results suggest that CCL11 is functional in all lagomorphs, and that the signatures of purifying and positive selection in mammalian CCL11 probably reflect the protein's biological roles. © The Author(s) 2016.

Severe Hemophilia A in a Male Old English Sheep Dog with a C→T Transition that Created a Premature Stop Codon in Factor VIII

PubMed Central

Lozier, Jay N; Kloos, Mark T; Merricks, Elizabeth P; Lemoine, Nathaly; Whitford, Margaret H; Raymer, Robin A; Bellinger, Dwight A; Nichols, Timothy C

2016-01-01

Animals with hemophilia are models for gene therapy, factor replacement, and inhibitor development in humans. We have actively sought dogs with severe hemophilia A that have novel factor VIII mutations unlike the previously described factor VIII intron 22 inversion. A male Old English Sheepdog with recurrent soft-tissue hemorrhage and hemarthrosis was diagnosed with severe hemophilia A (factor VIII activity less than 1% of normal). We purified genomic DNA from this dog and ruled out the common intron 22 inversion; we then sequenced all 26 exons. Comparing the results with the normal canine factor VIII sequence revealed a C→T transition in exon 12 of the factor VIII gene that created a premature stop codon at amino acid 577 in the A2 domain of the protein. In addition, 2 previously described polymorphisms that do not cause hemophilia were present at amino acids 909 and 1184. The hemophilia mutation creates a new TaqI site that facilitates rapid genotyping of affected offspring by PCR and restriction endonuclease analyses. This mutation is analogous to the previously described human factor VIII mutation at Arg583, which likewise is a CpG dinucleotide transition causing a premature stop codon in exon 12. Thus far, despite extensive treatment with factor VIII, this dog has not developed neutralizing antibodies (‘inhibitors’) to the protein. This novel mutation in a dog gives rise to severe hemophilia A analogous to a mutation seen in humans. This model will be useful for studies of the treatment of hemophilia. PMID:27780008

Mutation at Tyrosine in AMLRY (GILRY Like) Motif of Yeast eRF1 on Nonsense Codons Suppression and Binding Affinity to eRF3

PubMed Central

Akhmaloka; Susilowati, Prima Endang; Subandi; Madayanti, Fida

2008-01-01

Termination translation in Saccharomyces cerevisiae is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. Two regions in human eRF1, position at 281-305 and position at 411-415, were proposed to be involved on the interaction to eRF3. In this study we have constructed and characterized yeast eRF1 mutant at position 410 (correspond to 415 human eRF1) from tyrosine to serine residue resulting eRF1(Y410S). The mutations did not affect the viability and temperature sensitivity of the cell. The stop codons suppression of the mutant was analyzed in vivo using PGK-stop codon-LACZ gene fusion and showed that the suppression of the mutant was significantly increased in all of codon terminations. The suppression on UAG codon was the highest increased among the stop codons by comparing the suppression of the wild type respectively. In vitro interaction between eRF1 (mutant and wild type) to eRF3 were carried out using eRF1-(His)6 and eRF1(Y410S)-(His)6 expressed in Escherichia coli and indigenous Saccharomyces cerevisiae eRF3. The results showed that the binding affinity of eRF1(Y410S) to eRF3 was decreased up to 20% of the wild type binding affinity. Computer modeling analysis using Swiss-Prot and Amber version 9.0 programs revealed that the overall structure of eRF1(Y410S) has no significant different with the wild type. However, substitution of tyrosine to serine triggered the structural change on the other motif of C-terminal domain of eRF1. The data suggested that increasing stop codon suppression and decreasing of the binding affinity of eRF1(Y410S) were probably due to the slight modification on the structure of the C-terminal domain. PMID:18463713

Conserved nonsense-prone CpG sites in apoptosis-regulatory genes: conditional stop signs on the road to cell death.

PubMed

Zhao, Yongzhong; Epstein, Richard J

2013-01-01

Methylation-prone CpG dinucleotides are strongly conserved in the germline, yet are also predisposed to somatic mutation. Here we quantify the relationship between germline codon mutability and somatic carcinogenesis by comparing usage of the nonsense-prone CGA (→TGA) codons in gene groups that differ in apoptotic function; to this end, suppressor genes were subclassified as either apoptotic (gatekeepers) or repair (caretakers). Mutations affecting CGA codons in sporadic tumors proved to be highly asymmetric. Moreover, nonsense mutations were 3-fold more likely to affect gatekeepers than caretakers. In addition, intragenic CGA clustering nonrandomly affected functionally critical regions of gatekeepers. We conclude that human gatekeeper suppressor genes are enriched for nonsense-prone codons, and submit that this germline vulnerability to tumors could reflect in utero selection for a methylation-dependent capability to short-circuit environmental insults that otherwise trigger apoptosis and fetal loss.

Unusual AIP mutation and phenocopy in the family of a young patient with acromegalic gigantism.

PubMed

Imran, Syed Ali; Aldahmani, Khaled A; Penney, Lynette; Croul, Sidney E; Clarke, David B; Collier, David M; Iacovazzo, Donato; Korbonits, Márta

2018-01-01

Early-onset acromegaly causing gigantism is often associated with aryl-hydrocarbon-interacting receptor protein ( AIP ) mutation, especially if there is a positive family history. A15y male presented with tiredness and visual problems. He was 201 cm tall with a span of 217 cm. He had typical facial features of acromegaly, elevated IGF-1, secondary hypogonadism and a large macroadenoma. His paternal aunt had a history of acromegaly presenting at the age of 35 years. Following transsphenoidal surgery, his IGF-1 normalized and clinical symptoms improved. He was found to have a novel AIP mutation destroying the stop codon c.991T>C; p.*331R. Unexpectedly, his father and paternal aunt were negative for this mutation while his mother and older sister were unaffected carriers, suggesting that his aunt represents a phenocopy. Typical presentation for a patient with AIP mutation with excess growth and eunuchoid proportions.Unusual, previously not described AIP variant with loss of the stop codon.Phenocopy may occur in families with a disease-causing germline mutation.

Unusual AIP mutation and phenocopy in the family of a young patient with acromegalic gigantism

PubMed Central

Aldahmani, Khaled A; Penney, Lynette; Croul, Sidney E; Clarke, David B; Collier, David M; Iacovazzo, Donato; Korbonits, Márta

2018-01-01

Summary Early-onset acromegaly causing gigantism is often associated with aryl-hydrocarbon-interacting receptor protein (AIP) mutation, especially if there is a positive family history. A15y male presented with tiredness and visual problems. He was 201 cm tall with a span of 217 cm. He had typical facial features of acromegaly, elevated IGF-1, secondary hypogonadism and a large macroadenoma. His paternal aunt had a history of acromegaly presenting at the age of 35 years. Following transsphenoidal surgery, his IGF-1 normalized and clinical symptoms improved. He was found to have a novel AIP mutation destroying the stop codon c.991T>C; p.*331R. Unexpectedly, his father and paternal aunt were negative for this mutation while his mother and older sister were unaffected carriers, suggesting that his aunt represents a phenocopy. Learning points: Typical presentation for a patient with AIP mutation with excess growth and eunuchoid proportions. Unusual, previously not described AIP variant with loss of the stop codon. Phenocopy may occur in families with a disease-causing germline mutation. PMID:29472986

Structural insights into eRF3 and stop codon recognition by eRF1

PubMed Central

Cheng, Zhihong; Saito, Kazuki; Pisarev, Andrey V.; Wada, Miki; Pisareva, Vera P.; Pestova, Tatyana V.; Gajda, Michal; Round, Adam; Kong, Chunguang; Lim, Mengkiat; Nakamura, Yoshikazu; Svergun, Dmitri I.; Ito, Koichi; Song, Haiwei

2009-01-01

Eukaryotic translation termination is mediated by two interacting release factors, eRF1 and eRF3, which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. The crystal structures of human and Schizosaccharomyces pombe full-length eRF1 in complex with eRF3 lacking the GTPase domain revealed details of the interaction between these two factors and marked conformational changes in eRF1 that occur upon binding to eRF3, leading eRF1 to resemble a tRNA molecule. Small-angle X-ray scattering analysis of the eRF1/eRF3/GTP complex suggested that eRF1's M domain contacts eRF3's GTPase domain. Consistently, mutation of Arg192, which is predicted to come in close contact with the switch regions of eRF3, revealed its important role for eRF1's stimulatory effect on eRF3's GTPase activity. An ATP molecule used as a crystallization additive was bound in eRF1's putative decoding area. Mutational analysis of the ATP-binding site shed light on the mechanism of stop codon recognition by eRF1. PMID:19417105

A decamer duplication in the 3′ region of the BRI gene originates an amyloid peptide that is associated with dementia in a Danish kindred

PubMed Central

Vidal, Ruben; Révész, Tamas; Rostagno, Agueda; Kim, Eugene; Holton, Janice L.; Bek, Toke; Bojsen-Møller, Marie; Braendgaard, Hans; Plant, Gordon; Ghiso, Jorge; Frangione, Blas

2000-01-01

Familial Danish dementia (FDD), also known as heredopathia ophthalmo-oto-encephalica, is an autosomal dominant disorder characterized by cataracts, deafness, progressive ataxia, and dementia. Neuropathological findings include severe widespread cerebral amyloid angiopathy, hippocampal plaques, and neurofibrillary tangles, similar to Alzheimer's disease. N-terminal sequence analysis of isolated leptomeningeal amyloid fibrils revealed homology to ABri, the peptide originated by a point mutation at the stop codon of gene BRI in familial British dementia. Molecular genetic analysis of the BRI gene in the Danish kindred showed a different defect, namely the presence of a 10-nt duplication (795–796insTTTAATTTGT) between codons 265 and 266, one codon before the normal stop codon 267. The decamer duplication mutation produces a frame-shift in the BRI sequence generating a larger-than-normal precursor protein, of which the amyloid subunit (designated ADan) comprises the last 34 C-terminal amino acids. This de novo-created amyloidogenic peptide, associated with a genetic defect in the Danish kindred, stresses the importance of amyloid formation as a causative factor in neurodegeneration and dementia. PMID:10781099

Mucopolysaccharidosis IVA: Four new exonic mutations in patients with N-acetylgalactosamine-6-sulfate sulfatase deficiency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomatsu, Shunji; Fukuda, Seiji; Yamagishi, Atsushi

1996-05-01

We report four new mutations in Japanese patients with mucopolysaccharidosis IVA (MPSIVA) who were heterozygous for a common double gene deletion. A nonsense mutation of CAG to TAG at codon 148 in exon 4 was identified, resulting in a change of Q to a stop codon and three missense mutations: V (GTC) to A (GCC) at codon 138 in exon 4, P (CCC) to S (TCC) at codon 151 in exon 5, and P (CCC) to L (CTC) at codon 151 in exon 5. Introduction of these mutations into the normal GALNS cDNA and transient expression in cultured fibroblasts resultedmore » in a significant decrease in the enzyme activity. V138A and Q148X mutations result in changes of restriction site, which were analyzed by restriction-enzyme assay. P151S and P151L mutations that did not alter the restriction site were detected by direct sequencing or allele specific oligohybridization. Detection of the double gene deletion was initially done using Southern blots and was confirmed by PCR. Haplotypes were determined using seven polymorphisms to the GALNS locus in families with the double gene deletion. Haplotype analysis showed that the common double gene deletion occurred on a single haplotype, except for some variation in a VNTR-like polymorphism. This finding is consistent with a common founder for all individuals with this mutation. 48 refs., 5 figs., 1 tab.« less

Identification of ALK germline mutation (3605delG) in pediatric anaplastic medulloblastoma.

PubMed

Coco, Simona; De Mariano, Marilena; Valdora, Francesca; Servidei, Tiziana; Ridola, Vita; Andolfo, Immacolata; Oberthuer, André; Tonini, Gian Paolo; Longo, Luca

2012-10-01

The anaplastic lymphoma kinase (ALK) gene has been found either rearranged or mutated in several neoplasms such as anaplastic large-cell lymphoma, non-small-cell lung cancer, neuroblastoma and anaplastic thyroid cancer. Medulloblastoma (MB) is an embryonic pediatric cancer arising from nervous system, a tissue in which ALK is expressed during embryonic development. We performed an ALK mutation screening in 52 MBs and we found a novel heterozygous germline deletion of a single base in exon 23 (3605delG) in a case with marked anaplasia. This G deletion results in a frameshift mutation producing a premature stop codon in exon 25 of ALK tyrosine kinase domain. We also screened three human MB cell lines without finding any mutation of ALK gene. Quantitative expression analysis of 16 out of 52 samples showed overexpression of ALK mRNA in three MBs. In the present study, we report the first mutation of ALK found in MB. Moreover, a deletion of ALK gene producing a stop codon has not been detected in human tumors up to now. Further investigations are now required to elucidate whether the truncated form of ALK may have a role in signal transduction.

Two novel mutations in the alpha-galactosidase gene in Japanese classical hemizygotes with Fabry disease.

PubMed

Okumiya, T; Takenaka, T; Ishii, S; Kase, R; Kamei, S; Sakuraba, H

1996-09-01

Four alpha-galactosidase gene mutations were identified in Japanese male patients with Fabry disease who had no detectable alpha-galactosidase activity. Two of them were novel mutations, an 11-bp deletion in exon 2 and a g-1 to t substitution at the 3' end of the splice acceptor site in intron 1. The former caused a frameshift and led to the creation of a new stop codon at codon 118. The latter was predicted to provoke aberrant mRNA splicing followed by accelerated degradation of the mRNA. A nonsense mutation, R301X, and a 2-bp deletion starting at nucleotide position 718, which were reported previously, were also identified in unrelated patients.

Using Student Writing and Lexical Analysis to Reveal Student Thinking about the Role of Stop Codons in the Central Dogma.

PubMed

Prevost, Luanna B; Smith, Michelle K; Knight, Jennifer K

2016-01-01

Previous work has shown that students have persistent difficulties in understanding how central dogma processes can be affected by a stop codon mutation. To explore these difficulties, we modified two multiple-choice questions from the Genetics Concept Assessment into three open-ended questions that asked students to write about how a stop codon mutation potentially impacts replication, transcription, and translation. We then used computer-assisted lexical analysis combined with human scoring to categorize student responses. The lexical analysis models showed high agreement with human scoring, demonstrating that this approach can be successfully used to analyze large numbers of student written responses. The results of this analysis show that students' ideas about one process in the central dogma can affect their thinking about subsequent and previous processes, leading to mixed models of conceptual understanding. © 2016 L. B. Prevost et al. CBE—Life Sciences Education © 2016 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

[Novel CHST6 compound heterozygous mutations cause macular corneal dystrophy in a Chinese family].

PubMed

Qi, Yan-hua; Dang, Xiu-hong; Su, Hong; Zhou, Nan; Liang, Ting; Wang, Zheng; Huang, Shang-zhi

2010-02-01

The aim of this study was to identify mutations of CHST6 gene in a Chinese family with macular corneal dystrophy (MCD) and to investigate the histopathological changes of MCD. Corneal button of the proband was obtained from penetrating keratoplasty for the treatment of severe corneal dystrophy. The sections and ultrathin sections of this specimen were examined under light microscope and transmission electron microscope (TEM). Genomic DNA was extracted from leukocytes in peripheral blood from the family members. The coding region of CHST6 was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by direct sequencing and restriction enzyme digestion. Histochemical study revealed positive results of colloidal iron stain. TEM revealed enlargement of smooth endoplasmic reticulum and the presence of intracytoplasmic vacuoles. Two mutations, Q298X Y358H, were identified in exon 3 of CHST6. Three patients were compound heterozygotes of these two mutations. The C892T transversion occurred at codon 298 turned the codon of glutamine to a stop codon; the T1072C transversion occurred at codon 358 caused a missense mutation, tyrosine to histidine. All six unaffected family members were heterozygotes. These two mutations were not detected in any of the 100 control subjects. The novel compound heterozygous mutation results in loss of CHST6 function and causes the occurrence of MCD. This is the first report of this gene mutation.

OprD mutations and inactivation in imipenem-resistant Pseudomonas aeruginosa isolates from China.

PubMed

Fang, Zhi-Li; Zhang, Li-Yan; Huang, Ying-Min; Qing, Yun; Cao, Kai-Yuan; Tian, Guo-Bao; Huang, Xi

2014-01-01

To investigate the mechanisms involved in imipenem resistance of Pseudomonas aeruginosa in southern China, 61 imipenem-resistant P. aeruginosa clinical isolates were collected from 4 hospitals between October 2011 and June 2012. All isolates were resistant to imipenem, whereas 21.3% were susceptible or intermediate to meropenem. Variable degrees of resistance to other β-lactam and non-β-lactam antimicrobials were observed. PFGE revealed high-level of clonal diversity. Among the 61 isolates, 50 isolates had OprD loss by disrupted oprD mutations, including 43 with frameshift mutations of oprD and 7 with a premature stop codon by single point mutation. Six isolates were oprD-negative by PCR, suggestive of a major disruption of oprD genes. Five isolates had intact oprD but had reduced expression of oprD genes. In addition, only one isolate with disrupted oprD mutation by a premature stop codon was confirmed to be a metallo-β-lactamase producer (IMP-9). Our results show that the loss of OprD, as well as reduced expression of oprD and MBL production, were the predominant mechanisms of imipenem resistance in P. aeruginosa in southern China. Copyright © 2013 Elsevier B.V. All rights reserved.

Mitochondrial genetic codes evolve to match amino acid requirements of proteins.

PubMed

Swire, Jonathan; Judson, Olivia P; Burt, Austin

2005-01-01

Mitochondria often use genetic codes different from the standard genetic code. Now that many mitochondrial genomes have been sequenced, these variant codes provide the first opportunity to examine empirically the processes that produce new genetic codes. The key question is: Are codon reassignments the sole result of mutation and genetic drift? Or are they the result of natural selection? Here we present an analysis of 24 phylogenetically independent codon reassignments in mitochondria. Although the mutation-drift hypothesis can explain reassignments from stop to an amino acid, we found that it cannot explain reassignments from one amino acid to another. In particular--and contrary to the predictions of the mutation-drift hypothesis--the codon involved in such a reassignment was not rare in the ancestral genome. Instead, such reassignments appear to take place while the codon is in use at an appreciable frequency. Moreover, the comparison of inferred amino acid usage in the ancestral genome with the neutral expectation shows that the amino acid gaining the codon was selectively favored over the amino acid losing the codon. These results are consistent with a simple model of weak selection on the amino acid composition of proteins in which codon reassignments are selected because they compensate for multiple slightly deleterious mutations throughout the mitochondrial genome. We propose that the selection pressure is for reduced protein synthesis cost: most reassignments give amino acids that are less expensive to synthesize. Taken together, our results strongly suggest that mitochondrial genetic codes evolve to match the amino acid requirements of proteins.

Production of β-globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous β039 thalassemia patients

PubMed Central

Salvatori, Francesca; Breveglieri, Giulia; Zuccato, Cristina; Finotti, Alessia; Bianchi, Nicoletta; Borgatti, Monica; Feriotto, Giordana; Destro, Federica; Canella, Alessandro; Brognara, Eleonora; Lampronti, Ilaria; Breda, Laura; Rivella, Stefano; Gambari, Roberto

2013-01-01

In several types of thalassemia (including β039-thalassemia), stop codon mutations lead to premature translation termination and to mRNA destabilization through nonsense-mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal readthrough. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying β-thalassemia stop mutations have not yet been investigated. In this study, we have used a lentiviral construct containing the β039- thalassemia globin gene under control of the β-globin promoter and a LCR cassette. We demonstrated by fluorescence-activated cell sorting (FACS) analysis the production of β-globin by K562 cell clones expressing the β039-thalassemia globin gene and treated with G418. More importantly, after FACS and high-performance liquid chromatography (HPLC) analyses, erythroid precursor cells from β039-thalassemia patients were demonstrated to be able to produce β-globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal readthrough should be considered a strategy for developing experimental strategies for the treatment of β0-thalassemia caused by stop codon mutations. PMID:19810011

Analysis of four families with the Stickler syndrome by linkage studies. Identification of a new premature stop codon in the COL2A1 gene in a family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bonaventure, J.; Lasselin, C.; Toutain, A.

1994-09-01

The Stickler syndrome is an arthro-ophthalmopathy which associates progressive myopia with vitreal degeneration and retinal detachment. Cleft palate, cranio-facial abnormalities, deafness and osteoarthritis are often associated symptoms. Genetic heterogeneity of this autosomal dominant disease was consistent with its large clinical variability. Linkage studies have provided evidence for cosegregation of the disease with COL2A1, the gene coding for type II collagen, in about 50% of the families. Four additional families are reported here. Linkage analyses by using a VNTR located in the 3{prime} region of the gene were achieved. In three families, positive lod scores were obtained with a cumulative maximalmore » value of 3.5 at a recombination fraction of 0. In one of these families, single strand conformation analysis of 25 exons disclosed a new mutation in exon 42. Codon for glutamic acid at position a1-803 was converted into a stop codon. The mutation was detected in DNA samples from all the affected members of the family but not in the unaffected. This result confirms that most of the Stickler syndromes linked to COL2A1 are due to premature stop codons. In a second family, an abnormal SSCP pattern of exon 34 was detected in all the affected individuals. The mutation is likely to correspond to a splicing defect in the acceptor site of intron 33. In one family the disease did not segregate with the COL2A1 locus. Further linkage studies with intragenic dimorphic sites in the COL10A1 gene and highly polymorphic markers close to the COL9A1 locus indicated that this disorder did not result from defects in these two genes.« less

Modeling Treatment Response for Lamin A/C Related Dilated Cardiomyopathy in Human Induced Pluripotent Stem Cells.

PubMed

Lee, Yee-Ki; Lau, Yee-Man; Cai, Zhu-Jun; Lai, Wing-Hon; Wong, Lai-Yung; Tse, Hung-Fat; Ng, Kwong-Man; Siu, Chung-Wah

2017-07-28

Precision medicine is an emerging approach to disease treatment and prevention that takes into account individual variability in the environment, lifestyle, and genetic makeup of patients. Patient-specific human induced pluripotent stem cells hold promise to transform precision medicine into real-life clinical practice. Lamin A/C (LMNA)-related cardiomyopathy is the most common inherited cardiomyopathy in which a substantial proportion of mutations in the LMNA gene are of nonsense mutation. PTC124 induces translational read-through over the premature stop codon and restores production of the full-length proteins from the affected genes. In this study we generated human induced pluripotent stem cells-derived cardiomyocytes from patients who harbored different LMNA mutations (nonsense and frameshift) to evaluate the potential therapeutic effects of PTC124 in LMNA -related cardiomyopathy. We generated human induced pluripotent stem cells lines from 3 patients who carried distinctive mutations (R225X, Q354X, and T518fs) in the LMNA gene. The cardiomyocytes derived from these human induced pluripotent stem cells lines reproduced the pathophysiological hallmarks of LMNA -related cardiomyopathy. Interestingly, PTC124 treatment increased the production of full-length LMNA proteins in only the R225X mutant, not in other mutations. Functional evaluation experiments on the R225X mutant further demonstrated that PTC124 treatment not only reduced nuclear blebbing and electrical stress-induced apoptosis but also improved the excitation-contraction coupling of the affected cardiomyocytes. Using cardiomyocytes derived from human induced pluripotent stem cells carrying different LMNA mutations, we demonstrated that the effect of PTC124 is codon selective. A premature stop codon UGA appeared to be most responsive to PTC124 treatment. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Introduction of translation stop condons into the viral glycoprotein gene in a fish DNA vaccine eliminates induction of protective immunity

USGS Publications Warehouse

Garver, Kyle A.; Conway, Carla M.; Kurath, Gael

2006-01-01

A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was mutated to introduce two stop codons to prevent glycoprotein translation while maintaining the plasmid DNA integrity and RNA transcription ability. The mutated plasmid vaccine, denoted pIHNw-G2stop, when injected intramuscularly into fish at high doses, lacked detectable glycoprotein expression in the injection site muscle, and did not provide protection against lethal virus challenge 7 days post-vaccination. These results suggest that the G-protein itself is required to stimulate the early protective antiviral response observed after vaccination with the nonmutated parental DNA vaccine.

A novel mutation in the FGB: c.1105C>T turns the codon for amino acid Bβ Q339 into a stop codon causing hypofibrinogenemia.

PubMed

Marchi, Rita; Brennan, Stephen; Meyer, Michael; Rojas, Héctor; Kanzler, Daniela; De Agrela, Marisela; Ruiz-Saez, Arlette

2013-03-01

Routine coagulation tests on a 14year-old male with frequent epistaxis showed a prolonged thrombin time together with diminished functional (162mg/dl) and gravimetric (122mg/dl) fibrinogen concentrations. His father showed similar aberrant results and sequencing of the three fibrinogen genes revealed a novel heterozygous nonsense mutation in the FGB gene c.1105C>T, which converts the codon for residue Bβ 339Q to stop, causing deletion of Bβ chain residues 339-461. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and RP-HPLC (reverse-phase high-pressure liquid chromatography) of purified fibrinogen showed only normal Aα, Bβ, and γ chains, indicating that molecules with the truncated 37,990Da β chain were not secreted into plasma. Functional analysis showed impaired fibrin polymerization, fibrin porosity, and elasticity compared to controls. By laser scanning confocal microscopy the patient's fibers were slightly thinner than normal. Electrospray ionization mass spectrometry (ESI MS) presented normal sialylation of the oligosaccharide chains, and liver function tests showed no evidence of liver dysfunction that might explain the functional abnormalities. Copyright © 2012 Elsevier Inc. All rights reserved.

Congenital deficiency of alpha feto-protein.

PubMed

Sharony, Reuven; Zadik, Idit; Parvari, Ruti

2004-10-01

Alpha-fetoprotein (AFP) is the main fetus serum glycoprotein with a very low concentration in the adult. AFP deficiency is a rare phenomenon. We studied two families with congenital AFP deficiency and searched for mutations in the AFP gene. We identified one mutation of 2 base deletion in exon 8, in both families, that leads to the congenital deficiency of AFP. The mutation nt930-931delCT (T294fs25X) creates a frameshift after codon 294 that leads to a stop codon after 24 amino acids, thus truncating the normal length of AFP of 609 amino acids. All the affected children were found to be homozygous for the mutation as was one of the fathers. The affected individuals were asymptomatic and presented normal development. This first identification of a mutation in the AFP gene demonstrates for the first time that deficiency of AFP is compatible with human normal fetal development and further reproduction in males.

Mutations in the glucose-6-phosphatase gene that cause glycogen storage disease type 1a

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chou, J.Y.; Lei, K.J.; Shelly, L.L.

1994-09-01

Glycogen storage disease (GSD) type la (von Gierke disease) is caused by the deficiency of glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. The disease presents with clinical manifestations of severe hypoglycemia, hepatomegaly, growth retardation, lactic acidemia, hyperlipidemia, and hyperuricemia. We have succeeded in isolating a murine G6Pase cDNA from a normal mouse liver cDNA library by differentially screening method. We then isolated the human G6Pase cDNA and gene. To date, we have characterized the G6Pase genes of twelve GSD type la patients and uncovered a total of six different mutations. The mutations are comprised of R83C (an Arg atmore » codon 83 to a Cys), Q347X (a Gly at codon 347 to a stop codon), 459insTA (a two basepair insertion at nucleotide 459 yielding a truncated G6Pase of 129 residues), R295C (an Arg at codon 295 to a Cys), G222R (a Gly at codon 222 to an Arg) and {delta}F327 (a codon deletion for Phe-327 at nucleotides 1058 to 1060). The relative incidences of these mutations are 37.5% (R83C), 33.3% (Q347X), 16.6% (459insTA), 4.2% (G222R), 4.2% (R295C) and 4.2% ({delta}F327). Site-directed mutagenesis and transient expression assays demonstrated that the R83C, Q347X, R295C, and {delta}F327 mutations abolished whereas the G222R mutation greatly reduced G6Pase activity. We further characterized the structure-function requirements of amino acids 83, 222, and 295 in G6Pase catalysis. The identification of mutations in GSD type la patients has unequivocally established the molecular basis of the type la disorder. Knowledge of the mutations may be applied to prenatal diagnosis and opens the way for developing and evaluating new therapeutic approaches.« less

Characterization of six mutations in Exon 37 of neurofibromatosis type 1 gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Upadhyaya, M.; Osborn, M.; Maynard, J.

Neurofibromatosis type 1 (NF1) is one of the most common inherited disorders, with an incidence of 1 in 3,000. We screened a total of 320 unrelated NF1 patients for mutations in exon 37 of the NF1 gene. Six independent mutations were identified, of which three are novel, and these include a recurrent nonsense mutation identified in 2 unrelated patients at codon 2281 (G2281X), a 1-bp insertion (6791 ins A) resulting in a change of TAG (tyrosine) to a TAA (stop codon), and a 3-bp deletion (6839 del TAC) which generated a frameshift. Another recurrent nonsense mutation, Y2264X, which was detectedmore » in 2 unrelated patients in this study, was also previously reported in 2 NF1 individuals. All the mutations were identified within a contiguous 49-bp sequence. Further studies are warranted to support the notion that this region of the gene contains highly mutable sequences. 17 refs., 2 figs., 1 tab.« less

New mutations of DAX-1 genes in two Japanese patients with X-linked congenital adrenal hypoplasia and hypogonadotropic hypogonadism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yanase, Toshihiko; Takayanagi, Ryoichi; Oba, Koichi

Congenital adrenal hypoplasia, an X-linked disorder, is characterized by primary adrenal insufficiency and frequent association with hypogonadotropic hypogonadism. The X-chromosome gene DAX-1 has been most recently identified and shown to be responsible for this disorder. We analyzed the DAX-1 genes of two unrelated Japanese patients with congenital adrenal hypoplasia and hypogonadotropic hypogonadism by using PCR amplification of genomic DNA and its complete exonic sequencing. In a family containing several affected individuals, the proband male patient had a stop codon (TGA) in place of tryptophan (TGG) at amino acid position 171. As expected, his mother was a heterozygous carrier for themore » mutation, whereas his father and unaffected brother did not carry this mutation. In another male patient with noncontributory family history, sequencing revealed a 1-bp (T) deletion at amino acid position 280, leading to a frame shift and, subsequently a premature stop codon at amino acid position 371. The presence of this mutation in the patients` genome was further confirmed by digestion of genomic PCR product with MspI created by this mutation. Family studies using MspI digestion of genomic PCR products revealed that neither parent of this individual carried the mutation. These results clearly indicate that congenital adrenal hypoplasia and hypogonadotropic hypogonadism result from not only inherited but also de novo mutation in the DAX-1 gene. 31 refs., 4 figs., 2 tabs.« less

Culture adaptation of malaria parasites selects for convergent loss-of-function mutants.

PubMed

Claessens, Antoine; Affara, Muna; Assefa, Samuel A; Kwiatkowski, Dominic P; Conway, David J

2017-01-24

Cultured human pathogens may differ significantly from source populations. To investigate the genetic basis of laboratory adaptation in malaria parasites, clinical Plasmodium falciparum isolates were sampled from patients and cultured in vitro for up to three months. Genome sequence analysis was performed on multiple culture time point samples from six monoclonal isolates, and single nucleotide polymorphism (SNP) variants emerging over time were detected. Out of a total of five positively selected SNPs, four represented nonsense mutations resulting in stop codons, three of these in a single ApiAP2 transcription factor gene, and one in SRPK1. To survey further for nonsense mutants associated with culture, genome sequences of eleven long-term laboratory-adapted parasite strains were examined, revealing four independently acquired nonsense mutations in two other ApiAP2 genes, and five in Epac. No mutants of these genes exist in a large database of parasite sequences from uncultured clinical samples. This implicates putative master regulator genes in which multiple independent stop codon mutations have convergently led to culture adaptation, affecting most laboratory lines of P. falciparum. Understanding the adaptive processes should guide development of experimental models, which could include targeted gene disruption to adapt fastidious malaria parasite species to culture.

Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway

PubMed Central

Ge, Zhiyun; Quek, Bao Lin; Beemon, Karen L; Hogg, J Robert

2016-01-01

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing long 3'UTRs to perform dual roles in mRNA quality control and gene expression regulation. However, expansion of vertebrate 3'UTR functions has required a physical expansion of 3'UTR lengths, complicating the process of detecting nonsense mutations. We show that the polypyrimidine tract binding protein 1 (PTBP1) shields specific retroviral and cellular transcripts from NMD. When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs. PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression. Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD. DOI: http://dx.doi.org/10.7554/eLife.11155.001 PMID:26744779

Maple Syrup Urine Disease in Cypriot Families: Identification of Three Novel Mutations and Biochemical Characterization of the p.Thr211Met Mutation in the E1α Subunit

PubMed Central

Georgiou, Theodoros; Chuang, Jacinta L.; Wynn, R. Max; Stylianidou, Goula; Korson, Mark; Chuang, David T.

2009-01-01

We report five mutations, three of them novel, responsible for maple syrup urine disease in four unrelated Cypriot families. The five children studied are the first cases of classic maple syrup urine disease to be reported among Cypriots. The first novel mutation identified is a single-base deletion in exon 6 of the Elα gene (c.718delG), which leads to a frameshift after Ala240 and to a stop codon 89 residues further downstream. The other two novel mutations identified are in the Elβ subunit: a two-base deletion in exon 6, c.662_663delCC, which leads to a frameshift after Ala221 and creates a stop codon 17 residues further downstream, as well as a splice mutation, IVS3[+3]delA, which results in the skipping of exon 3. The two known mutations identified are in the Elα gene: the G > C transversion at the 3′-splice acceptor site, (IVS5-1G > C), which results in the deletion of the entire exon 6, and the missense mutation in exon 5 (c.632C > T), which corresponds to a p.Thr211Met substitution. The p.Thr211Met substitution is located in a potassium-ion pocket in the E1 component required for stability of the bound cofactor thiamine diphosphate. The mutant E1 protein harboring the p.Thr211Met substitution was shown unable to bind thiamine diphosphate, leading to undetectable E1 activity. PMID:19715473

Germline mutations of regulator of telomere elongation helicase 1, RTEL1, in Dyskeratosis congenita.

PubMed

Ballew, Bari J; Yeager, Meredith; Jacobs, Kevin; Giri, Neelam; Boland, Joseph; Burdett, Laurie; Alter, Blanche P; Savage, Sharon A

2013-04-01

Dyskeratosis congenita (DC) is an inherited bone marrow failure and cancer predisposition syndrome caused by aberrant telomere biology. The classic triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia is diagnostic of DC, but substantial clinical heterogeneity exists; the clinically severe variant Hoyeraal Hreidarsson syndrome (HH) also includes cerebellar hypoplasia, severe immunodeficiency, enteropathy, and intrauterine growth retardation. Germline mutations in telomere biology genes account for approximately one-half of known DC families. Using exome sequencing, we identified mutations in RTEL1, a helicase with critical telomeric functions, in two families with HH. In the first family, two siblings with HH and very short telomeres inherited a premature stop codon from their mother who has short telomeres. The proband from the second family has HH and inherited a premature stop codon in RTEL1 from his father and a missense mutation from his mother, who also has short telomeres. In addition, inheritance of only the missense mutation led to very short telomeres in the proband's brother. Targeted sequencing identified a different RTEL1 missense mutation in one additional DC proband who has bone marrow failure and short telomeres. Both missense mutations affect the helicase domain of RTEL1, and three in silico prediction algorithms suggest that they are likely deleterious. The nonsense mutations both cause truncation of the RTEL1 protein, resulting in loss of the PIP box; this may abrogate an important protein-protein interaction. These findings implicate a new telomere biology gene, RTEL1, in the etiology of DC.

Germline Mutations of Regulator of Telomere Elongation Helicase 1, RTEL1, In Dyskeratosis Congenita

PubMed Central

Ballew, Bari J.; Yeager, Meredith; Jacobs, Kevin; Giri, Neelam; Boland, Joseph; Burdett, Laurie; Alter, Blanche P.; Savage, Sharon A.

2013-01-01

Dyskeratosis congenita (DC) is an inherited bone marrow failure and cancer predisposition syndrome caused by aberrant telomere biology. The classic triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia is diagnostic of DC, but substantial clinical heterogeneity exists; the clinically severe variant Hoyeraal Hreidarsson syndrome (HH) also includes cerebellar hypoplasia, severe immunodeficiency, enteropathy, and intrauterine growth retardation. Germline mutations in telomere biology genes account for approximately one-half of known DC families. Using exome sequencing, we identified mutations in RTEL1, a helicase with critical telomeric functions, in two families with HH. In the first family, two siblings with HH and very short telomeres inherited a premature stop codon from their mother who has short telomeres. The proband from the second family has HH and inherited a premature stop codon in RTEL1 from his father and a missense mutation from his mother, who also has short telomeres. Additionally, inheritance of only the missense mutation led to very short telomeres in the proband’s brother. Targeted sequencing identified a different RTEL1 missense mutation in one additional DC proband who has bone marrow failure and short telomeres. Both missense mutations affect the helicase domain of RTEL1, and three in silico prediction algorithms suggest that they are likely deleterious. The nonsense mutations both cause truncation of the RTEL1 protein, resulting in loss of the PIP box; this may abrogate an important protein-protein interaction. These findings implicate a new telomere biology gene, RTEL1, in the etiology of DC. PMID:23329068

A novel alpha-thalassemia nonsense mutation in HBA2: C.382 A > T globin gene.

PubMed

Hamid, Mohammad; Bokharaei Merci, Hanieh; Galehdari, Hamid; Saberi, Ali Hossein; Kaikhaei, Bijan; Mohammadi-Anaei, Marziye; Ahmadzadeh, Ahmad; Shariati, Gholamreza

2014-07-01

In this study, a new alpha globin gene mutation on the α2-globin gene is reported. This mutation resulted in a Lys > stop codon substitution at position 127 which was detected in four individuals (three males and one female). DNA sequencing revealed this mutation in unrelated persons in Khuzestan province, Southwestern Iran of Lor ethnicity. This mutation caused no severe hematological abnormalities in the carriers. From the nature of substituted residues in α2-globin, it is widely expected that this mutation leads to unstable and truncated protein and should be detected in couples at risk for α-thalassemia.

Mutation discovery for Mendelian traits in non-laboratory animals: a review of achievements up to 2012

PubMed Central

Nicholas, Frank W; Hobbs, Matthew

2014-01-01

Within two years of the re-discovery of Mendelism, Bateson and Saunders had described six traits in non-laboratory animals (five in chickens and one in cattle) that show single-locus (Mendelian) inheritance. In the ensuing decades, much progress was made in documenting an ever-increasing number of such traits. In 1987 came the first discovery of a causal mutation for a Mendelian trait in non-laboratory animals: a non-sense mutation in the thyroglobulin gene (TG), causing familial goitre in cattle. In the years that followed, the rate of discovery of causal mutations increased, aided mightily by the creation of genome-wide microsatellite maps in the 1990s and even more mightily by genome assemblies and single-nucleotide polymorphism (SNP) chips in the 2000s. With sequencing costs decreasing rapidly, by 2012 causal mutations were being discovered in non-laboratory animals at a rate of more than one per week. By the end of 2012, the total number of Mendelian traits in non-laboratory animals with known causal mutations had reached 499, which was half the number of published single-locus (Mendelian) traits in those species. The distribution of types of mutations documented in non-laboratory animals is fairly similar to that in humans, with almost half being missense or non-sense mutations. The ratio of missense to non-sense mutations in non-laboratory animals to the end of 2012 was 193:78. The fraction of non-sense mutations (78/271 = 0.29) was not very different from the fraction of non-stop codons that are just one base substitution away from a stop codon (21/61 = 0.34). PMID:24372556

A novel mutation causing complete thyroxine-binding globulin deficiency (TBG-CD-Negev) among the Bedouins in southern Israel.

PubMed

Miura, Y; Hershkovitz, E; Inagaki, A; Parvari, R; Oiso, Y; Phillip, M

2000-10-01

T4-binding globulin (TBG) is the major thyroid hormone transport protein in human serum. Inherited TBG abnormalities do not usually alter the metabolic status and are transmitted in X-linked inheritance. A high prevalence of complete TBG deficiency (TBG-CD) has been reported among the Bedouin population in the Negev (southern Israel). In this study we report a novel single mutation causing complete TBG deficiency due to a deletion of the last base of codon 38 (exon 1), which led to a frame shift resulting in a premature stop at codon 51 and a presumed truncated peptide of 50 residues. This new variant of TBG (TBG-CD-Negev) was found among all of the patients studied. We conclude that a single mutation may account for TBG deficiency among the Bedouins in the Negev. This report is the first to describe a mutation in a population with an unusually high prevalence of TBG-CD.

Whole exome sequencing identifies a homozygous nonsense variation in ALMS1 gene in a patient with syndromic obesity.

PubMed

Das Bhowmik, Aneek; Gupta, Neerja; Dalal, Ashwin; Kabra, Madhulika

In the present study we report on genetic analysis in a patient with developmental delay, truncal obesity and vision problem, to find the causative mutation. Whole exome sequencing was performed on genomic DNA extracted from whole blood of the patient which revealed a homozygous nonsense variant (c.2816T>A) in exon 8 of ALMS1 gene that results in a stop codon and premature truncation at codon 939 (p.L939Ter) of the protein. The mutation was confirmed by Sanger sequencing. Exome sequencing was helpful in establishing diagnosis of Alstrom syndrome in this patient. This case highlights the utility of exome sequencing in clinical practice. Copyright © 2016 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.

Detection of a premature stop codon in the surface gene of hepatitis B virus from an HBsAg and antiHBc negative blood donor.

PubMed

Datta, Sibnarayan; Banerjee, Arup; Chandra, Partha K; Chakraborty, Subhasis; Basu, Subir Kumar; Chakravarty, Runu

2007-11-01

In blood donors, HBV infection is detected by the presence of serum hepatitis B surface antigen (HBsAg). However, some mutations in the surface gene region may result in altered or truncated HBsAg that can escape from immunoassay-based diagnosis. Such diagnostic escape mutants pose a potential risk for blood transfusion services. In the present study, we report a blood donor seronegative for HBsAg and antiHBc, but positive for antiHBs who was HBV DNA positive by PCR. Sequencing of the HBsAg gene revealed presence of a point mutation (T-A) at 207th nucleotide of the HBsAg ORF, which resulted in a premature stop codon at position 69. This results in a truncated HBsAg gene lacking the entire 'a' determinant region. However, follow-up of the donor after 2 years revealed clearance of HBV DNA from the serum. The case illustrates an unusual mutation, which causes HBsAg negativity. The finding emphasizes the importance of molecular assays in reducing the possibility of HBV transmission through blood transfusion. However, developing more sensitive serological assays, capable of detecting HBV mutants, is an alternative to expensive and complex amplification-based assays for developing countries.

Alignment-based and alignment-free methods converge with experimental data on amino acids coded by stop codons at split between nuclear and mitochondrial genetic codes.

PubMed

Seligmann, Hervé

2018-05-01

Genetic codes mainly evolve by reassigning punctuation codons, starts and stops. Previous analyses assuming that undefined amino acids translate stops showed greater divergence between nuclear and mitochondrial genetic codes. Here, three independent methods converge on which amino acids translated stops at split between nuclear and mitochondrial genetic codes: (a) alignment-free genetic code comparisons inserting different amino acids at stops; (b) alignment-based blast analyses of hypothetical peptides translated from non-coding mitochondrial sequences, inserting different amino acids at stops; (c) biases in amino acid insertions at stops in proteomic data. Hence short-term protein evolution models reconstruct long-term genetic code evolution. Mitochondria reassign stops to amino acids otherwise inserted at stops by codon-anticodon mismatches (near-cognate tRNAs). Hence dual function (translation termination and translation by codon-anticodon mismatch) precedes mitochondrial reassignments of stops to amino acids. Stop ambiguity increases coded information, compensates endocellular mitogenome reduction. Mitochondrial codon reassignments might prevent viral infections. Copyright © 2018 Elsevier B.V. All rights reserved.

Novel SIL1 nonstop mutation in a Chinese consanguineous family with Marinesco-Sjögren syndrome and Dandy-Walker syndrome.

PubMed

Gai, Nan; Jiang, Chen; Zou, Yong-Yi; Zheng, Yu; Liang, De-Sheng; Wu, Ling-Qian

2016-07-01

Marinesco-Sjögren syndrome (MSS) is a rare autosomal recessive disorder, which is characterized by congenital cataracts, cerebellar ataxia, progressive muscle weakness, and delayed psychomotor development. SIL1, which is located at 5q31.2, is the only gene known to cause MSS. Dandy-Walker syndrome (DWS) is defined by hypoplasia, upward rotation of the cerebellar vermis, and cystic dilation of the fourth ventricle; however, its genetic pathogeny remains unclear. Here, we report a Chinese consanguineous family with MSS and DWS. Whole exome sequencing identified a novel nonstop mutation in SIL1. Sanger sequencing revealed that the mutation was segregated in this family according to a recessive mode of inheritance. We found that the mutation changed a stop codon (TGA) to an arginine codon (CGA), and no in-frame termination codon in the 3' untranslated region (UTR) of SIL1 could be found. The mRNA levels of SIL1 were decreased by 56.6% and 37.5% in immortalized lymphoblasts of the patients respectively; the protein levels of SIL1 were substantially decreased. This case study is the first report on Chinese MSS patients, MSS complicated by DWS, and a nonstop mutation in SIL1. Our findings imply the pathogenetic association between DWS and MSS. Copyright © 2016 Elsevier B.V. All rights reserved.

Novel APC gene mutations associated with protein alteration in diffuse type gastric cancer.

PubMed

Ghatak, Souvik; Chakraborty, Payel; Sarkar, Sandeep Roy; Chowdhury, Biswajit; Bhaumik, Arup; Kumar, Nachimuthu Senthil

2017-06-02

The role of adenomatous polyposis coli (APC) gene in mitosis might be critical for regulation of genomic stability and chromosome segregation. APC gene mutations have been associated to have a role in colon cancer and since gastric and colon tumors share some common genetic lesions, it is relevant to investigate the role of APC tumor suppressor gene in gastric cancer. We investigated for somatic mutations in the Exons 14 and 15 of APC gene from 40 diffuse type gastric cancersamples. Rabbit polyclonal anti-APC antibody was used, which detects the wild-type APC protein and was recommended for detection of the respective protein in human tissues. Cell cycle analysis was done from tumor and adjacent normal tissue. APC immunoreactivity showed positive expression of the protein in stages I, II, III and negative expression in Stages III and IV. Two novel deleterious variations (g.127576C > A, g.127583C > T) in exon 14 sequence were found to generate stop codon (Y622* and Q625*)in the tumor samples. Due to the generation of stop codon, the APC protein might be truncated and all the regulatory features could be lost which has led to the down-regulation of protein expression. Our results indicate that aneuploidy might occurdue to the codon 622 and 625 APC-driven gastric tumorigenesis, in agreement with our cell cycle analysis. The APC gene function in mitosis and chromosomal stability might be lost and G1 might be arrested with high quantity of DNA in the S phase. Six missense somatic mutations in tumor samples were detected in exon 15 A-B, twoof which showed pathological and disease causing effects based on SIFT, Polyphen2 and SNPs & GO score and were not previously reported in the literature or the public mutation databases. The two novel pathological somatic mutations (g.127576C > A, g.127583C > T) in exon 14 might be altering the protein expression leading to development of gastric cancer in the study population. Our study showed that mutations in the APC gene alter the protein expression and cell cycle regulation in diffuse type gastric adenocarcinoma.

Stop codon readthrough generates a C-terminally extended variant of the human vitamin D receptor with reduced calcitriol response

PubMed Central

Loughran, Gary; Jungreis, Irwin; Tzani, Ioanna; Power, Michael; Dmitriev, Ruslan I.; Ivanov, Ivaylo P.; Kellis, Manolis; Atkins, John F.

2018-01-01

Although stop codon readthrough is used extensively by viruses to expand their gene expression, verified instances of mammalian readthrough have only recently been uncovered by systems biology and comparative genomics approaches. Previously, our analysis of conserved protein coding signatures that extend beyond annotated stop codons predicted stop codon readthrough of several mammalian genes, all of which have been validated experimentally. Four mRNAs display highly efficient stop codon readthrough, and these mRNAs have a UGA stop codon immediately followed by CUAG (UGA_CUAG) that is conserved throughout vertebrates. Extending on the identification of this readthrough motif, we here investigated stop codon readthrough, using tissue culture reporter assays, for all previously untested human genes containing UGA_CUAG. The readthrough efficiency of the annotated stop codon for the sequence encoding vitamin D receptor (VDR) was 6.7%. It was the highest of those tested but all showed notable levels of readthrough. The VDR is a member of the nuclear receptor superfamily of ligand-inducible transcription factors, and it binds its major ligand, calcitriol, via its C-terminal ligand-binding domain. Readthrough of the annotated VDR mRNA results in a 67 amino acid–long C-terminal extension that generates a VDR proteoform named VDRx. VDRx may form homodimers and heterodimers with VDR but, compared with VDR, VDRx displayed a reduced transcriptional response to calcitriol even in the presence of its partner retinoid X receptor. PMID:29386352

Stop codons in the hepatitis B surface proteins are enriched during antiviral therapy and are associated with host cell apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Colledge, Danielle; Soppe, Sally; Yuen, Lilly

Premature stop codons in the hepatitis B virus (HBV) surface protein can be associated with nucleos(t)ide analogue resistance due to overlap of the HBV surface and polymerase genes. The aim of this study was to determine the effect of the replication of three common surface stop codon variants on the hepatocyte. Cell lines were transfected with infectious HBV clones encoding surface stop codons rtM204I/sW196*, rtA181T/sW172*, rtV191I/sW182*, and a panel of substitutions in the surface proteins. HBsAg was measured by Western blotting. Proliferation and apoptosis were measured using flow cytometry. All three surface stop codon variants were defective in HBsAg secretion.more » Cells transfected with these variants were less proliferative and had higher levels of apoptosis than those transfected with variants that did not encode surface stop codons. The most cytopathic variant was rtM204I/sW196*. Replication of HBV encoding surface stop codons was toxic to the cell and promoted apoptosis, exacerbating disease progression. - Highlights: •Under normal circumstances, HBV replication is not cytopathic. •Premature stop codons in the HBV surface protein can be selected and enriched during nucleos(t)ide analogue therapy. •Replication of these variants can be cytopathic to the cell and promote apoptosis. •Inadequate antiviral therapy may actually promote disease progression.« less

A protocol for isolating insect mitochondrial genomes: a case study of NUMT in Melipona flavolineata (Hymenoptera: Apidae).

PubMed

Françoso, Elaine; Gomes, Fernando; Arias, Maria Cristina

2016-07-01

Nuclear mitochondrial DNA insertions (NUMTs) are mitochondrial DNA sequences that have been transferred into the nucleus and are recognized by the presence of indels and stop codons. Although NUMTs have been identified in a diverse range of species, their discovery was frequently accidental. Here, our initial goal was to develop and standardize a simple method for isolating NUMTs from the nuclear genome of a single bee. Subsequently, we tested our new protocol by determining whether the indels and stop codons of the cytochrome c oxidase subunit I (COI) sequence of Melipona flavolineata are of nuclear origin. The new protocol successfully demonstrated the presence of a COI NUMT. In addition to NUMT investigations, the protocol described here will also be very useful for studying mitochondrial mutations related to diseases and for sequencing complete mitochondrial genomes with high read coverage by Next-Generation technology.

Thiamine-responsive megaloblastic anemia: early diagnosis may be effective in preventing deafness.

PubMed

Onal, Hasan; Bariş, Safa; Ozdil, Mine; Yeşil, Gözde; Altun, Gürkan; Ozyilmaz, Isa; Aydin, Ahmet; Celkan, Tiraje

2009-01-01

Thiamine-responsive megaloblastic anemia syndrome is an autosomal recessive disorder characterized by diabetes mellitus, megaloblastic anemia and sensorineural hearing loss. Mutations in the SLC19A2 gene, encoding a high-affinity thiamine transporter protein, THTR-1, are responsible for the clinical features associated with thiamine-responsive megaloblastic anemia syndrome in which treatment with pharmacological doses of thiamine correct the megaloblastic anemia and diabetes mellitus. The anemia can recur when thiamine is withdrawn. Thiamine may be effective in preventing deafness if started before two months. Our patient was found homozygous for a mutation, 242insA, in the nucleic acid sequence of exon B, with insertion of an adenine introducing a stop codon at codon 52 in the high-affinity thiamine transporter gene, SLC19A2, on chromosome 1q23.3.

Novel Single-Base Deletional Mutation in Major Intrinsic Protein (MIP) in Autosomal Dominant Cataract

PubMed Central

Geyer, David D.; Spence, M. Anne; Johannes, Meriam; Flodman, Pamela; Clancy, Kevin P.; Berry, Rebecca; Sparkes, Robert S.; Jonsen, Matthew D.; Isenberg, Sherwin J.; Bateman, J. Bronwyn

2006-01-01

PURPOSE To further elucidate the cataract phenotype, and identify the gene and mutation for autosomal dominant cataract (ADC) in an American family of European descent (ADC2) by sequencing the major intrinsic protein gene (MIP), a candidate based on linkage to chromosome 12q13. DESIGN Observational case series and laboratory experimental study. METHODS We examined two at-risk individuals in ADC2. We PCR-amplified and sequenced all four exons and all intron-exon boundaries of the MIP gene from genomic and cloned DNA in affected members to confirm one variant as the putative mutation. RESULTS We found a novel single deletion of nucleotide (nt) 3223 (within codon 235) in exon four, causing a frameshift that alters 41 of 45 subsequent amino acids and creates a premature stop codon. CONCLUSIONS We identified a novel single base pair deletion in the MIP gene and conclude that it is a pathogenic sequence alteration. PMID:16564824

GATA3 mutation in a family with hypoparathyroidism, deafness and renal dysplasia syndrome.

PubMed

Zhu, Zi-Yang; Zhou, Qiao-Li; Ni, Shi-Ning; Gu, Wei

2014-08-01

The hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome is an autosomal dominant disorder primarily caused by GATA3 gene mutation. We report here a case that both of a Chinese boy and his father had HDR syndrome which caused by a novel mutation of GATA3. Polymerase chain reaction and DNA sequencing was performed to detect the exons of the GATA3 gene for mutation analysis. Sequence analysis of GATA3 revealed a heterozygous nonsense mutation in this family: a mutation of GATA3 at exon 2 (c.515C >A) that resulted in a premature stop at codon 172 (p.S172X) with a loss of two zinc finger domains. We identified a novel nonsense mutation which will expand the spectrum of HDR-associated GATA3 mutations.

Identification of a novel mutation in a patient with pseudohypoparathyroidism type Ia

PubMed Central

Lee, Ye Seung; Kim, Hui Kwon; Kim, Hye Rim; Lee, Jong Yoon; Choi, Joong Wan; Bae, Eun Ju; Oh, Phil Soo; Park, Won Il; Ki, Chang Seok

2014-01-01

Pseudohypoparathyroidism type Ia (PHP Ia) is a disorder characterized by multiform hormonal resistance including parathyroid hormone (PTH) resistance and Albright hereditary osteodystrophy (AHO). It is caused by heterozygous inactivating mutations within the Gs alpha-encoding GNAS exons. A 9-year-old boy presented with clinical and laboratory abnormalities including hypocalcemia, hyperphosphatemia, PTH resistance, multihormone resistance and AHO (round face, short stature, obesity, brachydactyly and osteoma cutis) which were typical of PHP Ia. He had a history of repeated convulsive episodes that started from the age of 2 months. A cranial computed tomography scan showed bilateral calcifications in the basal ganglia and his intelligence quotient testing indicated mild mental retardation. Family history revealed that the patient's maternal relatives, including his grandmother and 2 of his mother's siblings, had features suggestive of AHO. Sequencing of the GNAS gene of the patient identified a heterozygous nonsense mutation within exon 11 (c.637 C>T). The C>T transversion results in an amino acid substitution from Gln to stop codon at codon 213 (p.Gln213*). To our knowledge, this is a novel mutation in GNAS. PMID:25045367

Truncated variants of apolipoprotein B cause hypobetalipoproteinaemia.

PubMed Central

Collins, D R; Knott, T J; Pease, R J; Powell, L M; Wallis, S C; Robertson, S; Pullinger, C R; Milne, R W; Marcel, Y L; Humphries, S E

1988-01-01

Familial hypobetalipoproteinaemia is a rare autosomal dominant disorder in which levels of apo-B-containing plasma lipoproteins are approximately half-normal in heterozygotes and virtually absent in homozygotes. Here we describe mutations of the apo-B gene that cause two different truncated variants of apo-B in unrelated individuals with hypobetalipoproteinaemia. One variant, apo-B(His1795----Met-Trp-Leu-Val-Thr-Term) is predicted to be 1799 amino acids long and arises from deletion of a single nucleotide (G) from leucine codon 1794. This protein was found at low levels in very low density and low density lipoprotein fractions in the blood. The second, shorter variant, apo-B(Arg1306----Term), is caused by mutation of a CpG dinucleotide in arginine codon 1306 converting it to a stop codon and predicting a protein of 1305 residues. The product of this allele could not be detected in the circulation. The differences in size and behaviour of these two variants compared to apo-B100 or apo-B48 point to domains that may be important for the assembly, secretion or stability of apo-B-containing lipoproteins. Images PMID:2843815

Ocular phenotypes associated with two mutations (R121W, C126X) in the Norrie disease gene.

PubMed

Kellner, U; Fuchs, S; Bornfeld, N; Foerster, M H; Gal, A

1996-06-01

To describe the ocular phenotypes associated with 2 mutations in the Norrie disease gene including a manifesting carrier. Ophthalmological examinations were performed in 2 affected males and one manifesting carrier. Genomic DNA was analyzed by direct sequencing of the Norrie disease gene. Family I: A 29-year-old male had the right eye enucleated at the age of 3 years. His left eye showed severe temporal dragging of the retina and central scars. Visual acuity was 20/300. DNA analysis revealed a C-to-T transition of the first nucleotide in codon 121 predicting the replacement of arginine-121 by tryptophan (R121W). Both the mother and maternal grandmother carry the same mutation in heterozygous form. Family 2: A 3-month-old boy presented with severe temporal dragging of the retina on both eyes and subsequently developed retinal detachment. Visual acuity was limited to light perception. His mother's left eye was amaurotic and phthitic. Her right eye showed severe retinal dragging, visual acuity was reduced to 20/60. DNA analysis revealed a T-to-A transversion of the third nucleotide in codon 126 creating a stop codon (C126X). The mother and maternal grandmother were carriers. Mutations in the Norrie disease gene can lead to retinal malformations of variable severity both in hemizygous males and manifesting carriers.

Peeling skin syndrome: genetic defects in late terminal differentiation of the epidermis.

PubMed

Bowden, Paul E

2011-03-01

In this issue, Israeli and colleagues confirm that homozygous mutations in corneodesmosin (CDSN) cause type B peeling skin syndrome (PSS), an autosomal recessive skin disorder. The deletion mutation described resulted in a frameshift, producing a downstream premature stop codon and early truncation of the protein. The recently described CDSN nonsense mutation in another PSS family also resulted in protein truncation and nonsense-mediated mRNA decay. Type B generalized PSS can now be clearly distinguished from acral PSS, caused by mutations in transglutaminase 5. This directly affects cornified envelope cross-linking rather than corneodesmosome adherence. These observations provide new insight into the molecular defects underlying two closely related forms of PSS.

A novel two-nucleotide deletion in the ATP7A gene associated with delayed infantile onset of Menkes disease.

PubMed

Wada, Takahito; Haddad, Marie Reine; Yi, Ling; Murakami, Tomomi; Sasaki, Akiko; Shimbo, Hiroko; Kodama, Hiroko; Osaka, Hitoshi; Kaler, Stephen G

2014-04-01

Determining the relationship between clinical phenotype and genotype in genetic diseases is important in clinical practice. In general, frameshift mutations are expected to produce premature termination codons, leading to production of mutant transcripts destined for degradation by nonsense-mediated decay. In X-linked recessive diseases, male patients with frameshift mutations typically have a severe or even lethal phenotype. We report a case of a 17-month-old boy with Menkes disease (NIM #309400), an X-linked recessive copper metabolism disorder caused by mutations in the ATP7A copper transporter gene. He exhibited an unexpectedly late onset and experienced milder symptoms. His genomic DNA showed a de novo two-nucleotide deletion in exon 4 of ATP7A, predicting a translational frameshift and premature stop codon, and a classic severe phenotype. Characterization of his ATP7A mRNA showed no abnormal splicing. We speculate that translation reinitiation could occur downstream to the premature termination codon and produce a partially functional ATP7A protein. Study of the child's fibroblasts found no evidence of translation reinitiation; however, the possibility remains that this phenomenon occurred in neural tissues and influenced the clinical phenotype. Copyright © 2014 Elsevier Inc. All rights reserved.

Reassigning stop codons via translation termination: How a few eukaryotes broke the dogma.

PubMed

Alkalaeva, Elena; Mikhailova, Tatiana

2017-03-01

The genetic code determines how amino acids are encoded within mRNA. It is universal among the vast majority of organisms, although several exceptions are known. Variant genetic codes are found in ciliates, mitochondria, and numerous other organisms. All revealed genetic codes (standard and variant) have at least one codon encoding a translation stop signal. However, recently two new genetic codes with a reassignment of all three stop codons were revealed in studies examining the protozoa transcriptomes. Here, we discuss this finding and the recent studies of variant genetic codes in eukaryotes. We consider the possible molecular mechanisms allowing the use of certain codons as sense and stop signals simultaneously. The results obtained by studying these amazing organisms represent a new and exciting insight into the mechanism of stop codon decoding in eukaryotes. Also see the video abstract here. © 2017 WILEY Periodicals, Inc.

Novel mutation of OCRL1 in Lowe syndrome.

PubMed

Liu, Ting; Yue, Zhihui; Wang, Haiyan; Tong, Huajuan; Sun, Liangzhong

2015-01-01

Lowe syndrome is a rare, X-linked recessive genetic disease with multi-organ involvement. The pathogenic gene is OCRL1. The authors analyzed the OCRL1 mutation and summarized the clinical features of a Chinese child with Lowe syndrome. The patient is a 3 year 7 mo-old boy. He presented with hypotonia at birth and gradually presented with bilateral congenital cataracts, psychomotor retardation, hypophosphatemic rickets and renal tubular function disorder. Sequence analysis of OCRL1 revealed a novel insertion mutation, c.2367insA (p. Ala813X), in exon 22. This mutation was suspected to cause a premature stop codon of OCRL1 and truncation of the OCRL1 protein. His mother, who carried a heterozygous mutation, had no sign of abnormality.

Compound heterozygous HAX1 mutations in a Swedish patient with severe congenital neutropenia and no neurodevelopmental abnormalities.

PubMed

Carlsson, Göran; Elinder, Göran; Malmgren, Helena; Trebinska, Alicja; Grzybowska, Ewa; Dahl, Niklas; Nordenskjöld, Magnus; Fadeel, Bengt

2009-12-01

Kostmann disease or severe congenital neutropenia (SCN) is an autosomal recessive disorder of neutrophil production. Homozygous HAX1 mutations were recently identified in SCN patients belonging to the original family in northern Sweden described by Kostmann. Moreover, recent studies have suggested an association between neurological dysfunction and HAX1 deficiency. Here we describe a patient with a compound heterozygous HAX1 mutation consisting of a nonsense mutation (c.568C > T, p.Glu190X) and a frame-shift mutation (c.91delG, p.Glu31LysfsX54) resulting in a premature stop codon. The patient has a history of neutropenia and a propensity for infections, but has shown no signs of neurodevelopmental abnormalities.

A Novel Loss-of-Sclerostin Function Mutation in a First Egyptian Family with Sclerosteosis

PubMed Central

Fayez, Alaaeldin; Aglan, Mona; Esmaiel, Nora; El Zanaty, Taher; Abdel Kader, Mohamed; El Ruby, Mona

2015-01-01

Sclerosteosis is a rare autosomal recessive condition characterized by increased bone density. Mutations in SOST gene coding for sclerostin are linked to sclerosteosis. Two Egyptian brothers with sclerosteosis and their apparently normal consanguineous parents were included in this study. Clinical evaluation and genomic sequencing of the SOST gene were performed followed by in silico analysis of the resulting variation. A novel homozygous frameshift mutation in the SOST gene, characterized as one nucleotide cytosine insertion that led to premature stop codon and loss of functional sclerostin, was identified in the two affected brothers. Their parents were heterozygous for the same mutation. To our knowledge this is the first Egyptian study of sclerosteosis and SOST gene causing mutation. PMID:25984533

Macular corneal dystrophy in a Chinese family related with novel mutations of CHST6

PubMed Central

Dang, Xiuhong; Zhu, Qingguo; Wang, Li; Su, Hong; Lin, Hui; Zhou, Nan; Liang, Ting; Wang, Zheng; Huang, Shangzhi; Ren, Qiushi

2009-01-01

Purpose To identify mutations in the carbohydrate sulfotransferase gene (CHST6) for a Chinese family with macular corneal dystrophy (MCD) and to investigate the histopathological changes in the affected cornea. Methods A corneal button of the proband was obtained by penetrating keratoplasty. The half button and ultrathin sections from the other half button were examined with special stains under a light microscope (LM) and an electron microscope (EM) separately. Genomic DNA was extracted from peripheral blood of 11 family members, and the coding region of CHST6 was amplified by the polymerase chain reaction (PCR) method. The PCR products were analyzed by direct sequencing and restriction enzyme digestion. Results The positive reaction to colloidal iron stain (extracellular blue accumulations in the stroma) was detected under light microscopy. Transmission electron microscopy revealed the enlargement of smooth endoplasmic reticulum and the presence of intracytoplasmic vacuoles. The compound heterozygous mutations, c.892C>T and c.1072T>C, were identified in exon 3 of CHST6 in three patients. The two transversions resulted in the substitution of a stop codon for glutamine at codon 298 (p.Q298X) and a missense mutation at codon 358, tyrosine to histidine (p.Y358H). The six unaffected family individuals carried alternative heterozygous mutations. These two mutations were not detected in any of the 100 control subjects. Conclusions Those novel compound heterozygous mutations were thought to contribute to the loss of CHST6 function, which induced the abnormal metabolism of keratan sulfate (KS) that deposited in the corneal stroma. It could be proved by the observation of a positive stain reaction and the enlarged collagen fibers as well as hyperplastic fibroblasts under microscopes. PMID:19365571

A new nonsense mutation in the NF1 gene with neurofibromatosis-Noonan syndrome phenotype.

PubMed

Yimenicioğlu, Sevgi; Yakut, Ayten; Karaer, Kadri; Zenker, Martin; Ekici, Arzu; Carman, Kürşat Bora

2012-12-01

Neurofibromatosis-Noonan syndrome is a rare autosomal dominant disorder which combines neurofibromatosis type 1 (NF1) features with Noonan syndrome. NF1 gene mutations are reported in the majority of these patients. Sequence analysis of the established genes for Noonan syndrome revealed no mutation; a heterozygous NF1 point mutation c.7549C>T in exon 51, creating a premature stop codon (p.R2517X), had been demonstrated. Neurofibromatosis-Noonan syndrome recently has been considered a subtype of NF1 and caused by different NF1 mutations. We report the case of a 14-year-old boy with neurofibromatosis type 1 with Noonan-like features, who complained of headache with triventricular hydrocephaly and a heterozygous NF1 point mutation c.7549C>T in exon 51.

Analyses of clinicopathological, molecular, and prognostic associations of KRAS codon 61 and codon 146 mutations in colorectal cancer: cohort study and literature review

PubMed Central

2014-01-01

Background KRAS mutations in codons 12 and 13 are established predictive biomarkers for anti-EGFR therapy in colorectal cancer. Previous studies suggest that KRAS codon 61 and 146 mutations may also predict resistance to anti-EGFR therapy in colorectal cancer. However, clinicopathological, molecular, and prognostic features of colorectal carcinoma with KRAS codon 61 or 146 mutation remain unclear. Methods We utilized a molecular pathological epidemiology database of 1267 colon and rectal cancers in the Nurse’s Health Study and the Health Professionals Follow-up Study. We examined KRAS mutations in codons 12, 13, 61 and 146 (assessed by pyrosequencing), in relation to clinicopathological features, and tumor molecular markers, including BRAF and PIK3CA mutations, CpG island methylator phenotype (CIMP), LINE-1 methylation, and microsatellite instability (MSI). Survival analyses were performed in 1067 BRAF-wild-type cancers to avoid confounding by BRAF mutation. Cox proportional hazards models were used to compute mortality hazard ratio, adjusting for potential confounders, including disease stage, PIK3CA mutation, CIMP, LINE-1 hypomethylation, and MSI. Results KRAS codon 61 mutations were detected in 19 cases (1.5%), and codon 146 mutations in 40 cases (3.2%). Overall KRAS mutation prevalence in colorectal cancers was 40% (=505/1267). Of interest, compared to KRAS-wild-type, overall, KRAS-mutated cancers more frequently exhibited cecal location (24% vs. 12% in KRAS-wild-type; P < 0.0001), CIMP-low (49% vs. 32% in KRAS-wild-type; P < 0.0001), and PIK3CA mutations (24% vs. 11% in KRAS-wild-type; P < 0.0001). These trends were evident irrespective of mutated codon, though statistical power was limited for codon 61 mutants. Neither KRAS codon 61 nor codon 146 mutation was significantly associated with clinical outcome or prognosis in univariate or multivariate analysis [colorectal cancer-specific mortality hazard ratio (HR) = 0.81, 95% confidence interval (CI) = 0.29-2.26 for codon 61 mutation; colorectal cancer-specific mortality HR = 0.86, 95% CI = 0.42-1.78 for codon 146 mutation]. Conclusions Tumors with KRAS mutations in codons 61 and 146 account for an appreciable proportion (approximately 5%) of colorectal cancers, and their clinicopathological and molecular features appear generally similar to KRAS codon 12 or 13 mutated cancers. To further assess clinical utility of KRAS codon 61 and 146 testing, large-scale trials are warranted. PMID:24885062

Identification of novel mutations of the CHST6 gene in Vietnamese families affected with macular corneal dystrophy in two generations.

PubMed

Ha, Nguyen Thanh; Chau, Hoang Minh; Cung, Le Xuan; Thanh, Ton Kim; Fujiki, Keiko; Murakami, Akira; Hiratsuka, Yoshimune; Hasegawa, Nobuko; Kanai, Atsushi

2003-08-01

To report the clinical and genetic findings of Vietnamese families affected with macular corneal dystrophy (MCD) in 2 generations. Two families, including 7 patients and 3 unaffected members, were examined clinically. Blood samples were collected. Fifty normal Vietnamese individuals were used as controls. Genomic DNA was extracted from leukocytes. Analysis of the carbohydrate sulfotransferase (CHST6) gene was performed using polymerase chain reaction and direct sequencing. The typical form of MCD was recognized in family B, in which sequencing of CHST6 gene revealed an nt 1067-1068ins(GGCCGTG) mutation (frameshift after 125V) homozygously in MCD patients and heterozygously in the unaffected members. Family N also showed clinical features of MCD, moderate in the mother but severe in the affected son. Sequencing revealed a single heterozygous Arg211Gln in the mother, compound heterozygous Arg211Gln+ Gln82Stop in the affected son, and heterozygous Arg211Gln mutation in the unaffected members. The identified mutations in these pedigrees were excluded from normal controls. The novel frameshift and compound heterozygous mutations might be responsible for MCD in the families studied. The phenotypic variation between affected parents and offspring was unclear. In family N, severe MCD phenotype seen in the affected son may be due the fact that he had an early stop codon mutation (Gln82Stop).

Identification of a novel mutation in a Chinese family with Nance-Horan syndrome by whole exome sequencing.

PubMed

Hong, Nan; Chen, Yan-hua; Xie, Chen; Xu, Bai-sheng; Huang, Hui; Li, Xin; Yang, Yue-qing; Huang, Ying-ping; Deng, Jian-lian; Qi, Ming; Gu, Yang-shun

2014-08-01

Nance-Horan syndrome (NHS) is a rare X-linked disorder characterized by congenital nuclear cataracts, dental anomalies, and craniofacial dysmorphisms. Mental retardation was present in about 30% of the reported cases. The purpose of this study was to investigate the genetic and clinical features of NHS in a Chinese family. Whole exome sequencing analysis was performed on DNA from an affected male to scan for candidate mutations on the X-chromosome. Sanger sequencing was used to verify these candidate mutations in the whole family. Clinical and ophthalmological examinations were performed on all members of the family. A combination of exome sequencing and Sanger sequencing revealed a nonsense mutation c.322G>T (E108X) in exon 1 of NHS gene, co-segregating with the disease in the family. The nonsense mutation led to the conversion of glutamic acid to a stop codon (E108X), resulting in truncation of the NHS protein. Multiple sequence alignments showed that codon 108, where the mutation (c.322G>T) occurred, was located within a phylogenetically conserved region. The clinical features in all affected males and female carriers are described in detail. We report a nonsense mutation c.322G>T (E108X) in a Chinese family with NHS. Our findings broaden the spectrum of NHS mutations and provide molecular insight into future NHS clinical genetic diagnosis.

Primary hyperoxaluria type 1: a cluster of new mutations in exon 7 of the AGXT gene.

PubMed

von Schnakenburg, C; Rumsby, G

1997-06-01

Primary hyperoxaluria type 1 (PH1) is a severe autosomal recessive inborn error of glyoxylate metabolism caused by deficiency of the hepatic peroxisomal enzyme alanine:glyoxylate aminotransferase. This enzyme is encoded by the AGXT gene on chromosome 2q37.3. DNA samples from 79 PH1 patients were studied using single strand conformation polymorphism analysis to detect sequence variants, which were then characterised by direct sequencing and confirmed by restriction enzyme digestion. Four novel mutations were identified in exon 7 of AGXT: a point mutation T853C, which leads to a predicted Ile244Thr amino acid substitution, occurred in nine patients. Two other mutations in adjacent nucleotides, C819T and G820A, mutated the same codon at residue 233 from arginine to cysteine and histidine, respectively. The fourth mutation, G860A, introduced a stop codon at amino acid residue 246. Enzyme studies in these patients showed that AGT catalytic activity was either very low or absent and that little or no immunoreactive protein was present. Together with a new polymorphism in exon 11 (C1342A) these findings underline the genetic heterogeneity of the AGXT gene. The novel mutation T853C is the second most common mutation found to date with an allelic frequency of 9% and will therefore be of clinical importance for the diagnosis of PH1.

Primary hyperoxaluria type 1: a cluster of new mutations in exon 7 of the AGXT gene.

PubMed Central

von Schnakenburg, C; Rumsby, G

1997-01-01

Primary hyperoxaluria type 1 (PH1) is a severe autosomal recessive inborn error of glyoxylate metabolism caused by deficiency of the hepatic peroxisomal enzyme alanine:glyoxylate aminotransferase. This enzyme is encoded by the AGXT gene on chromosome 2q37.3. DNA samples from 79 PH1 patients were studied using single strand conformation polymorphism analysis to detect sequence variants, which were then characterised by direct sequencing and confirmed by restriction enzyme digestion. Four novel mutations were identified in exon 7 of AGXT: a point mutation T853C, which leads to a predicted Ile244Thr amino acid substitution, occurred in nine patients. Two other mutations in adjacent nucleotides, C819T and G820A, mutated the same codon at residue 233 from arginine to cysteine and histidine, respectively. The fourth mutation, G860A, introduced a stop codon at amino acid residue 246. Enzyme studies in these patients showed that AGT catalytic activity was either very low or absent and that little or no immunoreactive protein was present. Together with a new polymorphism in exon 11 (C1342A) these findings underline the genetic heterogeneity of the AGXT gene. The novel mutation T853C is the second most common mutation found to date with an allelic frequency of 9% and will therefore be of clinical importance for the diagnosis of PH1. Images PMID:9192270

Nonsense mutation in the phosphofructokinase muscle subunit gene associated with retention of intron 10 in one of the isolated transcripts in Ashkenazi Jewish patients with Tarui disease.

PubMed Central

Vasconcelos, O; Sivakumar, K; Dalakas, M C; Quezado, M; Nagle, J; Leon-Monzon, M; Dubnick, M; Gajdusek, D C; Goldfarb, L G

1995-01-01

Mutations in the human phosphofructokinase muscle subunit gene (PFKM) are known to cause myopathy classified as glycogenosis type VII (Tarui disease). Previously described molecular defects include base substitutions altering encoded amino acids or resulting in abnormal splicing. We report a mutation resulting in phosphofructokinase deficiency in three patients from an Ashkenazi Jewish family. Using a reverse transcription PCR assay, PFKM subunit transcripts differing by length were detected in skeletal muscle tissue of all three affected subjects. In the longer transcript, an insertion of 252 nucleotides totally homologous to the structure of the 10th intron of the PFKM gene was found separating exon 10 from exon 11. In addition, two single base transitions were identified by direct sequencing: [exon 6; codon 95; CGA (Arg) to TGA (stop)] and [exon 7; codon 172; ACC (Thr) to ACT (Thr)] in either transcript. Single-stranded conformational polymorphism and restriction enzyme analyses confirmed the presence of these point substitutions in genomic DNA and strongly suggested homozygosity for the pathogenic allele. The nonsense mutation at codon 95 appeared solely responsible for the phenotype in these patients, further expanding genetic heterogeneity of Tarui disease. Transcripts with and without intron 10 arising from identical mutant alleles probably resulted from differential pre-mRNA processing and may represent a novel message from the PFKM gene. Images Fig. 2 Fig. 4 Fig. 5 PMID:7479776

A novel homozygous stop-codon mutation in human HFE responsible for nonsense-mediated mRNA decay.

PubMed

Padula, Maria Carmela; Martelli, Giuseppe; Larocca, Marilena; Rossano, Rocco; Olivieri, Attilio

2014-09-01

HFE-hemochromatosis (HH) is an autosomal disease characterized by excessive iron absorption. Homozygotes for H63D variant, and still less H63D heterozygotes, generally do not express HH phenotype. The data collected in our previous study in the province of Matera (Basilicata, Italy) underlined that some H63D carriers showed altered iron metabolism, without additional factors. In this study, we selected a cohort of 10/22 H63D carriers with severe biochemical iron overload (BIO). Additional analysis was performed for studying HFE exons, exon-intron boundaries, and untranslated regions (UTRs) by performing DNA extraction, PCR amplification and sequencing. The results showed a novel substitution (NM_000410.3:c.847C>T) in a patient exon 4 (GenBankJQ478433); it introduces a premature stop-codon (PTC). RNA extraction and reverse-transcription were also performed. Quantitative real-time PCR was carried out for verifying if our aberrant mRNA is targeted for nonsense-mediated mRNA decay (NMD); we observed that patient HFE mRNA was expressed much less than calibrator, suggesting that the mutated HFE protein cannot play its role in iron metabolism regulation, resulting in proband BIO. Our finding is the first evidence of a variation responsible for a PTC in iron cycle genes. The genotype-phenotype correlation observed in our cases could be related to the additional mutation. Copyright © 2014 Elsevier Inc. All rights reserved.

Color-deficient cone mosaics associated with Xq28 opsin mutations: A stop codon versus gene deletions

PubMed Central

Wagner-Schuman, Melissa; Neitz, Jay; Rha, Jungtae; Williams, David R.; Neitz, Maureen; Carroll, Joseph

2010-01-01

Our understanding of the etiology of red-green color vision defects is evolving. While missense mutations within the long- (L-) and middle-wavelength sensitive (M-) photopigments and gross rearrangements within the L/M-opsin gene array are commonly associated with red-green defects, recent work using adaptive optics retinal imaging has shown that different genotypes can have distinct consequences for the cone mosaic. Here we examined the cone mosaic in red-green color deficient individuals with multiple X-chromosome opsin genes that encode L opsin, as well as individuals with a single X-chromosome opsin gene that encodes L opsin and a single patient with a novel premature termination codon in his M-opsin gene and a normal L-opsin gene. We observed no difference in cone density between normal trichomats and multiple or single gene dichromats. In addition, we demonstrate different phenotypic effects of a nonsense mutation versus the previously described deleterious polymorphism, (LIAVA), both of which differ from multiple and single gene dichromats. Our results help refine the relationship between opsin genotype and cone photoreceptor mosaic phenotype. PMID:20854834

Analysis of Hungarian patients with Rett syndrome phenotype for MECP2, CDKL5 and FOXG1 gene mutations.

PubMed

Hadzsiev, Kinga; Polgar, Noemi; Bene, Judit; Komlosi, Katalin; Karteszi, Judit; Hollody, Katalin; Kosztolanyi, Gyorgy; Renieri, Alessandra; Melegh, Bela

2011-03-01

Rett syndrome (RTT) is characterized by a relatively specific clinical phenotype. We screened 152 individuals with RTT phenotype. A total of 22 different known MECP2 mutations were identified in 42 subjects (27.6%). Of the 22 mutations, we identified 7 (31.8%) frameshift-causing deletions, 4 (18.2%) nonsense, 10 (45.5%) missense mutations and one insertion (4.5%). The most frequent pathologic changes were: p.Thr158Met (14.2%) and p.Arg133Cys (11.9%) missense, and p.Arg255Stop (9.5%) and p.Arg294Stop (9.5%) nonsense mutations. We also detected the c.925C >T (p.Arg309Trp) mutation in an affected patient, whose role in RTT pathogenesis is still unknown. Patients without detectable MECP2 defects were screened for mutations of cyclin-dependent kinase-like 5 (CDKL5) gene, responsible for the early-onset variant of RTT. We discovered two novel mutations: c.607G >T resulting in a termination codon at aa203, disrupting the catalytic domain, and c.1708G >T leading to a stop at aa570 of the C terminus. Both patients with CDKL5 mutation presented therapy-resistant epilepsy and a phenotype fitting with the diagnosis of early-onset variant of RTT. No FOXG1 mutation was detected in any of the remaining patients. A total of 110 (72.5%) patients remained without molecular genetic diagnosis that necessitates further search for novel gene mutations in this phenotype. Our results also suggest the need of screening for CDKL5 mutations in patients with Rett phenotype tested negative for MECP2 mutations.

Dural ectasia and FBN1 mutation screening of 40 patients with Marfan syndrome and related disorders: role of dural ectasia for the diagnosis.

PubMed

Attanasio, Monica; Pratelli, Elisa; Porciani, Maria Cristina; Evangelisti, Lucia; Torricelli, Elena; Pellicanò, Giannantonio; Abbate, Rosanna; Gensini, Gian Franco; Pepe, Guglielmina

2013-07-01

Marfan syndrome is an autosomal dominant disorder of connective tissue caused by mutations in the gene encoding fibrillin-1 (FBN1), a matrix component of microfibrils. Dural ectasia, i.e. enlargement of the neural canal mainly located in the lower lumbar and sacral region, frequently occurs in Marfan patients. The aim of our study was to investigate the role of dural ectasia in raising the diagnosis of Marfan syndrome and its association with FBN1 mutations. We studied 40 unrelated patients suspected for MFS, who underwent magnetic resonance imaging searching for dural ectasia. In all of them FBN1 gene analysis was also performed. Thirty-seven patients resulted affected by Marfan syndrome according to the '96 Ghent criteria; in 30 of them the diagnosis was confirmed when revaluated by the recently revised criteria (2010). Thirty-six patients resulted positive for dural ectasia. The degree of dural ectasia was grade 1 in 19 patients, grade 2 in 11 patients, and grade 3 in 6 patients. In 7 (24%) patients, the presence of dural ectasia allowed to reach a positive score for systemic feature criterion. Twenty-four patients carried an FBN1 mutation, that were represented by 13 missense (54%), and 11 (46%) mutations generating a premature termination codon (PTC, frameshifts and stop codons). No mutation was detected in the remaining 16 (6 patients with MFS and 10 with related disorders according to revised Ghent criteria). The prevalence of severe (grade 2 and grade 3) involvement of dura mater was higher in patients harbouring premature termination codon (PTC) mutations than those carrying missense-mutations (8/11 vs 2/13, P = 0.0111). Our data emphasizes the importance of dural ectasia screening to reach the diagnosis of Marfan syndrome especially when it is uncertain and indicates an association between PTC mutations and severe dural ectasia in Marfan patients. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Position-dependent termination and widespread obligatory frameshifting in Euplotes translation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lobanov, Alexei V.; Heaphy, Stephen M.; Turanov, Anton A.

2016-11-21

The ribosome can change its reading frame during translation in a process known as programmed ribosomal frameshifting. These rare events are supported by complex mRNA signals. However, we found that the ciliates Euplotes crassus and Euplotes focardii exhibit widespread frameshifting at stop codons. 47 different codons preceding stop signals resulted in either +1 or +2 frameshifts, and +1 frameshifting at AAA was the most frequent. The frameshifts showed unusual plasticity and rapid evolution, and had little influence on translation rates. The proximity of a stop codon to the 3' mRNA end, rather than its occurrence or sequence context, appeared tomore » designate termination. Thus, a ‘stop codon’ is not a sufficient signal for translation termination, and the default function of stop codons in Euplotes is frameshifting, whereas termination is specific to certain mRNA positions and probably requires additional factors.« less

RNA editing makes mistakes in plant mitochondria: editing loses sense in transcripts of a rps19 pseudogene and in creating stop codons in coxI and rps3 mRNAs of Oenothera.

PubMed Central

Schuster, W; Brennicke, A

1991-01-01

An intact gene for the ribosomal protein S19 (rps19) is absent from Oenothera mitochondria. The conserved rps19 reading frame found in the mitochondrial genome is interrupted by a termination codon. This rps19 pseudogene is cotranscribed with the downstream rps3 gene and is edited on both sides of the translational stop. Editing, however, changes the amino acid sequence at positions that were well conserved before editing. Other strange editings create translational stops in open reading frames coding for functional proteins. In coxI and rps3 mRNAs CGA codons are edited to UGA stop codons only five and three codons, respectively, downstream to the initiation codon. These aberrant editings in essential open reading frames and in the rps19 pseudogene appear to have been shifted to these positions from other editing sites. These observations suggest a requirement for a continuous evolutionary constraint on the editing specificities in plant mitochondria. Images PMID:1762921

Diverse growth hormone receptor gene mutations in Laron syndrome.

PubMed Central

Berg, M A; Argente, J; Chernausek, S; Gracia, R; Guevara-Aguirre, J; Hopp, M; Pérez-Jurado, L; Rosenbloom, A; Toledo, S P; Francke, U

1993-01-01

To better understand the molecular genetic basis and genetic epidemiology of Laron syndrome (growth-hormone insensitivity syndrome), we analyzed the growth-hormone receptor (GHR) genes of seven unrelated affected individuals from the United States, South America, Europe, and Africa. We amplified all nine GHR gene exons and splice junctions from these individuals by PCR and screened the products for mutations by using denaturing gradient gel electrophoresis (DGGE). We identified a single GHR gene fragment with abnormal DGGE results for each affected individual, sequenced this fragment, and, in each case, identified a mutation likely to cause Laron syndrome, including two nonsense mutations (R43X and R217X), two splice-junction mutations, (189-1 G to T and 71 + 1 G to A), and two frameshift mutations (46 del TT and 230 del TA or AT). Only one of these mutations, R43X, has been previously reported. Using haplotype analysis, we determined that this mutation, which involves a CpG dinucleotide hot spot, likely arose as a separate event in this case, relative to the two prior reports of R43X. Aside from R43X, the mutations we identified are unique to patients from particular geographic regions. Ten GHR gene mutations have now been described in this disorder. We conclude that Laron syndrome is caused by diverse GHR gene mutations, including deletions, RNA processing defects, translational stop codons, and missense codons. All the identified mutations involve the extracellular domain of the receptor, and most are unique to particular families or geographic areas. Images Figure 1 Figure 2 PMID:8488849

Recessive mutations in SPTBN2 implicate β-III spectrin in both cognitive and motor development.

PubMed

Lise, Stefano; Clarkson, Yvonne; Perkins, Emma; Kwasniewska, Alexandra; Sadighi Akha, Elham; Schnekenberg, Ricardo Parolin; Suminaite, Daumante; Hope, Jilly; Baker, Ian; Gregory, Lorna; Green, Angie; Allan, Chris; Lamble, Sarah; Jayawant, Sandeep; Quaghebeur, Gerardine; Cader, M Zameel; Hughes, Sarah; Armstrong, Richard J E; Kanapin, Alexander; Rimmer, Andrew; Lunter, Gerton; Mathieson, Iain; Cazier, Jean-Baptiste; Buck, David; Taylor, Jenny C; Bentley, David; McVean, Gilean; Donnelly, Peter; Knight, Samantha J L; Jackson, Mandy; Ragoussis, Jiannis; Németh, Andrea H

2012-01-01

β-III spectrin is present in the brain and is known to be important in the function of the cerebellum. Heterozygous mutations in SPTBN2, the gene encoding β-III spectrin, cause Spinocerebellar Ataxia Type 5 (SCA5), an adult-onset, slowly progressive, autosomal-dominant pure cerebellar ataxia. SCA5 is sometimes known as "Lincoln ataxia," because the largest known family is descended from relatives of the United States President Abraham Lincoln. Using targeted capture and next-generation sequencing, we identified a homozygous stop codon in SPTBN2 in a consanguineous family in which childhood developmental ataxia co-segregates with cognitive impairment. The cognitive impairment could result from mutations in a second gene, but further analysis using whole-genome sequencing combined with SNP array analysis did not reveal any evidence of other mutations. We also examined a mouse knockout of β-III spectrin in which ataxia and progressive degeneration of cerebellar Purkinje cells has been previously reported and found morphological abnormalities in neurons from prefrontal cortex and deficits in object recognition tasks, consistent with the human cognitive phenotype. These data provide the first evidence that β-III spectrin plays an important role in cortical brain development and cognition, in addition to its function in the cerebellum; and we conclude that cognitive impairment is an integral part of this novel recessive ataxic syndrome, Spectrin-associated Autosomal Recessive Cerebellar Ataxia type 1 (SPARCA1). In addition, the identification of SPARCA1 and normal heterozygous carriers of the stop codon in SPTBN2 provides insights into the mechanism of molecular dominance in SCA5 and demonstrates that the cell-specific repertoire of spectrin subunits underlies a novel group of disorders, the neuronal spectrinopathies, which includes SCA5, SPARCA1, and a form of West syndrome.

KRAS exon 2 codon 13 mutation is associated with a better prognosis than codon 12 mutation following lung metastasectomy in colorectal cancer

PubMed Central

Renaud, Stéphane; Guerrera, Francesco; Seitlinger, Joseph; Costardi, Lorena; Schaeffer, Mickaël; Romain, Benoit; Mossetti, Claudio; Claire-Voegeli, Anne; Filosso, Pier Luigi; Legrain, Michèle; Ruffini, Enrico; Falcoz, Pierre-Emmanuel; Oliaro, Alberto; Massard, Gilbert

2017-01-01

Introduction The utilization of molecular markers as routinely used biomarkers is steadily increasing. We aimed to evaluate the potential different prognostic values of KRAS exon 2 codons 12 and 13 after lung metastasectomy in colorectal cancer (CRC). Results KRAS codon 12 mutations were observed in 116 patients (77%), whereas codon 13 mutations were observed in 34 patients (23%). KRAS codon 13 mutations were associated with both longer time to pulmonary recurrence (TTPR) (median TTPR: 78 months (95% CI: 50.61–82.56) vs 56 months (95% CI: 68.71–127.51), P = 0.008) and improved overall survival (OS) (median OS: 82 months vs 54 months (95% CI: 48.93–59.07), P = 0.009). Multivariate analysis confirmed that codon 13 mutations were associated with better outcomes (TTPR: HR: 0.40 (95% CI: 0.17–0.93), P = 0.033); OS: HR: 0.39 (95% CI: 0.14–1.07), P = 0.07). Otherwise, no significant difference in OS (P = 0.78) or TTPR (P = 0.72) based on the type of amino-acid substitutions was observed among KRAS codon 12 mutations. Materials and Methods We retrospectively reviewed data from 525 patients who underwent a lung metastasectomy for CRC in two departments of thoracic surgery from 1998 to 2015 and focused on 150 patients that had KRAS exon 2 codon 12/13 mutations. Conclusions KRAS exon 2 codon 13 mutations, compared to codon 12 mutations, seem to be associated with better outcomes following lung metastasectomy in CRC. Prospective multicenter studies are necessary to fully understand the prognostic value of KRAS mutations in the lung metastases of CRC. PMID:27911859

Novel nonsense mutation of the endothelin-B receptor gene in a family with Waardenburg-Hirschsprung disease.

PubMed

Syrris, P; Carter, N D; Patton, M A

1999-11-05

Waardenburg syndrome (WS) comprises sensorineural hearing loss, hypopigmentation of skin and hair, and pigmentary disturbances of the irides. Four types of WS have been classified to date; in WS type IV (WS4), patients additionally have colonic aganglionosis (Hirschsprung disease, HSCR). Mutations in the endothelin-3 (EDN3), endothelin-B receptor (EDNRB), and Sox10 genes have been identified as causative for WS type IV. We screened a family with a combined WS-HSCR phenotype for mutations in the EDNRB locus using standard DNA mutation analysis and sequencing techniques. We have identified a novel nonsense mutation at codon 253 (CGA-->TGA, Arg-->STOP). This mutation leads to a premature end of the translation of EDNRB at exon 3, and it is predicted to produce a truncated and nonfunctional endothelin-B receptor. All affected relatives were heterozygous for the Arg(253)-->STOP mutation, whereas it was not observed in over 50 unrelated individuals used as controls. These data confirm the role of EDNRB in the cause of the Waardenburg-Hirschsprung syndrome and demonstrate that in WS-HSCR there is a lack of correlation between phenotype and genotype and a variable expression of disease even within the same family. Copyright 1999 Wiley-Liss, Inc.

A novel germline inactivating mutation in the CASR gene in an Italian kindred affected by familial hypocalciuric hypercalcemia.

PubMed

Falchetti, Alberto; Gozzini, Alessia; Terranegra, Annalisa; Soldati, Laura; Vezzoli, Giuseppe; Leoncini, Gigliola; Giusti, Francesca; Franceschelli, Francesco; Masi, Laura; Tanini, Annalisa; Cavalli, Loredana; Brandi, Maria Luisa

2012-05-01

Familial hypocalciuric hypercalcemia (FHH) syndrome is a rare benign condition, inherited as an autosomal dominant trait, in which inactivating mutations of the calcium-sensing receptor (CASR) gene affects the body's ability to regulate calcium homeostasis. Its outcome is featured by increased levels of serum calcium, moderate hypophosphatemia, and inadequately normal or elevated circulating parathyroid hormone levels. Affected patients are mostly asymptomatic and do not benefit from surgical resection of their mildly enlarged parathyroids. We evaluated for hypercalcemia an Italian family that was identified via a young adult male proband referred to our center for parathyroidectomy. The patients and the family members were evaluated both biochemically and genetically as suspected FHH subjects. An in vitro functional study was performed by site-directed mutagenesis, and CASR activity was monitored by measuring intracellular calcium ([Ca(2)(+)](i)). The patient had a novel germline heterozygous CASR mutation (c.361_364GATT; p.D121del/fsX122). The mutation caused a premature stop codon at codon 122, exiting a truncated protein. The biochemical phenotype of all family members carrying the heterozygous deletion was concordant with classic FHH syndrome. Our findings confirm the role of CASR gene mutational analysis to offer a valuable addition for the recognition of FHH in hypercalcemic patients not yet characterized for a positive familial history of hypercalcemia, the only condition that identifies CASR gene mutations in hypercalcemia.

Identification of a novel mutation in a Chinese family with Nance-Horan syndrome by whole exome sequencing*

PubMed Central

Hong, Nan; Chen, Yan-hua; Xie, Chen; Xu, Bai-sheng; Huang, Hui; Li, Xin; Yang, Yue-qing; Huang, Ying-ping; Deng, Jian-lian; Qi, Ming; Gu, Yang-shun

2014-01-01

Objective: Nance-Horan syndrome (NHS) is a rare X-linked disorder characterized by congenital nuclear cataracts, dental anomalies, and craniofacial dysmorphisms. Mental retardation was present in about 30% of the reported cases. The purpose of this study was to investigate the genetic and clinical features of NHS in a Chinese family. Methods: Whole exome sequencing analysis was performed on DNA from an affected male to scan for candidate mutations on the X-chromosome. Sanger sequencing was used to verify these candidate mutations in the whole family. Clinical and ophthalmological examinations were performed on all members of the family. Results: A combination of exome sequencing and Sanger sequencing revealed a nonsense mutation c.322G>T (E108X) in exon 1 of NHS gene, co-segregating with the disease in the family. The nonsense mutation led to the conversion of glutamic acid to a stop codon (E108X), resulting in truncation of the NHS protein. Multiple sequence alignments showed that codon 108, where the mutation (c.322G>T) occurred, was located within a phylogenetically conserved region. The clinical features in all affected males and female carriers are described in detail. Conclusions: We report a nonsense mutation c.322G>T (E108X) in a Chinese family with NHS. Our findings broaden the spectrum of NHS mutations and provide molecular insight into future NHS clinical genetic diagnosis. PMID:25091991

Confirmation of RAX gene involvement in human anophthalmia

PubMed Central

Lequeux, L.; Rio, Marlène; Vigouroux, Armelle; Titeux, Matthias; Etchevers, Heather; Malecaze, François; Chassaing, Nicolas; Calvas, Patrick

2008-01-01

Microphthalmia and anophthalmia are at the severe end of the spectrum of abnormalities in ocular development. Mutations in several genes have been involved in syndromic and non-syndromic anophthalmia. Previously, RAX recessive mutations were implicated in a single patient with right anophthalmia and left microphthalmia and sclerocornea. Here, we report the findings of novel compound heterozygous RAX mutations in a child with bilateral anophthalmia. Both mutations are located in exon 3. c.664delT is a frameshifting deletion predicted to introduce a premature stop codon (p.Ser222ArgfsX62), and c.909 C>G is a nonsense mutation with similar consequences (p.Tyr303X). This is the second report of a patient with anophthalmia caused by RAX mutations. These findings confirm that RAX plays a major role in the early stages of eye development and is involved in human anophthalmia. PMID:18783408

Confirmation of RAX gene involvement in human anophthalmia.

PubMed

Lequeux, L; Rio, M; Vigouroux, A; Titeux, M; Etchevers, H; Malecaze, F; Chassaing, N; Calvas, P

2008-10-01

Microphthalmia and anophthalmia are at the severe end of the spectrum of abnormalities in ocular development. Mutations in several genes have been involved in syndromic and non-syndromic anophthalmia. Previously, RAX recessive mutations were implicated in a single patient with right anophthalmia, left microphthalmia and sclerocornea. In this study, we report the findings of novel compound heterozygous RAX mutations in a child with bilateral anophthalmia. Both mutations are located in exon 3. c.664delT is a frameshifting deletion predicted to introduce a premature stop codon (p.Ser222ArgfsX62), and c.909C>G is a nonsense mutation with similar consequences (p.Tyr303X). This is the second report of a patient with anophthalmia caused by RAX mutations. These findings confirm that RAX plays a major role in the early stages of eye development and is involved in human anophthalmia.

A novel Werner Syndrome mutation: pharmacological treatment by read-through of nonsense mutations and epigenetic therapies

PubMed Central

Agrelo, Ruben; Sutz, Miguel Arocena; Setien, Fernando; Aldunate, Fabian; Esteller, Manel; Da Costa, Valeria; Achenbach, Ricardo

2015-01-01

Werner Syndrome (WS) is a rare inherited disease characterized by premature aging and increased propensity for cancer. Mutations in the WRN gene can be of several types, including nonsense mutations, leading to a truncated protein form. WRN is a RecQ family member with both helicase and exonuclease activities, and it participates in several cell metabolic pathways, including DNA replication, DNA repair, and telomere maintenance. Here, we reported a novel homozygous WS mutation (c.3767 C > G) in 2 Argentinian brothers, which resulted in a stop codon and a truncated protein (p.S1256X). We also observed increased WRN promoter methylation in the cells of patients and decreased messenger WRN RNA (WRN mRNA) expression. Finally, we showed that the read-through of nonsense mutation pharmacologic treatment with both aminoglycosides (AGs) and ataluren (PTC-124) in these cells restores full-length protein expression and WRN functionality. PMID:25830902

Year to year change in FEV1 in patients with cystic fibrosis and different mutation classes.

PubMed

De Boeck, K; Zolin, A

2017-03-01

In patients with cystic fibrosis, most treatments addressing the underlying basic defect are mutation or mutation class specific. These treatments are disease modifying if they lower the year to year change in lung function. We therefore calculated the current loss of lung function, measured by year to year change in forced expired volume in 1s in 11,417 patients included in the European Cystic Fibrosis Society Patient Registry. Whereas patients with at least one mutation of class IV or V have on average a lower year to year change, we did not find a difference between patients with a stop codon mutation, homozygous for F508del or at least one class III mutation. These data are useful background information to discuss the impact of different disease modifying treatments. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Four novel germline mutations in the MLH1 and PMS2 mismatch repair genes in patients with hereditary nonpolyposis colorectal cancer.

PubMed

Montazer Haghighi, Mahdi; Radpour, Ramin; Aghajani, Katayoun; Zali, Narges; Molaei, Mahsa; Zali, Mohammad Reza

2009-08-01

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common cause of early onset hereditary colorectal cancer. In the majority of HNPCC families, microsatellite instability (MSI) and germline mutation in one of the DNA mismatch repair (MMR) genes are found. The entire coding sequence of MMR genes (MLH1, MLH2, MLH6, and PMS2) was analyzed using direct sequencing. Also, tumor tests were done as MSI and immunohistochemistry testing. We were able to find three novel MLH1 and one novel PMS2 germline mutations in three Iranian HNPCC patients. The first was a transversion mutation c.346A>C (T116P) and happened in the highly conserved HATPase-c region of MLH1 protein. The second was a transversion mutation c.736A>T (I246L), which caused an amino acid change of isoleucine to leucine. The third mutation (c.2145,6 delTG) was frameshift and resulted in an immature stop codon in five codons downstream. All of these three mutations were detected in the MLH1 gene. The other mutation was a transition mutation, c.676G>A (G207E), which has been found in exon six of the PMS2 gene and caused an amino acid change of glycine to glutamic acid. MSI assay revealed high instability in microsatellite for two patients and microsatellite stable for one patient. In all patients, an abnormal expression of the MMR proteins in HNPCC was related to the above novel mutations.

Inhibition of Exotoxin Production by Mobile Genetic Element SCCmec-Encoded psm-mec RNA Is Conserved in Staphylococcal Species

PubMed Central

Saito, Yuki; Mao, Han; Sekimizu, Kazuhisa; Kaito, Chikara

2014-01-01

Staphylococcal species acquire antibiotic resistance by incorporating the mobile-genetic element SCCmec. We previously found that SCCmec-encoded psm-mec RNA suppresses exotoxin production as a regulatory RNA, and the psm-mec translation product increases biofilm formation in Staphylococcus aureus. Here, we examined whether the regulatory role of psm-mec on host bacterial virulence properties is conserved among other staphylococcal species, S. epidermidis and S. haemolyticus, both of which are important causes of nosocomial infections. In S. epidermidis, introduction of psm-mec decreased the production of cytolytic toxins called phenol-soluble modulins (PSMs) and increased biofilm formation. Introduction of psm-mec with a stop-codon mutation that did not express PSM-mec protein but did express psm-mec RNA also decreased PSM production, but did not increase biofilm formation. Thus, the psm-mec RNA inhibits PSM production, whereas the PSM-mec protein increases biofilm formation in S. epidermidis. In S. haemolyticus, introduction of psm-mec decreased PSM production, but did not affect biofilm formation. The mutated psm-mec with a stop-codon also caused the same effect. Thus, the psm-mec RNA also inhibits PSM production in S. haemolyticus. These findings suggest that the inhibitory role of psm-mec RNA on exotoxin production is conserved among staphylococcal species, although the stimulating effect of the psm-mec gene on biofilm formation is not conserved. PMID:24926994

Colistin-Resistant Acinetobacter baumannii Clinical Strains with Deficient Biofilm Formation

PubMed Central

Dafopoulou, Konstantina; Xavier, Basil Britto; Hotterbeekx, An; Janssens, Lore; Lammens, Christine; Dé, Emmanuelle; Goossens, Herman; Tsakris, Athanasios; Malhotra-Kumar, Surbhi

2015-01-01

In two pairs of clinical colistin-susceptible/colistin-resistant (Csts/Cstr) Acinetobacter baumannii strains, the Cstr strains showed significantly decreased biofilm formation in static and dynamic assays (P < 0.001) and lower relative fitness (P < 0.05) compared with those of the Csts counterparts. The whole-genome sequencing comparison of strain pairs identified a mutation converting a stop codon to lysine (*241K) in LpsB (involved in lipopolysaccharide [LPS] synthesis) in one Cstr strain and a frameshift mutation in CarO and the loss of a 47,969-bp element containing multiple genes associated with biofilm production in the other. PMID:26666921

Treacher Collins syndrome with a de Novo 5-bp deletion in the TCOF1 gene.

PubMed

Su, Pen-Hua; Chen, Jia-Yu; Chen, Suh-Jen; Yu, Ju-Shan

2006-06-01

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development with features including malar hypoplasia, micrognathia, microtia, downward slanting palpebral fissures, lower eyelid coloboma, conductive hearing loss, and cleft palate. TCS is caused by mutations in the TCOF1 gene, which encodes the nuclear phosphoprotein treacle. Here, we describe a 1-day-old male infant with classical TCS presentation. A 5-bp deletion in exon 22 of the TCOF1 gene (3469del ACTCT) was found to cause a premature stop codon. This is the first report of TCOF1 gene mutation in the Taiwanese population.

Codon 219 polymorphism of PRNP in healthy caucasians and Creutzfeldt-Jakob disease patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petraroli, R.; Pocchiari, M.

1996-04-01

A number of point and insert mutations of the PrP gene (PRNP) have been linked to familial Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker disease (GSS). Moreover, the methionine/valine homozygosity at the polymorphic codon 129 of PRNP may cause a predisposition to sporadic and iatrogenic CJD or may control the age at onset of familial cases carrying either the 144-bp insertion or codon 178, codon 198, and codon 210 pathogenic mutations in PRNP. In addition, the association of methionine or valine at codon 129 and the point mutation at codon 178 on the same allele seem to play an important role inmore » determining either fatal familial insomnia or CJD. However, it is noteworthy that a relationship between codon 129 polymorphism and accelerated pathogenesis (early age at onset or shorter duration of the disease) has not been seen in familial CJD patients with codon 200 mutation or in GSS patients with codon 102 mutation, arguing that other, as yet unidentified, gene products or environmental factors, or both, may influence the clinical expression of these diseases. 17 refs.« less

Turbidostat Culture of Saccharomyces cerevisiae W303-1A under Selective Pressure Elicited by Ethanol Selects for Mutations in SSD1 and UTH1

PubMed Central

Avrahami-Moyal, Liat; Engelberg, David; Wenger, Jared. W.; Sherlock, Gavin; Braun, Sergei

2012-01-01

We investigated the genetic causes of ethanol tolerance by characterizing mutations selected in Saccharomyces cerevisiae W303-1A under the selective pressure of ethanol. W303-1A was subjected to three rounds of turbidostat, in medium supplemented with increasing amounts of ethanol. By the end of selection, the growth rate of the culture has increased from 0.029 h-1 to 0.32 h-1. Unlike the progenitor strain, all yeast cells isolated from this population were able to form colonies on medium supplemented with 7% ethanol within six days, our definition of ethanol tolerance. Several clones selected from all three stages of selection were able to form dense colonies within two days on solid medium supplemented with 9% ethanol. We sequenced the whole genomes of 6 clones and identified mutations responsible for ethanol tolerance. Thirteen additional clones were tested for the presence of similar mutations. In 15 out of 19 tolerant clones the stop-codon in ssd1-d was replaced with an aminoacid-encoding codon. Three other clones contained one of two mutations in UTH1, and one clone did not contain mutations in either SSD1 or UTH1. We showed that the mutations in SSD1 and UTH1 increased tolerance of the cell wall to zymolyase and conclude that stability of the cell wall is a major factor in increased tolerance to ethanol. PMID:22443114

Waardenburg syndrome type II in a Chinese patient caused by a novel nonsense mutation in the SOX10 gene.

PubMed

Ma, Jing; Zhang, Tie-Song; Lin, Ken; Sun, Hao; Jiang, Hong-Chao; Yang, Yan-Li; Low, Fan; Gao, Ying-Qin; Ruan, Biao

2016-06-01

Waardenburg syndrome is a congenital genetic disorder. It is the most common type of syndromic hearing impairment with highly genetic heterogeneity and proved to be related by 6 genes as follows: PAX3, MITF, SNAI2, EDN3, EDNRB and SOX10. This article aims to identify the genetic causes of a Chinese WS child patient. A Chinese WS child was collected for clinical data collection by questionnaire survey. DNA samples of proband and his parents were extracted from peripheral blood samples. Six candidate genes were sequenced by the Trusight One sequencing panel on the illumina NextSeq 500 platform. A novel nonsense heterozygous mutation was found in the coding region of exon 2 in the SOX10 gene of proband. The novel nonsense heterozygous mutation could cause the replacement of the 55th lysine codon by stop codon (484T > C, C142R) and further more possibly cause terminating the protein translation in advance. However, both proband's parents had no mutation of genes above mentioned. The gene mutation of SOX10 [NM_006941.3 c.163A > T] is a novel nonsense mutation. No record of this mutation has been found in dbSNP, HGMD, 1000 Genomes Project, ClinVar and ESP6500 databases. It meets the condition of PS2 of strong evidence in 2015 ACMG Standards and Guidelines. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Inherited human complement C5 deficiency: Nonsense mutations in exons 1 (Gln{sup 1} to Stop) and 36 (Arg{sup 1458} to Stop) and compound heterozygosity in three African-American families

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, X.; Fleischer, D.T.; Whitehead, W.T.

1995-05-15

Hereditary C5 deficiency has been reported in several families of different ethnic backgrounds and from different geographic regions, but the molecular genetic defect causing C5 deficiency has not been delineated in any of them. To examine the molecular basis of C5 deficiency in the African-American population, the exons and intron/exon boundaries of the C5 structural genes from three C5-deficient (C5D) African-American families were sequenced, revealing two nonsense mutations. The nonsense mutations are located in exon 1 (C{sup 84}AG to TAG) in two of the C5D families (Rhode Island and North Carolina) and in exon 36 (C{sup 4521}GA to TGA) inmore » the third C5D family (New York). The exon 1 and 36 mutations are contained in codons that encode the first amino acid of the C5 {beta}-chain (Gln{sup 1} to Stop) and residue 1458 in the {alpha}-chain (Arg{sup 1458} to Stop), respectively. Allele-specific PCR and sequence analyses demonstrated that the exon 1 mutation is present in only one of the C5 null genes in both the Rhode Island and North Carolina families, and the exon 36 mutation is contained in only one C5 null gene in the New York family. Neither of the nonsense mutations was found in the European or Caucasian-American C5D individuals examined. Collectively, these data indicate that: (1) C5 deficiency is caused by several different molecular genetic defects, (2) C5 deficiency in the African-American population can be explained in part by two distinct nonsense mutations in exons 1 and 36, and (3) compound heterozygosity exists in all of the reported African-American C5D families. 44 refs., 5 figs., 1 tab.« less

Representation mutations from standard genetic codes

NASA Astrophysics Data System (ADS)

Aisah, I.; Suyudi, M.; Carnia, E.; Suhendi; Supriatna, A. K.

2018-03-01

Graph is widely used in everyday life especially to describe model problem and describe it concretely and clearly. In addition graph is also used to facilitate solve various kinds of problems that are difficult to be solved by calculation. In Biology, graph can be used to describe the process of protein synthesis in DNA. Protein has an important role for DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). Proteins are composed of amino acids. In this study, amino acids are related to genetics, especially the genetic code. The genetic code is also known as the triplet or codon code which is a three-letter arrangement of DNA nitrogen base. The bases are adenine (A), thymine (T), guanine (G) and cytosine (C). While on RNA thymine (T) is replaced with Urasil (U). The set of all Nitrogen bases in RNA is denoted by N = {C U, A, G}. This codon works at the time of protein synthesis inside the cell. This codon also encodes the stop signal as a sign of the stop of protein synthesis process. This paper will examine the process of protein synthesis through mathematical studies and present it in three-dimensional space or graph. The study begins by analysing the set of all codons denoted by NNN such that to obtain geometric representations. At this stage there is a matching between the sets of all nitrogen bases N with Z 2 × Z 2; C=(\\overline{0},\\overline{0}),{{U}}=(\\overline{0},\\overline{1}),{{A}}=(\\overline{1},\\overline{0}),{{G}}=(\\overline{1},\\overline{1}). By matching the algebraic structure will be obtained such as group, group Klein-4,Quotien group etc. With the help of Geogebra software, the set of all codons denoted by NNN can be presented in a three-dimensional space as a multicube NNN and also can be represented as a graph, so that can easily see relationship between the codon.

Emerging genetic therapies to treat Duchenne muscular dystrophy

PubMed Central

Nelson, Stanley F.; Crosbie, Rachelle H.; Miceli, M. Carrie; Spencer, Melissa J.

2010-01-01

Purpose of review Duchenne muscular dystrophy is a progressive muscle degenerative disease caused by dystrophin mutations. The purpose of this review is to highlight two emerging therapies designed to repair the primary genetic defect, called `exon skipping' and `nonsense codon suppression'. Recent findings A drug, PTC124, was identified that suppresses nonsense codon translation termination. PTC124 can lead to restoration of some dystrophin expression in human Duchenne muscular dystrophy muscles with mutations resulting in premature stops. Two drugs developed for exon skipping, PRO051 and AVI-4658, result in the exclusion of exon 51 from mature mRNA. They can restore the translational reading frame to dystrophin transcripts from patients with a particular subset of dystrophin gene deletions and lead to some restoration of dystrophin expression in affected boys' muscle in vivo. Both approaches have concluded phase I trials with no serious adverse events. Summary These novel therapies that act to correct the primary genetic defect of dystrophin deficiency are among the first generation of therapies tailored to correct specific mutations in humans. Thus, they represent paradigm forming approaches to personalized medicine with the potential to lead to life changing treatment for those affected by Duchenne muscular dystrophy. PMID:19745732

Functional Analyses of c.2268dup in Thyroid Peroxidase Gene Associated with Goitrous Congenital Hypothyroidism

PubMed Central

Harun, Fatimah; Jalaludin, Muhammad Yazid; Lim, Chor Yin; Ng, Khoon Leong

2014-01-01

The c.2268dup mutation in thyroid peroxidase (TPO) gene was reported to be a founder mutation in Taiwanese patients with dyshormonogenetic congenital hypothyroidism (CH). The functional impact of the mutation is not well documented. In this study, homozygous c.2268dup mutation was detected in two Malaysian-Chinese sisters with goitrous CH. Normal and alternatively spliced TPO mRNA transcripts were present in thyroid tissues of the two sisters. The abnormal transcript contained 34 nucleotides originating from intron 12. The c.2268dup is predicted to generate a premature termination codon (PTC) at position 757 (p.Glu757X). Instead of restoring the normal reading frame, the alternatively spliced transcript has led to another stop codon at position 740 (p.Asp739ValfsX740). The two PTCs are located at 116 and 201 nucleotides upstream of the exons 13/14 junction fulfilling the requirement for a nonsense-mediated mRNA decay (NMD). Quantitative RT-PCR revealed an abundance of unidentified transcripts believed to be associated with the NMD. TPO enzyme activity was not detected in both patients, even though a faint TPO band of about 80 kD was present. In conclusion, the c.2268dup mutation leads to the formation of normal and alternatively spliced TPO mRNA transcripts with a consequential loss of TPO enzymatic activity in Malaysian-Chinese patients with goitrous CH. PMID:24745015

Two recessive mutations in FGF5 are associated with the long-hair phenotype in donkeys.

PubMed

Legrand, Romain; Tiret, Laurent; Abitbol, Marie

2014-09-25

Seven donkey breeds are recognized by the French studbook. Individuals from the Pyrenean, Provence, Berry Black, Normand, Cotentin and Bourbonnais breeds are characterized by a short coat, while those from the Poitou breed (Baudet du Poitou) are characterized by a long-hair phenotype. We hypothesized that loss-of-function mutations in the FGF5 (fibroblast growth factor 5) gene, which are associated with a long-hair phenotype in several mammalian species, may account for the special coat feature of Poitou donkeys. To the best of our knowledge, mutations in FGF5 have never been described in Equidae. We sequenced the FGF5 gene from 35 long-haired Poitou donkeys, as well as from a panel of 67 short-haired donkeys from the six other French breeds and 131 short-haired ponies and horses. We identified a recessive c.433_434delAT frameshift deletion in FGF5, present in Poitou and three other donkey breeds and a recessive nonsense c.245G > A substitution, present in Poitou and four other donkey breeds. The frameshift deletion was associated with the long-hair phenotype in Poitou donkeys when present in two copies (n = 31) or combined with the nonsense mutation (n = 4). The frameshift deletion led to a stop codon at position 159 whereas the nonsense mutation led to a stop codon at position 82 in the FGF5 protein. In silico, the two truncated FGF5 proteins were predicted to lack the critical β strands involved in the interaction between FGF5 and its receptor, a mandatory step to inhibit hair growth. Our results highlight the allelic heterogeneity of the long-hair phenotype in donkeys and enlarge the panel of recessive FGF5 loss-of-function alleles described in mammals. Thanks to the DNA test developed in this study, breeders of non-Poitou breeds will have the opportunity to identify long-hair carriers in their breeding stocks.

Functional Versatility of AGY Serine Codons in Immunoglobulin Variable Region Genes

PubMed Central

Detanico, Thiago; Phillips, Matthew; Wysocki, Lawrence J.

2016-01-01

In systemic autoimmunity, autoantibodies directed against nuclear antigens (Ags) often arise by somatic hypermutation (SHM) that converts AGT and AGC (AGY) Ser codons into Arg codons. This can occur by three different single-base changes. Curiously, AGY Ser codons are far more abundant in complementarity-determining regions (CDRs) of IgV-region genes than expected for random codon use or from species-specific codon frequency data. CDR AGY codons are also more abundant than TCN Ser codons. We show that these trends hold even in cartilaginous fishes. Because AGC is a preferred target for SHM by activation-induced cytidine deaminase, we asked whether the AGY abundance was solely due to a selection pressure to conserve high mutability in CDRs regardless of codon context but found that this was not the case. Instead, AGY triplets were selectively enriched in the Ser codon reading frame. Motivated by reports implicating a functional role for poly/autoreactive specificities in antiviral antibodies, we also analyzed mutations at AGY in antibodies directed against a number of different viruses and found that mutations producing Arg codons in antiviral antibodies were indeed frequent. Unexpectedly, however, we also found that AGY codons mutated often to encode nearly all of the amino acids that are reported to provide the most frequent contacts with Ag. In many cases, mutations producing codons for these alternative amino acids in antiviral antibodies were more frequent than those producing Arg codons. Mutations producing each of these key amino acids required only single-base changes in AGY. AGY is the only codon group in which two-thirds of random mutations generate codons for these key residues. Finally, by directly analyzing X-ray structures of immune complexes from the RCSB protein database, we found that Ag-contact residues generated via SHM occurred more often at AGY than at any other codon group. Thus, preservation of AGY codons in antibody genes appears to have been driven by their exceptional functional versatility, despite potential autoreactive consequences. PMID:27920779

Molecular analysis of beta-globin gene mutations among Thai beta-thalassemia children: results from a single center study

PubMed Central

Boonyawat, Boonchai; Monsereenusorn, Chalinee; Traivaree, Chanchai

2014-01-01

Background Beta-thalassemia is one of the most common genetic disorders in Thailand. Clinical phenotype ranges from silent carrier to clinically manifested conditions including severe beta-thalassemia major and mild beta-thalassemia intermedia. Objective This study aimed to characterize the spectrum of beta-globin gene mutations in pediatric patients who were followed-up in Phramongkutklao Hospital. Patients and methods Eighty unrelated beta-thalassemia patients were enrolled in this study including 57 with beta-thalassemia/hemoglobin E, eight with homozygous beta-thalassemia, and 15 with heterozygous beta-thalassemia. Mutation analysis was performed by multiplex amplification refractory mutation system (M-ARMS), direct DNA sequencing of beta-globin gene, and gap polymerase chain reaction for 3.4 kb deletion detection, respectively. Results A total of 13 different beta-thalassemia mutations were identified among 88 alleles. The most common mutation was codon 41/42 (-TCTT) (37.5%), followed by codon 17 (A>T) (26.1%), IVS-I-5 (G>C) (8%), IVS-II-654 (C>T) (6.8%), IVS-I-1 (G>T) (4.5%), and codon 71/72 (+A) (2.3%), and all these six common mutations (85.2%) were detected by M-ARMS. Six uncommon mutations (10.2%) were identified by DNA sequencing including 4.5% for codon 35 (C>A) and 1.1% initiation codon mutation (ATG>AGG), codon 15 (G>A), codon 19 (A>G), codon 27/28 (+C), and codon 123/124/125 (-ACCCCACC), respectively. The 3.4 kb deletion was detected at 4.5%. The most common genotype of beta-thalassemia major patients was codon 41/42 (-TCTT)/codon 26 (G>A) or betaE accounting for 40%. Conclusion All of the beta-thalassemia alleles have been characterized by a combination of techniques including M-ARMS, DNA sequencing, and gap polymerase chain reaction for 3.4 kb deletion detection. Thirteen mutations account for 100% of the beta-thalassemia genes among the pediatric patients in our study. PMID:25525381

Ribosomal scanning past the primary initiation codon as a mechanism for expression of CTL epitopes encoded in alternative reading frames

PubMed Central

1996-01-01

An increasing amount of evidence has shown that epitopes restricted to MHC class I molecules and recognized by CTL need not be encoded in a primary open reading frame (ORF). Such epitopes have been demonstrated after stop codons, in alternative reading frames (RF) and within introns. We have used a series of frameshifts (FS) introduced into the Influenza A/PR/8 /34 nucleoprotein (NP) gene to confirm the previous in vitro observations of cryptic epitope expression, and show that they are sufficiently expressed to prime immune responses in vivo. This presentation is not due to sub-dominant epitopes, transcription from cryptic promoters beyond the point of the FS, or internal initiation of translation. By introducing additional mutations to the construct exhibiting the most potent presentation, we have identified initiation codon readthrough (termed scanthrough here, where the scanning ribosome bypasses the conventional initiation codon, initiating translation further downstream) as the likely mechanism of epitope production. Further mutational analysis demonstrated that, while it should operate during the expression of wild-type (WT) protein, scanthrough does not provide a major source of processing substrate in our system. These findings suggest (i) that the full array of self- and pathogen-derived epitopes available during thymic selection and infection has not been fully appreciated and (ii) that cryptic epitope expression should be considered when the specificity of a CTL response cannot be identified or in therapeutic situations when conventional CTL targets are limited, as may be the case with latent viral infections and transformed cells. Finally, initiation codon readthrough provides a plausible explanation for the presentation of exocytic proteins by MHC class I molecules. PMID:8879204

Complete mitochondrial genome of Palawan peacock-pheasant Polyplectron napoleonis (Galliformes, Phasianidae).

PubMed

Quach, Tommy; Brooks, Daniel M; Miranda, Hector C

2016-01-01

The complete mitochondrial genome of the Palawan peacock-pheasant Polyplectron napoleonis is 16,710 bp and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control-region. All protein-coding genes use the standard ATG start codon, except for cox1 which has GTG start codon. Seven out of 13 PCGs have TAA stop codons, two have AGG (cox1 and nd6), and three PCGs (nd2, cox2 and nd4) have incomplete stop codon of just T- - nucleotide.

The Role of +4U as an Extended Translation Termination Signal in Bacteria

PubMed Central

Wei, Yulong; Xia, Xuhua

2017-01-01

Termination efficiency of stop codons depends on the first 3′ flanking (+4) base in bacteria and eukaryotes. In both Escherichia coli and Saccharomyces cerevisiae, termination read-through is reduced in the presence of +4U; however, the molecular mechanism underlying +4U function is poorly understood. Here, we perform comparative genomics analysis on 25 bacterial species (covering Actinobacteria, Bacteriodetes, Cyanobacteria, Deinococcus-Thermus, Firmicutes, Proteobacteria, and Spirochaetae) with bioinformatics approaches to examine the influence of +4U in bacterial translation termination by contrasting highly- and lowly-expressed genes (HEGs and LEGs, respectively). We estimated gene expression using the recently formulated Index of Translation Elongation, ITE, and identified stop codon near-cognate transfer RNAs (tRNAs) from well-annotated genomes. We show that +4U was consistently overrepresented in UAA-ending HEGs relative to LEGs. The result is consistent with the interpretation that +4U enhances termination mainly for UAA. Usage of +4U decreases in GC-rich species where most stop codons are UGA and UAG, with few UAA-ending genes, which is expected if UAA usage in HEGs drives up +4U usage. In HEGs, +4U usage increases significantly with abundance of UAA nc_tRNAs (near-cognate tRNAs that decode codons differing from UAA by a single nucleotide), particularly those with a mismatch at the first stop codon site. UAA is always the preferred stop codon in HEGs, and our results suggest that UAAU is the most efficient translation termination signal in bacteria. PMID:27903612

L-MPZ, a Novel Isoform of Myelin P0, Is Produced by Stop Codon Readthrough*

PubMed Central

Yamaguchi, Yoshihide; Hayashi, Akiko; Campagnoni, Celia W.; Kimura, Akio; Inuzuka, Takashi; Baba, Hiroko

2012-01-01

Myelin protein zero (P0 or MPZ) is a major myelin protein (∼30 kDa) expressed in the peripheral nervous system (PNS) in terrestrial vertebrates. Several groups have detected a P0-related 36-kDa (or 35-kDa) protein that is expressed in the PNS as an antigen for the serum IgG of patients with neuropathy. The molecular structure and function of this 36-kDa protein are, however, still unknown. We hypothesized that the 36-kDa protein may be derived from P0 mRNA by stop codon readthrough. We found a highly conserved region after the regular stop codon in predicted sequences from the 3′-UTR of P0 in higher animals. MS of the 36-kDa protein revealed that both P0 peptides and peptides deduced from the P0 3′-UTR sequence were found among the tryptic fragments. In transfected cells and in an in vitro transcription/translation system, the 36-kDa molecule was also produced from the identical mRNA that produced P0. We designated this 36-kDa molecule as large myelin protein zero (L-MPZ), a novel isoform of P0 that contains an additional domain at the C terminus. In the PNS, L-MPZ was localized in compact myelin. In transfected cells, just like P0, L-MPZ was localized at cell-cell adhesion sites in the plasma membrane. These results suggest that L-MPZ produced by the stop codon readthrough mechanism is potentially involved in myelination. Since this is the first finding of stop codon readthrough in a common mammalian protein, detailed analysis of L-MPZ expression will help to understand the mechanism of stop codon readthrough in mammals. PMID:22457349

Dysfunctional growth hormone receptor in a strain of sex-linked dwarf chicken: evidence for a mutation in the intracellular domain.

PubMed

Agarwal, S K; Cogburn, L A; Burnside, J

1994-09-01

The sex-linked dwarf (dwdw) chicken represents a valuable animal model for studying GH insensitivity and the consequence of mutations in the GH receptor (GHR) gene. We have recently reported undetectable hepatic GH-binding activity and an aberrantly sized transcript in a strain of dwdw chickens obtained from Arbor Acre Farms, Inc. (Glastonbury, CT, USA). Southern blot analysis of the chicken GHR (cGHR) gene revealed a restriction-fragment length polymorphism in HindIII and EcoRI digests of genomic DNA in this strain of dwdw chicken. In order to localize the molecular mutation, we analysed the gene structure and determined the complete sequence of the 3' untranslated region (3' UTR) of the normal cGHR. With the use of this information, we located a large deletion in the 3' end of the cGHR gene of the Connecticut (CT) strain of dwdw chicken. This deletion (1773 bp) contained 27 highly conserved amino acids of the 3' end of the coding region, the in-frame stop codon, a less frequently used poly(A) signal that is normally found 445 bp downstream of the stop codon, and a large portion of the 3' UTR. Because of this deletion, 27 novel amino acids were substituted and the open reading frame was extended for an additional 26 amino acids before reaching the transcriptional termination site. The predicted amino acid sequence of the novel carboxyl-terminus of the dwdw cGHR is largely hydrophobic with a polylysine tail, whereas the carboxyl-terminus of the wild-type (DwDw) cGHR is composed of hydrophilic amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel BRCA1 splice-site mutation in ovarian cancer patients of Slavic origin.

PubMed

Krivokuca, Ana; Dragos, Vita Setrajcic; Stamatovic, Ljiljana; Blatnik, Ana; Boljevic, Ivana; Stegel, Vida; Rakobradovic, Jelena; Skerl, Petra; Jovandic, Stevo; Krajc, Mateja; Magic, Mirjana Brankovic; Novakovic, Srdjan

2018-04-01

Mutations in breast cancer susceptibility gene 1 (BRCA1) lead to defects in a number of cellular pathways including DNA damage repair and transcriptional regulation, resulting in the elevated genome instability and predisposing to breast and ovarian cancers. We report a novel mutation LRG_292t1:c.4356delA,p.(Ala1453Glnfs*3) in the 12th exon of BRCA1, in the splice site region near the donor site of intron 12. It is a frameshift mutation with the termination codon generated on the third amino acid position from the site of deletion. Human Splice Finder 3.0 and MutationTaster have assessed this variation as disease causing, based on the alteration of splicing, creation of premature stop codon and other potential alterations initiated by nucleotide deletion. Among the most important alterations are frameshift and splice site changes (score of the newly created donor splice site: 0.82). c.4356delA was associated with two ovarian cancer cases in two families of Slavic origin. It was detected by next generation sequencing, and confirmed with Sanger sequencing in both cases. Because of the fact that it changes the reading frame of the protein, novel mutation c.4356delA p.(Ala1453Glnfs*3) in BRCA1 gene might be of clinical significance for hereditary ovarian cancer. Further functional as well as segregation analyses within the families are necessary for appropriate clinical classification of this variant. Since it has been detected in two ovarian cancer patients of Slavic origin, it is worth investigating founder effect of this mutation in Slavic populations.

A Single Base Pair Mutation Encoding a Premature Stop Codon in the MIS type II receptor is Responsible for Canine Persistent Müllerian Duct Syndrome

PubMed Central

Wu, Xiufeng; Wan, Shengqin; Pujar, Shashikant; Haskins, Mark E.; Schlafer, Donald H.; Lee, Mary M.; Meyers-Wallen, Vicki N.

2008-01-01

Müllerian Inhibiting Substance (MIS), a secreted glycoprotein in the Transforming Growth Factor-beta (TGF-beta) family of growth factors, mediates regression of the Müllerian ducts during embryonic sex differentiation in males. In Persistent Müllerian Duct Syndrome (PMDS), rather than undergoing involution, the Müllerian ducts persist in males, giving rise to the uterus, Fallopian tubes, and upper vagina. Genetic defects in MIS or its receptor (MISRII) have been identified in patients with PMDS. The phenotype in the canine model of PMDS derived from the miniature schnauzer breed is strikingly similar to that of human patients. In this model, PMDS is inherited as a sex-limited autosomal recessive trait. Previous studies indicated that a defect in the MIS receptor or its downstream signaling pathway was likely to be causative of the canine syndrome. In this study the canine PMDS phenotype and clinical sequelae are described in detail. Affected and unaffected members of this pedigree are genotyped, identifying a single base pair substitution in MISRII that introduces a stop codon in exon 3. The homozygous mutation terminates translation at 80 amino acids, eliminating much of the extracellular domain and the entire transmembrane and intracellular signaling domains. Findings in this model may enable insights to be garnered from correlation of detailed clinical descriptions with molecular defects, which are not otherwise possible in the human syndrome. PMID:18723470

Identification of Mutations Underlying 20 Inborn Errors of Metabolism in the United Arab Emirates Population

PubMed Central

Ben-Rebeh, Imen; Hertecant, Jozef L.; Al-Jasmi, Fatma A.; Aburawi, Hanan E.; Al-Yahyaee, Said A.; Al-Gazali, Lihadh

2012-01-01

Inborn errors of metabolism (IEM) are frequently encountered by physicians in the United Arab Emirates (UAE). However, the mutations underlying a large number of these disorders have not yet been determined. Therefore, the objective of this study was to identify the mutations underlying a number of IEM disorders among UAE residents from both national and expatriate families. A case series of patients from 34 families attending the metabolic clinic at Tawam Hospital were clinically evaluated, and molecular testing was carried out to determine their causative mutations. The mutation analysis was carried out at molecular genetics diagnostic laboratories. Thirty-eight mutations have been identified as responsible for twenty IEM disorders, including in the metabolism of amino acids, lipids, steroids, metal transport and mitochondrial energy metabolism, and lysosomal storage disorders. Nine of the identified mutations are novel, including two missense mutations, three premature stop codons and four splice site mutations. Mutation analysis of IEM disorders in the UAE population has an important impact on molecular diagnosis and genetic counseling for families affected by these disorders. PMID:22106832

Identification of a novel exonic mutation at -13 from 5' splice site causing exon skipping in a girl with mitochondrial acetoacetyl-coenzyme A thiolase deficiency.

PubMed Central

Fukao, T; Yamaguchi, S; Wakazono, A; Orii, T; Hoganson, G; Hashimoto, T

1994-01-01

We identified a novel exonic mutation which causes exon skipping in the mitochondrial acetoacetyl-CoA thiolase (T2) gene from a girl with T2 deficiency (GK07). GK07 is a compound heterozygote; the maternal allele has a novel G to T transversion at position 1136 causing Gly379 to Val substitution (G379V) of the T2 precursor. In case of in vivo expression analysis, cells transfected with this mutant cDNA showed no evidence of restored T2 activity. The paternal allele was associated with exon 8 skipping at the cDNA level. At the gene level, a C to T transition causing Gln272 to termination codon (Q272STOP) was identified within exon 8, 13 bp from the 5' splice site of intron 8 in the paternal allele. The mRNA with Q272STOP could not be detected in GK07 fibroblasts, presumably because pre-mRNA with Q272STOP was unstable because of the premature termination. In vivo splicing experiments revealed that the exonic mutation caused partial skipping of exon 8. This substitution was thought to alter the secondary structure of T2 pre-mRNA around exon 8 and thus impede normal splicing. The role of exon sequences in the splicing mechanism is indicated by the exon skipping which occurred with an exonic mutation. Images PMID:7907600

A novel c.240_241insGG mutation in NDP gene in a family with Norrie disease.

PubMed

Andarva, Monavvar; Jamshidi, Javad; Ghaedi, Hamid; Daftarian, Narsis; Emamalizadeh, Babak; Alehabib, Elham; Taghavi, Shaghyegh; Pouriran, Ramin; Darvish, Hossein

2018-03-01

Norrie disease (ND) is a rare, X-linked recessive disorder with the main characteristic of early childhood blindness. The aim of the present study was to identify the genetic cause of the disease and the phenotypic characteristics of the patients in an Iranian family with four affected males with ND. Norrie disease pseudoglioma (NDP) gene was sequenced and clinical examination was performed on patients. A GG dinucleotide insertion in exon 3 (c.240_241insGG) of NDP was detected in all patients. The mutation caused a frameshift and an early stop codon (p.Phe81Glyfs*23). A novel mutation was found in the NDP gene in the affected males of the family. As the mutation was absent in the normal male members of the family, it should be the genetic cause of the disease. © 2017 Optometry Australia.

A chondrodysplasia family produced by mutations in the diastrophic dysplasia sulfate transporter gene: Genotype/phenotype correlations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Superti-Furga, A.; Steinmann, B.; Gitzelmann, R.

1996-05-03

Achondrogenesis type 1B (ACG-1B), atelosteogenesis type 2 (AO-2), and diastrophic dysplasia (DTD) are recessively inherited chondrodysplasia of decreasing severity caused by mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene on chromosome 5. In these conditions, sulfate transport across the cell membrane is impaired which results in insufficient sulfation of cartilage proteoglycans and thus in an abnormally low sulfate content of cartilage. The severity of the phenotype correlates well with the predicted effect of the underlying DTDST mutations: homozygosity or compound heterozygosity for stop codons or transmembrane domain substitutions mostly result in achondrogenesis type 1B, while other structural or regulatorymore » mutations usually result in one of the less severe phenotypes. The chondrodysplasia arising at the DTDST locus constitute a bone dysplasia family with recessive inheritance. 28 refs., 2 tabs.« less

Ataxia-telangiectasia: founder effect among north African Jews.

PubMed

Gilad, S; Bar-Shira, A; Harnik, R; Shkedy, D; Ziv, Y; Khosravi, R; Brown, K; Vanagaite, L; Xu, G; Frydman, M; Lavin, M F; Hill, D; Tagle, D A; Shiloh, Y

1996-12-01

The ATM gene is responsible for the autosomal recessive disorder ataxia-telangiectasia (A-T), characterized by cerebellar degeneration, immunodeficiency and cancer predisposition. A-T carriers were reported to be moderately cancer-prone. A wide variety of A-T mutations, most of which are unique to single families, were identified in various ethnic groups, precluding carrier screening with mutation-specific assays. However, a single mutation was observed in 32/33 defective ATM alleles in Jewish A-T families of North African origin, coming from various regions of Morocco and Tunisia. This mutation, 103C-->T, results in a stop codon at position 35 of the ATM protein. In keeping with the nature of this mutation, various antibodies directed against the ATM protein failed to defect this protein in patient cells. A rapid carrier detection assay detected this mutation in three out of 488 ATM alleles of Jewish Moroccan or Tunisian origin. This founder effect provides a unique opportunity for population-based screening for A-T carriers in a large Jewish community.

Deep intronic GPR143 mutation in a Japanese family with ocular albinism

PubMed Central

Naruto, Takuya; Okamoto, Nobuhiko; Masuda, Kiyoshi; Endo, Takao; Hatsukawa, Yoshikazu; Kohmoto, Tomohiro; Imoto, Issei

2015-01-01

Deep intronic mutations are often ignored as possible causes of human disease. Using whole-exome sequencing, we analysed genomic DNAs of a Japanese family with two male siblings affected by ocular albinism and congenital nystagmus. Although mutations or copy number alterations of coding regions were not identified in candidate genes, the novel intronic mutation c.659-131 T > G within GPR143 intron 5 was identified as hemizygous in affected siblings and as heterozygous in the unaffected mother. This mutation was predicted to create a cryptic splice donor site within intron 5 and activate a cryptic acceptor site at 41nt upstream, causing the insertion into the coding sequence of an out-of-frame 41-bp pseudoexon with a premature stop codon in the aberrant transcript, which was confirmed by minigene experiments. This result expands the mutational spectrum of GPR143 and suggests the utility of next-generation sequencing integrated with in silico and experimental analyses for improving the molecular diagnosis of this disease. PMID:26061757

Deep intronic GPR143 mutation in a Japanese family with ocular albinism.

PubMed

Naruto, Takuya; Okamoto, Nobuhiko; Masuda, Kiyoshi; Endo, Takao; Hatsukawa, Yoshikazu; Kohmoto, Tomohiro; Imoto, Issei

2015-06-10

Deep intronic mutations are often ignored as possible causes of human disease. Using whole-exome sequencing, we analysed genomic DNAs of a Japanese family with two male siblings affected by ocular albinism and congenital nystagmus. Although mutations or copy number alterations of coding regions were not identified in candidate genes, the novel intronic mutation c.659-131 T > G within GPR143 intron 5 was identified as hemizygous in affected siblings and as heterozygous in the unaffected mother. This mutation was predicted to create a cryptic splice donor site within intron 5 and activate a cryptic acceptor site at 41nt upstream, causing the insertion into the coding sequence of an out-of-frame 41-bp pseudoexon with a premature stop codon in the aberrant transcript, which was confirmed by minigene experiments. This result expands the mutational spectrum of GPR143 and suggests the utility of next-generation sequencing integrated with in silico and experimental analyses for improving the molecular diagnosis of this disease.

Identification of the Bovine Arachnomelia Mutation by Massively Parallel Sequencing Implicates Sulfite Oxidase (SUOX) in Bone Development

PubMed Central

Drögemüller, Cord; Tetens, Jens; Sigurdsson, Snaevar; Gentile, Arcangelo; Testoni, Stefania; Lindblad-Toh, Kerstin; Leeb, Tosso

2010-01-01

Arachnomelia is a monogenic recessive defect of skeletal development in cattle. The causative mutation was previously mapped to a ∼7 Mb interval on chromosome 5. Here we show that array-based sequence capture and massively parallel sequencing technology, combined with the typical family structure in livestock populations, facilitates the identification of the causative mutation. We re-sequenced the entire critical interval in a healthy partially inbred cow carrying one copy of the critical chromosome segment in its ancestral state and one copy of the same segment with the arachnomelia mutation, and we detected a single heterozygous position. The genetic makeup of several partially inbred cattle provides extremely strong support for the causality of this mutation. The mutation represents a single base insertion leading to a premature stop codon in the coding sequence of the SUOX gene and is perfectly associated with the arachnomelia phenotype. Our findings suggest an important role for sulfite oxidase in bone development. PMID:20865119

Molecular diagnosis of analbuminemia: a new case caused by a nonsense mutation in the albumin gene.

PubMed

Dagnino, Monica; Caridi, Gianluca; Haenni, Ueli; Duss, Adrian; Aregger, Fabienne; Campagnoli, Monica; Galliano, Monica; Minchiotti, Lorenzo

2011-01-01

Analbuminemia is a rare autosomal recessive disorder manifested by the absence, or severe reduction, of circulating serum albumin (ALB). We report here a new case diagnosed in a 45 years old man of Southwestern Asian origin, living in Switzerland, on the basis of his low ALB concentration (0.9 g/L) in the absence of renal or gastrointestinal protein loss, or liver dysfunction. The clinical diagnosis was confirmed by a mutational analysis of the albumin (ALB) gene, carried out by single-strand conformational polymorphism (SSCP), heteroduplex analysis (HA), and DNA sequencing. This screening of the ALB gene revealed that the proband is homozygous for two mutations: the insertion of a T in a stretch of eight Ts spanning positions c.1289 + 23-c.1289 + 30 of intron 10 and a c.802 G > T transversion in exon 7. Whereas the presence of an additional T in the poly-T tract has no direct deleterious effect, the latter nonsense mutation changes the codon GAA for Glu244 to the stop codon TAA, resulting in a premature termination of the polypeptide chain. The putative protein product would have a length of only 243 amino acid residues instead of the normal 585 found in the mature serum albumin, but no evidence for the presence in serum of such a truncated polypeptide chain could be obtained by two dimensional electrophoresis and western blotting analysis.

Nuclear sequestration of COL1A1 mRNA transcript associated with type I osteogenesis imperfecta (OI)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Primorac, D.; Stover, M.L.; McKinstry, M.B.

Previously we identified an OI type I patient with a splice donor mutation that resulted in intron 26 retention instead of exon skipping and sequestration of normal levels of the mutant transcript in the nuclear compartment. Intron retention was consistent with the exon definition hypothesis for splice site selection since the size of the exon-intron-exon unit was less than 300 bp. Furthermore, the retained intron contained in-frame stop codons which is thought to cause the mutant RNA to remain within the nucleus rather than appearing in the cytoplasm. To test these hypotheses, genomic fragments containing the normal sequence or themore » donor mutation were cloned into a collagen minigene and expressed in stably tansfected NIH 3T3 cells. None of the modifications to the normal intron altered the level of RNA that accumulated in the cytoplasm, as expected. However none of the modifications to the mutant intron allowed accumulation of normal levels of mRNA in the cytoplasm. Moreover, in contrast to our findings in the patient`s cells only low levels of mutant transcript were found in the nucleus; a fraction of the transcript did appear in the cytoplasm which had spliced the mutant donor site correctly. Nuclear run-on experiments demonstrated equal levels of transcription from each transgene. Expression of another donor mutation known to cause in-frame exon skipping in OI type IV was accurately reproduced in the minigene in transfected 3T3 cells. Our experience suggests that either mechanism can lead to formation of a null allele possibly related to the type of splicing events surrounding the potential stop codons. Understanding the rules governing inactivation of a collagen RNA transcript may be important in designing a strategy to inactivate a dominate negative mutation associated with the more severe forms of OI.« less

rpoB gene mutations among Mycobacterium tuberculosis isolates from extrapulmonary sites.

PubMed

Khosravi, Azar Dokht; Meghdadi, Hossein; Ghadiri, Ata A; Alami, Ameneh; Sina, Amir Hossein; Mirsaeidi, Mehdi

2018-03-01

The aim of this study was to analyze mutations occurring in the rpoB gene of Mycobacterium tuberculosis (MTB) isolates from clinical samples of extrapulmonary tuberculosis (EPTB). Seventy formalin-fixed, paraffin-embedded samples and fresh tissue samples from confirmed EPTB cases were analyzed. Nested PCR based on the rpoB gene was performed on the extracted DNAs, combined with cloning and subsequent sequencing. Sixty-seven (95.7%) samples were positive for nester PCR. Sequence analysis of the 81 bp region of the rpoB gene demonstrated mutations in 41 (61.2%) of 67 sequenced samples. Several point mutations including deletion mutations at codons 510, 512, 513 and 515, with 45% and 51% of the mutations in codons 512 and 513 respectively were seen, along with 26% replacement mutations at codons 509, 513, 514, 518, 520, 524 and 531. The most common alteration was Gln → His, at codon 513, presented in 30 (75.6%) isolates. This study demonstrated sequence alterations in codon 513 of the 81 bp region of the rpoB gene as the most common mutation occurred in 75.6% of molecularly confirmed rifampin-resistant strains. In addition, simultaneous mutation at codons 512 and 513 was demonstrated in 34.3% of the isolates. © 2018 APMIS. Published by John Wiley & Sons Ltd.

Differential Reprogramming of Isogenic Colorectal Cancer Cells by Distinct Activating KRAS Mutations

PubMed Central

2015-01-01

Oncogenic mutations of Ras at codons 12, 13, or 61, that render the protein constitutively active, are found in ∼16% of all cancer cases. Among the three major Ras isoforms, KRAS is the most frequently mutated isoform in cancer. Each Ras isoform and tumor type displays a distinct pattern of codon-specific mutations. In colon cancer, KRAS is typically mutated at codon 12, but a significant fraction of patients have mutations at codon 13. Clinical data suggest different outcomes and responsiveness to treatment between these two groups. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phosphoproteome of isogenic SW48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbor specific KRAS mutations (G12V, G12D, or G13D). Each mutation generates a distinct signature, with the most variability seen between G13D and the codon 12 KRAS mutants. One notable example of specific up-regulation in KRAS codon 12 mutant SW48 cells is provided by the short form of the colon cancer stem cell marker doublecortin-like Kinase 1 (DCLK1) that can be reversed by suppression of KRAS. PMID:25599653

The proto-oncogene KRAS and BRAF profiles and some clinical characteristics in colorectal cancer in the Turkish population.

PubMed

Ozen, Filiz; Ozdemir, Semra; Zemheri, Ebru; Hacimuto, Gizem; Silan, Fatma; Ozdemir, Ozturk

2013-02-01

The aim of the current study was to investigate the prevalence and predictive significance of the KRAS and BRAF mutations in Turkish patients with colorectal cancer (CRC). Totally, 53 fresh tumoral tissue specimens were investigated in patients with CRC. All specimens were obtained during routine surgery of patients who were histopathologically diagnosed and genotyped for common KRAS and BRAF point mutations. After DNA extraction, the target mutations were analyzed using the AutoGenomics INFINITI(®) assay, and some samples were confirmed by quantitative real-time polymerase chain reaction fluorescence melting curve analyses. KRAS mutations were found in 26 (49.05%) CRC samples. Twenty-seven samples (50.95%) had wild-type profiles for KRAS codon 12, 13, and 61 in the current cohort. In 17 (65.38%) samples, codon 12; in 7 (26.93%) samples, codon 13; and in 2 (7.69%) samples, codon 61 were found to be mutated, particularly in grade 2 of tumoral tissues. No point mutation was detected in BRAF codon Val600Glu for the studied CRC patients. Our study, based on a representative collection of human CRC tumors, indicates that KRAS gene mutations were detected in 49.05% of the samples, and the most frequent mutation was in the G12D codon. Results also showed that codons 12 and 13 of KRAS are relatively frequently without BRAF mutation in a CRC cohort from the Turkish population.

A novel nonsense mutation in CRYBB1 associated with autosomal dominant congenital cataract

PubMed Central

Yang, Juhua; Zhu, Yihua; Gu, Feng; He, Xiang; Cao, Zongfu; Li, Xuexi; Tong, Yi

2008-01-01

Purpose To identify the molecular defect underlying an autosomal dominant congenital nuclear cataract in a Chinese family. Methods Twenty-two members of a three-generation pedigree were recruited, clinical examinations were performed, and genomic DNA was extracted from peripheral blood leukocytes. All members were genotyped with polymorphic microsatellite markers adjacent to each of the known cataract-related genes. Linkage analysis was performed after genotyping. Candidate genes were screened for mutation using direct sequencing. Individuals were screened for presence of a mutation by restriction fragment length polymorphism (RFLP) analysis. Results Linkage analysis identified a maximum LOD score of 3.31 (recombination fraction [θ]=0.0) with marker D22S1167 on chromosome 22, which flanks the β-crystallin gene cluster (CRYBB3, CRYBB2, CRYBB1, and CRYBA4). Sequencing the coding regions and the flanking intronic sequences of these four candidate genes identified a novel, heterozygous C→T transition in exon 6 of CRYBB1 in the affected individuals of the family. This single nucleotide change introduced a novel BfaI site and was predicted to result in a nonsense mutation at codon 223 that changed a phylogenetically conserved amino acid to a stop codon (p.Q223X). RFLP analysis confirmed that this mutation co-segregated with the disease phenotype in all available family members and was not found in 100 normal unrelated individuals from the same ethnic background. Conclusions This study has identified a novel nonsense mutation in CRYBB1 (p.Q223X) associated with autosomal dominant congenital nuclear cataract. PMID:18432316

Different inactivating mutations of the mineralocorticoid receptor in fourteen families affected by type I pseudohypoaldosteronism.

PubMed

Sartorato, Paola; Lapeyraque, Anne-Laure; Armanini, Decio; Kuhnle, Ursula; Khaldi, Yasmina; Salomon, Rémi; Abadie, Véronique; Di Battista, Eliana; Naselli, Arturo; Racine, Alain; Bosio, Maurizio; Caprio, Massimiliano; Poulet-Young, Véronique; Chabrolle, Jean-Pierre; Niaudet, Patrick; De Gennes, Christiane; Lecornec, Marie-Hélène; Poisson, Elodie; Fusco, Anna Maria; Loli, Paola; Lombès, Marc; Zennaro, Maria-Christina

2003-06-01

We have analyzed the human mineralocorticoid receptor (hMR) gene in 14 families with autosomal dominant or sporadic pseudohypoaldosteronism (PHA1), a rare form of mineralocorticoid resistance characterized by neonatal renal salt wasting and failure to thrive. Six heterozygous mutations were detected. Two frameshift mutations in exon 2 (insT1354, del8bp537) and one nonsense mutation in exon 4 (C2157A, Cys645stop) generate truncated proteins due to premature stop codons. Three missense mutations (G633R, Q776R, L979P) differently affect hMR function. The DNA binding domain mutant R633 exhibits reduced maximal transactivation, although its binding characteristics and ED(50) of transactivation are comparable with wild-type hMR. Ligand binding domain mutants R776 and P979 present reduced or absent aldosterone binding, respectively, which is associated with reduced or absent ligand-dependent transactivation capacity. Finally, P979 possesses a transdominant negative effect on wild-type hMR activity, whereas mutations G633R and Q776R probably result in haploinsufficiency in PHA1 patients. We conclude that hMR mutations are a common feature of autosomal dominant PHA1, being found in 70% of our familial cases. Their absence in some families underscores the importance of an extensive investigation of the hMR gene and the role of precise diagnostic procedures to allow for identification of other genes potentially involved in the disease.

A Novel Frameshift Mutation at Codons 138/139 (HBB: c.417_418insT) on the β-Globin Gene Leads to β-Thalassemia.

PubMed

Jiang, Fan; Huang, Lv-Yin; Chen, Gui-Lan; Zhou, Jian-Ying; Xie, Xing-Mei; Li, Dong-Zhi

2017-01-01

We describe a new β-thalassemic mutation in a Chinese subject. This allele develops by insertion of one nucleotide (+T) between codons 138 and 139 in the third exon of the β-globin gene. The mutation causes a frameshift that leads to a termination codon at codon 139. In the heterozygote, this allele has the phenotype of classical β-thalassemia (β-thal) minor.

Codon 13 KRAS mutation predicts patterns of recurrence in patients undergoing hepatectomy for colorectal liver metastases.

PubMed

Margonis, Georgios A; Kim, Yuhree; Sasaki, Kazunari; Samaha, Mario; Amini, Neda; Pawlik, Timothy M

2016-09-01

Investigations regarding the impact of tumor biology after surgical management of colorectal liver metastasis have focused largely on overall survival. We investigated the impact of codon-specific KRAS mutations on the rates and patterns of recurrence in patients after surgery for colorectal liver metastasis (CRLM). All patients who underwent curative-intent surgery for CRLM between 2002 and 2015 at Johns Hopkins who had available data on KRAS mutation status were identified. Clinico-pathologic data, recurrence patterns, and recurrence-free survival (RFS) were assessed using univariable and multivariable analyses. A total of 512 patients underwent resection only (83.2%) or resection plus radiofrequency ablation (16.8%). Although 5-year overall survival was 64.6%, 284 (55.5%) patients recurred with a median RFS time of 18.1 months. The liver was the initial recurrence site for 181 patients, whereas extrahepatic recurrence was observed in 162 patients. Among patients with an extrahepatic recurrence, 102 (63%) had a lung recurrence. Although overall KRAS mutation was not associated with overall RFS (P = 0.186), it was independently associated with a worse extrahepatic (P = 0.004) and lung RFS (P = 0.007). Among patients with known KRAS codon-specific mutations, patients with codon 13 KRAS mutation had a worse 5-year extrahepatic RFS (P = 0.01), whereas codon 12 mutations were not associated with extrahepatic (P = 0.11) or lung-specific recurrence rate (P = 0.24). On multivariable analysis, only codon 13 mutation independently predicted worse overall extrahepatic RFS (P = 0.004) and lung-specific RFS (P = 0.023). Among patients undergoing resection of CRLM, overall KRAS mutation was not associated with RFS. KRAS codon 13 mutations, but not codon 12 mutations, were associated with a higher risk for overall extrahepatic recurrence and lung-specific recurrence. Cancer 2016. © 2016 American Cancer Society. Cancer 2016;122:2698-2707. © 2016 American Cancer Society. © 2016 American Cancer Society.

Genotyping of beta thalassemia trait by high-resolution DNA melting analysis.

PubMed

Saetung, Rattika; Ongchai, Siriwan; Charoenkwan, Pimlak; Sanguansermsri, Torpong

2013-11-01

Beta thalassemia is a common hereditary hemalogogical disease in Thailand, with a prevalence of 5-8%. In this study, we evaluated the high resolution DNA melting (HRM) assay to identify beta thalassemia mutation in samples from 143 carriers of the beta thalassemia traits in at risk couples. The DNA was isolated from venous blood samples and tested for mutation under a series of 5 PCR-HRM (A, B, C, D and E primers) protocols. The A primers were for detection of beta thalassemia mutations in the HBB promoter region, the B primers for mutations in exon I, the C primers for exon II, the D primers for exon III and the E primers for the 3.4 kb deletion mutation. The mutations were diagnosed by comparing the complete melting curve profiles of a wild type control with those for each mutant sample. With the PCR-HRM technique, fourteen types of beta thalassemia mutations were detected. Each mutation had a unique and specific melting profile. The mutations included 36.4% (52 cases) codon 41/42-CTTT, 26.6% (38 cases) codon 17 A-T, 11.2% (16 cases) IVS1-1 G-T, 8.4% (12 cases) codon 71/72 +A, 8.4% (12 cases) of the 3.4 kb deletion and 3.5% (5 cases) -28 A-G. The remainder included one instance each of -87 C-A, -31 A-C, codon 27/28 +C, codon 30 G-A, IVS1-5 G-C, codon 35 C-A, codon 41-C and IVSII -654 C-T. Of the total cases, 85.8% of the mutations could be detected by primers B and C. The PCR-HRM method provides a rapid, simple and highly feasible strategy for mutation screening of beta thalassemia traits.

The first de novo mutation of the connexin 32 gene associated with X linked Charcot-Marie-Tooth disease.

PubMed Central

Meggouh, F; Benomar, A; Rouger, H; Tardieu, S; Birouk, N; Tassin, J; Barhoumi, C; Yahyaoui, M; Chkili, T; Brice, A; LeGuern, E

1998-01-01

X linked Charcot-Marie-Tooth disease (CMTX) is a hereditary motor and sensory neuropathy caused by mutations in the connexin 32 gene (Cx32). Using the SSCP technique and direct sequencing of PCR amplified genomic DNA fragments of the Cx32 gene from a Moroccan patient and her relatives, we identified the first de novo mutation of the Cx32 gene, consisting of a deletion of a G residue at position 499 in the Cx32 open reading frame. This previously unreported mutation produces a frameshift at position 147 in the protein and introduces a premature stop codon (TAG) at nucleotide 643, which results in the production of a truncated Cx32 molecule. This mutation illustrates the risk of an erroneous diagnosis of autosomal recessive CMT, especially in populations where consanguineous unions are frequent, and its consequences for genetic counselling, which can be avoided by molecular analysis. Images PMID:9541114

Functional analysis of a nonstop mutation in MITF gene identified in a patient with Waardenburg syndrome type 2

PubMed Central

Sun, Jie; Hao, Ziqi; Luo, Hunjin; He, Chufeng; Mei, Lingyun; Liu, Yalan; Wang, Xueping; Niu, Zhijie; Chen, Hongsheng; Li, Jia-Da; Feng, Yong

2017-01-01

Waardenburg syndrome (WS) is an autosomal dominant inherited neurogenic disorder with the combination of various degrees of sensorineural deafness and pigmentary abnormalities affecting the skin, hair and eye. The four subtypes of WS were defined on the basis of the presence or absence of additional symptoms. Mutation of human microphthalmia-associated transcription factor (MITF) gene gives rise to WS2. Here, we identified a novel WS-associated mutation at the stop codon of MITF (p.X420Y) in a Chinese WS2 patient. This mutation resulted in an extension of extra 33 amino-acid residues in MITF. The mutant MITF appeared in both the nucleus and the cytoplasm, whereas the wild-type MITF was localized in the nucleus exclusively. The mutation led to a reduction in the transcriptional activities, whereas the DNA-binding activity was not altered. We show that the foremost mechanism was haploinsufficiency for the mild phenotypes of WS2 induced in X420Y MITF. PMID:28356565

Functional analysis of a nonstop mutation in MITF gene identified in a patient with Waardenburg syndrome type 2.

PubMed

Sun, Jie; Hao, Ziqi; Luo, Hunjin; He, Chufeng; Mei, Lingyun; Liu, Yalan; Wang, Xueping; Niu, Zhijie; Chen, Hongsheng; Li, Jia-Da; Feng, Yong

2017-07-01

Waardenburg syndrome (WS) is an autosomal dominant inherited neurogenic disorder with the combination of various degrees of sensorineural deafness and pigmentary abnormalities affecting the skin, hair and eye. The four subtypes of WS were defined on the basis of the presence or absence of additional symptoms. Mutation of human microphthalmia-associated transcription factor (MITF) gene gives rise to WS2. Here, we identified a novel WS-associated mutation at the stop codon of MITF (p.X420Y) in a Chinese WS2 patient. This mutation resulted in an extension of extra 33 amino-acid residues in MITF. The mutant MITF appeared in both the nucleus and the cytoplasm, whereas the wild-type MITF was localized in the nucleus exclusively. The mutation led to a reduction in the transcriptional activities, whereas the DNA-binding activity was not altered. We show that the foremost mechanism was haploinsufficiency for the mild phenotypes of WS2 induced in X420Y MITF.

Whole exome sequencing in recurrent early pregnancy loss.

PubMed

Qiao, Ying; Wen, Jiadi; Tang, Flamingo; Martell, Sally; Shomer, Naomi; Leung, Peter C K; Stephenson, Mary D; Rajcan-Separovic, Evica

2016-05-01

Exome sequencing can identify genetic causes of idiopathic recurrent pregnancy loss (RPL). We identified compound heterozygous deleterious mutations affecting DYNC2H1 and ALOX15 in two out of four families with RPL. Both genes have a role in early development. Bioinformatics analysis of all genes with rare and putatively pathogenic mutations in miscarriages and couples showed enrichment in pathways relevant to pregnancy loss, including the complement and coagulation cascades pathways. Next generation sequencing (NGS) is increasingly being used to identify known and novel gene mutations in children with developmental delay and in fetuses with ultrasound-detected anomalies. In contrast, NGS is rarely used to study pregnancy loss. Chromosome microarray analysis detects putatively causative DNA copy number variants (CNVs) in ∼2% of miscarriages and CNVs of unknown significance (predominantly parental in origin) in up to 40% of miscarriages. Therefore, a large number of miscarriages still have an unknown cause. Whole exome sequencing (WES) was performed using Illumina HiSeq 2000 platform on seven euploid miscarriages from four families with RPL. Golden Helix SVS v8.1.5 was used for data assessment and inheritance analysis for deleterious DNA variants predicted to severely disrupt protein-coding genes by introducing a frameshift, loss of the stop codon, gain of the stop codon, changes in splicing or the initial codon. Webgestalt (http://bioinfo.vanderbilt.edu/webgestalt/) was used for pathway and disease association enrichment analysis of a gene pool containing putatively pathogenic variants in miscarriages and couples in comparison to control gene pools. Compound heterozygous mutations in DYNC2H1 and ALOX15 were identified in miscarriages from two families with RPL. DYNC2H1 is involved in cilia biogenesis and has been associated with fetal lethality in humans. ALOX15 is expressed in placenta and its dysregulation has been associated with inflammation, placental, dysfunction, abnormal oxidative stress response and angiogenesis. The pool of putatively pathogenic single nucleotide variants (SNVs) and small insertions and deletions (indels) detected in the miscarriages showed enrichment in 'complement and coagulation cascades pathway', and 'ciliary motility disorders'. We conclude that CNVs, individual SNVs and pool of deleterious gene mutations identified by exome sequencing could contribute to RPL. The size of our sample cohort is small. The functional effect of candidate mutations should be evaluated to determine whether the mutations are causative. This is the first study to assess whether SNVs may contribute to the pathogenesis of miscarriage. Furthermore, our findings suggest that collective effect of mutations in relevant biological pathways could be implicated in RPL. The study was funded by Canadian Institutes of Health Research (grant MOP 106467) and Michael Smith Foundation of Health Research Career Scholar salary award to ERS. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Whole exome sequencing in recurrent early pregnancy loss

PubMed Central

Qiao, Ying; Wen, Jiadi; Tang, Flamingo; Martell, Sally; Shomer, Naomi; Leung, Peter C.K.; Stephenson, Mary D.; Rajcan-Separovic, Evica

2016-01-01

STUDY HYPOTHESIS Exome sequencing can identify genetic causes of idiopathic recurrent pregnancy loss (RPL). STUDY FINDING We identified compound heterozygous deleterious mutations affecting DYNC2H1 and ALOX15 in two out of four families with RPL. Both genes have a role in early development. Bioinformatics analysis of all genes with rare and putatively pathogenic mutations in miscarriages and couples showed enrichment in pathways relevant to pregnancy loss, including the complement and coagulation cascades pathways. WHAT IS KNOWN ALREADY Next generation sequencing (NGS) is increasingly being used to identify known and novel gene mutations in children with developmental delay and in fetuses with ultrasound-detected anomalies. In contrast, NGS is rarely used to study pregnancy loss. Chromosome microarray analysis detects putatively causative DNA copy number variants (CNVs) in ∼2% of miscarriages and CNVs of unknown significance (predominantly parental in origin) in up to 40% of miscarriages. Therefore, a large number of miscarriages still have an unknown cause. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Whole exome sequencing (WES) was performed using Illumina HiSeq 2000 platform on seven euploid miscarriages from four families with RPL. Golden Helix SVS v8.1.5 was used for data assessment and inheritance analysis for deleterious DNA variants predicted to severely disrupt protein-coding genes by introducing a frameshift, loss of the stop codon, gain of the stop codon, changes in splicing or the initial codon. Webgestalt (http://bioinfo.vanderbilt.edu/webgestalt/) was used for pathway and disease association enrichment analysis of a gene pool containing putatively pathogenic variants in miscarriages and couples in comparison to control gene pools. MAIN RESULTS AND THE ROLE OF CHANCE Compound heterozygous mutations in DYNC2H1 and ALOX15 were identified in miscarriages from two families with RPL. DYNC2H1 is involved in cilia biogenesis and has been associated with fetal lethality in humans. ALOX15 is expressed in placenta and its dysregulation has been associated with inflammation, placental, dysfunction, abnormal oxidative stress response and angiogenesis. The pool of putatively pathogenic single nucleotide variants (SNVs) and small insertions and deletions (indels) detected in the miscarriages showed enrichment in ‘complement and coagulation cascades pathway’, and ‘ciliary motility disorders’. We conclude that CNVs, individual SNVs and pool of deleterious gene mutations identified by exome sequencing could contribute to RPL. LIMITATIONS, REASONS FOR CAUTION The size of our sample cohort is small. The functional effect of candidate mutations should be evaluated to determine whether the mutations are causative. WIDER IMPLICATIONS OF THE FINDINGS This is the first study to assess whether SNVs may contribute to the pathogenesis of miscarriage. Furthermore, our findings suggest that collective effect of mutations in relevant biological pathways could be implicated in RPL. STUDY FUNDING AND COMPETING INTEREST(S) The study was funded by Canadian Institutes of Health Research (grant MOP 106467) and Michael Smith Foundation of Health Research Career Scholar salary award to ERS. PMID:26826164

Genomic analysis of codon usage shows influence of mutation pressure, natural selection, and host features on Marburg virus evolution.

PubMed

Nasrullah, Izza; Butt, Azeem M; Tahir, Shifa; Idrees, Muhammad; Tong, Yigang

2015-08-26

The Marburg virus (MARV) has a negative-sense single-stranded RNA genome, belongs to the family Filoviridae, and is responsible for several outbreaks of highly fatal hemorrhagic fever. Codon usage patterns of viruses reflect a series of evolutionary changes that enable viruses to shape their survival rates and fitness toward the external environment and, most importantly, their hosts. To understand the evolution of MARV at the codon level, we report a comprehensive analysis of synonymous codon usage patterns in MARV genomes. Multiple codon analysis approaches and statistical methods were performed to determine overall codon usage patterns, biases in codon usage, and influence of various factors, including mutation pressure, natural selection, and its two hosts, Homo sapiens and Rousettus aegyptiacus. Nucleotide composition and relative synonymous codon usage (RSCU) analysis revealed that MARV shows mutation bias and prefers U- and A-ended codons to code amino acids. Effective number of codons analysis indicated that overall codon usage among MARV genomes is slightly biased. The Parity Rule 2 plot analysis showed that GC and AU nucleotides were not used proportionally which accounts for the presence of natural selection. Codon usage patterns of MARV were also found to be influenced by its hosts. This indicates that MARV have evolved codon usage patterns that are specific to both of its hosts. Moreover, selection pressure from R. aegyptiacus on the MARV RSCU patterns was found to be dominant compared with that from H. sapiens. Overall, mutation pressure was found to be the most important and dominant force that shapes codon usage patterns in MARV. To our knowledge, this is the first detailed codon usage analysis of MARV and extends our understanding of the mechanisms that contribute to codon usage and evolution of MARV.

Differential functional readthrough over homozygous nonsense mutations contributes to the bleeding phenotype in coagulation factor VII deficiency.

PubMed

Branchini, A; Ferrarese, M; Lombardi, S; Mari, R; Bernardi, F; Pinotti, M

2016-10-01

Essentials Potentially null homozygous Factor(F)7 nonsense mutations are associated to variable bleeding symptoms. Readthrough of p.Ser112X (life-threatening) and p.Cys132X (moderate) stop codons was investigated. Readthrough-mediated insertion of wild-type or tolerated residues produce functional proteins. Functional readthrough over homozygous F7 nonsense mutations contributes to the bleeding phenotype. Background Whereas the rare homozygous nonsense mutations causing factor (F)VII deficiency may predict null conditions that are almost completely incompatible with life, they are associated with appreciable differences in hemorrhagic symptoms. The misrecognition of premature stop codons (readthrough) may account for variable levels of functional full-length proteins. Objectives To experimentally evaluate the basal and drug-induced levels of FVII resulting from the homozygous p.Cys132X and p.Ser112X nonsense mutations that are associated with moderate (132X) or life-threatening (112X) symptoms, and that are predicted to undergo readthrough with (132X) or without (112X) production of wild-type FVII. Methods We transiently expressed recombinant FVII (rFVII) nonsense and missense variants in human embryonic kidney 293 cells, and evaluated secreted FVII protein and functional levels by ELISA, activated FX generation, and coagulation assays. Results The levels of functional FVII produced by p.Cys132X and p.Ser112X mutants (rFVII-132X, 1.1% ± 0.2% of wild-type rFVII; rFVII-112X, 0.5% ± 0.1% of wild-type rFVII) were compatible with the occurrence of spontaneous readthrough, which was magnified by the addition of G418 - up to 12% of the wild-type value for the rFVII-132X nonsense variant. The predicted missense variants arising from readthrough abolished (rFVII-132Trp/Arg) or reduced (rFVII-112Trp/Cys/Arg, 22-45% of wild-type levels) secretion and function. These data suggest that the appreciable rescue of p.Cys132X function was driven by reinsertion of the wild-type residue, whereas the minimal p.Ser112X function was explained by missense changes permitting FVII secretion and function. Conclusions The extent of functional readthrough might explain differences in the bleeding phenotype of patients homozygous for F7 nonsense mutations, and prevent null conditions even for the most readthrough-unfavorable mutations. © 2016 International Society on Thrombosis and Haemostasis.

PCR-RFLP to Detect Codon 248 Mutation in Exon 7 of "p53" Tumor Suppressor Gene

ERIC Educational Resources Information Center

Ouyang, Liming; Ge, Chongtao; Wu, Haizhen; Li, Suxia; Zhang, Huizhan

2009-01-01

Individual genome DNA was extracted fast from oral swab and followed up with PCR specific for codon 248 of "p53" tumor suppressor gene. "Msp"I restriction mapping showed the G-C mutation in codon 248, which closely relates to cancer susceptibility. Students learn the concepts, detection techniques, and research significance of point mutations or…

The impact of KRAS mutations on VEGF-A production and tumour vascular network

PubMed Central

2013-01-01

Background The malignant potential of tumour cells may be influenced by the molecular nature of KRAS mutations being codon 13 mutations less aggressive than codon 12 ones. Their metabolic profile is also different, with an increased anaerobic glycolytic metabolism in cells harbouring codon 12 KRAS mutations compared with cells containing codon 13 mutations. We hypothesized that this distinct metabolic behaviour could be associated with different HIF-1α expression and a distinct angiogenic profile. Methods Codon13 KRAS mutation (ASP13) or codon12 KRAS mutation (CYS12) NIH3T3 transfectants were analyzed in vitro and in vivo. Expression of HIF-1α, and VEGF-A was studied at RNA and protein levels. Regulation of VEGF-A promoter activity was assessed by means of luciferase assays using different plasmid constructs. Vascular network was assessed in tumors growing after subcutaneous inoculation. Non parametric statistics were used for analysis of results. Results Our results show that in normoxic conditions ASP13 transfectants exhibited less HIF-1α protein levels and activity than CYS12. In contrast, codon 13 transfectants exhibited higher VEGF-A mRNA and protein levels and enhanced VEGF-A promoter activity. These differences were due to a differential activation of Sp1/AP2 transcription elements of the VEGF-A promoter associated with increased ERKs signalling in ASP13 transfectants. Subcutaneous CYS12 tumours expressed less VEGF-A and showed a higher microvessel density (MVD) than ASP13 tumours. In contrast, prominent vessels were only observed in the latter. Conclusion Subtle changes in the molecular nature of KRAS oncogene activating mutations occurring in tumour cells have a major impact on the vascular strategy devised providing with new insights on the role of KRAS mutations on angiogenesis. PMID:23506169

Molecular Diagnosis of Analbuminemia: A New Case Caused by a Nonsense Mutation in the Albumin Gene

PubMed Central

Dagnino, Monica; Caridi, Gianluca; Haenni, Ueli; Duss, Adrian; Aregger, Fabienne; Campagnoli, Monica; Galliano, Monica; Minchiotti, Lorenzo

2011-01-01

Analbuminemia is a rare autosomal recessive disorder manifested by the absence, or severe reduction, of circulating serum albumin (ALB). We report here a new case diagnosed in a 45 years old man of Southwestern Asian origin, living in Switzerland, on the basis of his low ALB concentration (0.9 g/L) in the absence of renal or gastrointestinal protein loss, or liver dysfunction. The clinical diagnosis was confirmed by a mutational analysis of the albumin (ALB) gene, carried out by single-strand conformational polymorphism (SSCP), heteroduplex analysis (HA), and DNA sequencing. This screening of the ALB gene revealed that the proband is homozygous for two mutations: the insertion of a T in a stretch of eight Ts spanning positions c.1289 + 23–c.1289 + 30 of intron 10 and a c.802 G > T transversion in exon 7. Whereas the presence of an additional T in the poly-T tract has no direct deleterious effect, the latter nonsense mutation changes the codon GAA for Glu244 to the stop codon TAA, resulting in a premature termination of the polypeptide chain. The putative protein product would have a length of only 243 amino acid residues instead of the normal 585 found in the mature serum albumin, but no evidence for the presence in serum of such a truncated polypeptide chain could be obtained by two dimensional electrophoresis and western blotting analysis. PMID:22174600

A Frameshift Mutation in the Cubilin Gene (CUBN) in Border Collies with Imerslund-Gräsbeck Syndrome (Selective Cobalamin Malabsorption)

PubMed Central

Owczarek-Lipska, Marta; Jagannathan, Vidhya; Drögemüller, Cord; Lutz, Sabina; Glanemann, Barbara

2013-01-01

Imerslund-Gräsbeck syndrome (IGS) or selective cobalamin malabsorption has been described in humans and dogs. IGS occurs in Border Collies and is inherited as a monogenic autosomal recessive trait in this breed. Using 7 IGS cases and 7 non-affected controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 3.53 Mb interval on chromosome 2. We re-sequenced the genome of one affected dog at ∼10× coverage and detected 17 non-synonymous variants in the critical interval. Two of these non-synonymous variants were in the cubilin gene (CUBN), which is known to play an essential role in cobalamin uptake from the ileum. We tested these two CUBN variants for association with IGS in larger cohorts of dogs and found that only one of them was perfectly associated with the phenotype. This variant, a single base pair deletion (c.8392delC), is predicted to cause a frameshift and premature stop codon in the CUBN gene. The resulting mutant open reading frame is 821 codons shorter than the wildtype open reading frame (p.Q2798Rfs*3). Interestingly, we observed an additional nonsense mutation in the MRC1 gene encoding the mannose receptor, C type 1, which was in perfect linkage disequilibrium with the CUBN frameshift mutation. Based on our genetic data and the known role of CUBN for cobalamin uptake we conclude that the identified CUBN frameshift mutation is most likely causative for IGS in Border Collies. PMID:23613799

Three cases of Creutzfeldt-Jakob disease with prion protein gene codon180 mutation presenting with pathological laughing and crying.

PubMed

Iwasaki, Yasushi

2012-08-15

Although there are no reports of pathological laughing and crying being observed in patients with Creutzfeldt-Jakob disease (CJD), the author experienced three patients with CJD with prion protein gene codon180 mutation (V180I CJD) who showed this characteristic clinical finding. This finding was observed from the early disease stage in all 3 patients and continued for several months. Startle reaction was also remarkable in all patients, although myoclonus was generally mild. The dissociation between the startle reaction and myoclonus was suspected to be another feature of V180I CJD. The pathological laughing and crying co-occured with the startle reaction and stopped right before the onset of akinetic mutism, and the degree of both symptoms was almost parallel during this period. On the basis of MRI and autopsy findings, pathological laughing and crying was suspected of being induced by the widespread cerebral cortical involvement that is characteristic of V180I CJD. From the present observations, the author speculated that pathological laughing and crying may be a comparatively frequent observation in V180I CJD patients. Copyright © 2012 Elsevier B.V. All rights reserved.

Molecular Decay of the Tooth Gene Enamelin (ENAM) Mirrors the Loss of Enamel in the Fossil Record of Placental Mammals

PubMed Central

Meredith, Robert W.; Gatesy, John; Murphy, William J.; Ryder, Oliver A.; Springer, Mark S.

2009-01-01

Vestigial structures occur at both the anatomical and molecular levels, but studies documenting the co-occurrence of morphological degeneration in the fossil record and molecular decay in the genome are rare. Here, we use morphology, the fossil record, and phylogenetics to predict the occurrence of “molecular fossils” of the enamelin (ENAM) gene in four different orders of placental mammals (Tubulidentata, Pholidota, Cetacea, Xenarthra) with toothless and/or enamelless taxa. Our results support the “molecular fossil” hypothesis and demonstrate the occurrence of frameshift mutations and/or stop codons in all toothless and enamelless taxa. We then use a novel method based on selection intensity estimates for codons (ω) to calculate the timing of iterated enamel loss in the fossil record of aardvarks and pangolins, and further show that the molecular evolutionary history of ENAM predicts the occurrence of enamel in basal representatives of Xenarthra (sloths, anteaters, armadillos) even though frameshift mutations are ubiquitous in ENAM sequences of living xenarthrans. The molecular decay of ENAM parallels the morphological degeneration of enamel in the fossil record of placental mammals and provides manifest evidence for the predictive power of Darwin's theory. PMID:19730686

Hearing loss in a patient with the myopathic form of mitochondrial DNA depletion syndrome and a novel mutation in the TK2 gene.

PubMed

Martí, Ramon; Nascimento, Andrés; Colomer, Jaume; Lara, Mari C; López-Gallardo, Ester; Ruiz-Pesini, Eduardo; Montoya, Julio; Andreu, Antoni L; Briones, Paz; Pineda, Mercè

2010-08-01

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is a devastating disorder of infancy caused by a significant reduction of the number of copies of mitochondrial DNA in one or more tissues. We report a Spanish patient with the myopathic form of MDS, harboring two mutations in the thymidine kinase 2 gene (TK2): a previously reported deletion (p.K244del) and a novel nucleotide duplication in the exon 2, generating a frameshift and premature stop codon. Sensorineural hearing loss was a predominant symptom in the patient and a novel feature of MDS due to TK2 mutations. The patient survived up to the age of 8.5 y, which confirms that survival above the age of 5 y is not infrequent in patients with MDS due to TK2 deficiency.

Negative and Translation Termination-Dependent Positive Control of FLI-1 Protein Synthesis by Conserved Overlapping 5′ Upstream Open Reading Frames in Fli-1 mRNA

PubMed Central

Sarrazin, Sandrine; Starck, Joëlle; Gonnet, Colette; Doubeikovski, Alexandre; Melet, Fabrice; Morle, François

2000-01-01

The proto-oncogene Fli-1 encodes a transcription factor of the ets family whose overexpression is associated with multiple virally induced leukemias in mouse, inhibits murine and avian erythroid cell differentiation, and induces drastic perturbations of early development in Xenopus. This study demonstrates the surprisingly sophisticated regulation of Fli-1 mRNA translation. We establish that two FLI-1 protein isoforms (of 51 and 48 kDa) detected by Western blotting in vivo are synthesized by alternative translation initiation through the use of two highly conserved in-frame initiation codons, AUG +1 and AUG +100. Furthermore, we show that the synthesis of these two FLI-1 isoforms is regulated by two short overlapping 5′ upstream open reading frames (uORF) beginning at two highly conserved upstream initiation codons, AUG −41 and GUG −37, and terminating at two highly conserved stop codons, UGA +35 and UAA +15. The mutational analysis of these two 5′ uORF revealed that each of them negatively regulates FLI-1 protein synthesis by precluding cap-dependent scanning to the 48- and 51-kDa AUG codons. Simultaneously, the translation termination of the two 5′ uORF appears to enhance 48-kDa protein synthesis, by allowing downstream reinitiation at the 48-kDa AUG codon, and 51-kDa protein synthesis, by allowing scanning ribosomes to pile up and consequently allowing upstream initiation at the 51-kDa AUG codon. To our knowledge, this is the first example of a cellular mRNA displaying overlapping 5′ uORF whose translation termination appears to be involved in the positive control of translation initiation at both downstream and upstream initiation codons. PMID:10757781

Mutational analysis of a patient with mucopolysaccharidosis type VII, and identification of pseudogenes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shipley, J.M.; Klinkenberg, M.; Wu, B.M.

1993-03-01

PCR of cDNA produced from patient fibroblasts allowed the authors to determine the paternal mutation in the first patient reported with [beta]-glucuronidase-deficiency mucopolysaccharidosis type VII (MPS VII). The G[r arrow]T transversion 1,881 bp downstream of the ATG translation initiation codon destroys an MboII restriction site and converts Trp627 to Cys (W627C). Digestion of genomic DNA PCR fragments with MboII indicated that the patient and the father were heterozygous for this missense mutation in exon 12. Failure to find cDNAs from patient RNA which did not contain this mutation suggested that the maternal mutation leads to greatly reduced synthesis or reducedmore » stability of mRNA from the mutant allele. In order to identify the maternal mutation, it was necessary to analyze genomic sequences. This approach was complicated by the finding of multiple unprocessed pseudogenes and/or closely related genes. Using PCR with a panel of human/rodent hybrid cell lines, the authors found that these pseudogenes were present over chromosomes 5-7, 20, and 22 and the Y chromosome. Conditions were defined which allowed them to amplify and characterize genomic sequences for the true [beta]-glucuronidase gene despite this background of related sequences. The patient proved to be heterozygous for a second mutation, in which a C[r arrow]T transition introduces a termination codon (R356STOP) in exon 7. The mother was also heterozygous for this mutation. Expression of a cDNA containing the maternal mutation produced no enzyme activity, as expected. Expression of the paternal mutation in COS-7 cells produced a surprisingly high (65% of control) level of activity. However, activity was 13% of control in transiently transfected murine MPS VII cells. The level of activity of this mutant allele appears to correlate with the level of overexpression. 39 refs., 5 figs., 1 tab.« less

Mutations in the Small GTPase Gene RAB39B Are Responsible for X-linked Mental Retardation Associated with Autism, Epilepsy, and Macrocephaly

PubMed Central

Giannandrea, Maila; Bianchi, Veronica; Mignogna, Maria Lidia; Sirri, Alessandra; Carrabino, Salvatore; D'Elia, Errico; Vecellio, Matteo; Russo, Silvia; Cogliati, Francesca; Larizza, Lidia; Ropers, Hans-Hilger; Tzschach, Andreas; Kalscheuer, Vera; Oehl-Jaschkowitz, Barbara; Skinner, Cindy; Schwartz, Charles E.; Gecz, Jozef; Van Esch, Hilde; Raynaud, Martine; Chelly, Jamel; de Brouwer, Arjan P.M.; Toniolo, Daniela; D'Adamo, Patrizia

2010-01-01

Human Mental Retardation (MR) is a common and highly heterogeneous pediatric disorder affecting around 3% of the general population; at least 215 X-linked MR (XLMR) conditions have been described, and mutations have been identified in 83 different genes, encoding proteins with a variety of function, such as chromatin remodeling, synaptic function, and intracellular trafficking. The small GTPases of the RAB family, which play an essential role in intracellular vesicular trafficking, have been shown to be involved in MR. We report here the identification of mutations in the small GTPase RAB39B gene in two male patients. One mutation in family X (D-23) introduced a stop codon seven amino acids after the start codon (c.21C > A; p.Y7X). A second mutation, in the MRX72 family, altered the 5′ splice site (c.215+1G > A) and normal splicing. Neither instance produced a protein. Mutations segregate with the disease in the families, and in some family members intellectual disabilities were associated with autism spectrum disorder, epileptic seizures, and macrocephaly. We show that RAB39B, a novel RAB GTPase of unknown function, is a neuronal-specific protein that is localized to the Golgi compartment. Its downregulation leads to an alteration in the number and morphology of neurite growth cones and a significant reduction in presynaptic buttons, suggesting that RAB39B is required for synapse formation and maintenance. Our results demonstrate developmental and functional neuronal alteration as a consequence of downregulation of RAB39B and emphasize the critical role of vesicular trafficking in the development of neurons and human intellectual abilities. PMID:20159109

JGH Foundation emerging leadership lecture. Significance of hepatitis B virus genotypes and mutations in the development of hepatocellular carcinoma in Asia.

PubMed

Chan, Henry Lik-Yuen

2011-01-01

Advances in molecular biology technology in the last two decades have allowed detailed study of the viral mutations and genomic heterogeneity of hepatitis B virus (HBV). The first mutant discovered was precore stop codon mutation. It was reported in HBeAg-negative patients and initially thought to associate with fulminant hepatitis. Subsequent studies have suggested that it is merely one of the mechanisms of losing HBeAg by the virus. Another mutation that can downregulate the production of HBeAg is the basal core promoter mutation, which is located in the X gene upstream of the precore region. Based on the configuration of codon 15 and the stability of the epsilon of the precore region, these two mutants will be differentially selected during the course of HBeAg seroconversion. The most common HBV genotypes in South-East Asia are genotype B and C HBV. The higher hepatocellular carcinoma (HCC) risk of genotype C HBV has been confirmed by longitudinal studies in Hong Kong and Taiwan. One possible carcinogenic mechanism is its association with basal core promoter mutation, which has also been found to be a risk factor of HCC. Within genotype C HBV, subgenotype Cs is predominant in South-East Asia and subgenotype Ce is predominant in East Asia. Subgenotype Ce HBV has been found to have the highest risk of HCC as compared with subgenotype Cs or genotype B HBV. The understanding of the carcinogenic mechanisms of these HBV strains may shed light into future therapeutics in the prevention and treatment of HBV-related HCC. © 2010 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.

The TREAT-NMD DMD Global Database: Analysis of More than 7,000 Duchenne Muscular Dystrophy Mutations

PubMed Central

Bladen, Catherine L; Salgado, David; Monges, Soledad; Foncuberta, Maria E; Kekou, Kyriaki; Kosma, Konstantina; Dawkins, Hugh; Lamont, Leanne; Roy, Anna J; Chamova, Teodora; Guergueltcheva, Velina; Chan, Sophelia; Korngut, Lawrence; Campbell, Craig; Dai, Yi; Wang, Jen; Barišić, Nina; Brabec, Petr; Lahdetie, Jaana; Walter, Maggie C; Schreiber-Katz, Olivia; Karcagi, Veronika; Garami, Marta; Viswanathan, Venkatarman; Bayat, Farhad; Buccella, Filippo; Kimura, En; Koeks, Zaïda; van den Bergen, Janneke C; Rodrigues, Miriam; Roxburgh, Richard; Lusakowska, Anna; Kostera-Pruszczyk, Anna; Zimowski, Janusz; Santos, Rosário; Neagu, Elena; Artemieva, Svetlana; Rasic, Vedrana Milic; Vojinovic, Dina; Posada, Manuel; Bloetzer, Clemens; Jeannet, Pierre-Yves; Joncourt, Franziska; Díaz-Manera, Jordi; Gallardo, Eduard; Karaduman, A Ayşe; Topaloğlu, Haluk; El Sherif, Rasha; Stringer, Angela; Shatillo, Andriy V; Martin, Ann S; Peay, Holly L; Bellgard, Matthew I; Kirschner, Jan; Flanigan, Kevin M; Straub, Volker; Bushby, Kate; Verschuuren, Jan; Aartsma-Rus, Annemieke; Béroud, Christophe; Lochmüller, Hanns

2015-01-01

Analyzing the type and frequency of patient-specific mutations that give rise to Duchenne muscular dystrophy (DMD) is an invaluable tool for diagnostics, basic scientific research, trial planning, and improved clinical care. Locus-specific databases allow for the collection, organization, storage, and analysis of genetic variants of disease. Here, we describe the development and analysis of the TREAT-NMD DMD Global database (http://umd.be/TREAT_DMD/). We analyzed genetic data for 7,149 DMD mutations held within the database. A total of 5,682 large mutations were observed (80% of total mutations), of which 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) were duplications (1 exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of all mutations), of which 358 (25%) were small deletions and 132 (9%) small insertions and 199 (14%) affected the splice sites. Point mutations totalled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic therapies for DMD including stop codon read-through therapies (10% of total mutations) and exon skipping therapy (80% of deletions and 55% of total mutations). PMID:25604253

Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6.

PubMed

Evans, Ben A; Smith, Olivia L; Pickerill, Ethan S; York, Mary K; Buenconsejo, Kristen J P; Chambers, Antonio E; Bernstein, Douglas A

2018-01-01

Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the Candida albicans UME6 Zn 2+ -binding domain, a known regulator of C. albicans filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type C. albicans . Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest.

Neutral lipid-storage disease with myopathy and extended phenotype with novel PNPLA2 mutation.

PubMed

Massa, Roberto; Pozzessere, Simone; Rastelli, Emanuele; Serra, Laura; Terracciano, Chiara; Gibellini, Manuela; Bozzali, Marco; Arca, Marcello

2016-04-01

Neutral lipid-storage disease with myopathy is caused by mutations in PNPLA2, which produce skeletal and cardiac myopathy. We report a man with multiorgan neutral lipid storage and unusual multisystem clinical involvement, including cognitive impairment. Quantitative brain MRI with voxel-based morphometry and extended neuropsychological assessment were performed. In parallel, the coding sequences and intron/exon boundaries of the PNPLA2 gene were screened by direct sequencing. Neuropsychological assessment revealed global cognitive impairment, and brain MRI showed reduced gray matter volume in the temporal lobes. Molecular characterization revealed a novel homozygous mutation in exon 5 of PNPLA2 (c.714C>A), resulting in a premature stop codon (p.Cys238*). Some PNPLA2 mutations, such as the one described here, may present with an extended phenotype, including brain involvement. In these cases, complete neuropsychological testing, combined with quantitative brain MRI, may help to characterize and quantify cognitive impairment. © 2016 Wiley Periodicals, Inc.

Identification of a novel NHS mutation in a Chinese family with Nance-Horan syndrome.

PubMed

Li, Aijun; Li, Bingzhen; Wu, Lemeng; Yang, Liping; Chen, Ningning; Ma, Zhizhong

2015-04-01

To identiy the disease causing mutation in a Chinese family presenting with early-onset cataract and dental anomalies. A specific Hereditary Eye Disease Enrichment Panel (HEDEP) (personalized customization by MyGenostics, Baltimore, MD) based on targeted exome capture technology was used to collect the protein coding regions of 30 early-onset cataract associated genes, and high throughput sequencing was done with Illumina HiSeq 2000 platform. The identified variant was confirmed with Sanger sequencing. A novel deletion in exon 4 (c.852delG) of NHS gene was identified; the identified 1 bp deletion altered the reading frame and was predicted to result in a premature stop codon after the addition of twelve novel amino acid (p.S285PfsX13). This mutation co-segregated in affected males and obligate female carriers, but was absent in 100 matched controls. Our findings broaden the spectrum of NHS mutations causing Nance-Horan syndrome and phenotypic spectrum of the disease in Chinese patients.

A de novo mutation in KIT causes white spotting in a subpopulation of German Shepherd dogs.

PubMed

Wong, A K; Ruhe, A L; Robertson, K R; Loew, E R; Williams, D C; Neff, M W

2013-06-01

Although variation in the KIT gene is a common cause of white spotting among domesticated animals, KIT has not been implicated in the diverse white spotting observed in the dog. Here, we show that a loss-of-function mutation in KIT recapitulates the coat color phenotypes observed in other species. A spontaneous white spotting observed in a pedigree of German Shepherd dogs was mapped by linkage analysis to a single locus on CFA13 containing KIT (pairwise LOD = 15). DNA sequence analysis identified a novel 1-bp insertion in the second exon that co-segregated with the phenotype. The expected frameshift and resulting premature stop codons predicted a severely truncated c-Kit receptor with presumably abolished activity. No dogs homozygous for the mutation were recovered from multiple intercrosses (P = 0.01), suggesting the mutation is recessively embryonic lethal. These observations are consistent with the effects of null alleles of KIT in other species. © 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.

Effect of KRAS codon13 mutations in patients with advanced colorectal cancer (advanced CRC) under oxaliplatin containing chemotherapy. Results from a translational study of the AIO colorectal study group

PubMed Central

2012-01-01

Background To evaluate the value of KRAS codon 13 mutations in patients with advanced colorectal cancer (advanced CRC) treated with oxaliplatin and fluoropyrimidines. Methods Tumor specimens from 201 patients with advanced CRC from a randomized, phase III trial comparing oxaliplatin/5-FU vs. oxaliplatin/capecitabine were retrospectively analyzed for KRAS mutations. Mutation data were correlated to response data (Overall response rate, ORR), progression-free survival (PFS) and overall survival (OS). Results 201 patients were analysed for KRAS mutation (61.2% males; mean age 64.2 ± 8.6 years). KRAS mutations were identified in 36.3% of tumors (28.8% in codon 12, 7.4% in codon 13). The ORR in codon 13 patients compared to codon 12 and wild type patients was significantly lower (p = 0.008). There was a tendency for a better overall survival in KRAS wild type patients compared to mutants (p = 0.085). PFS in all patients was not different in the three KRAS genetic groups (p = 0.72). However, we found a marked difference in PFS between patients with codon 12 and 13 mutant tumors treated with infusional 5-FU versus capecitabine based regimens. Conclusions Our data suggest that the type of KRAS mutation may be of clinical relevance under oxaliplatin combination chemotherapies without the addition of monoclonal antibodies in particular when overall response rates are important. Trial registration number 2002-04-017 PMID:22876876

Detection of EGFR Gene Mutation by Mutation-oriented LAMP Method.

PubMed

Matsumoto, Naoyuki; Kumasaka, Akira; Ando, Tomohiro; Komiyama, Kazuo

2018-04-01

Epidermal growth factor receptor (EGFR) is a target of molecular therapeutics for non-small cell lung cancer. EGFR gene mutations at codons 746-753 promote constitutive EGFR activation and result in worst prognosis. However, these mutations augment the therapeutic effect of EGFR-tyrosine kinase inhibitor. Therefore, the detection of EGFR gene mutations is important for determining treatment planning. The aim of the study was to establish a method to detect EGFR gene mutations at codons 746-753. EGFR gene mutation at codons 746-753 in six cancer cell lines were investigated. A loop-mediated isothermal amplification (LAMP)-based procedure was developed, that employed peptide nucleic acid to suppress amplification of the wild-type allele. This mutation-oriented LAMP can amplify the DNA fragment of the EGFR gene with codons 746-753 mutations within 30 min. Moreover, boiled cells can work as template resources. Mutation oriented-LAMP assay for EGFR gene mutation is sensitive on extracted DNA. This procedure would be capable of detecting EGFR gene mutation in sputum, pleural effusion, broncho-alveolar lavage fluid or trans-bronchial lung biopsy by chair side. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Decoding Mechanisms by which Silent Codon Changes Influence Protein Biogenesis and Function

PubMed Central

Bali, Vedrana; Bebok, Zsuzsanna

2015-01-01

Scope Synonymous codon usage has been a focus of investigation since the discovery of the genetic code and its redundancy. The occurrences of synonymous codons vary between species and within genes of the same genome, known as codon usage bias. Today, bioinformatics and experimental data allow us to compose a global view of the mechanisms by which the redundancy of the genetic code contributes to the complexity of biological systems from affecting survival in prokaryotes, to fine tuning the structure and function of proteins in higher eukaryotes. Studies analyzing the consequences of synonymous codon changes in different organisms have revealed that they impact nucleic acid stability, protein levels, structure and function without altering amino acid sequence. As such, synonymous mutations inevitably contribute to the pathogenesis of complex human diseases. Yet, fundamental questions remain unresolved regarding the impact of silent mutations in human disorders. In the present review we describe developments in this area concentrating on mechanisms by which synonymous mutations may affect protein function and human health. Purpose This synopsis illustrates the significance of synonymous mutations in disease pathogenesis. We review the different steps of gene expression affected by silent mutations, and assess the benefits and possible harmful effects of codon optimization applied in the development of therapeutic biologics. Physiological and medical relevance Understanding mechanisms by which synonymous mutations contribute to complex diseases such as cancer, neurodegeneration and genetic disorders, including the limitations of codon-optimized biologics, provides insight concerning interpretation of silent variants and future molecular therapies. PMID:25817479

Truncated ORF1 proteins can suppress LINE-1 retrotransposition in trans

PubMed Central

Sokolowski, Mark; Chynces, May; deHaro, Dawn; Christian, Claiborne M.

2017-01-01

Abstract Long interspersed element 1 (L1) is an autonomous non-LTR retroelement that is active in mammalian genomes. Although retrotranspositionally incompetent and functional L1 loci are present in the same genomes, it remains unknown whether non-functional L1s have any trans effect on mobilization of active elements. Using bioinformatic analysis, we identified over a thousand of human L1 loci containing at least one stop codon in their ORF1 sequence. RNAseq analysis confirmed that many of these loci are expressed. We demonstrate that introduction of equivalent stop codons in the full-length human L1 sequence leads to the expression of truncated ORF1 proteins. When supplied in trans some truncated human ORF1 proteins suppress human L1 retrotransposition. This effect requires the N-terminus and coiled-coil domain (C-C) as mutations within the ORF1p C-C domain abolish the suppressive effect of truncated proteins on L1 retrotransposition. We demonstrate that the expression levels and length of truncated ORF1 proteins influence their ability to suppress L1 retrotransposition. Taken together these findings suggest that L1 retrotransposition may be influenced by coexpression of defective L1 loci and that these L1 loci may reduce accumulation of de novo L1 integration events. PMID:28431148

Gene mutations and increased levels of p53 protein in human squamous cell carcinomas and their cell lines.

PubMed Central

Burns, J. E.; Baird, M. C.; Clark, L. J.; Burns, P. A.; Edington, K.; Chapman, C.; Mitchell, R.; Robertson, G.; Soutar, D.; Parkinson, E. K.

1993-01-01

Using immunocytochemical and Western blotting techniques we have demonstrated the presence of abnormally high levels of p53 protein in 8/24 (33%) of human squamous cell carcinomas (SCC) and 9/18 (50%) of SCC cell lines. There was a correlation between the immunocytochemical results obtained with eight SCC samples and their corresponding cell lines. Direct sequencing of PCR-amplified, reverse transcribed, p53 mRNA confirmed the expression of point mutations in six of the positive cell lines and detected in-frame deletions in two others. We also detected two stop mutations and three out-of-frame deletions in five lines which did not express elevated levels of p53 protein. Several of the mutations found in SCC of the tongue (3/7) were in a region (codons 144-166) previously identified as being a p53 mutational hot spot in non-small cell lung tumours (Mitsudomi et al., 1992). In 11/13 cases only the mutant alleles were expressed suggesting loss or reduced expression of the wild type alleles in these cases. Six of the mutations were also detected in the SCCs from which the lines were derived, strongly suggesting that the mutations occurred, and were selected, in vivo. The 12th mutation GTG-->GGG (valine-->glycine) at codon 216 was expressed in line SCC-12 clone B along with an apparently normal p53 allele and is to our knowledge a novel mutation. Line BICR-19 also expressed a normal p53 allele in addition to one where exon 10 was deleted. Additionally 15 of the SCC lines (including all of those which did not show elevated p53 protein levels) were screened for the presence of human papillomavirus types 16 and 18 and were found to be negative. These results are discussed in relation to the pathogenesis of SCC and the immortalisation of human keratinocytes in vitro. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8390283

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosatelli, M.C.; Faa, V.; Sardu, R.

This study reports the molecular characterization of [beta]-thalassemia in the Sardinian population. Three thousand [beta]-thalassemia chromosomes from prospective parents presenting at the genetic service were initially analyzed by dot blot analysis with oligonucleotide probes complementary to the most common [beta]-thalassemia mutations in the Mediterranean at-risk populations. The mutation which remained uncharacterized by this approach were defined by denaturing gradient gel electrophoresis (DGGE) followed by direct sequence analysis on amplified DNA. The authors reconfirmed that the predominant mutation in the Sardinian population is the codon 39 nonsense mutation, which accounts for 95.7% of the [beta]-thalassemia chromosomes. The other two relatively commonmore » mutations are frameshifts at codon 6 (2.1%) and at codon 76 (0.7%), relatively uncommon in other Mediterranean-origin populations. In this study they have detected a novel [beta]-thalassemia mutation, i.e., a frameshift at codon 1, in three [beta]-thalassemia chromosomes. The DGGE procedure followed by direct sequencing on amplified DNA is a powerful approach for the characterization of unknown mutations in this genetic system.« less

High incidence of biallelic point mutations in the Runt domain of the AML1/PEBP2 alpha B gene in Mo acute myeloid leukemia and in myeloid malignancies with acquired trisomy 21.

PubMed

Preudhomme, C; Warot-Loze, D; Roumier, C; Grardel-Duflos, N; Garand, R; Lai, J L; Dastugue, N; Macintyre, E; Denis, C; Bauters, F; Kerckaert, J P; Cosson, A; Fenaux, P

2000-10-15

The AML1 gene, situated in 21q22, is often rearranged in acute leukemias through t(8;21) translocation, t(12;21) translocation, or less often t(3;21) translocation. Recently, point mutations in the Runt domain of the AML1 gene have also been reported in leukemia patients. Observations for mutations of the Runt domain of the AML1 gene in bone marrow cells were made in 300 patients, including 131 with acute myeloid leukemia (AML), 94 with myelodysplastic syndrome (MDS), 28 with blast crisis chronic myeloid leukemia (CML), 3 with atypical CML, 41 with acute lymphoblastic leukemia (ALL), and 3 with essential thrombocythemia (ET). Forty-one of the patients had chromosome 21 abnormalities, including t(8;21) in 6 of the patients with AML, t(12;21) in 8 patients with ALL, acquired trisomy 21 in 17 patients, tetrasomy 21 in 7 patients, and constitutional trisomy 21 (Down syndrome) in 3 patients. A point mutation was found in 14 cases (4.7%), including 9 (22%) of the 41 patients with AML of the Mo type (MoAML) (none of them had detectable chromosome 21 rearrangement) and 5 (38%) of the 13 myeloid malignancies with acquired trisomy 21 (1 M1AML, 2 M2AML, 1 ET, and 1 atypical CML). In at least 8 of 9 mutated cases of MoAML, both AML alleles were mutated: 3 patients had different stop codon mutations of the 2 AML1 alleles, and 5 patients had the same missense or stop codon mutation in both AML1 alleles, which resulted in at least 3 of the patients having duplication of the mutated allele and deletion of the normal residual allele, as shown by FISH analysis and by comparing microsatellite analyses of several chromosome 21 markers on diagnosis and remission samples. In the remaining mutated cases, with acquired trisomy 21, a missense mutation of AML1, which involved 2 of the 3 copies of the AML1 gene, was found. Four of the 7 mutated cases could be reanalyzed in complete remission, and no AML1 mutation was found, showing that mutations were acquired in the leukemic clone. In conclusion, these findings confirm the possibility of mutations of the Runt domain of the AML1 gene in leukemias, mainly in MoAML and in myeloid malignancies with acquired trisomy 21. AML1 mutations, in MoAML, involved both alleles and probably lead to nonfunctional AML1 protein. As AML1 protein regulates the expression of the myeloperoxidase gene, the relationship between AML1 mutations and Mo phenotype in AML will have to be further explored. (Blood. 2000;96:2862-2869)

Vertebrate codon bias indicates a highly GC-rich ancestral genome.

PubMed

Nabiyouni, Maryam; Prakash, Ashwin; Fedorov, Alexei

2013-04-25

Two factors are thought to have contributed to the origin of codon usage bias in eukaryotes: 1) genome-wide mutational forces that shape overall GC-content and create context-dependent nucleotide bias, and 2) positive selection for codons that maximize efficient and accurate translation. Particularly in vertebrates, these two explanations contradict each other and cloud the origin of codon bias in the taxon. On the one hand, mutational forces fail to explain GC-richness (~60%) of third codon positions, given the GC-poor overall genomic composition among vertebrates (~40%). On the other hand, positive selection cannot easily explain strict regularities in codon preferences. Large-scale bioinformatic assessment, of nucleotide composition of coding and non-coding sequences in vertebrates and other taxa, suggests a simple possible resolution for this contradiction. Specifically, we propose that the last common vertebrate ancestor had a GC-rich genome (~65% GC). The data suggest that whole-genome mutational bias is the major driving force for generating codon bias. As the bias becomes prominent, it begins to affect translation and can result in positive selection for optimal codons. The positive selection can, in turn, significantly modulate codon preferences. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular Characterization of β-Thalassemia Mutations in Central Vietnam.

PubMed

Doro, Maria G; Casu, Giuseppina; Frogheri, Laura; Persico, Ivana; Triet, Le Phan Minh; Hoa, Phan Thi Thuy; Hoang, Nguyen Huy; Pirastru, Monica; Mereu, Paolo; Cucca, Francesco; Masala, Bruno

2017-03-01

The molecular basis of β-thalassemia (β-thal) mutations in North and in South Vietnam have been described during the past 15 years, whereas limited data were available concerning the central area of the country. In this study, we describe the molecular characterization and frequency of β-globin gene mutations in the Thua Thien Hue Province of Central Vietnam as the result of a first survey conducted in 22 transfusion-dependent patients, and four unrelated heterozygotes. Nine different known mutations were identified (seven of the β 0 and two of the β + type) in a total of 48 chromosomes. The most common was codon 26 (G>A) or Hb E (HBB: c.79 G>A) accounting for 29.2% of the total studied chromosomes, followed by codon 17 (A>T) (HBB: c.52 A>T) (25.0%), and codons 41/42 (-TTCT) (HBB: c.126_129delCTTT) (18.8%). Other mutations with appreciable frequencies (6.3-8.3%) were IVS-I-1 (G>T) (HBB: c.92+1 G>T), codon 26 (G>T) (HBB: c.79 G>T) and codons 71/72 (+A) (HBB: c.216_217insA). Relatively rarer (2.0%) were the promoter -28 (A>G) (HBB: c.78 A>G) mutation, the codon 95 (+A) (HBB: c.287_288insA), which is reported only in the Vietnamese, and the codons 14/15 (+G) (HBB: c.45_46insG) mutation, thus far observed only in Thailand. Results are relevant for implementing appropriate measures for β-thal prevention and control in the region as well as in the whole country.

Simultaneous identification of 36 mutations in KRAS codons 61and 146, BRAF, NRAS, and PIK3CA in a single reaction by multiplex assay kit

PubMed Central

2013-01-01

Background Retrospective analyses in the West suggest that mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA are negative predictive factors for cetuximab treatment in colorectal cancer patients. We developed a novel multiplex kit detecting 36 mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA using Luminex (xMAP) assay in a single reaction. Methods Tumor samples and clinical data from Asian colorectal cancer patients treated with cetuximab were collected. We investigated KRAS, BRAF, NRAS, and PIK3CA mutations using both the multiplex kit and direct sequencing methods, and evaluated the concordance between the 2 methods. Objective response, progression-free survival (PFS), and overall survival (OS) were also evaluated according to mutational status. Results In total, 82 of 83 samples (78 surgically resected specimens and 5 biopsy specimens) were analyzed using both methods. All multiplex assays were performed using 50 ng of template DNA. The concordance rate between the methods was 100%. Overall, 49 (59.8%) patients had all wild-type tumors, 21 (25.6%) had tumors harboring KRAS codon 12 or 13 mutations, and 12 (14.6%) had tumors harboring KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations. The response rates in these patient groups were 38.8%, 4.8%, and 0%, respectively. Median PFS in these groups was 6.1 months (95% confidence interval (CI): 3.1–9.2), 2.7 months (1.2–4.2), and 1.6 months (1.5–1.7); median OS was 13.8 months (9.2–18.4), 8.2 months (5.7–10.7), and 6.3 months (1.3–11.3), respectively. Statistically significant differences in both PFS and OS were found between patients with all wild-type tumors and those with KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations (PFS: 95% CI, 0.11–0.44; P < 0.0001; OS: 95% CI, 0.15–0.61; P < 0.0001). Conclusions Our newly developed multiplex kit is practical and feasible for investigation of a range of sample types. Moreover, mutations in KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA detected in Asian patients were not predictive of clinical benefits from cetuximab treatment, similar to the result obtained in European studies. PMID:24006859

Two novel mutations in thymidine kinase-2 cause early onset fatal encephalomyopathy and severe mtDNA depletion.

PubMed

Lesko, Nicole; Naess, Karin; Wibom, Rolf; Solaroli, Nicola; Nennesmo, Inger; von Döbeln, Ulrika; Karlsson, Anna; Larsson, Nils-Göran

2010-03-01

Deficiency of thymidine kinase-2 (TK2) has been described in children with early onset fatal skeletal myopathy. TK2 is a mitochondrial deoxyribonucleoside kinase required for the phosphorylation of deoxycytidine and deoxythymidine and hence is vital for the maintenance of a balanced mitochondrial dNTP pool in post-mitotic tissues. We describe a patient with two novel TK2 mutations, which caused disease onset shortly after birth and death at the age of three months. One mutation (219insCG) generated an early stop codon, thus preventing the synthesis of a functional protein. The second mutation (R130W) resulted in an amino acid substitution, which caused a severe reduction (<3%) of TK2 enzyme activity. These two novel TK2 mutations cause an extremely severe phenotype with overwhelming central nervous system symptoms not commonly seen in patients with TK2-deficiency. We conclude that the severe clinical presentation in this patient was due to a virtual lack of mitochondrial TK2 activity. Copyright 2009 Elsevier B.V. All rights reserved.

A novel non-sense mutation in the SLC2A10 gene of an arterial tortuosity syndrome patient of Kurdish origin.

PubMed

Zaidi, Syed H E; Meyer, Sascha; Peltekova, Vanya D; Lindinger, Angelika; Teebi, Ahmad S; Faiyaz-Ul-Haque, Muhammad

2009-07-01

Arterial tortuosity syndrome (ATS) is a rare autosomal recessive disorder in which patients display tortuosity of arteries in addition to hyperextensible skin, joint laxity, and other connective tissue features. This syndrome is caused by mutations in the SLC2A10 gene. In this article we describe an ATS girl of Kurdish origin who, in addition to arterial tortuosity and connective tissue features, displays stomach displacement within the thorax and bilateral hip dislocation. Clinical details of this patient have been reported previously. Sequencing of the SLC2A10 gene identified a novel homozygous non-sense c.756C>A mutation in this patient's DNA. This mutation in the SLC2A10 gene replaces a cysteine encoding codon with a stop signal. This is believed to cause a premature truncation of GLUT10 protein in this patient. We conclude that patients of Kurdish origin who display arterial tortuosity associated with skin hyperextensibility, joint hypermobility, and characteristic facial features may carry mutations in the SLC2A10 gene.

Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms

PubMed Central

Oh, Stephen T.; Simonds, Erin F.; Jones, Carol; Hale, Matthew B.; Goltsev, Yury; Gibbs, Kenneth D.; Merker, Jason D.; Zehnder, James L.; Nolan, Garry P.

2010-01-01

Dysregulated Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling due to activation of tyrosine kinases is a common feature of myeloid malignancies. Here we report the first human disease-related mutations in the adaptor protein LNK, a negative regulator of JAK-STAT signaling, in 2 patients with JAK2 V617F–negative myeloproliferative neoplasms (MPNs). One patient exhibited a 5 base-pair deletion and missense mutation leading to a premature stop codon and loss of the pleckstrin homology (PH) and Src homology 2 (SH2) domains. A second patient had a missense mutation (E208Q) in the PH domain. BaF3-MPL cells transduced with these LNK mutants displayed augmented and sustained thrombopoietin-dependent growth and signaling. Primary samples from MPN patients bearing LNK mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34+ early progenitors were abnormally abundant in both patients. These findings indicate that JAK-STAT activation due to loss of LNK negative feedback regulation is a novel mechanism of MPN pathogenesis. PMID:20404132

Termination and read-through proteins encoded by genome segment 9 of Colorado tick fever virus.

PubMed

Mohd Jaafar, Fauziah; Attoui, Houssam; De Micco, Philippe; De Lamballerie, Xavier

2004-08-01

Genome segment 9 (Seg-9) of Colorado tick fever virus (CTFV) is 1884 bp long and contains a large open reading frame (ORF; 1845 nt in length overall), although a single in-frame stop codon (at nt 1052-1054) reduces the ORF coding capacity by approximately 40 %. However, analyses of highly conserved RNA sequences in the vicinity of the stop codon indicate that it belongs to a class of 'leaky terminators'. The third nucleotide positions in codons situated both before and after the stop codon, shows the highest variability, suggesting that both regions are translated during virus replication. This also suggests that the stop signal is functionally leaky, allowing read-through translation to occur. Indeed, both the truncated 'termination' protein and the full-length 'read-through' protein (VP9 and VP9', respectively) were detected in CTFV-infected cells, in cells transfected with a plasmid expressing only Seg-9 protein products, and in the in vitro translation products from undenatured Seg-9 ssRNA. The ratios of full-length and truncated proteins generated suggest that read-through may be down-regulated by other viral proteins. Western blot analysis of infected cells and purified CTFV showed that VP9 is a structural component of the virion, while VP9' is a non-structural protein.

Novel insertion mutation in a non-Jewish Caucasian type 1 Gaucher disease patient

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choy, F.Y.M.; Humphries, M.L.; Ferreira, P.

1997-01-20

Gaucher disease is the most prevalent lysosomal storage disorder. It is autosomal recessive, resulting in lysosomal glucocerebrosidase deficiency. Three clinical forms of Gaucher disease have been described: type 1 (nonneuronopathic), type 2 (acute neuronopathic), and type 3 (subacute neuronopathic). We performed PCR-thermal cycle sequence analysis of glucocerebrosidase genomic DNA and identified a novel mutation in a non-Jewish type 1 Gaucher disease patient. It is a C insertion in exon 3 at cDNA nucleotide position 122 and genomic nucleotide position 1626. This mutation causes a frameshift and, subsequently, four of the five codons immediately downstream of the insertion were changed whilemore » the sixth was converted to a stop codon, resulting in premature termination of protein translation. The 122CC insertion abolishes a Cac81 restriction endonuclease cleavage site, allowing a convenient and reliable method for detection using RFLP analysis of PCR-amplified glucocerebrosidase genomic DNA. The mutation in the other Gaucher allele was found to be an A{r_arrow}G substitution at glucocerebrosidase cDNA nucleotide position 1226 that so far has only been reported among type 1 Gaucher disease patients. Since mutation 122CC causes a frameshift and early termination of protein translation, it most likely results in a meaningless transcript and subsequently no residual glucocerebrosidase enzyme activity. We speculate that mutation 122CC may result in a worse prognosis than mutations associated with partial activity. When present in the homozygous form, it could be a lethal allele similar to what has been postulated for the other known insertion mutation, 84GG. Our patient, who is a compound heterozygote 122CC/1226G, has moderately severe type 1 Gaucher disease. Her clinical response to Ceredase{reg_sign} therapy that began 31 months ago has been favorable, though incomplete. 30 refs., 3 figs., 2 tabs.« less

Mutation in the 5' alternatively spliced region of the XNP/ATR-X gene causes Chudley-Lowry syndrome.

PubMed

Abidi, Fatima E; Cardoso, Carlos; Lossi, Anne-Marie; Lowry, Robert Brian; Depetris, Danielle; Mattéi, Marie-Geneviève; Lubs, Herbert A; Stevenson, Roger E; Fontes, Michel; Chudley, Albert E; Schwartz, Charles E

2005-02-01

The Chudley-Lowry syndrome (ChLS, MIM 309490) is an X-linked recessive condition characterized by moderate to severe mental retardation, short stature, mild obesity, hypogonadism, and distinctive facial features characterized by depressed nasal bridge, anteverted nares, inverted-V-shaped upper lip, and macrostomia. The original Chudley-Lowry family consists of three affected males in two generations. Linkage analysis had localized the gene to a large interval, Xp21-Xq26 and an obligate carrier was demonstrated to have highly skewed X inactivation. The combination of the clinical phenotype, consistent with that of the patients with ATR-X syndrome, the skewed X-inactivation pattern in a carrier female, as well as the mapping interval including band Xq13.3, prompted us to consider the XNP/ATR-X gene being involved in this syndrome. Using RT-PCR analysis, we screened the entire XNP/ATR-X gene and found a mutation in exon 2 (c.109C > T) giving rise to a stop codon at position 37 (p.R37X). Western blot and immunocytochemical analyses using a specific monoclonal antibody directed against XNP/ATR-X showed the protein to be present in lymphoblastoid cells from one affected male, despite the premature stop codon. To explain these discordant results, we further analyzed the 5' region of the XNP/ATR-X gene and found three alternative transcripts, which differ in the presence or absence of exon 2, and the length of exon 1. Our data suggest that ChLS is allelic to the ATR-X syndrome with its less severe phenotype being due to the presence of some XNP/ATR-X protein.

dupA polymorphisms and risk of Helicobacter pylori-associated diseases.

PubMed

Queiroz, Dulciene M M; Rocha, Gifone A; Rocha, Andreia M C; Moura, Sílvia B; Saraiva, Ivan E B; Gomes, Luciana I; Soares, Taciana F; Melo, Fabrício F; Cabral, Mônica M D A; Oliveira, Celso A

2011-03-01

The dupA of Helicobacter pylori has been suggested as a virulence marker associated with the development of duodenal ulcer disease. However, the studies performed in different geographical areas have shown that there are variations in the prevalence of dupA and its association with H. pylori clinical outcomes. Our group did not observe associations between the presence of dupA and H. pylori clinical outcomes in Brazil. On the other hand, we observed 2 mutations in the sequence of dupA that lead to stop codons: a deletion of an adenine at position 1311 and an insertion of an adenine at position 1426 of the gene. Our aim was to evaluate associations of the presence of dupA with duodenal ulcer and gastric cancer, considering dupA-positive only those H. pylori strains that do not have the mutations in the gene sequence. We also evaluated the effect of infection with a strain carrying an intact dupA on the gastric mucosa histology and IL-8 gastric levels. Colonization with strains that had the intact dupA was negatively associated with gastric carcinoma (p=0.001, OR=0.32, 95% CI=0.16-0.66). The presence of dupA was also associated with an increased degree of antral mucosa inflammation (p=0.01) and with decreased corpus atrophy (p<0.01) as well as with increased gastric mucosa IL-8 levels (p=0.04). In conclusion, the infection with a H. pylori strain containing the dupA without the stop codon polymorphisms is associated with a lower risk of development of gastric carcinoma in Brazilian subjects. Copyright © 2010 Elsevier GmbH. All rights reserved.

Ineffectiveness of the presence of H-ras/p53 combination of mutations in squamous cell carcinoma cells to induce a conversion of a nontumorigenic to a tumorigenic phenotype.

PubMed

Lee, H; Li, D; Prior, T; Casto, B C; Weghorst, C M; Shuler, C F; Milo, G E

1997-10-01

Human tumor cells have properties in vitro or in surrogate hosts that are distinct from those of normal cells, such as immortality, anchorage independence, and tumor formation in nude mice. However, different cells from individual tumors may exhibit some, but not all of these features. In previous years, human tumor cell lines derived from different tumor and tissue types have been studied to determine those molecular changes that are associated with the in vitro properties listed above and with tumorigenicity in nude mice. In the present study, seven cell lines derived from human tumors were characterized for p53 and ras mutations that may occur in SCC tumor phenotypes and for tumor formation in nude mice. This investigation was designed to examine whether co-occurrence of mutated ras and p53 lead to a malignant stage in the progression process. None of the seven cell lines contained mutations in the recognized "hot spots" of the p53 tumor suppressor gene, but four had a nonsense/splice mutation in codon 126 and a mutation in codon 12 of the H-ras gene. The remaining three cell lines had p53 mutations in intron 5, in codon 193, and a missense mutation in codon 126, respectively. Four of seven cell lines were nontumorigenic; two of these cell lines contained a nonsense p53-126 mutation and mutated ras; one had a missense mutation at codon 126 but no mutated ras; the the fourth had only a p53 mutation at codon 193. Two of the nontumorigenic cell lines were converted to tumorigenicity after treatment with methyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine with no apparent additional mutations in either gene. Our analysis revealed that there was a high frequency of genetic diversity and mutations in both p53 and H-ras. There was also a lack of a causal relationship in the presence of mutations in p53 and the cells' ability to exhibit a malignant potential in nude mice.

[Mutation screening of MITF gene in patients with Waardenburg syndrome type 2].

PubMed

Chen, Jing; Yang, Shu-Zhi; Liu, Jun; Han, Bing; Wang, Guo-Jian; Zhang, Xin; Kang, Dong-Yang; Dai, Pu; Young, Wie-Yen; Yuan, Hui-Jun

2008-04-01

Warrgenburg syndrome type 2 (WS2) is the most common autosomal dominantly-inherited syndrome with hearing loss. MITF (microphthalmia associated transcription factor)is a basic-helix-loop-helix-luecine zipper (bHLHZip) factor which regulates expression of tyrosinase, and is involved in melanocyte differentiation. Mutations in MITF associated with WS2 have been identified in some but not all affected families. Here, we report a three-generation Chinese family with a point mutation in the MITF gene causing WS2. The proband exhibits congenital severe sensorineural hearing loss, heterochromia iridis and facial freckles. One of family members manifests sensorineural deafness, and the other patients show premature greying or/and freckles. This mutation, heterozygous deletion c.639delA, creates a stop codon in exon 7 and is predicted to result in a truncated protein lacking normal interaction with its target DNA motif. This mutation is a novel mutation and the third case identified in exon 7 of MITF in WS2. Though there is only one base pair distance between this novel mutation and the other two documented cases and similar amino acids change, significant difference is seen in clinical phenotype, which suggests genetic background may play an important role.

Identification a nonsense mutation of APC gene in Chinese patients with familial adenomatous polyposis.

PubMed

Li, Haishan; Zhang, Lingling; Jiang, Quan; Shi, Zhenwang; Tong, Hanxing

2017-04-01

Familial adenomatous polyposis (FAP; Mendelian of Inherintance in Man ID, 175100) is a rare autosomal dominant disorder characterized by the development of numerous adenomatous polyps throughout the colon and rectum associated with an increased risk of colorectal cancer. FAP is at time accompanied with certain extraintestinal manifestations such as congenital hypertrophy of the retinal pigment epithelium, dental disorders and desmoid tumors. It is caused by mutations in the adenomatous polyposis coli ( APC ) gene. The present study reported on a Chinese family with FAP. Polymerase chain reaction and direct sequencing of the full coding sequence of the APC gene were performed to identify the mutation in this family. A nonsense mutation of the APC gene was identified in this pedigree. It is a heterozygous G>T substitution at position 2,971 in exon 15 of the APC gene, which formed a premature stop codon at amino acid residue 991 (p.Glu991*). The resulting truncated protein lacked 1,853 amino acids. The present study expanded the database on APC gene mutations in FAP and enriched the spectrum of known germline mutations of the APC gene. Prophylactic proctocolectomy may be considered as a possible treatment for carriers of the mutation.

Mutation of the PAX6 gene in a sporadic patient with atypical aniridia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, D.; Li, Y.; Traboulsi, E.I.

1994-09-01

A 28 year-old man presented with poor vision since childhood and gradual further decline of several years duration. His visual acuity measures 20/200 OD with -11.50 + 0.50 x 150 and 20/100 OS with -12.25 + 0.25 x 35. He had a fine nystagmus. His visual fields were full. There was a circumferential pannus with areas of corneal stromal opacification. The iris was hypoplastic with atypical colobomatous defects. The lenses had scattered cortical opacities. The intraocular pressures were normal. The optic nerves had cup disk ratios of 0.6 OU. The family history was negative for similar defects. A diagnosis ofmore » aniridia was made and blood was drawn for analysis of the PAX6 gene. PCR amplification of exon 5 showed heterozygous fragments with one allele being larger than normal. Direct DNA sequencing of the individual heterozygous allele showed a 41 base pair insertion at nucleotide 483 in exon 5 of the paired domain. This frameshift mutation changed codon 71 to a stop codon. The diagnosis of aniridia was confirmed in this atypical patient, who will need to be monitored for his high risk of glaucoma. The risk of developing Wilms` tumor in patients with mutations within the aniridia gene is presumably negligible since the neighboring Wilms` tumor gene is unaffected. The identification of intragenic mutations of the PAX6 gene in patients with sporadic aniridia modifies the management of such patients because of recognition of the increased risk of glaucoma and by reducing the necessity for frequent monitoring for the presence of Wilms` tumor.« less

Multiple origins of the phenol reaction negative phenotype in foxtail millet, Setaria italica (L.) P. Beauv., were caused by independent loss-of-function mutations of the polyphenol oxidase (Si7PPO) gene during domestication.

PubMed

Inoue, Takahiko; Yuo, Takahisa; Ohta, Takeshi; Hitomi, Eriko; Ichitani, Katsuyuki; Kawase, Makoto; Taketa, Shin; Fukunaga, Kenji

2015-08-01

Foxtail millet shows variation in positive phenol color reaction (Phr) and negative Phr in grains, but predominant accessions of this crop are negative reaction type, and the molecular genetic basis of the Phr reaction remains unresolved. In this article, we isolated polyphenol oxidase (PPO) gene responsible for Phr using genome sequence information and investigated molecular genetic basis of negative Phr and crop evolution of foxtail millet. First of all, we searched for PPO gene homologs in a foxtail millet genome database using a rice PPO gene as a query and successfully found three copies of the PPO gene. One of the PPO gene homologs on chromosome 7 showed the highest similarity with PPO genes expressed in hulls (grains) of other cereal species including rice, wheat, and barley and was designated as Si7PPO. Phr phenotypes and Si7PPO genotypes completely co-segregated in a segregating population. We also analyzed the genetic variation conferring negative Phr reaction. Of 480 accessions of the landraces investigated, 87 (18.1 %) showed positive Phr and 393 (81.9 %) showed negative Phr. In the 393 Phr negative accessions, three types of loss-of-function Si7PPO gene were predominant and independently found in various locations. One of them has an SNP in exon 1 resulting in a premature stop codon and was designated as stop codon type, another has an insertion of a transposon (Si7PPO-TE1) in intron 2 and was designated as TE1-insertion type, and the other has a 6-bp duplication in exon 3 resulting in the duplication of 2 amino acids and was designated as 6-bp duplication type. As a rare variant of the stop codon type, one accession additionally has an insertion of a transposon, Si7PPO-TE2, in intron 2 and was designated as "stop codon +TE2 insertion type". The geographical distribution of accessions with positive Phr and those with three major types of negative Phr was also investigated. Accessions with positive Phr were found in subtropical and tropical regions at frequencies of ca. 25-67 % and those with negative Phr were broadly found in Europe and Asia. The stop codon type was found in 285 accessions and was broadly distributed in Europe and Asia, whereas the TE-1 insertion type was found in 99 accessions from Europe and Asia but was not found in India. The 6-bp duplication type was found in only 8 accessions from Nansei Islands (Okinawa Prefecture) of Japan. We also analyzed Phr in the wild ancestor and concluded that the negative Phr type was likely to have originated after domestication of foxtail millet. It was also implied that negative Phr of foxtail millet arose by multiple independent loss of function of PPO gene through dispersal because of some advantages under some environmental conditions and human selection as in rice and barley.

The Frequency and Type of K-RAS Mutations in Mexican Patients With Colorectal Cancer: A National Study.

PubMed

Cárdenas-Ramos, Susana G; Alcázar-González, Gregorio; Reyes-Cortés, Luisa M; Torres-Grimaldo, Abdiel A; Calderón-Garcidueñas, Ana L; Morales-Casas, José; Flores-Sánchez, Patricia; De León-Escobedo, Raúl; Gómez-Díaz, Antonio; Moreno-Bringas, Carmen; Sánchez-Guillén, Jorge; Ramos-Salazar, Pedro; González-de León, César; Barrera-Saldaña, Hugo A

2017-06-01

Current metastatic colorectal cancer (mCRC) therapy uses monoclonal antibodies against the epidermal growth factor receptor. This treatment is only useful in the absence of K-RAS gene mutations; therefore the study of such mutations is part of a personalized treatment. The aim of this work is to determine the frequency and type of the most common K-RAS mutations in Mexican patients with metastatic disease by nucleotide sequencing. We studied 888 patients with mCRC from different regions of Mexico. The presence of mutations in exon 2, codons 12 and 13, of the K-RAS gene was determined by nucleotide sequencing. Patients exhibited K-RAS gene mutations in 35% (310/888) of cases. Mutation frequency of codons 12 and 13 was 71% (221/310) and 29% (89/310), respectively. The most common mutation (45.7%) in codon 12 was c.35G>A (p.G12D), whereas the one in codon 13 was c.38G>A (p.G13D) (78.7%). Given the frequency of K-RAS mutations in Mexicans, making a genetic study before deciding to treat mCRC patients with monoclonal antibodies is indispensable.

Brooke-Spiegler syndrome: report of 10 patients from 8 families with novel germline mutations: evidence of diverse somatic mutations in the same patient regardless of tumor type.

PubMed

Sima, Radek; Vanecek, Tomas; Kacerovska, Denisa; Trubac, Pavel; Cribier, Bernard; Rutten, Arno; Vazmitel, Marina; Spagnolo, Dominic V; Litvik, Radek; Vantuchova, Yvetta; Weyers, Wolfgang; Pearce, Robert L; Pearn, John; Michal, Michal; Kazakov, Dmitry V

2010-06-01

Brooke-Spiegler syndrome (BSS) is an inherited autosomal dominant disease characterized by the development of multiple adnexal cutaneous neoplasms including spiradenoma, cylindroma, spiradenocylindroma, and trichoepithelioma (cribriform trichoblastoma). BSS patients have various mutations in the CYLD gene, a tumor suppressor gene located on chromosome 16q. Our search of the literature revealed 51 germline CYLD mutations reported to date. Somatic CYLD mutations have rarely been investigated. We studied 10 patients from 8 families with BSS. Analysis of germline mutations of the CYLD gene was performed using either peripheral blood or nontumorous tissue. In addition, 19 formalin-fixed paraffin-embedded tumor samples were analyzed for somatic mutations, including loss of heterozygosity studies. A total of 38 tumors were available for histopathologic review. We have identified 8 novel germline mutations, all of which consisted of substitutions, deletions, and insertions/duplications and all except one led to premature stop codons. The substitution mutation in a single case was also predicted to disrupt protein function and seems causally implicated in tumor formation. We demonstrate for the first time that somatic events, loss of heterozygosity, or sequence mutations may differ among multiple neoplasms even of the same histologic type, occurring in the same patient.

A Turkish family with Nance-Horan Syndrome due to a novel mutation.

PubMed

Tug, Esra; Dilek, Nihal F; Javadiyan, Shahrbanou; Burdon, Kathryn P; Percin, Ferda E

2013-08-01

Nance-Horan Syndrome (NHS) is a rare X-linked syndrome characterized by congenital cataract which leads to profound vision loss, characteristic dysmorphic features and specific dental anomalies. Microcornea, microphthalmia and mild or moderate mental retardation may accompany these features. Heterozygous females often manifest similarly but with less severe features than affected males. We describe two brothers who have the NHS phenotype and their carrier mother who had microcornea but not cataract. We identified a previously unreported frameshift mutation (c.558insA) in exon 1 of the NHS gene in these patients and their mother which is predicted to result in the incorporation of 11 aberrant amino acids prior to a stop codon (p.E186Efs11X). We also discussed genotype-phenotype correlation according to relevant literature. Copyright © 2013 Elsevier B.V. All rights reserved.

A unique TBX5 microdeletion with microinsertion detected in patient with Holt-Oram syndrome.

PubMed

Morine, Mikio; Kohmoto, Tomohiro; Masuda, Kiyoshi; Inagaki, Hidehito; Watanabe, Miki; Naruto, Takuya; Kurahashi, Hiroki; Maeda, Kazuhisa; Imoto, Issei

2015-12-01

Holt-Oram syndrome (HOS) is an autosomal dominant condition characterized by upper limb and congenital heart defects and caused by numerous germline mutations of TBX5 producing preterminal stop codons. Here, we report on a novel and unusual heterozygous TBX5 microdeletion with microinsertion (microindel) mutation (c.627delinsGTGACTCAGGAAACGCTTTCCTGA), which is predicted to synthesize a truncated TBX5 protein, detected in a sporadic patient with clinical features of HOS prenatally diagnosed by ultrasonography. This uncommon and relatively large inserted sequence contains sequences derived from nearby but not adjacent templates on both sense and antisense strands, suggesting two possible models, which require no repeat sequences, causing this complex microindel through the bypass of large DNA adducts via an error-prone DNA polymerase-mediated translesion synthesis. © 2015 Wiley Periodicals, Inc.

Whole Genome Sequencing Reveals a De Novo SHANK3 Mutation in Familial Autism Spectrum Disorder

PubMed Central

Nemirovsky, Sergio I.; Córdoba, Marta; Zaiat, Jonathan J.; Completa, Sabrina P.; Vega, Patricia A.; González-Morón, Dolores; Medina, Nancy M.; Fabbro, Mónica; Romero, Soledad; Brun, Bianca; Revale, Santiago; Ogara, María Florencia; Pecci, Adali; Marti, Marcelo; Vazquez, Martin; Turjanski, Adrián; Kauffman, Marcelo A.

2015-01-01

Introduction Clinical genomics promise to be especially suitable for the study of etiologically heterogeneous conditions such as Autism Spectrum Disorder (ASD). Here we present three siblings with ASD where we evaluated the usefulness of Whole Genome Sequencing (WGS) for the diagnostic approach to ASD. Methods We identified a family segregating ASD in three siblings with an unidentified cause. We performed WGS in the three probands and used a state-of-the-art comprehensive bioinformatic analysis pipeline and prioritized the identified variants located in genes likely to be related to ASD. We validated the finding by Sanger sequencing in the probands and their parents. Results Three male siblings presented a syndrome characterized by severe intellectual disability, absence of language, autism spectrum symptoms and epilepsy with negative family history for mental retardation, language disorders, ASD or other psychiatric disorders. We found germline mosaicism for a heterozygous deletion of a cytosine in the exon 21 of the SHANK3 gene, resulting in a missense sequence of 5 codons followed by a premature stop codon (NM_033517:c.3259_3259delC, p.Ser1088Profs*6). Conclusions We reported an infrequent form of familial ASD where WGS proved useful in the clinic. We identified a mutation in SHANK3 that underscores its relevance in Autism Spectrum Disorder. PMID:25646853

Whole genome sequencing reveals a de novo SHANK3 mutation in familial autism spectrum disorder.

PubMed

Nemirovsky, Sergio I; Córdoba, Marta; Zaiat, Jonathan J; Completa, Sabrina P; Vega, Patricia A; González-Morón, Dolores; Medina, Nancy M; Fabbro, Mónica; Romero, Soledad; Brun, Bianca; Revale, Santiago; Ogara, María Florencia; Pecci, Adali; Marti, Marcelo; Vazquez, Martin; Turjanski, Adrián; Kauffman, Marcelo A

2015-01-01

Clinical genomics promise to be especially suitable for the study of etiologically heterogeneous conditions such as Autism Spectrum Disorder (ASD). Here we present three siblings with ASD where we evaluated the usefulness of Whole Genome Sequencing (WGS) for the diagnostic approach to ASD. We identified a family segregating ASD in three siblings with an unidentified cause. We performed WGS in the three probands and used a state-of-the-art comprehensive bioinformatic analysis pipeline and prioritized the identified variants located in genes likely to be related to ASD. We validated the finding by Sanger sequencing in the probands and their parents. Three male siblings presented a syndrome characterized by severe intellectual disability, absence of language, autism spectrum symptoms and epilepsy with negative family history for mental retardation, language disorders, ASD or other psychiatric disorders. We found germline mosaicism for a heterozygous deletion of a cytosine in the exon 21 of the SHANK3 gene, resulting in a missense sequence of 5 codons followed by a premature stop codon (NM_033517:c.3259_3259delC, p.Ser1088Profs*6). We reported an infrequent form of familial ASD where WGS proved useful in the clinic. We identified a mutation in SHANK3 that underscores its relevance in Autism Spectrum Disorder.

Complete mitochondrial genome of the Yellownose skate: Zearaja chilensis (Rajiformes, Rajidae).

PubMed

Jeong, Dageum; Lee, Youn-Ho

2016-01-01

The complete sequence of mitochondrial DNA of a Yellownose skate, Zearaja chilensis was determined for the first time. It is 16,909 bp in length covering 2 rRNA, 22 tRNA and 13 protein coding genes with the identical gene order and structure as those of other Rajidae species. The nucleotide of L-strand is composed of low G (14.3%), and slightly high A + T (58.9%) nucleotides. The strong codon usage bias against the use of G (6.0%) is found at the third codon positions. Twelve of the 13 protein coding genes use ATG as the start codon while COX1 starts with GTG. As for the stop codon, only ND4 shows an incomplete stop codon TA. This is the first report of the mitogenome for a species in the genus Zearaja, providing a valuable source of genetic information on the evolution of the family Rajidae and the genus Zearaja as well as for establishment of a sustainble fishery management plan of the species.

Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6

PubMed Central

Evans, Ben A.; Smith, Olivia L.; Pickerill, Ethan S.; York, Mary K.; Buenconsejo, Kristen J.P.; Chambers, Antonio E.

2018-01-01

Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the Candida albicans UME6 Zn2+-binding domain, a known regulator of C. albicans filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type C. albicans. Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest. PMID:29892505

A detailed analysis of codon usage patterns and influencing factors in Zika virus.

PubMed

Singh, Niraj K; Tyagi, Anuj

2017-07-01

Recent outbreaks of Zika virus (ZIKV) in Africa, Latin America, Europe, and Southeast Asia have resulted in serious health concerns. To understand more about evolution and transmission of ZIKV, detailed codon usage analysis was performed for all available strains. A high effective number of codons (ENC) value indicated the presence of low codon usage bias in ZIKV. The effect of mutational pressure on codon usage bias was confirmed by significant correlations between nucleotide compositions at third codon positions and ENCs. Correlation analysis between Gravy values, Aroma values and nucleotide compositions at third codon positions also indicated some influence of natural selection. However, the low codon adaptation index (CAI) value of ZIKV with reference to human and mosquito indicated poor adaptation of ZIKV codon usage towards its hosts, signifying that natural selection has a weaker influence than mutational pressure. Additionally, relative dinucleotide frequencies, geographical distribution, and evolutionary processes also influenced the codon usage pattern to some extent.

Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.

PubMed

Ho, Joanne M; Reynolds, Noah M; Rivera, Keith; Connolly, Morgan; Guo, Li-Tao; Ling, Jiqiang; Pappin, Darryl J; Church, George M; Söll, Dieter

2016-02-19

Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.

Point mutation in the MITF gene causing Waardenburg syndrome type II in a three-generation Indian family.

PubMed

Lalwani, A K; Attaie, A; Randolph, F T; Deshmukh, D; Wang, C; Mhatre, A; Wilcox, E

1998-12-04

Waardenburg syndrome (WS) is an autosomal-dominant neural crest cell disorder phenotypically characterized by hearing impairment and disturbance of pigmentation. A presence of dystopia canthorum is indicative of WS type 1, caused by loss of function mutation in the PAX3 gene. In contrast, type 2 WS (WS2) is characterized by normally placed medial canthi and is genetically heterogeneous; mutations in MITF (microphthalmia associated transcription factor) associated with WS2 have been identified in some but not all affected families. Here, we report on a three-generation Indian family with a point mutation in the MITF gene causing WS2. This mutation, initially reported in a Northern European family, creates a stop codon in exon 7 and is predicted to result in a truncated protein lacking the HLH-Zip or Zip structure necessary for normal interaction with its target DNA motif. Comparison of the phenotype between the two families demonstrates a significant difference in pigmentary disturbance of the eye. This family, with the first documented case of two unrelated WS2 families harboring identical mutations, provides additional evidence for the importance of genetic background on the clinical phenotype.

Mutation in the Auxiliary Calcium-Channel Subunit CACNA2D4 Causes Autosomal Recessive Cone Dystrophy

PubMed Central

Wycisk, Katharina Agnes; Zeitz, Christina; Feil, Silke; Wittmer, Mariana; Forster, Ursula; Neidhardt, John; Wissinger, Bernd; Zrenner, Eberhart; Wilke, Robert; Kohl, Susanne; Berger, Wolfgang

2006-01-01

Retinal signal transmission depends on the activity of high voltage–gated l-type calcium channels in photoreceptor ribbon synapses. We recently identified a truncating frameshift mutation in the Cacna2d4 gene in a spontaneous mouse mutant with profound loss of retinal signaling and an abnormal morphology of ribbon synapses in rods and cones. The Cacna2d4 gene encodes an l-type calcium-channel auxiliary subunit of the α2δ type. Mutations in its human orthologue, CACNA2D4, were not yet known to be associated with a disease. We performed mutation analyses of 34 patients who received an initial diagnosis of night blindness, and, in two affected siblings, we detected a homozygous nucleotide substitution (c.2406C→A) in CACNA2D4. The mutation introduces a premature stop codon that truncates one-third of the corresponding open reading frame. Both patients share symptoms of slowly progressing cone dystrophy. These findings represent the first report of a mutation in the human CACNA2D4 gene and define a novel gene defect that causes autosomal recessive cone dystrophy. PMID:17033974

[Analysis of prevalence of point mutations in codon 12 of oncogene K-ras from non-cancerous samples of cervical cytology positive for type 16 or 18 PVH].

PubMed

Golijow, C D; Mourón, S A; Gómez, M A; Dulout, F N

1999-12-01

Ninety-one non cancerous samples from genital specimens positives for VPH 16 or 18 and 27 non-infected samples as controls were studied. Mutations at codon 12 in K-ras gene was analyzed using enriched alelic PCR technique. Among the samples studied 17.58% showed mutations in this codon. Significant differences were observed between the control group (negative DNA-HPV) and positives DNA-HPV samples (p < 0.01). No differences were found between both viral types in relation to the mutation frequency. The presence of mutations in the K-ras gene in non cancerous cytological samples point out new questions about the role of mutations in proto-oncogenes and the development of cervical cancer.

Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in α-spectrin–deficient red cells

PubMed Central

Robledo, Raymond F.; Lambert, Amy J.; Birkenmeier, Connie S.; Cirlan, Marius V.; Cirlan, Andreea Flavia M.; Campagna, Dean R.; Lux, Samuel E.

2010-01-01

Five spontaneous, allelic mutations in the α-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph1J, sph2J, sph2BC, sphDem). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph3J, a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sphIhj, a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent β-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph4J, a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, β-adducin. The severity of anemia in sph4J indicates that the highly conserved cysteine residue at the C-terminus of α-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3. PMID:20056793

Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in alpha-spectrin-deficient red cells.

PubMed

Robledo, Raymond F; Lambert, Amy J; Birkenmeier, Connie S; Cirlan, Marius V; Cirlan, Andreea Flavia M; Campagna, Dean R; Lux, Samuel E; Peters, Luanne L

2010-03-04

Five spontaneous, allelic mutations in the alpha-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph(1J), sph(2J), sph(2BC), sph(Dem)). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph(3J), a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sph(Ihj), a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent beta-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph(4J), a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, beta-adducin. The severity of anemia in sph(4J) indicates that the highly conserved cysteine residue at the C-terminus of alpha-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis.

PubMed

Gan, Qinglei; Fan, Chenguang

2017-11-01

Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNA Lys , tRNA Tyr , and tRNA Gln (CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2016 Elsevier B.V. All rights reserved.

A specific collagen type II gene (COL2A1) mutation presenting as spondyloperipheral dysplasia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zabel, B.; Hilbert, K.; Spranger, J.

1996-05-03

We report on a patient with a skeletal dysplasia characterized by short stature, spondylo-epiphyseal involvement, and brachydactyly E-like changes. This condition has been described as spondyloperipheral dysplasia and the few published cases suggest autosomal dominant inheritance with considerable clinical variability. We found our sporadic case to be due to a collagen type II defect resulting from a specific COL2A1 mutation. This mutation is the first to be located at the C-terminal outside the helical domain of COL2A1. A frameshift as consequence of a 5 bp duplication in exon 51 leads to a stop codon. The resulting truncated C-propeptide region seemsmore » to affect helix formation and produces changes of chondrocyte morphology, collagen type II fibril structure and cartilage matrix composition. Our case with its distinct phenotype adds another chondrodysplasia to the clinical spectrum of type II collagenopathies. 16 refs., 4 figs.« less

Ataluren treatment of patients with nonsense mutation dystrophinopathy.

PubMed

Bushby, Katharine; Finkel, Richard; Wong, Brenda; Barohn, Richard; Campbell, Craig; Comi, Giacomo P; Connolly, Anne M; Day, John W; Flanigan, Kevin M; Goemans, Nathalie; Jones, Kristi J; Mercuri, Eugenio; Quinlivan, Ros; Renfroe, James B; Russman, Barry; Ryan, Monique M; Tulinius, Mar; Voit, Thomas; Moore, Steven A; Lee Sweeney, H; Abresch, Richard T; Coleman, Kim L; Eagle, Michelle; Florence, Julaine; Gappmaier, Eduard; Glanzman, Allan M; Henricson, Erik; Barth, Jay; Elfring, Gary L; Reha, Allen; Spiegel, Robert J; O'donnell, Michael W; Peltz, Stuart W; Mcdonald, Craig M

2014-10-01

Dystrophinopathy is a rare, severe muscle disorder, and nonsense mutations are found in 13% of cases. Ataluren was developed to enable ribosomal readthrough of premature stop codons in nonsense mutation (nm) genetic disorders. Randomized, double-blind, placebo-controlled study; males ≥ 5 years with nm-dystrophinopathy received study drug orally 3 times daily, ataluren 10, 10, 20 mg/kg (N=57); ataluren 20, 20, 40 mg/kg (N=60); or placebo (N=57) for 48 weeks. The primary endpoint was change in 6-Minute Walk Distance (6MWD) at Week 48. Ataluren was generally well tolerated. The primary endpoint favored ataluren 10, 10, 20 mg/kg versus placebo; the week 48 6MWD Δ=31.3 meters, post hoc P=0.056. Secondary endpoints (timed function tests) showed meaningful differences between ataluren 10, 10, 20 mg/kg, and placebo. As the first investigational new drug targeting the underlying cause of nm-dystrophinopathy, ataluren offers promise as a treatment for this orphan genetic disorder with high unmet medical need. Copyright © 2014 Wiley Periodicals, Inc.

Mutations of the RTEL1 Helicase in a Hoyeraal-Hreidarsson Syndrome Patient Highlight the Importance of the ARCH Domain.

PubMed

Jullien, Laurent; Kannengiesser, Caroline; Kermasson, Laetitia; Cormier-Daire, Valérie; Leblanc, Thierry; Soulier, Jean; Londono-Vallejo, Arturo; de Villartay, Jean-Pierre; Callebaut, Isabelle; Revy, Patrick

2016-05-01

The DNA helicase RTEL1 participates in telomere maintenance and genome stability. Biallelic mutations in the RTEL1 gene account for the severe telomere biology disorder characteristic of the Hoyeraal-Hreidarsson syndrome (HH). Here, we report a HH patient (P4) carrying two novel compound heterozygous mutations in RTEL1: a premature stop codon (c.949A>T, p.Lys317*) and an intronic deletion leading to an exon skipping and an in-frame deletion of 25 amino-acids (p.Ile398_Lys422). P4's cells exhibit short and dysfunctional telomeres similarly to other RTEL1-deficient patients. 3D structure predictions indicated that the p.Ile398_Lys422 deletion affects a part of the helicase ARCH domain, which lines the pore formed with the core HD and the iron-sulfur cluster domains and is highly specific of sequences from the eukaryotic XPD family members. © 2016 WILEY PERIODICALS, INC.

A method for multi-codon scanning mutagenesis of proteins based on asymmetric transposons.

PubMed

Liu, Jia; Cropp, T Ashton

2012-02-01

Random mutagenesis followed by selection or screening is a commonly used strategy to improve protein function. Despite many available methods for random mutagenesis, nearly all generate mutations at the nucleotide level. An ideal mutagenesis method would allow for the generation of 'codon mutations' to change protein sequence with defined or mixed amino acids of choice. Herein we report a method that allows for mutations of one, two or three consecutive codons. Key to this method is the development of a Mu transposon variant with asymmetric terminal sequences. As a demonstration of the method, we performed multi-codon scanning on the gene encoding superfolder GFP (sfGFP). Characterization of 50 randomly chosen clones from each library showed that more than 40% of the mutants in these three libraries contained seamless, in-frame mutations with low site preference. By screening only 500 colonies from each library, we successfully identified several spectra-shift mutations, including a S205D variant that was found to bear a single excitation peak in the UV region.

Physical Model for the Evolution of the Genetic Code

NASA Astrophysics Data System (ADS)

Yamashita, Tatsuro; Narikiyo, Osamu

2011-12-01

Using the shape space of codons and tRNAs we give a physical description of the genetic code evolution on the basis of the codon capture and ambiguous intermediate scenarios in a consistent manner. In the lowest dimensional version of our description, a physical quantity, codon level is introduced. In terms of the codon levels two scenarios are typically classified into two different routes of the evolutional process. In the case of the ambiguous intermediate scenario we perform an evolutional simulation implemented cost selection of amino acids and confirm a rapid transition of the code change. Such rapidness reduces uncomfortableness of the non-unique translation of the code at intermediate state that is the weakness of the scenario. In the case of the codon capture scenario the survival against mutations under the mutational pressure minimizing GC content in genomes is simulated and it is demonstrated that cells which experience only neutral mutations survive.

Molecular basis of beta-thalassemia in the Maldives.

PubMed

Furuumi, H; Firdous, N; Inoue, T; Ohta, H; Winichagoon, P; Fucharoen, S; Fukumaki, Y

1998-03-01

We have systematically analyzed beta-thalassemia genes using polymerase chain reaction-related techniques, dot-blot hybridization with oligonucleotide probes, allele specific-polymerase chain reaction, and sequencing of amplified DNA fragments from 41 unrelated patients, including 37 beta-thalassemia homozygotes, three with beta-thalassemia/Hb E, and one with beta-thalassemia/Hb S. Four different beta-thalassemia mutations were detected in 78 alleles. These are the IVS-I-5 (G-->C), codon 30 (AGG-->ACG) [also indicated as IVS-I (-1)], IVS-I-1 (G-->A), and codons 41/42 (-TTCT) mutations. The distribution of the beta-thalassemia mutations in the Maldives is 58 alleles (74.3%) with the IVS-I-5 (G-->C) mutation, 12 (15.4%) with the codon 30 (AGG-->ACG) mutation, seven (9%) with the IVS-I-1 (G-->A) mutation, and one with the codons 41/42 (-TTCT) mutation. The first three mutations account for 98.7% of the total number of beta-thalassemia chromosomes studied. These mutations are clustered in the region spanning 6 bp around the junction of exon 1 and the first intervening sequence of the beta-globin gene. These observations have significant implications for setting up a thalassemia prevention and control program in the Maldives. Analysis of haplotypes and frameworks of chromosomes bearing each beta-thalassemia mutation suggested that the origin and spread of these mutations were reflected by the historical record.

Random codon re-encoding induces stable reduction of replicative fitness of Chikungunya virus in primate and mosquito cells.

PubMed

Nougairede, Antoine; De Fabritus, Lauriane; Aubry, Fabien; Gould, Ernest A; Holmes, Edward C; de Lamballerie, Xavier

2013-02-01

Large-scale codon re-encoding represents a powerful method of attenuating viruses to generate safe and cost-effective vaccines. In contrast to specific approaches of codon re-encoding which modify genome-scale properties, we evaluated the effects of random codon re-encoding on the re-emerging human pathogen Chikungunya virus (CHIKV), and assessed the stability of the resultant viruses during serial in cellulo passage. Using different combinations of three 1.4 kb randomly re-encoded regions located throughout the CHIKV genome six codon re-encoded viruses were obtained. Introducing a large number of slightly deleterious synonymous mutations reduced the replicative fitness of CHIKV in both primate and arthropod cells, demonstrating the impact of synonymous mutations on fitness. Decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. The wild-type and two re-encoded viruses were passaged 50 times either in primate or insect cells, or in each cell line alternately. These viruses were analyzed using detailed fitness assays, complete genome sequences and the analysis of intra-population genetic diversity. The response to codon re-encoding and adaptation to culture conditions occurred simultaneously, resulting in significant replicative fitness increases for both re-encoded and wild type viruses. Importantly, however, the most re-encoded virus failed to recover its replicative fitness. Evolution of these viruses in response to codon re-encoding was largely characterized by the emergence of both synonymous and non-synonymous mutations, sometimes located in genomic regions other than those involving re-encoding, and multiple convergent and compensatory mutations. However, there was a striking absence of codon reversion (<0.4%). Finally, multiple mutations were rapidly fixed in primate cells, whereas mosquito cells acted as a brake on evolution. In conclusion, random codon re-encoding provides important information on the evolution and genetic stability of CHIKV viruses and could be exploited to develop a safe, live attenuated CHIKV vaccine.

Intronic splicing mutations in PTCH1 cause Gorlin syndrome.

PubMed

Bholah, Zaynab; Smith, Miriam J; Byers, Helen J; Miles, Emma K; Evans, D Gareth; Newman, William G

2014-09-01

Gorlin syndrome is an autosomal dominant disorder characterized by multiple early-onset basal cell carcinoma, odontogenic keratocysts and skeletal abnormalities. It is caused by heterozygous mutations in the tumour suppressor PTCH1. Routine clinical genetic testing, by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to confirm a clinical diagnosis of Gorlin syndrome, identifies a mutation in 60-90 % of cases. We undertook RNA analysis on lymphocytes from ten individuals diagnosed with Gorlin syndrome, but without known PTCH1 mutations by exonic sequencing or MLPA. Two altered PTCH1 transcripts were identified. Genomic DNA sequence analysis identified an intron 7 mutation c.1068-10T>A, which created a strong cryptic splice acceptor site, leading to an intronic insertion of eight bases; this is predicted to create a frameshift p.(His358Alafs*12). Secondly, a deep intronic mutation c.2561-2057A>G caused an inframe insertion of 78 intronic bases in the cDNA transcript, leading to a premature stop codon p.(Gly854fs*3). The mutations are predicted to cause loss of function of PTCH1, consistent with its tumour suppressor function. The findings indicate the importance of RNA analysis to detect intronic mutations in PTCH1 not identified by routine screening techniques.

Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency

NASA Technical Reports Server (NTRS)

Kim, Chun-Hyung; Zabetian, Cyrus P.; Cubells, Joseph F.; Cho, Sonhae; Biaggioni, Italo; Cohen, Bruce M.; Robertson, David; Kim, Kwang-Soo

2002-01-01

Norepinephrine (NE), a key neurotransmitter of the central and peripheral nervous systems, is synthesized by dopamine beta-hydroxylase (DBH) that catalyzes oxidation of dopamine (DA) to NE. NE deficiency is a congenital disorder of unknown etiology, in which affected patients suffer profound autonomic failure. Biochemical features of the syndrome include undetectable tissue and circulating levels of NE and epinephrine, elevated levels of DA, and undetectable levels of DBH. Here, we report identification of seven novel variants including four potentially pathogenic mutations in the human DBH gene (OMIM 223360) from analysis of two unrelated patients and their families. Both patients are compound heterozygotes for variants affecting expression of DBH protein. Each carries one copy of a T-->C transversion in the splice donor site of DBH intron 1, creating a premature stop codon. In patient 1, there is a missense mutation in DBH exon 2. Patient 2 carries missense mutations in exons 1 and 6 residing in cis. We propose that NE deficiency is an autosomal recessive disorder resulting from heterogeneous molecular lesions at DBH. Copyright 2002 Wiley-Liss, Inc.

Quantitation of normal CFTR mRNA in CF patients with splice-site mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Z.; Olsen, J.C.; Silverman, L.M.

Previously we identified two mutations in introns of the CFTR gene associated with partially active splice sites and unusual clinical phenotypes. One mutation in intron 19 (3849+10 kb C to T) is common in CF patients with normal sweat chloride values; an 84 bp sequence from intron 19, which contains a stop codon, is inserted between exon 19 and exon 20 in most nasal CFTR transcripts. The other mutation in intron 14B (2789+5 G to A) is associated with elevated sweat chloride levels, but mild pulmonary disease; exon 14B (38 bp) is spliced out of most nasal CFTR transcipts. Themore » remaining CFTR cDNA sequences, other than the 84 bp insertion of exon 14B deletion, are identical to the published sequence. To correlate genotype and phenotype, we used quantitative RT-PCR to determine the levels of normally-spliced CFTR mRNA in nasal epithelia from these patients. CFTR cDNA was amplified (25 cycles) by using primers specific for normally-spliced species, {gamma}-actin cDNA was amplified as a standard.« less

Cloning and Characterization of a Critical Regulator for Preharvest Sprouting in Wheat

PubMed Central

Liu, Shubing; Sehgal, Sunish K.; Li, Jiarui; Lin, Meng; Trick, Harold N.; Yu, Jianming; Gill, Bikram S.; Bai, Guihua

2013-01-01

Sprouting of grains in mature spikes before harvest is a major problem in wheat (Triticum aestivum) production worldwide. We cloned and characterized a gene underlying a wheat quantitative trait locus (QTL) on the short arm of chromosome 3A for preharvest sprouting (PHS) resistance in white wheat using comparative mapping and map-based cloning. This gene, designated TaPHS1, is a wheat homolog of a MOTHER OF FLOWERING TIME (TaMFT)-like gene. RNA interference-mediated knockdown of the gene confirmed that TaPHS1 positively regulates PHS resistance. We discovered two causal mutations in TaPHS1 that jointly altered PHS resistance in wheat. One GT-to-AT mutation generates a mis-splicing site, and the other A-to-T mutation creates a premature stop codon that results in a truncated nonfunctional transcript. Association analysis of a set of wheat cultivars validated the role of the two mutations on PHS resistance. The molecular characterization of TaPHS1 is significant for expediting breeding for PHS resistance to protect grain yield and quality in wheat production. PMID:23821595

Autosomal Recessive Oculodentodigital Dysplasia: A Case Report and Review of the Literature.

PubMed

Taşdelen, Elifcan; Durmaz, Ceren D; Karabulut, Halil G

2018-06-15

Oculodentodigital dysplasia (ODDD) is a rare condition characterized by a typical facial appearance and variable findings of the eyes, teeth, and fingers. ODDD is caused by mutations in the GJA1 gene in chromosome 6q22 and inherited in an autosomal dominant manner in the majority of the patients. However, in recent clinical reports, autosomal recessive ODDD cases due to by GJA1 mutations were also described. Here, we report on a 14-year-old boy with microphthalmia, microcornea, narrow nasal bridge, hypoplastic alae nasi, prominent columnella, hypodontia, dental caries, and partial syndactyly of the 2nd and 3rd toes. These clinical findings were concordant with the diagnosis of ODDD, and a novel homozygous mutation (c.442C>T, p.Arg148Ter) was determined in the GJA1 gene leading to a premature stop codon. His phenotypically normal parents were found to be carriers of the same mutation. This is the third family in the literature in which ODDD segregates in an autosomal recessive manner. © 2018 S. Karger AG, Basel.

Pseudogenization of the tooth gene enamelysin (MMP20) in the common ancestor of extant baleen whales

PubMed Central

Meredith, Robert W.; Gatesy, John; Cheng, Joyce; Springer, Mark S.

2011-01-01

Whales in the suborder Mysticeti are filter feeders that use baleen to sift zooplankton and small fish from ocean waters. Adult mysticetes lack teeth, although tooth buds are present in foetal stages. Cladistic analyses suggest that functional teeth were lost in the common ancestor of crown-group Mysticeti. DNA sequences for the tooth-specific genes, ameloblastin (AMBN), enamelin (ENAM) and amelogenin (AMEL), have frameshift mutations and/or stop codons in this taxon, but none of these molecular cavities are shared by all extant mysticetes. Here, we provide the first evidence for pseudogenization of a tooth gene, enamelysin (MMP20), in the common ancestor of living baleen whales. Specifically, pseudogenization resulted from the insertion of a CHR-2 SINE retroposon in exon 2 of MMP20. Genomic and palaeontological data now provide congruent support for the loss of enamel-capped teeth on the common ancestral branch of crown-group mysticetes. The new data for MMP20 also document a polymorphic stop codon in exon 2 of the pygmy sperm whale (Kogia breviceps), which has enamel-less teeth. These results, in conjunction with the evidence for pseudogenization of MMP20 in Hoffmann's two-toed sloth (Choloepus hoffmanni), another enamel-less species, support the hypothesis that the only unique, non-overlapping function of the MMP20 gene is in enamel formation. PMID:20861053

Origin of noncoding DNA sequences: molecular fossils of genome evolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Naora, H.; Miyahara, K.; Curnow, R.N.

The total amount of noncoding sequences on chromosomes of contemporary organisms varies significantly from species to species. The authors propose a hypothesis for the origin of these noncoding sequences that assumes that (i) an approx. 0.55-kilobase (kb)-long reading frame composed the primordial gene and (ii) a 20-kb-long single-stranded polynucleotide is the longest molecule (as a genome) that was polymerized at random and without a specific template in the primordial soup/cell. The statistical distribution of stop codons allows examination of the probability of generating reading frames of approx. 0.55 kb in this primordial polynucleotide. This analysis reveals that with three stopmore » codons, a run of at least 0.55-kb equivalent length of nonstop codons would occur in 4.6% of 20-kb-long polynucleotide molecules. They attempt to estimate the total amount of noncoding sequences that would be present on the chromosomes of contemporary species assuming that present-day chromosomes retain the prototype primordial genome structure. Theoretical estimates thus obtained for most eukaryotes do not differ significantly from those reported for these specific organisms, with only a few exceptions. Furthermore, analysis of possible stop-codon distributions suggests that life on earth would not exist, at least in its present form, had two or four stop codons been selected early in evolution.« less

Rules of UGA-N decoding by near-cognate tRNAs and analysis of readthrough on short uORFs in yeast.

PubMed

Beznosková, Petra; Gunišová, Stanislava; Valášek, Leoš Shivaya

2016-03-01

The molecular mechanism of stop codon recognition by the release factor eRF1 in complex with eRF3 has been described in great detail; however, our understanding of what determines the difference in termination efficiencies among various stop codon tetranucleotides and how near-cognate (nc) tRNAs recode stop codons during programmed readthrough in Saccharomyces cerevisiae is still poor. Here, we show that UGA-C as the only tetranucleotide of all four possible combinations dramatically exacerbated the readthrough phenotype of the stop codon recognition-deficient mutants in eRF1. Since the same is true also for UAA-C and UAG-C, we propose that the exceptionally high readthrough levels that all three stop codons display when followed by cytosine are partially caused by the compromised sampling ability of eRF1, which specifically senses cytosine at the +4 position. The difference in termination efficiencies among the remaining three UGA-N tetranucleotides is then given by their varying preferences for nc-tRNAs. In particular, UGA-A allows increased incorporation of Trp-tRNA whereas UGA-G and UGA-C favor Cys-tRNA. Our findings thus expand the repertoire of general decoding rules by showing that the +4 base determines the preferred selection of nc-tRNAs and, in the case of cytosine, it also genetically interacts with eRF1. Finally, using an example of the GCN4 translational control governed by four short uORFs, we also show how the evolution of this mechanism dealt with undesirable readthrough on those uORFs that serve as the key translation reinitiation promoting features of the GCN4 regulation, as both of these otherwise counteracting activities, readthrough versus reinitiation, are mediated by eIF3. © 2016 Beznosková et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Characterization of codon usage pattern and influencing factors in Japanese encephalitis virus.

PubMed

Singh, Niraj K; Tyagi, Anuj; Kaur, Rajinder; Verma, Ramneek; Gupta, Praveen K

2016-08-02

Recently, several outbreaks of Japanese encephalitis (JE), caused by Japanese encephalitis virus (JEV), have been reported and it has become cause of concern across the world. In this study, detailed analysis of JEV codon usage pattern was performed. The relative synonymous codon usage (RSCU) values along with mean effective number of codons (ENC) value of 55.30 indicated the presence of low codon usages bias in JEV. The effect of mutational pressure on codon usage bias was confirmed by significant correlations of A3s, U3s, G3s, C3s, GC3s, ENC values, with overall nucleotide contents (A%, U%, G%, C%, and GC%). The correlation analysis of A3s, U3s, G3s, C3s, GC3s, with axis values of correspondence analysis (CoA) further confirmed the role of mutational pressure. However, the correlation analysis of Gravy values and Aroma values with A3s, U3s, G3s, C3s, and GC3s, indicated the presence of natural selection on codon usage bias in addition to mutational pressure. The natural selection was further confirmed by codon adaptation index (CAI) analysis. Additionally, relative dinucleotide frequencies, geographical distribution, and evolutionary processes also influenced the codon usage pattern to some extent. Copyright © 2016 Elsevier B.V. All rights reserved.

Mutation at codon 442 in the rpoB gene of Mycobacterium leprae does not confer resistance to rifampicin.

PubMed

Lavania, Mallika; Hena, Abu; Reja, Hasanoor; Nigam, Astha; Biswas, Nibir Kumar; Singh, Itu; Turankar, Ravindra P; Gupta, Ud; Kumar, Senthil; Rewaria, Latika; Patra, Pradip K R; Sengupta, Utpal; Bhattacharya, Basudeb

2016-03-01

Rifampicin is the major drug in the treatment of leprosy. The rifampicin resistance of Mycobacterium leprae results from a mutation in the rpoB gene, encoding the β subunit of RNA polymerase. As M. leprae is a non-cultivable organism observation of its growth using mouse food-pad (MFP) is the only Gold Standard assay used for confirmation of "in-vivo" drug resistance. Any mutation at molecular level has to be verified by MFP assay for final confirmation of drug resistance in leprosy. In the present study, M. leprae strains showing a mutation only at codon 442 Gln-His and along with mutation either at codon 424 Val-Gly or at 438 Gln-Val within the Rifampicin Resistance Determining Region (RRDR) confirmed by DNA sequencing and by high resolution melting (HRM) analysis were subjected for its growth in MFP. The M. leprae strain having the new mutation at codon 442 Gln-His was found to be sensitive to all the three drugs and strains having additional mutations at 424 Val-Gly and 438 Gln-Val were conferring resistance with Multi drug therapy (MDT) in MFP. These results indicate that MFP is the gold standard method for confirming the mutations detected by molecular techniques.

N-ras Mutation Detection by Pyrosequencing in Adult Patients with Acute Myeloid Leukemia at a Single Institution

PubMed Central

Jeong, Ji Hun; Park, Soon Ho; Park, Mi Jung; Kim, Moon Jin; Kim, Kyung Hee; Park, Pil Whan; Seo, Yiel Hea; Lee, Jae Hoon; Park, Jinny; Hong, Junshik

2013-01-01

Background N-ras mutations are one of the most commonly detected abnormalities of myeloid origin. N-ras mutations result in a constitutively active N-ras protein that induces uncontrolled cell proliferation and inhibits apoptosis. We analyzed N-ras mutations in adult patients with AML at a particular institution and compared pyrosequencing analysis with a direct sequencing method for the detection of N-ras mutations. Methods We analyzed 90 bone marrow samples from 83 AML patients. We detected N-ras mutations in codons 12, 13, and 61 using the pyrosequencing method and subsequently confirmed all data by direct sequencing. Using these methods, we screened the N-ras mutation quantitatively and determined the incidence and characteristic of N-ras mutation. Results The incidence of N-ras mutation was 7.2% in adult AML patients. The patients with N-ras mutations showed significant higher hemoglobin levels (P=0.022) and an increased incidence of FLT3 mutations (P=0.003). We observed 3 cases with N-ras mutations in codon 12 (3.6%), 2 cases in codon 13 (2.4%), and 1 case in codon 61 (1.2%). All the mutations disappeared during chemotherapy. Conclusions There is a low incidence (7.2%) of N-ras mutations in AML patients compared with other populations. Similar data is obtained by both pyrosequencing and direct sequencing. This study showed the correlation between the N-ras mutation and the therapeutic response. However, pyrosequencing provides quantitative data and is useful for monitoring therapeutic responses. PMID:23667841

Novel folliculin (FLCN) mutation and familial spontaneous pneumothorax.

PubMed

Zhu, J-F; Shen, X-Q; Zhu, F; Tian, L

2017-01-01

Familial spontaneous pneumothorax is one of the characteristics of Birt-Hogg-Dubé syndrome (BHDS), which is an autosomal dominant disease caused by the mutation of folliculin (FLCN). To investigate the mutation of FLCN gene in a familial spontaneous pneumothorax. Prospective case study. Clinical and genetic data of a Chinese family with four patients who presented spontaneous pneumothorax in the absence of skin lesions or renal tumors were collected. CT scan of patient's lung was applied for observation of pneumothorax. DNA sequencing of the coding exons (4-14 exons) of FLCN was performed for all 11 members of the family and 100 unrelated healthy controls. CT scan of patient's lung showed spontaneous pneumothorax. A mutation (c. 510C > G) that leads to a premature stop codon (p. Y170X) was found in the proband using DNA sequencing of coding exons (4-14 exons) of FLCN. This mutation was also observed in the other affected members of the family. A nonsense mutation of FLCN was found in a spontaneous pneumothorax family. Our results expand the mutational spectrum of FLCN in patients with BHDS. © The Author 2016. Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Two novel compound heterozygous mutations in the BCKDHB gene that cause the intermittent form of maple syrup urine disease.

PubMed

Guo, Yi; Liming, Liu; Jiang, Li

2015-12-01

Intermittent maple syrup urine disease (MSUD) is a potentially life-threatening metabolic disorder caused by a deficiency of branched chain α-ketoacid dehydrogenase (BCKD) complex. In contrast to classic MSUD, children with the intermittent form usually have an atypical clinical manifestation. Here, we describe the presenting symptoms and clinical course of a Chinese boy with intermittent MSUD. Mutation analysis identified two previously unreported mutations in exon 7 of the BCKDHB gene: c.767A > G (p.Y256C) and c.768C > G (p.Y256X); the parents were each heterozygous for one of these mutations. In silico analysis predicted Y256C probably affects protein structure; Y256X leads to a premature stop codon. This case demonstrates intermittent MSUD should be suspected in cases with symptoms of recurrent encephalopathy, especially ataxia or marked drowsiness, which usually present after the neonatal period and in conjunction with infection. symmetrical basal ganglia damage but normal myelination in the posterior limb will assist differential diagnosis; alloisoleucine is a useful diagnostic marker and mutation analysis may be of prognostic value. These novel mutations Y256C and Y256X result in the clinical manifestation of a variant form of MSUD, expanding the mutation spectrum of this disease.

Novel FAM20A mutation causes autosomal recessive amelogenesis imperfecta.

PubMed

Volodarsky, Michael; Zilberman, Uri; Birk, Ohad S

2015-06-01

To relate the peculiar phenotype of amelogenesis imperfecta in a large Bedouin family to the genotype determined by whole genome linkage analysis. Amelogenesis imperfecta (AI) is a broad group of inherited pathologies affecting enamel formation, characterized by variability in phenotypes, causing mutations and modes of inheritance. Autosomal recessive or compound heterozygous mutations in FAM20A, encoding sequence similarity 20, member A, have been shown to cause several AI phenotypes. Five members from a large consanguineous Bedouin family presented with hypoplastic amelogenesis imperfecta with unerupted and resorbed permanent molars. Following Soroka Medical Center IRB approval and informed consent, blood samples were obtained from six affected offspring, five obligatory carriers and two unaffected siblings. Whole genome linkage analysis was performed followed by Sanger sequencing of FAM20A. The sequencing unravelled a novel homozygous deletion mutation in exon 11 (c.1523delC), predicted to insert a premature stop codon (p.Thr508Lysfs*6). We provide an interesting case of novel mutation in this rare disorder, in which the affected kindred is unique in the large number of family members sharing a similar phenotype. Copyright © 2015 Elsevier Ltd. All rights reserved.

High-resolution melting analysis of gyrA codon 84 and grlA codon 80 mutations conferring resistance to fluoroquinolones in Staphylococcus pseudintermedius isolates from canine clinical samples.

PubMed

Loiacono, Monica; Martino, Piera A; Albonico, Francesca; Dell'Orco, Francesca; Ferretti, Manuela; Zanzani, Sergio; Mortarino, Michele

2017-09-01

Staphylococcus pseudintermedius is an opportunistic pathogen of dogs and cats. A high-resolution melting analysis (HRMA) protocol was designed and tested on 42 clinical isolates with known fluoroquinolone (FQ) susceptibility and gyrA codon 84 and grlA codon 80 mutation status. The HRMA approach was able to discriminate between FQ-sensitive and FQ-resistant strains and confirmed previous reports that the main mutation site associated with FQ resistance in S. pseudintermedius is located at position 251 (Ser84Leu) of gyrA. Routine, HRMA-based FQ susceptibility profiles may be a valuable tool to guide therapy. The FQ resistance-predictive power of the assay should be tested in a significantly larger number of isolates.

A novel silent deletion, an insertion mutation and a nonsense mutation in the TCOF1 gene found in two Chinese cases of Treacher Collins syndrome.

PubMed

Wang, Yan; Yin, Xiao-Juan; Han, Tao; Peng, Wei; Wu, Hong-Lin; Liu, Xin; Feng, Zhi-Chun

2014-12-01

Treacher Collins syndrome (TCS) is the most common and well-known craniofacial disorder caused by mutations in the genes involved in pre-rRNA transcription, which include the TCOF1 gene. This study explored the role of TCOF1 mutations in Chinese patients with TCS. Mutational analysis of the TCOF1 gene was performed in three patients using polymerase chain reaction and direct sequencing. Among these three patients, two additional TCOF1 variations, a novel 18 bp deletion and a novel 1 bp insertion mutation, were found in patient 1, together with a novel nonsense mutation (p.Ser476X) and a previously reported 4 bp deletion (c.1872_1875delTGAG) in other patients. Pedigree analysis allowed for prediction of the character of the mutation, which was either pathological or not. The 18 bp deletion of six amino acids, Ser-Asp-Ser-Glu-Glu-Glu (798*803), which was located in the CKII phosphorylation site of treacle, seemed relatively benign for TCS. By contrast, another novel mutation of c.1072_1073insC (p.Gln358ProfsX23) was a frameshift mutation and expected to result in a premature stop codon. This study provides insights into the functional domain of treacle and illustrates the importance of clinical and family TCS screening for the interpretation of novel sequence alterations.

Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP).

PubMed

Rowe, P S; Oudet, C L; Francis, F; Sinding, C; Pannetier, S; Econs, M J; Strom, T M; Meitinger, T; Garabedian, M; David, A; Macher, M A; Questiaux, E; Popowska, E; Pronicka, E; Read, A P; Mokrzycki, A; Glorieux, F H; Drezner, M K; Hanauer, A; Lehrach, H; Goulding, J N; O'Riordan, J L

1997-04-01

Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.

Unexpectedly low mutation rates in beta-myosin heavy chain and cardiac myosin binding protein genes in Italian patients with hypertrophic cardiomyopathy.

PubMed

Roncarati, Roberta; Latronico, Michael V G; Musumeci, Beatrice; Aurino, Stefania; Torella, Annalaura; Bang, Marie-Louise; Jotti, Gloria Saccani; Puca, Annibale A; Volpe, Massimo; Nigro, Vincenzo; Autore, Camillo; Condorelli, Gianluigi

2011-11-01

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. Fourteen sarcomeric and sarcomere-related genes have been implicated in HCM etiology, those encoding β-myosin heavy chain (MYH7) and cardiac myosin binding protein C (MYBPC3) reported as the most frequently mutated: in fact, these account for around 50% of all cases related to sarcomeric gene mutations, which are collectively responsible for approximately 70% of all HCM cases. Here, we used denaturing high-performance liquid chromatography followed by bidirectional sequencing to screen the coding regions of MYH7 and MYBPC3 in a cohort (n = 125) of Italian patients presenting with HCM. We found 6 MHY7 mutations in 9/125 patients and 18 MYBPC3 mutations in 19/125 patients. Of the three novel MYH7 mutations found, two were missense, and one was a silent mutation; of the eight novel MYBPC3 mutations, one was a substitution, three were stop codons, and four were missense mutations. Thus, our cohort of Italian HCM patients did not harbor the high frequency of mutations usually found in MYH7 and MYBPC3. This finding, coupled to the clinical diversity of our cohort, emphasizes the complexity of HCM and the need for more inclusive investigative approaches in order to fully understand the pathogenesis of this disease. Copyright © 2011 Wiley-Liss, Inc.

Prognostic impact of KRAS mutation subtypes in 677 patients with metastatic lung adenocarcinomas

PubMed Central

Yu, Helena A.; Sima, Camelia S.; Shen, Ronglai; Kass, Samantha; Gainor, Justin; Shaw, Alice; Hames, Megan; Iams, Wade; Aston, Jonathan; Lovly, Christine M.; Horn, Leora; Lydon, Christine; Oxnard, Geoffrey R.; Kris, Mark G.; Ladanyi, Marc; Riely, Gregory J.

2015-01-01

Background We previously demonstrated that patients with metastatic KRAS mutant lung cancers have a shorter survival compared to patients with KRAS wild type cancers. Recent reports have suggested different clinical outcomes and distinct activated signaling pathways depending on KRAS mutation subtype. To better understand the impact of KRAS mutation subtype, we analyzed data from 677 patients with KRAS mutant metastatic lung cancer. Methods We reviewed all patients with metastatic or recurrent lung cancers found to have KRAS mutations over a 6 year time period. We evaluated the associations between KRAS mutation type, clinical factors, and overall survival in univariate and multivariate analyses. Any significant findings were validated in an external multi-institution patient data set. Results Among 677 patients with KRAS mutant lung cancers (53 at codon 13, 624 at codon 12), there was no difference in overall survival for patients when comparing KRAS transition versus transversion mutations (p=0.99), smoking status (p=0.33) or when comparing specific amino acid substitutions (p=0.20). In our data set, patients with KRAS codon 13 mutant tumors (n=53) had shorter overall survival compared to patients with codon 12 mutant tumors (n=624)( 1.1 vs 1.3 years, respectively, p=0.009), and the findings were confirmed in a multivariate Cox model controlling for age, sex and smoking status (HR 1.52 95% CI 1.11-2.08, p=0.008). In an independent validation set of tumors from 682 patients with stage IV KRAS mutant lung cancers, there was no difference in survival between patients with KRAS codon 13 versus codon 12 mutations (1.0 vs 1.1 years respectively, p=0.41). Conclusions Among individuals with KRAS mutant metastatic lung cancers treated with conventional therapy, there are apparent differences in outcome based on KRAS mutation subtype PMID:25415430

Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster

PubMed Central

Dunn, Joshua G; Foo, Catherine K; Belletier, Nicolette G; Gavis, Elizabeth R; Weissman, Jonathan S

2013-01-01

Ribosomes can read through stop codons in a regulated manner, elongating rather than terminating the nascent peptide. Stop codon readthrough is essential to diverse viruses, and phylogenetically predicted to occur in a few hundred genes in Drosophila melanogaster, but the importance of regulated readthrough in eukaryotes remains largely unexplored. Here, we present a ribosome profiling assay (deep sequencing of ribosome-protected mRNA fragments) for Drosophila melanogaster, and provide the first genome-wide experimental analysis of readthrough. Readthrough is far more pervasive than expected: the vast majority of readthrough events evolved within D. melanogaster and were not predicted phylogenetically. The resulting C-terminal protein extensions show evidence of selection, contain functional subcellular localization signals, and their readthrough is regulated, arguing for their importance. We further demonstrate that readthrough occurs in yeast and humans. Readthrough thus provides general mechanisms both to regulate gene expression and function, and to add plasticity to the proteome during evolution. DOI: http://dx.doi.org/10.7554/eLife.01179.001 PMID:24302569

Influence of certain forces on evolution of synonymous codon usage bias in certain species of three basal orders of aquatic insects.

PubMed

Selva Kumar, C; Nair, Rahul R; Sivaramakrishnan, K G; Ganesh, D; Janarthanan, S; Arunachalam, M; Sivaruban, T

2012-12-01

Forces that influence the evolution of synonymous codon usage bias are analyzed in six species of three basal orders of aquatic insects. The rationale behind choosing six species of aquatic insects (three from Ephemeroptera, one from Plecoptera, and two from Odonata) for the present analysis is based on phylogenetic position at the basal clades of the Order Insecta facilitating the understanding of the evolution of codon bias and of factors shaping codon usage patterns in primitive clades of insect lineages and their subtle differences in some of their ecological and environmental requirements in terms of habitat-microhabitat requirements, altitudinal preferences, temperature tolerance ranges, and consequent responses to climate change impacts. The present analysis focuses on open reading frames of the 13 protein-coding genes in the mitochondrial genome of six carefully chosen insect species to get a comprehensive picture of the evolutionary intricacies of codon bias. In all the six species, A and T contents are observed to be significantly higher than G and C, and are used roughly equally. Since transcription hypothesis on codon usage demands A richness and T poorness, it is quite likely that mutation pressure may be the key factor associated with synonymous codon usage (SCU) variations in these species because the mutation hypothesis predicts AT richness and GC poorness in the mitochondrial DNA. Thus, AT-biased mutation pressure seems to be an important factor in framing the SCU variation in all the selected species of aquatic insects, which in turn explains the predominance of A and T ending codons in these species. This study does not find any association between microhabitats and codon usage variations in the mitochondria of selected aquatic insects. However, this study has identified major forces, such as compositional constraints and mutation pressure, which shape patterns of codon usage in mitochondrial genes in the primitive clades of insect lineages.

Novel mutation at the initiation codon in the Norrie disease gene in two Japanese families.

PubMed

Isashiki, Y; Ohba, N; Yanagita, T; Hokita, N; Doi, N; Nakagawa, M; Ozawa, M; Kuroda, N

1995-01-01

We have identified a new mutation of Norrie disease (ND) gene in two Japanese males from unrelated families; they showed typical ocular features of ND but no mental retardation or hearing impairment. A mutation was found in both patients at the initiation codon of exon 2 of the ND gene (ATG to GTG), with otherwise normal nucleotide sequences. Their mothers had the normal and mutant types of the gene, which was expected for heterozygotes of the disease. The mutation of the initiation codon would cause the failure of ND gene expression or a defect in translation thereby truncating the amino terminus of ND protein. In view of the rarity and marked heterogeneity of mutations in the ND gene, the present apparently unrelated Japanese families who have lived in the same area for over two centuries presumably share the origin of the mutation.

Phenotypic and genotypic evaluation of fluoroquinolone resistance in clinical isolates of Staphylococcus aureus in Tehran

PubMed Central

Aligholi, Marzieh; Mirsalehian, Akbar; Halimi, Shahnaz; Imaneini, Hossein; Taherikalani, Morovat; Jabalameli, Fereshteh; Asadollahi, Parisa; Mohajer, Babak; Abdollahi, Alireza; Emaneini, Mohammad

2011-01-01

Summary Background Fluoroquinolones are broad-spectrum antibiotics widely used in the treatment of bacterial infections such as Staphylococcus aureus isolates. Resistance to these antibiotics is increasing. Material/Methods The occurrence of mutations in the grlA and gyrA loci were evaluated in 69 fluoroquinolone-resistant S. aureus isolates from 2 teaching hospitals of Tehran University of Medical Sciences. Results Out of the 165 S. aureus isolates, 87 (52.7%) were resistant to methicillin and 69 (41.8%) were resistant to fluoroquinolone. Fluoroquinolone-resistant S. aureus isolates had a mutation at codon 80 in the grlA gene and different mutational combinations in the gyrA gene. These mutational combinations included 45 isolates at codons 84 and 86, 23 isolates at codons 84, 86 and 106 and 1 isolate at codons 84, 86 and 90. Fluoroquinolone-resistant S. aureus isolates were clustered into 33 PFGE types. Conclusions The findings of this study show that the fluoroquinolone-resistant S. aureus strains isolated in the teaching hospitals in Tehran had multiple mutations in the QRDRs region of both grlA and gyrA genes. PMID:21873957

Phenotypic and genotypic evaluation of fluoroquinolone resistance in clinical isolates of Staphylococcus aureus in Tehran.

PubMed

Aligholi, Marzieh; Mirsalehian, Akbar; Halimi, Shahnaz; Imaneini, Hossein; Taherikalani, Morovat; Jabalameli, Fereshteh; Asadollahi, Parisa; Mohajer, Babak; Abdollahi, Alireza; Emaneini, Mohammad

2011-09-01

Fluoroquinolones are broad-spectrum antibiotics widely used in the treatment of bacterial infections such as Staphylococcus aureus isolates. Resistance to these antibiotics is increasing. The occurrence of mutations in the grlA and gyrA loci were evaluated in 69 fluoroquinolone-resistant S. aureus isolates from 2 teaching hospitals of Tehran University of Medical Sciences. Out of the 165 S. aureus isolates, 87 (52.7%) were resistant to methicillin and 69 (41.8%) were resistant to fluoroquinolone. Fluoroquinolone-resistant S. aureus isolates had a mutation at codon 80 in the grlA gene and different mutational combinations in the gyrA gene. These mutational combinations included 45 isolates at codons 84 and 86, 23 isolates at codons 84, 86 and 106 and 1 isolate at codons 84, 86 and 90. Fluoroquinolone-resistant S. aureus isolates were clustered into 33 PFGE types. The findings of this study show that the fluoroquinolone-resistant S. aureus strains isolated in the teaching hospitals in Tehran had multiple mutations in the QRDRs region of both grlA and gyrA genes.

ACB-PCR measurement of spontaneous and furan-induced H-ras codon 61 CAA to CTA and CAA to AAA mutation in B6C3F1 mouse liver.

PubMed

Banda, Malathi; Recio, Leslie; Parsons, Barbara L

2013-10-01

Furan is a rodent liver carcinogen, but the mode of action for furan hepatocarcinogenicity is unclear. H-ras codon 61 mutations have been detected in spontaneous liver tumors of B6C3F1 mice, and the fraction of liver tumors carrying H-ras codon 61 CAA to AAA mutation increased in furan-treated mice. Allele-specific competitive blocker PCR (ACB-PCR) has been used previously to quantify early, carcinogen-induced increases in tumor-associated mutations. The present pilot study investigated whether furan drives clonal expansion of pre-existing H-ras mutant cells in B6C3F1 mouse liver. H-ras codon 61 CAA to CTA and CAA to AAA mutations were measured in DNA isolated from liver tissue of female mice treated with 0, 1, 2, 4, or 8 mg furan/kg body weight, five days per week for three weeks, using five mice per treatment group. Spontaneous levels of mutation were low, with two of five control mice having an H-ras codon 61 CTA or AAA mutant fraction (MF) greater than 10(-5) . Several furan-treated mice had H-ras codon 61 AAA or CTA MFs greater than those measured in control mice and lower bound estimates of induced MF were calculated. However, no statistically-significant differences were observed between treatment groups. Therefore, while sustained exposure to furan is carcinogenic, at the early stage of carcinogenesis examined in this study (three weeks), there was not a significant expansion of H-ras mutant cells. Copyright © 2013 Wiley Periodicals, Inc.

HMG CoA lyase deficiency: identification of five causal point mutations in codons 41 and 42, including a frequent Saudi Arabian mutation, R41Q.

PubMed Central

Mitchell, G A; Ozand, P T; Robert, M F; Ashmarina, L; Roberts, J; Gibson, K M; Wanders, R J; Wang, S; Chevalier, I; Plöchl, E; Miziorko, H

1998-01-01

The hereditary deficiency of 3-hydroxy-3-methylglutaryl (HMG) CoA lyase (HL; OMIM 246450 [http://www3.ncbi.nlm.nih. gov:80/htbin-post/Omim/dispmim?246450]) results in episodes of hypoketotic hypoglycemia and coma and is reported to be frequent and clinically severe in Saudi Arabia. We found genetic diversity among nine Saudi HL-deficient probands: six were homozygous for the missense mutation R41Q, and two were homozygous for the frameshift mutation F305fs(-2). In 32 non-Saudi HL-deficient probands, we found three R41Q alleles and also discovered four other deleterious point mutations in codons 41 and 42: R41X, D42E, D42G, and D42H. In purified mutant recombinant HL, all four missense mutations in codons 41 and 42 cause a marked decrease in HL activity. We developed a screening procedure for HL missense mutations that yields residual activity at levels comparable to those obtained using purified HL peptides. Codons 41 and 42 are important for normal HL catalysis and account for a disproportionate 21 (26%) of 82 of mutant alleles in our group of HL-deficient probands. PMID:9463337

High-level tetracycline resistance mediated by efflux pumps Tet(A) and Tet(A)-1 with two start codons.

PubMed

Wang, Weixia; Guo, Qinglan; Xu, Xiaogang; Sheng, Zi-ke; Ye, Xinyu; Wang, Minggui

2014-11-01

Efflux is the most common mechanism of tetracycline resistance. Class A tetracycline efflux pumps, which often have high prevalence in Enterobacteriaceae, are encoded by tet(A) and tet(A)-1 genes. These genes have two potential start codons, GTG and ATG, located upstream of the genes. The purpose of this study was to determine the start codon(s) of the class A tetracycline resistance (tet) determinants tet(A) and tet(A)-1, and the tetracycline resistance level they mediated. Conjugation, transformation and cloning experiments were performed and the genetic environment of tet(A)-1 was analysed. The start codons in class A tet determinants were investigated by site-directed mutagenesis of ATG and GTG, the putative translation initiation codons. High-level tetracycline resistance was transferred from the clinical strain of Klebsiella pneumoniae 10-148 containing tet(A)-1 plasmid pHS27 to Escherichia coli J53 by conjugation. The transformants harbouring recombinant plasmids that carried tet(A) or tet(A)-1 exhibited tetracycline MICs of 256-512 µg ml(-1), with or without tetR(A). Once the ATG was mutated to a non-start codon, the tetracycline MICs were not changed, while the tetracycline MICs decreased from 512 to 64 µg ml(-1) following GTG mutation, and to ≤4 µg ml(-1) following mutation of both GTG and ATG. It was presumed that class A tet determinants had two start codons, which are the primary start codon GTG and secondary start codon ATG. Accordingly, two putative promoters were predicted. In conclusion, class A tet determinants can confer high-level tetracycline resistance and have two start codons. © 2014 The Authors.

Pyviko: an automated Python tool to design gene knockouts in complex viruses with overlapping genes.

PubMed

Taylor, Louis J; Strebel, Klaus

2017-01-07

Gene knockouts are a common tool used to study gene function in various organisms. However, designing gene knockouts is complicated in viruses, which frequently contain sequences that code for multiple overlapping genes. Designing mutants that can be traced by the creation of new or elimination of existing restriction sites further compounds the difficulty in experimental design of knockouts of overlapping genes. While software is available to rapidly identify restriction sites in a given nucleotide sequence, no existing software addresses experimental design of mutations involving multiple overlapping amino acid sequences in generating gene knockouts. Pyviko performed well on a test set of over 240,000 gene pairs collected from viral genomes deposited in the National Center for Biotechnology Information Nucleotide database, identifying a point mutation which added a premature stop codon within the first 20 codons of the target gene in 93.2% of all tested gene-overprinted gene pairs. This shows that Pyviko can be used successfully in a wide variety of contexts to facilitate the molecular cloning and study of viral overprinted genes. Pyviko is an extensible and intuitive Python tool for designing knockouts of overlapping genes. Freely available as both a Python package and a web-based interface ( http://louiejtaylor.github.io/pyViKO/ ), Pyviko simplifies the experimental design of gene knockouts in complex viruses with overlapping genes.

Novel mutations of endothelin-B receptor gene in Pakistani patients with Waardenburg syndrome.

PubMed

Jabeen, Raheela; Babar, Masroor Ellahi; Ahmad, Jamil; Awan, Ali Raza

2012-01-01

Mutations in EDNRB gene have been reported to cause Waardenburg-Shah syndrome (WS4) in humans. We investigated 17 patients with WS4 for identification of mutations in EDNRB gene using PCR and direct sequencing technique. Four genomic mutations were detected in four patients; a G to C transversion in codon 335 (S335C) in exon 5 and a transition of T to C in codon (S361L) in exon 5, a transition of A to G in codon 277 (L277L) in exon 4, a non coding transversion of T to A at -30 nucleotide position of exon 5. None of these mutations were found in controls. One of the patients harbored two novel mutations (S335C, S361L) in exon 5 and one in Intronic region (-30exon5 A>G). All of the mutations were homozygous and novel except the mutation observed in exon 4. In this study, we have identified 3 novel mutations in EDNRB gene associated with WS4 in Pakistani patients.

SENCA: A Multilayered Codon Model to Study the Origins and Dynamics of Codon Usage

PubMed Central

Pouyet, Fanny; Bailly-Bechet, Marc; Mouchiroud, Dominique; Guéguen, Laurent

2016-01-01

Gene sequences are the target of evolution operating at different levels, including the nucleotide, codon, and amino acid levels. Disentangling the impact of those different levels on gene sequences requires developing a probabilistic model with three layers. Here we present SENCA (site evolution of nucleotides, codons, and amino acids), a codon substitution model that separately describes 1) nucleotide processes which apply on all sites of a sequence such as the mutational bias, 2) preferences between synonymous codons, and 3) preferences among amino acids. We argue that most synonymous substitutions are not neutral and that SENCA provides more accurate estimates of selection compared with more classical codon sequence models. We study the forces that drive the genomic content evolution, intraspecifically in the core genome of 21 prokaryotes and interspecifically for five Enterobacteria. We retrieve the existence of a universal mutational bias toward AT, and that taking into account selection on synonymous codon usage has consequences on the measurement of selection on nonsynonymous substitutions. We also confirm that codon usage bias is mostly driven by selection on preferred codons. We propose new summary statistics to measure the relative importance of the different evolutionary processes acting on sequences. PMID:27401173

Truncating Mutations of MAGEL2, a Gene within the Prader-Willi Locus, Are Responsible for Severe Arthrogryposis

PubMed Central

Mejlachowicz, Dan; Nolent, Flora; Maluenda, Jérome; Ranjatoelina-Randrianaivo, Hanitra; Giuliano, Fabienne; Gut, Ivo; Sternberg, Damien; Laquerrière, Annie; Melki, Judith

2015-01-01

Arthrogryposis multiplex congenita (AMC) is characterized by the presence of multiple joint contractures resulting from reduced or absent fetal movement. Here, we report two unrelated families affected by lethal AMC. By genetic mapping and whole-exome sequencing in a multiplex family, a heterozygous truncating MAGEL2 mutation leading to frameshift and a premature stop codon (c.1996delC, p.Gln666Serfs∗36) and inherited from the father was identified in the probands. In another family, a distinct heterozygous truncating mutation leading to frameshift (c.2118delT, p.Leu708Trpfs∗7) and occurring de novo on the paternal allele of MAGEL2 was identified in the affected individual. In both families, RNA analysis identified the mutated paternal MAGEL2 transcripts only in affected individuals. MAGEL2 is one of the paternally expressed genes within the Prader-Willi syndrome (PWS) locus. PWS is associated with, to varying extents, reduced fetal mobility, severe infantile hypotonia, childhood-onset obesity, hypogonadism, and intellectual disability. MAGEL2 mutations have been recently reported in affected individuals with features resembling PWS and called Schaaf-Yang syndrome. Here, we show that paternal MAGEL2 mutations are also responsible for lethal AMC, recapitulating the clinical spectrum of PWS and suggesting that MAGEL2 is a PWS-determining gene. PMID:26365340

A family of oculofaciocardiodental syndrome (OFCD) with a novel BCOR mutation and genomic rearrangements involving NHS.

PubMed

Kondo, Yukiko; Saitsu, Hirotomo; Miyamoto, Toshinobu; Nishiyama, Kiyomi; Tsurusaki, Yoshinori; Doi, Hiroshi; Miyake, Noriko; Ryoo, Na-Kyung; Kim, Jeong Hun; Yu, Young Suk; Matsumoto, Naomichi

2012-03-01

Oculofaciocardiodental syndrome (OFCD) is an X-linked dominant disorder associated with male lethality, presenting with congenital cataract, dysmorphic face, dental abnormalities and septal heart defects. Mutations in BCOR (encoding BCL-6-interacting corepressor) cause OFCD. Here, we report on a Korean family with common features of OFCD including bilateral 2nd-3rd toe syndactyly and septal heart defects in three affected females (mother and two daughters). Through the mutation screening and copy number analysis using genomic microarray, we identified a novel heterozygous mutation, c.888delG, in the BCOR gene and two interstitial microduplications at Xp22.2-22.13 and Xp21.3 in all the three affected females. The BCOR mutation may lead to a premature stop codon (p.N297IfsX80). The duplication at Xp22.2-22.13 involved the NHS gene causative for Nance-Horan syndrome, which is an X-linked disorder showing similar clinical features with OFCD in affected males, and in carrier females with milder presentation. Considering the presence of bilateral 2nd-3rd toe syndactyly and septal heart defects, which is unique to OFCD, the mutation in BCOR is likely to be the major determinant for the phenotypes in this family.

Using Microarrays to Facilitate Positional Cloning: Identification of Tomosyn as an Inhibitor of Neurosecretion

PubMed Central

Dybbs, Michael; Ngai, John; Kaplan, Joshua M

2005-01-01

Forward genetic screens have been used as a powerful strategy to dissect complex biological pathways in many model systems. A significant limitation of this approach has been the time-consuming and costly process of positional cloning and molecular characterization of the mutations isolated in these screens. Here, the authors describe a strategy using microarray hybridizations to facilitate positional cloning. This method relies on the fact that premature stop codons (i.e., nonsense mutations) constitute a frequent class of mutations isolated in screens and that nonsense mutant messenger RNAs are efficiently degraded by the conserved nonsense-mediated decay pathway. They validate this strategy by identifying two previously uncharacterized mutations: (1) tom-1, a mutation found in a forward genetic screen for enhanced acetylcholine secretion in Caenorhabditis elegans, and (2) an apparently spontaneous mutation in the hif-1 transcription factor gene. They further demonstrate the broad applicability of this strategy using other known mutants in C. elegans, Arabidopsis, and mouse. Characterization of tom-1 mutants suggests that TOM-1, the C. elegans ortholog of mammalian tomosyn, functions as an endogenous inhibitor of neurotransmitter secretion. These results also suggest that microarray hybridizations have the potential to significantly reduce the time and effort required for positional cloning. PMID:16103915

A new titinopathy

PubMed Central

De Cid, Rafael; Ben Yaou, Rabah; Roudaut, Carinne; Charton, Karine; Baulande, Sylvain; Leturcq, France; Romero, Norma Beatriz; Malfatti, Edoardo; Beuvin, Maud; Vihola, Anna; Criqui, Audrey; Nelson, Isabelle; Nectoux, Juliette; Ben Aim, Laurène; Caloustian, Christophe; Olaso, Robert; Udd, Bjarne; Bonne, Gisèle; Eymard, Bruno

2015-01-01

Objective: To identify the genetic defects present in 3 families with muscular dystrophy, contractures, and calpain 3 deficiency. Methods: We performed targeted exome sequencing on one patient presenting a deficiency in calpain 3 on Western blot but for which mutations in the gene had been excluded. The identification of a homozygous truncating mutation in the M-line part of titin prompted us to sequence this region in 2 additional patients presenting similar clinical and biochemical characteristics. Results: The 3 patients shared similar features: coexistence of limb-girdle weakness and early-onset diffuse joint contractures without cardiomyopathy. The biopsies showed rimmed vacuoles, a dystrophic pattern, and secondary reduction in calpain 3. We identified a novel homozygous mutation in the exon Mex3 of the TTN gene in the first patient. At protein level, this mutation introduces a stop codon at the level of Mex3. Interestingly, we identified truncating mutations in both alleles in the same region of the TTN gene in patients from 2 additional families. Molecular protein analyses confirm loss of the C-ter part of titin. Conclusions: Our study broadens the phenotype of titinopathies with the report of a new clinical entity with prominent contractures and no cardiac abnormality and where the recessive mutations lead to truncation of the M-line titin and secondary calpain 3 deficiency. PMID:26581302

Identification of a de novo mutation of SOX10 in a Chinese patient with Waardenburg syndrome type IV.

PubMed

Liang, Fenghe; Zhao, Min; Fan, Lynn; Zhang, Hongyan; Shi, Yang; Han, Rui; Qu, Chunyan

2016-12-01

Waardenburg syndrome is a rare genetic disorder, characterized by the association of sensorineural hearing loss and pigmentation abnormalities. Four subtypes have been classified. The present study aimed to analyze the clinical feature and investigate the genetic cause for a Chinese case of Waardenburg type IV (WS4). The patient and his family members were subjected to mutation detection in the candidate gene SOX10 by Sanger sequencing. The patient has the clinical features of WS4, including sensorineural hearing loss, bright blue irides, premature graying of the hair and Hirschsprung disease. A novel heterozygous frameshift mutation, c.752_753ins7 (p.Gly252Alafs*31) in the exon 5 of SOX10 was detected in the patient, but not found in the unaffected family members and 100 normal controls. This mutation results in a premature stop codon 31 amino acid downstream. The novel mutation c.752_753ins7 (p.Gly252Alafs*31) arose de novo and was considered as the cause of WS4 in the proband. This study further characterized the molecular complexity of WS4 and provided a clinical case for genotype-phenotype correlation studies of different phenotypes caused by SOX10 mutations. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Methylation of class I translation termination factors: structural and functional aspects.

PubMed

Graille, Marc; Figaro, Sabine; Kervestin, Stéphanie; Buckingham, Richard H; Liger, Dominique; Heurgué-Hamard, Valérie

2012-07-01

During protein synthesis, release of polypeptide from the ribosome occurs when an in frame termination codon is encountered. Contrary to sense codons, which are decoded by tRNAs, stop codons present in the A-site are recognized by proteins named class I release factors, leading to the release of newly synthesized proteins. Structures of these factors bound to termination ribosomal complexes have recently been obtained, and lead to a better understanding of stop codon recognition and its coordination with peptidyl-tRNA hydrolysis in bacteria. Release factors contain a universally conserved GGQ motif which interacts with the peptidyl-transferase centre to allow peptide release. The Gln side chain from this motif is methylated, a feature conserved from bacteria to man, suggesting an important biological role. However, methylation is catalysed by completely unrelated enzymes. The function of this motif and its post-translational modification will be discussed in the context of recent structural and functional studies. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

The complete mitochondrial genome of the Longnose skate: Raja rhina (Rajiformes, Rajidae).

PubMed

Jeong, Dageum; Lee, Youn-Ho

2015-02-01

The complete sequence of mitochondrial DNA of a longnose skate, Raja rhina was determined for the first time. It is 16,910 bp in length containing 2 rRNA, 22 tRNA and 13 protein coding genes with the same gene order and structure as those of other Rajidae species. The nucleotide of L-strand is composed of 30.1% A, 27.2% C, 28.5% T and 14.2% G, showing a slight A + T bias. The G is the least used base and markedly lower at the third codon position (5.4%). Twelve of the 13 protein coding genes use ATG as their start codon while the COX1 starts with GTG. As for stop codon, only ND4 shows incomplete stop codon TA. This mitogenome is the first report for a species of the genus Raja, and providing a valuable resource of genetic information for understanding the phylogenetic relationship and the evolution of the genus Raja as well as the family, Rajidae.

Codon usage bias in phylum Actinobacteria: relevance to environmental adaptation and host pathogenicity.

PubMed

Lal, Devi; Verma, Mansi; Behura, Susanta K; Lal, Rup

2016-10-01

Actinobacteria are Gram-positive bacteria commonly found in soil, freshwater and marine ecosystems. In this investigation, bias in codon usages of ninety actinobacterial genomes was analyzed by estimating different indices of codon bias such as Nc (effective number of codons), SCUO (synonymous codon usage order), RSCU (relative synonymous codon usage), as well as sequence patterns of codon contexts. The results revealed several characteristic features of codon usage in Actinobacteria, as follows: 1) C- or G-ending codons are used frequently in comparison with A- and U ending codons; 2) there is a direct relationship of GC content with use of specific amino acids such as alanine, proline and glycine; 3) there is an inverse relationship between GC content and Nc estimates, 4) there is low SCUO value (<0.5) for most genes; and 5) GCC-GCC, GCC-GGC, GCC-GAG and CUC-GAC are the frequent context sequences among codons. This study highlights the fact that: 1) in Actinobacteria, extreme GC content and codon bias are driven by mutation rather than natural selection; (2) traits like aerobicity are associated with effective natural selection and therefore low GC content and low codon bias, demonstrating the role of both mutational bias and translational selection in shaping the habitat and phenotype of actinobacterial species. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Novel and recurrent germline LEMD3 mutations causing Buschke-Ollendorff syndrome and osteopoikilosis but not isolated melorheostosis.

PubMed

Zhang, Y; Castori, M; Ferranti, G; Paradisi, M; Wordsworth, B P

2009-06-01

Mutations in the LEMD3 gene were recently incriminated in Buschke-Ollendorff syndrome (BOS) and osteopoikilosis, with or without melorheostosis. The relationship of this gene with isolated sporadic melorheostosis is less clear. We investigated LEMD3 in a two-generation BOS family showing an extremely variable expression of the disease, in a sporadic patient with skin features of BOS, and in an additional subject with isolated melorheostosis. We identified two different mutations, both resulting in a premature stop codon, in the two cases of BOS. The mutation (c.2564G>A) reported in the familial case is novel, while that observed in the sporadic case (c.1963C>T) has been previously reported in an American woman with osteopoikilosis and melorheostosis who had a family history of isolated osteopoikilosis. The search for mutations in DNA extracted from the peripheral blood, as well as skin and bone biopsies of the patient with melorheostosis failed to identify any pathogenic change. Our results further expand the LEMD3 mutation repertoire, corroborate the extreme interfamilial and intrafamilial clinical variability of LEMD3 mutations, and underline the lack of a clear phenotype-genotype correlation in BOS. The present study supports the general conclusion that LEMD3 mutations do not contribute to isolated sporadic melorheostosis. The genetic or epigenetic influences that are responsible for the development of melorheostosis require further investigation.

Compound Heterozygous Desmoplakin Mutations Result in a Phenotype with a Combination of Myocardial, Skin, Hair, and Enamel Abnormalities

PubMed Central

Mahoney, Mỹ G.; Sadowski, Sara; Brennan, Donna; Pikander, Pekka; Saukko, Pekka; Wahl, James; Aho, Heikki; Heikinheimo, Kristiina; Bruckner-Tuderman, Leena; Fertala, Andrzej; Peltonen, Juha; Uitto, Jouni; Peltonen, Sirkku

2014-01-01

Desmoplakin (DP) anchors the intermediate filament cytoskeleton to the desmosomal cadherins and thereby confers structural stability to tissues. In this study, we present a patient with extensive mucocutaneous blisters, epidermolytic palmoplantar keratoderma, nail dystrophy, enamel dysplasia, and sparse woolly hair. The patient died at the age of 14 years from undiagnosed cardiomyopathy. The skin showed hyperplasia and acantholysis in the mid- and lower epidermal layers, whereas the heart showed extensive fibrosis and fibrofatty replacement in both ventricles. Immunofluorescence microscopy showed a reduction in the C-terminal domain of DP in the skin and oral mucosa. Sequencing of the DP gene showed undescribed mutations in the maternal and paternal alleles. Both mutations affected exon 24 encoding the C-terminal domain. The paternal mutation, c.6310delA, leads to a premature stop codon. The maternal mutation, c.7964 C to A, results in a substitution of an aspartic acid for a conserved alanine residue at amino acid 2655 (A2655D). Structural modeling indicated that this mutation changes the electrostatic potential of the mutated region of DP, possibly altering functions that depend on intermolecular interactions. To conclude, we describe a combination of DP mutation phenotypes affecting the skin, heart, hair, and teeth. This patient case emphasizes the importance of heart examination of patients with desmosomal genodermatoses. PMID:19924139

Natural Variant of Collagen-Like Protein A in Serotype M3 Group A Streptococcus Increases Adherence and Decreases Invasive Potential

PubMed Central

Jewell, Brittany E.; Versalovic, Erika M.; Olsen, Randall J.; Bachert, Beth A.; Lukomski, Slawomir; Musser, James M.

2015-01-01

Group A Streptococcus (GAS) predominantly exists as a colonizer of the human oropharynx that occasionally breaches epithelial barriers to cause invasive diseases. Despite the frequency of GAS carriage, few investigations into the contributory molecular mechanisms exist. To this end, we identified a naturally occurring polymorphism in the gene encoding the streptococcal collagen-like protein A (SclA) in GAS carrier strains. All previously sequenced invasive serotype M3 GAS possess a premature stop codon in the sclA gene truncating the protein. The carrier polymorphism is predicted to restore SclA function and was infrequently identified by targeted DNA sequencing in invasive strains of the same serotype. We demonstrate that a strain with the carrier sclA allele expressed a full-length SclA protein, while the strain with the invasive sclA allele expressed a truncated variant. An isoallelic mutant invasive strain with the carrier sclA allele exhibited decreased virulence in a mouse model of invasive disease and decreased multiplication in human blood. Further, the isoallelic invasive strain with the carrier sclA allele persisted in the mouse nasopharynx and had increased adherence to cultured epithelial cells. Repair of the premature stop codon in the invasive sclA allele restored the ability to bind the extracellular matrix proteins laminin and cellular fibronectin. These data demonstrate that a mutation in GAS carrier strains increases adherence and decreases virulence and suggest selection against increased adherence in GAS invasive isolates. PMID:25561712

WES homozygosity mapping in a recessive form of Charcot-Marie-Tooth neuropathy reveals intronic GDAP1 variant leading to a premature stop codon.

PubMed

Masingue, Marion; Perrot, Jimmy; Carlier, Robert-Yves; Piguet-Lacroix, Guenaelle; Latour, Philippe; Stojkovic, Tanya

2018-05-01

Charcot-Marie-Tooth disease (CMT) refers to a group of clinically and genetically heterogeneous inherited neuropathies. Ganglioside-induced differentiation-associated protein 1 GDAP1-related CMT has been reported in an autosomal dominant or recessive form in patients presenting either axonal or demyelinating neuropathy. We report two Sri Lankan sisters born to consanguineous parents and presenting with a severe axonal sensorimotor neuropathy. The early onset of the disease, the distal and proximal weakness and atrophy leading to major disability, along with areflexia, and, most notably, vocal cord and diaphragm paralysis were highly evocative of a GDAP1-related CMT. However, sequencing of the coding regions of the gene was normal. Whole-exome sequencing (WES) was performed and revealed that the largest region of homozygosity was around GDAP1 with several variants, mostly in non-coding regions. In view of the high clinical suspicion of GDAP1 gene involvement, we examined the variants in this gene and this, along with functional studies, allowed us to identify an alternative splicing site revealing a cryptic in-frame stop codon in intron 4 responsible for a severe loss of wild-type GDAP1. This work is the first to describe a deleterious mutation in GDAP1 gene outside of coding sequences or intronic junctions and emphasizes the importance of interpreting molecular analysis, and in particular WES results, in light of the clinical and electrophysiological phenotype.

[Molecular genetic analysis for a pedigree with severe hereditary coagulation factor VII deficiency].

PubMed

Ding, Qiu-lan; Wang, Hong-li; Wang, Xue-feng; Wang, Ming-shan; Fu, Qi-hua; Wu, Wen-man; Hu, Yi-qun; Wang, Zhen-yi

2003-10-01

To identify the genetic mutations of a severe inherited coagulation factor VII (FVII) deficiency pedigree. The diagnosis was validated by coagulant and haemostatic parameters. FVII gene mutations were screened in the propositus and his family members by DNA direct sequencing and confirmed by digestions of the restriction enzymes of the PCR production. Two heterozygous missense mutations were found in the propositus of the pedigree: a G to T transversion at position 9482 in exon 6 and a C to T mutation at position 11348 in exon 8 resulting in the amino acid substitution of Arg152 with Leu and Arg304 with Trp, respectively. A heterozygous single nucleotide deletion (C) at position 11487-11489(CCC) within exon 8 was identified, which predicted the frameshift mutation at position His351 followed by the changes of six corresponding amino acids and appearance of a premature protein caused by stop codon. The heterozygous mutations identified in the proband were derived from his father (Arg152 to Leu) and his mother (Arg304 to Trp mutation) and a heterozygous deletion (C) at position 11487-9(CCC). By tracing the other pedigree members, it was found that his grandmother had a heterozygous mutation of Arg304Trp and a heterozygous polymorphism of Arg353Gln and his grandfather had a heterozygous Arg152Leu mutation. Three heterozygous mutations were found in a pedigree with hereditary coagulation factor VII deficiency. Arg152Leu and deletion C at position 11487-9(CCC) were novel mutations.

Capturing the metabolomic diversity of KRAS mutants in non-small-cell lung cancer cells

PubMed Central

Marabese, Mirko; Broggini, Massimo; Pastorelli, Roberta

2014-01-01

In non-small-cell lung cancer (NSCLC), one-fifth of patients have KRAS mutations, which are considered a negative predictive factor to first-line therapy. Evidence is emerging that not all KRAS mutations have the same biological activities and possible remodeling of cell metabolism by KRAS activation might complicate the scenario. An open question is whether different KRAS mutations at codon-12 affect cellular metabolism differently with possible implications for different responses to cancer treatments. We applied an explorative mass spectrometry-based untargeted metabolomics strategy to characterize the largest possible number of metabolites that might distinguish isogenic NSCLC cells overexpressing mutated forms of KRAS at codon-12 (G12C, G12D, G12V) and the wild-type. The glutamine deprivation assay and real-time PCR were used to confirm the involvement of some of the metabolic pathways highlighted. Cell clones indicated distinct metabolomic profiles in KRAS wild-type and mutants. Clones harboring different KRAS mutations at codon-12 also had different metabolic remodeling, such as a different redox buffering system and different glutamine-dependency not driven by the transcriptional state of enzymes involved in glutaminolysis. These findings indicate that KRAS mutations at codon-12 are associated with different metabolomic profiles that might affect the responses to cancer treatments. PMID:24952473

Genotyping of K-ras codons 12 and 13 mutations in colorectal cancer by capillary electrophoresis.

PubMed

Chen, Yen-Ling; Chang, Ya-Sian; Chang, Jan-Gowth; Wu, Shou-Mei

2009-06-26

Point mutations of the K-ras gene located in codons 12 and 13 cause poor responses to the anti-epidermal growth factor receptor (anti-EGFR) therapy of colorectal cancer (CRC) patients. Besides, mutations of K-ras gene have also been proven to play an important role in human tumor progression. We established a simple and effective capillary electrophoresis (CE) method for simultaneous point mutation detection in codons 12 and 13 of K-ras gene. We combined one universal fluorescence-based nonhuman-sequence primer and two fragment-oriented primers in one tube, and performed this two-in-one polymerase chain reaction (PCR). PCR fragments included wild type and seven point mutations at codons 12 and 13 of K-ras gene. The amplicons were analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE analysis was performed by using a 1x Tris-borate-EDTA (TBE) buffer containing 1.5% (w/v) hydroxyethylcellulose (HEC) (MW 250,000) under reverse polarity with 15 degrees C and 30 degrees C. Ninety colorectal cancer patients were blindly genotyped using this developed method. The results showed good agreement with those of DNA sequencing method. The SSCP-CE was feasible for mutation screening of K-ras gene in populations.

Characterization of the porcine epidemic diarrhea virus codon usage bias.

PubMed

Chen, Ye; Shi, Yuzhen; Deng, Hongjuan; Gu, Ting; Xu, Jian; Ou, Jinxin; Jiang, Zhiguo; Jiao, Yiren; Zou, Tan; Wang, Chong

2014-12-01

Porcine epidemic diarrhea virus (PEDV) has been responsible for several recent outbreaks of porcine epidemic diarrhea (PED) and has caused great economic loss in the swine-raising industry. Considering the significance of PEDV, a systemic analysis was performed to study its codon usage patterns. The relative synonymous codon usage value of each codon revealed that codon usage bias exists and that PEDV tends to use codons that end in T. The mean ENC value of 47.91 indicates that the codon usage bias is low. However, we still wanted to identify the cause of this codon usage bias. A correlation analysis between the codon compositions (A3s, T3s, G3s, C3s, and GC3s), the ENC values, and the nucleotide contents (A%, T%, G%, C%, and GC%) indicated that mutational bias plays role in shaping the PEDV codon usage bias. This was further confirmed by a principal component analysis between the codon compositions and the axis values. Using the Gravy, Aroma, and CAI values, a role of natural selection in the PEDV codon usage pattern was also identified. Neutral analysis indicated that natural selection pressure plays a more important role than mutational bias in codon usage bias. Natural selection also plays an increasingly significant role during PEDV evolution. Additionally, gene function and geographic distribution also influence the codon usage bias to a degree. Copyright © 2014 Elsevier B.V. All rights reserved.

Coexistence of two different mutations in codon 12 of the Kras gene in colorectal cancer: Report of a case supporting the concept of tumoral heterogeneity.

PubMed

Improta, Giuseppina; Zupa, Angela; Possidente, Luciana; Tartarone, Alfredo; Pedicini, Piernicola; Nappi, Antonio; Molinari, Sergio; Fraggetta, Filippo; Vita, Giulia

2013-05-01

Evaluation of the mutational status of KRAS is a crucial step for the correct therapeutic approach in treating advanced colorectal cancer as the identification of wild-type KRAS tumors leads to more specific and less toxic treatments for patients. Although several studies have highlighted the differences between primary and metastatic tumors, the possibility of two or more mutations in the same codon has seldom been reported. The present study reports an additional case of an advanced adenocarcinoma of the colon showing two somatic mutations (p.G12D and p.G12V) in the same codon (codon 12) of exon 2 of the KRAS gene, thus supporting the possibility of two differing clonal origins of the tumor. Although the clinical significance of multiple mutations remains unknown at present, based on the limited data available in the literature, this rare event appears to be associated with a more aggressive disease, as in the present case. This case report demonstrates the existence of intratumoral heterogeneity and the coexistence of distinct clones within a tumor that may have profound clinical implications for disease progression and therapeutic responses.

Hyperphenylalaninemia due to defects in tetrahydrobiopterin metabolism: Molecular characterization of mutations in 6-pyruvoyl-tetrahydropterin synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thoeny, B.; Leimbacher, W.; Blau, N.

1994-05-01

A variant type of hyperphenylalaninemia is caused by a deficiency of tetrahydrobiopterin (BH[sub 4]), the obligatory cofactor for phenylalanine hydroxylase. The most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (PTPS) activity, the second enzyme in the biosynthetic pathway for BH[sub 4]. The human liver cDNA for PTPS was previously isolated, and the recombinant protein was found to be active when expressed in Escherichia coli. The authors now have investigated two patients for their molecular nature of this autosomal recessive disorder. Both patients were diagnosed as PTPS deficient, one with the central and one withmore » the peripheral form, on the basis of an elevated serum phenylalanine concentration concomitant with lowered levels of urinary biopterin and PTPS activity in erythrocytes. Molecular analysis was performed on the patients' cultured primary skin fibroblasts. PTPS activities were found in vitro to be reduced to background activity. Direct cDNA sequence analysis using reverse transcriptase-PCR technology showed for the patient with the central form a homozygous G-to-A transition at codon 25, causing the replacement of an arginine by glutamine (R25Q). Expression of this mutant allele in E.coli revealed 14% activity when compared with the wild-type enzyme. The patient with the peripheral form exhibited compound heteroxygosity, having on one allele a C-to-T transition resulting in the substitution of arginine 16 for cysteine (R16C) in the enzyme and having on the second allele a 14-bp deletion ([Delta]14bp), leading to a frameshift at lysine 120 and a premature stop codon (K120[yields]Stop). Heterologous expression of the enzyme with the single-amino-acid exchange R16C revealed only 7% enzyme activity, whereas expression of the deletion allele [Delta]14bp exhibited no detectable activity. All three mutations result in reduced enzymatic activity when reconstituted in E. coli.« less

Use of ade1 and ade2 mutations for development of a versatile red/white colour assay of amyloid-induced oxidative stress in saccharomyces cerevisiae.

PubMed

Bharathi, Vidhya; Girdhar, Amandeep; Prasad, Archana; Verma, Meenkshi; Taneja, Vibha; Patel, Basant K

2016-12-01

Mutations in adenine biosynthesis pathway genes ADE1 and ADE2 have been conventionally used to score for prion [PSI + ] in yeast. If ade1-14 mutant allele is present, which contains a premature stop codon, [psi - ] yeast appear red on YPD medium owing to accumulation of a red intermediate compound in vacuoles. In [PSI + ] yeast, partial inactivation of the translation termination factor, Sup35 protein, owing to its amyloid aggregation allows for read-through of the ade1-14 stop codon and the yeast appears white as the red intermediate pigment is not accumulated. The red colour development in ade1 and ade2 mutant yeast requires reduced-glutathione, which helps in transport of the intermediate metabolite P-ribosylaminoimidazole carboxylate into vacuoles, which develops the red colour. Here, we hypothesize that amyloid-induced oxidative stress would deplete reduced-glutathione levels and thus thwart the development of red colour in ade1 or ade2 yeast. Indeed, when we overexpressed amyloid-forming human proteins TDP-43, Aβ-42 and Poly-Gln-103 and the yeast prion protein Rnq1, the otherwise red ade1 yeast yielded some white colonies. Further, the white colour eventually reverted back to red upon turning off the amyloid protein's expression. Also, the aggregate-bearing yeast have increased oxidative stress and white phenotype yeast revert to red when grown on media with reducing agent. Furthermore, the red/white assay could also be emulated in ade2-1, ade2Δ, and ade1Δ mutant yeast and also in an ade1-14 mutant with erg6 gene deletion that increases cell-wall permeability. This model would be useful tool for drug-screening against general amyloid-induced oxidative stress and toxicity. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Evolution of Nucleotide Punctuation Marks: From Structural to Linear Signals.

PubMed

El Houmami, Nawal; Seligmann, Hervé

2017-01-01

We present an evolutionary hypothesis assuming that signals marking nucleotide synthesis (DNA replication and RNA transcription) evolved from multi- to unidimensional structures, and were carried over from transcription to translation. This evolutionary scenario presumes that signals combining secondary and primary nucleotide structures are evolutionary transitions. Mitochondrial replication initiation fits this scenario. Some observations reported in the literature corroborate that several signals for nucleotide synthesis function in translation, and vice versa. (a) Polymerase-induced frameshift mutations occur preferentially at translational termination signals (nucleotide deletion is interpreted as termination of nucleotide polymerization, paralleling the role of stop codons in translation). (b) Stem-loop hairpin presence/absence modulates codon-amino acid assignments, showing that translational signals sometimes combine primary and secondary nucleotide structures (here codon and stem-loop). (c) Homopolymer nucleotide triplets (AAA, CCC, GGG, TTT) cause transcriptional and ribosomal frameshifts. Here we find in recently described human mitochondrial RNAs that systematically lack mono-, dinucleotides after each trinucleotide (delRNAs) that delRNA triplets include 2x more homopolymers than mitogenome regions not covered by delRNA. Further analyses of delRNAs show that the natural circular code X (a little-known group of 20 translational signals enabling ribosomal frame retrieval consisting of 20 codons {AAC, AAT, ACC, ATC, ATT, CAG, CTC, CTG, GAA, GAC, GAG, GAT, GCC, GGC, GGT, GTA, GTC, GTT, TAC, TTC} universally overrepresented in coding versus other frames of gene sequences), regulates frameshift in transcription and translation. This dual transcription and translation role confirms for X the hypothesis that translational signals were carried over from transcriptional signals.

Nuclear expression and gain-of-function β-catenin mutation in glomangiopericytoma (sinonasal-type hemangiopericytoma): insight into pathogenesis and a diagnostic marker.

PubMed

Lasota, Jerzy; Felisiak-Golabek, Anna; Aly, F Zahra; Wang, Zeng-Feng; Thompson, Lester D R; Miettinen, Markku

2015-05-01

Glomangiopericytoma (sinonasal-type hemangiopericytoma) is a rare mesenchymal neoplasm with myoid phenotype (smooth muscle actin-positive), which distinguishes this tumor from soft tissue hemangiopericytoma/solitary fibrous tumor. Molecular genetic changes underlying the pathogenesis of glomangiopericytoma are not known. In this study, 13 well-characterized glomangiopericytomas were immunohistochemically evaluated for β-catenin expression. All analyzed tumors showed strong expression and nuclear accumulation of β-catenin. Following this observation, β-catenin glycogen serine kinase-3 beta phosphorylation region, encoded by exon 3, was PCR amplified in all cases and evaluated for mutations using Sanger sequencing. Heterozygous mutations were identified in 12 of 13 tumors. All mutations consisted of single-nucleotide substitutions: three in codon 32 (c.94G>C (n=2) and c.95A>T), four in codon 33 (two each c.98C>G and c.98C>T), two in codon 37 (c.109T>G), one in codon 41 (c.121A>G), and two in codon 45 (c.133T>C). At the protein level, these substitutions would lead to p.D32H, p.D32V, p.S33C, p.S33F, p.S37A, p.T41A, and p.S45L mutations, respectively. Previously, similar mutations have been reported in different types of cancers and shown to trigger activation of β-catenin signaling. All analyzed glomangiopericytomas showed prominent nuclear expression of cyclin D1, as previously shown for tumors with nuclear expression of β-catenin as a sign of oncogenic activation. These results demonstrate that mutational activation of β-catenin and associated cyclin D1 overexpression may be central events in the pathogenesis of glomangiopericytoma. In additon, nuclear accumulation of β-catenin is a diagnostic marker for glomangiopericytoma.

Multiple Evolutionary Selections Involved in Synonymous Codon Usages in the Streptococcus agalactiae Genome.

PubMed

Ma, Yan-Ping; Ke, Hao; Liang, Zhi-Ling; Liu, Zhen-Xing; Hao, Le; Ma, Jiang-Yao; Li, Yu-Gu

2016-02-24

Streptococcus agalactiae is an important human and animal pathogen. To better understand the genetic features and evolution of S. agalactiae, multiple factors influencing synonymous codon usage patterns in S. agalactiae were analyzed in this study. A- and U-ending rich codons were used in S. agalactiae function genes through the overall codon usage analysis, indicating that Adenine (A)/Thymine (T) compositional constraints might contribute an important role to the synonymous codon usage pattern. The GC3% against the effective number of codon (ENC) value suggested that translational selection was the important factor for codon bias in the microorganism. Principal component analysis (PCA) showed that (i) mutational pressure was the most important factor in shaping codon usage of all open reading frames (ORFs) in the S. agalactiae genome; (ii) strand specific mutational bias was not capable of influencing the codon usage bias in the leading and lagging strands; and (iii) gene length was not the important factor in synonymous codon usage pattern in this organism. Additionally, the high correlation between tRNA adaptation index (tAI) value and codon adaptation index (CAI), frequency of optimal codons (Fop) value, reinforced the role of natural selection for efficient translation in S. agalactiae. Comparison of synonymous codon usage pattern between S. agalactiae and susceptible hosts (human and tilapia) showed that synonymous codon usage of S. agalactiae was independent of the synonymous codon usage of susceptible hosts. The study of codon usage in S. agalactiae may provide evidence about the molecular evolution of the bacterium and a greater understanding of evolutionary relationships between S. agalactiae and its hosts.

Multiple Evolutionary Selections Involved in Synonymous Codon Usages in the Streptococcus agalactiae Genome

PubMed Central

Ma, Yan-Ping; Ke, Hao; Liang, Zhi-Ling; Liu, Zhen-Xing; Hao, Le; Ma, Jiang-Yao; Li, Yu-Gu

2016-01-01

Streptococcus agalactiae is an important human and animal pathogen. To better understand the genetic features and evolution of S. agalactiae, multiple factors influencing synonymous codon usage patterns in S. agalactiae were analyzed in this study. A- and U-ending rich codons were used in S. agalactiae function genes through the overall codon usage analysis, indicating that Adenine (A)/Thymine (T) compositional constraints might contribute an important role to the synonymous codon usage pattern. The GC3% against the effective number of codon (ENC) value suggested that translational selection was the important factor for codon bias in the microorganism. Principal component analysis (PCA) showed that (i) mutational pressure was the most important factor in shaping codon usage of all open reading frames (ORFs) in the S. agalactiae genome; (ii) strand specific mutational bias was not capable of influencing the codon usage bias in the leading and lagging strands; and (iii) gene length was not the important factor in synonymous codon usage pattern in this organism. Additionally, the high correlation between tRNA adaptation index (tAI) value and codon adaptation index (CAI), frequency of optimal codons (Fop) value, reinforced the role of natural selection for efficient translation in S. agalactiae. Comparison of synonymous codon usage pattern between S. agalactiae and susceptible hosts (human and tilapia) showed that synonymous codon usage of S. agalactiae was independent of the synonymous codon usage of susceptible hosts. The study of codon usage in S. agalactiae may provide evidence about the molecular evolution of the bacterium and a greater understanding of evolutionary relationships between S. agalactiae and its hosts. PMID:26927064

Mechanisms responsible for imipenem resistance among Pseudomonas aeruginosa clinical isolates exposed to imipenem concentrations within the mutant selection window.

PubMed

Vassilara, Foula; Galani, Irene; Souli, Maria; Papanikolaou, Konstantinos; Giamarellou, Helen; Papadopoulos, Antonios

2017-07-01

The aim of this study was to determine the propensities of imipenem to select for resistant Pseudomonas aeruginosa mutants by determining the mutant prevention concentrations (MPCs) for 9 unrelated clinical isolates and the accession of any relationship with mechanisms of resistance development. The MPC/MIC ratios ranged from 4 to 16. Detection of resistance mechanisms in the mutant derivatives of the nine isolates mainly revealed inactivating mutations in the gene coding for outer membrane protein OprD. Point mutations leading to premature stop codons or amino acid substitution S278P, ≥1bp deletion leading to frameshift mutations and interruption of the oprD by an insertion sequence, were observed. MPC and mutant selection window (MSW) are unique parameters that may guide the implementation of antimicrobial treatment, providing useful information about the necessary imipenem concentration needed in the infection area, in order to avoid the emergence of resistance, especially in clinical situations with high bacterial load. Copyright © 2017 Elsevier Inc. All rights reserved.

Rhnull syndrome: identification of a novel mutation in RHce.

PubMed

Rosa, K A; Reid, M E; Lomas-Francis, C; Powell, V I; Costa, F F; Stinghen, S T; Watanabe, A M; Carboni, E K; Baldon, J P; Jucksch, M M F; Castilho, L

2005-11-01

The deficiency of Rh proteins on red blood cells (RBCs) from individuals of the Rh(null) amorph type are the result of homozygosity for a silent RHCE in cis with a deleted RHD. A novel mutation in RHce was identified in two Caucasian Brazilian girls with the amorph type of Rh(null) who were born to parents who were first cousins. RBCs from the Rh(null) sisters and from family members were analyzed by serology and flow cytometry with specific antibodies. Genomic DNA and transcripts were tested by polymerase chain reaction and sequence analysis. Rh(null) RBCs were nonreactive with anti-Rh and anti-LW. Molecular analyses showed a deletion of RHD and of one nucleotide (960/963; GGGG-->GGG) in exon 7 of the RHce. This deletion introduced a frameshift after Gly321, a new C-terminal sequence, and a premature stop codon, resulting in a shorter predicted protein with 357 amino acids. The detection of a unique RHce transcript indicated that the two sisters were homozygous, whereas the other family members were heterozygous for the mutation. A novel mutation resulting in the amorph Rh(null) with loss of Rh antigen expression is described.

A novel SMAD3 mutation caused multiple aneurysms in a patient without osteoarthritis symptoms.

PubMed

Courtois, Audrey; Coppieters, Wouter; Bours, Vincent; Defraigne, Jean-Olivier; Colige, Alain; Sakalihasan, Natzi

2017-04-01

Heterozygous mutations in the SMAD3 gene were recently described as the cause of a form of non-syndromic familial aortic thoracic aneurysm and dissection (FTAAD) transmitted as an autosomal dominant disorder and often associated with early-onset osteoarthritis. This new clinical entity, called aneurysms-osteoarthritis syndrome (AOS) or Loeys-Dietz syndrome 3 (LDS3), is characterized by aggressive arterial damages such as aneurysms, dissections and tortuosity throughout the arterial tree. We report, here, the case of a 45 year-old man presenting multiple visceral arteries and abdominal aortic aneurysms but without dissection of the thoracic aorta and without any sign of osteoarthritis. Exome-sequencing revealed a new frameshift heterozygous c.455delC (p.Pro152Hisfs*34) mutation in the SMAD3 gene. This deletion is located in the exon 3 coding for the linker region of the protein and causes a premature stop codon at positions 556-558 in the exon 4. The same mutation was found in the proband's mother and sister who had open surgery for abdominal aortic aneurysm and in one of his children who was 5 year-old and did not present aneurysm yet. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ion, A.; Telvi, L.; Galacteros, F.

We describe a pedigree presenting X-linked severe mental retardation associated with multiple congenital abnormalities and 46,XY gonadal dysgenesis, leading in one family member to female gender assignment. Female carriers are unaffected. The dysmorphic features are similar to those described in the {alpha}-thalassemia and mental retardation (ATR-X) syndrome, although there is no clinical evidence of {alpha}-thalassemia in this family. In addition, the family had other clinical features not previously observed in the ATR-X syndrome, including partial optic-nerve atrophy and partial ocular albinism. Mutations in a putative DNA helicase, termed XH2, have been reported to give rise to the ATR-X syndrome. Wemore » screened the YCH2 gene for mutations in affected members of the family and identified a 4-bp deletion at an intron/exon boundary that removes an invariant 3{prime} splice-acceptor site. The mutation cosegregates with the syndrome. The genomic deletion causes missplicing of the pre-mRNA, which results in the loss of 8 bp of coding sequence, thereby generating a frameshift and a downstream premature stop codon. Our finding increases the range of clinical features associated with mutations in the XH2 gene. 17 refs., 4 figs., 2 tabs.« less

Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

PubMed

Gagnon, James A; Valen, Eivind; Thyme, Summer B; Huang, Peng; Akhmetova, Laila; Ahkmetova, Laila; Pauli, Andrea; Montague, Tessa G; Zimmerman, Steven; Richter, Constance; Schier, Alexander F

2014-01-01

The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.

Large-scale analyses of synonymous substitution rates can be sensitive to assumptions about the process of mutation.

PubMed

Aris-Brosou, Stéphane; Bielawski, Joseph P

2006-08-15

A popular approach to examine the roles of mutation and selection in the evolution of genomes has been to consider the relationship between codon bias and synonymous rates of molecular evolution. A significant relationship between these two quantities is taken to indicate the action of weak selection on substitutions among synonymous codons. The neutral theory predicts that the rate of evolution is inversely related to the level of functional constraint. Therefore, selection against the use of non-preferred codons among those coding for the same amino acid should result in lower rates of synonymous substitution as compared with sites not subject to such selection pressures. However, reliably measuring the extent of such a relationship is problematic, as estimates of synonymous rates are sensitive to our assumptions about the process of molecular evolution. Previous studies showed the importance of accounting for unequal codon frequencies, in particular when synonymous codon usage is highly biased. Yet, unequal codon frequencies can be modeled in different ways, making different assumptions about the mutation process. Here we conduct a simulation study to evaluate two different ways of modeling uneven codon frequencies and show that both model parameterizations can have a dramatic impact on rate estimates and affect biological conclusions about genome evolution. We reanalyze three large data sets to demonstrate the relevance of our results to empirical data analysis.

A mutated hygromycin resistance gene is functional in the n-alkane-assimilating yeast Candida tropicalis.

PubMed

Hara, A; Ueda, M; Misawa, S; Matsui, T; Furuhashi, K; Tanaka, A

2000-03-01

Development of a transformation system in the n-alkane-assimilating diploid yeast Candida tropicalis requires an antibiotic resistance gene in order to establish a selectable marker. The resistance gene for hygromycin B has often been used as a selectable marker in yeast transformation. However, C. tropicalis harboring the hygromycin resistance gene (HYG) was as sensitive to hygromycin B as the wild-type strain. Nine CTG codons were found in the ORF of the HYG gene. This codon has been reported to be translated as serine rather than leucine in Candida species. Analysis of the tRNA gene in C. tropicalis with the anticodon CAG [tRNA(CAG) gene], which is complementary to the codon CTG, showed that the sequence was highly similar to that of the C. maltosa tRNA(CAG) gene. In C. maltosa, the codon CTG is read as serine and not leucine. These results suggested that the HYG gene was not functional due to the nonuniversal usage of the CTG codon. Each of the nine CTG codons in the ORF of the HYG gene was changed to a CTC codon, which is read as leucine, by site-directed mutagenesis. When a plasmid containing the mutated HYG gene (HYG#) was constructed and introduced into C. tropicalis, hygromycin-resistant transformants were successfully obtained. This mutated hygromycin resistance gene may be useful for direct selection of C. tropicalis transformants.

Base damage, local sequence context and TP53 mutation hotspots: a molecular dynamics study of benzo[a]pyrene induced DNA distortion and mutability

PubMed Central

Menzies, Georgina E.; Reed, Simon H.; Brancale, Andrea; Lewis, Paul D.

2015-01-01

The mutational pattern for the TP53 tumour suppressor gene in lung tumours differs to other cancer types by having a higher frequency of G:C>T:A transversions. The aetiology of this differing mutation pattern is still unknown. Benzo[a]pyrene,diol epoxide (BPDE) is a potent cigarette smoke carcinogen that forms guanine adducts at TP53 CpG mutation hotspot sites including codons 157, 158, 245, 248 and 273. We performed molecular modelling of BPDE-adducted TP53 duplex sequences to determine the degree of local distortion caused by adducts which could influence the ability of nucleotide excision repair. We show that BPDE adducted codon 157 has greater structural distortion than other TP53 G:C>T:A hotspot sites and that sequence context more distal to adjacent bases must influence local distortion. Using TP53 trinucleotide mutation signatures for lung cancer in smokers and non-smokers we further show that codons 157 and 273 have the highest mutation probability in smokers. Combining this information with adduct structural data we predict that G:C>T:A mutations at codon 157 in lung tumours of smokers are predominantly caused by BPDE. Our results provide insight into how different DNA sequence contexts show variability in DNA distortion at mutagen adduct sites that could compromise DNA repair at well characterized cancer related mutation hotspots. PMID:26400171

Codon 62 (GTG>GCG, Val→Ala) (α1) (HBA1: c.188T>C) causing nondeletional α-thalassemia in a Chinese family.

PubMed

Liao, Can; Tang, Hai-Shen; Li, Ru; Li, Dong-Zhi

2013-01-01

We report a novel α-globin gene point mutation detected during newborn screening for hemoglobinopathies. Sequence analyses identified a GTG>GCG substitution at codon 62 of the α1-globin gene. This mutation causes a silent α-thalassemia (α-thal).

Coffin-Siris syndrome with café-au-lait spots, obesity and hyperinsulinism caused by a mutation in the ARID1B gene

PubMed Central

Sonmez, Fatma Mujgan; Uctepe, Eyyup; Gunduz, Mehmet; Gormez, Zeliha; Erpolat, Seval; Oznur, Murat; Sagiroglu, Mahmut Samil; Demirci, Huseyin; Gunduz, Esra

2016-01-01

Summary Coffin-Siris syndrome (CSS) (MIM 135900) is characterized by developmental delay, severe speech impairment, distinctive facial features, hypertrichosis, aplasia or hypoplasia of the distal phalanx or nail of the fifth digit and agenesis of the corpus callosum. Recently, it was shown that mutations in the ARID1B gene are the main cause of CSS, accounting for 76% of identified mutations. Here, we report a 15 year-old female patient who was admitted to our clinic with seizures, speech problems, dysmorphic features, bilaterally big, large thumb, café-au-lait (CAL) spots, obesity and hyperinsulinism. First, the patient was thought to have an association of neurofibromatosis and Rubinstein Taybi syndrome. Because of the large size of the NF1 gene for neurofibromatosis and CREBBP gene for Rubinstein Taybi syndrome, whole exome sequence analysis (WES) was conducted and a novel ARID1B mutation was identified. The proband WES test identified a novel heterozygous frameshift mutation c.3394_3395insTA in exon 13 of ARID1B (NM_017519.2) predicting a premature stop codon p.(Tyr1132Leufs*67). Sanger sequencing confirmed the heterozygous c.3394_3395insTA mutation in the proband and that it was not present in her parents indicating de novo mutation. Further investigation and new cases will help to understand this phenomenon better. PMID:27672547

Coffin-Siris syndrome with café-au-lait spots, obesity and hyperinsulinism caused by a mutation in the ARID1B gene.

PubMed

Sonmez, Fatma Mujgan; Uctepe, Eyyup; Gunduz, Mehmet; Gormez, Zeliha; Erpolat, Seval; Oznur, Murat; Sagiroglu, Mahmut Samil; Demirci, Huseyin; Gunduz, Esra

2016-08-01

Coffin-Siris syndrome (CSS) (MIM 135900) is characterized by developmental delay, severe speech impairment, distinctive facial features, hypertrichosis, aplasia or hypoplasia of the distal phalanx or nail of the fifth digit and agenesis of the corpus callosum. Recently, it was shown that mutations in the ARID1B gene are the main cause of CSS, accounting for 76% of identified mutations. Here, we report a 15 year-old female patient who was admitted to our clinic with seizures, speech problems, dysmorphic features, bilaterally big, large thumb, café-au-lait (CAL) spots, obesity and hyperinsulinism. First, the patient was thought to have an association of neurofibromatosis and Rubinstein Taybi syndrome. Because of the large size of the NF1 gene for neurofibromatosis and CREBBP gene for Rubinstein Taybi syndrome, whole exome sequence analysis (WES) was conducted and a novel ARID1B mutation was identified. The proband WES test identified a novel heterozygous frameshift mutation c.3394_3395insTA in exon 13 of ARID1B (NM_017519.2) predicting a premature stop codon p.(Tyr1132Leufs*67). Sanger sequencing confirmed the heterozygous c.3394_3395insTA mutation in the proband and that it was not present in her parents indicating de novo mutation. Further investigation and new cases will help to understand this phenomenon better.

A novel RLBP1 gene geographical area-related mutation present in a young patient with retinitis punctata albescens.

PubMed

Scimone, Concetta; Donato, Luigi; Esposito, Teresa; Rinaldi, Carmela; D'Angelo, Rosalia; Sidoti, Antonina

2017-08-01

Autosomal recessive forms of retinitis punctata albescens (RPA) have been described. RPA is characterized by progressive retinal degeneration due to alteration in visual cycle and consequent deposit of photopigments in retinal pigment epithelium. Five loci have been linked to RPA onset. Among these, the retinaldehyde-binding protein 1 gene, RLBP1, is the most frequently involved and several founder mutations were reported. We report results of a genetic molecular investigation performed on a large Sicilian family in which appears a young woman with RPA. The proband is in homozygous condition for a novel RLBP1 single-pair deletion, and her healthy parents, both heterozygous, are not consanguineous. Thenovelc.398delC (p.P133Qfs*258) involves the exon 6 and leads to a premature stop codon, resulting in a truncated protein entirely missing of CRAL-TRIO lipid-binding domain. Pedigree analysis showed other non-consanguineous relatives heterozygous for the same mutation in the family. Extension of mutation research in the native town of the proband revealed its presence also in healthy subjects, in a heterozygous condition. A novel RLBP1 truncating mutation was detected in a young girl affected by RPA. Although her parents are not consanguineous, the mutation was observed in a homozygous condition. Being them native of the same small Sicilian town of Fiumedinisi, the hypothesis of a geographical area-related mutation was assessed and confirmed.

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli

PubMed Central

Standley, Melissa; Allen, Jennifer; Cervantes, Layla; Lilly, Joshua; Camps, Manel

2017-01-01

Mutagenesis in model organisms following exposure to chemicals is used as an indicator of genotoxicity. Mutagenesis assays are also used to study mechanisms of DNA homeostasis. The present article focuses on detection of mutagenesis in prokaryotes, which boils down to two approaches: reporter inactivation (forward mutation assay) and reversion of an inactivating mutation (reversion mutation assay). Both methods are labor-intensive, involving visual screening, quantification of colonies on solid media, or determining a Poisson distribution in liquid culture. Here we present two reversion reporters for in vivo mutagenesis that produce a quantitative output, and thus have the potential to greatly reduce the amount of test chemical and labor involved in these assays. This output is obtained by coupling a TEM β lactamase-based reversion assay with GFP fluorescence, either by placing the two genes on the same plasmid or by fusing them translationally and interrupting the N-terminus of the ORF with a stop codon. We also describe a reporter aimed at facilitating the monitoring of continuous mutagenesis in mutator strains. This reporter couples two reversion markers, allowing the temporal separation of mutation events in time, thus providing information about the dynamics of mutagenesis in mutator strains. Here, we describe these reporter systems, provide protocols for use, and demonstrate their key functional features using error-prone Pol I mutagenesis as a source of mutations. PMID:28645368

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli.

PubMed

Standley, Melissa; Allen, Jennifer; Cervantes, Layla; Lilly, Joshua; Camps, Manel

2017-01-01

Mutagenesis in model organisms following exposure to chemicals is used as an indicator of genotoxicity. Mutagenesis assays are also used to study mechanisms of DNA homeostasis. This chapter focuses on detection of mutagenesis in prokaryotes, which boils down to two approaches: reporter inactivation (forward mutation assay) and reversion of an inactivating mutation (reversion mutation assay). Both methods are labor intensive, involving visual screening, quantification of colonies on solid media, or determining a Poisson distribution in liquid culture. Here, we present two reversion reporters for in vivo mutagenesis that produce a quantitative output, and thus have the potential to greatly reduce the amount of test chemical and labor involved in these assays. This output is obtained by coupling a TEM β lactamase-based reversion assay with GFP fluorescence, either by placing the two genes on the same plasmid or by fusing them translationally and interrupting the N-terminus of the chimeric ORF with a stop codon. We also describe a reporter aimed at facilitating the monitoring of continuous mutagenesis in mutator strains. This reporter couples two reversion markers, allowing the temporal separation of mutation events in time, thus providing information about the dynamics of mutagenesis in mutator strains. Here, we describe these reporter systems, provide protocols for use, and demonstrate their key functional features using error-prone Pol I mutagenesis as a source of mutations. © 2017 Elsevier Inc. All rights reserved.

Molecular spectrum of c-KIT and PDGFRA gene mutations in gastro intestinal stromal tumor: determination of frequency, distribution pattern and identification of novel mutations in Indian patients.

PubMed

Ahmad, Firoz; Lad, Purnima; Bhatia, Simi; Das, Bibhu Ranjan

2015-01-01

KIT and PDGFRA gene mutations are the major genetic alterations seen in gastrointestinal stromal tumors (GISTs) and are being used clinically for predicting response to imatinib therapy. In the current study, we set out to explore the frequency and distribution pattern of c-KIT (exons 9, 11 and 13) and PDGFRA (exons 12 and 18) by direct sequencing in a series of 70 Indian GIST cases. Overall, 27 (38.5 %) and 4 (5.7 %) of the cases had c-KIT and PDGFRA mutations, respectively. Majority of KIT mutations involved exon 11 (85.7 %), followed by exon 9 (14.3 %), while none showed exon 13 mutation. Most exon 9 mutations showed Ala503-Tyr504 duplication, while one had novel point mutation at codon 476 (S476G). In contrast to exon 9 mutations, most exon 11 mutations were in-frame deletions (79 %, 19/24), predominantly at codons 550-560, while remaining exon 11 mutant cases were point mutations at codons 559, 560, 568, 573 and 575. Interestingly, P573T, Q556_V560delinsH, Q575H and Q575_P577 were novel variations observed in exon 11. The PDGFRA mutations were seen mostly in exon 18, which showed point mutation at codon 842 (D842V), while exon 12 showed a novel indel variation (V561_H570delinsT). No significant correlation between c-KIT/PDGFRA mutations and clinicopathological data was observed. In conclusion, this study highlights the frequency and distribution pattern of c-KIT/PDGFRA mutation in Indian cohort. The current study identified novel variations that added new insights into the genetic heterogeneity of GIST patients. Furthermore, this is the first study to report the presence of PDGFRA mutation from Indian subcontinent.

Molecular screening of the Hbs Constant Spring (codon 142, TAA>CAA, α2) and Paksé (codon 142, TAA>TAT, α2) mutations in Thailand.

PubMed

Pichanun, Dalad; Munkongdee, Thongperm; Klamchuen, Sumonmaln; Butthep, Punnee; Winichagoon, Pranee; Fucharoen, Suthat; Svasti, Saovaros

2010-01-01

Hb Constant Spring [Hb CS, α142(H19)Term] and Hb Paksé [α142(H19)Term] occur from the mutation in the termination codon of the α2-globin gene, TAA>CAA (→Gln) and TAA>TAT (→Tyr), respectively. They are the most common nondeletional α-thalassemia (α-thal) variants causing Hb H disease in Southeast Asia. In this study, 587 cord blood samples were screened for the Hb CS and Hb Paksé mutations by a dot-blot hybridization technique using oligonucleotide probes specific for each mutation. The results showed that the prevalence of Hb CS and Hb Paksé in Central Thailand are 5.80 and 0.51%, respectively, which is in concordance with the results from previous studies.

In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal, ochre, and amber suppressor tRNAs

PubMed Central

Gubbens, Jacob; Kim, Soo Jung; Yang, Zhongying; Johnson, Arthur E.; Skach, William R.

2010-01-01

Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNACys that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies. PMID:20581130

Generation of obese rat model by transcription activator-like effector nucleases targeting the leptin receptor gene.

PubMed

Chen, Yuting; Lu, Wenqing; Gao, Na; Long, Yi; Shao, Yanjiao; Liu, Meizhen; Chen, Huaqing; Ye, Shixin; Ma, Xueyun; Liu, Mingyao; Li, Dali

2017-02-01

The laboratory rat is a valuable mammalian model organism for basic research and drug discovery. Here we demonstrate an efficient methodology by applying transcription activator-like effector nucleases (TALENs) technology to generate Leptin receptor (Lepr) knockout rats on the Sprague Dawley (SD) genetic background. Through direct injection of in vitro transcribed mRNA of TALEN pairs into SD rat zygotes, somatic mutations were induced in two of three resulting pups. One of the founders carrying bi-allelic mutation exhibited early onset of obesity and infertility. The other founder carried a chimeric mutation which was efficiently transmitted to the progenies. Through phenotyping of the resulting three lines of rats bearing distinct mutations in the Lepr locus, we found that the strains with a frame-shifted or premature stop codon mutation led to obesity and metabolic disorders. However, no obvious defect was observed in a strain with an in-frame 57 bp deletion in the extracellular domain of Lepr. This suggests the deleted amino acids do not significantly affect Lepr structure and function. This is the first report of generating the Lepr mutant obese rat model in SD strain through a reverse genetic approach. This suggests that TALEN is an efficient and powerful gene editing technology for the generation of disease models.

A rare male patient with classic Rett syndrome caused by MeCP2_e1 mutation.

PubMed

Tokaji, Narumi; Ito, Hiromichi; Kohmoto, Tomohiro; Naruto, Takuya; Takahashi, Rizu; Goji, Aya; Mori, Tatsuo; Toda, Yoshihiro; Saito, Masako; Tange, Shoichiro; Masuda, Kiyoshi; Kagami, Shoji; Imoto, Issei

2018-03-01

Rett syndrome (RTT) is a severe neurodevelopmental disorder typically affecting females. It is mainly caused by loss-of-function mutations that affect the coding sequence of exon 3 or 4 of methyl-CpG-binding protein 2 (MECP2). Severe neonatal encephalopathy resulting in death before the age of 2 years is the most common phenotype observed in males affected by a pathogenic MECP2 variant. Mutations in MECP2 exon 1 affecting the MeCP2_e1 isoform are relatively rare causes of RTT in females, and only one case of a male patient with MECP2-related severe neonatal encephalopathy caused by a mutation in MECP2 exon 1 has been reported. This is the first reported case of a male with classic RTT caused by a 5-bp duplication in the open-reading frame of MECP2 exon 1 (NM_001110792.1:c.23_27dup) that introduced a premature stop codon [p.(Ser10Argfs*36)] in the MeCP2_e1 isoform, which has been reported in one female patient with classic RTT. Therefore, both males and females displaying at least some type of MeCP2_e1 mutation may exhibit the classic RTT phenotype. © 2018 Wiley Periodicals, Inc.

Murine knockin model for progranulin-deficient frontotemporal dementia with nonsense-mediated mRNA decay

PubMed Central

Nguyen, Thi A.; Zhang, Jiasheng; Devireddy, Swathi; Zhou, Ping; Karydas, Anna M.; Xu, Xialian; Miller, Bruce L.; Rigo, Frank; Ferguson, Shawn M.; Walther, Tobias C.; Farese, Robert V.

2018-01-01

Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized GrnR493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous GrnR493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in GrnR493X mice and cell lines and in fibroblasts from patients containing the GRNR493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation. PMID:29511098

Mutation Analysis of KRAS and BRAF Genes in Metastatic Colorectal Cancer: a First Large Scale Study from Iran.

PubMed

Koochak, Aghigh; Rakhshani, Nasser; Karbalaie Niya, Mohammad Hadi; Tameshkel, Fahimeh Safarnezhad; Sohrabi, Masoud Reza; Babaee, Mohammad Reza; Rezvani, Hamid; Bahar, Babak; Imanzade, Farid; Zamani, Farhad; Khonsari, Mohammad Reza; Ajdarkosh, Hossein; Hemmasi, Gholamreza

2016-01-01

The investigation of mutation patterns in oncogenes potentially can make available a reliable mechanism for management and treatment decisions for patients with colorectal cancer (CRC). This study concerns the rate of KRAS and BRAF genes mutations in Iranian metastatic colorectal cancer (mCRC) patients, as well as associations of genotypes with clinicopathological features. A total of 1,000 mCRC specimens collected from 2008 to 2012 that referred to the Mehr Hospital and Partolab center, Tehran, Iran enrolled in this cross sectional study. Using HRM, Dxs Therascreen and Pyrosequencing methods, we analyzed the mutational status of KRAS and BRAF genes in these. KRAS mutations were present in 33.6% cases (n=336). Of KRAS mutation positive cases, 85.1% were in codon 12 and 14.9% were in codon 13. The most frequent mutation at KRAS codon 12 was Gly12Asp; BRAF mutations were not found in any mCRC patients (n=242). In addition, we observed a strong correlation of KRAS mutations with some clinicopathological characteristics. KRAS mutations are frequent in mCRCs while presence of BRAF mutations in these patients is rare. Moreover, associations of KRAS genotypes with non-mucinous adenocarcinoma and depth of invasion (pT3) were remarkable.

Generation of Esr1-Knockout Rats Using Zinc Finger Nuclease-Mediated Genome Editing

PubMed Central

Dhakal, Pramod; Kubota, Kaiyu; Chakraborty, Damayanti; Lei, Tianhua; Larson, Melissa A.; Wolfe, Michael W.; Roby, Katherine F.; Vivian, Jay L.

2014-01-01

Estrogens play pivotal roles in development and function of many organ systems, including the reproductive system. We have generated estrogen receptor 1 (Esr1)-knockout rats using zinc finger nuclease (ZFN) genome targeting. mRNAs encoding ZFNs targeted to exon 3 of Esr1 were microinjected into single-cell rat embryos and transferred to pseudopregnant recipients. Of 17 live births, 5 had biallelic and 1 had monoallelic Esr1 mutations. A founder with monoallelic mutations was backcrossed to a wild-type rat. Offspring possessed only wild-type Esr1 alleles or wild-type alleles and Esr1 alleles containing either 482 bp (Δ482) or 223 bp (Δ223) deletions, indicating mosaicism in the founder. These heterozygous mutants were bred for colony expansion, generation of homozygous mutants, and phenotypic characterization. The Δ482 Esr1 allele yielded altered transcript processing, including the absence of exon 3, aberrant splicing of exon 2 and 4, and a frameshift that generated premature stop codons located immediately after the codon for Thr157. ESR1 protein was not detected in homozygous Δ482 mutant uteri. ESR1 disruption affected sexually dimorphic postnatal growth patterns and serum levels of gonadotropins and sex steroid hormones. Both male and female Esr1-null rats were infertile. Esr1-null males had small testes with distended and dysplastic seminiferous tubules, whereas Esr1-null females possessed large polycystic ovaries, thread-like uteri, and poorly developed mammary glands. In addition, uteri of Esr1-null rats did not effectively respond to 17β-estradiol treatment, further demonstrating that the Δ482 Esr1 mutation created a null allele. This rat model provides a new experimental tool for investigating the pathophysiology of estrogen action. PMID:24506075

Generation of Esr1-knockout rats using zinc finger nuclease-mediated genome editing.

PubMed

Rumi, M A Karim; Dhakal, Pramod; Kubota, Kaiyu; Chakraborty, Damayanti; Lei, Tianhua; Larson, Melissa A; Wolfe, Michael W; Roby, Katherine F; Vivian, Jay L; Soares, Michael J

2014-05-01

Estrogens play pivotal roles in development and function of many organ systems, including the reproductive system. We have generated estrogen receptor 1 (Esr1)-knockout rats using zinc finger nuclease (ZFN) genome targeting. mRNAs encoding ZFNs targeted to exon 3 of Esr1 were microinjected into single-cell rat embryos and transferred to pseudopregnant recipients. Of 17 live births, 5 had biallelic and 1 had monoallelic Esr1 mutations. A founder with monoallelic mutations was backcrossed to a wild-type rat. Offspring possessed only wild-type Esr1 alleles or wild-type alleles and Esr1 alleles containing either 482 bp (Δ482) or 223 bp (Δ223) deletions, indicating mosaicism in the founder. These heterozygous mutants were bred for colony expansion, generation of homozygous mutants, and phenotypic characterization. The Δ482 Esr1 allele yielded altered transcript processing, including the absence of exon 3, aberrant splicing of exon 2 and 4, and a frameshift that generated premature stop codons located immediately after the codon for Thr157. ESR1 protein was not detected in homozygous Δ482 mutant uteri. ESR1 disruption affected sexually dimorphic postnatal growth patterns and serum levels of gonadotropins and sex steroid hormones. Both male and female Esr1-null rats were infertile. Esr1-null males had small testes with distended and dysplastic seminiferous tubules, whereas Esr1-null females possessed large polycystic ovaries, thread-like uteri, and poorly developed mammary glands. In addition, uteri of Esr1-null rats did not effectively respond to 17β-estradiol treatment, further demonstrating that the Δ482 Esr1 mutation created a null allele. This rat model provides a new experimental tool for investigating the pathophysiology of estrogen action.

Mutation in and lack of expression of tyrosinase-related protein-1 (TRP-1) in melanocytes from an individual with brown oculocutaneous albinism: a new subtype of albinism classified as "OCA3".

PubMed Central

Boissy, R. E.; Zhao, H.; Oetting, W. S.; Austin, L. M.; Wildenberg, S. C.; Boissy, Y. L.; Zhao, Y.; Sturm, R. A.; Hearing, V. J.; King, R. A.; Nordlund, J. J.

1996-01-01

Most types of human oculocutaneous albinism (OCA) result from mutations in the gene for tyrosinase (OCA1) or the P protein (OCA2), although other types of OCA have been described but have not been mapped to specific loci. Melanocytes were cultured from an African-American with OCA, who exhibited the phenotype of Brown OCA, and his normal fraternal twin. Melanocytes cultured from the patient with OCA and the normal twin appeared brown versus black, respectively. Melanocytes from both the patient with OCA and the normal twin demonstrated equal amounts of NP-40-soluble melanin; however, melanocytes from the patient with OCA contained only 7% of the amount of insoluble melanin found from the normal twin. Tyrosinase- related protein-1 (TRP-1) was not detected in the OCA melanocytes by use of various anti-TRP-1 probes. Furthermore, transcripts for TRP-1 were absent in cultured OCA melanocytes. The affected twin was homozygous for a single-bp deletion in exon 6, removing an A in codon 368 and leading to a premature stop at codon 384. Tyrosine hydroxylase activity of the OCA melanocytes was comparable to controls when assayed in cell lysates but was only 30% of controls when assayed in intact cells. We conclude that this mutation of the human TRP-1 gene affects its interaction with tyrosinase, resulting in dysregulation of tyrosinase activity, promotes the synthesis of brown versus black melanin, and is responsible for a third genetic type of OCA in humans, which we classify as "OCA3." Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8651291

The complete mitochondrial genome of the Korean skate: Hongeo koreana (Rajiformes, Rajidae).

PubMed

Jeong, Dageum; Kim, Sung; Kim, Choong-Gon; Lee, Youn-Ho

2014-12-01

The complete mitochondrial genome of the Korean skate, Hongeo koreana, the sole member of its genus, is investigated for the first time. The genome consists of 16,906 bp in length including 2 rRNA, 22 tRNA and 13 protein coding genes with the same gene order and structure of the genome as those of other Rajidae species. The overall nucleotide composition of the L-strand is A = 29.8%, C = 27.9%, T = 27.9% and G = 14.3%, showing a high A + T bias. The anti-G bias (6.0%) is more significant in the third codon position. Twelve of the 13 protein-coding genes use ATG as their start codon while the COX1 gene starts with GTG. For stop codon, ND3 and ND4 genes show incomplete stop codon T. The mitogenome sequence of H. koreana will provide important information on the evolution and the phylogenetic relation of the genus Hongeo in relation to the other genera of the family Rajidae.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baserga, S.J.; Benz, E.J. Jr.

A number of premature translation termination mutations (nonsense mutations) have been described in the human {alpha}- and {beta}-globin genes. Studies on mRNA isolated from patients with {beta}{sup 0}-thalassemia have shown that for both the {beta}-17 and the {beta}-39 mutations less than normal levels of {beta}-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human {beta}-globin mRNA). In vitro studies using the cloned {beta}-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. Themore » authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human {beta}-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation.« less

The detection of pfcrt and pfmdr1 point mutations as molecular markers of chloroquine drug resistance, Pahang, Malaysia

PubMed Central

2012-01-01

Background Malaria is still a public health problem in Malaysia with chloroquine (CQ) being the first-line drug in the treatment policy of uncomplicated malaria. There is a scarcity in information about the magnitude of Plasmodium falciparum CQ resistance. This study aims to investigate the presence of single point mutations in the P. falciparum chloroquine-resistance transporter gene (pfcrt) at codons 76, 271, 326, 356 and 371 and in P. falciparum multi-drug resistance-1 gene (pfmdr1) at codons 86 and 1246, as molecular markers of CQ resistance. Methods A total of 75 P. falciparum blood samples were collected from different districts of Pahang state, Malaysia. Single nucleotide polymorphisms in pfcrt gene (codons 76, 271, 326, 356 and 371) and pfmdr1 gene (codons 86 and 1246) were analysed by using mutation-specific nested PCR and restriction fragment length polymorphism (PCR-RFLP) methods. Results Mutations of pfcrt K76T and pfcrt R371I were the most prevalent among pfcrt gene mutations reported by this study; 52% and 77%, respectively. Other codons of the pfcrt gene and the positions 86 and 1246 of the pfmdr1 gene were found mostly of wild type. Significant associations of pfcrt K76T, pfcrt N326S and pfcrt I356T mutations with parasitaemia were also reported. Conclusion The high existence of mutant pfcrt T76 may indicate the low susceptibility of P. falciparum isolates to CQ in Peninsular Malaysia. The findings of this study establish baseline data on the molecular markers of P. falciparum CQ resistance, which may help in the surveillance of drug resistance in Peninsular Malaysia. PMID:22853645

Analysis of transcriptome data reveals multifactor constraint on codon usage in Taenia multiceps.

PubMed

Huang, Xing; Xu, Jing; Chen, Lin; Wang, Yu; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

2017-04-20

Codon usage bias (CUB) is an important evolutionary feature in genomes that has been widely observed in many organisms. However, the synonymous codon usage pattern in the genome of T. multiceps remains to be clarified. In this study, we analyzed the codon usage of T. multiceps based on the transcriptome data to reveal the constraint factors and to gain an improved understanding of the mechanisms that shape synonymous CUB. Analysis of a total of 8,620 annotated mRNA sequences from T. multiceps indicated only a weak codon bias, with mean GC and GC3 content values of 49.29% and 51.43%, respectively. Our analysis indicated that nucleotide composition, mutational pressure, natural selection, gene expression level, amino acids with grand average of hydropathicity (GRAVY) and aromaticity (Aromo) and the effective selection of amino-acids all contributed to the codon usage in T. multiceps. Among these factors, natural selection was implicated as the major factor affecting the codon usage variation in T. multiceps. The codon usage of ribosome genes was affected mainly by mutations, while the essential genes were affected mainly by selection. In addition, 21codons were identified as "optimal codons". Overall, the optimal codons were GC-rich (GC:AU, 41:22), and ended with G or C (except CGU). Furthermore, different degrees of variation in codon usage were found between T. multiceps and Escherichia coli, yeast, Homo sapiens. However, little difference was found between T. multiceps and Taenia pisiformis. In this study, the codon usage pattern of T. multiceps was analyzed systematically and factors affected CUB were also identified. This is the first study of codon biology in T. multiceps. Understanding the codon usage pattern in T. multiceps can be helpful for the discovery of new genes, molecular genetic engineering and evolutionary studies.

Establishment of an Immortalized Skin Keratinocyte Cell Line Derived from the Animal Model Mastomys coucha.

PubMed

Hasche, Daniel; Stephan, Sonja; Savelyeva, Larissa; Westermann, Frank; Rösl, Frank; Vinzón, Sabrina E

2016-01-01

In the present report we describe the establishment of a spontaneous immortalized skin keratinocyte cell line derived from the skin of the multimammate rodent Mastomys coucha. These animals are used in preclinical studies for a variety of human diseases such as infections with nematodes, bacteria and papillomaviruses, especially regarding cutaneous manifestations such as non-melanoma skin cancer. Here we characterize the cells in terms of their origin and cytogenetic features. Searching for genomic signatures, a spontaneous mutation in the splicing donor sequence of Trp53 (G to A transition at the first position of intron 7) could be detected. This point mutation leads to alternative splicing and to a premature stop codon, resulting in a truncated and, in turn, undetectable form of p53, probably contributing to the process of immortalization. Mastomys coucha-derived skin keratinocytes can be used as an in vitro system to investigate molecular and immunological aspects of infectious agent interactions with their host cells.

Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakamura, Tomoyuki; Sato, Yuko; Watanabe, Daisuke

2010-03-15

To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal inmore » any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.« less

Analysis of codon usage bias of envelope glycoprotein genes in nuclear polyhedrosis virus (NPV) and its relation to evolution.

PubMed

Zhao, Yongchao; Zheng, Hao; Xu, Anying; Yan, Donghua; Jiang, Zijian; Qi, Qi; Sun, Jingchen

2016-08-24

Analysis of codon usage bias is an extremely versatile method using in furthering understanding of the genetic and evolutionary paths of species. Codon usage bias of envelope glycoprotein genes in nuclear polyhedrosis virus (NPV) has remained largely unexplored at present. Hence, the codon usage bias of NPV envelope glycoprotein was analyzed here to reveal the genetic and evolutionary relationships between different viral species in baculovirus genus. A total of 9236 codons from 18 different species of NPV of the baculovirus genera were used to perform this analysis. Glycoprotein of NPV exhibits weaker codon usage bias. Neutrality plot analysis and correlation analysis of effective number of codons (ENC) values indicate that natural selection is the main factor influencing codon usage bias, and that the impact of mutation pressure is relatively smaller. Another cluster analysis shows that the kinship or evolutionary relationships of these viral species can be divided into two broad categories despite all of these 18 species are from the same baculovirus genus. There are many elements that can affect codon bias, such as the composition of amino acids, mutation pressure, natural selection, gene expression level, and etc. In the meantime, cluster analysis also illustrates that codon usage bias of virus envelope glycoprotein can serve as an effective means of evolutionary classification in baculovirus genus.

B cell Variable genes have evolved their codon usage to focus the targeted patterns of somatic mutation on the complementarity determining regions

PubMed Central

Saini, Jasmine; Hershberg, Uri

2015-01-01

The exceptional ability of B cells to diversify through somatic mutation and improve affinity of the repertoire towards the antigens is the cornerstone of adaptive immunity. Somatic mutation is not evenly distributed and exhibits certain micro-sequence specificities. We show here that the combination of somatic mutation targeting and the codon usage in human B cell receptor (BCR) Variable (V) genes create expected patterns of mutation and post mutation changes that are focused on their complementarity determining regions (CDR). T cell V genes are also skewed in targeting mutations but to a lesser extent and are lacking the codon usage bias observed in BCRs. This suggests that the observed skew in T cell receptors is due to their amino acid usage, which is similar to that of BCRs. The mutation targeting and the codon bias allow B cell CDRs to diversify by specifically accumulating nonconservative changes. We counted the distribution of mutations to CDR in 4 different human datasets. In all four cases we found that the number of actual mutations in the CDR correlated significantly with the V gene mutation biases to the CDR predicted by our models. Finally, it appears that the mutation bias in V genes indeed relates to their long-term survival in actual human repertoires. We observed that resting repertoires of B cells overexpressed V genes that were especially biased towards focused mutation and change in the CDR. This bias in V gene usage was somewhat relaxed at the height of the immune response to a vaccine, presumably because of the need for a wider diversity in a primary response. However, older patients did not retain this flexibility and were biased towards using only highly skewed V genes at all stages of their response. PMID:25660968

B cell variable genes have evolved their codon usage to focus the targeted patterns of somatic mutation on the complementarity determining regions.

PubMed

Saini, Jasmine; Hershberg, Uri

2015-05-01

The exceptional ability of B cells to diversify through somatic mutation and improve affinity of the repertoire toward the antigens is the cornerstone of adaptive immunity. Somatic mutation is not evenly distributed and exhibits certain micro-sequence specificities. We show here that the combination of somatic mutation targeting and the codon usage in human B cell receptor (BCR) Variable (V) genes create expected patterns of mutation and post mutation changes that are focused on their complementarity determining regions (CDR). T cell V genes are also skewed in targeting mutations but to a lesser extent and are lacking the codon usage bias observed in BCRs. This suggests that the observed skew in T cell receptors is due to their amino acid usage, which is similar to that of BCRs. The mutation targeting and the codon bias allow B cell CDRs to diversify by specifically accumulating nonconservative changes. We counted the distribution of mutations to CDR in 4 different human datasets. In all four cases we found that the number of actual mutations in the CDR correlated significantly with the V gene mutation biases to the CDR predicted by our models. Finally, it appears that the mutation bias in V genes indeed relates to their long-term survival in actual human repertoires. We observed that resting repertoires of B cells overexpressed V genes that were especially biased toward focused mutation and change in the CDR. This bias in V gene usage was somewhat relaxed at the height of the immune response to a vaccine, presumably because of the need for a wider diversity in a primary response. However, older patients did not retain this flexibility and were biased toward using only highly skewed V genes at all stages of their response. Copyright © 2015 Elsevier Ltd. All rights reserved.

A common periodic table of codons and amino acids.

PubMed

Biro, J C; Benyó, B; Sansom, C; Szlávecz, A; Fördös, G; Micsik, T; Benyó, Z

2003-06-27

A periodic table of codons has been designed where the codons are in regular locations. The table has four fields (16 places in each) one with each of the four nucleotides (A, U, G, C) in the central codon position. Thus, AAA (lysine), UUU (phenylalanine), GGG (glycine), and CCC (proline) were placed into the corners of the fields as the main codons (and amino acids) of the fields. They were connected to each other by six axes. The resulting nucleic acid periodic table showed perfect axial symmetry for codons. The corresponding amino acid table also displaced periodicity regarding the biochemical properties (charge and hydropathy) of the 20 amino acids and the position of the stop signals. The table emphasizes the importance of the central nucleotide in the codons and predicts that purines control the charge while pyrimidines determine the polarity of the amino acids. This prediction was experimentally tested.

A universal method for the mutational analysis of K-ras and p53 gene in non-small-cell lung cancer using formalin-fixed paraffin-embedded tissue.

PubMed

Sarkar, F H; Valdivieso, M; Borders, J; Yao, K L; Raval, M M; Madan, S K; Sreepathi, P; Shimoyama, R; Steiger, Z; Visscher, D W

1995-12-01

The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized nonradioisotopic detection system using a single 6-microns tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non-small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes.

Three new PAX6 mutations including one causing an unusual ophthalmic phenotype associated with neurodevelopmental abnormalities.

PubMed

Dansault, Anouk; David, Gabriel; Schwartz, Claire; Jaliffa, Carolina; Vieira, Véronique; de la Houssaye, Guillaume; Bigot, Karine; Catin, Françise; Tattu, Laurent; Chopin, Catherine; Halimi, Philippe; Roche, Olivier; Van Regemorter, Nicole; Munier, Francis; Schorderet, Daniel; Dufier, Jean-Louis; Marsac, Cécile; Ricquier, Daniel; Menasche, Maurice; Penfornis, Alfred; Abitbol, Marc

2007-04-02

The PAX6 gene was first described as a candidate for human aniridia. However, PAX6 expression is not restricted to the eye and it appears to be crucial for brain development. We studied PAX6 mutations in a large spectrum of patients who presented with aniridia phenotypes, Peters' anomaly, and anterior segment malformations associated or not with neurological anomalies. Patients and related families were ophthalmologically phenotyped, and in some cases neurologically and endocrinologically examined. We screened the PAX6 gene by direct sequencing in three groups of patients: those affected by aniridia; those with diverse ocular manifestations; and those with Peters' anomaly. Two mutations were investigated by generating crystallographic representations of the amino acid changes. Three novel heterozygous mutations affecting three unrelated families were identified: the g.572T>C nucleotide change, located in exon 5, and corresponding to the Leucine 46 Proline amino-acid mutation (L46P); the g.655A>G nucleotide change, located in exon 6, and corresponding to the Serine 74 Glycine amino-acid mutation (S74G); and the nucleotide deletion 579delG del, located in exon 6, which induces a frameshift mutation leading to a stop codon (V48fsX53). The L46P mutation was identified in affected patients presenting bilateral microphthalmia, cataracts, and nystagmus. The S74G mutation was found in a large family that had congenital ocular abnormalities, diverse neurological manifestations, and variable cognitive impairments. The 579delG deletion (V48fsX53) caused in the affected members of the same family bilateral aniridia associated with congenital cataract, foveal hypolasia, and nystagmus. We also detected a novel intronic nucleotide change, IVS2+9G>A (very likely a mutation) in an apparently isolated patient affected by a complex ocular phenotype, characterized primarily by a bilateral microphthalmia. Whether this nucleotide change is indeed pathogenic remains to be demonstrated. Two previously known heterozygous mutations of the PAX6 gene sequence were also detected in patients affected by aniridia: a de novo previously known nucleotide change, g.972C>T (Q179X), in exon 8, leading to a stop codon and a heterozygous g.555C>A (C40X) recurrent nonsense mutation in exon 5. No mutations were found in patients with Peters' anomaly. We identified three mutations associated with aniridia phenotypes (Q179X, C40X, and V48fsX53). The three other mutations reported here cause non-aniridia ocular phenotypes associated in some cases with neurological anomalies. The IVS2+9G>A nucleotide change was detected in a patient with a microphthalmia phenotype. The L46P mutation was detected in a family with microphthalmia, cataract, and nystagmus. This mutation is located in the DNA-binding paired-domain and the crystallographic representations of this mutation show that this mutation may affect the helix-turn-helix motif, and as a consequence the DNA-binding properties of the resulting mutated protein. Ser74 is located in the PAX6 PD linker region, essential for DNA recognition and DNA binding, and the side chain of the Ser74 contributes to DNA recognition by the linker domain through direct contacts. Crystallographic representations show that the S74G mutation results in no side chain and therefore perturbs the DNA-binding properties of PAX6. This study highlights the severity and diversity of the consequences of PAX6 mutations that appeared to result from the complexity of the PAX6 gene structure, and the numerous possibilities for DNA binding. This study emphasizes the fact that neurodevelopmental abnormalities may be caused by PAX6 mutations. The neuro-developmental abnormalities caused by PAX6 mutations are probably still overlooked in the current clinical examinations performed throughout the world in patients affected by PAX6 mutations.

Three new PAX6 mutations including one causing an unusual ophthalmic phenotype associated with neurodevelopmental abnormalities

PubMed Central

Dansault, Anouk; David, Gabriel; Schwartz, Claire; Jaliffa, Carolina; Vieira, Véronique; de la Houssaye, Guillaume; Bigot, Karine; Catin, Françise; Tattu, Laurent; Chopin, Catherine; Halimi, Philippe; Roche, Olivier; Van Regemorter, Nicole; Munier, Francis; Schorderet, Daniel; Dufier, Jean-Louis; Marsac, Cécile; Ricquier, Daniel; Menasche, Maurice; Penfornis, Alfred

2007-01-01

Purpose The PAX6 gene was first described as a candidate for human aniridia. However, PAX6 expression is not restricted to the eye and it appears to be crucial for brain development. We studied PAX6 mutations in a large spectrum of patients who presented with aniridia phenotypes, Peters' anomaly, and anterior segment malformations associated or not with neurological anomalies. Methods Patients and related families were ophthalmologically phenotyped, and in some cases neurologically and endocrinologically examined. We screened the PAX6 gene by direct sequencing in three groups of patients: those affected by aniridia; those with diverse ocular manifestations; and those with Peters' anomaly. Two mutations were investigated by generating crystallographic representations of the amino acid changes. Results Three novel heterozygous mutations affecting three unrelated families were identified: the g.572T>C nucleotide change, located in exon 5, and corresponding to the Leucine 46 Proline amino-acid mutation (L46P); the g.655A>G nucleotide change, located in exon 6, and corresponding to the Serine 74 Glycine amino-acid mutation (S74G); and the nucleotide deletion 579delG del, located in exon 6, which induces a frameshift mutation leading to a stop codon (V48fsX53). The L46P mutation was identified in affected patients presenting bilateral microphthalmia, cataracts, and nystagmus. The S74G mutation was found in a large family that had congenital ocular abnormalities, diverse neurological manifestations, and variable cognitive impairments. The 579delG deletion (V48fsX53) caused in the affected members of the same family bilateral aniridia associated with congenital cataract, foveal hypolasia, and nystagmus. We also detected a novel intronic nucleotide change, IVS2+9G>A (very likely a mutation) in an apparently isolated patient affected by a complex ocular phenotype, characterized primarily by a bilateral microphthalmia. Whether this nucleotide change is indeed pathogenic remains to be demonstrated. Two previously known heterozygous mutations of the PAX6 gene sequence were also detected in patients affected by aniridia: a de novo previously known nucleotide change, g.972C>T (Q179X), in exon 8, leading to a stop codon and a heterozygous g.555C>A (C40X) recurrent nonsense mutation in exon 5. No mutations were found in patients with Peters' anomaly. Conclusions We identified three mutations associated with aniridia phenotypes (Q179X, C40X, and V48fsX53). The three other mutations reported here cause non-aniridia ocular phenotypes associated in some cases with neurological anomalies. The IVS2+9G>A nucleotide change was detected in a patient with a microphthalmia phenotype. The L46P mutation was detected in a family with microphthalmia, cataract, and nystagmus. This mutation is located in the DNA-binding paired-domain and the crystallographic representations of this mutation show that this mutation may affect the helix-turn-helix motif, and as a consequence the DNA-binding properties of the resulting mutated protein. Ser74 is located in the PAX6 PD linker region, essential for DNA recognition and DNA binding, and the side chain of the Ser74 contributes to DNA recognition by the linker domain through direct contacts. Crystallographic representations show that the S74G mutation results in no side chain and therefore perturbs the DNA-binding properties of PAX6. This study highlights the severity and diversity of the consequences of PAX6 mutations that appeared to result from the complexity of the PAX6 gene structure, and the numerous possibilities for DNA binding. This study emphasizes the fact that neurodevelopmental abnormalities may be caused by PAX6 mutations. The neuro-developmental abnormalities caused by PAX6 mutations are probably still overlooked in the current clinical examinations performed throughout the world in patients affected by PAX6 mutations. PMID:17417613

Both a frameshift and a missense mutation of the STRA6 gene observed in an infant with the Matthew-Wood syndrome.

PubMed

Sadowski, Samantha; Chassaing, Nicolas; Gaj, Zuzanna; Czichos, Ewa; Wilczynski, Jan; Nowakowska, Dorota

2017-03-01

The Matthew-Wood syndrome is associated with mutations of the STRA6 gene. It combines a pulmonary agenesis/hypoplasia; microphthalmia/anophthalmia; congenital cardiac, digestive, and urogenital malformations; and diaphragmatic defects. A 23-year-old nulliparous woman was referred to our center after a fetal ultrasound examination at 26 weeks of pregnancy revealed an abnormal head shape, a heart malformation, multiple cysts in both kidneys, and dilated ureters. A male baby (46, XY; 3600g; Apgar score 1) was delivered at 38 weeks of gestation and died 1 hr later due to respiratory failure. The diagnosis of Matthew-Wood syndrome was suspected given the association of bilateral anophthalmia, agenesis of the left lung, and heart and kidney defects. It was confirmed by the identification of two deleterious mutations of the STRA6 gene. The child was a compound heterozygote for two previously reported mutations, a paternally inherited missense mutation (c.878C>T [p.Pro293Leu] and a maternally inherited frameshift mutation (c.50_52delACTinsCC [p. Asp17Alafs*55]), producing a premature stop codon. The diagnosis of Matthew-Wood syndrome should be considered in all fetuses with microphthalmia/anophthalmia. It requires an extensive ultrasound/MRI examination of the lung, heart, and diaphragm. Birth Defects Research 109:251-253, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A novel LEMD3 mutation common to patients with osteopoikilosis with and without melorheostosis.

PubMed

Couto, Ana R; Bruges-Armas, Jacome; Peach, Chris A; Chapman, Kay; Brown, Matthew A; Wordsworth, B Paul; Zhang, Yun

2007-08-01

Recent studies have reported loss of function mutations in the LEMD3 gene, encoding an inner nuclear membrane protein that influences Smad signaling, as a cause of osteopoikilosis, Buschke-Ollendorff syndrome, and melorheostosis. We investigated LEMD3 in a three-generation family with osteopoikilosis from the Azores, an affected father and daughter from Ireland with osteopoikilosis (the daughter also had melorheostosis), and two other individuals from the UK with isolated melorheostosis. We found a novel C to T substitution at position 2032 bp (cDNA) in exon 8 of LEMD3, resulting in a premature stop codon at amino acid position 678. This mutation co-segregates with the osteopoikilosis phenotype in both the Azorean family and the Irish family. It was not detected in any of the six unaffected family members or in 342 healthy Caucasian individuals. No LEMD3 mutations were detected in the two patients with sporadic melorheostosis. The LEMD3 mutation reported was clearly the cause of osteopoikilosis in the two families but its relationship to melorheostosis in one of the family members is still unclear. Perhaps unsurprisingly in what is a segmental disease, we did not find LEMD3 mutations in peripheral-blood-derived DNA from the two other individuals with sporadic melorheostosis. The nature of the additional genetic and/or environmental influences required for the development of melorheostosis in those with osteopoikilosis requires further investigation.

A Novel Nonsense Mutation in Exon 5 of KIND1 Gene in an Iranian Family with Kindler Syndrome.

PubMed

Heidari, Mohammad Mehdi; Khatami, Mehri; Kargar, Saeed; Azari, Mojdeh; Hoseinzadeh, Hassan; Fallah, Hamedeh

2016-06-01

Kindler syndrome (KS) is an autosomal recessive skin disease characterized by actual blistering, photosensitivity and a progressive poikiloderma. The disorder results from rare mutations in the KIND1 gene. This gene contains 15 exons and expresses two kindlin-1 isoforms. The aim of this investigation was to analyze mutations in the exons 1 to 15 of KIND1 gene in an Iranian family clinically affected with Kindler syndrome. The mutations analysis of 15 coding exons of KIND1 gene was performed with PCR-SSCP and direct sequencing in 14 subjects from one Iranian family clinically affected with Kindler syndrome. We identified eight new nucleotide changes in KIND1 in this family. These changes were found in g.3892delA, g.3951T>C, g.3962T>G, g.4190G>T, g.7497G>A, g.11076T>C, g.11102C>T and g.13177C>T positions. Among them, the g.13177C>T mutation resulting in the formation of a premature stop codon (Q226X) was detected only in seven affected family individuals as homozygous but was not present in 100 unrelated healthy controls. This study suggests that nonsense mutation may lead to incomplete and non-functional protein products and is pathogenic and has meaningful implications for the diagnosis of patients with Kindler syndrome.

Diversified clinical presentations associated with a novel sal-like 4 gene mutation in a Chinese pedigree with Duane retraction syndrome.

PubMed

Yang, Ming-ming; Ho, Mary; Lau, Henry H W; Tam, Pancy O S; Young, Alvin L; Pang, Chi Pui; Yip, Wilson W K; Chen, LiJia

2013-01-01

To determine the underlying genetic cause of Duane retraction syndrome (DRS) in a non-consanguineous Chinese Han family. Detailed ophthalmic and physical examinations were performed on all members from a pedigree with DRS. All exons and their adjacent splicing junctions of the sal-like 4 (SALL4) gene were amplified with polymerase chain reaction and analyzed with direct sequencing in all the recruited family members and 200 unrelated control subjects. Clinical examination revealed a broad spectrum of phenotypes in the DRS family. Mutation analysis of SALL4 identified a novel heterozygous duplication mutation, c.1919dupT, which was completely cosegregated with the disease in the family and absent in controls. This mutation was predicted to cause a frameshift, introducing a premature stop codon, when translated, resulting in a truncated SALL4 protein, i.e., p.Met640IlefsX25. Bioinformatics analysis showed that the affected region of SALL4 shared a highly conserved sequence across different species. Diversified clinical manifestations were observed in the c.1919dupT carriers of the family. We identified a novel truncating mutation in the SALL4 gene that leads to diversified clinical features of DRS in a Chinese family. This mutation is predicted to result in a truncated SALL4 protein affecting two functional domains and cause disease development due to haploinsufficiency through nonsense-mediated mRNA decay.

X-linked nephrogenic diabetes insipidus mutations in North America and the Hopewell hypothesis.

PubMed Central

Bichet, D G; Arthus, M F; Lonergan, M; Hendy, G N; Paradis, A J; Fujiwara, T M; Morgan, K; Gregory, M C; Rosenthal, W; Didwania, A

1993-01-01

In X-linked nephrogenic diabetes insipidus (NDI) the urine of male patients is not concentrated after the administration of the antidiuretic hormone arginine-vasopressin. This disease is due to mutations in the V2 receptor gene that maps to chromosome region Xq28. In 1969, Bode and Crawford suggested that most NDI patients in North America shared common ancestors of Ulster Scot immigrants who arrived in Halifax in 1761 on the ship Hopewell. A link between this family and a large Utah kindred was also suggested. DNA was obtained from 17 affected male patients from the "Hopewell" kindred and from four additional families from Nova Scotia and New Brunswick who shared the same Xq28 NDI haplotype. The Utah kindred and two families (Q2, Q3) from Quebec were also studied. The "Hopewell" mutation, W71X, is a single base substitution (G-->A) that changes codon 71 from TGG (tryptophan) to TGA (stop). The W71X mutation was found in affected members of the Hopewell and of the four satellite families. The W71X mutation is the cause of X-linked NDI for the largest number of related male patients living in North America. Other families (Utah, Q2 and Q3) that are historically and ethnically unrelated bear other mutations in the V2 receptor gene. Images PMID:8104196

Stop Codon Reassignment in the Wild

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ivanova, Natalia; Schwientek, Patrick; Tripp, H. James

Since the discovery of the genetic code and protein translation mechanisms (1), a limited number of variations of the standard assignment between unique base triplets (codons) and their encoded amino acids and translational stop signals have been found in bacteria and phages (2-3). Given the apparent ubiquity of the canonical genetic code, the design of genomically recoded organisms with non-canonical codes has been suggested as a means to prevent horizontal gene transfer between laboratory and environmental organisms (4). It is also predicted that genomically recoded organisms are immune to infection by viruses, under the assumption that phages and their hostsmore » must share a common genetic code (5). This paradigm is supported by the observation of increased resistance of genomically recoded bacteria to phages with a canonical code (4). Despite these assumptions and accompanying lines of evidence, it remains unclear whether differential and non-canonical codon usage represents an absolute barrier to phage infection and genetic exchange between organisms. Our knowledge of the diversity of genetic codes and their use by viruses and their hosts is primarily derived from the analysis of cultivated organisms. Advances in single-cell sequencing and metagenome assembly technologies have enabled the reconstruction of genomes of uncultivated bacterial and archaeal lineages (6). These initial findings suggest that large scale systematic studies of uncultivated microorganisms and viruses may reveal the extent and modes of divergence from the canonical genetic code operating in nature. To explore alternative genetic codes, we carried out a systematic analysis of stop codon reassignments from the canonical TAG amber, TGA opal, and TAA ochre codons in assembled metagenomes from environmental and host-associated samples, single-cell genomes of uncultivated bacteria and archaea, and a collection of phage sequences« less

Application of temperature-gradient gel electrophoresis for detection of prion protein gene polymorphisms in Polish Swiniarka sheep.

PubMed

Jasik, Agnieszka; Reichert, Michal

2006-05-01

This study presents preliminary data on the polymorphism in the prion protein gene of Swiniarka sheep using temperature gradient gel electrophoresis (TGGE). Available data indicate that sensitivity to scrapie is associated with polymorphisms in three codons of prion protein gene: 136,154, and 171. The TGGE method was used to detect point mutations in these codons responsible for sensitivity or resistance to scrapie. This study revealed presence of an allele encoding valine (V) in codon 136, which is associated with high sensitivity to scrapie and occurred in the form of heterozygous allele together with alanine (AV). The highest variability was observed in codon 171, with presence of arginine (R) and glutamine (Q) in the homozygous (RR or QQ) as well as the heterozygous form (RQ). The results of examination of fifty sheep DNA samples with mutations in codons 136, 154, and 171 demonstrated that TGGE can be used as a simple and rapid method to detect mutations in the PrP gene of sheep. Several samples can be run at the same time, making TGGE ideal for the screening of large numbers of samples.

Phenotypic spectrum associated with mutations of the mitochondrial polymerase gamma gene.

PubMed

Horvath, Rita; Hudson, Gavin; Ferrari, Gianfrancesco; Fütterer, Nancy; Ahola, Sofia; Lamantea, Eleonora; Prokisch, Holger; Lochmüller, Hanns; McFarland, Robert; Ramesh, V; Klopstock, Thomas; Freisinger, Peter; Salvi, Fabrizio; Mayr, Johannes A; Santer, Rene; Tesarova, Marketa; Zeman, Jiri; Udd, Bjarne; Taylor, Robert W; Turnbull, Douglass; Hanna, Michael; Fialho, Doreen; Suomalainen, Anu; Zeviani, Massimo; Chinnery, Patrick F

2006-07-01

Mutations in the gene coding for the catalytic subunit of the mitochondrial DNA (mtDNA) polymerase gamma (POLG1) have recently been described in patients with diverse clinical presentations, revealing a complex relationship between genotype and phenotype in patients and their families. POLG1 was sequenced in patients from different European diagnostic and research centres to define the phenotypic spectrum and advance understanding of the recurrence risks. Mutations were identified in 38 cases, with the majority being sporadic compound heterozygotes. Eighty-nine DNA sequence changes were identified, including 2 predicted to alter a splice site, 1 predicted to cause a premature stop codon and 13 predicted to cause novel amino acid substitutions. The majority of children had a mutation in the linker region, often 1399G-->A (A467T), and a mutation affecting the polymerase domain. Others had mutations throughout the gene, and 11 had 3 or more substitutions. The clinical presentation ranged from the neonatal period to late adult life, with an overlapping phenotypic spectrum from severe encephalopathy and liver failure to late-onset external ophthalmoplegia, ataxia, myopathy and isolated muscle pain or epilepsy. There was a strong gender bias in children, with evidence of an environmental interaction with sodium valproate. POLG1 mutations cause an overlapping clinical spectrum of disease with both dominant and recessive modes of inheritance. 1399G-->A (A467T) is common in children, but complete POLG1 sequencing is required to identify multiple mutations that can have complex implications for genetic counselling.

Mutational spectrum of Xeroderma pigmentosum group A in Egyptian patients.

PubMed

Amr, Khalda; Messaoud, Olfa; El Darouti, Mohamad; Abdelhak, Sonia; El-Kamah, Ghada

2014-01-01

Xeroderma pigmentosum (XP) is a rare autosomal recessive hereditary disease characterized by hyperphotosensitivity, DNA repair defects and a predisposition to skin cancers. The most frequently occurring type worldwide is the XP group A (XPA). There is a close relationship between the clinical features that ranged from severe to mild form and the mutational site in XPA gene. The aim of this study is to carry out the mutational analysis in Egyptian patients with XP-A. This study was carried out on four unrelated Egyptian XP-A families. Clinical features were examined and direct sequencing of the coding region of XPA gene was performed in patients and their parents. Direct sequencing of the whole coding region of the XPA gene revealed the identification of two homozygous nonsense mutations: (c.553C >T; p.(Gln185)) and (c.331G>T; p.(Glu111)), which create premature, stop codon and a homodeletion (c.374delC: p.Thr125Ilefs 15) that leads to frameshift and premature translation termination. We report the identification of one novel XPA gene mutation and two known mutations in four unrelated Egyptian families with Xermoderma pigmentosum. All explored patients presented severe neurological abnormalities and have mutations located in the DNA binding domain. This report gives insight on the mutation spectrum of XP-A in Egypt. This would provide a valuable tool for early diagnosis of this severe disease. © 2013 Elsevier B.V. All rights reserved.

Disease-associated mitochondrial mutations and the evolution of primate mitogenomes

PubMed Central

Tavares, William Corrêa

2017-01-01

Several human diseases have been associated with mutations in mitochondrial genes comprising a set of confirmed and reported mutations according to the MITOMAP database. An analysis of complete mitogenomes across 139 primate species showed that most confirmed disease-associated mutations occurred in aligned codon positions and gene regions under strong purifying selection resulting in a strong evolutionary conservation. Only two confirmed variants (7.1%), coding for the same amino acids accounting for severe human diseases, were identified without apparent pathogenicity in non-human primates, like the closely related Bornean orangutan. Conversely, reported disease-associated mutations were not especially concentrated in conserved codon positions, and a large fraction of them occurred in highly variable ones. Additionally, 88 (45.8%) of reported mutations showed similar variants in several non-human primates and some of them have been present in extinct species of the genus Homo. Considering that recurrent mutations leading to persistent variants throughout the evolutionary diversification of primates are less likely to be severely damaging to fitness, we suggest that these 88 mutations are less likely to be pathogenic. Conversely, 69 (35.9%) of reported disease-associated mutations occurred in extremely conserved aligned codon positions which makes them more likely to damage the primate mitochondrial physiology. PMID:28510580

Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion

PubMed Central

O’Donoghue, Patrick; Prat, Laure; Heinemann, Ilka U.; Ling, Jiqiang; Odoi, Keturah; Liu, Wenshe R.; Söll, Dieter

2012-01-01

Over 300 amino acids are found in proteins in nature, yet typically only 20 are genetically encoded. Reassigning stop codons and use of quadruplet codons emerged as the main avenues for genetically encoding non-canonical amino acids (NCAAs). Canonical aminoacyl-tRNAs with near-cognate anticodons also read these codons to some extent. This background suppression leads to ‘statistical protein’ that contains some natural amino acid(s) at a site intended for NCAA. We characterize near-cognate suppression of amber, opal and a quadruplet codon in common Escherichia coli laboratory strains and find that the PylRS/tRNAPyl orthogonal pair cannot completely outcompete contamination by natural amino acids. PMID:23036644

Genome-wide analysis of codon usage bias in four sequenced cotton species.

PubMed

Wang, Liyuan; Xing, Huixian; Yuan, Yanchao; Wang, Xianlin; Saeed, Muhammad; Tao, Jincai; Feng, Wei; Zhang, Guihua; Song, Xianliang; Sun, Xuezhen

2018-01-01

Codon usage bias (CUB) is an important evolutionary feature in a genome which provides important information for studying organism evolution, gene function and exogenous gene expression. The CUB and its shaping factors in the nuclear genomes of four sequenced cotton species, G. arboreum (A2), G. raimondii (D5), G. hirsutum (AD1) and G. barbadense (AD2) were analyzed in the present study. The effective number of codons (ENC) analysis showed the CUB was weak in these four species and the four subgenomes of the two tetraploids. Codon composition analysis revealed these four species preferred to use pyrimidine-rich codons more frequently than purine-rich codons. Correlation analysis indicated that the base content at the third position of codons affect the degree of codon preference. PR2-bias plot and ENC-plot analyses revealed that the CUB patterns in these genomes and subgenomes were influenced by combined effects of translational selection, directional mutation and other factors. The translational selection (P2) analysis results, together with the non-significant correlation between GC12 and GC3, further revealed that translational selection played the dominant role over mutation pressure in the codon usage bias. Through relative synonymous codon usage (RSCU) analysis, we detected 25 high frequency codons preferred to end with T or A, and 31 low frequency codons inclined to end with C or G in these four species and four subgenomes. Finally, 19 to 26 optimal codons with 19 common ones were determined for each species and subgenomes, which preferred to end with A or T. We concluded that the codon usage bias was weak and the translation selection was the main shaping factor in nuclear genes of these four cotton genomes and four subgenomes.

Nevirapine resistance mutation at codon 181 of the HIV-1 reverse transcriptase confers stavudine resistance by increasing nucleotide substrate discrimination and phosphorolytic activity.

PubMed

Blanca, Giuseppina; Baldanti, Fausto; Paolucci, Stefania; Skoblov, Alexander Yu; Victorova, Lyubov; Hübscher, Ulrich; Gerna, Giuseppe; Spadari, Silvio; Maga, Giovanni

2003-05-02

Recombinant HIV-1 reverse transcriptase (RT) carrying non-nucleoside inhibitors (NNRTIs) resistance mutation at codon 181 showed reduced incorporation and high efficiency of phosphorolytic removal of stavudine, a nucleoside RT inhibitor. These results reveal a new mechanism for cross-resistance between different classes of HIV-1 RT inhibitors.

High resolution melting analytical platform for rapid prenatal and postnatal diagnosis of β-thalassemia common among Southeast Asian population.

PubMed

Prajantasen, Thanet; Fucharoen, Supan; Fucharoen, Goonnapa

2015-02-20

High resolution melting (HRM) analysis is a powerful technology for scanning sequence alteration. We have applied this HRM assay to screen common β-thalassemia mutations found among Southeast Asian population. Known DNA samples with 8 common mutations were used in initial development of the methods including -28 A-G, codon 17 A-T, IVSI-1G-T, IVSI-5G-C, codon 26G-A (Hb E), codons 41/42 -TTCT, codons 71/72+A and IVSII-654 C-T. Further validation was done on 60 postnatal and 6 prenatal diagnoses of β-thalassemia. Each mutation has specific HRM profile which could be used in rapid screening. Apart from those with DNA deletions, the results of HRM assay matched 100% with those of routine diagnosis made by routine allele specific PCR. In addition, the HRM assay could initially recognize three unknown mutations including a hitherto un-described one in Thai population. The established HRM assay should prove useful for rapid and high throughput platform for screening and prenatal diagnosis of β-thalassemia common in the region. Copyright © 2014 Elsevier B.V. All rights reserved.

The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code

PubMed Central

Hofhuis, Julia; Schueren, Fabian; Nötzel, Christopher; Lingner, Thomas; Gärtner, Jutta; Jahn, Olaf

2016-01-01

Translational readthrough gives rise to C-terminally extended proteins, thereby providing the cell with new protein isoforms. These may have different properties from the parental proteins if the extensions contain functional domains. While for most genes amino acid incorporation at the stop codon is far lower than 0.1%, about 4% of malate dehydrogenase (MDH1) is physiologically extended by translational readthrough and the actual ratio of MDH1x (extended protein) to ‘normal' MDH1 is dependent on the cell type. In human cells, arginine and tryptophan are co-encoded by the MDH1x UGA stop codon. Readthrough is controlled by the 7-nucleotide high-readthrough stop codon context without contribution of the subsequent 50 nucleotides encoding the extension. All vertebrate MDH1x is directed to peroxisomes via a hidden peroxisomal targeting signal (PTS) in the readthrough extension, which is more highly conserved than the extension of lactate dehydrogenase B. The hidden PTS of non-mammalian MDH1x evolved to be more efficient than the PTS of mammalian MDH1x. These results provide insight into the genetic and functional co-evolution of these dually localized dehydrogenases. PMID:27881739

Revelation of Influencing Factors in Overall Codon Usage Bias of Equine Influenza Viruses

PubMed Central

Bhatia, Sandeep; Sood, Richa; Selvaraj, Pavulraj

2016-01-01

Equine influenza viruses (EIVs) of H3N8 subtype are culprits of severe acute respiratory infections in horses, and are still responsible for significant outbreaks worldwide. Adaptability of influenza viruses to a particular host is significantly influenced by their codon usage preference, due to an absolute dependence on the host cellular machinery for their replication. In the present study, we analyzed genome-wide codon usage patterns in 92 EIV strains, including both H3N8 and H7N7 subtypes by computing several codon usage indices and applying multivariate statistical methods. Relative synonymous codon usage (RSCU) analysis disclosed bias of preferred synonymous codons towards A/U-ended codons. The overall codon usage bias in EIVs was slightly lower, and mainly affected by the nucleotide compositional constraints as inferred from the RSCU and effective number of codon (ENc) analysis. Our data suggested that codon usage pattern in EIVs is governed by the interplay of mutation pressure, natural selection from its hosts and undefined factors. The H7N7 subtype was found less fit to its host (horse) in comparison to H3N8, by possessing higher codon bias, lower mutation pressure and much less adaptation to tRNA pool of equine cells. To the best of our knowledge, this is the first report describing the codon usage analysis of the complete genomes of EIVs. The outcome of our study is likely to enhance our understanding of factors involved in viral adaptation, evolution, and fitness towards their hosts. PMID:27119730

Transcript profiling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of flax (Linum usitatissimum L.) and their role in linolenic acid accumulation.

PubMed

Banik, Mitali; Duguid, Scott; Cloutier, Sylvie

2011-06-01

Three genes encoding fatty acid desaturase 3 (fad3a, fad3b, and a novel fad3c) were cloned from four flax genotypes varying in linolenic acid content. Real-time PCR was used to quantify expression levels of the three fad3 genes during seed development. High amounts of both fad3a and fad3b transcripts were observed and reached their peak levels at 20 days after anthesis, except for fad3a from SP2047 where only low level expression was observed throughout seed development. Transcript accumulation of the novel fad3c gene was at similar background levels. The fatty acid composition was analysed for all genotypes and stages of development and compared with the fad3 gene expression patterns. α-Linolenic acid gradually accumulated during seed development, while linoleic acid was transient and decreased in M5791, UGG5-5, and AC McDuff. In contrast, the linolenic acid present in the early stages of development nearly completely disappeared in SP2047, while linoleic acid steadily accumulated. fad3a of the low linolenic acid line SP2047 encoded a truncated protein caused by a premature stop codon resulting from a single point mutation, and the low level of transcript accumulation in this genotype is likely due to nonsense-mediated mRNA decay caused by the premature termination of translation as a result of this early stop codon. Although substantial amounts of transcript accumulation occurred with fad3b of SP2047 genotype, cloning of the gene revealed a mutation in the first histidine box causing an amino acid change. Heterologous expression in yeast of the SP2047 and UGG5-5 fad3b genes showed that the mutation in the histidine box in SP2047 caused the enzyme inactivity. Taken together, these results showed that fad3a and fad3b are responsible for linolenic acid accumulation in flax seeds but did not support a major role for the novel fad3c. These observations were further supported by phenotypic and genotypic assessment of a doubled haploid population. Expression patterns of fad3a and fad3b were highly correlated with linolenic acid accumulation during seed development, with the exception of fad3b in SP2047 whose lack of activity was caused by the histidine box mutation despite its transcript accumulation being similar to that of the fad3b of the other genotypes.

HSJ1-related hereditary neuropathies: novel mutations and extended clinical spectrum.

PubMed

Gess, Burkhard; Auer-Grumbach, Michaela; Schirmacher, Anja; Strom, Tim; Zitzelsberger, Manuela; Rudnik-Schöneborn, Sabine; Röhr, Dominik; Halfter, Hartmut; Young, Peter; Senderek, Jan

2014-11-04

To determine the nature and frequency of HSJ1 mutations in patients with hereditary motor and hereditary motor and sensory neuropathies. Patients were screened for mutations by genome-wide or targeted linkage and homozygosity studies, whole-exome sequencing, and Sanger sequencing. RNA and protein studies of skin fibroblasts were used for functional characterization. We describe 2 additional mutations in the HSJ1 gene in a cohort of 90 patients with autosomal recessive distal hereditary motor neuropathy (dHMN) and Charcot-Marie-Tooth disease type 2 (CMT2). One family with a dHMN phenotype showed the homozygous splice-site mutation c.229+1G>A, which leads to retention of intron 4 in the HSJ1 messenger RNA with a premature stop codon and loss of protein expression. Another family, presenting with a CMT2 phenotype, carried the homozygous missense mutation c.14A>G (p.Tyr5Cys). This mutation was classified as likely disease-related by several automatic algorithms for prediction of possible impact of an amino acid substitution on the structure and function of proteins. Both mutations cosegregated with autosomal recessive inheritance of the disease and were absent from the general population. Taken together, in our cohort of 90 probands, we confirm that HSJ1 mutations are a rare but detectable cause of autosomal recessive dHMN and CMT2. We provide clinical and functional information on an HSJ1 splice-site mutation and report the detailed phenotype of 2 patients with CMT2, broadening the phenotypic spectrum of HSJ1-related neuropathies. © 2014 American Academy of Neurology.

Teaching the fluctuation test in silico by using mutate: a program to distinguish between the adaptive and spontaneous mutation hypotheses.

PubMed

Carvajal-Rodríguez, Antonio

2012-07-01

Mutate is a program developed for teaching purposes to impart a virtual laboratory class for undergraduate students of Genetics in Biology. The program emulates the so-called fluctuation test whose aim is to distinguish between spontaneous and adaptive mutation hypotheses in bacteria. The plan is to train students in certain key multidisciplinary aspects of current genetics such as sequence databases, DNA mutations, and hypothesis testing, while introducing the fluctuation test. This seminal experiment was originally performed studying Escherichia coli resistance to the infection by bacteriophage T1. The fluctuation test initiated the modern bacterial genetics that 25 years later ushered in the era of the recombinant DNA. Nowadays we know that some deletions in fhuA, the gene responsible for E. coli membrane receptor of T1, could cause the E. coli resistance to this phage. For the sake of simplicity, we will introduce the assumption that a single mutation generates the resistance to T1. During the practical, the students use the program to download some fhuA gene sequences, manually introduce some stop codon mutations, and design a fluctuation test to obtain data for distinguishing between preadaptative (spontaneous) and induced (adaptive) mutation hypotheses. The program can be launched from a browser or, if preferred, its executable file can be downloaded from http://webs.uvigo.es/acraaj/MutateWeb/Mutate.html. It requires the Java 5.0 (or higher) Runtime Environment (freely available at http://www.java.com). Copyright © 2012 Wiley Periodicals, Inc.

A new compound heterozygous frameshift mutation in the type II 3{beta}-hydroxysteroid dehydrogenase 3{beta}-HSD gene causes salt-wasting 3{beta}-HSD deficiency congenital adrenal hyperplasia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, L.; Sakkal-Alkaddour, S.; Chang, Ying T.

1996-01-01

We report a new compound heterozygous frameshift mutation in the type II 3{Beta}-hydroxysteroid dehydrogenase (3{beta}-HSD) gene in a Pakistanian female child with the salt-wasting form of 3{Beta}-HSD deficiency congenital adrenal hyperplasia. The etiology for her congenital adrenal hyperplasia was not defined. Although the family history suggested possible 3{beta}-HSd deficiency disorder, suppressed adrenal function caused by excess glucocorticoid therapy in this child at 7 yr of age did not allow hormonal diagnosis. To confirm 3{beta}-HSD deficiency, we sequenced the type II 3{beta}-HSD gene in the patient, her family, and the parents of her deceased paternal cousins. The type II 3{beta}-HSD genemore » region of a putative promotor, exons I, II, III, and IV, and exon-intron boundaries were amplified by PCR and sequenced in all subjects. The DNA sequence of the child revealed a single nucleotide deletion at codon 318 [ACA(Thr){r_arrow}AA] in exon IV in one allele, and two nucleotide deletions at codon 273 [AAA(Lys){r_arrow}A] in exon IV in the other allele. The remaining gene sequences were normal. The codon 318 mutation was found in one allele from the father, brother, and parents of the deceased paternal cousins. The codon 273 mutation was found in one allele of the mother and a sister. These findings confirmed inherited 3{beta}-HSD deficiency in the child caused by the compound heterozygous type II 3{beta}-HSD gene mutation. Both codons at codons 279 and 367, respectively, are predicted to result in an altered and truncated type II 3{beta}-HSD protein, thereby causing salt-wasting 3{beta}-HSD deficiency in the patient. 21 refs., 2 figs., 1 tab.« less

Identification of seven haplotypes of the caprine PrP gene at codons 127, 142, 154, 211, 222 and 240 in French Alpine and Saanen breeds and their association with classical scrapie.

PubMed

Barillet, F; Mariat, D; Amigues, Y; Faugeras, R; Caillat, H; Moazami-Goudarzi, K; Rupp, R; Babilliot, J M; Lacroux, C; Lugan, S; Schelcher, F; Chartier, C; Corbière, F; Andréoletti, O; Perrin-Chauvineau, C

2009-03-01

In sheep, susceptibility to scrapie is mainly influenced by polymorphisms of the PrP gene. In goats, there are to date few data related to scrapie susceptibility association with PrP gene polymorphisms. In this study, we first investigated PrP gene polymorphisms of the French Alpine and Saanen breeds. Based on PrP gene open reading frame sequencing of artificial insemination bucks (n=404), six encoding mutations were identified at codons 127, 142, 154, 211, 222 and 240. However, only seven haplotypes could be detected: four (GIH(154)RQS, GIRQ(211)QS, GIRRK(222)S and GIRRQP(240)) derived from the wild-type allele (G(127)I(142)R(154)R(211)Q(222)S(240)) by a single-codon mutation, and two (S(127)IRRQP(240) and GM(142)RRQP(240)) by a double-codon mutation. A case-control study was then implemented in a highly affected Alpine and Saanen breed herd (90 cases/164 controls). Mutations at codon 142 (I/M), 154 (R/H), 211 (R/Q) and 222 (Q/K) were found to induce a significant degree of protection towards natural scrapie infection. Compared with the baseline homozygote wild-type genotype I(142)R(154)R(211)Q(222)/IRRQ goats, the odds of scrapie cases in IRQ(211)Q/IRRQ and IRRK(222)/IRRQ heterozygous animals were significantly lower [odds ratio (OR)=0.133, P<0.0001; and OR=0.048, P<0.0001, respectively]. The heterozygote M(142)RRQ/IRRQ genotype was only protective (OR=0.243, P=0.0186) in goats also PP(240) homozygous at codon 240. However, mutated allele frequencies in French Alpine and Saanen breeds were low (0.5-18.5 %), which prevent us from assessing the influence of all the possible genotypes in natural exposure conditions.

Examination of food chain-derived Listeria monocytogenes strains of different serotypes reveals considerable diversity in inlA genotypes, mutability, and adaptation to cold temperatures.

PubMed

Kovacevic, Jovana; Arguedas-Villa, Carolina; Wozniak, Anna; Tasara, Taurai; Allen, Kevin J

2013-03-01

Listeria monocytogenes strains belonging to serotypes 1/2a and 4b are frequently linked to listeriosis. While inlA mutations leading to premature stop codons (PMSCs) and attenuated virulence are common in 1/2a, they are rare in serotype 4b. We observed PMSCs in 35% of L. monocytogenes isolates (n = 54) recovered from the British Columbia food supply, including serotypes 1/2a (30%), 1/2c (100%), and 3a (100%), and a 3-codon deletion (amino acid positions 738 to 740) seen in 57% of 4b isolates from fish-processing facilities. Caco-2 invasion assays showed that two isolates with the deletion were significantly more invasive than EGD-SmR (P < 0.0001) and were either as (FF19-1) or more (FE13-1) invasive than a clinical control strain (08-5578) (P = 0.006). To examine whether serotype 1/2a was more likely to acquire mutations than other serotypes, strains were plated on agar with rifampin, revealing 4b isolates to be significantly more mutable than 1/2a, 1/2c, and 3a serotypes (P = 0.0002). We also examined the ability of 33 strains to adapt to cold temperature following a downshift from 37°C to 4°C. Overall, three distinct cold-adapting groups (CAG) were observed: 46% were fast (<70 h), 39% were intermediate (70 to 200 h), and 15% were slow (>200 h) adaptors. Intermediate CAG strains (70%) more frequently possessed inlA PMSCs than did fast (20%) and slow (10%) CAGs; in contrast, 87% of fast adaptors lacked inlA PMSCs. In conclusion, we report food chain-derived 1/2a and 4b serotypes with a 3-codon deletion possessing invasive behavior and the novel association of inlA genotypes encoding a full-length InlA with fast cold-adaptation phenotypes.

HRAS mutations in Costello syndrome: detection of constitutional activating mutations in codon 12 and 13 and loss of wild-type allele in malignancy.

PubMed

Estep, Anne L; Tidyman, William E; Teitell, Michael A; Cotter, Philip D; Rauen, Katherine A

2006-01-01

Costello syndrome (CS) is a complex developmental disorder involving characteristic craniofacial features, failure to thrive, developmental delay, cardiac and skeletal anomalies, and a predisposition to develop neoplasia. Based on similarities with other cancer syndromes, we previously hypothesized that CS is likely due to activation of signal transduction through the Ras/MAPK pathway [Tartaglia et al., 2003]. In this study, the HRAS coding region was sequenced for mutations in a large, well-characterized cohort of 36 CS patients. Heterogeneous missense point mutations predicting an amino acid substitution were identified in 33/36 (92%) patients. The majority (91%) had a 34G --> A transition in codon 12. Less frequent mutations included 35G --> C (codon 12) and 37G --> T (codon 13). Parental samples did not have an HRAS mutation supporting the hypothesis of de novo heterogeneous mutations. There is phenotypic variability among patients with a 34G --> A transition. The most consistent features included characteristic facies and skin, failure to thrive, developmental delay, musculoskeletal abnormalities, visual impairment, cardiac abnormalities, and generalized hyperpigmentation. The two patients with 35G --> C had cardiac arrhythmias whereas one patient with a 37G --> T transversion had an enlarged aortic root. Of the patients with a clinical diagnosis of CS, neoplasia was the most consistent phenotypic feature for predicating an HRAS mutation. To gain an understanding of the relationship between constitutional HRAS mutations and malignancy, HRAS was sequenced in an advanced biphasic rhabdomyosarcoma/fibrosarcoma from an individual with a 34G --> A mutation. Loss of the wild-type HRAS allele was observed, suggesting tumorigenesis in CS patients is accompanied by additional somatic changes affecting HRAS. Finally, due to phenotypic overlap between CS and cardio-facio-cutaneous (CFC) syndromes, the HRAS coding region was sequenced in a well-characterized CFC cohort. No mutations were found which support a distinct genetic etiology between CS and CFC syndromes. (c) 2005 Wiley-Liss, Inc.

Prevalence of c-KIT Mutations in Gonadoblastoma and Dysgerminomas of Patients with Disorders of Sex Development (DSD) and Ovarian Dysgerminomas

PubMed Central

Hersmus, Remko; Stoop, Hans; van de Geijn, Gert Jan; Eini, Ronak; Biermann, Katharina; Oosterhuis, J. Wolter; DHooge, Catharina; Schneider, Dominik T.; Meijssen, Isabelle C.; Dinjens, Winand N. M.; Dubbink, Hendrikus Jan; Drop, Stenvert L. S.; Looijenga, Leendert H. J.

2012-01-01

Activating c-KIT mutations (exons 11 and 17) are found in 10–40% of testicular seminomas, the majority being missense point mutations (codon 816). Malignant ovarian dysgerminomas represent ∼3% of all ovarian cancers in Western countries, resembling testicular seminomas, regarding chromosomal aberrations and c-KIT mutations. DSD patients with specific Y-sequences have an increased risk for Type II Germ Cell Tumor/Cancer, with gonadoblastoma as precursor progressing to dysgerminoma. Here we present analysis of c-KIT exon 8, 9, 11, 13 and 17, and PDGFRA exon 12, 14 and 18 by conventional sequencing together with mutational analysis of c-KIT codon 816 by a sensitive and specific LightCycler melting curve analysis, confirmed by sequencing. The results are combined with data on TSPY and OCT3/4 expression in a series of 16 DSD patients presenting with gonadoblastoma and dysgerminoma and 15 patients presenting pure ovarian dysgerminomas without DSD. c-KIT codon 816 mutations were detected in five out of the total of 31 cases (all found in pure ovarian dysgerminomas). A synonymous SNP (rs 5578615) was detected in two patients, one DSD patient (with bilateral disease) and one patient with dysgerminoma. Next to these, three codon N822K mutations were detected in the group of 15 pure ovarian dysgerminomas. In total activating c-KIT mutations were found in 53% of ovarian dysgerminomas without DSD. In the group of 16 DSD cases a N505I and D820E mutation was found in a single tumor of a patient with gonadoblastoma and dysgerminoma. No PDGFRA mutations were found. Positive OCT3/4 staining was present in all gonadoblastomas and dysgerminomas investigated, TSPY expression was only seen in the gonadoblastoma/dysgerminoma lesions of the 16 DSD patients. This data supports the existence of two distinct but parallel pathways in the development of dysgerminoma, in which mutational status of c-KIT might parallel the presence of TSPY. PMID:22937135

Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis.

PubMed

Cheng, Feng; Xu, Jian-Miao; Xiang, Chao; Liu, Zhi-Qiang; Zhao, Li-Qing; Zheng, Yu-Guo

2017-04-01

To develop a practically simple and robust multi-site saturation mutagenesis (MSSM) method that enables simultaneously recombination of amino acid positions for focused mutant library generation. A general restriction enzyme-free and ligase-free MSSM method (Simple-MSSM) based on prolonged overlap extension PCR (POE-PCR) and Simple Cloning techniques. As a proof of principle of Simple-MSSM, the gene of eGFP (enhanced green fluorescent protein) was used as a template gene for simultaneous mutagenesis of five codons. Forty-eight randomly selected clones were sequenced. Sequencing revealed that all the 48 clones showed at least one mutant codon (mutation efficiency = 100%), and 46 out of the 48 clones had mutations at all the five codons. The obtained diversities at these five codons are 27, 24, 26, 26 and 22, respectively, which correspond to 84, 75, 81, 81, 69% of the theoretical diversity offered by NNK-degeneration (32 codons; NNK, K = T or G). The enzyme-free Simple-MSSM method can simultaneously and efficiently saturate five codons within one day, and therefore avoid missing interactions between residues in interacting amino acid networks.

Novel mutations of the AGXT gene causing primary hyperoxaluria type 1.

PubMed

Yuen, Yuet-Ping; Lai, Chi-Kong; Tong, Gensy Mei-Wah; Wong, Ping-Nam; Wong, Francis Kim-Ming; Mak, Siu-Ka; Lo, Kin-Yee; Wong, Andrew Kui-Man; Tong, Sui-Fan; Chan, Yan-Wo; Lam, Ching-Wan

2004-01-01

Primary hyperoxaluria type 1 (PH1), an inherited cause of nephrolithiasis, is due to a functional defect of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). A definitive PH1 diagnosis can be established by analyzing AGT activity in liver tissue or mutation analysis of the AGXT gene. The molecular basis of PH1 in three Chinese patients, two with adult-onset and one with childhood-onset recurrent nephrolithiasis, was established by analyzing the entire AGXT gene. Three novel mutations (c2T>C, c817insAG and c844C>T) and two previously reported mutations (c33insC and 679-IVS6+2delAAgt) were identified. c2T>C converts the initiation codon from ATG to ACG, which predicts significant reduction, if not complete abolition, of protein translation. c817insAG leads to a frameshift and changes the amino acid sequence after codon 274. c844C>T changes glutamine at codon 282 to a termination codon, resulting in protein truncation. This is the first report describing AGXT gene mutations in Chinese patients with PH1. AGXT genotypes cannot fully explain the clinical heterogeneity of PH1, and other factors involved in disease pathogenesis remain to be identified. Our experience emphasizes the importance of excluding PH1 in patients with recurrent nephrolithiasis to avoid delay or inappropriate management.

Molecular basis and clinical presentation of classic galactosemia in a Croatian population.

PubMed

Ramadža, Danijela Petković; Sarnavka, Vladimir; Vuković, Jurica; Fumić, Ksenija; Krželj, Vjekoslav; Lozić, Bernarda; Pušeljić, Silvija; Pereira, Hana; Silva, Maria João; Tavares de Almeida, Isabel; Barić, Ivo; Rivera, Isabel

2018-01-26

Classic galactosemia is an autosomal recessive disorder of galactose metabolism caused by severely decreased activity of galactose-1-phosphate uridylyltransferase (GALT) due to pathogenic mutations in the GALT gene. To date more than 330 mutations have been described, with p.Q188R and p.K285N being the most common in Caucasian populations. Although acute manifestations can be fully avoided by a galactose-restricted diet, chronic complications, such as neurological ones, cannot be prevented in a significant number of patients despite compliance with the dietary treatment. A cohort of 16 galactosemic Croatian patients, including one pair of siblings, was studied. Molecular characterization was performed by direct sequence analysis of the GALT gene. Sixteen patients were analyzed and only four different mutations were detected. As expected, p.Q188R and p.K285N were common, accounting for 40% and 37% of unrelated alleles, respectively. The third mutation accounting for 20% of mutant alleles was p.R123X causing a premature stop codon, is thus considered to be severe, which is in accordance with the phenotype presented by the homozygous patient described here. The fourth mutation p.E271D was found in a single allele. More than half of our patients manifested some chronic neurological complications. This is the first report on mutational and phenotypic spectra of classic galactosemia in Croatia that expands the knowledge on the mutational map of the GALT gene across Europe and reveals the genetic homogeneity of the Croatian population.

A new mutation within the porphobilinogen deaminase gene leading to a truncated protein as a cause of acute intermittent porphyria in an extended Indian family.

PubMed

Flachsová, E; Verma, I C; Ulbrichová, D; Saxena, R; Zeman, J; Saudek, V; Raman, C S; Martásek, P

2007-01-01

Based on Internet search, we were contacted by a 50-year-old man suffering from severe abdominal pain. Acute hepatic porphyria was considered from positive Watson-Schwartz test. He, not being a health professional, searched for centres with ability to do molecular diagnosis and for information about therapeutic possibilities. He asked his physician for haem-arginate (Normosang, Orphan Europe, Paris) treatment, arranged sending his blood to our laboratory and mediated genetic counselling for him and his family. Molecular analyses of the PBGD gene revealed a novel mutation in exon 15, the 973insG. Subsequently, genetic analysis was performed in 18 members of the proband's extensive family. In 12 members of the family, the same mutation was found. The mutation, which consisted of one nucleotide insertion, resulted in addition of four different amino acids leading to a protein that is prematurely truncated by the stop codon. The effect of this mutation was investigated by expression of the wildtype and mutated PBGD in a prokaryotic expression system. The mutation resulted in instability of the protein and loss of enzymatic function. The increasing access to a number of disease- and symptom-oriented web pages presents a new and unusual venue for gaining knowledge and enabling self-diagnosis and self-help. It is, therefore, important that diseaseoriented Internet pages for public use should be designed with clarity and accurate current knowledge based background.

TP53 mutation and human papilloma virus status of oral squamous cell carcinomas in young adult patients.

PubMed

Braakhuis, B J M; Rietbergen, M M; Buijze, M; Snijders, P J F; Bloemena, E; Brakenhoff, R H; Leemans, C R

2014-09-01

Little is known about the molecular carcinogenesis of oral squamous cell carcinoma (OSCC) in young adult patients. The aim of this study was to investigate the detailed TP53 mutation and human papilloma virus (HPV) status of OSCC in patients, younger than 45 years. TP53 mutations were determined with direct sequencing on paraffin-embedded carcinoma tissue from 31 young patients and compared with two older age OSCC reference groups: one from the same institute (N = 87) and an independent one (N = 675). Biologically active tumour HPV was detected by p16-immunohistochemistry followed by a HPV-DNA GP5 + /6 + -PCR. HPV16 was present in one OSCC (3%). TP53 mutations were found in 14 (45%) OSCC: five were missense and nine resulted in a truncated protein. Six of these latter were insertions or deletions of one or more nucleotides leading to frameshift, one was at a splice site and two resulted in a stop codon. The percentage of truncating mutations (64% of all mutations) was higher than that observed in the institute's reference group (44%, P = 0.23) and in the independent reference group (24%, P = 0.002). This study shows that TP53 mutations are common in OSCC of young adult patients; infection with biologically active HPV is rare. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Selenocysteine incorporation: A trump card in the game of mRNA decay

PubMed Central

Shetty, Sumangala P.; Copeland, Paul R.

2015-01-01

The incorporation of the 21st amino acid, selenocysteine (Sec), occurs on mRNAs that harbor in-frame stop codons because the Sec-tRNASec recognizes a UGA codon. This sets up an intriguing interplay between translation elongation, translation termination and the complex machinery that marks mRNAs that contain premature termination codons for degradation, leading to nonsense mediated mRNA decay (NMD). In this review we discuss the intricate and complex relationship between this key quality control mechanism and the process of Sec incorporation in mammals. PMID:25622574

PubMed Central

Bello, Luca

2016-01-01

Accurate definition of genetic mutations causing Duchenne muscular dystrophy (DMD) has always been relevant in order to provide genetic counseling to patients and families, and helps to establish the prognosis in the case where the distinction between Duchenne, Becker, or intermediate muscular dystrophy is not obvious. As molecular treatments aimed at dystrophin restoration in DMD are increasingly available as commercialized drugs or within clinical trials, genetic diagnosis has become an indispensable tool in order to determine eligibility for these treatments. DMD patients in which multiplex ligation-dependent probe amplification (MLPA) or similar techniques show a deletion suitable to exon skipping of exons 44, 45, 51, or 53, may be currently treated with AONs targeting these exons, in the context of clinical trials, or, as is the case for exon 51 skipping in the United States, with the first commercialized drug (eteplirsen). Patients who test negative at MLPA, but in whom DMD gene sequencing shows a nonsense mutation, may be amenable for treatment with stop codon readthrough compounds such as ataluren. Novel molecular approaches such as CRISPR-Cas9 targeting of specific DMD mutations are still in the preclinical stages, but appear promising. In conclusion, an accurate genetic diagnosis represents the entrance into a new scenario of personalized medicine in DMD. PMID:28484312

A patient with a unique frameshift mutation in GPC3, causing Simpson-Golabi-Behmel syndrome, presenting with craniosynostosis, penoscrotal hypospadias, and a large prostatic utricle.

PubMed

Villarreal, Diana D; Villarreal, Humberto; Paez, Ana Maria; Peppas, Dennis; Lynch, Jane; Roeder, Elizabeth; Powers, George C

2013-12-01

We present a Hispanic male with the clinical and molecular diagnosis of Simpson-Golabi-Behmel syndrome (SGBS). The patient was born with multiple anomalies not entirely typical of SGBS patients, including penoscrotal hypospadias, a large prostatic utricle, and left coronal craniosynostosis. In addition, he demonstrated endocrine anomalies including a low random cortisol level suspicious for adrenal insufficiency and low testosterone level. To our knowledge, this is the first report of a prostatic utricle in SGBS and the second report of craniosynostosis. The unique disease-causing mutation likely arose de novo in the mother. It is a deletion-insertion that leads to a frameshift at the p.p. S359 [corrected] residue of GPC3 and a premature stop codon after five more amino acids. p. S359 [corrected] is the same residue that is normally cleaved by the Furin convertase, although the significance of this novel mutation with respect to the patient's multiple anomalies is unknown. We present this case as the perinatal course of a patient with unique features of SGBS and a confirmed molecular diagnosis. © 2013 Wiley Periodicals, Inc.

Inherited biallelic CSF3R mutations in severe congenital neutropenia.

PubMed

Triot, Alexa; Järvinen, Päivi M; Arostegui, Juan I; Murugan, Dhaarini; Kohistani, Naschla; Dapena Díaz, José Luis; Racek, Tomas; Puchałka, Jacek; Gertz, E Michael; Schäffer, Alejandro A; Kotlarz, Daniel; Pfeifer, Dietmar; Díaz de Heredia Rubio, Cristina; Ozdemir, Mehmet Akif; Patiroglu, Turkan; Karakukcu, Musa; Sánchez de Toledo Codina, José; Yagüe, Jordi; Touw, Ivo P; Unal, Ekrem; Klein, Christoph

2014-06-12

Severe congenital neutropenia (SCN) is characterized by low numbers of peripheral neutrophil granulocytes and a predisposition to life-threatening bacterial infections. We describe a novel genetic SCN type in 2 unrelated families associated with recessively inherited loss-of-function mutations in CSF3R, encoding the granulocyte colony-stimulating factor (G-CSF) receptor. Family A, with 3 affected children, carried a homozygous missense mutation (NM_000760.3:c.922C>T, NP_000751.1:p.Arg308Cys), which resulted in perturbed N-glycosylation and aberrant localization to the cell surface. Family B, with 1 affected infant, carried compound heterozygous deletions provoking frameshifts and premature stop codons (NM_000760.3:c.948_963del, NP_000751.1:p.Gly316fsTer322 and NM_000760.3:c.1245del, NP_000751.1:p.Gly415fsTer432). Despite peripheral SCN, all patients had morphologic evidence of full myeloid cell maturation in bone marrow. None of the patients responded to treatment with recombinant human G-CSF. Our study highlights the genetic and morphologic SCN variability and provides evidence both for functional importance and redundancy of G-CSF receptor-mediated signaling in human granulopoiesis. © 2014 by The American Society of Hematology.

Severe congenital muscular dystrophy in a Mexican family with a new nonsense mutation (R2578X) in the laminin alpha-2 gene.

PubMed

Coral-Vazquez, Ramon M; Rosas-Vargas, Haydee; Meza-Espinosa, Pedro; Mendoza, Irma; Huicochea, Juan C; Ramon, Guillermo; Salamanca, Fabio

2003-01-01

The congenital muscular dystrophies (CMDs) are a heterogeneous group of autosomal recessive disorders. Approximately one half of cases diagnosed with classic CMD show primary deficiency of the laminin alpha2 chain of merosin. Complete absence of this protein is usually associated with a severe phenotype characterized by drastic muscle weakness and characteristic changes in white matter in cerebral magnetic resonance imaging (MRI). Here we report an 8-month-old Mexican female infant, from a consanguineous family, with classical CMD. Serum creatine kinase was elevated, muscle biopsy showed dystrophic changes, and there were abnormalities in brain MRI. Immunofluorescence analysis demonstrated the complete absence of laminin alpha2. In contrast, expression of alpha-, beta-, gamma-, and delta-sarcoglycans and dystrophin, all components of the dystrophin-glycoprotein complex, appeared normal. A homozygous C long right arrow T substitution at position 7781 that generated a stop codon in the G domain of the protein was identified by mutation analysis of the laminin alpha2 gene ( LAMA2). Sequence analysis on available DNA samples of the family showed that parents and other relatives were carriers of the mutation.

Inherited biallelic CSF3R mutations in severe congenital neutropenia

PubMed Central

Triot, Alexa; Järvinen, Päivi M.; Arostegui, Juan I.; Murugan, Dhaarini; Kohistani, Naschla; Dapena Díaz, José Luis; Racek, Tomas; Puchałka, Jacek; Gertz, E. Michael; Schäffer, Alejandro A.; Kotlarz, Daniel; Pfeifer, Dietmar; Díaz de Heredia Rubio, Cristina; Ozdemir, Mehmet Akif; Patiroglu, Turkan; Karakukcu, Musa; Sánchez de Toledo Codina, José; Yagüe, Jordi; Touw, Ivo P.; Unal, Ekrem

2014-01-01

Severe congenital neutropenia (SCN) is characterized by low numbers of peripheral neutrophil granulocytes and a predisposition to life-threatening bacterial infections. We describe a novel genetic SCN type in 2 unrelated families associated with recessively inherited loss-of-function mutations in CSF3R, encoding the granulocyte colony-stimulating factor (G-CSF) receptor. Family A, with 3 affected children, carried a homozygous missense mutation (NM_000760.3:c.922C>T, NP_000751.1:p.Arg308Cys), which resulted in perturbed N-glycosylation and aberrant localization to the cell surface. Family B, with 1 affected infant, carried compound heterozygous deletions provoking frameshifts and premature stop codons (NM_000760.3:c.948_963del, NP_000751.1:p.Gly316fsTer322 and NM_000760.3:c.1245del, NP_000751.1:p.Gly415fsTer432). Despite peripheral SCN, all patients had morphologic evidence of full myeloid cell maturation in bone marrow. None of the patients responded to treatment with recombinant human G-CSF. Our study highlights the genetic and morphologic SCN variability and provides evidence both for functional importance and redundancy of G-CSF receptor-mediated signaling in human granulopoiesis. PMID:24753537

Evidence that the recA441 (tif-1) mutant of Escherichia coli K-12 contains a thermosensitive intragenic suppressor of RecA constitutive protease activity.

PubMed Central

Wang, W B; Tessman, E S

1985-01-01

The recA441 mutant of Escherichia coli, which has been thought to have thermoinducible constitutive RecA protease activity, is known to have two mutations within recA. We show here that the mutation that alters codon 38 actually confers temperature-independent constitutive protease activity; the second mutation in recA441, which is at codon 298, appears to be acting as a temperature-sensitive suppressor of the protease activity. Images PMID:3891740

Evidence that the recA441 (tif-1) mutant of Escherichia coli K-12 contains a thermosensitive intragenic suppressor of RecA constitutive protease activity.

PubMed

Wang, W B; Tessman, E S

1985-07-01

The recA441 mutant of Escherichia coli, which has been thought to have thermoinducible constitutive RecA protease activity, is known to have two mutations within recA. We show here that the mutation that alters codon 38 actually confers temperature-independent constitutive protease activity; the second mutation in recA441, which is at codon 298, appears to be acting as a temperature-sensitive suppressor of the protease activity.

Nonsense mutations in the PAX3 gene cause Waardenburg syndrome type I in two Chinese patients.

PubMed

Yang, Shu-Zhi; Cao, Ju-Yang; Zhang, Rui-Ning; Liu, Li-Xian; Liu, Xin; Zhang, Xin; Kang, Dong-Yang; Li, Mei; Han, Dong-Yi; Yuan, Hui-Jun; Yang, Wei-Yan

2007-01-05

Waardenburg syndrome type I (WS1) is an autosomal dominant disorder characterized by sensorineural hearing loss, pigmental abnormalities of the eye, hair and skin, and dystopia canthorum. The gene mainly responsible for WS1 is PAX3 which is involved in melanocytic development and survival. Mutations of PAX3 have been reported in familiar or sporadic patients with WS1 in several populations of the world except Chinese. In order to explore the genetic background of Chinese WS1 patients, a mutation screening of PAX3 gene was carried out in four WS1 pedigrees. A questionnaire survey and comprehensive clinical examination were conducted in four Chinese pedigrees of WS1. Genomic DNA from each patient and their family members was extracted and exons of PAX3 were amplified by PCR. PCR fragments were ethanol-purified and sequenced in both directions on an ABI_Prism 3100 DNA sequencer with the BigDye Terminator Cycle Sequencing Ready Reaction Kit. The sequences were obtained and aligned to the wild type sequence of PAX3 with the GeneTool program. Two nonsense PAX3 mutations have been found in the study population. One is heterozygous for a novel nonsense mutation S209X. The other is heterozygous for a previously reported mutation in European population R223X. Both mutations create stop codons leading to truncation of the PAX3 protein. This is the first demonstration of PAX3 mutations in Chinese WS1 patients and one of the few examples of an identical mutation of PAX3 occurred in different populations.

Mutational analysis of the RB1 gene and the inheritance patterns of retinoblastoma in Jordan.

PubMed

Yousef, Yacoub A; Tbakhi, Abdelghani; Al-Hussaini, Maysa; AlNawaiseh, Ibrahim; Saab, Ala; Afifi, Amal; Naji, Maysa; Mohammad, Mona; Deebajah, Rasha; Jaradat, Imad; Sultan, Iyad; Mehyar, Mustafa

2018-04-01

Retinoblastoma (RB) is a childhood cancer developing in the retina due to RB1 pathologic variant. Herein we are evaluating the oncogenic mutations in the RB1 gene and the inheritance patterns of RB in the Jordanian patients. In this prospective study, the peripheral blood of 50 retinoblastoma patients was collected, genomic DNA was extracted, mutations were identified using Quantitative multiplex PCR (QM-PCR), Allele-specific PCR, Next Generation Sequencing analysis, and Sanger sequencing. In this cohort of 50 patients, 20(40%) patients had unilateral RB and 30(60%) were males. Overall, 36(72%) patients had germline disease, 17(47%) of whom had the same RB1 pathologic variant detected in one of the parents (inherited disease). In the bilateral group, all (100%) patients had germline disease; 13(43%) of them had inherited mutation. In the unilateral group, 6(30%) had germline disease, 4(20%) of them had inherited mutation. Nonsense mutation generating a stop codon and producing a truncated non-functional protein was the most frequent detected type of mutations (n = 15/36, 42%). Only one (2%) of the patients had mosaic mutation, and of the 17 inherited cases, 16(94%) had an unaffected carrier parent. In conclusion, in addition to all bilateral RB patients in our cohort, 30% of unilateral cases showed germline mutation. Almost half (47%) of germline cases had inherited disease from affected (6%) parent or unaffected carrier (94%). Therefore molecular screening is critical for the genetic counseling regarding the risk for inherited RB in both unilateral and bilateral cases including those with no family history.

Eight previously unidentified mutations found in the OA1 ocular albinism gene

PubMed Central

Mayeur, Hélène; Roche, Olivier; Vêtu, Christelle; Jaliffa, Carolina; Marchant, Dominique; Dollfus, Hélène; Bonneau, Dominique; Munier, Francis L; Schorderet, Daniel F; Levin, Alex V; Héon, Elise; Sutherland, Joanne; Lacombe, Didier; Said, Edith; Mezer, Eedy; Kaplan, Josseline; Dufier, Jean-Louis; Marsac, Cécile; Menasche, Maurice; Abitbol, Marc

2006-01-01

Background Ocular albinism type 1 (OA1) is an X-linked ocular disorder characterized by a severe reduction in visual acuity, nystagmus, hypopigmentation of the retinal pigmented epithelium, foveal hypoplasia, macromelanosomes in pigmented skin and eye cells, and misrouting of the optical tracts. This disease is primarily caused by mutations in the OA1 gene. Methods The ophthalmologic phenotype of the patients and their family members was characterized. We screened for mutations in the OA1 gene by direct sequencing of the nine PCR-amplified exons, and for genomic deletions by PCR-amplification of large DNA fragments. Results We sequenced the nine exons of the OA1 gene in 72 individuals and found ten different mutations in seven unrelated families and three sporadic cases. The ten mutations include an amino acid substitution and a premature stop codon previously reported by our team, and eight previously unidentified mutations: three amino acid substitutions, a duplication, a deletion, an insertion and two splice-site mutations. The use of a novel Taq polymerase enabled us to amplify large genomic fragments covering the OA1 gene. and to detect very likely six distinct large deletions. Furthermore, we were able to confirm that there was no deletion in twenty one patients where no mutation had been found. Conclusion The identified mutations affect highly conserved amino acids, cause frameshifts or alternative splicing, thus affecting folding of the OA1 G protein coupled receptor, interactions of OA1 with its G protein and/or binding with its ligand. PMID:16646960

The A31P missense mutation in cardiac myosin binding protein C alters protein structure but does not cause haploinsufficiency.

PubMed

van Dijk, Sabine J; Bezold Kooiker, Kristina; Mazzalupo, Stacy; Yang, Yuanzhang; Kostyukova, Alla S; Mustacich, Debbie J; Hoye, Elaine R; Stern, Joshua A; Kittleson, Mark D; Harris, Samantha P

2016-07-01

Mutations in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C), are a major cause of hypertrophic cardiomyopathy (HCM). While most mutations encode premature stop codons, missense mutations causing single amino acid substitutions are also common. Here we investigated effects of a single proline for alanine substitution at amino acid 31 (A31P) in the C0 domain of cMyBP-C, which was identified as a natural cause of HCM in cats. Results using recombinant proteins showed that the mutation disrupted C0 structure, altered sensitivity to trypsin digestion, and reduced recognition by an antibody that preferentially recognizes N-terminal domains of cMyBP-C. Western blots detecting A31P cMyBP-C in myocardium of cats heterozygous for the mutation showed a reduced amount of A31P mutant protein relative to wild-type cMyBP-C, but the total amount of cMyBP-C was not different in myocardium from cats with or without the A31P mutation indicating altered rates of synthesis/degradation of A31P cMyBP-C. Also, the mutant A31P cMyBP-C was properly localized in cardiac sarcomeres. These results indicate that reduced protein expression (haploinsufficiency) cannot account for effects of the A31P cMyBP-C mutation and instead suggest that the A31P mutation causes HCM through a poison polypeptide mechanism that disrupts cMyBP-C or myocyte function. Copyright © 2016 Elsevier Inc. All rights reserved.

Antimalarial drug susceptibility and point mutations associated with drug resistance in 248 Plasmodium falciparum isolates imported from Comoros to Marseille, France in 2004 2006.

PubMed

Parola, Philippe; Pradines, Bruno; Simon, Fabrice; Carlotti, Marie-Paule; Minodier, Philippe; Ranjeva, Marie-Pierre; Badiaga, Sékéné; Bertaux, Lionel; Delmont, Jean; Morillon, Marc; Silai, Ramatou; Brouqui, Philippe; Parzy, Daniel

2007-09-01

A total of 248 Plasmodium falciparum isolates were sampled in travelers with malaria who came to Marseille, France from Comoros to investigate in vitro activities of antimalarial drugs and molecular markers of drug resistance. Of the 248 isolates, 126 were maintained in culture. Of these, 53% were resistant to chloroquine, and 3% had reduced susceptibility to quinine, mefloquine, and atovaquone; 1% had reduced susceptibility to halofantrine and dihydroartemisinin; 7% had reduced susceptibility to monodesethylamodiaquine; 37% had reduced susceptibility to cycloguanil; and none had reduced susceptibility to lumefantrine. Resistance-associated point mutations were screened in 207 isolates. No mutations in the cytochrome b gene were found. Of the 207 isolates, 119 (58%) had a mutation in the P. falciparum dihydrofolate reductase (Pfdhfr) gene at codon 108, 6 (5%) had mutations in both Pfdhfr codon 108 and the P. falciparum dihydropteroate synthase codon 437, and 115 (56%) had the chloroquine resistance-associated K76T mutation in the P. falciparum chloroquine resistance transporter gene. This study represents a unique opportunity to improve surveillance of P. falciparum drug resistance in Comoros with consequences for treatment and chemoprophylaxis guidelines.

Two-Exon Skipping within MLPH Is Associated with Coat Color Dilution in Rabbits

PubMed Central

Lehner, Stefanie; Gähle, Marion; Dierks, Claudia; Stelter, Ricarda; Gerber, Jonathan; Brehm, Ralph; Distl, Ottmar

2013-01-01

Coat color dilution turns black coat color to blue and red color to cream and is a characteristic in many mammalian species. Matings among Netherland Dwarf, Loh, and Lionhead Dwarf rabbits over two generations gave evidence for a monogenic autosomal recessive inheritance of coat colour dilution. Histological analyses showed non-uniformly distributed, large, agglomerating melanin granules in the hair bulbs of coat color diluted rabbits. We sequenced the cDNA of MLPH in two dilute and one black rabbit for polymorphism detection. In both color diluted rabbits, skipping of exons 3 and 4 was present resulting in altered amino acids at p.QGL[37-39]QWA and a premature stop codon at p.K40*. Sequencing of genomic DNA revealed a c.111-5C>A splice acceptor mutation within the polypyrimidine tract of intron 2 within MLPH. This mutation presumably causes skipping of exons 3 and 4. In 14/15 dilute rabbits, the c.111-5C>A mutation was homozygous and in a further dilute rabbit, heterozygous and in combination with a homozygous frame shift mutation within exon 6 (c.585delG). In conclusion, our results demonstrated a colour dilution associated MLPH splice variant causing a strongly truncated protein (p.Q37QfsX4). An involvement of further MLPH-associated mutations needs further investigations. PMID:24376820

Genetic analysis of Chinese families reveals a novel truncation allele of the retinitis pigmentosa GTPase regulator gene

PubMed Central

Hu, Fang; Zeng, Xiang-Yun; Liu, Lin-Lin; Luo, Yao-Ling; Jiang, Yi-Ping; Wang, Hui; Xie, Jing; Hu, Cheng-Quan; Gan, Lin; Huang, Liang

2014-01-01

AIM To make comprehensive molecular diagnosis for retinitis pigmentosa (RP) patients in a consanguineous Han Chinese family using next generation sequencing based Capture-NGS screen technology. METHODS A five-generation Han Chinese family diagnosed as non-syndromic X-linked recessive RP (XLRP) was recruited, including four affected males, four obligate female carriers and eleven unaffected family members. Capture-NGS was performed using a custom designed capture panel covers 163 known retinal disease genes including 47 RP genes, followed by the validation of detected mutation using Sanger sequencing in all recruited family members. RESULTS Capture-NGS in one affected 47-year-old male reveals a novel mutation, c.2417_2418insG:p.E806fs, in exon ORF15 of RP GTPase regulator (RPGR) gene results in a frameshift change that results in a premature stop codon and a truncated protein product. The mutation was further validated in three of four affected males and two of four female carriers but not in the other unaffected family members. CONCLUSION We have identified a novel mutation, c.2417_2418insG:p.E806fs, in a Han Chinese family with XLRP. Our findings expand the mutation spectrum of RPGR and the phenotypic spectrum of XLRP in Han Chinese families, and confirms Capture-NGS could be an effective and economic approach for the comprehensive molecular diagnosis of RP. PMID:25349787

Zinc finger point mutations within the WT1 gene in Wilms tumor patients.

PubMed Central

Little, M H; Prosser, J; Condie, A; Smith, P J; Van Heyningen, V; Hastie, N D

1992-01-01

A proposed Wilms tumor gene, WT1, which encodes a zinc finger protein, has previously been isolated from human chromosome 11p13. Chemical mismatch cleavage analysis was used to identify point mutations in the zinc finger region of this gene in a series of 32 Wilms tumors. Two exonic single base changes were detected. In zinc finger 3 of a bilateral Wilms tumor patient, a constitutional de novo C----T base change was found changing an arginine to a stop codon. One tumor from this patient showed allele loss leading to 11p hemizygosity of the abnormal allele. In zinc finger 2 of a sporadic Wilms tumor patient, a C----T base change resulted in an arginine to cysteine amino acid change. To our knowledge, a WT1 gene missense mutation has not been detected previously in a Wilms tumor. By comparison with a recent NMR and x-ray crystallographic analysis of an analogous zinc finger gene, early growth response gene 1 (EGR1), this amino acid change in WT1 occurs at a residue predicted to be critical for DNA binding capacity and site specificity. The detection of one nonsense point mutation and one missense WT1 gene point mutation adds to the accumulating evidence implicating this gene in a proportion of Wilms tumor patients. Images PMID:1317572

Murine knockin model for progranulin-deficient frontotemporal dementia with nonsense-mediated mRNA decay.

PubMed

Nguyen, Andrew D; Nguyen, Thi A; Zhang, Jiasheng; Devireddy, Swathi; Zhou, Ping; Karydas, Anna M; Xu, Xialian; Miller, Bruce L; Rigo, Frank; Ferguson, Shawn M; Huang, Eric J; Walther, Tobias C; Farese, Robert V

2018-03-20

Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized Grn R493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous Grn R493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in Grn R493X mice and cell lines and in fibroblasts from patients containing the GRN R493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation. Copyright © 2018 the Author(s). Published by PNAS.

EPO Receptor Gain-of-Function Causes Hereditary Polycythemia, Alters CD34+ Cell Differentiation and Increases Circulating Endothelial Precursors

PubMed Central

Perrotta, Silverio; Cucciolla, Valeria; Ferraro, Marcella; Ronzoni, Luisa; Tramontano, Annunziata; Rossi, Francesca; Scudieri, Anna Chiara; Borriello, Adriana; Roberti, Domenico; Nobili, Bruno; Cappellini, Maria Domenica; Oliva, Adriana; Amendola, Giovanni; Migliaccio, Anna Rita; Mancuso, Patrizia; Martin-Padura, Ines; Bertolini, Francesco; Yoon, Donghoon; Prchal, Josef T.; Della Ragione, Fulvio

2010-01-01

Background Gain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined. Methodology/Principal Findings We describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G→T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34+ cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway. Conclusions/Significance Our data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis. PMID:20700488

Nonsense mutations in the alcohol dehydrogenase gene of Drosophila melanogaster correlate with an abnormal 3' end processing of the corresponding pre-mRNA.

PubMed Central

Brogna, S

1999-01-01

From bacteria to mammals, mutations that generate premature termination codons have been shown to result in the reduction in the abundance of the corresponding mRNA. In mammalian cells, more often than not, the reduction happens while the RNA is still associated with the nucleus. Here, it is reported that mutations in the alcohol dehydrogenase gene (Adh) of Drosophila melanogaster that generate premature termination codons lead to reduced levels of cytoplasmic and nuclear mRNA. Unexpectedly, it has been found that the poly(A) tails of Adh mRNAs and pre-mRNAs that carry a premature termination codon are longer than in the wild-type transcript. The more 5' terminal the mutation is, the longer is the poly(A) tail of the transcript. These findings suggest that the integrity of the coding region may be required for accurate mRNA 3' end processing. PMID:10199572

Is high pressure liquid chromatography an effective screening tool for characterization of molecular defects in hemoglobinopathies?

PubMed

Moorchung, Nikhil; Phillip, Joseph; Sarkar, Ravi Shankar; Prasad, Rupesh; Dutta, Vibha

2013-01-01

Hemoglobinopathies constitute entities that are generated by either abnormal hemoglobin or thalassemias. high pressure liquid chromatography (HPLC) is one of the best methods for screening and detection of various hemoglobinopathies but it has intrinsic interpretive problems. The study was designed to evaluate the different mutations seen in cases of hemoglobinopathies and compare the same with screening tests. 68 patients of hemoglobinopathies were screened by HPLC. Mutation studies in the beta globin gene was performed using the polymerase chain reaction (PCR)-based allele-specific Amplification Refractory Mutation System (ARMS). Molecular analysis for the sickle cell mutation was done by standard methods. The IVS 1/5 mutation was the commonest mutation seen and it was seen in 26 (38.23%) of the cases. This was followed by the IVS 1/1, codon 41/42, codon 8/9, del 22 mutation, codon 15 mutation and the -619 bp deletion. No mutation was seen in eight cases. There was a 100% concordance between the sickle cell trait as diagnosed by HPLC and genetic testing. Our study underlies the importance of molecular testing in all cases of hemoglobinopathies. Although HPLC is a useful screening tool, molecular testing is very useful in accurately diagnosing the mutations. Molecular testing is especially applicable in cases with an abnormal hemoglobin (HbD, HbE and HbS) because there may be a concomitant inheritance of a beta thalassemia mutation. Molecular testing is the gold standard when it comes to the diagnosis of hemoglobinopathies.

PTT analysis of polyps from FAP patients reveals a great majority of APC truncating mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luijt, R.B. van der; Khan, P.M.; Tops, C.M.J.

The adenomatous polyposis coli (APC) gene plays an important role in colorectal carcinogenesis. Germline APC mutations are associated with familial adenomatous polyposis (FAP), an autosomal dominantly inherited predisposition to colorectal cancer, characterized by the development of numerous adenomatous polyps in the large intestine. In order to investigate whether somatic inactivation of the remaining APC allele is necessary for adenoma formation, we collected multiple adenomatous polyps from individual FAP patients and investigated the presence of somatic mutations in the APC gene. The analysis of somatic APC mutations in these tumor samples was performed using a rapid and sensitive assay, called themore » protein truncation test (PTT). Chain-terminating somatic APC mutations were detected in the great majority of the tumor samples investigated. As expected, these mutations were mainly located in the mutation cluster region (MCR) in exon 15. Our results confirm that somatic mutation of the second APC allele is required for adenoma formation in FAP. Interestingly, in the polyps investigated in our study, the second APC allele is somatically inactivated through point mutation leading to a stop codon rather than by loss of heterozygosity. The observation that somatic second hits in APC are required for tumor development in FAP is in apparent accordance with the Knudson hypothesis for classical tumor suppressor genes. However, it is yet unknown whether chain-terminating APC mutations lead to a truncated protein exerting a dominant-negative effect or whether these mutations result in a null allele. Further investigation of this important issue will hopefully provide a better understanding of the mechanism of action of the mutated APC alleles in colorectal carcinogenesis.« less

Molecular investigations of β-thalassemic children in Erbil governorate

NASA Astrophysics Data System (ADS)

Hasan, Ahmad N.; Al-Attar, Mustafa S.

2017-09-01

The present work studies the molecular investigation of 40 thalassemic carriers using polymerase chain reaction. Forty thalassemic carriers who were registered and treated at Erbil thalassemic center and twenty apparently healthy children have been included in the present study. Ages of both groups ranged between 1-18 years. Four primers used to detect four different beta thalassemia mutations they were codon 8/9, codon 8, codon 41/42 and IVS-1-5. The two most common mutations detected among thalassemia group were Cd8/9 with 8 cases (20%) and Cd-8 with 6 cases (15%) followed by codon 41/42 with 4 cases (10%) which investigated and detected for the first time in Erbil governorate through the present study and finally IVS-1-5 with 3 cases (7.5%), while no any cases detected among control group.

Ribosomes slide on lysine-encoding homopolymeric A stretches

PubMed Central

Koutmou, Kristin S; Schuller, Anthony P; Brunelle, Julie L; Radhakrishnan, Aditya; Djuranovic, Sergej; Green, Rachel

2015-01-01

Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome ‘sliding’ represents an unexpected type of ribosome movement possible during translation. DOI: http://dx.doi.org/10.7554/eLife.05534.001 PMID:25695637

Characterization of the complete mitochondrial genome of the Grey-backed Shrike, Lanius tephronotus (Aves: Passeriformes): the first representative of the family Laniidae with a novel CAA stop codon at the end of cox2 gene.

PubMed

Qian, Chaoju; Yan, Xia; Guo, Zhichun; Wang, Yuanxiu; Li, Xixi; Yang, Jianke; Kan, Xianzhao

2013-08-01

The complete Grey-backed Shrike mitochondrial genome has been sequenced to be 16,820 bp in length, consisting of 37 encode genes: 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. In addition, a single control region was also observed. Compared with other reported Passeriformes mtgenome sequences, three bases CAA were detected at the end of Lanius tephronotus cox2 gene with the downstream adjacent base T. The first base of CAA probably occurred C to U transcript editing event resulting in a normal stop codon UAA.

Prion protein gene analysis in three kindreds with fatal familial insomnia (FFI): Codon 178 mutation and codon 129 polymorphism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medori, R.; Tritschler, H.J.

1993-10-01

Fatal familial insomnia (FFI) is a disease linked to a GAC(Asp) [yields] AAC(Asn) mutation in codon 178 of the prion protein (PrP) gene. FFI is characterized clinically by untreatable progressive insomnia, dysautonomia, and motor dysfunctions and is characterized pathologically by selective thalamic atrophy. The authors confirmed the 178[sup Asn] mutation in the PrP gene of a third FFI family of French ancestry. Three family members who are under 40 years of age and who inherited the mutation showed only reduced perfusion in the basal ganglia on single photon emission computerized tomography. Some FFI features differ from the clinical and neuropathologicmore » findings associated with 178[sup Asn] reported elsewhere. However, additional intragenic mutations accounting for the phenotypic differences were not observed in two affected individuals. In other sporadic and familial forms of Creutzfeldt-Jakob disease and Gerstmann-Straeussler syndrome, Met or Val homozygosity at polymorphic codon 129 is associated with a more severe phenotype, younger age at onset, and faster progression. In FFI, young and old individuals at disease onset had 129[sup Met/Val]. Moreover, of five 178[sup Asn] individuals who are above age-at-onset range and who are well, two have 129[sup Met] and three have 129[sup Met/Val], suggesting that polymorphic site 129 does not modulate FFI phenotypic expression. Genetic heterogeneity and environment may play an important role in inter- and intrafamilial variability of the 178[sup Asn] mutation. 32 refs., 5 figs., 1 tab.« less

Mucopolysaccharidosis type I: Identification and characterization of mutations affecting alpha-L-iduronidase activity.

PubMed

Lee-Chen, Guey-Jen; Lin, Shuan-Pei; Chen, I-Shen; Chang, Jui-Hung; Yang, Chyau-Wen; Chin, Yi-Wen

2002-06-01

Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA). MPS I covers a broad spectrum of clinical severity ranging from severe Hurler syndrome through intermediate Hurler/Scheie syndrome to mild Scheie syndrome. Mutation screening was performed in two unrelated Taiwanese MPS I patients. A Hurler/Scheie patient had A79V (C to T transition in codon 79) in exon 2 and R619G (C to G transversion in codon 619) in exon 14. R619G has been shown to cause disease. Expression of A79V in COS-7 cells showed trace amounts of IDUA activity, demonstrating the deleterious nature of the mutation. A79V mutation did not cause a reduction in IDUA mRNA levels. The reduced level of IDUA protein suggests increased degradation of the mutant enzyme. A Hurler patient had 134del12 (in-frame deletion of codons 16-19 in signal peptide) in exon 1 and Q584X (C to T transition in codon 584) in exon 13. Transfection of COS-7 cells with Q584X did not yield active enzyme. Q584X mutation caused an apparent reduction in the IDUA mRNA level and no IDUA protein was detected. Conversely, 134del12 showed 124.6% of normal activity in transfected cells and a 77-kDa precursor protein was observed on Western blot, suggesting biologic activity of precursor IDUA without posttranslational cleavage. These findings provide further evidence of the molecular heterogeneity in mutations in MPS I.

Sequence Analysis of Mitochondrial Genome of Toxascaris leonina from a South China Tiger.

PubMed

Li, Kangxin; Yang, Fang; Abdullahi, A Y; Song, Meiran; Shi, Xianli; Wang, Minwei; Fu, Yeqi; Pan, Weida; Shan, Fang; Chen, Wu; Li, Guoqing

2016-12-01

Toxascaris leonina is a common parasitic nematode of wild mammals and has significant impacts on the protection of rare wild animals. To analyze population genetic characteristics of T. leonina from South China tiger, its mitochondrial (mt) genome was sequenced. Its complete circular mt genome was 14,277 bp in length, including 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions. The nucleotide composition was biased toward A and T. The most common start codon and stop codon were TTG and TAG, and 4 genes ended with an incomplete stop codon. There were 13 intergenic regions ranging 1 to 10 bp in size. Phylogenetically, T. leonina from a South China tiger was close to canine T. leonina . This study reports for the first time a complete mt genome sequence of T. leonina from the South China tiger, and provides a scientific basis for studying the genetic diversity of nematodes between different hosts.

Definition of the mutation responsible for maple syrup urine disease in Poll Shorthorns and genotyping Poll Shorthorns and Poll Herefords for maple syrup urine disease alleles.

PubMed

Dennis, J A; Healy, P J

1999-08-01

The organisation of the E1alpha subunit of bovine branched-chain alpha-keto acid dehydrogenase gene was established. c DNA was cloned from Poll Shorthorn x Poll Hereford calves affected with Maple Syrup Urine Disease to identify the mutation responsible for the disease in Poll Shorthorns. Clones containing the c DNA sequences inherited from the Poll Shorthorn sire of the affected calves were identified. Paternal clones were sequenced and a cytidine to thymidine transition was found at nucleotide 1380. The mutation is predicted to substitute leucine in place of a highly conserved proline at codon 372. A polymerase chain reaction procedure was developed for detection of the 1380C-->T mutation in genomic DNA. Three Poll Shorthorn parents of affected calves and three affected Poll Shorthorn x Poll Hereford calves were heterozygous and an affected Poll Shorthorn calf was homozygous for this mutation. An improved polymerase chain reaction procedure was also devised to genotype Poll Herefords for the 248C-->T mutation. The procedures will facilitate disease prevention programs and assist in differential diagnosis of conditions in new-born calves that present with a rapid onset of progressive neurological disease and are characterised histologically by 'status spongiosus'. Maple Syrup Urine Disease (MSUD) is an autosomal recessive defect reported in humans (Danner and Elsas 1989), and in Poll Hereford (PH) and Poll Shorthorn (PS) calves (Harper et al 1986, Healy et al 1992). The clinical, biochemical and pathological manifestations of the disease are identical in the two breeds of cattle, and are characterised by the rapid onset of progressive neurological disease, leading to death within a few days of birth. The disease is caused by a deficiency of activity of the mitochondrial enzyme branched-chain alpha-keto acid dehydrogenase (BCKADH). This deficiency leads to elevated concentrations, in blood and tissues, of branched chain alpha-keto acids and their precursors, the branched chain amino acids, valine, leucine and isoleucine. BCKADH consists of four subunits E1alpha, E1beta, E2 and E3 that are encoded by separate genes, and MSUD may result from deficiency of any of the subunits. In PH s, the disease in caused by premature termination of translation, of the E1alpha subunit, that is induced by a cytidine to thymidine transition exon 2 (248C-->T), that converts the glutamine codon -6 to a stop codon (Q-6ST; Zhang et al 1990). We have shown that MSUD -affected PSxPH calves are heterozygous at the PH locus, illustrating molecular heterogeneity exists for bovine MSUD (Healy and Dennis 1994a). The fact that these crossbred calves are affected, indicates the PS, like the PH mutation, resides in the E1alpha subunit. Copyright 1999 Harcourt Publishers Ltd.

Pandemic influenza A virus codon usage revisited: biases, adaptation and implications for vaccine strain development

PubMed Central

2012-01-01

Background Influenza A virus (IAV) is a member of the family Orthomyxoviridae and contains eight segments of a single-stranded RNA genome with negative polarity. The first influenza pandemic of this century was declared in April of 2009, with the emergence of a novel H1N1 IAV strain (H1N1pdm) in Mexico and USA. Understanding the extent and causes of biases in codon usage is essential to the understanding of viral evolution. A comprehensive study to investigate the effect of selection pressure imposed by the human host on the codon usage of an emerging, pandemic IAV strain and the trends in viral codon usage involved over the pandemic time period is much needed. Results We performed a comprehensive codon usage analysis of 310 IAV strains from the pandemic of 2009. Highly biased codon usage for Ala, Arg, Pro, Thr and Ser were found. Codon usage is strongly influenced by underlying biases in base composition. When correspondence analysis (COA) on relative synonymous codon usage (RSCU) is applied, the distribution of IAV ORFs in the plane defined by the first two major dimensional factors showed that different strains are located at different places, suggesting that IAV codon usage also reflects an evolutionary process. Conclusions A general association between codon usage bias, base composition and poor adaptation of the virus to the respective host tRNA pool, suggests that mutational pressure is the main force shaping H1N1 pdm IAV codon usage. A dynamic process is observed in the variation of codon usage of the strains enrolled in these studies. These results suggest a balance of mutational bias and natural selection, which allow the virus to explore and re-adapt its codon usage to different environments. Recoding of IAV taking into account codon bias, base composition and adaptation to host tRNA may provide important clues to develop new and appropriate vaccines. PMID:23134595

Functional and splicing defect analysis of 23 ACVRL1 mutations in a cohort of patients affected by Hereditary Hemorrhagic Telangiectasia

PubMed Central

Alaa el Din, Ferdos; Patri, Sylvie; Thoreau, Vincent; Rodriguez-Ballesteros, Montserrat; Hamade, Eva; Bailly, Sabine; Gilbert-Dussardier, Brigitte; Abou Merhi, Raghida; Kitzis, Alain

2015-01-01

Hereditary Hemorrhagic Telangiectasia syndrome (HHT) or Rendu-Osler-Weber (ROW) syndrome is an autosomal dominant vascular disorder. Two most common forms of HHT, HHT1 and HHT2, have been linked to mutations in the endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1or ALK1) genes respectively. This work was designed to examine the pathogenicity of 23 nucleotide variations in ACVRL1 gene detected in more than 400 patients. Among them, 14 missense mutations and one intronic variant were novels, and 8 missense mutations were previously identified with questionable implication in HHT2. The functionality of missense mutations was analyzed in response to BMP9 (specific ligand of ALK1), the maturation of the protein products and their localization were analyzed by western blot and fluorescence microscopy. The splicing impairment of the intronic and of two missense mutations was examined by minigene assay. Functional analysis showed that 18 out of 22 missense mutations were defective. Splicing analysis revealed that one missense mutation (c.733A>G, p.Ile245Val) affects the splicing of the harboring exon 6. Similarly, the intronic mutation outside the consensus splicing sites (c.1048+5G>A in intron 7) was seen pathogenic by splicing study. Both mutations induce a frame shift creating a premature stop codon likely resulting in mRNA degradation by NMD surveillance mechanism. Our results confirm the haploinsufficiency model proposed for HHT2. The affected allele of ACVRL1 induces mRNA degradation or the synthesis of a protein lacking the receptor activity. Furthermore, our data demonstrate that functional and splicing analyses together, represent two robust diagnostic tools to be used by geneticists confronted with novel or conflicted ACVRL1 mutations. PMID:26176610

Stickler syndrome caused by COL2A1 mutations: genotype–phenotype correlation in a series of 100 patients

PubMed Central

Hoornaert, Kristien P; Vereecke, Inge; Dewinter, Chantal; Rosenberg, Thomas; Beemer, Frits A; Leroy, Jules G; Bendix, Laila; Björck, Erik; Bonduelle, Maryse; Boute, Odile; Cormier-Daire, Valerie; De Die-Smulders, Christine; Dieux-Coeslier, Anne; Dollfus, Hélène; Elting, Mariet; Green, Andrew; Guerci, Veronica I; Hennekam, Raoul C M; Hilhorts-Hofstee, Yvonne; Holder, Muriel; Hoyng, Carel; Jones, Kristi J; Josifova, Dragana; Kaitila, Ilkka; Kjaergaard, Suzanne; Kroes, Yolande H; Lagerstedt, Kristina; Lees, Melissa; LeMerrer, Martine; Magnani, Cinzia; Marcelis, Carlo; Martorell, Loreto; Mathieu, Michèle; McEntagart, Meriel; Mendicino, Angela; Morton, Jenny; Orazio, Gabrielli; Paquis, Véronique; Reish, Orit; Simola, Kalle O J; Smithson, Sarah F; Temple, Karen I; Van Aken, Elisabeth; Van Bever, Yolande; van den Ende, Jenneke; Van Hagen, Johanna M; Zelante, Leopoldo; Zordania, Riina; De Paepe, Anne; Leroy, Bart P; De Buyzere, Marc; Coucke, Paul J; Mortier, Geert R

2010-01-01

Stickler syndrome is an autosomal dominant connective tissue disorder caused by mutations in different collagen genes. The aim of our study was to define more precisely the phenotype and genotype of Stickler syndrome type 1 by investigating a large series of patients with a heterozygous mutation in COL2A1. In 188 probands with the clinical diagnosis of Stickler syndrome, the COL2A1 gene was analyzed by either a mutation scanning technique or bidirectional fluorescent DNA sequencing. The effect of splice site alterations was investigated by analyzing mRNA. Multiplex ligation-dependent amplification analysis was used for the detection of intragenic deletions. We identified 77 different COL2A1 mutations in 100 affected individuals. Analysis of the splice site mutations showed unusual RNA isoforms, most of which contained a premature stop codon. Vitreous anomalies and retinal detachments were found more frequently in patients with a COL2A1 mutation compared with the mutation-negative group (P<0.01). Overall, 20 of 23 sporadic patients with a COL2A1 mutation had either a cleft palate or retinal detachment with vitreous anomalies. The presence of vitreous anomalies, retinal tears or detachments, cleft palate and a positive family history were shown to be good indicators for a COL2A1 defect. In conclusion, we confirm that Stickler syndrome type 1 is predominantly caused by loss-of-function mutations in the COL2A1 gene as >90% of the mutations were predicted to result in nonsense-mediated decay. On the basis of binary regression analysis, we developed a scoring system that may be useful when evaluating patients with Stickler syndrome. PMID:20179744

Codon Usage Bias and Determining Forces in Taenia solium Genome.

PubMed

Yang, Xing; Ma, Xusheng; Luo, Xuenong; Ling, Houjun; Zhang, Xichen; Cai, Xuepeng

2015-12-01

The tapeworm Taenia solium is an important human zoonotic parasite that causes great economic loss and also endangers public health. At present, an effective vaccine that will prevent infection and chemotherapy without any side effect remains to be developed. In this study, codon usage patterns in the T. solium genome were examined through 8,484 protein-coding genes. Neutrality analysis showed that T. solium had a narrow GC distribution, and a significant correlation was observed between GC12 and GC3. Examination of an NC (ENC vs GC3s)-plot showed a few genes on or close to the expected curve, but the majority of points with low-ENC (the effective number of codons) values were detected below the expected curve, suggesting that mutational bias plays a major role in shaping codon usage. The Parity Rule 2 plot (PR2) analysis showed that GC and AT were not used proportionally. We also identified 26 optimal codons in the T. solium genome, all of which ended with either a G or C residue. These optimal codons in the T. solium genome are likely consistent with tRNAs that are highly expressed in the cell, suggesting that mutational and translational selection forces are probably driving factors of codon usage bias in the T. solium genome.

K-ras mutations and HLA-DR expression in large bowel adenomas.

PubMed Central

Norheim Andersen, S.; Breivik, J.; Løvig, T.; Meling, G. I.; Gaudernack, G.; Clausen, O. P.; Schjölberg, A.; Fausa, O.; Langmark, F.; Lund, E.; Rognum, T. O.

1996-01-01

A total of 72 sporadic colorectal adenomas in 56 patients were studied for the presence of point mutations in codons 12 and 13 of the K-ras gene and for HLA-DR antigen expression related to clinicopathological variables. Forty K-ras mutations in 39 adenomas were found (54%): 31 (77%) in codon 12 and nine (23%) in codon 13. There was a strong relationship between the incidence of K-ras mutations and adenoma type, degree of dysplasia and sex. The highest frequency of K-ras mutations was seen in large adenomas of the villous type with high-grade dysplasia. Fourteen out of 15 adenomas obtained from 14 women above 65 years of age carried mutations. HLA-DR positivity was found in 38% of the adenomas, large tumours and those with high-grade dysplasia having the strongest staining. Coexpression of K-ras mutations and HLA-DR was found significantly more frequently in large and highly dysplastic adenomas, although two-way analysis of variance showing size and grade of dysplasia to be the most important variable. None of the adenomas with low-grade dysplasia showed both K-ras mutation and HLA-DR positivity (P = 0.004). K-ras mutation is recognised as an early event in colorectal carcinogenesis. The mutation might give rise to peptides that may be presented on the tumour cell surface by class II molecules, and thereby induce immune responses against neoplastic cells. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8679466

Common founder mutation in the LDL receptor gene causing familial hypercholesterolaemia in the Icelandic population.

PubMed

Gudnason, V; Sigurdsson, G; Nissen, H; Humphries, S E

1997-01-01

Haplotype analysis in 18 apparently unrelated families with familial hypercholesterolaemia (FH) in Iceland has identified at least five different chromosomes cosegregating with hypercholesterolaemia. The most common haplotype was identified in 11 of the 18 families, indicating a responsible for FH in the Icelandic population. By using single-strand conformation polymorphism (SSCP) and direct sequencing of amplified DNA, we identified a novel mutation (a T to a C) in the second nucleotide in the 5' part of intron 4 in the LDL receptor gene. This mutation was present in approximately 60% of the FH families (10/18), supporting the prediction of a common founder. These families could be traced to a common ancestor in half of the cases by going back no further than the eighteenth century. The mutation was predicted to affect correct splicing of exon 4, and analysis at the cellular level demonstrated an abnormal mRNA containing intron 4 sequence in lymphoblastoid cells from a patient carrying this mutation. Translation of the mRNA would lead to a premature stop codon and a truncated nonfunctional protein of 285 amino acids. The novel sequence change created a new restriction site for the restriction endonuclease NlaIII, and using this assay, 29 unrelated individuals with possible FH attending a lipid clinic for treatment were examined for this mutation. Two individuals in this group of patients were found to be carriers of this mutation, supporting the suggestion of a founder mutation. Using this assay for the detection of FH in the Icelandic population should identify > 60% of these individuals.

A novel Dutch mutation in UNC13D reveals an essential role of the C2B domain in munc13-4 function.

PubMed

Elstak, Edo D; te Loo, Maroeska; Tesselaar, Kiki; van Kerkhof, Peter; Loeffen, Jan; Grivas, Dimitris; Hennekam, Eric; Boelens, Jaap Jan; Hoogerbrugge, Peter M; van der Sluijs, Peter; van Gijn, Marielle E; van de Corput, Lisette

2012-04-01

UNC13D, encoding the protein munc13-4, is essential in intracellular trafficking and exocytosis of lytic granules. Mutations in this gene are associated with familial hemophagocytic lymphohistiocytosis type 3 (FHL3), a genetically heterogeneous, rare autosomal recessive immune disorder. How mutations affect function of munc13-4 is poorly understood. Since 2006 we genetically identified seven FHL patients with mutations in UNC13D. Here, we report for the first time a c.2695C>T (p.Arg899X) mutation in exon 28 of UNC13D in three young unrelated Dutch patients. The mutation causes a premature stop codon and encodes munc13-4(1-899), which lacks the C-terminal C2 domain. Genealogical research and haplotyping of the patient families demonstrated that a single ancestral founder introduced the mutation in the Netherlands. We then characterized the mutant protein phenotypically in cell biological and immunological assays. Munc13-4(1-899) was correctly targeted to CD63-positive secretory lysosomes, although its stability was reduced and dynamic turnover on the granule membrane became uncoupled from receptor signaling. In accord, and in contrast to wild-type munc13-4, ectopically expressed mutant failed to rescue degranulation in cells with silenced endogenous munc13-4. The functional and clinical data showed that this novel Dutch founder mutation leads to severe early onset of FHL3 due to misfolding and degradation of munc13-4(1-899). Copyright © 2011 Wiley Periodicals, Inc.

A Homozygous Mutation in a Novel Zinc-Finger Protein, ERIS, Is Responsible for Wolfram Syndrome 2

PubMed Central

Amr, Sami ; Heisey, Cindy ; Zhang, Min ; Xia, Xia-Juan ; Shows, Kathryn H. ; Ajlouni, Kamel ; Pandya, Arti ; Satin, Leslie S. ; El-Shanti, Hatem ; Shiang, Rita 

2007-01-01

A single missense mutation was identified in a novel, highly conserved zinc-finger gene, ZCD2, in three consanguineous families of Jordanian descent with Wolfram syndrome (WFS). It had been shown that these families did not have mutations in the WFS1 gene (WFS1) but were mapped to the WFS2 locus at 4q22-25. A G→C transversion at nucleotide 109 predicts an amino acid change from glutamic acid to glutamine (E37Q). Although the amino acid is conserved and the mutation is nonsynonymous, the pathogenesis for the disorder is because the mutation also causes aberrant splicing. The mutation was found to disrupt messenger RNA splicing by eliminating exon 2, and it results in the introduction of a premature stop codon. Mutations in WFS1 have also been found to cause low-frequency nonsyndromic hearing loss, progressive hearing loss, and isolated optic atrophy associated with hearing loss. Screening of 377 probands with hearing loss did not identify mutations in the WFS2 gene. The WFS1-encoded protein, Wolframin, is known to localize to the endoplasmic reticulum and plays a role in calcium homeostasis. The ZCD2-encoded protein, ERIS (endoplasmic reticulum intermembrane small protein), is also shown to localize to the endoplasmic reticulum but does not interact directly with Wolframin. Lymphoblastoid cells from affected individuals show a significantly greater rise in intracellular calcium when stimulated with thapsigargin, compared with controls, although no difference was observed in resting concentrations of intracellular calcium. PMID:17846994

Mutation profile of all 49 exons of the human myosin VIIA gene, and haplotype analysis, in Usher 1B families from diverse origins.

PubMed

Adato, A; Weil, D; Kalinski, H; Pel-Or, Y; Ayadi, H; Petit, C; Korostishevsky, M; Bonne-Tamir, B

1997-10-01

Usher syndrome types I (USH1A-USH1E) are a group of autosomal recessive diseases characterized by profound congenital hearing loss, vestibular areflexia, and progressive visual loss due to retinitis pigmentosa. The human myosin VIIA gene, located on 11q14, has been shown to be responsible for Usher syndrome type 1B (USH1B). Haplotypes were constructed in 28 USH1 families by use of the following polymorphic markers spanning the USH1B locus: D11S787, D11S527, D11S1789, D11S906, D11S4186, and OMP. Affected individuals and members of their families from 12 different ethnic origins were screened for the presence of mutations in all 49 exons of the myosin VIIA gene. In 15 families myosin VIIA mutations were detected, verifying their classification as USH1B. All these mutations are novel, including three missense mutations, one premature stop codon, two splicing mutations, one frameshift, and one deletion of >2 kb comprising exons 47 and 48, a part of exon 49, and the introns between them. Three mutations were shared by more than one family, consistent with haplotype similarities. Altogether, 16 USH1B haplotypes were observed in the 15 families; most haplotypes were population specific. Several exonic and intronic polymorphisms were also detected. None of the 20 known USH1B mutations reported so far in other world populations were identified in our families.

Carcinogen-induced mutations in the mouse c-Ha-ras gene provide evidence of multiple pathways for tumor progression.

PubMed Central

Brown, K; Buchmann, A; Balmain, A

1990-01-01

A number of mouse skin tumors initiated by the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), 3-methylcholanthrene (MCA), and 7,12-dimethylbenz[a]anthracene (DMBA) have been shown to contain activated Ha-ras genes. In each case, the point mutations responsible for activation have been characterized. Results presented demonstrate the carcinogen-specific nature of these ras mutations. For each initiating agent, a distinct spectrum of mutations is observed. Most importantly, the distribution of ras gene mutations is found to differ between benign papillomas and carcinomas, suggesting that molecular events occurring at the time of initiation influence the probability with which papillomas progress to malignancy. This study provides molecular evidence in support of the existence of subsets of papillomas with differing progression frequencies. Thus, the alkylating agents MNNG and MNU induced exclusively G ---- A transitions at codon 12, with this mutation being found predominantly in papillomas. MCA initiation produced both codon 13 G ---- T and codon 61 A ---- T transversions in papillomas; only the G ---- T mutation, however, was found in carcinomas. These findings provide strong evidence that the mutational activation of Ha-ras occurs as a result of the initiation process and that the nature of the initiating event can affect the probability of progression to malignancy. Images PMID:2105486

Mutations in STRA6 Cause a Broad Spectrum of Malformations Including Anophthalmia, Congenital Heart Defects, Diaphragmatic Hernia, Alveolar Capillary Dysplasia, Lung Hypoplasia, and Mental Retardation

PubMed Central

Pasutto, Francesca; Sticht, Heinrich; Hammersen, Gerhard; Gillessen-Kaesbach, Gabriele; FitzPatrick, David R.; Nürnberg, Gudrun; Brasch, Frank; Schirmer-Zimmermann, Heidemarie; Tolmie, John L.; Chitayat, David; Houge, Gunnar; Fernández-Martínez, Lorena; Keating, Sarah; Mortier, Geert; Hennekam, Raoul C. M.; von der Wense, Axel; Slavotinek, Anne; Meinecke, Peter; Bitoun, Pierre; Becker, Christian; Nürnberg, Peter; Reis, André; Rauch, Anita

2007-01-01

We observed two unrelated consanguineous families with malformation syndromes sharing anophthalmia and distinct eyebrows as common signs, but differing for alveolar capillary dysplasia or complex congenital heart defect in one and diaphragmatic hernia in the other family. Homozygosity mapping revealed linkage to a common locus on chromosome 15, and pathogenic homozygous mutations were identified in STRA6, a member of a large group of “stimulated by retinoic acid” genes encoding novel transmembrane proteins, transcription factors, and secreted signaling molecules or proteins of largely unknown function. Subsequently, homozygous STRA6 mutations were also demonstrated in 3 of 13 patients chosen on the basis of significant phenotypic overlap to the original cases. While a homozygous deletion generating a premature stop codon (p.G50AfsX22) led to absence of the immunoreactive protein in patient’s fibroblast culture, structural analysis of three missense mutations (P90L, P293L, and T321P) suggested significant effects on the geometry of the loops connecting the transmembrane helices of STRA6. Two further variations in the C-terminus (T644M and R655C) alter specific functional sites, an SH2-binding motif and a phosphorylation site, respectively. STRA6 mutations thus define a pleiotropic malformation syndrome representing the first human phenotype associated with mutations in a gene from the “STRA” group. PMID:17273977

X-linked primary immunodeficiency associated with hemizygous mutations in the moesin (MSN) gene.

PubMed

Lagresle-Peyrou, Chantal; Luce, Sonia; Ouchani, Farid; Soheili, Tayebeh Shabi; Sadek, Hanem; Chouteau, Myriam; Durand, Amandine; Pic, Isabelle; Majewski, Jacek; Brouzes, Chantal; Lambert, Nathalie; Bohineust, Armelle; Verhoeyen, Els; Cosset, François-Loïc; Magerus-Chatinet, Aude; Rieux-Laucat, Frédéric; Gandemer, Virginie; Monnier, Delphine; Heijmans, Catherine; van Gijn, Marielle; Dalm, Virgil A; Mahlaoui, Nizar; Stephan, Jean-Louis; Picard, Capucine; Durandy, Anne; Kracker, Sven; Hivroz, Claire; Jabado, Nada; de Saint Basile, Geneviève; Fischer, Alain; Cavazzana, Marina; André-Schmutz, Isabelle

2016-12-01

We investigated 7 male patients (from 5 different families) presenting with profound lymphopenia, hypogammaglobulinemia, fluctuating monocytopenia and neutropenia, a poor immune response to vaccine antigens, and increased susceptibility to bacterial and varicella zoster virus infections. We sought to characterize the genetic defect involved in a new form of X-linked immunodeficiency. We performed genetic analyses and an exhaustive phenotypic and functional characterization of the lymphocyte compartment. We observed hemizygous mutations in the moesin (MSN) gene (located on the X chromosome and coding for MSN) in all 7 patients. Six of the latter had the same missense mutation, which led to an amino acid substitution (R171W) in the MSN four-point-one, ezrin, radixin, moesin domain. The seventh patient had a nonsense mutation leading to a premature stop codon mutation (R533X). The naive T-cell counts were particularly low for age, and most CD8 + T cells expressed the senescence marker CD57. This phenotype was associated with impaired T-cell proliferation, which was rescued by expression of wild-type MSN. MSN-deficient T cells also displayed poor chemokine receptor expression, increased adhesion molecule expression, and altered migration and adhesion capacities. Our observations establish a causal link between an ezrin-radixin-moesin protein mutation and a primary immunodeficiency that could be referred to as X-linked moesin-associated immunodeficiency. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

In vitro antibacterial activity of rifampicin in combination with imipenem, meropenem and doripenem against multidrug-resistant clinical isolates of Pseudomonas aeruginosa.

PubMed

Hu, Yi-Fan; Liu, Chang-Pan; Wang, Nai-Yu; Shih, Shou-Chuan

2016-08-24

Multidrug-resistant Pseudomonas aeruginosa has emerged as one of the most important healthcare-associated pathogens. Colistin is regarded as the last-resort antibiotic for multidrug-resistant Gram-negative bacteria, but is associated with high rates of acute kidney injury. The aim of this in vitro study is to search for an alternative treatment to colistin for multidrug-resistant P. aeruginosa infections. Multidrug and carbapenem-resistant P. aeruginosa isolates were collected between January 2009 and December 2012 at MacKay Memorial Hospital. Minimal inhibitory concentrations (MICs) were determined for various antibiotic combinations. Carbapenemase-producing genes including bla VIM, other β-lactamase genes and porin mutations were screened by PCR and sequencing. The efficacy of carbapenems (imipenem, meropenem, doripenem) with or without rifampicin was correlated with the type of porin mutation (frameshift mutation, premature stop codon mutation) in multidrug-resistant P. aeruginosa isolates without carbapenemase-producing genes. Of the 71 multidrug-resistant clinical P. aeruginosa isolates, only six harboured the bla VIM gene. Imipenem, meropenem and doripenem were significantly more effective (reduced fold-change of MICs) when combined with rifampicin in bla VIM-negative isolates, especially in isolates with porin frameshift mutation. Imipenem + rifampicin combination has a low MIC against multidrug-resistant P. aeruginosa, especially in isolates with porin frameshift mutation. The imipenem + rifampicin combination may provide an alternative treatment to colistin for multidrug -resistant P. aeruginosa infections, especially for patients with renal insufficiency.

A survey of liver pathology in needle biopsies from HBsAg and anti-HBe positive individuals.

PubMed

ter Borg, F; ten Kate, F J; Cuypers, H T; Leentvaar-Kuijpers, A; Oosting, J; Wertheim-van Dillen, P M; Honkoop, P; Rasch, M C; de Man, R A; van Hattum, J; Chamuleau, R A; Tytgat, G N; Jones, E A

2000-07-01

To use laboratory data and liver biopsies, prospectively obtained from hepatitis B surface antigen (HBsAg) and anti hepatitis B e antigen (anti-HBe) positive patients, for the assessment of: (1) the relation between biopsy length/number of portal tracts and sampling error; (2) the relation between the severity of piecemeal necrosis and the new grading terminology (minimal, mild, moderate, and severe chronic hepatitis); and (3) liver pathology, which has not been studied in patients with this specific serological profile. The study group (n = 174) included 104 patients with normal aminotransferase concentrations and no cases with clinically apparent cirrhosis. The specimen length and number of portal tracts were measured at light microscopy examination. Sampling error analysis was related to the discrepancies between aminotransferase concentrations versus histological grade. Detailed histological scorings were undertaken by the reference pathologist and compared with laboratory and hepatitis B virus (HBV) DNA precore sequence data. Sampling error seemed to be a constant feature, even for biopsies > or = 20 mm, but increased dramatically in biopsies < 5 mm long and/or containing less than four portal tracts. Between 25% and 30% of biopsies, graded as "mild" or "moderate" activity showed features of moderate and severe piecemeal necrosis, respectively. Ten per cent of the patients with normal aminotransferase values had stage III-IV hepatic fibrosis, and 20% had piecemeal necrosis. Only cytoplasmic, not nuclear, core antigen expression was a strong predictor of high hepatitis B viraemia. There was no association between precore stop codon mutations, grade/stage of liver disease, and hepatitis B core antigen (HBcAg) expression. The specimen available for light microscopical examination should be > 5 mm long and should contain more than four portal tracts. In addition, the new grading terminology might give the clinician an inappropriately mild impression of the severity of piecemeal necrosis. Furthermore, even in the presence of normal aminotransferase concentrations, considerable liver pathology can be found in 10-20% of HBsAg and anti-HBe positive individuals; such pathology is not associated with the occurrence of precore stop codon mutations.

Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon

PubMed Central

Jacinto-Loeza, Eva; Vivanco-Domínguez, Serafín; Guarneros, Gabriel; Hernández-Sánchez, Javier

2008-01-01

Rare AGA or AGG codons close to the initiation codon inhibit protein synthesis by a tRNA-sequestering mechanism as toxic minigenes do. To further understand this mechanism, a parallel analysis of protein synthesis and peptidyl-tRNA accumulation was performed using both a set of lacZ constructs where AGAAGA codons were moved codon by codon from +2, +3 up to +7, +8 positions and a series of 3–8 codon minigenes containing AGAAGA codons before the stop codon. β-Galactosidase synthesis from the AGAAGA lacZ constructs (in a Pth defective in vitro system without exogenous tRNA) diminished as the AGAAGA codons were closer to AUG codon. Likewise, β-galactosidase expression from the reporter +7 AGA lacZ gene (plus tRNA, 0.25 μg/μl) waned as the AGAAGAUAA minigene shortened. Pth counteracted both the length-dependent minigene effect on the expression of β-galactosidase from the +7 AGA lacZ reporter gene and the positional effect from the AGAAGA lacZ constructs. The +2, +3 AGAAGA lacZ construct and the shortest +2, +3 AGAAGAUAA minigene accumulated the highest percentage of peptidyl-tRNAArg4. These observations lead us to propose that hungry codons at early positions, albeit with less strength, inhibit protein synthesis by a minigene-like mechanism involving accumulation of peptidyl-tRNA. PMID:18583364

Activation of RAS family genes in urothelial carcinoma.

PubMed

Boulalas, I; Zaravinos, A; Karyotis, I; Delakas, D; Spandidos, D A

2009-05-01

Bladder cancer is the fifth most common malignancy in men in Western society. We determined RAS codon 12 and 13 point mutations and evaluated mRNA expression levels in transitional cell carcinoma cases. Samples from 30 human bladder cancers and 30 normal tissues were analyzed by polymerase chain reaction/restriction fragment length polymorphism and direct sequencing to determine the occurrence of mutations in codons 12 and 13 of RAS family genes. Moreover, we used real-time reverse transcriptase-polymerase chain reaction to evaluate the expression profile of RAS genes in bladder cancer specimens compared to that in adjacent normal tissues. Overall H-RAS mutations in codon 12 were observed in 9 tumor samples (30%). Two of the 9 patients (22%) had invasive bladder cancer and 7 (77%) had noninvasive bladder cancer. One H-RAS mutation (11%) was homozygous and the remaining 89% were heterozygous. All samples were WT for K and N-RAS oncogenes. Moreover, 23 of 30 samples (77%) showed over expression in at least 1 RAS family gene compared to adjacent normal tissue. K and N-RAS had the highest levels of over expression in bladder cancer specimens (50%), whereas 27% of transitional cell carcinomas demonstrated H-RAS over expression relative to paired normal tissues. Our results underline the importance of H-RAS activation in human bladder cancer by codon 12 mutations. Moreover, they provide evidence that increased expression of all 3 RAS genes is a common event in bladder cancer that is associated with disease development.

The Mitochondrial Genome and Transcriptome of the Basal Dinoflagellate Hematodinium sp.: Character Evolution within the Highly Derived Mitochondrial Genomes of Dinoflagellates

PubMed Central

Gornik, S. G.; Waller, R. F.

2012-01-01

The sister phyla dinoflagellates and apicomplexans inherited a drastically reduced mitochondrial genome (mitochondrial DNA, mtDNA) containing only three protein-coding (cob, cox1, and cox3) genes and two ribosomal RNA (rRNA) genes. In apicomplexans, single copies of these genes are encoded on the smallest known mtDNA chromosome (6 kb). In dinoflagellates, however, the genome has undergone further substantial modifications, including massive genome amplification and recombination resulting in multiple copies of each gene and gene fragments linked in numerous combinations. Furthermore, protein-encoding genes have lost standard stop codons, trans-splicing of messenger RNAs (mRNAs) is required to generate complete cox3 transcripts, and extensive RNA editing recodes most genes. From taxa investigated to date, it is unclear when many of these unusual dinoflagellate mtDNA characters evolved. To address this question, we investigated the mitochondrial genome and transcriptome character states of the deep branching dinoflagellate Hematodinium sp. Genomic data show that like later-branching dinoflagellates Hematodinium sp. also contains an inflated, heavily recombined genome of multicopy genes and gene fragments. Although stop codons are also lacking for cox1 and cob, cox3 still encodes a conventional stop codon. Extensive editing of mRNAs also occurs in Hematodinium sp. The mtDNA of basal dinoflagellate Hematodinium sp. indicates that much of the mtDNA modification in dinoflagellates occurred early in this lineage, including genome amplification and recombination, and decreased use of standard stop codons. Trans-splicing, on the other hand, occurred after Hematodinium sp. diverged. Only RNA editing presents a nonlinear pattern of evolution in dinoflagellates as this process occurs in Hematodinium sp. but is absent in some later-branching taxa indicating that this process was either lost in some lineages or developed more than once during the evolution of the highly unusual dinoflagellate mtDNA. PMID:22113794

The mitochondrial genome and transcriptome of the basal dinoflagellate Hematodinium sp.: character evolution within the highly derived mitochondrial genomes of dinoflagellates.

PubMed

Jackson, C J; Gornik, S G; Waller, R F

2012-01-01

The sister phyla dinoflagellates and apicomplexans inherited a drastically reduced mitochondrial genome (mitochondrial DNA, mtDNA) containing only three protein-coding (cob, cox1, and cox3) genes and two ribosomal RNA (rRNA) genes. In apicomplexans, single copies of these genes are encoded on the smallest known mtDNA chromosome (6 kb). In dinoflagellates, however, the genome has undergone further substantial modifications, including massive genome amplification and recombination resulting in multiple copies of each gene and gene fragments linked in numerous combinations. Furthermore, protein-encoding genes have lost standard stop codons, trans-splicing of messenger RNAs (mRNAs) is required to generate complete cox3 transcripts, and extensive RNA editing recodes most genes. From taxa investigated to date, it is unclear when many of these unusual dinoflagellate mtDNA characters evolved. To address this question, we investigated the mitochondrial genome and transcriptome character states of the deep branching dinoflagellate Hematodinium sp. Genomic data show that like later-branching dinoflagellates Hematodinium sp. also contains an inflated, heavily recombined genome of multicopy genes and gene fragments. Although stop codons are also lacking for cox1 and cob, cox3 still encodes a conventional stop codon. Extensive editing of mRNAs also occurs in Hematodinium sp. The mtDNA of basal dinoflagellate Hematodinium sp. indicates that much of the mtDNA modification in dinoflagellates occurred early in this lineage, including genome amplification and recombination, and decreased use of standard stop codons. Trans-splicing, on the other hand, occurred after Hematodinium sp. diverged. Only RNA editing presents a nonlinear pattern of evolution in dinoflagellates as this process occurs in Hematodinium sp. but is absent in some later-branching taxa indicating that this process was either lost in some lineages or developed more than once during the evolution of the highly unusual dinoflagellate mtDNA.

Evaluation of dihydrofolate reductase and dihydropteroate synthetase genotypes that confer resistance to sulphadoxine-pyrimethamine in Plasmodium falciparum in Haiti

PubMed Central

2012-01-01

Background Malaria caused by Plasmodium falciparum infects roughly 30,000 individuals in Haiti each year. Haiti has used chloroquine (CQ) as a first-line treatment for malaria for many years and as a result there are concerns that malaria parasites may develop resistance to CQ over time. Therefore it is important to prepare for alternative malaria treatment options should CQ resistance develop. In many other malaria-endemic regions, antifolates, particularly pyrimethamine (PYR) and sulphadoxine (SDX) treatment combination (SP), have been used as an alternative when CQ resistance has developed. This study evaluated mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes that confer PYR and SDX resistance, respectively, in P. falciparum to provide baseline data in Haiti. This study is the first comprehensive study to examine PYR and SDX resistance genotypes in P. falciparum in Haiti. Methods DNA was extracted from dried blood spots and genotyped for PYR and SDX resistance mutations in P. falciparum using PCR and DNA sequencing methods. Sixty-one samples were genotyped for PYR resistance in codons 51, 59, 108 and 164 of the dhfr gene and 58 samples were genotyped for SDX resistance codons 436, 437, 540 of the dhps gene in P. falciparum. Results Thirty-three percent (20/61) of the samples carried a mutation at codon 108 (S108N) of the dhfr gene. No mutations in dhfr at codons 51, 59, 164 were observed in any of the samples. In addition, no mutations were observed in dhps at the three codons (436, 437, 540) examined. No significant difference was observed between samples collected in urban vs rural sites (Welch’s T-test p-value = 0.53 and permutations p-value = 0.59). Conclusion This study has shown the presence of the S108N mutation in P. falciparum that confers low-level PYR resistance in Haiti. However, the absence of SDX resistance mutations suggests that SP resistance may not be present in Haiti. These results have important implications for ongoing discussions on alternative malaria treatment options in Haiti. PMID:22889367

Nonneutral GC3 and retroelement codon mimicry in Phytophthora.

PubMed

Jiang, Rays H Y; Govers, Francine

2006-10-01

Phytophthora is a genus entirely comprised of destructive plant pathogens. It belongs to the Stramenopila, a unique branch of eukaryotes, phylogenetically distinct from plants, animals, or fungi. Phytophthora genes show a strong preference for usage of codons ending with G or C (high GC3). The presence of high GC3 in genes can be utilized to differentiate coding regions from noncoding regions in the genome. We found that both selective pressure and mutation bias drive codon bias in Phytophthora. Indicative for selection pressure is the higher GC3 value of highly expressed genes in different Phytophthora species. Lineage specific GC increase of noncoding regions is reminiscent of whole-genome mutation bias, whereas the elevated Phytophthora GC3 is primarily a result of translation efficiency-driven selection. Heterogeneous retrotransposons exist in Phytophthora genomes and many of them vary in their GC content. Interestingly, the most widespread groups of retroelements in Phytophthora show high GC3 and a codon bias that is similar to host genes. Apparently, selection pressure has been exerted on the retroelement's codon usage, and such mimicry of host codon bias might be beneficial for the propagation of retrotransposons.

Four novel mutations in the lactase gene (LCT) underlying congenital lactase deficiency (CLD).

PubMed

Torniainen, Suvi; Freddara, Roberta; Routi, Taina; Gijsbers, Carolien; Catassi, Carlo; Höglund, Pia; Savilahti, Erkki; Järvelä, Irma

2009-01-22

Congenital lactase deficiency (CLD) is a severe gastrointestinal disorder of newborns. The diagnosis is challenging and based on clinical symptoms and low lactase activity in intestinal biopsy specimens. The disease is enriched in Finland but is also present in other parts of the world. Mutations encoding the lactase (LCT) gene have recently been shown to underlie CLD. The purpose of this study was to identify new mutations underlying CLD in patients with different ethnic origins, and to increase awareness of this disease so that the patients could be sought out and treated correctly. Disaccharidase activities in intestinal biopsy specimens were assayed and the coding region of LCT was sequenced from five patients from Europe with clinical features compatible with CLD. In the analysis and prediction of mutations the following programs: ClustalW, Blosum62, PolyPhen, SIFT and Panther PSEC were used. Four novel mutations in the LCT gene were identified. A single nucleotide substitution leading to an amino acid change S688P in exon 7 and E1612X in exon 12 were present in a patient of Italian origin. Five base deletion V565fsX567 leading to a stop codon in exon 6 was found in one and a substitution R1587H in exon 12 from another Finnish patient. Both Finnish patients were heterozygous for the Finnish founder mutation Y1390X. The previously reported mutation G1363S was found in a homozygous state in two siblings of Turkish origin. This is the first report of CLD mutations in patients living outside Finland. It seems that disease is more common than previously thought. All mutations in the LCT gene lead to a similar phenotype despite the location and/or type of mutation.

Molecular analysis of congenital goitres with hypothyroidism caused by defective thyroglobulin synthesis. Identification of a novel c.7006C>T [p.R2317X] mutation and expression of minigenes containing nonsense mutations in exon 7.

PubMed

Machiavelli, Gloria A; Caputo, Mariela; Rivolta, Carina M; Olcese, María C; Gruñeiro-Papendieck, Laura; Chiesa, Ana; González-Sarmiento, Rogelio; Targovnik, Héctor M

2010-01-01

Thyroglobulin (TG) deficiency is an autosomal-recessive disorder that results in thyroid dyshormonogenesis. A number of distinct mutations have been identified as causing human hypothyroid goitre. The purpose of this study was to identify and characterize new mutations in the TG gene in an attempt to increase the understanding of the genetic mechanism responsible for this disorder. A total of six patients from four nonconsanguineous families with marked impairment of TG synthesis were studied. Single-strand conformation polymorphism (SSCP) analysis, sequencing of DNA, genotyping, expression of chimeric minigenes and bioinformatic analysis were performed. Four different inactivating TG mutations were identified: one novel mutation (c.7006C>T [p.R2317X]) and three previously reported (c.886C>T [p.R277X], c.6701C>A [p.A2215D] and c.6725G>A [p.R2223H]). Consequently, one patient carried a compound heterozygous for p.R2223H/p.R2317X mutations; two brothers showed a homozygous p.A2215D substitution and the remaining three patients, from two families with typical phenotype, had a single p.R277X mutated allele. We also showed functional evidences that premature stop codons inserted at different positions in exon 7, which disrupt exonic splicing enhancer (ESE) sequences, do not interfere with exon definition and processing. In this study, we have identified a novel nonsense mutation p.R2317X in the acetylcholinesterase homology domain of TG. We have also observed that nonsense mutations do not interfere with the pre-mRNA splicing of exon 7. The results are in accordance with previous observations confirming the genetic heterogeneity of TG defects.

Erythrocytic Pyruvate Kinase Mutations Causing Hemolytic Anemia, Osteosclerosis, and Secondary Hemochromatosis in Dogs

PubMed Central

Gultekin, G. Inal; Raj, K.; Foureman, P.; Lehman, S.; Manhart, K.; Abdulmalik, O.; Giger, U.

2013-01-01

Background Erythrocytic pyruvate kinase (PK) deficiency, first documented in Basenjis, is the most common inherited erythroenzymopathy in dogs. Objectives To report 3 new breed-specific PK-LR gene mutations and a retrospective survey of PK mutations in a small and selected group of Beagles and West Highland White Terriers (WHWT). Animals Labrador Retrievers (2 siblings, 5 unrelated), Pugs (2 siblings, 1 unrelated), Beagles (39 anemic, 29 other), WHWTs (22 anemic, 226 nonanemic), Cairn Terrier (n = 1). Methods Exons of the PK-LR gene were sequenced from genomic DNA of young dogs (<2 years) with persistent highly regenerative hemolytic anemia. Results A nonsense mutation (c.799C>T) resulting in a premature stop codon was identified in anemic Labrador Retriever siblings that had osteosclerosis, high serum ferritin concentrations, and severe hepatic secondary hemochromatosis. Anemic Pug and Beagle revealed 2 different missense mutations (c.848T>C, c.994G>A, respectively) resulting in intolerable amino acid changes to protein structure and enzyme function. Breed-specific mutation tests were developed. Among the biased group of 248 WHWTs, 9% and 35% were homozygous (affected) and heterozygous, respectively, for the previously described mutation (mutant allele frequency 0.26). A PK-deficient Cairn Terrier had the same insertion mutation as the affected WHWTs. Of the selected group of 68 Beagles, 35% were PK-deficient and 3% were carriers (0.37). Conclusions and Clinical Importance Erythrocytic PK deficiency is caused by different mutations in different dog breeds and causes chronic severe hemolytic anemia, hemosiderosis, and secondary hemochromatosis because of chronic hemolysis and, an as yet unexplained osteosclerosis. The newly developed breed-specific mutation assays simplify the diagnosis of PK deficiency. PMID:22805166

Prevalence of the CHEK2 R95* germline mutation.

PubMed

Knappskog, Stian; Leirvaag, Beryl; Gansmo, Liv B; Romundstad, Pål; Hveem, Kristian; Vatten, Lars; Lønning, Per E

2016-01-01

While germline CHEK2 mutations have been linked to a moderately elevated cancer risk, to date, a limited number of such mutations have been identified. Recently, we reported a germline nonsense mutation (C283T; R95*), introducing an early stop-codon, in two Norwegian patients diagnosed with locally advanced breast cancer. Both patients were resistant to anthracycline therapy, resembling what has been observed for TP53 mutations. In the present study, we screened a large population based sample, including 3748 non-cancer individuals and 7081 incident cancer cases (breast cancer, n  = 1717; prostate cancer n  = 2501, lung cancer n  = 1331 and colorectal cancer n  = 1532), for the distribution of CHEK2 R95*. We found that 12 individuals (0.11 %) carried the R95* variant: 4 non-cancer individuals (0.11 %), 4 breast cancer cases (0.23 %), and 4 prostate cancer cases (0.16 %). Although the low number of observations precluded formal statistical assessment, our data may indicate an elevated risk for breast (OR: 2.19, 95 % CI: 0.55-8.75) and prostate cancer (OR: 1.5, 95 % CI: 0.36-6.00) associated with CHEK2 R95*. By mining international databanks, we found no individuals carrying the R95* mutation, indicating it to be restricted to the Norwegian population. We provide proof-of-concept that previously unknown CHEK2 germline mutations may be present in certain populations. Notably, germline mutations in tumours are in general missed by contemporary massive parallel sequencing strategies, since tumour mutations are usually filtered against the germline. The fact that the CHEK2 R95* mutation may be associated with resistance to anthracyclines in cancer patients emphasizes its possible clinical importance.

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis clinical strains isolated in the Philippines.

PubMed

Herrera, Laura; Valverde, Azucena; Saiz, Pilar; Sáez-Nieto, Juan A; Portero, José L; Jiménez, M Soledad

2004-06-01

The prevalence of mutations in the katG, inhA and oxyR-ahpC genes of isoniazid (INH)-resistant Mycobacterium tuberculosis isolates in the Philippines were determined. Of 306 M. tuberculosis isolates studied, 81 (26.5%) exhibited INH-resistance. Forty-four strains (54.3%) had mutations in the katG gene, eighteen strains (22.2%) had mutations in the putative inhA locus region, seven had mutations in both regions and five strains had mutations in the oxyR-ahpC operon. Only seven strains had no mutations. A total of 71 of the 81 (87.6%) resistant strains and 65 of the 72 (90.3%) INH sensitive randomly selected strains showed amino acid substitution in codon 463 (Arg to Leu) (88.9%). This fact supports the hypothesis that mutations at codon 463 are independent of INH-resistance and are linked to the geographical origins of the strains. Copyright 2004 Elsevier B.V.

Silent mutations at codons 65 and 66 in reverse transcriptase alleviate indel formation and restore fitness in subtype B HIV-1 containing D67N and K70R drug resistance mutations

PubMed Central

Telwatte, Sushama; Hearps, Anna C.; Johnson, Adam; Latham, Catherine F.; Moore, Katie; Agius, Paul; Tachedjian, Mary; Sonza, Secondo; Sluis-Cremer, Nicolas; Harrigan, P. Richard; Tachedjian, Gilda

2015-01-01

Resistance to combined antiretroviral therapy (cART) in HIV-1-infected individuals is typically due to nonsynonymous mutations that change the protein sequence; however, the selection of synonymous or ‘silent’ mutations in the HIV-1 genome with cART has been reported. These silent K65K and K66K mutations in the HIV-1 reverse transcriptase (RT) occur in over 35% of drug-experienced individuals and are highly associated with the thymidine analog mutations D67N and K70R, which confer decreased susceptibility to most nucleoside and nucleotide RT inhibitors. However, the basis for selection of these silent mutations under selective drug pressure is unknown. Using Illumina next-generation sequencing, we demonstrate that the D67N/K70R substitutions in HIV-1 RT increase indel frequency by 100-fold at RT codons 65–67, consequently impairing viral fitness. Introduction of either K65K or K66K into HIV-1 containing D67N/K70R reversed the error-prone DNA synthesis at codons 65–67 in RT and improved viral replication fitness, but did not impact RT inhibitor drug susceptibility. These data provide new mechanistic insights into the role of silent mutations selected during antiretroviral therapy and have broader implications for the relevance of silent mutations in the evolution and fitness of RNA viruses. PMID:25765644

PTEN/MMAC1 Mutations in Hepatocellular Carcinomas: Somatic Inactivation of Both Alleles in Tumors

PubMed Central

Kawamura, Naoki; Nagai, Hisaki; Bando, Koichi; Koyama, Masaaki; Matsumoto, Satoshi; Tajiri, Takashi; Onda, Masahiko; Fujimoto, Jiro; Ueki, Takahiro; Konishi, Noboru; Shiba, Tadayoshi

1999-01-01

Allelic loss of loci on chromosome 10q occurs frequently in hepatocellular carcinomas. Somatic mutations of the PTEN/MMAC1 gene on this chromosome at 10q23 were recently identified in sporadic cancers of the uterus, brain, prostate and breast. To investigate the potential role of PTEN/MMAC1 gene in the genesis of hepatocellular carcinomas, we examined 96 tumors for allelic loss on 10q and also for subtle mutations anywhere within the coding region of PTEN/MMAC1 gene. Allelic loss was identified in 25 of the 89 (27%) tumors that were informative for polymorphic markers in the region. Somatic mutations were identified in five of those tumors: three frameshift mutations, a 1‐bp insertion at codon 83–84 in exon 4 and two 4‐bp deletions, both at codon 318–319 in exon 8; two C‐to‐G transversion mutation, both at ‐9 bp from the initiation codon in the 5’non‐coding region of exon 1. No missense mutation was observed in this panel of tumors. In most of the informative tumors carrying intragenic mutations of one allele, we were able to detect loss of heterozygosity as well. These findings suggest that two alleles of the PTEN/MMAC1 gene may be inactivated by a combination of intragenic point mutation on one allele and loss of chromosomal material on the other allele in some of these tumors. PMID:10363579

Molecular Mechanisms of Fluoroquinolone Resistance in Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients

PubMed Central

Jalal, Shah; Ciofu, Oana; Høiby, Niels; Gotoh, Naomasa; Wretlind, Bengt

2000-01-01

Twenty P. aeruginosa isolates were collected from six cystic fibrosis (CF) patients, aged 27 to 33, in 1994 (9 isolates) and 1997 (11 isolates) at the CF Center, Copenhagen, Denmark, and were typed by pulse-field gel electrophoresis (PFGE) or ribotyping. Five of the patients had isolates with the same PFGE or ribotyping patterns in 1997 as in 1994, and ciprofloxacin had a two- to fourfold higher MIC for the isolates collected in 1997 than those from 1994. Genomic DNA was amplified for gyrA, parC, mexR, and nfxB by PCR and sequenced. Eleven isolates had mutations in gyrA, seven isolates had mutations at codon 83 (Thr to Ile), and four isolates had mutations at codon 87 (Asp to Asn or Tyr). Sixteen isolates had mutations in nfxB at codon 82 (Arg to Leu). Increased amounts of OprN were found in six isolates and OprJ in eight isolates as determined by immunoblotting. No isolates had mutations in parC or mexR. Six isolates had mutations in efflux pumps without gyrA mutations. The average number of mutations was higher in isolates from 1997 than in those from 1994. The results also suggested that efflux resistance mechanisms are more common in isolates from CF patients than in strains from urine and wounds from non-CF patients, in which mutations in gyrA and parC dominate (S. Jalal and B. Wretlind, Microb. Drug Resist. 4:257–261, 1998). PMID:10681343

Effects of mutations in the human uncoupling protein 3 gene on the respiratory quotient and fat oxidation in severe obesity and type 2 diabetes.

PubMed Central

Argyropoulos, G; Brown, A M; Willi, S M; Zhu, J; He, Y; Reitman, M; Gevao, S M; Spruill, I; Garvey, W T

1998-01-01

Human uncoupling protein 3 (UCP3) is a mitochondrial transmembrane carrier that uncouples oxidative ATP phosphorylation. With the capacity to participate in thermogenesis and energy balance, UCP3 is an important obesity candidate gene. A missense polymorphism in exon 3 (V102I) was identified in an obese and diabetic proband. A mutation introducing a stop codon in exon 4 (R143X) and a terminal polymorphism in the splice donor junction of exon 6 were also identified in a compound heterozygote that was morbidly obese and diabetic. Allele frequencies of the exon 3 and exon 6 splice junction polymorphisms were determined and found to be similar in Gullah-speaking African Americans and the Mende tribe of Sierra Leone, but absent in Caucasians. Moreover, in exon 6-splice donor heterozygotes, basal fat oxidation rates were reduced by 50%, and the respiratory quotient was markedly increased compared with wild-type individuals, implicating a role for UCP3 in metabolic fuel partitioning. PMID:9769326

Cryptic Amyloidogenic Elements in the 3′ UTRs of Neurofilament Genes Trigger Axonal Neuropathy

PubMed Central

Rebelo, Adriana P.; Abrams, Alexander J.; Cottenie, Ellen; Horga, Alejandro; Gonzalez, Michael; Bis, Dana M.; Sanchez-Mejias, Avencia; Pinto, Milena; Buglo, Elena; Markel, Kasey; Prince, Jeffrey; Laura, Matilde; Houlden, Henry; Blake, Julian; Woodward, Cathy; Sweeney, Mary G.; Holton, Janice L.; Hanna, Michael; Dallman, Julia E.; Auer-Grumbach, Michaela; Reilly, Mary M.; Zuchner, Stephan

2016-01-01

Abnormal protein aggregation is observed in an expanding number of neurodegenerative diseases. Here, we describe a mechanism for intracellular toxic protein aggregation induced by an unusual mutation event in families affected by axonal neuropathy. These families carry distinct frameshift variants in NEFH (neurofilament heavy), leading to a loss of the terminating codon and translation of the 3′ UTR into an extra 40 amino acids. In silico aggregation prediction suggested the terminal 20 residues of the altered NEFH to be amyloidogenic, which we confirmed experimentally by serial deletion analysis. The presence of this amyloidogenic motif fused to NEFH caused prominent and toxic protein aggregates in transfected cells and disrupted motor neurons in zebrafish. We identified a similar aggregation-inducing mechanism in NEFL (neurofilament light) and FUS (fused in sarcoma), in which mutations are known to cause aggregation in Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis, respectively. In summary, we present a protein-aggregation-triggering mechanism that should be taken into consideration during the evaluation of stop-loss variants. PMID:27040688

Two cloned β thalassemia genes are associated with amber mutations at codon 39

PubMed Central

Pergolizzi, Robert; Spritz, Richard A.; Spence, Sally; Goossens, Michel; Kan, Yuet Wai; Bank, Arthur

1981-01-01

Two β globin genes from patients with the β+ thalassemia phenotype have been cloned and sequenced. A single nucleotide change from CAG to TAG (an amber mutation) at codon 39 is the only difference from normal in both genes analyzed. The results are consistent with the assumption that both patients are doubly heterozygous for β+ and β° thalassemia, and that we have isolated and analyzed the β° thalassemia gene. Images PMID:6278453

Co-expression of the Thermotoga neapolitana aglB gene with an upstream 3'-coding fragment of the malG gene improves enzymatic characteristics of recombinant AglB cyclomaltodextrinase.

PubMed

Lunina, Natalia A; Agafonova, Elena V; Chekanovskaya, Lyudmila A; Dvortsov, Igor A; Berezina, Oksana V; Shedova, Ekaterina N; Kostrov, Sergey V; Velikodvorskaya, Galina A

2007-07-01

A cluster of Thermotoga neapolitana genes participating in starch degradation includes the malG gene of sugar transport protein and the aglB gene of cyclomaltodextrinase. The start and stop codons of these genes share a common overlapping sequence, aTGAtg. Here, we compared properties of expression products of three different constructs with aglB from T. neapolitana. The first expression vector contained the aglB gene linked to an upstream 90-bp 3'-terminal region of the malG gene with the stop codon overlapping with the start codon of aglB. The second construct included the isolated coding sequence of aglB with two tandem potential start codons. The expression product of this construct in Escherichia coli had two tandem Met residues at its N terminus and was characterized by low thermostability and high tendency to aggregate. In contrast, co-expression of aglB and the 3'-terminal region of malG (the first construct) resulted in AglB with only one N-terminal Met residue and a much higher specific activity of cyclomaltodextrinase. Moreover, the enzyme expressed by such a construct was more thermostable and less prone to aggregation. The third construct was the same as the second one except that it contained only one ATG start codon. The product of its expression had kinetic and other properties similar to those of the enzyme with only one N-terminal Met residue.

Codon usage bias and phylogenetic analysis of mitochondrial ND1 gene in pisces, aves, and mammals.

PubMed

Uddin, Arif; Choudhury, Monisha Nath; Chakraborty, Supriyo

2018-01-01

The mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1) gene is a subunit of the respiratory chain complex I and involved in the first step of the electron transport chain of oxidative phosphorylation (OXPHOS). To understand the pattern of compositional properties, codon usage and expression level of mitochondrial ND1 genes in pisces, aves, and mammals, we used bioinformatic approaches as no work was reported earlier. In this study, a perl script was used for calculating nucleotide contents and different codon usage bias parameters. The codon usage bias of MT-ND1 was low but the expression level was high as revealed from high ENC and CAI value. Correspondence analysis (COA) suggests that the pattern of codon usage for MT-ND1 gene is not same across species and that compositional constraint played an important role in codon usage pattern of this gene among pisces, aves, and mammals. From the regression equation of GC12 on GC3, it can be inferred that the natural selection might have played a dominant role while mutation pressure played a minor role in influencing the codon usage patterns. Further, ND1 gene has a discrepancy with cytochrome B (CYB) gene in preference of codons as evident from COA. The codon usage bias was low. It is influenced by nucleotide composition, natural selection, mutation pressure, length (number) of amino acids, and relative dinucleotide composition. This study helps in understanding the molecular biology, genetics, evolution of MT-ND1 gene, and also for designing a synthetic gene.

Correction of nonsense BMPR2 and SMAD9 mutations by ataluren in pulmonary arterial hypertension.

PubMed

Drake, Kylie M; Dunmore, Benjamin J; McNelly, Lauren N; Morrell, Nicholas W; Aldred, Micheala A

2013-09-01

Heritable pulmonary arterial hypertension (HPAH) is a serious lung vascular disease caused by heterozygous mutations in the bone morphogenetic protein (BMP) pathway genes, BMPR2 and SMAD9. One noncanonical function of BMP signaling regulates biogenesis of a subset of microRNAs. We have previously shown that this function is abrogated in patients with HPAH, making it a highly sensitive readout of BMP pathway integrity. Ataluren (PTC124) is an investigational drug that permits ribosomal readthrough of premature stop codons, resulting in a full-length protein. It exhibits oral bioavailability and limited toxicity in human trials. Here, we tested ataluren in lung- or blood-derived cells from patients with HPAH with nonsense mutations in BMPR2 (n = 6) or SMAD9 (n = 1). Ataluren significantly increased BMP-mediated microRNA processing in six of the seven cases. Moreover, rescue was achieved even for mutations exhibiting significant nonsense-mediated mRNA decay. Response to ataluren was dose dependent, and complete correction was achieved at therapeutic doses currently used in clinical trials for cystic fibrosis. BMP receptor (BMPR)-II protein levels were normalized and ligand-dependent phosphorylation of downstream target Smads was increased. Furthermore, the usually hyperproliferative phenotype of pulmonary artery endothelial and smooth muscle cells was reversed by ataluren. These results indicate that ataluren can effectively suppress a high proportion of BMPR2 and SMAD9 nonsense mutations and correct BMP signaling in vitro. Approximately 29% of all HPAH mutations are nonsense point mutations. In light of this, we propose ataluren as a potential new personalized therapy for this significant subgroup of patients with PAH.

Inferring Selection on Amino Acid Preference in Protein Domains

PubMed Central

Durbin, Richard

2009-01-01

Models that explicitly account for the effect of selection on new mutations have been proposed to account for “codon bias” or the excess of “preferred” codons that results from selection for translational efficiency and/or accuracy. In principle, such models can be applied to any mutation that results in a preferred allele, but in most cases, the fitness effect of a specific mutation cannot be predicted. Here we show that it is possible to assign preferred and unpreferred states to amino acid changing mutations that occur in protein domains. We propose that mutations that lead to more common amino acids (at a given position in a domain) can be considered “preferred alleles” just as are synonymous mutations leading to codons for more abundant tRNAs. We use genome-scale polymorphism data to show that alleles for preferred amino acids in protein domains occur at higher frequencies in the population, as has been shown for preferred codons. We show that this effect is quantitative, such that there is a correlation between the shift in frequency of preferred alleles and the predicted fitness effect. As expected, we also observe a reduction in the numbers of polymorphisms and substitutions at more important positions in domains, consistent with stronger selection at those positions. We examine the derived allele frequency distribution and polymorphism to divergence ratios of preferred and unpreferred differences and find evidence for both negative and positive selections acting to maintain protein domains in the human population. Finally, we analyze a model for selection on amino acid preferences in protein domains and find that it is consistent with the quantitative effects that we observe. PMID:19095755

Mutation at embB Codon 306, a Potential Marker for the Identification of Multidrug Resistance Associated with Ethambutol in Mycobacterium tuberculosis

PubMed Central

Cuevas-Córdoba, Betzaida; Juárez-Eusebio, Dulce María; Almaraz-Velasco, Raquel; Muñiz-Salazar, Raquel; Laniado-Laborin, Rafael

2015-01-01

Ethambutol inhibits arabinogalactan and lipoarabinomannan biosynthesis in mycobacteria. The occurrence of mutations in embB codon 306 in ethambutol-susceptible isolates and their absence in resistant isolates has raised questions regarding the utility of this codon as a potential marker for resistance against ethambutol. The characterization of mutations on embB 306 will contribute to a better understanding of the mechanisms of resistance to this drug; therefore, the purpose of this study was to investigate the association between embB 306 mutations and first-line drug resistance profiles in tuberculosis isolates. We sequenced the region surrounding the embB 306 codon in 175 tuberculosis clinical isolates, divided according to drug sensitivity, in three groups: 110 were resistant to at least one first-line drug, of which 61 were resistant to ethambutol (EMBr), 49 were sensitive to ethambutol (EMBs) but were resistant to another drug, and 65 were pansensitive isolates (Ps). The associations between embB 306 mutations and phenotypic resistance to all first-line drugs were determined, and their validity and safety as a diagnostic marker were assessed. One of the Ps isolates (1/65), one of the EMBs isolates (1/49), and 20 of the EMBr isolates (20/61) presented with an embB 306 mutation. Four different single-nucleotide polymorphisms (SNPs) at embB 306 were associated with simultaneous resistance to ethambutol, isoniazid, and rifampin (odds ratio [OR], 17.7; confidence interval [CI], 5.6 to 56.1) and showed a positive predictive value of 82%, with a specificity of 97% for diagnosing multidrug resistance associated with ethambutol, indicating its potential as a molecular marker for several drugs. PMID:26124153

hOGG1 Ser326Cys polymorphism and G:C-to-T:A mutations: no evidence for a role in tobacco-related non small cell lung cancer.

PubMed

Hu, Ying Chuan; Ahrendt, Steven A

2005-04-10

Human 8-oxoguanine DNA glycosylase 1 (hOGG1) plays a major role in the repair of 8-hydroxyguanine, one of the major forms of DNA damage generated by reactive oxygen species in tobacco smoke. If left unrepaired by hOGG1, 8-hydroxyguanine can produce G:C-to-T:A transversions. Recent studies have suggested that the hOGG1 Ser326Cys polymorphism is associated with both a decrease in enzyme activity and an increased risk of lung cancer. To define the interaction between tobacco carcinogens, hOGG1-mediated DNA repair and DNA damage, we examined the role of the hOGG1 Ser326Cys polymorphism in mutation of the p53 gene in non small cell lung cancer (NSCLC). Tumor and nonneoplastic DNA were collected from 141 cigarette smokers with NSCLC. p53 mutations were detected by direct dideoxy sequencing and/or the GeneChip p53 assay in 74 of the 141 (52%) tumors. hOGG1 codon 326 polymorphisms were identified by polymerase chain reaction-restriction fragment length polymorphism analysis. The distribution of hOGG1 codon 326 genotypes was Ser/Ser, 90 of 141 (64%); Ser/Cys, 45 of 141 (32%); and Cys/Cys, 6 of 141 (4%). p53 mutations were significantly (p = 0.04) less common in NSCLC from patients with codon 326 Ser/Cys or Cys/Cys genotypes (21 of 51; 41%) than in NSCLC from Ser/Ser homozygotes (53 of 90; 59%). The decrease in p53 mutation frequency among carriers of the Cys allele was more evident in lung squamous cell cancer [7 of 17 (41%) for Cys/Cys and Ser/Cys vs. 27 of 38 (71%) for Ser/Ser; p = 0.04] than in nonbronchoalveolar adenocarcinoma [11 of 26 (42%) for Cys/Cys and Ser/Cys vs. 20 of 35 (57%) for Ser/Ser; p = 0.25]. The prevalence of G:C-to-T:A transversions was similar among hOGG1 codon 326 genotypes. In summary, the hOGG1 codon 326 Cys allele was associated with a decrease in p53 mutations and no effect on G:C-to-T:A transversions in NSCLC. This decrease in p53 mutations in vivo is not consistent with a decrease in the repair of 8-hydroxyguanine among carriers of the hOGG1 codon 326 Cys allele in vitro. (c) 2004 Wiley-Liss, Inc.

Comparative Genomic Analysis MERS CoV Isolated from Humans and Camels with Special Reference to Virus Encoded Helicase.

PubMed

Alnazawi, Mohamed; Altaher, Abdallah; Kandeel, Mahmoud

2017-01-01

Middle East Respiratory Syndrome Coronavirus (MERS CoV) is a new emerging viral disease characterized by high fatality rate. Understanding MERS CoV genetic aspects and codon usage pattern is important to understand MERS CoV survival, adaptation, evolution, resistance to innate immunity, and help in finding the unique aspects of the virus for future drug discovery experiments. In this work, we provide comprehensive analysis of 238 MERS CoV full genomes comprised of human (hMERS) and camel (cMERS) isolates of the virus. MERS CoV genome shaping seems to be under compositional and mutational bias, as revealed by preference of A/T over G/C nucleotides, preferred codons, nucleotides at the third position of codons (NT3s), relative synonymous codon usage, hydropathicity (Gravy), and aromaticity (Aromo) indices. Effective number of codons (ENc) analysis reveals a general slight codon usage bias. Codon adaptation index reveals incomplete adaptation to host environment. MERS CoV showed high ability to resist the innate immune response by showing lower CpG frequencies. Neutrality evolution analysis revealed a more significant role of mutation pressure in cMERS over hMERS. Correspondence analysis revealed that MERS CoV genomes have three genetic clusters, which were distinct in their codon usage, host, and geographic distribution. Additionally, virtual screening and binding experiments were able to identify three new virus-encoded helicase binding compounds. These compounds can be used for further optimization of inhibitors.

Non-uniqueness of factors constraint on the codon usage in Bombyx mori.

PubMed

Jia, Xian; Liu, Shuyu; Zheng, Hao; Li, Bo; Qi, Qi; Wei, Lei; Zhao, Taiyi; He, Jian; Sun, Jingchen

2015-05-06

The analysis of codon usage is a good way to understand the genetic and evolutionary characteristics of an organism. However, there are only a few reports related with the codon usage of the domesticated silkworm, Bombyx mori (B. mori). Hence, the codon usage of B. mori was analyzed here to reveal the constraint factors and it could be helpful to improve the bioreactor based on B. mori. A total of 1,097 annotated mRNA sequences from B. mori were analyzed, revealing there is only a weak codon bias. It also shows that the gene expression level is related to the GC content, and the amino acids with higher general average hydropathicity (GRAVY) and aromaticity (Aromo). And the genes on the primary axis are strongly positively correlated with the GC content, and GC3s. Meanwhile, the effective number of codons (ENc) is strongly correlated with codon adaptation index (CAI), gene length, and Aromo values. However, the ENc values are correlated with the second axis, which indicates that the codon usage in B. mori is affected by not only mutation pressure and natural selection, but also nucleotide composition and the gene expression level. It is also associated with Aromo values, and gene length. Additionally, B. mori has a greater relative discrepancy in codon preferences with Drosophila melanogaster (D. melanogaster) or Saccharomyces cerevisiae (S. cerevisiae) than with Arabidopsis thaliana (A. thaliana), Escherichia coli (E. coli), or Caenorhabditis elegans (C. elegans). The codon usage bias in B. mori is relatively weak, and many influence factors are found here, such as nucleotide composition, mutation pressure, natural selection, and expression level. Additionally, it is also associated with Aromo values, and gene length. Among them, natural selection might play a major role. Moreover, the "optimal codons" of B. mori are all encoded by G and C, which provides useful information for enhancing the gene expression in B. mori through codon optimization.

Population-Based Genetic Study of β-Thalassemia Mutations in Mardan Division, Khyber Pakhtunkhwa Province, Pakistan.

PubMed

Muhammad, Raj; Shakeel, Muhammad; Rehman, Shoaib U; Lodhi, Muhammad A

2017-03-01

β-Thalassemia (β-thal) is the most prevalent hereditary blood disorder in Pakistan with a carrier rate of 5.0-8.0%. The homozygous affected children require frequent blood transfusions for their survival. This autosomal recessive disease can only be prevented through awareness programs, carrier screening, mutation detection, genetic counseling and prenatal diagnosis (PND). The present study aimed to determine the prevalence of various mutations causing β-thal and also to detect carriers of these mutations in families living in the Mardan Division, Khyber Pakhtunkhwa (KP) Province, Pakistan. The study was conducted at the Department of Biochemistry, Abdul Wali Khan University Mardan, Pakistan. Blood samples of β-thalassemic families were collected from various transfusion centers in Mardan Division. Using the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) technique, all samples were analyzed for the six most common mutations causing β-thal in this area. Six different mutant primers for the detection of different mutations were used. The most common mutations detected in thalassemic patients were frameshift codons (FSC) 8/9 (+G) (HBB: c.27_28insG), codons 41/42 (-TTCT) (HBB: c.126_129delCTTT), and IVS-I-5 (G>C) (HBB: c.92+5G>C). The predominant mutation for carrying the mutant genes for β-thal were FSC 8/9, IVS-I-5, codons 41/42, IVS-I-1. It was also found that 66.7% of marriages were consanguineous. The FSC 8/9 mutation was found to be the most common β-thal mutation with a frequency of 44.4%. This research project provides a strong incentive for the establishment of large scale mutation detection and PND services in the Mardan Division.

Whole genome sequencing based characterization of extensively drug-resistant Mycobacterium tuberculosis isolates from Pakistan.

PubMed

Ali, Asho; Hasan, Zahra; McNerney, Ruth; Mallard, Kim; Hill-Cawthorne, Grant; Coll, Francesc; Nair, Mridul; Pain, Arnab; Clark, Taane G; Hasan, Rumina

2015-01-01

Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91-94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.

Detecting Adaptation in Protein-Coding Genes Using a Bayesian Site-Heterogeneous Mutation-Selection Codon Substitution Model.

PubMed

Rodrigue, Nicolas; Lartillot, Nicolas

2017-01-01

Codon substitution models have traditionally attempted to uncover signatures of adaptation within protein-coding genes by contrasting the rates of synonymous and non-synonymous substitutions. Another modeling approach, known as the mutation-selection framework, attempts to explicitly account for selective patterns at the amino acid level, with some approaches allowing for heterogeneity in these patterns across codon sites. Under such a model, substitutions at a given position occur at the neutral or nearly neutral rate when they are synonymous, or when they correspond to replacements between amino acids of similar fitness; substitutions from high to low (low to high) fitness amino acids have comparatively low (high) rates. Here, we study the use of such a mutation-selection framework as a null model for the detection of adaptation. Following previous works in this direction, we include a deviation parameter that has the effect of capturing the surplus, or deficit, in non-synonymous rates, relative to what would be expected under a mutation-selection modeling framework that includes a Dirichlet process approach to account for across-codon-site variation in amino acid fitness profiles. We use simulations, along with a few real data sets, to study the behavior of the approach, and find it to have good power with a low false-positive rate. Altogether, we emphasize the potential of recent mutation-selection models in the detection of adaptation, calling for further model refinements as well as large-scale applications. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Impact of KRAS codon subtypes from a randomised phase II trial of selumetinib plus docetaxel in KRAS mutant advanced non-small-cell lung cancer.

PubMed

Jänne, P A; Smith, I; McWalter, G; Mann, H; Dougherty, B; Walker, J; Orr, M C M; Hodgson, D R; Shaw, A T; Pereira, J R; Jeannin, G; Vansteenkiste, J; Barrios, C H; Franke, F A; Crinò, L; Smith, P

2015-07-14

Selumetinib (AZD6244, ARRY-142886)+docetaxel increases median overall survival (OS) and significantly improves progression-free survival (PFS) and objective response rate (ORR) compared with docetaxel alone in patients with KRAS mutant, stage IIIB/IV non-small-cell lung cancer (NSCLC; NCT00890825). Retrospective analysis of OS, PFS, ORR and change in tumour size at week 6 for different sub-populations of KRAS codon mutations. In patients receiving selumetinib+docetaxel and harbouring KRAS G12C or G12V mutations there were trends towards greater improvement in OS, PFS and ORR compared with other KRAS mutations. Different KRAS mutations in NSCLC may influence selumetinib/docetaxel sensitivity.

Identification of a novel mutation in the human growth hormone receptor gene (GHR) in a patient with Laron syndrome.

PubMed

Gennero, Isabelle; Edouard, Thomas; Rashad, Mona; Bieth, Eric; Conte-Aurio, Françoise; Marin, Françoise; Tauber, Maithé; Salles, Jean Pierre; El Kholy, Mohamed

2007-07-01

Deletions and mutations in the growth hormone receptor (GHR) gene are the underlying etiology of Laron syndrome (LS) or growth hormone (GH) insensitivity syndrome (GHIS), an autosomal recessive disease. Most patients are distributed in or originate from Mediterranean and Middle-Eastern countries. Sixty mutations have been described so far. We report a novel mutation in the GHR gene in a patient with LS. Genomic DNA sequencing of exon 5 revealed a TT insertion at nucleotide 422 after codon 122. The insertion resulted in a frameshift introducing a premature termination codon that led to a truncated receptor. We present clinical, biochemical and molecular evidence of LS as the result of this homozygous insertion.

Meningocerebrovascular amyloidosis associated with a novel transthyretin mis-sense mutation at codon 18 (TTRD 18G)

PubMed Central

Vidal, R.; Garzuly, F.; Budka, H.; Lalowski, M.; Linke, R. P.; Brittig, F.; Frangione, B.; Wisniewski, T.

1996-01-01

We describe a novel transthyretin mutation at codon 18 where Asp is replaced by Gly (D18G) in a Hungarian kindred. This mutation is associated with meningocerebrovascular amyloidosis, producing dementia, ataxia, and spasticity. Fifty different transthyretin mutations are related to amyloid deposition, typically producing a peripheral neuropathy or cardiac dysfunction. These symptoms are absent in this family. Up to now, amyloid-beta (A beta), cystatin C, and prion proteins have been known to be deposited as amyloid in the brain, leading to stroke or dementia. With this report we establish that transthyretin amyloid deposition can also produce central nervous system dysfunction as the major clinical symptom. Images Figure 2 Figure 4 PMID:8579098

Adrenocorticotropin-dependent precocious puberty of testicular origin in a boy with X-linked adrenal hypoplasia congenita due to a novel mutation in the DAX1 gene.

PubMed

Domenice, S; Latronico, A C; Brito, V N; Arnhold, I J; Kok, F; Mendonca, B B

2001-09-01

Primary adrenal insufficiency is a rare condition in pediatric age, and its association with precocious sexual development is very uncommon. We report a 2-yr-old Brazilian boy with DAX1 gene mutation whose first clinical manifestation was isosexual gonadotropin-independent precocious puberty. He presented with pubic hair, enlarged penis and testes, and advanced bone age. T levels were elevated, whereas basal and GnRH-stimulated LH levels were compatible with a prepubertal pattern. Chronic GnRH agonist therapy did not reduce T levels, supporting the diagnosis of gonadotropin-independent precocious puberty. Testotoxicosis was ruled out after normal sequencing of exon 11 of the LH receptor gene. At age 3 yr he developed clinical and hormonal features of severe primary adrenal insufficiency. The entire coding region of the DAX1 gene was analyzed through direct sequencing. A nucleotide G insertion between nucleotides 430 and 431 in exon 1, resulting in a novel frameshift mutation and a premature stop codon at position 71 of DAX-1, was identified. Surprisingly, steroid replacement therapy induced a clear decrease in testicular size and T levels to the prepubertal range. These findings suggest that chronic excessive ACTH levels resulting from adrenal insufficiency may stimulate Leydig cells and lead to gonadotropin-independent precocious puberty in some boys with DAX1 gene mutations.

Recurrent elevated liver transaminases and acute liver failure in two siblings with novel bi-allelic mutations of NBAS.

PubMed

Regateiro, Frederico S; Belkaya, Serkan; Neves, Nélson; Ferreira, Sandra; Silvestre, Paula; Lemos, Sónia; Venâncio, Margarida; Casanova, Jean-Laurent; Gonçalves, Isabel; Jouanguy, Emmanuelle; Diogo, Luísa

2017-08-01

Acute liver failure (ALF) in children can be life-threatening. Although many causes are known, ALF remains unexplained in about half of the cases. Recently, bi-allelic mutations in NBAS were reported to underlie recurrent episodes of elevated liver transaminases (ELT) and ALF in the context of diverse extrahepatic phenotypes. We here describe two sisters, born to non-consanguineous Portuguese parents, who had short stature and presented with recurrent episodes of severe ELT triggered by febrile respiratory viral infections since early childhood. Patient 1 had mild facial dysmorphism and died during the second ELT crisis at 3-11/12 years of age. Patient 2, currently 9 years old, had multiple episodes of ELT (>30), twice with ALF, often accompanied by extensive urticaria and facial angioedema. Whole-exome and Sanger sequencing revealed that both patients carried previously undescribed compound heterozygous mutations of NBAS (NM_015909.3): c.680A > C (p.His227Pro), affecting an evolutionarily conserved residue, and c.1749G > A (p.Trp583*), causing a premature stop codon. Both mutations are predicted to be highly damaging. The parents and two younger siblings are healthy and heterozygous for one or another mutant allele. The multiplex kindred reported herein expands the genotypic and phenotypic spectrum of this recently described clinical syndrome due to autosomal recessive NBAS deficiency. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Respiratory chain complex I deficiency due to NDUFA12 mutations as a new cause of Leigh syndrome.

PubMed

Ostergaard, Elsebet; Rodenburg, Richard J; van den Brand, Mariël; Thomsen, Lise Lykke; Duno, Morten; Batbayli, Mustafa; Wibrand, Flemming; Nijtmans, Leo

2011-11-01

This study investigated a girl with Leigh syndrome born to first-cousin parents of Pakistani descent with an isolated respiratory chain complex I deficiency in muscle and fibroblasts. Her early development was delayed, and from age 2 years she started losing motor abilities. Cerebral MRI showed basal ganglia lesions typical of Leigh syndrome. A genome-wide search for homozygosity was performed with the Affymetrix GeneChip 50K Xba array. The analysis revealed several homozygous regions. Three candidate genes were identified, and in one of the genes, NDUFA12, a homozygous c.178C→T mutation leading to a premature stop codon (p.Arg60X) was found. Western blot analysis showed absence of NDUFA12 protein in patient fibroblasts and functional complementation by a baculovirus system showed restoration of complex I activity. NDUFA12 mutations are apparently not a frequent cause of complex I deficiency, since mutations were not found by screening altogether 122 complex I deficient patients in two different studies. NDUFA12 encodes an accessory subunit of complex I and is a paralogue of NDUFAF2. Despite the complete absence of NDUFA12 protein, a fully assembled and enzymatically active complex I could be found, albeit in reduced amounts. This suggests that NDUFA12 is required either at a late step in the assembly of complex I, or in the stability of complex I.

Molecular genetic analysis of macular corneal dystrophy patients from North India.

PubMed

Paliwal, Preeti; Sharma, Arundhati; Tandon, Radhika; Sharma, Namrata; Titiyal, Jeevan S; Sen, Seema; Vajpayee, Rasik B

2012-01-01

To identify underlying genetic defects in the carbohydrate sulfotransferase-6 (CHST6) gene in North Indian patients with macular corneal dystrophy (MCD). 30 clinically diagnosed MCD patients from 21 families and 50 healthy normal controls were recruited in the study. Detailed clinical evaluation in the patients was undertaken followed by histopathology and ultrastructural studies in corneal tissues. DNA from blood samples was amplified for the CHST6 coding and upstream region followed by direct sequencing and in silico analysis. We identified pathogenic mutations in 17 patients from 11 families. Of these 4 were novel (p.Ser54Tyr, p.Gln58Arg, p.Leu59His and p.Leu293Phe), 2 were previously reported (Arg93His and Glu274Lys) homozygous, 1 heterozygous stop codon (p.Trp123X) and 2 compound heterozygous (p.Arg93His + p.Arg97Pro; p.Leu22Arg + p.Gln58X) mutations. A missense single-nucleotide polymorphism was also identified in 11 patients. The novel mutations were conserved as shown by in silico analysis. Thirteen patients did not show any pathogenic CHST6 changes. This is the first report on molecular analysis of MCD in North Indian patients. All cases could not be explained by mutations in CHST6, suggesting that MCD may result from other changes in the regulatory elements of CHST6 or from genetic heterogeneity. Copyright © 2012 S. Karger AG, Basel.

Tissue- and case-specific retention of intron 40 in mature dystrophin mRNA.

PubMed

Nishida, Atsushi; Minegishi, Maki; Takeuchi, Atsuko; Niba, Emma Tabe Eko; Awano, Hiroyuki; Lee, Tomoko; Iijima, Kazumoto; Takeshima, Yasuhiro; Matsuo, Masafumi

2015-06-01

The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), comprises 79 exons that show multiple alternative splicing events. Intron retention, a type of alternative splicing, may control gene expression. We examined intron retention in dystrophin introns by reverse-transcription PCR from skeletal muscle, focusing on the nine shortest (all <1000 bp), because these are more likely to be retained. Only one, intron 40, was retained in mRNA; sequencing revealed insertion of a complete intron 40 (851 nt) between exons 40 and 41. The intron 40 retention product accounted for 1.2% of the total product but had a premature stop codon at the fifth intronic codon. Intron 40 retention was most strongly observed in the kidney (36.6%) and was not obtained from the fetal liver, lung, spleen or placenta. This indicated that intron retention is a tissue-specific event whose level varies among tissues. In two DMD patients, intron 40 retention was observed in one patient but not in the other. Examination of splicing regulatory factors revealed that intron 40 had the highest guanine-cytosine content of all examined introns in a 30-nt segment at its 3' end. Further studies are needed to clarify the biological role of intron 40-retained dystrophin mRNA.

A novel frameshift deletion in the albumin gene causes analbuminemia in a young Turkish woman.

PubMed

Dagnino, Monica; Caridi, Gianluca; Aydin, Zeki; Ozturk, Savas; Karaali, Zeynep; Kazancioglu, Rumeyza; Cefle, Kivanc; Gursu, Meltem; Campagnoli, Monica; Galliano, Monica; Minchiotti, Lorenzo

2010-11-11

Analbuminemia is a rare autosomal recessive disorder manifested by the absence, or severe reduction, of circulating serum albumin. The analbuminemic trait was diagnosed in a young Turkish woman on the basis of her clinical symptoms (bilateral lower limb edema) and biochemical findings (minimal albumin amount and variable increases in other protein fractions). Total DNA from the analbuminemic proband and her parents was PCR-amplified using oligonucleotide primers designed to amplify the 14 exons of the albumin gene (ALB) and the flanking intron regions. The products were screened for mutations by single-strand conformation polymorphism (SSCP) and heteroduplex analyses (HA). HA allowed the identification of the mutation site in exon 12. Direct DNA sequencing of this abnormal fragment revealed that the analbuminemic trait was caused by a homozygous CA deletion at nucleotide positions c. 1614-1615 in the codons for Cys538 and Thr539. The subsequent frameshift should give rise to a putative truncated albumin variant in which the sequence Cys(538)-Thr-Leu-Ser has been changed to Cys(538)-Thr-Phe-Stop. The parents were heterozygous for the same mutation. Gel-based mutation detection and DNA sequencing substantiate the clinical diagnosis of congenital analbuminemia in our patient and show that the condition is caused by a novel mutation within the ALB gene. These results contribute to shed light on the molecular basis of this rare condition. 2010 Elsevier B.V. All rights reserved.

A novel truncation mutation in CRYBB1 associated with autosomal dominant congenital cataract with nystagmus.

PubMed

Rao, Yan; Dong, Sufang; Li, Zuhua; Yang, Guohua; Peng, Chunyan; Yan, Ming; Zheng, Fang

2017-01-01

To identify the potential candidate genes for a large Chinese family with autosomal dominant congenital cataract (ADCC) and nystagmus, and investigate the possible molecular mechanism underlying the role of the candidate genes in cataractogenesis. We combined the linkage analysis and direct sequencing for the candidate genes in the linkage regions to identify the causative mutation. The molecular and bio-functional properties of the proteins encoded by the candidate genes was further explored with biophysical and biochemical studies of the recombinant wild-type and mutant proteins. We identified a c. C749T (p.Q227X) transversion in exon 6 of CRYBB1 , a cataract-causative gene. This nonsense mutation changes a phylogenetically conserved glutamine to a stop codon and is predicted to truncate the C-terminus of the wild-type protein by 26 amino acids. Comparison of the biophysical and biochemical properties of the recombinant full-length and truncated βB1-crystallins revealed that the mutation led to the insolubility and the phase separation phenomenon of the truncated protein with a changed conformation. Meanwhile, the thermal stability of the truncated βB1-crystallin was significantly decreased, and the mutation diminished the chaperoning ability of αA-crystallin with the mutant under heating stress. Our findings highlight the importance of the C-terminus in βB1-crystallin in maintaining the crystalline function and stability, and provide a novel insight into the molecular mechanism underlying the pathogenesis of human autosomal dominant congenital cataract.

The p16INK4alpha/p19ARF gene mutations are infrequent and are mutually exclusive to p53 mutations in Indian oral squamous cell carcinomas.

PubMed

Kannan, K; Munirajan, A K; Krishnamurthy, J; Bhuvarahamurthy, V; Mohanprasad, B K; Panishankar, K H; Tsuchida, N; Shanmugam, G

2000-03-01

Eighty-seven untreated primary oral squamous cell carcinomas (SCCs) associated with betel quid and tobacco chewing from Indian patients were analysed for the presence of mutations in the commonly shared exon 2 of p16INK4alpha/p19ARF genes. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing analysis were used to detect mutations. SSCP analysis indicated that only 9% (8/87) of the tumours had mutation in p16INK4alpha/p19ARF genes. Seventy-two tumours studied here were previously analysed for p53 mutations and 21% (15/72) of them were found to have mutations in p53 gene. Only one tumour was found to have mutation at both p53 and p16INK4alpha/p19ARF genes. Thus, the mutation rates observed were 21% for p53, 9% for p16INK4alpha/p19ARF, and 1% for both. Sequencing analysis revealed two types of mutations; i) G to C (GCAG to CCAG) transversion type mutation at intron 1-exon 2 splice junction and ii) another C to T transition type mutation resulting in CGA to TGA changing arginine to a termination codon at p16INK4alpha gene codon 80 and the same mutation will alter codon 94 of p19ARF gene from CCG to CTG (proline to leucine). These results suggest that p16INK4alpha/p19ARF mutations are less frequent than p53 mutations in Indian oral SCCs. The p53 and p16INK4alpha/p19ARF mutational events are independent and are mutually exclusive suggesting that mutational inactivation of either p53 or p16INK4alpha/p19ARF may alleviate the need for the inactivation of the other gene.

Comprehensive analysis of the codon usage patterns in the envelope glycoprotein E2 gene of the classical swine fever virus

PubMed Central

Chi, Xiaojuan; Wang, Song; Ma, Yanmei; Chen, Jilong

2017-01-01

The classical swine fever virus (CSFV), circulating worldwide, is a highly contagious virus. Since the emergence of CSFV, it has caused great economic loss in swine industry. The envelope glycoprotein E2 gene of the CSFV is an immunoprotective antigen that induces the immune system to produce neutralizing antibodies. Therefore, it is essential to study the codon usage of the E2 gene of the CSFV. In this study, 140 coding sequences of the E2 gene were analyzed. The value of effective number of codons (ENC) showed low codon usage bias in the E2 gene. Our study showed that codon usage could be described mainly by mutation pressure ENC plot analysis combined with principal component analysis (PCA) and translational selection-correlation analysis between the general average hydropathicity (Gravy) and aromaticity (Aroma), and nucleotides at the third position of codons (A3s, T3s, G3s, C3s and GC3s). Furthermore, the neutrality analysis, which explained the relationship between GC12s and GC3s, revealed that natural selection had a key role compared with mutational bias during the evolution of the E2 gene. These results lay a foundation for further research on the molecular evolution of CSFV. PMID:28880881

Comprehensive analysis of the codon usage patterns in the envelope glycoprotein E2 gene of the classical swine fever virus.

PubMed

Chen, Ye; Li, Xinxin; Chi, Xiaojuan; Wang, Song; Ma, Yanmei; Chen, Jilong

2017-01-01

The classical swine fever virus (CSFV), circulating worldwide, is a highly contagious virus. Since the emergence of CSFV, it has caused great economic loss in swine industry. The envelope glycoprotein E2 gene of the CSFV is an immunoprotective antigen that induces the immune system to produce neutralizing antibodies. Therefore, it is essential to study the codon usage of the E2 gene of the CSFV. In this study, 140 coding sequences of the E2 gene were analyzed. The value of effective number of codons (ENC) showed low codon usage bias in the E2 gene. Our study showed that codon usage could be described mainly by mutation pressure ENC plot analysis combined with principal component analysis (PCA) and translational selection-correlation analysis between the general average hydropathicity (Gravy) and aromaticity (Aroma), and nucleotides at the third position of codons (A3s, T3s, G3s, C3s and GC3s). Furthermore, the neutrality analysis, which explained the relationship between GC12s and GC3s, revealed that natural selection had a key role compared with mutational bias during the evolution of the E2 gene. These results lay a foundation for further research on the molecular evolution of CSFV.

Complete mitochondrial genome of Chocolate Pansy, Junonia iphita (Lepidoptera: Nymphalidae: Nymphalinae).

PubMed

Vanlalruati, Catherine; Mandal, Surajit De; Gurusubramanian, Guruswami; Senthil Kumar, Nachimuthu

2016-07-01

The complete mitochondrial genome of Junonia iphita was determined to be 15,433 bp in length, including 37 typical mitochondrial genes and an AT-rich region. All the protein coding genes (PCGs) are initiated by typical ATN codons, except cox1 gene that is by CGA codon. Eight genes use complete termination codon (TAA), whereas the cox1, cox2 and nad5 genes end with single T; nad4 and nad1 ends with stop codon TA. All the tRNA show secondary cloverleaf structures except trnS1 (AGN). The A + T rich region is 546 bp in length containing ATAGA motif followed by a 18 bp poly-T stretch, two microsatellite-like (TA)9 elements and 8 bp poly-A stretch immediately upstream of trnM gene.

Genome-wide comparative analysis of codon usage bias and codon context patterns among cyanobacterial genomes.

PubMed

Prabha, Ratna; Singh, Dhananjaya P; Sinha, Swati; Ahmad, Khurshid; Rai, Anil

2017-04-01

With the increasing accumulation of genomic sequence information of prokaryotes, the study of codon usage bias has gained renewed attention. The purpose of this study was to examine codon selection pattern within and across cyanobacterial species belonging to diverse taxonomic orders and habitats. We performed detailed comparative analysis of cyanobacterial genomes with respect to codon bias. Our analysis reflects that in cyanobacterial genomes, A- and/or T-ending codons were used predominantly in the genes whereas G- and/or C-ending codons were largely avoided. Variation in the codon context usage of cyanobacterial genes corresponded to the clustering of cyanobacteria as per their GC content. Analysis of codon adaptation index (CAI) and synonymous codon usage order (SCUO) revealed that majority of genes are associated with low codon bias. Codon selection pattern in cyanobacterial genomes reflected compositional constraints as major influencing factor. It is also identified that although, mutational constraint may play some role in affecting codon usage bias in cyanobacteria, compositional constraint in terms of genomic GC composition coupled with environmental factors affected codon selection pattern in cyanobacterial genomes. Copyright © 2016 Elsevier B.V. All rights reserved.

Codes in the codons: construction of a codon/amino acid periodic table and a study of the nature of specific nucleic acid-protein interactions.

PubMed

Benyo, B; Biro, J C; Benyo, Z

2004-01-01

The theory of "codon-amino acid coevolution" was first proposed by Woese in 1967. It suggests that there is a stereochemical matching - that is, affinity - between amino acids and certain of the base triplet sequences that code for those amino acids. We have constructed a common periodic table of codons and amino acids, where the nucleic acid table showed perfect axial symmetry for codons and the corresponding amino acid table also displayed periodicity regarding the biochemical properties (charge and hydrophobicity) of the 20 amino acids and the position of the stop signals. The table indicates that the middle (2/sup nd/) amino acid in the codon has a prominent role in determining some of the structural features of the amino acids. The possibility that physical contact between codons and amino acids might exist was tested on restriction enzymes. Many recognition site-like sequences were found in the coding sequences of these enzymes and as many as 73 examples of codon-amino acid co-location were observed in the 7 known 3D structures (December 2003) of endonuclease-nucleic acid complexes. These results indicate that the smallest possible units of specific nucleic acid-protein interaction are indeed the stereochemically compatible codons and amino acids.

Complete mitochondrial genome of Germain's Peacock-Pheasant Polyplectron germaini (Aves, Galliformes, Phasianidae).

PubMed

Omeire, Destiny; Abdin, Shaunte; Brooks, Daniel M; Miranda, Hector C

2015-04-01

The Germain's Peacock-Pheasant Polyplectron germaini (Aves, Galliformes, Phasianidae) is classified as Near Threatened on the IUCN Red List. The complete mitochondrial genome of P. germaini is 16,699 bp, consisting of 13 protein-coding genes, 2 rRNA, 22 tRNA genes and 1 control region. All of the 13 protein-coding genes have ATG as start codon. Eight of the 13 protein-coding genes have TAA as stop codon.

Autism-Associated Insertion Mutation (InsG) of Shank3 Exon 21 Causes Impaired Synaptic Transmission and Behavioral Deficits

PubMed Central

Speed, Haley E.; Kouser, Mehreen; Xuan, Zhong; Reimers, Jeremy M.; Ochoa, Christine F.; Gupta, Natasha; Liu, Shunan

2015-01-01

SHANK3 (also known as PROSAP2) is a postsynaptic scaffolding protein at excitatory synapses in which mutations and deletions have been implicated in patients with idiopathic autism, Phelan–McDermid (aka 22q13 microdeletion) syndrome, and other neuropsychiatric disorders. In this study, we have created a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3G). The resulting frameshift causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse. Shank3G/G mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, altered response to novelty, and sensory processing deficits. At the cellular level, Shank3G/G mice also exhibit impaired hippocampal excitatory transmission and plasticity as well as changes in baseline NMDA receptor-mediated synaptic responses. This work identifies clear alterations in synaptic function and behavior in a novel, genetically accurate mouse model of autism mimicking an autism-associated insertion mutation. Furthermore, these findings lay the foundation for future studies aimed to validate and study region-selective and temporally selective genetic reversal studies in the Shank3G/G mouse that was engineered with such future experiments in mind. PMID:26134648

WNT10A mutation results in severe tooth agenesis in a family of three sisters.

PubMed

Abid, M F; Simpson, M A; Barbosa, I A; Seppala, M; Irving, M; Sharpe, P T; Cobourne, M T

2018-06-21

To identify the genetic basis of severe tooth agenesis in a family of three affected sisters. A family of three sisters with severe tooth agenesis was recruited for whole-exome sequencing to identify potential genetic variation responsible for this penetrant phenotype. The unaffected father was tested for specific mutations using Sanger sequencing. Gene discovery was supplemented with in situ hybridization to localize gene expression during human tooth development. We report a nonsense heterozygous mutation in exon 2 of WNT10A c.321C>A[p.Cys107*] likely to be responsible for the severe tooth agenesis identified in this family through the creation of a premature stop codon, resulting in truncation of the amino acid sequence and therefore loss of protein function. In situ hybridization showed expression of WNT10A in odontogenic epithelium during the early and late stages of human primary tooth development. WNT10A has previously been associated with both syndromic and non-syndromic forms of tooth agenesis, and this report further expands our knowledge of genetic variation underlying non-syndromic forms of this condition. We also demonstrate expression of WNT10A in the epithelial compartment of human tooth germs during development. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Reengineering a transmembrane protein to treat muscular dystrophy using exon skipping.

PubMed

Gao, Quan Q; Wyatt, Eugene; Goldstein, Jeff A; LoPresti, Peter; Castillo, Lisa M; Gazda, Alec; Petrossian, Natalie; Earley, Judy U; Hadhazy, Michele; Barefield, David Y; Demonbreun, Alexis R; Bönnemann, Carsten; Wolf, Matthew; McNally, Elizabeth M

2015-11-02

Exon skipping uses antisense oligonucleotides as a treatment for genetic diseases. The antisense oligonucleotides used for exon skipping are designed to bypass premature stop codons in the target RNA and restore reading frame disruption. Exon skipping is currently being tested in humans with dystrophin gene mutations who have Duchenne muscular dystrophy. For Duchenne muscular dystrophy, the rationale for exon skipping derived from observations in patients with naturally occurring dystrophin gene mutations that generated internally deleted but partially functional dystrophin proteins. We have now expanded the potential for exon skipping by testing whether an internal, in-frame truncation of a transmembrane protein γ-sarcoglycan is functional. We generated an internally truncated γ-sarcoglycan protein that we have termed Mini-Gamma by deleting a large portion of the extracellular domain. Mini-Gamma provided functional and pathological benefits to correct the loss of γ-sarcoglycan in a Drosophila model, in heterologous cell expression studies, and in transgenic mice lacking γ-sarcoglycan. We generated a cellular model of human muscle disease and showed that multiple exon skipping could be induced in RNA that encodes a mutant human γ-sarcoglycan. Since Mini-Gamma represents removal of 4 of the 7 coding exons in γ-sarcoglycan, this approach provides a viable strategy to treat the majority of patients with γ-sarcoglycan gene mutations.

PLEKHG5 deficiency leads to an intermediate form of autosomal-recessive Charcot–Marie–Tooth disease

PubMed Central

Azzedine, Hamid; Zavadakova, Petra; Planté-Bordeneuve, Violaine; Vaz Pato, Maria; Pinto, Nuno; Bartesaghi, Luca; Zenker, Jennifer; Poirot, Olivier; Bernard-Marissal, Nathalie; Arnaud Gouttenoire, Estelle; Cartoni, Romain; Title, Alexandra; Venturini, Giulia; Médard, Jean-Jacques; Makowski, Edward; Schöls, Ludger; Claeys, Kristl G.; Stendel, Claudia; Roos, Andreas; Weis, Joachim; Dubourg, Odile; Leal Loureiro, José; Stevanin, Giovanni; Said, Gérard; Amato, Anthony; Baraban, Jay; LeGuern, Eric; Senderek, Jan; Rivolta, Carlo; Chrast, Roman

2013-01-01

Charcot–Marie–Tooth disease (CMT) comprises a clinically and genetically heterogeneous group of peripheral neuropathies characterized by progressive distal muscle weakness and atrophy, foot deformities and distal sensory loss. Following the analysis of two consanguineous families affected by a medium to late-onset recessive form of intermediate CMT, we identified overlapping regions of homozygosity on chromosome 1p36 with a combined maximum LOD score of 5.4. Molecular investigation of the genes from this region allowed identification of two homozygous mutations in PLEKHG5 that produce premature stop codons and are predicted to result in functional null alleles. Analysis of Plekhg5 in the mouse revealed that this gene is expressed in neurons and glial cells of the peripheral nervous system, and that knockout mice display reduced nerve conduction velocities that are comparable with those of affected individuals from both families. Interestingly, a homozygous PLEKHG5 missense mutation was previously reported in a recessive form of severe childhood onset lower motor neuron disease (LMND) leading to loss of the ability to walk and need for respiratory assistance. Together, these observations indicate that different mutations in PLEKHG5 lead to clinically diverse outcomes (intermediate CMT or LMND) affecting the function of neurons and glial cells. PMID:23777631

A nonsense mutation of PEPD in four Amish children with prolidase deficiency.

PubMed

Wang, Heng; Kurien, Biji T; Lundgren, David; Patel, Nisha C; Kaufman, K M; Miller, David L; Porter, Andrew C; D'Souza, Anil; Nye, Leah; Tumbush, John; Hupertz, Vera; Kerr, Douglas S; Kurono, S; Matsumoto, H; Scofield, R Hal

2006-03-15

Encoded by the peptidase D (PEPD) gene located at 19q12-q13.11, prolidase is a ubiquitous cytosolic enzyme that catalyzes hydrolysis of oligopeptides with a C-terminal proline or hydroxyproline. We describe here four Amish children with a severe phenotype of prolidase deficiency in the Geauga settlements of Ohio as the first report of prolidase deficiency in the Amish population as well as in the United States. The patients presented with infection, hepatosplenomegaly, or thrombocytopenia, in contrast to most cases previously reported in the literature, presenting with skin ulcers. All four patients had typical facial features, classic skin ulcers, and multisystem involvement. Recurrent infections, asthma-like chronic reactive airway disease, hyperimmunoglobulins, hepatosplenomegaly with mildly elevated aspartate transaminase (AST), anemia, and thrombocytopenia were common and massive imidodipeptiduria was universal. Prolidase activity in our patients is nearly undetectable. Direct sequencing of PCR-amplified genomic DNA for all of the exons from the four patients revealed the same homozygous single nucleotide mutation c.793 T > C in exon 11, resulting in a premature stop-codon at amino acid residue 265 (p.R265X). It is speculated that the severe phenotype in these patients might be associated with the type of the PEPD gene mutation. 2006 Wiley-Liss, Inc.

Genetic diagnosis as a tool for personalized treatment of Duchenne muscular dystrophy.

PubMed

Bello, Luca; Pegoraro, Elena

2016-12-01

Accurate definition of genetic mutations causing Duchenne muscular dystrophy (DMD) has always been relevant in order to provide genetic counseling to patients and families, and helps to establish the prognosis in the case where the distinction between Duchenne, Becker, or intermediate muscular dystrophy is not obvious. As molecular treatments aimed at dystrophin restoration in DMD are increasingly available as commercialized drugs or within clinical trials, genetic diagnosis has become an indispensable tool in order to determine eligibility for these treatments. DMD patients in which multiplex ligation-dependent probe amplification (MLPA) or similar techniques show a deletion suitable to exon skipping of exons 44, 45, 51, or 53, may be currently treated with AONs targeting these exons, in the context of clinical trials, or, as is the case for exon 51 skipping in the United States, with the first commercialized drug (eteplirsen). Patients who test negative at MLPA, but in whom DMD gene sequencing shows a nonsense mutation, may be amenable for treatment with stop codon readthrough compounds such as ataluren. Novel molecular approaches such as CRISPR-Cas9 targeting of specific DMD mutations are still in the preclinical stages, but appear promising. In conclusion, an accurate genetic diagnosis represents the entrance into a new scenario of personalized medicine in DMD.

Reengineering a transmembrane protein to treat muscular dystrophy using exon skipping

PubMed Central

Gao, Quan Q.; Wyatt, Eugene; Goldstein, Jeff A.; LoPresti, Peter; Castillo, Lisa M.; Gazda, Alec; Petrossian, Natalie; Earley, Judy U.; Hadhazy, Michele; Barefield, David Y.; Demonbreun, Alexis R.; Bönnemann, Carsten; Wolf, Matthew; McNally, Elizabeth M.

2015-01-01

Exon skipping uses antisense oligonucleotides as a treatment for genetic diseases. The antisense oligonucleotides used for exon skipping are designed to bypass premature stop codons in the target RNA and restore reading frame disruption. Exon skipping is currently being tested in humans with dystrophin gene mutations who have Duchenne muscular dystrophy. For Duchenne muscular dystrophy, the rationale for exon skipping derived from observations in patients with naturally occurring dystrophin gene mutations that generated internally deleted but partially functional dystrophin proteins. We have now expanded the potential for exon skipping by testing whether an internal, in-frame truncation of a transmembrane protein γ-sarcoglycan is functional. We generated an internally truncated γ-sarcoglycan protein that we have termed Mini-Gamma by deleting a large portion of the extracellular domain. Mini-Gamma provided functional and pathological benefits to correct the loss of γ-sarcoglycan in a Drosophila model, in heterologous cell expression studies, and in transgenic mice lacking γ-sarcoglycan. We generated a cellular model of human muscle disease and showed that multiple exon skipping could be induced in RNA that encodes a mutant human γ-sarcoglycan. Since Mini-Gamma represents removal of 4 of the 7 coding exons in γ-sarcoglycan, this approach provides a viable strategy to treat the majority of patients with γ-sarcoglycan gene mutations. PMID:26457733

A Frameshift Mutation within LAMC2 Is Responsible for Herlitz Type Junctional Epidermolysis Bullosa (HJEB) in Black Headed Mutton Sheep

PubMed Central

Mömke, Stefanie; Kerkmann, Andrea; Wöhlke, Anne; Ostmeier, Miriam; Hewicker-Trautwein, Marion; Ganter, Martin; Kijas, James; Distl, Ottmar

2011-01-01

Junctional epidermolysis bullosa (JEB) is a hereditary mechanobullous skin disease in humans and animals. A Herlitz type JEB was identified in German Black Headed Mutton (BHM) sheep and affected lambs were reproduced in a breeding trial. Affected lambs showed skin and mucous membranes blistering and all affected lambs died within the first weeks of life. The pedigree data were consistent with a monogenic autosomal recessive inheritance. Immunofluorescence showed a reduced expression of laminin 5 protein which consists of 3 subunits encoded by the genes LAMA3, LAMB3 and LAMC2. We screened these genes for polymorphisms. Linkage and genome-wide association analyses identified LAMC2 as the most likely candidate for HJEB. A two base pair deletion within exon 18 of the LAMC2 gene (FM872310:c.2746delCA) causes a frameshift mutation resulting in a premature stop codon (p.A928*) 13 triplets downstream of this mutation and in addition, introduces an alternative splicing of exon 18 LAMC2. This deletion showed a perfect co-segregation with HJEB in all 740 analysed BHM sheep. Identification of the LAMC2 deletion means an animal model for HJEB is now available to develop therapeutic approaches of relevance to the human form of this disease. PMID:21573221

Interest of proviral HIV-1 DNA genotypic resistance testing in virologically suppressed patients candidate for maintenance therapy.

PubMed

Allavena, C; Rodallec, A; Leplat, A; Hall, N; Luco, C; Le Guen, L; Bernaud, C; Bouchez, S; André-Garnier, E; Boutoille, D; Ferré, V; Raffi, F

2018-01-01

Switch of antiretroviral therapy in virologically suppressed HIV-infected patients is frequent, to prevent toxicities, for simplification or convenience reasons. Pretherapeutic genotypic resistance testing on RNA can be lacking in some patients, which could enhance the risk of virologic failure, if resistance-associated mutations of the new regimen are not taken into account. Proviral DNA resistance testing in 69 virologically suppressed patients on antiretroviral treatment with no history of virological failure were pair-wised compared with pre-ART plasma RNA resistance testing. The median time between plasma (RNA testing) and whole blood (proviral DNA testing) was 47 months (IQR 29-63). A stop codon was evidenced in 23% (16/69) of proviral DNA sequences; these strains were considered as defective, non-replicative, and not taken into consideration. Within the non defective strains, concordance rate between plasma RNA and non-defective proviral DNA was high both on protease (194/220 concordant resistance-associated mutations=88%) and reverse transcriptase (28/37 concordant resistance-associated mutations=76%) genes. This study supports that proviral DNA testing might be an informative tool before switching antiretrovirals in virologically suppressed patients with no history of virological failure, but the interpretation should be restricted to non-defective viruses. Copyright © 2017 Elsevier B.V. All rights reserved.

Linkage and mutational analysis of familial Alzheimer disease kindreds for the APP gene region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamino, K.; Anderson, L.; O'dahl, S.

1992-11-01

A large number of familial Alzheimer disease (FAD) kindreds were examined to determine whether mutations in the amyloid precursor protein (APP) gene could be responsible for the disease. Previous studies have identified three mutations at APP codon 717 which are pathogenic for Alzheimer disease (AD). Samples from affected subjects were examined for mutations in exons 16 and 17 of the APP gene. A combination of direct sequencing and single-strand conformational polymorphism analysis was used. Sporadic AD and normal controls were also examined by the same methods. Five sequence variants were identified. One variant at APP codon 693 resulted in amore » Glu[yields]Gly change. This is the same codon as the hereditary cerebral hemorrhage with amyloidosis-Dutch type Glu[yields]Gln mutation. Another single-base change at APP codon 708 did not alter the amino acid encoded at this site. Two point mutations and a 6-bp deletion were identified in the intronic sequences surrounding exon 17. None of the variants could be unambigously determined to be responsible for FAD. The larger families were also analyzed by testing for linkage of FAD to a highly polymorphic short tandem repeat marker (D21S210) that is tightly linked to APP. Highly negative LOD scores were obtained for the family groups tested, and linkage was formally excluded beyond [theta] = .10 for the Volga German kindreds, [theta] = .20 for early-onset non-Volga Germans, and [theta] = .10 for late-onset families. LOD scores for linkage of FAD to markers centromeric to APP (D21S1/S11, D21S13, and D21S215) were also negative in the three family groups. These studies show that APP mutations account for AD in only a small fraction of FAD kindreds. 49 refs., 6 figs., 4 tabs.« less

COL4A3/COL4A4 mutations and features in individuals with autosomal recessive Alport syndrome.

PubMed

Storey, Helen; Savige, Judy; Sivakumar, Vanessa; Abbs, Stephen; Flinter, Frances A

2013-12-01

Alport syndrome is an inherited disease characterized by hematuria, progressive renal failure, hearing loss, and ocular abnormalities. Autosomal recessive Alport syndrome is suspected in consanguineous families and when female patients develop renal failure. Fifteen percent of patients with Alport syndrome have autosomal recessive inheritance caused by two pathogenic mutations in either COL4A3 or COL4A4. Here, we describe the mutations and clinical features in 40 individuals including 9 children and 21 female individuals (53%) with autosomal recessive inheritance indicated by the detection of two mutations. The median age was 31 years (range, 6-54 years). The median age at end stage renal failure was 22.5 years (range, 10-38 years), but renal function was normal in nine adults (29%). Hearing loss and ocular abnormalities were common (23 of 35 patients [66%] and 10 of 18 patients [56%], respectively). Twenty mutation pairs (50%) affected COL4A3 and 20 pairs affected COL4A4. Of the 68 variants identified, 39 were novel, 12 were homozygous changes, and 9 were present in multiple individuals, including c.2906C>G (p.(Ser969*)) in COL4A4, which was found in 23% of the patients. Thirty-six variants (53%) resulted directly or indirectly in a stop codon, and all 17 individuals with early onset renal failure had at least one such mutation, whereas these mutations were less common in patients with normal renal function or late-onset renal failure. In conclusion, patient phenotypes may vary depending on the underlying mutations, and genetic testing should be considered for the routine diagnosis of autosomal recessive Alport syndrome.

COL4A3/COL4A4 Mutations and Features in Individuals with Autosomal Recessive Alport Syndrome

PubMed Central

Savige, Judy; Sivakumar, Vanessa; Abbs, Stephen; Flinter, Frances A.

2013-01-01

Alport syndrome is an inherited disease characterized by hematuria, progressive renal failure, hearing loss, and ocular abnormalities. Autosomal recessive Alport syndrome is suspected in consanguineous families and when female patients develop renal failure. Fifteen percent of patients with Alport syndrome have autosomal recessive inheritance caused by two pathogenic mutations in either COL4A3 or COL4A4. Here, we describe the mutations and clinical features in 40 individuals including 9 children and 21 female individuals (53%) with autosomal recessive inheritance indicated by the detection of two mutations. The median age was 31 years (range, 6–54 years). The median age at end stage renal failure was 22.5 years (range, 10–38 years), but renal function was normal in nine adults (29%). Hearing loss and ocular abnormalities were common (23 of 35 patients [66%] and 10 of 18 patients [56%], respectively). Twenty mutation pairs (50%) affected COL4A3 and 20 pairs affected COL4A4. Of the 68 variants identified, 39 were novel, 12 were homozygous changes, and 9 were present in multiple individuals, including c.2906C>G (p.(Ser969*)) in COL4A4, which was found in 23% of the patients. Thirty-six variants (53%) resulted directly or indirectly in a stop codon, and all 17 individuals with early onset renal failure had at least one such mutation, whereas these mutations were less common in patients with normal renal function or late-onset renal failure. In conclusion, patient phenotypes may vary depending on the underlying mutations, and genetic testing should be considered for the routine diagnosis of autosomal recessive Alport syndrome. PMID:24052634

A novel CYP27B1 mutation causes a feline vitamin D-dependent rickets type IA.

PubMed

Grahn, Robert A; Ellis, Melanie R; Grahn, Jennifer C; Lyons, Leslie A

2012-08-01

A 12-week-old domestic cat presented at a local veterinary clinic with hypocalcemia and skeletal abnormalities suggestive of rickets. Osteomalacia (rickets) is a disease caused by impaired bone mineralization leading to an increased prevalence of fractures and deformity. Described in a variety of species, rickets is most commonly caused by vitamin D or calcium deficiencies owing to both environmental and or genetic abnormalities. Vitamin D-dependent rickets type 1A (VDDR-1A) is a result of the enzymatic pathway defect caused by mutations in the 25-hydroxyvitamin D(3)-1-alpha-hydroxylase gene [cytochrome P27 B1 (CYP27B1)]. Calcitriol, the active form of vitamin D(3), regulates calcium homeostasis, which requires sufficient dietary calcium availability and correct hormonal function for proper bone growth and maintenance. Patient calcitriol concentrations were low while calcidiol levels were normal suggestive of VDDR-1A. The entire DNA coding sequencing of CYP27B1 was evaluated. The affected cat was wild type for previously identified VDDR-1A causative mutations. However, six novel mutations were identified, one of which was a nonsense mutation at G637T in exon 4. The exon 4 G637T nonsense mutation results in a premature protein truncation, changing a glutamic acid to a stop codon, E213X, likely causing the clinical presentation of rickets. The previously documented genetic mutation resulting in feline VDDR-1A rickets, as well as the case presented in this research, result from novel exon 4 CYP27B1 mutations, thus exon 4 should be the initial focus of future sequencing efforts.

Phenotypic Variation in Patients with Homozygous c.1678G>T Mutation in EVC Gene: Report of Two Mexican Families with Ellis-van Creveld Syndrome

PubMed Central

Ibarra-Ramirez, Marisol; Campos-Acevedo, Luis Daniel; Lugo-Trampe, Jose; Martínez-Garza, Laura E.; Martinez-Glez, Víctor; Valencia-Benitez, María; Lapunzina, Pablo; Ruiz-Peréz, Víctor

2017-01-01

Case series Patient: — Final Diagnosis: Ellis van Creveld syndrome Symptoms: Conical teeth • polydactyly • short stature Medication: — Clinical Procedure: — Specialty: Pediatrics and Neonatology Objective: Rare disease Background: Ellis-van Creveld syndrome is an autosomal recessive chondro-ectodermal dysplasia characterized by disproportionate short stature, limb shortening, narrow chest, postaxial polydactyly and dysplastic nails and teeth. In addition, 60% of cases present congenital heart defects. Ellis-van Creveld syndrome is predominantly caused by mutations in the EVC or EVC2 (4p16) genes, with only a few cases caused by mutations in WDR35. Case Report: Here, we report on two Mexican families with patients diagnosed with Ellis-van Creveld syndrome. Family 1 includes four patients: three females of 15, 18, and 23 years of age and a 7-year old male. Family 2 has only one affected newborn male. All patients exhibited multiple features including hypodontia, dysplastic teeth, extra frenula, mild short stature, distal limb shortening, postaxial polydactyly of hands and feet, nail dystrophy, and knee joint abnormalities. Only two patients had an atrial septal defect. In all cases, molecular analysis by Sanger sequencing identified the same homozygous mutation in exon 12 of EVC, c.1678G>T, which leads to a premature stop codon. Conclusions: The mutation c.1678G>T has been previously reported in another Mexican patient and it appears to be a recurrent mutation in Mexico which could represent a founder mutation. The large number of patients in this case allows the clinical variability and spectrum of manifestations present in individuals with Ellis-van Creveld syndrome even if they carry the same homozygous mutation in a same family. PMID:29229899

Within-host whole genome analysis of an antibiotic resistant Pseudomonas aeruginosa strain sub-type in cystic fibrosis.

PubMed

Sherrard, Laura J; Tai, Anna S; Wee, Bryan A; Ramsay, Kay A; Kidd, Timothy J; Ben Zakour, Nouri L; Whiley, David M; Beatson, Scott A; Bell, Scott C

2017-01-01

A Pseudomonas aeruginosa AUST-02 strain sub-type (M3L7) has been identified in Australia, infects the lungs of some people with cystic fibrosis and is associated with antibiotic resistance. Multiple clonal lineages may emerge during treatment with mutations in chromosomally encoded antibiotic resistance genes commonly observed. Here we describe the within-host diversity and antibiotic resistance of M3L7 during and after antibiotic treatment of an acute pulmonary exacerbation using whole genome sequencing and show both variation and shared mutations in important genes. Eleven isolates from an M3L7 population (n = 134) isolated over 3 months from an individual with cystic fibrosis underwent whole genome sequencing. A phylogeny based on core genome SNPs identified three distinct phylogenetic groups comprising two groups with higher rates of mutation (hypermutators) and one non-hypermutator group. Genomes were screened for acquired antibiotic resistance genes with the result suggesting that M3L7 resistance is principally driven by chromosomal mutations as no acquired mechanisms were detected. Small genetic variations, shared by all 11 isolates, were found in 49 genes associated with antibiotic resistance including frame-shift mutations (mexA, mexT), premature stop codons (oprD, mexB) and mutations in quinolone-resistance determining regions (gyrA, parE). However, whole genome sequencing also revealed mutations in 21 genes that were acquired following divergence of groups, which may also impact the activity of antibiotics and multi-drug efflux pumps. Comparison of mutations with minimum inhibitory concentrations of anti-pseudomonal antibiotics could not easily explain all resistance profiles observed. These data further demonstrate the complexity of chronic and antibiotic resistant P. aeruginosa infection where a multitude of co-existing genotypically diverse sub-lineages might co-exist during and after intravenous antibiotic treatment.

Efficient initiation of mammalian mRNA translation at a CUG codon.

PubMed Central

Dasso, M C; Jackson, R J

1989-01-01

Nucleotide substitutions were made at the initiation codon of an influenza virus NS cDNA clone in a vector carrying the bacteriophage T7 promoter. When capped mRNA transcripts of these constructs were translated in the rabbit reticulocyte lysate, a change in the initiation codon from...AUAAUGG...to...AUACUGG...reduced the in vitro translational efficiency by only 50-60%, and resulted in only a small increase in the yield of short products presumed to be initiated at downstream sites. Synthesis of the full-length product was initiated exclusively at the mutated codon, with negligible use either of in-frame upstream CUG or GUG codons, or of an in-frame downstream GUG codon. We conclude that CUG has the potential to function as an efficient initiation codon in mammalian systems, at least in certain contexts. Images PMID:2780285

Genome-wide analysis of codon usage bias in Ebolavirus.

PubMed

Cristina, Juan; Moreno, Pilar; Moratorio, Gonzalo; Musto, Héctor

2015-01-22

Ebola virus (EBOV) is a member of the family Filoviridae and its genome consists of a 19-kb, single-stranded, negative sense RNA. EBOV is subdivided into five distinct species with different pathogenicities, being Zaire ebolavirus (ZEBOV) the most lethal species. The interplay of codon usage among viruses and their hosts is expected to affect overall viral survival, fitness, evasion from host's immune system and evolution. In the present study, we performed comprehensive analyses of codon usage and composition of ZEBOV. Effective number of codons (ENC) indicates that the overall codon usage among ZEBOV strains is slightly biased. Different codon preferences in ZEBOV genes in relation to codon usage of human genes were found. Highly preferred codons are all A-ending triplets, which strongly suggests that mutational bias is a main force shaping codon usage in ZEBOV. Dinucleotide composition also plays a role in the overall pattern of ZEBOV codon usage. ZEBOV does not seem to use the most abundant tRNAs present in the human cells for most of their preferred codons. Copyright © 2014 Elsevier B.V. All rights reserved.

Quantitative Detection and Resolution of BRAF V600 Status in Colorectal Cancer Using Droplet Digital PCR and a Novel Wild-Type Negative Assay.

PubMed

Bidshahri, Roza; Attali, Dean; Fakhfakh, Kareem; McNeil, Kelly; Karsan, Aly; Won, Jennifer R; Wolber, Robert; Bryan, Jennifer; Hughesman, Curtis; Haynes, Charles

2016-03-01

A need exists for robust and cost-effective assays to detect a single or small set of actionable point mutations, or a complete set of clinically informative mutant alleles. Knowledge of these mutations can be used to alert the clinician to a rare mutation that might necessitate more aggressive clinical monitoring or a personalized course of treatment. An example is BRAF, a (proto)oncogene susceptible to either common or rare mutations in codon V600 and adjacent codons. We report a diagnostic technology that leverages the unique capabilities of droplet digital PCR to achieve not only accurate and sensitive detection of BRAF(V600E) but also all known somatic point mutations within the BRAF V600 codon. The simple and inexpensive two-well droplet digital PCR assay uses a chimeric locked nucleic acid/DNA probe against wild-type BRAF and a novel wild-type-negative screening paradigm. The assay shows complete diagnostic accuracy when applied to formalin-fixed, paraffin-embedded tumor specimens from metastatic colorectal cancer patients deficient for Mut L homologue-1. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Whole-Genome Sequencing of Sordaria macrospora Mutants Identifies Developmental Genes.

PubMed

Nowrousian, Minou; Teichert, Ines; Masloff, Sandra; Kück, Ulrich

2012-02-01

The study of mutants to elucidate gene functions has a long and successful history; however, to discover causative mutations in mutants that were generated by random mutagenesis often takes years of laboratory work and requires previously generated genetic and/or physical markers, or resources like DNA libraries for complementation. Here, we present an alternative method to identify defective genes in developmental mutants of the filamentous fungus Sordaria macrospora through Illumina/Solexa whole-genome sequencing. We sequenced pooled DNA from progeny of crosses of three mutants and the wild type and were able to pinpoint the causative mutations in the mutant strains through bioinformatics analysis. One mutant is a spore color mutant, and the mutated gene encodes a melanin biosynthesis enzyme. The causative mutation is a G to A change in the first base of an intron, leading to a splice defect. The second mutant carries an allelic mutation in the pro41 gene encoding a protein essential for sexual development. In the mutant, we detected a complex pattern of deletion/rearrangements at the pro41 locus. In the third mutant, a point mutation in the stop codon of a transcription factor-encoding gene leads to the production of immature fruiting bodies. For all mutants, transformation with a wild type-copy of the affected gene restored the wild-type phenotype. Our data demonstrate that whole-genome sequencing of mutant strains is a rapid method to identify developmental genes in an organism that can be genetically crossed and where a reference genome sequence is available, even without prior mapping information.

Whole-Genome Sequencing of Sordaria macrospora Mutants Identifies Developmental Genes

PubMed Central

Nowrousian, Minou; Teichert, Ines; Masloff, Sandra; Kück, Ulrich

2012-01-01

The study of mutants to elucidate gene functions has a long and successful history; however, to discover causative mutations in mutants that were generated by random mutagenesis often takes years of laboratory work and requires previously generated genetic and/or physical markers, or resources like DNA libraries for complementation. Here, we present an alternative method to identify defective genes in developmental mutants of the filamentous fungus Sordaria macrospora through Illumina/Solexa whole-genome sequencing. We sequenced pooled DNA from progeny of crosses of three mutants and the wild type and were able to pinpoint the causative mutations in the mutant strains through bioinformatics analysis. One mutant is a spore color mutant, and the mutated gene encodes a melanin biosynthesis enzyme. The causative mutation is a G to A change in the first base of an intron, leading to a splice defect. The second mutant carries an allelic mutation in the pro41 gene encoding a protein essential for sexual development. In the mutant, we detected a complex pattern of deletion/rearrangements at the pro41 locus. In the third mutant, a point mutation in the stop codon of a transcription factor-encoding gene leads to the production of immature fruiting bodies. For all mutants, transformation with a wild type-copy of the affected gene restored the wild-type phenotype. Our data demonstrate that whole-genome sequencing of mutant strains is a rapid method to identify developmental genes in an organism that can be genetically crossed and where a reference genome sequence is available, even without prior mapping information. PMID:22384404

Codon Usage Selection Can Bias Estimation of the Fraction of Adaptive Amino Acid Fixations.

PubMed

Matsumoto, Tomotaka; John, Anoop; Baeza-Centurion, Pablo; Li, Boyang; Akashi, Hiroshi

2016-06-01

A growing number of molecular evolutionary studies are estimating the proportion of adaptive amino acid substitutions (α) from comparisons of ratios of polymorphic and fixed DNA mutations. Here, we examine how violations of two of the model assumptions, neutral evolution of synonymous mutations and stationary base composition, affect α estimation. We simulated the evolution of coding sequences assuming weak selection on synonymous codon usage bias and neutral protein evolution, α = 0. We show that weak selection on synonymous mutations can give polymorphism/divergence ratios that yield α-hat (estimated α) considerably larger than its true value. Nonstationary evolution (changes in population size, selection, or mutation) can exacerbate such biases or, in some scenarios, give biases in the opposite direction, α-hat < α. These results demonstrate that two factors that appear to be prevalent among taxa, weak selection on synonymous mutations and non-steady-state nucleotide composition, should be considered when estimating α. Estimates of the proportion of adaptive amino acid fixations from large-scale analyses of Drosophila melanogaster polymorphism and divergence data are positively correlated with codon usage bias. Such patterns are consistent with α-hat inflation from weak selection on synonymous mutations and/or mutational changes within the examined gene trees. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Alexander Disease: A Novel Mutation in GFAP Leading to Epilepsia Partialis Continua.

PubMed

Bonthius, Daniel J; Karacay, Bahri

2016-06-01

Alexander disease is a genetically induced leukodystrophy, due to dominant mutations in the glial fibrillary acidic protein (GFAP ) gene, causing dysfunction of astrocytes. We have identified a novel GFAP mutation, associated with a novel phenotype for Alexander disease. A boy with global developmental delay and hypertonia was found to have a leukodystrophy. Genetic analysis revealed a heterozygous point mutation in exon 6 of the GFAP gene. The guanine-to-adenine change causes substitution of the normal glutamic acid codon (GAG) with a mutant lysine codon (AAG) at position 312 (E312 K mutation). At the age of 4 years, the child developed epilepsia partialis continua, consisting of unabating motor seizures involving the unilateral perioral muscles. Epilepsia partialis continua has not previously been reported in association with Alexander disease. Whether and how the E312 K mutation produces pathologic changes and clinical signs that are unique from other Alexander disease-inducing mutations in GFAP remain to be determined. © The Author(s) 2015.

Incidence of Ganciclovir Resistance in CMV-positive Renal Transplant Recipients and its Association with UL97 Gene Mutations.

PubMed

Aslani, Hamid Reza; Ziaie, Shadi; Salamzadeh, Jamshid; Zaheri, Sara; Samadian, Fariba; Mastoor-Tehrani, Shayan

2017-01-01

Human cytomegalovirus (CMV) remains the most common infection affecting organ transplant recipients. Despite advances in the prophylaxis and acute treatment of CMV, it remains an important pathogen affecting the short- and long-term clinical outcome of solid organ transplant recipient. The emergence of CMV resistance in a patient reduces the clinical efficacy of antiviral therapy, complicates therapeutic and clinical management decisions, and in some cases results in loss of the allograft and/or death of the patient. Common mechanisms of CMV resistance to ganciclovir have been described chiefly with the UL97 mutations. Here we evaluate Incidence of ganciclovir resistance in 144 CMV-positive renal transplant recipients and its association with UL97 gene mutations. Active CMV infection was monitored by viral DNA quantification in whole blood, and CMV resistance was assessed by UL97 gene sequencing. Six mutations in six patients were detected. Three patients (2.6%) of 112 patients with history of ganciclovir (GCV) treatment had clinical resistance with single UL97 mutations at loci known to be related to resistance (including mutations at codon 594, codon 460, and codon 520). three patients who were anti-CMV drug naïve had single UL97 mutations (D605E) without clinical resistance. Our results confirm and extend our earlier findings on the specific mutations in the UL97 phosphotransferase gene in loci that have established role in ganciclovir resistance and also indicate that clinical ganciclovir resistance due to UL97 gene mutations is an issue in subjects with history of with ganciclovir treatment. D605E mutations remains a controversial issue that needs further investigations.

Prognostic value of mutational characteristics in gastrointestinal stromal tumors: a single-center experience in 275 cases.

PubMed

Wang, Ming; Xu, Jia; Zhao, Wenyi; Tu, Lin; Qiu, Weiqing; Wang, Chaojie; Shen, Yangyin; Liu, Qiang; Cao, Hui

2014-01-01

The objective of this study was to investigate the impact of KIT/PDGFRA mutations on the prognosis of gastrointestinal stromal tumors (GISTs). In the present study, genotype analyses were performed based on GIST samples from 275 patients. The relationship between mutation and clinicopathological characteristics were explored. All factors were evaluated for their impacts on relapse-free survival (RFS). Briefly, the results of genotype analyses showed that mutations were identified in 258 (93.8 %) patients, and deletion was the most frequent type of mutation accounting for 47.3 % (122/258) of all mutation cases, followed by substitution (87/258, 33.7 %) and duplication (49/258, 19.0 %). Moreover, for KIT exon 11 mutation, the most frequently involved area was from codon 557 to 560. Deep analyses showed that the mutation types were correlated with tumor location (P = 0.005), tumor size (P = 0.022), mitosis rate (P < 0.001), risk grade (P < 0.001), and relapse (P = 0.004). Furthermore, delW557-K558 correlated with mitosis rate (P = 0.042) and relapse (P = 0.036), and delTyr568/570 correlated with tumor origin (P = 0.018). Most importantly, mitotic rate [HR = 2.901 (95 % CI 1.094-7.695), P = 0.032] and risk grade [HR = 9.629 (95 % CI 1.997-46.416), P = 0.005] would be the representative traditional prognostic factors, and deletion with >3 codons would be an novel independent predictor of poor outcome for RFS in GIST patients with deletion mutation of KIT exon 11 [HR = 7.970 (95 % CI 1.774-35.803), P = 0.007]. All results indicated that mutation determined clinicopathological features and prognosis of GISTs, and more than three codons involvement may be a novel adverse indicator.

Control of total GFP expression by alterations to the 3′ region nucleotide sequence

PubMed Central

2013-01-01

Background Previously, we distinguished the Escherichia coli type II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for unfolded and folded soluble target proteins. The translocation of folded protein to the periplasm for soluble expression via the Tat pathway was controlled by an N-terminal hydrophilic leader sequence. In this study, we investigated the effect of the hydrophilic C-terminal end and its nucleotide sequence on total and soluble protein expression. Results The native hydrophilic C-terminal end of GFP was obtained by deleting the C-terminal peptide LeuGlu-6×His, derived from pET22b(+). The corresponding clones induced total and soluble GFP expression that was either slightly increased or dramatically reduced, apparently through reconstruction of the nucleotide sequence around the stop codon in the 3′ region. In the expression-induced clones, the hydrophilic C-terminus showed increased Tat pathway specificity for soluble expression. However, in the expression-reduced clone, after analyzing the role of the 5′ poly(A) coding sequence with a substituted synonymous codon, we proved that the longer 5′ poly(A) coding sequence interacted with the reconstructed 3′ region nucleotide sequence to create a new mRNA tertiary structure between the 5′ and 3′ regions, which resulted in reduced total GFP expression. Further, to recover the reduced expression by changing the 3′ nucleotide sequence, after replacing selected C-terminal 5′ codons and the stop codon in the ORF with synonymous codons, total GFP expression in most of the clones was recovered to the undeleted control level. The insertion of trinucleotides after the stop codon in the 3′-UTR recovered or reduced total GFP expression. RT-PCR revealed that the level of total protein expression was controlled by changes in translational or transcriptional regulation, which were induced or reduced by the substitution or insertion of 3′ region nucleotides. Conclusions We found that the hydrophilic C-terminal end of GFP increased Tat pathway specificity and that the 3′ nucleotide sequence played an important role in total protein expression through translational and transcriptional regulation. These findings may be useful for efficiently producing recombinant proteins as well as for potentially controlling the expression level of specific genes in the body for therapeutic purposes. PMID:23834827

Impact of KRAS codon subtypes from a randomised phase II trial of selumetinib plus docetaxel in KRAS mutant advanced non-small-cell lung cancer

PubMed Central

Jänne, P A; Smith, I; McWalter, G; Mann, H; Dougherty, B; Walker, J; Orr, M C M; Hodgson, D R; Shaw, A T; Pereira, J R; Jeannin, G; Vansteenkiste, J; Barrios, C H; Franke, F A; Crinò, L; Smith, P

2015-01-01

Background: Selumetinib (AZD6244, ARRY-142886)+docetaxel increases median overall survival (OS) and significantly improves progression-free survival (PFS) and objective response rate (ORR) compared with docetaxel alone in patients with KRAS mutant, stage IIIB/IV non-small-cell lung cancer (NSCLC; NCT00890825). Methods: Retrospective analysis of OS, PFS, ORR and change in tumour size at week 6 for different sub-populations of KRAS codon mutations. Results: In patients receiving selumetinib+docetaxel and harbouring KRAS G12C or G12V mutations there were trends towards greater improvement in OS, PFS and ORR compared with other KRAS mutations. Conclusion: Different KRAS mutations in NSCLC may influence selumetinib/docetaxel sensitivity. PMID:26125448

X-linked Alport syndrome caused by splicing mutations in COL4A5.

PubMed

Nozu, Kandai; Vorechovsky, Igor; Kaito, Hiroshi; Fu, Xue Jun; Nakanishi, Koichi; Hashimura, Yuya; Hashimoto, Fusako; Kamei, Koichi; Ito, Shuichi; Kaku, Yoshitsugu; Imasawa, Toshiyuki; Ushijima, Katsumi; Shimizu, Junya; Makita, Yoshio; Konomoto, Takao; Yoshikawa, Norishige; Iijima, Kazumoto

2014-11-07

X-linked Alport syndrome is caused by mutations in the COL4A5 gene. Although many COL4A5 mutations have been detected, the mutation detection rate has been unsatisfactory. Some men with X-linked Alport syndrome show a relatively mild phenotype, but molecular basis investigations have rarely been conducted to clarify the underlying mechanism. In total, 152 patients with X-linked Alport syndrome who were suspected of having Alport syndrome through clinical and pathologic investigations and referred to the hospital for mutational analysis between January of 2006 and January of 2013 were genetically diagnosed. Among those patients, 22 patients had suspected splice site mutations. Transcripts are routinely examined when suspected splice site mutations for abnormal transcripts are detected; 11 of them showed expected exon skipping, but others showed aberrant splicing patterns. The mutation detection strategy had two steps: (1) genomic DNA analysis using PCR and direct sequencing and (2) mRNA analysis using RT-PCR to detect RNA processing abnormalities. Six splicing consensus site mutations resulting in aberrant splicing patterns, one exonic mutation leading to exon skipping, and four deep intronic mutations producing cryptic splice site activation were identified. Interestingly, one case produced a cryptic splice site with a single nucleotide substitution in the deep intron that led to intronic exonization containing a stop codon; however, the patient showed a clearly milder phenotype for X-linked Alport syndrome in men with a truncating mutation. mRNA extracted from the kidney showed both normal and abnormal transcripts, with the normal transcript resulting in the milder phenotype. This novel mechanism leads to mild clinical characteristics. This report highlights the importance of analyzing transcripts to enhance the mutation detection rate and provides insight into genotype-phenotype correlations. This approach can clarify the cause of atypically mild phenotypes in X-linked Alport syndrome. Copyright © 2014 by the American Society of Nephrology.

Codon-Resolution Analysis Reveals a Direct and Context-Dependent Impact of Individual Synonymous Mutations on mRNA Level

PubMed Central

Chen, Siyu; Li, Ke; Cao, Wenqing; Wang, Jia; Zhao, Tong; Huan, Qing; Yang, Yu-Fei; Wu, Shaohuan; Qian, Wenfeng

2017-01-01

Abstract Codon usage bias (CUB) refers to the observation that synonymous codons are not used equally frequently in a genome. CUB is stronger in more highly expressed genes, a phenomenon commonly explained by stronger natural selection on translational accuracy and/or efficiency among these genes. Nevertheless, this phenomenon could also occur if CUB regulates gene expression at the mRNA level, a hypothesis that has not been tested until recently. Here, we attempt to quantify the impact of synonymous mutations on mRNA level in yeast using 3,556 synonymous variants of a heterologous gene encoding green fluorescent protein (GFP) and 523 synonymous variants of an endogenous gene TDH3. We found that mRNA level was positively correlated with CUB among these synonymous variants, demonstrating a direct role of CUB in regulating transcript concentration, likely via regulating mRNA degradation rate, as our additional experiments suggested. More importantly, we quantified the effects of individual synonymous mutations on mRNA level and found them dependent on 1) CUB and 2) mRNA secondary structure, both in proximal sequence contexts. Our study reveals the pleiotropic effects of synonymous codon usage and provides an additional explanation for the well-known correlation between CUB and gene expression level. PMID:28961875

Codon Usage Patterns of Tyrosinase Genes in Clonorchis sinensis.

PubMed

Bae, Young-An

2017-04-01

Codon usage bias (CUB) is a unique property of genomes and has contributed to the better understanding of the molecular features and the evolution processes of particular gene. In this study, genetic indices associated with CUB, including relative synonymous codon usage and effective numbers of codons, as well as the nucleotide composition, were investigated in the Clonorchis sinensis tyrosinase genes and their platyhelminth orthologs, which play an important role in the eggshell formation. The relative synonymous codon usage patterns substantially differed among tyrosinase genes examined. In a neutrality analysis, the correlation between GC 12 and GC 3 was statistically significant, and the regression line had a relatively gradual slope (0.218). NC-plot, i.e., GC 3 vs effective number of codons (ENC), showed that most of the tyrosinase genes were below the expected curve. The codon adaptation index (CAI) values of the platyhelminth tyrosinases had a narrow distribution between 0.685/0.714 and 0.797/0.837, and were negatively correlated with their ENC. Taken together, these results suggested that CUB in the tyrosinase genes seemed to be basically governed by selection pressures rather than mutational bias, although the latter factor provided an additional force in shaping CUB of the C. sinensis and Opisthorchis viverrini genes. It was also apparent that the equilibrium point between selection pressure and mutational bias is much more inclined to selection pressure in highly expressed C. sinensis genes, than in poorly expressed genes.

Mutations in the ADAR1 gene in Chinese families with dyschromatosis symmetrica hereditaria.

PubMed

Zhang, G L; Shi, H J; Shao, M H; Li, M; Mu, H J; Gu, Y; Du, X F; Xie, P

2013-01-04

We investigated 2 Chinese families with dyschromatosis symmetrica hereditaria (DSH) and search for mutations in the adenosine deaminase acting on RNA1 (ADAR1) gene in these 2 pedigrees. We performed a mutation analysis of the ADAR1 gene in 2 Chinese families with DSH and reviewed all articles published regarding ADAR1 mutations reported since 2003 by using PubMed. By direct sequencing, a 2-nucleotide AG deletion, 2099-2100delAG, was found in family 1, and a C→T mutation was identified at nucleotide 1420 that changed codon 474 from arginine to a translational termination codon in family 2. Two different pathogenic mutations were identified, c.2099-2100delAG and c.1420C>T, the former being a novel mutation, and the latter previously reported in 3 other families with DSH. To date, a total of 110 mutations in the ADAR1 gene have been reported, and 10 of them were recurrent; the mutations R474X, R1083C, R1096X, and R1155W might be the DSH-related hotspots.

[Mutations of amyloid precursor protein in early-onset familial Alzheimer's disease].

PubMed

Naruse, S; Tsuji, S; Miyatake, T

1992-09-01

Genetic linkage studies of familial Alzheimer's disease (FAD) have suggested that some form of early-onset FAD is linked to proximal long arm of chromosome 21. It has been also suggested that some form of late-onset FAD is linked to long arm of chromosome 19. Goate et al have identified a mis-sense mutation (Val to Ile) in exon 17 of the amyloid precursor protein (APP) gene in 2 of 16 early-onset FAD families, and have shown that the FAD locus in an FAD family is tightly linked to the mis-sense mutation. To determine if the mis-sense mutation is observed in different ethnic origine, we have studied some early-onset FAD families. Two early-onset FAD families showed the existence of the mutation. As the mutation has been identified in different ethnic origine and the mutation has not been observed in normal individuals, it strengthen hypothesis that the mutation is pathogenic. Recently, Val to Phe and Val to Gly mutations have been also identified at the same codon (Codon 717) of the APP gene.

Atypical radiological findings in achondroplasia with uncommon mutation of the fibroblast growth factor receptor-3 (FGFR-3) gene (Gly to Cys transition at codon 375)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishimuri, Gen; Fukushima, Yoshimitsu; Ohashi, Hirofumi

1995-11-20

The recent discovery of mutations in the FGFR-3 (fibroblast growth factor receptor-3) gene (FGFR3) as the cause of achondroplasia has provided new insight into understanding genetic diseases. It was surprising from the viewpoint of molecular genetics that most patients with achondroplasia showed the same mutation at nucleotide 1138, leading to a single amino acid substitution from glycine to arginine at codon 380 (Gly380Arg). All 39 patients examined by two groups had the Gly380Arg; 38 patients and the other demonstrated a G to A and a G to C transition at nucleotide 1138, respectively. Subsequently another group disclosed a G tomore » A transition at the same nucleotide 1138 in 21/23 patients of diverse ethnic origin, although mutations were not identified in two patients. To date, a total of 193 patients with the mutation of the G380Arg have been reported; a single patient with another mutation resulting in a substitution from glycine to cysteine at codon 375 (Gly375Cys) has been described. The presence of this common mutation is consistent with the clinical fact that achondroplastic individuals show less phenotypic variability than is unusual for autosomal dominant diseases. We encountered a Japanese boy with the Gly375Cys. His mother with achondroplasia has the same mutation. The molecular investigation of these patients was reported elsewhere. Here we report the clinical and radiological findings in this boy who demonstrated some atypical manifestations from those of typical achondroplasia. 8 refs., 1 fig.« less

The Complete Mitochondrial Genome of the Rice Moth, Corcyra cephalonica

PubMed Central

Wu, Yu-Peng; Li, Jie; Zhao, Jin-Liang; Su, Tian-Juan; Luo, A-Rong; Fan, Ren-Jun; Chen, Ming-Chang; Wu, Chun-Sheng; Zhu, Chao-Dong

2012-01-01

The complete mitochondrial genome (mitogenome) of the rice moth, Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) was determined as a circular molecular of 15,273 bp in size. The mitogenome composition (37 genes) and gene order are the same as the other lepidopterans. Nucleotide composition of the C. cephalonica mitogenome is highly A+T biased (80.43%) like other insects. Twelve protein-coding genes start with a typical ATN codon, with the exception of coxl gene, which uses CGA as the initial codon. Nine protein-coding genes have the common stop codon TAA, and the nad2, cox1, cox2, and nad4 have single T as the incomplete stop codon. 22 tRNA genes demonstrated cloverleaf secondary structure. The mitogenome has several large intergenic spacer regions, the spacer1 between trnQ gene and nad2 gene, which is common in Lepidoptera. The spacer 3 between trnE and trnF includes microsatellite-like repeat regions (AT)18 and (TTAT)3. The spacer 4 (16 bp) between trnS2 gene and nad1 gene has a motif ATACTAT; another species, Sesamia inferens encodes ATCATAT at the same position, while other lepidopteran insects encode a similar ATACTAA motif. The spacer 6 is A+T rich region, include motif ATAGA and a 20-bp poly(T) stretch and two microsatellite (AT)9, (AT)8 elements. PMID:23413968

The complete mitochondrial genome of the rice moth, Corcyra cephalonica.

PubMed

Wu, Yu-Peng; Li, Jie; Zhao, Jin-Liang; Su, Tian-Juan; Luo, A-Rong; Fan, Ren-Jun; Chen, Ming-Chang; Wu, Chun-Sheng; Zhu, Chao-Dong

2012-01-01

The complete mitochondrial genome (mitogenome) of the rice moth, Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) was determined as a circular molecular of 15,273 bp in size. The mitogenome composition (37 genes) and gene order are the same as the other lepidopterans. Nucleotide composition of the C. cephalonica mitogenome is highly A+T biased (80.43%) like other insects. Twelve protein-coding genes start with a typical ATN codon, with the exception of coxl gene, which uses CGA as the initial codon. Nine protein-coding genes have the common stop codon TAA, and the nad2, cox1, cox2, and nad4 have single T as the incomplete stop codon. 22 tRNA genes demonstrated cloverleaf secondary structure. The mitogenome has several large intergenic spacer regions, the spacer1 between trnQ gene and nad2 gene, which is common in Lepidoptera. The spacer 3 between trnE and trnF includes microsatellite-like repeat regions (AT)18 and (TTAT)(3). The spacer 4 (16 bp) between trnS2 gene and nad1 gene has a motif ATACTAT; another species, Sesamia inferens encodes ATCATAT at the same position, while other lepidopteran insects encode a similar ATACTAA motif. The spacer 6 is A+T rich region, include motif ATAGA and a 20-bp poly(T) stretch and two microsatellite (AT)(9), (AT)(8) elements.

[DNA mutations associated to rifampicin or isoniazid resistance in M. tuberculosis clinical isolates from Sonora, Mexico].

PubMed

Bolado-Martínez, Enrique; Pérez-Mendoza, Ansix; Alegría-Morquecho, Francisco Monserrat; Candia-Plata, María del Carmen; Aguayo-Verdugo, María del Rosario; Alvarez-Hernández, Gerardo

2012-01-01

To perform the analysis of specific regions of the major genes associated with resistance to isoniazid or rifampin. Twenty two M. tuberculosis strains, isolated from human samples obtained in Sonora, Mexico. Specific primers for hotspots of the rpoB, katG, inhA genes and the ahpC-oxyR intergenic region were used. The purified PCR products were sequenced. Mutations in the promoter of inhA, the ahpC-oxyR region, and codon 315 of katG and in 451 or 456 codons of rpoB, were identified. Detection of mutations not previously reported requires further genotypic analysis of Mycobacterium tuberculosis isolates in Sonora.

Genome-Wide Identification and Mapping of NBS-Encoding Resistance Genes in Solanum tuberosum Group Phureja

PubMed Central

Lozano, Roberto; Ponce, Olga; Ramirez, Manuel; Mostajo, Nelly; Orjeda, Gisella

2012-01-01

The majority of disease resistance (R) genes identified to date in plants encode a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain containing protein. Additional domains such as coiled-coil (CC) and TOLL/interleukin-1 receptor (TIR) domains can also be present. In the recently sequenced Solanum tuberosum group phureja genome we used HMM models and manual curation to annotate 435 NBS-encoding R gene homologs and 142 NBS-derived genes that lack the NBS domain. Highly similar homologs for most previously documented Solanaceae R genes were identified. A surprising ∼41% (179) of the 435 NBS-encoding genes are pseudogenes primarily caused by premature stop codons or frameshift mutations. Alignment of 81.80% of the 577 homologs to S. tuberosum group phureja pseudomolecules revealed non-random distribution of the R-genes; 362 of 470 genes were found in high density clusters on 11 chromosomes. PMID:22493716

An Overview of Recent Therapeutics Advances for Duchenne Muscular Dystrophy.

PubMed

Mah, Jean K

2018-01-01

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy in childhood. Mutations of the DMD gene destabilize the dystrophin associated glycoprotein complex in the sarcolemma. Ongoing mechanical stress leads to unregulated influx of calcium ions into the sarcoplasm, with activation of proteases, release of proinflammatory cytokines, and mitochondrial dysfunction. Cumulative damage and reparative failure leads to progressive muscle necrosis, fibrosis, and fatty replacement. Although there is presently no cure for DMD, scientific advances have led to many potential disease-modifying treatments, including dystrophin replacement therapies, upregulation of compensatory proteins, anti-inflammatory agents, and other cellular targets. Recently approved therapies include ataluren for stop codon read-through and eteplirsen for exon 51 skipping of eligible individuals. The purpose of this chapter is to summarize the clinical features of DMD, to describe current outcome measures used in clinical studies, and to highlight new emerging therapies for affected individuals.

Balancing Selection of a Frame-Shift Mutation in the MRC2 Gene Accounts for the Outbreak of the Crooked Tail Syndrome in Belgian Blue Cattle

PubMed Central

Li, Wanbo; Dive, Marc; Tamma, Nico; Michaux, Charles; Druet, Tom; Huijbers, Ivo J.; Isacke, Clare M.; Coppieters, Wouter; Georges, Michel; Charlier, Carole

2009-01-01

We herein describe the positional identification of a 2-bp deletion in the open reading frame of the MRC2 receptor causing the recessive Crooked Tail Syndrome in cattle. The resulting frame-shift reveals a premature stop codon that causes nonsense-mediated decay of the mutant messenger RNA, and the virtual absence of functional Endo180 protein in affected animals. Cases exhibit skeletal anomalies thought to result from impaired extracellular matrix remodeling during ossification, and as of yet unexplained muscular symptoms. We demonstrate that carrier status is very significantly associated with desired characteristics in the general population, including enhanced muscular development, and that the resulting heterozygote advantage caused a selective sweep which explains the unexpectedly high frequency (25%) of carriers in the Belgian Blue Cattle Breed. PMID:19779552

Multiplex screening for RB1 germline mutations in 106 patients with hereditary retinoblastoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lohmann, D.R.; Brandt, B.; Passarge, E.

1994-09-01

The identification of germline mutations in the retinoblastoma susceptibility gene (RB1) is important for genetic counseling in hereditary retinoblastoma. Due to the complex genomic organization of this gene and the heterogeneity of mutations, efficient screening procedures are important for rapid mutation detection. We have developed methods based on simultaneous analysis of multiple regions of this gene in an ABI automated DNA fragment analyzer to examine 106 patients with hereditary retinoblastoma in which no alteration was identified by Southern blot hybridization. Primers for the amplification of all 27 exons of the RB1 gene as well as the promoter and poly(A) signalmore » sequences were labelled with distinct fluorescent dyes (FAM, HEX, TAMRA) to enable simultaneous electrophoretic analysis of PCR products with similar mobility. PCR fragments distinguishable by size or color were co-amplified by multiplex PCR and analyzed for length by GENESCAN analysis. Using this approach, small deletions ranging from 1 bp to 22 bp were identified in 24 patients (23%). Short sequence repeats or polypyrimidine runs were present in the vicinity of most of these deletions. In 4 patients (4%), insertions from 1 bp to 4 bp were found. The majority of length mutations resulted in a truncated gene product due to frameshift and premature termination. No mutation was identified in exons 25 to 27 possibly indicating that the encoded protein domains have minor functional importance. In order to screen for base substitutions that are not detectable by fragment length analysis, we adapted heteroduplex analysis for the use in the DNA fragment analyzer. During the optimization of this method we detected 10 single base substitutions most of which generated stop codons. Intriguingly, two identical missense mutations were identified in two unrelated families with a low-penetrance phenotype.« less

Molecular characterization of the breakpoints of a 12-kb deletion in the NF1 gene in a family showing germ-line mosaicism.

PubMed Central

Lázaro, C; Gaona, A; Lynch, M; Kruyer, H; Ravella, A; Estivill, X

1995-01-01

Neurofibromatosis type 1 (NF1) is caused by deletions, insertions, translocations, and point mutations in the NF1 gene, which spans 350 kb on the long arm of human chromosome 17. Although several point mutations have been described, large molecular abnormalities have rarely been characterized in detail. We describe here the molecular breakpoints of a 12-kb deletion of the NF1 gene, which is responsible for the NF1 phenotype in a kindred with two children affected because of germline mosaicism in the unaffected father, who has the mutation in 10% of his spermatozoa. The mutation spans introns 31-39, removing 12,021 nt and inserting 30 bp, of which 19 bp are a direct repetition of a sequence located in intron 31, just 4 bp before the 5' breakpoint. The 5' and 3' breakpoints contain the sequence TATTTTA, which could be involved in the generation of the deletion. The most plausible explanation for the mechanism involved in the generation of this 12-kb deletion is homologous/nonhomologous recombination. Since sperm of the father does not contain the corresponding insertion of the 12-kb deleted sequence, this deletion could have occurred within the NF1 chromosome through loop formation. RNA from lymphocytes of one of the NF1 patients showed similar levels of the mutated and normal transcripts, suggesting that the NF1-mRNA from mutations causing frame shifts of the reading frame or stop codons in this gene is not degraded during its processing. The mutation was not detected in fresh lymphocytes from the unaffected father by PCR analysis, supporting the case for true germ-line mosaicism. Images Figure 1 Figure 3 PMID:7485153

Identification of a novel dentin matrix protein-1 (DMP-1) mutation and dental anomalies in a kindred with autosomal recessive hypophosphatemia.

PubMed

Turan, Serap; Aydin, Cumhur; Bereket, Abdullah; Akcay, Teoman; Güran, Tülay; Yaralioglu, Betul Akmen; Bastepe, Murat; Jüppner, Harald

2010-02-01

An autosomal recessive form of hypophosphatemia (ARHP) was recently shown to be caused by homozygous mutations in DMP1, the gene encoding dentin matrix protein-1 (DMP-1), a non-collagenous bone matrix protein with an important role in the development and mineralization of bone and teeth. Here, we describe a previously not reported consanguineous ARHP kindred in which the three affected individuals carry a novel homozygous DMP-1 mutation. The index case presented at the age of 3 years with bowing of his legs and showed hypophosphatemia due to insufficient renal phosphate retention. Serum alkaline phosphatase activity was elevated, with initially normal PTH. FGF23 was inappropriately normal at an older age while being treated with oral phosphate and 1,25(OH)(2)D. Similar clinical and biochemical findings, except for elevated FGF23 levels, were present in his 16-month-old brother and his 12.5-year-old female cousin; the parents of the three affected children are first-degree cousins. Nucleotide sequence analysis was performed on PCR-amplified exons encoding DMP-1 and flanking intronic regions. A novel homozygous frame-shift mutation (c.485Tdel; p.Glu163ArgfsX53) in exon 6 resulting in a premature stop codon was identified in all effected individuals. The parents and available unaffected siblings were heterozygous for c.485Tdel. Tooth growth and shape were normal for the index case, his affected brother and cousin, but their permanent and deciduous teeth displayed enlarged pulp chambers. The identified genetic mutation underscores the importance of DMP-1 mutations in the pathogenesis of ARHP. Furthermore, DMP-1 mutations appear to contribute, through yet unknown mechanisms, to tooth development. (c) 2009 Elsevier Inc. All rights reserved.

Accurate, simple, and inexpensive assays to diagnose F8 gene inversion mutations in hemophilia A patients and carriers.

PubMed

Dutta, Debargh; Gunasekera, Devi; Ragni, Margaret V; Pratt, Kathleen P

2016-12-27

The most frequent mutations resulting in hemophilia A are an intron 22 or intron 1 gene inversion, which together cause ∼50% of severe hemophilia A cases. We report a simple and accurate RNA-based assay to detect these mutations in patients and heterozygous carriers. The assays do not require specialized equipment or expensive reagents; therefore, they may provide useful and economic protocols that could be standardized for central laboratory testing. RNA is purified from a blood sample, and reverse transcription nested polymerase chain reaction (RT-NPCR) reactions amplify DNA fragments with the F8 sequence spanning the exon 22 to 23 splice site (intron 22 inversion test) or the exon 1 to 2 splice site (intron 1 inversion test). These sequences will be amplified only from F8 RNA without an intron 22 or intron 1 inversion mutation, respectively. Additional RT-NPCR reactions are then carried out to amplify the inverted sequences extending from F8 exon 19 to the first in-frame stop codon within intron 22 or a chimeric transcript containing F8 exon 1 and the VBP1 gene. These latter 2 products are produced only by individuals with an intron 22 or intron 1 inversion mutation, respectively. The intron 22 inversion mutations may be further classified (eg, as type 1 or type 2, reflecting the specific homologous recombination sites) by the standard DNA-based "inverse-shifting" PCR assay if desired. Efficient Bcl I and T4 DNA ligase enzymes that cleave and ligate DNA in minutes were used, which is a substantial improvement over previous protocols that required overnight incubations. These protocols can accurately detect F8 inversion mutations via same-day testing of patient samples.

Identification of a Novel Dentin Matrix Protein-1 (DMP-1) Mutation and Dental Anomalies in a Kindred with Autosomal Recessive Hypophosphatemia

PubMed Central

Turan, Serap; Aydin, Cumhur; Bereket, Abdullah; Akcay, Teoman; Güran, Tülay; Yaralioglu, Betul Akmen; Bastepe, Murat; Jüppner, Harald

2009-01-01

An autosomal recessive form of hypophosphatemia (ARHP) was recently shown to be caused by homozygous mutations in DMP1, the gene encoding dentin matrix protein-1 (DMP-1), a non-collagenous bone matrix protein with an important role in the development and mineralization of bone and teeth. Here, we report a previously not reported consanguineous ARHP kindred in which the three affected individuals carry a novel homozygous DMP-1 mutation. The index case presented at the age of 3 years with bowing of his legs, and showed hypophosphatemia due to insufficient renal phosphate retention. Serum alkaline phosphatase activity was elevated, with initially normal PTH. FGF23 was inappropriately normal at an older age while being treated with oral phosphate and 1,25(OH)2D. Similar clinical and biochemical findings, except for elevated FGF23 levels, were present in his 16 month-old brother and his 12.5 year-old female cousin; the parents of the three affected children are first-degree cousins. Nucleotide sequence analysis was performed on PCR-amplified exons encoding DMP-1 and flanking intronic regions. A novel homozygous frame-shift mutation (c.485Tdel; p.Glu163ArgfsX53) in exon 6 resulting in a premature stop codon was identified in all effected individuals. The parents and available unaffected siblings were heterozygous for c.485Tdel. Tooth growth and shape were normal for the index case, his affected brother and cousin, but their permanent and deciduous teeth displayed enlarged pulp chambers. The identified genetic mutation underscores the importance of DMP-1 mutations in the pathogenesis of ARHP. Furthermore, DMP-1 mutations appear to contribute, through yet unknown mechanisms, to tooth development. PMID:19796717

Insights into Mutation Effect in Three Poikiloderma with Neutropenia Patients by Transcript Analysis and Disease Evolution of Reported Patients with the Same Pathogenic Variants.

PubMed

Colombo, Elisa A; Elcioglu, Nursel H; Graziano, Claudio; Farinelli, Pamela; Di Fede, Elisabetta; Neri, Iria; Facchini, Elena; Greco, Mariangela; Gervasini, Cristina; Larizza, Lidia

2018-05-16

Poikiloderma with neutropenia (PN) is a genodermatosis currently described in 77 patients, all presenting with early-onset poikiloderma, neutropenia, and several additional signs. Biallelic loss-of-function mutations in USB1 gene are detected in all molecularly tested patients but genotype-phenotype correlation remains elusive. Cancer predisposition is recognized among PN features and pathogenic variants found in patients who developed early in life myelodysplasia (n = 12), acute myeloid leukemia (n = 2), and squamous cell carcinoma (n = 2) should be kept into account in management and follow-up of novel patients. This will hopefully allow achieving data clustered on specific mutations relevant to oncological surveillance of the carrier patients. We describe the clinical features of three unreported PN patients and characterize their USB1 pathogenic variants by transcript analysis to get insights into the effect on the overall phenotype and disease evolution. A Turkish boy is homozygous for the c.531delA deletion, a recurrent mutation in Turkey; an adult Italian male is compound heterozygous for two nonsense mutations, c.243G>A and c.541C>T, while an Italian boy is homozygous for the splicing c.683_693+1del variant. The identified mutations have already been reported in PN patients who developed hematologic or skin cancer. Aberrant mRNAs of all four mutated alleles could be identified confirming that transcripts of USB1 main isoform either carrying stop codons or mis-spliced may at least partially escape nonsense-mediated decay. Our study addresses the need of gathering insights on genotype-phenotype correlations in newly described PN patients, by transcript analysis and information on disease evolution of reported patients with the same pathogenic variants.

Molecular evaluation of PIK3CA gene mutation in breast cancer: determination of frequency, distribution pattern and its association with clinicopathological findings in Indian patients.

PubMed

Ahmad, Firoz; Badwe, Anuya; Verma, Geeta; Bhatia, Simi; Das, Bibhu Ranjan

2016-07-01

Somatic mutations in the PIK3CA gene are common in breast cancer and represent a clinically useful marker for prognosis and therapeutic target. Activating mutations in the PI3K p110 catalytic subunit (PIK3CA) have been identified in 18-40 % of breast carcinomas. In this study, we evaluated PIK3CA mutation in 185 Indian breast cancer patients by direct DNA sequencing. PIK3CA mutations were observed in 23.2 % (43/185) of breast tumor samples. PIK3CA mutations were more frequent exon 30 (76.8 %) than in exon 9 (23.2 %). Mutations were mostly clustered within two hotspot region between nucleotides 1624 and 1636 or between 3129 and 3140. Sequencing analysis revealed four different missense mutations at codon 542 and 545 (E542K, E545K, E545A and E545G) in the helical domain and two different amino acid substitutions at codon 1047 (H1047R and H1047L) in the kinase domain. None of the cases harbored concomitant mutations at multiple codons. PIK3CA mutations were more frequent in older patients, smaller size tumors, ductal carcinomas, grade II tumors, lymph node-positive tumors and non-DCIS tumors; however, none of the differences were significant. In addition, PIK3CA mutations were common in ER+, PR+ and HER2+ cases (30 %), and a comparatively low frequency were noted in triple-negative tumors (13.6 %). In conclusion, to our knowledge, this is the largest study to evaluate the PIK3CA mutation in Indian breast cancer patients. The frequency and distribution pattern of PIK3CA mutations is similar to global reports. Furthermore, identification of molecular markers has unique strengths and can provide insights into the pathogenic process of breast carcinomas.

Four novel RS1 gene mutations in Polish patients with X-linked juvenile retinoschisis.

PubMed

Skorczyk, Anna; Krawczyński, Maciej R

2012-01-01

To determine the clinical features and to identify mutations in the retinoschisis gene (RS1) in ten patients with X-linked retinoschisis (XLRS). Ten male patients from nine Polish families were included in this study. Ophthalmologic examinations, including optical coherence tomography (OCT) and full-field electroretinography (ERG), were performed in all affected boys. The entire coding region encompassing six exons of the RS1 gene was amplified with PCR and directly sequenced in all the patients. All affected individuals showed typical retinoschisis signs and symptoms, and all appeared to have a mutation in the RS1 gene. Seven different mutations were identified, including two novel missense substitutions: c.176G>C (p.Cys59Ser), c.451T>A (p.Tyr151Asp); one novel nonsense substitution: c.218C>A (p.Ser73*); and one novel frameshift mutation: c.354_355delCA (p.Asp118Glufs*2). We also found two missense substitutions that had been previously described: c.214G>A (p.Glu72Lys) and c.626G>T (p.Arg209Leu) and one known splice site mutation in intron 5: c.522+1G>T (IVS5+1G>T). This study provides the first molecular genetic characteristics of patients with juvenile retinoschisis from the previously unexplored Polish population. We investigated the molecular background of XLRS in ten boys. The present study reports for the first time four novel mutations, including two missense substitutions, one nonsense substitution, and one frameshift deletion. One of these substitutions and 2-bp deletion created stop codons. Moreover, we described three substitutions that had been previously reported (one is a splicing mutation). Further genetic characterization of Polish patients with XLRS will be helpful in understanding the worldwide spectrum of RS1 mutations. Despite the mutation heterogeneity found in a small group of our patients, they presented a relatively uniform clinical picture. Identifying the causative mutation is helpful in confirming diagnosis and counseling, but cannot provide prognostic data.

Characterization of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Kuwait and Dubai.

PubMed

Ahmad, Suhail; Mokaddas, Eiman; Fares, Esther

2002-11-01

Mutations conferring resistance to rifampin in rifampin-resistant clinical Mycobacterium tuberculosis isolates occur mostly in the 81 bp rifampin-resistance-determining region (RRDR) of the rpoB gene. In this study, 29 rifampin-resistant and 12 -susceptible clinical M. tuberculosis isolates were tested for characterization of mutations in the rpoB gene by line probe (INNO-LiPA Rif. TB) assay and the results were confirmed and extended by DNA sequencing of the PCR amplified target DNA. The line probe assay identified all 12 susceptible strains as rifampin-sensitive and the DNA sequence of RRDR in the amplified rpoB gene from two isolates matched perfectly with the wild-type sequence. The line probe assay identified 28 resistant isolates as rifampin-resistant with specific detection of mutation in 22 isolates including one isolate that exhibited hetro-resistance containing both the wild-type pattern as well as a specific mutation within RRDR while one of the rifampin-resistant strain was identified as rifampin-susceptible. DNA sequencing confirmed these results and, in addition, led to the specific detection of mutations in 5 rifampin-resistant isolates in which specific base changes within RRDR could not be determined by the line probe assay. These analyses identified 8 different mutations within RRDR of the rpoB gene including one novel mutation (S522W) that has not been reported so far. The genotyping performed on the isolates carrying similar mutations showed that majority of these isolates were unique as they exhibited varying DNA banding patterns. Correlating the ethnic origin of the infected TB patients with the occurrence of specific mutations at three main codon positions (516, 526 and 531) in the rpoB gene showed that most patients (11 of 15) from South Asian region contained mutations at codon 526 while majority of isolates from patients (6 of 11) of Middle Eastern origin contained mutations at codon 531.

Whole Genome Sequencing Based Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Isolates from Pakistan

PubMed Central

Ali, Asho; Hasan, Zahra; McNerney, Ruth; Mallard, Kim; Hill-Cawthorne, Grant; Coll, Francesc; Nair, Mridul; Pain, Arnab; Clark, Taane G.; Hasan, Rumina

2015-01-01

Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91–94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates. PMID:25719196

GNAq mutations are not identified in papillary thyroid carcinomas and hyperfunctioning thyroid nodules.

PubMed

Cassol, Clarissa A; Guo, Miao; Ezzat, Shereen; Asa, Sylvia L

2010-12-01

Activating mutations of GNAq protein in a hotspot at codon 209 have been recently described in uveal melanomas. Since these neoplasms share with thyroid carcinomas a high frequency of MAP kinase pathway-activating mutations, we hypothesized whether GNAq mutations could also play a role in the development of thyroid carcinomas. Additionally, activating mutations of another subtype of G protein (GNAS1) are frequently found in hyperfunctioning thyroid adenomas, making it plausible that GNAq-activating mutations could also be found in some of these nodules. To investigate thyroid papillary carcinomas and thyroid hyperfunctioning nodules for GNAq mutations in exon 5, codon 209, a total of 32 RET/PTC, BRAF, and RAS negative thyroid papillary carcinomas and 13 hyperfunctioning thyroid nodules were evaluated. No mutations were identified. Although plausible, GNAq mutations seem not to play an important role in the development of thyroid follicular neoplasms, either benign hyperfunctioning nodules or malignant papillary carcinomas. Our results are in accordance with the literature, in which no GNAq hotspot mutations were found in thyroid papillary carcinomas, as well as in an extensive panel of other tumors. The molecular basis for MAP-kinase pathway activation in RET-PTC/BRAF/RAS negative thyroid carcinomas remains to be determined.

Identification of a splicing enhancer in MLH1 using COMPARE a new assay for determination of relative RNA splicing efficiencies

PubMed Central

Xu, Dong-Qing; Mattox, William

2006-01-01

Exonic splicing enhancers (ESEs) are sequences that facilitate recognition of splice sites and prevent exon-skipping. Because ESEs are often embedded within proteincoding sequences, alterations in them can also often be interpreted as nonsense, missense or silent mutations. To correctly interpret exonic mutations and their roles in disease, it is important to develop strategies that identify ESE mutations. Potential ESEs can be found computationally in many exons but it has proven difficult to predict if a given mutation will have effects on splicing based on sequence alone. Here we describe a flexible in vitro method that can be used to functionally compare the effects of multiple sequence variants on ESE activity in a single in vitro splicing reaction. We have applied this method in parallel with conventional splicing assays to test for a splicing enhancer in exon 17 of the human MLH1 gene. Point mutations associated with hereditary nonpolyposis colorectal cancer (HNPCC) have previously been found to correlate with exon-skipping in both lymphocytes and tumors from patients. We show that sequences from this exon can replace an ESE from the mouse IgM gene to support RNA splicing in HeLa nuclear extracts. ESE activity was reduced by HNPCC point mutations in codon 659 indicating that their primary effect is on splicing. Surprisingly the strongest enhancer function mapped to a different region of the exon upstream of this codon. Together our results indicate that HNPCC point mutations in codon 659 affect an auxillary element that augments the enhancer function to ensure exon inclusion. PMID:16357104

Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2',3'-dideoxycytidine.

PubMed Central

Fitzgibbon, J E; Howell, R M; Haberzettl, C A; Sperber, S J; Gocke, D J; Dubin, D T

1992-01-01

To investigate whether human immunodeficiency virus type 1 pol gene mutations are selected during prolonged 2',3'-dideoxycytidine (ddC) therapy, we used the polymerase chain reaction to amplify a portion of the reverse transcriptase segment of the pol gene from the peripheral blood mononuclear cell DNA of a patient with AIDS before and after an 80-week course of ddC therapy. The consensus sequence from the second sample contained a unique double mutation (ACT to GAT) in the codon for reverse transcriptase amino acid 69, causing substitution of aspartic acid (Asp) for the wild-type threonine (Thr). A mutation (ACA to ATA) also occurred in the codon for position 165, causing substitution of isoleucine (Ile) for Thr. The GAT (Asp) codon was introduced into the pol gene of a molecular clone of human immunodeficiency virus via site-directed mutagenesis. Following transfection, mutant and wild-type viruses were tested for susceptibility to ddC by a plaque reduction assay. The mutant virus was fivefold less susceptible to ddC than the wild type; cross-resistance to 3'-azido-3'-deoxythymidine or 2'3'-dideoxyinosine was not found. The Ile-165 mutation did not confer additional ddC resistance. The Asp-69 substitution may have contributed to the generation of resistant virus in this patient. Images PMID:1317143

Mycobacterium tuberculosis embB codon 306 mutations confer moderately increased resistance to ethambutol in vitro and in vivo.

PubMed

Plinke, Claudia; Walter, Kerstin; Aly, Sahar; Ehlers, Stefan; Niemann, Stefan

2011-06-01

Ethambutol (EMB) is a major component of the first-line therapy of tuberculosis. Mutations in codon 306 of embB (embB306) were suggested as a major resistance mechanism in clinical isolates. To directly analyze the impact of individual embB306 mutations on EMB resistance, we used allelic exchange experiments to generate embB306 mutants of M. tuberculosis H37Rv. The level of EMB resistance conferred by particular mutations was measured in vitro and in vivo after EMB therapy by daily gavage in a mouse model of aerogenic tuberculosis. The wild-type embB306 ATG codon was replaced by embB306 ATC, ATA, or GTG, respectively. All of the obtained embB306 mutants exhibited a 2- to 4-fold increase in EMB MIC compared to the wild-type H37Rv. In vivo, the one selected embB306 GTG mutant required a higher dose of ethambutol to restrict its growth in the lung compared to wild-type H37Rv. These experiments demonstrate that embB306 point mutations enhance the EMB MIC in vitro to a moderate, but significant extent, and reduce the efficacy of EMB treatment in the animal model. We propose that conventional EMB susceptibility testing, in combination with embB306 genotyping, may guide dose adjustment to avoid clinical treatment failure in these low-level resistant strains.

Neuropathology of Autosomal Dominant Alzheimer Disease in the National Alzheimer Coordinating Center Database.

PubMed

Ringman, John M; Monsell, Sarah; Ng, Denise W; Zhou, Yan; Nguyen, Andy; Coppola, Giovanni; Van Berlo, Victoria; Mendez, Mario F; Tung, Spencer; Weintraub, Sandra; Mesulam, Marek-Marsel; Bigio, Eileen H; Gitelman, Darren R; Fisher-Hubbard, Amanda O; Albin, Roger L; Vinters, Harry V

2016-03-01

Alzheimer disease (AD) represents a genetically heterogeneous entity. To elucidate neuropathologic features of autosomal dominant AD ([ADAD] due to PSEN1, APP, or PSEN2 mutations), we compared hallmark AD pathologic findings in 60 cases of ADAD and 120 cases of sporadic AD matched for sex, race, ethnicity, and disease duration. Greater degrees of neuritic plaque and neurofibrillary tangle formation and cerebral amyloid angiopathy (CAA) were found in ADAD (p values < 0.01). Moderate to severe CAA was more prevalent in ADAD (63.3% vs. 39.2%, p = 0.003), and persons with PSEN1 mutations beyond codon 200 had higher average Braak scores and severity and prevalence of CAA than those with mutations before codon 200. Lewy body pathology was less extensive in ADAD but was present in 27.1% of cases. We also describe a novel pathogenic PSEN1 mutation (P267A). The finding of more severe neurofibrillary pathology and CAA in ADAD, particularly in carriers of PSEN1 mutations beyond codon 200, warrants consideration when designing trials to treat or prevent ADAD. The finding of Lewy body pathology in a substantial minority of ADAD cases supports the assertion that development of Lewy bodies may be in part driven by abnormal β-amyloid protein precursor processing. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

New horizons for cystic fibrosis treatment.

PubMed

Fajac, Isabelle; De Boeck, Kris

2017-02-01

Cystic fibrosis is an inherited multi-system disease associated with chronic lung infection, malabsorption, salt loss syndromes, male infertility and leading to numerous comorbidities. The landscape in cystic fibrosis care has changed markedly with currently more adult patients than children in many countries. Over 2000 different mutations in the CFTR gene have been reported and the majority are extremely rare. Understanding how CFTR mutations translate to disturbed synthesis or function of the CFTR protein has opened the way to 'personalized' treatments to correct the basic defect. The first 2 drugs have reached the clinic: a CFTR potentiator to augment CFTR channel function, and the combination of this potentiator with a corrector to increase CFTR expression at the cell membrane. To obtain robust correction of CFTR expression at the cell membrane, combinations of correctors with additive efficacy are under investigation. Other mutation type-specific treatments under clinical investigation are premature stop codon-read through drugs and antisense oligonucleotides that correct the basic defect at the mRNA level. Restoring the defective gene by gene editing can already be achieved ex vivo. Mutation agnostic treatments are explored as well: stabilizing CFTR expression at the cell membrane, circumventing the CFTR channel by blocking or activating other ion channels, and gene therapy. Combinations of these therapies can be anticipated. The pipeline of corrective strategies under clinical investigation is increasing continuously and a rising number of pharmaceutical companies are entering the field. Copyright © 2016 Elsevier Inc. All rights reserved.

Single-Exome sequencing identified a novel RP2 mutation in a child with X-linked retinitis pigmentosa.

PubMed

Lim, Hassol; Park, Young-Mi; Lee, Jong-Keuk; Taek Lim, Hyun

2016-10-01

To present an efficient and successful application of a single-exome sequencing study in a family clinically diagnosed with X-linked retinitis pigmentosa. Exome sequencing study based on clinical examination data. An 8-year-old proband and his family. The proband and his family members underwent comprehensive ophthalmologic examinations. Exome sequencing was undertaken in the proband using Agilent SureSelect Human All Exon Kit and Illumina HiSeq 2000 platform. Bioinformatic analysis used Illumina pipeline with Burrows-Wheeler Aligner-Genome Analysis Toolkit (BWA-GATK), followed by ANNOVAR to perform variant functional annotation. All variants passing filter criteria were validated by Sanger sequencing to confirm familial segregation. Analysis of exome sequence data identified a novel frameshift mutation in RP2 gene resulting in a premature stop codon (c.665delC, p.Pro222fsTer237). Sanger sequencing revealed this mutation co-segregated with the disease phenotype in the child's family. We identified a novel causative mutation in RP2 from a single proband's exome sequence data analysis. This study highlights the effectiveness of the whole-exome sequencing in the genetic diagnosis of X-linked retinitis pigmentosa, over the conventional sequencing methods. Even using a single exome, exome sequencing technology would be able to pinpoint pathogenic variant(s) for X-linked retinitis pigmentosa, when properly applied with aid of adequate variant filtering strategy. Copyright © 2016 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved.

Loss of spatacsin function alters lysosomal lipid clearance leading to upper and lower motor neuron degeneration.

PubMed

Branchu, Julien; Boutry, Maxime; Sourd, Laura; Depp, Marine; Leone, Céline; Corriger, Alexandrine; Vallucci, Maeva; Esteves, Typhaine; Matusiak, Raphaël; Dumont, Magali; Muriel, Marie-Paule; Santorelli, Filippo M; Brice, Alexis; El Hachimi, Khalid Hamid; Stevanin, Giovanni; Darios, Frédéric

2017-06-01

Mutations in SPG11 account for the most common form of autosomal recessive hereditary spastic paraplegia (HSP), characterized by a gait disorder associated with various brain alterations. Mutations in the same gene are also responsible for rare forms of Charcot-Marie-Tooth (CMT) disease and progressive juvenile-onset amyotrophic lateral sclerosis (ALS). To elucidate the physiopathological mechanisms underlying these human pathologies, we disrupted the Spg11 gene in mice by inserting stop codons in exon 32, mimicking the most frequent mutations found in patients. The Spg11 knockout mouse developed early-onset motor impairment and cognitive deficits. These behavioral deficits were associated with progressive brain atrophy with the loss of neurons in the primary motor cortex, cerebellum and hippocampus, as well as with accumulation of dystrophic axons in the corticospinal tract. Spinal motor neurons also degenerated and this was accompanied by fragmentation of neuromuscular junctions and muscle atrophy. This new Spg11 knockout mouse therefore recapitulates the full range of symptoms associated with SPG11 mutations observed in HSP, ALS and CMT patients. Examination of the cellular alterations observed in this model suggests that the loss of spatacsin leads to the accumulation of lipids in lysosomes by perturbing their clearance from these organelles. Altogether, our results link lysosomal dysfunction and lipid metabolism to neurodegeneration and pinpoint a critical role of spatacsin in lipid turnover. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Fatal mitochondrial encephalopathy caused by fumarase deficiency: A molecular-genetic study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gellera, C.; Cavadini, P.; Baratta, S.

Fumarase deficiency is a rare autosomal recessive disorder of the citric acid cycle resulting in severe organic aciduria and encephalopathy. Mammalian cells contain two fumarase isoenzymes, one mitochondrial and one cytosolic. In rat, the two proteins are encoded by the same gene and are synthesized by alternative initiation of translation at two in-phase AUG codons. One single fumarase gene locus has been identified on human chromosome 1. In most of the patients so far described, the activities of both isozymes are severely affected, suggesting that mutations within a single gene may underlie the disease. Here, we report the molecular studymore » of fumarase deficiency in a patient exhibiting compound heterozygosity for two different allelic mutations affecting the amino acid composition of both isoforms. The proband, an Italian boy of nonconsanguineous parents, died at 7 months of age of a progressive encephalopathy. Immunoblot demonstrated absence of cross-reacting material in both cytosolic and mitochondrial fraction of all tissues examined. Molecular analysis of the patient`s fumarase cDNA amplified by RT-PCR showed the presence of two mutations affecting the amino acid composition of both isoforms, a missense mutation resulting in the nonconservative amino acid substitution at codon 190 (Arg190Cys) and an amino acid in-frame insertion at codon 434 (Lys434ins). SSCP analysis of genomic PCR fragments encompassing the mutations demonstrated that the patient was heterozygous for both mutations, having inherited the Arg-to-Cys substitution from the father and the in-frame insertion from the mother. Finally, the effects of the mutations on enzyme function were investigated by expressing both normal and mutated fumarase cDNAs in a fumarase-deficient ({delta}FUM1) S. cerevisiae strain.« less

Monoclonal origin of peritoneal implants and lymph node deposits in serous borderline ovarian tumors (s-BOT) with high intratumoral homogeneity.

PubMed

Horn, Lars-Christian; Höhn, Anne K; Einenkel, Jens; Siebolts, Udo

2014-11-01

Molecular studies have shown that the most prevalent mutations in serous ovarian borderline tumors (s-BOT) are BRAF and/or KRAS alterations. About one third of s-BOT represent peritoneal implants and/or lymph node involvement. These extraovarian deposits may be monoclonal or polyclonal in origin. To test both the hypotheses, mutational analyses using pyrosequencing for BRAF codon 600 and KRAS codon 12/13 and 61 of microdissected tissue was performed in 15 s-BOT and their invasive and noninvasive peritoneal implants. Two to 6 implants from different peritoneal sites were examined in 13 cases. Lymph node deposits were available for the analysis in 3 cases. Six s-BOT showed mutation in exon 2 codon 12 of the KRAS proto-oncogen. Five additional cases showed BRAF p.V600E mutation representing an overall mutation rate of 73.3%. Multiple (2-6) peritoneal implants were analyzed after microdissection in 13 of 15 cases. All showed identical mutational results when compared with the ovarian site of the disease. All lymph node deposits, including those with multiple deposits in different nodes, showed identical results, suggesting high intratumoral mutational homogeneity. The evidence presented in this study and the majority of data reported in the literature support the hypothesis that s-BOT with their peritoneal implants and lymph node deposits show identical mutational status of BRAF and KRAS suggesting a monoclonal rather than a polyclonal disease regarding these both tested genetic loci. In addition, a high intratumoral genetic homogeneity can be suggested. In conclusion, the results of the present study support the monoclonal origin of s-BOT and their peritoneal implants and lymph node deposits.

Co-amplification at lower denaturation-temperature PCR combined with unlabled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma cases.

PubMed

Wu, Jiong; Zhou, Yan; Zhang, Chun-Yan; Song, Bin-Bin; Wang, Bei-Li; Pan, Bai-Shen; Lou, Wen-Hui; Guo, Wei

2014-01-01

The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. It was found that the sensitivity of Sanger reached 0.5% with COLD- PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

Coinheritance of a novel mutation on the HBA1 gene: c.187delG (p.W62fsX66) [codon 62 (-G) (α1)] with the α212 patchwork allele and Hb S [β6(A3)Glu→Val, GAG>GTG; HBB: c.20A>T].

PubMed

Scheps, Karen G; De Paula, Silvia M; Bitsman, Alicia R; Freigeiro, Daniel H; Basack, F Nora; Pennesi, Sandra P; Varela, Viviana

2013-01-01

We describe a novel frameshift mutation on the HBA1 gene (c.187delG), causative of α-thalassemia (α-thal) in a Black Cuban family with multiple sequence variants in the HBA genes and the Hb S [β6(A3)Glu→Val, GAG>GTG; HBB: c.20A>T] mutation. The deletion of the first base of codon 62 resulted in a frameshift at amino acid 62 with a putative premature termination codon (PTC) at amino acid 66 on the same exon (p.W62fsX66), which most likely triggers nonsense mediated decay of the resulting mRNA. This study also presents the first report of the α212 patchwork allele in Latin America and the description of two new sequence variants in the HBA2 region (c.-614G>A in the promoter region and c.95+39 C>T on the first intron).

Spontaneous Loss of Virulence in Natural Populations of Listeria monocytogenes.

PubMed

Maury, Mylène M; Chenal-Francisque, Viviane; Bracq-Dieye, Hélène; Han, Lei; Leclercq, Alexandre; Vales, Guillaume; Moura, Alexandra; Gouin, Edith; Scortti, Mariela; Disson, Olivier; Vázquez-Boland, José A; Lecuit, Marc

2017-11-01

The pathogenesis of Listeria monocytogenes depends on the ability of this bacterium to escape from the phagosome of the host cells via the action of the pore-forming toxin listeriolysin O (LLO). Expression of the LLO-encoding gene ( hly ) requires the transcriptional activator PrfA, and both hly and prfA genes are essential for L. monocytogenes virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in L. monocytogenes Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed L. monocytogenes strains isolated from a variety of sources (0.1%). In most cases (56/60; 93.3%), the nonhemolytic phenotype resulted from nonsense, missense, or frameshift mutations in prfA Five strains carried hly mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both hly and gshF (encoding a glutathione synthase required for full PrfA activity) were missing due to genomic rearrangements likely caused by a transposable element. The PrfA/LLO loss-of-function (PrfA - /LLO - ) mutants belonged to phylogenetically diverse clades of L. monocytogenes , and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that prfA and hly are under purifying selection. Although occurring at a low frequency, PrfA - /LLO - mutational events in L. monocytogenes lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen. Copyright © 2017 Maury et al.

Spontaneous Loss of Virulence in Natural Populations of Listeria monocytogenes

PubMed Central

Maury, Mylène M.; Chenal-Francisque, Viviane; Bracq-Dieye, Hélène; Han, Lei; Leclercq, Alexandre; Vales, Guillaume; Moura, Alexandra; Gouin, Edith; Scortti, Mariela; Disson, Olivier

2017-01-01

ABSTRACT The pathogenesis of Listeria monocytogenes depends on the ability of this bacterium to escape from the phagosome of the host cells via the action of the pore-forming toxin listeriolysin O (LLO). Expression of the LLO-encoding gene (hly) requires the transcriptional activator PrfA, and both hly and prfA genes are essential for L. monocytogenes virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in L. monocytogenes. Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed L. monocytogenes strains isolated from a variety of sources (0.1%). In most cases (56/60; 93.3%), the nonhemolytic phenotype resulted from nonsense, missense, or frameshift mutations in prfA. Five strains carried hly mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both hly and gshF (encoding a glutathione synthase required for full PrfA activity) were missing due to genomic rearrangements likely caused by a transposable element. The PrfA/LLO loss-of-function (PrfA−/LLO−) mutants belonged to phylogenetically diverse clades of L. monocytogenes, and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that prfA and hly are under purifying selection. Although occurring at a low frequency, PrfA−/LLO− mutational events in L. monocytogenes lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen. PMID:28827366

Seventeen {alpha}-hydroxylase deficiency with one base pair deletion of the cytochrome P450c17 (CYP17) gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oshiro, Chikara; Takasu, Nobuyuki; Wakugami, Tamio

1995-08-01

Mutation of the cytochrome P450c17 (CYP17) gene causes 17{alpha}-hydroxylase deficiency (170HD). Recently, several researchers have elucidated the molecular basis of 170HD by gene analysis. We experienced a case of 170HD and intended to reveal the abnormality of the CYP17 gene in this Japanese female with 170HD. Leukocytes were obtained from the patient, her mother and sister, and normal control subjects. We amplified the CYP17 gene using polymerase chain reaction and performed the sequence analysis using the dideoxy terminator method and restriction enzyme analysis. We found that the patient had one base-pair deletion at the position of amino acid 438. Anmore » indentical result was obtained with restriction enzyme analysis. This G deletion altered the reading frame and resulted in a premature stop codon at position 443; the ligand of heme iron (Cys: cystine 442) was absent. This small mutation may account for the patient`s clinical manifestations of 170HD. This is the first case of 170HD with only one base pair deletion of the CYP17 gene. 18 refs., 3 figs.« less

Deletion in the EVC2 gene causes chondrodysplastic dwarfism in Tyrolean Grey cattle.

PubMed

Murgiano, Leonardo; Jagannathan, Vidhya; Benazzi, Cinzia; Bolcato, Marilena; Brunetti, Barbara; Muscatello, Luisa Vera; Dittmer, Keren; Piffer, Christian; Gentile, Arcangelo; Drögemüller, Cord

2014-01-01

During the summer of 2013 seven Italian Tyrolean Grey calves were born with abnormally short limbs. Detailed clinical and pathological examination revealed similarities to chondrodysplastic dwarfism. Pedigree analysis showed a common founder, assuming autosomal monogenic recessive transmission of the defective allele. A positional cloning approach combining genome wide association and homozygosity mapping identified a single 1.6 Mb genomic region on BTA 6 that was associated with the disease. Whole genome re-sequencing of an affected calf revealed a single candidate causal mutation in the Ellis van Creveld syndrome 2 (EVC2) gene. This gene is known to be associated with chondrodysplastic dwarfism in Japanese Brown cattle, and dwarfism, abnormal nails and teeth, and dysostosis in humans with Ellis-van Creveld syndrome. Sanger sequencing confirmed the presence of a 2 bp deletion in exon 19 (c.2993_2994ACdel) that led to a premature stop codon in the coding sequence of bovine EVC2, and was concordant with the recessive pattern of inheritance in affected and carrier animals. This loss of function mutation confirms the important role of EVC2 in bone development. Genetic testing can now be used to eliminate this form of chondrodysplastic dwarfism from Tyrolean Grey cattle.

Deletion in the EVC2 Gene Causes Chondrodysplastic Dwarfism in Tyrolean Grey Cattle

PubMed Central

Murgiano, Leonardo; Jagannathan, Vidhya; Benazzi, Cinzia; Bolcato, Marilena; Brunetti, Barbara; Muscatello, Luisa Vera; Dittmer, Keren; Piffer, Christian; Gentile, Arcangelo; Drögemüller, Cord

2014-01-01

During the summer of 2013 seven Italian Tyrolean Grey calves were born with abnormally short limbs. Detailed clinical and pathological examination revealed similarities to chondrodysplastic dwarfism. Pedigree analysis showed a common founder, assuming autosomal monogenic recessive transmission of the defective allele. A positional cloning approach combining genome wide association and homozygosity mapping identified a single 1.6 Mb genomic region on BTA 6 that was associated with the disease. Whole genome re-sequencing of an affected calf revealed a single candidate causal mutation in the Ellis van Creveld syndrome 2 (EVC2) gene. This gene is known to be associated with chondrodysplastic dwarfism in Japanese Brown cattle, and dwarfism, abnormal nails and teeth, and dysostosis in humans with Ellis-van Creveld syndrome. Sanger sequencing confirmed the presence of a 2 bp deletion in exon 19 (c.2993_2994ACdel) that led to a premature stop codon in the coding sequence of bovine EVC2, and was concordant with the recessive pattern of inheritance in affected and carrier animals. This loss of function mutation confirms the important role of EVC2 in bone development. Genetic testing can now be used to eliminate this form of chondrodysplastic dwarfism from Tyrolean Grey cattle. PMID:24733244

Chromosome VIII disomy influences the nonsense suppression efficiency and transition metal tolerance of the yeast Saccharomyces cerevisiae.

PubMed

Zadorsky, S P; Sopova, Y V; Andreichuk, D Y; Startsev, V A; Medvedeva, V P; Inge-Vechtomov, S G

2015-06-01

The SUP35 gene of the yeast Saccharomyces cerevisiae encodes the translation termination factor eRF3. Mutations in this gene lead to the suppression of nonsense mutations and a number of other pleiotropic phenotypes, one of which is impaired chromosome segregation during cell division. Similar effects result from replacing the S. cerevisiae SUP35 gene with its orthologues. A number of genetic and epigenetic changes that occur in the sup35 background result in partial compensation for this suppressor effect. In this study we showed that in S. cerevisiae strains in which the SUP35 orthologue from the yeast Pichia methanolica replaces the S. cerevisiae SUP35 gene, chromosome VIII disomy results in decreased efficiency of nonsense suppression. This antisuppressor effect is not associated with decreased stop codon read-through. We identified SBP1, a gene that localizes to chromosome VIII, as a dosage-dependent antisuppressor that strongly contributes to the overall antisuppressor effect of chromosome VIII disomy. Disomy of chromosome VIII also leads to a change in the yeast strains' tolerance of a number of transition metal salts. Copyright © 2015 John Wiley & Sons, Ltd.

Recurrent pulmonary embolism associated with deep venous thrombosis diagnosed as protein s deficiency owing to a novel mutation in PROS1: A case report.

PubMed

Huang, Xiaojie; Xu, Fangfang; Assa, Carmel Rebecca; Shen, Laigen; Chen, Bing; Liu, Zhenjie

2018-05-01

Protein S (PS) deficiency that can be inherited or acquired is an independent risk factor for venous thromboembolism (VTE). In this report, we present a case of recurrent pulmonary embolism (PE) and deep venous thrombosis (DVT) due to PS deficiency. A 32-year-old male patient with significant decrease in PS activity was detected by laboratory tests. Genetic examination of the PROS1 gene showed a transition of G to T in exon 14 (c.1792 G>T, p.E598X), which was a paternal inherited heterozygous G1792T substitution in the laminin G-type repeat domain, generating a premature stop codon at Glu598. We considered that the inherited PS deficiency due to a PROS1 gene mutation may associate with recurrent VTE. The patient was suggested to have an extended anticoagulant therapy to avoid a severe VTE event. The patient was discharged home with continued oral anticoagulants and was still seen in clinic for follow-up. It is necessary for the young patient with recurrent idiopathic thrombosis to perform an inherited PS deficiency test and receive anticoagulant therapy for an extended period.

Mutated cancer autoantigen implicated cause of paraneoplastic myasthenia gravis.

PubMed

Zekeridou, Anastasia; Griesmann, Guy E; Lennon, Vanda A

2018-05-09

Anti-tumor immune responses are postulated to initiate paraneoplastic neurological disorders when proteins normally restricted to neural cells are expressed as oncoproteins. Mutated oncopeptides could bypass self-tolerant T-cells to activate cytotoxic effector T-lymphocytes and requisite helper T-lymphocytes to stimulate autoantibody production by B-lymphocytes. We investigated muscle-type nicotinic acetylcholine receptor (AChR) antigen expression at transcriptional and protein levels in a small-cell lung cancer line (SCLC) established from a patient with AChR-IgG-positive myasthenia gravis. I We identified mRNA transcripts encoding the two AChR α1-subunit isoforms, and seven alternative-splicing products, three yielding premature stop codons. Despite detecting native muscle-type AChR pentamers in the tumor, we did not identify mutant α1-peptides. However, we found α1-subunit-derived peptides bound to tumor MHC1-protein. In a control SCLC from an ANNA-1(anti-Hu)-IgG-positive patient, we identified MHC1-complexed Hu protein-derived peptides, but not AChR peptides. Our findings support onconeural protein products as pertinent immunogens initiating paraneoplastic neurological autoimmunity. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Mutagenesis of the three bases preceding the start codon of the beta-galactosidase mRNA and its effect on translation in Escherichia coli.

PubMed Central

Hui, A; Hayflick, J; Dinkelspiel, K; de Boer, H A

1984-01-01

The effect on the translation efficiency of various mutations in the three bases (the -1 triplet) that precede the AUG start codon of the beta-galactosidase mRNA in Escherichia coli was studied. Of the 39 mutants examined, the level of expression varies over a 20-fold range. The most favorable combinations of bases in the -1 triplet are UAU and CUU. The expression levels in the mutants with UUC, UCA or AGG as the -1 triplet are 20-fold lower than those with UAU or CUU. In general, a U residue immediately preceding the start codon is more favorable for expression than any other base; furthermore, an A residue at the -2 position enhances the translation efficiency in most instances. In both cases, however, the degree of enhancement depends on its context, i.e. the neighboring bases. Although the rules derived from this study are complex, the results show that mutations in any of the three bases preceding the start codon can strongly affect the translational efficiency of the beta-galactosidase mRNA. PMID:6425057

AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo.

PubMed Central

Butler, J S; Springer, M; Grunberg-Manago, M

1987-01-01

We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo. PMID:2954162

AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo.

PubMed

Butler, J S; Springer, M; Grunberg-Manago, M

1987-06-01

We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo.

Characterization of the mutations in the glucose-6-phosphatase gene in Israeli patients with glycogen storage disease type 1a: R83C in six Jews and a novel V166G mutation in a Muslim Arab.

PubMed

Parvari, R; Moses, S; Hershkovitz, E; Carmi, R; Bashan, N

1995-01-01

Glycogen storage disease type 1a (GSD 1a), an autosomal recessive disease, is caused by the inactivity of glucose-6-phosphatase, the gene of which has been recently cloned. We report on the missense mutation C-->T at nucleotide 326 of the G6Pase gene, causing the change of the Arg codon at position 83 into a Cys codon, as the single mutation detected in six Jewish patients. This finding suggests that this mutation might be prevalent among the Jewish population. A new missense mutation T-->G at nucleotide 576 resulting in V166G was found in an Arab Muslim patient. These families may benefit now from pre- and postnatal diagnosis by analysis of DNA from blood and amniotic fluid or chorionic villus cells rather than liver biopsy. No mutations in the G6Pase gene were detected in two GSD 1b patients.

Germline Missense Changes in the APC Gene and Their Relationship to Disease.

PubMed

Scott, Rodney J; Crooks, Renee; Rose, Lindy; Attia, John; Thakkinstian, Ammarin; Thomas, Lesley; Spigelman, Allan D; Meldrum, Cliff J

2004-05-15

Familial adenomatous polyposis (FAP) is characterized by the presence of hundreds to thousands of adenomas that carpet the entire colon and rectum. Nonsense and frameshift mutations in the adenomatous polyposis coli (APC) gene account for the majority of mutations identified to date and predispose primarily to the typical disease phenotype. Some APC mutations are associated with a milder form of the disease known as attenuated FAP. Virtually all mutations that have been described in the APC gene result in the formation of a premature stop codon and very little is known about missense mutations apart from a common Ashkenazi Jewish mutation (1307 K) and a British E1317Q missense change. The incidence of missense mutations in the APC gene has been underreported since the APC gene lends itself to analysis using an artificial transcription and translation assay known as the Protein Truncation Test (PTT) or the In Vitro Synthetic Protein assay (IVSP).In this report we have used denaturing high performance liquid chromatography to analyse the entire coding sequence of the APC gene to determine if a cohort of patients adhering to the diagnostic criteria of FAP to assess the frequency of missense mutations in the APC gene. Altogether 112 patients were studied and 22 missense mutations were identified. From the total of 22 missense changes, 13 were silent changes and the remaining 9 resulted in amino acid substitutions. One or more of these changes were identified multiple times in 62.5% of the population under study.The results reveal that missense mutations in the APC gene appear not to radically alter protein function but may be associated with more subtle processing of RNA transcripts which in turn could result in the expression of differentially spliced forms of the APC gene which may interfere with the functional activity of the APC protein.

A novel loss-of-function mutation in Npr2 clarifies primary role in female reproduction and reveals a potential therapy for acromesomelic dysplasia, Maroteaux type.

PubMed

Geister, Krista A; Brinkmeier, Michelle L; Hsieh, Minnie; Faust, Susan M; Karolyi, I Jill; Perosky, Joseph E; Kozloff, Kenneth M; Conti, Marco; Camper, Sally A

2013-01-15

We discovered a new spontaneous mutant allele of Npr2 named peewee (pwe) that exhibits severe disproportionate dwarfism and female infertility. The pwe phenotype is caused by a four base-pair deletion in exon 3 that generates a premature stop codon at codon 313 (L313X). The Npr2(pwe/pwe) mouse is a model for the human skeletal dysplasia acromesomelic dysplasia, Maroteaux type (AMDM). We conducted a thorough analysis of the female reproductive tract and report that the primary cause of Npr2(pwe/pwe) female infertility is premature oocyte meiotic resumption, while the pituitary and uterus appear to be normal. Npr2 is expressed in chondrocytes and osteoblasts. We determined that the loss of Npr2 causes a reduction in the hypertrophic and proliferative zones of the growth plate, but mineralization of skeletal elements is normal. Mutant tibiae have increased levels of the activated form of ERK1/2, consistent with the idea that natriuretic peptide receptor type 2 (NPR2) signaling inhibits the activation of the MEK/ERK mitogen activated protein kinase pathway. Treatment of fetal tibiae explants with mitogen activated protein kinase 1 and 2 inhibitors U0126 and PD325901 rescues the Npr2(pwe/pwe) growth defect, providing a promising foundation for skeletal dysplasia therapeutics.

A novel loss-of-function mutation in Npr2 clarifies primary role in female reproduction and reveals a potential therapy for acromesomelic dysplasia, Maroteaux type

PubMed Central

Geister, Krista A.; Brinkmeier, Michelle L.; Hsieh, Minnie; Faust, Susan M.; Karolyi, I. Jill; Perosky, Joseph E.; Kozloff, Kenneth M.; Conti, Marco; Camper, Sally A.

2013-01-01

We discovered a new spontaneous mutant allele of Npr2 named peewee (pwe) that exhibits severe disproportionate dwarfism and female infertility. The pwe phenotype is caused by a four base-pair deletion in exon 3 that generates a premature stop codon at codon 313 (L313X). The Npr2pwe/pwe mouse is a model for the human skeletal dysplasia acromesomelic dysplasia, Maroteaux type (AMDM). We conducted a thorough analysis of the female reproductive tract and report that the primary cause of Npr2pwe/pwe female infertility is premature oocyte meiotic resumption, while the pituitary and uterus appear to be normal. Npr2 is expressed in chondrocytes and osteoblasts. We determined that the loss of Npr2 causes a reduction in the hypertrophic and proliferative zones of the growth plate, but mineralization of skeletal elements is normal. Mutant tibiae have increased levels of the activated form of ERK1/2, consistent with the idea that natriuretic peptide receptor type 2 (NPR2) signaling inhibits the activation of the MEK/ERK mitogen activated protein kinase pathway. Treatment of fetal tibiae explants with mitogen activated protein kinase 1 and 2 inhibitors U0126 and PD325901 rescues the Npr2pwe/pwe growth defect, providing a promising foundation for skeletal dysplasia therapeutics. PMID:23065701

Complex codon usage pattern and compositional features of retroviruses.

PubMed

RoyChoudhury, Sourav; Mukherjee, Debaprasad

2013-01-01

Retroviruses infect a wide range of organisms including humans. Among them, HIV-1, which causes AIDS, has now become a major threat for world health. Some of these viruses are also potential gene transfer vectors. In this study, the patterns of synonymous codon usage in retroviruses have been studied through multivariate statistical methods on ORFs sequences from the available 56 retroviruses. The principal determinant for evolution of the codon usage pattern in retroviruses seemed to be the compositional constraints, while selection for translation of the viral genes plays a secondary role. This was further supported by multivariate analysis on relative synonymous codon usage. Thus, it seems that mutational bias might have dominated role over translational selection in shaping the codon usage of retroviruses. Codon adaptation index was used to identify translationally optimal codons among genes from retroviruses. The comparative analysis of the preferred and optimal codons among different retroviral groups revealed that four codons GAA, AAA, AGA, and GGA were significantly more frequent in most of the retroviral genes inspite of some differences. Cluster analysis also revealed that phylogenetically related groups of retroviruses have probably evolved their codon usage in a concerted manner under the influence of their nucleotide composition.

Molecular Scanning of β-Thalassemia in the Southern Region of Central Java, Indonesia; a Step Towards a Local Prevention Program.

PubMed

Rujito, Lantip; Basalamah, Muhammad; Mulatsih, Sri; Sofro, Abdul Salam M

2015-08-03

Thalassemia is the most prevalent genetic blood disorder worldwide, and particularly prevalent in Indonesia. The purpose of this study was to determine the spectrum of β-thalassemia (β-thal) mutations found in the southern region of Central Java, Indonesia. The subjects of the study included 209 β-thal Javanese patients from Banyumas Residency, a southwest region of Central Java Province. DNA analysis was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), amplification refractory mutation system (ARMS), and the direct sequencing method. The results showed that 14 alleles were found in the following order: IVS-I-5 (G > C) (HBB: c.92 + 5G > C) 43.5%, codon 26 (Hb E; HBB: c.79G > A) 28.2%, IVS-I-1 (G > A) (HBB: c.92 + 1G > A) 5.0%, codon 15 (TGG > TAG) (HBB: c.47G > A) 3.8%, IVS-I-1 (G > T) (HBB: c.92 + 1G > T) 3.1%, codon 35 (-C) (HBB: c.110delC) 2.4%. The rest, including codons 41/42 (-TTCT) (HBB: c.126_129delCTTT), codons 8/9 (+G) (HBB: c.27_28insG), codon 19 (AAC > AGC) (HBB: c.59A > G), codon 17 (AAG > TAG) (HBB: c.52A > T), IVS-I-2 (T > C) (HBB: c.92 + 2T > C), codons 123/124/125 (-ACCCCACC) (HBB: c.370_378delACCCCACCA), codon 40 (-G) (HBB: c.123delG) and Cap +1 (A > C) (HBB: c.-50A > C), accounted for up to 1.0% each. The most prevalent alleles would be recommended to be used as part of β-thal screening for the Javanese, one of the major ethnic groups in the country.

Molecular Scanning of β-Thalassemia in the Southern Region of Central Java, Indonesia; a Step Towards a Local Prevention Program.

PubMed

Rujito, Lantip; Basalamah, Muhammad; Mulatsih, Sri; Sofro, Abdul Salam M

2015-01-01

Thalassemia is the most prevalent genetic blood disorder worldwide, and particularly prevalent in Indonesia. The purpose of this study was to determine the spectrum of β-thalassemia (β-thal) mutations found in the southern region of Central Java, Indonesia. The subjects of the study included 209 β-thal Javanese patients from Banyumas Residency, a southwest region of Central Java Province. DNA analysis was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), amplification refractory mutation system (ARMS), and the direct sequencing method. The results showed that 14 alleles were found in the following order: IVS-I-5 (G > C) (HBB: c.92 + 5G > C) 43.5%, codon 26 (Hb E; HBB: c.79G > A) 28.2%, IVS-I-1 (G > A) (HBB: c.92 + 1G > A) 5.0%, codon 15 (TGG > TAG) (HBB: c.47G > A) 3.8%, IVS-I-1 (G > T) (HBB: c.92 + 1G > T) 3.1%, codon 35 (-C) (HBB: c.110delC) 2.4%. The rest, including codons 41/42 (-TTCT) (HBB: c.126_129delCTTT), codons 8/9 (+G) (HBB: c.27_28insG), codon 19 (AAC > AGC) (HBB: c.59A > G), codon 17 (AAG > TAG) (HBB: c.52A > T), IVS-I-2 (T > C) (HBB: c.92 + 2T > C), codons 123/124/125 (-ACCCCACC) (HBB: c.370_378delACCCCACCA), codon 40 (-G) (HBB: c.123delG) and Cap +1 (A > C) (HBB: c.-50A > C), accounted for up to 1.0% each. The most prevalent alleles would be recommended to be used as part of β-thal screening for the Javanese, one of the major ethnic groups in the country.

Evolutionary and genetic analysis of the VP2 gene of canine parvovirus.

PubMed

Li, Gairu; Ji, Senlin; Zhai, Xiaofeng; Zhang, Yuxiang; Liu, Jie; Zhu, Mengyan; Zhou, Jiyong; Su, Shuo

2017-07-17

Canine parvovirus (CPV) type 2 emerged in 1978 in the USA and quickly spread among dog populations all over the world with high morbidity. Although CPV is a DNA virus, its genomic substitution rate is similar to some RNA viruses. Therefore, it is important to trace the evolution of CPV to monitor the appearance of mutations that might affect vaccine effectiveness. Our analysis shows that the VP2 genes of CPV isolated from 1979 to 2016 are divided into six groups: GI, GII, GIII, GIV, GV, and GVI. Amino acid mutation analysis revealed several undiscovered important mutation sites: F267Y, Y324I, and T440A. Of note, the evolutionary rate of the CPV VP2 gene from Asia and Europe decreased. Codon usage analysis showed that the VP2 gene of CPV exhibits high bias with an ENC ranging from 34.93 to 36.7. Furthermore, we demonstrate that natural selection plays a major role compared to mutation pressure driving CPV evolution. There are few studies on the codon usage of CPV. Here, we comprehensively studied the genetic evolution, codon usage pattern, and evolutionary characterization of the VP2 gene of CPV. The novel findings revealing the evolutionary process of CPV will greatly serve future CPV research.

Foamy virus reverse transcriptase is expressed independently from the Gag protein.

PubMed Central

Enssle, J; Jordan, I; Mauer, B; Rethwilm, A

1996-01-01

In the foamy virus (FV) subgroup of retroviruses the pol genes are located in the +1 reading frame relative to the gag genes and possess potential ATG initiation codons in their 5' regions. This genome organization suggests either a + 1 ribosomal frameshift to generate a Gag-Pol fusion protein, similar to all other retroviruses studied so far, or new initiation of Pol translation, as used by pararetroviruses, to express the Pol protein. By using a genetic approach we have ruled out the former possibility and provide evidence for the latter. Two down-mutations (M53 and M54) of the pol ATG codon were found to abolish replication and Pol protein expression of the human FV isolate. The introduction of a new ATG in mutation M55, 3' to the down-mutated ATG of mutation M53, restored replication competence, indicating that the pol ATG functions as a translational initiation codon. Two nonsense mutants (M56 and M57), which functionally separated gag and pol with respect to potential frame-shifting sites, were also replication-competent, providing further genetic evidence that FVs express the Pol protein independently from Gag. Our results show that during a particular step of the replication cycle, FVs differ fundamentally from all other retroviruses. Images Fig. 3 PMID:8633029

SLC3A1 and SLC7A9 mutations in autosomal recessive or dominant canine cystinuria: a new classification system.

PubMed

Brons, A-K; Henthorn, P S; Raj, K; Fitzgerald, C A; Liu, J; Sewell, A C; Giger, U

2013-01-01

Cystinuria, one of the first recognized inborn errors of metabolism, has been reported in many dog breeds. To determine urinary cystine concentrations, inheritance, and mutations in the SLC3A1 and SLC7A9 genes associated with cystinuria in 3 breeds. Mixed and purebred Labrador Retrievers (n = 6), Australian Cattle Dogs (6), Miniature Pinschers (4), and 1 mixed breed dog with cystine urolithiasis, relatives and control dogs. Urinary cystinuria and aminoaciduria was assessed and exons of the SLC3A1 and SLC7A9 genes were sequenced from genomic DNA. In each breed, male and female dogs, independent of neuter status, were found to form calculi. A frameshift mutation in SLC3A1 (c.350delG) resulting in a premature stop codon was identified in autosomal-recessive (AR) cystinuria in Labrador Retrievers and mixed breed dogs. A 6 bp deletion (c.1095_1100del) removing 2 threonines in SLC3A1 was found in autosomal-dominant (AD) cystinuria with a more severe phenotype in homozygous than in heterozygous Australian Cattle Dogs. A missense mutation in SLC7A9 (c.964G>A) was discovered in AD cystinuria in Miniature Pinschers with only heterozygous affected dogs observed to date. Breed-specific DNA tests were developed, but the prevalence of each mutation remains unknown. These studies describe the first AD inheritance and the first putative SLC7A9 mutation to cause cystinuria in dogs and expand our understanding of this phenotypically and genetically heterogeneous disease, leading to a new classification system for canine cystinuria and better therapeutic management and genetic control in these breeds. Copyright © 2013 by the American College of Veterinary Internal Medicine.

No evidence for involvement of SDHD in neuroblastoma pathogenesis

PubMed Central

De Preter, Katleen; Vandesompele, Jo; Hoebeeck, Jasmien; Vandenbroecke, Caroline; Smet, Jöel; Nuyts, Annick; Laureys, Geneviève; Combaret, Valérie; Van Roy, Nadine; Roels, Frank; Van Coster, Rudy; Praet, Marleen; De Paepe, Anne; Speleman, Frank

2004-01-01

Background Deletions in the long arm of chromosome 11 are observed in a subgroup of advanced stage neuroblastomas with poor outcome. The deleted region harbours the tumour suppressor gene SDHD that is frequently mutated in paraganglioma and pheochromocytoma, which are, like neuroblastoma, tumours originating from the neural crest. In this study, we sought for evidence for involvement of SDHD in neuroblastoma. Methods SDHD was investigated on the genome, transcriptome and proteome level using mutation screening, methylation specific PCR, real-time quantitative PCR based homozygous deletion screening and mRNA expression profiling, immunoblotting, functional protein analysis and ultrastructural imaging of the mitochondria. Results Analysis at the genomic level of 67 tumour samples and 37 cell lines revealed at least 2 bona-fide mutations in cell lines without allelic loss at 11q23: a 4bp-deletion causing skip of exon 3 resulting in a premature stop codon in cell line N206, and a Y93C mutation in cell line NMB located in a region affected by germline SDHD mutations causing hereditary paraganglioma. No evidence for hypermethylation of the SDHD promotor region was observed, nor could we detect homozygous deletions. Interestingly, SDHD mRNA expression was significantly reduced in SDHD mutated cell lines and cell lines with 11q allelic loss as compared to both cell lines without 11q allelic loss and normal foetal neuroblast cells. However, protein analyses and assessment of mitochondrial morphology presently do not provide clues as to the possible effect of reduced SDHD expression on the neuroblastoma tumour phenotype. Conclusions Our study provides no indications for 2-hit involvement of SDHD in the pathogenesis of neuroblastoma. Also, although a haplo-insufficient mechanism for SDHD involvement in advanced stage neuroblastoma could be considered, the present data do not provide consistent evidence for this hypothesis. PMID:15331017

Whole-exome sequencing and high throughput genotyping identified KCNJ11 as the thirteenth MODY gene.

PubMed

Bonnefond, Amélie; Philippe, Julien; Durand, Emmanuelle; Dechaume, Aurélie; Huyvaert, Marlène; Montagne, Louise; Marre, Michel; Balkau, Beverley; Fajardy, Isabelle; Vambergue, Anne; Vatin, Vincent; Delplanque, Jérôme; Le Guilcher, David; De Graeve, Franck; Lecoeur, Cécile; Sand, Olivier; Vaxillaire, Martine; Froguel, Philippe

2012-01-01

Maturity-onset of the young (MODY) is a clinically heterogeneous form of diabetes characterized by an autosomal-dominant mode of inheritance, an onset before the age of 25 years, and a primary defect in the pancreatic beta-cell function. Approximately 30% of MODY families remain genetically unexplained (MODY-X). Here, we aimed to use whole-exome sequencing (WES) in a four-generation MODY-X family to identify a new susceptibility gene for MODY. WES (Agilent-SureSelect capture/Illumina-GAIIx sequencing) was performed in three affected and one non-affected relatives in the MODY-X family. We then performed a high-throughput multiplex genotyping (Illumina-GoldenGate assay) of the putative causal mutations in the whole family and in 406 controls. A linkage analysis was also carried out. By focusing on variants of interest (i.e. gains of stop codon, frameshift, non-synonymous and splice-site variants not reported in dbSNP130) present in the three affected relatives and not present in the control, we found 69 mutations. However, as WES was not uniform between samples, a total of 324 mutations had to be assessed in the whole family and in controls. Only one mutation (p.Glu227Lys in KCNJ11) co-segregated with diabetes in the family (with a LOD-score of 3.68). No KCNJ11 mutation was found in 25 other MODY-X unrelated subjects. Beyond neonatal diabetes mellitus (NDM), KCNJ11 is also a MODY gene ('MODY13'), confirming the wide spectrum of diabetes related phenotypes due to mutations in NDM genes (i.e. KCNJ11, ABCC8 and INS). Therefore, the molecular diagnosis of MODY should include KCNJ11 as affected carriers can be ideally treated with oral sulfonylureas.

Activation of K-ras by codon 13 mutations in C57BL/6 X C3H F1 mouse tumors induced by exposure to 1,3-butadiene.

PubMed

Goodrow, T; Reynolds, S; Maronpot, R; Anderson, M

1990-08-01

1,3-Butadiene has been detected in urban air, gasoline vapors, and cigarette smoke. It has been estimated that 65,000 workers are exposed to this chemical in occupational settings in the United States. Lymphomas, lung, and liver tumors were induced in female and male C57BL/6 X C3H F1 (hereafter called B6C3F1) mice by inhalation of 6.25 to 625 ppm 1,3-butadiene for 1 to 2 years. The objective of this study was to examine these tumors for the presence of activated protooncogenes by the NIH 3T3 transfection and nude mouse tumorigenicity assays. Transfection of DNA isolated from 7 of 9 lung tumors and 7 of 12 liver tumors induced morphological transformation of NIH 3T3 cells. Southern blot analysis indicated that the transformation induced by 6 lung and 3 liver tumor DNA samples was due to transfer of a K-ras oncogene. Four of the 7 liver tumors that were positive upon transfection contained an activated H-ras gene. The identity of the transforming gene in one of the lung tumors has not been determined but was not a member of the ras family or a met or raf gene. Eleven 1,3-butadiene-induced lymphomas were examined for transforming genes using the nude mouse tumorigenicity assay. Activated K-ras genes were detected in 2 of the 11 lymphomas assayed. DNA sequencing of polymerase chain reaction-amplified ras gene exons revealed that 9 of 11 of the activating K-ras mutations were G to C transversions in codon 13. One liver tumor contained an activated K-ras gene with mutations in both codons 60 and 61. The activating mutation in one of the K-ras genes from a lymphoma was not identified but DNA sequence analysis of amplified regions in proximity to codons 12, 13, and 61 demonstrated that the mutation was not located in or near these codons. Activation of K-ras genes by codon 13 mutations has not been found in any lung or liver tumors or lymphomas from untreated B6C3F1 mice. Thus, the K-ras activation found in 1,3-butadiene-induced B6C3F1 mouse tumors probably occurred as a result of genotoxic effects of this chemical. The oncogenes most frequently detected in human pulmonary adenocarcinomas are K-ras genes. Activated K-ras genes have also been found in some human lymphomas. This suggest that activation of K-ras may be important in the induction of human pulmonary adenocarcinomas and lymphomas.(ABSTRACT TRUNCATED AT 400 WORDS)

Codon Optimization of the Human Papillomavirus E7 Oncogene Induces a CD8+ T Cell Response to a Cryptic Epitope Not Harbored by Wild-Type E7

PubMed Central

Lorenz, Felix K. M.; Wilde, Susanne; Voigt, Katrin; Kieback, Elisa; Mosetter, Barbara; Schendel, Dolores J.; Uckert, Wolfgang

2015-01-01

Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine. PMID:25799237

Codon usage in Chlamydia trachomatis is the result of strand-specific mutational biases and a complex pattern of selective forces

PubMed Central

Romero, Héctor; Zavala, Alejandro; Musto, Héctor

2000-01-01

The patterns of synonymous codon choices of the completely sequenced genome of the bacterium Chlamydia trachomatis were analysed. We found that the most important source of variation among the genes results from whether the sequence is located on the leading or lagging strand of replication, resulting in an over representation of G or C, respectively. This can be explained by different mutational biases associated to the different enzymes that replicate each strand. Next we found that most highly expressed sequences are located on the leading strand of replication. From this result, replicational-transcriptional selection can be invoked. Then, when the genes located on the leading strand are studied separately, the correspondence analysis detects a principal trend which discriminates between lowly and highly expressed sequences, the latter displaying a different codon usage pattern than the former, suggesting selection for translation, which is reinforced by the fact that Ks values between orthologous sequences from C.trachomatis and Chlamydia pneumoniae are much smaller in highly expressed genes. Finally, synonymous codon choices appear to be influenced by the hydropathy of each encoded protein and by the degree of amino acid conservation. Therefore, synonymous codon usage in C.trachomatis seems to be the result of a very complex balance among different factors, which rises the problem of whether the forces driving codon usage patterns among microorganisms are rather more complex than generally accepted. PMID:10773076

Codon usage in Chlamydia trachomatis is the result of strand-specific mutational biases and a complex pattern of selective forces.

PubMed

Romero, H; Zavala, A; Musto, H

2000-05-15

The patterns of synonymous codon choices of the completely sequenced genome of the bacterium Chlamydia trachomatis were analysed. We found that the most important source of variation among the genes results from whether the sequence is located on the leading or lagging strand of replication, resulting in an over representation of G or C, respectively. This can be explained by different mutational biases associated to the different enzymes that replicate each strand. Next we found that most highly expressed sequences are located on the leading strand of replication. From this result, replicational-transcriptional selection can be invoked. Then, when the genes located on the leading strand are studied separately, the correspondence analysis detects a principal trend which discriminates between lowly and highly expressed sequences, the latter displaying a different codon usage pattern than the former, suggesting selection for translation, which is reinforced by the fact that Ks values between orthologous sequences from C. trachomatis and Chlamydia pneumoniae are much smaller in highly expressed genes. Finally, synonymous codon choices appear to be influenced by the hydropathy of each encoded protein and by the degree of amino acid conservation. Therefore, synonymous codon usage in C.trachomatis seems to be the result of a very complex balance among different factors, which rises the problem of whether the forces driving codon usage patterns among microorganisms are rather more complex than generally accepted.

Synonymous codon usage of genes in polymerase complex of Newcastle disease virus.

PubMed

Kumar, Chandra Shekhar; Kumar, Sachin

2017-06-01

Newcastle disease virus (NDV) is pathogenic to both avian and non-avian species but extensively finds poultry as its primary host and causes heavy economic losses in the poultry industry. In this study, a total of 186 polymerase complex comprising of nucleoprotein (N), phosphoprotein (P), and large polymerase (L) genes of NDV was analyzed for synonymous codon usage. The relative synonymous codon usage and effective number of codons (ENC) values were used to estimate codon usage variation in each gene. Correspondence analysis (COA) was used to study the major trend in codon usage variation. Analyzing the ENC plot values against GC3s (at synonymous third codon position) we concluded that mutational pressure was the main factor determining codon usage bias than translational selection in NDV N, P, and L genes. Moreover, correlation analysis indicated, that aromaticity of N, P, and L genes also influenced the codon usage variation. The varied distribution of pathotypes for N, P, and L gene clearly suggests that change in codon usage for NDV is pathotype specific. The codon usage preference similarity in N, P, and L gene might be detrimental for polymerase complex functioning. The study represents a comprehensive analysis to date of N, P, and L genes codon usage pattern of NDV and provides a basic understanding of the mechanisms for codon usage bias. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Autism-Associated Insertion Mutation (InsG) of Shank3 Exon 21 Causes Impaired Synaptic Transmission and Behavioral Deficits.

PubMed

Speed, Haley E; Kouser, Mehreen; Xuan, Zhong; Reimers, Jeremy M; Ochoa, Christine F; Gupta, Natasha; Liu, Shunan; Powell, Craig M

2015-07-01

SHANK3 (also known as PROSAP2) is a postsynaptic scaffolding protein at excitatory synapses in which mutations and deletions have been implicated in patients with idiopathic autism, Phelan-McDermid (aka 22q13 microdeletion) syndrome, and other neuropsychiatric disorders. In this study, we have created a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3(G)). The resulting frameshift causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse. Shank3(G/G) mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, altered response to novelty, and sensory processing deficits. At the cellular level, Shank3(G/G) mice also exhibit impaired hippocampal excitatory transmission and plasticity as well as changes in baseline NMDA receptor-mediated synaptic responses. This work identifies clear alterations in synaptic function and behavior in a novel, genetically accurate mouse model of autism mimicking an autism-associated insertion mutation. Furthermore, these findings lay the foundation for future studies aimed to validate and study region-selective and temporally selective genetic reversal studies in the Shank3(G/G) mouse that was engineered with such future experiments in mind. Copyright © 2015 the authors 0270-6474/15/359648-18$15.00/0.

Human RHOH deficiency causes T cell defects and susceptibility to EV-HPV infections.

PubMed

Crequer, Amandine; Troeger, Anja; Patin, Etienne; Ma, Cindy S; Picard, Capucine; Pedergnana, Vincent; Fieschi, Claire; Lim, Annick; Abhyankar, Avinash; Gineau, Laure; Mueller-Fleckenstein, Ingrid; Schmidt, Monika; Taieb, Alain; Krueger, James; Abel, Laurent; Tangye, Stuart G; Orth, Gérard; Williams, David A; Casanova, Jean-Laurent; Jouanguy, Emmanuelle

2012-09-01

Epidermodysplasia verruciformis (EV) is a rare genetic disorder characterized by increased susceptibility to specific human papillomaviruses, the betapapillomaviruses. These EV-HPVs cause warts and increase the risk of skin carcinomas in otherwise healthy individuals. Inactivating mutations in epidermodysplasia verruciformis 1 (EVER1) or EVER2 have been identified in most, but not all, patients with autosomal recessive EV. We found that 2 young adult siblings presenting with T cell deficiency and various infectious diseases, including persistent EV-HPV infections, were homozygous for a mutation creating a stop codon in the ras homolog gene family member H (RHOH) gene. RHOH encodes an atypical Rho GTPase expressed predominantly in hematopoietic cells. Patients' circulating T cells contained predominantly effector memory T cells, which displayed impaired TCR signaling. Additionally, very few circulating T cells expressed the β7 integrin subunit, which homes T cells to specific tissues. Similarly, Rhoh-null mice exhibited a severe overall T cell defect and abnormally small numbers of circulating β7-positive cells. Expression of the WT, but not of the mutated RHOH, allele in Rhoh-/- hematopoietic stem cells corrected the T cell lymphopenia in mice after bone marrow transplantation. We conclude that RHOH deficiency leads to T cell defects and persistent EV-HPV infections, suggesting that T cells play a role in the pathogenesis of chronic EV-HPV infections.

Glycogen branching enzyme (GBE1) mutation causing equine glycogen storage disease IV.

PubMed

Ward, Tara L; Valberg, Stephanie J; Adelson, David L; Abbey, Colette A; Binns, Matthew M; Mickelson, James R

2004-07-01

Comparative biochemical and histopathological evidence suggests that a deficiency in the glycogen branching enzyme, encoded by the GBE1 gene, is responsible for a recently identified recessive fatal fetal and neonatal glycogen storage disease (GSD) in American Quarter Horses termed GSD IV. We have now derived the complete GBE1 cDNA sequences for control horses and affected foals, and identified a C to A substitution at base 102 that results in a tyrosine (Y) to stop (X) mutation in codon 34 of exon 1. All 11 affected foals were homozygous for the X34 allele, their 11 available dams and sires were heterozygous, and all 16 control horses were homozygous for the Y34 allele. The previous findings of poorly branched glycogen, abnormal polysaccharide accumulation, lack of measurable GBE1 enzyme activity and immunodetectable GBE1 protein, coupled with the present observation of abundant GBE1 mRNA in affected foals, are all consistent with the nonsense mutation in the 699 amino acid GBE1 protein. The affected foal pedigrees have a common ancestor and contain prolific stallions that are likely carriers of the recessive X34 allele. Defining the molecular basis of equine GSD IV will allow for accurate DNA testing and the ability to prevent occurrence of this devastating disease affecting American Quarter Horses and related breeds.