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‣ Positive Selection for Mutations Affecting Bioconversion of Aromatic Compounds in Agrobacterium tumefaciens: Analysis of Spontaneous Mutations in the Protocatechuate 3,4-Dioxygenase Gene

Parke, Donna
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2000 Português
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A positive selection method for mutations affecting bioconversion of aromatic compounds was applied to a mutant strain of Agrobacterium tumefaciens A348. The nucleotide sequence of the A348 pcaHGB genes, which encode protocatechuate 3,4-dioxygenase (PcaHG) and β-carboxy-cis,cis-muconate cycloisomerase (PcaB) for the first two steps in catabolism of the diphenolic protocatechuate, was determined. An omega element was introduced into the pcaB gene of A348, creating strain ADO2077. In the presence of phenolic compounds that can serve as carbon sources, growth of ADO2077 is inhibited due to accumulation of the tricarboxylate intermediate. The toxic effect, previously described for Acinetobacter sp., affords a powerful selection for suppressor mutations in genes required for upstream catabolic steps. By monitoring loss of the marker in pcaB, it was possible to determine that the formation of deletions was minimal compared to results obtained with Acinetobacter sp. Thus, the tricarboxylic acid trick in and of itself does not appear to select for large deletion mutations. The power of the selection was demonstrated by targeting the pcaHG genes of A. tumefaciens for spontaneous mutation. Sixteen strains carrying putative second-site mutations in pcaH or -G were subjected to sequence analysis. All single-site events...

‣ Gyrase mutations in laboratory-selected, fluoroquinolone-resistant mutants of Mycobacterium tuberculosis H37Ra.

Kocagöz, T; Hackbarth, C J; Unsal, I; Rosenberg, E Y; Nikaido, H; Chambers, H F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1996 Português
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To characterize mechanisms of resistance to fluoroquinolones by Mycobacterium tuberculosis, mutants of strain H37Ra were selected in vitro with ofloxacin. Their quinolone resistance-determining regions for gyrA and gyrB were amplified and sequenced to identify mutations in gyrase A or B. Three types of mutants were obtained: (i) one mutant (TKp1) had no mutations in gyrA or gyrB; (ii) mutants that had single missense mutations in gyrA, and (iii) mutants that had two missense mutations resulting in either two altered gyrase A residues or an altered residue in both gyrases A and B. The TKp1 mutant had slightly reduced levels of uptake of [14C]norfloxacin, which was associated with two- to fourfold increases in the MICs of ofloxacin, ciprofloxacin, and sparfloxacin. Gyrase mutations caused a much greater increase in the MICs of fluoroquinolones. For mutants with single gyrA mutations, the increases in the MICs were 4- to 16-fold, and for mutants with double gyrase mutations, the MICs were increased 32-fold or more compared with those for the parent. A gyrA mutation in TKp1 secondary mutants was associated with 32- to 128-fold increases in the MICs of ofloxacin and ciprofloxacin compared with the MICs for H37Ra and an eight-fold increase in the MIC of sparfloxacin. Sparfloxacin was the most active fluoroquinolone tested. No sparfloxacin-resistant single-step mutants were selected at concentrations of > 2.5 micrograms/ml...

‣ Adsorption of bacteriophage lambda on the LamB protein of Escherichia coli K-12: point mutations in gene J of lambda responsible for extended host range.

Werts, C; Michel, V; Hofnung, M; Charbit, A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1994 Português
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LamB is the cell surface receptor for bacteriophage lambda. LamB missense mutations yielding resistance to lambda group in two classes. Class I mutants block the growth of lambda with the wild-type host range (lambda h+) but support the growth of one-step host range mutants (lambda h). Class II mutants block lambda h but support the growth of two-step host range mutant (lambda hh*) phages. To identify amino acid residues in the J protein (the tail fiber of phage lambda) responsible for the extended host range phenotype of mutants of phage (lambda h+), we selected a series of one-step (lambda h) and two-step (lambda hh*) host range mutants and analyzed their corresponding J genes. Three different class I LamB missense mutants (mutations at sites 247, 245, and 148) were used to select 11 independent, new, one-step host range mutants (lambda h phages). DNA sequence analysis revealed a single-amino-acid change in each case. The 11 alterations affected only three residues in the distal part of J, corresponding to a Val-->Ala change at site 1077 in five cases, a Thr-->Met change at site 1040 in three cases, and a Leu-->Pro change at site 1127 in three cases. Recombination experiments confirmed that in the cases tested, the mutations identified were indeed responsible for the extended host range phenotype. The class II LamB mutant (Gly-->Asp at site 151) was used to select two-step extended host range mutants (lambda hh* phages) from three new lambda h phages...

‣ NSD1 Mutations Are the Major Cause of Sotos Syndrome and Occur in Some Cases of Weaver Syndrome but Are Rare in Other Overgrowth Phenotypes

Douglas, Jenny; Hanks, Sandra; Temple, I. Karen; Davies, Sally; Murray, Alexandra; Upadhyaya, Meena; Tomkins, Susan; Hughes, Helen E.; Cole, Trevor R. P.; Rahman, Nazneen
Fonte: The American Society of Human Genetics Publicador: The American Society of Human Genetics
Tipo: Artigo de Revista Científica
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Sotos syndrome is a childhood overgrowth syndrome characterized by a distinctive facial appearance, height and head circumference >97th percentile, advanced bone age, and developmental delay. Weaver syndrome is characterized by the same criteria but has its own distinctive facial gestalt. Recently, a 2.2-Mb chromosome 5q35 microdeletion, encompassing NSD1, was reported as the major cause of Sotos syndrome, with intragenic NSD1 mutations identified in a minority of cases. We evaluated 75 patients with childhood overgrowth, for intragenic mutations and large deletions of NSD1. The series was phenotypically scored into four groups, prior to the molecular analyses: the phenotype in group 1 (n=37) was typical of Sotos syndrome; the phenotype in group 2 (n=13) was Sotos-like but with some atypical features; patients in group 3 (n=7) had Weaver syndrome, and patients in group 4 (n=18) had an overgrowth condition that was neither Sotos nor Weaver syndrome. We detected three deletions and 32 mutations (13 frameshift, 8 nonsense, 2 splice-site, and 9 missense) that are likely to impair NSD1 functions. The truncating mutations were spread throughout NSD1, but there was evidence of clustering of missense mutations in highly conserved functional domains between exons 13 and 23. There was a strong correlation between presence of an NSD1 alteration and clinical phenotype...

‣ DAX1 mutations map to putative structural domains in a deduced three-dimensional model.

Zhang, Y H; Guo, W; Wagner, R L; Huang, B L; McCabe, L; Vilain, E; Burris, T P; Anyane-Yeboa, K; Burghes, A H; Chitayat, D; Chudley, A E; Genel, M; Gertner, J M; Klingensmith, G J; Levine, S N; Nakamoto, J; New, M I; Pagon, R A; Pappas, J G; Quigley, C A;
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1998 Português
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The DAX1 protein is an orphan nuclear hormone receptor based on sequence similarity in the putative ligand-binding domain (LBD). DAX1 mutations result in X-linked adrenal hypoplasia congenita (AHC). Our objective was to identify DAX1 mutations in a series of families, to determine the types of mutations resulting in AHC and to locate single-amino-acid changes in a DAX1 structural model. The 14 new mutations identified among our 17 families with AHC brought the total number of families with AHC to 48 and the number of reported mutations to 42; 1 family showed gonadal mosaicism. These mutations included 23 frameshift, 12 nonsense, and six missense mutations and one single-codon deletion. We mapped the seven single-amino-acid changes to a homology model constructed by use of the three-dimensional crystal structures of the thyroid-hormone receptor and retinoid X receptor alpha. All single-amino-acid changes mapped to the C-terminal half of the DAX1 protein, in the conserved hydrophobic core of the putative LBD, and none affected residues expected to interact directly with a ligand. We conclude that most genetic alterations in DAX1 are frameshift or nonsense mutations and speculate that the codon deletion and missense mutations give insight into the structure and function of DAX1.

‣ High prevalence of mutations in the microtubule-associated protein tau in a population study of frontotemporal dementia in the Netherlands.

Rizzu, P; Van Swieten, J C; Joosse, M; Hasegawa, M; Stevens, M; Tibben, A; Niermeijer, M F; Hillebrand, M; Ravid, R; Oostra, B A; Goedert, M; van Duijn, C M; Heutink, P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1999 Português
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Mutations in microtubule-associated protein tau recently have been identified in familial cases of frontotemporal dementia (FTD). We report the frequency of tau mutations in a large population-based study of FTD carried out in the Netherlands from January 1994 to June 1998. Thirty-seven patients had >/=1 first-degree relative with dementia. A mutation in the tau gene was found in 17.8% of the group of patients with FTD and in 43% of patients with FTD who also had a positive family history of FTD. Three distinct missense mutations (G272V, P301L, R406W) accounted for 15.6% of the mutations. These three missense mutations, and a single amino acid deletion (DeltaK280) that was detected in one patient, strongly reduce the ability of tau to promote microtubule assembly. We also found an intronic mutation at position +33 after exon 9, which is likely to affect the alternative splicing of tau. Tau mutations are responsible for a large proportion of familial FTD cases; however, there are also families with FTD in which no mutations in tau have been found, which indicates locus and/or allelic heterogeneity. The different tau mutations may result in disturbances in the interactions of the protein tau with microtubules, resulting in hyperphosphorylation of tau protein...

‣ Effects of novel maturity-onset diabetes of the young (MODY)-associated mutations on glucokinase activity and protein stability

Galán, María; Vincent, Olivier; Roncero, Isabel; Azriel, Sharona; Boix-Pallares, Pedro; Delgado-Alvarez, Elías; Díaz-Cadórniga, Francisco; Blázquez, Enrique; Navas, María-Angeles
Fonte: Portland Press Ltd. Publicador: Portland Press Ltd.
Tipo: Artigo de Revista Científica
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Glucokinase acts as the pancreatic glucose sensor and plays a critical role in the regulation of insulin secretion by the β-cell. Heterozygous mutations in the glucokinase-encoding GCK gene, which result in a reduction of the enzymatic activity, cause the monogenic form of diabetes, MODY2 (maturity-onset diabetes of the young 2). We have identified and functionally characterized missense mutations in the GCK gene in diabetic families that result in protein mutations Leu165→Phe, Glu265→Lys and Thr206→Met. The first two are novel GCK mutations that co-segregate with the diabetes phenotype in their respective families and are not found in more than 50 healthy control individuals. In order to measure the biochemical effects of these missense mutations on glucokinase activity, we bacterially expressed and affinity-purified islet human glucokinase proteins carrying the respective mutations and fused to GST (glutathione S-transferase). Enzymatic assays on the recombinant proteins revealed that mutations Thr206→Met and Leu165→Phe strongly affect the kinetic parameters of glucokinase, in agreement with the localization of both residues close to the active site of the enzyme. In contrast, mutation Glu265→Lys, which has a weaker effect on the kinetics of glucokinase...

‣ Disease mutations in RUNX1 and RUNX2 create nonfunctional, dominant-negative, or hypomorphic alleles

Matheny, Christina J; Speck, Maren E; Cushing, Patrick R; Zhou, Yunpeng; Corpora, Takeshi; Regan, Michael; Newman, Miki; Roudaia, Liya; Speck, Caroline L; Gu, Ting-Lei; Griffey, Stephen M; Bushweller, John H; Speck, Nancy A
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
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Monoallelic RUNX1 mutations cause familial platelet disorder with predisposition for acute myelogenous leukemia (FPD/AML). Sporadic mono- and biallelic mutations are found at high frequencies in AML M0, in radiation-associated and therapy-related myelodysplastic syndrome and AML, and in isolated cases of AML M2, M5a, M3 relapse, and chronic myelogenous leukemia in blast phase. Mutations in RUNX2 cause the inherited skeletal disorder cleidocranial dysplasia (CCD). Most hematopoietic missense mutations in Runx1 involve DNA-contacting residues in the Runt domain, whereas the majority of CCD mutations in Runx2 are predicted to impair CBFβ binding or the Runt domain structure. We introduced different classes of missense mutations into Runx1 and characterized their effects on DNA and CBFβ binding by the Runt domain, and on Runx1 function in vivo. Mutations involving DNA-contacting residues severely inactivate Runx1 function, whereas mutations that affect CBFβ binding but not DNA binding result in hypomorphic alleles. We conclude that hypomorphic RUNX2 alleles can cause CCD, whereas hematopoietic disease requires more severely inactivating RUNX1 mutations.

‣ Mutations in residues of TP53 that directly contact DNA predict poor outcome in human primary breast cancer.

Berns, E. M.; van Staveren, I. L.; Look, M. P.; Smid, M.; Klijn, J. G.; Foekens, J. A.
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
Publicado em /04/1998 Português
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The tumour-suppressor gene TP53 is frequently mutated in breast tumours, and the majority of the mutations are clustered within the core domain, the region involved in DNA binding. We searched for alterations in this central domain of the TP53gene in 222 human breast cancer specimens using polymerase chain reaction-single-strand conformation analysis (PCR-SSCA) followed by sequencing. TP53 gene mutations were observed in 66 tumours (31%), including three tumours that contain two mutations. Fifty-four (78%) of these mutations were missense point mutations, one was a nonsense mutation and four were deletions and/or insertions causing disruption of the protein reading frame, whereas four mutations were either silent or a polymorphism (at codon 213; n = 6). Interestingly, the majority of missense mutations were observed at codon 248. The outcome has been related with patient and tumour characteristics, and with prognosis in 177 patients who were eligible for analysis of both relapse-free and overall survival (median survival for patients alive was 115 months). There was no significant association between the frequency of TP53 mutations and menopausal or nodal status, or tumour size. In a Cox univariate analysis, TP53 gene mutation was significantly associated with poor relapse-free survival (RFS: P = 0.02) but not with overall survival (OS: P = 0.07). In a Cox multivariate analysis...

‣ p53 missense but not truncation mutations are associated with low levels of p21CIP1/WAF1 mRNA expression in primary human sarcomas

Mousses, S; Gokgoz, N; Wunder, J S; Ozcelik, H; Bull, S; Bell, R S; Andrulis, I L
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
Publicado em /06/2001 Português
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Many growth-suppressing signals converge to control the levels of the CDK inhibitor p21CIP1/WAF1. Some human cancers exhibit low levels of expression of p21CIP1/WAF1and mutations in p53 have been implicated in this down-regulation. To evaluate whether the presence of p53 mutations was related to the in vivo expression of p21CIP1/WAF1 mRNA in sarcomas we measured the p21CIP1/WAF1 mRNA levels for a group of 71 primary bone and soft tissue tumours with known p53 status. As expected, most tumours with p53 mutations expressed low levels of p21CIP1/WAF1 mRNA. However, we identified a group of tumours with p53 gene mutations that exhibited normal or higher levels of p21CIP1/WAF1 mRNA. The p53 mutations in the latter group were not the common missense mutations in exons 4–9, but were predominantly nonsense mutations predicted to result in truncation of the p53 protein. The results of this study suggest that different types of p53 mutations can have different effects on the expression of downstream genes such as p21CIP1/WAF1 in human sarcomas. © 2001 Cancer Research Campaign http://www.bjcancer.com

‣ Novel mutations affecting the Na, K ATPase alpha model complex neurological diseases and implicate the sodium pump in increased longevity

Ashmore, Lesley J.; Hrizo, Stacy L.; Paul, Sarah M.; Van Voorhies, Wayne A.; Beitel, Greg J.; Palladino, Michael J.
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
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Mutations affecting the Na+, K+ ATPase alpha subunit have been implicated in at least two distinct human diseases, rapid-onset dystonia Parkinsonism (RDP), and familial hemiplegic migraine (FHM). Over 40 mutations have been mapped to the human ATP1A2 and ATP1A3 genes and are known to result in RDP, FHM or a variant of FHM with neurological complications. To develop a genetically tractable model system for investigating the role of the Na+, K+ ATPase in neural pathologies we performed genetic screens in Drosophila melanogaster to isolate loss-of-function alleles affecting the Na+, K+ ATPase alpha subunit. Flies heterozygous for these mutations all exhibit reduced respiration, consistent with a loss-of-function in the major ATPase. However, these mutations do not affect all functions of the Na+, K+ ATPase alpha protein since embryos homozygous for these mutations have normal septate junction paracellular barrier function and tracheal morphology. Importantly, all of these mutations cause neurological phenotypes and, akin to the mutations that cause RDP and FHM, these new alleles are missense mutations. All of these alleles exhibit progressive stress-induced locomotor impairment suggesting neuromuscular dysfunction, yet neurodegeneration is observed in an allele-specific manner. Surprisingly...

‣ Identification and in silico analysis of 14 novel GJB1, MPZ and PMP22 gene mutations

Miltenberger-Miltenyi, Gabriel; Schwarzbraun, Thomas; Löscher, Wolfgang N; Wanschitz, Julia; Windpassinger, Christian; Duba, Hans-Christoph; Seidl, Rainer; Albrecht, Gerhard; Weirich-Schwaiger, Helga; Zoller, Heinz; Utermann, Gerd; Auer-Grumbach, Michael
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
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Duplication within the chromosome 17p11.2 (CMT1Adup), peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ) and gap junction β1-protein (GJB1) gene mutations are frequent causes of the Charcot-Marie-Tooth disease (CMT). A large number of mutations in these genes are listed in databases. Sequence variants identified in patients are frequently reported as mutations without further evaluation. We analyzed 250 consecutively recruited unrelated Austrian CMT patients for CMT1Adup by microsatellite marker typing, real-time PCR or MLPA, and found 79 duplications (31.6%). The coding regions of the PMP22, MPZ and GJB1 genes were analyzed by direct sequencing in the remaining patients; 28 patients showed mutations, 14 of which were novel. We scored the pathogenicity of novel missense mutations by segregation studies and by their exclusion in control samples. Our comprehensive literature study found that up to 60% of the reported mutations in these genes had not been evaluated regarding their pathogenicity, and the PANTHER bioinformatics tool was used to score novel and published missense variants. The PANTHER program scored known polymorphisms as such, but scored ∼82–88% only of the published and novel mutations as most likely deleterious. Mutations associated with axonal CMT were less likely to be classified as deleterious...

‣ Identification of c-kit gene mutations in primary adenoid cystic carcinoma of the salivary gland

Vila, Lizette; Liu, Hongyan; Al-Quran, Samer Z; Coco, Dominique P; Dong, Hui-Jia; Liu, Chen
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The CD117 (KIT) protein is overexpressed in many human neoplasms including adenoid cystic carcinoma of salivary glands. To evaluate the function of c-kit-activating mutations in adenoid cystic carcinoma of the salivary gland, we studied 14 cases (13 primary, 1 cervical lymph node metastasis) from our institution. KIT protein expression was evaluated by immunohistochemistry using formalin-fixed paraffin-embedded tissue. Mutational analyses of c-kit extracellular (exon 9), juxtamembrane (exon 11) and tyrosine kinase domains (exons 13 and 17) were performed by polymerase chain reaction, clonal selection and DNA sequencing. All 14 cases demonstrated strong KIT expression by immunohistochemistry. Molecular analysis was successful in 8 of 14 cases, and c-kit missense point mutations were detected in seven of eight cases (88%) including seven in exon 11, two in exon 9, two in exon 13 and two in exon 17. Eight silent point mutations were detected in five cases. Two cases contained missense mutations in more than one exon. Different mutations were found in the primary tumor and the cervical lymph node metastasis of one patient. Point mutations in domains similar to those described in gastrointestinal stromal tumors were detected, including Pro551Leu and Lys558Glu (5′ end of exon 11)...

‣ Identification and Glycerol-Induced Correction of Misfolding Mutations in the X-Linked Mental Retardation Gene CASK

LaConte, Leslie E. W.; Chavan, Vrushali; Mukherjee, Konark
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 05/02/2014 Português
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The overwhelming amount of available genomic sequence variation information demands a streamlined approach to examine known pathogenic mutations of any given protein. Here we seek to outline a strategy to easily classify pathogenic missense mutations that cause protein misfolding and are thus good candidates for chaperone-based therapeutic strategies, using previously identified mutations in the gene CASK. We applied a battery of bioinformatics algorithms designed to predict potential impact on protein structure to five pathogenic missense mutations in the protein CASK that have been shown to underlie pathologies ranging from X-linked mental retardation to autism spectrum disorder. A successful classification of the mutations as damaging was not consistently achieved despite the known pathogenicity. In addition to the bioinformatics analyses, we performed molecular modeling and phylogenetic comparisons. Finally, we developed a simple high-throughput imaging assay to measure the misfolding propensity of the CASK mutants in situ. Our data suggests that a phylogenetic analysis may be a robust method for predicting structurally damaging mutations in CASK. Mutations in two evolutionarily invariant residues (Y728C and W919R) exhibited a strong propensity to misfold and form visible aggregates in the cytosolic milieu. The remaining mutations (R28L...

‣ Loss-of-function HDAC8 mutations cause a phenotypic spectrum of Cornelia de Lange syndrome-like features, ocular hypertelorism, large fontanelle and X-linked inheritance

Kaiser, Frank J.; Ansari, Morad; Braunholz, Diana; Concepción Gil-Rodríguez, María; Decroos, Christophe; Wilde, Jonathan J.; Fincher, Christopher T.; Kaur, Maninder; Bando, Masashige; Amor, David J.; Atwal, Paldeep S.; Bahlo, Melanie; Bowman, Christine
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes...

‣ Targeted Next-Generation Sequencing Reveals Novel USH2A Mutations Associated with Diverse Disease Phenotypes: Implications for Clinical and Molecular Diagnosis

Chen, Xue; Sheng, Xunlun; Liu, Xiaoxing; Li, Huiping; Liu, Yani; Rong, Weining; Ha, Shaoping; Liu, Wenzhou; Kang, Xiaoli; Zhao, Kanxing; Zhao, Chen
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 18/08/2014 Português
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USH2A mutations have been implicated in the disease etiology of several inherited diseases, including Usher syndrome type 2 (USH2), nonsyndromic retinitis pigmentosa (RP), and nonsyndromic deafness. The complex genetic and phenotypic spectrums relevant to USH2A defects make it difficult to manage patients with such mutations. In the present study, we aim to determine the genetic etiology and to characterize the correlated clinical phenotypes for three Chinese pedigrees with nonsyndromic RP, one with RP sine pigmento (RPSP), and one with USH2. Family histories and clinical details for all included patients were reviewed. Ophthalmic examinations included best corrected visual acuities, visual field measurements, funduscopy, and electroretinography. Targeted next-generation sequencing (NGS) was applied using two sequence capture arrays to reveal the disease causative mutations for each family. Genotype-phenotype correlations were also annotated. Seven USH2A mutations, including four missense substitutions (p.P2762A, p.G3320C, p.R3719H, and p.G4763R), two splice site variants (c.8223+1G>A and c.8559-2T>C), and a nonsense mutation (p.Y3745*), were identified as disease causative in the five investigated families, of which three reported to have consanguineous marriage. Among all seven mutations...

‣ Phenotypic severity of homozygous GCK mutations causing neonatal or childhood-onset diabetes is primarily mediated through effects on protein stability

Raimondo, Anne; Chakera, Ali J.; Thomsen, Soren K.; Colclough, Kevin; Barrett, Amy; De Franco, Elisa; Chatelas, Alisson; Demirbilek, Huseyin; Akcay, Teoman; Alawneh, Hussein; ; Flanagan, Sarah E.; Van De Bunt, Martijn; Hattersley, Andrew T.; Gloyn, Anna L
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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Mutations in glucokinase (GCK) cause a spectrum of glycemic disorders. Heterozygous loss-of-function mutations cause mild fasting hyperglycemia irrespective of mutation severity due to compensation from the unaffected allele. Conversely, homozygous loss-of-function mutations cause permanent neonatal diabetes requiring lifelong insulin treatment. This study aimed to determine the relationship between in vitro mutation severity and clinical phenotype in a large international case series of patients with homozygous GCK mutations. Clinical characteristics for 30 patients with diabetes due to homozygous GCK mutations (19 unique mutations, including 16 missense) were compiled and assigned a clinical severity grade (CSG) based on birth weight and age at diagnosis. The majority (28 of 30) of subjects were diagnosed before 9 months, with the remaining two at 9 and 15 years. These are the first two cases of a homozygous GCK mutation diagnosed outside infancy. Recombinant mutant GCK proteins were analyzed for kinetic and thermostability characteristics and assigned a relative activity index (RAI) or relative stability index (RSI) value. Six of 16 missense mutations exhibited severe kinetic defects (RAI ≤ 0.01). There was no correlation between CSG and RAI (r2 = 0.05...

‣ Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

Wang, H.; Kong, F.; Sorrell, T.; Wang, B.; McNicholas, P.; Pantarat, N.; Ellis, D.; Xiao, M.; Widmer, F.; Chen, S.
Fonte: BioMed Central Ltd. Publicador: BioMed Central Ltd.
Tipo: Artigo de Revista Científica
Publicado em //2009 Português
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BACKGROUND Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing. RESULTS The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D...

‣ Identification of novel Bruton's tyrosine kinase mutations in 10 unrelated subjects with X linked agammaglobulinaemia.

Brooimans, R A; van den Berg, A J; Rijkers, G T; Sanders, L A; van Amstel, J K; Tilanus, M G; Grubben, M J; Zegers, B J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1997 Português
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Mutations of the Bruton's tyrosine kinase (Btk) gene cause X linked agammaglobulinaemia (XLA). This inherited immunodeficiency disease causes an arrest in B cell differentiation of pre-B cells to mature B cells. In this study we report the characterisation of mutations in the Btk gene in 10 unrelated XLA families. The screening approach we used was based on reverse transcriptase PCR and direct cycle sequencing of the amplified products followed by analysis of the observed mutations at the level of genomic DNA. The single strand confirmation polymorphism (SSCP) technique was used for assessment of the carriers in some of these families. Various mutations throughout the coding gene were observed, including missense and nonsense mutations, a deletion, and several splicing defects. None of the mutations except one has been previously described. There were three point mutations resulting in a single amino acid substitution. One of these missense mutations was observed in a conserved region of the PH domain, the other two were found in the src homology domain 2 that is involved in phosphotyrosyl peptide binding. Two mutations were single base pair substitutions resulting in premature stop codons. In four patients abnormal Btk transcripts were found that were the result of aberrant splicing. One small deletion was observed causing a frameshift and a secondary premature termination signal. Characterisation of the mutations responsible for XLA allowed us to diagnose the disease conclusively and identify the phenotypically normal female carriers.

‣ Germline and somatic cancer-associated mutations in the ATP-binding motifs of PTEN influence its subcellular localization and tumor suppressive function

Lobo, Glenn P.; Waite, Kristin A.; Planchon, Sarah M.; Romigh, Todd; Nassif, Najah T.; Eng, Charis
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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Germline and somatic PTEN mutations are found in Cowden syndrome (CS) and multiple sporadic malignancies, respectively. PTEN function appears to be modulated by subcellular compartmentalization, and mislocalization may affect function. We have shown that cellular ATP levels affect nuclear PTEN levels. Here, we examined the ATP-binding capabilities of PTEN and functional consequences, relevant to cancer-associated mutations. PTEN mutation analysis of CS patients and sporadic colorectal carcinomas and comparative aminoacid analysis were utilized to identify mutations in ATP-binding motifs. The ability of wild-type (WT) or mutant PTEN to bind ATP was assessed by ATP–agarose-binding assays. Subcellular fractionation, western blotting, confocal microscopy and growth assays were used to determine relative nuclear-cytoplasmic localization and function. Somatic colorectal carcinoma-derived PTEN missense mutations were associated with nuclear mislocalization. These mutations altered cellular proliferation, apoptosis and anchorage-dependent growth. Examination of PTEN's amino acid sequence revealed these mutations resided in previously undescribed ATP-binding motifs (c.60–73; c.122–136). In contrast to WT PTEN, both cancer-associated somatic and germline-derived PTEN missense mutations...