scholarly journals Pyrazinamide resistance associated with pncA gene mutation in Mycobacterium tuberculosis in Japan

2002 ◽  
Vol 128 (2) ◽  
pp. 337-342 ◽  
Author(s):  
T. ENDOH ◽  
A. YAGIHASHI ◽  
N. UEHARA ◽  
D. KOBAYASHI ◽  
N. TSUJI ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Direct Sequencing ◽  
Gene Mutations ◽  
Japanese Patients ◽  
Wild Type ◽  
Gene Encoding ◽  
Single Strand Conformational Polymorphism ◽  
Proportion Method ◽  
Polymerase Chain ◽  
Medium Method

Thirty Japanese clinical isolates of Mycobacterium tuberculosis were analysed by pyrazinamide susceptibility testing and pyrazinamidase assay, as well as polymerase chain reaction for single-strand conformational polymorphism and direct sequencing of the gene encoding pyrazinamidase (pncA). All sensitive isolates showed pyrazinamidase activity and a wild-type pncA gene, but three resistant isolates had pncA gene mutations and lacked pyrazinamidase activity. The latter isolates showed a minimum inhibitory concentration of at least 100 mg/l by the 7H10 agar proportion method and 400 mg/l by the 7H9 liquid medium method. Isolate 28 showed T-to-C change at position 11, leading to Leu4→Ser substitution; isolate 29 had an 8-bp deletion from position 382; and isolate 30 had A-to-C change at position 29, leading to Gln10→Pro substitution. The deletion has not been described previously. This is the first demonstration of pncA gene mutations in PZA-resistant M. tuberculosis strains isolated from Japanese patients.

scholarly journals Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

2009 ◽  
Vol 54 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Hassan Safi ◽  
Robert D. Fleischmann ◽  
Scott N. Peterson ◽  
Marcus B. Jones ◽  
Behnam Jarrahi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
In Vitro Selection ◽  
Gene Mutations ◽  
Wild Type ◽  
Mutant Selection ◽  
Preferential Growth ◽  
Allelic Exchange ◽  
High Level ◽  
Isogenic Mutants

ABSTRACT Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

scholarly journals Expression of Mycobacterium smegmatisPyrazinamidase in Mycobacterium tuberculosis Confers Hypersensitivity to Pyrazinamide and Related Amides

2000 ◽  
Vol 182 (19) ◽  
pp. 5479-5485 ◽  
Author(s):  
Helena I. M. Boshoff ◽  
Valerie Mizrahi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Intracellular Localization ◽  
Wild Type ◽  
Gene Encoding ◽  
Allelic Exchange ◽  
Mutant Strains ◽  
Established Role ◽  
Amidase Gene

ABSTRACT A pyrazinamidase (PZase)-deficient pncA mutant ofMycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 μg/ml), in accordance with the well-established role ofpncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662–667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809–5814, 1998) or the M. tuberculosis pncA gene into the pncAmutant complemented its PZase/nicotinamidase defect. In bothpzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. ThepzaA-complemented strain was hypersensitive to PZA (MIC, ≤10 μg/ml) and nicotinamide (MIC, ≥20 μg/ml) and was also sensitive to benzamide (MIC, 20 μg/ml), unlike the wild-type andpncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 μg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 μg/ml in all cases) and rendered Escherichia colihypersensitive for growth at low pH.

Novel ABCD1 gene mutations in Iranian pedigrees with X-linked adrenoleukodystrophy

2019 ◽  
Vol 32 (11) ◽  
pp. 1207-1215
Author(s):  
Babak Emamalizadeh ◽  
Yousef Daneshmandpour ◽  
Abbas Tafakhori ◽  
Sakineh Ranji-Burachaloo ◽  
Sajad Shafiee ◽  
...  
Keyword(s):  
Protein Structure ◽  
Direct Sequencing ◽  
Gene Mutations ◽  
Protein Product ◽  
Structure And Function ◽  
Novel Mutations ◽  
Chain Reaction ◽  
Abcd1 Gene ◽  
Polymerase Chain ◽  
And Function

Abstract Background X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disorder, is caused by mutations in the ABCD1 gene located on Xq28. X-ALD is characterized by a spectrum of different manifestations varying in patients and families. Methods Four pedigrees with X-ALD consisting of patients and healthy members were selected for investigation of ABCD1 gene mutations. The mutation analysis was performed by polymerase chain reaction (PCR) followed by direct sequencing of all exons. The identified mutations were investigated using bioinformatics tools to predict their effects on the protein product and also to compare the mutated sequence with close species. Results One previously known missense mutation (c.1978 C > T) and three novel mutations (c.1797dupT, c.879delC, c.1218 C > G) were identified in the ABCD1 gene, each in one family. Predicting the effects of the mutations on protein structure and function indicated the probable damaging effect for them with significant alterations in the protein structure. We found three novel mutations in the ABCD1 gene with damaging effects on its protein product and responsible for X-ALD.

First Place—Resident Clinical Science Award 1997: Germline screening of the NF-2 gene in families with unilateral vestibular schwannoma

1998 ◽  
Vol 119 (1) ◽  
pp. 1-6 ◽  
Author(s):  
Paul H. Bikhazi ◽  
Anil K. Lalwani ◽  
Eugene J. Kim ◽  
Nadim Bikhazi ◽  
Ali Attaie ◽  
...  
Keyword(s):  
Vestibular Schwannoma ◽  
Direct Sequencing ◽  
Gene Mutations ◽  
Neurofibromatosis Type ◽  
Germline Mutations ◽  
Type Ii ◽  
Chain Reaction ◽  
Neurofibromatosis Type Ii ◽  
Polymerase Chain ◽  
Head Neck

Vestibular schwannoma may present clinically in two forms: sporadic unilateral or hereditary bilateral. Familial transmission of vestibular schwannoma is known to occur only in neurofibromatosis type II (NF-2). We have previously described the clinical characteristics of unilateral vestibular schwannoma presenting in families, in the absence of ther criteria necessary for the diagnosis of NF-2. Polymerase chain reaction–single strand chain polymorphism was used to screen for germline NF-2 gene mutations in six families with unilateral vestibular schwannoma. Direct sequencing of DNA from blood was done in affected subjects from three families. No germline mutations were identified. Because NF-2 gene mutations are detected in only 33% of patients with NF-2, hereditary transmission of mutations cannot be entirely excluded. However, in the absence of germline mutations in the NF-2 gene, familial occurrence of unilateral vestibular schwannoma more likely represents either a chance somatic NF-2 gene mutation or originates from a separate genetic loci. (Otolaryngol Head Neck Surg 1998;119:1–6.)

Novel mutations in katG gene of a clinical isolate of isoniazid-resistant Mycobacterium tuberculosis

Biologia ◽  
2012 ◽  
Vol 67 (1) ◽  
Author(s):  
Purkan ◽  
Ihsanawati ◽  
Yana Syah ◽  
Debbie Retnoningrum ◽  
Achmad Noer ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Isolate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intramolecular Interactions ◽  
Wild Type ◽  
Katg Gene ◽  
Gene Encoding ◽  
Oxidation Activity ◽  
Catalase Peroxidase

AbstractMost of isoniazid-resistant Mycobacterium tuberculosis evolved due to mutation in the katG gene encoding catalase-peroxidase. A set of new mutations, namely T1310C, G1388T, G1481A, T1553C, and A1660G, which correspond to amino acid substitutions of L437P, R463L, G494D, I518T, and K554E, in the katG gene of the L10 clinical isolate M. tuberculosis was identified. The wild-type and mutant KatG proteins were expressed in Escherichia coli BL21(DE3) as a protein of 80 kDa based on sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. The mutant KatG protein exhibited catalase and peroxidase activities of 4.6% and 24.8% toward its wild type, respectively, and retained 19.4% isoniazid oxidation activity. The structure modelling study revealed that these C-terminal mutations might have induced formation of a new turn, perturbing the active site environment and also generated new intramolecular interactions, which could be unfavourable for the enzyme activities.

scholarly journals Identification of Novel ARSA Mutations in Chinese Patients with Metachromatic Leukodystrophy

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Li Chen ◽  
Huifang Yan ◽  
Binbin Cao ◽  
Ye Wu ◽  
Qiang Gu ◽  
...  
Keyword(s):  
Metachromatic Leukodystrophy ◽  
Direct Sequencing ◽  
Chinese Patients ◽  
Insertion Mutation ◽  
Inherited Disease ◽  
Wild Type ◽  
Novel Mutations ◽  
Genetic Characteristics ◽  
Developmental Problems ◽  
Polymerase Chain

Objective. Metachromatic leukodystrophy (MLD) is an inherited disease caused by a deficiency of the enzyme arylsulfatase A (ARSA) that leads to severe physiologic and developmental problems. Our study is aimed at elucidating the clinical and genetic characteristics of Chinese MLD patients. Methods. Clinical data of 21 MLD patients was collected. All coding exons of ARSA and their flanking intronic sequences were amplified by polymerase chain reaction and subjected to direct sequencing. Results. All 21 patients were diagnosed with MLD clinically and genetically, out of which 17 patients were late infantile and 4 were juvenile types. A total of 34 ARSA mutations, including 28 novel mutations (22 missense, 1 splicing, 1 nonsense, 3 small insertions, and 1 small deletion mutation) and 6 known mutations (5 missense and 1 small insertion mutation), were identified. Prenatal diagnosis was performed for four pedigrees. One fetus was a patient, two fetuses were carriers, and two were wild type. Conclusions. The present study discovered 28 novel ARSA mutations and widely expanded the mutation spectrum of ARSA. Four successful prenatal diagnoses provided critical information for MLD families.

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