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At least 217 records · Page 12Linked to original sources
PubMed2005

The Rv0805 gene from Mycobacterium tuberculosis encodes a 3',5'-cyclic nucleotide phosphodiesterase: biochemical and mutational analysis.

Mycobacterium tuberculosis is an important human pathogen and has developed sophisticated mechanisms to evade the host immune system. These could involve the use of cyclic nucleotide-dependent signaling systems, since the M. tuberculosis genome encodes a large number of functional adenylyl cyclases. Using bioinformatic approaches, we identify, clone, and biochemically characterize the Rv0805 gene product, the first cyclic nucleotide phosphodiesterase identified in M. tuberculosis and a homologue of the cAMP phosphodiesterase present in Escherichia coli (cpdA). The Rv0805 gene product, a class III phosphodiesterase, is a member of the metallophosphoesterase family, and computational modeling and mutational analyses indicate that the protein possesses interesting properties not reported earlier in this class of enzymes. Mutational analysis of critical histidine and aspartate residues predicted to be essential for metal coordination reduced catalytic activity by 90-50%, and several mutant proteins showed sigmoidal kinetics with respect to Mn in contrast to the wild-type enzyme. Mutation of an asparagine residue in the GNHD motif that is conserved throughout the metallophosphoesterase enzymes almost completely abolished catalytic activity, and these studies therefore represent the first mutational analysis of this class of phosphodiesterases. The Rv0805 protein hydrolyzes cAMP and cGMP in vitro, and overexpression in Mycobacterium smegmatis and E. coli reduces intracellular cAMP levels. The presence of an orthologue of Rv0805 in Mycobacterium leprae suggests that the Rv0805 protein could have an important role to play in regulating cAMP levels in these bacteria and adds an additional level of complexity to cyclic nucleotide signaling in this organism.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2005

Mutational analysis of the c-KIT AND PDGFRalpha in a series of molecularly well-characterized synovial sarcomas.

The c-KIT and the platelet-derived growth factor receptor alpha (PDGFRalpha) have been shown to be important for tumor growth and progression in several soft-tissue sarcomas, including synovial sarcomas (SSs). It has been suggested that these c-KIT-positive cases might benefit from a tyrosine kinase inhibitor therapy. In this study, we analyze a series of SSs to investigate the presence of c-KIT and PDGFRalpha mutations with the aim of selecting those for a more adequate and appropriate therapy. We analyzed fresh-frozen tissues from 12 SSs (8 primary tumors and 4 nude mice xenotransplants from primary tumors). RNA was extracted to identify the presence of the SYT-SSX gene fusion to confirm the SS diagnosis. Mutational analysis of exons 9, 11, 13, and 17 of c-KIT and exons 12 and 18 of PDGFRalpha was performed by direct sequencing. Immunohistochemical analysis of c-KIT, PDGFRalpha, and p-PDGFRalpha was also performed. All analyzed cases showed the presence of SYT-SSX gene fusion transcripts confirming the diagnosis of SS, 10 carried the SYT-SSX1 fusion, and 2 the SYT-SSX2. Immunohistochemical analysis showed expression of c-KIT in 3 cases in which no molecular alterations were detected. For the PDGFRalpha, we observed an in-frame deletion of codons 554 and 555 in a case which also showed a strong immunopositivity for the phosphorylated form of PDGFRalpha. PDGFRalpha expression was observed in 8 cases. We suggest that a more exhaustive mutational analysis of the c-KIT and PDGFRalpha genes should be performed to ascertain which cases would really benefit from a tyrosine kinase inhibitor therapy in SS.

Adolescent↗
PubMedDate not supplied

TGFBI gene mutation analysis in families with hereditary corneal dystrophies from Ukraine.

In our study, 5 previously reported mutations of the TGFBI gene - R124C, R124H, R124L (exon 4), R555W, R555Q (exon 12) - were analyzed using polymerase chain reaction followed by restriction digestion in 48 individuals from 19 unrelated families with different forms of corneal dystrophy from different regions of Ukraine. The R555W mutation was detected in 6 patients from 4 families with granular corneal dystrophy. The R124C mutation was detected in 1 unaffected 10-year-old individual and in 24 patients from 8 families with lattice corneal dystrophy. As far as the R124C mutation detected in 1 patient with clinically diagnosed Reis-Bucklers corneal dystrophy is concerned, we concluded that this patient was misdiagnosed. The obtained results show that TGFBI gene mutation analysis is important as well for the early differential diagnosis of corneal dystrophies and genetic consulting in high-risk families.

Child↗
PubMed1999

Prenatal diagnosis of glycogen storage disease type IV using PCR-based DNA mutation analysis.

Deficiency of glycogen branching enzyme activity causes glycogen storage disease type IV (GSD-IV). Clinically, GSD-IV has variable clinical presentations ranging from a fatal neonatal neuromuscular disease, to a progressive liver cirrhosis form, and to a milder liver disease without progression. Current methods for prenatal and postnatal diagnosis are based on an indirect method of measuring the enzyme activity, which has a limited sensitivity and cannot be used to distinguish patients with these variable clinical phenotypes. In this study, a GSD-IV family with a non-progressive hepatic form of the disease requested prenatal diagnosis. Determination of the branching enzyme activity in cultivated amniocytes showed 20 per cent residual activity overlapping with the level detected in the heterozygotes. Mutation analysis revealed that the fetus carried two mutant alleles, L224P and Y329S, the same as the proband of this family. The fetus was predicted to be affected and postnatally his clinical presentation is consistent with the diagnosis. We conclude that DNA mutation analysis should be used in the prenatal diagnosis of GSD-IV, especially in the situation of high residual enzyme activity.

Child, Preschool↗
PubMed2005

Mutation analysis in 51 patients with haemophilia A: report of 10 novel mutations and correlations between genotype and clinical phenotype.

We report the results of genetic analysis on a series of 51 patients attending this Haemophilia Comprehensive Care Centre. The most common cause of severe haemophilia A--the factor VIII intron 22 inversion was detected in eight families and the factor VIII intron 1 inversion in three families. Mutation analysis was carried out on the remaining patients by nucleotide sequencing of genomic DNA after screening with conformation-sensitive gel electrophoresis (CSGE) or denaturing high-performance liquid chromatography (dHPLC). A total of 27 different FVIII non-inversion mutations were detected. Severe haemophilia was associated with 12 null mutations (six nonsense, six frameshift) and four missense mutations. A further 11 different missense mutations were associated with moderate or mild disease. To our knowledge, six null mutations [1950del 4(tttg), 3270-75insA, 4416del 10, 6735-38delA, W1029X, Y1792X] and four missense mutations (E1682K, M1947V, P2048L, P2143L) have not been previously published. Each novel missense mutation occurred at a highly conserved residue, no other candidate mutation was detected on screening the entire coding region of the FVIII gene and they were not detected in a screen of individuals without haemophilia A. The genotype-phenotype correlations of the FVIII mutations detected will be discussed.

Base Sequence↗
PubMed2000

Prospective evaluation of the contribution of K-ras mutational analysis and CA 19.9 measurement to cytological diagnosis in patients with clinical suspicion of pancreatic cancer.

The aim of this study was to prospectively evaluate the diagnostic contribution of the detection of K-ras mutation and measurement of serum CA 19.9 concentrations to cytological diagnosis in patients with clinical suspicion of pancreatic cancer. These patients had either the presence or absence of a pancreatic mass as determined by imaging procedures. A total of 156 consecutive patients with clinical suspicion of pancreatic cancer or for confirmation and follow-up of their chronic pancreatitis disease were included: 84 patients presenting a pancreatic mass (group 1) and 72 patients without a pancreatic mass (group 2). K-ras mutations were detected by a restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) method and CA 19.9 by an immunoluminometric assay. When a pancreatic mass was present, cytology offered a high sensitivity, but with a significant number of inconclusive results and K-ras mutational analysis offered a highly specific test. In the absence of a pancreatic mass, CA 19.9 (cut-off 100 U/ml) increased the sensitivity of the diagnosis by cytology and K-ras mutational analysis did not add significant information. Thus both tests contribute to the clinical decision process when pancreatic cancer is clinically suspected and the cytological report is not conclusive.

Adult↗
PubMed2004

Homology modeling and mutational analysis of Ho endonuclease of yeast.

Ho endonuclease is a LAGLIDADG homing endonuclease that initiates mating-type interconversion in yeast. Ho is encoded by a free-standing gene but shows 50% primary sequence similarity to the intein (protein-intron encoded) PI-SceI. Ho is unique among LAGLIDADG endonucleases in having a 120-residue C-terminal putative zinc finger domain. The crystal structure of PI-SceI revealed a bipartite enzyme with a protein-splicing domain (Hint) and intervening endonuclease domain. We made a homology model for Ho on the basis of the PI-SceI structure and performed mutational analysis of putative critical residues, using a mating-type switch as a bioassay for activity and GFP-fusion proteins to detect nuclear localization. We found that residues of the N-terminal sequence of the Hint domain are important for Ho activity, in particular the DNA recognition region. C-terminal residues of the Hint domain are dispensable for Ho activity; however, the C-terminal putative zinc finger domain is essential. Mutational analysis indicated that residues in Ho that are conserved relative to catalytic, active-site residues in PI-SceI and other related homing endonucleases are essential for Ho activity. Our results indicate that in addition to the conserved catalytic residues, Hint domain residues and the zinc finger domain have evolved a critical role in Ho activity.

Amino Acid Sequence↗
PubMed2004

Mutational analysis of serotonin receptor genes: HTR3A and HTR3B in fibromyalgia patients.

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has been implicated in numerous human disorders. Dysfunction of serotonergic neurotransmission is thought to play a major role in the pathophysiology of the fibromyalgia syndrome (FMS) which is characterised by non-restorative sleep and severe pain. In our study, both serotonin receptor subunit genes, HTR3A and HTR3B, have been investigated for sequence variations in FMS patients in order to reveal a possible involvement in the aetiology of FMS. We examined DNA samples from 48 patients with FMS representing sporadic cases by single-strand conformation polymorphism (SSCP) and denaturing high-performance liquid chromatography (dHPLC) analysis, sequenced samples with conspicuous patterns and performed statistical calculations. HTR3A mutational analysis revealed one novel as well as five known sequence variations. Investigating HTR3B, we detected seven formerly described mutations and one novel sequence variant. Statistical computation rated all variants as probably non-disease-related polymorphisms. Nevertheless, one might speculate about an effect of the respective sequence variants on the severity of the disease. Sequence variants of the serotonin receptor subunit genes HTR3A and HTR3B indicate no obvious significance in the aetiology of fibromyalgia, yet they represent the basis for future studies on their pharmacogenetic relevance.

Chromatography, High Pressure Liquid↗
PubMed1995

Cystic fibrosis mutation analysis: report from 22 U.K. regional genetics laboratories.

We have collated the results of cystic fibrosis (CF) mutation analysis conducted in 22 laboratories in the United Kingdom. A total of 9,807 CF chromosomes have been analysed, demonstrating 56 different mutations so far observed and accounting for 86% of CF genes in the native Caucasian population of the United Kingdom. delta F508 is the most common at 75.3% of CF mutations (range 56.5-83.7%), followed by G551D (3.08%; range 0.71-7.60%), G542X (1.68%; range 0.85-3.66%), 621 + 1 (G > T) (0.93%; range 0.41-3.16%), 1717-1(G > A) (0.57%; range 0.17-1.14%), 1898 + 1)(G > A) (0.46%), R117H (0.46%), N1303K (0.46%), and R553X (0.46%). The data show a clear geographical variation in the distribution of some of the mutations, most notably a marked regional variation in the distribution of 621 + 1 (G > T) and 1989 + 1(G > A), which are both apparently more frequent in Wales. R560T and R117H appear to be more frequent in Ireland and Scotland, and G551D more frequent in Scotland. In summary, these data illustrate that the mutations present within a particular population need to be defined in order to provide meaningful carrier screening and testing for rare mutations in affected individuals. Furthermore, it is apparent that the ethnic origin of a patient, even within a small country such as the United Kingdom, should be taken into account.

Cystic Fibrosis↗
PubMed1998

Mutation analysis of the rearranged immunoglobulin heavy chain genes of marginal zone cell lymphomas indicates an origin from different marginal zone B lymphocyte subsets.

Marginal zone cell lymphoma is a recently described entity among the non-Hodgkin's lymphomas. It likely originates from the marginal zone B cells in the spleen and equivalent cells in the lymph node and extranodal tissues. Recent evidence indicates that marginal zone B cells are functionally heterogeneous and may differ with respect to the pattern of somatic hypermutation in their Ig variable genes. To test whether marginal zone lymphomas may originate from different subsets of marginal zone B cells, we performed a sequence and mutation analysis of the rearranged Ig heavy chain (IgH) variable genes (VH) of a series of 14 cases of marginal zone lymphoma, occurring in the spleen (4), the lymph node (4), the stomach (2), the orbit (2), the tongue (1), and the skin (1). Our data show that marginal zone cell lymphomas preferentially rearrange the VH4, VH3, and VH1 family genes, without preference for any particular VH gene. Somatic mutations are present in 13 cases; one case of marginal zone cell lymphoma of the skin showed a germline configuration of the rearranged VH gene. Mutation analysis shows evidence of antigen selection in three cases of marginal zone cell lymphoma, one of the spleen, stomach, and orbit, respectively. No evidence of antigen selection was present in the other cases. These data indicate that marginal zone cell lymphomas may arise from different subsets of marginal zone B cells. In addition, lymphomagenesis may not be triggered by antigen in all cases of marginal zone cell lymphoma.

Aged↗
PubMed2004

Diagnostic value of mitochondrial DNA mutation analysis in juvenile unilateral ptosis.

PURPOSE: To highlight the diagnostic relevance of mitochondrial DNA (mtDNA) mutation analysis in acquired juvenile unilateral upper eyelid ptosis. METHODS: A 13-year-old boy presented with acquired, slowly progressive unilateral ptosis. We performed ophthalmological and neurological examinations, laboratory testing, skeletal muscle biopsy including histological and histochemical investigations, biochemical analysis of respiratory chain enzymes in skeletal muscle homogenate and molecular genetic testing of skeletal muscle DNA. RESULTS: Though clinical, laboratory, histological and biochemical analyses did not reveal any hints suggesting a mitochondrial cytopathy, molecular genetic testing by Southern blot analysis of total DNA from skeletal muscle tissue showed a 5.8 kb mtDNA deletion thus proving the diagnosis of mitochondrial chronic progressive external ophthalmoplegia (CPEO). CONCLUSIONS: In patients with unexplained acquired juvenile unilateral ptosis, an underlying mitochondrial cytopathy should be considered even in cases of inconspicuous ancillary examinations comprising skeletal muscle histology and biochemistry. To establish the diagnosis, molecular genetic testing of DNA derived from skeletal muscle tissue is essential in those patients.

Adolescent↗
PubMed1998

Mutation analysis of the HLA-H gene in French hemochromatosis patients, and genetic counseling in families.

Mutation analysis of the HLA-H gene in French hemochromatosis patients and genetic counseling in families: Genotype analysis of 61 hemochromatosis patients living in France and 126 controls confirms that the disease is strongly associated with homozygosity for the mutation C282Y of the HLA-H gene: in our sample 67.2% patients, and none of the controls, carry two copies of the C282Y mutation; the frequency of the C282Y allele in a control population is 3.97%. A second variant (H63D) at the HLA-H gene is somehow more enriched (8.2%) in patient chromosomes that do not carry the C282Y mutation. The C282Y mutation was used in 10 hemochromatosis families for genetic counseling.

Amino Acid Substitution↗
PubMed1999

Glycogen storage disease type Ib: structural and mutational analysis of the microsomal glucose-6-phosphate transporter gene.

Glycogen storage disease type Ib is caused by a mutation in the gene encoding microsomal glucose-6-phosphate (G6P) transporter. We determined the exon/intron organization of the G6P transporter gene. Four overlapping genomic fragments containing the entire coding region of the gene were amplified by polymerase chain reaction (PCR) using exonic primers, and their nucleotide sequences were determined. The gene spans 4.5 kb and has eight exons. All exon/intron boundaries adhered to the canonical AG/GT rule. We then designed eight pairs of PCR primers to amplify all coding exons for a mutational analysis and studied five Japanese patients with the disease. Two novel homozygous mutations were identified in two families: a three-base deletion (delV235) in exon 2 in a consanguineous family and a splicing mutation (IVS7+1G-->T) in intron 7 in a nonconsanguineous family. Patient 3 was a compound heterozygote of W118R and IVS1+1G-->A, both of which we previously identified [Kure et al., 1998: Biochem Biophys Res Commun 248:426-431]. Patients 4 and 5 were homozygotes of W118R. Including our previous study, we found a total of ten W118R alleles in nine Japanese patients. The results support our previous suggestion that W118R is prevalent among Japanese patients. The genomic sequence data and mutation spectrum obtained from the Japanese patients will facilitate genetic diagnosis of glycogen storage disease type Ib.

Antiporters↗
PubMed2006

Mutational analysis of the NF2 gene in sporadic meningiomas by denaturing high-performance liquid chromatography.

The NF2 tumor suppressor gene, located in chromosome 22q12, is involved in the development of sporadic meningiomas of the nervous system. In order to evaluate the role of the NF2 gene in sporadic meningiomas, we analyzed the entire coding regions of the NF2 gene in a group of 42 sporadic meningiomas: 17 meningothelial, 11 transitional, 11 fibrous, one secretory, one atypical, and one malignant subtype, using denaturing high-performance liquid chromatography (DHPLC) and sequence analysis. Twenty-one mutations were identified in 20 patients with an overall mutation detection rate of 47.6%. The mutations included nine deletions (exons 1, 2, 5, 10, and 12), resulting in a frameshift, four non-sense mutations (exons 1, 2, and 7), four splice errors (exons 4, 5, 7, and 12), two missense mutations (exon 5) and two silent mutations (exon 11). Among these, 14 novel mutations were also identified in the present study. All mutations were noted in the first 12 exons, the region of homology with the ezrin-moesin-radixin protein. Furthermore, an association between NF2 mutations and histologic subtypes were observed; NF2 mutations were more frequent in fibrous meningiomas (8/11, 73%) and transitional meningiomas (6/11, 55%), than in meningothelial variant (5/17, 29%). These results provide evidence that mutations in the NF2 gene play an important role in the development of sporadic meningiomas as well as indicating a different tumorigenesis of these meningioma variants.

Adult↗
PubMed1998

Strategies for mutational analysis of the large multiexon ATM gene using high-density oligonucleotide arrays.

Mutational analysis of large genes with complex genomic structures plays an important role in medical genetics. Technical limitations associated with current mutation screening protocols have placed increased emphasis on the development of new technologies to simplify these procedures. High-density arrays of >90,000-oligonucleotide probes, 25 nucleotides in length, were designed to screen for all possible heterozygous germ-line mutations in the 9.17-kb coding region of the ATM gene. A strategy for rapidly developing multiexon PCR amplification protocols in DNA chip-based hybridization analysis was devised and implemented in preparing target for the 62 ATM coding exons. Improved algorithms for interpreting data from two-color experiments, where reference and test samples are cohybridized to the arrays, were developed. In a blinded study, 17 of 18 distinct heterozygous and 8 of 8 distinct homozygous sequence variants in the assayed region were detected accurately along with five false-positive calls while scanning >200 kb in 22 genomic DNA samples. Of eight heterozygous sequence changes found in more than one sample, six were detected in all cases. Five previously unreported sequence changes, not found by other mutational scanning methodologies on these same samples, were detected that led to either amino acid changes or premature truncation of the ATM protein. DNA chip-based assays should play a valuable role in high throughput sequence analysis of complex genes.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1996

PAH Mutation Analysis Consortium Database: a database for disease-producing and other allelic variation at the human PAH locus.

The PAH Mutation Analysis Consortium (81 investigators, 26 countries) is engaged in mutation detection at the human PAH locus. Ascertainment of probands occurs largely through newborn screening for hyperphenylalaninemia. A relational database records allelic variation (disease-producing and polymorphic) at the locus. Information is distributed by Newsletter, diskette (WINPAHDB software stand-alone executable on IBM compatible hardware), and at a 'real' site on the Worldwide Web (http://www.mcgill.ca/pahdb). The database presently records (Sept. 27, 1995) 248 alleles in 798 different associations (with polymorphic haplotype, geographic region and population) along with additional information. The database, as a record of human genetic diversity, at a particular locus, contributes to the study of human evolution and demic expansion; it also has medical relevance.

Alleles↗
PubMed2004

[X-linked adrenoleukodystrophy ABCD1 gene mutation analysis in China].

OBJECTIVE: To investigate mutations of ABCD1 gene in X- linked adrenoleukodystrophy (ALD) patients in China. METHODS: Polymerase chain reaction and DNA direct sequencing were employed to analyze the 10 exons of ABCD1 gene in 25 ALD patients. RESULTS: Seventeen mutations in different exons (except exons 4, 9 and 10) were identified in 18 of 25 patients. Most of the mutations were missense mutations, including R182P, G266R, H283D, S404P, N509I, R518G, L520Q, Q556R, S606L and R617C, four (H283D, S40 4P, N509I, R518G) of 10 missense mutations were novel. Also identified were 3 nonsense mutations (W132X, W242X, W595X), 1 dinucleotides deletion mutation (1414 del AG) resulting in frameshift, and 1 base pair deletion at splice acceptor site (IVS5-6 del C). Two synonymous mutations (L516L and V349V) appeared simultaneously in 2 unrelated patients, and no other mutations could be found with them in all 10 exons screened. CONCLUSION: There were no hot spot mutations in ABCD1 gene in China. Mutations in gene were found over 70% of patients with ALD in China. The ABCD1 gene mutations identified revealed no obvious correlation between the type of mutation and phenotype.

ATP Binding Cassette Transporter, Subfamily D, Mem↗
PubMed1995

Mutation analysis of Western Australian families affected by cystic fibrosis.

OBJECTIVE: To document the results of mutation analysis on 160 individuals with cystic fibrosis and 31 obligate carriers of the cystic fibrosis gene in 191 Western Australian families to facilitate accurate genetic counselling. METHODS: We tested for 17 mutations of the cystic fibrosis gene by either a variation of the polymerase chain reaction amplification refractory mutation system (PCR-ARMS) or with a series of restriction enzyme cuts and dot blots using chemiluminescent probes. RESULTS: At least one of the two intragenic mutations causing cystic fibrosis was identified in 98% of affected individuals and both were detected in 68%. The delta F508 deletion occurred in 89.8% of patients: 51% were homozygous for this defect. In carriers, 85% of the mutations were detected with a panel of 16 probes, identifying 17 intragenic defects: the delta F508 deletion occurred in 72.4%. Both cystic fibrosis mutations were detected in 68% of cystic fibrosis families. CONCLUSIONS: By analysis with 16 intragenic cystic fibrosis genomic probes, we have documented the frequencies of various mutations in the Western Australian population. These data will be useful in accurate genetic counselling for affected families and carrier screening for the general population.

Adult↗