Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Point Mutation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 451 records · Page 25Linked to original sources
PubMed1994

Detection of point mutations in human genes by the solid-phase minisequencing method.

The increased understanding of the molecular defects causing human genetic diseases has created a need for diagnostic methods to detect these defects at the DNA level. We have developed a new method, denoted solid-phase minisequencing, for the detection of previously known point mutations. Because of its convenient format, the method is well suited for routine use in the clinical laboratory. We have applied it for diagnosis and identification of carriers of the recessively inherited disease aspartylglucosaminura, for diagnosis of dominantly inherited amyloidosis of the Finnish type and for detecting polymorphic nucleotides of the genome. The solid-phase minisequencing method allows accurate and sensitive quantitation of two sequences which differ from each other by one nucleotide and are present as a mixture in a sample. This feature of the method is an advantage in the diagnosis of mitochondrial disorders caused by heteroplasmic point mutations and for the detection of minimal residual cells carrying somatic point mutations in samples from patients with myeloid malignancies.

DNA Mutational Analysis↗
PubMed1995

Extra-pancreatic manifestations in diabetes secondary to mitochondrial DNA point mutation within the tRNALeu(UUR) gene.

OBJECTIVE: A point mutation in the mitochondrial genome has been identified as a cause of diabetes and deafness. We report two pedigrees with and A-to-G transition at nucleotide 3243 of mtDNA within the tRNALeu(UUR) gene and focus our investigations on other localizations of the anomaly, particularly muscle and retina. RESEARCH DESIGN AND METHODS: Muscular localization has been studied in probands by invasive and noninvasive methods, including muscle biopsy (evaluation of the proportion of mutated mtDNA in comparison to blood cells, measurement of respiratory chain complex activities and histological and histochemical aspects) and 31P-nuclear magnetic resonance (NMR) spectroscopy. Ophthalmic and angiographic examination of retina, electroretinography, and visual evoked potentials were performed in five subjects. RESULTS: This mutation, previously described in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), was expressed more abundantly in muscle than in nucleated blood cells. This low expression in blood cells could hamper the diagnosis for some patients. In addition, despite poor clinical expression, muscle was found to be highly affected. Ragged red fibers and dystrophic mitochondria were observed in muscle biopsy. Histochemical assays showed decreased activity of respiratory chain complexes, and 31P-NMR in vivo data further confirmed the defect of muscle oxidative processes. Exercise-induced lactate production was increased. Finally, in both families, an atypical "salt and pepper" pigmentary retinopathy was observed without consequences on visual acuity. CONCLUSIONS: In diabetes secondary to 3243 mtDNA mutation, infraclinical muscular and ocular lesions are frequent. These two locations of the disease, which are easily investigated by simple methods, can help in the diagnosis of this new type of diabetes.

Adult↗
PubMed1996

Point mutation in the alpha-galactosidase A gene of atypical Fabry disease with only nephropathy.

A point mutation in exon 6 of the alpha-galactosidase A gene (alpha-GAL A) was found in a Japanese hemizygous male without typical manifestations of Fabry disease other than renal involvement. This 45-year-old man developed moderate proteinuria and was diagnosed with Fabry disease on the basis of renal histologic findings and prominent decreases in alpha-GAL A activity in his plasma, urine, leukocytes, and skin fibroblasts. Determination of the cDNA sequence of his alpha-GAL A gene revealed substitution of a G to A in codon 301, resulting in a glutamine rather than an arginine residue. Our case is unique in that this patient only demonstrated renal manifestations while all other reported patients with atypical Fabry disease, including a case with the identical point mutation, present with a cardiomyopathy. Direct DNA sequencing of exon 6 and measurement of alpha-GAL A activity among the patient's family confirmed that the mutation was transmitted from his mother.

Biopsy↗
PubMed1996

Effects of Denys-Drash syndrome point mutations on the DNA binding activity of the Wilms' tumor suppressor protein WT1.

A number of point mutations in the zinc finger domain of the Wilms' tumor suppressor protein WT1 have been isolated from the DNA of patients with Denys-Drash syndrome, an association of Wilms' tumor, nephropathy, and genital anomalies. To date, five different mutations that alter amino acids predicted to interact specifically with nucleotides in the target DNA sequence have been described. Two of these mutations are located in zinc finger 2 (R366H, R366C), and three are located in finger 3 (R394W, D396G, D396N). These five Denys-Drash mutations were introduced into WT1-ZFP, a recombinant polypeptide containing the zinc finger domain of WT1, and the effects of these mutations on DNA sequence specificity were determined using a selection, amplification, and binding (SAAB) assay. The SAAB assay was carried out using two different DNA templates, one with a randomized finger 2 subsite (GCG TGG NNN TGT) and one with a randomized finger 3 subsite (GCG NNN GCG TGT). A comparison of the DNA sequences selected by WT1-ZFP and by Denys-Drash mutants suggests that the point mutations reduce the sequence selectivity of the zinc finger protein. With the exception of the R394W mutant, the other Denys-Drash mutations selected one alternative sequence in addition to the wild-type DNA subsite sequence. The binding affinities of these proteins for their selected sequences were determined using a quantitative nitrocellulose filter binding assay. These results revealed that the wild-type WT1 binds with slightly higher affinity to sequences with GAG in the finger 2 subsite than sequences with the EGR-1 consensus GCG finger 2 subsite. With the exception of R394W, which appears to lack specific DNA binding activity, the Denys-Drash mutants bound to selected DNAs with 1.4-14-fold lower affinities than the wild-type WT1-ZFP. These results suggest that the clinical phenotype of Denys-Drash syndrome can be associated with a modest reduction in the DNA binding affinity of WT1.

Amino Acid Sequence↗
PubMed1993

Dejerine-Sottas syndrome associated with point mutation in the peripheral myelin protein 22 (PMP22) gene.

Dejerine-Sottas syndrome is a hypertrophic, demyelinating neuropathy which appears to demonstrate autosomal recessive inheritance in most pedigrees. Clinical symptoms are similar but more severe than Charcot-Marie-Tooth disease type 1 (CMT1), of which the major subtype, CMT1A, results either from duplication of a 1.5-megabase DNA region in chromosome 17p11.2-p12 containing the myelin gene PMP22, or from PMP22 point mutation. Mutational analysis of the PMP22 coding region in two unrelated Dejerine-Sottas patients identified individual missense point mutations present in the heterozygous state. These findings suggest that Dejerine-Sottas syndrome can result from dominant point mutation alleles of PMP22.

Amino Acid Sequence↗
PubMed1996

Detection of K-ras point mutations in mesenteric venous blood from colorectal cancer patients by enriched polymerase chain reaction and single-strand conformation polymorphism analysis.

In order to confirm the presence of cancer cells in mesenteric venous blood and to examine their relationship with the occurrence of liver metastases, we attempted to detect K-ras codon 12 point mutations in perioperative mesenteric blood using enriched polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis in 25 patients with primary colorectal tumors carrying K-ras point mutations. Among these patients, three with synchronous liver metastases were included. The same K-ras point mutation (substitution of GAT for GGT) was detected in both the blood and the primary tumor in a Dukes' C patient. We confirmed this result by colony hybridization and estimated the tumor-to-normal cell ratio to be 1:400. This patient has no liver metastases two years after surgery and her carcinoembryonic antigen (CEA) level remains normal. We demonstrated that considerable numbers of cancer cells can be found in mesenteric venous blood during colorectal cancer surgery. However, their potential role in the formation of liver metastases remains unclear.

Colorectal Neoplasms↗
PubMed2001

Genome quality control: RIP (repeat-induced point mutation) comes to Podospora.

RIP (repeat-induced point mutation) is a silencing process discovered in Neurospora crassa and so far clearly established only in this species as a currently occurring process. RIP acts premeiotically on duplicated sequences, resulting in C-G to T-A mutations, with a striking preference for CpA/TpG dinucleotides. In Podospora anserina, an RIP-like event was observed after several rounds of sexual reproduction in a strain with a 40 kb tandem duplication resulting from homologous integration of a cosmid in the mating-type region. The 9 kb sequenced show 106 C-G to T-A transitions, with 80% of the replaced cytosines located in CpA dinucleotides. This led to the alteration of at least six genes, two of which were unidentified. This RIP-like event extended to single-copy genes between the two members of the repeat. The overall data show that the silencing process is strikingly similar to a light form of RIP, unaccompanied by C-methylation. Interestingly, the N. crassa zeta-eta sequence, which acts as a potent de novo C-methylation RIP signal in this species, is weakly methylated when introduced into P. anserina. These results demonstrate that RIP, at least in light forms, can occur beyond N. crassa.

Ascomycota↗
PubMed1992

Zinc finger point mutations within the WT1 gene in Wilms tumor patients.

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.

Amino Acid Sequence↗
PubMed2000

Interferon regulatory factor-2 point mutations in human pancreatic tumors.

Interferon regulatory factor (IRF)-2, a member of the IRF family, is a transcription factor involved in the regulation of various interferon and virus-stimulated genes and other genes. For example, IRF-2 is an activator of the interferon (IFN)-gamma-inducible MHC class II transactivator (CIITA) type IV promoter. It cooperates with IRF-1 in the activation of the CIITA type IV promoter and can co-occupy the IRF-E of the promoter with IRF-1. In a previous study, we identified an inactivating point mutation in the DNA binding domain of IRF-2 expressed in a human pancreatic tumor cell line that does not express CIITA or MHC class II in response to IFN-gamma. To further assess the potential impact of IRF-2 mutations in tumorigenesis, we screened fresh pancreatic tumor explants and identified 2 IRF-2 point mutations in the 2 alleles of IRF-2 from a single tumor specimen. Both mutations occurred in the DNA binding domain of IRF-2. DNA binding assays demonstrated that the IRF-2 point mutations impaired IRF-2 DNA binding. The transactivation function of the mutant IRF-2s was similarly impaired. This is the first report of IRF-2 mutations in human tumor explants.

Alleles↗
PubMed1998

Compensatory point mutations in the human immunodeficiency virus type 1 Gag region that are distal from deletion mutations in the dimerization initiation site can restore viral replication.

The dimerization initiation site (DIS), downstream of the long terminal repeat within the human immunodeficiency virus type 1 (HIV-1) genome, can form a stem-loop structure (SL1) that has been shown to be involved in the packaging of viral RNA. In order to further determine the role of this region in the virus life cycle, we deleted the 16 nucleotides (nt) at positions +238 to +253 within SL1 to generate a construct termed BH10-LD3 and showed that this virus was impaired in viral RNA packaging, viral gene expression, and viral replication. Long-term culture of these mutated viruses in MT-2 cells, i.e., 18 passages, yielded revertant viruses that possessed infectivities similar to that of the wild type. Cloning and sequencing showed that these viruses retained the original 16-nt deletion but possessed two additional point mutations, which were located within the p2 and NC regions of the Gag coding region, respectively, and which were therefore named MP2 and MNC. Site-directed mutagenesis studies revealed that both of these point mutations were necessary to compensate for the 16-nt deletion in BH10-LD3. A construct with both the 16-nt deletion and the MP2 mutation, i.e., LD3-MP2, produced approximately five times more viral protein than BH10-LD3, while the MNC mutation, i.e., construct LD3-MNC, reversed the defects in viral RNA packaging. We also deleted nt +261 to +274 within the 3' end of SL1 and showed that the diminished infectivity of the mutated virus, termed BH10-LD4, could also be restored by the MP2 and MNC point mutations. Therefore, compensatory mutations within the p2 and NC proteins, distal from deletions within the DIS region of the HIV genome, can restore HIV replication, viral gene expression, and viral RNA packaging to control levels.

Amino Acid Sequence↗
PubMed1997

Non-isocratic capillary electrophoresis for detection of DNA point mutations.

Capillary electrophoresis under non-isocratic conditions is reviewed here. In particular, these conditions are elicited by programming the temperature over the run, just as in temperature-programmed gas chromatography. There is at least one major instance in which this technique is particularly useful: detection of DNA point mutations in PCR-amplified DNA fragments. In this case, migration of DNA homo- and hetero-duplexes against a denaturing gradient (either thermal or chemical) is the only technique capable of developing a pattern of four zones, indicative of the presence of a point mutation in genomic DNA, and due to the formation of two homo- (Wt/Wt, M/M, where Wt = wild type and M = mutant) and two hetero- (Wt/M and Wt/M) duplexes, formed by fully denaturing and then re-annealing the Wt and M filaments in solution. It is demonstrated that it is possible to obtain such gradients (which do not exist in space, i.e., along the length of the capillary, but in time), simply by producing voltage ramps, which in turn generate temperature ramps from within the capillary lumen, rather than from without, by circulating liquids and thermostats. This technique can be applied to any possible type of mutation, from low- to intermediate to high-melters, i.e., from 40 degrees C up to 60-65 degrees C. Examples of separations of point mutations in the cystic fibrosis transmembrane regulator gene and in the beta-globin chain gene are shown.

DNA↗
PubMed1998

Single point mutations distributed in 10 soluble and membrane regions of the Nicotiana plumbaginifolia plasma membrane PMA2 H+-ATPase activate the enzyme and modify the structure of the C-terminal region.

The Nicotiana plumbaginifolia pma2 (plasma membrane H+-ATPase) gene is capable of functionally replacing the H+-ATPase genes of the yeast Saccharomyces cerevisiae, provided that the external pH is kept above 5.0. Single point mutations within the pma2 gene were previously identified that improved H+-ATPase activity and allowed yeast growth at pH 4.0. The aim of the present study was to identify most of the PMA2 positions, the mutation of which would lead to improved growth and to determine whether all these mutations result in similar enzymatic and structural modifications. We selected additional mutants in total 42 distinct point mutations localized in 30 codons. They were distributed in 10 soluble and membrane regions of the enzyme. Most mutant PMA2 H+-ATPases were characterized by a higher specific activity, lower inhibition by ADP, and lower stimulation by lysophosphatidylcholine than wild-type PMA2. The mutants thus seem to be constitutively activated. Partial tryptic digestion and immunodetection showed that the PMA2 mutants had a conformational change making the C-terminal region more accessible. These data therefore support the hypothesis that point mutations in various H+-ATPase parts displace the inhibitory C-terminal region, resulting in enzyme activation. The high density of mutations within the first half of the C-terminal region suggests that this part is involved in the interaction between the inhibitory C-terminal region and the rest of the enzyme.

Enzyme Activation↗
PubMed1999

Proteolipoprotein gene analysis in 82 patients with sporadic Pelizaeus-Merzbacher Disease: duplications, the major cause of the disease, originate more frequently in male germ cells, but point mutations do not. The Clinical European Network on Brain Dysmyelinating Disease.

Pelizaeus-Merzbacher Disease (PMD) is an X-linked developmental defect of myelination affecting the central nervous system and segregating with the proteolipoprotein (PLP) locus. Investigating 82 strictly selected sporadic cases of PMD, we found PLP mutations in 77%; complete PLP-gene duplications were the most frequent abnormality (62%), whereas point mutations in coding or splice-site regions of the gene were involved less frequently (38%). We analyzed the maternal status of 56 cases to determine the origin of both types of PLP mutation, since this is relevant to genetic counseling. In the 22 point mutations, 68% of mothers were heterozygous for the mutation, a value identical to the two-thirds of carrier mothers that would be expected if there were an equal mutation rate in male and female germ cells. In sharp contrast, among the 34 duplicated cases, 91% of mothers were carriers, a value significantly (chi2=9. 20, P<.01) in favor of a male bias, with an estimation of the male/female mutation frequency (k) of 9.3. Moreover, we observed the occurrence of de novo mutations between parental and grandparental generations in 17 three-generation families, which allowed a direct estimation of the k value (k=11). Again, a significant male mutation imbalance was observed only for the duplications. The mechanism responsible for this strong male bias in the duplications may involve an unequal sister chromatid exchange, since two deletion events, responsible for mild clinical manifestations, have been reported in PLP-related diseases.

Cystic Fibrosis Transmembrane Conductance Regulato↗
PubMed1998

The EAAT2 (GLT-1) gene in motor neuron disease: absence of mutations in amyotrophic lateral sclerosis and a point mutation in patients with hereditary spastic paraplegia.

OBJECTIVES: To investigate if sequence alterations of the excitatory amino acid transporter gene EAAT2 (GLT-1) may be a contributory factor to the pathogenesis of motor system degeneration. EAAT2 serves as a candidate gene as its reduced expression was reported in patients with amyotrophic lateral sclerosis (ALS). Furthermore, neurolathyrism, a motor neuron disease clinically related to hereditary spastic paraplegia (HSP), has been associated with an exogenous excitotoxin. METHODS: Sequence alterations were screened for in the coding region of EAAT2 in 55 patients with ALS and one family with autosomal dominant HSP (AD-HSP). RESULTS: In ALS, no sequence alteration in the EAAT2 gene have been found. Interestingly, a heterozygous A79G mutation of the EAAT2 gene was detected in two of seven affected patients with AD-HSP in the same kindred. The absence of cosegregation with the familial disease showed that the detected variant was not the cause of disease. The A79G sequence variant was not found in 55 patients with ALS or in 50 non-neurological controls. CONCLUSION: The allelic variant of the EAAT2 gene in conjunction with the primary gene defect may be a modifying factor for the highly variable AD-HSP phenotype.

Adult↗
PubMed1998

Isoleucylation properties of native human mitochondrial tRNAIle and tRNAIle transcripts. Implications for cardiomyopathy-related point mutations (4269, 4317) in the tRNAIle gene.

A growing number of mutated mitochondrial tRNA genes have been found associated with severe human diseases. To investigate the potential interference of such mutations with the primordial function of tRNAs, i.e. their aminoacylation by cognate aminoacyl-tRNA synthetases, a human mitochondrial in vitro aminoacylation system specific for isoleucine has been established. Both native tRNAIleand isoleucyl-tRNA synthetase activity have been recovered from human placental mitochondria and the kinetic parameters of tRNA aminoacylation determined. The effect of pathological point mutations present in the mitochondrial gene encoding tRNAIlehas been tackled by investigating the isoleucylation properties of wild-type and mutated in vitro transcripts. Data show that: (i) modified nucleotides contribute to efficient isoleucylation; (ii) point mutation A4269G in the gene (A-->G at nt 7 in the tRNA), associated with a cardiomyopathy, does not affect aminoacylation significantly; (iii) point mutation A4317G (A-->G at nt 59 in the tRNA), reported in a case of fatal infantile cardiomyopathy, induces a small but significant decrease in isoleucylation. The potential implications of these findings on the understanding of the molecular mechanisms involved in the expression of pathology are discussed.

Adenine↗
PubMed2001

A new method for generating point mutations in bacterial artificial chromosomes by homologous recombination in Escherichia coli.

Bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs), which contain large fragments of genomic DNA, have been successfully used as transgenes to create mouse models of dose-dependent diseases. They are also potentially valuable as transgenes for dominant diseases given that point mutations and/or small rearrangements can be accurately introduced. Here, we describe a new method to introduce small alterations in BACs, which results in the generation of point mutations with high frequency. The method involves homologous recombination between the original BAC and a shuttle vector providing the mutation. Each recombination step is monitored using positive and negative selection markers, which are the Kanamycin-resistance gene, the sacB gene and temperature-sensitive replication, all conferred by the shuttle plasmid. We have used this method to introduce four different point mutations and the insertion of the ss-galactosidase gene in a BAC, which has subsequently been used for transgenic animal production.

Animals↗
PubMed2001

[Gene point mutation in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum].

OBJECTIVE: To investigate the gene point mutation in the dihydrofolate reductase-thymidylate synthase(dhfr) gene of Plasmodium falciparum isolate from Yunnan Province strongly associated with pyrimethamine and cycloguanil resistance. METHODS: Nested PCR and restriction endonuclease digestion were applied to detect the gene mutation using dried blood filter paper collected from the fields in Yunnan Province. RESULTS: Different mutations were found in 4 amino acids at positions 16, 51, 108 and 164 of dhfr gene, particularly, Asn-108 and Ile-51, the mutation frequency being 94.1% and 90.1%, respectively. The frequency of the wild-type genotype (3D7-type) Ser-108 appeared lower (9.1%), while the frequency of the Ala-16 was high(61.8%); the mutation-type was very high, the ratio of HB3-type, 7G8-type/FCR3-type and Cambodian-type was 1:21:7.5. CONCLUSION: The investigation first demonstrated that Plasmodium falciparum Yunnan isolate dihydrofolate reductase-thymidylate synthase gene (dhfr) at positions 16, 51, 108 and 164 exhibited different degrees of point mutation. The frequency of mutation of the 7D8-type involved in pyrimethamine resistance was higher, while that of the FCR3-type involved in cycloquanil resistance was lower.

Animals↗
PubMed2005

Point mutation detection with the sandwich method employing hydrogel nanospheres by the surface plasmon resonance imaging technique.

We propose a surface modification procedure to construct DNA arrays for use in surface plasmon resonance (SPR) imaging studies for the highly sensitive detection of a K-ras point mutation, enhanced with hydrogel nanospheres. A homobifunctional alkane dithiol was adsorbed on Au film to obtain the thiol surface, and ethyleneglycol diglycidylether (EGDE) was reacted to insert the ethyleneglycol moiety, which can suppress nonspecific adsorption during SPR analysis. Then streptavidin (SA) was immobilized on EGDE using tosyl chloride activation. Biotinylated DNA ligands were bound to the SA surface via biotin-SA interaction to fabricate DNA arrays. In SPR analysis, the DNA analyte was exposed on the DNA array and hybridized with the immobilized DNA probes. Subsequently, the hydrogel nanospheres conjugated with DNA probes were bound to the DNA analytes in a sandwich configuration. The DNA-carrying nanospheres led to SPR signal enhancement and enabled us to discriminate a K-ras point mutation in the SPR difference image. The application of DNA-carrying hydrogel nanospheres for SPR imaging assays was a promising technique for high throughput and precise detection of point mutations.

Base Sequence↗