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At least 739 records · Page 41Linked to original sources
PubMed2005

[Identification of point mutations in the 21-hydroxylase gene in patients affected with congenital adrenal hyperplasia].

INTRODUCTION: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder due to impaired cortisol secretion. Approximately 95% of CAH cases are caused by defects in the 21-hydrodylase2 (CYPA2) gene. The spectrum of clinical manifestations includes a severe and mild form of expression. The frequency of the following point mutations was determined: P30L, IVS2-12A/C-G splice, Del 8pb, I172N, cluster Ex6, V281L, Q318X, R356W and P453S. MATERIALS AND METHODS: The 58 patients consisted of 48 with the severe form of CAH and 10 with the mild form. Point mutations in the hydroxylase gene were isolated by allele-specific PCR and PCR-ACRS (amplification created restriction site), and their frequency was determined. RESULTS: Alternate alleles were identified in 82.8% of the samples. The most frequent mutations were IVS2-12A/C-G splice (26.7%), Q318X (21.5%), V281L (12.1%) and I172N (12.1%). DISCUSSION: The most frequent mutations were similar to those observed in other countries, except for Q318X. Although its frequency was higher but similar to that observed in Latin American countries, it contrasted with those of other continents and indicated the possible influence of genetic background in its expression. Several of the mutations were associated with specific clinical forms related to the enzyme activity. In the milder forms of CAH, several alleles were detected. These were important because these patients can have children with the virilizing and salt wasting forms. Recognition of the allelic forms of CAH will permit more specific genetic counseling and prenatal diagnosis.

Adrenal Hyperplasia, Congenital↗
PubMed1989

A single point mutation of Ala-25 to Asp in the 14,000-Mr envelope protein of vaccinia virus induces a size change that leads to the small plaque size phenotype of the virus.

The molecular defect responsible for a structural and functional abnormality of the 14,000-molecular-weight (14K) envelope protein of vaccinia virus has been identified. Through DNA sequence analysis of the entire 14K gene from wild-type vaccinia virus and three vaccinia virus mutants, a single base change of C to A was found that resulted in the substitution of Asp for Ala-25. This mutation is responsible for protein size abnormality, as documented by cell-free translation in a rabbit reticulocyte lysate of in vitro mRNA transcripts. In addition, through marker rescue experiments we show that this mutation is responsible for the small plaque size phenotype of vaccinia virus mutants. The structural consequence of the point mutation is a possible turn in an alpha-helix domain with destabilization of a hydrophobic interaction at the N terminus, resulting in monomers and trimers of vaccinia virus 14K protein with decreased electrophoretic mobilities. The functional consequence of the point mutation is a reduction in virulence of the virus.

Alanine↗
PubMedDate not supplied

Charcot-Marie-Tooth disease type 1A: molecular mechanisms of gene dosage and point mutation underlying a common inherited peripheral neuropathy.

Charcot-Marie-Tooth disease type 1A is a demyelinating, inherited peripheral neuropathy which is associated with a DNA duplication in chromosome 17p11.2-p12 in over 70% of patients with CMT1A. The CMT1A duplication is not detected cytogenetically, and constitutes a tandem duplication of a 1.5-Mb region of DNA flanked by homologous sequences designated as CMT1A-REP. Detection of the CMT1A duplication by molecular methods is a valuable diagnostic test for the majority of CMT1A cases. This duplication mutation shows stable inheritance through multiple generations, and may also arise as a new mutation in sporadic patients. The CMT1A duplication leads to the disease phenotype apparently through increased dosage of a gene(s) within the duplicated segment. A disease gene associated with CMT1A has been identified in the form of PMP22, which maps within the CMT1A duplication region, and encodes a myelin protein of the peripheral nerve. Point mutations in the PMP22 gene have been identified in CMT1A patients, including one case of a new mutation in PMP22 which coincided with the onset of the disease. Thus, two alternative molecular mechanisms are responsible for CMT1A: DNA duplication leading to increased gene dosage, and point mutation of the PMP22 gene.

Charcot-Marie-Tooth Disease↗
PubMed1997

Gain or loss of diabetogenicity resulting from a single point mutation in recombinant encephalomyocarditis virus.

Molecular pathogenic mechanisms for virus-induced disease have received considerable attention. Encephalomyocarditis (EMC) virus-induced diabetes in mice has been extensively studied to elucidate the cellular and molecular mechanisms involved in the development of this disease. In this study, we report for the first time that a single point mutation at nucleotide position 3155 or 3156 of the recombinant EMC viral genome, located on the major capsid protein VP1, which causes an amino acid change, results in the gain or loss of viral diabetogenicity. A G base at nucleotide position 3155 (alanine at amino acid position 776 of the EMC virus polyprotein [Ala776]; GCC) results in viral diabetogenicity, whereas the substitution of other bases at the same or next position results in a loss of viral diabetogenicity. This finding provides clear evidence that a point mutation at a critical site in a viral genome affects the ability of the virus to cause a cell-specific disease.

Alanine↗
PubMed1994

High rates of Ki-ras point mutation in both intra- and extra-hepatic cholangiocarcinomas.

The presence of the Ki-ras gene mutations in 14 cases of intrahepatic cholangiocarcinoma (IHC) and nine cases of extrahepatic cholangiocarcinoma (EHC) were investigated by polymerase chain reaction-single strand conformation polymorphism analysis. To obtain enriched tumor cell DNA, the microdissection method was used on formalin-fixed paraffin-embedded tissue sections. Point mutations at codon 12 of the Ki-ras gene were detected in seven (50%) of the 14 cases of IHC and six (67%) of the nine EHC cases. In all but one of the ras gene mutation cases, G to A transitions in the second position of codon 12 were detected, the exception being a G to T transition in the same position in one IHC. No point mutations were detected at codon 13 or 61 in either IHC or EHC. Furthermore, there was no demonstrable correlation between Ki-ras mutation and patient age, tumor Stage, histological findings or prognosis. The present results demonstrated a higher participation of Ki-ras gene mutations in EHC than found in previous studies, and provided a confirmation and extension of the results earlier reported by Tada et al. and Tsuda et al. for IHC.

Adult↗
PubMed2002

Conversion of a transmembrane to a water-soluble protein complex by a single point mutation.

Proteins exist in one of two generally incompatible states: either membrane associated or soluble. Pore-forming proteins are exceptional because they are synthesized as a water-soluble molecule but end up being located in the membrane -- that is, they are nonconstitutive membrane proteins. Here we report the pronounced effect of the single point mutation Y221G of the pore-forming toxin aerolysin. This mutation blocks the hemolytic activity of the toxin but does not affect its initial structure, its ability to bind to cell-surface receptors or its capacity to form heptamers, which constitute the channel-forming unit. The overall structure of the Y221G protein as analyzed by cryo-negative staining EM and three-dimensional reconstruction is remarkably similar to that of the wild type heptamer. The mutant protein forms a mushroom-shaped complex whose stem domain is thought to be within the membrane in the wild type toxin. In contrast to the wild type heptamer, which is a hydrophobic complex, the Y221G heptamer is fully hydrophilic. This point mutation has, therefore, converted a normally membrane-embedded toxin into a soluble complex.

Aeromonas hydrophila↗
PubMed1996

The vacuolar ATPase of Neurospora crassa is indispensable: inactivation of the vma-1 gene by repeat-induced point mutation.

To analyze the phenotype of cells lacking the vacuolar ATPase, we inactivated the vma-1 gene, which encodes the catalytic subunit of the enzyme. Because preliminary experiments suggested the vma-1 gene was essential, we developed a method of simultaneously inactivating the gene and complementing it with a functional copy. We call this method repeat-induced point mutation (RIP) & Rescue. Two strains, both of which contained an extra copy of the vma-1 gene, were mated. Progeny that had inherited a functional copy of the gene at an ectopic site in the genome were selected. In some of these progeny the endogenous vma-1 gene had been altered by the RIP process. Sequencing showed the endogenous vma-1 gene had been inactivated by multiple point mutations. Progeny from strains with an inactive endogenous vma-1 gene were inviable unless a functional copy of the gene cosegregated, indicating that the vacuolar ATPase is essential in Neurospora crassa.

Base Sequence↗
PubMed1997

[Absence of point mutation in the third intracellular loop of alpha 1B- and alpha 1D adrenergic receptor in the aortae of spontaneously hypertensive rats].

Our previous experiments showed that the basal level of inositol phosphates (IPs) accumulation in the aorta was higher in spontaneously hypertensive rats (SHR) than in WKY rats. According to the constitutive activity hypothesis for G-protein coupling receptors, we inferred that point mutations might possibly exsit in the third intracellular loop of alpha 1B- and/or alpha 1D-adrenergic receptor (AR), the main subtypes of alpha 1-AR mediating phosphatidylinositol hydrolysis in SHR aortae. This inference, however, was not demonstrated by the results of our PCR-SSCP analyses, which indicated that the increased basal level of IPs accumulation in the isolated aortae from SHR does not result from constitutive activity of alpha 1B- and alpha 1D-AR induced by point mutations of the third intracellular loop of the genes.

Animals↗
PubMed1996

RET proto-oncogene point mutations in sporadic neuroendocrine tumors.

We investigated the possible role of the RET proto-oncogene, which has recently been identified as the susceptibility gene for multiple endocrine neoplasia type 2, in the development of sporadic neuroendocrine tumors from different locations. DNA extracted from paraffin-embedded specimens of 112 neuroendocrine tumors was screened for somatic RET point mutations in exons 10, 11, 13, 15, and 16, where recently oncogenic mutations have been described in a subset of sporadic medullary thyroid carcinomas and pheochromocytomas. Methods employed included nonisotopic PCR-based single strand conformation polymorphism (PCR-SSCP) analysis, heteroduplex gel electrophoresis, and restriction enzyme digestion. The nucleotide sequence of samples with aberrant band patterns was identified by nonisotopic direct sequencing of PCR-amplified DNA. Forty-four percent (7/16) of sporadic medullary thyroid carcinomas and 15% (3/20) of pheochromocytomas contained a somatic, heterozygous point mutation at codon 918 of exon 16 (ATG --> ACG) causing a Met --> Thr substitution. None of the remaining 4 parathyroid adenomas, 8 pituitary adenomas, 17 pancreatic neuroendocrine tumors, 11 pulmonary and 10 gastrointestinal carcinoids, 7 small cell lung carcinomas, 5 neuroblastomas, 10 malignant melanomas, or 4 schwannomas contained mutations in any of the five RET exons tested. Although the numbers of each investigated neuroendocrine tumor type are small, our data indicate that oncogenic RET proto-oncogene mutations are involved in the formation of a subset of sporadically occurring medullary thyroid carcinomas and pheochromocytomas but do not appear to be generally important in the formation of other types of sporadically occurring neuroendocrine tumors.

Adrenal Gland Neoplasms↗
PubMed1994

Inclusion of synthetic DNA templates of similar length and base composition to PCR-amplified products in restriction enzyme digestions: an efficient aid in characterization of point mutations.

Because of a subtle anomaly we encountered upon an analytical gel while characterizing a point mutation in an exon of a patient, we decided to perform expensive and time-consuming procedures to characterize the anomaly. Although initial and subsequent Southern blots and PCR analyses of this patient's mutation suggested that his mutation lay directly within a TaqI recognition site, further characterization revealed that the mutation actually lay in a base immediately outside the recognition site. Had we included an appropriate double-stranded DNA control in the restriction enzyme digestion of this patient's PCR-amplified exon, we could have arrived at the correct conclusion as to the location of the mutation without incurring high costs and time loss. This brief report depicts the use of DNA controls of appropriate length and base composition as a means of avoiding erroneous conclusions and expense in routine mutational analyses in the clinical setting.

Alleles↗
PubMed1995

A missense point mutation (Ser515Phe) in the adrenoleukodystrophy gene in a family with adrenomyeloneuropathy: a clinical, biochemical, and genetic study.

A 36 year old male patient with adrenomyeloneuropathy (AMN) developed progressive spastic paraparesis and sensory ataxia from the age of 18. Biochemical studies showed increased plasma concentrations of saturated very long chain fatty acids (VLCFAs), subclinical evidence of adrenal insufficiency, and primary hypogonadism. Three female family members had increased plasma concentrations of VLCFAs, suggesting carrier status of adrenoleukodystrophy (ALD). Molecular genetic analysis detected a missense point mutation (C1930T) in exon 6 within the ALD gene, which predicts substitution of an amino acid (Ser515Phe) that is conserved between the deduced amino acid sequence of the peroxisomal membrane protein PMP70 and ALD protein. Detection of this point mutation allows diagnosis of ALD or AMN, identification of heterozygotes, and prenatal diagnosis of ALD.

Adrenoleukodystrophy↗
PubMed2001

Partial restoration of replication of simian immunodeficiency virus by point mutations in either the dimerization initiation site (DIS) or Gag region after deletion mutagenesis within the DIS.

We used the simian immunodeficiency virus (SIV) molecular clone SIVmac239 to generate a deletion construct, termed SD2, in which we eliminated 22 nucleotides at positions +398 to +418 within the putative dimerization initiation site (DIS) stem. This SD2 deletion severely impaired viral replication, due to adverse effects on the packaging of viral genomic RNA, the processing of Gag proteins, and viral protein patterns. However, long-term culture of SD2 in either C8166 or CEMx174 cells resulted in restoration of replication capacity, due to two different sets of three compensatory point mutations, located within both the DIS and Gag regions. In the case of C8166 cells, both a K197R and a E49K mutation were identified within the capsid (CA) protein and the p6 protein of Gag, respectively, while the other point mutation (A423G) was found within the putative DIS loop. In the case of CEMx174 cells, two compensatory mutations were present within the viral nucleocapsid (NC) protein, E18G and Q31K, in addition to the same A423G substitution as observed with C8166 cells. A set of all three mutations was required in each case for restoration of replication capacity, and either set of mutations could be substituted for the other in both the C8166 and CEMx174 cell lines.

Base Sequence↗
PubMed1997

Heterozygosity for a point mutation in an invariant splice donor site of dihydropyrimidine dehydrogenase and severe 5-fluorouracil related toxicity.

Dihydropyrimidine dehydrogenase (DPD) is responsible for the breakdown of the widely used antineoplastic agent 5-fluorouracil (5-FU), thereby limiting the efficacy of the therapy. It has been suggested that patients suffering from 5-FU toxicities due to a low activity of DPD are genotypically heterozygous for a mutant allele of the gene encoding DPD. In this study we investigated the cDNA and a genomic region of the DPD gene of a cancer patient experiencing severe toxicity following 5-FU treatment for the presence of mutations. Although normal activity of DPD was observed in fibroblasts, the DPD activity in leucocytes of the cancer patient proved to be in the heterozygous range. Analysis of the DPD cDNA showed heterozygosity for a 165bp deletion that results from exon skipping. Sequence analysis of the genomic region encompassing the skipped exon showed that the tumour patient was heterozygous for a G-->A point mutation in the invariant GT splice donor sequence in the intron downstream of the skipped exon. So far, the G-->A point mutation has also been found in 8 out of 11 patients suffering from a complete deficiency of DPD. Considering the frequent use of 5-FU in the treatment of cancer patients, the severe 5-FU-related toxicities in patients with a low activity of DPD and the high frequency of the G-->A mutation in DPD deficient patients, analysis of the DPD activity and screening for the G-->A mutation should be routinely carried out prior to the start of the treatment with 5-FU.

Antimetabolites, Antineoplastic↗
PubMed1996

A heteroplasmic point mutation of mitochondrial tRNALeu(CUN) in non-lymphoid haemopoietic cell lineages from a patient with acquired idiopathic sideroblastic anaemia.

Acquired idiopathic sideroblastic anaemia (AISA) has been proposed to be a disorder of mitochondrial DNA (mtDNA). The hallmark of mitochondrial iron overload may be attributable to a respiratory chain defeat leading to impaired reduction of ferric iron (Fe3+) to ferrous iron (Fe2+), which is essential to the last step of mitochondrial haem biosynthesis. In a 71-year-old patient we identified a point mutation in one of the two mitochondrial transfer-RNAs coding for leucine (tRNA(leu)(CUN)). The mutation involves a G --> A transition in the anticodon loop, immediately adjacent to the anticodon triplet (mtDNA position 12301). The mutated guanine is highly conserved in a wide range of species. The mutation is heteroplasmic, i.e. there is a mixture of normal and mutated mitochondrial genomes (ratio c. 50:50). Heteroplasmy of mtDNA is not found in normal individuals, but is a typical feature of mitochondrial cytopathies. The point mutation was present in the patient's bone marrow and whole blood samples, in purified platelets, and in the granulocyte/erythrocyte pellet after mononuclear cell separation by density gradient centrifugation. The mutation was not found in T- and B-lymphocytes isolated by immunomagnetic bead separation. It was also absent from buccal mucosa cells and cultured skin fibroblasts. This pattern of involvement suggests that the mutation occurred in a self-renewing myeloid stem cell of the CFU-GEMM type.

Aged↗
PubMed1997

An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription.

Equine arteritis virus (EAV) is a positive-strand RNA virus that uses a discontinuous transcription mechanism to generate a nested set of six subgenomic mRNAs from which its structural genes are expressed. A stable bacterial plasmid (pEAV030) containing a full-length cDNA copy of the 12.7-kb EAV genome was constructed. After removal of a single point mutation in the replicase gene, RNA transcripts generated in vitro from pEAV030 were shown to be infectious upon electroporation into BHK-21 cells. A genetic marker mutation was introduced at the cDNA level and recovered from the genome of the progeny virus. The potential of pEAV030 as a tool to express foreign genes was demonstrated by the efficient expression of the chloramphenicol acetyltransferase (CAT) reporter gene from two different subgenomic mRNAs. The point mutation that initially rendered the full-length clone noninfectious was found to result in a particularly intriguing phenotype: RNA carrying this mutation can replicate efficiently but does not produce the subgenomic mRNAs required for structural protein expression. To our knowledge, this mutant provides the first evidence that the requirements for arterivirus genome replication and discontinuous mRNA synthesis are, at least partially, different and that these processes may be separated experimentally.

Animals↗
PubMed1995

Tyrosine phosphorylation of Shc proteins and formation of Shc/Grb2 complex correlate to the transformation of NIH3T3 cells mediated by the point-mutation activated neu.

PLC-gamma, ras-GAP and Shc have been proposed to be in vivo substrates for the neu-encoded p185neu receptor tyrosine kinases. We compared the tyrosine phosphorylation levels of PLC-gamma, ras-GAP and Shc in two NIH3T3 derived cell lines, transformed B104-1-1 and non-transformed DHFR/G8 cells in which point-mutation activated and normal rat neu genes were transfected and expressed, respectively. We found that tyrosine phosphorylation of Shc and formation of Shc/Grb2 complex were more significant in B104-1-1 cells than in DHFR/G8 cells, while no obvious difference could be detected for the tyrosine phosphorylation levels of ras-GAP and PLC-gamma between these two cell lines. Furthermore, we observed that association with Shc was severely impaired by deletion of most of the major autophosphorylation sites of the point-mutated neu. The truncated neu product, however, fully retained its ability to transform NIH3T3 cells, induce Shc tyrosine phosphorylation and Shc/Grb2 complex formation. Our results suggest that tyrosine phosphorylation of Shc which allows formation of Shc/Grb2 complex may play an important role for cell transformation induced by the point mutation-activated neu, and that stable binding to mutant p185neu may not be necessary for Shc to mediate this signaling pathway.

3T3 Cells↗
PubMed1996

A point mutation in glycoprotein IX coding sequence (Cys73 (TGT) to Tyr(TAT)) causes impaired surface expression of GPIb/IX/V complex in two families with Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS) is a rare inherited bleeding disorder which is caused by abnormal expression or function of the glycoprotein (GP) Ib/IX/V complex, a platelet major receptor for von Willebrand factor. We studied four BSS patients in two unrelated families in which the same and novel mutation was found. Flow cytometric analysis showed that GPIX was completely absent but residual amounts of GPIb alpha and GPV were detectable in these patients. We analyzed all coding regions of GPIb alpha, GPIb beta, GPV and GPIX which were amplified from the patients' genomic DNA by the polymerase chain reaction (PCR). In all four cases, we identified a point mutation in the GPIX coding region that changes the codon for cysteine 73 (TGT) to a codon for tyrosine (TAT). Furthermore, we confirmed by a transient expression study that the mutation caused the loss of adequate surface expression of GPIX. Since cysteine might be important for the secondary structure, this mutation of GPIX gene would lead to a dramatic conformational change of GPIX protein, resulting in the reduced surface expression. We concluded that this novel point mutation of the GPIX gene was responsible for BSS in these families.

Adult↗
PubMed2005

Prevalence of pfcrt point mutations and level of chloroquine resistance in Plasmodium falciparum isolates from Africa.

The development in Plasmodium falciparum of the resistance to chloroquine (CQ) constitutes a public health priority, due to its direct influence in childhood mortality. The molecular basis for CQ resistance (CQR) is still unclear but, recently, a new relevant gene, named pfcrt, with several point mutations was identified in P. falciparum. Two mutations, K76T and A220S, have been considered crucial for CQR in further studies, making the pfcrt a good candidate as determinant for CQR in P. falciparum. To contribute to this topic, we have undertaken a molecular screening on 164 P. falciparum isolates from Africa: 120 isolates were Italian imported malaria cases, 27 and 17 isolates were from a school-children survey from Congo and Tanzania, respectively. In vitro tests (pLDH and WHO-Mark III tests) for CQ sensitivity have been also carried out on 28 plasmodial isolates and results compared to those obtained by molecular analysis in the same isolates. The SVIET pfcrt haplotype has been identified in the samples from Congo, and this is the first time that this haplotype is detected in Africa. Our results give further evidence to the reliability of the 76T (and the linked 74I-75E) pfcrt point mutation as molecular marker for CQR.

Adolescent↗