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PubMed2001

Mutation analysis in Chariot-Marie Tooth disease type 1: point mutations in the MPZ gene and the GJB1 gene cause comparable phenotypic heterogeneity.

Charcot-Marie-Tooth disease type 1 (CMT1) is a demyelinating peripheral neuropathy most commonly caused by a DNA duplication on chromosome 17p11.2 including the peripheral myelin protein 22 (PMP22). Point mutations in the myelin protein zero gene (MPZ) and gap junction protein, beta-1 gene (GJB1) are also found in association with CMT1 or the subclass of CMT type X (CMTX), respectively. Recently point mutations in these genes have been found in patients showing the axonal variant of CMT, CMT type 2 (CMT2). We here describe the clinical and electro-physiological findings caused by two novel and two recently described MPZ mutations and six GJB1 mutations. Different MPZ and GJB1 mutations were associated with different grades of severity in CMT1 and CMTX. The novel MPZ Glu141st op mutation was associated with the axonal CMT2. We conclude that the clinical and electrophysiological heterogeneity among CMT patients carrying point mutations in MPZ and GJB1 is similar. Thus for clinical purposes CMT1 and CMT2 patients should be screened for mutations in these two genes after duplication on chromosome 17p11.2 has been excluded as the disease causing mutation.

Adolescent↗
PubMed1992

Mutational spectrometry: a general approach for hot-spot point mutations in selectable genes.

To observe point mutational spectra with a high degree of precision, independent large cultures of human lymphoblastoid cells were treated with a mutagen, benzo[a]pyrene diol epoxide, and mutants at the HPRT gene were selected en masse by 6-thioguanine resistance. An average of 1.6 x 10(4) 6-thioguanine-resistant mutants were created per experiment and the kinds, positions, and numbers of the most frequent mutations were examined in exon 3 of the HPRT gene by using a high-fidelity polymerase chain reaction and denaturing gradient gel electrophoresis. Sixteen exon 3-specific mutations were found to be predominantly G----T transversions and corresponded to an average of 3500 induced mutants per experiment. Of these mutations, 6 occurred within a run of 6 guanines and 5 occurred in the sequence 5'-GAAGAG-3'. The variation among independent experiments is consistent with the numerical expectation that all 16 mutations fulfill reasonable statistical criteria for mutational hot spots. The agreement with data from various systems using clone-by-clone analysis shows that the protocol reported herein can be a useful tool to study hot-spot point mutational spectra for DNA sequences for which phenotypic selection systems exist.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2003

Detection of point mutations of the Axin1 gene in colorectal cancers.

Axin is a recently identified tumor suppressor that plays an important role in liver and colon cancers. To gain further insights into the structure and function of Axin in controlling cell growth, we analyzed 54 colorectal cancer tissues for mutations in AXIN1 gene. We employed PCR amplification with 23 sets of primers against introns that encompassed the whole coding region of AXIN1 followed by single-strand conformation polymorphism (SSCP) analysis. After subcloning and sequencing analysis of the reamplified DNA from the aberrant bands, we found, in addition to 3 silent mutations, 6 missense point mutations in different functionally important regions. The missense mutation rate is hence 11%, suggesting that Axin deficiency may contribute to the onset of colorectal tumorigenesis.

Adenocarcinoma↗
PubMed1988

Base substitutions, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells.

The relative role of point mutations and large genomic rearrangements in ionizing radiation-induced mutagenesis has been an issue of long-standing interest. Recent studies using Southern blotting analysis permit the partitioning of ionizing radiation-induced mutagenesis in mammalian cells into detectable deletions and major genomic rearrangements and into point mutations. The molecular nature of these point mutations has been left unresolved; they may include base substitutions as well as small deletions, insertions, and frame-shifts below the level of resolution of Southern blotting analysis. In this investigation, we have characterized a collection of ionizing radiation-induced point mutations at the endogenous adenine phosphoribosyltransferase (aprt) locus of Chinese hamster ovary cells at the DNA sequence level. Base substitutions represented approximately equal to 2/3 of the point mutations analyzed. Although the collection of mutants is relatively small, every possible type of base substitution event has been recovered. These mutations are well distributed throughout the coding sequence with only one multiple occurrence. Small deletions represented the remainder of characterized mutants; no insertions have been observed. Sequence-directed mechanisms mediated by direct repeats could account for some of the observed deletions, while others appear to be directly attributable to radiation-induced strand breakage.

Adenine Phosphoribosyltransferase↗
PubMed1989

Rare occurrence of N-ras point mutations in Philadelphia chromosome positive chronic myeloid leukemia.

Point mutations of the N-ras oncogene are relatively common in acute myelogenous leukemia (AML) cells, occurring in some 25% to 50% of patient samples. We used a technique involving the direct nucleotide sequencing of in vitro amplified N-ras genomic fragments to determine the frequency of N-ras point mutations in chronic myeloid leukemia (CML) cells at various stages of the disease. This approach will detect N-ras point mutations in a mixed population of cells if the mutation is present in 25% or more of the cells. We could not demonstrate any point mutation at N-ras codons 12,13 or 59-63 in any of the 44 CML cases analyzed, which included 21 blast crisis samples. In contrast with AML N-ras point mutations are exceedingly rare in CML.

Gene Rearrangement↗
PubMed1992

Direct determination of the point mutation rate of a murine retrovirus.

The point mutation rate of a murine leukemia virus (MuLV) genome (AKV) was determined under conditions in which the number of replicative cycles was carefully controlled and the point mutation rate was determined by direct examination of the RNA genomes of progeny viruses. A clonal cell line infected at a low multiplicity of infection (2 x 10(-3)) was derived to provide a source of virus with high genetic homogeneity. Virus stocks from this cell line were used to infect cells at a low multiplicity of infection, and the cells were seeded soon after infection to obtain secondary clonal cell lines. RNase T1-oligonucleotide fingerprinting analyses of virion RNAs from 93 secondary lines revealed only 3 base changes in nearly 130,000 bases analyzed. To obtain an independent assessment of the mutation rate, we directly sequenced virion RNAs by using a series of DNA oligonucleotide primers distributed across the genome. RNA sequencing detected no mutations in over 21,000 bases analyzed. The combined fingerprinting and sequencing analyses yielded a mutation rate for infectious progeny viruses of one base change per 50,000 (2 x 10(-5)) bases per replication cycle. Our results suggest that over 80% of infectious progeny MuLVs may be replicated with complete fidelity and that only a low percentage undergo more than one point mutation during a replication cycle. Previous estimates of retroviral mutation rates suggest that the majority of infectious progeny viruses have undergone one or more point mutations. Recent studies of the mutation rates of marker genes in spleen necrosis virus-based vectors estimate a base substitution rate lower than estimates for infectious avian retroviruses and nearly identical to our determinations with AKV. The differences between mutation rates observed in studies of retroviruses may reflect the imposition of different selective conditions.

Animals↗
PubMed1994

[Molecular oncological study on DNA ploidy, Ki-ras point mutation, and p21 expression in colorectal cancer].

We studied DNA ploidy, point mutation of Ki-ras oncogene codon 12, and p21 expression using paraffin embedded materials from 42 cases of colorectal cancer. DNA ploidy was measured by the method of Hedley et al. flow cytometrically. Point mutation of Ki-ras oncogene was examined by the method of Bos et al. using a dot-blot screening procedure, and p21 expression was examined immunohistochemically. Incidence of aneuploidy, Ki-ras point mutation, and p21 expression was 71.4%, 26.2%, 40.5%, respectively. There was a very weak correlation between p21 expression and pathologic findings, but there was no correlation between pathologic findings and DNA ploidy, as well as Ki-ras point mutation. Patients who showed aneuploidy tended to have more point mutation of Ki-ras oncogene. There was no correlation between p21 expression and Ki-ras point mutation, as well as DNA ploidy. Although there was no correlation between Ki-ras point mutation and survival, a significant correlation between survival and DNA ploidy, as well as p21 expression was recognized. Patients who had tumors with diploidy or p21 expression tended to have better prognosis.

Adult↗
PubMed2000

A pathogenic point mutation reduces stability of mitochondrial mutant tRNA(Ile).

Point mutations in mitochondrial tRNA genes are responsible for individual subgroups of mitochondrial encephalomyopathies. We have recently reported that point mutations in the tRNA(Leu)(UUR) and tRNA(Lys) genes cause a defect in the normal modification at the first nucleotide of the anticodon. As part of a systematic analysis of pathogenic mutant mitochondrial tRNAs, we purified tRNA(Ile) with a point mutation at nucleotide 4269 to determine its nucleotide sequence, including modified nucleotides. We found that, instead of causing a defect in the post-transcriptional modification, a pathogenic point mutation in the mitochondrial tRNA(Ile) reduced the stability of the mutant tRNA molecule, resulting in a low steady-state level of aminoacyl-tRNA. The reduced stability was confirmed by examining the life-span of the mutant tRNA(Ile) both in vitro and in vivo, as well as by monitoring its melting profile. Our finding indicates that the mutant tRNA(Ile) itself is intrinsically unstable.

Acylation↗
PubMed1998

[Failure of mifepristone induced interruption of pregnancy: point mutation at genetic codon 722 in human progesterone receptor gene].

OBJECTIVE: To detect the point mutation of hormone binding domain (HBD) of human progesterone receptor (hPR) gene by molecular biological methods in women with insensitivity to mifepristone. METHODS: 26 women in 5 hospitals were consented in the study. 16 women were as study group: continuing pregnancy in 11 and missed abortion in 5. 10 women with successful abortion were as control group. Decidual tissue was collected in each woman and RNA was extracted from it and purified. Reverse transcription-polymerase chain reaction (RT-PCR), single strand conformation polymorphism(SSCP) and restriction enzyme reaction were performed. All women were followed up. RESULT: No point mutation of HBD of hPR gene was detected in control group except one with point mutation in trace. Point mutation of HBD of hPR gene was detected in 7 of women with continuing pregnancy. Mifepristone was administrated in midluteal phase in two of these women and no vaginal bleeding was observed. No mutation of hPR gene in the other women with continuing pregnancy. Three women had pregnancy again in 6 months and had successful abortion. The other one administrated mifepristion in midluteal phase and had second vaginal bleeding. No point mutation of hPR gene was detected in women with missed abortion. CONCLUSION: There is point mutation of HBD of hPR gene in women with insensitivity to mifepristone, and women with point mutation of hPR gene detected by molecular biological methods are really insensitive to mifepristone.

Abortifacient Agents, Steroidal↗
PubMed1995

p53 point mutation and survival in colorectal cancer patients.

We have examined the relationship between point mutation of the p53 tumor suppressor gene and survival in colorectal cancer patients. We found that patients with tumors harboring mutated p53 genes showed a significantly poorer prognosis than did those patients with genes without point mutations, and, moreover, patient response to postoperative therapies depended significantly on mutation status in both adjuvant and palliative treatment cohorts. However, not all point mutations were the same functionally; point mutations within the conserved domains of the p53 tumor suppressor gene were inherently more aggressive than tumors with point mutations outside of these domains, and mutations of codon 175 were particularly aggressive. These results suggest that knowledge of a patient's p53 status, both with respect to the presence of point mutations and to the specific nature of the lesion, may be required to accurately predict both the course of the disease and the response of the disease to postoperative therapeutic interventions, especially those therapies based on the induction of apoptosis in the neoplastic cell.

Adenocarcinoma↗
PubMed1996

A detailed analysis of K-ras point mutations in relation to tumor progression and survival in colorectal cancer patients.

Point mutations in codon 12, 13, and 61 of the K-ras gene are an early event in tumorigenesis of colorectal cancer, but the impact of number, type, and position of such mutations on the progression of adenomas as well as the clinical behaviour of colorectal carcinomas is not clearly established. A series of 35 adenomas and 117 carcinomas at various stages was subjected to single-strand conformation polymorphism (SSCP) to analyse type, position and number of exon-I K-ras point mutations and to relate the results with patients survival. From our data we conclude that the number of K-ras point mutated tumors shows a trend to increase with tumor progression. The number of multiple K-ras point mutations, however, significantly increases with stage. Most mutations occur in the 1st or 2nd base of codon 12, whereas point mutations in the 3rd base are rare. In adenomas mutations, particularly G-T transversions, in the K-ras gene could indicate a propensity to malignant transformation. G-A transitions and G-C transversions of the second base are associated with metastasized tumors. Regarding survival, patients with K-ras point mutated tumors did worse than their non-mutated counterparts. G-A transitions in the 1st and 2nd base and G-C transversions in the 2nd base were associated with a poor prognosis as compared with G-T transversions in both the 1st and 2nd base. Patient survival therefore is related to the occurrence and type, but not the location, of K-ras point mutations.

Adenoma↗
PubMed1990

Use of denaturing gradient gel electrophoresis to detect point mutations in the factor VIII gene.

Point mutations in the factor VIII gene are responsible for the majority of cases of hemophilia A, and only a small fraction of these mutations can be recognized by restriction endonuclease analysis. We have now used polymerase chain reaction and denaturing gradient gel electrophoresis to characterize single nucleotide substitutions in the factor VIII gene. Five regions of the gene were studied: exon 8, the 3' end of exon 14, exon 17, exon 18, and exon 24. A GC clamp was attached to the 5' PCR primer to allow detection of the majority of single base changes in DNA fragments ranging from 249 to 356 bp. Ten of eleven known point mutations were definitively separated. Fifty-two patients with unknown mutations were then studied by these methods, and the disease-producing mutation was found in three. First, we identified a new missense mutation in exon 14 which is the likely cause of hemophilia A in one patient (tyrosine changed to cysteine at amino acid residue 1709). Second, we found a new missense mutation in exon 18 in one patient (asparagine to aspartic acid at amino acid residue 1922). Third, a previously described mutation in exon 24 was detected (arginine changed to glutamine at amino acid residue 2209). In addition, a new polymorphic nucleotide substitution was found in intron 7. Moreover, these mutations can be detected when the GC-clamped PCR products from all five regions are run in the same denaturing gel. Our results indicate that denaturing gradient gel electrophoresis can be successfully applied to the analysis of point mutations in large genes whose transcripts are not readily available.

Base Sequence↗
PubMed1996

Homology modeling of human methylmalonyl-CoA mutase: a structural basis for point mutations causing methylmalonic aciduria.

Point mutations in the human gene encoding coenzyme B12 (adenosylcobalamin)-dependent methylmalonyl-CoA mutase give rise to an inherited disorder of propionic acid metabolism termed mut methylmalonic aciduria. Almost all such mutations alter amino acids in the homodimeric human enzyme that are identical to residues in the catalytic alpha-subunit of the heterodimeric methylmalonyl-CoA mutase from the bacterium Propionibacterium shermanii, to which the mature human enzyme shows an overall 65% sequence identity. To explore how specific mutations might cause the observed clinical phenotype, 12 known mutations were mapped onto a three-dimensional homology model of the subunit of the human enzyme, generated using the program MODELLER on the basis of the recently published 2.0 A X-ray crystal structure of the P. shermanii methylmalonyl-CoA mutase. Eight mutations are found in the C-terminal B12-binding domain, of which 4 (G623R, G626C, G630E, G703R) are in direct contact with the corrin and are clustered around the histidine ligand (H627) provided by the protein to coordinate the cobalt atom of the B12 cofactor. Introduction of a side chain, particularly one that is charged, at any of these positions is expected to disrupt the flavodoxin-like fold and severely impair its binding of B12. Mutation at either of two other highly conserved glycine residues in this domain (G648D, G717V) also disrupts critical elements in the fold as would the introduction of an additional positive charge in the mutation H678R. Mutation of an arginine in a solvent-exposed loop to a hydrophobic residue (R694W) is also pathogenic. The remaining mutations have been mapped to the N-terminal region of the mutase, two of which introduce a buried, uncompensated charge, either near the subunit interface (A377E), or near the narrow channel through which acyl-CoA esters gain access to the active site (W105R). The extreme N-terminus of methylmalonyl-CoA mutase is predicted to make extensive contacts with the other subunit, and a mutant in this region (R93H) may prevent the correct assembly of the dimer.

Amino Acid Metabolism, Inborn Errors↗
PubMed1992

p53 point mutations in primary human gastric carcinomas.

p53 point mutations in primary gastric carcinomas were analyzed by performing cDNA deoxynucleotide sequencing of the gene. Out of 16, 9 (56.3%) primary gastric carcinoma cases, including early cancer, showed one or more p53 point mutations in their open-reading frame, and 4 out of 9 cases had a p53 point mutation within highly conserved domains. The characteristics of the p53 mutation spectrum observed in primary tumors were (a) frequent mutation at an A:T pair (50%, 7 out of 14 mutations), (b) high transversion incidence (29%, 4 out of 14 mutations), (c) no transition at CpG, and (d) no G:C to T:A transversion. Our results suggest that p53 mutation is a common event in gastric carcinoma occurring from the early stage of progression with its specific mutation spectrum.

Adult↗
PubMed2003

Genetic analysis of wild-isolated Neurospora crassa strains identified as dominant suppressors of repeat-induced point mutation.

Repeat-induced point mutation (RIP) in Neurospora results in inactivation of duplicated DNA sequences. RIP is thought to provide protection against foreign elements such as retrotransposons, only one of which has been found in N. crassa. To examine the role of RIP in nature, we have examined seven N. crassa strains, identified among 446 wild isolates scored for dominant suppression of RIP. The test system involved a small duplication that targets RIP to the easily scorable gene erg-3. We previously showed that RIP in a small duplication is suppressed if another, larger duplication is present in the cross, as expected if the large duplication competes for the RIP machinery. In two of the strains, RIP suppression was associated with a barren phenotype--a characteristic of Neurospora duplications that is thought to result in part from a gene-silencing process called meiotic silencing by unpaired DNA (MSUD). A suppressor of MSUD (Sad-1) was shown not to prevent known large duplications from impairing RIP. Single-gene duplications also can be barren but are too short to suppress RIP. RIP suppression in strains that were not barren showed inheritance that was either simple Mendelian or complex. Adding copies of the LINE-like retrotransposon Tad did not affect RIP efficiency.

Blotting, Southern↗
PubMed2003

Analysis of haemophilia B database and strategies for identification of common point mutations in the factor IX gene.

Haemophilia B is an X-linked recessively inherited bleeding disorder caused by heterogeneous mutations spanning the entire factor IX gene. As spontaneous germ-line mutations are known to occur mostly at CpG dinucleotides in the FIX gene, control of the disease would require continuous carrier detection and mutation screening. Identification of point mutations, the most common type of mutation in FIX gene, is more challenging compared with deletion and insertion mutations. We examined the haemophilia B database to identify specific nucleotides in the FIX gene that are mutated in relatively large number of patients and the variability (if any) in the mutational hotspots at CpG dinucleotides. It was found that while mutations responsible to account for all 2348 haemophilia B patients covered 20% of the FIX cDNA, only 1% of the cDNA involving mostly CpG dinucleotides accounted for mutation in 42.41% of the patient pool. Thus, only 27 nucleotides need to be investigated to identify the common point mutations, among which 15 are predicted to undergo change in restriction sites on mutation. It is interesting to note that seven nucleotides occurring in CpG dinucleotides do not have any reported mutation despite each of those being predicted to harbour mutation as a result of transition and having mutations recorded in the database for the neighbouring nucleotides. Strikingly large number of mutation in codon 296 causing T to M change in catalytic domain originally proposed to be the result of the founder effect also contains largest number of haplotype suggesting recurrence of de novo mutation.

DNA, Complementary↗
PubMed1999

Phylogenetic signals from point mutations and polymorphic Alu insertions.

Allelic frequency data derived from five polymorphic Alu insertion loci and five point mutation polymorphic loci were compared to determine their ability to infer phylogenetic relationships among human populations. While point mutation polymorphisms inferred a monophyletic Caucasian clade that is corroborated by other studies, these data failed to support the generally accepted monophyly of Orientals with native Americans. In addition, there is less statistical bootstrap support for the maximum-likelihood tree derived from the point mutation polymorphisms as compared to those generated from either the Alu insertion data or the combined Alu insertion + point mutation data. The Alu data and the combined Alu insertion + point mutation data inferred a monophyletic relationship among the Oriental and native American populations. The Alu insertion data and the combined Alu insertion + point mutation data also displayed two separate, well defined, tight clusters of the Caucasian and the Oriental + native American populations which was not inferred from the point mutation data. These findings indicate greater phylogenetic information contained in Alu insertion frequencies than in allelic frequencies derived from point-mutations.

Alleles↗
PubMed1997

Direct detection of multiple point mutations in mitochondrial DNA.

Mitochondrial defects can be caused by mutations in nuclear or mitochondrial DNA. Large deletion/duplication and point mutations are the two major types of mitochondrial DNA (mtDNA) mutations. Comprehensive molecular diagnosis requires the analysis of multiple point mutations. We developed an effective multiplex PCR/allele-specific oligonucleotide (ASO) method to simultaneously screen multiple point mutations in mtDNA. The system involved three pairs of primers to amplify mutation "hot spots" at tRNA(leu(UUR)), tRNA(lys)/ATPase, and ND4 regions, followed by detection of point mutations with ASO probes. Over 2000 specimens were analyzed and the results were compared with those from previous studies with the PCR/restriction fragment length polymorphism method. Our data demonstrate that the multiplex PCR/ASO method is much more sensitive in the detection of low mutant heteroplasmy. It is simple and cost effective, especially if a large number of samples are to be screened for multiple point mutations.

DNA, Mitochondrial↗