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PubMed1988

Evidence that a point mutation in dihydrofolate reductase-thymidylate synthase confers resistance to pyrimethamine in falciparum malaria.

Analysis of a genetic cross of Plasmodium falciparum and of independent parasite isolates from Southeast Asia, Africa, and South America indicates that resistance to pyrimethamine, an antifolate used in the treatment of malaria, results from point mutations in the gene encoding dihydrofolate reductase-thymidylate synthase (EC 1.5.1.3 and EC 2.1.1.45, respectively). Parasites having a mutation from Thr-108/Ser-108 to Asn-108 in DHFR-TS are resistant to the drug. The Asn-108 mutation occurs in a region analogous to the C alpha-helix bordering the active site cavity of bacterial, avian, and mammalian enzymes. Additional point mutations (Asn-51 to Ile-51 and Cys-59 to Arg-59) are associated with increased pyrimethamine resistance and also occur at sites expected to border the active site cavity. Analogies with known inhibitor/enzyme structures from other organisms suggest that the point mutations occur where pyrimethamine contacts the enzyme and may act by inhibiting binding of the drug.

Animals↗
PubMed2003

The G-to-T point mutation in codon 34 of the factor XIII gene and the risk of pre-eclampsia.

A G-to-T point mutation in exon 2 of the FXIII A-subunit gene results in a leucine rather than valine at amino acid position 34 of the factor XIII molecule. The presence of leucine has been associated with a reduced risk of both arterial and venous thrombosis. We examined the prevalence of this point mutation in 102 cases of pre-eclampsia and 208 matched control subjects, as inherited and acquired risk factors for arterial and venous thrombosis are associated with an increased risk of pre-eclampsia. The GT genotype was observed in 38% of controls and 29.4% of cases and the TT genotype was observed in 6.7% of controls and 5.9% of cases. In subjects heterozygous for the T genotype (GT) the relative risk of pre-eclampsia was 0.7 [95% confidence interval (CI95 ) 0.4-1.1] when compared with the GG genotype. For subjects homozygous for the T allele the relative risk for pre-eclampsia was 0.8 (CI95 0.3-2.1) when compared with the GG genotype. The risk associated with the T allele in the heterozygous and homozygous forms compared with the GG genotype was 0.7 (CI95 0.4-1.1). We conclude that the presence of leucine at this site is not associated with a protection against pre-eclampsia to the magnitude of that reported in other thrombotic disease.

Adult↗
PubMed1997

Aromatase deficiency in a female who is compound heterozygote for two new point mutations in the P450arom gene: impact of estrogens on hypergonadotropic hypogonadism, multicystic ovaries, and bone densitometry in childhood.

We report on a female who is compound heterozygote for two new point mutations in the CYP19 gene. The allele inherited from her mother presented a base pair deletion (C) occurring at P408 (CCC, exon 9), causing a frameshift that results in a nonsense codon 111 bp (37 aa) further down in the CYP19 gene. The allele inherited from her father showed a point mutation from G-->A at the splicing point (canonical GT to mutational AT) between exon and intron 3. This mutation ignores the splice site and a stop codon 3 bp downstream occurs. Aromatase deficiency was already suspected because of the marked virilization occurring prepartum in the mother, and the diagnosis was confirmed shortly after birth. Extremely low levels of serum estrogens were found in contrast to high levels of androgens. Ultrasonographic follow-up studies revealed persistently enlarged ovaries (19.5-22 mL) during early childhood (2 to 4 yr) which contained numerous large cysts up to 4.8 x 3.7 cm and normal-appearing large tertiary follicles already at the age of 2 yr. In addition, both basal and GnRH-induced FSH levels remained consistently strikingly elevated. Low-dose estradiol (E2) (0.4 mg/day) given for 50 days at the age of 3 6/12 yr resulted in normalization of serum gonadotropin levels, regression of ovarian size, and increase of whole body and lumbar spine (L1-L4) bone mineral density. The FSH concentration and ovarian size returned to pretreatment levels shortly (150 days) after cessation of E2 therapy. Therefore, we recommend that affected females be treated with low-dose E2 in amounts sufficient to result in physiological prepubertal E2 concentrations using an ultrasensitive estrogen assay. However, E2 replacement needs to be adjusted throughout childhood and puberty to ensure normal skeletal maturation and adequate adolescent growth spurt, normal accretion of bone mineral density, and, at the appropriate age, female secondary sex maturation.

Adult↗
PubMed1992

Precise excision of transposons and point mutations induced by chemicals.

The ability of 23 chemicals (carcinogens and non-carcinogens) to induce precise excision of Tn10 and point mutations was studied in experiments with a single strain. The mutation assay was shown to detect a wider spectrum of genotoxic agents than the assay of Tn10 precise excision. The latter was induced only by potent SOS mutagens, which is in accordance with data on the SOS dependence of the induction of precise excision of Tn10. The precise excision assay as an additional test contributing to the knowledge of particular features of the action of a tested mutagen is discussed. The induction of precise excision of Tn10 by pyrene (and its failure to induce point mutations in this strain) demonstrates the value of using the transposon excision assay in cases of 'problem' mutagens.

Carcinogenicity Tests↗
PubMed1995

Crouzon syndrome: mutations in two spliceoforms of FGFR2 and a common point mutation shared with Jackson-Weiss syndrome.

Dominant mutations in the fibroblast growth factor receptor 2 (FGFR2) gene have been recently identified as causes of four phenotypically distinct craniosynostosis syndromes, including Crouzon, Jackson-Weiss, Pfeiffer, and Apert syndromes. These data suggest that the genetics of the craniosynostosis syndromes is more complex than would be expected from their simple autosomal-dominant inheritance pattern. Identical mutations in the FGFR2 gene have been reported to cause both Pfeiffer and Crouzon syndrome phenotypes. We now report the finding of a mutation in exon IIIc of the FGFR2 gene in a kindred affected with Crouzon syndrome (C1043 to G; Ala344Gly) that is identical to the mutation previously associated with Jackson-Weiss syndrome. We also report finding in a Crouzon kindred a mutation in the 3' end of exon IIIu (formerly referred to as exon 5, exon 7, or exon U) (A878 to C; Gln289Pro) which encodes the amino terminal portion of the Ig-like III domain of the FGFR2 protein. This exon is common to both the FGFR2 and the KGFR spliceoforms of the FGFR2 gene, unlike all previously reported Crouzon mutations, which have been found only in the FGFR2 spliceoform. These findings reveal further unexpected complexity in the molecular genetics of these craniosynostosis syndromes. The data implies that second-site mutations in FGFR2 itself (outside of exon IIIc) or in other genes may determine specific aspects of the phenotypes of craniosynostosis syndromes.

Base Sequence↗
PubMed1997

Sporadic and Thorotrast-induced angiosarcomas of the liver manifest frequent and multiple point mutations in K-ras-2.

Hepatic angiosarcoma (HA) is an uncommon neoplasm associated with known etiologic factors in 25% to 42% of cases. It is, however, one of the most common sarcomas found in the liver. The aim of this study was to find was to find mutations in the K-ras-2 oncogene in sporadic and Thorotrast (TT)-induced HA. Point mutations in K-ras-2 were sought in archival, formalin-fixed tissue blocks from 24 patients with angiosarcoma. Of these, 19 cases were sporadic and 5 were TT-induced. Mutational analysis was performed by topographic microdissection with PCR amplification followed by genotyping. Specific mutations were determined by two independent methods: (a) direct sequencing of the PCR product confirmed by rePCR and by using a different sequencing primer, and (b) PCR-based selective enrichment of mutant DNA by endonuclease digestion followed by heteroduplex DNA analysis using denaturing gradient gel electrophoresis. Eleven K-ras-2 point mutations were detected in 7 of 24 (29%) tumors, including 5 of 19 (26%) sporadic HA and 2 of 5 (40%) TT-induced HA. There were seven G:C > A:T and four G:C > T:A mutations. All seven mutated tumors contained a codon 12-aspartate amino acid substitution. In addition, a second codon 12-cysteine mutant cell population was present in one of two codon 12-aspartate mutated TT-induced HA and in three of five codon 12-aspartate sporadic tumors. Of these four tumors, three contained both aspartate and cysteine mutations and were composed of multiple nodules; the fourth was a single mass. Seventeen tumors had multiple nodules; whereas 5 had a K-ras-2 mutation, 12 were wild-type. The molecular pathology of both sporadic and TT-induced HA is characterized by a high rate of K-ras-2 mutations characteristic of oxidative damage (ie, G:C > A:T and G:C > T:A mutations) resulting in two mutated population sets: codon 12 GGT > GAT and GGT > TGT (glycine to aspartic acid and cysteine). This is, to date, the first study to characterize the K-ras-2 gene mutations within human sporadic and TT-induced HA by direct sequence analysis and denaturing gradient gel electrophoresis. These data further support the hypothesis linking adduct-forming vinyl chloride exposure to HA containing a much higher frequency of K-ras-2 mutations and a mutational spectrum characteristic of chloroethylene oxide, a carcinogenic metabolite of vinyl chloride.

Adult↗
PubMed1995

Further elucidation of the genomic structure of PAX3, and identification of two different point mutations within the PAX3 homeobox that cause Waardenburg syndrome type 1 in two families.

Waardenburg syndrome is an autosomal dominant disorder characterized by sensorineural deafness and pigmentary disturbances. Previous work has linked the disease to PAX3 on chromosome 2, and several mutations within the highly conserved paired-box and octapeptide motifs, but not the homeobox, have been reported. In this report, we have used the published cDNA sequence to further define the genomic structure of PAX3, using inverse PCR. We have identified exon/intron boundaries between exons 5 and 6 and between exons 6 and 7. Further, we have identified the first two mutations within the homeobox in two different families with type 1 Waardenburg syndrome. The first is a point mutation (G-->T) at the first base of exon 6, which substitutes phenylalanine for valine. In another family, we have identified a point mutation (C-->G) within the homebox, in exon 6, which substitutes a glycine for arginine at a highly conserved site. The homeodomain is important in binding of DNA and in effecting transcriptional control. These mutations likely result in structural change within the homeodomain that either change the DNA-binding specificity of the homedomain or reduce the affinity of the PAX3 protein for DNA. These homeodomain mutations should aid in elucidating the role of the homeodomain in the function of the PAX3 protein.

Amino Acid Sequence↗
PubMed2000

Point mutations of the mtDNA control region in normal and neurodegenerative human brains.

Recent observations in cultured human fibroblasts suggest that the accumulation of point mutations in the noncoding control region of mtDNA may be important in human aging. We studied the mtDNA control region in brain tissue from 31 normal elderly individuals, from 35 individuals who had Alzheimer disease, and from 47 individuals who had dementia with Lewy bodies. We found no evidence that these somatic mtDNA point mutations accumulate either in the brains of normal elderly individuals or in the brains of individuals with neurodegenerative disease.

Aged↗
PubMed1998

Point mutations (Thr240Arg and Gln311Stop) [correction of Thr240Arg and Ala311Stop] in the Parkin gene.

Autosomal recessive juvenile parkinsonism (AR-JP) is a distinct clinical and genetic entity characterized by selective degeneration of nigral neurons. Recently, the parkin gene responsible for AR-JP has been identified. To date, we found two different deletional mutations including single and multiple exonic deletions. In the present study, we identified two types of point mutations (Thr240Arg and Gln311Stop) involving exons 6 and 8 in the parkin gene of the AR-JP patients from two Turkish families. This is the first report on point mutations for the parkin gene. Furthermore, the Thr240Arg mutation was located on a consensus sequence for the site of phosphorylation by casein kinase II. Identification of its mutation provides an important clue as to the role of the Parkin protein in degeneration of the substantia nigra in the brain of AR-JP patients.

Adolescent↗
PubMed2000

[Experimental study on the influence of Gynostemma pentaphyllam Mak upon point mutation of Ha-ras oncogene in blocking leukoplakia from canceration].

OBJECTIVE: To study the influence of Gynostemma pentaphyllam Mak (GP) upon point mutation of Ha-ras oncogene the molecular mechanism of GP in blocking canceration, and the relationship between Ha-ras oncogene and canceration of leukoplakia. METHODS: DMBA induced carcinogeneses of the buccal pouch mucosa of the hamsters were divided into leukoplakia model group(LKG) and GP treated group(GPG). Point mutation (codon 61) of Ha-ras oncogene was detected by PCR-SSCP in both groups. RESULTS: The mutation incidence of LKG was 22.96%, while the mean incidence of GPG was 7.59% (The incidences of C20-OH group, glucoside group and aglucon group were 5.32%, 6.25% and 11.00% respectively). When treated with DMBA for 4 to 8 weeks, the mutation incidence of LKG ranged from 11.10% to 40.00%, while GPG ranged from 0 to 10.70%. CONCLUSIONS: GP has outstanding cancer blocking effect chiefly affected by free C20-OH. Mutation of Ha-ras oncogene plays an important role in leukoplakia canceration.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2003

Large-scale discovery of induced point mutations with high-throughput TILLING.

TILLING (Targeting Induced Local Lesions in Genomes) is a general reverse-genetic strategy that provides an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and low-cost discovery of induced point mutations in populations of chemically mutagenized individuals. As chemical mutagenesis is widely applicable and mutation detection for TILLING is dependent only on sufficient yield of PCR products, TILLING can be applied to most organisms. We have developed TILLING as a service to the Arabidopsis community known as the Arabidopsis TILLING Project (ATP). Our goal is to rapidly deliver allelic series of ethylmethanesulfonate-induced mutations in target 1-kb loci requested by the international research community. In the first year of public operation, ATP has discovered, sequenced, and delivered >1000 mutations in >100 genes ordered by Arabidopsis researchers. The tools and methodologies described here can be adapted to create similar facilities for other organisms.

Alkylating Agents↗
PubMed2001

A new approach for point mutation detection based on a ligase chain reaction.

A new method for the identification of point mutations is proposed. The method is based on ligase chain reaction (LCR) and it includes a procedure for correction of ligation by Cleavase. Reaction products are detected by a colorimetric method after adsorption of the resulting DNA duplexes to the solid phase. One strand of LCR products carries biotin to be bound on a streptavidin-coated microwell. Another strand contains a single-stranded region that is to be coupled with an oligonucleotide carrying a substrate for colorimetric detection. The suggested method has two advantages: (i) use of Cleavase increases the accuracy of ligation and (ii) a template independent ligation does not occur in LCR due to a special design of primers.

Base Sequence↗
PubMed1989

Designed diagnostic restriction fragment length polymorphisms for the detection of point mutations in ras oncogenes.

The polymerase chain reaction (PCR) technique has greatly facilitated the identification of ras oncogenes by allele-specific hybridization of the PCR-amplified DNA to radioactively labelled oligonucleotide probes. In this study, we describe a different method which employs designed mismatch primers that create diagnostic restriction fragment length polymorphisms (RFLPs). This procedure allows the identification of point mutations in the amplified DNA without the use of any radioactive probes. We apply this method to the detection of specific point mutations in the rat H- and K-ras oncogenes in carcinogen-induced tumors. We also suggest strategies for the diagnostic RFLP analysis of most point mutations in the 12th and 61st codons of human ras oncogenes. This simple method is especially suitable for analyzing minuscule amounts of tissue samples where only a fraction of cells may carry activated oncogenes.

Alleles↗
PubMed1993

Malignant transformation of a mouse liver epithelial cell line by transfection of an activated c-H-ras gene with a point mutation at codon 12.

In order to scrutinize the reason why in mouse liver system only activated H-ras gene with a point mutation at codon 61 but not codon 12 is frequently seen although the latter mutation is highly frequent in methylnitrosourea-induced rat mammary tumors, transforming activity of these two types of mutated H-ras gene was investigated utilizing an immortalized but not fully transformed mouse liver epithelial cell line MLE-10, established in our laboratory. MLE-10 cells were transfected with activated human c-H-ras gene having a point mutation at either codon 12 (PT24) or 61 (PSK2), together with PSV2neo, or with PSV2neo only. G418 resistant colonies, propagated separately, gave rise to 6, 3 and 6 lines respectively. All the PT24 and PSK2-transfected cell lines were growth capable in both soft agar and nude mouse subcutis, with similar growth rate and morphological features whereas none of the cell lines transfected with the PSV2neo only revealed such growth capability. The results thus revealed that c-H-ras with a mutation at codon 12 has oncogenic activity to the mouse hepatocyte, although after immortalization, at the degree similar to the same gene with a mutation at codon 61.

Animals↗
PubMed1996

Three new mutations of the pyruvate dehydrogenase alpha subunit: a point mutation (M181V), 3 bp deletion (-R282), and 16 bp insertion/frameshift (K358SVS-->TVDQS).

Three novel mutations in the coding region of E1 alpha gene were found in three PDC-deficient male patients, including a missense mutation (M181V), a 3 bp deletion (AGA, corresponding to R282), and a 16 bp insertion (CAGTGGATCAAGTTTA), causing a frameshift starting with lysine 358 and resulting in decrease of both E1 subunits.

Cells, Cultured↗
PubMed2001

A point mutation in the LIM domain of Lhx3 reduces activation of the glycoprotein hormone alpha-subunit promoter.

Lhx3, a member of the LIM homeodomain family of transcription factors, is required for development of the pituitary in mice. A recent report has described a point mutation in the human LHX3 gene that is associated with a combined pituitary hormone disorder. The mutation is predicted to lead to the replacement of a tyrosine residue with a cysteine in the second LIM domain of LHX3. We have characterized the effects of this point mutation (Y114C) when analyzed in the context of the mouse Lhx3 coding sequence. Mobility shift assays demonstrated that the Lhx3 Y114C mutant is capable of binding DNA, although a decrease in the formation of a specific complex was observed. Transfection assays using an expression vector for either full-length Lhx3 or a GAL4-Lhx3 LIM domain fusion provided evidence that the Lhx3 Y114C mutant has a decreased ability to stimulate transcription. In particular, a GAL4-Lhx3 Y114C LIM mutant was unable to support Ras responsiveness of a modified glycoprotein hormone alpha-subunit reporter gene. Protein interaction studies suggest that the Y114C mutation may modestly reduce binding to the POU transcription factor, Pit-1. Interestingly, the Y114C mutation essentially abrogated binding to the putative co-activator/adapter, selective LIM-binding protein. The findings provide insights into the mechanisms mediating transcriptional activation by Lhx3 and suggest that the observed phenotype of the human mutation probably involves reduced transcriptional activity of the mutant LHX3.

Binding Sites↗
PubMed1993

Optimization of the single-strand conformation polymorphism (SSCP) technique for detection of point mutations.

The efficiency of detection of single base substitutions by single-stranded conformation polymorphism (SSCP) analysis was tested on 86 randomly distributed point mutations in a 193-bp-long DNA fragment of the mouse beta-globin gene. Multiple parameters were varied, including electrophoresis temperature, buffer concentration, gel concentration, acrylamide-to-bis-acrylamide ratio, and/or addition of different compounds to the gel matrix. Gels with a higher concentration of acrylamide and lower crosslinking gave optimal separation, and all 86 mutations can be clearly distinguished from the wild type on a 5% or 7.5% (2.6% C) acrylamide gel at 4 degrees C. Most of the mutations are also resolvable from wild type on gels with 5% urea or formamide, or 10% dimethylsulfoxide or sucrose. The relative position of the purine and pyrimidine-rich single strands were followed by an asymmetric PCR-SSCP technique. We found that most of the informativity comes from the purine rich strand, which appears to be much more sensitive to changes in the gel. The position or type of mutation showed no correlation with its ability to be detected. However, the neighboring base sequence around the mutation appears to have an effect on mobility. For example, A-->G substitutions in GC-rich regions significantly increase the mobility shift of the purine-rich strand, while most G-->A changes decrease it. We conclude that SSCP is a very efficient method for the detection of point mutations, if the parameters that effect the separation are optimized for a particular DNA fragment.

Animals↗
PubMed1995

Rapid restriction fragment analysis for screening four point mutations of the low-density lipoprotein receptor gene in French Canadians.

Familial hypercholesterolemia (FH) has an estimated frequency of 1:154 among French Canadians in Northeastern Quebec, compared with 1:500 in most other populations. FH is caused by numerous mutations of the low-density lipoprotein (LDL) receptor gene, but only six well-characterized mutations are known to cause FH in French Canadians. High prevalence of the phenotype, along with a limited number of mutations in this population, provides a unique opportunity to study genotype-phenotype variation. Since the current methods for detection of point mutations in this population use complicated approaches, we report polymerase chain reaction (PCR)-based restriction fragment analysis to detect all four point mutations. This approach provides a rapid diagnosis and is suitable to screen large number of samples for studies in genetic epidemiology; it should be useful in identifying FH in other populations bearing the same mutations.

Base Sequence↗