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PubMed1995

Point mutation in the second phosphatase domain of CD45 abrogates tyrosine phosphatase activity.

CD45 is a transmembrane protein tyrosine phosphatase that possesses two phosphatase domains in its cytoplasmic region. Whether both domains function independently as phosphatase enzymes or whether both domains interact to form an active enzyme is unclear. A point mutation of a critical cysteine residue in domain I is known to abolish CD45 activity, implying that the catalytic activity resides in domain I. In this report, mutational analysis of purified, recombinant CD45 cytoplasmic domain protein was performed. It was found that a single amino acid change in domain II (glutamine 1180 to a glycine) resulted in an inactive phosphatase enzyme, whereas two other point mutations in the membrane proximal region of the molecule had no effect on activity. Deletion of the region linking the two phosphatase domains also abolished enzymatic activity. Amino acids crucial for phosphatase activity thus reside in both phosphatase domains of CD45, illustrating that the phosphatase domains of CD45 do not act independently, but are both required for the phosphatase activity of CD45.

Amino Acid Sequence↗
PubMed1997

Point mutation flanking a CTL epitope ablates in vitro and in vivo recognition of a full-length viral protein.

CD8+ T cells (T(CD8+)) recognize viral Ags as short peptides (epitopes) displayed at the cell surface by MHC class I molecules. Using a panel of recombinant vaccinia viruses, we show that single-point mutations flanking either side of an H-2Kd-restricted epitope, residues 147-155, within full-length influenza nucleoprotein (NP) can impact, even ablate, presentation of that epitope, while having no effect on presentation of distal epitopes. The most severe blocking mutation (Ala to Pro at position 146) did not inhibit NP(147-155) presentation in the context of a truncated minigene, implying that this peptide is not a functional processing intermediate. An amino-terminal proline replacement also significantly reduced presentation of NP(50-57) (H-2Kk restricted), while the same mutation did not affect a third NP epitope. Thus, while trends in processing specificity may exist, the epitope itself contributes to flanking sequence effects. These findings were paralleled by in vivo priming experiments in which, depending on viral dose, subtle in vitro blocking effects were absolute. Proteasome/synthetic peptide coincubation studies support a role for enhanced epitope destruction in preventing presentation, as did the effect of the peptide aldehyde, LLnL, which restored presentation of NP(147-155) from the mutated constructs. This reagent did not inhibit epitope presentation, even from wild-type NP, suggesting that its production may be proteasome independent. These results support the notion that point mutation of epitope flanking sequence can serve as a mechanism for viral immune evasion, shed light on the mechanisms involved, and suggest that in vitro assays may not be sensitive indicators of flanking sequence effects.

Amino Acid Sequence↗
PubMed1990

Point mutations in four hemophilia B patients from China.

Point mutations in factor IX genes of four unrelated Chinese patients with hemophilia B have been identified by direct sequencing of amplified genomic DNA fragments. These four mutations occur in exon 8 of the factor IX gene. A C to T transition at nucleotide 30,863 changes codon 248 from Arg (CGA) to a new Stop codon (TGA), described in a previous family as factor IXMalmo3 (Green P M et al., EMBO J 1989; 8: 1067). A G to A transition at nucleotide 31,051 changes codon 310 from Trp (TGG) to a nonsense or Stop codon (TGA; factor IXChongquing2). A G to A transition at nucleotide 31,119 changes codon 333 which is for Arg (CGA) in normal factor IX, to one for Gln (CAA) in the variant previously described as factor IXLondon2 (Tsang T C et al., EMBO J 1988; 7: 3009) in a patient with moderately severe hemophilia B. The fourth patient has a novel C to A transversion at nucleotide 31,290, which corresponds to replacement of codon 390 which is for Ala (GCA) in normal factor IX, to one for Glu (GAA) in a patient with moderately severe hemophilia B (factor IXChongquing3). DNA sequences of amplified fragments from mothers of three showed both their son's variant and a normal nucleotide at the appropriate position, indicating that they are carriers. The fourth patient's (factor IXMalmo3) mother, whose DNA was not evaluable, was most probably a carrier because of her low plasma factor IX levels.

Amino Acid Sequence↗
PubMed1993

An alternate nonisotopic technique of PCR-mediated allele-specific oligonucleotide analysis for the detection of a point mutation.

We report a simple protocol for the allele analysis of a point mutation using polymerase chain reaction. Conditions have been determined to use probes prepared for the conventional allele-specific oligonucleotide hybridization method directly. After amplification of the genomic region containing the mutation site, the nested polymerase chain reaction was performed using an allele-specific oligonucleotide probe as one side of the primer. Specificity of the amplification was achieved by decreasing the primer concentration in a two segment thermal cycling pattern. This protocol does not require additional special primers and an allelic mutation can be determined non-isotopically.

Alleles↗
PubMed1995

Variable clinical symptoms in familial amyotrophic lateral sclerosis with a novel point mutation in the Cu/Zn superoxide dismutase gene.

We report a novel missense point mutation in exon 4 of the Cu/Zn superoxide dismutase (SOD) gene of affected members of a Japanese kindred segregating familial amyotrophic lateral sclerosis (FALS) through at least three successive generations. The mutation, which is predicted to cause the replacement of isoleucine at codon 104 by phenylalanine (I104F), is associated with a significant reduction in Cu/Zn SOD enzyme activity but results in a highly variable clinical phenotype. Age at onset varied from 6 to 55; the initial symptoms occurred in either the lower or upper extremities in different family members. The duration of the disease varied from 3 to 38 years. Two subjects, aged 59 and 34, remained asymptomatic until their death from other causes, although their offspring carrying the same mutation have already developed clinical evidence of the disease. These results suggest that FALS from this novel I104F mutation shows considerable clinical variation.

Adult↗
PubMed1992

A pituitary specific point mutation of codon 201 of the Gs alpha gene in a pituitary adenoma of a patient with multiple endocrine neoplasia (MEN) type 1.

The DNA from a pituitary adenoma of a patient with multiple endocrine neoplasia (MEN) type 1 was analyzed to detect a point mutation of the Gs alpha gene (gsp) by the PCR direct-sequencing method. The patient had galactorrhea, amenorrhea and acromegalic features. Hormonal examination revealed high serum levels of PRL and GH. The tumor was histologically diagnosed as a mixed GH cell-PRL cell adenoma in which GH and PRL were produced by different cells. Sequence analysis of the DNAs extracted from paraffin sections of pituitary, parathyroid, and pancreas tumors demonstrated the substitution of thymidine for cytidine in codon 201 of the Gs alpha gene that resulted in replacement of arginine (CGT) with cysteine (TGT) only in the pituitary adenoma, but not in the parathyroid and pancreas tumors. These results suggest that a pituitary specific point mutational activation of the Gs alpha gene may be involved in the development of the pituitary adenoma in this patient.

Adenoma↗
PubMed1997

Significance of individual point mutations, T202C and C314T, in the human Lewis (FUT3) gene for expression of Lewis antigens by the human alpha(1,3/1,4)-fucosyltransferase, Fuc-TIII.

The Lewis alpha(1,3/1,4)-fucosyltransferase, Fuc-TIII, encoded by the FUT3 gene is responsible for the final synthesis of Lea and Leb antigens. Various point mutations have been described explaining the Lewis negative phenotype, Le(a-b-), on erythrocytes and secretions. Two of these, T202C and C314T originally described in a Swedish population, have not been found as single isolated point mutations so far. To define the relative contribution of each of these two mutations to the Lewis negative phenotype, we cloned and made chimeric FUT3 constructs separating the T202C mutation responsible for the amino acid change Trp68 --> Arg, from the C314T mutation leading to the Thr105 --> Met shift. COS-7 cells were transfected and the expression of Fuc-TIII enzyme activity and the presence of Lewis antigens were determined. There was no decrease in enzyme activity nor of immunofluorescence staining on cells transfected with the construct containing the isolated C314T mutation compared with cells transfected with a wild type FUT3 allele control. No enzyme activity nor immunoreactivity for Lewis antigens was detected in FUT3 constructs containing both mutations in combination. The T202C mutation alone decreased the enzyme activity to less than 1% of the activity of the wild type FUT3 allele. These results demonstrate, that the Trp68 --> Arg substitution in human Fuc-TIII is the capital amino acid change responsible for the appearance of the Le(a-b-) phenotype on human erythrocytes in individuals homozygous for both the T202C and C314T mutations.

Alleles↗
PubMed2006

Detection and quantification of the age-related point mutation A189G in the human mitochondrial DNA.

Mutation analysis in the mitochondrial DNA (mtDNA) control region is widely used in population genetic studies as well as in forensic medicine. Among the difficulties linked to the mtDNA analysis, one can find the detection of heteroplasmy, which can be inherited or somatic. Recently, age-related point mutation A189G was described in mtDNA and shown to accumulate with age in muscles. We carried out the detection of this 189 heteroplasmic point mutation using three technologies: automated DNA sequencing, Southern blot hybridization using a digoxigenin-labeled oligonucleotide probe, and peptide nucleic acid (PNA)/real-time PCR combined method on different biological samples. Our results give additional information on the increase in mutation frequency with age in muscle tissue and revealed that the PNA/real-time PCR is a largely more sensitive method than DNA sequencing for heteroplasmy detection. These investigations could be of interest in the detection and interpretation of mtDNA heteroplasmy in anthropological and forensic studies.

Adolescent↗
PubMed2001

Rapid evolution of cis-regulatory sequences via local point mutations.

Although the evolution of protein-coding sequences within genomes is well understood, the same cannot be said of the cis-regulatory regions that control transcription. Yet, changes in gene expression are likely to constitute an important component of phenotypic evolution. We simulated the evolution of new transcription factor binding sites via local point mutations. The results indicate that new binding sites appear and become fixed within populations on microevolutionary timescales under an assumption of neutral evolution. Even combinations of two new binding sites evolve very quickly. We predict that local point mutations continually generate considerable genetic variation that is capable of altering gene expression.

Animals↗
PubMed1994

Identification of a point mutation in the cDNA of the catalytic subunit of DNA polymerase alpha from a temperature-sensitive mouse FM3A cell line.

DNA polymerase alpha is a key enzyme in eukaryotic chromosomal DNA replication. tsFT20 is a temperature-sensitive mutant cell line derived from mouse mammary carcinoma FM3A cells, and the cells contain heat-labile DNA polymerase alpha and are arrested at the S phase at the nonpermissive temperature. We isolated cDNA of the catalytic subunit of DNA polymerase alpha from tsFT20 cells. DNA sequence analysis revealed that the cDNA from tsFT20 has a single mutation, a cytosine to thymine substitution that changes amino acid 1180 from serine to phenylalanine. We have also shown that tsFT20 cells could be rescued by transfection with the wild-type cDNA. These results demonstrate that the point mutation in the gene of DNA polymerase alpha causes the temperature-sensitive phenotype of tsFT20 cells and provide additional evidence that DNA polymerase alpha is essential for chromosomal replication in mammalian cells. We also detected mutation sites in one spontaneous and six N-methyl-N'-nitro-N-nitrosoguanidine-induced growth revertants of tsFT20 cells by single strand conformation polymorphism analyses and direct sequencing. All revertant cell lines had a second point mutation adjacent to the first mutation site in tsFT20 cells.

Amino Acid Sequence↗
PubMed2000

X inactivation phenotype in carriers of Pelizaeus-Merzbacher disease: skewed in carriers of a duplication and random in carriers of point mutations.

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive disease caused by coding sequence mutations in the PLP gene, sub-microscopic duplications of variable sizes including the PLP gene or very rarely deletions of the PLP gene. We analysed the X inactivation pattern in blood of PMD female carriers with duplications and with point mutations. In the majority of duplication carriers (7/11), the X chromosome bearing the duplication was preferentially inactivated, whereas a random pattern of X inactivation was detected in point mutation carriers (3/3), a deletion carrier (1/1), affected females (4/4) who did not have a recognised mutation and normal control females. However 2/5 non-carrier female relatives of patients with a duplication, had skewed X inactivation. The skewed pattern of inactivation observed in most duplication carriers and not in mutation carriers suggests a) that there is selection against those cells in which the duplicated X chromosome is active and b) other expressed sequences within the duplicated region rather than mutant PLP may be responsible. Since the skewed X inactivation did not segregate with the disease in two families and the pattern of X inactivation was variable among the duplication carriers, the pattern X inactivation is an unsuitable diagnostic tool for female carriers of PMD.

DNA-Binding Proteins↗
PubMed1997

Occurrence of repeat induced point mutation in long segmental duplications of Neurospora.

Previous studies of repeat induced point mutation (RIP) have typically involved gene-size duplications resulting from insertion of transforming DNA at ectopic chromosomal positions. To ascertain whether genes in larger duplications are subject to RIP, progeny were examined from crosses heterozygous for long segmental duplications obtained using insertional or quasiterminal translocations. Of 17 distinct mutations from crossing 11 different duplications, 13 mapped within the segment that was duplicated in the parent, one was closely linked, and three were unlinked. Half of the mutations in duplicated segments were at previously unknown loci. The mutations were recessive and were expressed both in haploid and in duplication progeny from Duplication x Normal, suggesting that both copies of the wild-type gene had undergone RIP. Seven transition mutations characteristic of RIP were found in 395 base pairs (bp) examined in one ro-11 allele from these crosses and three were found in approximately 750 bp of another. A single chain-terminating C to T mutation was found in 800 bp of arg-6. RIP is thus responsible. These results are consistent with the idea that the impaired fertility that is characteristic of segmental duplications is due to inactivation by RIP of genes needed for progression through the sexual cycle.

Crosses, Genetic↗
PubMed1995

High incidence of point mutation in K-ras codon 12 in carcinoma of the fallopian tube.

BACKGROUND: Adenocarcinoma of the fallopian tube is a rare tumor with a poor prognosis. Whether these carcinomas possess any genetic changes that contribute to their malignant behavior is unknown and to date few studies regarding the molecular pathogenesis of these tumors have been reported. In adenocarcinoma of the endometrium, mutations in the first exon of K-ras, although relatively infrequent, were observed to be an independent risk factor for poor clinical outcome. METHODS: Eight patients with adenocarcinoma of the fallopian tube were examined for mutations in the 12th codon of K-ras. DNA was obtained from single sections of paraffin embedded tumor tissue and the first exon of K-ras was amplified by the polymerase chain reaction. Point mutations were assayed using a nonradioactive restriction fragment length polymorphism technique. RESULTS: The eight patients in this study varied in clinical stage from I-IV and were all treated with surgery and chemotherapy. Six of eight of the patients died and one of the surviving patients had metastases in the vertebrae. K-ras point mutations were detected at codon 12 in seven of the eight tumors (87.5%). CONCLUSIONS: K-ras mutations occurred with high frequency in this series of eight patients with fallopian tube carcinoma, suggesting that mutations of this protooncogene could play an important role in the molecular pathogenesis of this lesion.

Adenocarcinoma↗
PubMed1999

Identification of LMX1B gene point mutations in italian patients affected with Nail-Patella syndrome.

Nail-Patella syndrome, or osteo-onychodysplasia, is an autosomal dominant disorder characterized by nail dysplasia, absent or hypoplastic patellae, iliac horns and nephropathy. Previous studies have demonstrated linkage of the Nail-Patella locus with polymorphic markers on human chromosome 9q34. Recently, point mutations in the LMX1B gene have been identified in Nail-Patella patients and in families with recurrence of Nail-Patella syndrome and open-angle glaucoma. We describe here the identification of additional point mutations in the LMX1B gene in a set of Italian patients affected with Nail-Patella syndrome: two deletions of 1 and 2 bp causing a frameshift in two sporadic patients and nonsense mutations in two familial and one sporadic cases have been identified. All the mutations affect the homeodomain of the LMX1B protein and could cause the Nail-Patella syndrome through a loss of function as well as a dominant negative effect. Haplotype analysis in the two familial cases carrying the same stop codon mutation suggests the presence of a founder effect. Finally, analysis of cDNA clones obtained from human fetal kidney has revealed the existence of two different transcripts of LMX1B gene likely due to an alternative splicing.

Alternative Splicing↗
PubMed1995

Six different point mutations in seven Danish families with symptomatic protein C deficiency.

Six different point mutations of the protein C gene are described in seven Danish families with protein C deficiency associated with an increased risk of venous thromboembolism. All affected family members are heterozygotes for the mutated protein C genotype. One mutation is a G2992-->A transition at position +5 in the 5' splice site of intron D. The other five mutations affect the protein coding region. One is a C1432-->T transition in exon III converting the highly conserved Arg15 to Trp in the Gla-domain. Another mutation is a G3157-->C transversion in exon V converting the non-conserved Gly72 to Arg in the epidermal growth factor domain. The remaining three mutations are located in non-conserved amino acid positions in exon IX and affect the serine proteinase domain. The first is a G8559-->C transversion converting Gly282 to Arg. The second is a C8571-->T transition (present in two families) converting Arg286 to Cys. The third is a C8695-->T transition converting Pro327 to Leu. In each family the protein C deficiency cosegregates or probably cosegregates (one family, G8559-->C) with the mutation. All affected family members exhibit a reduction of both the antigen and the functional plasma concentration of protein C to approximately 50% of normal indicating that the mutated protein C is not present (type 1 deficiency) or only present in low amounts in plasma. Agarose gel electrophoresis followed by Western blotting shows that the Arg15-->Trp substitution is associated with a normal as well as an abnormal migrating plasma protein C band.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed2006

Prothrombin Saint-Denis: a natural variant with a point mutation resulting in Asp to Glu substitution at position 552 in prothrombin.

A new prothrombin variant, with a point mutation at nucleotide 20 029 resulting in Asp 552 to Glu substitution (prothrombin numbering), has been identified in a male newborn. Plasma prothrombin level was <3%, 16% and 60% when measured by clotting, chromogenic and immunological assays respectively. The substitution did not affect the rate of prothrombin conversion to thrombin but altered thrombin activity. Amino acid 552 has been reported to be involved in the allosteric transition, which is induced by sodium binding to thrombin. This is the first known amino acid substitution at this site to result in dysprothrombinaemia.

Base Sequence↗
PubMed1994

[Point mutation of T3 receptor-beta gene and syndrome of inappropriate secretion of TSH].

Thyroid hormone receptors (TRs) are bound to the T3-responsive element of DNA as dimers and heterodimerization with TR-auxiliary protein (TRAP) strongly enhances this binding. In affected members of families with the thyroid hormone resistance syndrome, significant point mutations have been identified in the hormone-binding domain of one of the two alleles of the TR beta gene, especially in two "hot spot" regions, avoiding the regions required for DNA binding, dimerization or binding to TRAP. These findings suggested that DNA-binding and receptor dimerization are important for dominant negative inhibition by the mutant TR, which is responsible for thyroid hormone resistance in a heterozygous patient with a point mutation in the TR beta gene.

DNA↗
PubMed1993

Variation in K-ras codon 12 point mutation rate with histological atypia within individual colorectal tumors.

To elucidate genetic alteration in relation to morphology and also to confirm more directly the proposed adenoma-carcinoma sequence, we analyzed thirty-eight colorectal "cancer in adenoma" lesions exhibiting areas of different atypia, in terms of K-ras codon 12 point mutation. The mutation incidence was 26.3% (10/38) for all cancerous areas. Well-differentiated and very well-differentiated carcinoma exhibited values of 17.6% (3/17) and 30.4% (7/23), respectively (statistically not significant). Positive cases of adenoma with severe atypia and adenoma with moderate or slight atypia were 26.7% (8/30) and 8.3% (3/36) respectively (statistically significant). Thus, K-ras point mutation, as indicated previously, may play an important role in the early stages of colorectal tumorigenesis. As for the nature of the mutation, GGT(Gly) to GAT(Asp) was the most frequent (80%). Eight cases had mutations concurrently in different areas of the same tumor and in all of these the mutation was homogeneous (6 cases to GAT, 1 case to TGT and 1 case to GTT). This provides genetic support for the "adenoma-carcinoma sequence" theory proposed on the basis of morphological considerations. All lesions with a mutation were of polypoid type, and no mutation was found in the flat type.

Adenocarcinoma↗