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PubMed2002

Three novel point mutations causing haemophilia A.

Haemophilia A is an X-linked bleeding disorder caused by mutations in the factor VIII gene. In our efforts to elucidate molecular defects in the haemophilia A patients from the Republic of Macedonia, we employed nonradioactive single-strand conformation polymorphism analysis followed by direct sequencing, for identifying point mutations in the factor VIII gene. In the present study we report the detection of three novel missense mutations: Met19 --> Arg; Ala78 --> Pro and Cys2174 --> Gly, all causing haemophilia A.

Adolescent↗
PubMed1996

A novel point mutation in the McLeod syndrome gene in neuroacanthocytosis.

McLeod syndrome is an X-linked recessive disorder, characterized by neuromuscular and hematopoietic dysfunction. Two cases of McLeod syndrome were reported in a family with neuroacanthocytosis and, remarkably, 1 of them was female. Direct sequence analysis of the McLeod gene in 12 members of the family revealed a novel point mutation in exon 2 that creates a frameshift and results in premature termination of translation. There was marked skewing of X inactivation in the severely affected female.

Acanthocytes↗
PubMed2001

[SSCP (single strand conformation polymorphism) analysis for detection of point mutations. A technique and its limits exemplified by dominantly inherited forms of diabetes (MODY)].

BACKGROUND: Molecular biological techniques of mutation detection become more and more a focus of interest in clinical medicine because the responsible genes are now known for an increasing number of diseases and the detection is essential to enable reliable predictions and to design individual therapies. Therefore the SSCP (Single Strand Conformation Polymorphism) analysis is one of the most commonly applied methods in detection of point mutations. SSCP ANALYSIS: The use of SSCP for detection of the pathogenic mutations of MODY (Maturity Onset Diabetes in the Young) is described as an example. Handling, efficiency and costs are critically analyzed. In a blind test, this method was only partly suitable for reliable analysis. Particularly diseases without a distinct hotspot and with a widespread distribution pattern of mutations throughout the coding sequence require sequencing as the gold standard.

DNA Mutational Analysis↗
PubMed1996

Point mutations abolish 11 beta-hydroxysteroid dehydrogenase type II activity in three families with the congenital syndrome of apparent mineralocorticoid excess.

The 11 beta-hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) converts cortisol into mineralocorticoid receptor inactive cortisone, thus preventing occupation of the non-selective mineralocorticoid receptor by glucocorticoids in the kidney. Mutations generating inactive enzymes have been described in the HSD11B2 gene in the congenital syndrome of apparent mineralocorticoid excess (AME), although proof of mutant protein synthesis was not provided. In the present study we have examined the metabolism of cortisol in mammalian cells transfected with plasmids expressing the wild type and mutant enzymes from three additional families of patients with mutations in the HSD11B2 gene. These studies revealed that the mutants were enzymatically inactive in intact mammalian cells expressing significant levels of both full length and truncated proteins. This is the first study to definitively show that point mutations in the HSD11B2 gene abolish 11 beta HSD2 enzymatic activity in the syndrome of AME.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

Heteroplasmic point mutations in the human mtDNA control region.

As part of an investigation of the fixation mechanisms of mtDNA mutations in humans, we sequenced the first hypervariable segment of the control region in 180 twin pairs and found evidence of site heteroplasmy in 4 pairs. Significant levels of two mitochondrial haplotypes differing by a single point mutation were found in two MZ pairs, and within each pair, both members had similar levels of heteroplasmy. Two DZ pairs were found in which the predominant mitochondrial haplotype differed within the pair. We measured proportions of mitochondrial haplotypes within two twin pairs and their maternal relatives, using primer extension. In both maternal lineages, most family members were heteroplasmic, and the proportions of each genotype varied widely in different individuals. We used the changes in haplotype proportions within mother-offspring pairs to calculate the size range of potential bottlenecks in mitochondrial numbers occurring during development of the offspring. In most individuals, the most likely effective bottleneck sizes ranged from 3 to 20 segregating units, though in two individuals a small bottleneck was very unlikely and there was no upper limit on its possible size. We also used the data from this study, together with unpublished data from other populations, to estimate the frequency of site heteroplasmy in normal human populations. From this, we calculated that the rate of mutation and fixation in the first hypervariable segment of the human mtDNA control region is between 1.2 x 10(-6) and 2.7 x 10(-5) per site per generation. This range is in good agreement with published estimates calculated by other methods.

DNA, Mitochondrial↗
PubMed1999

Generated single point-mutations can considerably dismantle the lymphocyte overstimulation induced by Yersinia pseudotuberculosis superantigen.

The superantigenic Yersinia pseudotuberculosis-derived mitogen (YPM) may contribute to severe complications in Y. pseudotuberculosis infections. Since the pathogenic mechanism of a superantigen (SAg) is based on its capability for T-cell overstimulation, by introducing point mutations into YPM an attempt was made to abrogate this effect. Six mutants studied exhibited a variety of T-cell proliferating responses. Two had activity reduced by 80-90%, three had activity reduced by approximately 50%, and one mutant showed almost no attenuation. The SAg-associated in vitro pathogenic functions, cytotoxic activation and the production of proinflammatory cytokines, were also diminished, in parallel. Since these mutants were confirmed to be defective in TCR Vbeta binding, it was possible to compare them with native YPM. Our results suggested that the intensity of TCR Vbeta binding is a crucial factor determining the severity of pathogenesis and that single amino acid alterations might be useful for producing immunotherapeautical agents from native YPM.

Animals↗
PubMed2003

Molecular pathology of haemophilia B in Turkish patients: identification of a large deletion and 33 independent point mutations.

Heterogeneous mutations in the coagulation factor IX (FIX) gene result in a bleeding tendency known as haemophilia B. The haemophilia B mutation database has a total of 2353 patient entries, including 10 of the estimated 1000 Turkish patients. In this study, a more comprehensive analysis of the molecular pathology of haemophilia B in Turkey revealed one large deletion and 33 point mutations in the FIX gene of 34 unrelated patients. Haplotype analysis using six polymorphic sites showed that the mutations identified in a total of 45 patients occurred on 13 different haplotypes and that each mutation was family specific.

Base Sequence↗
PubMed1999

Identification of a point mutation in the para-type sodium channel gene from a pyrethroid-resistant cattle tick.

To investigate the molecular mechanism of resistance to pyrethroids in the southern cattle tick, Boophilus microplus, we have obtained and sequenced a partial para-homologous sodium channel cDNA from susceptible and pyrethroid-resistant tick strains. A point mutation that results in an amino acid change from Phe to Ile was identified in the highly conserved domain IIIS6 of the homologous sodium channel from ticks that are highly resistant to pyrethroid acaricides. This mutation is at a location different from those reported in the same gene in pyrethroid-resistant insects.

Amino Acid Sequence↗
PubMed2006

Consequences of point mutations in trout anion exchanger 1 (tAE1) transmembrane domains: evidence that tAE1 can behave as a chloride channel.

In this study, we have shown that, when expressed in Xenopus oocytes, trout anion exchanger 1 (tAE1) was able to act as a bifunctional protein, either an anion exchanger or a chloride conductance. Point mutations of tAE1 were carried out and their effect on Cl- conductance and Cl- unidirectional flux were studied. We have shown that mutations made in transmembrane domain 7 had dramatic effects on tAE1 function. Indeed, when these residues were mutated, either individually or together (mutants E632K, D633G, and ED/KG), Cl- conductance was reduced to 28-44% that of wild-type tAE1. Moreover, ion substitution experiments showed that anion selectivity was altered. However, the exchanger function was unchanged, as evidenced by the fact that Cl- influx and K(m) were identical for each of these mutants and similar to the wild-type protein parameters. By contrast, mutations made in the C-terminal domains of the protein (R819M, Q829K) affected both transport functions. Cl- conductance was increased by approximately 200% with respect to tAE1 and anion selectivity was impaired. Likewise, Cl- influx was increased by approximately 260% and was no longer saturable. These and other mutations carried out in transmembrane domains 7, 8, 12-14 of tAE1 allow us to demonstrate without doubt that, in addition to its anion exchanger activity, tAE1 can also function as a chloride channel. Above all, this work led us to identify amino acids involved in this double function organization.

Amino Acid Sequence↗
PubMed1999

Aging-dependent large accumulation of point mutations in the human mtDNA control region for replication.

Progressive damage to mitochondrial DNA (mtDNA) during life is thought to contribute to aging processes. However, this idea has been difficult to reconcile with the small fraction of mtDNA so far found to be altered. Here, examination of mtDNA revealed high copy point mutations at specific positions in the control region for replication of human fibroblast mtDNA from normal old, but not young, individuals. Furthermore, in longitudinal studies, one or more mutations appeared in an individual only at an advanced age. Some mutations appeared in more than one individual. Most strikingly, a T414G transversion was found, in a generally high proportion (up to 50 percent) of mtDNA molecules, in 8 of 14 individuals above 65 years of age (57 percent) but was absent in 13 younger individuals.

Adolescent↗
PubMed2000

Resistance to multiple fluoroquinolones in a clinical isolate of Streptococcus pyogenes: identification of gyrA and parC and specification of point mutations associated with resistance.

A strain of Streptococcus pyogenes resistant to multiple fluoroquinolones was isolated from the blood of an immunocompromised patient. Resistance to fluoroquinolones in S. pyogenes has not been previously studied. Compared to 10 sensitive strains of S. pyogenes, the fluoroquinolone-resistant clinical isolate of S. pyogenes presented point mutations in gyrA, predicting that serine-81 was changed to phenylalanine and that methionine-99 was changed to leucine, and in parC, predicting that serine-79 was changed to tyrosine. The mechanism of fluoroquinolone resistance in this isolate of S. pyogenes appears to be analogous to previously reported mechanisms for Streptococcus pneumoniae.

Anti-Infective Agents↗
PubMed1999

An azoospermic man with a de novo point mutation in the Y-chromosomal gene USP9Y.

In humans, deletion of any one of three Y-chromosomal regions- AZFa, AZFb or AZFc-disrupts spermatogenesis, causing infertility in otherwise healthy men. Although candidate genes have been identified in all three regions, no case of spermatogenic failure has been traced to a point mutation in a Y-linked gene, or to a deletion of a single Y-linked gene. We sequenced the AZFa region of the Y chromosome and identified two functional genes previously described: USP9Y (also known as DFFRY) and DBY (refs 7,8). Screening of the two genes in 576 infertile and 96 fertile men revealed several sequence variants, most of which appear to be heritable and of little functional consequence. We found one de novo mutation in USP9Y: a 4-bp deletion in a splice-donor site, causing an exon to be skipped and protein truncation. This mutation was present in a man with nonobstructive azoospermia (that is, no sperm was detected in semen), but absent in his fertile brother, suggesting that the USP9Y mutation caused spermatogenic failure. We also identified a single-gene deletion associated with spermatogenic failure, again involving USP9Y, by re-analysing a published study.

Base Sequence↗
PubMed1992

A rapid and simple procedure for the routine detection of ras point mutations in formalin-fixed, paraffin-embedded tissues.

The use of the polymerase chain reaction (PCR) to detect specific DNA sequences in small amounts of tissues or cells has become a widespread tool in the field of molecular biology. With the better understanding of the clinical significance of oncogene activations in human tumors, the application of PCR in a routine setting is rapidly gaining importance. We have developed a rapid and simple procedure for the detection of mutated ras oncogenes in routinely fixed, paraffin-embedded tissue samples. DNA is isolated from three 10 microns tissue sections by incubation with a nonionic detergent and proteinase K, and can be directly used for amplification by PCR. The amplified DNA fragments are then dot-blotted onto nylon membranes and are hybridized to radioactively labeled oligodeoxynucleotides, specific for each of the mutated ras sequences. After a selective washing procedure, only fully matched oligodeoxynucleotides remain bound to the membrane, thus revealing the nature of the sequences that were present in the starting material. With this method, the detection of point mutations in ras genes can be performed in a routine setting, and the results of the analyses can be available in as few as 3-4 days.

Base Sequence↗
PubMed2001

Point mutations in the post-M2 region of human alpha-ENaC regulate cation selectivity.

We tested the hypothesis that an arginine-rich region immediately following the second transmembrane domain may constitute part of the inner mouth of the epithelial Na+ channel (ENaC) pore and, hence, influence conduction and/or selectivity properties of the channel by expressing double point mutants in Xenopus oocytes. Double point mutations of arginines in this post-M2 region of the human alpha-ENaC (alpha-hENaC) led to a decrease and increase in the macroscopic conductance of alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, respectively, but had no effect on the single-channel conductance of either double point mutant. However, the apparent equilibrium dissociation constant for Na+ was decreased for both alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, and the maximum amiloride-sensitive Na+ current was decreased for alphaR586E,R587Ebetagamma-hENaC and increased for alphaR589E,R591Ebetagamma-hENaC. The relative permeabilities of Li+ and K+ vs. Na+ were increased 11.25- to 27.57-fold for alphaR586E,R587Ebetagamma-hENaC compared with wild type. The relative ion permeability of these double mutants and wild-type ENaC was inversely related to the crystal diameter of the permeant ions. Thus the region of positive charge is important for the ion permeation properties of the channel and may form part of the pore itself.

Animals↗
PubMed1995

Familial amyotrophic lateral sclerosis with a point mutation of SOD-1: intrafamilial heterogeneity of disease duration associated with neurofibrillary tangles.

Mutations of SOD-1 have recently been associated with autosomal dominant familial amyotrophic lateral sclerosis (ALS). A patient is described with a 20 year duration of motor neuron disease, with clinical features of ALS, who was heterozygous for a point mutation ATT to ACT leading to substitution of isoleucine for threonine at codon 113 in exon 4 of SOD-1. This mutation has previously been described in two families with ALS and three apparently sporadic cases of ALS. The patient described here had a family history suggestive of autosomal dominant inheritance of this genetic mutation; other members of the family having a more typical disease duration. Unusual pathological features included neurofibrillary tangles in neurons of the globus pallidus, substantia nigra, locus coeruleus, and inferior olivary nuclei, and absence of ubiquitin immunoreactive inclusions in motor neurons. This may reflect the slow progression of the neurodegeneration associated with the SOD-1 mutation in this patient. The prolonged survival, of over 20 years, with other family members having a more typical survival of two to three years, has important implications for genetic counselling in families with ALS in addition to the fundamental biological questions concerning the influence of these mutations on disease expression.

Amyotrophic Lateral Sclerosis↗
PubMed1993

Paris I dysfibrinogenemia: a point mutation in intron 8 results in insertion of a 15 amino acid sequence in the fibrinogen gamma-chain.

Paris I dysfibrinogenemia results in the production of a fibrinogen molecule containing a functionally abnormal gamma-chain. We determined the basis of the molecular defect using polymerase chain reaction (PCR) to amplify the gamma-chain region of the Paris I subject's genomic DNA. Comparative sequence analysis of cloned PCR segments of normal and Paris I genomic DNA revealed only an A-->G point mutation occurring at nucleotide position 6588 within intron 8 of the Paris I gamma-chain gene. We examined six normal individuals and found only normal sequence in this region, indicating that this change is not likely to represent a normal polymorphism. This nucleotide change leads to a 45 bp fragment being inserted between exons 8 and 9 in the mature gamma Paris I chain mRNA, and encodes a 15 amino acid insert after gamma 350 [M-C-G-E-A-L-P-M-L-K-D-P-C-Y]. Alternative splicing of this region from intron 8 into the mature Paris I gamma-chain mRNA also results after translation into a substitution of S for G at position gamma 351. Biochemical studies of 14C-iodoacetamide incorporation into disulfide-reduced Paris I and normal fibrinogen corroborated the molecular biologic predictions that two additional cysteine residues exist within the gamma Paris I chain. We conclude that the insertion of this amino acid sequence leads to a conformationally-altered, and dysfunctional gamma-chain in Paris I fibrinogen.

Afibrinogenemia↗
PubMed1996

A point mutation in the catalytic domain of c-kit induces growth factor independence, tumorigenicity, and differentiation of mast cells.

The murine W and Steel loci encode the Kit receptor tyrosine kinase and its ligand, Steel factor, respectively. Loss of function mutations at either the W or Sl loci lead to a variety of pleiotropic developmental defects, including mast cell deficiency and severe macrocytic anemia. In addition to these loss-of-function mutations, gain-of-function mutations in c-kit, leading to constitutive activation of the Kit receptor, have also been identified in both rodent and human mastocytomas. In this study, we have examined the transforming potential and biologic effects of a point mutation that results in substitution of the aspartic acid at codon 814 in the cytoplasmic kinase domain to tyrosine (D814Y) by introducing either wild-type (Kit) or mutant KitD814Y (KDY) cDNA into an interleukin-3-dependent mast cell line IC2. Stimulation of cells expressing the wild-type Kit receptor (IC2/Kit) with Steel factor in vitro resulted in a short-term growth response, whereas IC2/KDY cells were capable of sustained proliferation in a ligand-independent manner. In addition, expression of KDY resulted in the oncogenic transformation of IC2 cells, as determined by colony formation in vitro in the absence of exogenous growth factors and the formation of mastocytomas in vivo in syngeneic DBA/2 mice. Surprisingly, KDY expression in IC2 cells triggered dramatic changes in cell size and the extent of granulation. In addition, KDY induced the expression of mouse mast cell protease-4 (MMCP-4) and MMCP-6. In contrast, neither of these molecular or cellular changes was observed in IC2/Kit cells treated with Steel factor. These results show that the D814Y mutation in the cytoplasmic kinase domain of the Kit receptor induces ligand-independent mast cell growth in vitro, tumorigenicity in vivo, and mast cell differentiation.

Animals↗
PubMed2001

[A point mutation at Arg169 (CGG-->TGG) in hereditary protein C deficiency].

We investigated a 56-year-old Japanese man with protein C deficiency, who was referred to our hospital because of venous sinus thrombosis and pulmonary thromboembolism. Protein C (PC) activity and the corresponding antigen level in plasma were 66% and 106% of the normal values, respectively. Both the activity and antigen levels of protein C were reduced by approximately 50% in plasma from the patient's mother. All exons and their flanking intron regions were amplified by PCR from genomic DNA. Sequencing analysis of the PCR fragments revealed that the patient was heterozygous for a C to T substitution at nucleotide position 6218, resulting in a single amino acid substitution of arginine (CGG) by tryptophan (TGG) at codon 169 of the heavy chain. We analyzed the patient, his mother, and normal controls by a Sac II digestion study of exon 7 and found that the patient and his mother had the same C to T point mutation at base 6218. This mutation could have been responsible for the defective activation of the molecule and the resulting thrombotic disorder. The patient is now being treated with warfarin, and so far no further clinical thrombotic episode has occurred.

DNA Mutational Analysis↗