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PubMed2004

Mutational analysis of transmembrane regions 3 and 4 of SecY, a central component of protein translocase.

The SecYEG heterotrimeric membrane protein complex functions as a channel for protein translocation across the Escherichia coli cytoplasmic membrane. SecY is the central subunit of the SecYEG complex and contains 10 transmembrane segments (TM1 to TM10). Previous mutation studies suggested that TM3 and TM4 are particularly important for SecY function. To further characterize TM3 and TM4, we introduced a series of cysteine-scanning mutations into these segments. With one exception (an unstable product), all the mutant proteins complemented the cold-sensitive growth defect of the secY39 mutant. A combination of this secY mutation and the secG deletion resulted in synthetic lethality, and the TM3 and TM4 SecY cysteine substitution mutations were examined for their ability to complement this lethality. Although they were all positive for complementation, some of the complemented cells exhibited significant retardation of protein export. The substitution-sensitive residues in TM3 can be aligned to one side of the alpha-helix, and those in TM4 revealed a tendency for residues closer to the cytosolic side of the membrane to be more severely affected. Disulfide cross-linking experiments identified a specific contact point for TM3 and SecG TM2 as well as for TM4 and SecG TM1. Thus, although TM3 and TM4 do not contain any single residue that is absolutely required, they include functionally important helix surfaces and specific contact points with SecG. These results are discussed in light of the structural information available for the SecY complex.

Adenosine Triphosphatases↗
PubMed1999

Microplate array diagonal gel electrophoresis for mutation research in DNA banks.

Molecular genetic epidemiology, association and linkage studies in populations, human or other species, is now yielding powerful new insights into disease and susceptibility genes. Inter alia, the subject requires laboratory analytical methodologies focused on achieving high throughput. Here we review one suite of methodology suitable for such laboratories. Microplate array diagonal gel electrophoresis (MADGE) was invented for molecular genetic epidemiological studies. It combines direct compatibility with microplates, convenient polyacrylamide gel electrophoresis and economy of time and reagents, at minimal capital cost, and enables one user to run up to several thousand gel lanes per day for direct assay of single-base variations. Melt-MADGE combines temporal thermal ramp apparatus to achieve similar throughput for de novo mutation scanning.

Animals↗
PubMed1997

Identification of two novel missense mutations in the human OB gene.

OBJECTIVE: To clone the human OB gene and to investigate if mutations in the OB gene are related to juvenile onset obesity in Caucasians. DESIGN: Case-cohort study with mutational scanning of the OB gene in an obese cohort and in a population sample of young Caucasians. SUBJECTS: An obese cohort of 156 subjects with juvenile onset obesity and a population sample of 380 healthy young Caucasians. MEASUREMENTS: Various anthropometric and biochemical measures of obesity and insulin sensitivity and single strand conformation polymorphism scanning and nucleotide sequencing. RESULTS: Analysis of the coding region of the OB gene in the 536 participants revealed that one obese subject was heterozygous for a mutation at codon Phe17Leu and one normal weight subject was heterozygous for a mutation at codon Val110Met. The phenotypes of the carriers were not different from matched non-mutation carrying subjects. CONCLUSIONS: Mutations exist in the OB gene among obese as well as lean subjects although they are rare. However, it is unlikely that mutations in the coding region of the OB gene are a common cause of juvenile onset obesity among Caucasians.

Adult↗
PubMed1995

Theoretical basis of one-dimensional genome scanning: a direct method to identify the site of a mutation.

Genome scanning is a technique designed to uncover a net genetic difference between otherwise identical DNA samples. As such, it can be used to directly identify the site of a gene mutation, facilitating the cloning of DNA fragments from that site. Unlike other conventional positional cloning methods, one-dimensional genome scanning does not require prior knowledge of the location of the gene or mutation nor does it require closely linked markers. Rather, this method can directly identify the site of a net genomic change, such as a deletion or duplication caused by a mutation. Thus, the genome scanning method can be used in place of classic positional cloning strategies because prior positioning or mapping of the objective gene is unnecessary. By using this approach, we have identified and cloned a DNA fragment duplicated in the p(un) mutation of the mouse pink-eyed dilution locus (Brilliant et al., Science 1991, 252, 566-569). However, no other similar attempt using one-dimensional genome scanning has been reported so far, in spite of the simplicity of the procedure and its success in identifying and ultimately characterizing the pink-eyed dilution gene of the mouse. The lack of other reports of its success are perhaps not because of the practical difficulties of this method, but may be due to the false presumption that the probability for directly identifying the mutation site using genome scanning is extremely low. The theoretical probability was calculated and is presented here.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

The protein truncation test: A review.

Only changes in the DNA sequence manifesting deleterious effects at a functional level provide "disease-causing" mutations. Consequently, mutation-scanning techniques applied on a protein level would be most informative. However, because of a lack of functional knowledge and powerful methods, most currently applied techniques try to resolve mutations at the DNA level. The protein truncation test (PTT) provides a rare exception, targeting mutations that generate shortened proteins, mainly premature translation termination. PTT has several attractive characteristics, including pinpointing the site of a mutation, good sensitivity, a low false-positive rate, and, more importantly, the near-exclusive highlighting of disease-causing mutations. In addition, PTT facilitated the detection of a new mutation type, i.e., a sequence change generating a hypermutable region surfacing in the RNA. The main technical problems are related to the fact that PTT generally uses an RNA target, including the difficulties that arise from the potential differential expression and stability of the transcripts derived from the two alleles present. The PTT has hardly evolved from the method originally described, with multiplexing and N-terminal protein tagging forming the only innovating modifications. To implement high-throughput screens using PTT, major improvements of the basic procedure will be required.

Base Sequence↗
PubMed2002

High throughput mutation screening of the factor VIII gene (F8C) in hemophilia A: 37 novel mutations and genotype-phenotype correlation.

Hemophilia A (HEMA) is an X-linked bleeding disorder caused by mutations in the factor VIII gene (F8C). Molecular genetic testing for the factor VIII gene is challenging due to its large size. Here we present results of high throughput mutation scanning based on Southern blot analysis and direct sequencing of all PCR amplified coding exons and the exon-intron boundaries of the factor VIII gene. The results of mutation analysis on 89 hemophiliac males showed presence of a disease-causing mutation in 80 individuals (90%, 95% CI of 82%-95%). Seven out of nine mutation-negative individuals were severe cases of hemophilia A with < 1% factor VIII protein in the blood. The correlation of phenotype with genotype as observed in this study was not absolute. This finding is supported by similar observations in the international database for hemophilia A mutations (HAMSTeRS). This issue raises the importance of genotypes at other loci that can act as modifiers for the phenotype. Thirty-four novel mutations and three novel substitutions for previously reported amino acid residues were identified in this series of 80 mutations. The mutations cover the full spectrum including rearrangements, deletions, frameshift, and point mutations. The novel missense mutations require careful evaluation. Prediction of a mutation as the disease-causing allele was made from the nature of the substitution and the degree of conservation of the mutated amino acid among species that have diverged in evolution. In some cases segregation analysis of the mutation with disease condition was performed when other family members were available.

Child↗
PubMed2005

Mutation rates in the dystrophin gene: a hotspot of mutation at a CpG dinucleotide.

An analysis of mutations was performed in 141 Duchenne muscular dystrophy (DMD) patients previously found to be negative for large deletions by standard multiplex PCR assays. Comprehensive mutation scanning of all coding exons, adjacent intronic splice regions, and promoter sequences was performed by DOVAM-S, a robotically enhanced, high throughput method that detects essentially all point mutations. Samples negative for point mutations were further analyzed for duplications by multiplex amplifiable probe hybridization (MAPH). Presumptive causative mutations were detected in 90% of the patients (70% protein truncating point mutations, 13% duplications, and 7% deletions not detected by the standard multiplex screening method). A total of 40 of the mutations are putatively novel. Most duplications involve multiple exons with an average and median size of about 160 and 153 kb, respectively. This is the first analysis of the absolute and relative rates of point mutations in the dystrophin gene. Relative to microdeletions (0.68 x 10(-9) per bp per generation), transitions at CpG dinucleotides are enhanced 150-fold while complex indels, the least common mutation type, are less frequent than microdeletions by a factor of five. The frequency of microdeletions and microinsertions at mononucleotide repeats increases exponentially with length. When compared to the well-studied human factor IX gene (F9), the results are similar, with two exceptions: a hotspot of mutation in the dystrophin gene (c.8713C>T/p.R2905X) at a CpG dinucleotide and an altered size distribution of microdeletions. The hotspot reflects a difference in the underlying pattern of mutation, while the altered size distribution of microdeletions reflects certain abundant sequence motifs within the dystrophin coding sequence (relative to factor IX).

Adolescent↗
PubMedDate not supplied

Identification of new mutations of hepcidin and hemojuvelin in patients with HFE C282Y allele.

HFE-hemochromatosis is the most common form of hereditary hemochromatosis. The disorder is associated with the homozygous C282Y mutation and has variable phenotype, being modulated by environmental and genetic factors. Candidate modifier genes are hemojuvelin and hepcidin, which are responsible for juvenile hemochromatosis. We used DHPLC to scan mutations in these genes in a cohort of unrelated patients with C282Y mutation. They consisted of 136 C282Y homozygous, 43 heterozygous, and 42 C282Y/H63D compound heterozygous, plus 62 controls subjects. Mutations and polymorphisms were found in 16 patients and 4 controls. Abnormally high indices of iron status were found in subjects C282Y/H63D heterozygous for the N196K hemojuvelin mutation and the -72C > T hepcidin substitution. The already described G71D mutation of hepcidin did not induce evident modification of the C282Y/H63D phenotype. The data show that heterozygous mutations of the hemojuvelin gene contribute like those of hepcidin to the phenotypic heterogeneity of hemochromatosis. However, they are rare and explain only a minor portion of the variable penetrance of the disorder.

Alleles↗
PubMed2004

RNA analysis reveals splicing mutations and loss of expression defects in MLH1 and BRCA1.

The classical paradigm of mutation screening seeks to relate alterations in DNA sequence to their effect at the protein level. However, the majority of missense mutations are problematic as their pathological significance is often unclear. In order to test the hypothesis that many missense mutations primarily cause defects at the RNA rather than the protein level, we have performed retrospective RNA analysis of 12 individuals carrying missense mutations in the cancer predisposition genes APC, BRCA1, BRCA2, MLH1, and MSH2. RNA was extracted from peripheral blood samples and RT-PCR performed in order to assess the splicing and expression of the mutant allele in each case. Four of the 12 missense mutations analysed were associated with RNA defects. We detected two cases of exon skipping and one case of partial intron inclusion with activation of a cryptic intronic splice site in MLH1. A fourth case was associated with monoallelic expression of BRCA1. In addition, allele-specific analysis of common coding polymorphisms identified a further case of monoallelic BRCA1 expression in one of two individuals who had previously screened as mutation-negative. Although we were unable to identify the underlying cause of this loss of expression, it strongly suggests the presence of a pathogenic defect in BRCA1 in this case, highlighting the use of allelic expression studies as a method of mutation scanning. Finally, we used our dataset to test the ability of several in silico sequence analysis tools to identify splicing defects. Our results suggest that a significant number of missense mutations in cancer predisposition genes are associated with defects of RNA splicing, and that the use of gene- and splice site prediction software can aid in identifying such mutations.

Adaptor Proteins, Signal Transducing↗
PubMed2007

A common polymorphism in SCN5A is associated with lone atrial fibrillation.

The cardiac sodium channel (SCN5A) is a target for the treatment of arrhythmias. We hypothesized that vulnerability to atrial fibrillation (AF) could be caused by genetic variation in SCN5A. We recruited 157 patients with early-onset AF who lacked traditional risk factors, and 314 matched controls. SCN5A was subject to targeted genotyping of a common loss-of-function H558R polymorphism and comprehensive mutation scanning. Genotype frequencies in the AF cohort vs controls were as follows: HH, 50 vs 63%; HR, 40 vs 33%; and RR, 10 vs 4% (P=0.008). Additional coding sequence mutations were ruled out. The R558 allele was more common in patients than in controls (30 vs 21%, P=0.002), conferring an odds ratios for AF of 1.6 (95% confidence interval 1.2-2.2). The SCN5A R558 allele, present in one-third of the population, thus constitutes a risk factor for lone AF and may increase susceptibility to sodium channel blocker-induced proarrhythmia.

Atrial Fibrillation↗
PubMed2001

Linkage of the human inducible nitric oxide synthase gene to type 1 diabetes.

Exposure of human pancreatic islets to a mixture of cytokines induces expression of the inducible nitric oxide synthase (iNOS), impairs beta-cell function, and induces apoptosis. We performed a mutational scanning of all 27 exons of the human NOS2 gene and linkage transmission disequilibrium testing of identified NOS2 polymorphisms in a Danish nationwide type 1 diabetes mellitus (IDDM) family collection. Mutational screening was performed using PCR-amplified exons, followed by single stranded conformation polymorphism and verification of potential polymorphisms by sequencing. The transmission disequilibrium test was performed in an IDDM family material comprising 257 Danish families; 154 families were affected sibling pair families, and 103 families were simplex families. In total, 10 polymorphisms were identified in 8 exons, of which 4 were tested in the family material. A C/T single nucleotide polymorphism in exon 16 resulting in an amino acid substitution, Ser(608)Leu, showed linkage to IDDM in human leukocyte antigen DR3/4-positive affected offspring (P = 0.008; corrected P = 0.024). No other distorted transmission patterns were found for any other tested single nucleotide polymorphism or constructed haplotypes with the exception of those including data from exon 16. In conclusion, linkage of the human NOS2 gene to IDDM in a subset of patients supports a pathogenic role of nitric oxide in human IDDM.

Diabetes Mellitus, Type 1↗
PubMed1988

Mutational analysis of upstream activation sequence 2 of the CYC1 gene of Saccharomyces cerevisiae: a HAP2-HAP3-responsive site.

We analyzed upstream activation sequence 2 (UAS2), one of two independent UAS elements in the CYC1 gene of Saccharomyces cerevisiae. Deletions and linker scanning mutations across the 87 base pairs previously defined as UAS2 showed two separate functional elements required for full activity. Region 1, from -230 to -200, contains the principal activation site and responds to the trans-acting regulatory loci HAP2 and HAP3. A portion of region 1 is homologous to two other HAP2-HAP3-responsive UASs and includes the G----A transition mutation UP1, which increases UAS2 activity. This consensus sequence TNATTGGT bears striking similarity to several CAAT box sequences of higher cells. Region 2, from -192 to -178, substantially enhances the activity of region 1, yet has little activity by itself. These regions bind distinct proteins found in crudely fractionated yeast extracts.

Base Sequence↗
PubMed1997

Detection of somatic mutations in the mitochondrial DNA control region of colorectal and gastric tumors by heteroduplex and single-strand conformation analysis.

Each entire hypervariable region of the mitochondrial DNA control region was screened for mutations from paired normal and tumor DNA corresponding to a group of 21 patients (13 colorectal and 8 gastric adenocarcinomas) using both heteroduplex analysis and single-strand conformation analysis. These two mutation scanning strategies allowed the identification of sequence alterations in 3/13 (23%) colorectal tumors and in 3/8 (37%) gastric tumors. Heteroduplex analysis showed the heteroplasmic state of the majority of these tumor mutations. Sequence analysis revealed two A:T/G:C transitions (nucleotide positions: 16241 and 16166) in hypervariable region 1 (HV1) and two C:G/T:A transitions (nucleotide positions: 76 and 312), one A:T/G:C transition (nucleotide position: 93), a 1-basepair C:G deletion (nucleotide position: 309), and a 2-base-pair CC:GG insertion (nucleotide position: 309) in the HV 2 region. A considerable proportion of these mutations was found in homopolymeric regions which are highly polymorphic among humans. Different mechanisms (clonal expansion, increased oxidative damage, and nuclear mutator mutations) were suggested to explain the increased mitochondrial DNA mutation rate observed in cancer.

Adenocarcinoma↗
PubMed2003

Mutational analysis of the ATRX gene by DGGE: a powerful diagnostic approach for the ATRX syndrome.

Molecular defects affecting the ATRX gene lead to the ATRX syndrome (alpha thalassemia/mental retardation syndrome, X-linked), characterized by severe mental retardation, microcephaly, distinct facial dysmorphism, and genital abnormalities, as well as a wide spectrum of other pathological features. Alpha thalassemia is frequent but does not represent a constant characteristic of the syndrome. An expanding phenotype of the ATRX gene (a RAD54 homologue encoding a putative zinc-finger helicase) has been demonstrated as a result of the association of single mutations with specific X-linked mental retardation syndromes. To date, mutational analysis of the gene has been based on direct DNA sequencing or using methods with a lower detection rate. In this paper, we present a broad-range DGGE method for single-step mutation scanning of the entire open reading frame (ORF) and canonical splice sites of the gene. Using this method, we successfully identified five novel sequence changes in the ATRX gene, including four missense mutations (K1733E, R2085C, D2136N, T2169A) and one polymorphism (IVS5+35G>A).

Abnormalities, Multiple↗
PubMed1996

Human adenosine A2a receptor (A2aAR) gene: systematic mutation screening in patients with schizophrenia.

Several lines of evidence suggest an involvement of adenosine A2a receptor (A2aAR) mediated adenosinergic neuromodulation in the etiopathogenesis of schizophrenia. We therefore performed a systematic mutation scan of the complete coding region of the human A2aAR gene in a sample of 42 schizophrenic patients. We detected one rare naturally occurring receptor variant (Gly-340-Ser) and two silent mutations (405C/T and 1083C/T). To our knowledge the Gly-340-Ser substitution is the first naturally occurring molecular variant of the A2aAR identified. Determining the frequency of the three variants in 42 unrelated healthy controls, we observed a significant trend towards an overrepresentation of the 1083T variant in patients when compared to controls (p = 0.041). This trend was followed up in a large independent replication sample. However, we were not able to confirm the original trend in the second sample (p = 0.367). The Ser-340 variant was found in a single schizophrenic individual. Investigation of the patient's family revealed independent segregation between the Ser-340 variant and psychiatric illness. Our data suggest that genetically determined structural variation of the A2aAR does not play a major role in the development of schizophrenia.

Alleles↗
PubMed1994

Mutational analyses of the metal ion and substrate binding sites of phosphorylase kinase gamma subunit.

Phosphorylase kinase (PhK) and truncated gamma subunit, denoted gamma 1-300, can phosphorylate seryl and tyrosyl residues dependent on the metal ion [Yuan, C.-J., Huang, C. F., & Graves, D. J. (1993) J. Biol. Chem. 268, 17683-17686]. Recombinant gamma 1-300 was used to explore its dual specificity and the location of the metal ion binding sites by using site-directed mutagenesis. Two approaches were taken to generate 26 mutants. First, on the basis of the crystal structure of cAMP-dependent protein kinase (cAPK), the invariant Asn155 and highly conserved Asp168-Phe169-Gly170 residues were mutated. Changes included production of N155H, D168E, D168N, F169R, G170V, G170I, G170L (less than 1% of enzymatic activities were found in these mutants), F169W, and G170A mutants. Second, charge to alanine and charge reversal scanning mutations were used to probe the metal ion binding sites. Two mutants, E111K and E154R, showed very different metal ion response compared to wild-type gamma and were further characterized. The mutants F169W, G170A, E111K, and E154R had 15%, 5%, 8%, and 25% specific activity relative to wild-type gamma, respectively. The folding pattern of wild-type and mutated enzyme forms of gamma was determined by photoacoustic infrared spectroscopy. Conformational disruptions were found in G170V, G170I, and G170L mutants, but the conformation of the rest of the mutants was similar to that of wild-type gamma, suggesting that the loss of enzymatic activities of these mutants was not because of incorrect refolding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1999

DNA-binding factors assemble in a sequence-specific manner on the maize mitochondrial atpA promoter.

The maize mitochondrial atpA promoter has been well-characterized using in vitro transcription. The functional elements of this promoter comprise a central domain extending from -7 to +5 relative to the transcription start site, and an upstream domain of 1-3 bp that is purine-rich and centered around positions -11 to -12. As a first step in characterizing the transcriptional machinery, exonuclease-III mapping (toeprinting) was used to map the borders of DNA-protein interactions using either a 107-bp wild-type template or transcriptionally-inactive templates containing linker-scanning mutations. These experiments revealed that, with a wild-type promoter, protein factors occupy as much as 36 bp, from positions -20 to +16 relative to the transcription initiation site. Protein-binding patterns were altered when the linker-scanning mutants were used, suggesting that either the number or conformation of DNA-binding proteins could account for their inability to promote transcription initiation.

Base Sequence↗
PubMed2007

A versatile denaturing HPLC approach for human beta-globin gene mutation screening.

Hemoglobinopathies represent the most common genetic disorder worldwide, with a higher prevalence among populations with a history of malaria endemicity. More than 690 mutations in the human beta-globin gene are usually the cause of beta-type hemoglobinopathies. Here, we report a rapid and highly sensitive beta-globin gene mutation screening approach based on denaturing high-performance liquid chromatography (DHPLC), which contrary to the previously described ones can be used in every HPLC apparatus. The sensitivity and specificity of the method were tested in 120 healthy Greek subjects and 25 beta-thalassemia heterozygotes and homozygotes, in which 11 different beta-globin sequence variations had been previously characterized by denaturing gradient gel electrophoresis. Using this method, we were able to rapidly identify the commonest beta-globin gene mutations, accounting for more than 90% of the mutant beta-globin alleles reported for the Hellenic population. Compared to classical mutation screening approaches, our DHPLC approach provides the means for rapid, highly sensitive, cost-effective, and semi-automated simultaneous mutational scanning of a large number of samples.

Chromatography, High Pressure Liquid↗