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PubMed1999

Direct detection of common mutations in the familial Mediterranean fever gene (MEFV) using naturally occurring and primer mediated restriction fragment analysis. Mutation in brief no. 257. Online.

The MEFV gene involved in familial Mediterranean fever was recently cloned and four distinct sequence alterations (M680I, M694V, M6941 and V726A) were identified at the 3'-most exon. We genotyped 170 unrelated FMF patients from various ethnic groups in Israel and found that mutation M694V predominates in North African Jews, that mutation V726A is common in Jewish patients other than North African Jews and that all four mutations occur in patients of Arabian origin, namely, Moslems, Christians and Druze. Since these four distinct sequence alterations seem to account for the majority of mutations identified in FMF patients from the middle east, we have devised a simple protocol using PCR mediated site directed mutagenesis or naturally occurring recognition sites to scan for these mutations.

Cytoskeletal Proteins↗
PubMed1992

Detection of mutations in human DNA.

Efficient methods for the detection of mutations are of fundamental importance in research and in diagnostics. By detection of a DNA sequence alteration that cosegregates with a clinical phenotype in an affected family, the gene at fault may be identified and assigned a function. Mutation detection methods are also a rate-limiting factor for the clinical application of DNA diagnostics. Currently a large number of techniques are in use to scan for new mutations and to distinguish among previously established sequence variants. Here, some of the problems connected with mutation detection are discussed together with principles on which current and future mutation detection assays can be based.

Alleles↗
PubMed1995

Superoxide dismutase mutations in an unselected cohort of Scottish amyotrophic lateral sclerosis patients.

Mutations in the Cu/Zn superoxide dismutase (SOD1) gene are responsible for some cases of familial amyotrophic lateral sclerosis (ALS). We have shown that SOD1 mutations can also occur in apparently sporadic ALS. To establish how often this happens we have undertaken a study of the prevalence of SOD1 mutations in an unselected cohort of Scottish ALS patients, with both sporadic (n = 57) and familial (n = 10) disease. Single strand conformation polymorphism analysis was used to scan for new mutations, and selective restriction enzyme digestion to screen for 11 of the 20 SOD1 mutations published to date. We detected mutations in five (50%) of the familial ALS patients and also in four (7%) of the sporadic patients. One mutation, ile113thr, seems to be particularly prevalent in the Scottish population since it was detected in a total of 6/67 (9%) unrelated cases.

Aged↗
PubMed2003

[A rare transcription mutation (-90 C-->T) in a Chinese family with beta-thalassemia].

OBJECTIVE: To identify a rare transcription mutation (C-->T) at position -90 of the beta-globin gene previously unreported in the beta-thalassemia carriers from a Chinese family. METHODS: In phenotype analysis, standard hematological techniques were used to measure RBC counts and Hb concentration. Reverse dot blot (RDB) analysis, which can simultaneously detect 18 known types of beta-thalassemia mutations in Chinese, was used to scan beta-globin gene mutations. DNA sequence analysis of the entire human beta-globin gene was performed to characterize the underlying causative mutation of the sample and to identify its genotype. A semi-quantitative RT-PCR method was used to measure beta-globin gene expression in the form of mRNA from the subjects. RESULTS: The proband, his brother and his mother presented a typical beta-thalassemic trait with reduced mean corpuscular volume (MCV, 68.2-73.6 fL) and elevated level of Hb A(2) (5.7%-6.4%) but no known beta-thalassemia mutations were found in the samples by RDB analysis. DNA sequencing of the beta-gene region of these three samples revealed heterozygosity for the C-->T substitution at position -90 within proximal CACCC box of the beta-globin gene promoter element, which was previously unreported in the Chinese population. Analysis of mRNA from the positive carriers demonstrated that the mutant beta-globin gene significantly reduced beta-globin transcription (mutants: 2.233 +/- 0.01 vs normal: 3.779+/-1.19; 95%CI: 3.060, 4.499), showing a level comparable with that of the other beta-thalassemia heterozygotes (2.110+/-0.53, 95%CI: 1.732, 2.488). CONCLUSION: A rare transcriptional mutation that led to beta-thalassemia in Chinese population has been characterized. The findings enrich knowledge of the mutation spectrum of beta-thalassemia.

Adult↗
PubMed2004

A region in the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+.

Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2+ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile819-Glu837 of the 7TM domain represents a "hot spot" for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro823, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca2+. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu837, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile819-Glu837 plays a key role in CaR activation by Ca2+. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed.

Alanine↗
PubMed2002

Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis.

Shotgun scanning combinatorial mutagenesis was used to study the antigen-binding site of Fab2C4, a humanized monoclonal antibody fragment that binds to the extracellular domain of the human oncogene product ErbB2. Essentially all the residues in the Fab2C4 complementarity determining regions (CDRs) were alanine-scanned using phage-displayed libraries that preferentially allowed side-chains to vary as the wild-type or alanine. A separate homolog-scan was performed using libraries that allowed side-chains to vary only as the wild-type or a similar amino acid residue. Following binding selections to isolate functional clones, DNA sequencing was used to determine the wild-type/mutant ratios at each varied position, and these ratios were used to assess the contributions of each side-chain to antigen binding. The alanine-scan revealed that most of the side-chains that contribute to antigen binding are located in the heavy chain, and the Fab2C4 three-dimensional structure revealed that these residues fall into two groups. The first group consists of solvent-exposed residues which likely make energetically favorable contacts with the antigen and thus comprise the functional-binding epitope. The second group consists of buried residues with side-chains that pack against other CDR residues and apparently act as scaffolding to maintain the functional epitope in a binding-competent conformation. The homolog-scan involved subtle mutations, and as a result, only a subset of the side-chains that were intolerant to alanine substitutions were also intolerant to homologous substitutions. In particular, the 610 A2 functional epitope surface revealed by alanine-scanning shrunk to only 369 A2 when mapped with homologous substitutions, suggesting that this smaller subset of side-chains may be involved in more precise contacts with the antigen. The results validate shotgun scanning as a rapid and accurate method for determining the functional contributions of individual side-chains involved in protein-protein interactions.

Alanine↗
PubMed1994

Two-dimensional DNA electrophoresis in mutation detection.

Accurate detection of gene mutations is important in many areas of biology and medicine. In fundamental studies of mutagenesis it is often necessary to assess all possible mutations, either spontaneous or induced by genotoxic agents, in a particular gene or gene sequence to explain a given cellular or physiological endpoint. In molecular medicine comprehensive detection of all possible mutations in a disease gene is required before clinical genetic testing becomes feasible. Of the many mutation detection methods currently available none is capable of scanning for all possible mutations in a cost-effective manner. Here we show that by two-dimensional DNA electrophoretic separation, on the basis of both size and base pair sequence, in principle all mutations in a given gene can be detected. This is illustrated by some data on 2-D electrophoresis of 10 exons of the cystic fibrosis gene.

Cystic Fibrosis↗
PubMed2000

Highly selective isolation of unknown mutations in diverse DNA fragments: toward new multiplex screening in cancer.

Cancer research would greatly benefit from technologies that allow simultaneous screening of several unknown gene mutations. Lack of such methods currently hampers the large-scale detection of genetic alterations in complex DNA samples. We present a novel mismatch-capture methodology for the highly efficient isolation and amplification of mutation-containing DNA from diverse nucleic acid fragments of unknown sequence. To demonstrate the potential of this method, heteroduplexes with a single A/G mismatch are formed via cross-hybridization of mutant (T-->G) and wild-type DNA-fragment populations. Aldehydes are uniquely introduced at the position of mismatched adenines via the Escherichia coli glycosylase, MutY. Subsequent treatment with a biotinylated hydroxylamine results in highly specific and covalent biotinylation of the site of mismatch. For PCR amplification, synthetic linkers are then ligated to the DNA fragments. Biotinylated DNA is then isolated and PCR amplified. Mutation-containing DNA fragments can subsequently be sequenced to identify type and position of mutation. This method correctly detects a single T-->G transversion introduced into a 7-kb plasmid containing full-length cDNA from the p53 gene. In the presence of a high excess wild-type DNA (1:1000 mutant:normal plasmids) or in the presence of diverse DNA fragment sizes, the DNA fragments containing the mutation are readily detectable and can be isolated and amplified. The present Aldehyde-Linker-Based Ultrasensitive Mismatch Scanning has a current limit of detection of one base substitution in 7 Mb of DNA and increases the limit for unknown mutation scanning by two to three orders of magnitude. Homozygous and heterozygous p53 regions (G-->T, exon 4) from genomic DNA are also examined, and correct identification of mutations is demonstrated. This method should allow large-scale detection of genetic alterations in cancer samples without any assumption as to the genes of interest.

Aldehydes↗
PubMed2001

Double-gradient denaturing gradient gel electrophoresis assay for identification of L-ferritin iron-responsive element mutations responsible for hereditary hyperferritinemia-cataract syndrome: identification of the new mutation C14G.

BACKGROUND: Hereditary hyperferritinemia-cataract syndrome is an autosomic dominant disorder caused by heterogeneous mutations on the iron-responsive element (IRE) of ferritin L-chain mRNA. The mutations described to date were identified by direct sequencing of DNA from probands with hyperferritinemia often associated to bilateral cataracts. A direct genetic approach on a large population is useful to recognize polymorphisms in the DNA region and the prevalence of mutations associated with minor increases in serum ferritin and subclinical cataracts. We developed a rapid DNA scanning technique to detect mutations in a single electrophoretic analysis. METHODS: The double-gradient denaturing gradient gel electrophoresis (DG-DGGE) method consisted of PCR amplification of the target genomic DNA with GC-clamped oligonucleotides. The sequence encoded the 5' untranslated flanking region of ferritin L-chain mRNA, which includes an IRE stem-loop structure. The product was subjected to DG-DGGE (8.5-15% polyacrylamide and 50-95% denaturant) to separate the homo- and heteroduplexes. RESULTS: The method clearly identified all eight accessible mutations, including C-G transversions, which are the most difficult to detect. The method was applied to scan DNA samples from 50 healthy subjects and from 230 subjects with serum ferritin >400 microg/L. The new mutation G14C was identified. CONCLUSIONS: The DG-DGGE method detects all the mutations in the L-ferritin IRE sequence, is rapid and economical, and can be applied to scan large populations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized.

Base Sequence↗
PubMed2002

Comprehensive scanning of the entire mitochondrial genome for mutations.

BACKGROUND: Definitive molecular diagnosis of mitochondrial disorders has been greatly hindered by the tremendous clinical and genetic heterogeneity, the heteroplasmic condition of pathogenic mutations, and the presence of numerous homoplasmic mitochondrial DNA (mtDNA) variations with unknown significance. We used temporal temperature gradient gel electrophoresis (TTGE) to detect heteroplasmic mutations from homoplasmic variations in the whole mitochondrial genome. METHODS: We screened 179 unrelated patients by TTGE with use of 32 overlapping primer pairs. Mutations were identified by direct sequencing of the PCR products and confirmed by PCR with allele-specific oligonucleotide or restriction fragment length polymorphism analysis. RESULTS: We detected 71 heteroplasmic and 647 homoplasmic banding patterns. Sequencing of the heteroplasmic fragments identified 68 distinct novel mutations and 132 reported sequence variations and mutations; most of them occurred only once. The deleterious nature of some of the novel mutations was established by analyzing the asymptomatic family members and the biochemical and molecular characteristics of the mutation. When the number of mutations was normalized to the size of the region, the occurrence of mutations was 2.4 times more frequent in the tRNA genes than in the mRNA (protein coding) regions. CONCLUSIONS: Screening by TTGE detects low proportions of mutant mtDNA and distinguishes heteroplasmic from homoplasmic variations. Results from comprehensive molecular analysis should be followed up with clinical correlation to establish a guideline for complete mutational analysis of the entire mitochondrial genome and to facilitate the diagnosis of mitochondrial disorders.

Child↗
PubMed2004

Crystal structure of the human RORalpha Ligand binding domain in complex with cholesterol sulfate at 2.2 A.

The retinoic acid-related orphan receptor alpha (RORalpha) is an orphan member of the subfamily 1 of nuclear hormone receptors. Our recent structural and functional studies have led to the hypothesis that cholesterol or a cholesterol derivative is the natural ligand of RORalpha. We have now solved the x-ray crystal structure of the ligand binding domain of RORalpha in complex with cholesterol-3-O-sulfate following a ligand exchange experiment. In contrast to the 3-hydroxyl of cholesterol, the 3-O-sulfate group makes additional direct hydrogen bonds with three residues of the RORalpha ligand binding domain, namely NH-Gln(289), NH-Tyr(290), and NH1-Arg(370). When compared with the complex with cholesterol, seven well ordered water molecules have been displaced, and the ligand is slightly shifted toward the hydrophilic part of the ligand binding pocket, which is ideally suited for interactions with a sulfate group. These additional ligand-protein interactions result in an increased affinity of cholesterol sulfate when compared with cholesterol, as shown by mass spectrometry analysis done under native conditions and differential scanning calorimetry. Moreover, mutational studies show that the higher binding affinity of cholesterol sulfate translates into an increased transcriptional activity of RORalpha. Our findings suggest that cholesterol sulfate could play a crucial role in the regulation of RORalpha in vivo.

Animals↗
PubMed2002

Rapid automated simultaneous screening of (G1691A) Factor V, (G20210A) prothrombin, and (C677T) methylenetetrahydrofolate reductase variants by multiplex PCR using fluorescence scanning technology.

The Factor V Leiden mutation (G1691A), and mutations in the prothrombin (G20210A) and 5,10-methylenetetrahydrofolate reductase (C677T) genes are common hereditary risk factors associated with venous thrombosis. The aim of this study was to develop an automated, PCR-based genotyping assay for rapid simultaneous screening of these three mutations. We adapted multiplex PCR, using primer modifications to introduce cleavage sites for restriction endonucleases into the fragments bearing each of the mutations. The three mutations were analyzed in a single tube by fluorescence scanning. An internal digestion control was introduced to prevent false-negative results due to incomplete digestion or a total lack of digestion. DNA fragment analysis was carried out using an automated capillary electrophoresis instrument (ABI310). This reliable, efficient, easy-to-use assay can be applied to specimens from large clinical trials and epidemiological surveys.

Factor V↗
PubMed2002

Novel mutations in the BRCA1 and BRCA2 genes in Iranian women with early-onset breast cancer.

BACKGROUND: Breast cancer is the most common female malignancy and a major cause of death in middle-aged women. So far, germline mutations in the BRCA1 and BRCA2 genes in patients with early-onset breast and/or ovarian cancer have not been identified within the Iranian population. METHODS: With the collaboration of two main centres for cancer in Iran, we obtained clinical information, family history and peripheral blood from 83 women under the age of 45 with early-onset breast cancer for scanning of germline mutations in the BRCA1 and BRCA2 genes. We analysed BRCA1 exons 11 and BRCA2 exons 10 and 11 by the protein truncation test, and BRCA1 exons 2, 3, 5, 13 and 20 and BRCA2 exons 9, 17, 18 and 23 with the single-strand conformation polymorphism assay on genomic DNA amplified by polymerase chain reaction. RESULTS: Ten sequence variants were identified: five frameshifts (putative mutations - four novel); three missense changes of unknown significance and two polymorphisms, one seen commonly in both Iranian and British populations. CONCLUSIONS: Identification of these novel mutations suggests that any given population should develop a mutation database for its programme of breast cancer screening. The pattern of mutations seen in the BRCA genes seems not to differ from other populations studied. Early-onset breast cancer (less than 45 years) and a limited family history is sufficient to justify mutation screening with a detection rate of over 25% in this group, whereas sporadic early-onset breast cancer (detection rate less than 5%) is unlikely to be cost-effective.

Adult↗
PubMed2006

Determination of essential and variable residues in pediocin PA-1 by NNK scanning.

Pediocin PA-1 is an antimicrobial peptide (called bacteriocin) that shows inhibitory activity against the food-borne pathogen Listeria monocytogenes. To elucidate which residue(s) is responsible for this function, the antimicrobial activities of pediocin PA-1 mutants were evaluated and compared. Each of the 44 native codons was replaced with the NNK triplet oligonucleotide in a technique termed NNK scanning, and 35 mutations at each position were examined for antimicrobial activities using a modified colony overlay screening method. As a consequence, the functional responsibility of each residue was estimated by counting the number of active mutants, allowing us to identify candidate essential/variable residues. Activity was abrogated by many of the mutations at residues Y2, G6, C9, C14, C24, W33, G37, and C44, indicating that these residues may be essential. In contrast, activity was retained by almost all versions harboring mutations at K1, T8, G10, S13, G19, N28, and N41, indicating that these are functionally redundant residues. Sequence analysis revealed that only the wild type was active and 14 and 11 substitutions were inactive at G6 and C14, respectively, while 12 and 11 substitutions were active and 2 and 0 substitutions were inactive at T8 and K1, respectively. These findings suggest that NNK scanning is effective for determining essential and variable residues in pediocin PA-1, leading to an elucidation of structure-function relationships and to improvements in the antimicrobial function efficiently by peptide engineering.

Amino Acid Sequence↗
PubMed2004

Feline polycystic kidney disease mutation identified in PKD1.

Autosomal dominant polycystic kidney disease (ADPKD) is a commonly inherited disorder in humans that causes the formation of fluid-filled renal cysts, often leading to renal failure. PKD1 mutations cause 85% of ADPKD. Feline PKD is autosomal dominant and has clinical presentations similar to humans. PKD affects approximately 38% of Persian cats worldwide, which is approximately 6% of cats, making it the most prominent inherited feline disease. Previous analyses have shown significant linkage between the PKD phenotype and microsatellite markers linked to the feline homolog for PKD1. In this report, the feline PKD1 gene was scanned for causative mutations and a C>A transversion was identified at c.10063 (human ref NM_000296) in exon 29, resulting in a stop mutation at position 3284, which suggests a loss of approximately 25% of the C-terminus of the protein. The same mutation has not been identified in humans, although similar regions of the protein are truncated. The C>A transversion has been identified in the heterozygous state in 48 affected cats examined, including 41 Persians, a Siamese, and several other breeds that have been known to outcross with Persians. In addition, the mutation is segregating concordantly in all available PKD families. No unaffected cats have been identified with the mutation. No homozygous cats have been identified, supporting the suggestion that the mutation is embryonic lethal. These data suggest that the stop mutation causes feline PKD, providing a test to identify cats that will develop PKD and demonstrating that the domestic cat is an ideal model for human PKD.

Animals↗
PubMed2003

Ha-ras oncogene mutation associated to progression of papillomavirus induced lesions of uterine cervix.

BACKGROUND: Epidemiological and virological surveys suggest that the HPV presence is not enough condition to generate anogenital cancer, others factors (genetic, environmental, hormonal, etc) may have an important role. Mutations of ras genes were observed in several human neoplasias, including cervical cancer. OBJECTIVE: The aim of the study was to assess the frequency of Ha-ras oncogene mutations in cervical intraepithelial neoplasia (CIN) grade III and invasive squamous cell carcinomas and to examine this genetic factor in relation to HPV infection and the clinical evolution of cervical lesions. STUDY DESIGN: They were selected for (a) evaluation of the frequency of Ha-ras mutations: 39 cases of invasive carcinomas (InCa), 47 CIN III and 12 normal tissues taken from areas adjacent to the tumor (NT). (b) Retrospective follow-up: 18 cases of lesion progression; 9 cases of persistence and 12 of regression to mature or immature metaplasia after specific treatment. All biopsies obtained from each patient during the follow-up done between 5 and 10 years were included. HPV typing and scanning of possible mutations in Ha-ras were made by single-strand conformation polymorphism analysis/polymerase chain reaction. RESULTS: HPV-DNA was detected in 95% of InCa and 84% of CIN III; HPV 16/18 was found in 65% of patients, mainly associated with persistent infection and lesion progression. The undetermined HPV types (18%) could indicate the circulation in our country of types other than those screened (6, 11, 16, 18, 31 and 33). Twenty percent of CIN III and 41% of InCa had patterns compatible with Ha-ras mutations. Mutated Ha-ras was detected in 61 and 44% of progression and persistence cases, respectively, including early stages of progression. CONCLUSIONS: Ha-ras mutations were detected in CIN II-III lesions; in mutated cases, the progression took place in under 2 years, then this detection may be an early predictive marker of rapid progression.

Carcinoma, Squamous Cell↗
PubMed1997

Recurrent PIG-A mutation (IVS5+1G-->A) in a paediatric case of paroxysmal nocturnal haemoglobinuria: detection by the protein truncation test.

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemopoietic stem cell disorder caused by the absence of glycosyl phosphatidylinositol (GPI)-anchored surface proteins due to a deficient biosynthesis of GPI-anchor. The disease occurs predominantly in adults, and very few cases have been described in children and adolescents. Recent analyses have shown that null mutations in the X-linked PIG-A (phosphatidylinositol glycan-class A) gene are responsible for GPI-anchor deficiency in most PNH adult patients analysed. We report a young male from southern France who was diagnosed with PNH at 12 years of age during follow-up of aplastic anaemia. To further elucidate the molecular basis of PNH occurring in childhood, we used the powerful and rapid protein truncation test to scan for truncative mutations in the entire PIG-A mRNA reverse transcribed and amplified from blood mononuclear cells. The somatic defect responsible for PNH in the patient was found to be a splicing mutation. IVS5+1G-->A, which has previously been described in two Asiatic adults with PNH.

Child↗
PubMedDate not supplied

Detection of mutations in PCR products from clinical samples by surface plasmon resonance.

Two different strategies for scanning and screening of mutations in polymerase chain reaction (PCR) products by hybridization analysis are described, employing real-time biospecific interaction analysis (BIA) for detection. Real-time BIA was used to detect differences in hybridization responses between PCR products and different 17-mer oligonucleotide probes. For the analysis using a biosensor instrument, two different experimental formats were investigated based on immobilization of either biotinylated PCR products or oligonucleotide probes onto a sensor chip. Applied on the human tumour suppressor p53 gene, differences in hybridization levels for full-match and mismatch situations employing both formats allowed the detection of point mutations in exon 6 PCR products, derived from a breast tumour biopsy sample. In addition, a mutant sample sequence could be detected in a 50/50 background of wild type exon 6 sequence. The suitability of the different formats for obtaining a regenerable system and a high throughput of samples is discussed.

Bacterial Proteins↗