Detection of virtually all mutations-SSCP (DOVAM-S): a rapid method for mutation scanning with virtually 100% sensitivity.

Dideoxy fingerprinting (ddF) was used as a tool to search for a generic set of conditions with sufficient power to detect virtually all mutations. For each condition tested, a very large sample of mutation-containing, single-stranded segments (about 1500) were analyzed with ddF. Correlation coefficients identified pairs of conditions in which single-strand conformation polymorphism (SSCP) mobilities were poorly correlated. The data strongly suggest that tertiary structure (e.g., base-sugar and sugar-sugar interactions) rather than secondary structure is the predominant determinant of mobility shifts by SSCP. Five conditions were selected with sufficient redundancy to detect all the mutations. The sensitivity of detection of virtually all mutations-SSCP (DOVAM-S) was determined by blinded analyses on samples containing additional mutations scattered throughout the eight exons and splice junctions in the factor IX gene. The factor IX gene sequence (2.5 kb) was scanned in one lane by 15 PCR-amplified segments (125 kb of sequence scanned per gel). All of the 84 single-base substitutions were detected in the blinded analyses, the first consisting of 50 hemizygous mutant and wild-type (WT) samples and the second consisting of 50 heterozygous mutant and WT samples. DOVAM-S is estimated to be five times faster than fluorescent DNA sequencing for the detection of virtually all mutations when the five conditions are applied.     

Determination of cell concentration in a plant cell suspension using a fluorescence microplate reader

Microscopic counting of plant cells is a very tedious and time-consuming process and is therefore seldom used to evaluate plant cell number on a routine basis. This study describes a fast and simple method to evaluate cell concentration in a plant cell suspension using a fluorescence microplate reader. Eschscholtzia californica cells were fixed in a mix of methanol and acetic acid (3:1) and stained with a fluorescent DNA binding dye (Hoechst 33258). Readings were done in a fluorescence microplate reader at 360/465 nm. Specific binding of the dye to double-stranded DNA was significantly favored over unspecific binding when 1.0 M Tris buffer at pH 7.5 containing 1.0 M NaCl and 75 microg ml(-1) of Hoechst 33258 was used. Fluorescence readings must be done between 4 min and 12 min following the addition of the staining solution to the sample. The microplate counting method provides a convenient, rapid and sensitive procedure for determining the cell concentration in plant cell suspensions. The assay has a linear detection range from 0.2 x 10(6) cells to 10.0 x 10(6) cells per milliliter (actual concentration in the tested cell suspension). The time needed to perform the microplate counting was 10% of that needed for the microscopic enumeration. However, this microplate counting method can only be used on genetically stable cell lines and on asynchronous cell suspensions.     

Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: Identification of novel mutations

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene.     

Development of a micelle‐enhanced high‐throughput fluorometric method for determination of niclosamide using a microplate reader

The present paper describes the development and validation of a simple and sensitive micelle‐enhanced high‐throughput fluorometric method for the determination of niclosamide (NIC) in 96‐microwell plates. The proposed method is based on the reduction of the nitro group of niclosamide to an amino group using Zn/HCl to give a highly fluorescent derivative that was developed simultaneously and measured at λ_em 444 nm after excitation at λ_ex 275 nm. Tween‐80 and carboxymethylcellulose (CMC) have been used as fluorescence enhancers and greatly enhanced the fluorescence by factors of 100–150%. The different experimental conditions affecting the fluorescence reaction were carefully investigated and optimized. The proposed method showed good linearity (r²≥ 0.9997) over the concentration ranges of 1–5 and 0.5–5 μg/ml with lower detection limits of 0.01 and 0.008 μg/ml and lower quantification limits of 0.04 and 0.03 μg/ml on using Tween‐80 and or CMC, respectively. The developed high‐throughput method was successfully applied for the determination of niclosamide in both tablets and spiked plasma. The capability of the method for measuring microvolume samples made it convenient for handling a very large number of samples simultaneously. In addition, it is considered an environmentally friendly method with lower consumption of chemicals and solvents.     

Detection of a novel missense mutation and second recurrent mutation in the CACNA1A gene in individuals with EA-2 and FHM

Mutations in the brain specific P/Q type Ca2+ channel alpha1 subunit gene, CACNA1A, have been identified in three clinically distinct disorders, viz. episodic ataxia type 2 (EA-2), familial hemiplegic migraine (FHM) and spinocerebellar ataxia 6 (SCA6). For individuals with EA-2, the mutations described thus far are presumed to result in a truncated protein product. Several different missense mutations have been identified in patients with FHM. At least two of these mutations have been identified on two different chromosome 19p13 haplotypes and thus represent recurrent mutations. In the present study, we have screened several individuals for mutations in all 47 exons in the CACNA1A gene by single-strand conformation analysis. We have characterised a novel missense mutation, G5260A, in exon 32 in a family segregating for EA-2. The consequence of this mutation is an amino acid substitution at a highly conserved position within the CACNA1A gene. This represents the first point mutation not resulting in a proposed truncated protein. Furthermore, this mutation has been detected in a family member with mild clinical signs including only migraine. Additionally, a second previously identified recurrent muta tion, C2272T, in exon 16 has been discovered in a patient with FHM.     

Use of a microplate reader in an assay of glutathione reductase using 5,5'-dithiobis(2-nitrobenzoic acid)

The use of 96-well microtiter plates and a programmable microplate reader to measure glutathione reductase in an assay based on reduction of 5,5'-dithiobis(2-nitrobenzoic acid) by GSH generated from an excess of GSSG is described. Samples are prepared in 96-well plates and absorbance at 415 nm with a reference wavelength of 595 is determined every 30 s for 3 min. The rate of increase in absorbance is directly proportional to the amount of glutathione reductase in the sample. Activity in an unknown sample is determined from a standard curve. The assay is rapid and allows many small samples to be analyzed in replicates of two or more at the same time.     

The spectrum of beta-thalassemia mutations in Taiwan: identification of a novel frameshift mutation.

Seventy-four beta-thalassemia genes from 37 unrelated beta-thalassemia-major patients were systematically characterized by using PCR, dot-blot hybridization, and direct sequencing of amplified genomic DNA. We found that six mutations--namely, II-654, 41/42, -28, 17 beta, -29, and 27/28--were prevalent, accounting, respectively, for 45.9%, 28.4%, 10.8%, 10.8%, 1.4%, and 2.7% of studied patients. The 27/28 mutation has at codon 27-28 a cytosine insertion which has never been reported before. These results indicate that four oligo-probes (II-654, 41/42, -28, and 17 beta) allow allele-mutant determination by oligonucleotide analysis in 95.9% of this group of patients, and direct sequencing can be carried out for other samples. These data will facilitate the prenatal diagnosis of this disease by DNA analysis in Taiwan.     

Multiple dispersed spontaneous mutations: a novel pathway of mutation in a malignant human cell line.

We analyzed the nature of spontaneous mutations at the autosomal locus coding for adenine phosphoribosyltransferase in the human colorectal carcinoma cell line SW620 to establish whether distinctive mutational pathways exist that might underlie the more complex genome rearrangements arising in tumor cells. Point mutations occur at a low rate in aprt hemizygotes derived from SW620, largely as a result of base substitutions at G.C base pairs to yield transversions and transitions. However, a novel pathway is evident in the form of multiple dispersed mutations in which two errors, separated by as much as 1,800 bp, fall in the same mutant gene. Such mutations could be the result of error-prone DNA synthesis occurring during normal replication or during long-patch excision-repair of spontaneously arising DNA lesions. This process could also contribute to the chromosomal instability evident in these tumor cells.     

Ratiometric and nonratiometric Ca2+ indicators for the assessment of intracellular free Ca2+ in a breast cancer cell line using a fluorescence microplate reader

Transporters of Ca2+ are potential drug targets and Ca2+ is a useful signal in the assessment of G-protein-coupled receptor activation. Assays involving the assessment of intracellular Ca2+ using microplate readers most often use Ca2+ indicators which do not exhibit a spectra shift on Ca2+ binding (e.g. fluo-3). Indicators that do exhibit a spectral shift upon Ca2+ binding (e.g. fura-2) offer potential advantages for the calibration of intracellular Ca2+ levels. However, experimental limitations may limit the use of ratiometric dyes in microplate readers capable of screening. In this study, we compared the assessment of intracellular Ca2+ in adherent breast cancer cells using ratiometric and nonratiometric Ca2+ indicators. Our results demonstrate that both fluo-3 and fura-2 detect ATP dose-dependent increases in intracellular Ca2+ in the MCF-7 breast cancer cell line and that some of the limitations in the use of fura-2 appear to be overcome by the use of glass bottom microplates. The calibrated intracellular Ca2+ levels derived using fura-2 are consistent with those from microscopy and cuvette-based studies. Fura-2 may be useful in microplate studies, where cell lines with different properties are compared or where screening treatments lead to differences in the number of cells or dye loading.     

Detection of a novel mutation in exon 20 of the BRCA1 gene

Hereditary breast cancer constitutes 5–10% of all breast cancer cases. Inherited mutations in the BRCA1 and BRCA2 tumor-suppressor genes account for the majority of hereditary breast cancer cases. The BRCA1 C-terminal region (BRCT) has a functional duplicated globular domain, which helps with DNA damage repair and cell cycle checkpoint protein control. More than 100 distinct BRCA1 missense variants with structural and functional effects have been documented within the BRCT domain. Interpreting the results of mutation screening of tumor-suppressor genes that can have high-risk susceptibility mutations is increasingly important in clinical practice. This study includes a novel mutation, p.His1746 Pro (c.5237A>C), which was found in BRCA1 exon 20 of a breast cancer patient. In silico analysis suggests that this mutation could alter the stability and orientation of the BRCT domain and the differential binding of the BACH1 substrate.     

Search for mutations in pancreatic sufficient cystic fibrosis Italian patients: detection of 90% of molecular defects and identification of three novel mutations

A cohort of 31 cystic fibrosis patients showing pancreatic sufficiency and bearing an unidentified mutation on at least one chromosome was analyzed through denaturing gradient gel electrophoresis of the whole coding region of the cystic fibrosis transmembrane conductance regulator gene, including intron-exon boundaries. Three new and 19 previously described mutations were detected. The combination of these with known mutations detected by other methods, allowed the characterization of mutations on 56/62 (90.3%) chromosomes. Among those identified, 17 can be considered responsible for pancreatic sufficiency, since they were found in patients carrying a severe mutation on the other chromosome. Among these presumed mild mutations, eight were detected more than once, R352Q being the most frequent in this sample (4.83%). Intragenic microsatellite analysis revealed that the six chromosomes still bearing unidentified mutations are associated with five different haplotypes. This may indicate that these chromosomes bear different mutations, rarely occurring among cystic fibrosis patients, further underlying the molecular heterogeneity of the genetic defects present in patients having pancreatic sufficiency.     

Deletion loop mutagenesis: a novel method for the construction of point mutations using deletion mutants

Deletion loop mutagenesis is a new, general method for site-directed mutagenesis that allows point mutations to the introduced within a sequence of DNA defined by a previously isolated deletion mutant. Wild type and deletion mutant DNA are cloned into a bacterial plasmid and each is cleaved with a different single cut restriction enzyme. Heteroduplexes are formed between the two DNAs to produce circular molecules containing a nick in each strand and a single-stranded deletion loop. The deletion loops are mutagenised using sodium bisulphite and the DNA transfected directly into a uracil repair deficient strain of Escherichia coli. Up to half of the resultant clones contain DNA produced by replication of the wild-type length strand and bear mutations exclusively within the target area. An example is given in which a deletion mutant lacking 21 nucleotides from the region coding for SV40 large-T was used. Eight of the possible nine target cytosine residues were mutagenised. The method described is specific, efficient and simple.     

Detection of the most common G6PD gene mutations in Chinese using amplification refractory mutation system.

Glucose-6-phosphate dehydrogenase (G6PD) is the most common human enzymopathy. To date more than 122 mutations in the G6PD gene have been discovered, among which 12 point mutations are found in the Chinese. The 2 most common mutations, G1388A and G1376T, account for more than 50% of mutations representing various regions and ethnic groups in China. Setting up a simple and accurate method for detecting these mutations is not only useful for studying the frequency of the G6PD genotypes, but also for finding new mutations. The purpose of this study was to find a simple, inexpensive and accurate method for detecting these common mutations. The amplification refractory mutation system (ARMS) method was used in this study. Samples from 28 G6PD-deficient males were investigated. The natural and mismatched amplification and restriction enzyme digestion method was used as a standard method to evaluate the nature of the point mutations. Sixteen cases were found carrying the G1388A mutation and 12 the G1376T mutation. Fourteen cases of G1388A and 10 cases of G1376T were confirmed by ARMS. Four cases were not in concordance with the results obtained by the mismatched amplification-restriction enzyme digestion. These 4 cases were then judged by direct PCR sequencing at exon 12. The DNA sequencing data supported the results obtained by ARMS. Thus we concluded that the ARMS is a rapid, simple, inexpensive and accurate method for detecting the most common G6PD gene mutations among the Chinese.     

Tree scanning: a method for using haplotype trees in phenotype/genotype association studies

We use evolutionary trees of haplotypes to study phenotypic associations by exhaustively examining all possible biallelic partitions of the tree, a technique we call tree scanning. If the first scan detects significant associations, additional rounds of tree scanning are used to partition the tree into three or more allelic classes. Two worked examples are presented. The first is a reanalysis of associations between haplotypes at the Alcohol Dehydrogenase locus in Drosophila melanogaster that was previously analyzed using a nested clade analysis, a more complicated technique for using haplotype trees to detect phenotypic associations. Tree scanning and the nested clade analysis yield the same inferences when permutation testing is used with both approaches. The second example is an analysis of associations between variation in various lipid traits and genetic variation at the Apolipoprotein E (APOE) gene in three human populations. Tree scanning successfully identified phenotypic associations expected from previous analyses. Tree scanning for the most part detected more associations and provided a better biological interpretative framework than single SNP analyses. We also show how prior information can be incorporated into the tree scan by starting with the traditional three electrophoretic alleles at APOE. Tree scanning detected genetically determined phenotypic heterogeneity within all three electrophoretic allelic classes. Overall, tree scanning is a simple, powerful, and flexible method for using haplotype trees to detect phenotype/genotype associations at candidate loci.     

Identification of a novel mitochondrial mutation in Dupuytren's disease using multiplex DHPLC

Dupuytren's disease is a familial fibroproliferative disorder of late onset affecting the hands. It is extremely common in individuals of Northern European extraction. Genetic studies have yet to identify the genes involved in the formation of the disease. Mitochondria play a critical role in cell metabolism and apoptosis. It is known that defective mitochondria generate abnormally high levels of reactive oxygen species by means of electron leak and that antioxidant enzyme activities decrease with age in skin fibroblasts. Respiratory function of mitochondria is also impaired in aging human tissues. Oxidative stress and production of free radicals may be important factors in the pathogenesis of Dupuytren's disease. Mitochondrial genes are also included in the regulation of apoptosis. Diseased tissue contains large numbers of myo- fibroblasts, which disappear by apoptosis during normal wound healing. High numbers of mitochondria have been observed in fibroblasts derived from diseased tissue. In the light of this evidence, the mitochondrial genome represents a potential location for candidate susceptibility genes for this late-onset disorder. In this study, the authors investigated the presence of mutations within the mitochondrial genome in 40 subjects; 20 Caucasian Dupuytren's disease patients with a maternally transmitted inheritance pattern and 20 control subjects were matched for age, sex, and race using a multiplex denaturing high-performance liquid chromatography approach. A hitherto unknown heteroplasmic mutation located within the mitochondrial 16s rRNA region was evident in 90 percent of patients and absent from all control subjects (p < 0.001; chi2 = 16.1). This mutation may be important in the pathogenesis of Dupuytren's disease.     

Persistent response to pneumococcal vaccine in individuals supplemented with a novel water soluble extract of Uncaria tomentosa, C-Med-100

A human intervention study was carried out using male volunteers attending a General Practice Clinic in New York City involving comparison of individuals supplemented with 350 mg x 2 C-Med-100 daily dose for two months with untreated controls for their abilities to respond to a 23 valent pneumococcal vaccine. C-Med-100 is a novel nutraceutical extract from the South American plant Uncaria tomentosa or Cat's Claw which is known to possess immune enhancing and antiinflammatory properties in animals. There were no toxic side effects observed as judged by medical examination, clinical chemistry and blood cell analysis. However, statistically significant immune enhancement for the individuals on C-Med-100 supplement was observed by (i) an elevation in the lymphocyte/neutrophil ratios of peripheral blood and (ii) a reduced decay in the 12 serotype antibody titer responses to pneumococcal vaccination at 5 months.     

WT1 mutations in patients with Denys-Drash syndrome: a novel mutation in exon 8 and paternal allele origin

Denys-Drash syndrome (DDS) is characterized by early onset nephropathy, pseudohermaphroditism in males and a high risk for developing Wilms' tumour (WT). The exact cause of DDS is unknown but germline mutations in the Wilms' tumour suppressor gene (WT1) have recently been described in the majority of DDS patients studied. These mutations occur de novo and are clustered around the zinc finger (ZF) coding exons of the WT1 gene. Analysis of exons 2–10 of the WT1 gene in constitutional DNA from five patients with DDS was carried out using the polymerase chain reaction (PCR) and direct DNA sequencing. In four out of the five patients, heterozygous germline mutations were found: a novel point mutation in exon 8 (ZF₂) at codon 377 altering the wild-type histidine to arginine, and three previously described point mutations in exon 9 (ZF₃) in the codons corresponding to amino acids ³⁹⁴Arg and ³⁹⁶Asp. In one patient, no mutations could be demonstrated. In three patients where parental DNA was available, the mutations were shown to have occurred de novo. Furthermore, since tumour DNA in two of these cases had lost the wild-type allele, polymorphic markers from the short arm of chromosome 11 were used to determine the parental origin of the mutant chromosome. In both cases, the mutant chromosome was shown to be of paternal origin. Since the majority of published WT1 mutations in DDS patients alter a RsrII restriction site in exon 9, we were able to perform PCR-based diagnosis in a female patient with early renal insufficiency and normal external genitalia.     

Detection of point mutations in DNA using capillary electrophoresis in a polymer network

The use of capillary electrophoresis (CE) in a polymer network for single-strand conformation polymorphism (SSCP) is investigated. SSCP is a method to detect DNA point mutations, essential in the diagnosis of several diseases. The PCR (polymerase chain reaction) amplified p53 gene, a tumour suppressor gene known to be frequently mutated in malignant cells, was subjected to CE analysis. Two single-strand DNA fragments of 372 bp in length differing in only one nucleotide could be separated. We conclude that SSCP using CE in a polymer network is a powerful method for the detection of point mutations in DNA sequences.