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Biomedical subjects

Richard G H Cotton

Publications and source records attributed to Richard G H Cotton.

At least 19 recordsLinked to original sources
PubMed2006

Detection of 100% of mutations in 124 individuals using a standard UV/Vis microplate reader: a novel concept for mutation scanning.

We report the development of a simple and inexpensive assay for the detection of DNA polymorphisms and mutations that is based on the modification of mismatched bases by potassium permanganate. Unlike the chemical cleavage of mismatch assay, which also exploits the reactivity of potassium permanganate to detect genomic variants, the assay we describe here does not require a cleavage manipulation and therefore does not require expensive or toxic chemicals or a separation step, as mismatches are detected using direct optical methods in a microplate format. Studies with individual deoxynucleotides demonstrated that the reactivity with potassium permanganate resulted in a specific colour change. Furthermore, studies with synthetic oligonucleotide heteroduplexes demonstrated that this colour change phenomenon could be applied to detect mismatched bases spectrophotometrically. A collection of plasmids carrying single point mutations in the mouse beta-globin promoter region was used as a model system to develop a functional mutation detection assay. Finally, the assay was validated as 100% effective in detecting mismatches in a blinded manner using DNA from patients previously screened for mutations using established techniques, such as sequencing, SSCP and denaturing high-performance liquid chromatography (DHPLC) analysis in DNA fragments up to 300 bp in length.

Animals↗
PubMed2006

Scanning the ocular albinism 1 (OA1) gene for polymorphisms in congenital nystagmus by DHPLC.

BACKGROUND: Nystagmus is common to all types of albinism. Some subjects with nystagmus lack convincing signs of albinism, have no other visual pathway disease, and are classified as possessing congenital idiopathic nystagmus (CN). It has been postulated that CN may be a form of ocular albinism. METHODS: The presence of nystagmus, iris transillumination, and visual acuity were recorded in 39 CN and albino patients and their families. Physical characteristics were also noted. DNA from buccal swabs was obtained for use in denaturing high performance liquid chromatography (DHPLC) and chemical cleavage of mismatch (CCM) to scan several hotspots for X-linked ocular albinism (OA1) mutations. RESULTS: Two previously reported polymorphisms were confirmed: neither was found to be a causative mutation. CONCLUSION: No correlation was identified between nystagmus and OA1.

Albinism, Ocular↗
PubMed2006

Spontaneous DNA-DNA interaction of homologous duplexes and factors affecting the result of heteroduplex formation.

Mutation detection and mismatch repair investigations based on heteroduplex formation require a linear DNA structure. DNA branching, described previously under physiological conditions, has been analysed in the heteroduplex formation process. Symmetrical chi-structures were detected after heteroduplex formation by gel electrophoresis and electron microscopy. Buffer composition, DNA concentration and duplex end-sequences influence DNA branching. Duplexes with homologous central regions but non-complementary ends do not form hybrid heteroduplexes or hybrid Holliday junctions. Our results explain the requirements for efficient heteroduplex formation, which were previously determined empirically: special solution composition, optimal DNA concentration and GC clamps. This provides the theoretical background for further optimisation of the procedure.

Animals↗
PubMed2005

Locus-specific databases: from ethical principles to practice.

Locus-specific databases (LSDBs) play an essential role in clinical care and research. They differ from traditional genetic databases in that they propose to place the mutations of "anonymized" patients directly on the World Wide Web. The proliferation of ethical guidelines and legal requirements affects the rapid and free transmission of clinical data, which is vital for both the daily management of patients and research into better diagnostics and treatment. This paper proposes a review of ethical principles endorsed by international instruments that are of particular relevance to LSDBs. It aims to translate them into 12 proposed practical guidelines that LSDB curators can use in collecting data for clinical research. Perhaps these guideposts will serve as a first step toward translating principles into practice.

Databases, Nucleic Acid↗
PubMed2004

Expression of truncated presenilin 2 splice variant in Alzheimer's disease, bipolar disorder, and schizophrenia brain cortex.

We have analysed the expression of a truncated variant presenilin 2 protein (PS2V) in frontal cortex from subjects with Alzheimer's disease (AD) and age-matched controls, and compared these results with cortex from bipolar disorder (BP), schizophrenia (SZ) and controls in a second brain bank collection. PS2V protein was detected as a 14 kDa species with antibodies directed to the PS2 N-terminal region and to the new C-terminus created by alternative transcription. PS2V protein levels were significantly increased by two-fold in AD cortex, as compared to age-matched controls. In tissue from the second collection, levels of PS2V were markedly elevated in some BP and SZ cases, but there was no overall difference between diagnostic groups. Our findings support previous evidence for increased expression of this variant PS2 isoform in sporadic AD and suggest this isoform may contribute to neurodegeneration.

Adult↗
PubMed2004

UV-visible spectral identification of the solution-phase and solid-phase permanganate oxidation reactions of thymine acetic acid.

Solution-phase and solid-phase permanganate oxidation reactions of thymine acetic acid were investigated by spectroscopy. The spectral data showed the formation of a stable organomanganese intermediate, which was responsible for the rise in the absorbance at 420 nm. This result enables unambiguous interpretation of the absorbance change at 420 nm, as the intermediate permanganate ions could be isolated on the solid supports.

Acetic Acid↗
PubMed2004

The challenge of documenting mutation across the genome: the human genome variation society approach.

New methods for the detection of mutations and the completion of the human genome sequencing project have contributed to an exponential rise in variation information that must be collected, quality controlled, documented, and stored safely to ensure future availability to health care professionals, researchers, and others. There may be anywhere from one to more than 1,000 mutations in any given gene. To date, this information has been collected by general databases such as Online Mendelian Inheritance in Man (OMIM) or the Human Gene Mutation Database (HGMD), which collect only published mutations and, in the case of OMIM, selected published mutations. Unpublished mutations have made their way into Locus Specific Databases (LSDBs), and these can often contain as many unpublished mutations as published ones, in addition to other more detailed gene-specific information. LSDBs, however, do not exist for all genes at this time. Through their interactions, a number of members of the Human Genome Variation Society (HGVS) have developed nomenclature, standard software to curate mutations in gene specific databases, a WayStation to collect and review new mutations from research and diagnostic laboratories, and central databases to store and display these mutations and their associated phenotypes. Nomenclature is now well defined for the commonest types of mutation, with work continuing on systematically naming the more complex types. Other projects, such as dedicated specialized software for LSDBs, are in the early stages of development.

Computational Biology↗
PubMed2003

Spectroscopic study of permanganate oxidation reactions of oligonucleotides containing single base mismatches.

The electrophoretic gel-based chemical cleavage of the mismatch method gives an incomplete view of the DNA conformational changes induced by a single base mismatch. This spectroscopic study investigates the permanganate oxidation reactions with matched and mismatched DNA under constant and variable temperature conditions. The results, which include the oxidation levels, reaction patterns with isosbestic points, color changes, thermal spectra, spectroscopy derivative, and gel separation and melting temperatures, provide a fundamental background for identification of oligonucleotides containing single base mismatches by chemical means.

Base Pair Mismatch↗
PubMed2003

Variations of the human glucocorticoid receptor gene (NR3C1): pathological and in vitro mutations and polymorphisms.

Glucocorticoid (GC) resistance can occur in a number of diseases. It can be either generalized (i.e., familial glucocorticoid resistance) or localized (i.e., asthma). In many cases, a reason for this resistance to steroids lies with mutations or polymorphisms present in the glucocorticoid receptor gene (GR/NR3C1) that belongs to a large family of nuclear receptors. A number of GC-resistant cell lines have been isolated in vitro, some of which arose or may have arisen in vivo. These and the mutations defined in them are included in this review as well as mutations engineered in plasmids and expressed in vitro. It also lists polymorphisms and the individual studies where association-related studies have been performed. NR3C1 is located on chromosome 5q31 and contains 10 exons that code for a 777 amino acid protein. There are two naturally occurring isoforms of the NR3C1, GRalpha (functional) and GRbeta (no hormone-binding ability). A total of 15 missense, three nonsense, three frameshift, one splice site, and two alternative spliced mutations have been reported in the NR3C1 gene associated with glucocorticoid resistance as well as 16 polymorphisms. Mutation and polymorphism data for NR3C1 will soon be found on the newly created locus-specific database.

Animals↗
PubMed2003

Permanganate oxidation reactions of DNA: perspective in biological studies.

KMnO4 has been well known as a powerful chemical probe for numerous applications in biological fields, particularly for those used in conformational studies of DNA. The KMnO4 assay provides essential information for understanding biochemical processes and detecting aberrant DNA, which is associated with many genetic diseases. Elegant examples are sequencing techniques, foot-printing assays for transcriptional studies, an interference method for hormone receptor binding assays as well as DNA conformational studies of Z-DNA, Z-Z junctions, hairpins, curvatures, short nucleotide base repeats, binding of intercalators and groove binders, etc. Recently, KMnO4 has been successfully applied to detect single base changes and mutations in DNA (chemical cleavage of mismatch method, CCM) as well as other types of base damage (8-oxoguanine and thymine dimers). This paper aims to review the usefulness and limitations of the permanganate oxidation reaction used in various biological studies of DNA.

Base Composition↗
PubMed2002

Alternative transcripts of presenilin-1 associated with frontotemporal dementia.

We have analyzed the expression of Alzheimer's disease-associated presenilin 1 (PS1) in various neurodegenerative disorders. Western blotting identified PS1 N- and C-terminal fragments similarly in the cortex of controls, Parkinson, Huntington and schizophrenia subjects. Additional PS1 immunoreactive species of 42 and 46 kDa were present in six out of seven cases of sporadic frontotemporal dementia (FTD) and these were particularly prominent in two cases. RT-PCR analysis using nested primers showed the presence of PS1 gene products with deletions within the exon 4-8 region. Our results suggest that alternative transcription of PS1 may be associated with FTD.

Adult↗
PubMed2002

Alternative transcripts of presenilin-1 associated with frontotemporal dementia.

We have analyzed the expression of Alzheimer's disease-associated presenilin 1 (PS1) in various neurodegenerative disorders. Western blotting identified PS1 N- and C-terminal fragments similarly in the cortex of controls, Parkinson, Huntington and schizophrenia subjects. Additional PS1 immunoreactive species of 42 and 46 kDa were present in six out of seven cases of sporadic frontotemporal dementia (FTD) and these were particularly prominent in two cases. RT-PCR analysis using nested primers showed the presence of PS1 gene products with deletions within the exon 4-8 region. Our results suggest that alternative transcription of PS1 may be associated with FTD.

Alternative Splicing↗