Biomedical subjects

Robert Williamson

Publications and source records attributed to Robert Williamson.

At least 19 recordsLinked to original sources

PubMed2006

A humanized BAC transgenic/knockout mouse model for HbE/beta-thalassemia.

Hemoglobin E (HbE) is caused by a G-->A mutation at codon 26 of the beta-globin gene, which substitutes Glu-->Lys. This mutation gives rise to functional but unstable hemoglobin and activates a cryptic splice site causing mild anemia. HbE reaches a carrier frequency of 60-80% in some Southeast Asian populations. HbE causes serious disease when co-inherited with a beta-thalassemia mutation. In this study, we report the creation and evaluation of humanized transgenic mice containing the beta(E) mutation in the context of the human beta-globin locus. Developmental expression of the human beta(E) locus transgene partially complements the hematological abnormalities in heterozygous knockout mice ((mu)beta(th-3/+)) and rescues the embryonic lethality of homozygous knockout mice ((mu)beta(th-3/th-3)). The phenotype of rescued mice was dependent on the transgene copy number. This mouse model displays hematological abnormalities similar to HbE/beta-thalassemia patients and represent an ideal in vivo model system for pathophysiological studies and evaluation of novel therapies.

Animals↗

PubMed2006

Humanized beta-thalassemia mouse model containing the common IVSI-110 splicing mutation.

Splicing mutations are common causes of beta-thalassemia. Some splicing mutations permit normal splicing as well as aberrant splicing, which can give a reduced level of normal beta-globin synthesis causing mild disease (thalassemia intermedia). For other mutations, normal splicing is reduced to low levels, and patients are transfusion-dependent when homozygous for the disease. The development of therapies for beta-thalassemia will require suitable mouse models for preclinical studies. In this study, we report the generation of a humanized mouse model carrying the common IVSI-110 splicing mutation on a BAC including the human beta-globin ((hu)beta-globin) locus. We examined heterozygous murine beta-globin knock-out mice ((mu)beta(th-3/+)) carrying either the IVSI-110 or the normal (hu)beta-globin locus. Our results show a 90% decrease in (hu)beta-globin chain synthesis in the IVSI-110 mouse model compared with the mouse model carrying the normal (hu)beta-globin locus. This notable difference is attributed to aberrant splicing. The humanized IVSI-110 mouse model accurately recapitulates the splicing defect found in comparable beta-thalassemia patients. This mouse model is available as a platform for testing strategies for the restoration of normal splicing.

Alternative Splicing↗

PubMed2006

Gross genomic rearrangements involving deletions in the CFTR gene: characterization of six new events from a large cohort of hitherto unidentified cystic fibrosis chromosomes and meta-analysis of the underlying mechanisms.

Gross genomic rearrangements involving deletions in the CFTR gene have recently been found to account for approximately 20% of unidentified cystic fibrosis (CF) chromosomes in both French and Italian patients. Using QMPSF and walking quantitative DHPLC, six novel mutations (three simple deletions, two complex deletions with short insertions of 3-6 bp, and a complex deletion with a 182 bp inverted downstream sequence) were characterized by screening 274 unidentified CF chromosomes from 10 different countries. These lesions increase the total number of fully characterized large CFTR genomic rearrangements involving deletions to 21. Systematic analysis of the 42 associated breakpoints indicated that all 21 events were caused by nonhomologous recombination. Whole gene complexity analysis revealed a significant correlation between regions of low sequence complexity and the locations of the deletion breakpoints. Known recombination-promoting motifs were noted in the vicinity of the breakpoints. A total of 11 simple deletions were potentially explicable in terms of the classical model of replication slippage. However, the complex deletions appear to have arisen via multiple mechanisms; three of the five complex deletions with short insertions and both examples of large inverted insertions (299 and 182 bp, respectively) can be explained by either a model of serial replication slippage in cis (SRScis) or SRS in trans (SRStrans). Finally, the nature and distribution of large genomic rearrangements in the CFTR gene were compared and contrasted with those of two other genes, DMD and MSH2, with a view to gaining a broader understanding of DNA sequence context in mediating the diverse underlying mutational mechanisms.

Base Sequence↗

PubMed2006

Chromatin-binding regions of EBNA1 protein facilitate the enhanced transfection of Epstein-Barr virus-based vectors.

Epstein-Barr virus (EBV)-based vectors can stably maintain large genomic fragments in mammalian cells, offering great potential for the treatment/correction of many acquired and inherited disorders. Numerous studies report marked increases in the transfection efficiency of EBV-based vectors after delivery into cell lines constitutively expressing Epstein-Barr nuclear antigen-1 (EBNA1), compared with cells not expressing EBNA1. We employ a novel strategy, involving the transfection of mRNA encoding EBNA1, to transiently express EBNA1 protein in human cells. Subsequently we show that the transfection efficiency of a 21-kb EBVbased vector is improved significantly when codelivered with mRNA encoding EBNA1. Similar increases in transfection efficiency were observed after delivery of the plasmid into cells constitutively expressing EBNA1. We also investigate the mechanism by which EBNA1 facilitates the transfection of EBV-based vectors, using mRNA encoding modified versions of the protein. Previous studies suggest that the EBNA1 DNA-binding domain (DBD), together with the nuclear localization signal (NLS), may enhance transfection of EBV plasmids by facilitating their nuclear transport. We demonstrate that an EBNA1 derivative comprising only the NLS and DBD does not facilitate transfection of EBV-based vectors. However, cells expressing an EBNA1 derivative devoid of a functional NLS but retaining the chromatin-binding regions, domains A and B, enhances plasmid transfection efficiency by up to 10-fold. Moreover, a variant of EBNA1 comprising two copies of domain A fused to the DBD enhances DNA transfection to an even greater extent than wild-type EBNA1. We therefore propose that EBNA1-mediated transfection of EBV-based vectors is dependent on the presence of chromatin- binding regions and the DBD, but not the NLS.

Active Transport, Cell Nucleus↗

PubMed2005

A 191-kb genomic fragment containing the human alpha-globin locus can rescue alpha-thalassemic mice.

A 191-kb human bacterial artificial chromosome (BAC) containing the human alpha-globin genomic locus was used to generate transgenic mice that express, exclusively, human alpha-globin ((hu)alpha-globin). Expression of (hu)alpha-globin reaches a level of 36% of that of endogenous mouse alpha-globin ((mu)alpha-globin) on a heterozygous mouse alpha-thalassemia background ((mu)alpha-globin knockout, (mu)alpha(+/-)). Hemizygous transgenic mice carrying the (hu)alpha-globin locus on a heterozygous knockout background ((hu)alpha(+/0), (mu)alpha(++/--)) demonstrated complementation of most hematologic parameters. By crossing (hu)alpha(+/0), (mu)alpha(++/--) mice, we were able to generate mice entirely dependent on (hu)alpha-globin synthesis. Breeding and fluorescent in situ hybridization studies demonstrate that only mice homozygous for the transgene were able to rescue embryonic lethal homozygous (mu)alpha-globin knockout embryos ((mu)alpha(--/--)). Adult rescued mice produce hemoglobin at levels similar to wild-type mice, with partial red cell complementation based on mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and red cell distribution width (RDW) measurements. Significant erythrocythemia above wild-type levels seems to be the main compensatory mechanism for the normalization of the hemoglobin levels in the rescued animals. Our studies demonstrate that the (hu)alpha-globin locus in the 191-kb transgene contains all the necessary elements for the regulated expression of (hu)alpha-globin in transgenic mice. This animal model should be valuable for studying the mechanisms regulating (hu)alpha-globin production and for development of therapeutic strategies for beta-thalassemia based on downregulation of alpha-globin expression.

Animals↗

PubMed2005

Transgene copy number-dependent rescue of murine beta-globin knockout mice carrying a 183 kb human beta-globin BAC genomic fragment.

We report the generation and characterisation of the first transgenic mice exclusively expressing normal human beta-globin ((hu)beta-globin) from a 183 kb genomic fragment. Four independent lines were generated, each containing 2-6 copies of the (hu)beta-globin locus at a single integration site. Steady state levels of (hu)beta-globin protein were dependent on transgene copy number, but independent of the site of integration. Hemizygosity for the transgene on a heterozygous knockout background ((hu)beta(+/0), (mu)beta(th-3/+)) complemented fully the hematological abnormalities associated with the heterozygous knockout mutation in all four lines. Importantly, the rescue of the embryonic lethal phenotype that is characteristic of homozygosity for the knockout mutation was also demonstrated in two transgenic lines that were homozygous for two copies of the (hu)beta-globin locus, and in one transgenic line, which was hemizygous for six copies of the (hu)beta-globin locus. Our results illustrate the importance of transgene copy number determination and of the hemizygosity/homozygosity status in phenotypic complementation studies of transgenic mice containing large heterologous transgenes. Transgenic mouse colonies with 100% (hu)beta-globin production from the intact (hu)beta-globin locus have been established and will be invaluable in comparative and gene therapy studies with mouse models containing specific beta-thalassemia mutations in the (hu)beta-globin locus.

Animals↗

PubMed2005

A humanized mouse model for a common beta0-thalassemia mutation.

Accurate animal models that recapitulate the phenotype and genotype of patients with beta-thalassemia would enable the development of a range of possible therapeutic approaches. Here we report the generation of a mouse model carrying the codons 41-42 (-TTCT) beta-thalassemia mutation in the intact human beta-globin locus. This mutation accounts for approximately 40% of beta-thalassemia mutations in southern China and Thailand. We demonstrate a low level of production of gamma-globins from the mutant locus in day 18 embryos, as well as production of mutant human beta-globin mRNA. However, in contrast to transgenic mice carrying the normal human beta-globin locus, 4-bp deletion mice fail to show any phenotypic complementation of the knockout mutation of both murine beta-globin genes. Our studies suggest that this is a valuable model for gene correction in hemopoietic stem cells and for studying the effects of HbF inducers in vivo in a "humanized" thalassemic environment.

Animals↗

PubMed2005

Association between the COMT Val158Met polymorphism and propensity to anxiety in an Australian population-based longitudinal study of adolescent health.

OBJECTIVES: Catechol-O-methyltransferase plays a central role in the metabolism of biogenic amines such as norepinephrine, dopamine and serotonin. Functional studies have demonstrated a dose relationship between ValMet genotypes and catechol-O-methyltransferase activity. Compared with the ValVal genotype, the ValMet and MetMet genotypes result in two- and four-fold reductions in catechol-O-methyltransferase activity, respectively. Two recent reports have observed the association between the MetMet genotype and risk of anxiety in adult populations. We examined the association between the ValMet genotypes and propensity to anxiety across adolescence. METHODS: Participants were drawn from an eight-wave study of the mental and behavioural health of over 2000 young Australians followed from 14 to 24 years of age (Victorian Adolescent Health Cohort Study, 1992 to present). DNA was received from 962 participants using a cheek swab collection method. RESULTS: The odds of reporting persistent episodic anxiety (phobic avoidance, panic attacks) were doubled among carriers of the MetMet genotype (odds ratio 2.0, 95% confidence interval 1.1-3.4, P=0.014). A dose relationship between additional copies of the Met allele and persistent episodic anxiety was also observed (1.5, 1.1-1.94, P=0.007). Stratification by sex showed that the risk effect of the Met allele was among females only. No association was observed for measures of neuroticism, persistent generalized anxiety, or a composite measure of psychiatric distress. CONCLUSION: These data replicate previous findings suggesting association between the ValMet polymorphism and specific expressions of anxiety among females.

Adolescent↗

PubMed2004

Synthesis and biological activity of analogues of the antimicrotubule agent N,beta,beta-trimethyl-L-phenylalanyl-N(1)-[(1S,2E)-3-carboxy-1-isopropylbut-2-enyl]- N(1),3-dimethyl-L-valinamide (HTI-286).

Hemiasterlin, a tripeptide isolated from marine sponges, induces microtubule depolymerization and mitotic arrest in cells. HTI-286, an analogue from an initial study of the hemiasterlins, is presently in clinical trials. In addition to its potent antitumor effects, 2 has the advantage of circumventing the P-glycoprotein-mediated resistance that hampers the efficacy of other antimicrotubule agents such as paclitaxel and vincristine in animal models. This paper describes an in-depth study of the structure--activity relationships of analogues of 2, their effects on microtubule polymerization, and their in vitro and in vivo anticancer activity. Regions of the molecule necessary for potent activity are identified. Groups tolerant of modification, leading to novel analogues, are reported. Potent analogues identified through in vivo studies in tumor xenograft models include one superior analogue, HTI-042.

Amines↗

PubMed2004

Human BAC-mediated rescue of the Friedreich ataxia knockout mutation in transgenic mice.

Three independent transgenic mouse lines were generated with the human Friedreich ataxia gene, FRDA, in an 188-kb bacterial artificial chromosome (BAC) genomic sequence. Three copies of the transgene per diploid mouse genome were integrated in a single site in each mouse line. Transgenic mice were mated with mice heterozygous for a knockout mutation of the murine Frda gene, to generate mice homozygous for the Frda knockout mutation and hemizygous or homozygous for the human transgene. Rescue of the embryonic lethality that is associated with homozygosity for the Frda knockout mutation was observed in all three lines. Rescued mice displayed normal behavioral and biochemical parameters. RT-PCR analysis demonstrated that human FRDA mRNA is expressed in all the lines. The relative expression of the human FRDA and mouse Frda genes showed a similar pattern in different tissues in all three lines, indicating position-independent control of expression of the human FRDA transgene. However, large differences in the human:mouse mRNA ratio were observed between different tissues in all three lines. The human transgene is expressed at much higher levels in the brain, liver, and skeletal muscle than the endogenous gene, while expression of the human transgene in blood is only 25-30% of the mouse gene. These studies will facilitate the development of humanized mouse models of Friedreich ataxia through introduction of a GAA trinucleotide expansion or specific known point mutations in the normal human FRDA locus and the study of the regulation of gene expression from the FRDA locus.

Animals↗

PubMed2004

Association between dependent smoking and a polymorphism in the tyrosine hydroxylase gene in a prospective population-based study of adolescent health.

This study reports pilot data on an association between tobacco dependence and a five-allele tetranucleotide repeat polymorphism in the first intron of the tyrosine hydroxylase (TH) gene. One hundred and twenty-six Australian adolescents who had participated in the Health in Transition Study (1993-1997), and who showed patterns of either dependent or nondependent smoking across four waves of data collection, consented to participation in the pilot study. The smoking status of those recruited was confirmed using a telephone-administered drug use questionnaire during 2000. Tobacco dependence was defined as smoking more than 6 days per week and more than 10 cigarettes per day during wave 5 (year 2000) and at least one prior wave ( n = 58). A second, more stringent phenotype included smoking within an hour of waking ( n = 37). The control group comprised adolescents who had used tobacco but had remained low-level social smokers across each wave of data ( n = 56). DNA was collected using a mouthwash procedure. Using the more strictly defined tobacco dependence phenotype, and after adjusting for sex, a significant protective association was found between the K4 allele and tobacco dependence (OR 0.27, 95% confidence interval [CI] 0.09, 0.82). No association was found using the liberal criteria of tobacco dependence (OR 0.51, 95% confidence interval [CI] 0.23, 1.2). These preliminary results replicate a previous association between tobacco use and the K4 allele of the TH gene (Lerman et al., 1997). The potential significance of including time to first cigarette in definitions of tobacco dependence and the possible role that these TH variants might play in tobacco dependence are discussed.

Adolescent↗

PubMed2004

Complementation of alpha-thalassaemia in alpha-globin knockout mice with a 191 kb transgene containing the human alpha-globin locus.

alpha-thalassaemia is an inherited blood disorder caused by a decrease in the synthesis of alpha-globin due to mutations in one or both of the alpha-globin genes located on human chromosome 16. A 191 kb transgene derived from a sequenced bacterial artificial chromosome (BAC) clone carrying the human alpha-globin gene cluster, together with about 100 kb of sequence upstream of DNase1 hypersensitive site HS-40 and 30 kb downstream of the alpha1-globin gene, was introduced into fertilised mouse oocytes by pronuclear microinjection. Three transgenic founder mice were obtained. Analysis of one transmitting line by fluorescent in situ hybridisation and quantitative PCR demonstrated a single copy integration of the human alpha-globin transgene on chromosome 1. Analysis of haemoglobins from the peripheral blood by cellulose acetate electrophoresis and high performance liquid chromatography (HPLC) demonstrated synthesis of human alpha-globin to about 36% of the level of each mouse alpha-globin locus. Breeding of transgenic mice with mice heterozygous for a knockout (KO) deletion of both murine alpha-globin genes showed that the human alpha-globin locus restored haemoglobin levels and red cell distribution width to normal in double heterozygous mice and significantly normalised other haematological parameters. Interestingly the human transgene also induced a significant increase in red cell production and haematocrit above wild type values. This is the first report demonstrating complementation of a murine alpha-globin KO mutation by human alpha-globin gene expression from an intact human alpha-globin locus. The transgenic mouse model described in this report should be very useful for the study of human alpha-globin gene regulation and for the development of strategies to down regulate alpha-globin production as a means of ameliorating the severity of beta-thalassaemia.

Animals↗

PubMed2004

Nicotine dependence in a prospective population-based study of adolescents: the protective role of a functional tyrosine hydroxylase polymorphism.

Dopamine is a key neurotransmitter of the mesolimbic reward pathway in the human brain, and tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis. Consequently, the gene encoding TH is a strong candidate for involvement in the genetic component of addiction. The importance of this gene in nicotine dependence is supported by many studies showing a link between nicotine administration and TH expression. A functional tetranucleotide repeat polymorphism within intron 1 of the TH gene (HUMTH01-VNTR) has been shown to modify tobacco use in two independent Caucasian samples from the USA and Australia. Using information drawn from an eight-wave Australian population-based longitudinal study of adolescent health, we tested the effect of the HUMTH01-VNTR on nicotine dependence. Comparisons were made between dependent smokers and non-dependent smokers. These data provide further support for a protective association between the K4 allele and dependent smoking (odds ratio 0.54, 95% confidence interval 0.28-1.0). No associations were observed at any of three other common TH polymorphisms (rs6356, rs6357 and HUMTH01-PstI). Including these data, three independent studies, two of which use identical phenotypes, have now identified a protective relationship between the K4 allele of the functional HUMTH01-VNTR polymorphism and high-level smoking.

Adolescent↗

PubMed2004

Liver cell transplantation leads to repopulation and functional correction in a mouse model of Wilson's disease.

BACKGROUND AND AIM: The toxic milk (tx) mouse is a non-fatal animal model for the metabolic liver disorder, Wilson's disease. The tx mouse has a mutated gene for a copper-transporting protein, causing early copper accumulation in the liver and late accumulation in other tissues. The present study investigated the efficacy of liver cell transplantation (LCT) to correct the tx mouse phenotype. METHODS: Congenic hepatocytes were isolated and intrasplenically transplanted into 3-4-month-old tx mice, which were then placed on various copper-loaded diets to examine its influence on repopulation by transplanted cells. The control animals were age-matched untransplanted tx mice. Liver repopulation was determined by comparisons of restriction fragment length polymorphism ratios (DNA and mRNA), and copper levels were measured by atomic absorption spectroscopy. RESULTS: Repopulation in recipient tx mice was detected in 11 of 25 animals (44%) at 4 months after LCT. Dietary copper loading (whether given before or after LCT, or both) provided no growth advantage for donor cells, with similar repopulation incidences in all copper treatment groups. Overall, liver copper levels were significantly lower in repopulated animals (538 +/- 68 microg/g, n = 11) compared to non-repopulated animals (866 +/- 62 microg/g, n = 14) and untreated controls (910 +/- 103 microg/g, n = 6; P < 0.05). This effect was also seen in the kidney and spleen. Brain copper levels remained unchanged. CONCLUSION: Transplanted liver cells can proliferate and correct a non-fatal metabolic liver disease, with some restoration of hepatic copper homeostasis after 4 months leading to reduced copper levels in the liver and extrahepatic tissues, but not in the brain.

Adenosine Triphosphatases↗

PubMed2004

The potential of bone marrow stem cells to correct liver dysfunction in a mouse model of Wilson's disease.

Metabolic liver diseases are excellent targets for correction using novel stem cell, hepatocyte, and gene therapies. In this study, the use of bone marrow stem cell transplantation to correct liver disease in the toxic milk (tx) mouse, a murine model for Wilson's disease, was evaluated. Preconditioning with sublethal irradiation, dietary copper loading, and the influence of cell transplantation sites were assessed. Recipient tx mice were sublethally irradiated (4 Gy) prior to transplantation with bone marrow stem cells harvested from normal congenic (DL) littermates. Of 46 transplanted tx mice, 11 demonstrated genotypic repopulation in the liver. Sublethal irradiation was found to be essential for donor cell engraftment and liver repopulation. Dietary copper loading did not improve cell engraftment and repopulation results. Both intravenously and intrasplenically transplanted cells produced similar repopulation successes. Direct evidence of functionality and disease correction following liver repopulation was observed in the 11 mice where liver copper levels were significantly reduced when compared with mice with no liver repopulation. The reversal of copper loading with bone marrow cells is similar to the level of correction seen when normal congenic liver cells are used. Transplantation of bone marrow cells partially corrects the metabolic phenotype in a mouse model for Wilson's disease.

Animals↗

PubMed2003

Cellular genomic reporter assays for screening and evaluation of inducers of fetal hemoglobin.

Reactivation of fetal hemoglobin (HbF) expression using pharmacological agents represents a potential strategy for the therapy of beta-thalassemia, sickle cell disease, HbE and other beta-hemoglobinopathies. However, the drugs currently available have low efficacy and specificity and are associated with high toxicity. We describe the development of stable cellular genomic reporter assays (GRAs) based on the green fluorescence protein (EGFP) gene under the Ggamma-globin promoter in the intact human beta-globin locus. We show that human erythroleukemic cell lines stably transfected with a Ggamma-EGFP beta-globin locus construct can maintain a uniform basal level of EGFP expression over long periods of continuous culture and that induction of EGFP expression parallels the induction of the endogenous globin genes. We compared the EGFP-induction potency of a number of chemotherapeutic agents, including histone deacetylase inhibitors and DNA-binding agents. We show that hydroxyurea and butyrate result in moderate levels of induction (70-80%) but with an additive inductive effect. Among the DNA-binding agents tested, cisplatin was the most potent inducer of HbF expression, (442+/-32%), a level which is comparable to hemin (764+/-145%). These results indicate that cellular GRAs containing Ggamma-EGFP-modified beta-globin locus constructs can be used to develop novel inducers of HbF synthesis for the therapy of beta-hemoglobinopathies.

Butyrates↗

PubMed2003

Ethical and legal issues and the "new genetics".

Although uniquely personal, the information from gene analyses impacts on parents, siblings, children and even entire ethnic groups. Doctors need to carefully balance the right of a patient to privacy against the wider family and society interests, consistent with ethical standards and their legal obligations. Doctors also need to be in a position to advise their patients of potential risks that may result from obtaining predictive genetic information, such as discrimination by third parties. While these issues are not new, they occur with new sharpness in the "new genetics", where clinicians have to be familiar not only with clinical significance, but also the ethical and legal implications of genetic analyses and information.

Australia↗

PubMed2003

The "new genetics" and clinical practice.

A "new genetics" has emerged driven by knowledge gained at the DNA level. In clinical practice, a practical application of the new genetics is DNA testing, which can be expected to expand with the completion of the Human Genome Project as the functions of new genes are discovered. Genetic DNA testing scenarios include diagnostic DNA testing, prenatal DNA testing, predictive (presymptomatic) DNA testing and screening DNA testing. The challenge for genetic DNA testing and clinical practice will be to define the roles to be played by the general practitioner, the specialist, and other healthcare professionals. From the patients' and families' perspective, the new genetics will best be implemented if a planned approach is adopted in the ordering of DNA tests and the associated counselling and support processes.

DNA↗