Current Pharmacogenomics - Volume 2, Issue 3, 2004

The arylamine N-acetyltransferases (NATs) are a unique family of enzymes that catalyse the transfer of an acetyl group from acetyl CoA to the terminal nitrogen of hydrazine and arylamine xenobiotics. They have well characterised roles in drug detoxification and carcinogen activation and NAT homologues are present in numerous species from bacteria to humans. The importance of human NAT in xenobiotic metabolism is well established and much interest has focussed on the significance of the high degree of polymorphism present in the human isozymes. Recent advances, not least of all the availability of genomic information for species as diverse as Mycobacterium smegmatis to Danio Ranio, have seen an expansion in the NAT field. NAT in Mycobacterium tuberculosis has recently been linked to the inactivation of the forefront anti-tubercular agent, isoniazid. The availability of the three-dimensional structures of NAT from Salmonella typhimurium and Mycobacterium smegmatis is aiding investigations into the endogenous role of these and other NAT homologues. There is much speculation as to possible endogenous roles for NAT in both prokaryotes and eukaryotes and the everexpanding depth of genomic information seems likely to provide important clues in this investigation as well as allowing quicker advances in the identification of NAT substrates and inhibitors. Here we review to what extent the availability of genomic information has enabled the identification of NAT homologues in a variety of species and how this is aiding the investigation of the relevance of N-acetylation by NATs both pharmacologically and endogenously.

Genomic imprinting is a process that determines differential expression of genes according to their parental origin. Most imprinted genes play roles in growth, development and tumour suppression. Angelman syndrome is one of the most studied human diseases related to a gene that is expressed on the maternal chromosome only (at least in certain brain cells). It is caused by inactivation of the UBE3A gene in the brain due to various abnormalities of chromosome 15q11-q13 inherited from the mother. Its phenotype includes developmental delay, absent speech, motor impairment, a typical electroencephalogram, seizures and a peculiar behaviour. Lack of UBE3A expression may result from deletion of the 15q11-q13 region where this gene and GABRB3 are located, paternal uniparental disomy, imprinting defect or UBE3A mutation. Animal models corresponding to the different molecular classes have been generated. An integrative hypothesis for the molecular pathophysiology of the syndrome suggests dysregulation of synaptic neurotransmission through UBE3A-related modulation of functional GABAA receptors and GABRB3-related amount of β3 sub-unit in these receptors. This would account for developmental changes as well as for the differences in severity between deletion and nondeletion cases. In addition to rehabilitation programmes adapted to the patients' individual needs, promising management approaches may include pharmacological agents interfering with GABAA receptors, increasing GABRB3 expression or altering DNA methylation.

The field of pharmacogenomics is continuously progressing with the development and availability of molecular biotechnologies such as protein and DNA chips. These applications are pushing forward the frontiers towards individualised medicines and advanced predictions of an individual's predisposition to diseases such as asthma, cardiovascular disease and cancer. The cytochrome P450s are an essential group of enzymes involved in metabolism of many currently administered drugs as well as foreign compounds and endogenous substrates. Inter-individual variation in response to cancer chemotherapy is often attributed to genetic alterations in this particular group of drug metabolising enzymes. Such alterations can impact on the pharmacokinetics and pharmacodynamics of anti-cancer drugs, especially those drugs with a narrow therapeutic index. The identification of variations is therefore, key in the areas of drug selection and dosage. Polymorphisms have been identified in many P450s including the CYP2C and CYP3A families, as well as CYP2D6 and CYP1B1. Polymorphisms in CYP2C and CYP3A family members have demonstrated altered cytotoxic drug potential while polymorphisms in CYP1B1 are considered potentially important in the etiology of a range of human cancers. However, the functional significance of P450 variants in tumours is largely unknown. Importantly, the identification of differential cytochrome P450 expression between tumour and corresponding normal tissue are key to the development of novel drugs and therapeutic strategies that can utilise the overexpression of these enzymes to their benefit. It is for these reasons that variants in the cytochrome P450 multigene family have become the focus of current research in this area.

Here, we focus on how endocrine disruptors having estrogen activity can be analysed based on gene expression profiles. First, a comprehensive survey of the assays for detecting estrogen activity including ligand-binding assays, reporter gene assays, ELISA, and cell growth assays gives the current status in the evaluation of endocrine disruptors and the advantages of using DNA microarrays. Second, a database consisting of gene expression data from various cell types in response to various chemicals is needed for profiling the effects of chemicals as well as for clustering the genes for specific outcomes as a result of such effects. A useful interpretation of clustering requires functional annotations of the genes. Third, one promising approach to the functional characterisation of genes is proteomics and when this is combined with DNA microarray data, analyses of signal transduction pathways will be most effective. A number of genes related with estrogen signalling, signalling via receptors, Ras superfamily members, MAPK family members and AP-1 family members for example, are explained as part of a working hypothesis for such characterisations.

Site-specific DNA recombinases (SSRs) are very versatile genetic tools which have found widespread application in modern molecular biology. Although mostly prokaryotic in origin, these enzymes catalyse a wide variety of reactions, like DNA deletions, inversions, translocations or insertions also in the eukaryotic system. This versatility made one of the members of this family of enzymes, the Cre recombinase from phage P1 the tool of choice for gene rearrangements in mouse transgenics. Several other recombinases, including the yeast Flp or the C31 integrase from the Streptomyces phage, followed thereafter and offered a plethora of new options especially when combined with other SSRs. With ongoing sequencing projects, the list of available recombinases to choose from may grow tremendously in the near future. Combined with approaches such as in vitro evolution, this development could provide researchers with countless tailor-made tools which would even allow speculating about therapeutic applications. This review aims at giving an introduction to site-specific recombination as exemplified by the Cre recombinase. Recent progress in the field will be outlined (e.g. cassette exchange technologies, large chromosomal rearrangements, SSRs as cloning devices) and its potential for future applications in basic and clinical research will be discussed. Furthermore, promising novel SSR candidates for genome engineering, which have not been applied extensively so far, will be presented.

Nonsyndromic cleft of the lip with or without palate (CLP) derives from an embryopathy with failure of the nasal processes and / or fusion of the palatal shelves. This severe birth defect is one of the most common malformations among live births. Human cleft is composed of two separate entities: cleft lip and / or palate (CLP) and cleft palate only (CPO). Both have a genetic origin, whereas environmental factors contribute to these congenital malformations. In this review we analyse the role of drugs, environmental factors, genes and loci related to the onset of cleft. The data were obtained from (i) epidemiologic studies, (ii) animal models and (iii) human genetic investigations. Epidemiologic studies have demonstrated a relation between certain environmental factors (alcohol, cigarette smoking, steroids and anticonvulsants) during pregnancy and a higher risk of generating offspring with CLP. On the contrary, folic acid intake seems to have a protective effect. No clear relation has been demonstrated between aspirin and CLP. Murine models were developed to investigate drug-induced embryopathy and, more recently, to obtain information regarding genes and biochemical pathways. Steroids and anticonvulsants are the most widely studied clefting drugs, whereas TGFs and retinoic acids are the most thoroughly investigated among biochemical pathways. Human genetic studies on CLP have identified several loci and, in one case, also a specific gene. In CPO, one gene has been identified, though many more are probably involved. Among the identified chromosomal regions, there are some carrying genes with functions related to environmental factors with a clefting or protective effect.

The primary route for membrane transport of reduced folates into mammalian cells and tissues is the ubiquitously expressed reduced folate carrier (RFC). RFC is also involved in specialized tissue functions related to folates, including absorption across the intestinal epithelium and transplacental transport of folates. This chapter summarizes the current understanding of the major human RFC gene and transcript variants, best typified by G80A that results in a Arg to His substitution at position 27, a functional 61 bp deletion in promoter A, and a CATG insertion at position 191 that results in loss of functional carrier. The occurrence of RFC gene and transcript sequence variants might alter levels of tetrahydrofolate cofactor transport into cells and tissues at the level of modified or decreased RFC, resulting in effects on folate absorption, or downstream effects on folate-dependent biosynthetic pathways. These may contribute to inter-individual differences in susceptibilities to cardiovascular disease, fetal abnormalities, or cancer, particularly in combination with low serum folates. For patients with cancer, treated with antifolate chemotherapy, RFC variants may alter drug pharmacokinetics and antifolate uptake by both tumor and normal cells, thus influencing antitumor activities and toxicities associated with the administration of chemotherapy. Transport defects resulting from changes in RFC structure or expression may be compounded by changes in the catalytic activities of folate-dependent interconverting and biosynthetic enzymes (e.g., 5,10-methylene tetrahydrofolate reductase) that impact cellular distributions of individual tetrahydrofolate forms. By identifying and better understanding naturally occurring RFC gene and transcript variants, it may be possible to develop genetic screens to identify particular groups of patients who may be predisposed to pathologies resulting from folate deficiencies, or who may be subject to unacceptable toxicities or enhanced antitumor effects of antifolate therapeutics.

Huntington's disease (HD) is a devastating neurodegenerative disorder for which there is no effective treatment yet. The disease presents motor, psychiatric and cognitive symptoms, and it is caused by the expansion of CAG repeats in the huntingtin (htt) gene, which codes for a polyglutamine tract in the htt protein. The repeat expansion causes the preferential degeneration of the medium spiny neurons in the caudate-putamen of patients, and the symptoms of the disease normally appear between the third and the fifth decades of life. Despite important advances in the study of this disease, the mechanism(s) by which the mutated protein induces the degeneration of the striatal neurons remains elusive. Thus, current therapeutic approaches are symptomatic, and directed to relieve specific aspects of the disease, but cannot detain neuronal degeneration, or induce functional recovery. In this review, we will first describe the existing knowledge of the molecular basis of the disease obtained both from the study of patients and from animal models, with particular attention to transgenic models. Finally, we will discuss novel and rational therapies designed to control the transcriptional changes associated with the disease, the apoptotic death of neurons, the alterations of energy metabolism and oxidative damage, and neuroprotective strategies, with a special focus on gene therapy, as well as restorative therapies.