Pharmacogenetics and psychopharmacology

For more than 40 years, epidemiological studies have clearly demonstrated a tendency for diseases such as schizophrenia, bipolar disorder and autism to run in families. Thus it has been shown that such disorders are much more frequent in close relatives of patients than in the general population. For example, estimates of the increased risk of suffering from the disorder if the patient has a sibling with the disorder range from nine- to eleven-fold for schizophrenia and about sevenfold for those with bipolar disorder. These major psychiatric disorders show a significantly greater concordance rate in genetically identical twins. Thus the concordance rate for monozygotic twins in schizophrenia is approximately three times that observed in dizygotic twins. In bipolar disorder, the corresponding concordance rate is approximately eight times greater in monozygotic than in dizygotic twins. From such studies it has been calculated that between 60 and 80% of the liability of these two disorders is genetic in origin. However, it must be emphasized that these calculations do not identify specific genetic causes for the conditions but they do demonstrate that the genetic, as well as environmental components, play a significant role.

The question arises regarding how the genes that contribute to major psychiatric disorders can be identified. Many of the medical conditions for which a genetic component has been identified follow a genetic pattern that clearly follows classical Mendelian inheritance. For example, in cases of Huntington's disease and cystic fibrosis, the two parents who carry the recessive gene give rise to offspring in the ratio of 1:2:1, one expressing the disease, two not showing the symptoms but carrying the gene and one not carrying the gene for the disorder. Such conditions follow the Mendelian pattern of inheritance because the condition is caused by a mutation of a single gene. While the locating of the underlying gene in such situations is often difficult and time-consuming, the techniques of classical molecular genetic analysis, and linkage studies followed by positional cloning, are now well established. For monogenic diseases such as Huntington's, a common pattern emerges in which a single gene, or small number of genes, that may harbour a number of rare mutations can be identified. Each mutation alone is then sufficient to produce a phenotype of the disease.

Regarding bipolar disorder and schizophrenia, while there is some evidence that some families transmit the risk of the disease in a Mendelian fashion, the overall pattern of disease transmission is complex and it is unlikely that these conditions are due to a single gene. This suggests that there may be multiple genes involved, either many genes with strong alleles or common variants in many genes, each of which increases the risk of the disease in a modest way. An example of this would be Alzheimer's disease, in which significant associations have been demonstrated between apolipoprotein (APO) E4 and the occurrence of the disease. An account of the genetic basis of Alzheimer's disease can provide a useful example of the relationship between the genetic basis and the expression of the disease.

Alzheimer's disease exists in two major forms, the so-called early and late onset types. The former follows typical Mendelian inheritance while the latter shows a more complex, non-Mendelian, pattern of inheritance. The early onset form of the disease has permitted the identification of several genes which are causally related to the condition.

In the elderly, in which Alzheimer's disease has been estimated to occur in up to 20% of those aged 80 years, it has been shown that one allelic form of APO E is associated with an increased risk for developing the disease. Of the three APO E allelic forms in man, APO E4 is associated with the late onset form of the disease; this may account for up to 50% of the genetic risk for the late onset form whereas those carrying the less frequent E2 allele appear to be protected from the disease.

In the early onset familial form of the disease, affecting approximately 5% of cases, there is a clear autosomal dominant pattern of inheritance. Mutations in three genes have been identified involving the beta amyloid precursor protein, presenilin-1 and presenilin-2. The function of these proteins is described in more detail in the chapter on the dementias (Chapter 14). It has been estimated that mutations in these genes account for approximately 50% of the cases of the early onset disease.

Summary of methods used to detect possible genetic defects in psychiatric disorders

The genetic basis of late onset Alzheimer's disease conforms to the common disease-common variant hypothesis which states that genetic susceptibility is attributable to common variants present in the population at a high frequency. Significant associations have been demonstrated between several common polymorphisms such as APO E4 and Alzheimer's disease, already referred to. Individuals differ from each other at many positions across the genome and a variation at a particular nucleotide is called a polymorphism. It has been calculated that approximately one nucleotide base in every 1000-2000 nucleotides differs between chromosomes. If, as seems likely, the common disease-common variant hypothesis applies to schizophrenia and bipolar disorder then it may be essential to undertake association studies which follow the inheritance of a gene within a population rather than within families. Association studies test if a polymorphism is more frequently found in those with the disease (called ''cases'') than in normal individuals (controls). In such tests, the transmission of a polymorphism from a heterozygous parent to an affected offspring is followed. If the polymorphisms are not associated with the disease, then the rate of transmission from parent to affected offspring in a population will be 50%. Significant deviations from this predicted transmission rate indicate a possible association with the disease. Such studies are an attractive means to understanding complex psychiatric diseases because they have the power to identify causal associations between a particular allele and a heterogeneous disease and do not require the collection of large pedigrees containing multiple affected individuals. However, for such a method to be applied, the polymorphisms used in the assessments must have been identified. This necessitates the collection of a large group of polymorphisms to form a library of potential disease-causing alleles. Traditionally techniques for the detection of polymorphisms have involved gel-based screening methods or direct DNA sequencing of numerous individuals. Such methods are laborious and time-consuming. Microarray technology has revolutionized the approach by developing DNA microchips that contain a high density of oligonucleotides which are capable of rapidly detecting variations in nucleotide sequences. Micro-arrays consist of microchips of DNA attached to the surface of a solid support that may vary according to the density and the form, or size, of the DNA on the surface. There are different commercially available forms of microarray chips and the presence of many independent DNA molecules on the chip surface allows hybridization of many different species simultaneously and for the results to be similarly detected on a microscope slide. By labelling the final products formed with a fluorescent dye, the resulting hybridization pattern can be detected by a confocal microscope.

The major question which arises now that the technology for rapid screening has become available is which genes should be chosen? If the possible cause of the disease is known then the answer is clear. However, for most of the major psychiatric disorders the causes are unknown although neurochemical, neurodevelopmental, autoimmune and environmental factors are probably involved. For this reason, association studies in schizophrenia and bipolar disorder have focused on genes related to the dopaminergic and serotonergic systems. Unfortunately, polymorphisms related to these genes have not convincingly been found to be associated with schizophrenia or bipolar disorder. However, several other lines of evidence suggest that schizophrenia may be a neurodevelopmental disorder in which the frontal association cortex, an area responsible for several important high-level functions, has been shown to be malfunctional. Thus schizophrenia may result from a disorder of neuronal migration. If so, the discovery of polymorphisms in genes that regulate cortical development could prove to be a useful area of investigation. This approach has already identified the gene dsh (dishevelled, named after a gene identified in the fruit fly, Drosophila) in the mouse which, when absent, leads to a reduction in the startle response. Prepulse inhibition, the reduction in the startle that the animal shows to a previous stimulus, has been shown to be diminished in patients with schizophrenia. While it is premature to propose that dsh deficient mice are a model for schizophrenia, such findings do suggest that such genes may play an important role in the early differentiation of the brain. During the normal development of the brain, a large number of genes are activated and become involved at different times and have different functions. In the early pattern formation of the brain, during the development of the neural tube, the homeotic genes (the so-called hox family genes) encode for transcriptional factors that bind to DNA and thereby regulate the expression of other genes. This process is believed to convey information on dorsoventral positioning to the cells of the developing neural tube. However, there is evidence that a mutation in a homeobox gene is associated with gross brain defects which makes it unlikely that a mutation in the early genes could be responsible for the subtle changes found in schizophrenia.

There is evidence that the cortex of the brain of schizophrenic patients contains neurons that are in abnormal positions when compared to non-schizophrenic individuals. This suggests that the migration of neurons may be abnormal during the developmental period. The neuronal cell adhesion molecule (NCAM) is an immunoglobulin that mediates adhesion between neurons, thereby exerting a key role in morphogenesis, differentiation and the migration of neurons. NCAM is encoded by a single gene that undergoes alternative splicing to generate several alleles which exhibit different spatial and temporal patterns of expression in vertebrates. The NCAM gene is located on chromosome 11 and two polymorphisms are known to occur in the NCAM gene region. However linkage analysis of 71 families has failed to confirm that an abnormality in the NCAM gene occurs in schizophrenia.

During brain development, neurons send out numerous dendrites in which growth factors play a prominent part. The neurotrophins comprise a family of structurally and functionally related growth factors that include nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and the neurotrophins 3 and 4/5. These peptides cause neuronal growth, increase the size of the body of the neuron and maintain the survival of the neurons of the dopaminergic, serotonergic and glutaminergic systems. As a decrease of 20-30% in BDNF mRNA has been reported to occur in the hippocampi of schizophrenic patients, it seems possible that a defect in this growth factor could be responsible for the occurrence of smaller neurons in these patients. The BDNF gene is located on chromosome 11 and preliminary findings from 72 nuclear families suggest that the frequency of the A2 allele is significantly higher, and that of the A1 allele significantly lower, in schizophrenic patients. The NT-3 gene, located on chromosome 12, and the promoter region of this gene contain a highly polymorphic marker yielding at least 11 dinucleotide repeat alleles. Of the case-controlled association studies undertaken so far, abnormal alleles have been reported in two of the five studies.

Lastly, studies on the different polymorphic forms of the synapsins, that organize the mobilization of neurotransmitter vesicles thereby regulating neurotransmitter release, could account for some of the subtle changes in neurotransmission that occur in schizophrenia. However, to date linkage analysis studies have failed to reveal any positive associations between the various polymorphisms of the synapsin gene and schizophrenia.

The genetic basis of obsessive-compulsive disorder (OCD)

There have been over 18 family studies of OCD during the past 70 years but, due to methodological differences, the familial aspects of the disorder remain controversial. Family studies look for the prevalence of the disorder among the biological relatives of the probands (i.e. individuals who are affected by OCD) and the prevalence is then compared with that seen in the general population or in a control group. The latter are usually unaffected subjects or relatives of those with OCD. Despite the limitations of the studies, most have found a significant increase in the rates of OCD among the first-degree relatives of the probands when compared with the general population. Similarly, there is evidence from family studies of patients with chronic motor tics and Tourette syndrome that the rates of these conditions, and OCD, are higher among the relatives of patients with Tourette syndrome. More recent studies have suggested that some of these cases are familial but unrelated to tics while in other cases there appears to be no family history of either OCD or tics.

Twin studies comparing mono- and dizygotic twins have shown concordance rates of between 25 and 87%, depending on the study. Since none of the studies show a concordance rate of 100%, it is clear that non-genetic factors must influence the expression of OCD.

Linkage analysis studies, in which the OCD is linked to a known polymorphic marker, are used to determine if there is a single gene locus causing susceptibility for the disorder. Using such an approach for a battery of candidate genes coding for receptors and enzymes involved in dopaminergic and serotonergic transmission (i.e. for the two neurotransmitters most likely to be involved in OCD), no evidence was found for a link.

Association studies have proven to be more fruitful. In such studies, the allelic frequencies for specific marker genes are compared with a control population. When OCD patients were investigated for the association of tics with the dopamine receptor 2 marker it was shown that there was an increased frequency of homozygosity for the allele A2 of TaqIA at the D2 receptor locus. Not all investigators could replicate this finding however. No positive associations have been found between OCD and the D3 gene while there was some association found between OCD with tics and the D4

gene. Two other catecholaminergic markers have been investigated in patients with OCD. Thus a positive association has been reported between the low activity allele of catechol-O-methyl transferase in male patients and a higher frequency of the low activity allele of monoamine oxidase type A gene in female patients. There is also evidence that the serotonin transporter gene is abnormal. In conclusion, evidence from genetic studies lends support to the view that a single gene defect occurs in patients with OCD while association studies of candidate genes, in spite of methodological difficulties, have highlighted the loci for D2 and D4 receptor genes together with those for catechol-O-methyl-transferase and monoamine oxidase A.

Genetics of panic disorder

Studies assessing the diagnosis of panic disorder by the direct interview of family members, a method commonly employed in the 1980s, showed that the risk of developing the disorder is higher in the first-degree probands. This has been estimated to be 17% for first-degree relatives compared to 2% for the control population. In addition, relatives of probands had a higher prevalence of generalized anxiety disorder and major depression. This was confirmed in another study of anxiety disorders in female relatives and showed a morbidity risk for all anxiety disorders to be 32% among first-degree relatives of agoraphobic patients with panic attacks and 33% in those with panic disorder. The risk was found to be even greater in the offspring when both had anxiety disorders. Overall, family studies have shown that first-degree relatives of probands with panic disorder have a three- to 21fold higher risk of developing panic disorder than first-degree relatives of healthy probands.

The relationships between panic disorder and other types of anxiety disorder have also been the subject of study. Thus some investigators have reported that relatives of those with agoraphobia have a higher risk for the disorder than relatives of patients with panic disorder, leading to the suggestion that agoraphobia should be considered a more severe form of panic disorder which has an independent genetic transmission. Other studies, however, have found that the diagnosis of separation anxiety and agoraphobia in probands increased the risk of both panic disorder and agoraphobia in the relatives of the patients. Such observations support the hypothesis that panic disorder and agoraphobia are two phenotypic expressions of the same condition due to a different degree of genetic penetrance.

The analysis of the relationship between panic disorder and major depression has produced conflicting results. The possible link between these disorders has been provided by the frequent occurrence of major depression in patients with panic disorder and agoraphobia, both conditions responding to antidepressant treatments. Whereas most of the genetic analyses available suggest that there is an independent genetic basis for these conditions, it is noteworthy that relatives of patients with both panic disorder and agoraphobia are more likely to develop depression, phobias and alcoholism when compared with relatives of probands with panic disorder alone. There does not appear to be an association between generalized anxiety disorder and panic disorder, suggesting that they are separate entities. Generalized anxiety disorder appears to be more prevalent (approximately 20%) in relatives of probands with the disorder than in those of relatives with panic disorder (5%) or agoraphobia (4%).

Twin studies have demonstrated only a moderate concordance in monozygotic versus dizygotic pairs. Using the hypersensitivity to carbon dioxide as a challenge test, it has been shown that there is a significantly higher concordance rate in monozygotic (56%) than in dizygotic (13%) twins.

In recent years, emphasis has tended to switch from family and twin studies to molecular genetics. Linkage analysis in families with a psychiatric disorder has often proven to be a fruitful method to determine the degree of inheritance of a disorder. The method of parametric linkage estimates the likelihood of the distribution of genetic markers of an illness by a predetermined model and expressed as the logarithm of the odds score (the so-called lod score). For simple Mendelian inheritance of a disease, a lod score of 3.0 or greater, obtained by scanning the genome for markers of the disease, is considered to be a statistically significant linkage. However, most psychiatric disorders do not follow classical Mendelian genetics so such an approach is of limited value. For this reason, other linkage methods have been developed. For example, the allelic-sharing method is based on detecting the frequency of the inheritance of the same genetic marker from each parent. The presence of a gene that causes the disorder is revealed when the allele that is shared between the siblings is greater than 50%. To date, all the linkage studies in panic disorder have been inconclusive. Neither has the search for candidate genes for neurotransmitters (for example, GABA, tyrosine hydroxylase, serotonin receptors, dopamine receptors, adrenoceptors, opioid receptors) proven to be any more fruitful. However, there is some preliminary evidence for an association between the cholecytokinin (CCK) promoter gene and panic disorder which, if replicated, would support the hypothesis that the CCK-B receptor is hypersensitive in panic disorder. This is discussed in more detail in Chapter 9.

Genetics of attention deficit/hyperactivity disorder (ADHD)

ADHD is the most common psychiatric disorder with onset in childhood. The condition is characterized by inattention and/or hyperactivity and impulsivity which is associated with cognitive, social and academic impairments. It has been estimated that in up to 60% of patients, these impairments persist into adulthood. Males are affected more than females (4:1).

ADHD is a familial disorder with a population-based prevalence of about 10% and a prevalence rate in siblings of approximately 25%. Detailed studies of the disorder suggest that there is a five- to sixfold increase in first-degree relatives of affected persons. However, as with other psychiatric disorders, the finding of familial aggregation alone does not necessarily lead to the conclusion that the disorder is of genetic origin as such studies do not separate genetic from environmental factors.

Twin studies have been useful in discriminating the genetic from environmental factors. The concordance rate for monozygotic versus dizygotic twins has been estimated at 51% and 33% respectively, and it has been estimated that approximately half the variance in the trait factors of hyperactivity and inattentiveness are accounted for by the genetic basis of the disorder. There is evidence that, in ADHD, there is an incompletely penetrant autosomal-dominant gene; the penetrance of the gene being calculated as 46% in boys and 31% in girls.

Molecular genetic studies have been particularly fruitful in evaluating the neurochemical basis of the disorder. Genes involved in the dopaminergic system were considered to be important as the most effective symptomatic treatment of the condition has been methylphenidate and dextroampheta-mine, drugs which potentiate the release, and inhibit the reuptake, of dopamine. In addition, the involvement of dopamine in ADHD is further implicated by the increased vulnerability of these patients to drug abuse. As discussed in Chapter 15, the dopaminergic system has been implicated in reward mechanisms so the search for abnormalities in the genes encoding for different aspects of them seems a reasonable start.

Of the genes for the dopamine receptors which have been studied, the candidate gene for the dopamine D4 receptor has been shown to be positively associated with ADHD. However, not all investigators have verified this finding.

One of the major limitations in studies of the genetics of behavioural disorders in children arises from the overlap with other conditions. For example, nearly 50% of the patients with ADHD also have co-morbid conduct disorders. In addition, a subtype of the disorder may exist in those children in which the disorder persists into adulthood. An additional problem arises from the overlap between ADHD and bipolar disorder; this has been estimated to be as high as 16%.

In CONCLUSION, although positive genetic studies have been reported and subsequently replicated, the results must be treated with caution as they are based on small sample sizes with restricted statistical power and complicated by co-morbid illnesses. Nevertheless, preliminary evidence suggests that ADHD, like many major psychiatric disorders, does have a genetic basis.

The impact of molecular neurobiology on psychopharmacology: from genes to drugs

About 150 years ago, Charles Darwin observed that ''those who make many species are the 'splitters', and those who make few are the 'lumpers' ''. Today, the ''splitters'' dominate research in the life sciences. Such researchers can generate massive quantities of data on genes and their networks, proteins and their pathways and the numerous cascades of messenger molecules that ultimately result in a physiological response. Technological progress in recent years has enabled the genome of species as diverse as the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster to the mouse and man to be unravelled, thereby opening up the possibility not only of identifying genes that are responsible for physiological processes but also those that are aberrant and cause genetically based diseases.

Few would deny the importance of such research, but the very success of the ''splitters'' has had a seriously detrimental effect on the equally important role of the ''lumpers'', who attempt to integrate the molecular/cellular approach with the behavioural/psychological consequences. As a consequence, the ''lumpers'' are becoming a threatened species of researchers. There are several reasons for this, not the least of which is the widespread opposition to vivisection and the lack of training in behavioural pharmacology in university courses. As a consequence, research (and funding for behavioural research) has declined in prestige. This has had an adverse impact not only in areas of basic life science research but also in the pharmaceutical industry where the ultimate validation of the therapeutic potential of a new molecule depends on behavioural pharmacology. As a senior neuro-pharmacologist has recently remarked ''Many can genotype but few can phenotype''.

Despite this unfortunate disparity between molecular neurobiology and behavioural pharmacology, it is essential that the neuropharmacologist and biological psychiatrist are fully conversant with the basic concepts of the subject in order to appreciate both its success and limitations.

An outline of the terminology

Pick up any intelligent newspaper or magazine, view any television programme dealing with the life sciences or medicine, and the impact of cloning is likely to be discussed. To understand the basis of cloning, it is necessary to consider how bacteria have evolved to resist infection by external sources of genetic material. It has long been recognized that if a virus could infect one strain of bacteria, it could then also infect other bacteria of the same strain but not those of a different strain. Thus virus infection was shown to be restricted to a particular strain, a restriction now known to be due to two classes of enzyme, namely the methylases, which modify bacterial DNA marking them as ''self'', and the destruction enzymes, which act as molecular ''scissors'' and can destroy foreign DNA.

Restriction enzymes are sequence-specific in that they cut DNA at specific locations along the nucleotide chain. While some of these enzymes yield ''blunt'' ends to the resulting DNA fragment, others make staggered cuts in the DNA chain to produce ''sticky'' ends. Over 250 restriction enzymes are now commercially available.

Cloning would not be possible without restriction enzymes. DNA chains with a ''sticky'' end act like molecular ''Velcro'', thereby enabling two pieces of DNA with complementary nucleotide sequences to be joined together. The linking of the DNA strands is brought about by the enzyme DNAligase which permanently joins the assembled DNA sequences with covalent bonds, thereby producing a recombinant DNA molecule.

The next stage is to ensure that the recombinant DNA molecule is copied by the enzymes which synthesize nucleic acids. These DNA and RNA polymerases synthesize an exact copy of either DNA or RNA from a pre-existing molecule. In this way the DNA polymerase duplicates the chromosome before each cell division such that each daughter cell will have a complete set of genetic instructions which are then passed to the newly formed RNA by RNA polymerase. While both DNA and RNA polymerase require a preformed DNA template, some viruses (such as HIV) have an RNA genome. To duplicate that genome, and incorporate it into a bacterial or mammalian cell, the viruses encode a reverse transcriptase enzyme which produces a DNA copy from an RNA template.

Thermostable DNA polymerases have now been produced for polymerase chain reaction (PCR) studies in which specific segments of the DNA molecule can be mass produced from minute quantities of material. RNA polymerases are then used to create RNA transcripts from cloned genes in vitro. Reverse transcriptases have their specific uses in molecular biology. These enzymes are used to form ''cDNA libraries'' which are batteries of molecules each one representing a single gene expression. Such DNA libraries can then be analysed to determine which genes are active under different conditions and in different tissues. cDNA libraries are now used experimentally in microarray assemblies to detect gene changes following drug treatment. This will be discussed further later in this chapter.

In a typical experimental situation, the gene of interest is incorporated into a plasmid, which is a natural vector used by either a bacterium or other cell type. To transfer the DNA fragment of a gene, the plasmids are digested with one or two restriction enzymes and the desired fragment joined into a single DNA recombinant molecule using DNA ligase. To express the new gene in vitro, the plasmid containing the recombinant DNA is then incubated with an RNA polymerase to form new RNA which is then used to programme an in vitro system which translates the information necessary for the synthesis of a new protein.

The foregoing is only intended to give a brief overview of the mechanisms behind cloning. So far, the impact on diseases in man has been limited to experimental approaches to the treatment of cystic fibrosis and rare conditions in which a recessive gene is responsible. However, cloning techniques have provided important information in producing animals, usually mice, which have been manipulated to express or remove genes that are implicated in psychiatric disorders. Such ''knock-out'' and ''knock-in'' mice now provide important information in which specific genes can be studied for their effects on behaviour, which may ultimately be an important contribution to understanding the genetic basis of psychiatric and neurological diseases.

Genetically modified mice and their importance in psychopharmacology

Just as adding genes from a complex to a simpler organism (for example, from man to a fruit fly) may be helpful in understanding the function of a gene, so it may help to understand how a gene functions by eliminating it. To date, most gene ''knock-out'' studies have been undertaken in mice because of:

(a) the relative ease with which genes can be manipulated and eliminated;

(b) the relatively rapid rate at which mice breed;

(c) their well established and relatively complex behaviour.

The success, and also the limitations of the gene elimination strategy can be illustrated by studies on the molecular basis of memory and learning. In the early 1980s it had been shown that the glutamate NMDA receptor was an essential component of memory formation, the term ''long-term potentia-tion'' (LTP) being applied to the molecular mechanism involved. The drugs which were then available were limited in their specificity for the NMDA receptor but by selectively deleting genes thought to be involved in memory it was possible to identify the precise components of the NMDA-linked messenger complex located in the hippocampus. Further studies enabled genes ranging from those encoding neurotransmitter receptors, protein kinases and transcription factors to be identified. However, there are limitations to these techniques which should be considered.

A major problem with ''knock-out'' technology relates to the need to delete the gene at the very early stage of embryonic development. Often this results in the death of the neonate. Even if the gene is not essential for survival, it could have a key role to play in development that is unrelated to neuronal plasticity. Thus the deficits in learning and memory seen in the mature mouse could be the result of a developmental defect rather than a specific abnormality in the NMDA receptor complex. Alternatively, the deletion of a gene that from experimental studies might be expected to have a major effect on learning and memory in practice may have no apparent effect. This is due to the mechanism of compensation whereby other genes take over the function of the deleted gene.

Thus developing ''knock-out'' mice to understand the function of a particular gene gives little information on the timing when the gene becomes active. Nor does it necessarily reflect the location of the gene in the intact (wild-type) mouse or indeed, the long-term effect of the nervous system on its function. Nevertheless, these are largely technical drawbacks that will undoubtedly shortly be solved. In principle, studying the actions of psychotropic drugs on genetically modified animals will allow the detrimental effects of a deleted gene on the general health of the animal to be avoided. Such an approach will also allow investigations of the interactions between neuronal signalling pathways by assessing the synergistic interactions between the behavioural and other biological effects of the deleted gene and drugs.

The importance of genomics to psychopharmacology

Virtually every physical and psychiatric disorder has a genetic component. However, the vast majority of these diseases have a complex pattern of inheritance and there is no evidence that a single genetic locus is responsible for any of the major psychiatric disorders. Rather it appears that multiple alleles (gene products) occurring at multiple sites within the genome interact to produce a vulnerability to the disorder. The enthusiastic reception for the unravelling of the human genome rests largely on the promise that it will soon lead to an understanding of the pathological basis of most diseases which, in turn, will aid the development of more effective therapeutic treatments.

Following the sequencing of the human genome it was found that there were between 30 000 and 40 000 genes that code for proteins, only twice as many as occur in the fruit fly or the nematode worm! However, it does appear that human genes are more complex than those of flies and worms in that they generate a large number of proteins due to the alternative ways of splicing the molecules. Hopefully knowledge of the human genome will enable genes to be identified that convey a risk for psychiatric diseases in addition to those genes which are linked to a therapeutic response to drug treatment. Knowledge of the latter forms the basis of pharmacogenomics which, hopefully, will eventually lead to the development of specific treatments for the individual patient.

The potential value of pharmacogenomics can be illustrated by two examples involving the response of individual patients to antidepressants. In this approach, the potential importance of the cDNA microarray technique for identifying changes in thousands of individual genes that are expressed in the mouse brain is now widely accepted. Experimental studies have indicated that different antidepressants exert distinct effects on gene expression in the mouse brain, these differences becoming more marked as the duration of the treatment increased. Such findings may eventually lead to an individualized treatment strategy for depressed patients based upon their cDNA analysis.

At the practical clinical level, individual differences in the pharmaco-kinetic characteristics of antidepressant drugs have been more successful. It is well established that the enzymatic activity of different allelic forms of the cytochrome P450 oxidase system in the liver is particularly important in the metabolism of many psychotropic and non-psychotropic drugs (see pp. 91-94). Of the major forms of cytochrome P450 in man, the 2D6 isozyme is particularly important in the metabolism of antidepressants and a potential cause of drug interactions. Three of the five commonly available SSRI antidepressants (fluoxetine, paroxetine and sertraline) undergo autoinhibition of this isozyme and can therefore increase the tissue concentration of a more toxic drug (for example, an antiarrhythmic or beta-blocker) should it be given concurrently.

Over 50 allelic variants of the cytochrome P450 2D6 gene have been identified, including individuals who lack the gene and others who have multiple copies of the gene. This means that an individual (the functional genotype) can either be normal, a slow or an ultra-fast metabolizer of a drug that passes through the 2D6 pathway in the liver. Slow metabolizers will therefore be at an increased risk for adverse effects while the rapid metabolizers will have little benefit from the normal doses. Thus genotyping the enzymes that metabolize the commonly used psychotropic drugs could help to optimize the response, and to indicate the potential for adverse drug effects, of the individual patient.

A new term has recently been introduced to cover the application of pharmacogenomics to the design of drugs for the individual patient, namely theranostics (from therapeutics+diagnostics). This approach involves creating tests that can identify which patients are most suited to a particular therapy and also to provide information on how effective this drug is in optimizing the treatment. Theranostics is said to adopt a broad dynamic and integrated approach to therapeutics which may be of practical relevance in differentiating diseases which are closely associated diagnostically (for example, Alzheimer's disease and Lewy body dementia) by applying a combination of immunoassays that enhance the differential diagnosis. Several biotechnological companies now specialize in designing immunoassays for application to infectious diseases such as hepatitis by genotyping the hepatitis C virus for example. There are six genotypes of the virus known: genotype 1 is more resistant to standard therapy (requiring at least one year of continuous therapy) whereas the other genotypes usually respond to treatment within 6 months. Clearly a knowledge of which viral genotype is present is important in determining the duration of treatment in the individual patient and hopefully it will soon be possible to extend such approaches to the drug treatment of central nervous system disorders.

Applying pharmacogenomics to the pharmacodynamic aspects of psychopharmacology is still at a very early stage of development, largely because so little is known of the psychopathological basis of the major psychiatric disorders or of the mechanisms whereby psychotropic drugs work. In depression, for example, it is widely assumed that the inhibition of the serotonin transporter on the neuronal membrane is ultimately responsible for the enhanced serotonin function caused by the SSRI antidepressants. The serotonin transporter is structurally complex. The promoter region of the transporter, to which serotonin is linked before it is transported back into the neuron following its release into the synaptic cleft, exists in several polymorphic forms which are broadly categorized into the long and short forms. It is known that when the polymorphic form occurs in which an additional 44 pairs of nucleotide bases are inserted, there is a higher transcription rate and a greater degree of binding of serotonin to the promoter region. The practical importance of this finding is that depressed patients with the long form of the transporter show a better response to SSRIs than those with the short form. In bipolar patients, there is an indication that the short form of the promoter is more likely to result in the precipitation of a manic episode if given an SSRI during the depressive phase of the disorder. There is also some evidence that the short and long forms of the transporter may be correlated with the frequency of extrapyramidal side effects and akathisia, which is sometimes caused by SSRIs.

There are two caveats that should be taken into account with regard to the application of pharmacogenomics. Drug response is as complex as the underlying genetic basis of the disease due not only to the genotypic variation taking place at mostly unknown chromosomal loci, but also from variations in gene expression, post-translational modification of proteins, pharmacokinetic features of the drugs, the effect of diet, drug interactions, etc. One would therefore anticipate that the effects of individual genes on the drug response are relatively slight. Thus it has been shown in studies of pharmacogenetic markers that they only confer a twofold increased likelihood of predicting drug response. However, the widespread application of microarray technology, whereby information on thousands of genes can be determined simultaneously, may help to overcome the limitations of the candidate gene approach, the method which until now has been used to obtain information on a few genes presumed to be involved in the underlying pathology of a disease or its response to drug treatment.

Another aspect requiring attention concerns the statistical evaluation of the results. For example, recently it has been shown that in a study of asthma one genotype had a 100% positive predictive value for non-response to a drug. However, because the susceptibility genotype only occurs in less than 9% of patients, in practice less than 10% of the non-response to treatment can be attributed to this abnormal genotype. In this case, it has been calculated that avoidance of the drug as a result of pharmacogenomic profiling would only improve its efficacy from 46% to 51%. Thus the reliance on candidate gene variation, which ranges from 2% to 7%, is currently not in the range for practical application.

In CONCLUSION, molecular genetics is providing important tools that enable the physiological role of specific receptors and enzymes to be elucidated. In situ hybridization is a powerful technique for locating receptors in specific brain regions and in studying the influence of drug treatment on these receptors. The accuracy, specificity and sensitivity of such a technique is substantially greater than any other available technique. Transgenic mice and ''knock-out'' mice are also providing valuable models of human disease that have not been obtained by other methods. With regard to psychiatric illnesses, molecular genetic techniques are being used in human genome screening which are designed to locate those genes that may be responsible for bipolar disorder and schizophrenia. By collecting cell lines from family pedigrees, it may be possible to determine the genes that contribute to alcoholism and Alzheimer's disease in addition to those involved in schizophrenia and the affective disorders. This may eventually lead to the identification of new methods for the pharmacotherapy of such conditions.

Anxiety and Depression 101

Anxiety and Depression 101

Everything you ever wanted to know about. We have been discussing depression and anxiety and how different information that is out on the market only seems to target one particular cure for these two common conditions that seem to walk hand in hand.

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