Anorexia Nervosa

ANOREXIA NERVOSA: Biological data focused in the genetic field Giotakos

Orestis (MD, MSc)

SUMMARY Anorexia nervosa is a chronic and severe psychiatric disorder, which occurs 10-20 times more often in women, is estimated to occurs in about 0.5-1% of adolescent girls, is characterized by a profound disturbance of body image and a relentless pursuit of thinness, and has a range of mortality 5-18%. Biological factors, such as genetic background, neurotransmitter and neuroendocrine systems, social factors, such as, society's emphasis on thinness, and psychological factors, such as, personality traits and ego defense mechanisms, contribute to the pathogenesis of anorexia nervosa. Abnormalities of both 5HT neurotransmission and HPA axis activity are found in depression, OCD, and eating disorders, but abnormal 5HT neurotransmission seems to be the key factor in anorexia nervosa. Serotonin has also implicated in the pathogenesis of depressive and obsessive-compulsive symptoms which are common features in anorexia nervosa, has been found to participate in the caloric-requirement system and the appetite system, to demonstrate its action in physical animal model, and to provide therapeutic modulations in anorexic patients. Some resent genetic studies have reported positive association between the -1438/A allele of the -1438A/G 5HT-2A-promoter receptor gene polymorphism and anorexia nervosa, but some others have failed to replicate this finding. Tryptophan hydroxylase gene, serotonin transporter gene, dopamine type 1 to 4 receptor genes, noradrenergic α-3 receptor gene, CRH gene, and Ob gene, provide a hopeful area for future genetic research in patients suffering from anorexia nervosa. Key Words: Anorexia Nervosa, Dopamine, Serotonin, receptor gene, genetic association study.

1. CLINICAL AND ETIOLOGICAL ISSUES IN PATIENTS WITH ANOREXIA NERVOSA

1.1. HISTORY Official history has it that the French psychiatrist Ernest-Charles Lasegue (1816-1833) and the English physician Sir William Withey Gull (1816-1890) simultaneously and independently presented descriptions of a new syndrome of eating disorders among adolescent women. Lasegue described his clinical and therapeutical experiences with eight female patients whose eating habits were severely disturbed in a paper which appeared in 'Archives Generales de Medicine' in April 1873. Lasegue named this syndrome 'Anorexie hysterique'. A month after Lasegue's paper appeared in English medical press, the British physician Sir William Gall gave a lecture at the Clinical Society in London. Accounts of this lecture by listeners were published shortly afterwards in 1873. When the lecture itself was published in 1874, its title introduced the term since conferred on the syndrome: 'Anorexia Nervosa (Anorexia Hysterica, Apepsia Hysterica)' [1].

1.2. CLINICAL FEATURES Anorexia Nervosa is characterized by a profound disturbance of body image and the relentless pursuit of thinness, often to the point of starvation. In DSM-IV (1994), the criteria consist of a persistent refusal to maintain body weight at or above a minimum expected weight (< 85% of expected weight) or a failure to gain the expected weight during a period of growth. The patient exhibits disturbance of body image, fear of becoming fat, and peculiar behavior regarding food. They hide food all over the house and frequently carry large quantities of candies in their pockets. Ritualistic exercising, extensive cycling, walking, jogging, and running are common activities [2].The onset of anorexia nervosa usually occurs between the ages of 10 and 30 years, with the maximum frequency at 17 to18 years of age. About 85% of all anorexia nervosa patients have the onset of the illness between the ages of 13 and 20 years. Amenorrhoea has been included as a diagnostic criterion for females in DSM III R (1987) and DSM IV (1994) [1]. However, Garfinkel et al [3], using data from a large community epidemiological survey, found that amenorrhoea did not discriminate between women with anorexia nervosa and women with all the features except amenorrhoea across a number of relevant variables. Unfortunately, the term "anorexia", meaning loss of appetite, is a misnomer, because the loss of appetite is usually rare until late in the disorder. Evidence that the patients are constantly thinking about food is their passion for collecting recipes and preparing elaborate meals for others. Some patients cannot continuously control their voluntary restriction of food intake, and so they have eating binges [1].

1.3. SUBTYPES DSM-IV (1994) introduced a new item related to the identification of two types of anorexia nervosa, the 'restricting type' and the 'binge eating-purging type'. Binge eating-purging is common among anorexia nervosa patients and it develops in up to 50% of them. They share many features with persons who have bulimia nervosa, they tend to have families in which some members are obese, and they are likely to be associated with substance abuse, impulse control disorders, and personality disorders. Restricting anorexia nervosa persons limit their food selection, take in as few calories as possible, and often have obsessive-compulsive traits. Both types of persons are preoccupied with weight and body image, they may exercise for hours every day, exhibit bizarre eating behaviors, and may be socially isolated, depressive and with diminished sexual interest. Some anorexia nervosa persons purge but not binge [1].

1.4. COURSE AND THERAPY The course of anorexia nervosa varies greatly : spontaneous recovery without treatment, recovery after a variety of treatments, a fluctuating course of weight gains followed by relapses, a gradually deteriorating course resulting in death caused by complications of starvation. Studies have shown a range of mortality rates from 5 to 18 percent. Dynamic expressive-supportive psychotherapy is sometimes used in the treatment of anorexia nervosa patients. Many clinicians prefer cognitive-behavioral approaches to monitor weight gain and maintenance and to address eating behaviors. Pharmacological studies have not yet identified any medication resulting in definitive improvement. Some reports support the use of cyproheptadine, a drug with antihistaminic and antiserotonergic properties, in the restricting type of anorexia nervosa. Amitriptiline (mainly noradrenergic antidepressant), has been reported to have some benefit. Clomipramine (mainly serotonergic antidepressant), pimozide, and chorpromazine (classical antipsychotics), have not yielded positive responses. Uncontrolled trials of fluoxetine (a selective serotonin reuptake inhibitor), have resulted in some reports of weight gain [1].

1.5. EPIDEMIOLOGY Eating disorders of various kinds have been reported in up to 4% of adolescent and young adult students. Anorexia nervosa is estimated to occur in about 0.5 to 1% of adolescent girls (less than 0.1% of the general population). It occurs 10 to 20 times more often in females than in males. Anorexia nervosa, although less common in men than in women, appear to display striking similar features in affected individuals of the two genders [5]. Although the disorder was initially reported most often among upper classes, recent epidemiological studies do not show that distribution. A recently reported case controlled study from the Aberdeen psychiatric case register found no class differences between cases of affective disorders and cases of anorexia nervosa [6]. It seems to be most frequent in developed countries, and it may be seen with greatest frequency among young women in professions that require thinness, such as modelling and ballet. In a large epidemiological study in the female population of northeast Scotland, it was found the that rates of refferal for female subjects with anorexia nervosa have been greatly increased since the 1960s. These rates likely reflect a genuine increase in incidence, but the data suggest that less severely ill patients are now being referred [7]. However, Willi et al [8], did not find an increase in incidence between 1973-1975 and 1983-1985, in the canton of Zurich in Switzerland. 1.6. FAMILY AND TWIN STUDIES: A genetic component to anorexia nervosa was first considered when Theander [9] in 1970, noted the increased prevalence in the sisters of sufferers. There is a significantly higher frequency of anorexia nervosa in first-degree relatives of anorexic patients (3.22%) than in the relatives of controlled patients (0.02%) [10]. Twin studies indicate a greater concordance of anorexia nervosa in monozygotic twins (33 to 56%) than dizygotic twins (0 to 11%) [11, 12]. Using genetic modelling techniques on these data, Treasure & Holland [13] in 1991, estimated a heritability of 76% for anorexia nervosa. The same authors used the Eating Disorder Inventory to explore the heritability of anorexia nervosa in a female twin sample in which at least one twin was affected. They obtained high values for two of the subscales; the 'drive for thinness' and 'body dissatisfaction' subscales had heritability estimated of almost 100%. Rutherford et al [14] administered the Eating Disorder Inventory to 147 normal monozygotic and 99 normal dizygotic twin pairs. A heritability value of 41% was obtained for the overall scores, while the "body dissatisfaction" and "drive for thinness" subscales had heritability values of 52% and 44% respectively. Walters et al [15] in 1995, explored the epidemiological characteristics and risk factors of anorexia nervosa administering structured interviews to a population based on a sample of 2.163 female twin. Lifetime prevalence estimates of 0.51%, 1.62%, and 3.70% were obtained for the computer narrow, clinical narrow, and clinical broad diagnoses, respectively. Co-twins of twins with anorexia nervosa were at significantly higher risk for lifetime anorexia nervosa, bulimia nervosa, major depression, and current low body mass index. Significant comorbidity was found between anorexia nervosa and major depression, bulimia nervosa, generalized anxiety disorder, alcoholism, phobias, and panic disorder. Finally, Gorwood et al [16] in 1998, in a meta-analysis study, found 55 non-systematically recruited twin studies (with at least one patient affected with anorexia nervosa), describing 66 monozygotic and 24 dizygotic twins. Concordance rates were 44% for monozygotic and 12.5% for dizygotic twins. The four systematically recruited twin studies showed that 20 out of 40 monozygotic twins (50%) were concordant for anorexia nervosa versus one out of 32 dizygotic twins (3%).

1.7. ETIOLOGY Biological, social, and psychological factors are implicated in the causes of anorexia nervosa.

1.7.1. BIOLOGICAL FACTORS: Twin studies (have already discussed), caloric requirements, neurotransmitter dysregulations (are discussed extensively bellow), CT & PET findings, and hormonal (FSH, LH,TSH,CRH) suppression indicate the biological component of etiology. It seems that anorexic patients may have abnormal caloric requirements. To maintain stable weight after weight restoration, restricting anorexic patients require a significantly higher caloric intake than do bulimic anorexic patients. Elevated caloric requirements, when coupled with reduced food intake, may particularly contribute to relapse in these anorexic patients [17]. Furthermore, CT studies reveal enlarged sulci and ventricles in anorexia nervosa patients during starvation, a finding that is reversed by weight gain [18]. In one PET- scan study, caudate nucleus metabolism was higher in the anorectic state than after the episode [1]. The hypothalamic-pituitary-gonadal axis (HPG) has excited particular interest because amenorrhea is a central feature of anorexia nervosa and its onset and recovery show some dissociation from weight; as many as 25% of cases develop amenorrhea prior to significant weight loss and even following weight restoration there may be considerable delays in the re-establishment of normal menses. The levels of gonadotropins, luteinizing hormon (LH) and folicle-stimulating hormone (FSH), are uniformly low which increase with weight recovery in most but not all patients. The levels and the circadian pattern of LH, FSH and oestrogens, in the emaciated anorexic is similar to that in the pre-pubertal state. In addition, anorexia nervosa mainly occurs in the age of puberty. The onset of mammalian female puberty requires the functional activation of a specialized group of hypothalamic neurons that produce luteinizing hormone-releasing hormone (LHRH), the neurohormone governing sexual maturation and reproductive function. A combination of a decrease in transynaptic inhibitory tone (via GABA and opioid peptides) and an increase in excitatory inputs (via norepinephrine and neuropeptide Y) to LHRH neurons is thought to be instrumental in bringing about the initiation of puberty. Recent studies have raised the possibility that glial cells may also play a role in the control of LHRH. The finding that about 50% of glial cells have estrogen receptors further supports the notion of a direct action of estrogen on hypothalamic astrocytes [19]. The hypothalamic-pituitary-adrenal axis (HPA) has also been the subject of much study particularly in view of its disturbance in affective disorder as well as in anorexia nervosa. An elevation of 24h mean plasma cortisol concentrations is well established and also raised levels of free-cortisol in the urine. The cortisol production rate of patients with anorexia nervosa declines with slight weight gain, a finding opposite to that noted when malnourished non-anorexic persons gain weight on refeeding. The majority of anorexics fail to suppress cortisol normally following the administration of dexamethasone. Elevated CSF levels of corticotropin-releasing hormon (CRH), further support the hypothesis that the hypercortisolism of anorexics reflects a defect at or above the hypothalamus. A hyperdynamic HPA axis is not a feature of simple malnutrition but its significance in anorexia nervosa is still unclear. Finally, many anorexic patients demonstrate the low triiodothironine (T3) syndrome which is also seen in malnutrition. This syndrome probably contributes to a number of clinical features of anorexics including the low basal metabolic rate, dry skin, tachycardia and elevated serum cholesterol [1].

1.7.2. SOCIAL FACTORS: Anorexia nervosa patients find support for their practices in society's emphasis on thinness and exercise. No family constellations are specific to anorexia nervosa.

1.7.3. PSYCHOLOGICAL FACTORS: Psychoanalytic clinicians who treat patients with anorexia nervosa generally agree that those young patients have been unable to separate psychologically from their mothers. The body may be perceived as though it were inhabited by an introject of an intrusive and unempathic mother. Starvation may have the unconscious meaning of arresting the growth of that intrusive internal object and thereby destroying it. The multifactorial model of anorexia nervosa Biological Level Psychological Level Socio-cultural Level Predisposing Factors Genetic factors, Female sex, Caucasian race Female gender Identity and autonomy problems Middle and upper class, Industrializa- tion, Pressures to be slim Precipitating Factors Neurochemi- cal and endocrinologi- cal activations Sexual maturation, Stressful events Pressure to achieve and to be slim. Riskful occu- pation (mode- lling, ballet) Perpetuating Factors Neurochemi-cal and endocrinologi-cal changes Isolation, Fear of loss control As above

2. NEUROTRANSMITTER SYSTEMS WHICH MAY BE INVOLVED IN GENETIC SUSCEPTIBILITY TO ANOREXIA NERVOSA

2.1. DOPAMINERGIC SYSTEM: The first argument for the involvement of the dopaminergic system is suggested by the findings that Apomorphine (D2 agonist), L-Dopa (enhancing dopamine synthesis), and amphetamine (releasing dopamine from presynaptic stores) have been shown to be anorexigenic , an action selectively blocked by dopamine antagonists such as haloperidol and chlorpromazine. Second, weight loss of the anorectic patient is often associated with hyperactivity and distortion of body image. Dopamine agonists (such as amphetamine) lead to both loss of appetite and hyperactivity. Third, in amenorrhoea, a decrease in blood oestrogen and luteinizing hormone (LH) and the absence of cyclical secretion of LH has been observed in female anorectic patients. Dopamine agonists inhibit LH-RH and stimulate the secretion of prolactin. Fourth, some clinicians [20] consider anorexia nervosa as a pathology belonging to the spectrum of 'addictive behaviors'. Dopamine is released in anticipation of feeding, and many experimental data show the implication of mesolimbic dopaminergic systems in behavior reward and reinforcement. Furthermore, Ebstein R P et al [21] in 1997, found that, both Dopaminergic and serotonergic (5HT-2C) receptor gene polymorphisms are associated with the human personality trait of 'reward dependence' assuming that the relationship between 5HT and dopaminergic activity presumed to exist for the regulation of drug-induced motor activity extends to the regulation of brain stimulation-induced reward. Finally, knock-out mice give many interesting informations: Mice with an inactivated tyrosine hydroxylase gene are born hypoactive and stop feeding a week after birth [22]. Absence of D2 receptors leads to animals that are akinetic and bradykinetic in behavioral tests, but absence of D3 receptors leads to mice that are hyperactive in exploratory tests [23, 24]. Lastly, dopamine transporter (DAT) lacking mice result in hyperlocomotion, low weight, and no effect of amphetamine and cocaine [25]. The dopaminergic system may thus intervene not only in the general scheme of anorexia nervosa (eg the anorexigenic effects of dopamine agonists), but also in the more specific characteristics such as amenorrhoea, hyperactivity, distortion of body image, and in traits suggesting a possible addictive tendency in behavior. 2.2. NORADRENERGIC SYSTEM : The possible role of catecholamines in hypothalamic regulation as well as affective disorders suggest that noradrenaline could participate in the genesis of anorexia nervosa [26]. For example, amphetamine, which block reuptake of NA, reduces hunger sensation and food intake in a dose dependent manner in humans. Hypothalamus is largely innervated by noradrenergic fibers. Furthermore, it is known that NA is involved in secretion of gonadotrophines, reduces the secretion of CRH, participates in the hypothalamic control of TRH secretion, and it is also regulates the hypothalamic liberation of vasopressin.

2.3. OPIATE, AND OTHER SYSTEMS : Evidences that endogenous opiate activity could contribute to a deregulation of eating behavior exist. Administration of opiate agonists increases food intake in rats whereas antagonists decrease feeding behavior. Similarly, administration of opiate antagonists, such as naloxone, in humans diminish food intake. Different peptides appear to influence appetite control. Neuropeptide Y (NPY), for example, infused in the CNS of rats leads to significant food consumption, while infusion of cholocystokinin (CCK) decreases food intake in both animals and man. Genetic factors appear to be also involved in the deposition to store fat and the way body uses dietary energy. The ob gene encodes for leptin, a fat-secreted hormone secreted by adipocytes which regulates ingestive behavior and energy balance. Ob gene thus also belongs to candidate genes in anorexia nervosa, and has been recently localized in humans on chromosome 2p21 [27]. Leptin emerges as a hormone that may not only control food intake and energy expenditure but may also regulate neuroendocrine function and certain aspects of human behavior. Moreover, falling leptin concentrations may contribute to the pathophysiological implications of restrained eating and binge eating [28]. Von Prittwitz et al [29] in 1997, in the only study which identify a relationship between a score on a psychometric scale and leptin levels, determined the serum leptin levels in 136 underweight and 49 overweight students, who completed the Three-Factor Eating Questionnaire. They found that individuals with high restraint scores have lower serum leptin concentrations than low scoring subjects .

2.4. SEROTONERGIC SYSTEM

2.4.1. Neurochemical evidences : It seems clear that 5-HT is involved in the regulation of human eating, as it is in animals. Fenfluramine, a serotonin agonist, reduces appetite and increases satiety, while ritanserin, a 5-HT2 antagonist neutralize this anorexigenic effect [30]. Intraventricular injections of neurotoxins that block or destroy serotonergic neurons also provoke hyperphagy. 5-HIAA concentrations in the CNS of anorectic patients showed reduced levels and normal levels after weight gain. Brewerton et al [31] found that anorectic women showed a decrease in prolactin response after stimulation with l-tryptophane or mCPP (serotonin agonists)in the acute phase as well as after weight gain compared to controls who did not show a decrease prolactin response. Increased tryptophan levels in plasma and CSF of anorectic cancer, uremic, and cirrhotic patients have been reported. It is conceivable that the increased brain tryptophan concentrations, and the consequent increased brain serotonergic activity, may mediate the onset of anorexia in these patients [32]. Ingestion of glucose (pure carbohydrate) will lower plasma tryptophan but increase the ratio of tryptophan to other large neutral amino acids (LNAA). This rise in the tryptophan ratio is blocked by the addition of protein. People with severe obesity have lower plasma tryptophan ratios than lean subjects. In underweight anorexics a carbohydrate meal fails to raise the plasma tryptophan level in comparison with the level in normal controls, while a protein meal causes a marked drop in the tryptophan ratio. These data would imply a reduced availability of tryptophan of the brain. In keeping with these findings, there is a low level of the serotonin metabolite 5HIAA in the CSF of underweight anorexics, which normalize after weight gain. These findings are in different direction with the above Laviano's hypothesis but they also support the suggestion that serotonin neurons in the brain are somehow involved in physiological and behavioral responses to diatery adjustments. Treasure J [33] in 1997, proposed a physical animal model which indicate the central role of 5-HT in anorexia nervosa. She described a syndrome in pigs which resemble that of anorexia nervosa in humans. The affected animals restrict their intake of normal food, and spend more time on nonnutritive hyperactive behavior. Interestingly, the psychoactive drug amperozide (a 5-HT receptor antagonist) prevents and successfully treats these condition [34]. Serotonin may also be involved in important symptoms of anorexia nervosa, such as the initial onset of anorexia nervosa which can be associated with a stressful event, the psychosexual immaturity, the predominance in females, the obsessional premorbid personality, and the comorbidity between anorexia nervosa and depressive disorders. 2.4.2. Serotonergic system and Stress, in patients with Anorexia Nervosa : Brain 5-HT neurons discharge in a slow and rhythmic manner that is a manifestation of their endogenous pacemaker activity. This regulation firing during waking creates a steady synaptic release of 5-HT which provides a tonic excitatory drive that modulates motor system neuronal activity. The anticipation of motor activity by 5-HT neurons suggest that they serve a priming function for motor output. Furthermore, 5-HT's involvement in autonomic and neuroendocrine regulation serves a support function for the demands of changes in the level of motor output, such as increased oxygenation of the blood and increased carbohydrate consumption for maintaining a stable glucose supply to the brain. Illness onset of anorexia nervosa is frequently marked by stressful events, such as in many psychiatric disorders, with serotonin playing a critical role in the response to stress [35]. 2.4.3. Serotonin and psychosexual immaturity in anorexia nervosa: Psychosexual immaturity as well as the experience of conflicting sexual feelings are, according to many authors, at the heart of anorexia nervosa. Serotonin has been shown to inhibit sexual behavior in certain species, for example in the rat after destruction of serotonergic pathways in the ventro-medial hypothalamus. Furthermore, the penile erection caused by 5-HT receptor agonists is mediated by 5-HT-2C receptors, and 5HT-2A receptor stimulation suppresses sexual performance. Bagdy et al [36], found that stimulation of 5HT-2C receptors in rats induces excessive grooming, penile erection and increased oxytocin secretion, and that stimulation of 5HT-2A receptors causes a further increase in plasma oxytocin concentration, but inhibits grooming and penile erection. 2.4.4. Sex-dependent effects of serotonin and oestrogens and their relation with anorexia nervosa : The hypothesis of serotonin involvement in anorexia nervosa has been further supported from the fact that serotonin circuits show variations that are partially sex-dependent. For example, d-fenfluramine and mCPP have greater hypophagic effect in the female rats. Variations during menstrual cycle and secretion of prolactin during food restriction exist only in females. Serotonergic mechanisms are implicated in behaviors (aggression, sexual behavior, impulse control) and psychiatric disorders (depression, eating disorders, suicide) which are related to gender. Some data are available about gender differences in serotonargic activity: High-density [3H]imipramine binding sites, which are located on the presynaptic serotonergic terminals, are more concentrated in female than in male rats. Tryptophan, serotonin, and 5-HIAA are present in higher levels in females than in males in several rat brain regions. There are similar gender differences in the level of 5-HIAA in the CSF of patients with chronic pain or depression [37]. Some investigators report concentrations of serotonin reuptake sites in platelets which are higher in healthy women that man, and in post-mortem studies seems that gender influences frontal [3H]imipramine binding sites, particularly in the right orbital cortex [38]. These animal and human data support the hypothesis of a higher serotonergic activity, at least at the presynaptic level, in females compared to males. Biver's et al [39] PET findings, support the evidence for the high presynaptic activity of the female serotonin system and the consequent apparent 'down-regulation' of 5HT2 receptors in women compared to men. These results support the view that distinct liability for men and women to suffer from some psychiatric diseases responding to serotonergic agents may be related to differences in brain serotonin receptors. Furthermore, oestrogen is thought to exert powerful effects on mood, mental state and behavior in women. The role of oestrogen in affective disorders is suggested by the fact that menopausal and postnatal depression are associated with a massive drop in plasma oestrogen concentrations, and oestrogen has been reported to be effective in the treatment of Depression and Alzheimer's disease in women. Differences in the age of onset and symptoms of schizophrenia in women compared with men has also implicated oestrogen in schizophrenia. Recent reports have been shown that oestrogen can stimulate a threefold increase in the amount of 5HT-2A receptor messenger RNA in the dorsal raph nucleus of the female rat. In addition, the binding sites were increased in anterior frontal, anterior cingulate and the primary olfactory cortex and nucleus accubens, essential brain regions for cognition, emotion, and neuroendocrine control [40]. These findings indicate that the acute effects of oestrogen on mood and mental state are mediated at least in part by an increase in density of the 5HT-2A receptors. 2.4.5. Serotonin and obsessional tendencies in patients with anorexia nervosa : The personality trait more frequently found in anorectic patients is 'neurotic perfectionism' as well as 'obsessional tendencies', which could both be considered similar to the 'pain-avoidance' dimension described by Cloninger [41], a dimension defined by the author as a marker for an abnormal serotonin function. Obsessional personality traits and symptoms have been reported in between 3% to 83% of eating-disordered cases, the results being dependent on the criteria [42]. Up to 30% of anorexia nervosa patients have been reported to have significant obsessional personality features at first presentation. Hudson et al [43] found that a diagnosis of Obsessive-Compulsive Disorder (OCD) can be made in as many as 69% of restrictor and 44% of bulimic anorexics. Recent studies have found a high rate of OCD in anorexics several years after the reported onset of the eating disorder, and 20% continued to meet criteria for OCD after weight restoration. In addition, anxiety disorders are common in all anorexics with rates of 39% to75%. The hypothesis that 5HT is related to the pathobiology of OCD was initially derived from the observation that the potent serotonin reuptake inhibitor (SRI) chlomipramine, and the later introduced serotonin selective reuptake inhibitor (SSRI) fluoxetine, was effective in relieving OC symptoms. The results of pharmacological treatment trials and studies of biological markers indicate that dysregulation of the 5HT system may be involved in the pathogenesis of OC symptoms. Blunting of neuroendocrine responses to 5HT agonists and increase of 5HT functioning after treatment with SSRIs (eg. fluoxetine), suggest that, the primary deficit in OCD is a decreased 5HT responsivity. Exacerbation of OC symptoms after mCPP challenge is not entirely consistent with this hypothesis, however, behavioral hypersensitivity coupled with neuroendocrine hyposensitivity to serotonergic stimulation might characterize the serotonergic dysfunction of OCD [44]. Taken together, malnutrition and weight loss are associated with a severe increase in the intensity of depressive, anxious, and obsessive symptoms in underweight anorexics. Whether persistent symptoms after long-term weight restoration from anorexia nervosa reveal an underlying psychobiological trait, or are related to good outcome remains uncertain. In any event, the major comorbidities of anorexia nervosa (depression, anxiety, and obsessionality) may contribute to starvation and weight loss, which in turn, may exacerbate these symptoms. That is, anorexics may enter a vicious cycle in which the symptoms make the malnutrition worse, and the malnutrition makes the symptoms worse [45]. 2.4.6. Serotonin and depression in patients with anorexia nervosa : The comorbidity between anorexia nervosa and depressive disorder (in which serotonin may be implicated) also underlines the potential role of serotonin in the genesis of anorexia nervosa. These two disorders both exhibit weight loss, insomnia, loss of libido, depressed mood, disturbance in concentration, and also demonstrate abnormalities in sleep architecture and endocrine function. In fact, as many as 91% of malnourished anorexics suffer from depressive disorders. In comparison, 15% to 58% of patients continue to exhibit some degree of depressive disturbance after weight restoration . Abnormalities of both serotonergic neurotransmission and hypothalamic-pituitary-adrenal (HPA) axis activity are found in both depression and eating disorders and may play important pathogenic roles. Both conditions are frequently associated with elevated plasma cortisol levels and insensitivity to glucocorticoid (dexamethasone) feedback, presumed to be caused by increased central drive on the HPA axis. Abnormal 5HT neurotransmission is also believed to be a key factor in depression. Many clinically effective antidepressants alter 5HT neurotransmission, improving both mood and hypercortisolemia in depressive patients. Serotonergic drugs, like chlomipramine and fluoxetine, also provide therapeutic effects in patients with anorexia nervosa supporting the evidence that serotonergic system is a key factor in the pathogenesis of anorexia nervosa [43, 45]. 2.4.7. Involvement of serotonin in impulsivity, aggression and suicidality. What's the common in anorexia nervosa? Suicidal behavior is a prevalent symptom associated with depression. A number of reports have suggested that an abnormality in central serotonergic activity may be associated with suicidality. Studies of suicide attempters have demonstrated low CSF 5HIAA in patients with a history of violent suicide attempts [46]. Post-mortem studies have demonstrated decreased levels of serotonin and decreased imipramine binding in the brains of suicide victims as compared to accident victims. A significant decreased prolactin response to d,l-fenfluramine, interpreted as reduced net central serotonergic activity, is found in depressed as well as personality disordered patients compared to controls and associated with suicide in both cohorts [47]. Type 2 serotonin receptor (5HT-2) are widespread in the human cortex and appear modified in the frontal lobe of patients who suffer from depression or who die from suicide [48, 49]. Studies of non-suicidal self-injurious behavior have demonstrated low CSF 5HIAA in depressed patients with self-injurious behavior compared to those without. New et al [50] in 1997, have hypothesized that the abnormality in central serotonergic activity that has been associated with suicidal behavior might be associated with the more broadly defined category of 'self-directed aggression', not necesserily with suicidal intent. The main clinical feature of patients with anorexia nervosa (limit of food intake) can be viewed as a self-directed aggression which may mediated by the serotonergic system. There is a recognized association between various types of self-mutilation (e.g. cutting and burning) and eating disorders [51]. In addition, evidences suggest that impulsive aggression is both partially heritable and associated with abnormalities in serotonergic functioning [52]. The selection of genes involved in serotonin functioning as candidate genes for influencing these behaviors comes from the observation that decreased serotonergic activity has been associated with 'impulsive aggression'. Nielsen et al [53], found a significant association between tryptophan hydroxylase genotype and CSF 5HIAA concentration in one impulsive group, but no association of tryptophan hydroxylase genotype with impulsive behavior was detected. New et al [54] in 1998, assessed the relationship between 'impulsive aggression' and tryptophan hydroxylase genotype in a clinical sample with personality disorders. It was conclude that 'impulsive-aggressive' behavior in male personality disorder patients may be associated with the tryptophane hydroxylase genotype. 2.4.8. Serotonin and Schizophrenia. Are there common pathways in anorexia nervosa? 5HT-2A receptors are involved in the actions of hallucinogenic drugs and have been implicating in the pathogenesis and treatment of schizophrenia. An important property of clozapine's atypicality is that this antipsychotic drug is an effective 5HT-2A antagonist. Clozapine administration decreases 5HT-2A mRNA and ketanserin binding sites. Some recent genetic association studies have found a positive association between schizophrenia and the 102T/C polymorphism of the 5HT-2A receptor gene [55, 56]. Jakab et al [57] in 1998, studying in primates, found that 5HT-2A receptors accumulated in the apical dendritic 'bottle-neck' segment of pyramidal cells have a strategic role in controlling the rate of dendritic currents. This apical dendritic gating mechanism may play an important role in working memory and latent inhibition. Excessive 5HT-2A receptor activation, in this region, may be disruptive. The 5HT-2A agonist 'Ecstasy' can induce hallucinations and delusions because of the excessive rate of dendritic currents reaching the soma. It was hypothesized that hyperactive pyramidal cells may produce the 'positive' hallucination-like symptoms observed in schizophrenia due to a dysfunctional 5HT-2A receptor system in their apical dendrites. In the case of anorexia nervosa, the main clinical feature 'disturbance of body image' as well as the secondary symptoms 'fear of becoming fat' and 'peculiar behavior regarding food' seem to have a delusional dimension or even a schizophrenic-like dimension. Similar symptoms with disturbance in body image and generally peculiar behavior are common in schizophrenics. In conclusion, 5HT-2A receptor system may provide common pathways, in the pathogenesis of both anorexia nervosa and schizophrenia. Clozapine's efficacy in schizophrenia and serotonergic antidepressant's efficacy in anorexia nervosa support this hypothesis. 2.4.9. Obesity, Anorexia Nervosa, and Serotonergic system : The regulation of body weight involves coordination of intake and expenditure of calories. Control over this process is highly complex , however, and involves multiple neural circuits with specific neuropeptides, neurotransmitters, all influenced by peripheral signals, such as leptin, and input of higher cortical centers. Identification of molecules that participate in these central regulatory circuits is accelerating and the list now includes leptin, the leptin receptor, melanin concentrating hormone, the melanocortin 4-receptor , urocortin, and neuropeptide Y and its type 5-receptor [58]. In addition, there are many indications that obesity is a genetic disease which results when a variety of environmental factors act on multiple genes to influence our eating, metabolism and energy expenditure. During the past years, researchers have linked mutations in five different genes (ob, db, tub, A-y, and fat) to obesity in mice [59]. Isolated cases of obesity in humans have recently been identified which also result from a mutation in some of these genes [60]. All these known genes regulate body fat mainly by affecting appetite. Mutations in any of these five genes cause a significant increase in food intake. For example, ob mice eat 62% more food than normal mice, and agouti yellow (A-y) mice consume 36% more food than controls. However, Dong & Wagner [61] in 1998, found that ICAM-1 -/- mice develop a maturity-onset obesity without an obvious increase of appetite. Recent data have suggested that adipocytes synthesize and secrete a 16 kDa peptide (leptin) which acts centrally to regulate weight gain by suppressing appetite. To exert such effects, it may acts as an endogenous ligand in the CNS, since specific receptors (Ob receptors) have been recently reported to be widely distributed in the brain. On this basis, Costa et al [62] suggested that leptin may act to regulate appetite at least in part by directly modulating the secretion of CRH from the hypothalamus. Most of the research in the neurotransmitter area has focused on the serotonergic system. Weight gain is a relevant side-effect of treatment with the atypical neuroleptic drug clozapine. Some clinicians found that weight gain on clozapine tender to be greatest in patients who had the best clinical response [63]. The mechanisms of the weight gain are poorly understood, but some hypothesis have been suggested. Neuroleptics (mainly the low potency antipsychotics, chlorpromazine, and thioridazine as well as the atypical clozapine) block serotonergic transmission [64]. Patients frequently report an inability to suppress appetite after a full meal, suggesting a change in satiety perception. Bromel et al [65] in 1998, found that overeating and weight gain in predisposed schizophrenics treated with clozapine was related with elevated leptin secretion. Leptin, has been shown to correlate with the body mass index, and is presumed to signal the size of the adipose depot to brain and peripheral tissues. The authors of the above study assumed that in predisposed individuals central effects of clozapine induce increased appetite and as a consequence overeating. Both overeating and the ensuing gain in fat mass induce the increased leptin secretion. In addition, hyperglucemia and ketoacidosis, in predisposed patients, are some rare relative side-effects of clozapine [66]. In women with anorexia nervosa serum leptin levels are reduced significantly in association with low body fat and weight, suggesting that the regulation of leptin is maintained even at an extreme of low body weight and fat in subjects with anorexia nervosa [67]. Summarizing, energy balance, appetite, food intake, weight gain and the extreme condition of obesity share at least some pathogenic pathways regulating by the serotonergic system. Anorexia nervosa is another extreme condition in relation to weight, appetite, and food intake. It is possible that some common pathogenic pathways, involving serotonergic system, may participate in the pathogenesis of both these conditions. 3.

GENETIC ASSOCIATION STUDIES IN PATIENTS WITH ANOREXIA NERVOSA : Association studies are case control studies based on a comparison of unrelated affected and unaffected individuals from a population. An allele A at a gene of interest is said to be associated with the trait if it occurs at a significantly higher frequency among affected compared with control individuals. Positive association can occur if the allele A is actually a cause of the disease. In this case, the same positive association would be to expected to occur in all populations. Positive association can also occur if allele A does not cause the trait but is in linkage disequilibrium with the actual cause, that is, A tends to occur on the chromosomes that also carry a trait causing mutation. Linkage disequilibrium will arise in a population when two conditions are met: most cases of the trait are due to relatively few distinct ancestral mutations at a trait-causing locus, and the marker allele A was present on one of these ancestral chromosomes and lies close enough to the trait-causing locus that the correlation has not yet been eroded by recombination during the population's history. Linkage disequilibrium is most likely to occur in young and isolated populations. It has been already found a relation of serotonin system in the pathogenesis of anorexia nervosa. Until now five association studies have analyzed the 5HT-2A gene promoter polymorphism in patients with anorexia nervosa. Collier et al [68] in 1997, studied the 5HT-2A promoter polymorphism (-1438-G/A) in 81 white females with anorexia nervosa and 88 females controls. The frequency of the -1438/A allele was increased in anorexia nervosa patients compared with the controls ( p=0.022) as was the -1438/A genotype frequency (p=0.026). The odds ratio for the -1438/A allele was 1.52 (95% CI 1.04-2.21). Sorbi et al. [69] in 1998, analyzed the segregation of the same polymorphism in 77 female patients with anorexia nervosa, 43 restricted type and 34 purging type, and in 107 normal females controls. The restricted type showed a high frequency of the -1438A/A genotype , 41.8 in anorexic patients vs 9.3in controls; p< 0.0001. Patients with purging type had a genotype distribution similar to the control group. These results suggest a role of the 5HT-2A gene in anorexia nervosa and also that restricting and purging types have a different involvement of this gene. This finding may lead to a better understanding of the two types of this psychiatric diseases and suggest a different implication of the serotonergic system in the pathogenesis and possibly in treatment of anorexia nervosa. Enoch et al [70] in 1998, genotyped three independent sets of unrelated white people for the -1438G/A 5HT-2A polymorphism. One set was from the USA, with 68 anorexics, 22 bulimics and 69 normal controls; one set was from Italy with 20 anorexics and 37 bulimics; and another set was from USA with 62 patients with OCD and 144 normal controls. -1438/A allele was more common in US anorexics than in controls, 0.51 vs 0.36, p<0.005 . The genotype frequencies were also different, p<0.025. However, in US bulimia patients the -1438A 5HT-2A allele frequency did not differ from controls and was lower than in patients with anorexia nervosa, p<0.005. The 1438 allele frequency in Italian bulimics was 0.38 compared with 0.65 in anorexics, p<0.005, and genotype frequencies also differed, p<0.05. Finally, there was an increase in the frequency of the -1438A allele in patients with OCD, compared with controls, p<0.05, although, genotype frequencies did not differ. The author suggested that the 5HT-2A - 1438G/A promoter polymorphism, or a variant with which it is closely linked, may contribute to a behavioral trait, for example perfectionism or obsessionality, common to both anorexia nervosa and OCD but uncommon to bulimia. All these data suggest a role of 5HT-2A in anorexia nervosa. However, three groups have failed to replicate these findings: Campbell et al [71] in 1998, genotyped 152 patients with anorexia nervosa and 160 controls, for the -1438G/A 5HT-2A promoter without finding significant difference. Hinney et al [72] genotyped 100 patients with anorexia nervosa, 101 underweight patients and 254 obese patients for the -1438 G/A , Thr-25-Asn, and His-452-Tyr 5HT-2A polymorphism without finding significant difference. Finally, preliminary results of a large genome scan linkage study (400 markers at 20 cM intervals) in 192 probands with restricting-type anorexia nervosa, and 240 pairs of affected relatives (190 siblings) showed that analysis of 13q14-21, where association to 5HT-2A receptor gene has been reported, revealed no evidence for linkage [73]. 4. FUTURE PROSPECTS 4.1. Serotonergic system: Understanding the role of 5HT function may hold promise for the therapy of this treatment-resistant disorder. Future research should study the effects of serotonergic drugs on the prevention of relapse in anorexic patients after weight restoration. Differences in 5HT function between restricting and bulimic anorexic patients need to be understood. Recent genetic association studies [68] found positive association between the 5HT-2A promoter region gene polymorphism and restricting type anorexic patients. In one of them [70], seemed that this polymorphism may contribute to the behavioral trait of perfectionism or even obsessionality, common feature of anorexia nervosa and OCD but uncommon in bulimia. Other studies [71, 72] have failed to replicate positive association. Possible explanation for the discrepant results are false-negative and false-positive results, stratification effects, and genetic heterogenity. In view of the possible multiple genes of low and similar size effect that may be involved in the pathogenesis of this disorder, some points might to be addressed: More association studies with large samples should be performed detecting the known polymorphisms. If possible, association studies should be performed within relatively homogenous populations. Family trios studies and the use of haplotype-relative-risk method and transmission disequilibrium test can outrun the difficulty of selecting a control group that is perfectly matched for ethnic ancestry. Systematic genome scan is necessary to pick up unexpected or missed QTLs. Until this time, some studies have tried to detect a relation between personality traits and serotonergic system. Ebstein et al [21], found an association between the 5HT-2C receptor gene polymorphism and the human personality trait of 'reward dependence'. Polymorphisms in the serotonin transporter gene (5HTT) have been reported to be associated with neuroticism (emotionality) and with depression [74]. Impulsive-aggression behavior and suicidality have found to be associated with tryptophan hydroxylase gene polymorphism [53, 54]. Patients with anorexia nervosa present high level of neurotic symptoms, depression, and impulsive-(auto)aggressive behavior which may underlie suicidality. In view of these findings, tryptophan hydroxylase gene and serotonin transporter gene (adding the 5HT-2A and 5HT-2C receptor genes) may serve as strong candidate genes in anorexic patients for future genetic research. In fact, recent findings on the 5HT-2A -promoter region - receptor gene polymorphism in Japanese eating disordered patients [75], as well as preliminary findings on a functional 5HT transporter gene polymorphism in anorectic patients [76, 77] support all the above suggestions. 4.2. Dopaminergic system: Pharmacological experiments tend to support a role for dopamine in that dopamine agonists stimulate, whereas antagonists inhibit feeding and drinking. Treasure [20], considered anorexia nervosa as a pathology belonging to a spectrum of 'addictive disorders'. Dopamine is released in anticipation of feeding and mesolimbic-mesocortical system is involved in reward-reinforcement behavior. Ebstein et al [21], found that both dopamine and serotonin (5HT-2C) gene polymorphisms are associated with the human personality trait of 'reward-dependence'. Furthermore, studies with transgenic mice [22] showed that mice lacking dopamine-2 receptors leads to animals hypoactive, adipsic, and aphagic and mice lacking dopamine transporter (DAT) result in hyperlocomotion and low weight. In conclusion, dopamine receptors genes, dopamine transporter gene, and tyrosine hydroxylase gene may serve as candidate genes for future genetic research in patients with anorexia nervosa. 4.3. Noradrenergic system: It is known that hypothalamus is largely innervated by noradrenergic fibres, and noradrenaline is involved in the secretion of gonadotrophins, TRH, and vassopressin, as well as in the suppression of CRH. In addition, a3 adrenergic receptor, located mainly in adipose tissue, is involved in the regulation of lipolysis and thermogenesis. Clement et al [78], found that the frequency of the Trp-64-Arg a3 noradrenergic receptor allele was similar in the morbidity obese patients and the normal subjects, but the patients with morbid obesity who were heterozygous for the Trp-64-Arg mutation had an increased capacity to gain weight. Thus, noradrenergic receptors genes, possibly in combination with CRH gene, can serve as candidate genes in the different subgroups of both anorexia and bulimia nervosa. 4.4. Other systems: Different peptides have been found to involve in appetite-energy balance. Opiate agonists as well as neuropeptide Y administration can increase food intake, and cholocystocinine (CCK) infusion can decrease food intake in animals. In addition, the Ob gene, encoded for leptin, a fat secreted hormone secreted by adipocytes regulates ingestion behavior and energy balance. Ob gene, thus, belongs to candidate genes in anorexia nervosa, and has been recently localized in human chromosome 2p21 [27]. The importance of Ob gene and Ob gene product (leptin) is supported by the recent finding which indicate a relation between Ob gene and the hypothalamic hormone CRH. Adypocytes synthesize and secrete a 16 kDa peptide(leptin), which acts centrally to regulate weight gain by suppressing appetite. To exert such effects, it may function as an endogenous ligand in the CNS, since specific receptors (Ob receptors) have been recently reported to be widely distributed in the brain. Costa et al [62], suggested that leptin ( the Ob gene product) may act to regulate appetite at least in part by directly modulating the secretion of CRH from the hypothalamus. Holsboer et al [79], in 1998, produced two transgenic mouse with specific alterations of the HPA system. The mouse with functionally impaired glucocorticoid receptors showed HPA hyperdrive and cognitive depression-like disturbances. The mouse with a CRH-1 receptor knock-out had low ACTH in corticosterone secretory activity, exhibited increased exploratory activity and reduced anxiety related behavior. These findings support the notion that defunct glucocorticoid signaling accounts for HPA overactivity and associated behavioral alterations, and that, the CRH-1 receptor is target for treatment of such clinical conditions. Recent reports showed positive a association of the COMT-high activity allele [80], as well as an agouti-related protein gene polymorphism [81], in anorexic patients. The study of the addictive effects of several genes will be informative. If on average each candidate gene contributes to only 1-8% of the variance of a given quantitative trait, the results may be modest, borderline or negative significance. However, the examination of the additive or epistatic effect of two or more candidate genes may show a much more significant effect. Comings et al [82], have observed this for the additive effect of the Ob and Dopamine receptor genes. Finally, more epidemiological and cultural studies will provide useful information about the subtypes, the inter-ethnic differences, and the exact influences of the social-cultural factors in the etiology of anorexia nervosa [83].

ΠΕΡΙΛΗΨΗ Η ψυχογενής ή νευρογενής ανορεξία είναι μία χρόνια και βαρειά ψυχιατρική διαταραχή, παρατηρείται 10-20 φορές στις γυναίκες, προσβάλλει το 0,5-1% των θηλέων εφήβων, χαρακτηρίζεται από σημαντική διαταραχή αντίληψης της εικόνας του σώματος με συνεχή προσπάθεια για απίσχναση, και η θνησιμότητά της κυμαίνεται από 5-18%. Στην παθογένεια της διαταραχής αυτής φαίνεται να συνεισφέρει η γενετική υποδομή, το ενδοκρινικό και νευρομεταβιβαστικό σύστημα, ψυχολογικοί παράγοντες, όπως η προσωπικότητα και οι μηχανισμοί άμυνας, και κοινωνικοί παράγοντες, όπως η έμφαση της κοινωνίας στο λεπτό σώμα. Ανωμαλίες τόσο στο σεροτονεργικό σύστημα όσο και στον άξονα υποθάλαμος-υπόφυση-επινεφρίδια έχουν φανεί σε παθήσεις όπως κατάθληψη, ιδεοψυχαναγκαστική διαταραχή, και διαταραχές στην πρόσληψη τροφής. Οι ανωμαλίες όμως στη σεροτονεργική νευρομεταβίβαση φαίνεται να είναι ο παράγοντας-κλειδί στην ανάπτυξη της ψυχογενούς ανορεξίας. Η σεροτονίνη πιθανόν να ενέχεται στην καταθλιπτική και ιδεοληπτική συμπτωματολογία των ανορεξικών ασθενών, ενώ συγχρόνως ενέχεται στο σύστημα θερμιδικών αναγκών του οργανισμού και της όρεξης. Επίσης, μοντέλα της νόσου σε ζώα καθώς και η θεραπευτική ικανότης των σεροτονεργικών φαρμάκων, ενισχύουν τη θέση της στην παθογένεια της νόσου. Κάποιες πρόσφατες γενετικές μελέτες έδειξαν τη συσχέτιση μεταξύ του -1438/Α αλληλίου του -1438Α/G σεροτονικού πολυμορφισμού και της ψυχογενούς ανορεξίας, αλλά κάποιες άλλες απέτυχαν να το επαναλάβουν. Τα γονίδια της υδροξυλάσης της τρυπτοφάνης, του μεταφορέα σεροτονίνης, της ντοπαμίνης τύπου 1 και 4, του α3-νοραδρενεργικού, της CRH, και του Ob, φαίνεται να προσφέρουν ένα ενθαρυντικό πεδίο έρευνας για μελλοντικές γενετικές μελέτες στους ασθενείς που πάσχουν από ψυχογενή ανορεξία.