Elena Salvatore

PhD Carlo Rinaldi

Tecla Tucci

Luigi Di Maio

Carolina Di Somma

PhD Silvia Savastano

PhD Gaetano Lombardi

Alessandro Filla

Annamaria Colao

PhD Giuseppe De Michele
email: demichel@unina.it

Prof Giuseppe De Michele

Md ; Luigi

Di Maio

GROWTH HORMONE RESPONSE TO ARGININE TEST DIFFERENTIATES TWO SUBGROUPS OF HUNTINGTON'S DISEASE PATIENTS

Keywords: Huntington's Disease, Neuroendocrinology, Growth Hormone, arginine test

published or not. The documents may come

INTRODUCTION

Huntington's disease (HD) is an autosomal dominant, neurodegenerative disorder caused by an expanded CAG repeat within the HD gene. [START_REF]The Huntington's Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes[END_REF] It is characterized by the triad of motor disturbances, personality changes, and cognitive decline. Hypothalamic dysfunction may also contribute to the clinical spectrum of HD, as several studies, both in animals and humans, have underlined. Hypothalamic atrophy has been shown to occur in the early stages of the disease by voxel-based MRI studies. [START_REF] Kassubek | Topography of cerebral atrophy in early Huntington's disease: a voxel based morphometric MRI study[END_REF] Loss of cells, especially in the somatostatin-containing neurons of the NTL (nucleus tuberalis lateralis) of the hypothalamus, has also been demonstrated. [START_REF] Kremer | The hypothalamic lateral tuberal nucleus and the characteristics of neuronal loss in Huntington's disease[END_REF][START_REF] Timmers | Somatostatin 1-12 immunoreactivity is decreased in the hypothalamic lateral tuberal nucleus of Huntington's disease patients[END_REF] Hypothalamic dysfunction in HD may account for several well known manifestations of the disease such as weight loss, [START_REF] Morales | Nutritional evaluation of Huntington disease patients[END_REF] sleep disorders, [START_REF] Arnulf | Rapid eye movement sleep disturbances in Huntington disease[END_REF][START_REF] Morton | Disintegration of the sleep-wake cycle and circadian timing in Huntington's disease[END_REF] hyperactivity of the hypothalamic-pituitary-adrenal axis, [START_REF] Aziz | Increased hypothalamic-pituitary-adrenal axis activity in Huntington's disease[END_REF] and decreased levels of luteinizing hormone and testosterone in male patients. [START_REF] Markianos | Plasma testosterone in male patients with Huntington's disease: relations to severity of illness and dementia[END_REF] Circulating growth hormone (GH) and insulin-like growth factor-1 (IGF-1) levels have recently been found higher in HD patients. [START_REF] Saleh | Neuroendocrine disturbances in Huntington's disease[END_REF] GH secretion from the pituitary gland is stimulated by the GH-Releasing Hormone (GHRH) and inhibited by Somatostatin (SS): both these hypothalamic neurohormones have a pulsatile secretion modulated mainly by noradrenergic and cholinergic networks. [START_REF] Muller | Neuroendocrine control of growth hormone secretion[END_REF][START_REF] Giustina | Pathophysiology of the neuroregulation of growth hormone secretion in experimental animals and the human[END_REF] Both clonidine, an α2-adrenoceptor agonist, and arginine, an amino acid activating the cholinergic system, induce GH release, the former by stimulation of GHRH, the latter by inhibition of SS release, and they have been widely used to evaluate pituitary GH secretion and reserve. [START_REF] Hormone | Consensus guidelines for the diagnosis and treatment of adults with growth hormone deficiency: summary statement of the Growth Hormone Research Society workshop on adult growth hormone deficiency[END_REF] The aim of our study was to evaluate the GH response to arginine in a cohort of molecularly confirmed HD patients, in order to look for an in vivo demonstration of hypothalamic dysfunction in HD. We also investigated the relationship between the GH response and clinical, metabolic, and endocrine variables, to understand if GH response abnormalities may represent a biomarker of disease progression.

MATERIALS AND METHODS

Study participants (n=17, 10 males, 7 females) were recruited among consecutive patients afferent to the HD Clinics at the Department of Neurological Sciences, from February 2008 to February 2009. Disease specific inclusion criteria were: age between 18-70 years, manifest signs and symptoms of the disease, and positive molecular test for the presence of a CAG triplet repeat number > 35 in the HD gene. Exclusion criteria were: history of endocrine disorders, chronic systemic diseases, substance abuse, or inability to cooperate to the study. Eleven patients were drug-naïve. To avoid possible interactions, administration of antipsychotic agents or any other dopamine receptor blockers was interrupted at least 2 weeks before the test in further four patients. One patient was allowed to take 25 mg/day of tetrabenazine and another 5 mg/day of olanzapine. The age of onset was considered the time when motor symptoms were first noticed.

The phenotype at onset was also defined as psychiatric, if psychiatric disturbances were present prior to motor onset, as motor, if only motor symptoms were present at onset, and mixed, if motor and psychiatric symptoms appeared together. Motor symptoms and Total Function Capacity (TFC) were evaluated by one experienced neurologist using the Unified Huntington's Disease Rating Scale (UHDRS). [START_REF] Study | Unified Huntington's Disease Rating Scale: reliability and consistency[END_REF] The motor component of the UHDRS scale consists of 31 questions rated on a 0-to 4-point scale with 4 indicating the most severe impairment. The TFC is a standardized scale (range 0-13) most sensitive to early changes in disability with higher scores indicating better function. [START_REF] Shoulson | A controlled clinical trial of baclofen as protective therapy in early Huntington's disease[END_REF] Disease stage was determined according to TFC: stage I represents scores from 11 to 13; stage II, from 7 to 10; stage III, from 3 to 6; stage IV, from 1 to 2; and stage V, a score of 0. [START_REF] Marder | Rate of functional decline in Huntington's disease[END_REF] Control subjects (n=17, 10 males and 7 females) were recruited among healthy volunteers matched to the patients according to age (± 2 yrs), sex, and body mass index (BMI; ± 2). The only exception was for a male patient aged 67 yrs with BMI of 16.0, who was matched with a man with a BMI of 20.3, as it was not possible to find a healthy control of that age with a similar BMI. Control subjects were free from any neurological and endocrine disease and none of them was taking medications. The study protocol was approved by the local Ethic Committee and informed consent was obtained from all the study participants. For HD patients lacking capacity of making decisions, the informed consent was obtained from the relatives.

In the early morning, after a 12-hour fasting, patients and control subjects underwent routine investigations and an endocrine study, inclusive of basal insulin, total cholesterol, LDL and arginine (arginine hydrochloride, 30% solution, Salf, Italy) were infused intravenously over 30 minutes, and blood was sampled every 30 minutes for 1 hour (T30, T60, and T90) thereafter.

Blood pressure and heart rate were monitored through the test. Blood samples were centrifugated immediately after sampling and the supernatants stored at -80°C until analysis. Assays were performed according to the manufacturer's instructions. Serum GH was measured with commercially available immunoradiometric kit (Chematil, Italy). The sensitivity of the assay is 0.015 ng/mL. A peak serum GH lower than 1.4 ng/mL at T60 or T90 time points was considered abnormal, as for guidelines for adult GH deficiency (GHD) diagnosis. [START_REF] Biller | Sensitivity and specificity of six tests for the diagnosis of adult GH deficiency[END_REF] Basal IGF-1 was also measured, using a chemoluminescent IGF-1 assay (Immunolite 2000, Medical System, Italy) with the limit of detection of 20 μg/L. Homeostatis model assessment of insulin resistance (HOMA-IR) was computed as follows: fasting insulin (μIU/ml) x fasting glucose (mmol/ml)/22.5. Inter-and intragroup analyses were performed using Yates chi-square test and t-test (paired or unpaired), when appropriated. Relationships between variables are expressed by the Pearson correlation coefficient. Differences were considered statistically significant when p<0.01. All analyses were conducted using the STATA 10 for Windows Package (Stata Corporation, College Station, TX, USA).

RESULTS

Demographic, clinical, and laboratory data of the patients and the controls are summarized in table 1. Disease severity was mild to moderate in most. Compared with control subjects, the HD patients showed higher baseline GH and lower IGF-1 and SDS IGF-1 serum levels. During arginine infusion no changes in blood pressure or heart rate were observed in any group. The arginine test induced a GH peak higher than 1.4 ng/mL in all control subjects (mean peak value ± SD: 16.6 ng/mL ± 8.3; 35% of the subjects at T30, 65% at T60) and in 9 patients (53%) (16.4 ng/mL ± 8.8; 67% at T60, 33% at T90). In the remaining 8 patients (47%) the GH response to arginine stimulation test was blunted (0.6 ng/mL ± 0.4) (figure 1).

Among the two subgroups of patients (with normal GH response to arginine, Arg+, and with absent GH response, Arg-) there were no differences in age, sex distribution, CAG triplet repeats, phenotype at onset, disease duration and stage, and UHDRS motor score; a slightly lower TFC score (p=0.04) and BMI (p=0.02) value were observed in Arg+ patients (table 2). Previous exposition to neuroleptics did not affect GH response to arginine since six drug-naïve patients were Arg+ and five were Arg-. The patient who was still taking olanzapine had a normal GH peak, whereas the patient who was still taking tetrabenazine showed a blunted response. Arg-patients showed higher LDL cholesterol and lower HDL cholesterol compared to Arg+ patients (table 2).

Arg+ patients showed lower insulin and HOMA-IR compared to Arg-(table 2). Table 3 shows the relationship between clinical and laboratory variables in the 17 HD patients. As expected, BMI showed to correlate positively with total cholesterol and insulin and negatively with baseline GH. A positive correlation between baseline and peak value GH was found. Also expected was the negative correlation between TFC score and UHDRS motor score.

DISCUSSION

Several signs of a hypothalamic-endocrine dysfunction have been reported in HD. [5-7, 10, 18] Hypothalamic involvement in HD has been confirmed by neuropathological studies, showing a dramatic neuronal loss primarily of somatostatin-containing neurons in the NTL. [START_REF] Kremer | The hypothalamic lateral tuberal nucleus and the characteristics of neuronal loss in Huntington's disease[END_REF][START_REF] Timmers | Somatostatin 1-12 immunoreactivity is decreased in the hypothalamic lateral tuberal nucleus of Huntington's disease patients[END_REF] Recently, higher levels of circulating GH in HD patients have been found in a large case-control study, showing a correlation with disease severity and weight loss. [START_REF] Saleh | Neuroendocrine disturbances in Huntington's disease[END_REF] GH is a protein hormone synthesized and secreted by the somatotroph cells of the anterior pituitary gland, under two hypothalamic hormones controllers: GHRH and SS. It is a major participant in several complex physiologic processes, including growth and metabolism, exerting its role either directly on target cells or mediated by its peripheral effector, IGF-1. [START_REF] Muller | Neuroendocrine control of growth hormone secretion[END_REF][START_REF] Giustina | Pathophysiology of the neuroregulation of growth hormone secretion in experimental animals and the human[END_REF] The present study was aimed to investigate the metabolic/endocrine profile and the GH response after the arginine stimulation test in a cohort of HD patients and controls. The arginine test was chosen, because it induces GH release by inhibition of somatostatin release, thus potentially representing a marker of hypothalamic dysfunction in vivo. Moreover it is a safe test and has been widely used to evaluate pituitary GH secretion and reserve. [START_REF] Hormone | Consensus guidelines for the diagnosis and treatment of adults with growth hormone deficiency: summary statement of the Growth Hormone Research Society workshop on adult growth hormone deficiency[END_REF][START_REF] Biller | Sensitivity and specificity of six tests for the diagnosis of adult GH deficiency[END_REF] As patients not fully cooperative and those under heavy drug treatment were excluded from the study, recruitment was biased for the inclusion mostly of patients in early disease stages. HD patients showed higher basal GH values, as already shown in previous reports, [START_REF] Saleh | Neuroendocrine disturbances in Huntington's disease[END_REF][START_REF] Phillipson | Plasma growth hormone concentrations in Huntington's chorea[END_REF] and lower IGF-1 and SDS IGF-1 levels in comparison with age-, sex-and BMI-matched healthy controls. The last finding does not confirm the results of a recent paper, [START_REF] Saleh | Neuroendocrine disturbances in Huntington's disease[END_REF] which described a slight IGF-1 increase in HD patients. It is noteworthy that IGF-1 levels of the French HD cohort [START_REF] Saleh | Neuroendocrine disturbances in Huntington's disease[END_REF] are comparable to ours (153 ± 48 vs. 159 ± 55 μg/L), but the values of their control subjects, recruited among patients' spouses and relatives, are lower than ours, which are healthy volunteers (141 ± 42 vs. 216 ± 39 μg/L). As previously reported, [START_REF] Saleh | Neuroendocrine disturbances in Huntington's disease[END_REF] BMI was significantly related with baseline GH levels and was independent from motor and functional score.

The most relevant result of our study was that almost half of our patients showed no GH response to arginine infusion. The remaining patients had a normal peak, although somewhat delayed (at T90 in a third of them) in comparison with controls. Differences in insulin and cholesterol metabolism were also observed in the two groups.

These findings are unexpected and puzzling. We could speculate that the hypothalamic dysfunction primarily in somatostatin-containing neurons may determine the lack of response of GH to arginine stimulus in the Arg-patients. The unbalance in GH/IGF-1 axis regulation may also be responsible for the impaired lipid profile in this subgroup of patients.

On the other side, in Arg+ patients, a situation of negative energy balance appears to be likely for the following reasons: 1) high GH levels with reduced IGF-1, insulin and HOMA-IR levels are a frequent finding in critical illness and catabolic state and it has been proposed that GH hypersecretion may be driven by decreased circulating IGF-1 through a feedback mechanism in states of chronic starvation, [START_REF] Grottoli | Hormonal diagnosis of GH hypersecretory states[END_REF][START_REF] Botfield | The role of IGFs in catabolism[END_REF] 2) IGF-1 levels are a good index of nutritional status per se, [START_REF] Hawker | Relationship of somatomedin-C/insulin-like growth factor I levels to conventional nutritional indices in critically ill patients[END_REF] 3) BMI values are slightly lower than Arg-patients. The clinical picture of this subgroup of patients also appears to be slighlty more severe, according to the TFC score, suggesting that GH response is not a useful progression biomarker.

The evidence of these two sharply outlined endocrine/metabolic profiles in HD is difficult to understand. These results may reflect two different stages of the disease. Otherwise, we found no differences in age, sex distribution, CAG triplet size, phenotype at onset, and disease stage between the two groups. Another possible explanation is that the GH response to arginine infusion may disclose two different patterns of hypothalamic involvement in HD. It has been recently assessed in an in vivo PET study that hypothalamic changes in HD occur in almost 50% of HD patients and pre-manifest carriers, without correlation with disease duration and striatal dysfunction. [START_REF] Politis | Hypothalamic involvement in Huntington's disease: an in vivo PET study[END_REF] In this view, the degree of hypothalamic dysfunction may be different among patients, as well as the motor, cognitive or psychiatric phenotype may be largely heterogeneous in HD.

In conclusion, our data clearly demonstrate a dysfunction in the somatotropic axis in HD.

According to the GH responses to arginine stimulation, we observed two distinct metabolic/endocrine profiles in our cohort of patients. The main limitation of our study is the relative small number of patients, partially due to the rigorous inclusion criteria. To confirm the data, replication of the study with larger numbers of patients together with further neuropathological studies and extensively neuroendocrine assessment should be performed.

Prospective studies are also necessary to determine whether these changes occur at an early stage and whether they are linked to disease progression.

TABLES

Figure 1 .

 1 Figure 1. Serum concentration of GH (mean ± SEM) at baseline, 30, 60, and 90 minutes after arginine infusion. According to the results of the stimulation test, HD patients can be divided in two groups: Arg+ (n=9), with a normal GH response, and Arg-(n=8), with a blunted response compared to controls (n=17).

Figure 1 .

 1 Figure 1. GH response to arginine infusion

Table 1 . Clinical features and baseline characteristics of Huntington's disease patients and control subjects

 1 Motor; Ps, Psychiatric; Mx, Mixed (motor and psychiatric); UHDRS, Unified Huntington Disease Rating Scale; TFC, Total Functional Composite; BMI, Body Mass Index; HOMA-IR, Homeostasis Model Assessment-Insulin Resistance; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; GH, Growth Hormone; IGF-1, Insuline Growth Factor-1; SDS IGF-1, Standard Deviation Score of IGF-1 a Data are means ± SD (range) * p < 0.01; ** p < 0.001 (Asterisks mark values which differ also from normal controls).

		Patients	Control subjects
	Number	17	17
	M/F	10/7	10/7
	Age, years a	48.8 ± 11.6 (27-67)	49.2 ± 12.3 (27-68)
	CAG, triplets a	44.8 ± 4.9 (41-61)	NA
	Disease duration, months a	64.2 ± 41.3 (24-142)	NA
	Disease stage	6-I, 5-II, 5-III, 1-IV	NA
	UHDRS motor score a	26.2 ± 15.0 (9-57)	NA
	TFC score a	8.3 ± 3.7 (1-13)	NA
	BMI a	23.8 ± 4.4 (16.0-33.9)	23.8 ± 3.3 (19.2-32.1)
	Glucose, mg/dL a	82.7 ± 12.9 (59-105)	82.9 ± 8.8 (68-102)
	Insulin, µIU/mL a	5.4 ± 3.4 (2-11)	6.6 ±2.0 (3-10)
	HOMA-IR a	1.0 ± 0.9 (0-2.5)	1.4 ± 0.5 (0.6-2.2)
	Total Cholesterol, mg/dL a	203 ± 39 (125-272)	183 ± 26 (150-228)
	LDL-C, mg/dL a	129 ± 47 (63-214)	103 ± 24 (75-149)
	HDL-C, mg/dL a	56 ± 23 (32-88)	60 ± 5 (51-70)
	GH baseline, ng/mL a	0.76 ± 0.94 (0.07-3.40)* 0.06 ± 0.05 (0.01-0.20)
	IGF-1, µg/L a	159 ± 55 (70-263)*	216 ± 39 (139-288)
	SDS IGF-1 a	-0.36 ± 1.0 (-2.6-1.77)*	0.70 ± 0.58 (-0.3-1.6)
	Abbreviations: UHDRS, Unified Huntington Disease Rating Scale; TFC,
	Total Functional Composite; BMI, Body Mass Index; HOMA-IR,
	Homeostasis Model Assessment-Insulin Resistance; LDL-C, low-density
	lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; GH,
	Growth Hormone; IGF-1, Insulin Growth Factor 1; SDS IGF-1, Standard
	Deviation Score of IGF-1; NA, not applicable.

a Data are means ± SD (range) * p < 0.01

Table 3 . Relationships between clinical and laboratory variables

 3 Unified Huntington's Disease Rating Scale; TFC, Total Functional Capacity; BMI, body mass index; GH, growth hormone; IGF-1, Insulin Growth Factor 1. Data are correlation coefficients (r). * p < 0,01

		UHDRS motor score	TFC score	BMI	GH (T0)	GH (peak)	IGF-1	Total cholesterol	Insulin
	UHDRS motor
	score
	TFC score	-0,71*
	BMI	-0,22	0,52
	GH (T0)	0,42	-0,38	-0,71*
	GH (peak)	0,21	-0,34	-0,54	0,56*
	IGF-1	-0,22	-0,08	0,17	-0,42	-0,40
	Total cholesterol	-0,27	0,51	0,69* -0,52	-0,66	0,23
	Insulin	0,03	0,19	0,65*	-0,41	-0,51	0,12	0,48
	UHDRS,

ACKNOWLEDGEMENTS

We would like to thank all patients and families for participation to the study and the European Huntington's Disease Network (Euro-HD) for the support.

FINANCIAL DISCLOSURES RELATED TO RESEARCH COVERED IN THIS ARTICLE:

All authors report no disclosures.

COPYRIGHT LICENCE STATEMENT

I Giuseppe De Michele, the Corresponding Author of this article has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Group and co-owners or contracting owning societies (where published by the BMJ Group on their behalf), and its Licensees to permit this article (if accepted) to be published in Journal of Neurology, Neurosurgery & Psychiatry and any other BMJ Group products and to exploit all subsidiary rights, as set out in our licence set out at: http://jnnp.bmj.com/site/about/licence.pdf.


Emma O'keeffe

Osman Mukhtar

Dr Shaun T O'keeffe
email: s.okeeffe@hse.ie

ORIENTATION TO TIME AS A GUIDE TO THE PRESENCE AND SEVERITY OF COGNITIVE IMPAIRMENT IN OLDER HOSPITAL PATIENTS Revision JNNP/2010/214817

Keywords: Temporal orientation, delirium, dementia, elderly, diagnostic test. Word Count: 3, 426

Background: Testing of orientation to time is an important part of mental status examination.

We examined the validity of errors in different aspects of temporal orientation in older hospital patients as a guide to the presence of dementia or delirium and as a measure of the severity of dementia as defined by the Global Deterioration Scale.

Methods: Inpatients and outpatients attending an acute hospital underwent independent assessments by two doctors on the same day to determine orientation to time and cognitive status. Optimum cut-offs for error scores on the different aspects of temporal orientation were calculated to maximise the sum of sensitivity and specificity for detection of dementia or delirium.

Results: Of the 262 patients assessed, 62 (23.7%) had dementia or delirium. The best cutoffs for detection of these disorders were: any error in identifying the year, month, day of the month or day of the week; and an error of more than one hour in identifying the time of day. Failure to identify the year correctly was the most valuable single sign of dementia or delirium (sensitivity 86% and specificity 94%); failure to identify either year or month correctly was 95% sensitive and 86.5% specific or the detection of cognitive impairment. Severity of temporal disorientation, measured using a number of approaches, was strongly associated with severity of dementia.

Conclusion: Disorientation to time is a useful guide to the presence and severity of dementia or delirium in older hospital patients. Failure to identify the year or month correctly is a sufficiently sensitive and specific indicator of dementia or delirium to warrant more detailed cognitive assessment in this population.

Disorientation to time is a very common feature of delirium and dementia, and assessment of temporal orientation is a well-established and routine part of mental status testing. [START_REF] Berrios | Orientation failures in medicine and psychiatry: discussion paper[END_REF] There is no difficulty in recognizing abnormality in patients with gross temporal disorientation. However, clinicians are sometimes uncertain about the significance of more minor orientation errors, such as misstating the day of the month or the week by a day or two, as a guide to possible delirium or dementia in older people. [START_REF] Molloy | A guide to the standardized Mini-Mental State Examination[END_REF] Studies in young and middle-aged subjects show that it is rare for a normal individual to misidentify the year or the month. [START_REF] Benton | Temporal orientation. Contributions to Neuropsychological Assessment[END_REF] The most common error is to misidentify the day of the month. However, less than 5% misidentify the day of the month by more than 3 days.

Benton and colleagues reported that there was 'no evidence of a decline' in temporal orientation with age in a study of 162 community volunteers aged between 65 and 84 years (1981). [START_REF] Benton | Normative observations on neuropsychological test performances in old age[END_REF] Nevertheless, the frequency of temporal disorientation in their study was 1% in 90 patients aged 65 to 74 years and 6% in 72 patients aged 75 to 84 years.

Brotchie and colleagues used a different questionnaire to study temporal orientation in 235 hospital visitors aged 50-84 years. [START_REF] Brotchie | Temporal orientation in the pre-senium and old age[END_REF] They noted that less than 2% misstated the year or month but that errors in identifying the day of the month became more common with increased age. Studies in patients with possible dementia suggest that temporal orientation is a useful screening test for this disorder. [START_REF] Tractenberg | Benchmarking a test of temporal orientation with data from American and Taiwanese persons with Alzheimer's disease and American normal elderly[END_REF][START_REF] Fayers | Which mini-mental state exam items can be used to screen for delirium and cognitive impairment?[END_REF] Furthermore, studies in head injury [START_REF] High | Recovery of orientation following closed-head injury[END_REF], alcohol withdrawal [START_REF] Varney | Predicting short-term memory on the basis of temporal orientation[END_REF] and dementia [START_REF] Ryan | Predicting neuropsychological test performance on the basis of temporal orientation[END_REF] indicate that the magnitude of errors on orientation tests is predictive of the severity of impairment on more detailed neuropsychological testing.

In the only previous study, to our knowledge, to examine the significance of errors in different aspects of temporal orientation in older hospital patients, all errors were sensitive to cognitive impairment but only failure to identify the year, month or day of the week correctly had a specificity greater than 70% for identification of dementia or delirium. [START_REF] O'keeffe | Interpreting errors in temporal orientation in older hospital patients[END_REF] However, this fact that patients were studied just after admission may have exaggerated the specificity of abnormalities since it seems possible that orientation to time might worsen, even in normal subjects, with prolonged admission. Also, the fact that the same examiner assessed cognitive status and temporal orientation was a possible source of bias.

In this study, we examined the validity of errors in different aspects of temporal orientation as a guide to the presence or absence of cognitive impairment (dementia, delirium or both) in older hospital patients. We also examined whether the severity of temporal disorientation might provide a useful measure of the severity of dementia in this population.

METHODS

Patients and setting

Subjects recruited for this study were a convenience sample of patients aged 65 years or more present during a series of cross-sectional surveys of inpatients on the general medical wards of a University teaching hospital and among those attending the geriatric outpatient clinics during September to November 2009. Subjects with major communication difficulties (unable to speak English, severe deafness or such severe cognitive impairment that communication was impossible), those considered by their doctors or nurses to be too sick to be questioned and those who were unwilling to participate were excluded. The study was approved by the local Clinical Research Ethics Committee.

Assessment

Subjects were assessed separately by two different doctors on the same day. The first doctor explained the study and sought written consent or witnessed verbal assent to proceed. This doctor then examined temporal orientation by asking for the year, the month, the date (day of the month), the day of the week, and, making sure the patient could not look at a watch or clock, the time of day. Answers were recorded in the patients' own words. Patients who said they could not answer or were unsure about any item were encouraged to provide their best estimate. Demographic information, including age, sex, date of birth and length of hospital stay were obtained from the medical notes.

Subsequently, all patients were interviewed independently by an experienced clinician who determined whether they had cognitive impairment (delirium or dementia or both). Details of prior cognitive function were sought from all available sources including carers, health care professionals and medical and nursing notes. Delirium was defined according to the Confusion Assessment Method in the presence of: (1) acute onset and fluctuating course; and (2) difficulty focusing or maintaining attention; and either [START_REF] Benton | Temporal orientation. Contributions to Neuropsychological Assessment[END_REF] disorganised thinking or (4) altered level of consciousness. [START_REF] Inouye | Clarifying confusion: the confusion assessment method. A new method for detection of delirium[END_REF] Dementia was staged according to the Global Deterioration Scale, a well-established and validated clinical rating system comprising detailed descriptions of seven stages ranging from normal cognition to very severe dementia. [START_REF] Reisberg | The Global Deterioration Scale for assessment of primary degenerative dementia[END_REF][START_REF] Overall | Empirical scaling of the stages of cognitive decline in senile dementia[END_REF] For the purpose of analysis in this study, these seven stages were reduced to five: absent (stage 1 and 2), questionable (stage 3), mild (stage 4), moderate (stage 5) or severe (stage 6 and 7)

Sample size calculation

Sample size calculation was based on the approach recommended by Guyatt and colleagues for diagnostic test studies. [START_REF] Guyatt | Evaluating diagnostic tests[END_REF] We assumed that at least 20% of subjects would have cognitive impairment and that the best test would have a sensitivity of 85% for detection of cognitive impairment. [START_REF] O'keeffe | Interpreting errors in temporal orientation in older hospital patients[END_REF] The required sample size to measure this sensitivity with a 95% confidence interval of no more than 10% was 246.

Analyses

All analyses were performed using SPSS 15.0 for windows. Data were inspected to determine the frequency and nature of errors, including non-responses, in those without cognitive impairment. The hypothesis that orientation errors became more common with increased length of hospital stay was assessed using non-parametric tests.

This study was concerned primarily with two potential uses of temporal orientation: [START_REF] Berrios | Orientation failures in medicine and psychiatry: discussion paper[END_REF] predicting cognitive impairment (delirium or dementia or both) from temporal disorientation; and (2) using severity of temporal disorientation as a guide to the severity of dementia.

Predicting cognitive impairment from temporal disorientation

Error scores for the different components of temporal orientation (year, date, day, month, and time) were examined in patients with and without cognitive impairment. A single error point was given for each 30 minutes, day of week, day of month, month or year removed from the correct answer. It was considered likely that failure to provide an answer for any aspect of temporal orientation would be most likely in those with cognitive impairment; hence, non-responses received two error points. The area under receiver-operating characteristic (ROC) curves relating the different aspects of temporal orientation to a diagnosis of cognitive impairment were examined; this measure can take values between 0 and 1, where 1 is a perfect screening test and 0.5 is a test equal to chance. Examination of the ROC curves was used to select the cutoff points in error scores for each aspect that maximised the sum of sensitivity and specificity. Sensitivity, specificity and positive and negative likelihood ratios (with ninety five percent confidence intervals (CI)) were calculated for each cutoff.

Stepwise logistic regression was used to determine the optimal combination of orientation items with the intention of deriving a simple model for predicting the presence or absence of cognitive impairment from temporal orientation items alone. The Nagelkerke R square, which ranges between 0 (no association) and 1 (perfect association), was used to provide a guide to the power (effect size) of the model; [START_REF] Field | Discovering Statistics Using SPSS[END_REF] this can be regarded as an approximation of the ordinary least squares R square in linear regression which measures the proportion of variance in the outcome explained by the regression model.

Severity of temporal disorientation as a measure of severity of cognitive impairment

The relationships between dementia stage, as a measure of severity of cognitive impairment, and errors in different aspects of orientation were examined using the Jonckheere-Terpstra test, a non-parametric equivalent of chi-square for trend. [START_REF] Field | Discovering Statistics Using SPSS[END_REF][START_REF] Mehotcheva | The Kruskal-Wallis Test[END_REF] The same test was used to assess the relationship between dementia stage and simple additive and weighted approaches to measuring temporal orientation error scores. In the additive approach, a single error point was given for any error in year, month, date, day of the week or time, and these were summed to give an overall score from 0 to 5. In the weighted approach, we calculated the Temporal Orientation Scale (TOS) described by Benton and colleagues with, as before, a double score being given for non-responses in any domain (Table 1). [START_REF] Benton | Temporal Orientation in Cerebral Disease[END_REF] Subjects with delirium, with or without coexisting dementia, were excluded from all of these analyses.

Table 1. Scoring system for Benton's Temporal Orientation Scale (TOS)

Time of day: 1 point for each 30 minutes removed from correct time (maximum 5 points).

Day of week: 1 point for each day removed from correct day (maximum 3 points).

Day of month: 1 point for each day removed from correct day (maximum 15 points).

Month: 5 points for each month removed from correct month, but if date is within 15 days of correct date, no additional points are scored for the incorrect month; for example, July 28 th instead of August 2 nd gets 5 points.

Year: 10 error points for each year removed from correct year (maximum 60 points), but , if date is within 15 days of correct date, no additional points are scored for the incorrect year; for example, December 28 th 2008 for January 2 nd 2009 gets 5 error points.

The total number of points equals the patient's score on the test.

RESULTS

Of the 262 patients included in the study, 169 were inpatients and 93 outpatients. There were 151women and 111 men; median (range) age was 76 (65 -101) years. Overall, 62 (23.7%) patients (15 (16.1%) outpatients and 47 (27.8%) inpatients) had cognitive impairment: 15 (all inpatients) had delirium (11 of whom also had dementia) and 47 had dementia (mild in 26, moderate in 12, severe in 9); an additional 18 subjects (6.9% of study population) had questionable dementia.

The frequency of non-responses for different aspects of temporal orientation among the 262 patients were 25 (9.5%) for date, 12 (4.6%) for time of day, 10 (3.8%) for day of the week, 8 (3.1%) for month and 7 (2.7%) for year. Inspection of the data showed that nonresponses were more common with increasing cognitive impairment. Among the 200 patients without cognitive impairment, the frequency of non-responses were 8 (4.0%) for date, 4 (2.0%) for time of day, 3 (1.5%) for day of the week, 1 (0.5%) for month and 0% for year.

In the 200 patients without cognitive impairment, the following was the frequency of error (or failure to respond): date in 123 (61.5%); time (30 minutes or more) in 98 (49%); day of the week in 36 (18%); month in 28 (13%); and year in 12 (6%). Most errors were relatively minor: thus, again including failure to respond among the errors, 76 (38%) had the date wrong by 3 days or more, 61 (30.5%) had the time wrong by an hour or more, 12 (6%) had the day of the week wrong by more than one day, 4 (2%) had the month wrong by more than one month and 5 (2.5%) had the year wrong by more than one year. Also, of the 28 people who made an error in identifying the month, 9 made their error when the date of testing was one or two days either side of the start of the month.

Visual inspection and analysis of data in those without cognitive impairment showed a significant relationship between the presence and the magnitude of errors in identifying the date and the day of the week and increased length of hospital stay (LOS). For date, median LOS was 0 (range 0 -78) for those with no error and 6 (0 -67) for those with an error, p (Mann -Whitney U test) < 0.0001; there was a positive correlation (Spearman's rho 0.28, p<0.0001) between the size of the error and LOS (outpatients being given a LOS of zero). For day of the week, median LOS was 3 (range 0 -169) for those with no error and 7 (0 -67) for those with an error, p = 0.002; Spearman's rho was 0.21 (p=0.003). For year, median LOS was 3 (range 0 -133) for those with no error and 6 (0 -169) for those with an error, p = 0.08. For month, median LOS was 4 (range 0 -78) for those with no error and 4 (0 -169) for those with an error, p = 0.65. For time of day, median LOS was 3 (range 0 -78) for those with no error and 4 (0 -169) for those with an error, p = 0.1.

Predicting cognitive impairment from temporal disorientation

The best cut-offs for detection of cognitive impairment were: any error in identifying the year, month, day of the week or date and an error of 1 hour or more in identifying the time of day. The test characteristics associated with these cut-offs are shown in Table 2.

Areas under the ROC curve (95% CI, significance level) for detection of cognitive impairment were: 0.92 (0.86 -0.97, p<0.0001) for error in year, 0.80 (0.72 -0.87, p<0.0001) for error in month, 0.73 (0.67 -0.80, p<0.0001) for error in time of day, 0.70 (0.62 -0.78, p<0.0001) for error in day of the week and 0.61 (0.54 -0.69, p=0.006) for error in date. (If the definition of error in identifying month was changed to exclude errors within two days of a change in month, sensitivity of an error decreased to 0.66 (41/62) while specificity increased to 0.95 (190/200); the area under the ROC curve was almost identical at 0.79 (0.72-0.87)).

Of the 15 subjects with delirium, all misidentified (or failed to respond to) the year and the day of the month, 14 (93.3%) did not know the time and 12 (80.0%) did not know the month or day of the week.

In a backward stepwise logistic regression, not knowing the year (odds ratio (OR) 37.2 (95% confidence interval (CI) 13.1 -101.3), not knowing the month (OR 3.85 (1.43 -10.38) and error of 1 hour or more in time of day (OR 2.76 (1.0 -7.46) were independent predictors of cognitive impairment (Nagelkerke R square 0.70). On examination of the different possible combinations of these three factors, the highest sum of sensitivity and specificity for the detection of cognitive impairment was provided by the presence of either error in year or month ( 95% sensitivity (59/62) and 86.5% specificity (173/200)).

DISCUSSION

Our results confirm the value of testing temporal orientation in older hospital patients and establish useful cut-offs for identifying dementia or delirium in this group. Failure to identify the year correctly was the most valuable single sign of cognitive impairment, combining high sensitivity and specificity, while the combination of either error in identifying year or month provided the best combination of sensitivity and specificity.

Molloy and colleagues recommended in their standardized version of the MMSE that the responses to the month item should be accepted as correct if, on the first day of a new month or the last day of the previous month, people say either of these months. [START_REF] Molloy | Reliability of a Standardized Mini-Mental State Examination compared with the traditional Mini-Mental State Examination[END_REF] In the current study, many errors in identifying the month in cognitively normal people occurred within two days either side of the new month; however, improved specificity with using this diagnosis of error in identifying the month was more than offset by a reduction in sensitivity to cognitive impairment.

Even when using optimal cut-offs, assessment of the day of the month, the day of the week and the time of day were less helpful tests for identifying cognitive impairment in this study, although, because inability to identify the day of the month was so common in those with cognitive impairment, the absence of such an error might be useful in effectively ruling out a significant cognitive problem. There was a strong relationship between the duration of hospital stay and both the presence and the magnitude of errors in identifying date of the month and day of the week. This is not surprising given the relative absence of usual social and environmental cues during prolonged hospitalization.

The assumption that errors in knowing the year or the month are more likely to be significant than mistakes such as misstating the day of the month by a day or two is reflected in the TOS which has differently weighted scores for different elements of orientation, with errors in month and particularly year attracting higher error scores than errors in day or date. [START_REF] Benton | Temporal Orientation in Cerebral Disease[END_REF] However, in the Mini Mental State Examination (MMSE), [START_REF] Tombaugh | The mini-mental state examination: a comprehensive review[END_REF] questions regarding the season, day, month, year or date are scored equally as correct or incorrect, and a similar approach is taken in other brief cognitive tests. [START_REF] Jitapunkul | The abbreviated mental test: its use and validity[END_REF][START_REF] Nasreddine | The Montreal Cognitive Assessment, MoCA: a brief screening tool for mild cognitive impairment[END_REF] Both approaches to measuring severity of temporal disorientation have been moderately

predictive of performance on more detailed cognitive testing in a variety of patient groups. [START_REF] Varney | Predicting short-term memory on the basis of temporal orientation[END_REF][START_REF] Ryan | Predicting neuropsychological test performance on the basis of temporal orientation[END_REF][START_REF] Desmond | Disorientation following stroke: frequency, course, and clinical correlates[END_REF].

In the current study, both the additive scores and the TOS increased significantly with increasing severity of dementia. However, the greater effect size of the TOS supports the value of using a weighted scoring system. Further support for this approach comes from the finding that higher weighting of year error scores in the Modified Mini-Mental State Examination (3MS), [START_REF] Teng | The Modified Mini-Mental State (3MS) examination[END_REF] a revision of the MMSE, results in increased discrimination between normal cognitive function and Alzheimer's disease patients. [START_REF] Tombaugh | Test-retest reliable coefficients and 5-year change scores for the MMSE and 3MS[END_REF] Although the TOS remains the best standardized psychometric test of temporal orientation, the complexity of the scoring system is a disadvantage. The current study also suggests that the capping of year error scores when a 6 year error is reached is too restrictive; in our older hospital population, errors of some decades were not uncommon and occurred mainly in those with moderate to severe dementia. Indeed our results

suggest that reliance on year error scores alone might be a simple alternative in an older population.

Although the number of patients with delirium in the current study was small, orientation errors were very sensitive to delirium and the severity of disorientation in delirious patients was comparable to that seen in those with severe dementia. The relationship between the severity of temporal disorientation and of dementia raises the question of whether severity of disorientation might also be a simple marker of the severity of delirium and, as Varney and Shephard suggested, [START_REF] Varney | Predicting short-term memory on the basis of temporal orientation[END_REF] whether serial assessment of temporal orientation might be useful in monitoring change in cognitive status in patients with delirium. Studies in head-injury patients support the value of serial assessment of orientation [START_REF] High | Recovery of orientation following closed-head injury[END_REF][START_REF] Bode | Measurement properties of the Galveston Orientation and Amnesia Test (GOAT) and improvement patterns during inpatient rehabilitation[END_REF], but this requires further assessment in delirium.

The independent assessment of temporal orientation and of cognitive status on the same day in all subjects is an advantage of this study. The use of a convenience sample of patients is a major disadvantage of the study since this approach gives rise to concern about sampling bias and limits the generalizability of the study. Furthermore, we did not assess educational status of patients, although there is evidence that errors are more common in those with less education. [START_REF] Natelson | Temporal orientation and education. A direct relationship in normal people[END_REF] The evidence in this study for the superiority of the weighted approach to assessing severity of disorientation is indirect, and numbers were inadequate to examine the relative ability of different scoring systems to distinguish different stages of dementia.

Recognition of dementia and, more importantly, delirium in hospital patients by medical and nursing staff is notoriously poor. [START_REF] Inouye | Nurses' recognition of delirium and its symptoms: comparison of nurse and researcher ratings[END_REF] Orientation is often the only aspect of

Figure 1 .

 1 Figure 1. Boxplot relating error in year to dementia stage.

Figure 1 .A

 1 Figure 1. Boxplot relating error in year to dementia stage.

Table 2 . Sensitivity, specificity and likelihood ratios (with 95% CIs) of temporal orientation tests for the diagnosis of cognitive impairment.

 2

	Test	Sensitivity	Specificity	+LR	-LR
	Year	0.86	0.94	14.25	0.15
	(any error)	(0.78 -0.91) (0.92 -0.96) (9.15 -21.33) (0.09 -0.25)
	Month	0.69	0.86	4.95	0.36
	(any error)	(0.60 -0.78) (0.83 -0.89) (3.50 -6.82) (0.25 -0.49)
	Date	0.95	0.38	1.55	0.13
	(any error)	(0.88 -0.98) (0.36 -0.39) (1.36 -1.27) (0.04 -0.35)
	Day of week	0.58	0.82	3.23	0.51
	(any error)	(0.48 -0.68) (0.79 -0.85) (2.26 -4.48) (0.38 -0.66)
	Time of day	0.77	0.70	2.54	0.33
	(≥ 1 hour error)	(0.67 -0.86) (0.66 -0.72) (2.00 -3.05) 0.20 -0.50)
	LR = likelihood ratio.

Funding:

This study received no funding.

Severity of temporal disorientation as a measure of severity of cognitive impairment

The relationship between dementia stage and the magnitude of orientation errors are shown in Table 3; results for delirious patients are included for comparison. Standardised Jonckheere-Terpstra (J-T) statistics showed that scores on all measures, except error in identifying the date, increased significantly with increasing severity of dementia. Effect size provides a useful metric for comparing the relative magnitude of the differences between the groups with each measure of severity: as a rough guide, a large effect size is one that is equal to or greater than 0.80, a medium effect is one that is equal to or greater than 0.50 but less than 0.80, and a small effect is one that is equal to or greater than 0.20 but less than 0.50. These results suggest that while the TOS is slightly more responsive than the additive test for assessing severity of temporal orientation, assessing the magnitude of error in year on its own was an even better measure in this population. The boxplot relating error in year to dementia stage is shown in Figure 1. is not an adequate substitute for proper mental status testing or for knowledge of cognitive problems. However, if cognitive testing is to be restricted to a single domain, orientation to time is one of the most efficient measures for discriminating between those with and without dementia and in predicting cognitive decline with time. [START_REF] Tractenberg | Benchmarking a test of temporal orientation with data from American and Taiwanese persons with Alzheimer's disease and American normal elderly[END_REF][START_REF] Rockwood | Specific symptomatic changes following donepezil treatment of Alzheimer's disease: a multi-centre, primary care, open-label study[END_REF][START_REF] Lou | Postoperative cognitive changes among older Taiwanese patients[END_REF] Furthermore, the reliance on assessment of orientation has proven relatively resistant to educational interventions. [START_REF] Rockwood | Increasing the recognition of delirium in elderly patients[END_REF] Our results suggest that temporal orientation, properly recorded and interpreted, may provide a useful screening test for dementia or delirium in older hospital patients. In particular, failure to identify the year or month correctly is a sufficiently sensitive and specific indicator of dementia or delirium in older hospital patients to warrant specialist referral for more detailed assessment in this population.

Competing interests:

The authors have no competing interests to declare

Copyright declaration

The Corresponding Author (Dr Shaun O'Keeffe) has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article to be published in the Journal of Neurology, Neurosurgery & Psychiatry editions and any other BMJPGL products to exploit all subsidiary rights, as set out in http://jnnp .bmjjournals.com/ifora/licence.pdf.


Diego ; Forero

Diego A Forero

Karlijn Van Der Ven

Patrick Callaerts

Jurgen Del-Favero
email: jurgen.delfavero@ua.ac.be

AND THE BRAIN: IMPLICATIONS FOR PSYCHIATRIC DISORDERS

Keywords: Brain disorders, Psychiatric diseases, schizophrenia, miRNA, non coding RNA Brain disorders, psychiatric diseases, schizophrenia, microRNAs, non-coding RNA

MicroRNAs (miRNAs) are a class of non-protein coding genes with a growing importance in regulatory mechanisms of gene expression related to brain function and plasticity. Considering the relative lack of success of the analysis of variations in candidate protein coding genes and of genome-wide association studies to identify strong risk factors for common psychiatric disorders (PDs), miRNA genes are of particular interest for the field of psychiatric genetics as deregulation of the rate of transcription or translation of a normal gene may be phenotypically similar to disruption of the gene itself. In this paper, we review the current knowledge on the contribution of miRNAs in basic mechanisms of brain development and plasticity and their possible involvement in the pathogenesis of several PDs. Since future functional and genomic explorations of brain expressed miRNAs, and other types of non-coding RNAs, may identify additional candidate genes and pathways for common PDs, we believe that implementing additional strategies to further elucidate the role of miRNAs in the etiology of common PDs is of great importance.

INTRODUCTION Molecular genetics of psychiatric disorders

Psychiatric disorders (PDs) are debilitating diseases characterized by the dysfunction of complex cognitive and emotional processes and the presence of multiple behavioural symptoms. PDs have been classified in different categories depending on the clustering of their associated symptoms: mood disorders (bipolar disorder -BP-and major depressive disorder -MDD-), psychotic disorders (schizophrenia -SZ-), anxiety disorders (panic disorder and generalized anxiety disorder); pervasive developmental disorders (autism spectrum disorders -ASD-), among others [START_REF] Kendler | Reflections on the relationship between psychiatric genetics and psychiatric nosology[END_REF]. As five out of ten leading causes of disability in the world are related to PDs (being MDD, Alcohol use, BP, SZ and obsessive-compulsive disorders) it is obvious that PDs present a large burden on society [START_REF] Ramocki | Failure of neuronal homeostasis results in common neuropsychiatric phenotypes[END_REF].

Multiple lines of evidence show that some PDs have a strong genetic basis. For example, the heritability for BP, SZ and Attention Deficit Hyperactivity Disorder -ADHD-is estimated to be 0.59, 0.64 and 0.76, respectively [START_REF] Lichtenstein | Common genetic determinants of schizophrenia and bipolar disorder in Swedish families: a population-based study[END_REF][START_REF] Faraone | Molecular genetics of attentiondeficit/hyperactivity disorder[END_REF]. In addition, there is evidence suggesting shared genetic susceptibility for some of these PDs, such as between BP and SZ [START_REF] Lichtenstein | Common genetic determinants of schizophrenia and bipolar disorder in Swedish families: a population-based study[END_REF]. Despite this reported high heritability, joint efforts for the identification of susceptibility genes for PDs have shown little success. Meta-analyses of genomewide linkage studies [START_REF] Ng | Meta-analysis of 32 genome-wide linkage studies of schizophrenia[END_REF][START_REF] Mcqueen | Combined analysis from eleven linkage studies of bipolar disorder provides strong evidence of susceptibility loci on chromosomes 6q and 8q[END_REF] have identified significant signals in several large genomic regions containing thousands of genes. However, the specific underlying genetic alterations in those regions remain to be identified [START_REF] Burmeister | Psychiatric genetics: progress amid controversy[END_REF]. Even the recent introduction of high density SNP arrays enabling genome wide association studies (GWAS) in thousands of patients and control individuals, has failed to identify strong and consistent risk factors for common PDs. There are published reports of 8 GWAS for BP and 10 GWAS for SZ [START_REF] Alaerts | Searching genetic risk factors for schizophrenia and bipolar disorder: learn from the past and back to the future[END_REF]. In addition, there are several GWAS reports for MDD [START_REF] Muglia | Genome-wide association study of recurrent major depressive disorder in two European case-control cohorts[END_REF][START_REF] Sullivan | Genome-wide association for major depressive disorder: a possible role for the presynaptic protein piccolo[END_REF], ADHD [START_REF] Neale | Genome-wide association scan of attention deficit hyperactivity disorder[END_REF][START_REF] Lesch | Molecular genetics of adult ADHD: converging evidence from genome-wide association and extended pedigree linkage studies[END_REF] and ASD [START_REF] Ma | A genome-wide association study of autism reveals a common novel risk locus at 5p14.1[END_REF][START_REF] Wang | Common genetic variants on 5p14.1 associate with autism spectrum disorders[END_REF] with the top SNPs in these studies having low odd ratios (ORs) with the lower confidence intervals very close to 1 [START_REF] Scott | Genome-wide association and meta-analysis of bipolar disorder in individuals of European ancestry[END_REF][START_REF] Stefansson | Common variants conferring risk of schizophrenia[END_REF] , 2009). A detailed analysis of the top results from these GWAS for major PDs reveals that there is little consistency between studies. In the case of BP and SZ, only 5 (out of 174) and 5 (out of 194) genes had support from more than one GWAS, respectively (Supp. Tables S1 andS2). For MDD, ADHD and ASD the general picture was even worse, with not a single gene being supported from more than one study (Supp. Table S3). Even the most recent studies carrying out meta-analyses of GWAS in tens of thousands of patients and controls do not provide strong candidate genes for PDs.

Because of these apparent inconsistencies in genetic studies the complexity of PDs are assumed to be caused by genetic heterogeneity, where defects in multiple risk genes -either in combination or not -cause the same disorder. Apart from this 'locus heterogeneity model', another explanation is that not the coding DNA of PD risk genes is disrupted but their regulation [START_REF] Perkins | Expanding the 'central dogma': the regulatory role of nonprotein coding genes and implications for the genetic liability to schizophrenia[END_REF].

The fact that only a minor part of the top ~1,500 GWAS SNPs were located in protein coding regions (2.1% located in exons, 1.5% being nsSNPs) supports this hypothesis. The majority of identified SNPs were located in intergenic regions (55.1%), with 7.2% of the SNPs located in conserved regions in vertebrates. In addition, 4% of the top SNPs were located in UTR regions (Supp. Table S4). Therefore, the exploration of additional categories of non-coding genes and molecules is a potentially interesting approach for the identification of genetic factors implicated in PDs.

In recent years, microRNAs (miRNAs) have emerged as a new important class of non-coding RNA (ncRNA) regulatory molecules, playing a role in many major cellular processes related to brain function [START_REF] Fiore | MicroRNA function in neuronal development, plasticity and disease[END_REF]. Because deregulation of the rate of transcription and/or translation of a normal gene may be phenotypically similar to disruption of the gene itself, the exploration of these ncRNA molecules might provide candidate genes for common PDs and give additional clues about the possible role of miRNA-dependent pathways in the pathogenesis of PDs [START_REF] Perkins | Expanding the 'central dogma': the regulatory role of nonprotein coding genes and implications for the genetic liability to schizophrenia[END_REF]. In this review we provide an overview of current knowledge on the contribution of miRNAs in basic mechanisms of brain development and plasticity and their possible involvement in the pathogenesis of several PDs. The first papers describing miRNAs in humans appeared in [START_REF] Pasquinelli | Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA[END_REF][START_REF] Lagos-Quintana | Identification of novel genes coding for small expressed RNAs[END_REF][START_REF] Lagos-Quintana | Identification of novel genes coding for small expressed RNAs[END_REF][START_REF] Pasquinelli | Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA[END_REF]. Currently, there are already 706 known miRNAs for humans (miRBase release 13.0; 03/2009) [START_REF] Griffiths-Jones | miRBase: tools for microRNA genomics[END_REF]. These miRNA genes can be divided in two main categories based on their location and transcription patterns: intronic miRNAs and intergenic miRNAs [START_REF] Bartel | MicroRNAs: target recognition and regulatory functions[END_REF]. RNA polymerase II transcribes a major part of miRNAs [START_REF] Lee | MicroRNA genes are transcribed by RNA polymerase II[END_REF] and the resulting primary miRNAs (pri-miRNAs) are similar to classical mRNAs, being several kb long, polyadenylated and regulated by networks of transcription factors [START_REF] Cai | Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs[END_REF][START_REF] Lee | MicroRNA genes are transcribed by RNA polymerase II[END_REF][START_REF] Saini | Genomic analysis of human microRNA transcripts[END_REF] (Figure 1).

After the pri-miRNAs are transcribed, they are processed by the Microprocessor complex, composed of the Rnase III enzyme Drosha and Pasha (also known as DGCR8) [START_REF] Denli | Processing of primary microRNAs by the Microprocessor complex[END_REF][START_REF] Gregory | The Microprocessor complex mediates the genesis of microRNAs[END_REF]) into ~70 nucleotide precursor-miRNA (pre-miRNA) which are exported to the cytoplasm by Exportin 5 [START_REF] Lund | Nuclear export of microRNA precursors[END_REF] (Figure 1). In the cytoplasm, Dicer cleaves pre-miRNAs in a transient double-stranded RNA fragment of about 18-24 nucleotides. The duplex is loaded into the miRNA associated multiprotein RNA-induced silencing complex (RISC), composed of Argonaute proteins [START_REF] Liu | Argonaute2 is the catalytic engine of mammalian RNAi[END_REF][START_REF] Diederichs | Dual Role for Argonautes in MicroRNA Processing and Posttranscriptional Regulation of MicroRNA Expression[END_REF] and other recently described molecules [START_REF] Gregory | Human RISC couples microRNA biogenesis and posttranscriptional gene silencing[END_REF], such as TARBP2, EIF6, FXR1 and MOV10 [START_REF] Chendrimada | MicroRNA silencing through RISC recruitment of eIF6[END_REF][START_REF] Liu | Dicer-1, but not Loquacious, is critical for assembly of miRNA-induced silencing complexes[END_REF][START_REF] Macrae | In vitro reconstitution of the human RISC-loading complex[END_REF][START_REF] Jin | Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway[END_REF]) (Supp. Table S1).

One strand of the miRNA is preferentially retained in this complex and becomes the mature miRNA while the opposite strand, known as the passenger strand is eliminated from the complex.

It has been demonstrated that a 5' region in the miRNA, called the "seed", is the most important part, in terms of binding to the 3'UTR region of the target genes [START_REF] Bartel | MicroRNAs: target recognition and regulatory functions[END_REF]. There are 3 main types of binding profiles: canonical sites (binding of only the seed region), 3' supplementary sites (binding of the 3' region of the miRNA, in addition to the seed region) and 3' compensatory sites (binding of the 3' region of the miRNA with presence of mismatches in the seed region) [START_REF] Bartel | MicroRNAs: target recognition and regulatory functions[END_REF]. In animals the main consequence of miRNA targeting appears to be 6 the deadenylation of the mRNA leading to its instability [START_REF] Forstemann | Drosophila microRNAs Are Sorted into Functionally Distinct Argonaute Complexes after Production by Dicer-1[END_REF][START_REF] Karginov | A biochemical approach to identifying microRNA targets[END_REF] and/or the repression of RNA translation [START_REF] Filipowicz | Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?[END_REF][START_REF] Pillai | Inhibition of Translational Initiation by Let-7 MicroRNA in Human Cells[END_REF].

miRNAS AND THE CENTRAL NERVOUS SYSTEM

miRNAs regulate the expression of over 10,000 genes in a cell. It is therefore not surprising that miRNAs are involved in the regulation of almost all major cellular functions, such as cell differentiation, proliferation/growth, mobility, and apoptosis [START_REF] Zhang | Comparative profiling of genes and miRNAs expressed in the newborn, young adult, and aged human epididymides[END_REF]. In recent years, the interest in miRNAs and their possible roles in regulatory molecular mechanisms underlying the functional and structural complexity of the brain have grown rapidly [START_REF] Fiore | MicroRNA function in neuronal development, plasticity and disease[END_REF] (Table 1). The identification of brain-derived miRNAs has provided an initial catalogue of miRNAs of potential relevance for brain function [START_REF] Kim | Identification of many microRNAs that copurify with polyribosomes in mammalian neurons[END_REF][START_REF] Miska | Microarray analysis of microRNA expression in the developing mammalian brain[END_REF][START_REF] Wheeler | Identification of new central nervous system specific mouse microRNAs[END_REF][START_REF] He | Cloning and Identification of Novel MicroRNAs from Rat Hippocampus[END_REF]. Moreover, more systematic analysis of miRNA expression in brain tissues in different developmental stages in humans and rodents have been useful to identify those miRNAs that are brain-specific (expressed only in brain) or brain-enriched (expressed at higher levels in brain than in other tissues) [START_REF] Bak | MicroRNA expression in the adult mouse central nervous system[END_REF][START_REF] Kim | Identification of many microRNAs that copurify with polyribosomes in mammalian neurons[END_REF][START_REF] Liang | Characterization of microRNA expression profiles in normal human tissues[END_REF][START_REF] Miska | Microarray analysis of microRNA expression in the developing mammalian brain[END_REF][START_REF] Olsen | MicroRNAs show mutually exclusive expression patterns in the brain of adult male rats[END_REF][START_REF] Zhao | Genome-wide microRNA profiling in human fetal nervous tissues by oligonucleotide microarray[END_REF]) (Supp. Table S2 and supp. Figure S2). miRNAs are believed to play a critical role in brain development and structural plasticity.

Evidence for this comes from the identification of the CREB (cAMP response element binding) and BDNF (brain derived neurotrophic factor) dependent activation of miR-132 and miR-219 [START_REF] Vo | A cAMP-response element binding proteininduced microRNA regulates neuronal morphogenesis[END_REF][START_REF] Cheng | microRNA modulation of circadian-clock period and entrainment[END_REF][START_REF] Klein | Homeostatic regulation of MeCP2 expression by a CREBinduced microRNA[END_REF][START_REF] Wayman | An activity-regulated microRNA controls dendritic plasticity by down-regulating p250GAP[END_REF]. Other studies have provided evidence supporting a role of proteins involved in miRNA processing (such as Dicer, DGCR8, FXR1 or MOV10) in mechanisms of brain plasticity [START_REF] Xu | The fragile X-related gene affects the crawling behavior of Drosophila larvae by regulating the mRNA level of the DEG/ENaC protein pickpocket1[END_REF][START_REF] Lin | First in vivo evidence of microRNA-induced fragile X mental retardation syndrome[END_REF][START_REF] Karr | Regulation of glutamate receptor subunit availability by microRNAs[END_REF][START_REF] Jin | Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway[END_REF][START_REF] Giraldez | MicroRNAs regulate brain morphogenesis in zebrafish[END_REF][START_REF] Davis | Conditional loss of Dicer disrupts cellular and tissue morphogenesis in the cortex and hippocampus[END_REF][START_REF] Ashraf | Synaptic Protein Synthesis Associated with Memory Is Regulated by the RISC Pathway in Drosophila[END_REF]. Down regulation of Dicer led to molecular, morphological and physiological alterations in brains of zebrafish, fly and mouse models [START_REF] Giraldez | MicroRNAs regulate brain morphogenesis in zebrafish[END_REF] [START_REF] Davis | Conditional loss of Dicer disrupts cellular and tissue morphogenesis in the cortex and hippocampus[END_REF] [START_REF] Karr | Regulation of glutamate receptor subunit availability by microRNAs[END_REF]. [START_REF] Lin | First in vivo evidence of microRNA-induced fragile X mental retardation syndrome[END_REF][START_REF] Jin | Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway[END_REF][START_REF] Ashraf | Synaptic Protein Synthesis Associated with Memory Is Regulated by the RISC Pathway in Drosophila[END_REF]. Knockout mice for Dgcr8 presented structural brain changes and behavioural alterations [START_REF] Stark | Altered brain microRNA biogenesis contributes to phenotypic deficits in a 22q11-deletion mouse model[END_REF]. miRNAs have also been implicated in mechanisms underlying different behavioural paradigms in animal models (e.g. circadian rhythms, olfactory memory or sleep deprivation) [START_REF] Cheng | microRNA modulation of circadian-clock period and entrainment[END_REF][START_REF] Ashraf | Synaptic Protein Synthesis Associated with Memory Is Regulated by the RISC Pathway in Drosophila[END_REF][START_REF] Davis | Sleep loss changes microRNA levels in the brain: a possible mechanism for state-dependent translational regulation[END_REF]. In mice, for instance, antagomirs (i.e. small synthetic RNA molecule perfectly complementary to the miRNA target with either mispairing at the Ago2 cleavage site or base modification to inhibit Ago2 cleavage) against miR-219 and miR-132 regulated the circadian period length and resetting of the clock, respectively [START_REF] Cheng | microRNA modulation of circadian-clock period and entrainment[END_REF].

Mutations in

miRNAS AND PSYCHIATRIC DISORDERS

The data summarized in the previous section demonstrate how alterations of specific brainexpressed miRNAs may lead to altered expression levels of many genes involved in mechanisms of neural and synaptic plasticity, which are associated with the pathophysiology of several major neuropsychiatric disorders [START_REF] Forero | Synaptic dysfunction and oxidative stress in Alzheimer's disease: emerging mechanisms[END_REF][START_REF] Ramocki | Failure of neuronal homeostasis results in common neuropsychiatric phenotypes[END_REF]. Although genomewide expression and proteomics experiments in post-mortem brains of patients with major PDs have not provided protein coding genes with consistent deregulation between studies [START_REF] Choi | Putative psychosis genes in the prefrontal cortex: combined analysis of gene expression microarrays[END_REF][START_REF] Elashoff | Meta-analysis of 12 genomic studies in bipolar disorder[END_REF][START_REF] Higgs | An online database for brain disease research[END_REF], there is evidence that several signalling networks related to synaptic and brain plasticity are altered in these disorders [START_REF] Ramocki | Failure of neuronal homeostasis results in common neuropsychiatric phenotypes[END_REF][START_REF] Carter | Schizophrenia susceptibility genes converge on interlinked pathways related to glutamatergic transmission and long-term potentiation, oxidative stress and oligodendrocyte viability[END_REF][START_REF] Conner | Functional polymorphisms in the serotonin 1B receptor gene (HTR1B) predict self-reported anger and hostility among young men[END_REF][START_REF] Mudge | Genomic convergence analysis of schizophrenia: mRNA sequencing reveals altered synaptic vesicular transport in postmortem cerebellum[END_REF][START_REF] Ryan | Gene expression analysis of bipolar disorder reveals downregulation of the ubiquitin cycle and alterations in synaptic genes[END_REF][START_REF] Schloesser | Cellular plasticity cascades in the pathophysiology and treatment of bipolar disorder[END_REF]. These findings, in addition to expanding the repertoire of molecules and basic mechanisms related to brain plasticity; provide useful insights to explore potential roles of miRNA-dependent mechanisms in the pathogenesis of PDs. An overview of miRNA-regulated signalling networks important for brain plasticity and involved in PDs is shown in Figure 2.

Thus far, only a limited number of studies have addressed the role of miRNA dysfunction in PDs [START_REF] Hunsberger | MicroRNAs in mental health: from biological underpinnings to potential therapies[END_REF]. Systematic expression studies of post-mortem brains of psychiatric patients and of animal models have provided initial insights about specific miRNAs potentially involved in the pathogenesis of PDs [START_REF] Hunsberger | MicroRNAs in mental health: from biological underpinnings to potential therapies[END_REF]. In addition, exploration of common Considering the difficulties of getting access to brain tissues of patients, cell based models (especially lymphoblasts) and animal models of PDs (both genetic and pharmacological) are potential approaches to study of miRNA alterations in psychiatric disorders. An overview of miRNAs known to be deregulated in tissues of patients and/or animal and cell models for PDs (SZ, BP, ASD, mood disorders and drug abuse) [START_REF] Abu-Elneel | Heterogeneous dysregulation of microRNAs across the autism spectrum[END_REF][START_REF] Beveridge | Schizophrenia is associated with an increase in cortical microRNA biogenesis[END_REF][START_REF] Chandrasekar | microRNAs miR-124, let-7d and miR-181a regulate Cocaine-induced Plasticity[END_REF][START_REF] Chen | MicroRNA expression changes in lymphoblastoid cell lines in response to lithium treatment[END_REF][START_REF] Kocerha | MicroRNA-219 modulates NMDA receptor-mediated neurobehavioral dysfunction[END_REF][START_REF] Meerson | Changes in Brain MicroRNAs Contribute to Cholinergic Stress Reactions[END_REF][START_REF] Mellios | Molecular Determinants of Dysregulated GABAergic Gene Expression in the Prefrontal Cortex of Subjects with Schizophrenia[END_REF][START_REF] Perkins | Expanding the 'central dogma': the regulatory role of nonprotein coding genes and implications for the genetic liability to schizophrenia[END_REF][START_REF] Stark | Altered brain microRNA biogenesis contributes to phenotypic deficits in a 22q11-deletion mouse model[END_REF][START_REF] Talebizadeh | Feasibility and relevance of examining lymphoblastoid cell lines to study role of microRNAs in autism[END_REF][START_REF] Uchida | Characterization of the vulnerability to repeated stress in Fischer 344 rats: possible involvement of microRNAmediated down-regulation of the glucocorticoid receptor[END_REF][START_REF] Zhou | Evidence for selective microRNAs and their effectors as common long-term targets for the actions of mood stabilizers[END_REF][START_REF] Zhu | A MicroRNA gene is hosted in an intron of a schizophrenia-susceptibility gene[END_REF] is shown in Table 2.

Thus far, three miRNA expression studies in post-mortem brains of SZ patients have been performed, which have led to several interesting insights [START_REF] Beveridge | Dysregulation of miRNA 181b in the temporal cortex in schizophrenia[END_REF][START_REF] Perkins | miRNA expression in the prefrontal cortex of individuals with schizophrenia and schizoaffective disorder[END_REF][START_REF] Beveridge | Schizophrenia is associated with an increase in cortical microRNA biogenesis[END_REF]. In a first study, [START_REF] Perkins | miRNA expression in the prefrontal cortex of individuals with schizophrenia and schizoaffective disorder[END_REF] used prefrontal cortex tissue of 13 schizophrenia patients and 21 controls to analyze the expression of 265 mature miRNAs. 16 miRNAs were differentially expressed (15 down-regulated, 1 up-regulated) and 4 out of 8 downregulated miRNAs were confirmed by RT-PCR. Several signalling pathways related to mechanisms of synaptic function, such as calcium-dependent gene regulation, were enriched in the predicted target genes of these deregulated miRNAs [START_REF] Perkins | miRNA expression in the prefrontal cortex of individuals with schizophrenia and schizoaffective disorder[END_REF]. [START_REF] Beveridge | Dysregulation of miRNA 181b in the temporal cortex in schizophrenia[END_REF] used RNA isolated from the superior temporal gyrus of 7 SZ-control pairs to study 262 miRNAs and they confirmed the up-regulation of two miRNAs. The over expression of one of these miRNAs -miR-181b -was also shown by northern blot and RT-PCR. Here also, functional categories related to synaptic function, such as axon guidance, were enriched among the predicted targets of miR-181b [START_REF] Beveridge | Dysregulation of miRNA 181b in the temporal cortex in schizophrenia[END_REF]. The same group expanded their results and studied miRNA expression in a larger group of 17 SZ/control pairs for superior temporal gyrus and 15 SZ/control pairs for dorsolateral prefrontal cortex. In patients, 21 and 9.5 percent, respectively, of the expressed miRNAs in those tissues were up regulated. Members of the miR-15 family were particularly prominent among these up-regulated miRNAs. Again, deregulation of signalling pathways related to mechanisms of synaptic function was implicated, as shown by the enrichment of predicted target genes of the miR-15 family in these functional categories. Because these increased pre-miRNA expression levels were accompanied by normal values for pri-miRNAs expression levels, the authors suggested a global alteration in miRNA processing in SZ brains which was correlated with increased mRNA levels for Drosha, DGCR8 and DICER1. Four predicted targets of the miR-15 family of relevance for psychiatric genetics were experimentally validated (RGS4, GRM7, GRIN3A and RELN) [START_REF] Beveridge | Schizophrenia is associated with an increase in cortical microRNA biogenesis[END_REF].

Further evidence supporting a role for miRNA alterations in PDs comes from the use of genetic and pharmacological animal models. [START_REF] Stark | Altered brain microRNA biogenesis contributes to phenotypic deficits in a 22q11-deletion mouse model[END_REF] generated a mouse model with a hemizygous 1.3 Mb deficiency of a genomic region that is syntenic to the 1.5 Mb region missing in the 22q11.2 microdeletion found in SZ patients; it contains 27 genes, including Dgcr8. These animals showed behavioural alterations in several domains of relevance for PDs: locomotor hyperactivity, anxiety-like behaviours and deficits in prepulse inhibition, cued and contextual fear conditioning and working memory. Microarray analysis of hippocampus and prefrontal cortex of these animals showed alterations in the expression levels of multiple genes related to synaptic function. Expression levels of Dgcr8 were reduced, while no differences were seen in the expression levels of other genes involved in the miRNA processing machinery (Rnasen, Dicer1, among others). When further investigating the effect of this down regulation on miRNA processing, levels of pri-miRNAs were increased while mature miRNAs had decreased levels, which was also confirmed in heterozygous knockout mice for Dgcr8. This study is one of the first to suggest a potential link between a genetic alteration found in SZ patients (22q11.2 microdeletion) and a broad dysfunction in miRNA-dependent mechanisms of transcriptional regulation mediated by alterations in a gene involved in miRNA processing (DGCR8) [START_REF] Stark | Altered brain microRNA biogenesis contributes to phenotypic deficits in a 22q11-deletion mouse model[END_REF]. [START_REF] Zhou | Evidence for selective microRNAs and their effectors as common long-term targets for the actions of mood stabilizers[END_REF] treated rats with lithium and valproic acid and found respectively 37 and 31 responsive miRNAs with 9 miRNAs in common. GRM7, a glutamate metabotropic receptor and 10 one of the targets of one of these miRNAs (miR-34a) had increased protein levels in hippocampal regions of the treated animals [START_REF] Zhou | Evidence for selective microRNAs and their effectors as common long-term targets for the actions of mood stabilizers[END_REF]. In another study, mice were treated with a selective phencyclidine-like NMDA antagonist (dizolcipine) and found that miR-219 was downregulated in the prefrontal cortex [START_REF] Kocerha | MicroRNA-219 modulates NMDA receptor-mediated neurobehavioral dysfunction[END_REF]. Possible involvement of this miR-219 gene in PDs was suggested by the observation that this dizolcipine-induced down-regulation could be prevented by the treatment with antipsychotics (haloperidol and clozapine) and the use of an antagomir against miR-219 reduced the behavioural effects of dizolcipine treatment.

Although miRNA genes are usually highly conserved through evolution, there is evidence of intra individual variation in some of this conserved non-protein coding regions in the human genome [START_REF] Drake | Conserved noncoding sequences are selectively constrained and not mutation cold spots[END_REF][START_REF] Quach | Signatures of purifying and local positive selection in human miRNAs[END_REF][START_REF] Adams | The micro-ribonucleic acid (miRNA) miR-206 targets the human estrogen receptoralpha (ERalpha) and represses ERalpha messenger RNA and protein expression in breast cancer cell lines[END_REF][START_REF] Duan | Single nucleotide polymorphism associated with mature miR-125a alters the processing of pri-miRNA[END_REF]Muinos-Gimeno et al., 2009b;[START_REF] Saunders | Human polymorphism at microRNAs and microRNA target sites[END_REF]. Variations in candidate miRNA genes or in their binding sites have been analyzed in several PDs, such as Tourette syndrome [START_REF] Abelson | Sequence variants in SLITRK1 are associated with Tourette's syndrome[END_REF], schizophrenia [START_REF] Burmistrova | MicroRNA in schizophrenia: genetic and expression analysis of miR-130b (22q11)[END_REF][START_REF] Hansen | Brain Expressed microRNAs Implicated in Schizophrenia Etiology[END_REF][START_REF] Feng | Evidence for Xchromosomal schizophrenia associated with microRNA alterations[END_REF], mental retardation [START_REF] Chen | Mutation screening of brain-expressed X-chromosomal miRNA genes in 464 patients with nonsyndromic X-linked mental retardation[END_REF][START_REF] Zhang | A microdeletion in Xp11.3 accounts for cosegregation of retinitis pigmentosa and mental retardation in a large kindred[END_REF], conduct disorder-related behaviours [START_REF] Jensen | A common polymorphism in serotonin receptor 1B mRNA moderates regulation by miR-96 and associates with aggressive human behaviors[END_REF][START_REF] Conner | Functional polymorphisms in the serotonin 1B receptor gene (HTR1B) predict self-reported anger and hostility among young men[END_REF], nicotine dependence [START_REF] Huang | Differential allelic expression of dopamine D1 receptor gene (DRD1) is modulated by microRNA miR-504[END_REF], panic disorder (Muinos-Gimeno et al., 2009a) and obsessive compulsive disorder (Muinos-Gimeno et al., 2009a). An overview of published genetic variations in PD associated miRNA genes and 3'UTR miRNA binding sites is provided in tables 3 and 4.

As an example, Chen et al. ( 2007) screened brain-expressed miRNA genes located on chromosome X in 464 patients with X linked non-syndromic mental retardation and found 3 patient-specific variants (two in pre-miR-222 and one in pre-miR-363, each being present in only one patient). The two variations in miR-222 co-segregated with the affection status [START_REF] Chen | Mutation screening of brain-expressed X-chromosomal miRNA genes in 464 patients with nonsyndromic X-linked mental retardation[END_REF].

A few studies uncovered genetic variations in miRNA binding sites. Abelson et al [START_REF] Abelson | Sequence variants in SLITRK1 are associated with Tourette's syndrome[END_REF] identified a variation in the binding site for miR-189 in the 3' UTR region of SLITRK1 in 2 unrelated patients (with Tourette syndrome and obsessive compulsive symptoms) not found in Muinos-Gimeno et al (Muinos-Gimeno et al., 2009a) sequenced the 3' UTR region of NTRK3 (a neurotrophin receptor) and identified two variants in 212 Spanish patients with panic disorder which led to allele-dependent effects on gene expression.

OTHER ncRNAs AND THE BRAIN

It is evident that miRNAs are part of a bigger picture of ncRNA dependent regulation of gene expression [START_REF] Kapranov | Genome-wide transcription and the implications for genomic organization[END_REF][START_REF] Rigoutsos | Short blocks from the noncoding parts of the human genome have instances within nearly all known genes and relate to biological processes[END_REF]. Unfortunately, few studies have performed to elucidate the role of other types of ncRNAs, such as long ncRNAs, on brain function. A comprehensive analysis of the transcriptional landscape in mice (FANTOM3 project) identified a total number of 181.047 independent transcripts, from which 34.030 corresponded to potential long ncRNAs [START_REF] Katayama | Antisense transcription in the mammalian transcriptome[END_REF]. These long ncRNAs are suspected to be involved in the regulation of several target genes, possibly through anti-sense based mechanisms [START_REF] Katayama | Antisense transcription in the mammalian transcriptome[END_REF]. Mercer et al. (2009) studied the expression of long ncRNAs in different mouse brain regions using high-throughput RNA in situ hybridization and found 849 long ncRNAs with expression levels above background. Of these 239 have conserved secondary structures; 60 showed ubiquitous expression and 513 showed enriched expression profiles in specific regions [START_REF] Mercer | Specific expression of long noncoding RNAs in the mouse brain[END_REF]. Another microarray based study by Mercer et al. (2010) for systematic analysis of expression profiles of long ncRNAs during differentiation of mice embryonic forebrain derived neural stem cells showed several ncRNA expression patterns associated with the differentiation mechanisms from stem cells to neural or glial cell types. In addition, they showed that brain-expressed long ncRNAs are preferentially located in genomic regions close to protein coding genes involved in brain development and function indicating spatiotemporal co-expression. A specific long ncRNA (Evf2) was shown to modulate the expression of several transcription factors (such as MECP2 and DLX5) in developing neurons of mice [START_REF] Bond | Balanced gene regulation by an embryonic brain ncRNA is critical for adult hippocampal GABA circuitry[END_REF]. Evf2 mutants showed reduced numbers of GABAergic interneurons during brain development. Sone et al ( 2007) identified a novel ncRNA molecule (called Gomafu) with a characteristic expression pattern in the mouse nervous system localized in the cell nucleus.

IMPLICATIONS FOR FUTURE WORK

An increasing amount of data indicates that miRNAs act as key regulators in a large number of differential cellular processes [START_REF] Fiore | MicroRNA function in neuronal development, plasticity and disease[END_REF]. Furthermore, a variety of miRNAs has already been found in the CNS, of which the list is still expanding. Due to their crucial role in brain development and plasticity and broad genetic regulatory influence, growing evidence suggests that miRNAs may prove to be an important key to our understanding of the pathophysiology and -ultimately -the therapeutics of PD. Investigating the role of miRNAs in disease biology is a new frontier in biomedical research and further exploration of these ncRNAs in the etiology of PDs will benefit from the implementation of additional high throughput experimental approaches.

In this context, there are three key complementary issues to consider for further studies.

First, at present, the systematic analysis of miRNA expression has been carried out in a limited number of types and models of PDs. Comprehensive analyses of miRNA expression profiles in post-mortem brains of PD patients of epidemiological relevance (bipolar disorder and major depression, for example) will accelerate the identification of additional deregulated miRNAs [START_REF] Fiore | MicroRNA function in neuronal development, plasticity and disease[END_REF], and further exploration of miRNA expression profiles in pharmacological and genetic models of PDs will help to elucidate the transcriptional networks underlying PDs.

Apart from expanding the different PDs under investigation, considering the commondisease/common-variant and the common-disease/rare-variants hypotheses [START_REF] Bodmer | Common and rare variants in multifactorial susceptibility to common diseases[END_REF], further systematic studies of novel rare variations (using DNA sequencing) and common known variations (using DNA genotyping) in miRNA genes and their target gene binding sites is a second key issue that will help to identify additional ncRNA based risk factors for PDs. Recent developments in novel high-throughput methodologies for DNA sequencing and genotyping have created the opportunity to implement such approaches. These studies can be complemented by investigating variations in genes involved in the miRNA processing machinery and of CNVs in miRNA regions. In this context, the recent observations that miRNAs can also regulate the function of target genes through binding to their promoter and coding regions [START_REF] Place | MicroRNA-373 induces expression of genes with complementary promoter sequences[END_REF]; 13 [START_REF] Tay | MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation[END_REF], have important implications for the design and interpretation of these genetic studies.

As a final issue to fully comprehend the involvement of miRNAs in the etiology of PDs, we wish to emphasize the importance to confirm predicted functional effects of the deregulation of PDassociated miRNAs. This confirmation will greatly depend on the implementation of systematic analyses of behavioural and molecular profiles of other animal models (e.g. mice and flies) with genetic manipulations in candidate brain-expressed miRNAs and other genes involved in miRNA processing. A promising approach there is the targeted knock-down of specific disease related miRNAs using anti-miRNA oligos (miRNA inhibitors) [START_REF] Zhang | MicroRNomics: a newly emerging approach for disease biology[END_REF].

Although only a limited number of candidate miRNAs and UTRs have been studied in PD patients previously, these results clearly highlight the importance of exploring additional nonprotein-coding regions. We propose that future functional and genomic explorations of brain expressed miRNAs, and of other types of non-coding RNAs, will facilitate the identification of additional candidate genes and pathways for common PDs.

ACKNOWLEDGEMENTS:

Work in the laboratory of the authors was supported by grants from the Fund for Scientific Research Flanders (FWO-F), the Industrial Research Fund (IWT) and the Special Research Fund of the University of Antwerp, Belgium. DAF is supported by a fellowship from the VIB International PhD Program.

  Drosophila dfmr1 (homolog of the human FXR1 gene) and armitage (homolog of the human MOV10 gene) led to structural brain changes and behavioural abnormalities(Xu et al.,

  in miRNA genes and in their binding sites has given additional candidate genetic factors associated with several PDs. In the following paragraphs we present an overview of recent studies that have examined the biological role of miRNAs in PDs.

Figure 1 .Figure 2 :

 12 Figure 1. Known molecular mechanisms implicated in miRNA generation and processing. RNA pol II: RNA polymerase II; TU: Transcriptional Unit; TF : Transcription Factor.

Table 3 :

 3 Overview of published genetic variants in human microRNA genes associated with psychiatric disorders.

						Frequency
	3'UTR (miR)	Location	Variation	Phenotype	Population	(%)	Functional effects of mt allele	Reference
						Cases/Controls
	SLITRK1 (miR-189)	Chr 13q31.1	G/A (+689)	Tourette's syndrome (174 cases, 2148 controls)	USA	0.57/0.0	Higher inhibition of gene expression	(Abelson et al., 2005)
	HTR1B (miR-96)	Chr 6q14.1	A/G (rs13212041)	Quantitative measures of conduct-disorder-related behaviours (359 subjects)	USA	-/16	Lower inhibition of gene expression	(Jensen et al., 2009)
	DRD1 (miR-504)	Chr 5q35.2	A/G (rs686)	Nicotine dependence (2037 individuals)	USA	-/40.5	Lower activation of gene expression	(Huang and Li, 2009)
	NTRK3 (miR-485-3p)	Chr 15 q25.3	C/G rs28521337	Obsessive compulsive disorder (153 patients and 324 controls)	Spain	40/47	No differences in inhibition of gene expression	(Muinos-Gimeno et al., 2009)
	NTRK3 (miR-128 and miR-509)	Chr 15 q25.3	T/C (ss102661458)	Panic Disorder (212 patients and 324 controls)	Spain	1 patient/0	Lower inhibition of gene expression	(Muinos-Gimeno et al., 2009)
	NTRK3 (miR-765)	Chr 15 q25.3	G/C (ss102661460)	Panic Disorder (212 patients and 324 controls)	Spain	1 patient/0	Lower inhibition of gene expression	(Muinos-Gimeno et al., 2009)
	HTR1B (miR-96)	Chr 6q14.1	A/G (rs13212041)	Self-reports of anger and hostility in young men (361 subjects)	USA	-/16	Lower inhibition of gene expression	(Conner et al., 2009)

Table 4 :

 4 Overview of published genetic variants in miRNA binding sites in 3'UTR regions of candidate genes associated with psychiatric disorders in humans.

John Wiley & Sons, Inc.Human Mutation

microRNAs

Model Phenotype Reference miR-132 Neonatal rat cortical neurons Over-expression led to enhancement of neurite outgrowth; via p250GAP, miR-132 is regulated by CREB [START_REF] Vo | A cAMP-response element binding proteininduced microRNA regulates neuronal morphogenesis[END_REF][START_REF] Wayman | An activity-regulated microRNA controls dendritic plasticity by down-regulating p250GAP[END_REF] miR-134

Cultured rat hippocampal neurons Over-expression led to a diminution of dendritic spine volume and width; it regulates Limk1 (Schratt et al., 2006) miR-124 Neuronal cell lines Over-expression led to enhancement of neuronal differentiation and incorporation of neural-specific exons in some genes (Makeyev et al., 2007) miR-138 Rat hippocampal neurons Inhibition led to an increase in dendritic spine volume (Siegel et al., 2009) Circadian rhythm in mouse An antagomiR shortened the circadian period length [START_REF] Cheng | microRNA modulation of circadian-clock period and entrainment[END_REF] miR-30a, miR-195 Rat neurons and human prefrontal cortex

These miRNAs down-regulated BDNF and showed inverse correlations with levels of BDNF protein in human prefrontal cortex (Mellios et al., 2008b) let-7b, let-7d Rats Up-regulated in hippocampus of sleep deprived rats (Davis et al., 2007) miRNA-200 Family Rats Specific expression in olfactory neurons (Choi et al., 2008) miR-24, miR-92, miR-204, miR-325, miR-293, miR-181-b, miR-214, miR-292- 2007).