Johannes Hebebrand
email: johannes.hebebrand@uni-duisburg-essen.de

Andre Scherag

Benno G Schimmelmann

Anke Hinney

Á A Hinney

Child and adolescent psychiatric genetics

Keywords: Candidate gene, Linkage, Rett syndrome, Gene-environment interaction, Genome-wide association study

The current status of child and adolescent psychiatric genetics appears promising in light of the initiation of genome-wide association studies (GWAS) for diverse polygenic disorders and the molecular elucidation of monogenic Rett syndrome, for which recent functional studies provide hope for pharmacological treatment strategies. Within the last 50 years, tremendous progress has been made in linking genetic variation to behavioral phenotypes and psychiatric disorders. We summarize the major findings of the Human Genome Project and dwell on largely unsuccessful candidate gene and linkage studies. GWAS for the first time offer the possibility to detect single nucleotide polymorphisms and copy number variants without a priori hypotheses as to their molecular etiology. At the same time it is becoming increasingly clear that very large sample sizes are required in order to enable genome wide significant findings, thus necessitating further largescaled ascertainment schemes for the successful elucidation of the molecular genetics of childhood and adolescent psychiatric disorders. We conclude by reflecting on different scenarios for future research into the molecular basis of early onset psychiatric disorders. This review represents

Introduction

Over the past 50 years, substantial progress has been made in linking specific behavioral and psychiatric phenotypes to chromosomal aberrations or genetic variation at the DNA level. Prerequisites of this development were significant conceptual, methodological and technical advances in both molecular and statistical genetics.

Advances in cytogenetics allowed the identification of specific syndromes based on quantitative chromosomal imbalances of complete chromosomes as in trisomy 21 in 1959 [START_REF] Lejeune | E ´tude des chromosomes somatiques de neuf enfants mongoliens[END_REF] and later [START_REF] Lejeune | 3 cases of partial deletion of the short arm of a 5 chromosome[END_REF] of chromosomal regions as in the cri du chat syndrome due to monosomy 5p [START_REF] Lejeune | 3 cases of partial deletion of the short arm of a 5 chromosome[END_REF]. Approximately 5 Mb represents the resolution limits of banding techniques; fluorescent in situ hybridization (FISH) allows detection of deletions as small as 1.5 Mb. Micro-deletions can result in syndromes with distinct behavioral features such as the Prader-Willi syndrome (paternal 15q11), Angelman syndrome (maternal 15q11), and the velocardiofacial/DiGeorge syndrome (22q11). Because a single gene on average encompasses 10-15 kb, the phenotypes of micro-deletion syndromes result from the loss of a number of genes (partial monosomy). Quantitative imbalances can functionally result in overexpression (e.g., trisomy or partial trisomy) or underexpression (monosomy or partial monosomy) of those genes located on a chromosome or within a specific chromosomal region. Due to the high proportion of genes (&80% in mouse brain; [START_REF] Lein | Genome-wide atlas of gene expression in the adult mouse brain[END_REF]) expressed centrally, brain function is almost always perturbed in such disorders, thus entailing the cognitive and behavioral phenotypes.

Findings in Down syndrome have revealed that of the approximately 200 genes on chromosome 21 only a subset is responsible for the characteristic phenotype. One wellknown example is the early onset of dementia in subjects with Down syndrome, which can partially be attributed to the over-expression of the amyloid-ß precursor protein gene (APP) located at 21q21-22. In addition to the quantitative imbalance, the Down syndrome phenotype is affected by allelic variation; for example, differences in length of a tetranucleotide repeat in intron 7 of the APP locus explain substantial variation in age at onset of dementia in subjects with trisomy 21 [104].

The first large scaled and systematic genotype-phenotype studies with a primary behavioral, psychological and psychiatric focus centered on sex chromosome (gonosome) disorders; due to the low frequencies of the X0 (Turner Syndrome), XXX (Triple X Syndrome), XXY (Klinefelter Syndrome) and XYY syndromes thousands of newborns had to be screened to detect low numbers of individuals with such gonosomal disorders; the long-term follow-up of their development into adulthood revealed that these disorders are characterized by subtle neuropsychiatric and neuropsychological symptoms such as an IQ distribution shifted slightly to the left and elevated rates of attention problems, speech and reading difficulties and reduced impulse-control; nevertheless, the dissection of a highly specific behavioral phenotype associated with any one of these sex chromosome disorders was not possible [START_REF] Hebebrand | Genetic aspects of brain maturation and behavior[END_REF].

The ability to detect variation at the DNA level formed the basis for the successful elucidation of the molecular mechanisms underlying many monogenic disorders, which can entail more or less specific behavioral phenotypes (Table 1). Most frequently, such variation is detectable in exons of genes underlying such disorders and simplistically either entails that the respective gene product is structurally altered or not formed at all. For example, missense mutations entail the substitution of the regular amino acid at a specific position of the protein with another; this alteration of the amino acid sequence of the respective protein can have functional implications at the levels of the cell, tissue and organism. Below, we focus on the Rett syndrome (RS), because it is the only psychiatric disorder listed in DSM-IV TR which in the majority of cases results from mutations in a defined gene. Fascinatingly, within a 10-year period, the molecular analysis of this disorder is beginning to provide insight into potential future treatment venues and to contribute to our understanding of pathways involved in pervasive developmental disorders, in general.

All other DSM-IV TR psychiatric disorders are most likely complex implying that they do not at all or only Psychiatric symptoms (personality changes, depression, psychosis) [START_REF] Debie | Molecular pathogenesis of Wilson and Menkes disease: correlation of mutations with molecular defects and disease phenotypes[END_REF] Neurofibromatosis Autosomal dominant Neurofibromin (NF1) Mental retardation hyperactivity ADHD [START_REF] Acosta | Neurofibromatosis type 1: new insights into neurocognitive issues[END_REF] infrequently result from single gene mutations. Instead, it is assumed that several gene variants interact in a complex manner with environmental factors to produce the phenotype [START_REF] Kendler | A gene for'': the nature of gene action in psychiatric disorders[END_REF]. Complex disorders typically entail higher concordance rates in monozygotic than dizygotic twins; concordance rates in monozygotic twins are typically below 1 implying that environmental factors play a role in the manifestation of the disorder. Family studies have shown that complex psychiatric disorders are characterized by elevated recurrence risks in first and second degree relatives which are below those expected for classical monogenic dominant or recessive traits. Moreover, for the complex psychiatric disorders, a steep decline in recurrence risks is observable between first and second degree family members. In third degree relatives recurrence risks are usually only minimally elevated above population-based rates of the respective disorder [164]. Within child and adolescent psychiatry, the most twin, family and adoption studies have been carried out for attention deficit/hyperactivity disorder (ADHD; [START_REF] Freitag | Phenotypic and measurement influences on heritability estimates in childhood ADHD[END_REF]). ADHD is also noteworthy because large population-based twin studies have analyzed quantitative dimensions of the disorder; complex gene-environment twin studies have also been performed. For many personality and behavioral traits and developmental milestones heritability estimates based on categorical or dimensional (quantitative) data indicate that overall approximately half of the variance is explained by genetic factors [124], the other half by the environment (Tables 2,3). In their seminal review, Plomin and Daniels [125] pointed out that after controlling for genetic similarity, siblings often appear no more alike than individuals selected at random from the general population. The source of this dissimilarity is a variance component termed 'non-shared environment'. For many traits, the nonshared environment has been found to be of greater relevance than the shared environment.

For most psychiatric disorders, which are usually assessed categorically, genetic factors have also been shown to play an important role (Table 4). Heritability estimates typically exceed 0.5. ADHD has been shown to be one of the most highly heritable child and adolescent psychiatric disorders [18,[START_REF] Freitag | Phenotypic and measurement influences on heritability estimates in childhood ADHD[END_REF][START_REF] Heiser | Molecular genetic aspects of attention-deficit/hyperactivity disorder[END_REF]. Knowledge of the magnitude of the genetic basis of a particular disorder is valuable for interpreting psychiatric findings within a patient's family and for probing for specific disorders in relatives of the index patient.

Until recently, candidate gene and linkage studies dominated the attempts to uncover genetic variation underlying such complex disorders. However, viewed in retrospect, it can be concluded that these large-scaled efforts were largely unsuccessful; progress in the molecular dissection of complex psychiatric phenotypes proved to be exceedingly slow for a period of over 20 years (reviewed in [START_REF] Burmeister | Psychiatric genetics: progress amid controversy[END_REF]). Candidate gene studies could only be performed for those genes for which an a priori hypothesis existed as to their relevance for the respective disorder; obviously for each disorder this represented only a very limited number in light of the totally known number of human genes. In addition, candidate gene studies in different psychiatric disorders frequently focussed on the same set of genes of a particular neurotransmitter system, such as dopamine and serotonin transporters and receptors (e.g., for ADHD, obsessive compulsive disorder, and eating disorders see [START_REF] Banaschewski | Molecular genetics of attention-deficit/hyperactivity disorder: an overview[END_REF]141,167]). In other words, the candidate gene studies reflected the paucity of hypotheses as to the underlying pathways involved in complex psychiatric disorders.

Until recently, molecular genetic analyses of complex psychiatric disorders were based on low numbers of cases and controls or families. It has now become evident that for many complex disorders thousands of cases and controls are required to pick up gene variants with small effect sizes. In 1996, Risch and Merikangas [130] had already calculated that thousands of sib-pairs would be required to detect linkage if the effect sizes of the relevant gene variants are small; linkage studies only infrequently included more than 500 sib-pairs.

In 2006, the first genome-wide association studies (GWAS) based on DNA chip technology were introduced (reviewed in [START_REF] Hardy | Genomewide association studies and human disease[END_REF]; see Table 5 for an overview of GWAS in selected neuropsychiatric disorders), whereas it is still too early to judge the total insight that this novel technology will provide into the pathogenesis of complex disorders, we can nevertheless already conclude that GWAS have entailed a paradigm shift, thus justifying the nomination as ''breakthrough of the year'' by Science magazine in 2007 [122]. For many complex somatic and neuropsychiatric disorders, novel genes have been detected which provide initial insights into frequently unknown pathways involved in the respective disorders. For many disorders, different groups pooled their GWAS to come up with several thousand cases and controls, such numbers had almost never been analyzed in the pre-GWAS era. A major finding has been that the effect sizes of validated trait or disease-related SNPs are modest to small; according to a recent synopsis [START_REF] Hindorff | Potential etiologic and functional implications of genome-wide association loci for human diseases and traits[END_REF], the median odds ratio was 1.33 with an interquartile range of 1.2-1.61. Despite this recent progress, the molecular genetic basis for complex disorders remains largely unknown. For each disorder, the variance explained by the single newly identified gene variants is uniformly small. Developmental aspects represent a key feature of child and adolescent psychiatry. The unfolding of gene expression provides virtually all of the information necessary to guide the orderly succession of events underlying the development of any organism and the central nervous system, in particular [START_REF] Leckman | From genes to brain: developmental neurobiology[END_REF]. A behavioral trait or the symptoms of any given mental disorder are more uniform for a specific developmental stage than across all of infancy, childhood and adolescence; in addition, comorbidity is dependent on developmental stage [18]. Because developmental milestones, many traits and disorders must be viewed in the context of brain development, elucidation of the underlying molecular mechanisms will contribute to the identification of genes involved in normal development of the central nervous system and its function. Dyslexia genes, which are involved in global braindevelopment processes such as neural migration and axonal guidance represent just one such example [106,143]. Partially heritable somatic developmental traits such as age at menarche [158] and timing of puberty [120] have successfully been subjected to GWAS.

Specific disorders run their course during specific developmental phases. Examples include enuresis nocturna which at age 7 affects approximately 10% and at 18 only 1% [START_REF] Gontard | The genetics of enuresis: a review[END_REF]. The frequent reduction of hyperactivity in ADHD during adolescence is another example. Both anorexia and bulimia nervosa rarely start in childhood and only infrequently persist beyond age 30; in Tourette's disorder, both comorbid disorders and the development of the tics show age-related patterns. It appears probable that alterations in expression levels of specific genes partially account for disorder-specific manifestation ages and the symptom development over time (Tables 2,3, 4).

The Human Genome Project (HGP), other relevant international projects and interindividual variation at the DNA level

The completion of the HGP launched in the late 1980s in the USA [117] has provided the basis for a more rapid discovery of novel candidate genes for complex disorders. The first comprehensive analysis of the human draft sequence(s) was published in February 2001 by the International Human Genome Sequencing Consortium (IHGSC) in Nature [START_REF]Initial sequencing and analysis of the human genome[END_REF] and the private enterprise Celera Genomics (CG) in Science [166]. Most striking was the small number of estimated human genes, which had previously been thought to range up to over 100,000. Also, it became apparent that only *1.5% of the human genome contains coding information. About *50% is composed of repetitive elements. Hence, human complexity is based on diversity and finely tuned interaction of gene products such as RNA and proteins rather than gene numbers. Consistent with this, *50% of human protein coding genes exhibit alternative splicing [START_REF] Brett | EST comparison indicates 38% of human mRNAs contain possible alternative splice forms[END_REF]111,112] creating a proteome of [90,000 proteins [START_REF] Harrison | A question of size: the eukaryotic proteome and the problems in defining it[END_REF]. Gene expression is regulated by the complex interaction of a wide variety of transcription factors [START_REF] Fickett | Discovery and modeling of transcriptional regulatory regions[END_REF]151].

In April 2003, in the 50th anniversary year of the discovery of the double-helical structure of DNA [171], the human DNA sequence was virtually completely elucidated. It represented *99% of the euchromatic portion of the human genome (2.85 Gb) with 99,999% accuracy [START_REF]Finishing the euchromatic sequence of the human genome[END_REF]. Of main interest is the identification of all genes and a comprehensive genome annotation. Currently, 31,315 genes (including 9,899 pseudogenes, which are not transcribed) are listed in the human gene catalogue (Ensemble Database version 54.36p). The total number of protein coding genes is estimated at 20,000-25,000, which is consistent with data from cross species comparisons [115,132]. The finished sequence also provides the basis for the identification of potentially all genes causing or predisposing to disease The ''Big Five'' Extraversion NEO-PI-R 53 [START_REF] Jang | Heritability of the big five personality dimensions and their facets: a twin study[END_REF] Neuroticism NEO-PI-R 41 [START_REF] Jang | Heritability of the big five personality dimensions and their facets: a twin study[END_REF] Openness NEO-PI-R 61 [START_REF] Jang | Heritability of the big five personality dimensions and their facets: a twin study[END_REF] Agreeableness NEO-PI-R 41 [START_REF] Jang | Heritability of the big five personality dimensions and their facets: a twin study[END_REF] Conscientiousness NEO-PI-R 44 [START_REF] Jang | Heritability of the big five personality dimensions and their facets: a twin study[END_REF] EPQ-R Eysenck Personality Questionaire [START_REF] Eysenck | A revised version of the Psychoticism Scale[END_REF], TCI Temperament and Character Inventory [START_REF] Cloninger | The tridemisional personality questionnaire: U.S. normative data[END_REF], NEO-PI-R NEO Personality Inventory [START_REF] Costa | Personality disorders and the fivefactor model of personality[END_REF] as well as genetic variations affecting individual responses to medication and environmental factors. To detect variation in a DNA region of interest (e.g., a gene repeatedly or unambiguously identified in GWAS) in individuals with a specific disorder, the respective regions are commonly resequenced.

Interspecies comparison is helpful to identify regulatory regions and functional motifs, and so sequencing of many prokaryotic as well as eukaryotic organisms including mammals (mouse, rat, cat, chimpanzee, cow and dog) is completed or well under way. Comparison of highly accurate genome sequences enables the study of genome evolution, i.e., lineage-specific gene birth [START_REF] Emes | Comparative evolutionary genomics of androgen-binding protein genes[END_REF]116,121,135]. Chimpanzee is the closest relative to humans having DNA sequences 98% identical to each other. Of special medical interest is the high proportion of recent segmental duplications [START_REF] Bailey | Recent segmental duplications in the human genome[END_REF] and inversions [START_REF] Feuk | Discovery of human inversion polymorphisms by comparative analysis of human and chimpanzee DNA sequence assemblies[END_REF] in the human genome. The respective chromosomal regions are prone to rearrangements and/or deletions potentially resulting in phenotypic effects and can now be reliably analyzed [START_REF] Cheung | Genome-wide detection of segmental duplications and potential assembly errors in the human genome sequence[END_REF]108].

To systematically identify all genetic variations in the human population, the international HapMap Project was initiated in 2002 [START_REF]International HapMap Project[END_REF]. Populations with African, Asian, and European ancestry are studied to identify and catalogue genetic similarities and differences in humans. Genotyping a subset of these in the three populations generates an invaluable resource for the discovery of genes related to complex disorders via GWAS.

The Human Epigenome Project [START_REF]Human Epigenome Project[END_REF] aims to identify, catalogue and study genome-wide DNA methylation patterns. DNA methylation is a natural modification of the nucleotide cytosine via which gene expression is controlled. It is tissue-specific and changes over time in response to environmental factors. DNA methylation thus represents a direct link between environment and an individual's state. Epigenetic differences between monozygous twins potentially account for phenotypical variation despite an identical genome at the DNA level. Although twins have been found to be epigenetically indistinguishable during the early years of life, older monozygous twins exhibited remarkable differences in their overall content and genomic distribution of 5-methylcytosine DNA and histone acetylation, affecting their gene-expression portrait [START_REF] Fraga | Epigenetic differences arise during the lifetime of monozygotic twins[END_REF]. These findings indicate how an appreciation of epigenetics is currently largely missing from our understanding of the origin of different phenotypes from the same genotype.

The international Human Brain Proteome Organization Project (HBPP) is concerned with the brain proteome in health, aging and neuropsychiatric disorders [109]. The Encyclopedia of DNA Elements Project (ENCODE) aims at identifying all functional elements in the human genome [36]. Together with these and future initiatives, the HGP The comparison of whole genome sequences between individuals has allowed the detection of substantially more variation than expected [START_REF] Levy | The diploid genome sequence of an individual human[END_REF]169]. It had been assumed that most variation is due to single nucleotide polymorphisms (SNPs; exchanges at the level of a single base). Any two individuals differ by only one in 1,000 DNA bases or at approximately 3 million bases out of the total of 3 billion in the human genome. However, the comparison of a single sequenced genome (that of Craig Venter) with the National Center for Biotechnology Information human reference assembly identified more than 4 million variants (almost 1.3 million were novel) encompassing more than 12 Mb; roughly 1 million variants were not due to SNPs; instead these variants encompassed block substitutions, insertion/ deletion events (indels), 90 inversions, as well as numerous segmental duplications and copy number variation (CNV) regions. In total, non-SNP DNA variation accounted for 22% of all events identified in the genome of Craig Venter, which however accounted for 74% of all variant bases, suggesting an important role for non-SNP genetic alterations in defining the diploid genome structure [START_REF] Levy | The diploid genome sequence of an individual human[END_REF]. 44% of Venter's genes were heterozygous for one or more variants.

Due to their importance in neuropsychiatric genetics [START_REF] Cook | Copy-number variations associated with neuropsychiatric conditions[END_REF], CNVs deserve particular notice. These are defined as segments of DNA as large as several megabases in which copy number differences due to genomic rearrangements such as inversions, deletions, duplications and translocations have been found via representational oligonucleotide microarray analysis [145] and comparison of genomes (e.g., [START_REF] Kidd | Mapping and sequencing of structural variation from eight human genomes[END_REF][START_REF] Levy | The diploid genome sequence of an individual human[END_REF]); CNVs are either inherited or de novo. It is estimated that approximately 0.4% of the genomes of unrelated people typically differ with respect to copy number [START_REF] Kidd | Mapping and sequencing of structural variation from eight human genomes[END_REF].

RS as a monogenic psychiatric disorder

Originally, Rett [128] delineated the features of this serious neuropsychiatric disorder in three females; 17 years later, Hagberg et al. [START_REF] Hagberg | A progressive syndrome of autism, dementia, ataxia, and loss of purposeful hand use in girls: Rett's syndrome: report of 35 cases[END_REF] reported on 35 females with 'progressive autism, loss of purposeful hand movements, ataxia, and acquired microcephaly', which represent the cardinal symptoms of the disorder with an incidence of approximately 1 out of 10,000 female live births. After apparently normal development for 6-18 months, girls with RS lose acquired cognitive, social, and motor skills and develop autistic behavior accompanied by stereotypic hand movements. As the disorder progresses severe mental retardation and motor impairments, including ataxia, apraxia, and tremors ensue. Seizures, hyperventilation, apnea and feeding difficulties are also common. The disorder is sporadic in 99% of all cases [START_REF] Chahrour | The story of Rett syndrome: from neurobiology to clinic[END_REF]. The RS brain is small for age and height of the patient. Dendritic trees in pyramidal neurons of layers III and V in selected lobes are small. The RS brain has small neurons with an increased neuronal packing density; it exhibits a changing pattern of neurotransmitter receptors with an apparent reduction in many neurotransmitters [START_REF] Armstrong | Neuropathology of Rett syndrome[END_REF].

In 1999, different mutations in the methyl-CpG binding protein 2 gene (MECP2; human genes are commonly abbreviated in italic capital letters) were detected in RS females [START_REF] Amir | Rett syndrome is caused by mutations in Xlinked MECP2, encoding methyl-CpG-binding protein 2[END_REF]. This discovery marked a turning point in psychiatric genetics: a DSM-IV (and ICD-10) psychiatric disorder was found to be due to mutations within a single gene. About 95% of classical RS patients have one of[300 known pathogenic MECP2 mutations, the spectrum of which includes missense, nonsense, and frameshift mutations and deletions [START_REF] Chahrour | The story of Rett syndrome: from neurobiology to clinic[END_REF], the last three entailing a more severe phenotype. The X-chromosomal location (Xq28) of MECP2 explains why mostly only females are affected: males, who have only one copy of MECP2, are frequently not viable, if the gene is mutated. In those alive at birth, a much more severe clinical phenotype results; death mostly occurs prior to age 2.

Seemingly, both a reduced and an elevated expression of MECP2 lead to a similar phenotype: duplications of Xq28 including MECP2 were detected in males with clinical features similar to RS. Transgenic mice that express wildtype Mecp2 (only the first letter of murine genes is capitalized) at twice the normal level also have a progressive neurological phenotype similar to that observed in human patients [START_REF] Collins | Mild overexpression of MeCP2 causes a progressive neurological disorder in mice[END_REF].

MECP2 and the RS phenotype

MECP2 is expressed in all tissues. In the adult mouse, Mecp2 (protein encoded by Mecp2) is high in brain, lung and spleen, lower in heart and kidney, and barely detectable in liver, stomach and small intestine. The timing of the expression of murine Mecp2 and human MECP2 in humans has been shown to correlate with the maturation of the central nervous system, with the ontogenetically older structures such as the spinal cord and brainstem becoming positive before newer structures such as the hippocampus and cerebral cortex. In the cortex, MeCP2 first appears in the Cajal-Retzius cells, then in the neurons of the deeper, more mature cortical layers, and finally in the neurons of the more superficial layers [146]. Both timing and localization of MECP2 expression should explain developmental aspects of the RS phenotype.

MECP2 is subject to alternative splicing (different processing of the mRNA results in proteins of different sizes with partially identical domains). The B isoform has the highest expression in brain, and mutations specific to the exons encoding this isoform are sufficient to cause RS. The respective protein MeCP2 includes four functional domains, one of which is the methyl-CpG binding domain (MBD). This protein domain binds to symmetrically methylated CpGs located in the upstream region of many genes; binding of MeCP2 to methylated CpGs has an influence on the transcription rate of such a gene and is thus an important epigenetic mechanism in the regulation of distinct genes. The three other functional domains include an arginine-glycine repeat RNA-binding domain, a transcriptional repression domain that interacts with a co-repressor complex involving mSin3A and histone deacetylases and a RNA splicing factor binding region [165].

MeCP2 seemingly influences the transcription of a limited number of specific genes as either a repressor or activator, which is not only due to MeCP2 binding to CpGislands but also to periodic MeCP2 binding outside gene boundaries, entailing the organization of chromatin into functionally important domains or loops of imprinted regions [START_REF] Hite | Recent advances in MeCP2 structure and function[END_REF]178], which, in turn, modulate gene expression. If due to a non-functional (or over-expression of) MeCP2, the expression of target genes continues or is not initiated, more and more RS symptoms ensue over time [START_REF] Ben-Shachar | Mouse models of MeCP2 disorders share gene expression changes in the cerebellum and hypothalamus[END_REF]. Future research will potentially reveal which RS symptoms are related to activation or repression of specific genes due to the loss of function of MeCP2. For example, the frequent feeding problems in RS might be related to the persistent expression of brain-derived neurotrophic factor (BDNF), which aside from being involved in the regulation of neural survival, development, function, and plasticity in the brain also has an influence on feeding behavior and body weight regulation [START_REF] Hashimoto | Role of brain-derived neurotrophic factor in eating disorders: recent findings and its pathophysiological implications[END_REF].

Transgenic and knockout mice models and comparative sequence analysis [127] have helped considerably to elucidate the function of MeCP2, to assess the effect of specific mutations on the phenotype and to enable the identification of evolutionary conserved regions. If Mecp2 is completely knocked out in male mice, a mild RS-like phenotype ensues (female mice remain healthy into adulthood). A conditional knockout of Mecp2 in postnatal neurons of restricted regions in the brain leads to a similar although delayed neuronal phenotype, suggesting that the gene plays a role in post-mitotic neurons. Abnormalities in social interaction and home-cage behavior were identified in mice, in which a mutation similar to common RS causing alleles had been introduced [113]. The resultant phenotype was reminiscent of the sleep/wake dysfunction and autistic features of RS patients. Interestingly, transgenic expression of Mecp2 in Mecp2 knockout mice largely rescues the RS phenotype [101].

Although it is thought that the primary cause of RS results from a lack of functional MeCP2 in neurons (cell autonomous), non-cell autonomous factors also contribute to the disease. Thus, loss of MeCP2 also occurs in glial cells of RS brains [START_REF] Ballas | Non-cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology[END_REF]102]. Because mutant astrocytes from a RS mouse model, and their conditioned medium, failed to support normal dendritic morphology of either wild-type or mutant hippocampal neurons, astrocytes in the RS brain carrying MECP2 mutations may have a non-cell autonomous effect on neuronal properties, probably as a result of aberrant secretion of a soluble factor(s) [START_REF] Ballas | Non-cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology[END_REF].

MeCP2 deficiency in astrocytes causes significant abnormalities in BDNF regulation, cytokine production, and neuronal dendritic induction, which may contribute to abnormal neurodevelopment. In addition, the MeCP2 deficiency state can progressively spread at least in part via gap junction communications between mosaic Mecp2 -/? astrocytes in a novel non-cell-autonomous mechanism, which may lead to the pronounced loss of MeCP2 observed selectively in astrocytes in mouse Mecp2 -/? brain, which is coincident with phenotypic regression characteristic of RS. Based on these findings, Maezawa et al. [102] suggest that astrocytes are viable therapeutic targets for RS and perhaps regressive forms of autism.

A broader role of MECP2 in neurodevelopmental disorders

Significant differences in MeCP2 expression have been detected between brain samples of individuals with related neurodevelopmental disorders, including autism, pervasive developmental disorder, Prader-Willi and Angelman syndromes and age-matched controls [136,137]. Hence, the elucidation of the molecular mechanisms underlying RS has led to the theory that multiple pathways regulate the complex developmental expression of MECP2 and are not only defective in RS but also in other autism spectrum disorders. The common features of human neuro-developmental disorders caused by loss or increase of MeCP2 function suggest that even modest alterations of MeCP2 levels result in neurodevelopmental problems. In support of this hypothesis, a 50% reduction of the level of wild-type MeCP2 in mice was shown to result in a spectrum of subtle abnormalities such as learning and motor deficits, decreased anxiety, altered social behavior and nest building, decreased pain recognition and disrupted breathing patterns [138]. Seemingly, the precise control of MeCP2 expression level is critical for normal behavior.

A series of studies have aimed at assessing the role of MECP2 and related genes in autistic disorder and mental retardation. MECP2, MBD1, MBD2, MBD3, and MBD4 comprise a nuclear protein family sharing the methyl-CpG binding domain (MBD) and are related to transcriptional repression. However, mutations in MECP2 have only infrequently been detected in patients with autistic features; mutations in the other genes have not systematically been found to play a role in the etiology of autistic disorders [START_REF] Li | Mutation analysis of methyl-CpG binding protein family genes in autistic patients[END_REF]. Future candidate gene studies in autism spectrum disorders can focus on those genes whose expression levels are regulated by MeCP2 (see, e.g., [165]). The investigation of the role of frequent MECP2 polymorphisms in polygenic autism also merits consideration: in two family samples a three-marker SNP haplotype was associated with autism and autism spectrum disorders suggesting that one or more functional variants of MECP2 existing at significant frequencies in the population may confer increased risk [100].

Therapeutic implications

The finding that transgenic expression of Mecp2 in Mecp2 knockout mice largely rescues the RS phenotype [101] suggests that RS might be amenable to treatment. Because treatment would typically only be possible after the diagnosis of RS and thus after development of clinical symptoms, this rescue in the mouse model appears promising for the treatment of RS patients. As systemic treatment of MeCP2 mutant mice with an active peptide fragment of Insulin-like Growth Factor 1 (IGF-1) extends life span, improves locomotor function, ameliorates breathing patterns, and reduces irregularity in heart rate IGF-1 has been suggested as a prime candidate for pharmacological treatment of RS and other disorders caused by a deficit in synapse maturation in the brain [163]. Treatment with IGF-1 peptide was also shown to increase brain weight of the mutant mice; additionally, IGF-1 partially restored spine density and stabilized cortical plasticity to wild-type levels [163]. In Mecp2 mutants, BDNF overexpression extended the lifespan, rescued a locomotor defect, and reversed an electrophysiological deficit [START_REF] Chang | The disease progression of Mecp2 mutant mice is affected by the level of BDNF expression[END_REF].

Histone deacetylases (HDACs) are enzymes that affect the acetylation status of histones and other cellular proteins. Pharmacological manipulations using small-molecule HDAC inhibitors, which may restore transcriptional balance to neurons, modulate cytoskeletal function, affect immune responses and enhance protein degradation pathways, have proven beneficial in various experimental models of brain diseases [START_REF] Kazantsev | Therapeutic application of histone deacetylase inhibitors for central nervous system disorders[END_REF] and could thus prove beneficial in diverse neurodevelopmental disorders including RS. In other genetic disorders, it has been shown that aminoglycosides can cause a read-through of nonsense mutations with an efficiency of up to 20%. Brendel et al. [START_REF] Brendel | Suppression of nonsense mutations in Rett Syndrome by aminoglycoside antibiotics[END_REF] showed that this read-through of MECP2 nonsense mutations can be achieved in vitro with efficiency comparable to that seen in other disorders.

Identification of predisposing alleles in complex disorders

In conclusion, within a ten year period, substantial progress has been made in elucidating the molecular mechanisms underlying RS and deducing strategies for potential treatment. The situation in polygenic disorders is obviously much more complex. In the following, we examine how gene variation can be picked up in complex disorders.

Association studies

The comparison of genetic variants between cases and controls is the most common approach taken to identify variants predisposing to the respective disorder. In association studies, a familial loading is per se not required for classification as a case. However, requirement of such a loading would be expected to increase the probability that genetic factors indeed contribute to the disorder of the index patient. Controls are frequently screened for the respective disorder; a positive screen entails exclusion of the proband as a control. Use of controls with a phenotype that quantitatively differs as much as possible from cases can theoretically enhance the probability of detecting genes. Controls should be matched to cases for wellestablished, strong risk factors (e.g., ethnic group, sex, socio-economic status and intelligence) to ensure that significant results are not caused by a confounding risk factor differing between the two groups other than the trait of interest [START_REF] Kupper | Matching[END_REF]. Such confounders would bias the study if they interact with the considered candidate gene. Overmatching, on the other hand, e.g., for many potential, small risk factors, will lead to substantial loss of efficiency. Matching has to be accounted for in the analysis as well.

One of the most important confounders in genetic studies could be ethnicity in ethnically admixed or structured populations [START_REF] Lander | Genetic dissection of complex traits[END_REF]. This will cause serious bias if the studied candidate gene differs in allele frequencies between ethnically defined sub-populations (population stratification). It has been proposed to genotype several markers that are thought to bear no relationship to the disorder of interest in both cases and controls to potentially enable adjustment for systematic genetic differences [START_REF] Devlin | Analysis of multilocus models of association[END_REF]126]; this has become a standard requirement for GWAS (see below, [START_REF] Attia | How to use an article about genetic association. B: are the results of the study valid?[END_REF]).

Another popular approach to circumvent the potential confounding population stratification effects of case-control studies are family-based association studies, which include family members of the cases to use as (ethnically) matched controls. These can be unaffected sibs or pseudocontrols constructed from non-transmitted parental alleles. The first statistical tests for such case-parent trios were the haplotype relative risk method (HRR; [START_REF] Falk | Haplotype relative risks: an easy reliable way to construct a proper control sample for risk calculations[END_REF]) and the haplotype-based haplotype relative risk method (HHRR; [161]). These tests influenced the development of the most widespread test-the transmission disequilibrium test (TDT; [150]) which is unaffected by population stratification phenomena and a valid test for both association and linkage. The TDT test statistic is based on the comparison of the number of times the allele of interest is transmitted versus non-transmitted by heterozygous parents to an affected child. The ascertainment of parents is usually readily possible in disorders with a childhood onset.

Family-based association studies have certain disadvantages; in particular, they are less efficient than casecontrol studies. Sibs share on average one allele identical by descent, so there is effectively only one allele that can differ instead of two for unrelated cases and controls. Also, sibs tend to be more similar in many possible risk factors which imply over-matching with its associated loss in efficiency. In the trio design, three subjects have to be genotyped to yield approximately the similar information as two subjects in the case-control design. And finally, for diseases with a later age-of-onset, it will be difficult to ascertain parents of cases. Therefore, family-based association studies are more expensive and complex than case-control studies.

Effect size

In polygenic disorders, each gene variant (subsequently also referred to as an allele) has only a minor to minimal effect. Typically the effect size of a particular allele or genotype is indicated via its relative risk or the odds ratio in case-control designs. In diploid cells, humans have two copies of every gene (with the exception of most genes located on the X and Y chromosomes in males); in this context, the term genotype refers to both gene variants; typically the genotype relative risk is given for heterozygotes (AB) and homozygotes (BB) for the predisposing allele, it is set to 1 in individuals homozygous for the wildtype allele (AA). Low relative risks imply that (1) the variant (or genotype) in itself is by no means sufficient to explain the disorder of an affected individual and (2) many individuals without the respective disorder harbor the same allele (or genotype).

Given a limited number of cases and controls (e.g., 1,000 each), detection of predisposing alleles becomes more difficult (a) the lower the population frequency of such a variant and (b) the lower its effect size. A study is underpowered if a significant difference cannot reliably be detected between cases and controls. Recent GWAS for height, weight and body mass index (BMI in kg/m 2 ) based on ten thousands of individuals have picked up single variants which in the heterozygous state on average entail a 3 mm increased height [START_REF] Gudbjartsson | Many sequence variants affecting diversity of adult human height[END_REF] and a 180 g heavier weight [175], respectively. Because presumably only those gene variants with the most pronounced effects have currently been detected, effect sizes of most as yet undetected alleles may well be below 2 mm or 100 g. The implications for psychiatric disorders are evident: whereas previous molecular genetic studies frequently at best included only a few hundred individuals, current studies already include thousands of cases and controls. Many more are needed to confirm initial true positive or to rule out false negative findings in independent samples.

Common variants versus private mutations

The common disease-common variant hypothesis predicts that specific common alleles or variants predispose to common disorders and that such alleles/variants will be found in all human populations, in which the respective disease occurs. Indeed, for several complex traits and disorders common variants, mostly SNPs, have been detected in coding and regulatory sequences of genes (e.g., [START_REF] Hindorff | Potential etiologic and functional implications of genome-wide association loci for human diseases and traits[END_REF][START_REF] Iles | What can genome-wide association studies tell us about the genetics of common disease?[END_REF]); however, in many cases the localization of such a SNP does not allow any conclusions as to how this variation affects the function of the gene; it appears likely that in many cases, the respective SNP in itself merely tags a functionally relevant SNP or variant (linkage disequilibrium) in close proximity. In addition, because such SNPs can be located far from a gene it frequently is not even possible to definitely determine which gene (e.g., 3 0 or 5 0 ) is altered in its function. It appears likely that many of the functional variants influence gene expression levels.

The identification of variants with moderate effect sizes has been successful in single non-psychiatric disorders with complex inheritance such as Crohn's disease [START_REF] Hugot | Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease[END_REF]119], breast cancer [110, 177], and type 2 diabetes mellitus [START_REF] Grant | Variant of transcription factor 7-like 2 (TCF7L2) gene confers risk of type 2 diabetes[END_REF]. However, for complex psychiatric disorders, common gene variants with modest and robust effect sizes have not been detected. Even upon use of large-scaled GWAS (see below), the total number of validated common variants is rather low and their effect sizes are uniformly small.

Despite the recent advances, the explained variance of a quantitative phenotype remains low even if heritability estimates are high. Thus, even though heritability estimates exceed 0.8 for body height, less than 5% of the total variation in height has been uncovered despite the detection of over 40 validated gene variants [START_REF] Gudbjartsson | Many sequence variants affecting diversity of adult human height[END_REF][START_REF] Hirschhorn | Progress in genome-wide association studies of human height[END_REF]. Similarly, only about 1% of the variance in BMI has currently been found to be due to about 20 common variants [START_REF] Hinney | Polygenic obesity in humans[END_REF][START_REF] Hinney | From monogenic to polygenic obesity: recent advances[END_REF]175] despite heritability estimates of roughly 0.4-0.8.

The common disease-common variant hypothesis has been challenged. Based on molecular genetic findings, a recent discussion has focused on the relevance of ''private'' mutations defined as rare mutations found only in single families in whom a specific complex disorder occurs. Particularly for disorders with reduced fecundity, associated with severe mental disorders, a negative selection pressure conceivably acts on risk alleles, thus potentially explaining why common variants have not been readily detected in disorders such as autism, schizophrenia and mental retardation. Accordingly, rare variants may account for a larger fraction of the overall genetic risk than previously assumed. Indeed, rare CNVs have been detected in schizophrenia [153], autism (reviewed in [START_REF] Freitag | Genetics of autistic disorders: review and clinical implications[END_REF]) and mental retardation [107]. For example, Stefansson et al. in a genome-wide search for CNVs associating with schizophrenia used a population-based sample to identify de novo CNVs by analyzing 9,878 transmissions from parents to offspring. The 66 de novo CNVs identified were tested for association in a sample of 1,433 schizophrenia cases and 33,250 controls. Three deletions at chromosomes 1q21.1, 15q11.2 and 15q13.3 showing a nominally significant association with schizophrenia in the first sample were followed up in a second sample of 3,285 cases and 7,951 controls. All three deletions were significantly associated with schizophrenia and related psychoses in the combined sample.

In complex disorders, different sets of gene variants are operative in different affected individuals. Assume that 100 genes each occurring as the two alleles A and B in the population in total account for all genes predisposing to a particular disorder; all of these variants would have the same effect size and would act in an additive manner. Let us also assume that at least 50 predisposing alleles must be present in an individual for the disorder to break out, if predisposing environmental factors are abundant-more predisposing alleles would be necessary if only a limited number of negative environmental factors are operative. Accordingly, an affected subject could theoretically be homozygous for the predisposing variant at 25 loci, another could be heterozygous at 50 loci. Most affected individuals would be heterozygous at some loci and homozygous at others. Clearly in this scenario, any two affected individuals would not necessarily share a single predisposing variant; there is substantial genetic heterogeneity.

In reality, the frequencies and effect sizes of such alleles differ. Despite the fact that for the two quantitative phenotypes height and BMI effect sizes of currently detected variants have been shown to be additive, non-additive effects appear likely in complex disorders. Also consider that complex interactions of specific sets of gene variants presumably occur with environmental factors. Thus, a particular (set of) environmental factor(s) might only be relevant for a subgroup of individuals characterized by specific genotypes at diverse loci. Yet in current studies, all the individuals of such subgroups are analyzed as if they were a homogeneous sample.

We briefly dwell on the different strategies to detect gene variants in complex disorders.

Candidate gene studies

For many years, the candidate gene approach formed the most frequent attempt to discover genetic variation underlying psychiatric disorders. The choice of a specific candidate gene was commonly based on pharmacological, physiological, biochemical, anatomical and/or genetic data such as chromosomal localization within a linkage region or chromosomal breakpoints in chromosome aberrations in individuals with a psychiatric phenotype. However, in light of the estimated 21,000 human genes and our poor knowledge of the molecular basis of any psychiatric disorder, the a priori probability for the involvement of a particular gene is low, unless the underlying hypothesis is well founded. Most of the respective genes were originally selected because they belong to neurotransmitter systems assumed to be involved in psychiatric disorders (e.g., dopaminergic or serotonergic system). Typically, originally positive findings were not unequivocally confirmed in subsequent independent association studies; meta-analyses have been used to assess such conflicting findings. While meta-analyses may be useful to circumvent the problem of limited statistical power due to small samples, they are no means to cope with poor study quality (e.g., [START_REF] Little | Strengthening the reporting of genetic association studies (STREGA): an extension of the STROBE statement[END_REF]). The advent of GWAS has led to an almost complete halt of classical candidate gene studies; in retrospect, their contribution to the elucidation of genetic mechanisms in psychiatric disorders has been minimal. Because, as has been pointed out, case numbers were frequently low, it remains to be seen if some classical candidate genes for diverse psychiatric disorders will be picked up in large GWAS.

To illustrate the candidate gene approach we refer to a study conducted by Abelson et al. [START_REF] Abelson | Sequence variants in SLITRK1 are associated with Tourette's syndrome[END_REF], who identified a patient presenting with Tourette's disorder (TD) and ADHD carrying a de novo chromosome 13 inversion. Because the Slit and Trk-like family member 1 gene (SLITRK1) is one of three genes located within 500 kb of the two breakpoints, this gene was considered as a candidate gene for TD. Accordingly, SLITRK1 was screened in 174 patients with TD. One subject with TD and ADHD harbored a frameshift mutation; two unrelated patients with TD and obsessive-compulsive symptoms had an identical non-coding variant. Both this variant and the frameshift mutation were absent in 4,926 and 3,600 control chromosomes, respectively. Abelson et al. concluded that SLIT-RK1 mutations underlie TD in a small subgroup of patients affected with this tic disorder. However, caution is warranted because in contrast to the 174 TD patients, SLITRK1 was not screened for mutations in controls; hence, it cannot be excluded that healthy controls might also harbor other infrequent mutations.

Genome-wide linkage studies DNA sequences at specific loci are inherited together as a consequence of their physical proximity on a single chromosome. The closer the loci are to each other at the DNA level, the lower the probability that they will be separated during meiosis, and hence the greater the probability that they will be inherited together. By analyzing genetic meiotic recombination frequencies between specific loci, genetic linkage analysis can be used to localize susceptibility genes within a framework map of genetic markers with known positions in the genome. Genome scans have typically been based on 350-1,000 microsatellite markers spaced rather evenly throughout the genome with marker distances of about 10-3 cM [114]. Fine mapping with additional markers is frequently performed in an attempt to narrow in the chromosomal region which initially can span over a large region of a chromosome encompassing several genes. A priori hypotheses as to functional candidate genes that could influence the phenotype are not required. In addition, no a priori assumptions about mode of inheritance, frequency of the disease allele in the general population and penetrance are required; such analyses are also termed non-parametric or model-free. For parametric linkage analyses, the search space is usually restricted to for instance specific modes of inheritance; thus, a priori assumptions are made.

Linkage studies proved to be very successful for monogenic Mendelian disorders; nevertheless, progress usually was slow: for example, it took from 1983 to 1993 to proceed from the initial linkage finding on chromosome 4p in large pedigrees to the identification of the Huntingtin gene and the molecular mechanism underlying Huntington's disease [START_REF]Huntington's Disease Collaborative Research Group (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes[END_REF]. In complex disorders, linkage studies were originally also based on large, multiply affected pedigrees based on the (potentially incorrect) assumption that a single or only a small number of disease genes segregate in each of these families. Sibling pairs and small nuclear families formed the basis of more recent linkage studies [129].

With single exceptions (e.g., see [143], for linkage findings in dyslexia leading to the detection of doublecortin domain containing 2 gene) linkage scans for psychiatric disorders have failed. Potential explanations include: (1) sample sizes typically ranged well below 500 sib-pairs and thus had (very) low power. Meta-analyses of linkage studies have been performed in an attempt to cope with the small number of sib-pairs investigated in single studies; for example, for ADHD a genome-wide significant linkage was detected on chromosome 16 [179], in contrast no significant linkage finding was detected for obesity [140].

(2) If infrequent/rare gene variants (private mutations) with large effect sizes (major genes) exist in psychiatric disorders, their detection via linkage studies is exceedingly difficult or impossible. It remains to be seen to what extent GWAS will pick up genes located within linkage peaks detected via previous linkage studies; we are aware of one such example [140].

Genome-wide association studies (GWAS)

The possibility to place hundreds of thousand oliogonucleotides on small chips in combination with the advances of the Human Genome and related projects has revolutionized molecular genetic studies of complex traits and disorders. This transition to SNP-based technology has been rendered possible by the construction of a map of naturally occurring polymorphisms [START_REF]A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms[END_REF] ). Furthermore, the construction of a haplotype map of the human genome has greatly facilitated comprehensive genetic association studies of human disease [START_REF] Gabriel | The structure of haplotype blocks in the human genome[END_REF][START_REF] Goodstadt | Sequence variation and disease in the wake of the draft human genome[END_REF]103].

SNP associations are currently being detected at an unprecedented pace for many neuropsychiatric disorders [START_REF] Hindorff | Potential etiologic and functional implications of genome-wide association loci for human diseases and traits[END_REF][START_REF] Hindorff | A catalog of published genome-wide association studies[END_REF][START_REF] Khoury | Genome-wide association studies, field synopses, and the development of the knowledge base on genetic variation and human diseases[END_REF]103; see also Table 5). The chips allow the determination of genotypes for the number of SNPs that are detected with the respective oligonucleotides; currently, determination of 1 million SNP genotypes in every individual tested is feasible; only very small amounts of DNA are required. If 2,000 cases and 2,000 controls are analyzed in an association study, a total of 2 9 2,000 9 1 million genotypes are generated. Both allele and genotype frequencies for every single SNP can be compared; P values of 0.05 divided by 10 6 (simple Bonferroni correction for the 1 million tests), i.e., P values smaller than 0.00000005 (5 9 10 -8 ), indicate genome-wide significant differences between cases and controls for the respective SNP. However, a Bonferroni correction is too conservative because many SNPs are in linkage disequilibrium with other SNPs implying that the respective alleles are not independent. Nevertheless, in light of the hypothesized small effect sizes of variants underlying complex disorders the necessity to analyze very large samples of both cases and controls is readily evident. Thus, the absence of significant findings in ADHD GWAS most likely reflects the too low number of cases and controls or trios (index patient and both parents) in the currently published studies (see [START_REF] Banaschewski | Molecular genetics of attention-deficit/hyperactivity disorder: an overview[END_REF]).

The future and a word of caution

What can realistically be expected for the future? Two extreme scenarios appear possible: (1) only a limited number of novel gene variants are unambiguously identified within the next 5 years. This finding would imply that the effect sizes of most predisposing alleles are so small that they cannot be picked up. The ultimate step would be to re-sequence the whole genome (or at least all known genes) in a substantial number of cases and controls in an attempt to statistically identify those loci in which variations cluster in cases [105]. Genome-wide re-sequencing is currently, however, not yet feasible in a large number of individuals. Moreover, special statistical methodology for this data is still in its infancy. The accumulating costs for the search for alleles with very small effect sizes at some time point will need to be put into perspective to the expected outcome. ( 2) GWAS lead to the identification of at least ten novel gene variants for each analyzed psychiatric disorder. These genetic results are robust, implying that there is a consensus that indeed the respective genes contribute to the etiology of the respective disorder; (repeated) replications of each single finding are the prerequisite for the attainment of such a consensus. This knowledge will allow us to identify (a) the molecular genetic mechanisms relevant for psychiatric disorders (e.g., variation primarily within regulatory versus coding regions), (b) novel systems and pathways relevant in specific disorders, which may or may not include pharmacological targets, (c) the extent of overlap in the genetic predisposition to different psychiatric disorders (e.g., identification of genetic variation predisposing to both ADHD and autism; see [133]), (d) gene-gene and geneenvironment interactions and (e) developmental relationships between genotype and phenotype.

The crucial prerequisite for the use of sophisticated molecular technology is that several large and well-characterized samples of patients with a given disorder exist to allow repeated confirmations of a single finding and subsequent meta-analyses. By large, we imply that the respective samples should encompass well over 1,000 cases and an equal number of controls, thus providing sufficient power to detect alleles with a modest to weak effect. Undoubtedly, the need to analyze large samples and to confirm original findings will lead to extensive collaborations which need to also address ethnic aspects; the contribution of single researchers and groups to novel results will become small; thus, a total of 146 authors contributed to a recent GWAS for BMI [175]. Family-based association and linkage studies will also be helpful in specific situations such as the involvement of imprinted genes in the etiology of a given disorder.

As the field of molecular genetics of psychiatric disorders advances, we as child and adolescent psychiatrists need to stay at the forefront of these developments. To achieve our goals we depend on an interdisciplinary approach also encompassing molecular geneticists and biologists, biostatisticians, and several other specialists. We will profit by extensively integrating these disciplines into our research; a fundamental issue is to establish a sufficient amount of cross-talk to ensure that the full potential of this interdisciplinary approach can bear full profit. We as psychiatrists will also be responsible for the integration of novel molecular findings into our clinical routine. We need to be aware of ethical and societal implications of molecular genetic findings. We need to have training programs which provide us with the capability to grasp the molecular findings and to integrate them into novel research (e.g., analysis of gene-environment interactions) and even more important into our daily clinical routine if the evidence is strong enough. If a sufficiently large number of predisposing alleles for a given disorder become known, genetic analyses will most likely for the first time provide us with genetic markers useful for diagnostic purposes, prediction of the clinical course of a disorder and its treatment (personalized medicine). We will learn that the elucidation of the molecular puzzle of psychiatric disorders has the potential to alter our current phenomenological basis for the definition of neuropsychiatric disorders.

Despite these exiting potential implications of the discovery of predisposing alleles, we nevertheless should remain critical. Due to the necessity to reach a diagnosis via explicit diagnostic criteria, we as psychiatrists have become extremely critical of our own research work; with good reason we have come to expect a solid diagnostic procedure based on standard criteria. We would recommend that this critical approach is extended to the field of psychiatric genetics, where at times novel results are either extended beyond the evidence or are uncritically taken for granted.

For example, in 1993, Brunner and co-workers [START_REF] Brunner | Abnormal behavior associated with a point mutation in the structural gene for monoamine oxidase A[END_REF][START_REF] Brunner | X-linked borderline mental retardation with prominent behavioral disturbance: phenotype, genetic localization, and evidence for disturbed monoamine metabolism[END_REF] reported that a mutation in the MAO-A gene underlies a phenotype characterized by 'borderline mental retardation' and 'aggression'. This study is widely held to indicate that human aggression can be caused by a mutation in a single gene. However, the phenotype depicted in the respective publications is anything but straight forward: the mutation carriers were reported to have been involved in voyeurism, exhibitionism, arson and/or rape. The affected males were described as '…withdrawn and shy, being often without friends. All have shown aggressive outbursts of some sort, usually with little or no provocation. A number of males exhibit sexually aberrant behavior… Aggressive behavior tended to cluster in periods of 1-3 days, during which the affected male would sleep very little and would experience frequent night terrors.' No attempt was made to classify the symptoms according to a psychiatric classification scheme. The common trait appears to be 'aggressive outbursts of some sort', which without further specification could well apply to a substantial minority of the general population. This criticism all the more applies because the investigators deduced the presence of borderline mental retardation in nine males of the pedigree from information compiled 30 years ago by an unaffected family member. The only information provided for those mutation carriers whom the investigators themselves were able to phenotypically assess was as follows: 'A typically affected male showed a fullscale IQ of 85'. It was not specified how many of the males were psychologically tested and what IQ test was used (for a more detailed critical evaluation of the Brunner et al. studies [START_REF] Brunner | Abnormal behavior associated with a point mutation in the structural gene for monoamine oxidase A[END_REF][START_REF] Brunner | X-linked borderline mental retardation with prominent behavioral disturbance: phenotype, genetic localization, and evidence for disturbed monoamine metabolism[END_REF], see [START_REF] Hebebrand | Specification of the phenotype required for men with monoamine oxidase type A deficiency[END_REF], and the response of Brunner et al. [START_REF] Brunner | Specification of the phenotype required for men with monoamine oxidase type A deficiency. Reply to Hebebrand and Klug[END_REF]). Conceivably, the MAOA mutation entails symptoms compatible with the diagnosis of a personality disorder, which, in turn, could be associated with aggressive behavior in specific circumstances. In our opinion, there is no reason for us to be lenient when molecular geneticists uncover genetic variation underlying interindividual differences in behavioral phenotypes, particularly if they are as complex as aggression or intelligence. This is particularly important because the media frequently simplify and exploit such findings, thus conveying false or largely imprecise information to the lay public.

The delineation of the 'aggression gene' (term used by the media; see e.g., [156]) paved the way for another sensational finding, which pertains to a genotype-environment interaction, only few of which have been studied in psychiatry. MAO-A alleles can be classified as leading to low or high activity of its gene product; a study published in Science in 2003 [START_REF] Caspi | Influence of life stress on depression: moderation by a polymorphism in the 5-HTT gene[END_REF], which was purportedly independently confirmed [START_REF] Foley | Childhood adversity, monoamine oxidase a genotype, and risk for conduct disorder[END_REF], showed that maltreated children with a genotype conferring low levels of MAO-A were less likely to develop conduct disorders.

Why should such an exciting finding be subjected to a critical evaluation? Critical issues include (for a more detailed and general discussion of gene 9 environment interactions see also [174]): (1) solid molecular genetic findings in complex disorders are as yet scarce. There is no a priori evidence to indicate that a switch to gene-environment interactions will make the elucidation of predisposing alleles easier. [START_REF] Achenbach | Manual for the Child Behavior Checklist/4-18 and 1991 profile[END_REF] The delineation of a single specific hypothesis pertaining to a gene-environment interaction implies that just this one out of very many different possible ones is tested; both the gene (e.g., MAO-A) and the environmental condition (e.g., maltreatment) must be selected. If, however, different gene-environment interactions are tested, the effects of multiple testing need to be considered. [START_REF] Acosta | Neurofibromatosis type 1: new insights into neurocognitive issues[END_REF] The quality of the underlying hypothesis needs to be assessed. The investigation of MAO-A in conduct disorders to a considerable extent rests on the aforementioned study of Brunner et al. [START_REF] Brunner | Abnormal behavior associated with a point mutation in the structural gene for monoamine oxidase A[END_REF][START_REF] Brunner | X-linked borderline mental retardation with prominent behavioral disturbance: phenotype, genetic localization, and evidence for disturbed monoamine metabolism[END_REF], which as delineated above was not satisfactory in defining a psychiatric phenotype. (4) Confirmatory studies in geneenvironment analyses become almost impossible if the original assessment procedure for the relevant environmental factor is not employed in subsequent studies. Foley et al. [START_REF] Foley | Childhood adversity, monoamine oxidase a genotype, and risk for conduct disorder[END_REF], who used different variables to construct a maltreatment index than Caspi et al. [START_REF] Caspi | Role of genotype in the cycle of violence in maltreated children[END_REF], elegantly discuss this problem by speculating that their own positive molecular genetic results suggest that the measures used by the two different groups are 'intercorrelated and they may indicate an overlapping set of environmental risks'. A direct comparison of the variables assessed in the two studies is not too convincing to make this circular reasoning plausible. To the contrary, it seems probable that the two distinct sets of variables used to construct the maltreatment indices tap on different environmental and genetic factors; the extent of overlapping is subject to debate. (5) Finally, as Foley and co-workers [START_REF] Foley | Childhood adversity, monoamine oxidase a genotype, and risk for conduct disorder[END_REF] note, most of the power to detect the interaction stemmed from the extremes of the distribution. Only 15 and 5 subjects out of a total of 514 were ranked to be in the second highest and highest level of exposure to childhood adversity. The genotypes of these 20 subjects in total accounted for the significant effect of the genotype-environment interaction and thus for the confirmation of the original Caspi et al. [START_REF] Caspi | Influence of life stress on depression: moderation by a polymorphism in the 5-HTT gene[END_REF] study.

In the context of such gene-environment interactions, a recent meta-analysis deserves notice: despite previous reports to the contrary, the serotonin transporter genotype alone or in interaction with stressful life events was not found to be associated with an elevated risk of depression in men alone, women alone, or in both sexes combined; the number of stressful life events was however associated with depression [131].

There are no easy solutions to these problems. The complexity of the issues at hand requires that psychiatrists apply all their knowledge to adequately conduct genetic studies and to assess novel genetic findings. We believe that the elucidation of genes involved in psychiatric disorders has a tremendous potential to advance our understanding of their aetiologies and to contribute to future therapies. At the same time we need to remain humble in light of the complexity inherent to genetic and environmental factors in psychiatric disorders.

Table 1

 1 Examples of the influence of single gene disorders on cognition and behavior

	Monogenic	Mode of inheritance Gene	Cognitive phenotype Behavioral phenotype	Reference
	disorder
	Lesch-Nyhan	X-chromosomal	Hypoxanthine guanine	Mental retardation	Self-mutilative biting of fingers and lips [83]
	syndrome	recessive	phosphoribosyl
			transferase (HPRT)
	Tuberous	Autosomal	Tuberous sclerosis-1 and	Learning difficulties,	Behavioral problems, autistic disorder [97]
	sclerosis	dominant	-2 (TSC1/-2)	low IQ, mental
				retardation
	Fragile X	X-chromosomal,	Fragile X mental	Mental retardation,	Autism	[32]
	syndrome	triplet repeat	retardation (FMR1)	hyperactivity
		expansion
	Leptin deficiency Autosomal	Leptin (LEP)		Diminished perception of food reward,	[39]
		recessive			decreased response to satiety signals,
					hyperphagia
	Chorea	Autosomal	Huntingtin (HTT)	Dementia	Depression, psychosis, personality	[168]
	Huntington	dominant, triplet			change, obsessive-compulsive
		repeat expansion			behavior
	Phenylketonuria	Autosomal	Phenylalanine	Mental retardation	Autism	[144]
		recessive	hydroxylase (PAH)
	Wilson's disease Autosomal	ATPase, Cu??
		recessive	transporting, beta
			polypeptide (ATP7B)

Table 2

 2 Selected heritability estimates of personality dimensions

		Assessments	Heritability estimates		Reference
			Female (%)	Male (%)
	Extraversion	EPQ-R	57	57	[85]
	Neuroticism	EPQ-R	54	49	[85]
	Lie	EPQ-R	44	35	[85]
	Psychoticism	EPQ-R	39	43	[85]
	Harm Avoidance	TCI	53	57	[85]
	Novelty Seeking	TCI	55	55	[85]
	Reward depending	TCI	56	51	[85]
	Persistence	TCI	55	55	[85]

Table 4

 4 Heritability estimates of selected psychiatric disorders

	Disorder	Heritability estimates (%)	Reference
	PDD	90	[139]
	Enuresis	67-70	[54]
	Conduct disorder	53	[51]
	OCD	47	[25]
	Anxiety disorders	30-40	[33]
	ADHD	60-80	[64]
	Anorexia nervosa	48-88	[141]
	Bulimia nervosa	28-83	[141]
	Schizophrenia	73-90	[160]
	Bipolar disorder	60-85	[149]
	Major depression	31-42	[159]

OCD Obsessive Compulsive Disorder; PDD Pervasive Developmental Disorders (including autistic disorder, Asperger disorder, disintegrative disorder, and PDD not otherwise specified); ADHD Attention Deficit/Hyperactivity Disorder

Table 3

 3 Heritability estimates of selected behaviors and developmental milestones

		Assessments	Heritability	Reference
			estimates (%)
	Behaviors
	Dieting	EAT	42		[134]
	Body	EDI	52		[134]
	dissatisfaction
	Drive for thinness EDI	44		[134]
	Disinhibition of	TFEQ	40		[155]
	eating
	Restrained eating TFEQ	28		[155]
	Hunger	TFEQ	28		[155]
	Obsessive	CBCL	45-58		[71]
	compulsive
	behav.
	Developmental milestones
	Motor	Crawling, sitting	90		[53]
	development	standing, walking
		Crawling, sitting,	22-33		[123]
		walking
		Standing	0		[123]
			Heritability
			estimates (%)
			Female Male
	Expressive	MCD-I-R	8	20	[73]
	language
	''vocabulary''
	''Two-word-	MCD-I-R	28	10	[73]
	combination-use''

EAT Eating Attitudes Test

[START_REF] Garner | The eating attitudes test: psychometric features and clinical correlates[END_REF]

, EDI Eating Disorder Inventory

[START_REF] Garner | Development and validation of a multidimensional eating disorder inventory for anorexia nervosa and bulimia[END_REF]

, MCD-I-R MacArthur Communicative Development Inventories-short form

[START_REF] Fenson | MacArthur Communicative Development Inventories[END_REF]

, CBCL Child Behavior Checklist

[START_REF] Achenbach | Manual for the Child Behavior Checklist/4-18 and 1991 profile[END_REF]

, TFEQ Three Factor Eating Questionnaire

[157]

Table 5

 5 Examples of single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) detected in genome wide association studies (GWAS) for selected neuropsychiatric disorders ASTN2 astrotactin 2 demonstrates the immense power lying in coordinated efforts to provide the foundation of biological and biomedical research at a new, more global but at the same time intertwined level. More recently, the 1,000 Genomes Project was initiated to re-sequence the genomes of at least 1,000 individuals from around the world to create the most detailed and medically useful picture to date of human genetic variation[162].

	Disorder Gene(s) or region	Marker-risk allele	P value	Sample (cases/controls)	Reference
	Restless legs syndrome
	GWAS PTPRD	rs4626664-A	6 9 10 -10	628/1,644; replication in 1,835/3,111 [142]
			rs1975197-T	6 9 10 -9
		BTBD9	rs3923809-A	3 9 10 -14	306/15,633; replication in 311/1,895 [152]
		MEIS1	rs2300478-G	3 9 10 -28	401/1,644; replication in 1,158/1,178 [176]
		BTBD9	rs9296249-T	4 9 10 -18
		MAP2K5, LBXCOR1	rs12593813-G	1 9 10 -15
	ADHD
	CNV	CTNND2, GRM5, GRM7, PARK2	Deletions	9.48 9 10 -3 335 parent-child trios/2,026	[34]
	Schizophrenia
	GWAS ZNF804A	rs3923809-A	1.61 9 10 -7 479/2,937; replication in 6,666/	[118]
					19,897
		CSF2RA, IL3RA	rs4129148-C	4 9 10 -7	178/144	[95]
		MHC/HIST1H2BJ	rs6913660-C	1.1 9 10 -9	2,663/13,498; follow-up in 4,999/	[154]
		MHC/PRSS16	rs13219354-T	1.3 9 10 -10	15,555
			rs6932590-T	1.4 9 10 -12
		MHC/PGBD1	rs13211507-T	8.3 9 10 -11
		MHC/NOTCH4	rs3131296-G	2.3 9 10 -10
		NRGN	rs12807809-T	2.4 9 10 -9
		TCF4	rs9960767-C	4.11 9 10 -9
		MHC class 1 region	rs13194053-C	9.5 9 10 -9	3,322/3,587; meta-analysis with	[80]
					8,008/19,077
		6p22.1		9.54 9 10 -9 Meta-analysis of 8,008/19,077	[147]
	CNV	1q21.1		2.9 9 10 -5	1,433/33,250; replication in 3,285/	[153]
		15q11.2		6 9 10 -4	7,951
		15q13.3		5.3 9 10 -4
	Bipolar disorder
	GWAS ANK3	rs10994336-T	9 9 10 -9	4,387/6,209	[41]
		CACNA1C	rs1006737-A	7 9 10 -8
		DGKH	rs1012053-A	2 9 10 -8	461/563	[10]
		PALB2, NDUFAB1, DCTN5	rs420259-A	6 9 10 -8	1,868/2,938	[173]
	Autism
	GWAS CDH10, CDH9	rs4307059-T	2 9 10 -10	1,204/6,491; 3,101 family members;	[170]
					replication in 1,390 family
					members;
					108/540
	CNV	MDGA2	Exonic deletions	1.3 9 10 -4	912 families/1,488; replication in	[17]
		BZRAP1	Exonic deletions and duplications 2.3 9 10 -5	859/1051
		AK123120	Duplication	3.57 9 10 -6 859/1,409; replication in 1,336/	[52]
		PARK2, UBE3A, RFWD2, FBXO40 Duplication or deletion	3.3 9 10 -3	1,110 cc 1
		NLGN1, ASTN2	Duplication or deletion	9.5 9 10 -3
		16p11.2	Microdeletions/microduplications		751 families/4,234; replication in	[172]
					811/19,268

PTPRD protein tyrosine phosphatase, receptor type, D; BTBD9 BTB (POZ) domain containing 9; MEIS1 Meis homeobox 1; MAP2K5 mitogen-activated protein kinase kinase 5; LBXCOR1 LBXCOR1 homolog; CTNND2 catenin delta-2; GRM5 glutamate receptor metabotropic 5; GRM7 glutamate receptor metabotropic 7; ZNF804A zinc finger protein 804A; RELN reelin; CSF2RA colony stimulating factor, receptor 2 alpha; IL3RA interleukin-3 receptor subunit alpha; MHC major histocompatibility complex; HIST1H2BJ histone cluster 1 H2bj; PRSS16 thymus-specific serine protease; HIST1H2BJ histone H2B type 1-J; PGBD1 PiggyBac transposable element-derived protein 1; NOTCH4 notch homolog 4; NRGN neurogranin; NLGN1 neuroligin 1; TCF4 neurogranin, transcription factor 4; ANK3 ankyrin 3; CACNA1C calcium channel, voltage-dependent, L type, alpha 1C subunit; DGKH diacylglycerol kinase eta; PALB2 partner and localizer of BRCA2; NDUFAB1 NADH dehydrogenase (ubiquinone) 1, alpha/beta subcomplex, 1; DCTN5 dynactin subunit 5; CDH10 Cadherin-10 precursor; CDH9 cadherin-9 precursor; MDGA2 MAM domain containing glycosylphosphatiddylinositol anchor 2; BZRAP1 benzodiazapine receptor (peripheral) associated protein 1; AK123120 cDNA; PARK2 Parkinson disease 2; RFWD2 ring finger and WD repeat domain 2; UBE3A ubiquitin-protein ligase E3A; FBXO40 F-box protein 40;

  : 1.4 million unique SNPs were built in a *2-kb-resolution map placing *2-4 SNPs per human gene. Currently, there are *79 million submitted, 18 million referenced and 6.6 million validated SNPs known (latest update: 30 April 2009 [148]

Eur Child Adolesc Psychiatry (2010) 19:259-279

Acknowledgments This work was supported by grants from the Bundesministerium fu ¨r Bildung und Forschung (NGFNplus

01GS0820) and the Deutsche Forschungsgemeinschaft (HE144/9-1). The authors wish to thank Dr. Carla Vogel for her contribution to the paper.


Philippe Marguerès

Jean-Noël Périé

Julian Gutierrez Perez

Francis Collombet

Yves-Henri Grunevald

Characterization of a

Keywords: RFI, NDT, multi-instrumentation, strain field measurement

teaching and research institutions in France or abroad, or from public or private research centers.

INTRODUCTION

The work presented herein takes part in a more global study [START_REF] Torres | Characterization of composite material: HexFIT TM[END_REF] aiming at the characterization and the monitoring of the behaviour of composites structures from manufacture to complete ruin. This is done taking into account the effect of meso-or macro-scale variabilities and the process conditions. A first goal step is to propose a specific experimental protocol leading to define accurate investigation techniques. Here is considered a composite structure made of HexFIT® E-glass/M9.6FL. This semi-preg is dedicated to Resin Film Infusion process and supplied by Hexcel Composites.

RAW MAETRIAL AND MANUFACTURE

Resin Film Infusion (RFI) is a mix between RTM (Resin Transfer Moulding) and RIM (Resin Infusion

Moulding) offering relatively fast and low-cost manufacturing of large composite parts [START_REF] Torres | Characterization of composite material: HexFIT TM[END_REF][START_REF]Resin film infusion composites cost reducer[END_REF]. The HexFIT® semipreg considered in this study is made of preimpregnated 2x2 twill-weave E-glass fabrics combined with two dry ±45 E-glass reinforcements stitched together. The mass per unit area of the reinforcements reaches 1200 g/m² (Figure 1). The studied structural part is a 900 x 900 x 5 mm plate manufactured using a vacuum bag oven curing (Figure 2) and following the supplier cure cycle (Figure 3).

The used consumable supplies are described in Figure 4. The composite part is a quasi-isotropic and symmetric laminate made from only four plies (Figure 5). It is important to mention here that there's a specific mesoscale structure in the ply due to the different roving sizes used in the reinforcements. On one hand the 2x2 twill-weave fabric shows a 20x20 mm repetitive cell containing large yarns and on the other hand ±45 reinforcements containing thinner yarns and stitching (Figure 6).

INITIAL STATE AND INFLUENCE OF PROCESS

Two cartographies intented to define the initial state of the structural part and realized using ultrasonic echography (Figure 7) and infra-red method (Figure 8). Ultrasonic C-Scan was obtained through 2000 C-Scan measures performed on the upper face of the plate while it was still on the mould. This non destructive investigation highlights the thickness variation (macroscale) owing to process conditions. In fact, the thickness variation is mostly controlled by the location of the connector to the vacuum pump and the sealant tape and also the flexibility of the vacuum bag for the considered cure cycle:

-thickness near the sealant tape is smaller than the one located near the vacuum sniffer; -there is a symmetrical variation of the thickness both sides of a line joining the vacuum sniffer location and the median axis of the plate.

ACCEPTED MANUSCRIPT

The IR method required more attention because of the dimensions of the plate. In fact misinterpretation on the thickness variation can appear when using the IR measures obtained on the edges of the investigation areas used (numbered from 1 to 48). But ultrasonic or IR information are mainly similar.

An additional inspection using X-Ray technology was undertaken on two calibration test blocks respectively made from four plies of HexFIT® containing only 2x2 twill-weave E-glass fabrics and four plies of HexFIT® containing only ±45 E-glass reinforcements (Figure 9). It confirms the mesoscale effect of the reinforcements (different yarn sizes) but also highlights the macroscale effect of the 2x2 twll weave E-glass fabrics showing a spacing of about 0.5 mm between the rovings.

MECHANICAL CHARACTERIZATION

A sampling was undertaken on the plate in order to obtain standard specimens according to NFT57-101 (Figure 10). This characterization aims at highlighting effect of the variability of the specimens (thickness variation, dimensions) to help at defining future mechanical modelling. First results are given for specimens with similar thickness (same colour code on C-Scan) but different widths according to NFT57-101: 10, 25 and 50 mm [START_REF] Torres | Characterization of composite material: HexFIT TM[END_REF]. Figure 11 shows slightly differences in the obtained moduli and the rupture behaviour of the 10-mm-wide specimens.

In order to study the effect of the mesostructure on the global behaviour of such laminates multiinstrumented tensile tests were undertaken using 50-mm-wide specimens. (Figure 12). Three techniques were used:

-longitudinal and transverse strain gauges (Kyowa; K = 2,08; 119.6 ± 0.4 Ω) bounded on both sides of the specimens; gauges with 10-mm et de 6-mm lengths were compared; -80-mm-high-resolution-Zwick extensometer; -Digital Image Correlation (DIC) for full field strain measurements [START_REF] Hild | Multiscale displacement field measurements of compressed mineral-wool samples by digital image correlation[END_REF].

For DIC measurements, a random white speckle pattern is sprayed onto the initially black painted surface of the samples..Images were grabbed at various loadings and up to rupture with a 14 bit CCD PCO.1600 camera. The analysis performed using the software Correli LMT focused on local and average strains in order to compare with the other techniques and both strain gauges lengths. No significant difference was observed for the different gauges lengths chosen (Figure 13 and Figure 14).

However, the effect of the reinforcement clearly appeared on the surface deformation of the tested specimens: periodic patterns appeared during loading. Although strain gauges give local information and can break early, full field strain analysis allows monitoring the local and global information and discriminating the very beginning of the rupture.

CONCLUSION

This preliminary study allowed us to propose a first experimental protocol to characterize the HexFIT® material used in a large composite plate. US and IR cartographies were used to sample specimens in the for mechanical testing taking into account the thickness variation due to process conditions. Tensile tests undertaken on specimens with different widths and multi-instrumented tensile tests on 50-mm-wide specimens allowed us to highlight limitation in classical tensile tests. Specimens with 10-mm width seam not to be suitable for the characterization of the HexFIT® used in the study. Despite no significant difference between the information obtained through the use of 6-or 10-mm-long gauges and the 80-mm extensometer, the strain full field analysis highlights mesoscale structure influence on the deformation obtained on the inspected surface. A farther comparison between multi-instrumented specimens varying the thickness will be undertaken to complete this first approach. Once done, discrimination of damage mechanisms could be undertaken in function of width and thickness.