Malgorzata Arnaud Fernandez
email: fernandez.a@pediatrie-chulenval-nice.fr

Marta Drozd

Susanne Thümmler

Emmanuelle Dor

Maria Capovilla

Florence Askenazy

Barbara Bardoni
email: barbarabardonibardoni@ipmc.cnrs.fr

Patrick Dion

Malgorzata Marta Drozd

Childhood-Onset Schizophrenia: A Systematic Overview of Its Genetic Heterogeneity From Classical Studies to the Genomic Era

Keywords: childhood-onset schizophrenia, autism spectrum disorder, genetics, copy number variations, single nucleotide polymorphisms, exome sequencing

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INtRODUCtION

Childhood-onset schizophrenia (COS) is a rare (< 1/40,000) and severe chronic psychiatric condition that shares with adultonset schizophrenia (AOS) positive symptoms (delusions, hallucinations, and disorganized speech or behavior), but presents an early onset (before the age of 13) [START_REF] Burd | A North Dakota prevalence study of schizophrenia presenting in childhood[END_REF][START_REF] Nicolson | Childhood-onset schizophrenia: rare but worth studying[END_REF]. It remains considered by many authors as an early and severe variant of AOS [START_REF] Nicolson | Childhood-onset schizophrenia: rare but worth studying[END_REF][START_REF] Biswas | Comparative study of neuropsychological correlates in schizophrenia with onset in childhood, adolescence and adulthood[END_REF]. In COS, neurodevelopmental abnormalities (deficits in cognition, communication, or neuromotor impairments) and premorbid dysfunction are more frequent compared with AOS [START_REF] Vourdas | Increased developmental deviance and premorbid dysfunction in early onset schizophrenia[END_REF] and a clinical overlap exists with other neurodevelopmental disorders: 28% of patients with COS in the US cohort of the National Institute of Mental Health Child Psychiatry Branch met criteria for comorbid autism spectrum disorder (ASD) (Rapoport et al., 2009). In addition, more than 80% of children with schizophrenia or schizoaffective disorder present comorbid attention deficit and hyperactivity disorder (ADHD) [START_REF] Ross | High rates of comorbidity are found in childhood-onset schizophrenia[END_REF]. Few genetic studies of COS were reported, due to the very low prevalence (<1/40,000) [START_REF] Burd | A North Dakota prevalence study of schizophrenia presenting in childhood[END_REF] and to nosographic difficulties, which made it hard to obtain a consensual clinical definition of this disorder and to carry out etiological studies [START_REF] Maier | Diagnostic classification of psychiatric disorders and familialgenetic research[END_REF][START_REF] Gochman | Childhood-onset schizophrenia: the challenge of diagnosis[END_REF]. Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) classification provides recent clarification in this area with schizophrenia no longer excluding the diagnosis of ASD [START_REF] Petty | Autistic children who become schizophrenic[END_REF]American Psychiatric Association, 2013). Thus, clinical overlap between COS and ASD is now formally accepted. Surprisingly, DSM-5 still does not recognize the existence of COS, which therefore remains considered an adult clinical presentation (AOS) (American Psychiatric Association, 2013). Indeed, COS is a very rare complex disorder related to other neurodevelopmental disorders, and it represents a real challenge for clinical diagnosis with, to date, no objective test based on genetics [START_REF] Petty | Autistic children who become schizophrenic[END_REF]. However, a high heritability rate of COS (> 80%) has been suggested in early adoption/twin studies [START_REF] Kallmann | Genetic aspects of preadolescent schizophrenia[END_REF] and has been confirmed by familial aggregation studies [START_REF] Asarnow | Genetics of childhood-onset schizophrenia[END_REF]. To determine the etiology of COS, it is indispensable to start by reviewing the publications that have linked COS to DNA changes.

Macro-lesional cytogenetic abnormalities such as copy number variations (CNVs), including the 22q11.21 deletion, are more frequent in COS than in AOS [10.6% of patients with COS (DSM-III-R, onset <13 years) vs. 2-5%, in AOS, p < 0.0001]. These anomalies in the general population would concern only 0.86% of newborns (Nicolson et al., 1999).

Recently, Next Generation Sequencing (NGS) or "high throughput sequencing" allowed, with unprecedented scalability and speed, to determine the DNA sequence of a given individual. This tool opened up new perspectives to understand complex neurodevelopmental disorders, with particular attention to de novo single nucleotide variations (SNVs) occurring in genes involved in brain development [START_REF] Veltman | De novo mutations in human genetic disease[END_REF]. Only one study used whole exome sequencing (WES), a NGS method, in a cohort of patients with COS. This study identified 20 de novo variants in 17 COS probands (rate: 1.17) in genes previously linked to neuronal function or to psychiatric disorders [START_REF] Ambalavanan | De novo variants in sporadic cases of childhood onset schizophrenia[END_REF]. These arguments (phenotypic overlap with other neurodevelopmental disorders, high heritability, disease-related CNVs, and de novo SNV rates) strongly support the neurodevelopmental and genetic bases of COS [START_REF] Rapoport | Neurodevelopmental model of schizophrenia: update 2012[END_REF]. In this context, the main aim of this study is to describe the COS genomic variation (CNVs and SNVs) in the scientific literature to identify interesting genes or genetic pathways in both clinical practice and research.

mEtHODS

We carried out a systematic review of the MEDLINE database accessible via the search engine PubMed (www.ncbi.nlm.nih. gov/pubmed/) with the following key words: "childhood-onset schizophrenia" or "childhood-onset psychosis" or "early-onset schizophrenia" or "early-onset psychosis" and "genetics" or "genetic" or "gene" or "genes" or "genomic" or "genomics. " Our search terms were not limited by language or date of publication and were manually reviewed. According to inclusion criteria, we considered all genomic changes occurring in COS patients (age of onset before 13). We excluded all abnormalities at RNA or protein levels (regardless the age of onset). Genomic variations were classified based on cytogenetic position (Table 1) and candidate gene names (Table 2).

All CNVs were manually annotated using the University of California Santa Cruz (UCSC) Genome Browser (UCSC Mar. 2006 (NCBI36/hg18 or NCBI37/hg19) assembly; http://genome.ucsc. edu/). Regarding their type (gain or loss), their size, their genomic content, and making comparisons with external databases, we ranked each CNV as "pathogenic, " "uncertain clinical significance, " or "benign" (according to the American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants). For each CNV, we checked on the Simons Foundation Autism Research Initiative (SFARI) Gene database (autism/genetic database, http://sfari.org) which CNV involved in COS was also involved in ASD. For each gene, we checked on the Phenocarta Database (https://gemma. association of different genetic variations (two or more CNVs or CNVs and SNVs) as well as association with early acquired brain lesions such as infection, hypoxia, or early childhood trauma. [START_REF] Gordon | Brief report: translocation involving chromosomes 1 and 7 in a boy with childhood-onset schizophrenia[END_REF]Nicolson et al., 1999;[START_REF] Yan | Childhood-onset schizophrenia/autistic disorder and t(1;7) reciprocal translocation: identification of a BAC contig spanning the translocation breakpoint at 7q21[END_REF][START_REF] Idol | Sequencing and analyzing the t(1;7) reciprocal translocation breakpoints associated with a case of childhood-onset schizophrenia/autistic disorder[END_REF][START_REF] Eckstrand | Sex chromosome anomalies in childhood onset schizophrenia: an update[END_REF] 2). Two articles were added after the freezing of the inclusion process (41; 52). Mutations were identified in 21 chromosomes. The results were ranked either in ascending order of their chromosomal position for structural variants (cytogenetic abnormalities) (Table 1) or in alphabetical order according to their gene name for genetic variants (lesions at gene level) (Table 2).

RESULtS

I) Cytogenetic Abnormalities Associated with COS

The following cytogenetic abnormalities (n = 72, including 66 CNVs) were identified in 16 autosomes (1, 2, 3, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, and 22) and two sex chromosomes (X and Y) of 46 patients (17-40). The results were ranked in ascending order of their chromosomal position (Table 1) and were summarized in a genomic map (Figure 3). 2p25.3,3p21.31,3p25.3,5q12.3,7p13,8p22,8q11.23,8q24.3,10p11.23,10p13,15q13.3,16p11.2,16p13.11,16q23.3,17q21.31,18q22.1,22q13.32,Xp22.31,Xq28,and Yq11.221.

Only six CNVs (9%) of our study are described in COS only: del2q31.2-q31.3 (smaller than the CNV described in ASD), del3p12.2-p12.1 (described in ASD as a duplication), delXq24ter (larger than the CNV described in ASD), dup7p13 (described in ASD as a deletion), dup16q22.2-ter (larger than the CNV described in ASD), and dup16q22.3-q24.3 (larger than the CNV described in ASD).

In an on-site case series and literature review focusing on Childhood-Onset Schizophrenia Spectrum Disorders (SSDs; larger spectrum than COS), [START_REF] Giannitelli | An overview of medical risk factors for childhood psychosis: Implications for research and treatment[END_REF] showed that some CNVs, previously un-described in COS patients, are associated to childhood psychosis: 1q21.1 deletion, 1q21.1 duplication, Williams-Beuren region (7q11.23) duplication and 16p11.2 deletion [START_REF] Giannitelli | An overview of medical risk factors for childhood psychosis: Implications for research and treatment[END_REF].

The phenotypes of only 15 out of the 46 patients were fully described (33%). The neurodevelopmental disorders that have been presented are: motor impairments (fine or growth milestones delay, coordination disability, or tics) in 11/15 patients, language retardation in 7/15 patients, intellectual disability (IQ < 70) in two patients, and ASD in five patients [(including 1 Pervasive Developmental Disorder-Not Otherwise Specified (PDD-NOS)]. Social impairment was present in six other patients with COS and ADHD in three patients. Inattention impairment was specified in only one patient. The psychiatric comorbidities that have been highlighted are: two cases with mood disorders (major depressive disorder or dysthymia) and two cases with anxiety disorders. The somatic comorbidities detected are: dysmorphia in four cases and epilepsy in two cases. Sporadic cases of hypospadias, ureteric reflux, congenital ichthyosis, Chiari type 1, or celiac disease were also described.

2) Other DNA Lesions a) Aneuploidy One case of Trisomy X (47XXX) and two cases of Turner syndrome (45X0), one atypical form (46,X,del(X)(q24-ter) and one with mosaicism 46,X,i(X)(q10)(22%)/45,X(78%) [START_REF] Eckstrand | Sex chromosome anomalies in childhood onset schizophrenia: an update[END_REF][START_REF] Addington | The genetics of childhood-onset schizophrenia: when madness strikes the prepubescent[END_REF]); b) Uniparental and Segmental isoDisomy (iUSD)

In isodisomy, both copies of a chromosomal set are inherited from one parent (the mother or the father). An iUSD on 5q32-ter (35Mb) was described in a patient with COS [START_REF] Eckstrand | Sex chromosome anomalies in childhood onset schizophrenia: an update[END_REF][START_REF] Addington | The genetics of childhood-onset schizophrenia: when madness strikes the prepubescent[END_REF][START_REF] Seal | Segmental uniparental isodisomy on 5q32-qter in a patient with childhood-onset schizophrenia[END_REF]; c) Translocation 1, 7: p22q22 [START_REF] Gordon | Brief report: translocation involving chromosomes 1 and 7 in a boy with childhood-onset schizophrenia[END_REF]Nicolson et al., 1999;[START_REF] Yan | Childhood-onset schizophrenia/autistic disorder and t(1;7) reciprocal translocation: identification of a BAC contig spanning the translocation breakpoint at 7q21[END_REF][START_REF] Eckstrand | Sex chromosome anomalies in childhood onset schizophrenia: an update[END_REF][START_REF] Idol | Sequencing and analyzing the t(1;7) reciprocal translocation breakpoints associated with a case of childhood-onset schizophrenia/autistic disorder[END_REF][START_REF] Addington | The genetics of childhood-onset schizophrenia: when madness strikes the prepubescent[END_REF]) d) Trinucleotide Repeat Expansions i) CGG Expansions: Although a link between Fragile X syndrome (FXS) and COS has never been reported, [START_REF] Vantalon | Fragile X syndrome and very early onset schizophrenia: a female case study[END_REF] described a 1.5 Kb expansion and complete methylation of the CpG island upstream of FMR1 in a 9 yearsold girl with COS, dysmorphia, learning and social impairments, and mild mental retardation [START_REF] Vantalon | Fragile X syndrome and very early onset schizophrenia: a female case study[END_REF]. This abnormality is inherited from the mother who carries an FXS premutation. Interestingly, instead of being unaffected or weakly affected as most patients carrying a premutation, the mother presents schizotypal personality. It seems that the severity of the schizophrenic spectrum disorder, which affects both mother and daughter with gradual severity, is linked to the CGG expansion degree. Effect of the hazard could not be excluded in this case [START_REF] Vantalon | Fragile X syndrome and very early onset schizophrenia: a female case study[END_REF]. ii) CAG/CTG Expansion: a longer repeat expansion on chromosome 18 was found in a COS cohort and in a male subclass, with a significant p-value especially for the males (0.036 and 0.002, respectively; Wilcoxon-Mann-Whitney U test) [START_REF] Burgess | Large CAG/CTG repeats are associated with childhood-onset schizophrenia[END_REF]). e) Genetic Syndromes

In their recent review, [START_REF] Giannitelli | An overview of medical risk factors for childhood psychosis: Implications for research and treatment[END_REF] showed that some genetic syndromes, previously un-described in COS, are associated to childhood-onset SSDs: juvenile Huntington disease, Prader-Willi syndrome, Steinert myotonia, Ondine syndrome, Rubinstein-Taybi syndrome, and GLUT1 deficiency syndrome [START_REF] Giannitelli | An overview of medical risk factors for childhood psychosis: Implications for research and treatment[END_REF].

II) Genes Associated with COS

In total, 32 candidate genes have been described on 12 autosomes (1, 2, 3, 6, 7, 8, 11, 13, 15, 17, 19, and 22) and 1 sex chromosome (X) (Tables 1 and2) through the following studies [START_REF] Addington | Polymorphisms in the 13q33.2 gene G72/G30 are associated with childhood-onset schizophrenia and psychosis not otherwise specified[END_REF][START_REF] Sekizawa | Childhood-onset schizophrenia and tryptophan hydroxylase gene polymorphism[END_REF][START_REF] Gornick | Dysbindin (DTNBP1, 6p22.3) is associated with childhood-onset psychosis and endophenotypes measured by the Premorbid Adjustment Scale (PAS)[END_REF][START_REF] Addington | GAD1 (2q31.1), which encodes glutamic acid decarboxylase (GAD67), is associated with childhood-onset schizophrenia and cortical gray matter volume loss[END_REF][START_REF] Addington | Neuregulin 1 (8p12) and childhood-onset schizophrenia: susceptibility haplotypes for diagnosis and brain developmental trajectories[END_REF][START_REF] Pakhomova | Molecular-genetic study of earlyonset schizophrenia[END_REF][START_REF] Addington | A novel frameshift mutation in UPF3B identified in brothers affected with childhood onset schizophrenia and autism spectrum disorders[END_REF][START_REF] Raznahan | Catechol-o-methyl transferase (COMT) val158met polymorphism and adolescent cortical development in patients with childhood-onset schizophrenia, their non-psychotic siblings, and healthy controls[END_REF][START_REF] Smedemark-Margulies | A novel de novo mutation in ATP1A3 and childhoodonset schizophrenia[END_REF][START_REF] Chaumette | Missense variants in ATP1A3 and FXYD gene family are associated with childhood-onset schizophrenia[END_REF][START_REF] Ambalavanan | Exome sequencing of sporadic childhood-onset schizophrenia suggests the contribution of X-linked genes in males[END_REF]: i) DAOA and DAOA-AS1 [START_REF] Addington | Polymorphisms in the 13q33.2 gene G72/G30 are associated with childhood-onset schizophrenia and psychosis not otherwise specified[END_REF]: three Single Nucleotide Polymorphisms (SNPs) are associated with COS (rs1935058, rs3916967, and rs2391191 (p = 0.5, 0.015, 0.3, respectively)). The most significant SNP (p = 0.015) is rs3916967 (genomic sequence reference: NG_012694.1:g.4133T > C); ii) DTNBP1 [START_REF] Gornick | Dysbindin (DTNBP1, 6p22.3) is associated with childhood-onset psychosis and endophenotypes measured by the Premorbid Adjustment Scale (PAS)[END_REF]: one SNP, rs11558324 (NG_009309.1:g.5154A > G), is associated with COS (p = 0.014) and two twomarker haplotypes (containing rs11558324) are also associated with COS (p = 0.021, 0.008), iii) GAD 1 [START_REF] Addington | GAD1 (2q31.1), which encodes glutamic acid decarboxylase (GAD67), is associated with childhood-onset schizophrenia and cortical gray matter volume loss[END_REF]: three four-marker haplotypes are associated with COS (p = 0.005); iv) NRG1 [START_REF] Addington | Neuregulin 1 (8p12) and childhood-onset schizophrenia: susceptibility haplotypes for diagnosis and brain developmental trajectories[END_REF]: individual markers show association with COS (rs35753505, rs2881272, 420M9-1395 (microsatellite), and rs327417, with p-value between 0.009 and 0.05).

1) Genetic Association Studies (
The most significant SNP (p = 0.009) is rs327417 (NG_012005.2:g.341913G > A). Further, several novel four-marker haplotypes are associated with COS (lowest p = 0.0004). b. Population-Based "Case vs. Control" Studies (From 2004 to 2011) A polymorphism (VAL66MET) in the BDNF (11p13) gene was associated with COS in a 65 patient cohort (10.5 ± 3.7 years old at onset) vs. 111 controls (p = 0.03; χ² test) [START_REF] Pakhomova | Molecular-genetic study of earlyonset schizophrenia[END_REF]. A mutation (VAL158MET) in COMT (22q11.21) that increases protein activity levels in the brain accelerated adolescent cortical thinning (MRI findings) in both schizophrenia probands and their siblings (with resolution after a certain age for siblings), illustrating the influence of dopaminergic disruption on brain cortical maturation. Authors analyzed data from an 83 COS patient cohort vs. 208 healthy controls [START_REF] Raznahan | Catechol-o-methyl transferase (COMT) val158met polymorphism and adolescent cortical development in patients with childhood-onset schizophrenia, their non-psychotic siblings, and healthy controls[END_REF]. A mutation (A218C) in the TPH1 (11p15.3-p14) gene (p = 0.0058; χ² test) is described in 51 patients (early adolescent onset cohort before 16 years old) vs. 148 controls [START_REF] Sekizawa | Childhood-onset schizophrenia and tryptophan hydroxylase gene polymorphism[END_REF]. 2) Gene Sequencing Studies (2011 to Present) Finally, five studies by DNA sequencing (candidate genes or whole exome sequencing) have allowed identifying the following 18 mutations in 11 genes [START_REF] Addington | A novel frameshift mutation in UPF3B identified in brothers affected with childhood onset schizophrenia and autism spectrum disorders[END_REF][START_REF] Ambalavanan | De novo variants in sporadic cases of childhood onset schizophrenia[END_REF][START_REF] Smedemark-Margulies | A novel de novo mutation in ATP1A3 and childhoodonset schizophrenia[END_REF][START_REF] Chaumette | Missense variants in ATP1A3 and FXYD gene family are associated with childhood-onset schizophrenia[END_REF][START_REF] Ambalavanan | Exome sequencing of sporadic childhood-onset schizophrenia suggests the contribution of X-linked genes in males[END_REF]. i. Six SNVs inherited from a healthy mother:

-Five X-linked recessive variants: LUZP4 (arg278fs*10), OPHN1 (met461val), PCDH19 (leu1022ile), RPS6KA3 (arg723his), and UPF3B gene (gln228fsX18) that also segregates in the sibling. -One SNV (ala813val) in ATP1A3 (a boy with onset at 10 years old and co-morbid ASD). ii. Three SNVs inherited (missenses variants) in the FXYD gene family. FXYD1 (arg90cys), FXYD6-FXYD2 (val101ala), and FXYD6 (gly73arg) genes. Only the patient with the mutation in FXYD1 presents a co-morbid ASD (Asperger's disorder). iii. Nine de novo SNVs in the following genes: ATP1A3 (val129met; asp801asn; glu815lys), GPR153 (arg73cys), GTF2IRD1 (arg357cys), ITGA6 (glu1063del), RYR2 (glu746tyr), SEZ6 (thr229_thr231del), and TTBK1 (arg258gln).

The ATPase Na + /K + transporting Alpha-3 Polypeptide (ATP1A3 gene) encodes the alpha-3 catalytic subunit of the Na + /K + -ATPase transmembrane ion pump mapping at 19q12-q13.2 [START_REF] Harley | Localization of a human Na+,K+-ATPase alpha subunit gene to chromosome 19q12-q13.2 and linkage to the myotonic dystrophy locus[END_REF]. The ATP1A3 isoform is exclusively expressed in neurons of various brain regions, including the basal ganglia, hippocampus, and cerebellum (summary by [START_REF] Rosewich | Heterozygous de-novo mutations in ATP1A3 in patients with alternating hemiplegia of childhood: a whole-exome sequencing gene-identification study[END_REF]. Mutations in this gene have been associated with a spectrum of disorders depending on the domain they affect in the corresponding protein. The majority of mutations associated with rapid-onset dystonia parkinsonism, or dystonia-12 (DYT12), were located in exons 8 and 14 whereas those with alternating hemiplegia of childhood-2 (AHC2) were located in exons 17 and 18 and in general they seem to affect transmembrane and functional domains, being the most severe dysfunctions. By genetic analysis of clinical data from 155 patients with AHC2, 132 confirmed to have ATP1A3 mutations. Among those with AHC2, the most frequent mutations were D801N (in 43%), E815K (in 16%) and G947R (in 11%). E815K was associated with a severe phenotype, with greater intellectual and motor disability; D801N appeared to confer a milder phenotype and G947R correlated with the most favorable prognosis. For those with epilepsy, the age at seizure onset was earlier for patients with the E815K or G947R mutations than for those with the D801N mutation [START_REF] Panagiotakaki | Clinical profile of patients with ATP1A3 mutations in Alternating Hemiplegia of Childhood-a study of 155 patients[END_REF]. In 10 patients from three unrelated families with cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS; 601338) [START_REF] Demos | A novel recurrent mutation in ATP1A3 causes CAPOS syndrome[END_REF], the same heterozygous missense mutation in the ATP1A3 gene was identified (E818K; OMIM 182350.0014).

The G Protein-coupled Receptor 153 (GPR153) gene, located on 1p36.31, belongs to the large rhodopsin (RHO; OMIM 180380) family of GPCRs [START_REF] Gloriam | Nine new human Rhodopsin family G-protein coupled receptors: identification, sequence characterization and evolutionary relationship[END_REF] and shows a highest similarity to serotonin receptors, [START_REF] Gloriam | Nine new human Rhodopsin family G-protein coupled receptors: identification, sequence characterization and evolutionary relationship[END_REF]. Furthermore, knockdown of GPR153 in mice showed reduction in food intake and increased anxiety according to the elevated plus Maze test [START_REF] Sreedharan | The G protein coupled receptor Gpr153 shares common evolutionary origin with Gpr162 and is highly expressed in central regions including the thalamus, cerebellum and the arcuate nucleus[END_REF].

The InTeGrin Alpha-6 (ITGA6) gene is located on 2q31.1 [START_REF] Hogervorst | Molecular cloning of the human alpha 6 integrin subunit. Alternative splicing of alpha 6 mRNA and chromosomal localization of the alpha 6 and beta 4 genes[END_REF]. While functional absence of ITGA6 has been associated with epidermolysis bullosa [START_REF] Hogervorst | Molecular cloning of the human alpha 6 integrin subunit. Alternative splicing of alpha 6 mRNA and chromosomal localization of the alpha 6 and beta 4 genes[END_REF][START_REF] Georges-Labouesse | Absence of integrin alpha 6 leads to epidermolysis bullosa and neonatal death in mice[END_REF], a few works addressed the role of ITGA6 in neurons. Alpha-6 integrin was initially reported to be involved in neural migration (Yao et al., 2018). In addition, recent data suggested that α6 and β1 integrins may play a role in mediating Schwann cell interactions with axons and promote axonal regeneration [START_REF] Chang | alpha6 and beta1 Integrin heterodimer mediates schwann cell interactions with axons and facilitates axonal regeneration after peripheral nerve injury[END_REF].

The RYanodine Receptor 2 (RYR2) gene-located on chromosome 1 between q42.1 et q43-encodes a calcium channel that is located in the sarcoplasmic reticulum and is the major source of calcium required for cardiac muscle excitationcontraction coupling. [START_REF] Bhuiyan | Expanding spectrum of human RYR2related disease: new electrocardiographic, structural, and genetic features[END_REF]. Ryr2-/-mice die at approximately embryonic day 10 with morphologic abnormalities in the heart tube. Ca 2+ signaling has been associated with ASD [START_REF] Kabir | L-type Ca(2+) channels in mood, cognition and addiction: integrating human and rodent studies with a focus on behavioural endophenotypes[END_REF][START_REF] Stephenson | A novel human CAMK2A mutation disrupts dendritic morphology and synaptic transmission, and causes ASD-related behaviors[END_REF][START_REF] Castagnola | New Insights Into the Role of Cav2 Protein Family in Calcium Flux Deregulation in Fmr1-KO Neurons[END_REF] and with other psychiatric and neurological diseases [START_REF] Heyes | Genetic disruption of voltage-gated calcium channels in psychiatric and neurological disorders[END_REF]. It is not surprising that RYR2 was linked to ASD by genetic studies [START_REF] Lu | Allowing for sex differences increases power in a GWAS of multiplex Autism families[END_REF][START_REF] Soueid | RYR2, PTDSS1 and AREG genes are implicated in a Lebanese population-based study of copy number variation in autism[END_REF][START_REF] Chen | Developmental neurogenetics and multimodal neuroimaging of sex differences in autism[END_REF]. However, it is very interesting to underline that an SNP in this gene was associated with ASD in families with only affected males in contrast with those with affected females [START_REF] Lu | Allowing for sex differences increases power in a GWAS of multiplex Autism families[END_REF] suggesting that RYR2 is a sex-related genetic factor for ASD.

The SEiZure-related 6 (SEZ6) gene is located on the 17q11.2 chromosome. Sez6 types 1 and 2 have an N-terminal signal sequence, followed by a threonine-rich region, a Short Consensus Repeat (SCR), a CUB-like domain, a second SCR, a second CUB-like domain, three tandem SCRs, a transmembrane domain, and a cytoplasmic C-terminal tail. They differ only in the region between the last SCR and the transmembrane domain. SEZ6 was predicted to be involved in neuronal maturation and plasticity [START_REF] Miyazaki | Disturbance of cerebellar synaptic maturation in mutant mice lacking BSRPs, a novel brain-specific receptor-like protein family[END_REF]. Recently mutations and altered expression of this gene have been associated with Alzheimer's and Niemann-Pick disease [START_REF] Causevic | BACE1-cleavage of Sez6 and Sez6L is elevated in Niemann-Pick type C disease mouse brains[END_REF][START_REF] Paracchini | Exome sequencing in an Italian family with Alzheimer's disease points to a role for seizure-related gene 6 (SEZ6) rare variant R615H[END_REF].

The Tau TuBulin Kinase 2 (TTBK2) gene, located on 15q15.2, encodes a member of the casein kinase (CK1) group of eukaryotic protein kinases. TTBK1 has been implicated in Alzheimer's disease (OMIM 104300) and in neurofibrillary tangles formation [START_REF] Sato | Tau-tubulin kinase 1 (TTBK1), a neuron-specific tau kinase candidate, is involved in tau phosphorylation and aggregation[END_REF]. Mutations in this gene also cause spinocerebellar ataxia 11 (SCA11; 604432). SCA11 is a pure progressive cerebellar ataxia that has been linked to 15q14-q21 [START_REF] Worth | Autosomal dominant cerebellar ataxia type III: linkage in a large British family to a 7.6-cM region on chromosome 15q14-21.3[END_REF][START_REF] Houlden | Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11[END_REF]. In an 8-generation English family they found a one-base insertion in the TTBK2 gene creating a premature stop codon and a truncation of the normal protein (OMIM 611695.0001). In a second family of Pakistani ancestry, a different mutation was found (OMIM 611695.0002). [START_REF] Goetz | The spinocerebellar ataxiaassociated gene Tau tubulin kinase 2 controls the initiation of ciliogenesis[END_REF] concluded that TTBK2 is required for removal of CP110 for the initiation of ciliogenesis [START_REF] Goetz | The spinocerebellar ataxiaassociated gene Tau tubulin kinase 2 controls the initiation of ciliogenesis[END_REF].

3) Candidate Genes From Cytogenetic Studies (Table 1)

Interesting candidate genes deleted, duplicated, or truncated by the CNVs have also been found in cytogenetic studies (see above). These genes have brain expression and are mostly described in other neurodevelopmental or psychiatric disorders (Figure 1). Nine genes are described as putative COS-causing genes: CHRNA7 (15q13.3), DGCR6 (22q11.2), FMR1 (Xq27.3), MYT1L (2p25.3), NRXN1 (2p16.3), PRODH (22q11.2), PTK2 (8q24.3), STS (Xp22.31), and SRGAP3 (3p25.3) [START_REF] Yan | Chromosome 22q11.2 interstitial deletions among childhood-onset schizophrenics and "multidimensionally impaired[END_REF]Nicolson et al., 1999;[START_REF] Usiskin | Velocardiofacial syndrome in childhood-onset schizophrenia[END_REF][START_REF] Liu | Genetic variation at the 22q11 PRODH2/DGCR6 locus presents an unusual pattern and increases susceptibility to schizophrenia[END_REF][START_REF] Sporn | 22q11 deletion syndrome in childhood onset schizophrenia: an update[END_REF][START_REF] Vantalon | Fragile X syndrome and very early onset schizophrenia: a female case study[END_REF][START_REF] Eckstrand | Sex chromosome anomalies in childhood onset schizophrenia: an update[END_REF][START_REF] Walsh | Rare structural variants disrupt multiple genes in neurodevelopmental pathways in schizophrenia[END_REF][START_REF] Addington | The genetics of childhood-onset schizophrenia: when madness strikes the prepubescent[END_REF]Rapoport et al., 2009;[START_REF] Wilson | A novel microduplication in the neurodevelopmental gene SRGAP3 that segregates with psychotic illness in the family of a COS proband[END_REF][START_REF] Lee | Microduplications disrupting the MYT1L gene (2p25.3) are associated with schizophrenia[END_REF][START_REF] Ahn | High rate of disease-related copy number variations in childhood onset schizophrenia[END_REF][START_REF] Duong | Two rare deletions upstream of the NRXN1 gene (2p16.3) affecting the non-coding mRNA AK127244 segregate with diverse psychopathological phenotypes in a family[END_REF][START_REF] Zhou | 15q13.3 duplication in two patients with childhood-onset schizophrenia[END_REF][START_REF] Malik | X-linked ichthyosis associated with psychosis and behavioral abnormalities: a case report[END_REF].

CONCLUSIONS

COS is a neurodevelopmental disorder with several degrees of complexity (clinical and genetic heterogeneity). Clinically, getting the diagnostic is very challenging (severe disorder, comorbidities, and association with other neurodevelopmental disorders) [START_REF] Gochman | Childhood-onset schizophrenia: the challenge of diagnosis[END_REF]. The clinical overlap with ASD is well documented and in our study we found a co-morbidity rate (33%) nearly equal to the National Institute of Mental Health (NIMH) COS cohort rate (28%) (Rapoport et al., 2009). The genetic overlap with ASD is also well documented and we show that 91% of described CNVs are also described in ASD (SFARI). In the literature, we found only 20% of COS patients with co-morbid ADHD vs. 84% according to Ross et al. and we hypothesize that this trouble was under-diagnosed in schizophrenia studies [START_REF] Ross | High rates of comorbidity are found in childhood-onset schizophrenia[END_REF]. All intellectual, motor, communication, and learning impairments are also frequently observed in COS [START_REF] Ross | High rates of comorbidity are found in childhood-onset schizophrenia[END_REF]Nicolson et al., 1999). Psychiatric comorbidities were rarely described (two cases of mood disorders and two cases of anxiety disorders), which was an unexpected outcome given the published literature [START_REF] Ross | High rates of comorbidity are found in childhood-onset schizophrenia[END_REF]. Here, we highlight that only one-third of the full phenotypes associated with the mutations published in the literature are described, which constitutes a significant loss of information for researchers. Therefore, it appears fundamental to carry out preliminary work before genetic testing: perform a rigorous and homogeneous phenotypic characterization using International Classification of Disease (ICD-10 and DSM-5) with standardized and internationally validated psychiatric categorical assessments and in accordance with medical history (including perinatology), biography (with significant life event and trauma), and environmental factors (such as toxic exposure).

COS is characterized by a complex genetic architecture with both inherited and de novo mutations distributed in almost all chromosomes. Most of the genes causing COS are unknown yet. It is interesting that, the few that have been already proposed (see before) are involved both in neurodevelopmental and neurodegenerative disorders such as Parkinson, Alzheimer, or ataxia. Moreover, schizophrenia has been shown to have complex genetic traits with high polygenic risk [START_REF] Ahn | Common polygenic variation and risk for childhood-onset schizophrenia[END_REF]. Thus, a second hit (or more), in addition to CNV, is probably essential to explain the phenotypes. It includes de novo SNVs, other CNVs and/ or environmental factors (e.g., trauma at early childhood, central nervous system infections or injuries) [START_REF] Davis | A review of vulnerability and risks for schizophrenia: beyond the two hit hypothesis[END_REF]. At the interplay between genetic and environmental factors, epigenetics opens new perspectives to understand biological mechanisms of psychosis. In fact, recent findings suggest that pangenomic methylation changes during adolescence accompany conversion to psychosis [START_REF] Kebir | Epigenetic variability in conversion to psychosis: novel findings from an innovative longitudinal methylomic analysis[END_REF]. In clinical practice, as suggested by Szego et al. for ASD [START_REF] Szego | Whole genome sequencing as a genetic test for autism spectrum disorder: from bench to bedside and then back again[END_REF], it would seem useful to propose to COS patients genetic sequencing instead or in addition to microarrays [START_REF] Anagnostou | Autism spectrum disorder: advances in evidence-based practice[END_REF][START_REF] Soden | Effectiveness of exome and genome sequencing guided by acuity of illness for diagnosis of neurodevelopmental disorders[END_REF] to improve genetic testing and to allow de novo SNV detection.

In research, the major challenge of the upcoming years will be the analysis of big data from NGS (prioritization and interpretation of DNA variations) [START_REF] Richards | Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[END_REF] and the experimental validation of putative mutations. Sharing data with other teams around the world will be helpful to unravel the molecular pathology of COS and its underlying causes, paving the way for an early therapeutic intervention.

AUtHOR CONtRIBUtIONS

AF, FA, and BB: contributed to the conceptualization of the study and drafted the first version of the manuscript. All other authors MD, ST, ED, and MC have revised first version of the manuscript critically for important intellectual content and approved the final version.

FIGURE 1 |

 1 FIGURE 1 | Venn diagram: evidence for genetic overlap in neurodevelopmental disorders (Phenocarta Database).

  (CNVs) from 15Kb to 43Mb: 2p16.3, 3q29, 6p22.31, 9p24.2, 10q22.3, 15q11.2, 15q13.3, 16p12.1, 16p13.11, 16q24.1, 19p12, 20p12.1, 22q11.21, Xp22.31, b) Duplications (CNVs) from 120Kb to 17Mb: 1q21.3, 2p14,

FIGURE 3 |

 3 FIGURE 3 | Genomic map of CNVs identified in COS. Circles indicate their position and color represents type of abnormality (Red circle, deletion; Blue circle, duplication).

tABLE 1 |

 1 Cytogenetic abnormalities (including copy number variations) in COS patients with their localization, length and type of inheritance.

	CHR	CNv region and	Sex, age of onset	Neurodevelopmental	Inheritance	coordinates	SFARI	Clinical	Bibliography
		type (length)		disorders,		(hg18-hg19)		significance
				Comorbidities
	1	DUP 1q21.3	Male, 12 y	-	inherited	chr1:151,514,380-	+	UCS	Walsh et al., 2008
		(248 kb)				151,762,871
	1 ; 7	t(1;7) (p22;q22)	Male, 9 y	ASD? -ADHD?	inherited (father)	-	-	-
				-language,
				intellectual and
				motor impairments,
				dysmorphia,
				supraventricular
				tachycardia

tABLE 2 |

 2 Genomic microlesions (including single nucleotide variations) in COS patients with their localization, length, and type of inheritance.

	Gene name	Gene	SNv ID number/	Population / Phenotypes	Inheritance	p-value	Bibliography
		localization	mutation (protein level)
	FAmILy BASED ASSOCIAtION StUDy
	DAOA (G72) /	13q33.2	rs1935058, rs3916967,	n=64 (53 COS trios, 11 COS	-	0.015 to 0.5	Addington et al., 2004
	DAOA-AS1 (G30)		rs2391191	dyads)
	DtNBP1	6p22.3	rs11558324	n=92 (73 COS + PDD-NOS trios,	-	0.014	Gornick et al., 2005
				19 COS + PDD-NOS dyads)
	GAD1	2q31.1	rs3749034, rs2270335,	n=66 (55 COS + PDD-NOS trios,	-	0.005	Addington et al., 2005
			rs2241165	11 COS + PDD-NOS dyads)
	NRG1	8p12	rs35753505, rs2881272,	n=70 (59 COS + PDD-NOS trios,	-	0.009 to 0.05	Addington et al., 2007
			rs327417	11 COS + PDD-NOS dyads)
	CASE CONtROL StUDy
	BDNF	11p13	val66met	65 patients (10.5 ± 3.7 y) vs	-	0.03	Pakhomova et al.,
				111 controls			2010
	COmt	22q11.21	val158met	83 patients (<13 y) vs	-	-	Raznahan et al., 2011
				208 controls
	tPH1	11p15.3-p14	ala218cys	51 patients (<16 y) vs	-	0.0058	Sekizawa et al., 2004
				148 controls
	GENES SEQUENCING (candidate genes or full exome)
	AtP1A3	19q13.2	val129met	Male, onset: 6 y, motor delay	de novo	-	Smedemark-
							Margulies et al., 2016
			asp801asn	Male, onset: 10 y, ASD,		-	Chaumette et al.,
				dysmophia, motor, intellectual			2018
				and learning delays. Recurent
				MDD
			glu815lys	Male, onset: 12 y, motor and		-
				communication impairments,
				dysmophia, ASD
			ala813val	Male, onset: 10 y, ASD, motor	mother	-
				and intellectual delays
	FXyD1	19q13.12	arg90cys	Male, onset: 7 y, Asperger's	inherited	-
				disorder
	FXyD6-FXyD2	11q23.3	val101ala	Male, onset: 13 y		-
	FXyD6	11q23.3	gly73arg	Female, onset: 12 y		-
	GPR153	1p36.31	arg73cys	Male, onset: 12 y	de novo	-	Ambalavanan et al.,
							2016
	GtF2IRD1	7q11.23	arg357cys	Female, onset: 12 y		-
	ItGA6	2q31.1	glu1063del	Female, onset: 12 y		-
	LUZP4	Xq23	arg278fs*10	-	mother	-	Ambalavanan et al.,
							2019
	OPHN1	Xq12	met461val	IQ 88, PDDNOS		-
	PCDH19	Xq22.1	leu1022ile	-		-
	RPS6KA3	Xp22.12	arg723his	IQ 64, PDDNOS		-
	RyR2	1q43	glu746tyr	Male, onset: 8 y, PDDNOS,	de novo	-	Ambalavanan et al.,
				separation anxiety disorder,			2016
				Asperger's disorder
	SEZ6	17q11.2	thr229_thr231del	Male, onset: 11 y		-
	ttBK1	6p21.1	arg258gln	Male, onset: 7 y, Asperger's		-
				disorder
	UPF3B	Xq24	gln228fsX18	Male, 10 y, ADHD, PDDNOS,	mother	-	Addington et al., 2011
				ASD
	ADHD, Attention Deficit Hyperactivity Disorder; ASD, Autism Spectrum Disorder; CNV, Copy number variation; COS, Childhood Onset Schizophrenia; DEL, Deletion; DUP,
	Duplication; MDD, Major Depressive Disorder; PDD-NOS, Pervasive developmental disorder not otherwise specified; SNV, Single Nucleotide Variation; Y, Years. When available,
	phenotypes (sex, age of onset, other neurodevelopmental disorders, and comorbidities) are described.

Table 2

 2

	)

Frontiers in Genetics | www.frontiersin.org December 2019 | Volume 10 | Article 1137

Frontiers in Genetics | www.frontiersin.org December 2019 | Volume 10 | Article 1137

When available, phenotypes (sex, age of onset, other neurodevelopmental disorders and comorbidities) are described. Frontiers in Genetics | www.frontiersin.org December 2019 | Volume 10 | Article 1137

FUNDING

This study was supported by INSERM, CNRS, Université Côte d' Azur and Hôpitaux pédiatriques de Nice CHU-Lenval; ANR-11-LABX-0028-01 and ANR-15-CE16-0015 to BB; Monaco Against Autism (MONAA) Foundation to AF, MC, FA, and BB. MD is recipient of a Signalife-LabEx Program international Ph.D.

ACKNOwLEDGmENtS

The authors are indebted to T. Maurin for discussion and to F. Aguila for artwork.

Conflict of Interest:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.