Willy M Nillesen

Helger G Yntema

Marco Moscarda

Nienke E Verbeek

Louise C Wilson

Frances Cowan

Marga Schepens

Annick Raas-Rothschild

Orly Gafni-Weinstein

Marcella Zollino

Raymon Vijzelaar

Giovanni Neri

Marcel Nelen

Hans Van Bokhoven

Jacques Giltay

MD Tjitske Kleefstra

Characterization of a novel transcript of the EHMT1 gene reveals important diagnostic implications for Kleefstra Syndrome

Keywords: Kleefstra syndrome, Chromosome 9q34 microdeletion, 9qSTDS, EHMT1 Kleefstra syndrome, Chromosome 9q, Subtelomere deletion, 9qSTDS, EHMT1

The core phenotype of Kleefstra syndrome (KS) is characterized by intellectual disability, childhood hypotonia and a characteristic facial appearance. This can be caused by either submicroscopic 9q34 deletions or loss of function mutations of the EHMT1 gene. Remarkably, in three patients with a clinical suspicion of KS, molecular cytogenetic analysis revealed an interstitial 9q34 microdeletion proximal to the coding region of the EHMT1 gene based on the NM_024757.3 transcript. Since we found a mono-allelic EHMT1 transcript suggestive for haploinsufficiency of EHMT1 in two of these patients tested, we hypothesized that a deletion of regulatory elements or so far unknown coding sequences in the 5' region of the EHMT1 gene, might result in a phenotype compatible with KS. We further characterized the molecular content of deletions proximal to the transcript NM_024757.3 and confirmed presence of a novel predicted open reading frame comprising 27 coding exons (NM_024757.4). Further analysis showed that all three deletions included the presumed novel first exon of the EHMT1 gene and subsequent testing of 75 individuals without previously detectable EHMT1 aberrations, showed one additional case with a deletion comprising only this 5' part of the gene.

These results have important implications for the genetic screening of KS and for studies of the functional significance of EHMT1.

INTRODUCTION

Kleefstra syndrome (MIM# 610253) also known as 9q Subtelomeric Deletion Syndrome; 9qSTDS, is characterized by intellectual disability, childhood hypotonia, a characteristic facial appearance and in some patients, by congenital heart and renal defects, (brachy)microcephaly, epilepsy, obesity and behavioural problems as well [START_REF] Stewart | Subtelomeric deletions of chromosome 9q: a novel microdeletion syndrome[END_REF][START_REF] Yatsenko | Deletion 9q34.3 syndrome: genotype-phenotype correlations and an extended deletion in a patient with features of Opitz C trigonocephaly[END_REF][START_REF] Stewart | The chromosome 9q subtelomere deletion syndrome[END_REF][START_REF] Kleefstra | Disruption of the gene Euchromatin Histone Methyl Transferase1 (Eu-HMTase1) is associated with the 9q34 subtelomeric deletion syndrome[END_REF][START_REF] Kleefstra | Loss-of-function mutations in euchromatin histone methyl transferase 1 (EHMT1) cause the 9q34 subtelomeric deletion syndrome[END_REF][START_REF] Kleefstra | Further clinical and molecular delineation of the 9q Subtelomeric Deletion Syndrome supports a major contribution of EHMT1 haploinsufficiency to the core phenotype[END_REF][START_REF] Verhoeven | Behavioral Phenotype in the 9q Subtelomeric Deletion Syndrome: A report about two adult patients[END_REF]. The syndrome can be caused by either submicroscopic deletions of the distal long arm of chromosome 9q or intragenic loss of function mutations in the EHMT1 (Euchromatic Histone Methyltransferase 1) gene (MIM# 607001), both leading to haploinsufficiency of the EHMT1 gene. Since the identification of this gene, we have provided regular diagnostic services for mutational analysis comprising direct sequencing of the coding region and screening for deletions by Multiplex Ligation-dependent Probe Amplification (MLPA) of the transcript NM_024757.3 (http://www.ncbi.nlm.nih.gov/).

So far, we have published information about 27 cases with EHMT1 deletions or mutations [START_REF] Kleefstra | Loss-of-function mutations in euchromatin histone methyl transferase 1 (EHMT1) cause the 9q34 subtelomeric deletion syndrome[END_REF][START_REF] Kleefstra | Further clinical and molecular delineation of the 9q Subtelomeric Deletion Syndrome supports a major contribution of EHMT1 haploinsufficiency to the core phenotype[END_REF].

Remarkably, in three patients with a clinical suspicion of Kleefstra syndrome (KS), molecular cytogenetic analysis revealed a submicroscopic interstitial 9q deletion proximal to the coding region of the EHMT1 gene. We therefore hypothesized that regulatory elements or so far unknown coding sequences in the 5' region of the EHMT1 gene, might be affected by the deletion resulting in a phenotype compatible with KS. In this study we aimed to further characterize the molecular content of deletions proximal to the transcript NM_024757.3 causing KS and test a large group of patients suspected of having KS with this hitherto negative findings.

PATIENTS AND METHODS

Patients

All patients were referred to our centre for further genetic analysis because of the clinical suspicion of Kleefstra syndrome. Informed consent was obtained for further publication of data and photographs.

Patient 1 (Figure 1A,B) was born by vaginal delivery at 40 weeks gestation after a pregnancy complicated by first trimester bleedings, treated with progesterone, and contractions at 17 weeks, treated with isoxsuprine. Her birth weight was 3020 g (25-50 th centile), birth length 47 cm (10-25 th centile) and head circumference 32 cm (10-25 th centile). Her APGAR scores were 8/9 at 1/5 minutes. During the first day after birth, she was bradycardic and generally hypotonic. She suffered from gastroesophageal reflux until 8 months of age. Reduced head circumference centile was noted at 5 months. Sleep, feeding and bowel transit and an EEG and echocardiography were normal.

At 16 months her weight was 10 kg (~25 th centile), height 80 cm (~50 th centile), and head circumference 43 cm (<2 nd centile). She had mild synophrys, upslanting palpebral fissures, hypoplasic nasal saddle, normal ears with folded helix (also in the mother), a narrow palate and persistent fetal pads on her fingers. She had normal female external genitalia and a normal anus. She remained generally hypotonic and had joint hyperlaxity.

At three years of age she did not use complete words, but communicated with gestures (i.e. bringing the parent's hand to the item she wanted). She could walk but Patient 2 (Figure 1 C,D) was born at term after an uneventful pregnancy. Delivery was induced because pregnancy was overdue. Her birth weight was 2460 g (< 2 nd centile) , length 49 cm (25 th centile) and head circumference 32 cm (10-25 th centile), and her APGAR scores were 5/8 at 1/5 min. There were neonatal feeding problems and hypotonia.

Her developmental milestones were delayed. She could sit at one year, stand at two years and walk without support at three years of age. She was first referred to us at 15 months for investigation of her psychomotor retardation, hypotonia and joint hypermobility. At five years moderate to severe psychomotor retardation and autism were diagnosed. At this time her height was on the 50 th centile, her length on the 10 th centile and head circumference just <3 rd centile but at latest examination at 12 years it was at -1/-2 SD. There were facial dysmorphisms such as pronounced eyebrows, small mouth, irregularly placed teeth, full lips, anteverted nostrils, protruding ears with a thick ear helix in addition to generalized hypotonia and joint hypermobility which her mother also had. 6 months after birth she had feeding difficulties and needed nasogastric tube feeding.

Her parents were healthy

At 23 months the patient presented with hypotonia and psychomotor delay, left hydronephrosis, and a patent foramen ovale. She had an abnormal hearing test (ABER was positive only at 60db). Her brain MRI showed agenesis of the corpus callosum. She sat at 24 months. At 34 months her height was 91 cm (10 th centile), weight was 13.5 kg (12th centile), head circumference 45 cm (<3 rd centile), palm length 6.2 cm (25 th centile), third finger 4 cm (3 rd centile). She was brachycephalic and had a low frontal hairline, synophrys, hypertrichosis, adenoid hypertrophy, a bifid uvula and a protruding tongue. At 3 years 9 months her height was 91.8 cm( 3 rd centile), weight 13.5 kg (25 th centile) and head circumference 45.5 cm ( -3SD). At this age she had no behavioral problems, could walk with help but did not say any words.

Routine chromosomal analysis showed a normal female karyotype. Molecular analysis of the SNRP gene region allowed ruling out of uniparental disomy and methylation abnormality compatible with Angelman syndrome and transferrin immunoelectrophoresis was negative for carbohydrate deficient glycoprotein syndromes.

Patient 4 (Figure 1 F,G) was the second child of healthy unrelated parents. The pregnancy was unremarkable until 22 weeks when routine ultrasound scans showed borderline cerebral ventriculomegaly, choroid plexus cysts and echogenic bowel. Amniocentesis and screening for congenital infections were normal. Rescanning at 27 weeks gestation showed greater dilatation of the ventricles and a fetal MRI at 32 weeks gestation showed bilateral marked ventriculomegaly. She was delivered by elective caesarean section at 39 weeks gestation weighing 2094 g (9-25 th centile) with a head circumference of 35cm (50-75 th centile) and did not require resuscitation -her Apgar scores were 7/8 (after 1/5 minutes). She had global hypotonia, feeding difficulties secondary to a disorganised suck, gastro-esophageal reflux which was treated medically, and laryngomalacia. She had some dysmorphic facial features as described below. Her development was delayed. She walked at around 18 months of age. At 3 years her cognitive abilities were assessed at around a 13 month level. At 4 years she had around 100 words and used some 2-3 word phrases although her articulation was poor. She was sociable and good natured but had poor concentration. She has never had seizures. She has had recurrent middle ear infections and had mild-moderate conductive hearing loss and glue ear for which she has had grommets. She was long-sighted with a strabismus for which she wears glasses. She had 3 urinary tract infections but a renal ultrasound and DMSA scan did not show any significant abnormality.

When last formally examined at 3 years 3 months her height was 91.8cm (9-25 th centile), weight 14.0 kg (25-50 th centile) and head circumference 50.9 cm (50-75 th centile). She had up-slanting palpebral fissures, mild ptosis, a flattened mid face and nose with a rounded down-turned nasal tip and short columella, a tented mouth with down turned corners, a smallish chin and a protruding tongue. She had fine hair with a frontal upsweep and slightly sparse eyebrows. Her ears appeared normal. She had marked dimpling over both her sacroiliac joints and behind her shoulders. She had increased joint laxity and her fifth toes were curled under. An MRI brain at 7 weeks of age confirmed bilateral lateral ventricular dilation, right greater than left, unusually shaped posterior horns, a relatively small cisterna magna, a thinned corpus callosum and a slightly thickened cortex in the medial temporal region. Investigations have included a postnatal karyotype, telomere FISH, genome wide array-CGH analysis at approximately 1Mb resolution, Prader-Willi methylation testing, full blood count, urea, electrolytes, lactate, creatinine kinase, thyroid function, very long chain fatty acids, transferrin isoelectric focussing, plasma amino-acids, urine organic acids and glycosaminoglycans, CSF lactate, pyruvate, glucose and neurotransmitters, all of which were normal. There was a slightly low CSF/blood glucose ratio of 46% that was not investigated further in the absence of seizures. An EEG did not show any significant abnormalities and echocardiography was normal.

EHMT1 mutation negative patient cohort

An additional cohort of 75 patients, who were referred to the DNA diagnostic service of the Department of Human Genetics in Nijmegen, the Netherlands with a clinical suspicion of Kleefstra syndrome, were incorporated in this study. No mutations had been identified by sequencing and MLPA analysis of the coding region of EHMT1 based on the NM_024757.3 transcript. Furthermore, all patients have been screened for clinically relevant Copy Number Variations as part of their diagnostic routine by using high-density single nucleotide polymorphism (SNP) microarray platforms.

MLPA analysis was performed using a home-designed MLPA kit containing synthetic probes in all coding EHMT1 exons, except for exon 21 [START_REF] Kleefstra | Loss-of-function mutations in euchromatin histone methyl transferase 1 (EHMT1) cause the 9q34 subtelomeric deletion syndrome[END_REF][START_REF] Kleefstra | Further clinical and molecular delineation of the 9q Subtelomeric Deletion Syndrome supports a major contribution of EHMT1 haploinsufficiency to the core phenotype[END_REF].

Genomic and Molecular analysis

Genome wide array studies

Several array platforms were used to identify chromosomal abnormalities.

In patient 1, Array-CGH was performed using Agilent oligonucleotide-array (Human Genome CGH microarray 4x44K, Agilent Technologies Santa Clara, CA), with an average resolution of 75 kb, following the manufacturer's instructions. The array was analyzed using GenePix 4000B scanner (Axon, Union City, CA) and Feature Extraction V.9.5.1 software. A graphical overview of the results was obtained using CGH Analytics V.3.5.14 software.

In patient 2, the 105K custom Agilent oligo array (Oxford design, AMADID 019015) containing over 99,000 unique in situ synthesized 60-mer oligonucleotides, with a resolution of 21.7 kb, was applied. Hybridizations were performed according to the manufacturer's protocols (Agilent Technologies, Santa Clara CA). Images of the arrays were acquired using a G2505B microarray scanner (Agilent Technologies, Santa Clara CA). Data-analysis was done using the CGH-analytics software (Agilent Technologies, Santa Clara CA).

In patient 3, the Affymetrix Cytogenetics Whole-Genome 2.7M Array (Affymetrix, Santa Clara, CA) was used to screen for Copy Number Variations (CNVs). Genomewide SNP-array hybridization was performed following the manufacturer's instructions. Genome-wide copy number analysis was performed using the Affymetrix Chromosome Analysis Suite software (ChAS) and a pooled reference DNA sample data set.

Expression analysis

Expression studies were performed in patients 1 and 3 in order to investigate if the deletion had an effect on the expression level of EHMT1. Lymphoblastoid cell lines were established by Epstein-Barr virus transformation from peripheral blood lymphocytes of the patient and healthy controls. Lymphoblasts were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37°C with 5% CO 2 . Cell medium was changed every 48h.

Total RNA was extracted with the single-step acid phenol method using Trizol (Invitrogen). Two independent RNA extractions were performed from lymphoblastoid cell lines established from both patients. RNA was reverse-transcribed by MoMLVreverse transcriptase (Invitrogen). PCR products containing a coding SNP were

In silico analysis and expression studies of a novel EHMT1 transcript

A search for novel coding exons in the EHMT1 gene was performed at the UCSC

RESULTS

Genome wide array studies

The genome wide array studies in patients 1-3 are based on different array platforms.

Expression analysis

Expression analysis of EHMT1 performed in cases 1 and 3, showed mono-allelic expression of the EHMT1 gene. On the DNA level, heterozygosity for a coding SNP is shown (figures 2A and 2C). On the cDNA level only one of the alleles is expressed (figures 2B and 2D). This indicated haploinsufficiency for EHMT1, though this gene did not seem to be deleted on the basis of array results.

In silico analysis and RT-PCR

According to the UCSC Genome Browser database, February 2009 release To confirm that the novel exons are expressed we performed RT-PCR analysis with primers specific for the novel EHMT1 exons and obtained a PCR product corresponding in size and sequence to NM_024757.4 (data not shown). The EHMT1 protein therefore, might appear to be 31 amino acids larger comprising in total 1299 amino acids. This presumed new EHMT1 N-terminus does not contain any predicted domains, repeats, motifs or other features. Both the old (NM_024757.3 ) and new transcript (NM_024757.4) are indicated in figure 3.

MLPA

The MLPA probes located in exons 1 and 2 of C9orf37 and in intron 1 of EHMT1, clearly confirmed the presence of a heterozygous deletion in patients 1-3 whereas the distally located probe in novel exon 2 and the other distal exons of EHMT1

showed normal results (Figure 4).

To screen for additional deletions covering only this extended 5' part of the EHMT1 gene, we screened 75 patients who had been previously referred for diagnostic analysis of EHMT1 but with normal results based on sequencing and MLPA analysis of transcript NM_024757. The finding of 3 cases with Kleefstra syndrome harbouring interstitial 9q microdeletions encompassing only the novel first exon in the EHMT1 sequence (encoding 7 amino acids) in addition to several proximally located genes, prompted us to investigate this additional stretch in 75 cases with the clinical suspicion of Kleefstra syndrome but without any EHMT1 abnormalities found after molecular diagnostics of the previous transcript. Interestingly, in one patient (patient 4), we observed the presence of a small deletion of only the novel first EHMT1 exon and the first exon of the proximal flanking C9orf37 gene. Strikingly, in all 4 patients described here the distal breakpoint is located in intron 1 of the EHMT1 gene. This breakpoint is located between the MLPA probes in intron 1 and exon 2 (chr9: 139,633,920-139,725,201; hg18). This 91 kb region harbours several repetitive sequences, such as LINE and SINE. This locus could be considered as locus 4, in addition to the 3 loci described by Yatsenko et al (2009). We hypothesize that this region is the most common distal breakpoint locus involved in KS, but detailed analysis of the breakpoint sequences in all patients should be performed in order to prove this statement. Though the size of deletions differed in all 4 patients, no significant differences in the degree of intellectual disability or severity of medical problems was seen. This is in agreement with previous observations that haploinsufficiency of the EHMT1 gene is responsible for the core phenotype of KS [START_REF] Kleefstra | Loss-of-function mutations in euchromatin histone methyl transferase 1 (EHMT1) cause the 9q34 subtelomeric deletion syndrome[END_REF][START_REF] Kleefstra | Further clinical and molecular delineation of the 9q Subtelomeric Deletion Syndrome supports a major contribution of EHMT1 haploinsufficiency to the core phenotype[END_REF]Yatsenko et al., 2009). The transcript of C9orf37 with a so far unknown function, is located antisense to EHMT1 (head-to-head gene orientation). It is suspected that the EHMT1

and C9orf37 genes are activated by the same (bidirectional) promoter. It is not certain whether a deletion of the C9orf37 transcript has any molecular or clinical consequences. We and others published many cases with 9q deletions encompassing this gene and other proximal flanking genes and they do not have a phenotype significant different from those having deletions located more distally [START_REF] Kleefstra | Loss-of-function mutations in euchromatin histone methyl transferase 1 (EHMT1) cause the 9q34 subtelomeric deletion syndrome[END_REF][START_REF] Kleefstra | Further clinical and molecular delineation of the 9q Subtelomeric Deletion Syndrome supports a major contribution of EHMT1 haploinsufficiency to the core phenotype[END_REF]Yatsenko et al., 2009). However, a deletion of only C9orf37, has not been identified today but might have an effect on the expression of EHMT1. In addition, sequencing of the coding region of C9orf37 in 30 patients with suggestive clinical Kleefstra syndrome phenotype but negative for EHMT1 mutations did not reveal any mutations (unpublished observation).

The finding of novel or modified transcripts by in silico analyses having important clinical genetic consequences has been recognised before. A significant example is the identification of a previously unidentified MECP2 open reading frame encoding a new protein isoform relevant to Rett syndrome [START_REF] Kriaucionis | The major form of MeCP2 has a novel N-terminus generated by alternative splicing[END_REF][START_REF] Mnatzakanian | A previously unidentified MECP2 open reading frame defines a new protein isoform relevant to Rett syndrome[END_REF]. These and our studies emphasise the need to adjust routine diagnostics whenever such modifications of known genes arise. The update of transcripts in the databases however, requires the renaming of the identified , 2000). A significant departure from these recommendations is to designate the "A" of the ATG startcodon as the number 1 position. Since the new EHMT1 transcript contains 31 novel amino acids, the previously described mutations should be adjusted by adding 93 nucleotides to the cDNA position and 31 amino acids to the protein position of the mutation.

Consequently, the identification of deletions unique for the previously unknown 5' part of the EHMT1 transcript has implications for the genetic screening of patients suspected of having KS and for studies of the functional significance of EHMT1.

  climb stairs independently. She had sporadic stereotypic movements. She was longsighted (+7.5 dioptres).

  Auditory brainstem evoked responses (ABER), routine metabolic screening of plasma and urine, cardiac examination including cardiac echography, brain MRI and repeated chromosome analysis and FISH 7q11.21-23 (Williams syndrome) did not reveal any abnormalities. Patient 3 (Figure1E) was born at 40 weeks gestation with a birth weight of 2878 g (10 th centile). Polyhydramnios had been diagnosed at week 30. During the first

  interpretation, CNVs in the patient were compared to those previously identified in more than 840 control individuals and those reported in the Database of Genomic Variants (http://projects.tcag.ca).

  designed. The cDNA of Patient 1 was amplified by primers surrounding the SNP g.139,731,389G>A (UCSC Genome Browser database, March 2006 release (http://genome.ucsc.edu; hg18). The cDNA of patient 3 was amplified by primers surrounding the SNP rs1129768. Primer sequences and PCR conditions are available upon request.

(

  http://genome.ucsc.edu), and the consensus CDS project (CCDS) database at NCBI (http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi), the coding region of the EHMT1 gene has been updated to extend the N-terminus with an extra 5' exon. The previously defined EHMT1 gene transcript (NM_024757.3) contained 26 exons, the translation start site being located in exon 2. Diagnostic testing so far has been directed towards the 25 coding exons of the EHMT1 gene, however the novel open reading frame comprises 27 coding exons (NM_024757.4). The translation start site is located in a "novel" exon 1, 97.6 kb proximal to the 'old' ATG start codon and surrounded by a more than 75% CG rich region (Figure3). The previously determined non-coding first exon is now a coding exon (exon 2) in the new transcript.

  3. One case (patient 4) showed a deletion of C9orf37 exon 1 (chr9: 139,632,979) and the EHMT1 intron 1 probe (chr9:139,633,958) in MRC Holland kit P340-A1. The proximally located probe C9orf37 exon 2 (chr9; 139,632,295) and the distally located probe in EHMT1 exon 2 (chr9:139,725,213) showed a normal dosage. the deletion mapping, including array and MLPA probes, of patients 1-4 are schematically shown in figure 5. All aberrations comprised de novo interstitial deletions ranging from 1.0 kb to 386 kb. For probe alignments: build NCBI 36, UCSC hg18, Mar 2006 build was used. The deletion in patient 1 was spanning at least 226 kb, based on the first deleted array probe (A_14_P122901) and last deleted MLPA probe in EHMT1 intron 1. The maximal size of the deletion is 275 kb, based on the first and last preserved array probes (A_14_P125110 and A_14_P105992, respectively).In patient 2, the smallest deleted region is about 275 kb in size, and the maximal size of the deletion is 307 kb, based on the array results (deleted A_14_P125110-A_14_P105992; preserved A_1618802737-A_16_P02195868). The results of the MLPA did not add to the deletion size.The deletion in patient 3 ranges minimally from the MLPA probe in NELF exon 12 to array probe S-2JMMP, and maximally from array probes C-1CK64 to S-2IGFE. The deletion is therefore between 185 kb and 386 kb in size.Patient 4 has one of the smallest interstitial deletions described in Kleefstra syndrome so far: between 1.0 and 93 kb in size (based on the deleted MLPA probes C9orf37 exon 1 and EHMT1 intron 1 and the preserved probes C9orf37 exon 2 and EHMT1 exon 2.1). Since the results in this patient proves that a deletion of only the novel EHMT1 exon 1 can be causative of Kleefstra syndrome, we wanted to investigate whether point mutation in the novel part of the transcript are present in Kleefstra syndrome. We therefore PCR amplified and sequenced exons 1 and 2 in the remaining 74 individuals with Kleefstra syndrome, but this revealed no mutations. the identification and important clinical relevance of an elongated EHMT1 transcript. This novel transcript might contribute 31 additional amino acids to the 5' coding region of the previous transcript which would result in a novel EHMT1 protein of 1299 amino acids. So far, it is not known what biological properties this additional part possesses. No recognisable protein domain sequences could be traced within this transcript. However, there is large homology with transcripts in other species, as mouse and rat (Protein blast search at http://blast.ncbi.nlm.nih.gov).

  based on the recommendations for the description of sequence variants (Human Genome Variation Society (HGVS), Den Dunnen and Antonarakis

Figure 1 :

 1 Figure 1: photographs of patient 1 (A,B) at the age of 3 years, patient 2 (C,D) at the age of 12 years, patient 3 (E) at the age of 3 years, and patient 4 (F at the age of 3 years; G at the age of 3 months). A variety of facial dysmorphisms characteristic for Kleefstra syndrome are seen comprising midface hypoplasia, synophrys, upslanting palpebral fissures, eversion of lower lip, protruding tongue and downturned corners of the mouth.

Figure 2 :

 2 Figure 2: RT-PCR analysis of EHMT1 performed in lymphoblastoid cell lines of patients 1 and 3, showing mono-allelic expression of the EHMT1 transcript in RNA. Sequencing results in patient 1 shows heterozygosity for variant g.139,731,389G>A in DNA (A) , whereas the RNA only shows the A allele (B). Sequencing results in patient 3 show heterozygosity for rs1129768 in DNA (C) and only one allele in RNA (D).

Figure 3 :

 3 Figure 3: Part of the 9q subtelomeric region indicating schematically the different EHMT1 transcripts; The translation start site of the novel EHMT1 trancript (NM_024757.4) is located 97.6 kb proximal to the 'old' ATG start codon of transcript (NM_024757.3).

Figure 4 :

 4 Figure 4: MLPA analysis with MRC-Holland kit P340-A1 in patients 1-4 (filled symbols) and in 3 control individuals (open symbols). The deletion in patients 1-3 comprises C9orf37 and the first exon of EHMT1. The proximal part of the deletion (detected by array) is not tested in this MLPA analysis. Patient 4 shows the smallest

Figure 5 :

 5 Figure 5: The 9q subtelomeric region, with a schematical overview of the deletions identified in patients 1-4 (figure not to scale). For all patients, the first preserved and deleted probes are indicated (A). Agilent probes are shown in bold, and MLPA probes in italics. The remainder of the probes are from the Affymetrix platform. The position of the 5' and 3' ends of the genes, as well as the location of the probes (the latter in bold) on build NCBI 36, UCSC hg18, Mar 2006 is depicted in figure B. The 5' parts of the reference genes in the region are also depicted in build NCBI 37, UCSC hg19, Feb 2009 (C). The orientation and location of the genes (D), and the minimal size (filled grey) and maximum size (shaded grey) and location of the deletion in patients 1-4 (E) is schematically drawn (not to scale).

Figure 2 :Figure 3 :Figure 4 :

 234 photographs of patient 1 (A,B) at the age of 3 years, patient 2 (C,D) at the age of 12 years, patient 3 (E) at the age of 3 years, and patient 4 (F at the age of 3 years; G at the age of 3 months). A variety of facial dysmorphisms characteristic for Kleefstra syndrome are seen comprising midface hypoplasia, synophrys, upslanting palpebral fissures, eversion of lower lip, protruding tongue and downturned corners of the mouth. 184x127mm (400 x 400 DPI)

  //www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi). Since the high C/G content in exon 1 makes it impossible to design an MLPA probe, a probe in intron 1 is included. In this MLPA kit also two probes in the proximally located gene C9orf37 (exons 1 and 2) and one probe in the distally located gene CACNA1B (exon 3) are present. Tests were carried out according to the manufacturer's protocols (Version 2007, MRC-Holland, The Netherlands).

	and	the	consensus	CDS	project	(CCDS)	database	at	NCBI
	(http:MLPA and Sequencing
	Multiplex ligation dependent probe amplification was performed by applying MLPA kit
	P340-A1, which was developed by MRC-Holland (www.mrc-holland.com) comprising
	31 probes in EHMT1, covering almost the entire coding region, based on transcript
	F o r NM_024757.4. Analysis was done using the MLPA module of Genemapper software, version 4.0 F o r
	(Applied Biosystems).		P P
	e Direct sequencing of EHMT1 exons 1 and 2, based on transcipt NM_024757.4, was e
	performed by standard procedures.	e r e r
						R R
						e e
							v i e v i e
							w w
	Genome Browser database, February 2009 release (http://genome.ucsc.edu; hg19),
									11 12

  The probe locations mentioned below are based on the UCSC Genome Browser database, build NCBI 36, UCSC hg18, Mar 2006 (http://genome.ucsc.edu).

	Page 13 of 29	Human Mutation
	NELF exon 12 (chr9: 139,464,509). The distal deletion breakpoint is located between
	the probes S-2JMMP (chr9:139,649,316) and S-2IGFE (chr9: 139,659,097). The
	deletion in patient 3 contains (at least) the genes NELF, PNPLA7, MRPL41, WDR85,
	ZMYN19, ARRDC1 and C9orf37.
	In patient 1, the first deleted probe (A_14_P122901) is located on chromosome 9
	position 139,407,449, the last deleted probe (A_14_P100411) on position
	139,633,014. The first preserved probes (A_14_P125110 (chr9:139,405,999) proximally, A_14_P105992 (chr9:139,680,613 distally)). The deletion included the following genes: partially EXD3, NOXA1, ENTPD8, NELF, PNPLA7, MRPL41, F o r
	WDR85, ZMYND19, ARRDC1, and C9orf37. P
	e In patient 2, the first deleted probe is A_14_P125110, the last one A_14_P105992 (chr9:139,405,999-139,680,613). The first preserved probes are A_16_P18802737 and A_16_P02195868 (chr9: 139,390,002-139,697,348), indicating that the deletion e r
	contains 11 known genes (partially EXD3, NOXA1, ENTPD8, NELF, PNPLA7, R
	MRPL41, WDR85, ZMYND19, ARRDC1 and C9orf37).	e
	In patient 3, probe C-1CKEK (chr9: 139,609,502) was found to be deleted by SNP v i e
	array analysis, while the proximal probe C-1CK64 showed a normal ratio (139,273,509). Fine mapping of the proximal deletion breakpoint by MLPA analysis w
	with MRC Holland Kit P286 (www.mrcholland.com) showed that the proximal deletion
	breakpoint is located between the proximal probe C-1CK64 and the deleted probe in
			13
		John Wiley & Sons, Inc.

  Since the deletions identified in this paper are not detected by array CGH, MLPA analysis is highly recommended, and we would suggest this is done in those cases previously not found to have any EHMT1 abnormalities. MLPA analysis covering the

	Page 23 of 29	Human Mutation Human Mutation	Page 20 of 29
	This work was supported by grants from GENCODYS, a EU FP7 large-scale
	integrating project grant (Grant agreement no. 241995) (to T.K). Yatsenko SA, Brundage EK, Roney EK, Cheung SW, Chinault AC, Lupski JR. 2009.
			Field Code Changed
	Molecular mechanisms for subtelomeric rearrangements associated with the 9q34.3
	microdeletion syndrome. Hum Mol Genet 18:1924-36.
	F o r F o r F o r F o r F o r
	P P P P P
	C9orf37 gene and the novel 5' part of EHMT1 is now commercially available through e e e e e
	MRC-Holland (kit P340, www. MRC-Holland.com). Routine DNA diagnostic testing for new patients with clinical suspicion of Kleefstra syndrome should involve e r e r e r e r e r
	sequencing and, more importantly, MLPA analysis of the entire coding region of the R R R R R
	EHMT1 transcript NM_024757.4.	e e e e e
		v i e v i e v i e v i e v i e
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	John Wiley & Sons, Inc. John Wiley & Sons, Inc.

John Wiley & Sons, Inc.Human Mutation

ACKNOWLEDGEMENTS

The authors wish to thank the participating patients and parents. They also thank Martina Ruiterkamp-Versteeg and Gaby van de Ven-Schobers for expert technical assistance, and Nicole de Leeuw for performing additional array analysis in patient 3.


Dr John D Harrison
email: john.harrison@kcl.ac.uk

Manuel Rodriguez-Justo

Commentary on IgG4-related sialadenitis

Keywords: chronic sclerosing sialadenitis, IgG4-related sialadenitis, Küttner's tumour, Mikulicz's disease, salivary gland disease chronic sclerosing sialadenitis, IgG4-related sialadenitis, Küttner's tumour, Mikulicz's disease, salivary gland disease

) is the first description of IgG4related sialadenitis from Central Europe, and it seems likely that the disease will be found worldwide. Although the use of the terms Küttner's tumour and Mikulicz's disease as eponyms for IgG4-related sialadenitis is widespread, it is confusing and better avoided. It appears prudent to immunostain in order to estimate the number and fraction of plasma cells with IgG4 in cases of sialadenitis that show severe fibrosis and atrophy and a heavy lymphoid infiltrate in order that cases of IgG4-related sialadenitis may be confidently diagnosed. Laco et al. found that a lower limit of 50 IgG4-positive plasma cells per 1 high-power field produces a sensitivity and specificity of 100%, and that the ratio of IgG4-positive plasma cells to IgG-positive plasma cells is at least 0.40 in cases of IgG4-related sialadenitis and no more than 0.26 in control cases.

IgG4-related sialadenitis has only been established as an entity this century, and the reports include 30 patients in Japan, [START_REF] Kitagawa | Abundant IgG4-positive plasma cell infiltration characterizes chronic sclerosing sialadenitis (Küttner's tumor)[END_REF][START_REF] Yamamoto | Clinical and pathological differences between Mikulicz's disease and Sjögren's syndrome[END_REF][START_REF] Kamisawa | IgG4-related sclerosing disease incorporating sclerosing pancreatitis, cholangitis, sialadenitis and retroperitoneal fibrosis with lymphadenopathy[END_REF][START_REF] Aoki | A case of Mikulicz's disease complicated with severe interstitial nephritis associated with IgG4[END_REF][START_REF] Abe | Immunoglobulin G4-related sclerosing sialadenitis: report of two cases and review of the literature[END_REF][START_REF] Sakuma | Analysis of VH gene rearrangement and somatic hypermutation in Sjogren's syndrome and IgG4-related sclerosing sialadenitis[END_REF] 2 Chinese patients in New Zealand, [START_REF] Gill | A case of hyperIgG4 disease or IgG4-related sclerosing disease presenting as retroperitoneal fibrosis, chronic sclerosing sialadenitis and mediastinal lymphadenopathy[END_REF][START_REF] Gill | Salivary duct carcinoma arising in IgG4related autoimmune disease of the parotid gland[END_REF] 13 patients in the United States of America, [START_REF] Geyer | Chronic sclerosing sialadenitis (Küttner tumor) is an IgG4-associated disease[END_REF] and 1 South Asian patient in England, [START_REF] Deheragoda | The use of immunoglobulin G4 immunostaining in diagnosing pancreatic and extrapancreatic involvement in autoimmune pancreatitis[END_REF] The article by Laco et al. 11 is the first description of IgG4-related sialadenitis from Central Europe. It adds 6 cases to make a total of 52 cases, and it seems likely that the disease will be found worldwide.

The term Küttner's tumour has been used as an eponym for IgG4-related sialadenitis that involves one or more salivary glands and Mikulicz's disease as an eponym when the lacrimal glands are also involved, [START_REF] Kitagawa | Abundant IgG4-positive plasma cell infiltration characterizes chronic sclerosing sialadenitis (Küttner's tumor)[END_REF][START_REF] Yamamoto | Clinical and pathological differences between Mikulicz's disease and Sjögren's syndrome[END_REF][START_REF] Aoki | A case of Mikulicz's disease complicated with severe interstitial nephritis associated with IgG4[END_REF][START_REF] Abe | Immunoglobulin G4-related sclerosing sialadenitis: report of two cases and review of the literature[END_REF][START_REF] Sakuma | Analysis of VH gene rearrangement and somatic hypermutation in Sjogren's syndrome and IgG4-related sclerosing sialadenitis[END_REF][START_REF] Geyer | Chronic sclerosing sialadenitis (Küttner tumor) is an IgG4-associated disease[END_REF] However, this has introduced confusion and is unjustified, as is clearly shown by an appraisal of the work of Küttner and Mikulicz.

Küttner [START_REF] Küttner | Ueber entzündliche Tumoren der Submaxillar-Speicheldrüse[END_REF] in 1896 described 2 cases of chronic submandibular sialadenitis that were unilateral and presented as firm swellings, and in one of the cases there was an associated sialolith, which he considered to be secondary to the inflammation. He did not publish those with minimal change to those with extensive fibrosis, inflammation and atrophy. Seifert and Donath 13 coined the term 'chronic sclerosing sialadenitis', which had not been used by Küttner, but which from his histological description would be applicable to his cases. The terms 'Küttner tumour' and 'chronic sclerosing sialadenitis' were only introduced to the English-speaking medical profession in 1991 in the second edition of the WHO 'Histological typing of salivary gland tumours', [START_REF] Seifert | Chronic sclerosing sialadenitis of submandibular gland (Küttner tumour)[END_REF] of which the principal editor was Seifert. This was followed by a spate of case reports of a purportedly rare disease, namely 'Küttner's tumour', without the realization that it was no more than mundane chronic submandibular sialadenitis. [START_REF] Harrison | Causes, natural history, and incidence of salivary stones and obstructions[END_REF] The extent of the current classificatory confusion is evident in the article by Laco et al. [START_REF] Laco | Chronic sclerosing sialadenitis (Küttner tumor) as a member of IgG4-related diseases: a clinicopathological study of 6 cases from Central Europe[END_REF] when they quote from a major textbook on tumours of salivary glands [START_REF] Ellis | Tumors of the salivary glands[END_REF] that describes chronic sclerosing sialadenitis or Küttner's tumour as the most common disease of the submandibular gland, yet the disease to which Laco et al. refer is IgG4-related sialadenitis.

Although the incidence of IgG4-related sialadenitis among cases of chronic sialadenitis is unknown because no large series have been examined, it appears likely to be low if comparable to IgG4-related pancreatitis. [START_REF] Deheragoda | The use of immunoglobulin G4 immunostaining in diagnosing pancreatic and extrapancreatic involvement in autoimmune pancreatitis[END_REF] The textbook refers to all cases of chronic submandibular sialadenitis, many of which are associated with sialolithiasis, [START_REF] Ellis | Tumors of the salivary glands[END_REF] and does not mention IgG4-related sialadenitis. However, the presence of sialolithiasis does not exclude the possibility of IgG4-related sialadenitis, as a lith was present in one of the cases of Laco et al. [START_REF] Laco | Chronic sclerosing sialadenitis (Küttner tumor) as a member of IgG4-related diseases: a clinicopathological study of 6 cases from Central Europe[END_REF] Mikulicz [START_REF] Mikulicz | Ueber eine eigenartige symmetrische Erkrankung der Thränen-und Mundspeicheldrüsen[END_REF] as a uniform infiltrate of small round cells was recently examined and found to show the features of a MALT lymphoma. [START_REF] Ihrler | Mikulicz's disease and Mikulicz's syndrome: analysis of the original case report of 1892 in the light of current knowledge identifies a MALT lymphoma[END_REF] Deheragoda et al. [START_REF] Deheragoda | The use of immunoglobulin G4 immunostaining in diagnosing pancreatic and extrapancreatic involvement in autoimmune pancreatitis[END_REF] found that IgG4 immunostaining added to the diagnostic accuracy that was possible by morphological examination alone, and this is confirmed by the following example of two cases of chronic submandibular sialadenitis.

Case A was a female of 79 years with a painless submandibular swelling for 2 months, and case B was a male of 69 years with a painless submandibular swelling for 1 month. Both patients were white British. There was no associated sialolithiasis. Both glands were very fibrotic, for which reason immunohistochemistry for CD138, IgG and IgG4 was applied. The appearance in sections routinely stained with haematoxylin and eosin was similar. There was severe atrophy, severe fibrosis with cellular interlobular fibrous tissue, and a heavy lymphoid infiltrate with large, geographical germinal centres in most of each of the glands (Fig. 1A1 and 1B1). Phlebitis was not found in either gland. Immunostaining revealed that there were many plasma cells, which were stained for CD138 and IgG, in both glands (Fig. 1A2 and1B2). Only a very small minority of these was stained for IgG4 in the gland from case A (Fig. 1A3), whereas most were stained for IgG4 in the gland from case B (Fig. 1B3).

Thus only one case is of IgG4-related sialadenitis, although both are of the appearance described by Küttner, [START_REF] Küttner | Ueber entzündliche Tumoren der Submaxillar-Speicheldrüse[END_REF] and both would have attracted a diagnosis of Küttner's tumour and chronic sclerosing sialadenitis by application of the definition in the second edition of the WHO 'Histological typing of salivary gland tumours'. [START_REF] Seifert | Chronic sclerosing sialadenitis of submandibular gland (Küttner tumour)[END_REF] As IgG4-related sialadenitis can be part of a hyper-IgG4 multiorgan disease and can be successfully treated with steroids, it is important to diagnose it, which necessitates immunostaining for CD138, IgG and IgG4.

There is clearly no justification for using the eponyms Mikulicz's disease and Küttner's tumour for cases of IgG4-related sialadenitis, and since such use is misleading and likely to lead to confusion and mismanagement, it is to be deprecated. [START_REF] Mikulicz | Ueber eine eigenartige symmetrische Erkrankung der Thränen-und Mundspeicheldrüsen[END_REF] and Küttner. [START_REF] Küttner | Ueber entzündliche Tumoren der Submaxillar-Speicheldrüse[END_REF] Several articles that refer to Küttner's article of 1896 have similarly and incorrectly stated the page numbers, [START_REF] Kitagawa | Abundant IgG4-positive plasma cell infiltration characterizes chronic sclerosing sialadenitis (Küttner's tumor)[END_REF][START_REF] Abe | Immunoglobulin G4-related sclerosing sialadenitis: report of two cases and review of the literature[END_REF][START_REF] Geyer | Chronic sclerosing sialadenitis (Küttner tumor) is an IgG4-associated disease[END_REF] and one of them incorrectly stated that Küttner 'reported a "hard swelling" of 1 or both submandibular glands', [START_REF] Geyer | Chronic sclerosing sialadenitis (Küttner tumor) is an IgG4-associated disease[END_REF] whereas Küttner [START_REF] Küttner | Ueber entzündliche Tumoren der Submaxillar-Speicheldrüse[END_REF] had reported two cases of unilateral submandibular swelling. These errors indicate that these authors had not read Küttner's article but had correctly copied the same incorrect reference from other articles. 'Dwell on the past and you'll lose an eye. Forget the past and you'll lose both eyes.' [START_REF] Solzhenitsyn | The Gulag Archipelago, 1918-1956[END_REF] In conclusion, it appears prudent to immunostain in order to estimate the number and fraction of plasma cells with IgG4 in cases of sialadenitis that show severe fibrosis and atrophy and a heavy lymphoid infiltrate in order that cases of IgG4-related sialadenitis may be confidently diagnosed. Laco et al. 11 found that a lower limit of 50 IgG4-positive plasma cells per 1 high-power field produces a sensitivity and specificity of 100%, and that the ratio of IgG4-positive plasma cells to IgG-positive plasma cells is at least 0.40 in cases of IgG4related sialadenitis and no more than 0.26 in control cases. and B2 are medium-power photomicrographs of sections stained for IgG, which is present in similarly dense infiltrates of plasma cells. A3 and B3 are medium-power photomicrographs of sections adjacent to the above and stained for IgG4, which is present in a very small minority of the plasma cells in A3, but in B3 is present in a similar density of plasma cells to the IgG of B2. 169x215mm (400 x 400 DPI)

2 Abstract:

 2 The paper by Laco et al. (HISTOP-04-10-0251.R2

  published a detailed coloured illustration of the histological features of one of the swollen submandibular glands of his patient, who suffered from bilateral swollen major and minor salivary and lacrimal glands. The original illustration of what Mikulicz described

  eponymy could have been avoided by reading the original articles of Mikulicz

Figure 1 .

 1 FIGURE LEGEND

Figure 1 .

 1 Figure 1. A1, A2 and A3 are from the submandibular gland of case A. B1, B2 and B3 are from the submandibular gland of case B. A1 and B1 are scans of sections stained with haematoxylin and eosin in which much of each is very fibrotic, very atrophic, and with a heavy lymphoid infiltrate with large germinal centres, some of which are geographical. The left part of each of the sections is not fibrosed or atrophic, but contains focal lymphoid infiltrates with conspicuous germinal centres. A2and B2 are medium-power photomicrographs of sections stained for IgG, which is present in similarly dense infiltrates of plasma cells. A3 and B3 are medium-power photomicrographs of sections adjacent to the above and stained for IgG4, which is present in a very small minority of the plasma cells in A3, but in B3 is present in a similar density of plasma cells to the IgG of B2. 169x215mm (400 x 400 DPI)

Published on behalf of the British Division of the International Academy of Pathology Histopathology


SIPBS Benjamin Simon Pickard
email: benjamin.pickard@strath.ac.uk

Sha Li

Lynsey Macintyre

Michele P Kelly

David J Porteous

Nick Brandon

Walter John Muir

Douglas H Blackwood

David G Watson

Steven J Clapcote

Judith A Machell

Nicholas J Brandon

Andrew Hamnett Wing

Transcriptional regulation of neurodevelopmental and metabolic pathways by NPAS3

Keywords: schizophrenia, bipolar disorder, transcriptomics, metabolomics, glycolysis, SOX transcription factor

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Schizophrenia and bipolar disorder are common, lifelong psychiatric illnesses affecting mood, perception and cognition. A strong genetic contribution to these conditions is indicated by family and epidemiological studies.

A role for the NPAS3 gene in psychiatric illness risk was suggested through the study of a mother and daughter diagnosed with schizophrenia and mild learning disability who carried a chromosomal abnormality disrupting the gene [START_REF] Kamnasaran | Disruption of the neuronal PAS3 gene in a family affected with schizophrenia[END_REF][START_REF] Pickard | Disruption of a brain transcription factor, NPAS3, is associated with schizophrenia and learning disability[END_REF]. Subsequent gene-specific and genome-wide case-control association studies have linked single nucleotide polymorphisms at the NPAS3 locus with increased risk of schizophrenia [START_REF] Pickard | Interacting haplotypes at the NPAS3 locus alter risk of schizophrenia and bipolar disorder[END_REF][START_REF] Huang | Cross-Disorder Genomewide Analysis of Schizophrenia, Bipolar Disorder, and Depression[END_REF], bipolar disorder [START_REF] Pickard | Interacting haplotypes at the NPAS3 locus alter risk of schizophrenia and bipolar disorder[END_REF][START_REF] Huang | Cross-Disorder Genomewide Analysis of Schizophrenia, Bipolar Disorder, and Depression[END_REF][START_REF] Ferreira | Collaborative genome-wide association analysis supports a role for ANK3 and CACNA1C in bipolar disorder[END_REF] and major depression [START_REF] Huang | Cross-Disorder Genomewide Analysis of Schizophrenia, Bipolar Disorder, and Depression[END_REF]. Three common NPAS3 exonic variants have also been recently associated with increased risk of schizophrenia [START_REF] Macintyre | Association of NPAS3 exonic variation with schizophrenia[END_REF]. Genetic variation at the NPAS3 locus has also been linked with response to treatment with the antipsychotic drug, iloperidone [START_REF] Lavedan | Association of the NPAS3 gene and five other loci with response to the antipsychotic iloperidone identified in a whole genome association study[END_REF].

NPAS3 encodes a member of the bHLH-PAS (basic helix-loop-helix -Per, Arnt, Sim) domain transcription factor family that form functional heterodimers [START_REF] Brunskill | Characterization of npas3, a novel basic helix-loop-helix PAS gene expressed in the developing mouse nervous system[END_REF][START_REF] Gilles-Gonzalez | Signal transduction by hemecontaining PAS-domain proteins[END_REF]. Npas3 knockout mice display neuroanatomical, memory and behavioral phenotypes typical of a model of human psychiatric disorders [START_REF] Brunskill | Abnormal neurodevelopment, neurosignaling and behaviour in Npas3-deficient mice[END_REF][START_REF] Erbel-Sieler | Behavioral and regulatory abnormalities in mice deficient in the NPAS1 and NPAS3 transcription factors[END_REF]. They also exhibit a deficit in adult hippocampal neurogenesis [START_REF] Pieper | The neuronal PAS domain protein 3 transcription factor controls FGFmediated adult hippocampal neurogenesis in mice[END_REF] -a potential cellular pathology of schizophrenia and depression [START_REF] Kempermann | The contribution of failing adult hippocampal neurogenesis to psychiatric disorders[END_REF][START_REF] Reif | Neurogenesis and schizophrenia: dividing neurons in a divided mind?[END_REF]. Importantly, a novel compound acting on mitochondria, P7C3, reverses the neurogenesis, neuroanatomical, electrophysiological and behavioural phenotypes of the Npas3 knockout [START_REF] Pieper | Discovery of a proneurogenic, neuroprotective chemical[END_REF].

Here we report results from parallel experimental approaches designed to identify the function of NPAS3 in health and mental illness. The hippocampal neurogenesis deficit associated with Npas3 deletion suggested NPAS3 might directly regulate the developmental pathway associated with new neuron production and maturation and As a transcription factor, NPAS3 is particularly amenable to a transcriptomics approach because observed expression changes are likely to represent direct activity rather than secondary or homeostatic reactions. We employ an in vitro system; over-expression of full-length (FLNPAS3) and artificially truncated (ΔNPAS3) forms of NPAS3 in the human embryonic kidney cell line, HEK293, followed by microarray analysis not to model psychiatric illness but rather to efficiently identify target genes. We compare NPAS3 targets with those of a known family of neurodevelopmental regulators, the SOX family of transcription factors [START_REF] Azim | SOX6 controls dorsal progenitor identity and interneuron diversity during neocortical development[END_REF][START_REF] Haslinger | Expression of Sox11 in adult neurogenic niches suggests a stage-specific role in adult neurogenesis[END_REF][START_REF] Lefebvre | The SoxD transcription factors -Sox5, Sox6, and Sox13are key cell fate modulators[END_REF]. We also describe an immunofluorescence study to determine NPAS3's spatio-temporal contribution to hippocampal neurogenesis. The observation that the related NPAS2 protein acts as a functional equivalent of the CLOCK circadian regulator in certain brain regions [START_REF] Reick | NPAS2: an analog of clock operative in the mammalian forebrain[END_REF] prompted us to examine NPAS3 targets in cells stimulated to commence synchronous circadian cycling. Finally, we assess the biological relevance of the in vitro microarray data to the in vivo pathologies in the Npas3 knockout mouse. This was achieved by high-resolution mass spectrometry-based metabonomic comparison of wild-type and mutant brain tissue.

Our findings indicate that NPAS3 contributes to both neurodevelopmental transcription factor networks and the regulation of brain glucose metabolism.

Materials and methods

Immunofluorescence

Immunofluorescence of frozen brain sections was carried out as previously described [START_REF] Knight | A cytogenetic abnormality and rare coding variants identify ABCA13 as a candidate gene in schizophrenia, bipolar disorder, and depression[END_REF]. Antibodies against Npas3, Gfap, Dcx and Nestin were all obtained from Santa Cruz Biotechnology. 1:400 donkey secondary antibodies against goat or rabbit IgG, conjugated to Alexa Fluor 594 were applied for red fluorescence, or to Alexa Fluor 488 or FITC for green fluorescence (Invitrogen Life Technologies, Paisley, UK). Sections were mounted with Prolong antifade reagent with DAPI nuclear stain (Invitrogen). For circadian induction, cells were maintained in DMEM alone for 36 hours. At the zero hour time-point, the cell medium was replaced with DMEM supplemented with 50% horse serum plus tetracycline in order to induce circadian cycling/NPAS3 over-expression [START_REF] Balsalobre | A serum shock induces circadian gene expression in mammalian tissue culture cells[END_REF][START_REF] Huang | Induction of circadian rhythm in cultured human mesenchymal stem cells by serum shock and cAMP analogs in vitro[END_REF]. At +2 hours, cells were washed with DMEM and then incubated with the same plus tetracycline for the remaining period of the experiment. At either +12 or +24 hours, cells were removed, washed and frozen.

NPAS3 and SOX expression constructs and standard/circadian cell culture

Illumina microarray analysis and data normalization

For baseline and circadian NPAS3 over-expression experiments parental HEK293 [T-REx-293] cells were used as negative controls. Samples (n=2) were assessed for FLNPAS3 over-expression, ΔNPAS3 over-expression, and parental negative controls. In the SOX microarray studies, DNA-free Lipofectamine2000 transfections (n=4) were used as shared negative controls compared to transfections with each of the SOX expression constructs (n=3).

RNA extraction (RNeasy kit, Qiagen) and synthesised microarray probes (Illumina® TotalPrep RNA Amplification Kit, Ambion) were quantified and quality checked using an Agilent Bioanalyzer. An Illumina Beadstation platform was used in conjunction with Sentrix® HumanRef-8 v2 chips [START_REF] Reisz-Porszasz | Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT)[END_REF]500 gene transcripts) to detect gene expression profiles. This work was carried out in the Wellcome Trust Clinical Research Facility at the Western General Hospital, Edinburgh. Microarray data analysis was carried out using BRB-ArrayTools 3.8.0 freeware. Regulated genes were categorised using Ingenuity Pathway Analysis and GeneCodis2 for over-representation of particular gene ontologies or biological processes. ProSim Ternary Diagram freeware (http://www.prosim.net/en/resources/download.html) was used to visualise normalized/de-transformed microarray expression data.

Confirmation by QPCR

Oligo-dT-primed cDNA synthesis of microarray sample RNA was performed (Roche) followed by triplicate QPCR with SYBR green QPCR Master Mix (Invitrogen) and a Real-Time QPCR machine (BIO-RAD). For relative quantification of mRNA expression, geometric means were calculated using the comparative double delta method. Primers used in QPCR are included in Supplementary Information Table 9. The housekeeping gene (18sRNA) was selected as the endogenous references in this QPCR studies.

Dual luciferase assay

5' human VGF sequence (2029kb), containing promoter, exon1, intron1 and part of exon2, was amplified by PCR and cloned into digested pGL3 reporter vectors. HEK293 and SHSY-5Y cell samples were collected at 24 hours after transfection. The activities of Firefly luciferase (expressed from all pGL-3 reporter vectors) and Renilla luciferase (expressed from co-transfected pRL-TK vector) were measured sequentially from each sample by using the Dualluciferase reporter (DLR TM) Assay System with the Plate-reader (Synergy HT, Bio-Tech). For each sample, the Firefly luciferase activity was normalised by the Renilla luciferase activity and then compared with the mean value of control samples. Dual luciferase assay results were statistically analysed using SigmaPlot© (Bruxton Corp., Seattle, WA). One-way ANOVA tests test was used to compare the VGF promoter activity of different cells among treatment groups.

Metabonomics

Extraction of polar metabolites from brain tissue homogenates for LC-MS analysis The deleted mouse Npas3 locus (on a 129S6/SvEvTac background) was backcrossed with mice from the C57BL/6J strain such that, at the stage of metabonomic analysis, the mice were on average 62.5% C57BL/6J. Sagitally dissected half-brains from homozygote Npas3 knockout (n=4) and wild-type (n=4) littermates were snap frozen in isopentane and stored at -80ºC. A twostep metabolite extraction method using methanol, water and chloroform as extraction solvents produced a biphasic solution comprising polar and nonpolar fractions [START_REF] Wu | High-throughput tissue extraction protocol for NMR-and MS-based metabolomics[END_REF]. Only the polar fractions were analysed in this study. 200µl of collected polar extract was added to 600µl of 1:1 acetonitrile:water.

Samples were diluted to combat salt interference and to prevent ion suppression. Samples were filtered using Acrodisc 13 mm syringe filters with 0.2 µm nylon membrane (Sigma Aldrich) before LC-MS analysis.

LC-MS Analysis of Polar Metabolites

Analysis was carried out using a Finnigan LTQ-Orbitrap fitted with a Surveyor HPLC pump (Thermo Fisher, Hemel Hempstead, UK) using 30 000 resolution.

The software programme XCalibur (version 2.0) was used to acquire the LC-MS data. Analyses were carried out in positive and negative modes over mass range of 60-1 000 m/z. The capillary temperature was set at 250 °C and in positive ionisation mode the ion spray voltage was 4.5 kV, the capillary voltage 30 V and the tube lens voltage 105 V. In negative ionisation mode the ion spray voltage was -4.5 kV, the capillary voltage -25 V, the tube lens voltage -95.0 V. The sheath and auxiliary gas flow rates were 45 and 15, respectively (manufacturer units). A ZIC-HILIC column (5 µm, 150 x 4.6 mm; HiChrom, Reading, UK) was used with a binary gradient. Solvent A was 0.1% v/v formic acid in HPLC water and B was 0.1% formic acid in acetonitrile. A flow rate of 0.3 ml/min was used and injection volume was 10 µl. The gradient programme used was 80% B at 0 min to 50% B at 12 min to 20% B at 28 min to 80% B at 37 min with total run time of 45 minutes. The instrument was externally calibrated before analysis and internally calibrated using lock masses at m/z 83.06037 and m/z 195.08625. Samples were analysed sequentially and the vial tray temperature was set at a constant temperature of 4 °C. Raw XCalibur data files from wild-type and Npas3 knockout samples were processed using Sieve™ version 1.2 (Thermo Fisher, Hemel Hempstead, UK).

Results

Npas3 expression in the dentate gyrus supports its role in neurogenesis

We observed the strongest NPAS3 immunofluorescence originating in the subgranular zone of the dentate gyrus with immuno-positive processes radiating into the granule cell layer proper (Fig. 1j). Additional expression included nuclear distribution in ependymal cells and axo-dendritic staining adjacent to the cell soma in many cortical neurons. To define the stage of Npas3 action in newly forming neurons we co-stained with antibodies against Nestin, Gfap and Dcx (Fig. 1a-i). Only Dcx showed an overlapping expression pattern indicating that Npas3 acts during neuronal maturation/differentiation.

Identifying NPAS3 target genes in standard culture conditions

Unsupervised hierarchical clustering of gene expression profiles using centred correlation and average linkage showed reproducible transcriptional changes after FLNPAS3 or ΔNPAS3 over-expression (Supplementary Information Fig. 1a). Using the SAM algorithm (Significance Analysis of Microarrays) with a 0.01 target proportion of false discoveries and 100 permutations, 3 476 genes were found to discriminate between FLNPAS3 and control. 282 genes showed >=1.5-fold up-regulation by FLNPAS3, and 359 genes were similarly downregulated (Table 1a,b and Fig. 2a). NPAS3, as a consequence of experimental over-expression, was the most up-regulated gene (62-fold) but was only detected in FLNPAS3 arrays as the chip's oligonucleotide probe is complementary to 3' sequence absent in ΔNPAS3.

There was substantial correlation of target gene fold-change between FLNPAS3 and ΔNPAS3 cells in standard culture conditions, with linear regression r 2 values of 0.59 (up-regulated genes) and 0.78 (down-regulated genes) (SI Fig. 1b,c). A similar C-terminal deletion of a related bHLH transcription factor, ARNT (HIF1 beta), also largely preserved regulatory function [START_REF] Reisz-Porszasz | Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT)[END_REF]. QPCR directed against four selected regulated genes, VGF, HK2, ENO2 and SOX11 confirmed the microarray findings (SI Fig. 2a).

NPAS3 and SOX transcription factors share target genes, including VGF

The up-regulated set of genes (Table 1a) was analysed for overrepresentation of particular biological processes using online tool GeneCodis2 [START_REF] Nogales-Cadenas | GeneCodis: interpreting gene lists through enrichment analysis and integration of diverse biological information[END_REF]. Thirty-six of the 282 up-regulated genes (corrected hypergeometric pvalue = 2.07 X 10 -8 ) were categorised by the gene ontology term 'transcription' (GO:0006350) being either transcription factors or DNA-binding proteins with regulatory function (SI Table 3). The most highly up-regulated NPAS3 target gene was VGF (2.92-fold) which encodes a group of processed and secreted neuropeptides including TLQP-21. The human VGF promoter containing an 'E box' motif (consensus bHLH transcription factor binding site), was cloned into the pGL3 luciferase reporter plasmid. In HEK293 cells, luciferase activity linked to the VGF promoter was significantly increased upon FLNPAS3 or ΔNPAS3 co-expression compared to control vector transfection (SI Fig. 4,a). In SH-SY5Y neuroblastoma cells, luciferase activity was only significantly increased after FLNPAS3 over-expression (SI Fig. 4,b).

We compared microarray data from NPAS3 over-expression with those from SOX transcription factor over-expression (SI Tables 11a-d) in order to identify shared neurodevelopmental pathways. SOX genes within the same functional [START_REF] Haslinger | Expression of Sox11 in adult neurogenic niches suggests a stage-specific role in adult neurogenesis[END_REF][START_REF] Axell | A method for rapid derivation and propagation of neural progenitors from human embryonic stem cells[END_REF] were confirmed as directly down-regulated by NPAS3 over-expression, further supporting interaction of NPAS3 with SOX transcriptional networks (SI Fig. 2b).

The set of NPAS3 down-regulated genes is enriched for metabolic function NPAS3-repressed genes were queried using Ingenuity Pathway Analysis (IPA, SI Table 6), GeneCodis2 (SI Table 3) and a search for genomic clustering of targets (SI Fig. 7). A cluster of chromosome 1 histone genes was down-regulated by NPAS3 suggesting global alterations in chromatin statepotentially relating to the neuronal differentiation process. IPA identified significant enrichment of the glycolysis/gluconeogenesis pathway in the gene set (16 genes, p=6.14 X 10 -6 ). The glycolysis link (8 genes) was confirmed using GeneCodis2, (corrected hypergeometric p-value = 5.21 X 10 -7 ).

Additionally, 21 genes participating in the 'oxidation:reduction' biological process (GO:0055114) were also significantly down-regulated (p = 6.30 X 10 - 7 ). Visual inspection and comparison with published data (27-30) suggested many genes were targets for the hypoxia response pathway (Table 1b).

Glycolysis regulation can also be considered part of the hypoxia response.

Together, these findings strongly suggest that, in addition to a neurodevelopmental role, NPAS3 regulates the anaerobic catabolism of glucose into pyruvate and, more generally, enzymatic redox processes.

An interaction between NPAS3 activity and the circadian rhythm

A circadian rhythm was successfully induced in HEK293 cells (SI Fig. between +12 and +24hrs), 14 NPAS3-regulated glycolysis genes, and histone gene clusters located on chromosome 1 and 6. The randomly selected genes showed no deviation, indicating no systemic bias between the three experimental conditions (Fig. 3a). The circadian rhythm impacted on FLNPAS3 and ΔNPAS3 activities in three ways. Firstly, global NPAS3 activity was almost completely inhibitory at +12 hours unlike +24 hours (Fig. 2b,c).

Secondly, significant differences between FLNPAS3 and ΔNPAS3 activities on circadian (and glycolysis, to a lesser extent) target genes were exposed at +12 hours (Fig. 3b,c and SI Table 11f) but not at +24 hours. This was evident as reduced ΔNPAS3 potency at +12 hours as highlighted in Fig. 2d. Thirdly, the sets of genes regulated by FL/ΔNPAS3 at +12 hours (SI Table S11g) and +24 hours (SI Table 11h) showed considerable differences; for example, substantial down-regulation of the chromosome 1 histone cluster was observed at +12 but not +24 hours (Fig. 3d).

Altered metabolite levels in brain tissue from Npas3 knockout mice

To determine the in vivo actions of Npas3, we applied high-resolution mass spectrometry to homozygote knockout (KO) and wild-type littermate brain tissue. In both positive ionisation mode (SI Table 10a,b) and negative ionisation mode (SI Table 10c,d) statistically significant changes in multiple polar metabolites were observed. Of particular note were decreased levels of dihydroxyacetone phosphate/glycerone phosphate and octulose-1,8bisphosphate in the KO (6% and 5% of wild-type levels, respectively) and increased levels of NAD + (SI Fig. 9), cystathione and dTDP-glucose/galactose (12.4-, 3.7-and 3.4-fold increases, respectively). Many perturbations relate to glycolysis, the tricarboxylic acid cycle and the urea cycle (Fig. 4).

Discussion

Our experimental approaches produced novel and complementary insights into the normal and pathological activity of NPAS3. We concede that NPAS3 over-expression in the HEK293 cell line will not fully replicate the in vivo neuronal state. However, it may avoid confounding in vivo factors such as developmental/homeostatic compensation, cell-type complexity and variable physiological states. An additional reason for the approach taken was the failure to identify, using the Gene Expression Atlas at ArrayExpress (http://www.ebi.ac.uk/arrayexpress/), a laboratory neuronal cell line expressing endogenous NPAS3 at levels high enough to warrant a 'knockdown' strategy. We believe that certain observations suggest the in vitro data are biologically relevant. Firstly, overlaps in SOX target gene sets reflect evolutionary relatedness. Secondly, many up-regulated NPAS3/SOX targets are neuronal specific. The laboratory that originally derived the cell line in 1973 has speculated that HEK293 cells might actually be of neuronal origin (http://www.fda.gov/ohrms/dockets/ac/01/transcripts/3750t1_01.pdf). Finally, exogenous transcription factors (including SOX genes) can show dominant effects on cell gene transcription [START_REF] Takahashi | Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[END_REF].

The target similarity between NPAS3 and SOX5/6 may reflect a shared role in neuronal differentiation [START_REF] Azim | SOX6 controls dorsal progenitor identity and interneuron diversity during neocortical development[END_REF][START_REF] Lefebvre | The SoxD transcription factors--Sox5, Sox6, and Sox13-are key cell fate modulators[END_REF]. The co-localisation of Npas3 and Doublecortin indicates participation in a later, post-proliferative, stage of neuronal differentiation [START_REF] Brown | Transient expression of doublecortin during adult neurogenesis[END_REF] matching the observations of Pieper and colleagues [START_REF] Pieper | Discovery of a proneurogenic, neuroprotective chemical[END_REF]. This may refine our understanding of the link between adult neurogenesis pathologies and psychiatric illness [START_REF] Kempermann | The contribution of failing adult hippocampal neurogenesis to psychiatric disorders[END_REF].

The top NPAS3, SOX5 and SOX6 up-regulated gene, VGF, encodes a precursor for a number of processed and secreted signalling peptides such as TLQP-21. VGF is a highly relevant target gene as it shows; regulation by circadian rhythm [START_REF] Cirelli | Gene expression in the brain across the sleepwaking cycle[END_REF], involvement in metabolic control [START_REF] Bartolomucci | The role of the vgf gene and VGF-derived peptides in nutrition and metabolism[END_REF][START_REF] Jethwa | Role of VGF-derived peptides in the control of food intake, body weight and reproduction[END_REF], contribution to activity-related adult neurogenesis [START_REF] Thakker-Varia | The neuropeptide VGF produces antidepressant-like behavioral effects and enhances proliferation in the hippocampus[END_REF], and association with neurological diseases [START_REF] Pasinetti | Identification of potential CSF biomarkers in ALS[END_REF][START_REF] Selle | Identification of novel biomarker candidates by differential peptidomics analysis of cerebrospinal fluid in Alzheimer's disease[END_REF][START_REF] Ruetschi | Identification of CSF biomarkers for frontotemporal dementia using SELDI-TOF[END_REF][START_REF] Altar | Target identification for CNS diseases by transcriptional profiling[END_REF] including depression [START_REF] Thakker-Varia | The neuropeptide VGF produces antidepressant-like behavioral effects and enhances proliferation in the hippocampus[END_REF][START_REF] Malberg | VGF, a new player in antidepressant action?[END_REF] and psychosis [START_REF] Huang | Disease biomarkers in cerebrospinal fluid of patients with first-onset psychosis[END_REF].

Importantly, VGF and the related SCG2 protein (up-regulated here by SOX9/10/11) have been identified as discriminating cerebrospinal fluid biomarkers for schizophrenia and depression [START_REF] Huang | Disease biomarkers in cerebrospinal fluid of patients with first-onset psychosis[END_REF][START_REF] Huang | CSF metabolic and proteomic profiles in patients prodromal for psychosis[END_REF] suggesting they may be reporting neurogenic pathologies. NPAS3 regulates hypoxia/glycolysis pathways in a circadian-dependent context. NPAS2 has also been implicated in the redox-sensitive transcription of a metabolic gene, lactate dehydrogenase (LDH) [START_REF] Rutter | Metabolism and the control of circadian rhythms[END_REF][START_REF] Rutter | Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors[END_REF]. NPAS3 target gene sets show substantial differences between +12 and +24 hours. Binding partner combinations (via the bHLH domains that mediate DNA-binding and dimerization) may underlie the 'cross-talk' between NPAS3, circadian and hypoxia pathways. The observed ΔNPAS3-specific regulation of circadian targets at +12 hours suggests truncation permits abnormal interaction with circadian transcription factor partners present at +12hrs but absent at +24 hours. Altered circadian biology, manifest as sleep or metabolic dysfunction, has been described in individuals diagnosed with depression and bipolar disorder [START_REF] Lamont | The role of circadian clock genes in mental disorders[END_REF].

Despite differing experimental paradigms and contrasting detection

technologies, the in vitro and in vivo actions of NPAS3 converged at the glycolysis pathway. Perhaps most striking was the 12-fold increase in abundance of oxidised coenzyme nicotinamide adenine dinucleotide (NAD + ). This may reflect a slow-down in glycolysis (which generates the reduced form, NADH) or abnormally oxidative conditions in the cell. The decrease in fructose 1,6-bisphosphate and dihydroxyacetone phosphate (DHAP) concentrations provides direct evidence for glycolysis abnormalities in the knockout. Both glucose and the glycogen synthesis intermediate, UDP-glucose/galactose, were increased in knockout brain, potentially indicating glycogen storage in response to ineffective glycolysis (TDP-glucose/-galactose level changes might be similarly explained). Altered concentrations of sedoheptulose and octulose-1,8-bisphosphate may also indicate a compensatory impact on the pentose phosphate pathway initiating from glucose-6-phosphate. We speculate that the proposed mitochondrial deficit in Npas3 knockout mice [START_REF] Pieper | Discovery of a proneurogenic, neuroprotective chemical[END_REF] may cause the increases in Kreb's cycle intermediates 2-Oxoglutarate/α-Ketoglutaric acid and Succinate and, additionally, may inhibit the mitochondrial glycerol-3-phosphate (G3P) shuttle (Fig. 4) contributing to the altered DHAP, G3P and NAD + concentrations.

These findings may have bearing on the metabolic syndrome that can occur in psychiatric patients. This is most frequently encountered as a medication sideeffect [START_REF] Ma | Antipsychotic treatment alters protein expression associated with presynaptic function and nervous system development in rat frontal cortex[END_REF][START_REF] Mcquillin | A microarray gene expression study of the molecular pharmacology of lithium carbonate on mouse brain mRNA to understand the neurobiology of mood stabilization and treatment of bipolar affective disorder[END_REF]. However, schizophrenia has been repeatedly associated with an increased predisposition to metabolic dysfunction and type II diabetes in medication-naïve individuals (50). Studies suggest glycolytic dysfunction [START_REF] Herberth | Impaired glycolytic response in peripheral blood mononuclear cells of first-onset antipsychotic-naive schizophrenia patients[END_REF][START_REF] Martins-De-Souza | Proteomic analysis of dorsolateral prefrontal cortex indicates the involvement of cytoskeleton, oligodendrocyte, energy metabolism and new potential markers in schizophrenia[END_REF], mitochondrial failure, and oxidative stress (53) may be innate metabolic pathologies in schizophrenia and bipolar disorder.

We have revealed unsuspected global metabolic deficits in the Npas3 knockout brain. Other rodent models of psychiatric illness merit similar biochemical profiling as a means to uncover novel pathologies and opportunities for rational therapeutic development. 11e,f). FLNPAS3 and ΔNPAS3 inhibit the expression of 15 glycolysis genes at +24 hrs (c), although FLNPAS3 activity is much stronger than ΔNPAS3 at +12 hrs (see Fig. 2d). Chromosome 1 histones

  The full-length NPAS3 open reading frame (acc. NM_001164749) was cloned into a TET-inducible expression plasmid (pT-REx-DEST30, Invitrogen, UK) using a restriction digest, Gateway (Invitrogen) cloning linker ligation and the BP/LR reactions (Invitrogen). The truncated form, ΔNPAS3, was generated by cleavage and removal of sequence between internal and multiple cloning site XhoI sites thus deleting the second PAS domain and the putative transactivation domain. Plasmids were stably integrated into HEK293 [T-REx-293] cells (Invitrogen), using Geneticin and Blasticidin. HEK293 cells were maintained in DMEM supplemented with 10% Foetal Bovine Serum ('standard culture conditions'). SOX expression constructs were generously gifted by Chuanju Liu (SOX5/SOX6/SOX9) and Fabien Murisier/Friedrich Beerman (SOX10). SOX11 was cloned from human cDNA as described in Li et al (submitted). All SOX microarray experiments were carried out as transient transfections of HEK293 cells using Optimem/Lipofectamine 2000/plasmid DNA (Invitrogen) incubation for 6 hours followed by 24 hours in standard culture conditions.

  group (SOX5/6; SOX D group, SOX 9/10; SOX E group) displayed closely related target profiles, confirming their established functional conservation and redundancy. NPAS3 target profiles closely matched those of SOX5/6 with the VGF gene being most highly up-regulated by all three transcription factors. NPAS3-SOX target overlaps are summarised in SI Fig.5. Three members of the SOX family of transcription factors, SOX3, SOX11 and SOX12 involved in neuronal proliferation and differentiation

  8) in order to monitor NPAS3 activity in the context of a daily rhythm. A comparison of +12 and +24 hour microarray data in control cells (no NPAS3) identified a set of genes (SI Table 11e) showing putative circadian regulation including FOS, FOSB, EGR1, EGR2, EGR3 and PER2, all with established circadian rhythm roles. Circadian microarray data were visualised using ternary plots to highlight the influence of FLNPAS3, ΔNPAS3 and control conditions on individual gene expression levels. Expression data assessed comprised: 109 randomly selected genes, 200 genes showing normal circadian regulation in the parental control cells (100 increasing and 100 decreasing in expression

Fig. 1

 1 Fig.1 Npas3 expression in the mouse dentate gyrus. Npas3, Gfap, Nestin and Dcx protein expression was compared using immunofluorescence on frozen mouse brain sections. Npas3 (green; a, d and g) expression was mainly nonnuclear and localized to the inner face/subgranular zone of the dentate gyrus. Anti-Gfap, -Nestin and -Dcx/Doublecortin antibodies (red; b,e,h) were used in co-immunofluorescence studies (c,f,i). Only Dcx colocalised with Npas3 in the

Fig. 2

 2 Fig.2 Scatter-plots of microarray data. Diagonals represent equal expression in compared experimental groups. Genes in red show at least 1.5-fold expression difference between the two conditions. Control and FLNPAS3expressing HEK293 cell lines are shown in (a). FLNPAS3 displayed predominantly inhibitory activity at +12 hours after circadian induction (b) but not at +24 hours (c). Only the +12 hour time-point exposed functional differences between FLNPAS3 and ΔNPAS3 (d). The arrowed gene indicates exogenous FLNPAS3.

Fig. 3

 3 Fig.3 Ternary plots illustrate influences on target gene expression. 109 randomly selected genes are plotted for +12hrs (green) and +24hrs (red) time-points (a). The absence of substantial deviation from the centre indicates the no systemic bias between the three conditions -arrows indicate how inhibitory action of an experimental condition would directionally skew the distribution of points. 100 up-regulated genes (circles) and 100 downregulated genes (squares) between +12 and +24 hrs (b). The substantially deviating group (green squares) reflects strong and specific inhibition by ΔNPAS3 of the group of circadian genes down-regulated between +12 hrs and +24hrs (Supplementary Table11e,f). FLNPAS3 and ΔNPAS3 inhibit the

Table 1

 1 The 51 genes most up-regulated (a) and 50 most down-regulated (b) by FLNPAS3 in standard cell culture conditions. Enrichment for glycolysis and hypoxia gene targets is evident in the latter group.

	a
	Illumina ID	Accession	Symbol	Description	Fold up-regulation	Glycolysis	Hypoxia	Circadian
	ILMN_1752550	NM_022123.1	NPAS3	neuronal PAS domain protein 3	62.49
	ILMN_1757497	NM_003378.2	VGF	VGF nerve growth factor inducible	2.92
	ILMN_1705153	NM_021076.2	NEFH	neurofilament, heavy polypeptide 200kDa	2.58
	ILMN_1716264	NM_014391.2	ANKRD1	ankyrin repeat domain 1 (cardiac muscle)	2.40
	ILMN_1660841	NM_00100187 0.1	MGC14376	hypothetical protein MGC14376	2.36
	ILMN_1730818	NM_174947.2	C19orf30	chromosome 19 open reading frame 30	2.33
	ILMN_1655741	NM_181617.1	KRTAP21-2	keratin associated protein 21-2	2.31
	ILMN_1789096	NM_152672.4	OSTalpha	organic solute transporter alpha	2.23
	ILMN_1734011	NM_00101086 6.1	RP13-15M17.2	hypothetical protein LOC199953	2.20
	ILMN_1725524	NM_003727.1	DNAH17	dynein, axonemal, heavy chain 17	2.15
	ILMN_1680085	NM_004881.2	TP53I3	tumor protein p53 inducible protein 3	2.12
	ILMN_1657148	NM_152480.1	C19orf23	chromosome 19 open reading frame 23	2.08
	ILMN_1684886	NM_013452.2	VCX	variable charge, X-linked	2.07
	ILMN_1669410	NM_001275.3	CHGA	chromogranin A (parathyroid secretory protein 1)	2.07
	ILMN_1695199	NM_003543.3	HIST1H4H	histone cluster 1, H4h	2.05
	ILMN_1667295	NM_138440.2	VASN	vasorin	2.01
				phospholipase A2, group IVC
	ILMN_1810191	NM_003706.1	PLA2G4C	(cytosolic, calcium-	1.98
				independent)
	ILMN_1747506	NM_014681.4	DHX34	DEAH (Asp-Glu-Ala-His) box polypeptide 34	1.92
	ILMN_1754894	NM_174896.2	C1orf162	chromosome 1 open reading frame 162	1.92
	ILMN_1810514	NM_014655.1	SLC25A44	solute carrier family 25, member 44	1.92
				solute carrier family 9
	ILMN_1800425	NM_003047.2	SLC9A1	(sodium/hydrogen	1.92
				exchanger), member 1
	ILMN_1754061	NM_022917.4	NOL6	nucleolar protein family 6 (RNA-associated)	1.92
	ILMN_1667194	NM_144684.1	ZNF480	zinc finger protein 480	1.89
	ILMN_1658800	NM_015695.2	BRPF3	bromodomain and PHD finger containing, 3	1.88
	ILMN_1666690	NM_052957.3	ACRC	acidic repeat containing	1.83
	ILMN_1669113	NM_012068.3	ATF5	activating transcription factor 5	1.83
	ILMN_1769931	NM_005066.1	SFPQ	splicing factor proline/glutamine-rich	1.82
	ILMN_1710544	NM_002589.2	PCDH7	protocadherin 7	1.80
	ILMN_1786024	NM_00101805 2.1	POLR3H	polymerase (RNA) III (DNA (22.9kD) directed) polypeptide H	1.80
	ILMN_1718977	NM_015675.2	GADD45B	growth arrest and DNA-damage-inducible, beta	1.80		yes	yes
	ILMN_1694731	NM_001287.3	CLCN7	chloride channel 7	1.79
	ILMN_1697268	NM_032048.2	EMILIN2	elastin microfibril interfacer 2	1.79
	ILMN_1757406	NM_005319.3	HIST1H1C	histone cluster 1, H1c	1.77			yes
	ILMN_1725510	NM_014762.3	DHCR24	24-dehydrocholesterol reductase	1.75
	ILMN_1807719	NM_004937.2	CTNS	cystinosis, nephropathic	1.74

Funding: Royal Society of Physicians in Edinburgh Sim Fellowship to BSP and a China Council Studentship to LS. SJC was supported in part by a NARSAD Young Investigator Award.

Conflict of interest

The authors declare that no financial or other conflicts of interest will arise as a consequence of being named as an author on this paper.

Supplementary information

Supplementary information is available at Molecular Psychiatry's website.

NPAS3 microarray data has been deposited at ArrayExpress with accession

numbers: E-TABM-1078 and E-TABM-1079.

(squares), but not chromosome 6 histones (circles), are substantially inhibited by both FLNPAS3 and ΔNPAS3 at +12 hrs but not +24 hrs (d).