Fanny Kortüm

Soma Das

Max Flindt

Deborah J Morris-Rosendahl

Irina Stefanova

Amy Goldstein

Denise Horn

Eva Klopocki

Gerhard Kluger

Peter Martin

Anita Rauch

Agathe Roumer

Sulagna Saitta

Laurence E Walsh

Dagmar Wieczorek

Gökhan Uyanik

PhD Kerstin Kutsche
email: kkutsche@uke.de

MD William B Dobyns

The core FOXG1 syndrome phenotype consists of postnatal microcephaly, severe mental retardation, absent language, dyskinesia, and corpus callosum hypogenesis

Keywords: Rett syndrome, FOXG1, mental retardation, microcephaly

Background: Submicroscopic deletions in 14q12 spanning FOXG1 or intragenic mutations have been reported in patients with a developmental disorder described as a congenital variant of Rett syndrome. We aimed to further characterize and delineate the phenotype of FOXG1mutation positive patients.

Method:

We mapped the breakpoints of a 2;14 translocation by fluorescence in situ hybridization and analyzed three chromosome rearrangements in 14q12 by cytogenetic analysis and/or array CGH. We sequenced the FOXG1 gene in 210 patients, including 129 patients with unexplained developmental disorders and 81 MECP2-mutation negative individuals.

Results: We report one known mutation, seen in 2 patients, and 9 novel mutations of FOXG1 including 2 deletions, 2 chromosome rearrangements disrupting or displacing putative cisregulatory elements from FOXG1, and 7 sequence changes. Analysis of our 11 patients, and further 15 patients reported in the literature, demonstrates a complex constellation of features including mild postnatal growth deficiency, severe postnatal microcephaly, severe mental retardation with absent language development, deficient social reciprocity resembling autism, combined stereotypies and frank dyskinesias, epilepsy, poor sleep patterns, irritability in infancy, unexplained episodes of crying, recurrent aspiration, and gastroesophageal reflux.

Brain imaging studies reveal simplified gyral pattern and reduced white matter volume in the frontal lobes, corpus callosum hypogenesis, and variable mild frontal pachgyria.

Conclusions:

We significantly expanded the number of FOXG1 mutations and identified two affecting possible cis-regulatory elements. While the phenotype of the patients overlaps both classic and congenital Rett syndrome, extensive clinical evaluation demonstrates a distinctive and clinically recognizable phenotype which we suggest to designate as the FOXG1 syndrome.

INTRODUCTION

Recently, a series of reports have implicated deletions or sequence alterations of FOXG1 as the cause of a developmental disorder described as a congenital variant of Rett syndrome (RTT; MIM 312750). Small de novo interstitial deletions in 14q12 were detected by chromosome microarrays in four children [START_REF] Bisgaard | Additional chromosomal abnormalities in patients with a previously detected abnormal karyotype, mental retardation, and dysmorphic features[END_REF][START_REF] Jacob | Atypical Rett syndrome with selective FOXG1 deletion detected by comparative genomic hybridization: case report and review of literature[END_REF][START_REF] Mencarelli | 14q12 Microdeletion syndrome and congenital variant of Rett syndrome[END_REF][START_REF] Papa | A 3 Mb deletion in 14q12 causes severe mental retardation, mild facial dysmorphisms and Rett-like features[END_REF] and a complex chromosome rearrangement consisting of a translocation and adjacent small inversion in another. [START_REF] Shoichet | Haploinsufficiency of novel FOXG1B variants in a patient with severe mental retardation, brain malformations and microcephaly[END_REF] The common deleted region includes the telomeric breakpoint of the inversion in the latter patient and contains FOXG1, a strong candidate gene based on high and specific expression in developing brain and complex malformations of the telencephalon in both homozygous and heterozygous mouse mutants. [START_REF] Eagleson | Disruption of Foxg1 expression by knock-in of cre recombinase: effects on the development of the mouse telencephalon[END_REF][START_REF] Siegenthaler | Foxg1 haploinsufficiency reduces the population of cortical intermediate progenitor cells: effect of increased p21 expression[END_REF][START_REF] Xuan | Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres[END_REF] Subsequently, sequencing of FOXG1 in five small cohorts of patients with overlapping phenotypes but negative results for MECP2 testing identified 11 patients with intragenic mutations including 5 nonsense, 4 frameshift and 2 missense mutations. [START_REF] Ariani | FOXG1 is responsible for the congenital variant of Rett syndrome[END_REF][START_REF] Bahi-Buisson | Revisiting the phenotype associated with FOXG1 mutations: two novel cases of congenital Rett variant[END_REF][START_REF] Guen | A FOXG1 mutation in a boy with congenital variant of Rett syndrome[END_REF][START_REF] Mencarelli | Novel FOXG1 mutations associated with the congenital variant of Rett syndrome[END_REF][START_REF] Philippe | Phenotypic variability in Rett syndrome associated with FOXG1 mutations in females[END_REF] Common clinical features in all these children included decelerating head circumference from 3 months that resulted in severe postnatal microcephaly, severe mental retardation with absent language, apraxia, jerky movements and generalized seizures. Brain imaging in several patients revealed hypogenesis of the corpus callosum. 9 10 12 The phenotype was interpreted as resembling Rett syndrome except for no early period of normal development, which we view as one of several significant differences. Recently, duplications of FOXG1 have been associated with developmental epilepsy, mental retardation, and severe speech impairment in eight patients. 14 15 After mapping a de novo chromosome translocation found in a severely mentally retarded girl to this region, we became interested in the types of mutations involving FOXG1 and the nature of the associated developmental disorder. Is the phenotype accurately described as a congenital variant of Rett syndrome, or is it a specific and recognizable syndrome in its own right? Here we report 2 microdeletions, 2 chromosome rearrangements most likely causing dysregulated FOXG1 expression, and 7 intragenic sequence changes among 210 patients tested. This cohort includes 129 patients referred to us directly because of unexplained developmental disorders, and 81 ascertained following negative testing for MECP2.

Our data and review of prior reports confirm mutations or dysregulation of FOXG1 as the cause of the developmental phenotype, which differs from classic Rett syndrome and other so-called Rett variants based on a specific and recognizable combination of developmental and brain imaging features. We prefer to designate this as the FOXG1 syndrome, which has overlapping features with a rapidly growing group of developmental encephalopathies that already includes Angelman (UBE3A-associated), Rett (MECP2-associated), Pitt-Hopkins (TCF4-associated), CNTNAP2-associated, NRXN1-associated and SLC9A6-associated syndromes. [START_REF] Amiel | Mutations in TCF4, encoding a class I basic helix-loop-helix transcription factor, are responsible for Pitt-Hopkins syndrome, a severe epileptic encephalopathy associated with autonomic dysfunction[END_REF][START_REF] Amir | Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2[END_REF][START_REF] Gilfillan | SLC9A6 mutations cause X-linked mental retardation, microcephaly, epilepsy, and ataxia, a phenotype mimicking Angelman syndrome[END_REF][START_REF] Kishino | UBE3A/E6-AP mutations cause Angelman syndrome[END_REF][START_REF] Matsuura | De novo truncating mutations in E6-AP ubiquitin-protein ligase gene (UBE3A) in Angelman syndrome[END_REF][START_REF] Zweier | CNTNAP2 and NRXN1 are mutated in autosomal-recessive Pitt-Hopkins-like mental retardation and determine the level of a common synaptic protein in Drosophila[END_REF][START_REF] Zweier | Haploinsufficiency of TCF4 causes syndromal mental retardation with intermittent hyperventilation (Pitt-Hopkins syndrome)[END_REF]

PATIENTS AND METHODS

Patients

We obtained clinical data, brain imaging studies and results of laboratory testing, as well as blood or DNA samples from 129 patients with mental retardation, microcephaly, frontal pachygyria or hypogenesis of the corpus callosum who were assessed by experienced clinical geneticists or neurologists. Two patients were recruited through the MRNET consortium (www.german-mrnet.de). We received 81 DNA samples from patients tested in a diagnostic lab for MECP2 and found to be negative. All available brain imaging studies from patients with a FOXG1 mutation were interpreted by one of the authors (W.B.D.) with selected images shown in Figures 1 and2. The clinical data and samples were obtained with informed consent, including consent to use the photographs and videos in this report, under protocols approved by Institutional Review Boards at all participating institutions.

Fluorescence in situ hybridization (FISH)

Metaphase spreads from peripheral blood lymphocytes were prepared by standard procedure.

FISH experiments for delineation of the 2q11.2 and 14q12 breakpoints were performed with BAC and fosmid clones. FISH with BAC clones was also performed to confirm array CGH and molecular karyotyping data. BACs (RP11 Human BAC Library) were obtained from the Resource Center for Genome Research at the Max-Planck-Institute for Molecular Genetics, Berlin, Germany, or the BACPAC Resource Center, Children's Hospital Oakland, CA, USA.

Fosmid clones (WIBR-2 Human Fosmid Library [G248P8]) were received from the BACPAC Resource Center, Children's Hospital Oakland, CA, USA. BAC and fosmid DNA was prepared using the NucleoBond Xtra Midi kit (Machery-Nagel, Düren, Germany) or the Qiagen Large-Construct kit (Qiagen, Hilden, Germany). DNA was labeled by nick translation using the CGH Nick Translation Kit and Spectrum Green-dUTP (Vysis, Downers Grove, IL, USA) according to the protocol provided. Chromosomes were counterstained using 4',6diamidino-2-phenylindole (DAPI) (Serva Feinbiochemica, Heidelberg, Germany) and mounted in antifading solution (Vector Labs, Burlingame, CA, USA). Slides were analyzed with a Leica Axioscope fluorescence microscope. Images were merged using a cooled CCD camera (Pieper, Schwerte, Germany) and Cyto Vision software (Applied Imaging, San Jose, CA, USA).

Molecular karyotyping and array comparative genomic hybridization (CGH)

Molecular karyotyping using the Affymetrix Mapping GeneChip 250 K Mapping SNP-Array (Nsp) (Affymetrix, Santa Clara, CA, USA) was performed in patient 3 according to the supplier's instructions. Data were analyzed using Nexus and CNVF2.2 software. [START_REF] Hoyer | Molecular karyotyping in patients with mental retardation using 100K single-nucleotide polymorphism arrays[END_REF] Array CGH in patient 4 was performed using a whole genome oligonucleotide array (244K, Agilent Technologies, Santa Clara, CA, USA). 244K image data were analyzed using Feature Extraction 9.5.3.1 and CGH Analytics 3.4.40 software (Agilent, Santa Clara, CA, USA) with the following analysis settings: aberration algorithm ADM-2; threshold: 6.0; window size: 0.2 Mb; filter: 5probes, log2ratio = 0.29.

Mutation analysis

We amplified the coding region of the FOXG1 gene (1 exon; GenBank accession no. NM_005249) from genomic DNA with five primer pairs that yielded overlapping amplicons.

Primer sequences are available on request. Amplicons were directly sequenced using the ABI BigDye Terminator Sequencing Kit (Applied Biosystems, Darmstadt, Germany) and an automated capillary sequencer (ABI 3130; Applied Biosystems). Sequence electropherograms were analyzed using Sequence Pilot software (JSI medical systems, Kippenheim, Germany).

Where mutations were shown to have arisen de novo, we verified self-reported relationships by genotyping both parents and the patient at ten microsatellite loci.

Results

Mapping of the chromosomal breakpoint in 14q12 by fluorescence in situ hybridization

Patient 1, a 12 year-old girl, was referred at 7 years because of severe mental retardation and postnatal microcephaly. She is the fourth child of healthy parents and her three older sisters are healthy. She was born at 40 weeks gestation by natural delivery with birth weight 3350 g (0 SD), length 51 cm (0 SD), and OFC 32.5 cm (-1 SD). At 6 months, she was able to turn, but she has never been able to sit or stand. She did not react to visual and acoustic stimuli in infancy, but this improved at later ages. Ophthalmologic examination revealed convergent strabismus. At 7 months she developed seizures. EEG revealed spikes and sharp waves over the left centroparietal and both centrotemporal regions. She could speak single words at 1 year, but lost this ability by 2 years. She had gastroesophageal reflux and constipation. Her brain MRI demonstrated striking reduced volume of white matter in the anterior frontal lobes with mildly thick cortex, striking hypoplasia and dysplasia of the anterior corpus callosum resembling a sharp hook, and probably dysplastic hippocampus (figure 1E-H).

Prenatal chromosome analysis revealed an apparently balanced chromosome translocation: 46,XX,t(2;14)(q13;q13). Chromosome analysis was normal in both parents, indicating that it occurred de novo (paternity confirmed). FISH analysis with fosmid F-1425A7 from chromosome 2 identified signals on 2q11.2 as well as the der(2) and der (14), indicating that this clone spanned the breakpoint (figure 3A and data not shown). No gene was directly disrupted by the breakpoint, although INPP4A is located 5-10 kb telomeric to the breakpoint (data not shown). A 1-bp deletion in Inpp4a encoding inositol polyphosphate 4phosphatase type I occurred in the spontaneous weeble mouse mutant, which has severe locomotor instability and significant neuronal loss in the cerebellum and hippocampal CA1 sector. [START_REF] Nystuen | A null mutation in inositol polyphosphate 4-phosphatase type I causes selective neuronal loss in weeble mutant mice[END_REF] In addition, patient 1 is heterozygous for the breakpoint close to INPP4A, and further the weeble phenotype is a recessive condition that differs significantly from the phenotype in this girl. [START_REF] Nystuen | A null mutation in inositol polyphosphate 4-phosphatase type I causes selective neuronal loss in weeble mutant mice[END_REF] We therefore fine-mapped the 14q breakpoint, finding fosmid probes that hybridized proximal (F-4069H4) and distal (F-2653A9) to the breakpoint, as well as a single fosmid clone that spanned the breakpoint (F-9677B10, figure 3A). We narrowed the breakpoint to a ~10 kb gene-poor region in 14q12 located ~265 kb downstream of FOXG1 (figure 3A), suggesting that a position effect could cause transcriptional misregulation of this gene. In silico analysis using the Regulatory Potential score identified several candidate cis-regulatory elements that are evolutionary conserved throughout a 1-Mb region downstream of FOXG1 (figure 3C). Two of these noncoding elements located at genomic positions chr14:28,930,280-28,932,099 and chr14:28,754,647-28,756,495 (NCBI Build 36.1; figure 3C) have been analysed for gene enhancer activity as assessed in transgenic mice. Both DNA sequence elements are evolutionary highly conserved and were found to drive expression of a reporter gene (lacZ) in mouse forebrain (http://enhancer.lbl.gov/frnt_page.shtml). These data suggest that translocation of either or both of these putative cis-regulatory elements to chromosome 2 may have caused dysregulated expression of FOXG1 in the translocation patient.

Identification of deletions in 14q12 by cytogenetic and array CGH analysis

Patient 2 presented with developmental delay, slow head growth and severe feeding problems.

Brain MRI showed a short corpus callosum (figure 2M-P). Routine chromosome analysis revealed a de novo deletion in chromosome 14 (46,XX,del(14)(q13.1q13.2)). We confirmed the deletion by FISH with two BAC clones, RP11-412H8 and RP11-148E7, flanking FOXG1 as well as BAC RP11-330O19 hybridizing proximal and RP11-908D14 and RP11-116N8 hybridizing distal to the deletion, respectively (data not shown and figure 3B). We estimate the maximum size of the deletion as ~8.4 Mb in 14q12-q13.1 including FOXG1.

Genome-wide array CGH was performed on a clinical basis on two patients with postnatal microcephaly and severe mental retardation. We detected a ~5.4-Mb heterozygous deletion in 14q12-q13.1 in patient 3 that encompasses at least 13 genes including FOXG1, and confirmed the deletion by FISH with BAC clone RP11-329K12 (figure 3B and data not shown). Neither parent carried the deletion (data not shown; paternity confirmed). We identified a ~1.09-Mb microdeletion downstream of FOXG1 in patient 4 that removes a large part of the next gene, PRKD1. PRKD1 encodes protein kinase D1 (also known as PKD or PKD1), a serine/threonine kinase that regulates a variety of cellular functions, including membrane receptor signalling, transport processes, protection from oxidative stress, transcriptional regulation, and F-actin reorganization. 25 26 FISH with BAC clones RP11-30H9 and RP11-419C10 yielded only one signal on metaphase spreads, whereas two signals were obtained on metaphase spreads of his parents (figure 3B and data not shown; paternity confirmed). By quantitative PCR we confirmed the presence of two copies of the FOXG1 gene in patient 4 (data not shown), suggesting that similar to patient 1 a position effect might have caused altered expression of FOXG1. In line with this finding, the deletion spans the two putative long-range cis-regulatory elements for FOXG1 expression (figure 3C).

Sequence analysis of FOXG1

We next selected 125 patients with phenotypes overlapping patients 1-4, including mental retardation, postnatal microcephaly, mild frontal pachygyria, and callosal abnormalities. We sequenced FOXG1 in this cohort, and detected four intragenic mutations in the heterozygous state, including the transitions c.757A>G (p.N253D) in patient 5 and c.700T>C (p.S234P) in patient 11, the frameshift mutation c.460dupG (p.E154GfsX301) in patient 6, and the nonsense mutation c.256C>T (p.R86X) in patient 9. The missense mutations affect invariant amino acids, asparagine at position 253 and serine at position 234. Both residues are located in the DNA binding (forkhead) domain of FOXG1 suggesting that these amino acid exchanges may alter DNA binding properties of FOXG1. The c.757A>G mutation was not detected in the parents of patient 5 or in 204 control chromosomes. Similarly, the c.700T>C mutation was not found in the parents of patient 11. The 1-bp duplication was not identified in the parents of patient 6 or in 200 alleles, and the nonsense mutation was absent from the DNA sample of the mother of patient 9 (the father was deceased). Thus, three of four intragenic FOXG1 mutations occurred de novo (data not shown; all parental identities confirmed).

We also sequenced FOXG1 in a cohort of 81 patients (55 females and 26 males) tested for MECP2 mutations in a diagnostic lab with negative results. We detected the intragenic insertion-deletion mutation c.505_506delGGinsT (p.G168SfsX23) in patient 7 and the frameshift mutation c.263_278del16 (p.R88PfsX99) in patient 10, both in the heterozygous state. The parents of patients 7 and 10 did not carry the respective mutation (paternity confirmed), which was not detected in 200 control chromosomes. We also identified the same c.460dupG mutation in patient 8 as detected in patient 6. Sequencing of FOXG1 in patients 1-4 was negative.

Phenotype of the FOXG1-mutation positive patients

We reviewed clinical (table 1) and brain imaging (table 2) data in all eleven patients, and videotapes in four of the eleven patients (supplementary videos 1-4). Detailed results are listed in supplementary table 1 for all patients, and comparable information is collated for 16 previously reported patients in supplementary table 2. All of our patients had normal body measurements at birth with mildly slow growth thereafter leading to low weight (-1.5-2.5 SD) and low normal stature (0-2 SD) by 1-2 years. Birth head size was low normal (-0-2 SD) in 5/7 and borderline small (-2-3 SD) in 2/7 children. Severe postnatal microcephaly (MIC) was apparent by 2 years (-4-6 SD) in 6/8 patients.

All eleven patients had early global developmental delay and later severe mental retardation. Only 3/10 learned to sit independently and only 1/11 learned to walk. None developed speech. All had deficient social interactions, especially poor eye contact. Several improved enough to allow use of pointing and assistive-communication devices (but not speech), and limited social interaction. Two children had a history of possible regression, but this was subtle and difficult to document. The remainder had no history suggesting regression.

Exams reported muscular hypotonia in 10/10 and spasticity in 7/9 patients. We found stereotypies especially of the hands in 8/11, and dyskinesias in 8/10 with features of chorea, athetosis and dystonia. However, we observed dyskinesia in 6/6 patients reviewed during the course of this study with videos available of four patients (see supplementary videos 1-4), which suggests that this may be seen in all patients.

Dysfunction in several vegetative functions was apparent in all children. Sleep was disrupted from infancy in at least 8/11 children, with frequent nighttime waking. This usually improved with age, or after gastrostomy placement in one child. Prominent irritability or crying was reported in 6/10 patients, especially in infancy. Gastroesophageal reflux was diagnosed in 9/10 children, and was often severe, becoming a prominent part of the overall phenotype. Aspiration was documented in 6/10. A gastrostomy tube was recommended for feeding in 2/7 children but only placed in one.

Seizures were documented in 8/10 patients with onset between 3 months and 6 years.

They consisted of tonic, generalized tonic-clonic, and partial complex seizures. None had infantile spasms. EEG findings were variable from reports, but the original studies were not reviewed. Several other features are noted in supplementary table 1.

Brain MRI studies were reviewed in all eleven patients (figures 1 and 2). The forehead was usually low, although this was subjective. All eleven had a simplified gyral pattern defined as a reduced number of gyri separated by shallow sulci, and significantly reduced volume of white matter that appears most severe -by far -in the frontal lobes. We observed mild frontal pachygyria in 3/11 patients; our recognition of subtle frontal pachygyria in patient 1 led directly to testing and detection of an intragenic mutation in patient 5. We found hypogenesis of the corpus callosum in 9/11 patients including three with a novel defect in which the frontal portion of the corpus callosum narrows to a sharp point or hook, with an absent rostrum and very small genu (figure 1E, M andQ). One child with a large 14q12 deletion had complete agenesis of the corpus callosum (figure 2I). The remaining patients had a short or thin corpus callosum with dysmorphic or absent rostrum and narrow or absent splenium. The two patients with a normal corpus callosum were those with a 14q12 deletion 35 kb distant from FOXG1 (figure 2Q) and the patient with the missense mutation p.S234P (figure 2U).

The two children with large deletions (patients 2 and 3) had mild dysmorphic facial features in addition to the MIC, including wide and round face, flat midface, large ears and small jaw. The remaining children had obvious MIC with subtle and non-specific dysmorphic features (figure 4).

DISCUSSION

Foxg1 is a transcription factor that regulates development of the telencephalon from early embryonic to adult stages by multiple diverse mechanisms. 6 7 27 Not surprisingly, it was recognized as a strong candidate gene to explain the developmental disorder associated with deletion 14q12 and as a single gene cause of human developmental disorders. 5 9 Our study extends several previous reports, demonstrating that heterozygous loss of FOXG1 results in a consistent and severe developmental phenotype with emerging genotype-phenotype correlations.

Foxg1 function

Foxg1 is a conserved transcriptional repressor that is strongly expressed in progenitors of the ventricular zone and early postmitotic neurons in the developing neuroepithelium of the telencephalon and visual structures. [START_REF] Bourguignon | XBF-1, a winged helix transcription factor with dual activity, has a role in positioning neurogenesis in Xenopus competent ectoderm[END_REF][START_REF] Murphy | Human brain factor 1, a new member of the fork head gene family[END_REF][START_REF] Tao | Telencephalon-restricted expression of BF-1, a new member of the HNF-3/fork head gene family, in the developing rat brain[END_REF][START_REF] Zhao | Distinct expression of two foxg1 paralogues in zebrafish[END_REF] It is involved in the timing of neuronal differentiation and in the specification of subdomain identity, regulating dorsal-ventral and subsequently rostral-lateral regional differentiation and later neural progenitor versus Cajal-Retzius cell fate, expansion of the neural progenitor pool, and progenitor cell cycle length. 8 30 32-35 Homozygous Foxg1 -/-mutants die at birth with a dramatic reduction in the size of the cerebral hemispheres affecting ventral more severely than dorsal structures, and depletion of neural progenitor pools. 8 30 32-35 The cortical plate is poorly organized with loss of layerspecific markers, and appears relatively thick in lateral cortical regions as a result of accelerated neuronal differentiation (see figure 1 corticogenesis and, to a lesser extent, from a prolonged cell cycle in late corticogenesis. 6 7 These data predict that humans with FOXG1 haploinsufficiency should have microcephaly, thin cortex with abnormal cortical cytoarchitecture (but with a thicker cortex possible in lateral cortical regions), and cognitive plus behavioral defects.

FOXG1 mutations

Combining our series and prior reports, 18 intragenic sequence changes of FOXG1 have been reported including 14 null -6 nonsense and 8 frameshift -and 4 missense mutations (supplementary tables 1 and 2). The missense mutations (p.F215L, p.N227K, p.S234P, and p.N253D) all affect evolutionary highly conserved amino acid residues in the forkhead domain involved in DNA binding. One group hypothesized that amino acid substitutions in functional domains of FOXG1 and late-truncating mutations may lead to a milder phenotype including typical and atypical Rett syndrome because the resulting FOXG1 proteins may retain some functions, especially their ability to bind corepressors. [START_REF] Philippe | Phenotypic variability in Rett syndrome associated with FOXG1 mutations in females[END_REF] However, the p.Y400X and p.S323fsX325 mutations, identified in patients with a severe phenotype leave one FOXG1 domain intact which interacts with the Groucho corepressor family. [START_REF] Yao | The winged-helix protein brain factor 1 interacts with groucho and hes proteins to repress transcription[END_REF] Thus, missense as well as clear loss-of-function mutations can have a severe impact on FOXG1 function suggesting that other factors may modulate the phenotypic outcome.

The recurrent c.460dupG mutation causing duplication of a guanine after 7 subsequent guanines in FOXG1 was identified twice in our patient cohort and in one reported patient. [START_REF] Bahi-Buisson | Revisiting the phenotype associated with FOXG1 mutations: two novel cases of congenital Rett variant[END_REF] These findings suggest that this guanine stretch may be prone to replication errors and represent a mutational hot spot in FOXG1.

We identified the 14q12 breakpoint in patient 1 about 265 kb downstream of the FOXG1 gene. The finding of de novo FOXG1 mutations in patients with a developmental encephalopathy overlapping Rett syndrome strongly suggests that altered expression of FOXG1 due to displacement of cis-regulatory elements important for its spatial and temporal expression caused the disease phenotype in patient 1. The 14q12 breakpoint of a different de novo 2;14 translocation was mapped ~5 kb downstream of the FOXG1 gene. [START_REF] Shoichet | Haploinsufficiency of novel FOXG1B variants in a patient with severe mental retardation, brain malformations and microcephaly[END_REF] The authors postulated that the breakpoint disrupted novel FOXG1 splice variants between exons 3 and 4 with a brain-specific expression pattern, represented by the EST sequence BX248251.

However, the non-coding RNA gene C14orf23 maps distal to FOXG1 (figure 3A), and the reference sequence of this gene was derived from BX248251, which suggests that the putative FOXG1 exons 2-5 more likely represent exons of C14orf23. In line with this, no mutations in these exons have yet been reported. [START_REF] Philippe | Phenotypic variability in Rett syndrome associated with FOXG1 mutations in females[END_REF] Thus, we suggest that both 2;14 translocations resulted in removal of long-range regulatory elements of FOXG1, located in the gene desert between FOXG1 and PRKD1.

Further evidence supporting the existence of sequences regulating FOXG1 expression comes from our identification of an ~1-Mb microdeletion not covering FOXG1 in patient 4.

The phenotypes of both patients 1 and 4 are very similar to FOXG1-mutation positive individuals, suggesting that dysregulated FOXG1 expression may be responsible for their clinical features. In addition, two more patients with small overlapping microdeletions of 0.14 -1.8 Mb that do not seem to include FOXG1 have been reported and are associated with a phenotype fitting with that of FOXG1-mutation positive patients. [START_REF] Mencarelli | 14q12 Microdeletion syndrome and congenital variant of Rett syndrome[END_REF] Our in silico analysis revealed two highly conserved non-coding elements of 1819 bp and 1848 bp between FOXG1 and PRKD1 that drive expression of a reporter gene specifically in the forebrain of transgenic mice (http://enhancer.lbl.gov/frnt_page.shtml). Thus, we propose that regulatory mutations, i.e. displacement and deletion of the two putative cis-regulatory elements by the translocation and the ~1-Mb microdeletion, respectively, cause a phenotype similar to that caused by mutations directly affecting FOXG1. Nonetheless, the phenotype of patient 4 is less severe than our other patients. This suggests disrupted function of a spatiotemporally specific subset of the normal expression pattern. [START_REF] Kleinjan | Long-range gene control and genetic disease[END_REF] The FOXG1 syndrome

We report the largest series of patients with FOXG1 mutations to date. Analysis of our eleven patients and 16 patients from the literature 1-5 9 10 12 13 reveals a complex and consistent developmental phenotype that affects many areas of neurologic and behavioural function (thus classified as a developmental encephalopathy). By recognizing classic dyskinesias and a novel and specific pattern of brain malformations in our cohort of patients we also expand the phenotype.

The characteristic features of the FOXG1 syndrome include (1) moderate postnatal growth deficiency, (2) severe postnatal microcephaly, (3) severe mental retardation with (4) absent or minimal language development, (5) deficient social interactions including poor eye contact denoting a syndromic form of autism, (6) combined stereotypies and frank dyskinesias with mixed features of athetosis, chorea and dystonia, (7) epilepsy, (8) poor sleep patterns, (9) irritability especially in infancy, (10) excessive episodes of crying, (11) recurrent aspiration, and ( 12) frequent gastroesophageal reflux. While a few prior reports described developmental regression in some children, the history was vague as it was in two of our patients, leading us to exclude this as a diagnostic feature. Several other clinical problems were reported in a few patients, such as clubfeet, scoliosis, bruxism, and constipation.

Brain imaging studies reveal frontal predominant simplified gyral pattern, reduced white matter volume and callosal hypogenesis, with variable appearance of mild frontal pachygyria.

Complete agenesis of the corpus callosum was observed in only 3/24 patients overall, but in 2/7 patients with deletion 14q12, which suggests modifying genes in the region. Considering the thin cortex observed in the Foxg1 +/-mouse, it seems possible that the mild pachygyria seen in a few patients is an artifact resulting from image averaging of the cortical ribbon due to the very thin frontal white matter. However, the Foxg1 -/-mouse has a dysplastic cortex, so we prefer to reserve judgment on this issue until higher resolution brain scans or human brain tissue become available.

FOXG1 genotype-phenotype analysis

The number of reported mutations is now large enough to begin genotype-phenotype correlation. Combining our data and several literature reports, the core phenotype is seen in individuals with mutations causing protein truncation and in some with missense and other mutations. Patients with large deletions of 14q12 that include FOXG1 and other genes may have more severe phenotypes, adding complete agenesis of the corpus callosum and mild facial dysmorphism to the core phenotype.

Also, some patients with missense or putative regulatory mutations appear to have less The association between FOXG1 and developmental abnormalities in humans is further supported by recent reports of duplication 14q12 including the FOXG1 in 8 patients. [START_REF] Brunetti-Pierri | Duplications of FOXG1 in 14q12 are associated with developmental epilepsy, mental retardation, and severe speech impairment[END_REF]

The FOXG1 syndrome or congenital Rett syndrome variant

This disorder was first described as a congenital variant of Rett syndrome. While many features do overlap with Rett syndrome, several features beyond the congenital onset differ as well. For example, the presence of true dyskinesias and brain imaging abnormalities, as well as the lack of regression and lack of respiratory arrhythmia differ from classic Rett syndrome.

We believe that the combination of developmental and brain imaging features observed in individuals with FOXG1 mutations are sufficiently distinct to allow clinical recognition of this disorder, which we therefore prefer to designate simply as the FOXG1 syndrome.

Further, the phenotypic overlap with other complex developmental syndromes extends far beyond similarity to Rett syndrome to include at least nine different syndromes and genes:

Angelman syndrome, classic Rett and other MECP2-associated syndromes, the ARX-, CDKL5-, CNTNAP2-, NRXN1-, SLC9A6-, and TCF4-associated syndromes, and maternal isodicentric and duplication 15q11.2q13 syndromes. 16 18 21 22 38-42 Most are single gene disorders, and all present with a wide spectrum of cognitive, social, affective, autonomic and motor deficits, as well as epilepsy. Considering the increasing number and phenotypic heterogeneity of these disorders, we have begun to use the term complex or mixed "developmental encephalopathies" to refer to the group as a whole.

In conclusion, we present the first evidence for mutations of cis-regulatory elements required for spatially, temporally, and quantitatively correct activity of FOXG1 in patients with the same phenotype as individuals with intragenic FOXG1 lesions, as well as several novel intragenic mutations. The specific pattern of developmental features and brain malformations observed in individuals with FOXG1 mutations should allow clinical recognition of the FOXG1 syndrome and prompt genetic testing of the gene.

increased cortical thickness was not seen in the remaining patients (R-T and Figure 2). Also, three patients had mildly enlarged lateral ventricles, with one having abnormally narrow anterior body and frontal horns (S). These images come from patients 1 (E-H), 5 (I-L), 7 (M-P) and 9 (Q-T), and a normal control (A-D).

  severe phenotypes. Combining our series and literature reports, only 6/23 patients were able to sit, and 4 of the 6 had less severe mutations including a late truncation (p.S323fsX325), a regulatory mutation (deletion 34 kb downstream of FOXG1 in patient 4), and two missense mutations (p.F215L, and p.N253D in patient 5). Only 2/26 patients learned to walk and both had less severe mutations including one late truncation (p.Y400X) and the same missense mutation (patient 5 with p.N253D).

Figure 2

 2 Figure 2 Brain imaging in another six patients with FOXG1 mutations. Five have mildly

Figure 3

 3 Figure 3 Deletions including FOXG1 and regulatory mutations. (A) Physical map of the

Figure 4

 4 Figure 4 Patients with FOXG1 mutations. Two patients with 14q12 deletions (top row, A-B)

Table 1 .

 1 Clinical data for patients with deletion 14q12 or FOXG1 mutations

		This report N=11	Literature N=15 (ref. 1-5,9-13)
	FOXG1 mechanism	4 CYTO, 7 intragenic	5 CYTO, 10 intragenic
	Subject demographics
	Sex	5F:6M	14F:1M
	Age last follow-up	2y to 31y	10mo to 22y
	Growth
	Weight birth (%)	10/10 normal (10-90%)	15/15 (10-90%)
	Length birth (%)	9/9 normal (10-75%)	14/14 (25-90%)
	OFC birth (SD)	5/7 (-0-2 SD); 2/7 (-2.5 SD)	4/14 (+1-0 SD); 10/13 (-0-2)
	Weight age 1-2 years (SD), age	5/6 (-1.5-2.6 SD); 1/6 25%	insufficient data
	Length age 1-2 years (SD), age	5/6 (-0-2 SD); 1/6 (-3.5 SD)	insufficient data
	OFC age 1-2 years (SD), age	8/8 (-2.5-6 SD)	insufficient data
	Weight most recent (SD), age	4/10 (-1-2 SD); 6/10 (-2-5.5 SD)	6/9 (-1-2 SD); 3/9 (-3 SD)
	Length most recent (SD), age	2/8 (-1-2 SD); 5/8 (-2-5 SD)	6/9 (-0-2 SD); 2/9 (-3.5-4)
	OFC most recent (SD), age	9/9 (-2.5-5.5 SD)	12/12 (-2-5 SD)
	Development
	Developmental delay (DD)	11/11 DD
	Mental retardation (MR)	11/11 MR	15/15 MR
	Mental retardation (severity)	11/11 severe	5/5 severe
	Speech-language development	11/11 no language	13/14 no language
	Social interaction (eye contact)	10/10 poor	7/9 poor
	Sitting (age)	3/10 sit, all late	3/13 sit
	Walking (age)	1/11 walk, late	1/15 walk
	Loss of psychomotor skills (age)	2/11 vague history loss skills	8-11 regression
	Behavior, sleep, autonomic
	Sleep pattern	8/11 poor sleep pattern	4/8 poor sleep pattern
	Irritability	5/10 excess irritability	insufficient data
	Crying / weeping (inconsolable)	6/10 excess crying	3/6 excess crying
	Laughing (inappropriate)	0/11 excess laughing	insufficient data
	Motor and dyskinesias
	Hypotonia	10/10 hypotonia	6/6 hypotonia
	Spasticity	7/9 spasticity	1/2 insufficient data
	Functional hand use	8/11 no hand use	8/10 no hand use
	Stereotypic movements	8/11 stereotypies	10/10 stereotypies
	Dyskinesias (chorea, dystonia)	8/10 dyskinesia (or all?)	10/10 dyskinesia
	Strabismus	insufficient data	9/9 strabismus
	Bruxism	5/8 bruxism	10/13 bruxism
	Drooling (sialorhea)	no data	7/9 drooling
	Epilepsy
	Seizures (age onset)	8/10 seizures	10/15 SZ (6mo to 14y)
	Seizure types (specify)	CPS, FTCS, GTCS	CPS, GTCS, myoclonic
	GI and respiratory systems
	Feeding difficulties	9/11 poor feeding	5/6 poor feed
	Aspiration	6/10 aspiration	insufficient data
	Gastroesophageal reflux (GER)	9/10 GER often severe	5/5 GER
	Constipation	insufficient data	6/6 constipation
	Breathing abnormalities	non-specific	3/5 abnormal respirations
	Other abnormalities
	Facial dysmorphism	2/3 with deletions	4/4 with deletions
	Scoliosis	insufficient data	4/11 scoliosis
	Abbreviations: CPS, complex partial seizures; CYTO, cytogenetic abnormality; FTCS, focal tonic clonic seizures;
	GER, gastroesophageal reflux; GTCS, generalized tonic clonic seizures; rec, recommended; SZ, seizures

Table 2 .

 2 Brain imaging data for patients with deletion 14q12 or FOXG1 mutations

	This report N=11	Literature N=15 (ref. 1-5,9-13)

  in Hanashima et al 2004). Heterozygous Foxg1 +/-(especially Foxg1 +/cre ) mutants have less severe developmental defects including reduced volumes of the cerebral cortex, striatum and hippocampus, and thin cortex due to reduced thickness of the superficial layers (II-III) of the cortex. The latter results from loss of neuron-generating intermediate progenitor cells during mid-and late

  /8 patients, and walking in at least 3/8 patients. Based on limited data, brain imaging features appear to be similar but less severe including subtle hypogenesis of the corpus callosum in at least 3 patients, and reduced white matter volume in at least 2 patients (this interpretation is based partly on our interpretation of Figure2in[START_REF] Brunetti-Pierri | Duplications of FOXG1 in 14q12 are associated with developmental epilepsy, mental retardation, and severe speech impairment[END_REF] ).

	15 The
	phenotype resembles the FOXG1 haploinsufficiency syndrome described above, including
	severe developmental delay, mental retardation and absent or minimal language in 8/8,
	seizures in 5/8, hypotonia in 2/8, stereotyped behaviour especially perseverative hand
	movements in at least 4/8, and inappropriate laughing and sleep disturbance in at least 1/8
	patients. However, the seizures were more severe, with typical infantile spasms reported in
	4/8 patients. Other differentiating features include normal head circumference by age 1-3
	years in 7

Acknowledgments

We are grateful to the patients and their families who contributed to this study. Some of the results summarized here form part of the diploma work of Fanny Kortüm and the MD thesis of Max Flindt at the University of Hamburg. We are grateful to Birgit Schulze (Sozialpädiatrisches Zentrum, Frankfurt, Germany) and Christoph Korenke (Zentrum für Kinder-und Jugendmedizin, Klinikum Oldenburg, Germany) for their clinical assessment of patient 6, and Rainer Gabriel (Gemeinschaftspraxis Neuropädiatrie, Lüneburg, Germany) for clinical assessment of patient 11. We thank Inka Jantke, Stefanie Dora, and Fabienne Trotier for skillful technical assistance.

Funding

This work was supported by grants from the Werner Otto-Stiftung [WOS 3/76 to G.U.]; the Deutsche Forschungsgemeinschaft (DFG) [GRK1459 to K.K.]; and the National Institutes of Health (NIH) [1R01-NS058721 to W.B.D]. This work was part of the German Mental

Competing interest

None.

Patient consent

Parental consents obtained. mutations (E-T). Here and in figure 2, each row shows four images from the same patient.

The columns contain midline sagittal (left column) and parasagittal (2nd column) images, and axial images through the lateral ventricles (3rd column) and high convexity (right column).

All four patients have a low forehead that reflects underlying microcephaly, foreshortened frontal lobes and reduced white matter volume that appears severe in the frontal lobes (asterisks in 12/16 images) and subtle in posterior regions. All four have hypogenesis of the anterior corpus callosum with a "pointed hook" appearance in three of them (arrows in E, M, Q) that consists of striking narrowing of the anterior body, genu and rostrum with the tiny rostrum forming a pointed tip, and relatively normal posterior callosum. In the remaining patient (arrow in I), the entire body of the corpus callosum is narrow and the genu small but less so than the others, leaving it dysmorphic but different from the "pointed hook" appearance. Three patients have subtle pachygyria over the frontal lobes only that consists of mildly short and wide gyri and mildly thick cortex (arrowheads in F-H, J-L, N-P), a subtle abnormality that may be accentuated by the reduced volume of the white matter. The Licence for Publication statement:

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in JMG and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence (http://group.bmj.com/products/journals/instructions-for-authors/licence-forms).


Dr Theo Lorenc
email: theo.lorenc@lshtm.ac.uk

Isaac Marrero-Guillamón

Peter Aggleton

Chris Cooper

Alexis Llewellyn

Angela Lehmann

Keywords: AIDS Serodiagnosis/psychology Health Behavior HIV Infections/diagnosis Homosexuality, Male Systematic Review AEGIS, ASSIA, BL Direct, BNI, Centre for Reviews and Dissemination, Cochrane Database of Systematic Reviews, CINAHL, Current Contents Connect, EconLit, EMBASE, ERIC, HMIC, Medline, Medline In-Process, NRR, PsychINFO, Scopus, SIGLE, Social Policy

HAL is

Promoting the uptake of HIV testing among men who have sex with men

(MSM): systematic review of effectiveness and cost-effectiveness

There are estimated to be 33,300 MSM living with HIV in the UK, of whom 8,950 are undiagnosed (2008 figures). [START_REF]HIV in the United Kingdom: 2009 report[END_REF] Strategies to prevent HIV in this population include public health education and community-based awareness-raising about the risks of HIV, and the promotion of safer sex through condom distribution, outreach work and other means. Increasing the uptake of voluntary counselling and testing (VCT) also has a potentially important role to play in reducing the incidence of HIV. Of MSM attending genitourinary medicine (GUM) clinics in 2008, 3.1% were found to have a previously undiagnosed HIV infection. [START_REF]HIV in the United Kingdom: 2009 report[END_REF] Promoting HIV testing has the potential to reduce undiagnosed infections, hence improving individual outcomes and reducing transmission. It may also be of value in raising awareness of HIV more broadly, and engaging MSM with sexual health services.

Promoting HIV testing may be of particular value in reducing late diagnoses. Of new HIV diagnoses among MSM in 2008, 43% had CD4 cell counts below the threshold at which treatment should normally be commenced (<350 per mm 3 within three months of diagnosis), and 20% had CD4 counts less than 200 per mm 3 . [START_REF]HIV in the United Kingdom: 2009 report[END_REF] Late HIV diagnosis is an important predictor of morbidity and mortality. Those who start treatment at a more advanced stage of disease respond less well to treatment, and remain at increased risk of death for many years following treatment initiation. [START_REF] May | Prognosis of HIV-1 infected patients up to 5 years after initiation of HAART: collaborative analysis of prospective studies[END_REF] Rates of HIV testing among MSM vary widely between countries, [START_REF] Sullivan | Reemergence of the HIV epidemic among men who have sex with men in North America, Western Europe, and Australia, 1996-2005[END_REF] and between subgroups of MSM within the UK. [START_REF] Hickson | Tactical dangers: Findings from the Gay Men's Sex Survey[END_REF] Rates have varied over time; survey data show a steady upward trend in numbers ever tested for HIV in samples of MSM in the UK since 2000. [START_REF] Hickson | Testing targets: Findings from the Gay Men's Sex Survey[END_REF] Testing policies also vary internationally, although there is a general trend towards recommending more routine testing. [START_REF] Mounier-Jack | HIV testing strategies across European countries[END_REF] Current UK guidelines recommend that HIV testing should be offered to MSM annually, and more frequently if symptoms indicative of seroconversion or high risk exposure are present; [START_REF]UK National Guidelines for HIV Testing[END_REF] US guidelines recommend annual screening for MSM who themselves or whose sex partners have had more than one sex partner since their most recent HIV test. [START_REF] Branson | Revised recommendations for HIV testing of adults, adolescents, and pregnant women in health-care settings[END_REF] 'Optout' testing policies in STI clinics are now widely implemented, having been recommended by WHO and UNAIDS in 2004, [START_REF]Policy Statement on HIV Testing[END_REF] and by UK guidelines in 2006. [START_REF] Rogstad | UK national guidelines on HIV testing 2006[END_REF] Current UK guidelines also recommend opt-out testing in a much wider range of healthcare settings. [START_REF]UK National Guidelines for HIV Testing[END_REF] We aimed to systematically review the evidence on the effectiveness and costeffectiveness of interventions to increase uptake of HIV testing among MSM. The study presented here is part of a larger review, which also included relevant qualitative evidence, commissioned by NICE (the review protocol has not been published). The full methods and results are available at http://www.nice.org.uk/guidance/index.jsp?action=download&o=50931. This review includes only interventions which aimed to promote HIV testing, although it is clear that the promotion of HIV testing should form part of an integrated risk-reduction strategy aiming to change a range of sexual behaviours. [START_REF]Intensifying HIV prevention : a UNAIDS policy position paper[END_REF][START_REF] Unaids | Practical guidelines for intensifying HIV prevention: Towards universal access[END_REF] To our knowledge, no systematic review with this scope has previously been conducted. Two high-quality systematic reviews whose scope partially overlaps with that of the present review were located by our searches. One of these focused on mass media interventions to promote HIV testing, but included all populations, not only MSM. [START_REF] Vidanapathirana | Mass media interventions for promoting HIV testing[END_REF] The other included HIV health promotion interventions for MSM in general, but did not have a primary focus on HIV testing, and did not include a separate synthesis of evidence relating to interventions promoting HIV testing. [START_REF] Rees | HIV Health Promotion and Men who have Sex with Men (MSM): A systematic review of research relevant to the development and implementation of effective and appropriate interventions[END_REF] Where possible, searches were limited to studies of humans published in English. The Medline search strategy is presented in web-only supplement 1. Full search strategies are available in the complete review report. [START_REF] Lorenc | MSM): Increasing the uptake of HIV testing. Systematic review of effectiveness, cost-effectiveness and qualitative evidence[END_REF] Searching was conducted simultaneously for this review and the review of qualitative evidence (the strategy was designed to locate both types of evidence, and all references were screened for inclusion in both reviews).

METHODS

Identification of studies

In addition, we hand-searched nine key journals from January 2005 to December 2009; scanned the citation lists of included studies and those of systematic reviews which met all other criteria for inclusion; and conducted 'forward' citation chasing on included studies using ISI Web of Knowledge. Finally, a call for evidence to experts and stakeholders was issued; all references arising from the call were screened for inclusion.

Inclusion criteria

The inclusion criteria were as follows:

1) Does the study include men who have sex with men (MSM), or focus on services aimed at MSM?

2) Does the study relate to HIV testing?

3) Was the study conducted in a country which is a current member of the OECD?

4) Was the study published in 1996 or later?

5) Does the study include MSM who are HIV-negative or do not know their HIV status, and are competent to consent to an HIV test?

6) Does the study relate to an intervention that aims to increase the uptake of HIV testing?

7) Is the study a prospective outcome evaluation (randomised or non-randomised controlled trial, or one-group before-and-after study), or a cost-benefit or costeffectiveness analysis, or any other type of economic evaluation?

For criterion [START_REF]HIV in the United Kingdom: 2009 report[END_REF], studies which either had a sample more than 50% of whom were MSM, or presented disaggregated outcome data on the MSM within the sample, or focused on services aimed at MSM, were included. Studies of the general population which did not present outcome data on MSM were excluded. Studies published prior to 1996 were excluded (criterion 4) due to the lesser relevance of data collected before the introduction of effective anti-retroviral treatment for HIV.

For criterion [START_REF]UK National Guidelines for HIV Testing[END_REF], effectiveness studies were included if they either contained a comparison group receiving different interventions (randomised or non-randomised)

and/or presented outcome data for both before and after the intervention, or both. Any economic evaluation was included regardless of study design. Systematic reviews were not included; however, systematic reviews meeting criteria ( 1)-( 6) were retained and their lists of included primary studies scanned for inclusion.

A random sample of 10% of abstracts was screened by two reviewers independently.

Inter-rater reliability (Cohen's kappa) was κ=0.627. Subsequently all abstracts were screened by one reviewer alone. For all references included on abstract, the full text was retrieved and re-screened independently by two reviewers, with disagreements resolved by discussion and reference to a third reviewer if necessary.

Study quality

Study quality was assessed using a standardised tool. [START_REF]Methods for the development of NICE public health guidance[END_REF] The full quality assessment tool is presented in web-only supplement 2. Both quality assessment and data extraction were conducted for a sample of 10% of studies (N=2) by two reviewers independently; subsequently all references were quality-assessed by one reviewer and then checked in detail by a second reviewer. On the basis of this quality assessment, each study was assigned a rating: high (++), medium (+) or low (-). Quality ratings are presented for each study below; studies were not formally weighted by quality in the synthesis.

Data extraction and synthesis

Data were extracted from the studies using a standardised tool. [START_REF]Methods for the development of NICE public health guidance[END_REF] The list of variables for which data were extracted is presented in web-only supplement 2.

Outcome data were only extracted regarding the uptake of HIV testing and the number of new diagnoses; data on other outcomes (e.g. condom use) were not extracted. Due to the heterogeneity of intervention types, quantitative meta-analysis was not carried out, and studies were synthesized narratively.

RESULTS

A total of 4,342 unique references were located by our searches (this figure includes studies for the qualitative review as well as those for this review). After screening, a total of 14 study reports on effectiveness and one on cost-effectiveness were retained in the review. Two of these were linked reports presenting data from the same study.

Hence, a total of 12 effectiveness studies and one cost-effectiveness study were included. The flow of literature through the review is presented in Figure 1.

Basic characteristics of the study settings and populations are set out in Table 1. Table 1 also shows the results of the quality assessment process (full details are given in the full report). [START_REF] Lorenc | MSM): Increasing the uptake of HIV testing. Systematic review of effectiveness, cost-effectiveness and qualitative evidence[END_REF]

Type of test and testing protocol

One study from the USA, published in 2006, compared the uptake of HIV tests included with a package of other tests (for other STIs, alcohol and drug dependence, and depression) offered in outreach settings (bars) with that of HIV tests alone. [START_REF] Galvan | Increasing HIV testing among Latinos by bundling HIV testing with other tests[END_REF] This study found no significant difference in uptake between the two groups.

However, there were some promising but non-significant trends among high-risk subgroups towards greater acceptance of the packaged tests compared to the HIV test alone.

One study, also from the USA and conducted in 1999-2000, compared traditional serum testing and counselling with rapid serum testing and oral fluid testing offered in a gay bathhouse. [START_REF] Spielberg | Choosing HIV counseling and testing strategies for outreach settings: a randomized trial[END_REF] This study found that both rapid serum testing and oral fluid testing were significantly more likely to be accepted than traditional testing. This study provides evidence that rapid testing modalities can increase uptake in an outreach setting compared to traditional testing.

A further US study, conducted in 1996-97, compared oral fluid and blood spot home testing kits, finding high levels of adherence to both types of test. [START_REF] Spielberg | Home collection for frequent HIV testing: acceptability of oral fluids, dried blood spots and telephone results[END_REF]

Peer education and recruitment

Two studies from the UK, both from the late 1990s (1996-99), used non-randomised designs to investigate the effectiveness of peer-led community-based risk-reduction campaigns. [START_REF] Flowers | Does bar-based, peer-led sexual health promotion have a community-level effect amongst gay men in Scotland?[END_REF][START_REF] Elford | Peer education has no significant impact on HIV risk behaviours among gay men in London[END_REF] Both of these studies found such campaigns not to be effective in increasing HIV testing among a community sample of MSM. However, there are limitations in the design and reporting of both these studies (see Discussion), so they arguably do not provide conclusive evidence for the ineffectiveness of peer-led strategies.

One RCT conducted in 2005-07 evaluated an intensive weekend residential programme for Black MSM in the USA led by trained MSM peers. [START_REF] Wilton | Efficacy of an HIV/STI prevention intervention for black men who have sex with men: findings from the Many Men, Many Voices (3MV) Project[END_REF] This study found a small but significant improvement in HIV testing rates among participants at six-month follow-up.

One economic analysis, conducted in 2002-05, looked at a peer recruitment programme to increase rates of HIV testing, finding a cost per new case of HIV identified of US$4,929. [START_REF] Golden | Peer referral for HIV case-finding among men who have sex with men[END_REF] According to the study authors, this compares favourably with costs per case for other interventions, although the analysis presented falls considerably short of a full cost-effectiveness analysis.

Media and web-based campaigns

Two non-comparative studies in Australia and England investigated communitybased media campaigns to promote HIV testing among MSM, including components such as display advertising and websites and print media distributed to gay venues and organisations. [START_REF] Guy | No increase in HIV or sexually transmissible infection testing following a social marketing campaign among men who have sex with men[END_REF][START_REF] Mcowan | Can targeted HIV testing campaigns alter health-seeking behaviour?[END_REF] One of these studies, conducted in 2004-06, found no increase in the numbers of MSM in the targeted location reporting an HIV test in the previous year. [START_REF] Guy | No increase in HIV or sexually transmissible infection testing following a social marketing campaign among men who have sex with men[END_REF] The other, conducted in 1999-2000, found a substantial increase in HIV tests conducted in the targeted clinic. [START_REF] Mcowan | Can targeted HIV testing campaigns alter health-seeking behaviour?[END_REF] However, there are flaws in the design of both these studies (see Discussion). Overall, the evidence for media campaigns must be regarded as inconclusive.

One further non-comparative study in the USA, conducted in 2005-06, evaluated an educational video which was accessed through a banner advert on a gay sexual meeting website. [START_REF] Chiasson | Increased HIV disclosure three months after an online video intervention for men who have sex with men (MSM)[END_REF] This study found a significant increase in HIV testing at 3month follow-up (and a substantial number of new diagnoses were made as a result). This finding is promising, although since the study is non-comparative, it does not permit strong conclusions to be drawn.

STI clinic service delivery

Two studies from the mid-2000s (2003-07), both non-comparative, investigated the introduction of 'opt-out' policies in STI clinics in the Netherlands, such that all clients attending the clinic received an HIV test unless they requested not to have one. [START_REF] Dukers-Muijrers | Effectiveness of an opting-out strategy for HIV testing: evaluation of 4 years of standard HIV testing in a STI clinic[END_REF][START_REF] Heijman | Opting out increases HIV testing in a large sexually transmitted infections outpatient clinic[END_REF] Both these studies found substantial increases in HIV tests among MSM after the introduction of the opt-out policy. A finding of concern in both these studies was that certain groups, particularly older MSM and MSM with potentially STI-related symptoms, were more likely to opt out of testing after the introduction of the policy.

One study from 2000-02, also non-comparative, found that the implementation of guidelines promoting regular HIV testing in an STI clinic resulted in an increase in the proportion of MSM being tested, although it is unclear if the increase reached significance. [START_REF] Ryder | Clinical audit: adherence to sexually transmitted infection screening guidelines for men who have sex with men[END_REF]

DISCUSSION

This review indicates that several promising strategies are available to increase the uptake of HIV testing among MSM. Opt-out policies in STI clinics can increase testing rates, although there remain concerns about high rates of refusal among certain high-risk subgroups (as well as about those MSM who do not attend STI clinics).

Offering rapid testing in outreach settings can also increase the uptake of testing relative to traditional testing methods. However, there is limited evidence for the effectiveness of community-level strategies. One study shows that a holistic group intervention, led by trained peers, can increase testing among a minority ethnic, socio-economically disadvantaged group of MSM. While this is a promising result, such interventions are resourceintensive and may be challenging to implement. Nonetheless, further research on such strategies would be valuable. There are also promising findings, warranting further research, regarding a Web-based intervention.

Evidence regarding media campaigns and peer-led outreach strategies is inconclusive.

Methodologically, evaluations of existing programmes would benefit from measuring individual-level outcomes among men exposed to the intervention, as well as population-level outcomes, and from indexing outcome measures to the timeframe of the intervention (i.e. recent testing rather than lifetime testing). With regard to intervention content, more intensive programmes, sustained over longer timeframes and reaching a larger proportion of the population, are likely to have more impact, as studies of media campaigns in other fields have found. [START_REF] Wakefield | Use of mass media campaigns to change health behaviour[END_REF] Using formative research, or the published qualitative literature, could also be of value in targeting the content of such interventions.

Several limitations in the evidence should be noted. The findings show that, even where interventions succeeded in increasing uptake, numbers of new diagnoses were generally modest. Baseline rates of recent HIV testing in the targeted populations were often high: for example, Spielberg and colleagues' study excluded 35% of potential participants because they had already had an HIV test within the last three months. [START_REF] Spielberg | Choosing HIV counseling and testing strategies for outreach settings: a randomized trial[END_REF] In addition, most studies recruited primarily from gay venues and/or MSM-oriented services or organisations, and were unlikely to include less gayidentified MSM. These points raise the question as to whether evaluated interventions reach those MSM who are most at risk of having undiagnosed HIV.

Other limitations relate to the methods of the primary studies, particularly those evaluating community-level interventions. First, several studies used non-comparative designs and were conducted over several years. This makes it difficult to distinguish the effects of the intervention from longer-term upward trends in testing behaviour.

Second, several studies measured outcomes at a population level (i.e. different individuals were sampled at pre-and post-test), meaning that any measurement of effectiveness may be diluted by changes in the population. This is particularly an issue when the reach of interventions is limited: two studies found that relatively few of the targeted populations could recall direct contact with the intervention at post-test (29% [START_REF] Flowers | Does bar-based, peer-led sexual health promotion have a community-level effect amongst gay men in Scotland?[END_REF] and 3% [START_REF] Elford | Peer education has no significant impact on HIV risk behaviours among gay men in London[END_REF]). A third limitation relates to the measurement of outcomes: the use of lifetime HIV testing rather than recent testing, [START_REF] Flowers | Does bar-based, peer-led sexual health promotion have a community-level effect amongst gay men in Scotland?[END_REF][START_REF] Elford | Peer education has no significant impact on HIV risk behaviours among gay men in London[END_REF] or the total number of HIV tests conducted by specific services, [START_REF] Mcowan | Can targeted HIV testing campaigns alter health-seeking behaviour?[END_REF] may not provide an accurate picture of changes in testing behaviour.

There are also some limitations in the review itself, primarily because of its intentionally restricted scope. This review did not consider evidence relating to HIV testing in populations other than MSM, although this evidence is potentially relevant; for example, there is evidence that mass media interventions are effective in the general population. [START_REF] Vidanapathirana | Mass media interventions for promoting HIV testing[END_REF] It also did not consider the considerable evidence on other risk-reduction strategies among MSM. This evidence would be valuable in providing the context needed to understand the longer-term impacts of successful interventions to promote HIV testing among MSM. For example, the role of testing in serosorting strategies, which remain prevalent among some groups of MSM and may involve substantial risk of infection, [START_REF] Eaton | Serosorting sexual partners and risk for HIV among men who have sex with men[END_REF][START_REF] Hart | Sexual risk behaviour of men who have sex with men: emerging patterns and new challenges[END_REF] suggests that the relation between the uptake of testing, infection rates, and health status, is complex and unpredictable. Hence, interventions to promote testing, in isolation, may not have a substantial impact on rates of infection. Such interventions are more likely to be effective as part of an integrated programme for reducing HIV infections and promoting access to antiretroviral treatment among MSM.

  Promoting the uptake of HIV testing among men who have sex with men (MSM): systematic review of effectiveness and cost-effectiveness Theo Lorenc, Isaac Marrero-Guillamón, Peter Aggleton, Chris Cooper, Alexis Llewellyn, Angela Lehmann, Catriona Lindsay To cite this version: Theo Lorenc, Isaac Marrero-Guillamón, Peter Aggleton, Chris Cooper, Alexis Llewellyn, et al.. Promoting the uptake of HIV testing among men who have sex with men (MSM): systematic review of effectiveness and cost-effectiveness. Sexually Transmitted Infections, 2011, 87 (4), pp.272. 10.1136/sti.2010.048280. hal-00616886

*

  Interventions to promote the uptake of HIV testing among men who have sex with men have the potential to reduce HIV risk and late diagnoses * Opt-out testing policies, and offering rapid testing, can increase the uptake of HIV testing among MSM * Evidence concerning the effectiveness of community-level strategies, such as media campaigns or peer education and recruitment, is inconclusive * Strategies to promote testing should be seen in the broader context of a comprehensive approach to HIV prevention

Table 1 .

 1 Table 2 sets out the interventions evaluated and the findings of the studies. We have divided the interventions into four categories: Summary of included studies: population characteristics

	1) offering different types of test or testing protocols in outreach settings;[17-19]
	2) peer education or recruitment programmes, including intensive residential
	programmes[20] and community-based programmes;[21-23]
	3) community-based media campaigns[24-25] and web-based educational
	interventions;[26] and
	4) changes to the way in which services are delivered in STI clinics, including the
	introduction of opting-out schemes[27-28] and the implementation of guidelines
	recommending regular screening.[29]
	Broadly, these four categories can be grouped as service delivery interventions (1 and
	4) and community-level interventions (2 and 3).

Table 2 .

 2 Summary of included studies: interventions and outcomes

	Study	Intervention and comparison	Follow-up	HIV testing outcome HIV testing findings	New
	reference		(measured			diagnoses:
			from start of			N (% of
			intervention			completed
			delivery)			tests)
	Type of test / testing protocol
	Spielberg et	Four testing protocols offered in gay	Immediate	Acceptance of testing;	Acceptance. (1): 15.8%; (2):	(1 and 4):
	al. 2005[18]	bathhouses. (1) Traditional serum testing and		completion of testing;	21.2% *; (3): 22.8% *; (4): 13.6%	13 (6.1%)
		a return visit to receive results, with standard		receipt of test results	Completion. (1): 12.4%; (2):	(2 and 3): 2
		face-to-face counselling before testing; (2)			16.2% *; (3): 17.3% *; (4): 11.2%	(0.9%)
		rapid serum testing with same-day test results			Receipt. (1): 9.1%; (2): 16.0% *;
		and single-session counselling; (3) oral fluid			(3): 12.5% *; (4): 8.4%
		testing with standard counselling; (4)			[significance tests by comparison
		traditional serum testing with the choice of			with group (1)]
		pre-test written materials or standard
		counselling.
	Galvan et	HIV tests bundled with other tests (for other	Immediate	Acceptance of testing I (bundled): 10.2%	I (bundled):
	al. 2006[17]	STIs, alcohol and drug dependence and			C (HIV alone): 8.9%	5 (3.4%)
		depression) offered in bars. Comparison: HIV				C (HIV
		tests alone.				alone): 10
						(5.1%)
	Spielberg et	Two types of HIV tests for home testing: (1)	20 weeks	Adherence to home	(1) 92%; (2) 99% [not tested for	NR
	al. 2000[19]	dried blood spot home collection, 3 cycles,		specimen collection	significance by study authors]
		bimonthly. (2) oral fluid home collection, 3		schedule
		cycles, bimonthly.
	Peer education and recruitment
	Flowers et	Risk-reduction education in bars delivered by 15 months	Self-reported ever	I: 47.0% baseline, 47.9% at 15	NR

Acknowledgements: This review was funded by the National Institute for Health and Clinical Excellence (NICE). The authors would like to thank the NICE project team (Chris Carmona, Catherine Swann, Daniel Tuvey and Linda Sheppard) for their invaluable support and guidance throughout the review. The views and opinions expressed are those of the authors alone and do not reflect the official opinion of NICE, or any guidance issued by NICE. Contributions: TL led the design and conduct of the review. Literature searches were conceived and executed by CC. Screening was carried out by TL, IMG, AL (Alexis Llewellyn), AAL (Angela Lehmann), and CL. Quality assessment and data extraction was carried out by TL and IMG. Data analysis and interpretation were carried out by TL, IMG and PA. TL wrote the first draft of the paper with input from IMG and PA.

Conclusions: Findings indicate several promising approaches to increasing HIV testing among MSM. However, there is limited evidence overall, and evidence for the effectiveness of key intervention types (particularly peer outreach and media campaigns) remains lacking.

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