Abstract

Rett syndrome is a severe neurodevelopmental disease caused by mutations in the X-linked gene encoding for the methyl-CpG-binding protein MeCP2. Here, we report the identification of FOXG1-truncating mutations in two patients affected by the congenital variant of Rett syndrome. FOXG1 encodes a brain-specific transcriptional repressor that is essential for early development of the telencephalon. Molecular analysis revealed that Foxg1 might also share common molecular mechanisms with MeCP2 during neuronal development, exhibiting partially overlapping expression domain in postnatal cortex and neuronal subnuclear localization. Rett syndrome is a severe neurodevelopmental disease caused by mutations in the X-linked gene encoding for the methyl-CpG-binding protein MeCP2. Here, we report the identification of FOXG1-truncating mutations in two patients affected by the congenital variant of Rett syndrome. FOXG1 encodes a brain-specific transcriptional repressor that is essential for early development of the telencephalon. Molecular analysis revealed that Foxg1 might also share common molecular mechanisms with MeCP2 during neuronal development, exhibiting partially overlapping expression domain in postnatal cortex and neuronal subnuclear localization. In the classic form of Rett syndrome (RTT [MIM 312750]), females are heterozygous for mutations in the X-linked MECP2 gene (MIM 300005) and the few reported males have an XXY karyotype or MECP2 mutations in a mosaic state.1Chahrour M. Zoghbi H.Y. The story of Rett syndrome: from clinic to neurobiology.Neuron. 2007; 56: 422-437Abstract Full Text Full Text PDF PubMed Scopus (930) Google Scholar A number of variants have been described including the congenital, the early-onset seizures, and the preserved speech variant.2Hagberg B.A. Skjeldal O.H. Rett variants: A suggested model for inclusion criteria.Pediatr. Neurol. 1994; 11: 5-11Abstract Full Text PDF PubMed Scopus (192) Google Scholar Soon after the discovery of MECP2 as the RTT gene, we demonstrated that the preserved speech variant is allelic to the classic form.3De Bona C. Zappella M. Hayek G. Meloni I. Vitelli F. Bruttini M. Cusano R. Loffredo P. Longo I. Renieri A. Preserved speech variant is allelic of classic Rett syndrome.Eur. J. Hum. Genet. 2000; 8: 325-330Crossref PubMed Scopus (109) Google Scholar More recently, we and others showed that CDKL5 (MIM 300203) is responsible for atypical RTT, namely the early-onset seizures variant.4Tao J. Van Esch H. Hagedorn-Greiwe M. Hoffmann K. Moser B. Raynaud M. Sperner J. Fryns J. Schwinger E. Gecz J. et al.Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5/STK9) gene are associated with severe neurodevelopmental retardation.Am. J. Hum. Genet. 2004; 75: 1149-1154Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 5Scala E. Ariani F. Mari F. Caselli R. Pescucci C. Longo I. Meloni I. Giachino D. Bruttini M. Hayek G. et al.CDKL5/STK9 is mutated in Rett syndrome variant with infantile spasms.J. Med. Genet. 2005; 42: 103-107Crossref PubMed Scopus (209) Google Scholar The congenital variant was initially described by Rolando in 1985.6Rolando S. Rett syndrome: report of eight cases.Brain Dev. 1985; 7: 290-296Abstract Full Text PDF PubMed Scopus (58) Google Scholar In this form, girls are floppy and retarded from the very first months of life. The majority of congenital variants do not bear MECP2 or CDKL5 mutations,7Erlandson A. Samuelsson L. Hagberg B. Kyllerman M. Vujic M. Wahlstrom J. Multiplex ligation-dependent probe amplification (MLPA) detects large deletions in the MECP2 gene of Swedish Rett syndrome patients.Genet. Test. 2003; 7: 329-332Crossref PubMed Scopus (63) Google Scholar, 8Scala E. Longo I. Ottimo F. Speciale C. Sampieri K. Katzaki E. Artuso R. Mencarelli M.A. D'Ambrogio T. Vonella G. et al.MECP2 deletions and genotype-phenotype correlation in Rett syndrome.Am. J. Med. Genet. A. 2007; 143: 2775-2784Crossref Scopus (43) Google Scholar with only four cases being reported with MECP2 mutations.9Huppke P. Laccone F. Kramer N. Engel W. Hanefeld F. Rett syndrome: Analysis of MECP2 and clinical characterization of 31 patients.Hum. Mol. Genet. 2000; 9: 1369-1375Crossref PubMed Scopus (196) Google Scholar, 10Monros E. Armstrong J. Aibar E. Poo P. Canos I. Pineda M. Rett syndrome in Spain: Mutation analysis and clinical correlations.Brain Dev. 2001; 1: S251-S253Abstract Full Text Full Text PDF Scopus (73) Google Scholar, 11Smeets E. Schollen E. Moog U. Matthijs G. Herbergs J. Smeets H. Curfs L. Schrander-Stumpel C. Fryns J.P. Rett syndrome in adolescent and adult females: Clinical and molecular genetic findings.Am. J. Med. Genet. A. 2003; 122: 227-233Crossref Scopus (49) Google Scholar Using oligo array CGH, we recently identified a de novo 3 Mb interstitial deletion of chromosome 14q12 in a 7 year-old girl.12Papa F.T. Mencarelli M.A. Caselli R. Katzaki E. Sahpieri K. Meloni I. Ariani F. Longo I. Maggio A. Balestri P. et al.A 3 Mb deletion in 14912 causes severe mental retardation, mild facial dysmorphisms and Rett-like features.Am. J. Med. Genet. A. 2008; (in press)Google Scholar She showed dysmorphic features and a Rett-like clinical course, including normal perinatal period, postnatal microcephaly, seizures, and severe mental retardation. The deleted region was gene poor and contained only five genes. Among them, FOXG1 (MIM 164874) turned out to be a very interesting gene because it encodes a brain-specific transcriptional repressor. We analyzed this gene with a combination of both DHPLC and real-time quantitative PCR in a cohort of 53 MECP2/CDKL5 mutation-negative RTT patients, including seven classic, 21 preserved speech, seven early-onset seizures, one “forme fruste,” two congenital variants and 15 Rett-like cases.13Sampieri K. Meloni I. Scala E. Ariani F. Caselli R. Pescucci C. Longo I. Artuso R. Bruttini M. Mencarelli M.A. et al.Italian Rett database and biobank.Hum. Mutat. 2007; 28: 329-335Crossref PubMed Scopus (22) Google Scholar For real-time qPCR analysis, we designed primers and TaqMan probe complementary to a segment located in the middle of the single exon of the gene using Primer Express software (Applied Biosystems). Sequences of primers and probe (FAM labeled) are listed in Table S1 available online. We used an RNAase P kit as an internal reference (VIC-labeled probe, Applied Biosystems). PCR was carried out as previously described.14Ariani F. Mari F. Pescucci C. Longo I. Bruttini M. Meloni I. Hayek G. Rocchi R. Zappella M. Renieri A. Real-time quantitative PCR as a routine method for screening large rearrangements in Rett syndrome: Report of one case of MECP2 deletion and one case of MECP2 duplication.Hum. Mutat. 2004; 24: 172-177Crossref PubMed Scopus (91) Google Scholar The starting copy number of the unknown samples was determined with the comparative Ct method, as reported by Livak.15Livak, K. (1997). ABI Prism 7700 Sequence Detection System.Google Scholar By DHPLC, we identified a different de novo FOXG1 truncating mutation in the two congenital variant patients. Real-time qPCR failed to identify any microdeletion in the 53 patients. FOXG1 encodes forkhead box protein G1, FoxG1 (formerly brain factor 1 [BF-1]), a transcriptional factor with expression restricted to fetal and adult brain and testis. FoxG1 interacts with the transcriptional repressor JARID1B and with global transcriptional corepressors of the Groucho family. The interaction with these proteins is of functional importance for early brain development.16Tan K. Shaw A.L. Madsen B. Jensen K. Taylor-Papadimitriou J. Freemont P.S. Human PLU-1 Has transcriptional repression properties and interacts with the developmental transcription factors BF-1 and PAX9.J. Biol. Chem. 2003; 278: 20507-20513Crossref PubMed Scopus (83) Google Scholar, 17Yao J. Lai E. Stifani S. The winged-helix protein brain factor 1 interacts with groucho and hes proteins to repress transcription.Mol. Cell. Biol. 2001; 21: 1962-1972Crossref PubMed Scopus (105) Google Scholar Like MeCP2, FoxG1 also indirectly associates with the histone deacetylase 1 protein.1Chahrour M. Zoghbi H.Y. The story of Rett syndrome: from clinic to neurobiology.Neuron. 2007; 56: 422-437Abstract Full Text Full Text PDF PubMed Scopus (930) Google Scholar, 17Yao J. Lai E. Stifani S. The winged-helix protein brain factor 1 interacts with groucho and hes proteins to repress transcription.Mol. Cell. Biol. 2001; 21: 1962-1972Crossref PubMed Scopus (105) Google Scholar Both mutations disrupted the protein at different levels (Figure 1). In case 1, a stop-codon mutation p.W255X (c.765G→A) impaired the DNA binding because of the disruption of the forkhead domain (Figure 1D, left). Case 2 showed a 1 bp deletion c.969 delC (p.S323fsX325) causing the loss of JARID1B-interacting domain and the misfolding of the motif responsible for the Groucho binding (Figure 1D, right). Lastly, both FOXG1 mutations affected all the four brain fetal isoforms that lack the last 37 amino acids and have different C-terminal domains.18Shoichet S.A. Kunde S.A. Viertel P. Schell-Apacik C. von Voss H. Tommerup N. Ropers H.H. Kalscheuer V.M. Haploinsufficiency of novel FOXG1B variants in a patient with severe mental retardation, brain malformations and microcephaly.Hum. Genet. 2005; 117: 536-544Crossref PubMed Scopus (79) Google Scholar The two mutated individuals, aged 22 (case 1) and 7 years (case 2), fulfilled the international criteria for RTT variants.19Hagberg B. Hanefeld F. Percy A. Skjeldal O. An update on clinically applicable diagnostic criteria in Rett syndrome. Comments to Rett Syndrome Clinical Criteria Consensus Panel Satellite to European Paediatric Neurology Society Meeting, Baden Baden, Germany, 11 September 2001.Eur. J. Paediatr. Neurol. 2002; 6: 293-297Abstract Full Text PDF PubMed Scopus (346) Google Scholar Pregnancy, delivery, and auxological parameters at birth were normal. Neurological and behavioral neonatal evaluations were reported as normal, but at three months, an abnormal head-circumference growing was noticed in the patients. These patients appeared to weep inconsolably, and they did not respond when called and were unable to lift their heads. Case 1 was never able to sit unaided and laid permanently in bed, whereas case 2 was barely able to sit. They were always apraxic and from 1 year of age, they showed peculiar jerky movements of the upper limbs and midline stereotypic activities, typical of RTT syndrome (Figure 2). They never acquired spoken language. Generalized convulsions appeared at 14 years in case 1 and at 2 1/2 years in case 2. Ever since cases 1 and 2 were 3 and 5 years old, respectively, an EEG showed features often found in RTT patients: a multifocal pattern with spikes and sharp waves and occasional paroxysmal activity. In both patients brain MRI showed corpus callosum hypoplasia, a finding which has already been reported in RTT.20Murakami J.W. Courchesne E. Haas R.H. Press G.A. Yeung-Courchesne R. Cerebellar and cerebral abnormalities in Rett syndrome: a quantitative MR analysis.AJR Am. J. Roentgenol. 1992; 159: 177-183Crossref PubMed Scopus (89) Google Scholar Currently, they show microcephaly (OFC of 49 cm in case 1, and 47 cm in case 2). They have occasional periods of deep breathing with exaggerated inspirations. Sialorrhoea, bruxism, scoliosis, and cold lower extremities as well as stypsis are present in both patients who are currently fed by mouth. These two girls show neurological and neurovegetative symptoms as well as somatic features consistent with a diagnosis of congenital RTT variant. It should only be noted that a retrospective assessment concerning the possible presence of a regression was not feasible. We attempted to compare their phenotype with the four other MECP2-mutated girls described as congenital variants.9Huppke P. Laccone F. Kramer N. Engel W. Hanefeld F. Rett syndrome: Analysis of MECP2 and clinical characterization of 31 patients.Hum. Mol. Genet. 2000; 9: 1369-1375Crossref PubMed Scopus (196) Google Scholar, 10Monros E. Armstrong J. Aibar E. Poo P. Canos I. Pineda M. Rett syndrome in Spain: Mutation analysis and clinical correlations.Brain Dev. 2001; 1: S251-S253Abstract Full Text Full Text PDF Scopus (73) Google Scholar, 11Smeets E. Schollen E. Moog U. Matthijs G. Herbergs J. Smeets H. Curfs L. Schrander-Stumpel C. Fryns J.P. Rett syndrome in adolescent and adult females: Clinical and molecular genetic findings.Am. J. Med. Genet. A. 2003; 122: 227-233Crossref Scopus (49) Google Scholar However, they have been reported with very little detail, thereby hampering a posteriori clinical re-evaluation according to the revised criteria.19Hagberg B. Hanefeld F. Percy A. Skjeldal O. An update on clinically applicable diagnostic criteria in Rett syndrome. Comments to Rett Syndrome Clinical Criteria Consensus Panel Satellite to European Paediatric Neurology Society Meeting, Baden Baden, Germany, 11 September 2001.Eur. J. Paediatr. Neurol. 2002; 6: 293-297Abstract Full Text PDF PubMed Scopus (346) Google Scholar According to the new criteria, in the classic form, psychomotor development may have been delayed from birth; thus, a re-evaluation of these four patients would have lead to their reclassification as classic form. Alternatively, the disruption of either MeCP2 or FoxG1 may lead to a phenotype, namely the congenital variant, indistinguishable at the level of the clinical and instrumental investigations performed. A translocation with inversion affecting fetal isoforms of FOXG1 was recently described in a 7-year-old girl.18Shoichet S.A. Kunde S.A. Viertel P. Schell-Apacik C. von Voss H. Tommerup N. Ropers H.H. Kalscheuer V.M. Haploinsufficiency of novel FOXG1B variants in a patient with severe mental retardation, brain malformations and microcephaly.Hum. Genet. 2005; 117: 536-544Crossref PubMed Scopus (79) Google Scholar She had acquired microcephaly, alalia, inability to sit and walk, and epilepsy in common with the present cases. Corpus callosum was absent, whereas in our cases, it was hypoplasic. Stereotypic hand activities were not mentioned, and tetraplegia was described.18Shoichet S.A. Kunde S.A. Viertel P. Schell-Apacik C. von Voss H. Tommerup N. Ropers H.H. Kalscheuer V.M. Haploinsufficiency of novel FOXG1B variants in a patient with severe mental retardation, brain malformations and microcephaly.Hum. Genet. 2005; 117: 536-544Crossref PubMed Scopus (79) Google Scholar The clinical features of this patient have something in common with a RTT phenotype. The impairment of only fetal FOXG1 isoforms and the possible contribution of genes at the other two breakpoints of the complex rearrangement might explain the phenotypic differences. The mouse ortholog Foxg1 has a restricted expression domain in the central nervous system coinciding with the emergence of the telencephalic structures of the brain. Its function has been extensively characterized and found to promote telencephalon development by sustaining proliferation of the progenitor pool and preventing premature cortical neural differentiation.21Hanashima C. Shen L. Li S.C. Lai E. Brain factor-1 controls the proliferation and differentiation of neocortical progenitor cells through independent mechanisms.J. Neurosci. 2002; 22: 6526-6536Crossref PubMed Google Scholar, 22Seoane J. Le H.V. Shen L. Anderson S.A. Massague J. Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation.Cell. 2004; 117: 211-223Abstract Full Text Full Text PDF PubMed Scopus (807) Google Scholar In agreement, FoxG1 expression is found in the proliferating neuroepithelium starting from early development onward.23Tao W. Lai E. Telencephalon-restricted expression of BF-1, a new member of the HNF-3/fork head gene family, in the developing rat brain.Neuron. 1992; 8: 957-966Abstract Full Text PDF PubMed Scopus (343) Google Scholar This expression profile might explain the particular early onset of the neurological symptoms displayed by the patients. Despite its early expression in telencephalon development, in this study we found that Foxg1 expression is detectable in the differentiating cortical compartment in the postnatal stages, although at lower levels with respect to the early embryonic phases (Figure 3A). This expression profile overlaps with the described MeCP2 expression domain in cortical tissues, in differentiating and mature neurons (Figures 3A and 3B). Foxg1 homozygote-mutant mice die shortly after birth with severe brain defects.24Martynoga B. Morrison H. Price D.J. Mason J.O. Foxg1 is required for specification of ventral telencephalon and region-specific regulation of dorsal telencephalic precursor proliferation and apoptosis.Dev. Biol. 2005; 283: 113-127Crossref PubMed Scopus (281) Google Scholar, 25Xuan S. Baptista C.A. Balas G. Tao W. Soares V.C. Lai E. Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres.Neuron. 1995; 14: 1141-1152Abstract Full Text PDF PubMed Scopus (462) Google Scholar, 26Hanashima C. Fernandes M. Hebert J.M. Fishell G. The role of Foxg1 and dorsal midline signaling in the generation of Cajal-Retzius subtypes.J. Neurosci. 2007; 27: 11103-11111Crossref PubMed Scopus (106) Google Scholar Unfortunately, the severe compromised development of Foxg1 mutant telencephali has prevented the analysis of its function in more differentiated neurons. At the single-cell level, FoxG1 localizes in the nuclear compartment but is excluded from the MeCP2-positive heterochromatic foci both in nonneural and primary neurons (Figures 3C–3J). These findings suggest that, differently from MeCP2, FoxG1 is not a transcriptional repressor stably associated with heterochromatin. However, both proteins have a large colocalization domain in other nuclear compartments (Figure 3I). Overall, these data suggest that FoxG1 may exert some additional functions in differentiating and mature neurons, thus sharing similarities with those described for MeCP2. These findings may provide some biological evidence for the development of similar clinical manifestations in disorders affecting the two genes. However, it is also possible that the two transcriptional regulators act on different stages of the process that leads to proper cortical development, from early cell-fate decisions to later circuit connectivity and dendritic development. FoxG1 shares some interesting analogies with MeCP2 in its molecular functions, raising the question whether the two protein networks may interact in some circumstances and on selective common targets. Future studies will address this intriguing hypothesis. Recently, heterozygous Foxg1+/− mice were found to display subtler defects including a reduction in size of the corpus callosum together with specific patterning defects.25Xuan S. Baptista C.A. Balas G. Tao W. Soares V.C. Lai E. Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres.Neuron. 1995; 14: 1141-1152Abstract Full Text PDF PubMed Scopus (462) Google Scholar, 27Shen Q. Wang Y. Dimos J.T. Fasano C.A. Phoenix T.N. Lemischka I.R. Ivanova N.B. Stifani S. Morrisey E.E. Temple S. The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells.Nat. Neurosci. 2006; 9: 743-751Crossref PubMed Scopus (468) Google Scholar Furthermore, heterozygous Foxg1+/− exhibit learning deficits based on fear-condition behavioral tests associated with a loss of postnatal neurogenesis in the hippocampus.27Shen Q. Wang Y. Dimos J.T. Fasano C.A. Phoenix T.N. Lemischka I.R. Ivanova N.B. Stifani S. Morrisey E.E. Temple S. The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells.Nat. Neurosci. 2006; 9: 743-751Crossref PubMed Scopus (468) Google Scholar These mice represent a very interesting animal model for further investigation about how Foxg1 haploinsufficiency may impact on brain development and neuronal maturation and function. In conclusion, we demonstrated that FOXG1 is a previously unrecognized gene responsible for variant Rett syndrome. It is worth noting that in the revised criteria for Rett syndrome the female sex is no longer present as inclusion criteria.19Hagberg B. Hanefeld F. Percy A. Skjeldal O. An update on clinically applicable diagnostic criteria in Rett syndrome. Comments to Rett Syndrome Clinical Criteria Consensus Panel Satellite to European Paediatric Neurology Society Meeting, Baden Baden, Germany, 11 September 2001.Eur. J. Paediatr. Neurol. 2002; 6: 293-297Abstract Full Text PDF PubMed Scopus (346) Google Scholar This seemed to open the door to the discovery of an autosomal gene. We would first like to thank the Rett patients and their families. This work was supported by Telethon grants GTB07001 to A.R. and GGP07181 to V.B., by the EuroRETT E-RARE network to A.R. and to V.B, and by the Emma and Ernesto Rulfo Foundation to A.R. Download .pdf (.02 MB) Help with pdf files Document S1. One Table The URLs for data presented herein are as follows:Italian Rett database and biobank, http://www.biobank.unisi.it/Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/