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Novel mutation in Forkhead box G1 (FOXG1) gene in an Indian patient with Rett syndrome
Gene 538 (2014) 109–112
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Novel mutation in Forkhead box G1 (FOXG1) gene in an Indian patient with Rett syndrome Dhanjit Kumar Das a,⁎, Vaishali Jadhav a, Vikas C. Ghattargi a, Vrajesh Udani b a b
Genetic Research Centre, National Institute for Research in Reproductive Health (ICMR), Jahangir Merwanji Street, Parel, Mumbai 400 012, India Department of Pediatric Neurology, Hinduja National Hospital and Research Centre, Mahim, Mumbai 400 016, India
a r t i c l e
i n f o
Article history: Accepted 24 December 2013 Available online 9 January 2014 Keywords: Rett syndrome FOXG1 Mutation DNA sequencing MECP2
a b s t r a c t Rett syndrome (RTT) is a severe neurodevelopmental disorder characterized by the progressive loss of intellectual functioning, fine and gross motor skills and communicative abilities, deceleration of head growth, and the development of stereotypic hand movements, occurring after a period of normal development. The classic form of RTT involves mutation in MECP2 while the involvement of CDKL5 and FOXG1 genes has been identified in atypical RTT phenotype. FOXG1 gene encodes for a fork-head box protein G1, a transcription factor acting primarily as transcriptional repressor through DNA binding in the embryonic telencephalon as well as a number of other neurodevelopmental processes. In this report we have described the molecular analysis of FOXG1 gene in Indian patients with Rett syndrome. FOXG1 gene mutation analysis was done in a cohort of 34 MECP2/CDKL5 mutation negative RTT patients. We have identified a novel mutation (p. D263VfsX190) in FOXG1 gene in a patient with congenital variant of Rett syndrome. This mutation resulted into a frameshift, thereby causing an alteration in the reading frames of the entire coding sequence downstream of the mutation. The start position of the frameshift (Asp263) and amino acid towards the carboxyl terminal end of the protein was found to be well conserved across species using multiple sequence alignment. Since the mutation is located at forkhead binding domain, the resultant mutation disrupts the secondary structure of the protein making it non-functional. This is the first report from India showing mutation in FOXG1 gene in Rett syndrome. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Rett syndrome (RTT; OMIM #312750) is a progressive neurodevelopmental disorder primarily found only in girls. Clinically it can be classified into classic and atypical phenotype. Classic Rett syndrome is characterized by 6 to 18 months of apparently normal development before developing severe problems with communication, language, learning, coordination and other brain functions. The affected children develop microcephaly and tend to grow more slowly than other normal children. They also loss purposeful use of their hands and begin making repeated hand wringing, washing or clapping motions. Other signs and symptoms include breathing abnormalities, seizures and sleep disturbances. There are many variants of this disorder and those are collectively called atypical forms. The severe types of variants are known as the congenital variant, which has no period of normal development, and an early-onset seizure variant characterized by seizures that begin in infancy. In these atypical variants, deceleration of head growth starts at the age of four months and leads to severe microcephaly. There is absence of voluntary hand use and severe impairments of motor Abbreviations: RTT, Rett syndrome; MECP2, Methyl CpG Binding Protein 2; CDKL5, Cyclin Dependent Protein Kinase like 5; FOXG1, Forkhead box G1. ⁎ Corresponding author. Tel.: +91 22 24192108; fax: +91 22 24147843. E-mail address: [email protected] (D.K. Das). 0378-1119/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.12.063
development are observed. Previous reports on brain imaging showed corpus callosum hypoplasia in most of the cases (Guerrini and Parrini, 2012; Mencarelli et al., 2009, 2010). Genetic analysis revealed mutations primarily in the MECP2 gene, located at Xq28 comprising almost 95% of classic RTT; while the early onset seizure variant results from mutations in the CDKL5 gene, located at Xp22.5. In some severe RTT congenital variant cases, having no mutations in MECP2 and CDKL5 genes, Ariani et al. (2008) had shown the involvement of FOXG1 gene, located at 14q12 using combination of arrayCGH and candidate gene approach (Ariani et al., 2008), In this variant, the affected girls present with the same clinical features as in classic RTT; however, in addition they are flaccid and postnatal growth retardation from the very first months of life (Mencarelli et al., 2010). FoxG1 (MIM# 164874) is a DNA-binding transcription factor and is composed of 489 amino acids. The protein has 3 functional domains; a forkhead binding domain (FBD) that spans from amino acid residue 181 to residue 275; Groucho-binding domain (GBD) spans from amino acid residue 307 to residue 317 and the JARID-1B binding domain (JBD) spans from amino acid residue 383 to residue 406. The FBD causes repression of target genes by recruiting transcriptional co-repressor proteins during brain development. Abnormalities in this gene affect the early brain development, thereby impairing the cognitive abilities of the affected individuals. The first report stating the involvement of FOXG1 was seen in a female patient exhibiting a severe cognitive
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disability associated with complete agenesis of corpus callosum and microcephaly and was reported with a de-novo translocation of t(2,14) (p22,q12) that disrupts FOXG1 (Shoichet et al., 2005). Later on Ariani et al. (2008) showed the involvement of FOXG1 in RTT using oligo array-CGH where a de-novo 3 Mb interstitial deletion of chromosome 14q12 was present in a 7 year-old girl (Ariani et al., 2008). They have further shown that mutations in FOXG1 gene are responsible for RTT using combination of DHPLC and real-time quantitative PCR in a cohort of 53 MECP2/CDKL5 mutation negative RTT patients, including 7 classic, 21 preserved speech, 7 early-onset seizures, 1 “forme fruste”, 2 congenital variants and 15 Rett like cases. Almost all mutations that occurred in FOXG1 gene are de-novo. Eight point mutations have been identified in 15 cases including translocation, duplication and large deletion (Mencarelli et al., 2010) along with two de novo mutations (p.Y416X and p.E154GfsX300) in two unrelated girls (Bahi-Buisson et al., 2010; Philippe et al., 2010). We have reported the spectrum of mutations in MECP2 (Das et al., 2013b) and CDKL5 (Das et al., 2013a) genes in large cohorts of individuals suffering from typical and variant forms of RTT. Here, we report the molecular screening of the FOXG1 gene in a cohort of 34 female patients with atypical and classic forms including early congenital variant of RTT. This is the first report from India showing the involvement of FOXG1 gene in Indian patients with Rett Syndrome.
2. Materials and methods 2.1. Patients The study was approved by the Institutional Ethics Committee. Written informed consent was obtained from the parents and blood samples were collected for genetic analysis from the patients and controls. All patients screened in this study were sporadic. A total of 34 patients with Rett syndrome were analyzed in this study. The children were divided into classic and atypical Rett disorders according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition) (DSM IV).
2.2. Genomic DNA isolation & PCR amplification Whole blood samples from above patients were collected in EDTA tubes. Genomic DNA was isolated from 2.0 ml of blood collected from the above patients using QIAmp DNA extraction kit (Qiagen, GmBH, Germany). After isolation, the integrity was checked by running a 0.8% agarose gel electrophoresis. PCR amplification was performed in 50 μl of 10 mM Tris HCl (pH 8.3) containing 50 mM KCl, 1.5 mM MgCl2, 200 mM of each dNTPs (dATP, dCTP, dGTP and dTTP), 20 pmol primers, 250 ng template DNA and 1.5 units of Taq DNA polymerase (MBIFermantas, MD). The single coded exon was amplified in two fragments using specific primers (Table 1) designed from the wild type FOXG1 sequence (GenBank Accession: NM_005249.4). PCR was cycled 35 times; each cycle consisted of denaturation for 1 min at 94 °C, annealing for 45 s at 60–68 °C and extension for 1 min at 72 °C and final extension for 10 min. After amplification, 3 μl of PCR product was subjected to electrophoresis on 1% agarose gel for 45 min at 100 V in TAE buffer and bands were stained with ethidium bromide (0.5 mg/ml).
2.3. Sequencing and sequence analysis PCR amplified products were purified using ExoSAP-IT (a PCR clean up product — Affimatrix, Santa Clara, CA, USA), a specially formulated enzyme for the removal of unwanted primers and dNTPs from a PCR product mixture with no interference in sequencing. The PCR purified products of patients were sequenced using gene specific primers on ABI PRISM 3130xl version 3.1 DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The sequences were analyzed for the presence of mutations in the coding region of FOXG1 compared with the reference sequence (NM_005249.4) using Lasergen program (DNASTAR, Inc., Madison, USA). Multiple sequence alignment was carried out using MegAlign program of DNASTAR and WebLogo was created using online software (http://weblogo.berkeley.edu/WebLogo: A Sequence Logo Generator). 3. Results In this study, we have screened 34 female patients (Patients 1–34) with features of classic and atypical Rett syndrome for presence of mutations in the FOXG1 gene, who were negative for MECP2/CDKL5 gene mutation. Patients were classified based on clinical symptoms according to the DSM-IV criteria. The entire coding region of FOXG1 gene was amplified in two fragments using gene specific primers. Fragment 1 amplified a product of 771 bp and Fragment 2 amplified 1239 bp. Sequence analysis revealed a deletion of 5 bp ranging from position 788 to 792 (c.788_ 792delACGTG; p.D263VfsX190) in patient 17 (Fig. 1). This deletion is a frameshift mutation, thereby causing an alteration in the reading frames of the entire coding sequence downstream of the mutation. This mutation generates a stop codon at amino acid position 453 resulting into truncated FoxG1 protein. Absence of this mutation in parent sample indicated the de-novo nature of the mutation in patient 17. Moreover, the possibility of polymorphism was ruled out by mutation analyses in 25 control samples. The mutation positive female patient presented at 2 years of age with delayed development and a few stereotypies. She was born after an uneventful pregnancy to non-consanguineous parents with birth weight of 2.45 kg. Unlike in the classic Rett syndrome, she did not have a normal period of development. There was overall delay in all her developmental milestones. Head control was achieved at 8 months, sitting unaided at 14 months and standing with support by 2 years of age. She could say disyllables, trouble in speaking; looked when called and had poor eye contact. Additionally she had recurrent respiratory problems, starting at the age of 2 months. Epilepsy was evident at the age of 9 months. She did not show any purposeful hand use and developed midline stereotypies such as hand wringing and mouthing and tactile hypersensitivity such as brushing aversion. The differences in clinical presentation in patients were listed at supplementary Table 1. Complete physical and neurological evaluation showed presence of microcephaly (less than 5th percentile); generalized hypotonia. EEG at around 1 year demonstrated slow and epileptiform abnormalities, bilateral interictal epileptiform discharges (IED) at fronto-central regions over the central regions. MRI revealed progressive cortical and subcortical atrophy. Multiple sequence alignment was carried out for this mutation using MegAlign program of DNASTAR with its homologs retrieved from the NCBI database and those are amino acid sequences of Homo sapiens
Table 1 Primer sequence for amplification of the FOXG1 gene fragments. Primer name
Sequence of primer
Annealing temperature
Fragment length
FOXG1-Frag1F FOXG1-Frag1R FOXG1-Frag2F FOXG1-Frag2R
5′ GGT GGT TGC TGC TTT TGC TA 3′ 5′ ATC ATG ATG AGC GCG TTG TA 3′ 5′ CCG GAC GAG AAG GAG AAG G 3′ 5′ TGT ATT CTC CCC ACA TTG CAC 3′
60 °C 68 °C
771 bp 1239 bp
D.K. Das et al. / Gene 538 (2014) 109–112
111
1), Agama atra (ABD38852.1), Psammobates geometricus (ABD38851.1), Danio rerio (NP_001038680.1), Epomophorus gambianus (ABD38847.1), Pipistrellus rusticus (ABD38846.1), Xenopus laevis (NP_001079165.1), Bombyx mori (NP_001170880.1). Sequence alignment revealed that the sequences ranging from the frameshift start position (Asp263) to the carboxyl terminal end of the mutated protein were found to be well conserved across species (Fig. 2a). WebLogo was generated using online software (http://weblogo.berkeley.edu/ WebLogo: A Sequence Logo Generator). Analysis of WebLogo has demonstrated that these frameshift sequences were conserved among all the species studied (Fig. 2b). 4. Discussion Fig. 1. DNA sequence chromatogram showing the presence of deletion mutation p. D263VfsX190 in FOXG1 gene.
(Accession No. NP_005240.3), Gorilla gorilla gorilla (XP_004055090.1), Nomascus leucogenys (XP_003261005.2), Macaca mulatta (AFE71616.1), Cebus capucinus (ABD38844.1), Chlorocebus pygerythrus (ABD38845.1), Callithrix jacchus (XP_002753744.2), Bos taurus (DAA17496.1), Equus burchellii (ABD38848.1), Ovis aries (XP_004018155.1), Ceratotherium simum (ABD38849.1), Canis lupus familiaris (XP_547758.3), Felis catus (XP_003987623.1), Crocodylus niloticus (ABD38850.1), Gallus gallus (NP_ 990523.1), Rattus norvegicus (NP_036692.1), Mus musculus (NP_032267.
Rett syndrome is one of the leading causes of mental retardation and developmental regression in females. There is apparently normal development during the first months of life in patients with RTT. They show characteristic clinical features such as microcephaly, hand wringing, autism, seizures and loss of speech (Ariani et al., 2008). The disorder is a dysfunction of the aminergic neurons of the brain stem, starting during early infancy. Besides the classic form, several RTT variants have also been described (Hagberg and Skjeldal, 1994). Mutations in the MECP2 have been identified in almost 95% of classic RTT cases. Only 5% of patients affected by RTT variants show genetic heterogeneity, with the existence of at least one other locus (Guerrini and
Fig. 2. The extent of sequence conservation in FoxG1 compared with its homologs. (a) Multiple sequence alignment of p.D263VfsX190 with homologous sequence from different species. The Aspartic acid (D) residue at position 263, along with the frame-shift sequences caused due to the presence of mutation was shaded with light pink color. (b) WebLogo showing the conservation of deletion mutation p.D263VfsX190, showing the conserved aspartic acid (D) residue along with the frame-shift sequence across species indicated by light pink shade.
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Parrini, 2012). The involvement of CDKL5 and FOXG1 gene mutations has been identified in atypical form of RTT (Neul et al., 2010). Our study was focused to identify mutation in FOXG1 gene in a cohort of 34 MECP2/CDKL5 mutation negative RTT patients. FoxG1 primarily acts as a transcriptional repressor through DNA binding and belongs to the winged-helix or forkhead family of transcription factors. FoxG1 is expressed selectively in rapidly proliferating cells during early brain development, and recruits other proteins to regulate the rate of neurogenesis by keeping cells in a proliferative state and by inhibiting their differentiation into neurons (Dastidar et al., 2011). The FoxG1 protein consists of a highly conserved 100 bp residue forkhead DNAbinding domain (FBD). FoxG1 recruits transcriptional co-repressor proteins, a histone demethylase and Groucho (Gro), via two proteinbinding domains; the 10 residue JARID1B binding domain (JBD) and 23 residues Gro-binding domain (GBD). Mutations in these binding domains lead to disruption of FoxG1 protein. The position of mutation determines the extent and sites of disruption of FoxG1 protein (Florian et al., 2012). Here we have identified a 5 bp deletion mutation (c.788_ 792delACGTG) in FOXG1 located in the forkhead binding domain (p.D263VfsX190). This mutation caused a frameshift at 263 position of the protein, thereby, affecting all the downstream amino acid residues. The frameshift mutation introduced a stop codon at 453 position of mRNA, resulting in a truncated protein that lacks the GBD and JBD. This non-functional protein cannot recruit co-repressor proteins, thereby, affecting the normal functions of brain cells resulting in severe cognitive related symptoms in the patient. The conservation of deletion mutation (c.788_792delACGTG) was verified by using multiple sequence alignment and WebLogo. Analysis revealed that the altered sequences caused by this mutation was phylogenetically conserved across species (Arthropods to Mammals) indicating the importance of this residue. The extent of conservation of this region defined the importance on its structure and function of the protein. The genotype–phenotype correlation suggests that the truncated protein led to more severe symptoms in patient 17. All the patients had microcephaly and the age of onset of symptoms was variable. However, the mutation positive patient (P17) had no period of normal development and all her development milestones had been delayed. She also had neurological symptoms, hypotonia, severe microcephaly (less than 5th percentile), along with wringing of hands and with the most severe symptoms than any other patient. In conclusion, we have identified a deletion mutation in FOXG1 gene in one patient. No mutations have been identified in the other 33 cases. Mutations in FOXG1 are relatively common in patients with severe congenital variant of Rett syndrome. The phenotypes of Rett syndrome
have many differentials; however, a genetically based confirmed diagnosis would help in management and counseling. The molecular diagnosis approach described in this study can be a basis for further development in clinical use. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2013.12.063.
Conflict of interest The authors declare no conflict of interest. Acknowledgment We thank all the patients and their families for their contribution in this study. The authors are also thankful to the Director, NIRRH for providing the necessary facilities; the Indian Council of Medical Research (ICMR); the Department of Science & Technology (SR/FT/LS-87/2010), Govt. of India for providing financial grants for the study. References Ariani, F., et al., 2008. FOXG1 is responsible for the congenital variant of Rett syndrome. Am. J. Hum. Genet. 83, 89–93. Bahi-Buisson, N., et al., 2010. Revisiting the phenotype associated with FOXG1 mutations: two novel cases of congenital Rett variant. Neurogenetics 11, 241–249. Das, D.K., Mehta, B., Menon, S.R., Raha, S., Udani, V., 2013a. Novel mutations in cyclindependent kinase-like 5 (CDKL5) gene in Indian cases of Rett Syndrome. Neuromol. Med. 15, 218–225. Das, D.K., Raha, S., Sanghavi, D., Maitra, A., Udani, V., 2013b. Spectrum of MECP2 gene mutations in a cohort of Indian patients with Rett syndrome: report of two novel mutations. Gene 515, 78–83. Dastidar, S.G., Landrieu, P.M.Z., D'Mello, S.R., 2011. FoxG1 promotes the survival of postmitotic neurons. J. Neurosci. Off. J. Soc. Neurosci. 31, 402–413. Florian, C., Bahi-Buisson, N., Bienvenu, T., 2012. FOXG1-related disorders: from clinical description to molecular genetics. Mol. Syndromol. 2, 153–163. Guerrini, R., Parrini, E., 2012. Epilepsy in Rett syndrome, and CDKL5- and FOXG1-generelated encephalopathies. Epilepsia 53, 2067–2078. Hagberg, B.A., Skjeldal, O.H., 1994. Rett variants: a suggested model for inclusion criteria. Pediatr. Neurol. 11, 5–11. Mencarelli, M.A., et al., 2009. 14q12 Microdeletion syndrome and congenital variant of Rett syndrome. Eur. J. Med. Chem. Genet. 52, 148–152. Mencarelli, M.A., et al., 2010. Novel FOXG1 mutations associated with the congenital variant of Rett syndrome. J. Med. Genet. 47, 49–53. Neul, J.L., et al., 2010. Rett syndrome: revised diagnostic criteria and nomenclature. Ann. Neurol. 68, 944–950. Philippe, C., et al., 2010. Phenotypic variability in Rett syndrome associated with FOXG1 mutations in females. J. Med. Genet. 47, 59–65. Shoichet, S.A., et al., 2005. Haploinsufficiency of novel FOXG1B variants in a patient with severe mental retardation, brain malformations and microcephaly. Hum. Genet. 117, 536–544.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Novel mutation in Forkhead box G1 (FOXG1) gene in an Indian patient with Rett syndrome Dhanjit Kumar Das a,⁎, Vaishali Jadhav a, Vikas C. Ghattargi a, Vrajesh Udani b a b
Genetic Research Centre, National Institute for Research in Reproductive Health (ICMR), Jahangir Merwanji Street, Parel, Mumbai 400 012, India Department of Pediatric Neurology, Hinduja National Hospital and Research Centre, Mahim, Mumbai 400 016, India
a r t i c l e
i n f o
Article history: Accepted 24 December 2013 Available online 9 January 2014 Keywords: Rett syndrome FOXG1 Mutation DNA sequencing MECP2
a b s t r a c t Rett syndrome (RTT) is a severe neurodevelopmental disorder characterized by the progressive loss of intellectual functioning, fine and gross motor skills and communicative abilities, deceleration of head growth, and the development of stereotypic hand movements, occurring after a period of normal development. The classic form of RTT involves mutation in MECP2 while the involvement of CDKL5 and FOXG1 genes has been identified in atypical RTT phenotype. FOXG1 gene encodes for a fork-head box protein G1, a transcription factor acting primarily as transcriptional repressor through DNA binding in the embryonic telencephalon as well as a number of other neurodevelopmental processes. In this report we have described the molecular analysis of FOXG1 gene in Indian patients with Rett syndrome. FOXG1 gene mutation analysis was done in a cohort of 34 MECP2/CDKL5 mutation negative RTT patients. We have identified a novel mutation (p. D263VfsX190) in FOXG1 gene in a patient with congenital variant of Rett syndrome. This mutation resulted into a frameshift, thereby causing an alteration in the reading frames of the entire coding sequence downstream of the mutation. The start position of the frameshift (Asp263) and amino acid towards the carboxyl terminal end of the protein was found to be well conserved across species using multiple sequence alignment. Since the mutation is located at forkhead binding domain, the resultant mutation disrupts the secondary structure of the protein making it non-functional. This is the first report from India showing mutation in FOXG1 gene in Rett syndrome. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Rett syndrome (RTT; OMIM #312750) is a progressive neurodevelopmental disorder primarily found only in girls. Clinically it can be classified into classic and atypical phenotype. Classic Rett syndrome is characterized by 6 to 18 months of apparently normal development before developing severe problems with communication, language, learning, coordination and other brain functions. The affected children develop microcephaly and tend to grow more slowly than other normal children. They also loss purposeful use of their hands and begin making repeated hand wringing, washing or clapping motions. Other signs and symptoms include breathing abnormalities, seizures and sleep disturbances. There are many variants of this disorder and those are collectively called atypical forms. The severe types of variants are known as the congenital variant, which has no period of normal development, and an early-onset seizure variant characterized by seizures that begin in infancy. In these atypical variants, deceleration of head growth starts at the age of four months and leads to severe microcephaly. There is absence of voluntary hand use and severe impairments of motor Abbreviations: RTT, Rett syndrome; MECP2, Methyl CpG Binding Protein 2; CDKL5, Cyclin Dependent Protein Kinase like 5; FOXG1, Forkhead box G1. ⁎ Corresponding author. Tel.: +91 22 24192108; fax: +91 22 24147843. E-mail address: [email protected] (D.K. Das). 0378-1119/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.12.063
development are observed. Previous reports on brain imaging showed corpus callosum hypoplasia in most of the cases (Guerrini and Parrini, 2012; Mencarelli et al., 2009, 2010). Genetic analysis revealed mutations primarily in the MECP2 gene, located at Xq28 comprising almost 95% of classic RTT; while the early onset seizure variant results from mutations in the CDKL5 gene, located at Xp22.5. In some severe RTT congenital variant cases, having no mutations in MECP2 and CDKL5 genes, Ariani et al. (2008) had shown the involvement of FOXG1 gene, located at 14q12 using combination of arrayCGH and candidate gene approach (Ariani et al., 2008), In this variant, the affected girls present with the same clinical features as in classic RTT; however, in addition they are flaccid and postnatal growth retardation from the very first months of life (Mencarelli et al., 2010). FoxG1 (MIM# 164874) is a DNA-binding transcription factor and is composed of 489 amino acids. The protein has 3 functional domains; a forkhead binding domain (FBD) that spans from amino acid residue 181 to residue 275; Groucho-binding domain (GBD) spans from amino acid residue 307 to residue 317 and the JARID-1B binding domain (JBD) spans from amino acid residue 383 to residue 406. The FBD causes repression of target genes by recruiting transcriptional co-repressor proteins during brain development. Abnormalities in this gene affect the early brain development, thereby impairing the cognitive abilities of the affected individuals. The first report stating the involvement of FOXG1 was seen in a female patient exhibiting a severe cognitive
110
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disability associated with complete agenesis of corpus callosum and microcephaly and was reported with a de-novo translocation of t(2,14) (p22,q12) that disrupts FOXG1 (Shoichet et al., 2005). Later on Ariani et al. (2008) showed the involvement of FOXG1 in RTT using oligo array-CGH where a de-novo 3 Mb interstitial deletion of chromosome 14q12 was present in a 7 year-old girl (Ariani et al., 2008). They have further shown that mutations in FOXG1 gene are responsible for RTT using combination of DHPLC and real-time quantitative PCR in a cohort of 53 MECP2/CDKL5 mutation negative RTT patients, including 7 classic, 21 preserved speech, 7 early-onset seizures, 1 “forme fruste”, 2 congenital variants and 15 Rett like cases. Almost all mutations that occurred in FOXG1 gene are de-novo. Eight point mutations have been identified in 15 cases including translocation, duplication and large deletion (Mencarelli et al., 2010) along with two de novo mutations (p.Y416X and p.E154GfsX300) in two unrelated girls (Bahi-Buisson et al., 2010; Philippe et al., 2010). We have reported the spectrum of mutations in MECP2 (Das et al., 2013b) and CDKL5 (Das et al., 2013a) genes in large cohorts of individuals suffering from typical and variant forms of RTT. Here, we report the molecular screening of the FOXG1 gene in a cohort of 34 female patients with atypical and classic forms including early congenital variant of RTT. This is the first report from India showing the involvement of FOXG1 gene in Indian patients with Rett Syndrome.
2. Materials and methods 2.1. Patients The study was approved by the Institutional Ethics Committee. Written informed consent was obtained from the parents and blood samples were collected for genetic analysis from the patients and controls. All patients screened in this study were sporadic. A total of 34 patients with Rett syndrome were analyzed in this study. The children were divided into classic and atypical Rett disorders according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition) (DSM IV).
2.2. Genomic DNA isolation & PCR amplification Whole blood samples from above patients were collected in EDTA tubes. Genomic DNA was isolated from 2.0 ml of blood collected from the above patients using QIAmp DNA extraction kit (Qiagen, GmBH, Germany). After isolation, the integrity was checked by running a 0.8% agarose gel electrophoresis. PCR amplification was performed in 50 μl of 10 mM Tris HCl (pH 8.3) containing 50 mM KCl, 1.5 mM MgCl2, 200 mM of each dNTPs (dATP, dCTP, dGTP and dTTP), 20 pmol primers, 250 ng template DNA and 1.5 units of Taq DNA polymerase (MBIFermantas, MD). The single coded exon was amplified in two fragments using specific primers (Table 1) designed from the wild type FOXG1 sequence (GenBank Accession: NM_005249.4). PCR was cycled 35 times; each cycle consisted of denaturation for 1 min at 94 °C, annealing for 45 s at 60–68 °C and extension for 1 min at 72 °C and final extension for 10 min. After amplification, 3 μl of PCR product was subjected to electrophoresis on 1% agarose gel for 45 min at 100 V in TAE buffer and bands were stained with ethidium bromide (0.5 mg/ml).
2.3. Sequencing and sequence analysis PCR amplified products were purified using ExoSAP-IT (a PCR clean up product — Affimatrix, Santa Clara, CA, USA), a specially formulated enzyme for the removal of unwanted primers and dNTPs from a PCR product mixture with no interference in sequencing. The PCR purified products of patients were sequenced using gene specific primers on ABI PRISM 3130xl version 3.1 DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The sequences were analyzed for the presence of mutations in the coding region of FOXG1 compared with the reference sequence (NM_005249.4) using Lasergen program (DNASTAR, Inc., Madison, USA). Multiple sequence alignment was carried out using MegAlign program of DNASTAR and WebLogo was created using online software (http://weblogo.berkeley.edu/WebLogo: A Sequence Logo Generator). 3. Results In this study, we have screened 34 female patients (Patients 1–34) with features of classic and atypical Rett syndrome for presence of mutations in the FOXG1 gene, who were negative for MECP2/CDKL5 gene mutation. Patients were classified based on clinical symptoms according to the DSM-IV criteria. The entire coding region of FOXG1 gene was amplified in two fragments using gene specific primers. Fragment 1 amplified a product of 771 bp and Fragment 2 amplified 1239 bp. Sequence analysis revealed a deletion of 5 bp ranging from position 788 to 792 (c.788_ 792delACGTG; p.D263VfsX190) in patient 17 (Fig. 1). This deletion is a frameshift mutation, thereby causing an alteration in the reading frames of the entire coding sequence downstream of the mutation. This mutation generates a stop codon at amino acid position 453 resulting into truncated FoxG1 protein. Absence of this mutation in parent sample indicated the de-novo nature of the mutation in patient 17. Moreover, the possibility of polymorphism was ruled out by mutation analyses in 25 control samples. The mutation positive female patient presented at 2 years of age with delayed development and a few stereotypies. She was born after an uneventful pregnancy to non-consanguineous parents with birth weight of 2.45 kg. Unlike in the classic Rett syndrome, she did not have a normal period of development. There was overall delay in all her developmental milestones. Head control was achieved at 8 months, sitting unaided at 14 months and standing with support by 2 years of age. She could say disyllables, trouble in speaking; looked when called and had poor eye contact. Additionally she had recurrent respiratory problems, starting at the age of 2 months. Epilepsy was evident at the age of 9 months. She did not show any purposeful hand use and developed midline stereotypies such as hand wringing and mouthing and tactile hypersensitivity such as brushing aversion. The differences in clinical presentation in patients were listed at supplementary Table 1. Complete physical and neurological evaluation showed presence of microcephaly (less than 5th percentile); generalized hypotonia. EEG at around 1 year demonstrated slow and epileptiform abnormalities, bilateral interictal epileptiform discharges (IED) at fronto-central regions over the central regions. MRI revealed progressive cortical and subcortical atrophy. Multiple sequence alignment was carried out for this mutation using MegAlign program of DNASTAR with its homologs retrieved from the NCBI database and those are amino acid sequences of Homo sapiens
Table 1 Primer sequence for amplification of the FOXG1 gene fragments. Primer name
Sequence of primer
Annealing temperature
Fragment length
FOXG1-Frag1F FOXG1-Frag1R FOXG1-Frag2F FOXG1-Frag2R
5′ GGT GGT TGC TGC TTT TGC TA 3′ 5′ ATC ATG ATG AGC GCG TTG TA 3′ 5′ CCG GAC GAG AAG GAG AAG G 3′ 5′ TGT ATT CTC CCC ACA TTG CAC 3′
60 °C 68 °C
771 bp 1239 bp
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1), Agama atra (ABD38852.1), Psammobates geometricus (ABD38851.1), Danio rerio (NP_001038680.1), Epomophorus gambianus (ABD38847.1), Pipistrellus rusticus (ABD38846.1), Xenopus laevis (NP_001079165.1), Bombyx mori (NP_001170880.1). Sequence alignment revealed that the sequences ranging from the frameshift start position (Asp263) to the carboxyl terminal end of the mutated protein were found to be well conserved across species (Fig. 2a). WebLogo was generated using online software (http://weblogo.berkeley.edu/ WebLogo: A Sequence Logo Generator). Analysis of WebLogo has demonstrated that these frameshift sequences were conserved among all the species studied (Fig. 2b). 4. Discussion Fig. 1. DNA sequence chromatogram showing the presence of deletion mutation p. D263VfsX190 in FOXG1 gene.
(Accession No. NP_005240.3), Gorilla gorilla gorilla (XP_004055090.1), Nomascus leucogenys (XP_003261005.2), Macaca mulatta (AFE71616.1), Cebus capucinus (ABD38844.1), Chlorocebus pygerythrus (ABD38845.1), Callithrix jacchus (XP_002753744.2), Bos taurus (DAA17496.1), Equus burchellii (ABD38848.1), Ovis aries (XP_004018155.1), Ceratotherium simum (ABD38849.1), Canis lupus familiaris (XP_547758.3), Felis catus (XP_003987623.1), Crocodylus niloticus (ABD38850.1), Gallus gallus (NP_ 990523.1), Rattus norvegicus (NP_036692.1), Mus musculus (NP_032267.
Rett syndrome is one of the leading causes of mental retardation and developmental regression in females. There is apparently normal development during the first months of life in patients with RTT. They show characteristic clinical features such as microcephaly, hand wringing, autism, seizures and loss of speech (Ariani et al., 2008). The disorder is a dysfunction of the aminergic neurons of the brain stem, starting during early infancy. Besides the classic form, several RTT variants have also been described (Hagberg and Skjeldal, 1994). Mutations in the MECP2 have been identified in almost 95% of classic RTT cases. Only 5% of patients affected by RTT variants show genetic heterogeneity, with the existence of at least one other locus (Guerrini and
Fig. 2. The extent of sequence conservation in FoxG1 compared with its homologs. (a) Multiple sequence alignment of p.D263VfsX190 with homologous sequence from different species. The Aspartic acid (D) residue at position 263, along with the frame-shift sequences caused due to the presence of mutation was shaded with light pink color. (b) WebLogo showing the conservation of deletion mutation p.D263VfsX190, showing the conserved aspartic acid (D) residue along with the frame-shift sequence across species indicated by light pink shade.
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Parrini, 2012). The involvement of CDKL5 and FOXG1 gene mutations has been identified in atypical form of RTT (Neul et al., 2010). Our study was focused to identify mutation in FOXG1 gene in a cohort of 34 MECP2/CDKL5 mutation negative RTT patients. FoxG1 primarily acts as a transcriptional repressor through DNA binding and belongs to the winged-helix or forkhead family of transcription factors. FoxG1 is expressed selectively in rapidly proliferating cells during early brain development, and recruits other proteins to regulate the rate of neurogenesis by keeping cells in a proliferative state and by inhibiting their differentiation into neurons (Dastidar et al., 2011). The FoxG1 protein consists of a highly conserved 100 bp residue forkhead DNAbinding domain (FBD). FoxG1 recruits transcriptional co-repressor proteins, a histone demethylase and Groucho (Gro), via two proteinbinding domains; the 10 residue JARID1B binding domain (JBD) and 23 residues Gro-binding domain (GBD). Mutations in these binding domains lead to disruption of FoxG1 protein. The position of mutation determines the extent and sites of disruption of FoxG1 protein (Florian et al., 2012). Here we have identified a 5 bp deletion mutation (c.788_ 792delACGTG) in FOXG1 located in the forkhead binding domain (p.D263VfsX190). This mutation caused a frameshift at 263 position of the protein, thereby, affecting all the downstream amino acid residues. The frameshift mutation introduced a stop codon at 453 position of mRNA, resulting in a truncated protein that lacks the GBD and JBD. This non-functional protein cannot recruit co-repressor proteins, thereby, affecting the normal functions of brain cells resulting in severe cognitive related symptoms in the patient. The conservation of deletion mutation (c.788_792delACGTG) was verified by using multiple sequence alignment and WebLogo. Analysis revealed that the altered sequences caused by this mutation was phylogenetically conserved across species (Arthropods to Mammals) indicating the importance of this residue. The extent of conservation of this region defined the importance on its structure and function of the protein. The genotype–phenotype correlation suggests that the truncated protein led to more severe symptoms in patient 17. All the patients had microcephaly and the age of onset of symptoms was variable. However, the mutation positive patient (P17) had no period of normal development and all her development milestones had been delayed. She also had neurological symptoms, hypotonia, severe microcephaly (less than 5th percentile), along with wringing of hands and with the most severe symptoms than any other patient. In conclusion, we have identified a deletion mutation in FOXG1 gene in one patient. No mutations have been identified in the other 33 cases. Mutations in FOXG1 are relatively common in patients with severe congenital variant of Rett syndrome. The phenotypes of Rett syndrome
have many differentials; however, a genetically based confirmed diagnosis would help in management and counseling. The molecular diagnosis approach described in this study can be a basis for further development in clinical use. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2013.12.063.
Conflict of interest The authors declare no conflict of interest. Acknowledgment We thank all the patients and their families for their contribution in this study. The authors are also thankful to the Director, NIRRH for providing the necessary facilities; the Indian Council of Medical Research (ICMR); the Department of Science & Technology (SR/FT/LS-87/2010), Govt. of India for providing financial grants for the study. References Ariani, F., et al., 2008. FOXG1 is responsible for the congenital variant of Rett syndrome. Am. J. Hum. Genet. 83, 89–93. Bahi-Buisson, N., et al., 2010. Revisiting the phenotype associated with FOXG1 mutations: two novel cases of congenital Rett variant. Neurogenetics 11, 241–249. Das, D.K., Mehta, B., Menon, S.R., Raha, S., Udani, V., 2013a. Novel mutations in cyclindependent kinase-like 5 (CDKL5) gene in Indian cases of Rett Syndrome. Neuromol. Med. 15, 218–225. Das, D.K., Raha, S., Sanghavi, D., Maitra, A., Udani, V., 2013b. Spectrum of MECP2 gene mutations in a cohort of Indian patients with Rett syndrome: report of two novel mutations. Gene 515, 78–83. Dastidar, S.G., Landrieu, P.M.Z., D'Mello, S.R., 2011. FoxG1 promotes the survival of postmitotic neurons. J. Neurosci. Off. J. Soc. Neurosci. 31, 402–413. Florian, C., Bahi-Buisson, N., Bienvenu, T., 2012. FOXG1-related disorders: from clinical description to molecular genetics. Mol. Syndromol. 2, 153–163. Guerrini, R., Parrini, E., 2012. Epilepsy in Rett syndrome, and CDKL5- and FOXG1-generelated encephalopathies. Epilepsia 53, 2067–2078. Hagberg, B.A., Skjeldal, O.H., 1994. Rett variants: a suggested model for inclusion criteria. Pediatr. Neurol. 11, 5–11. Mencarelli, M.A., et al., 2009. 14q12 Microdeletion syndrome and congenital variant of Rett syndrome. Eur. J. Med. Chem. Genet. 52, 148–152. Mencarelli, M.A., et al., 2010. Novel FOXG1 mutations associated with the congenital variant of Rett syndrome. J. Med. Genet. 47, 49–53. Neul, J.L., et al., 2010. Rett syndrome: revised diagnostic criteria and nomenclature. Ann. Neurol. 68, 944–950. Philippe, C., et al., 2010. Phenotypic variability in Rett syndrome associated with FOXG1 mutations in females. J. Med. Genet. 47, 59–65. Shoichet, S.A., et al., 2005. Haploinsufficiency of novel FOXG1B variants in a patient with severe mental retardation, brain malformations and microcephaly. Hum. Genet. 117, 536–544.