De novo mutations in FOXP1 in cases with intellectual disability, autism, and language impairment

Fadi F Hamdan, Hussein Daoud, Daniel Rochefort, Amélie Piton, Julie Gauthier, Mathieu Langlois, Gila Foomani, Sylvia Dobrzeniecka, Marie-Odile Krebs, Ridha Joober, Ronald G Lafrenière, Jean-Claude Lacaille, Laurent Mottron, Pierre Drapeau, Miriam H Beauchamp, Michael S Phillips, Eric Fombonne, Guy A Rouleau, Jacques L Michaud, Fadi F Hamdan, Hussein Daoud, Daniel Rochefort, Amélie Piton, Julie Gauthier, Mathieu Langlois, Gila Foomani, Sylvia Dobrzeniecka, Marie-Odile Krebs, Ridha Joober, Ronald G Lafrenière, Jean-Claude Lacaille, Laurent Mottron, Pierre Drapeau, Miriam H Beauchamp, Michael S Phillips, Eric Fombonne, Guy A Rouleau, Jacques L Michaud

Abstract

Heterozygous mutations in FOXP2, which encodes a forkhead transcription factor, have been shown to cause developmental verbal dyspraxia and language impairment. FOXP2 and its closest homolog, FOXP1, are coexpressed in brain regions that are important for language and cooperatively regulate developmental processes, raising the possibility that FOXP1 may also be involved in developmental conditions that are associated with language impairment. In order to explore this possibility, we searched for mutations in FOXP1 in patients with intellectual disability (ID; mental retardation) and/or autism spectrum disorders (ASD). We first performed array-based genomic hybridization on sporadic nonsyndromic ID (NSID) (n = 30) or ASD (n = 80) cases. We identified a de novo intragenic deletion encompassing exons 4-14 of FOXP1 in a patient with NSID and autistic features. In addition, sequencing of all coding exons of FOXP1 in sporadic NSID (n = 110) or ASD (n = 135) cases, as well as in 570 controls, revealed the presence of a de novo nonsense mutation (c.1573C>T [p.R525X]) in the conserved forkhead DNA-binding domain in a patient with NSID and autism. Luciferase reporter assays showed that the p.R525X alteration disrupts the activity of the protein. Formal assessments revealed that both patients with de novo mutations in FOXP1 also show severe language impairment, mood lability with physical aggressiveness, and specific obsessions and compulsions. In conclusion, both FOXP1 and FOXP2 are associated with language impairment, but decrease of the former has a more global impact on brain development than that of the latter.

Copyright © 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1
Figure 1
De Novo FOXP1 Deletion at 3p14.1 in Patient A (A) The Affymetrix Genome-Wide Human SNP 6.0 Array result for patient A is shown above the ideogram of chromosome 3 (Netaffx version 28). Each dot represents a SNP or a copy number marker, with normal copy number having a log2 ratio of ∼0 and deleted regions less than −1. The CNV analysis was performed according to the manufacturer's procedure with the use of the Affymetrix reference library, GenomeWideSNP_6.hapmap270.na30r1.a5, with a regional GC correction. The de novo deletion (chr3: 71109689–71508061; hg18) is boxed, and the only gene affected with this deletion, FOXP1, is shown. (B) Mapping of FOXP1 deleted exons in patient A. Using MLPA analysis, we mapped the deletion breakpoints between exon 4 and exon 14 of FOXP1. MLPA probes targeting 14 exons (Ex) of FOXP1 were custom designed with the ProSeek software. These 14 probes were compared to two control probes on chromosome X and chromosome 1. MLPA was performed on 50 ng of genomic DNA. Probe amplification products were run on an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The data were analyzed with the GeneMapper software version 4.0 (Applied Biosystems). The dosage ratio (DR) was calculated as follows: DR = (peak height FOXP1 / peak height chromosome 1 probe) in the patient carrying the deletion / (peak height FOXP1 / peak height chromosome 1 probe) in her unaffected mother, who has no copy number variation in FOXP1. The DR has a theoretical value of ≤ 0.7 for a deletion, between 0.7 and 1.3 for a normal situation, and ≥ 1.3 for a duplication. Values represent mean of samples ± standard deviation of samples run in triplicate. Data show that exons 4–14 are deleted in patient A.
Figure 2
Figure 2
Localization and Characterization of FOXP1 De Novo Mutations (A) Localization of the de novo mutations identified herein with respect to FOXP1 known protein domains (Uniprot no. Q9H334; 677 amino acids [aa]). The deletion in patient A is predicted to affect the first half of FOXP1, including important functional domains such as a zinc finger (ZF; aa 306–331), a leucine zipper (LZ; aa 348–369), and CTBP1-binding (amino acids 382–386) domains. The nonsense p.R525X alteration affects the conserved forkhead DNA-binding domain (FHD, aa 465–555). Also shown are two glutamine rich (Q-rich) regions (aa 55–77, aa 110–194) at the N terminus of the protein and an acidic-rich region (aa 637–677) at its C terminus. The positions of the two nuclear localization signals (NLS; aa 434–440, aa 543–546) are indicated. (B). Alignment of the conserved FHD region of the four members of the human FOXP family. Highlighted and underlined in red are residues that when mutated lead to human disease: p.R525X (current study) in NSID, autism, and language impairment; p.R553H in FOXP2 in severe congenital speech disorder; and p.I363V, p.F371C/L, p.A384T, and p.R397T in FOXP3 in IPEX syndrome. Amino acid identity and similarity are represented by asterisks (∗) and dots (.), respectively. (C) Chromatograms corresponding to the FOXP1 de novo mutation (c.1573C>T [p.R525X]) identified in patient B. Wild-type (WT) and mutant (MT) FOXP1 DNA sequences are shown along with the corresponding amino acids. (D) Luciferase reporter assay assessing the impact of p.R525X on FOXP1 transactivation activity in transfected HEK293 cells. FOXP1 significantly inhibited pGL3-promoter (SV40) transcriptional activity (p Renilla luciferase) constructs. (E) Immunoblot performed on total protein extracts from HEK293 cells transfected with pcDNA4-HisMax-based constructs expressing FOXP1 or FOXP1-R525X in frame with an N-terminal Xpress tag, as described in the text. Proteins were resolved on SDS-PAGE, and FOXP1 and FOXP1-R525X were detected with the use of a monoclonal antibody against the N-terminal Xpress tag (Invitrogen). The blot was stripped and probed with an anti-alpha-tubulin antibody (Abcam) as an internal loading control.

References

    1. Fisher S.E., Lai C.S., Monaco A.P. Deciphering the genetic basis of speech and language disorders. Annu. Rev. Neurosci. 2003;26:57–80.
    1. Fisher S.E., Scharff C. FOXP2 as a molecular window into speech and language. Trends Genet. 2009;25:166–177.
    1. Enard W., Gehre S., Hammerschmidt K., Hölter S.M., Blass T., Somel M., Brückner M.K., Schreiweis C., Winter C., Sohr R. A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice. Cell. 2009;137:961–971.
    1. Haesler S., Rochefort C., Georgi B., Licznerski P., Osten P., Scharff C. Incomplete and inaccurate vocal imitation after knockdown of FoxP2 in songbird basal ganglia nucleus Area X. PLoS Biol. 2007;5:e321.
    1. Konopka G., Bomar J.M., Winden K., Coppola G., Jonsson Z.O., Gao F., Peng S., Preuss T.M., Wohlschlegel J.A., Geschwind D.H. Human-specific transcriptional regulation of CNS development genes by FOXP2. Nature. 2009;462:213–217.
    1. Hannenhalli S., Kaestner K.H. The evolution of Fox genes and their role in development and disease. Nat. Rev. Genet. 2009;10:233–240.
    1. Ferland R.J., Cherry T.J., Preware P.O., Morrisey E.E., Walsh C.A. Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain. J. Comp. Neurol. 2003;460:266–279.
    1. Lai C.S., Gerrelli D., Monaco A.P., Fisher S.E., Copp A.J. FOXP2 expression during brain development coincides with adult sites of pathology in a severe speech and language disorder. Brain. 2003;126:2455–2462.
    1. Shu W., Yang H., Zhang L., Lu M.M., Morrisey E.E. Characterization of a new subfamily of winged-helix/forkhead (Fox) genes that are expressed in the lung and act as transcriptional repressors. J. Biol. Chem. 2001;276:27488–27497.
    1. Teramitsu I., Kudo L.C., London S.E., Geschwind D.H., White S.A. Parallel FoxP1 and FoxP2 expression in songbird and human brain predicts functional interaction. J. Neurosci. 2004;24:3152–3163.
    1. Li S., Weidenfeld J., Morrisey E.E. Transcriptional and DNA binding activity of the Foxp1/2/4 family is modulated by heterotypic and homotypic protein interactions. Mol. Cell. Biol. 2004;24:809–822.
    1. Wang B., Lin D., Li C., Tucker P. Multiple domains define the expression and regulatory properties of Foxp1 forkhead transcriptional repressors. J. Biol. Chem. 2003;278:24259–24268.
    1. Shu W., Lu M.M., Zhang Y., Tucker P.W., Zhou D., Morrisey E.E. Foxp2 and Foxp1 cooperatively regulate lung and esophagus development. Development. 2007;134:1991–2000.
    1. Vernes S.C., MacDermot K.D., Monaco A.P., Fisher S.E. Assessing the impact of FOXP1 mutations on developmental verbal dyspraxia. Eur. J. Hum. Genet. 2009;17:1354–1358.
    1. Kaufman L., Ayub M., Vincent J.B. The genetic basis of non-syndromic intellectual disability: a review. J. Neurodev. Disord. 2010 Published online July 28 2010.
    1. Hamdan F.F., Gauthier J., Spiegelman D., Noreau A., Yang Y., Pellerin S., Dobrzeniecka S., Côté M., Perreau-Linck E., Perreault-Linck E., Synapse to Disease Group Mutations in SYNGAP1 in autosomal nonsyndromic mental retardation. N. Engl. J. Med. 2009;360:599–605.
    1. Gauthier J., Champagne N., Lafrenière R.G., Xiong L., Spiegelman D., Brustein E., Lapointe M., Peng H., Côté M., Noreau A., S2D Team De novo mutations in the gene encoding the synaptic scaffolding protein SHANK3 in patients ascertained for schizophrenia. Proc. Natl. Acad. Sci. USA. 2010;107:7863–7868.
    1. Zhang J., Feuk L., Duggan G.E., Khaja R., Scherer S.W. Development of bioinformatics resources for display and analysis of copy number and other structural variants in the human genome. Cytogenet. Genome Res. 2006;115:205–214.
    1. Banham A.H., Beasley N., Campo E., Fernandez P.L., Fidler C., Gatter K., Jones M., Mason D.Y., Prime J.E., Trougouboff P. The FOXP1 winged helix transcription factor is a novel candidate tumor suppressor gene on chromosome 3p. Cancer Res. 2001;61:8820–8829.
    1. Vernes S.C., Nicod J., Elahi F.M., Coventry J.A., Kenny N., Coupe A.M., Bird L.E., Davies K.E., Fisher S.E. Functional genetic analysis of mutations implicated in a human speech and language disorder. Hum. Mol. Genet. 2006;15:3154–3167.
    1. Stroud J.C., Wu Y., Bates D.L., Han A., Nowick K., Paabo S., Tong H., Chen L. Structure of the forkhead domain of FOXP2 bound to DNA. Structure. 2006;14:159–166.
    1. Bennett C.L., Christie J., Ramsdell F., Brunkow M.E., Ferguson P.J., Whitesell L., Kelly T.E., Saulsbury F.T., Chance P.F., Ochs H.D. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat. Genet. 2001;27:20–21.
    1. Wildin R.S., Ramsdell F., Peake J., Faravelli F., Casanova J.L., Buist N., Levy-Lahad E., Mazzella M., Goulet O., Perroni L. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat. Genet. 2001;27:18–20.
    1. Lai C.S., Fisher S.E., Hurst J.A., Vargha-Khadem F., Monaco A.P. A forkhead-domain gene is mutated in a severe speech and language disorder. Nature. 2001;413:519–523.
    1. MacDermot K.D., Bonora E., Sykes N., Coupe A.M., Lai C.S., Vernes S.C., Vargha-Khadem F., McKenzie F., Smith R.L., Monaco A.P., Fisher S.E. Identification of FOXP2 truncation as a novel cause of developmental speech and language deficits. Am. J. Hum. Genet. 2005;76:1074–1080.
    1. Carr C.W., Moreno-De-Luca D., Parker C., Zimmerman H.H., Ledbetter N., Martin C.L., Dobyns W.B., Abdul-Rahman O.A. Chiari I malformation, delayed gross motor skills, severe speech delay, and epileptiform discharges in a child with FOXP1 haploinsufficiency. Eur. J. Hum. Genet. 2010 Published online June 23, 2010.
    1. Pariani M.J., Spencer A., Graham J.M., Jr., Rimoin D.L. A 785kb deletion of 3p14.1p13, including the FOXP1 gene, associated with speech delay, contractures, hypertonia and blepharophimosis. Eur. J. Med. Genet. 2009;52:123–127.
    1. Jin Y., Birlea S.A., Fain P.R., Mailloux C.M., Riccardi S.L., Gowan K., Holland P.J., Bennett D.C., Wallace M.R., McCormack W.T. Common variants in FOXP1 are associated with generalized vitiligo. Nat. Genet. 2010;42:576–578.
    1. Vernes S.C., Newbury D.F., Abrahams B.S., Winchester L., Nicod J., Groszer M., Alarcón M., Oliver P.L., Davies K.E., Geschwind D.H. A functional genetic link between distinct developmental language disorders. N. Engl. J. Med. 2008;359:2337–2345.
    1. Alarcón M., Abrahams B.S., Stone J.L., Duvall J.A., Perederiy J.V., Bomar J.M., Sebat J., Wigler M., Martin C.L., Ledbetter D.H. Linkage, association, and gene-expression analyses identify CNTNAP2 as an autism-susceptibility gene. Am. J. Hum. Genet. 2008;82:150–159.
    1. Arking D.E., Cutler D.J., Brune C.W., Teslovich T.M., West K., Ikeda M., Rea A., Guy M., Lin S., Cook E.H., Chakravarti A. A common genetic variant in the neurexin superfamily member CNTNAP2 increases familial risk of autism. Am. J. Hum. Genet. 2008;82:160–164.
    1. Bakkaloglu B., O'Roak B.J., Louvi A., Gupta A.R., Abelson J.F., Morgan T.M., Chawarska K., Klin A., Ercan-Sencicek A.G., Stillman A.A. Molecular cytogenetic analysis and resequencing of contactin associated protein-like 2 in autism spectrum disorders. Am. J. Hum. Genet. 2008;82:165–173.
    1. Brown E.C., Aman M.G., Havercamp S.M. Factor analysis and norms for parent ratings on the Aberrant Behavior Checklist-Community for young people in special education. Res. Dev. Disabil. 2002;23:45–60.
    1. Bodfish J.W., Symons F.J., Parker D.E., Lewis M.H. Varieties of repetitive behavior in autism: comparisons to mental retardation. J. Autism Dev. Disord. 2000;30:237–243.
    1. Schouten J.P., McElgunn C.J., Waaijer R., Zwijnenburg D., Diepvens F., Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 2002;30:e57.
    1. Pantano L., Armengol L., Villatoro S., Estivill X. ProSeeK: a web server for MLPA probe design. BMC Genomics. 2008;9:573.

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