De Novo Mutations in FOXP1 in Cases with Intellectual Disability, Autism, and Language Impairment
2010; Elsevier BV; Volume: 87; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2010.09.017
ISSN1537-6605
AutoresFadi 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 Phillips, Éric Fombonne, Guy A. Rouleau, Jacques L. Michaud,
Tópico(s)interferon and immune responses
ResumoHeterozygous 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. 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. Developmental language disorders represent a heterogeneous group of conditions that are frequent but poorly understood. Although these disorders show high heritability, very little is known about the causative genes. FOXP2 (MIM 605317) represents an important entry point into the molecular basis of developmental language disorders.1Fisher S.E. Lai C.S. Monaco A.P. Deciphering the genetic basis of speech and language disorders.Annu. Rev. Neurosci. 2003; 26: 57-80Crossref PubMed Scopus (116) Google Scholar, 2Fisher S.E. Scharff C. FOXP2 as a molecular window into speech and language.Trends Genet. 2009; 25: 166-177Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar Patients heterozygous for mutations in FOXP2 show verbal dyspraxia (MIM 602081), which is characterized by impaired coordinated mouth movements that are required for speech, as well as variable impairment of expressive and receptive language, without consistent nonverbal cognitive deficits. Recent studies suggest that FOXP2 is also required for vocal learning in birds and that it might have played a role during the evolution of human language.3Enard W. Gehre S. Hammerschmidt K. Hölter S.M. Blass T. Somel M. Brückner M.K. Schreiweis C. Winter C. Sohr R. et al.A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice.Cell. 2009; 137: 961-971Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 4Haesler 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: e321Crossref PubMed Scopus (303) Google Scholar, 5Konopka 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: 213-217Crossref PubMed Scopus (271) Google Scholar FOXP2 belongs to a subgroup of the FOX family of winged-helix/forkhead transcription factors, which also includes FOXP1 (MIM 605515), FOXP3 (MIM 300292), and FOXP4 (MIM 608924).6Hannenhalli S. Kaestner K.H. The evolution of Fox genes and their role in development and disease.Nat. Rev. Genet. 2009; 10: 233-240Crossref PubMed Scopus (420) Google Scholar Several observations suggest that FOXP2 and its closest relative, FOXP1, may regulate common processes. For instance, FOXP1 and FOXP2 are expressed in distinct but also in overlapping regions of the developing bird, mouse, and human brain, including areas associated with the production and processing of vocalization and language.7Ferland 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: 266-279Crossref PubMed Scopus (350) Google Scholar, 8Lai 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: 2455-2462Crossref PubMed Scopus (272) Google Scholar, 9Shu 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: 27488-27497Crossref PubMed Scopus (269) Google Scholar, 10Teramitsu 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: 3152-3163Crossref PubMed Scopus (253) Google Scholar FOXP proteins act as transcriptional repressors by forming homo- or heterodimers.9Shu 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: 27488-27497Crossref PubMed Scopus (269) Google Scholar, 11Li 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: 809-822Crossref PubMed Scopus (229) Google Scholar, 12Wang 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: 24259-24268Crossref PubMed Scopus (171) Google Scholar Interestingly, FOXP1 and FOXP2 can physically interact in vitro, can repress the transcription of common targets in vivo by occupying the same binding sites, and cooperatively regulate lung and esophageal development in mice.11Li 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: 809-822Crossref PubMed Scopus (229) Google Scholar, 13Shu 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: 1991-2000Crossref PubMed Scopus (205) Google Scholar Because FOXP1 and FOXP2 cooperate to control developmental processes, it was hypothesized that mutations in FOXP1 could likewise be associated with language impairment.10Teramitsu 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: 3152-3163Crossref PubMed Scopus (253) Google Scholar, 14Vernes 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: 1354-1358Crossref PubMed Scopus (32) Google Scholar However, sequencing of FOXP1 in a cohort of individuals with developmental verbal dyspraxia did not reveal any pathogenic mutations.14Vernes 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: 1354-1358Crossref PubMed Scopus (32) Google Scholar Another possibility is that FOXP1 disruption causes other developmental conditions that are associated with language impairment, such as intellectual disability (ID; mental retardation) and autism spectrum disorders (ASD [MIM 209850]).15Kaufman 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)https://doi.org/10.1007/s11689-010-9055-2Crossref PubMed Scopus (170) Google Scholar In order to explore this possibility, we performed mutation analyses of FOXP1 in patients with ID and/or ASD. Because FOXP2 heterozygous mutations are sufficient to disrupt language development, we decided, likewise, to focus our attention on heterozygous mutations, which in the case of ID are more likely to arise de novo than to be transmitted. By analogy to the FOXP2 paradigm, we also restricted our analysis to patients without specific morphological abnormalities (referred to herein as having the nonsyndromic form of ID and ASD). We studied sporadic cases with nonsyndromic ID (NSID), ASD, or both NSID and ASD, most of which were of French Canadian origin. We chose to study sporadic cases to increase the likelihood of identifying de novo mutations. The NSID cases were selected with the use of previously described criteria.16Hamdan F.F. Gauthier J. Spiegelman D. Noreau A. Yang Y. Pellerin S. Dobrzeniecka S. Côté M. Perreau-Linck E. Perreault-Linck E. et al.Synapse to Disease GroupMutations in SYNGAP1 in autosomal nonsyndromic mental retardation.N. Engl. J. Med. 2009; 360: 599-605Crossref PubMed Scopus (209) Google Scholar The sporadic ASD patients were diagnosed according to DSM-IV criteria and were selected on the basis of a positive Autism Diagnostic Interview-Revised (ADI-R) and/or Autism Diagnostic Observation Schedule-Generic (ADOS-G). We used 570 healthy individuals as controls, including 285 French Canadians and 285 European individuals who were evaluated and found not to have cognitive dysfunction, neuropsychiatric symptoms, or family history of neuropsychiatric problems, as detailed elsewhere.17Gauthier J. Champagne N. Lafrenière R.G. Xiong L. Spiegelman D. Brustein E. Lapointe M. Peng H. Côté M. Noreau A. et al.S2D TeamDe novo mutations in the gene encoding the synaptic scaffolding protein SHANK3 in patients ascertained for schizophrenia.Proc. Natl. Acad. Sci. USA. 2010; 107: 7863-7868Crossref PubMed Scopus (287) Google Scholar Blood samples were collected from all members of these cohorts and from their parents, with informed consent and after approval of the study by institutional ethics committees. Genomic DNA was extracted from blood samples with the Puregene kit (Gentra). Paternity and maternity of each NSID and ASD patient proband trio were confirmed with the use of six informative microsatellite markers. We initially determined whether FOXP1 is affected by copy number changes in 80 individuals from the sporadic ASD cohort (including 27 with documented ID), using Genome-Wide Human Affymetrix 5.0 SNP arrays, and in 30 individuals from the sporadic NSID cohort, using Affymetrix 6.0 SNP arrays. Both parents of each case were also studied with these arrays. We identified a de novo intragenic FOXP1 deletion in an NSID female patient (R0031608) (patient A). This ∼390 kb deletion (genomic region on chromosome 3:71114875–71504640; hg18) encompasses exons 4–14 of the longest isoform of FOXP1 (FOXP1a; Refseq no. NM_032682.4), including sequence corresponding to its translation initiation site as well as to leucine zipper and zinc finger domains that are important for FOXP1 dimerization and regulation of transcriptional activity (Figure 1A ).11Li 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: 809-822Crossref PubMed Scopus (229) Google Scholar, 13Shu 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: 1991-2000Crossref PubMed Scopus (205) Google Scholar Multiplex ligation-dependent probe amplification (MLPA) analysis of FOXP1 in patient A confirmed the loss of these exons (Figure 1B). No deletions encompassing FOXP1 exons were identified in the other tested patients or their parents, nor have any been reported in the Database of Genomic Variants, a large public repository of structural variations in population controls.18Zhang 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: 205-214Crossref PubMed Scopus (167) Google Scholar We next sequenced all the coding exons and intron-exon boundaries of the longest FOXP1 isoform (FOXP1a; 16 coding exons) in 110 cases with NSID, 84 cases with ASD, and 51 cases with both NSID and ASD, as well as in 570 controls. We identified a de novo nonsense mutation, c.1573C>T (p.R525X), in a male patient with NSID and autism (R0024121) (patient B). This alteration abolishes the last 152 amino acids of FOXP1, including part of the forkhead DNA-binding domain (FHD) and a conserved nuclear localization signal (NLS) (Figure 2A ).19Banham A.H. Beasley N. Campo E. Fernandez P.L. Fidler C. Gatter K. Jones M. Mason D.Y. Prime J.E. Trougouboff P. et al.The FOXP1 winged helix transcription factor is a novel candidate tumor suppressor gene on chromosome 3p.Cancer Res. 2001; 61: 8820-8829PubMed Google Scholar, 20Vernes 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: 3154-3167Crossref PubMed Scopus (126) Google Scholar The FHDs of other FOXP proteins share more than 90% sequence identity with that of FOXP1 (Figure 2B).21Stroud 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: 159-166Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar Alteration of the corresponding arginine residue in FOXP3 (c.1189C>T [p.R397W]), as well as several other residues in the FOXP3 FHD, has been reported to cause a lethal X-linked neonatal autoimmune disease known as IPEX (MIM 304790).22Bennett 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: 20-21Crossref PubMed Scopus (2476) Google Scholar, 23Wildin R.S. Ramsdell F. Peake J. Faravelli F. Casanova J.L. Buist N. Levy-Lahad E. Mazzella M. Goulet O. Perroni L. et al.X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy.Nat. Genet. 2001; 27: 18-20Crossref PubMed Scopus (1399) Google Scholar Furthermore, high-resolution analysis of the crystal structure of the human FOXP2 FHD revealed that the residue homologous to p.R525, as well as residues downstream of it, plays an important role in DNA binding.21Stroud 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: 159-166Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar Indeed, pathogenic alterations in FOXP2 that abolish the FHD (p.R328X) or that are located in proximity to the residue homologous to p.R525 (p.R553H) have been shown to cause verbal dyspraxia (Figure 2B).24Lai 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: 519-523Crossref PubMed Scopus (1341) Google Scholar, 25MacDermot 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: 1074-1080Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar Sequencing also revealed a heterozygous missense mutation, c.643C>A (p.P215A), in both an NSID patient (n = 1/110) and a healthy individual (n = 1/570). In the case of the NSID patient, the variant was transmitted from an unaffected parent, further supporting the notion that it is not pathogenic. This variant was also previously reported in an individual with developmental verbal dyspraxia and in a control sample.14Vernes 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: 1354-1358Crossref PubMed Scopus (32) Google Scholar Three additional heterozygous missense variants (c.1333G>A [p.V445M], n = 1/570; c.1838C>A [p.T613N], n = 1/570; c.1709A>G [p.N570S], n = 2/570) were detected in FOXP1, but these did not affect any known functional domains and were present only in our cohort of controls, indicating that they are not pathogenic. No other amino-acid-altering variants were identified in any of the screened individuals, including the 570 healthy controls. In order to test the effect of the p.R525X mutation on FOXP1 activity, we took advantage of the well-documented ability of FOXP1 to repress transcription from the SV40 promoter, which can be easily monitored with the use of a standard luciferase assay.9Shu 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: 27488-27497Crossref PubMed Scopus (269) Google Scholar, 20Vernes 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: 3154-3167Crossref PubMed Scopus (126) Google Scholar The p.R525X mutation was introduced into the full-length coding sequence of the FOXP1 longest isoform (FOXP1a; obtained from Kazusa DNA Research Institute, Japan) and subcloned into the mammalian expression vector pcDNA4HisMax (Invitrogen). Human embryonic kidney (HEK) 293 cells were then cotransfected with the use of Fugene 6 (Roche) in 24-well plates, with 400 ng of pcDNA4HisMax without an insert or containing either the wild-type (WT) FOXP1 or the FOXP1-R525X cDNA, along with 50 ng of pGL3-promoter construct (Promega) (in which the SV40 promoter drives a firefly luciferase reporter). In order to account for variations in transfection efficiency and variation in cell number, cells were also cotransfected with 50 ng of a Renilla luciferase construct (pRL-TK; Promega) driven by the HSV-thymidine kinase promoter, which is not affected by FOXP1. Cells were lysed 48 hr after transfection and quantified for firefly and Renilla luciferase activities with the use of the Dual Luciferase Reporter Assay System (Promega). As shown in Figure 2D, WT FOXP1 was found to significantly inhibit the firefly luciferase signal (∼50% reduction; p < 0.001), as compared with an empty vector, whereas FOXP1-R525X failed to induce any reduction in luciferase levels, indicating that the p.R525X alteration impaired the FOXP1 ability to repress the SV40 promoter. Collectively, these results, along with the position of the p.R525X alteration in the FHD, strongly suggest that the de novo p.R525X alteration disrupts FOXP1 function. The mechanism(s) underlying this loss of FOXP1 activity could involve altered nuclear localization, as suggested by the disruption of one of the NLS in the FOXP1 mutant protein, impaired DNA binding, as suggested from the crystal structure of the FOXP2 FHD, or a combination of both. Although mutant and WT proteins were detected at comparable levels in transfected HEK293 cells, we cannot also exclude the possibility that the p.R525X mutation induces nonsense-mediated decay of FOXP1 mRNA in the patient's cells (Figure 2E). Additional work is needed to address these mechanistic questions. The phenotypes of both patients with de novo mutations in FOXP1 are summarized in Table 1. Patients A and B were born from nonconsanguineous French Canadian parents after uneventful pregnancies and deliveries. Patient B was operated on for atresia of the midjejunum and midileum gut during the neonatal period. Their initial development was characterized by global delay, with severe language impairment. They started to walk between the ages of 18 and 20 mo. Patient A was not clearly pronouncing any word until 3 yrs of age and started to associate words at 4 yrs of age, whereas patient B said his first words at the age of 6 yrs. The patients exhibited no deficits of oromotor coordination that could explain this delay in learning to talk.Table 1Clinical Phenotype of the Patients with De Novo FOXP1 MutationsPatient A (R0031608)Patient B (R0024121)FOXP1 de novo mutationdel (exons 4–14)c.1573C>T (p.R525X)Age (yrs: mo) / sex15:11 / F9:11 / MADI-R / ADOS-G−/−+/+Leiter International Performance Scale-RBRIEF IQ: standard score (percentile)58 (0.3)48 (0.1)Pre-school Language Scale: Age Equivalent (Yrs: Mo)Auditory comprehensionND3:7Expressive communicationND1:11Total language scoreND2:7Clinical Evaluation of Language Fundamentals: Age Equivalent (Yrs:Mo)Expressive subtestsMorphology< 4:0NDRecalling sentences< 4:0NDExpressive vocabulary< 4:5NDReceptive subtestsConcepts and following directions4:3NDBasic concepts> 4:0NDSentence structure< 4:0NDVineland Adaptive Behavior Scales: Percentile RankCommunication1< 1Daily living2< 1Socialization< 11Adaptive behavior composite1< 1Internalizing behavioral abnormalitiesclinically significantaClinically significant behavior problems on these scales correspond to the extreme top 2% of children of the same age.NDExternalizing behavioral abnormalitiesclinically significantaClinically significant behavior problems on these scales correspond to the extreme top 2% of children of the same age.NDMaladaptive behavior indexclinically significantaClinically significant behavior problems on these scales correspond to the extreme top 2% of children of the same age.NDAberrant Behavior Checklist: Raw Score (SD above Normal Mean)bClinically elevated: 1.5–1.9 SDs, significantly elevated: ≥ 2.0 SDs, where reported SDs are standard deviations from the normative ABC mean (community versus score).33Irritability subscale26 (2.3)31 (2.9)Lethargy subscale19 (1.7)8 (0.2)Stereotypy subscale7 (1.6)14 (2.9)Hyperactivity subscale24 (1.6)42 (2.6)Inappropriate speech subscale6 (1.6)6 (1.5)Repetitive Behavior Scale-Revised: Raw ScorescAll subscale scores correspond to significant case identification, except for the “self-injurious behavior” for patient A, as previously outlined.34Stereotyped behavior37Self-injurious behavior116Compulsive behavior36Ritualistic behavior66Sameness behavior611Restricted behavior410Other TestsFragile X testingnegativenegativeAGHdel FOXP1 (exons 4–14)normalCT scannormalNDND, not determined; AGH, array genomic hybridization. Affymetrix SNP arrays (5.0 or 6.0) were used for AGH.a Clinically significant behavior problems on these scales correspond to the extreme top 2% of children of the same age.b Clinically elevated: 1.5–1.9 SDs, significantly elevated: ≥ 2.0 SDs, where reported SDs are standard deviations from the normative ABC mean (community versus score).33Brown 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: 45-60Crossref PubMed Scopus (105) Google Scholarc All subscale scores correspond to significant case identification, except for the “self-injurious behavior” for patient A, as previously outlined.34Bodfish 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: 237-243Crossref PubMed Scopus (717) Google Scholar Open table in a new tab ND, not determined; AGH, array genomic hybridization. Affymetrix SNP arrays (5.0 or 6.0) were used for AGH. Patients A and B were assessed with the ADI-R and the ADOS-G at 6 yrs 8 mo and at 2 yrs 7 mo, respectively. Despite some autistic features in reciprocal social interaction (social avoidance with peers and decreased inhibition with adults) and restricted interests and repetitive behaviors (delayed echolalia and stereotyped language, self-injury, perceptual fixations), patient A would not have been considered as being autistic because of subthreshold scores in the communication area. In contrast, patient B showed scores above the diagnostic threshold for autism in all areas. Patients A and B were evaluated with a series of cognitive and behavioral assessment tools at 15 and 9 yrs of age, respectively. Assessment with the Leiter International Performance Scale-R (Brief IQ), an estimate of nonverbal intellectual functioning, revealed ID in the mild to moderate range. Results from the Vineland Adaptive Behavior Scale were consistent with the presence of ID in both patients, showing severe deficits in adaptive behaviors across the range of subscales (communication, daily living skills, socialization). Formal testing of patient A via the Clinical Evaluation for Language Fundamentals confirmed the presence of severe language impairment. Specifically, expressive language was severely limited, with oral communication characterized by single words or very short, simple sentences. Expressive vocabulary, equivalent to about 4.5 yrs, was slightly better than other expressive skills, such as morphology and repetition of increasingly complex sentences (both below 4 yrs) but was nonetheless mostly limited to relatively simple, frequent words. In several tests, she gave responses to expressive language items by miming the required words, indicating good comprehension of the question but an inability to produce the answer verbally. In support of this, receptive language abilities were somewhat more developed, as indicated by her understanding of a number of more complex relational and numerical concepts. Assessment of patient B with the Preschool Language Scale also confirmed severe language impairment. In terms of expressive communication, he scored at an age equivalence of 1 yr 11 mo. He had a range of about 100 words in his vocabulary and communicated with single words or short sentences (two to three words). In contrast, he was able to understand more complex relational concepts and performed at an age equivalence of 3 yrs 7 mo in auditory comprehension tasks. Both patients also showed a particular behavioral profile. Clinically significant problems were noted on the Vineland Maladaptive Behavior scale for patient A and were characterized by both internalizing (e.g., social withdrawal, anxiety) and externalizing (e.g., impulsivity, mood lability, temper tantrums, sulking, disobedience, physical aggression) behaviors. Parent responses on the Aberrant Behavior Checklist also indicated the presence of behavioral problems in both patients. In patient A, irritability was rated as the most significant problem, with all other problem scales (lethargy, stereotypy, hyperactivity, inappropriate speech) reaching clinically significant levels. Similarly, patient B had significantly elevated problems on the irritability, stereotypy, and hyperactivity subscales and clinically significant speech problems. A number of specific obsessions and compulsions (e.g., looking in mirrors, touching people's hair, nail biting, removing silicone in the house, hoarding objects) were described by the parents and confirmed via the Repetitive Behavior Scale. On this scale, ritualistic, restricted, and “sameness” behavior were most often reported for patient A (e.g., mealtime and bedtime rituals, rigid routines, listens to the same music continuously, strong attachment to specific objects). Stereotyped and self-injurious behavior (e.g., turning in circles, spinning objects, hitting and biting self) were also reported for patient B. In summary, we report two patients with de novo deleterious intragenic mutations in FOXP1. These patients share strikingly similar phenotypes, including mild to moderate ID with severe language impairment, autism and /or autistic features, mood lability with physical aggressiveness, and specific obsessions and compulsions. The patients with FOXP1 disruption described herein do not show verbal dyspraxia, but their phenotype nevertheless overlaps with that of patients with FOXP2 disruption to the extent that they also show language impairment. Two other patients with de novo deletions encompassing FOXP1 were recently described.26Carr 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)https://doi.org/10.1038/ejhg.2010.96Crossref PubMed Scopus (61) Google Scholar, 27Pariani M.J. Spencer A. Graham Jr., J.M. 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: 123-127Crossref PubMed Scopus (66) Google Scholar These patients, who were less than 4 yrs of age when studied, showed developmental and speech delay, but formal cognitive or behavioral evaluation could not be performed. In one case, the deletion spans 750 kb and affects three other genes whereas in the other case, the deletion spans 1 Mb and extends over 500 kb on the 3′ end of FOXP1. Even though this latter deletion does not affect any other known genes, the possibility that it encompasses elements that are important for the expression of genes located in its vicinity cannot be ruled out. Nevertheless, these cases reinforce our conclusion that FOXP1 haploinsufficiency affects cognitive development. Although an association between vitiligo (MIM 193200), an autoimmune form of skin depigmentation, and an intronic variant in FOXP1 was recently reported,28Jin 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. et al.Common variants in FOXP1 are associated with generalized vitiligo.Nat. Genet. 2010; 42: 576-578Crossref PubMed Scopus (75) Google Scholar none of our patients displayed signs of such a condition. Although FOXP1 and FOXP2 can cooperate to regulate transcription and are both associated with language impairment, FOXP1 disruption appears to have a more global impact on brain development than FOXP2 disruption. The difference between the phenotypic consequence of FOXP1 and FOXP2 haploinsufficiency might be explained at least in part by some difference in their expression patterns.7Ferland 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: 266-279Crossref PubMed Scopus (350) Google Scholar, 9Shu 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: 27488-27497Crossref PubMed Scopus (269) Google Scholar Alternatively, FOXP heterodimers and homodimers may have different biochemical properties. For instance, FOXP1-FOXP2 heterodimers may regulate targets that are involved in language impairment, whereas the respective FOXP homodimers may regulate additional targets that are associated with other developmental processes. Vernes et al. have found that FOXP2 directly regulates the expression of CNTNAP2, a gene that encodes a member of the neurexin superfamily of transmembrane proteins implicated in neuronal recognition and cell adhesion.29Vernes 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. et al.A functional genetic link between distinct developmental language disorders.N. Engl. J. Med. 2008; 359: 2337-2345Crossref PubMed Scopus (469) Google ScholarCNTNAP2 has been associated with language impairment and autism.29Vernes 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. et al.A functional genetic link between distinct developmental language disorders.N. Engl. J. Med. 2008; 359: 2337-2345Crossref PubMed Scopus (469) Google Scholar, 30Alarcón 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. et al.Linkage, association, and gene-expression analyses identify CNTNAP2 as an autism-susceptibility gene.Am. J. Hum. Genet. 2008; 82: 150-159Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, 31Arking 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: 160-164Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 32Bakkaloglu 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. et al.Molecular cytogenetic analysis and resequencing of contactin associated protein-like 2 in autism spectrum disorders.Am. J. Hum. Genet. 2008; 82: 165-173Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar It is tempting to speculate that FOXP1 homodimers or FOXP1-FOXP2 heterodimers also regulate CNTNAP2 expression and that FOXP1 haploinsufficiency affects language development and possibly causes ID and autism by disrupting this regulatory interaction. FOXP1 and FOXP2 thus represent interesting entry points for dissecting the molecular mechanisms underlying neurodevelopmental disorders such as ID, autism, and language impairment. This work was supported by grants from the Canadian Institute of Health Research (CIHR) (J.L.M., G.A.R., J.-C.L.), Réseau de Génétique Médicale Appliquée (RMGA) / Fonds de la Recherche en Santé du Québec (FRSQ) (J.L.M. and M.S.P.), Genome Canada and Genome Quebec, and cofunding by Université de Montréal for the Synapse to Diseases (S2D) project (G.A.R. and P.D.). J.L.M. is the recipient of a Clinical Investigatorship Award of the CIHR (Institute of Genetics) and a Senior Scientist Award from FRSQ. E.F. holds the CIHR-funded Canada Research Chair in Child Psychiatry. H.D. is a recipient of a CIHR postdoctoral fellowship award. We thank the patients and their parents for participating in this study. We are grateful for the dedicated work of members of the S2D team (CHUM Research Center, Montreal), including Management (Claude Marineau), Bioinformatics (Edouard Henrion, Ousmane Diallo, and Dan Spiegelman), and the Genetic Screening divisions (Annie Levert, Annie Raymond, Pascal Thibodeau, Sandra Laurent, and Karine Lachapelle). We are thankful for the efforts of the members of McGill University and Génome Québec Innovation Centre Sequencing (Pierre Lepage, Sébastien Brunet, and Hao Fan Yam) and Bioinformatics (Louis Létourneau and Louis Dumond Joseph) groups. The URLs for data presented herein are as follows:Database of Genomic Variants, http://projects.tcag.ca/variation/Online Mendelian Inheritance of Man (OMIM), http://www.ncbi.nlm.nih.gov/omim/UCSC Genome Browser, http://www.genome.ucsc.edu/Uniprot, http://www.uniprot.org/
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