Mutations in the BAF-Complex Subunit DPF2 Are Associated with Coffin-Siris Syndrome
2018; Elsevier BV; Volume: 102; Issue: 3 Linguagem: Inglês
10.1016/j.ajhg.2018.01.014
ISSN1537-6605
AutoresGeorgia Vasileiou, Silvia Vergarajauregui, Sabine Endele, Bernt Popp, Christian Büttner, Arif B. Ekici, Marion Gérard, Nuria C. Bramswig, Beate Albrecht, Jill Clayton‐Smith, Jenny Morton, Susan Tomkins, Karen Low, Astrid Weber, Maren Wenzel, Janine Altmüller, Yun Li, Bernd Wollnik, George Hoganson, Maria-Renée Plona, Megan T. Cho, Christian T. Thiel, Hermann‐Josef Lüdecke, Tim M. Strom, Eduardo Calpena, Andrew O.M. Wilkie, Dagmar Wieczorek, Felix B. Engel, André Reis,
Tópico(s)Protein Degradation and Inhibitors
ResumoVariants affecting the function of different subunits of the BAF chromatin-remodelling complex lead to various neurodevelopmental syndromes, including Coffin-Siris syndrome. Furthermore, variants in proteins containing PHD fingers, motifs recognizing specific histone tail modifications, have been associated with several neurological and developmental-delay disorders. Here, we report eight heterozygous de novo variants (one frameshift, two splice site, and five missense) in the gene encoding the BAF complex subunit double plant homeodomain finger 2 (DPF2). Affected individuals share common clinical features described in individuals with Coffin-Siris syndrome, including coarse facial features, global developmental delay, intellectual disability, speech impairment, and hypoplasia of fingernails and toenails. All variants occur within the highly conserved PHD1 and PHD2 motifs. Moreover, missense variants are situated close to zinc binding sites and are predicted to disrupt these sites. Pull-down assays of recombinant proteins and histone peptides revealed that a subset of the identified missense variants abolish or impaire DPF2 binding to unmodified and modified H3 histone tails. These results suggest an impairment of PHD finger structural integrity and cohesion and most likely an aberrant recognition of histone modifications. Furthermore, the overexpression of these variants in HEK293 and COS7 cell lines was associated with the formation of nuclear aggregates and the recruitment of both wild-type DPF2 and BRG1 to these aggregates. Expression analysis of truncating variants found in the affected individuals indicated that the aberrant transcripts escape nonsense-mediated decay. Altogether, we provide compelling evidence that de novo variants in DPF2 cause Coffin-Siris syndrome and propose a dominant-negative mechanism of pathogenicity. Variants affecting the function of different subunits of the BAF chromatin-remodelling complex lead to various neurodevelopmental syndromes, including Coffin-Siris syndrome. Furthermore, variants in proteins containing PHD fingers, motifs recognizing specific histone tail modifications, have been associated with several neurological and developmental-delay disorders. Here, we report eight heterozygous de novo variants (one frameshift, two splice site, and five missense) in the gene encoding the BAF complex subunit double plant homeodomain finger 2 (DPF2). Affected individuals share common clinical features described in individuals with Coffin-Siris syndrome, including coarse facial features, global developmental delay, intellectual disability, speech impairment, and hypoplasia of fingernails and toenails. All variants occur within the highly conserved PHD1 and PHD2 motifs. Moreover, missense variants are situated close to zinc binding sites and are predicted to disrupt these sites. Pull-down assays of recombinant proteins and histone peptides revealed that a subset of the identified missense variants abolish or impaire DPF2 binding to unmodified and modified H3 histone tails. These results suggest an impairment of PHD finger structural integrity and cohesion and most likely an aberrant recognition of histone modifications. Furthermore, the overexpression of these variants in HEK293 and COS7 cell lines was associated with the formation of nuclear aggregates and the recruitment of both wild-type DPF2 and BRG1 to these aggregates. Expression analysis of truncating variants found in the affected individuals indicated that the aberrant transcripts escape nonsense-mediated decay. Altogether, we provide compelling evidence that de novo variants in DPF2 cause Coffin-Siris syndrome and propose a dominant-negative mechanism of pathogenicity. Coffin-Siris syndrome (CSS [MIM: 135900]) is a neurodevelopmental disorder characterized by mild to severe intellectual disability, speech impairment, growth deficiency, feeding difficulties, coarse facial characteristics, sparse hair, hypoplastic or absent finger- and/or toenails, and brain anomalies, the most prominent of which is hypoplasia or agenesis of the corpus callosum.1Kosho T. Okamoto N. Ohashi H. Tsurusaki Y. Imai Y. Hibi-Ko Y. Kawame H. Homma T. Tanabe S. Kato M. et al.Clinical correlations of mutations affecting six components of the SWI/SNF complex: detailed description of 21 patients and a review of the literature.Am. J. Med. Genet. A. 2013; 161A: 1221-1237Crossref PubMed Scopus (78) Google Scholar, 2Coffin G.S. Siris E. Mental retardation with absent fifth fingernail and terminal phalanx.Am. J. Dis. Child. 1970; 119: 433-439PubMed Google Scholar The rather broad and highly variable clinical spectrum of CSS individuals often confounds the clinical diagnosis.3Kosho T. Miyake N. Carey J.C. Coffin-Siris syndrome and related disorders involving components of the BAF (mSWI/SNF) complex: historical review and recent advances using next generation sequencing.Am. J. Med. Genet. C. Semin. Med. Genet. 2014; 166C: 241-251Crossref PubMed Scopus (71) Google Scholar In recent years, germline de novo mutations in several subunits of the human BRG1-associated factor (BAF) chromatin-remodelling complex (also known as the SWI/SNF-A complex) have been associated with CSS. Heterozygous loss-of-function variants (frameshifts, nonsense variants, and microdeletions) have been identified in ARID1A (MIM: 603024) and ARID1B (MIM: 614556), whereas variants with dominant-negative or gain-of-function effects (missense and in-frame deletions) have been detected in SMARCA4 (BRG1 [MIM: 603254]), SMARCB1 (SNF5 [MIM: 601607]), and SMARCE1 (MIM: 603111).1Kosho T. Okamoto N. Ohashi H. Tsurusaki Y. Imai Y. Hibi-Ko Y. Kawame H. Homma T. Tanabe S. 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Genet. 2017; 136: 297-305Crossref PubMed Scopus (41) Google Scholar Here, we report de novo variants in DPF2 (also known as REQ, UBID4, or BAF45d [MIM: 601671]), encoding a subunit of the BAF chromatin-remodelling complex, which has not previously been associated with neurodevelopmental syndromes. In total, we identified eight unrelated individuals (four females and four males) with features of CSS. Written informed consent was obtained from all participants or their legal guardians, and the study was approved by the ethical review board of the University Erlangen-Nürnberg and the respective institutions, as well as the East of England-Cambrige South committee of the National Research Ethics Service for the UK Deciphering Developmental Disorders (DDD) Study. The study received UK research ethics committee (REC) approval (10/H0305/83 granted by the Cambridge South REC and GEN/284/12 granted by the Republic of Ireland REC). DPF2 is located in chromosomal region 11q13.1 and shows a ubiquitous expression pattern. DPF2, together with DPF1 and DPF3, belongs to the d4 protein family and functions as a non-catalytic subunit of the BAF chromatin-remodelling complex.14Chestkov A.V. Baka I.D. Kost M.V. Georgiev G.P. Buchman V.L. The d4 gene family in the human genome.Genomics. 1996; 36: 174-177Crossref PubMed Scopus (29) Google Scholar, 15Lessard J. Wu J.I. Ranish J.A. Wan M. Winslow M.M. Staahl B.T. Wu H. Aebersold R. Graef I.A. Crabtree G.R. An essential switch in subunit composition of a chromatin remodeling complex during neural development.Neuron. 2007; 55: 201-215Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar, 16Tando T. Ishizaka A. Watanabe H. Ito T. Iida S. Haraguchi T. Mizutani T. Izumi T. Isobe T. Akiyama T. et al.Requiem protein links RelB/p52 and the Brm-type SWI/SNF complex in a noncanonical NF-kappaB pathway.J. Biol. 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The PHD finger: a versatile epigenome reader.Trends Biochem. Sci. 2011; 36: 364-372Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar DPF2 was first identified as an early-apoptosis gene in mouse myeloid cells.26Gabig T.G. Mantel P.L. Rosli R. Crean C.D. Requiem: a novel zinc finger gene essential for apoptosis in myeloid cells.J. Biol. Chem. 1994; 269: 29515-29519PubMed Google Scholar Recently, its inhibitory role in myeloid differentiation via interaction with RUNX1, possibly mediated by the BAF complex, was reported.23Huber F.M. Greenblatt S.M. Davenport A.M. Martinez C. Xu Y. Vu L.P. Nimer S.D. Hoelz A. Histone-binding of DPF2 mediates its repressive role in myeloid differentiation.Proc. Natl. Acad. Sci. 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Acta. 2015; 1853: 3279-3293Crossref PubMed Scopus (17) Google Scholar The index subject (individual 1), a 10-year-old boy from France, was clinically suspected to have CSS on the basis of his coarse facial features, global developmental delay, mild intellectual disability, speech delay (first words at 48 months and whole sentences at 84 months), stereotypic behavior, feeding problems for 1–2 years starting at the age of 6 months, and muscular hypotonia. The boy was able to walk after the age of 17 months. Hypoplasia of the fourth and fifth toenails and brachydactyly of the fifth fingers were also observed (Figure 2, Table 1, and Table S1). Chromosomal microarray analysis did not reveal any pathogenic copy-number variants (CNVs), and Sanger sequencing of six genes (SMARCA4, SMARCB1, SMARCE1, ARID1B, ARID1A, and SMARCA2 [MIM: 600014]) associated with CSS and Nicolaides-Baraitser syndrome (MIM: 601358) showed no variant explaining the observed phenotype (Supplemental Note and Table S2). Subsequent trio exome sequencing on an Illumina HiSeq 2500 system after enrichment with SureSelect Target Enrichment V5 technology (Agilent Technologies) revealed the previously unreported heterozygous de novo missense variant c.827G>T (p.Cys276Phe) in exon 8 of DPF2. The nucleotide change affects a highly conserved amino acid in the PHD1 finger (Figure 1). The variant has not been described in the Genome Aggregation Database (gnomAD) and is computationally predicted to be deleterious (Table S3).Table 1Summary of the Clinical and Genetic Findings in Individuals with De Novo DPF2 VariantsIndividual 1Individual 2Individual 3Individual 4Individual 5Individual 6Individual 7Individual 8De novo DPF2 variant (GenBank: NM_006268.4)c.827G>T (p.Cys276Phe)c.990C>G (p.Cys330Trp)c.1049G>A (p.Arg350His)c.1037A>G (p.Asp346Gly)c.1105T>C (p.Trp369Arg)c.904+1G>T(p.?)c.1099+1G>A (p.Asp340Glufs∗12)c.1066_1073del (p.Cys356Profs∗5)Exon or intronexon 8exon 9exon 10exon 10exon 11intron 8intron 10exon 10LocalizationPHD1PHD2PHD2PHD2PHD2PHD1PHD2PHD2SexmalefemalefemalemalemalefemalefemalemaleAge at last clinical assessment10 years16 years18 years, 6 monthsNA15 years3 years, 2 months7 years, 5 months3 years, 9 monthsHeight<3rd %50th % (<3rd % until puberty)20th–50th %<3rd %<0.4th %11th %15th %<3rd %Weight<3rd %<3rd %50th–75th %<3rd % 97th %50th %>97th %96th %50th %Brain anomaliesNA+NANANA++NADevelopmentDevelopmental delayglobalmoderate globalmoderate globalmildglobalmild to moderatemild globalglobalCognition statusmild IDmoderate IDmoderate IDborderline IDmoderate IDprecise estimation not possible (the individual is too young)borderline IDmild IDSpeech delay++++++++Motor delay−+++−−++Behavioral anomalies+−+−+−−−Feeding problems+++−+−−+Muscular hypotonia+−−NA−+++Hearing loss−+++−−−+CSS-like facial features+−++−coarsecoarse+Skeletal AnomaliesCraniosynostosis−−−sagittalsagittal−trigonocephaly (radiographic imaging was not performed)−Brachydactylyonly fifth finger−−generalonly fifth fingeronly fifth finger−generalClinodactyly−only fifth finger−−only fifth finger−−only fifth fingerEctodermal AnomaliesSparse scalp hair+in childhood++−NA++Nail hypoplasia or aplasiaforth and fifth toenailsright fifth toenail, small left fifth toenailright fifth toenail, fourth and fifth fingernails, dysplasia of all nailsall toenails, fifth and index fingernailsfifth toenails, small thickened toenailsall toenails, fifth fingernailsfifth toenailsall toenails, fifth fingernailsCardiac anomalies−+−+−++−Constipation−++−++++Recurrent otitis+−+NA−NA++Accordance with HGVS variant nomenclature was checked with Mutalyzer on September 10, 2017. The following abbreviations and symbols are used: +, present; −, absent; %, percentile; NA, not analyzed; OFC, occipitofrontal circumference; and PHD1 and PHD2, plant homeodomains. Open table in a new tab Accordance with HGVS variant nomenclature was checked with Mutalyzer on September 10, 2017. The following abbreviations and symbols are used: +, present; −, absent; %, percentile; NA, not analyzed; OFC, occipitofrontal circumference; and PHD1 and PHD2, plant homeodomains. Additional evidence for the correlation between the identified DPF2 variant and the clinical phenotype was provided by a large exome-wide trio study on 4,293 children with severe, undiagnosed developmental disorders (DDD Study).28Deciphering Developmental Disorders S. Deciphering Developmental Disorders StudyPrevalence and architecture of de novo mutations in developmental disorders.Nature. 2017; 542: 433-438Crossref PubMed Scopus (765) Google Scholar From the de novo variants reported, we extracted two additional heterozygous DPF2 missense variants in female individuals originating from England: c.990C>G (p.Cys330Trp) in exon 9 (individual 2 [DDD4K.02108]) and c.1049G>A (p.Arg350His) in exon 10 (individual 3 [DDD4K.03804]). Further interrogation of the DDD resource at a later stage for subjects with craniosynostosis (Datafreeze 3: 7,833 trios and 1,792 singletons) revealed DPF2 missense mutations in two further individuals, tested by trio exome sequencing: c.1037A>G (p.Asp346Gly) in exon 10 (individual 4 [DDDP126054]) and c.1105T>C (p.Trp369Arg) in exon 11 (individual 5 [DDDP100312]). All four variants are located in the PHD2 finger and affect highly conserved amino acids (Figure 1). They are likewise not present in gnomAD, and the computer-based prediction programmes classified them as deleterious (Table S3). On the basis of further collaborations and by employing the web-based matching platform GeneMatcher,29Sobreira N. Schiettecatte F. Valle D. Hamosh A. GeneMatcher: a matching tool for connecting investigators with an interest in the same gene.Hum. Mutat. 2015; 36: 928-930Crossref PubMed Scopus (820) Google Scholar we identified another three individuals with de novo DPF2 likely gene-disrupting (LGD) variants, all located in one of the PHD domains. These individuals underwent trio exome sequencing at their respective institutions.30Bramswig N.C. Lüdecke H.J. Alanay Y. Albrecht B. Barthelmie A. Boduroglu K. Braunholz D. Caliebe A. Chrzanowska K.H. Czeschik J.C. et al.Exome sequencing unravels unexpected differential diagnoses in individuals with the tentative diagnosis of Coffin-Siris and Nicolaides-Baraitser syndromes.Hum. 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The third possible LGD variant was the de novo splice-donor mutation c.904+1G>T in individual 6 (Figure 1A). An RNA sample from this individual was not available. This variant is located >50 bp upstream of the last exon-exon junction. However, it is predicted to disrupt the affected canonical splice-donor site of intron 8 (Table S3) and most likely lead to the skipping of exon 8 (r.776_904del). The predicted skipping is likely to result in a stable, in-frame insertion or deletion (p.Ser259_Gly302delinsTrp) affecting several highly conserved amino acids of PHD1. Apart from the DPF2 variants, the broad genetic evaluation revealed additional rare single-nucleotide variants or CNVs in all individuals except individuals 4 and 8. However, these could not explain the clinical phenotype because of either their functional and spatial properties or the fact that they were inherited from an unaffected parent. Additional information about these variants is presented in the Supplemental Note (also see Table S2). According to the pLI (probability of loss-of-function intolerance) value in the ExAC Browser (version 0.3.1; accessed on September 10, 2017), DPF2 seems to be highly intolerant to heterozygous loss-of-function variants (pLI = 1.00) and missense variants (Z score = 3.30).35Lek M. Karczewski K.J. Minikel E.V. Samocha K.E. Banks E. Fennell T. O’Donnell-Luria A.H. Ware J.S. Hill A.J. Cummings B.B. et al.Exome Aggregation ConsortiumAnalysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Scopus (6554) Google Scholar, 36Samocha K.E. Robinson E.B. Sanders S.J. Stevens C. Sabo A. McGrath L.M. Kosmicki J.A. Rehnström K. Mallick S. Kirby A. et al.A framework for the interpretation of de novo mutation in human disease.Nat. Genet. 2014; 46: 944-950Crossref PubMed Scopus (611) Google Scholar Moreover, missense variants reported in the ExAC Browser are located mainly in the N-terminal domain of DPF2, whereas their frequency in PHD fingers is markedly lower (Figure 1A). Microdeletions encompassing DPF2 have not been associated with a clinical phenotype to date.37Nacinovich R. Villa N. Redaelli S. Broggi F. Bomba M. Stoppa P. Scatigno A. Selicorni A. Dalprà L. Neri F. Interstitial 11q deletion: genomic characterization and neuropsychiatric follow up from early infancy to adolescence and literature review.BMC Res. Notes. 2014; 7: 248Crossref PubMed Scopus (12) Google Scholar In the Database of Genomic Variants (DGV),38MacDonald J.R. Ziman R. Yuen R.K. Feuk L. Scherer S.W. The Database of Genomic Variants: a curated collection of structural variation in the human genome.Nucleic Acids Res. 2014; 42: D986-D992Crossref PubMed Scopus (766) Google Scholar two 149.5 and 242.1 kb deletions including DPF2 have been described. The clinical phenotype of individuals 2–8 was similar to that of individual 1 and consistent with the phenotypic spectrum of CSS (Table 1 and Table S1). Global developmental delay was present in all individuals. Three (individuals 2, 3, and 5), one (individual 8), and two (individuals 4 and 7) individuals exhibited moderate, mild, and borderline intellectually disability, respectively. Individual 6 was 3 years and 2 months old at the last clinical evaluation, so a precise estimation of intellectual development was not possible. Speech delay was prominent among all affected children. In five (individuals 2–4, 7, and 8), motor milestones were delayed. Notably, hypoplasia of the fifth toenails, a central feature of CSS diagnosis, was observed in all individuals. Some individuals displayed additional skeletal and ectodermal CSS features, including hypoplasia of further toenails (individuals 4, 6, and 8) and/or fingernails (individuals 3, 4, 6, and 8), generalized or fifth-finger brachydactyly (individuals 4 and 8 or 5 and 6, respectively), and fifth-finger clinodactyly (individuals 2, 5, and 8) (see also Figure 2). As is known about individuals with ARID1B variants, the CSS facial phenotype can be highly variable.39Santen G.W. Clayton-Smith J. ARID1B-CSS consortiumThe ARID1B phenotype: what we have learned so far.Am. J. Med. Genet. C. Semin. Med. Genet. 2014; 166C: 276-289Crossref PubMed Scopus (72) Google Scholar In our study, most individuals bearing DPF2 variants presented with coarse facies. The most consistent facial features were sparse scalp hair (6/8), down-slanting palpebral fissures (6/8), thick or small alae nasi (6/8), a short or broad philtrum (6/8), large, prominent, low-set and/or posteriorly rotated ears (6/8), a prominent forehead (5/8), a broad nose (4/8), a wide mouth (4/8), a thin upper lip (4/8), a thick lower vermillion (4/8), and thick eyebrows (3/8) (Figure 2). Evaluation of growth parameters revealed that five of eight children presented with short stature, although height normalized during puberty in individual 2. In total, four individuals showed evidence of muscular hypotonia, and five manifested feeding problems. Behavioral anomalies—including stereotypic movements (individual 1); temper tantrums, obsessive-compulsive behavior, hyperactivity, poor sleep pattern, and stereotypic hand movements (individual 3); and fixations, temper tantrums, competitivity, and clinically suspected autism (individual 5)—varied across individuals. Furthermore, hearing impairment was described in four affected children. Notably, two individuals were diagnosed with sagittal craniosynostosis (individuals 4 and 5). Individual 7 was clinically diagnosed with trigonocephaly, but no radiographic imaging was performed to confirm a metopic craniosynostosis. To our knowledge, craniosynostosis is extremely rare in CSS, given that it was previously described in only two individuals with an ARID1B variant or a 2p25 deletion encompassing SOX11.12Hempel A. Pagnamenta A.T. Blyth M. Mansour S. McConnell V. Kou I. Ikegawa S. Tsurusaki Y. Matsumoto N. Lo-Castro A. et al.DDD CollaborationDeletions and de novo mutations of SOX11 are associated with a neurodevelopmental disorder with features of Coffin-Siris syndrome.J. Med. Genet. 2016; 53: 152-162Crossref PubMed Scopus (52) Google Scholar, 40Mignot C. Moutard M.L. Rastetter A. Boutaud L. Heide S. Billette T. Doummar D. Garel C. Afenjar A. Jacquette A. et al.ARID1B mutations are the major genetic cause of corpus callosum anomalies in patients with intellectual disability.Brain. 2016; 139: e64Crossref PubMed Scopus (20) Google Scholar However, it is unclear whether it is a consistent manifestation in individuals bearing DPF2 variants, given that both individuals 4 and 5 were selected from the DDD Study on the basis of this anomaly. Other common features included broad thumbs (3/8) and prominent fetal fingertip pads (3/8). Finally, mild to severe constipation and recurrent otitis media were diagnosed in six and four of eight affected children, respectively. Three individuals underwent cerebral magnetic resonance imaging (MRI), which showed atrophy i
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