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Missense Variants in the Histone Acetyltransferase Complex Component Gene TRRAP Cause Autism and Syndromic Intellectual Disability

2019; Elsevier BV; Volume: 104; Issue: 3 Linguagem: Inglês

10.1016/j.ajhg.2019.01.010

ISSN

1537-6605

Autores

Benjamin Cogné, Sophie Ehresmann, Éliane Beauregard‐Lacroix, Justine Rousseau, Thomas Besnard, Thomas X. Garcia, Slavé Petrovski, Shiri Avni, Kirsty McWalter, Patrick R. Blackburn, Stephan Sanders, Kévin Uguen, Jacqueline Harris, Julie S. Cohen, Moira Blyth, Anna Lehman, Jonathan Berg, Mindy Li, Usha Kini, Shelagh Joss, Charlotte von der Lippe, Christopher T. Gordon, Jennifer Humberson, Laurie Robak, Daryl A. Scott, V. Reid Sutton, Cara Skraban, Jennifer J. Johnston, Annapurna Poduri, Magnus Nordenskjöld, Vandana Shashi, Erica H. Gerkes, Ernie M.H.F. Bongers, Christian Gilissen, Yuri A. Zárate, Malin Kvarnung, Kevin P. Lally, Peggy Kulch, Brina Daniels, Andrés Hernández, Nicholas Stong, Julie McGaughran, Kyle Retterer, Kristian Tveten, Jennifer A. Sullivan, Madeleine R. Geisheker, Asbjørg Stray‐Pedersen, Jennifer Tarpinian, Eric W. Klee, Julie C. Sapp, Jacob Zyskind, Øystein L. Holla, Emma Bedoukian, Francesca Filippini, Anne Guimier, A. Picard, Øyvind L. Busk, Jaya Punetha, Rolph Pfundt, Anna Lindstrand, Ann Nordgren, Fayth M. Kalb, Megha Desai, Ashley H. Ebanks, Shalini N. Jhangiani, Tammie Dewan, Zeynep Coban‐Akdemir, Aida Telegrafi, Elaine H. Zackai, Amber Begtrup, Xiaofei Song, Annick Toutain, Ingrid M. Wentzensen, Sylvie Odent, Dominique Bonneau, Xénia Latypova, Wallid Deb, Sylvia Redon, Frédéric Bilan, Marine Legendre, Caitlin Troyer, Kerri Whitlock, Oana Caluseriu, Marine I. Murphree, Pavel N. Pichurin, Katherine Agre, Ralitza H. Gavrilova, Tuula Rinne, Meredith Park, Catherine Shain, Erin L. Heinzen, Rui Xiao, Jeanne Amiel, Stanislas Lyonnet, Bertrand Isidor, Leslie G. Biesecker, Dan Lowenstein, Jennifer E. Posey, Anne‐Sophie Denommé‐Pichon, Claude Férec, Xiang‐Jiao Yang, Jill A. Rosenfeld, Brigitte Gilbert‐Dussardier, Séverine Audebert‐Bellanger, Richard Redon, Holly A.F. Stessman, Christoffer Nellåker, Yaping Yang, James R. Lupski, David B. Goldstein, Evan E. Eichler, François V. Bolduc, Stéphane Bézieau, Sébastien Küry, Philippe M. Campeau,

Tópico(s)

Autism Spectrum Disorder Research

Resumo

Acetylation of the lysine residues in histones and other DNA-binding proteins plays a major role in regulation of eukaryotic gene expression. This process is controlled by histone acetyltransferases (HATs/KATs) found in multiprotein complexes that are recruited to chromatin by the scaffolding subunit transformation/transcription domain-associated protein (TRRAP). TRRAP is evolutionarily conserved and is among the top five genes intolerant to missense variation. Through an international collaboration, 17 distinct de novo or apparently de novo variants were identified in TRRAP in 24 individuals. A strong genotype-phenotype correlation was observed with two distinct clinical spectra. The first is a complex, multi-systemic syndrome associated with various malformations of the brain, heart, kidneys, and genitourinary system and characterized by a wide range of intellectual functioning; a number of affected individuals have intellectual disability (ID) and markedly impaired basic life functions. Individuals with this phenotype had missense variants clustering around the c.3127G>A p.(Ala1043Thr) variant identified in five individuals. The second spectrum manifested with autism spectrum disorder (ASD) and/or ID and epilepsy. Facial dysmorphism was seen in both groups and included upslanted palpebral fissures, epicanthus, telecanthus, a wide nasal bridge and ridge, a broad and smooth philtrum, and a thin upper lip. RNA sequencing analysis of skin fibroblasts derived from affected individuals skin fibroblasts showed significant changes in the expression of several genes implicated in neuronal function and ion transport. Thus, we describe here the clinical spectrum associated with TRRAP pathogenic missense variants, and we suggest a genotype-phenotype correlation useful for clinical evaluation of the pathogenicity of the variants. Acetylation of the lysine residues in histones and other DNA-binding proteins plays a major role in regulation of eukaryotic gene expression. This process is controlled by histone acetyltransferases (HATs/KATs) found in multiprotein complexes that are recruited to chromatin by the scaffolding subunit transformation/transcription domain-associated protein (TRRAP). TRRAP is evolutionarily conserved and is among the top five genes intolerant to missense variation. Through an international collaboration, 17 distinct de novo or apparently de novo variants were identified in TRRAP in 24 individuals. A strong genotype-phenotype correlation was observed with two distinct clinical spectra. The first is a complex, multi-systemic syndrome associated with various malformations of the brain, heart, kidneys, and genitourinary system and characterized by a wide range of intellectual functioning; a number of affected individuals have intellectual disability (ID) and markedly impaired basic life functions. Individuals with this phenotype had missense variants clustering around the c.3127G>A p.(Ala1043Thr) variant identified in five individuals. The second spectrum manifested with autism spectrum disorder (ASD) and/or ID and epilepsy. Facial dysmorphism was seen in both groups and included upslanted palpebral fissures, epicanthus, telecanthus, a wide nasal bridge and ridge, a broad and smooth philtrum, and a thin upper lip. RNA sequencing analysis of skin fibroblasts derived from affected individuals skin fibroblasts showed significant changes in the expression of several genes implicated in neuronal function and ion transport. Thus, we describe here the clinical spectrum associated with TRRAP pathogenic missense variants, and we suggest a genotype-phenotype correlation useful for clinical evaluation of the pathogenicity of the variants. Post-translational modifications including acetylation, methylation, phosphorylation, and ubiquitination, of core histones directly alter DNA-histone and histone-histone interactions and thus influence nucleosome dynamics.1Bowman G.D. Poirier M.G. Post-translational modifications of histones that influence nucleosome dynamics.Chem. Rev. 2015; 115: 2274-2295Crossref PubMed Scopus (280) Google Scholar Tight regulation of these marks is required by cells for proper gene transcription,2Venkatesh S. Workman J.L. Histone exchange, chromatin structure and the regulation of transcription.Nat. Rev. Mol. Cell Biol. 2015; 16: 178-189Crossref PubMed Scopus (604) Google Scholar DNA repair,3Hunt C.R. Ramnarain D. Horikoshi N. Iyengar P. Pandita R.K. Shay J.W. Pandita T.K. Histone modifications and DNA double-strand break repair after exposure to ionizing radiations.Radiat. Res. 2013; 179: 383-392Crossref PubMed Scopus (99) Google Scholar and DNA replication. One major activator of transcription is the acetylation of histone tails, which act by neutralizing the positive charges of lysine residues or by recruiting chromatin remodelers and transcription factors.4Legube G. Trouche D. Regulating histone acetyltransferases and deacetylases.EMBO Rep. 2003; 4: 944-947Crossref PubMed Scopus (199) Google Scholar This tightly regulated process is performed by histone acetyltransferases (HATs) and reversed by histone deacetylases (HDACs). There are three major families of HATs: Gcn5-related N-acetyltrasnferase (GNAT), MYST (MOZ, SAS2, SAS3—also known as YBF2—and TIP60), and p300 (EP300-CREBBP).5Berndsen C.E. Denu J.M. Catalysis and substrate selection by histone/protein lysine acetyltransferases.Curr. Opin. Struct. Biol. 2008; 18: 682-689Crossref PubMed Scopus (164) Google Scholar The activity and localization of most HATs, such as TIP60 or GCNL5, depend on a multiprotein assembly that contains the scaffolding protein transformation/transcription domain-associated protein (TRRAP). TRRAP is a large protein of 3,859 amino acids and is conserved from yeast to humans. It is an ataxia-telangiectasia mutated (ATM) related member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family.6McMahon S.B. Van Buskirk H.A. Dugan K.A. Copeland T.D. Cole M.D. The novel ATM-related protein TRRAP is an essential cofactor for the c-Myc and E2F oncoproteins.Cell. 1998; 94: 363-374Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar Like other ATM-related members, it contains FAT (FRAP, ATM, and TRRAP) and FATC (FRAP, ATM, and TRRAP, C terminus) domains flanking a PI3/PI4-kinase domain. The kinase domain of TRRAP does not engage in catalytic activity7Vassilev A. Yamauchi J. Kotani T. Prives C. Avantaggiati M.L. Qin J. Nakatani Y. The 400 kDa subunit of the PCAF histone acetylase complex belongs to the ATM superfamily.Mol. Cell. 1998; 2: 869-875Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar but is required for the proper recruitment of HAT complexes.8Park J. Kunjibettu S. McMahon S.B. Cole M.D. The ATM-related domain of TRRAP is required for histone acetyltransferase recruitment and Myc-dependent oncogenesis.Genes Dev. 2001; 15: 1619-1624Crossref PubMed Scopus (107) Google Scholar TRRAP has been shown to be involved in P53-, E2F-, and c-MYC-dependent gene transcription and oncogenic transformation.6McMahon S.B. Van Buskirk H.A. Dugan K.A. Copeland T.D. Cole M.D. The novel ATM-related protein TRRAP is an essential cofactor for the c-Myc and E2F oncoproteins.Cell. 1998; 94: 363-374Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar, 9Zhao L.-J. Loewenstein P.M. Green M. Enhanced MYC association with the NuA4 histone acetyltransferase complex mediated by the adenovirus E1A N-terminal domain activates a subset of MYC target genes highly expressed in cancer cells.Genes Cancer. 2017; 8: 752-761PubMed Google Scholar, 10Jethwa A. Słabicki M. Hüllein J. Jentzsch M. Dalal V. Rabe S. Wagner L. Walther T. Klapper W. Bohnenberger H. et al.MMML Network ProjectTRRAP is essential for regulating the accumulation of mutant and wild-type p53 in lymphoma.Blood. 2018; 131: 2789-2802Crossref PubMed Scopus (19) Google Scholar As stressed in cancer studies, TRRAP plays an important role in cell-cycle regulation. A recurrent somatic TRRAP variant, c.2165C>T p.(Ser722Phe),11Wei X. Walia V. Lin J.C. Teer J.K. Prickett T.D. Gartner J. Davis S. Stemke-Hale K. Davies M.A. Gershenwald J.E. et al.NISC Comparative Sequencing ProgramExome sequencing identifies GRIN2A as frequently mutated in melanoma.Nat. Genet. 2011; 43: 442-446Crossref PubMed Scopus (385) Google Scholar has been identified in melanoma, and the oncogenic potential of TRRAP has been identified in glioblastoma multiforme,12Wurdak H. Zhu S. Romero A. Lorger M. Watson J. Chiang C.-Y. Zhang J. Natu V.S. Lairson L.L. Walker J.R. et al.An RNAi screen identifies TRRAP as a regulator of brain tumor-initiating cell differentiation.Cell Stem Cell. 2010; 6: 37-47Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar pancreatic adenocarcinoma,13Loukopoulos P. Shibata T. Katoh H. Kokubu A. Sakamoto M. Yamazaki K. Kosuge T. Kanai Y. Hosoda F. Imoto I. et al.Genome-wide array-based comparative genomic hybridization analysis of pancreatic adenocarcinoma: identification of genetic indicators that predict patient outcome.Cancer Sci. 2007; 98: 392-400Crossref PubMed Scopus (126) Google Scholar and lymphoma.10Jethwa A. Słabicki M. Hüllein J. Jentzsch M. Dalal V. Rabe S. Wagner L. Walther T. Klapper W. Bohnenberger H. et al.MMML Network ProjectTRRAP is essential for regulating the accumulation of mutant and wild-type p53 in lymphoma.Blood. 2018; 131: 2789-2802Crossref PubMed Scopus (19) Google Scholar Furthermore, Trrap knockout leads to early embryonic lethality in mice through errors in the cell cycle and a failure to arrest at the mitotic checkpoint.14Herceg Z. Hulla W. Gell D. Cuenin C. Lleonart M. Jackson S. Wang Z.Q. Disruption of Trrap causes early embryonic lethality and defects in cell cycle progression.Nat. Genet. 2001; 29: 206-211Crossref PubMed Scopus (105) Google Scholar In mouse embryonic stem cells (ESCs), Trrap is indispensable for self-renewal as well as correct differentiation,15Sawan C. Hernandez-Vargas H. Murr R. Lopez F. Vaissière T. Ghantous A.Y. Cuenin C. Imbert J. Wang Z.-Q. Ren B. Herceg Z. Histone acetyltransferase cofactor Trrap maintains self-renewal and restricts differentiation of embryonic stem cells.Stem Cells. 2013; 31: 979-991Crossref PubMed Scopus (21) Google Scholar suggesting an essential role in embryonic development and morphogenesis. Moreover, brain-specific Trrap knockout in mice leads to premature differentiation of neural progenitors and abnormal brain development through a decrease in the expression of cell-cycle regulators. This decreased expression results in brain atrophy and microcephaly.16Tapias A. Zhou Z.-W. Shi Y. Chong Z. Wang P. Groth M. Platzer M. Huttner W. Herceg Z. Yang Y.-G. Wang Z.Q. Trrap-dependent histone acetylation specifically regulates cell-cycle gene transcription to control neural progenitor fate decisions.Cell Stem Cell. 2014; 14: 632-643Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar TRRAP has previously been associated with neuropsychiatric disorders such as schizophrenia in a few patients.17Gupta A.R. Westphal A. Yang D.Y.J. Sullivan C.A.W. Eilbott J. Zaidi S. Voos A. Vander Wyk B.C. Ventola P. Waqar Z. et al.Neurogenetic analysis of childhood disintegrative disorder.Mol. Autism. 2017; 8: 19Crossref PubMed Scopus (14) Google Scholar, 18Xu B. Ionita-Laza I. Roos J.L. Boone B. Woodrick S. Sun Y. Levy S. Gogos J.A. Karayiorgou M. De novo gene mutations highlight patterns of genetic and neural complexity in schizophrenia.Nat. Genet. 2012; 44: 1365-1369Crossref PubMed Scopus (328) Google Scholar, 19Takata A. Xu B. Ionita-Laza I. Roos J.L. Gogos J.A. Karayiorgou M. Loss-of-function variants in schizophrenia risk and SETD1A as a candidate susceptibility gene.Neuron. 2014; 82: 773-780Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 20Geisheker M.R. Heymann G. Wang T. Coe B.P. Turner T.N. Stessman H.A.F. Hoekzema K. Kvarnung M. Shaw M. Friend K. et al.Hotspots of missense mutation identify neurodevelopmental disorder genes and functional domains.Nat. Neurosci. 2017; 20: 1043-1051Crossref PubMed Scopus (102) Google Scholar We herein provide data showing that TRRAP pathogenic variants are associated with a variable neurodevelopmental disorder. Through an international collaboration and aided by the web-based tool GeneMatcher,21Sobreira 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 (821) Google Scholar we identified 17 distinct missense TRRAP variants with strong clinical and/or molecular evidence for pathogenicity in 24 individuals with neurodevelopmental disorders (Table 1, Figure 1A). These variants were identified either by trio or solo exome sequencing (ES) from research and clinical cohorts. All affected individuals or their guardians gave appropriate consent for research procedures. This study was approved by the CHU de Nantes ethics committee (comité consultatif sur le traitement de l'information en matière de recherche no. 14.556). Methods are described in Table S1.Table 1De Novo TRRAP Variants Identified in 24 IndividualscDNAProteinInheritanceCpGgnomADCADD Phred Score (v1.3)SIFTPolyPhen2 HVARNumber of Individualsc.2413C>Tp.Leu805Phede novonoabsent28.2deleterious (0)probably damaging (0.998)1c.2580C>Gp.Phe860Leude novonoabsent27.6deleterious (0.03)possibly damaging (0.867)1c.2678G>Tp.Arg893Leuapparently de novoyesabsent34deleterious (0)probably damaging (0.986)1c.3093T>Gp.Ile1031Metde novonoabsent23.4deleterious (0.02)benign (0.308)1c.3104G>Ap.Arg1035Glnde novoyesabsent23.9tolerated (0.09)benign (0.404)1c.3111C>Ap.Ser1037Argde novoyesabsent23.7tolerated (0.14)possibly damaging (0.656)1c.3127G>Ap.Ala1043Thrde novoyesabsent23.2tolerated (0.27)benign (0.066)5c.3311A>Gp.Glu1104Glyde novonoabsent24.6deleterious (0.04)probably damaging (0.91)1c.3316G>Ap.Glu1106Lysde novoanoabsent27.7deleterious (0)possibly damaging (0.816)2c.3331G>Tp.Gly1111Trpapparently de novoyesabsent34deleterious (0)probably damaging (0.999)1c.3475G>Ap.Gly1159Argde novonoabsent33deleterious (0)probably damaging (0.999)1c.5575C>Tp.Arg1859Cysde novoyesabsent34deleterious (0)probably damaging (0.997)1c.5596T>Ap.Trp1866Argde novonoabsent28.7deleterious (0)probably damaging (0.999)1c.5598G>Tp.Trp1866Cysde novonoabsent33deleterious (0)probably damaging (0.999)1c.5647G>Ap.Gly1883Argde novoyesabsent33deleterious (0)probably damaging (1)2c.5795C>Tp.Pro1932Leugermline mosaicismyesabsent35deleterious (0)probably damaging (0.997)2c.11270G>Ap.Arg3757Glnde novoyesabsent28.6deleterious (0.01)benign (0.269)1The RefSeq transcript used for TRRAP is RefSeq: NM_001244580.1. Apparently de novo was mentioned when paternity and maternity were not checked. a. For one individual with p.(Glu1106Lys), father was unavailable, paternal grandparents were tested and did not carry the variant. Open table in a new tab The RefSeq transcript used for TRRAP is RefSeq: NM_001244580.1. Apparently de novo was mentioned when paternity and maternity were not checked. a. For one individual with p.(Glu1106Lys), father was unavailable, paternal grandparents were tested and did not carry the variant. These 17 variants were absent from ExAC and gnomAD22Lek 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 (6555) Google Scholar and were found de novo or apparently de novo (maternity and paternity not checked) in all individuals, except for two sisters who had inherited a variant from a mother with low-level mosaicism (Figure S1) and an individual whose father was unavailable but whose paternal grandparents did not carry the variant. Three variants were recurrently observed: p.Ala1043Thr was identified in five individuals, and p.Glu1106Lys and p.Gly1883Arg were each identified in two individuals. All the variants were predicted to be deleterious by CADD23Kircher M. Witten D.M. Jain P. O'Roak B.J. Cooper G.M. Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants.Nat. Genet. 2014; 46: 310-315Crossref PubMed Scopus (3676) Google Scholar (scaled C scores were over 20), and they were variously predicted to be pathogenic by SIFT24Kumar P. Henikoff S. Ng P.C. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.Nat. Protoc. 2009; 4: 1073-1081Crossref PubMed Scopus (5006) Google Scholar and PolyPhen-2 HVAR.25Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. Kondrashov A.S. Sunyaev S.R. A method and server for predicting damaging missense mutations.Nat. Methods. 2010; 7: 248-249Crossref PubMed Scopus (9290) Google Scholar As shown in Figure 2A, the 17 variants seen in the individuals we studied had significantly increased CADD scores compared to the scores for singleton missense variants reported in gnomAD. The 17 variants all occurred at residues conserved among vertebrates (Figure 1B) and in regions depleted in missense variants in gnomAD. Indeed, when we assessed missense tolerance ratios for TRRAP, we observed that most of the 17 variants were in regions intolerant to missense variants (Figure 2B). Nine out of the 17 variants occurred at highly mutable CpG sites, including one within the codon that leads to the recurrent p.Ala1043Thr variant observed in five individuals. Six missense variants with lesser evidence for pathogenicity were found in another six unrelated individuals (individuals 25 to 30 in Table S1). These variants might be deleterious but were not clearly pathogenic: perhaps the inheritance pattern could not be determined; the variant was present in gnomAD or led to another missense change at the same residue as a variant reported in gnomAD; or the variant was located in a less conserved region of TRRAP (Table S2). Given the number of de novo variants identified, the enrichment for TRRAP de novo variants in our study was calculated as (p = 4.2 × 10−6) on the basis of denovolyzer.26Ware J.S. Samocha K.E. Homsy J. Daly M.J. Interpreting de novo variation in human disease using denovolyzeR.Curr. Protoc. Hum. Genet. 2015; 87: 7.25.1-7.25.15Crossref Scopus (69) Google Scholar Nevertheless, the current number of 22 detected de novo variants in TRRAP is not of genome-wide significance (p = 0.08) after correction for the following: (a) ∼19,000 protein-coding genes, (b) 22,898 trios studied, and (c) the underlying mutability of the full-length protein-coding TRRAP transcript. However, this statistical calculation does not take into account the spatial distribution of the variants. Indeed, three-dimensional modeling of human TRRAP structure inferred from the orthologous Saccharomyces cerevisiae protein Tra1 (Figure 2C) suggested a clustering of the variants in different regions of TRRAP. The most important clustering was observed for 13 variants between codons 1031 and 1159. Interestingly, when visualized in 3D, these variants localized near one another (Figure 1C), revealing a domain of TRRAP with a potentially novel specific function, although this domain has not yet been characterized. We performed a statistical clustering analysis comparing the mean distance between observed variants to ten million permutations of random variants, as previously described.27Lelieveld S.H. Wiel L. Venselaar H. Pfundt R. Vriend G. Veltman J.A. Brunner H.G. Vissers L.E.L.M. Gilissen C. Spatial clustering of de novo missense mutations identifies candidate neurodevelopmental disorder-associated genes.Am. J. Hum. Genet. 2017; 101: 478-484Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar This analysis revealed a significant clustering of variants along the primary sequence of TRRAP (p value = 9 × 10−8), suggesting a model in which specific domains are affected and haploinsufficiency is unlikely, at least for clustering variants. Among the 24 individuals who carried pathogenic variants, 19 presented with facial dysmorphisms. Recurrent features that were noted among these individuals included upslanted palpebral fissures, epicanthus, telecanthus, a wide nasal bridge and ridge, a broad and smooth philtrum, and a thin upper lip (Figure 3). We performed a computer-assisted facial gestalt visualization,28Ferry Q. Steinberg J. Webber C. FitzPatrick D.R. Ponting C.P. Zisserman A. Nellåker C. Diagnostically relevant facial gestalt information from ordinary photos.eLife. 2014; 3: e02020Crossref PubMed Google Scholar, 29Reijnders M.R.F. Janowski R. Alvi M. Self J.E. van Essen T.J. Vreeburg M. Rouhl R.P.W. Stevens S.J.C. Stegmann A.P.A. Schieving J. et al.PURA syndrome: clinical delineation and genotype-phenotype study in 32 individuals with review of published literature.J. Med. Genet. 2018; 55: 104-113Crossref PubMed Scopus (40) Google Scholar which highlighted several of these features, particularly for individuals with variants clustering with the recurrent p.Ala1043Thr variant (Figure 3R). All the individuals had developmental delay, although the severity of intellectual disability (ID) was highly variable. Whereas most individuals had apparent ID with markedly impaired basic life functions, some of them presented with mild ID or even no cognitive deficits (Table 2 and Table S3). Peripheral neuropathy was also noted; it was severe in one individual and consisted of lower-limb hyperreflexia in five other individuals.Table 2Clinical Description of Individuals with Variants Inside or Outside the 1031–1159 ClusterSymptomsAll IndividualsCluster 1031–1159Variants Outside the ClusterGlobal developmental delay24/24 (100%)13/13 (100%)11/11 (100%)Intellectual disability17/20 (85%)11/11 (100%)6/9 (67%)Facial dysmorphisms19/24 (79%)11/13 (85%)8/11 (73%)Autism spectrum disorder5/24 (21%)0/13 (0%)5/11 (45%)Microcephaly ( G (p.Ile1031Met), c.3104G>A (p.Arg1035Gln), c.3111C>A (p.Ser1037Arg), c.3127G>A (p.Ala1043Thr), c.3311A>G (p.Glu1104Gly), c.3316G>A (p.Glu1106Lys), c.3331G>T (p.Gly1111Trp), and c.3475G>A (p.Gly1159Arg). In contrast, individuals with variants residing outside of this region had less malformation and presented mainly with autism spectrum disorder (ASD) and/or ID, sometimes associated with epilepsy. Variants in these individuals were more dispersed along the protein, although some, including c.5575C>T (p.Arg1859Cys), c.5596T>A (p.Trp1866Arg), c.5598G>T (p.Trp1866Cys), c.5647G>A (p.Gly1883Arg), and c.5795C>T (p.Pro1932Leu), apparently aggregated in another region. 13 individuals with variants in the codon 1031–1159 region had global developmental delay and apparent ID, ranging from speech delay and learning difficulties to markedly impaired basic life functions (Table 2 and Table S3). The last available occipitofrontal-circumference measurements revealed microcephaly (ranging from −2.8 to −5 standard deviations [SDs]) in 46% (6/13) of individuals. Cerebral magnetic resonance imaging (MRI) had been performed in 10 out of 13 individuals, and seven of those 10 (70%) showed structural brain anomalies, including cerebellar vermis hypoplasia (6/10), ventricular enlargement (3/10), cortical atrophy (2/10), brainstem atrophy (2/10), polymicrogyria (1/10), focal gliosis (1/10), delayed myelination (1/10), and corpus callosum hypoplasia (1/10). Neurological examination revealed hypotonia in 31% (4/13) of individuals. Only one individual was reported to have epilepsy. Seven individuals (54%) were reported to require feeding exclusively by gastrostomy tube. Among the 10 individuals who were examined by echocardiography, 70% (7/10) had abnormal results, 50% (5/10) had ventricular septal defects, 30% (3/10) had patent ductus arteriosus, 30% (3/10) had patent foramen ovale, 20% (2/10) had pulmonary hypertension, and 20% (2/10) had aortic coarctation. Abdominal ultrasound revealed anomalies in 70% (7/10) of individuals in which it was performed. Abnormal renal morphology, namely multicystic dysplastic kidney, hydronephrosis, a duplicate kidney, and/or a small kidney, was described in 60% (6/10) of individuals, and vesicoureteral reflux was also observed in 30% (3/10) of these individuals. Individual 15 presented with a large left-sided posterolateral congenital diaphragmatic hernia (Table S3). Hernias of the abdominal wall were also found in 23% (3/13) of individuals and included an umbilical hernia, an omphalocele, and an inguinal hernia. Three males (3/6; 50%) had external-genitalia anomalies, including microphallus, hypoplastic scrotum, and cryptorchidism, and two females (2/7; 29%) had a duplicated vagina and/or uterus. Other observed anomalies included dysplastic nails (8/13; 62%), cleft lip and palate (5/13; 38%), clinodactyly of the fifth finger (4/13; 31%), laryngotracheomalacia (3/13), accessory nipple (3/13; 23%), bilateral cutaneous syndactyly of the second and third toe (2/13; 15%), and anomalies of the lacrimal glands (1/13; 8%; see also below with regard to individuals 1 and 19). Four individuals (4/13; 31%) had visual impairment, and three (3/13; 23%) had hearing impairment. Hearing impairment was associated with inner-ear malformations in two cases. Recurrent infections, mainly respiratory and urinary-tract infections, affected three out of 13 (23%) individuals. Individual 9 died at 12 years of age in the context of multiple co-morbidities, including renal failure with acute fluid fluctuations, tracheostomy for severely obstructive laryngotracheomalacia, intermittent supraventricular tachycardia, arterial insufficiency, and polyendocrinopathy (insulin-dependent diabetes, adrenal insufficiency, and hypothyroidism). Among individuals with variants falling outside of the 1031–1159 region, 5/11 (45%) were diagnosed with ASD, and another three individuals (3/11; 27%) had some findings of ASD but no formal diagnosis. 8/11 (73%) had developmental delay and mild-to-severe ID, and three had speech delay, but their IQs were measured above 70, and two of these IQs were in the normal range. Four individuals (4/11; 36%) had various types of epilepsy, namely absence and tonic-clonic seizures, or Lennox-Gastaut syndrome. The age of seizure onset ranged from 2 to 10 years old. Malformations were infrequent in this group overall, although individual 2 had microcephaly and heart malformations, individual 1 had lacrimal duct aplasia, individual 19 had lacrimal duct aplasia and optic disc colobomas, and individual 21 had a postaxial polydactyly of one hand. TRRAP-associated chromatin remodeling complexes are generally associated with gene activation,30Murr R. Vaissière T. Sawan C. Shukla V. Herceg Z. Orchestration of chromatin-based processes: Mind the TRRAP.Oncogene. 2007; 26: 5358-5372Crossref PubMed Scopus (124) Google Scholar which is consistent with their HAT activity. Nevertheless, the NuA4 complex has been shown to have a gene-repression activity necessary for ESC pluripotency.31Chen P.B. Hung J.-H. Hickman T.L. Coles A.H. Carey J.F. Weng Z. Chu F. Fazzio T.G. Hdac6 regulates Tip60-p400 function in stem cells.eLife. 2013; 2: e01557Crossref PubMed Scopus (41) Google Scholar, 32Fazzio T.G. Huff J.T. Panning B. An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity.Cell. 2008; 134: 162-174Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar This gene-repression activity seems to be independent from its lysine acetyltransferase activity.33Acharya D. Hainer S.J. Yoon Y. Wang F. Bach I. Rivera-Pérez J.A. Fazzio T.G. KAT-independent gene regulation by Tip60 promotes ESC self-renewal but not pluripotency.Cell Rep. 2017; 19: 671-679Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar To test the hypothesis that TRRAP variants alter gene expression, we obtained skin fibroblasts from two individuals, individual 1, with p.Leu805Phe, and individual 19, with p.Trp1866Cys and performed next-generation sequencing with technical replicates of RNA (i.e., separately prepared libraries from the same samples). The RNA library preparation and sequencing as well as bioinformatics analysis methods can be found in the Supplemental Data. We found that, in comparison to two typically developing individuals (controls), both individuals with TRRAP variants had remarkably different gene expression patterns (Figure S2A). Interestingly, most differentially expressed genes (DEGs) analyzed with DESeq2 were upregulated in affected individuals compared to controls (Figure S2B). Moreover, the individual with p.Leu805

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