Mutations in DDX3X Are a Common Cause of Unexplained Intellectual Disability with Gender-Specific Effects on Wnt Signaling
2015; Elsevier BV; Volume: 97; Issue: 2 Linguagem: Inglês
10.1016/j.ajhg.2015.07.004
ISSN1537-6605
AutoresLot Snijders Blok, Erik Madsen, Jane Juusola, Christian Gilissen, Diana Baralle, Margot R.F. Reijnders, Hanka Venselaar, Céline Helsmoortel, Megan T. Cho, Alexander Hoischen, Lisenka E.L.M. Vissers, Tom S. Koemans, W.M. Wissink-Lindhout, Evan E. Eichler, Corrado Romano, Hilde Van Esch, Connie T. R. M. Stumpel, Maaike Vreeburg, Eric Smeets, Karin Oberndorff, Bregje W.M. van Bon, Marie Shaw, Jozef Gécz, Eric Haan, Melanie Bienek, Corinna Jensen, Bart Loeys, Anke Van Dijck, A. Micheil Innes, Hilary Racher, Sascha Vermeer, Nataliya Di Donato, Andreas Rump, Katrina Tatton‐Brown, Michael Parker, Alex Henderson, Sally Ann Lynch, Alan Fryer, Alison Ross, Pradeep Vasudevan, Usha Kini, Ruth Newbury‐Ecob, Kate Chandler, Alison Male, Sybe Dijkstra, Jolanda Schieving, Jacques C. Giltay, Koen L.I. van Gassen, Janneke Schuurs-Hoeijmakers, Perciliz L. Tan, Igor Pediaditakis, Stefan A. Haas, Kyle Retterer, Patrick Reed, Kristin G. Monaghan, Eden Haverfield, Marvin R. Natowicz, Angela Myers, Michael C. Kruer, Quinn Stein, Kevin A. Strauss, Karlla W. Brigatti, Katherine E. Keating, Barbara K. Burton, Katherine H. Kim, Joel Charrow, Jennifer Norman, Audrey Foster‐Barber, Antonie D. Kline, Amy Kimball, Elaine H. Zackai, Margaret Harr, Joyce E. Fox, Julie McLaughlin, Kristin Lindstrom, Katrina Haude, Kees van Roozendaal, Han G. Brunner, Wendy K. Chung, R. Frank Kooy, Rolph Pfundt, Vera M. Kalscheuer, Sarju Mehta, Nicholas Katsanis, Tjitske Kleefstra,
Tópico(s)Congenital heart defects research
ResumoIntellectual disability (ID) affects approximately 1%–3% of humans with a gender bias toward males. Previous studies have identified mutations in more than 100 genes on the X chromosome in males with ID, but there is less evidence for de novo mutations on the X chromosome causing ID in females. In this study we present 35 unique deleterious de novo mutations in DDX3X identified by whole exome sequencing in 38 females with ID and various other features including hypotonia, movement disorders, behavior problems, corpus callosum hypoplasia, and epilepsy. Based on our findings, mutations in DDX3X are one of the more common causes of ID, accounting for 1%–3% of unexplained ID in females. Although no de novo DDX3X mutations were identified in males, we present three families with segregating missense mutations in DDX3X, suggestive of an X-linked recessive inheritance pattern. In these families, all males with the DDX3X variant had ID, whereas carrier females were unaffected. To explore the pathogenic mechanisms accounting for the differences in disease transmission and phenotype between affected females and affected males with DDX3X missense variants, we used canonical Wnt defects in zebrafish as a surrogate measure of DDX3X function in vivo. We demonstrate a consistent loss-of-function effect of all tested de novo mutations on the Wnt pathway, and we further show a differential effect by gender. The differential activity possibly reflects a dose-dependent effect of DDX3X expression in the context of functional mosaic females versus one-copy males, which reflects the complex biological nature of DDX3X mutations. Intellectual disability (ID) affects approximately 1%–3% of humans with a gender bias toward males. Previous studies have identified mutations in more than 100 genes on the X chromosome in males with ID, but there is less evidence for de novo mutations on the X chromosome causing ID in females. In this study we present 35 unique deleterious de novo mutations in DDX3X identified by whole exome sequencing in 38 females with ID and various other features including hypotonia, movement disorders, behavior problems, corpus callosum hypoplasia, and epilepsy. Based on our findings, mutations in DDX3X are one of the more common causes of ID, accounting for 1%–3% of unexplained ID in females. Although no de novo DDX3X mutations were identified in males, we present three families with segregating missense mutations in DDX3X, suggestive of an X-linked recessive inheritance pattern. In these families, all males with the DDX3X variant had ID, whereas carrier females were unaffected. To explore the pathogenic mechanisms accounting for the differences in disease transmission and phenotype between affected females and affected males with DDX3X missense variants, we used canonical Wnt defects in zebrafish as a surrogate measure of DDX3X function in vivo. We demonstrate a consistent loss-of-function effect of all tested de novo mutations on the Wnt pathway, and we further show a differential effect by gender. The differential activity possibly reflects a dose-dependent effect of DDX3X expression in the context of functional mosaic females versus one-copy males, which reflects the complex biological nature of DDX3X mutations. Intellectual disability (ID) affects approximately 1%–3% of humans with a gender bias toward males.1Roeleveld N. Zielhuis G.A. Gabreëls F. The prevalence of mental retardation: a critical review of recent literature.Dev. Med. Child Neurol. 1997; 39: 125-132Crossref PubMed Scopus (337) Google Scholar, 2Leonard H. Wen X. The epidemiology of mental retardation: challenges and opportunities in the new millennium.Ment. Retard. Dev. Disabil. Res. Rev. 2002; 8: 117-134Crossref PubMed Scopus (478) Google Scholar, 3Maulik P.K. Mascarenhas M.N. Mathers C.D. Dua T. Saxena S. Prevalence of intellectual disability: a meta-analysis of population-based studies.Res. Dev. Disabil. 2011; 32: 419-436Crossref PubMed Scopus (912) Google Scholar, 4Van Naarden Braun K. Christensen D. Doernberg N. Schieve L. Rice C. Wiggins L. Schendel D. Yeargin-Allsopp M. Trends in the prevalence of autism spectrum disorder, cerebral palsy, hearing loss, intellectual disability, and vision impairment, metropolitan Atlanta, 1991-2010.PLoS ONE. 2015; 10: e0124120Crossref Scopus (184) Google Scholar It is characterized by serious limitations in intellectual functioning and adaptive behavior, starting before the age of 18 years.5Schalock R.L. Borthwick-Duffy S.A. Bradley V.J. Buntinx W.H.E. Coulter D.L. Craig E.M. Gomez S.C. Lachapelle Y. Luckasson R. Reeve A. et al.Intellectual Disability: Definition, Classification, and Systems of Supports. American Association on Intellectual and Developmental Disabilities, 2010Google Scholar Though mutations causing monogenic recessive X-linked intellectual disability (XLID) have been reported in more than 100 genes,6Piton A. Redin C. Mandel J.L. XLID-causing mutations and associated genes challenged in light of data from large-scale human exome sequencing.Am. J. Hum. Genet. 2013; 93: 368-383Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 7Hu H. Haas S.A. Chelly J. Van Esch H. Raynaud M. de Brouwer A.P. Weinert S. Froyen G. Frints S.G. Laumonnier F. et al.X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes.Mol. Psychiatry. 2015; (Published online February 3, 2015)https://doi.org/10.1038/mp.2014.193Crossref Scopus (185) Google Scholar the identification of conditions caused by de novo mutations on the X chromosome affecting females only is limited.8Dobyns W.B. Filauro A. Tomson B.N. Chan A.S. Ho A.W. Ting N.T. Oosterwijk J.C. Ober C. Inheritance of most X-linked traits is not dominant or recessive, just X-linked.Am. J. Med. Genet. A. 2004; 129A: 136-143Crossref PubMed Scopus (132) Google Scholar, 9Morleo M. Franco B. Dosage compensation of the mammalian X chromosome influences the phenotypic variability of X-linked dominant male-lethal disorders.J. Med. Genet. 2008; 45: 401-408Crossref PubMed Scopus (47) Google Scholar By undertaking a systematic analysis of whole exome sequencing (WES) data on 820 individuals (461 males, 359 females) with unexplained ID or developmental delay (from the Department of Human Genetics Nijmegen, the Netherlands), we identified de novo variants in DDX3X (MIM: 300160; GenBank: NM_001356.4) in seven females (1.9% of females). Exome sequencing and data analysis were performed essentially as previously described,10Neveling K. Feenstra I. Gilissen C. Hoefsloot L.H. Kamsteeg E.J. Mensenkamp A.R. Rodenburg R.J. Yntema H.G. Spruijt L. Vermeer S. et al.A post-hoc comparison of the utility of sanger sequencing and exome sequencing for the diagnosis of heterogeneous diseases.Hum. Mutat. 2013; 34: 1721-1726Crossref PubMed Scopus (252) Google Scholar and sequencing was performed in the probands and their unaffected parents (trio approach).11de Ligt J. Willemsen M.H. van Bon B.W. Kleefstra T. Yntema H.G. Kroes T. Vulto-van Silfhout A.T. Koolen D.A. de Vries P. Gilissen C. et al.Diagnostic exome sequencing in persons with severe intellectual disability.N. Engl. J. Med. 2012; 367: 1921-1929Crossref PubMed Scopus (1130) Google Scholar To replicate these findings, we examined a second cohort of 957 individuals (543 males, 414 females) with intellectual disability or developmental delay from GeneDx (sequencing methods as previously published12Retterer K. Scuffins J. Schmidt D. Lewis R. Pineda-Alvarez D. Stafford A. Schmidt L. Warren S. Gibellini F. Kondakova A. et al.Assessing copy number from exome sequencing and exome array CGH based on CNV spectrum in a large clinical cohort.Genet. Med. 2014; (Published online November 6, 2014)https://doi.org/10.1038/gim.2014.160Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and a third cohort of 4,295 individuals with developmental disorders (2,409 males, 1,886 females) from the Deciphering Developmental Disorders (DDD) study (UK).13Deciphering Developmental Disorders StudyLarge-scale discovery of novel genetic causes of developmental disorders.Nature. 2015; 519: 223-228PubMed Google Scholar In most individuals in these cohorts, a SNP array or array CGH had been performed as well and, based on the clinical findings, in several individuals additional metabolic testing, Fragile X testing, or targeted Sanger analysis of different genes associated with ID was completed previously. None of these prior analyses revealed the genetic cause in these individuals. We therefore refer to the individuals in these cohorts as individuals with unexplained ID. We identified 12 de novo alleles in DDX3X in the second cohort (2.9% of females) and 20 de novo alleles in the DDD cohort (1.1% of females). Consequently, based on our findings, mutations in DDX3X are one of the more common causes of ID, accounting for 1%–3% of unexplained ID in females. Altogether, 39 females with de novo variants in DDX3X were identified in our three cohorts and no de novo variants in DDX3X were identified in males (Fisher’s exact test: p = 4.815 × 10−9). To further define this neurodevelopmental disorder, additional females with de novo DDX3X variants were collected from other clinical and diagnostic centers in the Netherlands, Belgium, Germany, Italy, and Canada. In total, we obtained the complete clinical and molecular details of 38 females from across cohorts, which we present in this study. All legal representatives provided informed consent for the use of the data and photographs, and the procedures followed are in accordance with relevant institutional and national guidelines and regulations. The 38 females had 35 distinct de novo variants in DDX3X (Table 1). 19 of the 35 different alleles are predicted to be loss-of-function alleles (9 frameshift mutations leading to premature stop codon, 6 nonsense mutations, and 4 splice site mutations that possibly cause exon skipping), suggesting haploinsufficiency as the most likely pathological mechanism. The other variants, 15 missense variants and 1 in-frame deletion, are all located in the helicase ATP-binding domain or helicase C-terminal domain (Figure 1). One recurrent missense mutation was present in three females (c.1126C>T [p.Arg376Cys]), and in two females the same frameshift mutation, c.1535_1536del (p.His512Argfs∗5), was identified.Table 1Mutation CharacteristicsNucleotide Change (GenBank: )Amino Acid ChangeSIFTPolyPhen-2CohortPreviously ReportedFemalesIndividual 1c.1126C>Tp.Arg376Cysnot toleratedprobably damagingNijmegen–Individual 2c.233C>Gp.Ser78∗NANANijmegen–Individual 3c.1126C>Tp.Arg376Cysnot toleratedprobably damagingDDD StudyDDD Study13Deciphering Developmental Disorders StudyLarge-scale discovery of novel genetic causes of developmental disorders.Nature. 2015; 519: 223-228PubMed Google ScholarIndividual 4c.136C>Tp.Arg46∗NANADDD StudyDDD Study13Deciphering Developmental Disorders StudyLarge-scale discovery of novel genetic causes of developmental disorders.Nature. 2015; 519: 223-228PubMed Google ScholarIndividual 5c.1601G>Ap.Arg534Hisnot toleratedprobably damagingDDD StudyDDD Study13Deciphering Developmental Disorders StudyLarge-scale discovery of novel genetic causes of developmental disorders.Nature. 2015; 519: 223-228PubMed Google ScholarIndividual 6c.641T>Cp.Ile214Thrnot toleratedprobably damagingDDD StudyDDD Study13Deciphering Developmental Disorders StudyLarge-scale discovery of novel genetic causes of developmental disorders.Nature. 2015; 519: 223-228PubMed Google ScholarIndividual 7c.1520T>Cp.Ile507Thrnot toleratedprobably damagingother–Individual 8c.977G>Ap.Arg326Hisnot toleratedprobably damagingother–Individual 9c.868delp.Ser290Hisfs∗31NANAotherRauch et al.41Rauch A. Wieczorek D. Graf E. Wieland T. Endele S. Schwarzmayr T. Albrecht B. Bartholdi D. Beygo J. Di Donato N. et al.Range of genetic mutations associated with severe non-syndromic sporadic intellectual disability: an exome sequencing study.Lancet. 2012; 380: 1674-1682Abstract Full Text Full Text PDF PubMed Scopus (764) Google ScholarIndividual 10c.1229_1230dupp.Thr411Leufs∗10NANANijmegen–Individual 11c.1105dupp.Thr369Asnfs∗14NANANijmegen–Individual 12c.865−2A>Gp.?NANANijmegen–Individual 13c.1600dupp.Arg534Profs∗13NANAother–Individual 14c.269dupp.Ser90Argfs∗8NANANijmegen–Individual 15c.1440A>Tp.Arg480Sernot toleratedprobably damagingNijmegen–Individual 16c.873C>Ap.Tyr291∗NANANijmegen–Individual 17c.1693C>Tp.Gln565∗NANANijmegen–Individual 18c.1535_1536delp.His512Argfs∗5NANADDD Study–Individual 19c.766−1G>Cp.?NANAother–Individual 20c.599dupp.Tyr200∗NANAUSA–Individual 21c.1321delp.Asp441Ilefs∗3NANAUSA–Individual 22c.1383dupp.Tyr462Ilefs∗3NANAUSA–Individual 23c.1384_1385dupp.His463Thrfs∗34NANAUSA–Individual 24c.1535_1536delp.His512Argfs∗5NANAUSA–Individual 25c.1541T>Cp.Ile514Thrnot toleratedprobably damagingUSA–Individual 26c.704T>Cp.Leu235Pronot toleratedprobably damagingUSA–Individual 27c.1175T>Cp.Leu392Pronot toleratedprobably damagingUSA–Individual 28c.1463G>Ap.Arg488Hisnot toleratedpossibly damagingUSA–Individual 29c.1126C>Tp.Arg376Cysnot toleratedprobably damagingUSA–Individual 30c.1250A>Cp.Gln417Pronot toleratedprobably damagingUSA–Individual 31c.698C>Tp.Ala233Valnot toleratedprobably damagingDDD Study–Individual 32c.931C>Tp.Arg311∗NANADDD Study–Individual 33c.46-2A>Cp.?NANADDD Study–Individual 34c.1678_1680delp.Leu560delNANADDD Study–Individual 35c.1423C>Gp.Arg475Glynot toleratedprobably damagingDDD Study–Individual 36c.46-2A>Gp.?NANADDD Study–Individual 37c.1703C>Tp.Pro568Leunot toleratedprobably damagingDDD Study–Individual 38c.1526A>Tp.Asn509Ilenot toleratedprobably damagingDDD Study–MalesFamily 1c.1084C>Tp.Arg362Cysnot toleratedprobably damaging––Family 2c.1052G>Ap.Arg351Glnnot toleratedpossibly damaging–Hu et al.7Hu H. Haas S.A. Chelly J. Van Esch H. Raynaud M. de Brouwer A.P. Weinert S. Froyen G. Frints S.G. Laumonnier F. et al.X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes.Mol. Psychiatry. 2015; (Published online February 3, 2015)https://doi.org/10.1038/mp.2014.193Crossref Scopus (185) Google ScholarFamily 3c.898G>Tp.Val300Phenot toleratedprobably damaging––“Other” indicates additional females with de novo DDX3X mutations collected from different diagnostic centers in the Netherlands, Belgium, Germany, Italy, and Canada. Open table in a new tab “Other” indicates additional females with de novo DDX3X mutations collected from different diagnostic centers in the Netherlands, Belgium, Germany, Italy, and Canada. Analysis of the clinical data suggested a syndromic disorder with variable clinical presentation. The females (age range 1–33 years) showed varying degrees of ID (mild to severe) or developmental delay with associated neurological abnormalities, including hypotonia (29/38, 76%), movement disorders comprising dyskinesia, spasticity, and a stiff-legged or wide-based gait (17/38, 45%), microcephaly (12/38, 32%), behavior problems including autism spectrum disorder (ASD), hyperactivity, and aggression (20/38, 53%), and epilepsy (6/38, 16%). Several recurrent additional features were noted, including joint hyperlaxity, skin abnormalities (mosaic-like pigmentary changes in some females), cleft lip and/or palate, hearing and visual impairment, and precocious puberty. Abnormal brain MRIs were reported in various females, with corpus callosum hypoplasia (13/37, 35%), ventricular enlargement (13/37, 35%), and evidence of cortical dysplasia (e.g., polymicrogyria) in four individuals. A summary of the clinical data is presented in Tables 2 and S1, and facial profiles of 30 of the 38 females are shown in Figure 2. Although common dysmorphic features are reported, there is no consistent recognizable phenotype. Based on these clinical and molecular data, there is no evidence for an obvious genotype-phenotype correlation between the different types of mutations and degree of ID.Table 2Clinical Features of Females with De Novo DDX3X MutationsPercentageNumberDevelopmentIntellectual disability or developmental delay100%38/38Mild or mild-moderate disability26%10/38Moderate or moderate-severe disability26%10/38Severe disability40%15/38Developmental delay8%3/38GrowthLow weight32%12/38Microcephaly32%12/38NeurologyHypotonia76%29/38Epilepsy16%6/38Movement disorder (including spasticity)45%17/38Behavior problems53%20/38Brain MRICorpus callosum hypoplasia35%13/37Cortical malformation11%4/37Ventricular enlargement35%13/37OtherSkin abnormalities37%14/38Hyperlaxity37%14/38Visual problems34%13/38Hearing loss8%3/38Cleft lip or palate8%3/38Precocious puberty13%5/38Scoliosis11%4/38 Open table in a new tab DDX3X is known as one of the genes that are able to escape X inactivation.14Lahn B.T. Page D.C. Functional coherence of the human Y chromosome.Science. 1997; 278: 675-680Crossref PubMed Scopus (720) Google Scholar, 15Cotton A.M. Price E.M. Jones M.J. Balaton B.P. Kobor M.S. Brown C.J. Landscape of DNA methylation on the X chromosome reflects CpG density, functional chromatin state and X-chromosome inactivation.Hum. Mol. Genet. 2015; 24: 1528-1539Crossref PubMed Scopus (161) Google Scholar X-linked dominant conditions often show a remarkable variability among affected females which in particular holds true for genes that escape X inactivation.9Morleo M. Franco B. Dosage compensation of the mammalian X chromosome influences the phenotypic variability of X-linked dominant male-lethal disorders.J. Med. Genet. 2008; 45: 401-408Crossref PubMed Scopus (47) Google Scholar, 16Carrel L. Willard H.F. X-inactivation profile reveals extensive variability in X-linked gene expression in females.Nature. 2005; 434: 400-404Crossref PubMed Scopus (1482) Google Scholar It is known that most of the transcripts escaping X inactivation are not fully expressed from the inactivated X chromosome, which means that the escape is often partial and incomplete.9Morleo M. Franco B. Dosage compensation of the mammalian X chromosome influences the phenotypic variability of X-linked dominant male-lethal disorders.J. Med. Genet. 2008; 45: 401-408Crossref PubMed Scopus (47) Google Scholar Based on this, phenotypic severity might be influenced by the amount of gene expression of DDX3X in females, which could be affected by possibly skewed X inactivation or incompleteness of the escape. Different expression of DDX3X in different tissues could also be a contributing factor. To further explore the possible skewing of X inactivation, we determined X inactivation via the androgen receptor gene (AR) methylation assay17Allen R.C. Zoghbi H.Y. Moseley A.B. Rosenblatt H.M. Belmont J.W. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.Am. J. Hum. Genet. 1992; 51: 1229-1239PubMed Google Scholar on DNA from lymphocytes in 15 females. We found an almost complete skewing (>95%) of X inactivation in seven individuals and random skewing in the remainder. This is more than would be expected by chance, because it is known that a high degree of skewing of X inactivation is a statistically rare event in young women.18Busque L. Mio R. Mattioli J. Brais E. Blais N. Lalonde Y. Maragh M. Gilliland D.G. Nonrandom X-inactivation patterns in normal females: lyonization ratios vary with age.Blood. 1996; 88: 59-65PubMed Google Scholar However, in our affected females, there is no evidence of correlation of skewing of X inactivation with disease severity. Given the high frequency (1%–2%) of DDX3X mutations in females with unexplained ID, we sought to determine whether males carry deleterious alleles. We identified no de novo variants in DDX3X males in any of our cohorts. However, upon sequencing of the X chromosome (X-exome) of ID-affected families with apparent X-linked inheritance pattern,7Hu H. Haas S.A. Chelly J. Van Esch H. Raynaud M. de Brouwer A.P. Weinert S. Froyen G. Frints S.G. Laumonnier F. et al.X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes.Mol. Psychiatry. 2015; (Published online February 3, 2015)https://doi.org/10.1038/mp.2014.193Crossref Scopus (185) Google Scholar we identified two families with segregating missense variants in DDX3X. Moreover, one additional family was identified by diagnostic whole exome sequencing in Antwerp, Belgium. In these three families, males with the DDX3X variant have borderline to severe ID and carrier females are unaffected. Pedigrees of these three families are shown in Figure S1, and a summary of the clinical features of the affected males is presented in Table S2. All three missense mutations were predicted to be deleterious by prediction programs PolyPhen-2 and SIFT (Table 1) and map within the helicase ATP-binding domain (Figure 2). With three-dimensional protein analysis, we could not discern any clear difference between the missense mutations found in affected males and the de novo mutations found in females that could possibly explain the gender-specific pathogenicity (Figure S2, Table S3). Although in the first two families with affected males the phenotype consisted mainly of intellectual disability, family 3 was more complex. The male proband had severe ID and various other features such as a dysplastic pulmonary valve, hypertonia, and strabismus. In this male a SNP-array analysis was performed, as well as DNA analysis of PTEN (MIM: 601728) and FMR1 (MIM: 309550) and methylation studies on Angelman syndrome (MIM: 105830), all without abnormalities. With exome sequencing no other candidate genes were found. His mother had recurrent miscarriages of unknown gender. A second initially viable pregnancy was terminated because of ultrasound anomalies that had also been noted in the proband, including a thickened nuchal fold and absent nasal bone. After termination of the pregnancy, the male fetus was tested and found to have the same missense mutation in DDX3X as his brother. Sequencing of other family members demonstrated that the mutation arose de novo in the proband’s mother. X-inactivation studies in this mother demonstrated a random X-inactivation pattern (68/32). X-inactivation studies in female carriers in the other families showed that in family 1, the obligate carrier female (II-2) had highly skewed X inactivation (>95%), whereas X-inactivation studies in family 2 were not informative. None of the three DDX3X variants found in these families with affected males were reported in the ExAC database or in the Exome Variant Server (ESP), nor was one of the de novo variants found in females reported in these databases. Moreover, none of the DDX3X mutations identified in males were detected in affected females. As far as we are aware, in addition to the de novo missense mutation identified in family 3, no other de novo mutations in DDX3X are reported in healthy individuals or control cohorts. We downloaded all variants from the ExAC database, containing exome data of 60,706 individuals, and calculated per gene the number of missense and synonymous variants. These numbers were then normalized by dividing through the total number of possible missense and synonymous variants per gene. The ratio of corrected missense over synonymous variants was then used as a measure for tolerance of the gene to normal variation, similarly as was done previously.19Gilissen C. Hehir-Kwa J.Y. Thung D.T. van de Vorst M. van Bon B.W. Willemsen M.H. Kwint M. Janssen I.M. Hoischen A. Schenck A. et al.Genome sequencing identifies major causes of severe intellectual disability.Nature. 2014; 511: 344-347Crossref PubMed Scopus (783) Google Scholar When genes were ranked according to their tolerance score, DDX3X was among the most intolerant genes (1.09% of genes, rank 194 out of 17,856), showing that normal variation in this gene is extremely rare. DDX3X encodes a conserved DEAD-box RNA helicase important in a variety of fundamental cellular processes that include transcription, splicing, RNA transport, and translation.20Abdelhaleem M. RNA helicases: regulators of differentiation.Clin. Biochem. 2005; 38: 499-503Crossref PubMed Scopus (104) Google Scholar, 21Garbelli A. Beermann S. Di Cicco G. Dietrich U. Maga G. 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Determination of the role of DDX3 a factor involved in mammalian RNAi pathway using an shRNA-expression library.PLoS ONE. 2013; 8: e59445Crossref PubMed Scopus (25) Google Scholar and it is a key regulator of the Wnt/β-catenin pathway, acting as a regulatory subunit of CSNK1E and stimulating its kinase activity.25Cruciat C.M. Dolde C. de Groot R.E. Ohkawara B. Reinhard C. Korswagen H.C. Niehrs C. RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-β-catenin signaling.Science. 2013; 339: 1436-1441Crossref PubMed Scopus (156) Google Scholar Notably, of the 35 different de novo variants, 19 alleles are predicted to give a loss-of-function effect. Antisense-based knockdown of the DDX3X-ortholog PL10 in zebrafish (72% identical, 78% similar) is already described and shows a reduced brain size and head size in zebrafish embryos at 2 days postfertilization (dpf).13Deciphering Developmental Disorders StudyLarge-scale discovery of novel genetic causes of developmental disorders.Nature. 2015; 519: 223-228PubMed Google Scholar The missense changes in affected females were located in the same protein domain as the missense changes in affected males, so we explored the pathogenic mechanisms accounting for the differences in disease transmission and phenotype between affected females and affected males with DDX3X missense variants. We therefore employed a combination of in vitro and in vivo assays based on the known role of DDX3X in the regulation of Wnt/β-catenin signaling.25Cruciat C.M. Dolde C. de Groot R.E. Ohkawara B. Reinhard C. Korswagen H.C. Niehrs C. 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