De Novo Loss-of-Function Mutations in USP9X Cause a Female-Specific Recognizable Syndrome with Developmental Delay and Congenital Malformations
2016; Elsevier BV; Volume: 98; Issue: 2 Linguagem: Inglês
10.1016/j.ajhg.2015.12.015
ISSN1537-6605
AutoresMargot R.F. Reijnders, Vasilios Zachariadis, Brooke Latour, Lachlan A. Jolly, Grazia M.S. Mancini, Rolph Pfundt, Ka Man Wu, Conny M.A. van Ravenswaaij‐Arts, Hermine E. Veenstra‐Knol, Britt‐Marie Anderlid, Stephen A. Wood, Sau Wai Cheung, Angela Barnicoat, Frank J. Probst, Pilar Magoulas, Alice S. Brooks, Helena Malmgren, Arja Harila‐Saari, Carlo Marcelis, Maaike Vreeburg, Emma Hobson, V. Reid Sutton, Zornitza Stark, Julie Vogt, Nicola Cooper, Jiin Ying Lim, Sue Price, Angeline Lai, Deepti Domingo, Bruno Reversade, Jozef Gécz, Christian Gilissen, Han G. Brunner, Usha Kini, Ronald Roepman, Ann Nordgren, Tjitske Kleefstra,
Tópico(s)Genetic and Clinical Aspects of Sex Determination and Chromosomal Abnormalities
ResumoMutations in more than a hundred genes have been reported to cause X-linked recessive intellectual disability (ID) mainly in males. In contrast, the number of identified X-linked genes in which de novo mutations specifically cause ID in females is limited. Here, we report 17 females with de novo loss-of-function mutations in USP9X, encoding a highly conserved deubiquitinating enzyme. The females in our study have a specific phenotype that includes ID/developmental delay (DD), characteristic facial features, short stature, and distinct congenital malformations comprising choanal atresia, anal abnormalities, post-axial polydactyly, heart defects, hypomastia, cleft palate/bifid uvula, progressive scoliosis, and structural brain abnormalities. Four females from our cohort were identified by targeted genetic testing because their phenotype was suggestive for USP9X mutations. In several females, pigment changes along Blaschko lines and body asymmetry were observed, which is probably related to differential (escape from) X-inactivation between tissues. Expression studies on both mRNA and protein level in affected-female-derived fibroblasts showed significant reduction of USP9X level, confirming the loss-of-function effect of the identified mutations. Given that some features of affected females are also reported in known ciliopathy syndromes, we examined the role of USP9X in the primary cilium and found that endogenous USP9X localizes along the length of the ciliary axoneme, indicating that its loss of function could indeed disrupt cilium-regulated processes. Absence of dysregulated ciliary parameters in affected female-derived fibroblasts, however, points toward spatiotemporal specificity of ciliary USP9X (dys-)function. Mutations in more than a hundred genes have been reported to cause X-linked recessive intellectual disability (ID) mainly in males. In contrast, the number of identified X-linked genes in which de novo mutations specifically cause ID in females is limited. Here, we report 17 females with de novo loss-of-function mutations in USP9X, encoding a highly conserved deubiquitinating enzyme. The females in our study have a specific phenotype that includes ID/developmental delay (DD), characteristic facial features, short stature, and distinct congenital malformations comprising choanal atresia, anal abnormalities, post-axial polydactyly, heart defects, hypomastia, cleft palate/bifid uvula, progressive scoliosis, and structural brain abnormalities. Four females from our cohort were identified by targeted genetic testing because their phenotype was suggestive for USP9X mutations. In several females, pigment changes along Blaschko lines and body asymmetry were observed, which is probably related to differential (escape from) X-inactivation between tissues. Expression studies on both mRNA and protein level in affected-female-derived fibroblasts showed significant reduction of USP9X level, confirming the loss-of-function effect of the identified mutations. Given that some features of affected females are also reported in known ciliopathy syndromes, we examined the role of USP9X in the primary cilium and found that endogenous USP9X localizes along the length of the ciliary axoneme, indicating that its loss of function could indeed disrupt cilium-regulated processes. Absence of dysregulated ciliary parameters in affected female-derived fibroblasts, however, points toward spatiotemporal specificity of ciliary USP9X (dys-)function. X-linked intellectual disability (ID) with presumed recessive inheritance pattern is shown to be caused by mutations in more than a hundred genes.1Lubs H.A. Stevenson R.E. Schwartz C.E. Fragile X and X-linked intellectual disability: four decades of discovery.Am. J. Hum. Genet. 2012; 90: 579-590Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 2Piton 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 Most families display a clear X-linked segregation pattern, in which males are affected and females are unaffected or mildly affected carriers.3Tarpey P.S. Smith R. Pleasance E. Whibley A. Edkins S. Hardy C. O’Meara S. Latimer C. Dicks E. Menzies A. et al.A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation.Nat. Genet. 2009; 41: 535-543Crossref PubMed Scopus (467) Google Scholar, 4Hu 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. 2016; 21: 133-148Crossref PubMed Scopus (185) Google Scholar, 5Whibley A.C. Plagnol V. Tarpey P.S. Abidi F. Fullston T. Choma M.K. Boucher C.A. Shepherd L. Willatt L. Parkin G. et al.Fine-scale survey of X chromosome copy number variants and indels underlying intellectual disability.Am. J. Hum. Genet. 2010; 87: 173-188Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar In contrast, the number of identified X-linked genes in which de novo mutations cause ID specifically in females is limited. Using whole-exome sequencing (WES), SNP array, array CGH, and CytoScan HD array in a diagnostic setting as described before,6de 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, 7Vulto-van Silfhout A.T. Hehir-Kwa J.Y. van Bon B.W. Schuurs-Hoeijmakers J.H. Meader S. Hellebrekers C.J. Thoonen I.J. de Brouwer A.P. Brunner H.G. Webber C. et al.Clinical significance of de novo and inherited copy-number variation.Hum. Mutat. 2013; 34: 1679-1687Crossref PubMed Scopus (88) Google Scholar, 8Stevens-Kroef M.J. van den Berg E. Olde Weghuis D. Geurts van Kessel A. Pfundt R. Linssen-Wiersma M. Benjamins M. Dijkhuizen T. Groenen P.J. Simons A. Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling.Mol. Cytogenet. 2014; 7: 3Crossref PubMed Scopus (19) Google Scholar, 9Brett M. McPherson J. Zang Z.J. Lai A. Tan E.S. Ng I. Ong L.C. Cham B. Tan P. Rozen S. Tan E.C. Massively parallel sequencing of patients with intellectual disability, congenital anomalies and/or autism spectrum disorders with a targeted gene panel.PLoS ONE. 2014; 9: e93409Crossref PubMed Scopus (31) Google Scholar, 10Wright C.F. Fitzgerald T.W. Jones W.D. Clayton S. McRae J.F. van Kogelenberg M. King D.A. Ambridge K. Barrett D.M. Bayzetinova T. et al.DDD studyGenetic diagnosis of developmental disorders in the DDD study: a scalable analysis of genome-wide research data.Lancet. 2015; 385: 1305-1314Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 11Wiszniewska J. Bi W. Shaw C. Stankiewicz P. Kang S.H. Pursley A.N. Lalani S. Hixson P. Gambin T. Tsai C.H. et al.Combined array CGH plus SNP genome analyses in a single assay for optimized clinical testing.Eur. J. Hum. Genet. 2014; 22: 79-87Crossref PubMed Scopus (96) Google Scholar we identified 13 de novo loss-of-function mutations in USP9X (Ubiquitin-specific protease 9 [MIM: 300072; GenBank: NM_001039590.2]) in females with ID/developmental delay (DD) and multiple congenital malformations (Figures 1A and 1B ; Table S1). Female 7 was previously reported as part of a large study sequencing individuals with ID, congenital anomalies, and/or autism with a targeted gene panel.9Brett M. McPherson J. Zang Z.J. Lai A. Tan E.S. Ng I. Ong L.C. Cham B. Tan P. Rozen S. Tan E.C. Massively parallel sequencing of patients with intellectual disability, congenital anomalies and/or autism spectrum disorders with a targeted gene panel.PLoS ONE. 2014; 9: e93409Crossref PubMed Scopus (31) Google Scholar Written consent was obtained from the legal guardians for all females and the study was given IRB approval. We recognized a similar pattern of facial characteristics, congenital malformations, and brain abnormalities in these females. Four additional affected females were identified because their phenotype was suggestive for USP9X mutations. Subsequently, de novo protein-truncating mutations and intragenic USP9X deletions were duly demonstrated by Sanger sequencing, WES, or CytoScan HD array (Figures 1A and 1B; Table S1), illustrating the clinical recognizability of this new syndrome. All females (age ranging 2 years, 7 months to 23 years) with de novo mutations shared a distinct phenotype. They showed mild to moderate ID with motor and language delay, short stature, hearing loss, and distinct congenital malformations, notably choanal atresia, asymmetric hypomastia, cleft palate/bifid uvula, heart defects, progressive scoliosis, post-axial polydactyly, and anal abnormalities (Tables 1 and S2; case studies in Supplemental Note). Shared facial characteristics included prominent forehead, low nasal bridge, prominent nose with flared alae nasi, thin upper lip, smooth and long philtrum, and ears that were low set, posteriorly rotated, and dysplastic (Figure 2A). In addition to the USP9X variant, female 5 also harbored a de novo variant in PTPN11 (MIM: 176876), which has previously been reported to cause Noonan syndrome (MIM: 163950).12Lepri F.R. Scavelli R. Digilio M.C. Gnazzo M. Grotta S. Dentici M.L. Pisaneschi E. Sirleto P. Capolino R. Baban A. et al.Diagnosis of Noonan syndrome and related disorders using target next generation sequencing.BMC Med. Genet. 2014; 15: 14Crossref PubMed Scopus (50) Google Scholar Though all features that were observed in this female could potentially be explained by the USP9X variant itself (Table S2), a contribution of aberrant PTPN11 to phenotypic features such as intellectual disability, short stature, and heart defect in this female is likely. Neuroimaging reports were available for 13 out of 17 females (Table S2). Detailed evaluation of brain images of five of these females (females 1, 2, 3, 7, and 16) showed asymmetric hypoplasia of the cerebellar vermis and hemisphere with a retrocerebellar cyst, short and thin corpus callosum, thin brainstem, and mildly abnormal frontal gyration pattern (Figure 3). Notably, we observed thyroid hormone abnormalities in six of the females, requiring medical treatment in three of them.Table 1Clinical Features of Females with De Novo USP9X Loss-of-Function MutationsPercentageNumberDevelopmentIntellectual disability or developmental delay100%17/17GrowthShort stature53%9/17Congenital AbnormalitiesEye abnormality59%10/17Choanal atresia35%6/17Cleft palate/bifid uvula29%5/17Dental abnormality71%12/17Asymmetric hypomastia29%5/17Heart defect44%7/16Urogenital abnormality29%5/17Sacral dimple29%5/17Scoliosis65%11/17Hip dysplasia47%8/17Post-axial polydactyly53%9/17Abdominal wall abnormality12%2/17Anal atresia53%9/17NeurologySeizures24%4/17Hypotonia47%8/17Brain AbnormalitiesDandy walker malformation (variant)38%5/13Hypoplastic corpus callosum62%8/13(Asymmetric) cerebellar hypoplasia55%6/11(Asymmetric) enlarged ventricles73%8/11Thin brain stem30%3/10Abnormal gyration pattern frontal lobe50%5/10OtherHearing loss65%11/17(Blaschko) pigment abnormality65%11/17Hypertrichosis29%5/17Leg length discrepancy41%7/17Malignancy12%2/17Recurrent respiratory tract infections53%9/17Thyroid hormone abnormality35%6/17 Open table in a new tab Figure 2Clinical Characteristics of Females with De Novo USP9X Loss-of-Function MutationsShow full caption(A) Frontal and lateral photographs of females with de novo mutations in USP9X. Shared facial characteristics include facial asymmetry, prominent forehead, bitemporal narrowing, short palpebral fissures, low nasal bridge, prominent nose with flared alae nasi from adolescence age, thin upper lip, smooth and long philtrum, hanging full cheeks in early childhood, and low-set, posteriorly rotated, and dysplastic ears with attached lobule.(B) Photographs of the hands of seven affected females. Shared characteristics include ulnar deviation of 5th digit, tapered fingers, short 4th and 5th metacarpals, and post-axial polydactyly (simian crease present but not shown).(C) Photographs of the feet of five affected females. Shared characteristics include hallux valgus and sandal gap (pes cavus present but not shown).(D) Observed Blaschko lines of female 3, indicative for 11 of the affected females, suggestive of different X-inactivation pattern between tissues (functional mosaicism).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Representative MRI Images from Females 1, 2, 3, 7, and 16 with De Novo USP9X Loss-of-Function MutationsShow full caption(A–D) Female 1 (2 years): MRI T2 axial (A, B) and sagittal (C) and T1 axial (D) sections show brachycephaly, mild enlargement of the lateral and 3rd ventricles; mild hypoplasia of cerebellar vermis and left cerebellar hemisphere; enlarged IV ventricle and cisterna magna with small retrocerebellar cyst; thin brain stem and mesencephalon; relatively small frontal lobes with somewhat simplified gyration; and short hypoplastic corpus callosum (both rostrum and splenium).(E–H) Female 2 (1.5 years): MRI T2 axial (E, F) and T1 sagittal (G) and coronal (H) sections show enlargement of the lateral ventricles, mild hypoplasia of cerebellar vermis and left cerebellar hemisphere; enlarged cistern magna; thin corpus callosum, pons, mesencephalon, and brain stem; and broader and underdeveloped frontal gyri.(I–L) Female 3 (11 years): MRI T2 axial (I, J) and T1 sagittal (K) and axial (L) sections show asymmetric enlargement of the lateral ventricles; simplified convolutions of the frontal lobe gyri; hypoplasia of cerebellar vermis and left hemisphere; large cisterna magna and retrocerebellar cyst; and thin corpus callosum with hypoplasia of the rostrum.(M–P) Female 7: MRI T2 axial (M), T1 axial (N), T1 sagittal (O), and coronal FLAIR (P) sections show macrocephaly; enlargement of the lateral and 3rd ventricles with an interhemispheric cyst; dysplastic cerebellar hemispheres; dysplasia of the cerebellar vermis which is uplifted, with a high position of the tentorium and a large posterior fossa, typical of Dandy-Walker malformation; and thin and hypoplastic corpus callosum.(Q–T) Female 16 (2 years): MRI T2 axial (Q, R, S) and T1 coronal (T) sections show enlarged lateral ventricles; irregular gyri of the cerebral cortex with irregular depth of the sulci in frontal and perisylvian areas; small heterotopic nodule of gray matter (arrow) and thin and hypoplastic corpus callosum (both rostrum and splenium); hypoplasia of the anterior cerebellar vermis and left cerebellar hemisphere; enlarged cisterna magna and arachnoidal cyst surrounding the cerebellum, especially at the left side; and mild hypoplasia of pons and brain stem. This female was identified with Sanger sequencing based on these brain abnormalities in combination with ID, dysmorphic features, and congenital abnormalities.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Frontal and lateral photographs of females with de novo mutations in USP9X. Shared facial characteristics include facial asymmetry, prominent forehead, bitemporal narrowing, short palpebral fissures, low nasal bridge, prominent nose with flared alae nasi from adolescence age, thin upper lip, smooth and long philtrum, hanging full cheeks in early childhood, and low-set, posteriorly rotated, and dysplastic ears with attached lobule. (B) Photographs of the hands of seven affected females. Shared characteristics include ulnar deviation of 5th digit, tapered fingers, short 4th and 5th metacarpals, and post-axial polydactyly (simian crease present but not shown). (C) Photographs of the feet of five affected females. Shared characteristics include hallux valgus and sandal gap (pes cavus present but not shown). (D) Observed Blaschko lines of female 3, indicative for 11 of the affected females, suggestive of different X-inactivation pattern between tissues (functional mosaicism). (A–D) Female 1 (2 years): MRI T2 axial (A, B) and sagittal (C) and T1 axial (D) sections show brachycephaly, mild enlargement of the lateral and 3rd ventricles; mild hypoplasia of cerebellar vermis and left cerebellar hemisphere; enlarged IV ventricle and cisterna magna with small retrocerebellar cyst; thin brain stem and mesencephalon; relatively small frontal lobes with somewhat simplified gyration; and short hypoplastic corpus callosum (both rostrum and splenium). (E–H) Female 2 (1.5 years): MRI T2 axial (E, F) and T1 sagittal (G) and coronal (H) sections show enlargement of the lateral ventricles, mild hypoplasia of cerebellar vermis and left cerebellar hemisphere; enlarged cistern magna; thin corpus callosum, pons, mesencephalon, and brain stem; and broader and underdeveloped frontal gyri. (I–L) Female 3 (11 years): MRI T2 axial (I, J) and T1 sagittal (K) and axial (L) sections show asymmetric enlargement of the lateral ventricles; simplified convolutions of the frontal lobe gyri; hypoplasia of cerebellar vermis and left hemisphere; large cisterna magna and retrocerebellar cyst; and thin corpus callosum with hypoplasia of the rostrum. (M–P) Female 7: MRI T2 axial (M), T1 axial (N), T1 sagittal (O), and coronal FLAIR (P) sections show macrocephaly; enlargement of the lateral and 3rd ventricles with an interhemispheric cyst; dysplastic cerebellar hemispheres; dysplasia of the cerebellar vermis which is uplifted, with a high position of the tentorium and a large posterior fossa, typical of Dandy-Walker malformation; and thin and hypoplastic corpus callosum. (Q–T) Female 16 (2 years): MRI T2 axial (Q, R, S) and T1 coronal (T) sections show enlarged lateral ventricles; irregular gyri of the cerebral cortex with irregular depth of the sulci in frontal and perisylvian areas; small heterotopic nodule of gray matter (arrow) and thin and hypoplastic corpus callosum (both rostrum and splenium); hypoplasia of the anterior cerebellar vermis and left cerebellar hemisphere; enlarged cisterna magna and arachnoidal cyst surrounding the cerebellum, especially at the left side; and mild hypoplasia of pons and brain stem. This female was identified with Sanger sequencing based on these brain abnormalities in combination with ID, dysmorphic features, and congenital abnormalities. The X-linked USP9X encodes a structurally and functionally highly conserved deubiquitinating enzyme, containing a UBL (ubiquitin-like) and a catalytic ubiquitin specific protease (USP) domain.13Faesen A.C. Luna-Vargas M.P. Sixma T.K. The role of UBL domains in ubiquitin-specific proteases.Biochem. Soc. Trans. 2012; 40: 539-545Crossref PubMed Scopus (49) Google Scholar, 14Murtaza M. Jolly L.A. Gecz J. Wood S.A. La FAM fatale: USP9X in development and disease.Cell. Mol. Life Sci. 2015; 72: 2075-2089Crossref PubMed Scopus (119) Google Scholar, 15Khut P.Y. Tucker B. Lardelli M. Wood S.A. Evolutionary and expression analysis of the zebrafish deubiquitylating enzyme, usp9.Zebrafish. 2007; 4: 95-101Crossref PubMed Scopus (9) Google Scholar It is known to play an important role in neural development of both humans and mice and is required for fetal development.16Jolly L.A. Taylor V. Wood S.A. USP9X enhances the polarity and self-renewal of embryonic stem cell-derived neural progenitors.Mol. Biol. Cell. 2009; 20: 2015-2029Crossref PubMed Scopus (44) Google Scholar, 17Jones M.H. Furlong R.A. Burkin H. Chalmers I.J. Brown G.M. Khwaja O. Affara N.A. The Drosophila developmental gene fat facets has a human homologue in Xp11.4 which escapes X-inactivation and has related sequences on Yq11.2.Hum. Mol. Genet. 1996; 5: 1695-1701Crossref PubMed Scopus (125) Google Scholar, 18Wood S.A. Pascoe W.S. Ru K. Yamada T. Hirchenhain J. Kemler R. Mattick J.S. Cloning and expression analysis of a novel mouse gene with sequence similarity to the Drosophila fat facets gene.Mech. Dev. 1997; 63: 29-38Crossref PubMed Scopus (73) Google Scholar USP9X is highly expressed during embryogenesis and expression declines as cell fates become restricted.18Wood S.A. Pascoe W.S. Ru K. Yamada T. Hirchenhain J. Kemler R. Mattick J.S. Cloning and expression analysis of a novel mouse gene with sequence similarity to the Drosophila fat facets gene.Mech. Dev. 1997; 63: 29-38Crossref PubMed Scopus (73) Google Scholar The USP9X ortholog in Drosophila, fat facets (faf), has been shown to be important in cell polarity and cell fate of the developing eye in Drosophila.19Fischer-Vize J.A. Rubin G.M. Lehmann R. The fat facets gene is required for Drosophila eye and embryo development.Development. 1992; 116: 985-1000Crossref PubMed Google Scholar A range of signaling proteins involved in different neurodevelopmental pathways including Notch, Wnt, TGF-β, and mTOR have been shown to interact with USP9X.14Murtaza M. Jolly L.A. Gecz J. Wood S.A. La FAM fatale: USP9X in development and disease.Cell. Mol. Life Sci. 2015; 72: 2075-2089Crossref PubMed Scopus (119) Google Scholar, 20Dupont S. Mamidi A. Cordenonsi M. Montagner M. Zacchigna L. Adorno M. Martello G. Stinchfield M.J. Soligo S. Morsut L. et al.FAM/USP9x, a deubiquitinating enzyme essential for TGFbeta signaling, controls Smad4 monoubiquitination.Cell. 2009; 136: 123-135Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 21Friocourt G. Kappeler C. Saillour Y. Fauchereau F. Rodriguez M.S. Bahi N. Vinet M.C. Chafey P. Poirier K. Taya S. et al.Doublecortin interacts with the ubiquitin protease DFFRX, which associates with microtubules in neuronal processes.Mol. Cell. Neurosci. 2005; 28: 153-164Crossref PubMed Scopus (36) Google Scholar, 22Xie Y. Avello M. Schirle M. McWhinnie E. Feng Y. Bric-Furlong E. Wilson C. Nathans R. Zhang J. Kirschner M.W. et al.Deubiquitinase FAM/USP9X interacts with the E3 ubiquitin ligase SMURF1 protein and protects it from ligase activity-dependent self-degradation.J. Biol. Chem. 2013; 288: 2976-2985Crossref PubMed Scopus (86) Google Scholar, 23Agrawal P. Chen Y.T. Schilling B. Gibson B.W. Hughes R.E. Ubiquitin-specific peptidase 9, X-linked (USP9X) modulates activity of mammalian target of rapamycin (mTOR).J. Biol. Chem. 2012; 287: 21164-21175Crossref PubMed Scopus (38) Google Scholar, 24Taya S. Yamamoto T. Kanai-Azuma M. Wood S.A. Kaibuchi K. The deubiquitinating enzyme Fam interacts with and stabilizes beta-catenin.Genes Cells. 1999; 4: 757-767Crossref PubMed Scopus (119) Google Scholar, 25Taya S. Yamamoto T. Kano K. Kawano Y. Iwamatsu A. Tsuchiya T. Tanaka K. Kanai-Azuma M. Wood S.A. Mattick J.S. Kaibuchi K. The Ras target AF-6 is a substrate of the fam deubiquitinating enzyme.J. Cell Biol. 1998; 142: 1053-1062Crossref PubMed Scopus (103) Google Scholar, 26Mouchantaf R. Azakir B.A. McPherson P.S. Millard S.M. Wood S.A. Angers A. The ubiquitin ligase itch is auto-ubiquitylated in vivo and in vitro but is protected from degradation by interacting with the deubiquitylating enzyme FAM/USP9X.J. Biol. Chem. 2006; 281: 38738-38747Crossref PubMed Scopus (98) Google Scholar, 27Al-Hakim A.K. Zagorska A. Chapman L. Deak M. Peggie M. Alessi D.R. Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)-linked polyubiquitin chains.Biochem. J. 2008; 411: 249-260Crossref PubMed Scopus (169) Google Scholar USP9X also has been described to act as both an oncogene and tumor-suppressor gene and is frequently found to be dysregulated in human cancer.14Murtaza M. Jolly L.A. Gecz J. Wood S.A. La FAM fatale: USP9X in development and disease.Cell. Mol. Life Sci. 2015; 72: 2075-2089Crossref PubMed Scopus (119) Google Scholar, 28Luise C. Capra M. Donzelli M. Mazzarol G. Jodice M.G. Nuciforo P. Viale G. Di Fiore P.P. Confalonieri S. An atlas of altered expression of deubiquitinating enzymes in human cancer.PLoS ONE. 2011; 6: e15891Crossref PubMed Scopus (79) Google Scholar, 29Schwickart M. Huang X. Lill J.R. Liu J. Ferrando R. French D.M. Maecker H. O’Rourke K. Bazan F. Eastham-Anderson J. et al.Deubiquitinase USP9X stabilizes MCL1 and promotes tumour cell survival.Nature. 2010; 463: 103-107Crossref PubMed Scopus (497) Google Scholar Two of the affected females developed malignancy at a young age (22 and 8 years). Both acute lymphoblastic leukemia and osteosarcoma were treated successfully and have not reoccurred. To determine the risk and nature of particular malignancies in this new syndrome, further studies are required. We observed pigment abnormalities along Blaschko lines and facial asymmetry, asymmetric abnormalities of the brain and breast, and asymmetric length of the legs (Figures 2A, 2D, and 3), all suggestive of a pattern of post-zygotic mosaicism or differential X-inactivation (XCI) between tissues (functional mosaicism).30Firth H.V. Hurst J.A. Hall J.G. Clinical Genetics. Oxford University Press, New York2009Google Scholar USP9X is one of the genes shown to escape XCI.31Deng X. Berletch J.B. Nguyen D.K. Disteche C.M. X chromosome regulation: diverse patterns in development, tissues and disease.Nat. Rev. Genet. 2014; 15: 367-378Crossref PubMed Scopus (196) Google Scholar, 32Cotton 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 However, it is known that most of the genes that escape from XCI are not fully expressed from the inactivated X chromosome and instead show a partial escape.33Berletch J.B. Yang F. Xu J. Carrel L. Disteche C.M. Genes that escape from X inactivation.Hum. Genet. 2011; 130: 237-245Crossref PubMed Scopus (230) Google Scholar, 34Yang C. Chapman A.G. Kelsey A.D. Minks J. Cotton A.M. Brown C.J. X-chromosome inactivation: molecular mechanisms from the human perspective.Hum. Genet. 2011; 130: 175-185Crossref PubMed Scopus (46) Google Scholar, 35Morleo 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 Moreover, there is accumulating evidence for tissue-specific and developmental-stage-dependent differences in XCI and variability of escape of USP9X.34Yang C. Chapman A.G. Kelsey A.D. Minks J. Cotton A.M. Brown C.J. X-chromosome inactivation: molecular mechanisms from the human perspective.Hum. Genet. 2011; 130: 175-185Crossref PubMed Scopus (46) Google Scholar, 36Carrel 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, 37Talebizadeh Z. Simon S.D. Butler M.G. X chromosome gene expression in human tissues: male and female comparisons.Genomics. 2006; 88: 675-681Crossref PubMed Scopus (70) Google Scholar, 38Zweier C. Kraus C. Brueton L. Cole T. Degenhardt F. Engels H. Gillessen-Kaesbach G. Graul-Neumann L. Horn D. Hoyer J. et al.A new face of Borjeson-Forssman-Lehmann syndrome? De novo mutations in PHF6 in seven females with a distinct phenotype.J. Med. Genet. 2013; 50: 838-847Crossref PubMed Scopus (38) Google Scholar, 39Peeters S.B. Cotton A.M. Brown C.J. Variable escape from X-chromosome inactivation: identifying factors that tip the scales towards expression.BioEssays. 2014; 36: 746-756Crossref PubMed Scopus (70) Google Scholar In the partial escaping genes, non-random XCI or skewing, as observed often in female carriers of an X-linked mutation, will only partially restore a normal phenotype.35Morleo 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 Consistent with this hypothesis, XCI was found to be skewed >90% in fibroblasts in three of the five of the tested females, but skewing was not related to disease severity (Table S3). We note that a similar skewing pattern of XCI was observed recently in females with de novo mutations in DDX3X (MIM: 300160), another X-chromosomal gene that has been suggested to escape XCI and in which de novo mutations cause ID specifically in females.40Snijders Blok L. Madsen E. Juusola J. Gilissen C. Baralle D. Reijnders M.R. Venselaar H. Helsmoortel C. Cho M.T. Hoischen A. et al.DDD StudyMutations in DDX3X are a common cause of unexplained intellectual disability with gender-specific effects on Wnt signaling.Am. J. Hum. Genet. 2015; 97: 343-352Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar In one of the affected females, a predicted splice site mutation was identified. To evaluate whether this mutation indeed results in an aberrant transcript, we synthesized cDNA from RNA extracted from primary skin fibroblasts of both the affected female and a control. We amplified a fragment of 576 base pairs (bp) covering exon 20 to exon 22 by PCR. Electrophoretic separation showed two products of 576 and 455 bp in the sample from the affected female, and a single 576-bp product in the control. Sequencing of the smaller product revealed that this cDNA transcript from the affected female indeed lacked exon 21, confirming the truncating effect of the splice site mutation. Importantly, the level of the transcript was increased 4-fold when fibroblasts were treated with
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