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• W2759095162 abstract "Bromodomain PHD finger transcription factor (BPTF) is the largest subunit of nucleosome remodeling factor (NURF), a member of the ISWI chromatin-remodeling complex. However, the clinical consequences of disruption of this complex remain largely uncharacterized. BPTF is required for anterior-posterior axis formation of the mouse embryo and was shown to promote posterior neuroectodermal fate by enhancing Smad2-activated wnt8 expression in zebrafish. Here, we report eight loss-of-function and two missense variants (eight de novo and two of unknown origin) in BPTF on 17q24.2. The BPTF variants were found in unrelated individuals aged between 2.1 and 13 years, who manifest variable degrees of developmental delay/intellectual disability (10/10), speech delay (10/10), postnatal microcephaly (7/9), and dysmorphic features (9/10). Using CRISPR-Cas9 genome editing of bptf in zebrafish to induce a loss of gene function, we observed a significant reduction in head size of F0 mutants compared to control larvae. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and phospho-histone H3 (PH3) staining to assess apoptosis and cell proliferation, respectively, showed a significant increase in cell death in F0 mutants compared to controls. Additionally, we observed a substantial increase of the ceratohyal angle of the craniofacial skeleton in bptf F0 mutants, indicating abnormal craniofacial patterning. Taken together, our data demonstrate the pathogenic role of BPTF haploinsufficiency in syndromic neurodevelopmental anomalies and extend the clinical spectrum of human disorders caused by ablation of chromatin remodeling complexes. Bromodomain PHD finger transcription factor (BPTF) is the largest subunit of nucleosome remodeling factor (NURF), a member of the ISWI chromatin-remodeling complex. However, the clinical consequences of disruption of this complex remain largely uncharacterized. BPTF is required for anterior-posterior axis formation of the mouse embryo and was shown to promote posterior neuroectodermal fate by enhancing Smad2-activated wnt8 expression in zebrafish. Here, we report eight loss-of-function and two missense variants (eight de novo and two of unknown origin) in BPTF on 17q24.2. The BPTF variants were found in unrelated individuals aged between 2.1 and 13 years, who manifest variable degrees of developmental delay/intellectual disability (10/10), speech delay (10/10), postnatal microcephaly (7/9), and dysmorphic features (9/10). Using CRISPR-Cas9 genome editing of bptf in zebrafish to induce a loss of gene function, we observed a significant reduction in head size of F0 mutants compared to control larvae. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and phospho-histone H3 (PH3) staining to assess apoptosis and cell proliferation, respectively, showed a significant increase in cell death in F0 mutants compared to controls. Additionally, we observed a substantial increase of the ceratohyal angle of the craniofacial skeleton in bptf F0 mutants, indicating abnormal craniofacial patterning. Taken together, our data demonstrate the pathogenic role of BPTF haploinsufficiency in syndromic neurodevelopmental anomalies and extend the clinical spectrum of human disorders caused by ablation of chromatin remodeling complexes. Chromatin remodeling, an essential process regulating DNA accessibility and transcriptional activation, is controlled by covalent histone modifications and ATP-dependent nucleosome translocation which involves five conserved protein complexes: (1) SWI/SNF (a.k.a. BRG1-associated factors [BAF]), (2) ISWI (imitation switch), (3) CHD (chromatin helicase DNA-binding), (4) INO80/SWR1, and (5) ATRX.1Bartholomew B. Regulating the chromatin landscape: structural and mechanistic perspectives.Annu. Rev. Biochem. 2014; 83: 671-696Crossref PubMed Scopus (142) Google Scholar Thus far, pathogenic variants in 11 chromatin remodeling genes involving SWI/SNF (BAF), CHD, and ATRX have been implicated in congenital disorders as well as cancer development, i.e., ARID1A (Coffin-Siris syndrome 2 [MIM: 614607]), ARID1B (Coffin-Siris syndrome 1 [MIM: 135900]), SMARCA2 (Nicolaides-Baraitser syndrome [MIM: 601358]), SMARCA4 (Coffin-Siris syndrome 4 [MIM: 614609] and Rhabdoid tumors [MIM: 613325]), SMARCB1 (Coffin-Siris syndrome 3 [MIM: 614608], Rhabdoid tumors [MIM: 609322], and Schwannomatosis [MIM: 162091]), SMARCD2 (specific granule deficiency 2 [MIM: 617475]), SMARCE1 (Coffin-Siris syndrome 5 [MIM: 616938]), CHD2 (epileptic encephalopathy, childhood-onset [MIM: 615369]), CHD4 (Sifrim-Hitz-Weiss syndrome [MIM: 617159]), CHD7 (CHARGE syndrome [MIM: 214800] and hypogonadotropic hypogonadism 5 with or without anosmia [MIM: 612370]), and ATRX (alpha-thalassemia/mental retardation syndrome [MIM: 301040]). To date, neither INO80/SWR1 nor ISWI genes have been associated with disease in humans. The ISWI family member NURF (nucleosome remodeling factor) is an evolutionarily conserved key transcriptional regulator of development2Badenhorst P. Voas M. Rebay I. Wu C. Biological functions of the ISWI chromatin remodeling complex NURF.Genes Dev. 2002; 16: 3186-3198Crossref PubMed Scopus (174) Google Scholar in a locus-specific manner3Bai X. Larschan E. Kwon S.Y. Badenhorst P. Kuroda M.I. Regional control of chromatin organization by noncoding roX RNAs and the NURF remodeling complex in Drosophila melanogaster.Genetics. 2007; 176: 1491-1499Crossref PubMed Scopus (33) Google Scholar, 4Kwon S.Y. Xiao H. Glover B.P. Tjian R. Wu C. Badenhorst P. The nucleosome remodeling factor (NURF) regulates genes involved in Drosophila innate immunity.Dev. Biol. 2008; 316: 538-547Crossref PubMed Scopus (55) Google Scholar by virtue of the complex’s chromatin remodeling activity.5Hamiche A. Sandaltzopoulos R. Gdula D.A. Wu C. ATP-dependent histone octamer sliding mediated by the chromatin remodeling complex NURF.Cell. 1999; 97: 833-842Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 6Tsukiyama T. Wu C. Purification and properties of an ATP-dependent nucleosome remodeling factor.Cell. 1995; 83: 1011-1020Abstract Full Text PDF PubMed Scopus (515) Google Scholar In vertebrates, NURF consists of SNF2L (ISWI homolog encoded by SMARCA1), pRBAP46/48, and the largest subunit BPTF (bromodomain PHD finger transcription factor).7Alkhatib S.G. Landry J.W. The nucleosome remodeling factor.FEBS Lett. 2011; 585: 3197-3207Crossref PubMed Scopus (56) Google Scholar, 8Barak O. Lazzaro M.A. Lane W.S. Speicher D.W. Picketts D.J. Shiekhattar R. Isolation of human NURF: a regulator of Engrailed gene expression.EMBO J. 2003; 22: 6089-6100Crossref PubMed Scopus (131) Google Scholar, 9Xiao H. Sandaltzopoulos R. Wang H.M. Hamiche A. Ranallo R. Lee K.M. Fu D. Wu C. Dual functions of largest NURF subunit NURF301 in nucleosome sliding and transcription factor interactions.Mol. Cell. 2001; 8: 531-543Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar Human BPTF contains two PHD finger domains followed proximally by a bromodomain (BRD). The second PHD finger of BPTF mediates binding of the NURF complex to chromatin with trimethylation of histone H3 lysine 4 (H3K4me3).10Jones M.H. Hamana N. Shimane M. Identification and characterization of BPTF, a novel bromodomain transcription factor.Genomics. 2000; 63: 35-39Crossref PubMed Scopus (48) Google Scholar In a combinatorial manner via multivalent interactions together with the PHD finger, the BRD binds to acetylated lysine 16 in histone H4 (H4K16ac), enabling the selective targeting of BPTF to chromatin that contains both histone marks, thereby increasing its selectivity.11Filippakopoulos P. Picaud S. Mangos M. Keates T. Lambert J.P. Barsyte-Lovejoy D. Felletar I. Volkmer R. Müller S. Pawson T. et al.Histone recognition and large-scale structural analysis of the human bromodomain family.Cell. 2012; 149: 214-231Abstract Full Text Full Text PDF PubMed Scopus (1102) Google Scholar, 12Li H. Ilin S. Wang W. Duncan E.M. Wysocka J. Allis C.D. Patel D.J. Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF.Nature. 2006; 442: 91-95Crossref PubMed Scopus (183) Google Scholar, 13Ruthenburg A.J. Li H. Milne T.A. Dewell S. McGinty R.K. Yuen M. Ueberheide B. Dou Y. Muir T.W. Patel D.J. Allis C.D. Recognition of a mononucleosomal histone modification pattern by BPTF via multivalent interactions.Cell. 2011; 145: 692-706Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 14Wysocka J. Swigut T. Xiao H. Milne T.A. Kwon S.Y. Landry J. Kauer M. Tackett A.J. Chait B.T. Badenhorst P. et al.A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling.Nature. 2006; 442: 86-90Crossref PubMed Scopus (875) Google Scholar Heterozygous Bptf mutant mice are apparently normal and fertile with no obvious phenotype.15Goller T. Vauti F. Ramasamy S. Arnold H.H. Transcriptional regulator BPTF/FAC1 is essential for trophoblast differentiation during early mouse development.Mol. Cell. Biol. 2008; 28: 6819-6827Crossref PubMed Scopus (32) Google Scholar Analyses of homozygous mice have revealed that BPTF is essential for the formation of mesoderm, endoderm, and differentiated ectoderm lineages and is required for the establishment of the embryonal anterior-posterior axis during early development; homozygotes show lethality at embryonic day (E)7.5 to E8.5 with 100% penetrance.16Landry J. Sharov A.A. Piao Y. Sharova L.V. Xiao H. Southon E. Matta J. Tessarollo L. Zhang Y.E. Ko M.S. et al.Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells.PLoS Genet. 2008; 4: e1000241Crossref PubMed Scopus (108) Google Scholar BPTF was also found to be important for trophoblast differentiation during early mouse development (E6.5).15Goller T. Vauti F. Ramasamy S. Arnold H.H. Transcriptional regulator BPTF/FAC1 is essential for trophoblast differentiation during early mouse development.Mol. Cell. Biol. 2008; 28: 6819-6827Crossref PubMed Scopus (32) Google Scholar Additionally, depletion of Bptf has enhanced T cell-mediated antitumor immunity in two syngeneic mouse models of cancer.17Mayes K. Alkhatib S.G. Peterson K. Alhazmi A. Song C. Chan V. Blevins T. Roberts M. Dumur C.I. Wang X.Y. Landry J.W. BPTF depletion enhances T-cell-mediated antitumor immunity.Cancer Res. 2016; 76: 6183-6192Crossref PubMed Scopus (20) Google Scholar Consistent with murine mutant data, Bptf was shown previously to promote neuroectodermal posteriorization in zebrafish embryos. RNA in situ hybridization and protein analyses demonstrated that bptf is expressed ubiquitously during zebrafish early embryonic development, and it is expressed abundantly in the zebrafish head through 30 hr post fertilization (hpf).18Ma Y. Liu X. Liu Z. Wei S. Shang H. Xue Y. Cao Y. Meng A. Wang Q. The chromatin remodeling protein Bptf promotes posterior neuroectodermal fate by enhancing Smad2-activated wnt8a expression.J. Neurosci. 2015; 35: 8493-8506Crossref PubMed Scopus (14) Google Scholar Morpholino-based suppression of bptf results in abnormal anterior patterning and defects in neural posteriorization; these phenotypes are attributed to misregulated TGF-β/Smad2 signaling and concomitant restriction of wnt8a expression.18Ma Y. Liu X. Liu Z. Wei S. Shang H. Xue Y. Cao Y. Meng A. Wang Q. The chromatin remodeling protein Bptf promotes posterior neuroectodermal fate by enhancing Smad2-activated wnt8a expression.J. Neurosci. 2015; 35: 8493-8506Crossref PubMed Scopus (14) Google Scholar The TGF-β pathway is critical for cellular growth, differentiation, and apoptosis and was shown to be an important driver in neurogenesis and central nervous system development.19Dobolyi A. Vincze C. Pál G. Lovas G. The neuroprotective functions of transforming growth factor beta proteins.Int. J. Mol. Sci. 2012; 13: 8219-8258Crossref PubMed Scopus (174) Google Scholar Moreover, zebrafish wnt8a was shown to function in early-stage mesoderm patterning and posteriorization of the neuroectoderm.20Erter C.E. Wilm T.P. Basler N. Wright C.V. Solnica-Krezel L. Wnt8 is required in lateral mesendodermal precursors for neural posteriorization in vivo.Development. 2001; 128: 3571-3583PubMed Google Scholar In humans, BPTF is ubiquitously expressed.10Jones M.H. Hamana N. Shimane M. Identification and characterization of BPTF, a novel bromodomain transcription factor.Genomics. 2000; 63: 35-39Crossref PubMed Scopus (48) Google Scholar Amplification and overexpression of BPTF were reported in a variety of cancers including breast, lung, and brain.21Buganim Y. Goldstein I. Lipson D. Milyavsky M. Polak-Charcon S. Mardoukh C. Solomon H. Kalo E. Madar S. Brosh R. et al.A novel translocation breakpoint within the BPTF gene is associated with a pre-malignant phenotype.PLoS ONE. 2010; 5: e9657Crossref PubMed Scopus (47) Google Scholar, 22Dai M. Lu J.J. Guo W. Yu W. Wang Q. Tang R. Tang Z. Xiao Y. Li Z. Sun W. et al.BPTF promotes tumor growth and predicts poor prognosis in lung adenocarcinomas.Oncotarget. 2015; 6: 33878-33892Crossref PubMed Scopus (39) Google Scholar, 23Gong Y.C. Liu D.C. Li X.P. Dai S.P. BPTF biomarker correlates with poor survival in human NSCLC.Eur. Rev. Med. Pharmacol. Sci. 2017; 21: 102-107PubMed Google Scholar, 24Lee J.H. Kim M.S. Yoo N.J. Lee S.H. BPTF, a chromatin remodeling-related gene, exhibits frameshift mutations in gastric and colorectal cancers.APMIS. 2016; 124: 425-427Crossref PubMed Scopus (10) Google Scholar, 25Richart L. Carrillo-de Santa Pau E. Río-Machín A. de Andrés M.P. Cigudosa J.C. Lobo V.J. Real F.X. BPTF is required for c-MYC transcriptional activity and in vivo tumorigenesis.Nat. Commun. 2016; 7: 10153Crossref PubMed Scopus (79) Google Scholar, 26Xiao S. Liu L. Fang M. Zhou X. Peng X. Long J. Lu X. BPTF associated with EMT indicates negative prognosis in patients with hepatocellular carcinoma.Dig. Dis. Sci. 2015; 60: 910-918Crossref PubMed Scopus (21) Google Scholar BPTF was shown as essential for T cell homeostasis and function.27Wu B. Wang Y. Wang C. Wang G.G. Wu J. Wan Y.Y. BPTF is essential for T cell homeostasis and function.J. Immunol. 2016; 197: 4325-4333Crossref PubMed Scopus (17) Google Scholar BPTF also inhibits fNK cell activity and the abundance of natural cytotoxicity receptor co-ligands.28Mayes K. Elsayed Z. Alhazmi A. Waters M. Alkhatib S.G. Roberts M. Song C. Peterson K. Chan V. Ailaney N. et al.BPTF inhibits NK cell activity and the abundance of natural cytotoxicity receptor co-ligands.Oncotarget. 2017; https://doi.org/10.18632/oncotarget.17834Crossref PubMed Scopus (21) Google Scholar Lastly, BPTF maintains chromatin accessibility and the self-renewal capacity of mammary gland stem cells.29Frey W.D. Chaudhry A. Slepicka P.F. Ouellette A.M. Kirberger S.E. Pomerantz W.C.K. Hannon G.J. Dos Santos C.O. BPTF maintains chromatin accessibility and the self-renewal capacity of mammary gland stem cells.Stem Cell Reports. 2017; 9: 23-31Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Here, we describe phenotypic manifestations of germline loss-of-function (LoF) variants in BPTF in ten unrelated individuals with an autosomal-dominant neurodevelopmental disorder and show with an in vivo zebrafish model that bptf is relevant to brain development and craniofacial patterning. The study cohort consists of ten unrelated case subjects. Individuals 1–3 were found in the exome database of 9,056 individuals referred for clinical whole-exome sequencing (WES) at Baylor Genetics (BG). Individuals 4 and 5 were identified in the clinical database of 75,795 subjects referred for clinical chromosomal microarray analysis (CMA) at BG. These five subjects were chosen through filtering for potentially LoF variants in previously unsolved case subjects with overlapping neurological phenotypes. Subsequently, we identified three additional individuals: via DECIPHER30Firth H.V. Richards S.M. Bevan A.P. Clayton S. Corpas M. Rajan D. Van Vooren S. Moreau Y. Pettett R.M. Carter N.P. DECIPHER: Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources.Am. J. Hum. Genet. 2009; 84: 524-533Abstract Full Text Full Text PDF PubMed Scopus (1167) Google Scholar individuals 6 (DECIPHER 275557) and 7 (DECIPHER 264215) from the Deciphering Developmental Disorders (DDD) Study cohort31Deciphering Developmental Disorders StudyLarge-scale discovery of novel genetic causes of developmental disorders.Nature. 2015; 519: 223-228Crossref PubMed Scopus (713) Google Scholar and individual 8 from the University of Groningen, the Netherlands. Through the online matchmaker platform GeneMatcher,32Sobreira 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 individuals 9 and 10 were identified from the Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg in Erlangen, Germany and Virginia Commonwealth University in Richmond, respectively. Written informed consent (for individuals 1–3, 6–10) was obtained in accordance with protocols approved by the appropriate human subject ethics committees; individuals 4 and 5 were covered by a protocol (with waiver of consent) approved by Baylor College of Medicine. The study has UK Research Ethics Committee approval (10/H0305/83, granted by the Cambridge South REC, and GEN/284/12 granted by the Republic of Ireland REC). Individuals 1–3 were analyzed at BG Laboratories by trio WES (individual 1) or proband-only WES (individuals 2 and 3) using the capture design based on VCRome by NimbleGen.33Yang Y. Muzny D.M. Xia F. Niu Z. Person R. Ding Y. Ward P. Braxton A. Wang M. Buhay C. et al.Molecular findings among patients referred for clinical whole-exome sequencing.JAMA. 2014; 312: 1870-1879Crossref PubMed Scopus (965) Google Scholar The mean coverage of target bases was >120×, and >96% target bases were covered at >20×. PCR amplification and Sanger sequencing to verify all candidate variants were done according to standard procedures in the proband and the parents when available, and candidate variants were annotated using the BPTF RefSeq transcript GenBank: NM_004459.6. The two copy-number variant (CNV) deletions in individuals 4 and 5 were detected at BG using customized exon-targeted oligo arrays (OLIGO V8.1.1 and V11.2),34Boone P.M. Bacino C.A. Shaw C.A. Eng P.A. Hixson P.M. Pursley A.N. Kang S.H. Yang Y. Wiszniewska J. Nowakowska B.A. et al.Detection of clinically relevant exonic copy-number changes by array CGH.Hum. Mutat. 2010; 31: 1326-1342Crossref PubMed Scopus (209) Google Scholar, 35Wiszniewska 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 which cover more than 1,700 and 4,800 disease-associated genes, respectively, with exon-level resolution. CNV deletion junction fragments were amplified using long-range PCR with LA Taq DNA polymerase (TaKaRa Bio) and primers designed by Primer3 software. Individuals 6 and 7 were recruited and analyzed by WES in the DDD Study.31Deciphering Developmental Disorders StudyLarge-scale discovery of novel genetic causes of developmental disorders.Nature. 2015; 519: 223-228Crossref PubMed Scopus (713) Google Scholar The variant in individual 8 was detected in the Department of Human Genetics, Radboud University Medical Center, in Nijmegen, the Netherlands. The mutation in individual 9 was identified within an exome Pool-Seq screening approach which will be published elsewhere (B. Popp, unpublished data); validation and segregation analysis was performed by Sanger sequencing followed by genetic fingerprinting using the PowerPlex 21 system (Promega) to confirm de novo occurrence. For individual 10, parent-proband trio WES was performed at Ambry Genetics using the IDT xGen Exome Research Panel and analyzed as previously described.36Farwell K.D. Shahmirzadi L. El-Khechen D. Powis Z. Chao E.C. Tippin Davis B. Baxter R.M. Zeng W. Mroske C. Parra M.C. et al.Enhanced utility of family-centered diagnostic exome sequencing with inheritance model-based analysis: results from 500 unselected families with undiagnosed genetic conditions.Genet. Med. 2015; 17: 578-586Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 37Farwell Hagman K.D. Shinde D.N. Mroske C. Smith E. Radtke K. Shahmirzadi L. El-Khechen D. Powis Z. Chao E.C. Alcaraz W.A. et al.Candidate-gene criteria for clinical reporting: diagnostic exome sequencing identifies altered candidate genes among 8% of patients with undiagnosed diseases.Genet. Med. 2017; 19: 224-235Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar On average, ∼96.6% of the target bases were covered at >20× for the trio. The de novo frameshift alteration identified in this patient by WES was confirmed by Sanger sequencing and interpreted as a (suspected) candidate gene finding.37Farwell Hagman K.D. Shinde D.N. Mroske C. Smith E. Radtke K. Shahmirzadi L. El-Khechen D. Powis Z. Chao E.C. Alcaraz W.A. et al.Candidate-gene criteria for clinical reporting: diagnostic exome sequencing identifies altered candidate genes among 8% of patients with undiagnosed diseases.Genet. Med. 2017; 19: 224-235Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar We identified a single zebrafish (Danio rerio) ortholog of BPTF using reciprocal BLAST (Ensembl ID: ENSDART00000109601; GRCz10; 51% similarity to human BPTF; Figure S1). The CRISPR single-guide (sgRNA) target was identified with ChopChop software38Montague T.G. Cruz J.M. Gagnon J.A. Church G.M. Valen E. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing.Nucleic Acids Res. 2014; 42: W401-W407Crossref PubMed Scopus (677) Google Scholar and synthesized using the GeneArt Precision gRNA Synthesis Kit (Invitrogen) according to manufacturer’s instructions as described.39Küry S. Besnard T. Ebstein F. Khan T.N. Gambin T. Douglas J. Bacino C.A. Craigen W.J. Sanders S.J. Lehmann A. et al.De novo disruption of the proteasome regulatory subunit PSMD12 causes a syndromic neurodevelopmental disorder.Am. J. Hum. Genet. 2017; 100: 352-363Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar To generate F0 mutants, 75 pg of sgRNA and 150 pg of CAS9 protein (PNA bio, CP01) were injected directly into the cell of 1-cell stage zebrafish embryos. The efficiency of the sgRNA was determined by extracting genomic DNA from F0 embryos at 2 dpf by proteinase K digestion (Life technologies, AM2548). The sgRNA genome editing site was PCR amplified and resulting PCR products were denatured and slowly reannealed (denaturing at 95°C for 5 min, ramped down to 85°C at −1°C/s and then to 25°C at −0.1°C/s) to facilitate heteroduplex formation. Heteroduplexes were detected by 15% polyacrylamide gel electrophoresis38Montague T.G. Cruz J.M. Gagnon J.A. Church G.M. Valen E. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing.Nucleic Acids Res. 2014; 42: W401-W407Crossref PubMed Scopus (677) Google Scholar (n = 6 F0 embryos and 1 uninjected control embryo) followed by cloning and sequencing of PCR amplicons to estimate mosaicism. Zebrafish embryos were collected from natural matings of -1.4col1a1:egfp40Zhu X. Xu Y. Yu S. Lu L. Ding M. Cheng J. Song G. Gao X. Yao L. Fan D. et al.An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system.Sci. Rep. 2014; 4: 6420Crossref PubMed Scopus (180) Google Scholar heterozygous adults. For CRISPR experiments, one-cell stage embryos were injected with 1 nL, with the investigator masked to injection cocktail. Embryos were maintained in fresh embryo media (0.3 g/L NaCl, 75 mg/L CaSO4, 37.5 mg/L NaHCO3, 0.003% methylene blue) at 28°C until phenotypic endpoints. Larvae were phenotyped for cell death and cell proliferation at 2 days post fertilization (dpf); and for head size and craniofacial patterning at 3 dpf. Apoptotic cell death was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay as described.41Kague E. Gallagher M. Burke S. Parsons M. Franz-Odendaal T. Fisher S. Skeletogenic fate of zebrafish cranial and trunk neural crest.PLoS ONE. 2012; 7: e47394Crossref PubMed Scopus (141) Google Scholar Briefly, 2 dpf embryos were dechorionated and fixed in 4% paraformaldehyde (PFA) at 4°C overnight and then in 100% methanol at −20°C for 2 hr. After rehydration in PBS, embryos were permeabilized with proteinase K (10 μg/mL) and postfixed with 4% PFA. Embryos were incubated in equilibration buffer and then with TdT enzyme followed by anti-digoxigenin provided in ApopTag Red In Situ Apoptosis Detection Kit (Millipore) as suggested by the manufacturer. We imaged the dorsal anterior aspect of whole-mounts with Z stack image capture using a Nikon AZ100 fluorescent microscope. TUNEL stain was quantified by counting positive cells in defined regions of the head with ImageJ software. At 2 dpf, embryos were dechorionated and fixed in Dent’s solution overnight at 4°C. Embryos were rehydrated in PBS with a stepwise reduced concentration of methanol, and then bleached, postfixed with 4% PFA, and permeabilized using proteinase-K. Embryos were then washed twice in IF buffer (1% BSA, 0.1% Tween-20 in 1× PBS) and incubated overnight with anti-p-histone H3 (PH3; 1:500, Santa Cruz Biotechnology, sc-8656-R). Following two washes in IF buffer, embryos were placed in secondary antibody solution containing Alexa Fluor 488 goat anti-rabbit IgG (1:500; Invitrogen) in blocking solution for 1 hr at room temperature. Z stacked images were captured using a Nikon AZ100 fluorescent microscope. Immunostained cells in defined regions of the dorsal aspect of the head were counted with ImageJ software. We positioned and imaged live 3 dpf larvae with the Vertebrate Automated Screening Technology (VAST; software v.1.2.3.6) to capture dorsal (head size; bright field) or ventral (craniofacial skeleton; fluorescent excitation at 470 nm) images. Larvae were anesthetized, pattern recognition templates were created, and all VAST operational settings were similar to those described.42Isrie M. Breuss M. Tian G. Hansen A.H. Cristofoli F. Morandell J. Kupchinsky Z.A. Sifrim A. Rodriguez-Rodriguez C.M. Dapena E.P. et al.Mutations in either TUBB or MAPRE2 cause circumferential skin creases Kunze type.Am. J. Hum. Genet. 2015; 97: 790-800Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Once recognized inside the 600 μm borosilicate capillary, each larva was rotated to capture images using a 5× fluar objective and Axiocam 503 monochromatic camera (Zen Pro software; Zeiss). Head size and ceratohyal cartilage angle were measured in respective images using ImageJ software and pairwise comparisons to determine statistical significance were made via a Student’s t test. We identified 10 unrelated individuals with unique apparent non-mosaic variants in BPTF. The affected individuals included six males and four females, aged 2.1 to 13 years at the last clinical assessment (Table 1, Figure 1A). Common features included developmental delay (DD)/intellectual disability (ID) (10/10), speech delay (10/10), microcephaly (7/9), motor delay (8/10), hypotonia (5/10), and dysmorphic features (9/10), which included prominent nose (7/10), up-slanting or short palpebral fissures (4/10), and flaring of eyebrows (2/10), 5th finger clinodactyly (3/10), and bulbous halluces/broad great toes/sandal foot (5/10) (Table 1). Of note, individual 3 had clinical WES as part of an NIH-supported pediatric cancer study due to a diagnosis of pheochromocytoma, and WES resulted in a VHL pathogenic heterozygous variant c.499C>T (p.Arg167Trp) (GenBank: NM_000551.3) (Table S1).43Golzio C. Willer J. Talkowski M.E. Oh E.C. Taniguchi Y. Jacquemont S. Reymond A. Sun M. Sawa A. Gusella J.F. et al.KCTD13 is a major driver of mirrored neuroanatomical phenotypes of the 16p11.2 copy number variant.Nature. 2012; 485: 363-367Crossref PubMed Scopus (275) Google Scholar The medical record and WES requisition form had also noted mild developmental delay and speech delay.Table 1Clinical and Genetic Findings in Individuals with BPTF VariantsSubject 1Subject 2Subject 3Subject 4Subject 5Subject 6Subject 7Subject 8Subject 9Subject 10Variant (GRCh37; GeneBank NM_004459.6)chr17: g.65890220dupchr17: g.65908838_65908839delchr17: g.65972049A>Tchr17: g.65700188-65896330del (196 kb)chr17: g.65898399-65986981del (89 kb)chr17: g.65905867_65905878delchr17: g.65914918G>Achr17: g.65971957T>Gchr17: g.65850431delchr17: g.65889796delc.2860dupc.5216_5217delc.8650A>Tc.-121653_2922-3575delc.2922-1506_∗8577delc.3360_3370+1delc.5770G>Ac.8558T>Gc.989delc.2744delp.Glu954Glyfs∗5p.Val1739Glyfs∗96p.Lys2884∗CNV deletionCNV deletionr.spl?p.Ala1924Thrp.Met2853Argp.Leu330Argfs∗28p.Asn915Thrfs∗36Exon913291–910–3012142928Variant typeframeshiftframeshiftnonsenseCNV deletionCNV deletionsplicing & frameshiftmissensemissenseframeshiftframeshiftVariant inheritancede novomother negative, father unavailablede novode novounknownde novode novode novode novode novoEthnicitywhiteLatinoLatinoLatinoLatinowhitewhitewhitewhitewhiteGenderMMMFFMMFMFAge at last assessment (years)2.17.910.9104.31311117.1112Weight at birth (kg) (Z score)2.7 (Z = −1.42)2.63 (Z = −0.8)ND3.4 (Z = 0.00)2.4 (Z = −3.6)4.39 (Z = 0.84)1.89 (Z = −3.44)2.27 (Z = −3.5)3.46 (Z = −0.06)2.69 (Z = −1.38)Length at birth (cm) (Z score)ND47 (Z = −0.7)ND50 (Z = 0.28)48 (Z = −3.82)NDND45 (Z = −3.8)51 (Z = −0.39)48.3 (Z = 0.46)Head circumference at birth (cm) (Z score)33 (Z = −1.13)33 (Z = −0.3)ND33 (Z = −1.11)ND36 (Z = 0.11)33 (Z = −1.17)ND34.5 (Z = −0.62)33 (Z = −0.74)Weight at assessment (kg) (Z score)10.1 (Z = −2.29)20.6 (Z = −1.58)30.1 (Z = −0.98)" @default.
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• W2759095162 date "2017-10-01" @default.
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• W2759095162 title "Haploinsufficiency of the Chromatin Remodeler BPTF Causes Syndromic Developmental and Speech Delay, Postnatal Microcephaly, and Dysmorphic Features" @default.
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