De Novo Truncating Variants in ASXL2 Are Associated with a Unique and Recognizable Clinical Phenotype
2016; Elsevier BV; Volume: 99; Issue: 4 Linguagem: Inglês
10.1016/j.ajhg.2016.08.017
ISSN1537-6605
AutoresVandana Shashi, Loren D.M. Peña, Katherine Kim, Barbara K. Burton, Maja Hempel, Kelly Schoch, Magdalena Walkiewicz, Heather M. McLaughlin, Megan Cho, Nicholas Stong, Scott E. Hickey, Christine Shuss, Michael Freemark, Jane S. Bellet, Martha Ann Keels, Melanie J. Bonner, Maysantoine A. El-Dairi, Megan W. Butler, Peter G. Kranz, Constance T. R. M. Stumpel, Sylvia Klinkenberg, Karin Oberndorff, Malik Alawi, René Santer, Slavé Petrovski, Outi Kuismin, Satu Korpi-Heikkilä, Olli Pietiläinen, Aarno Palotie, Mitja Kurki, Alexander Hoischen, Anna C. Need, David B. Goldstein, Fanny Kortüm, A. Bacino, Brendan Lee, Ashok Balasubramanyam, Lindsay C. Burrage, Gary Clark, William J. Craigen, Shweta U. Dhar, Lisa Emrick, Brett H. Graham, Mahim Jain, Seema R. Lalani, Richard A. Lewis, Paolo Moretti, Sarah K. Nicholas, Jordan S. Orange, Jennifer E. Posey, Lorraine Potocki, Jill A. Rosenfeld, Daryl A. Scott, Neil A. Hanchard, Tran A. Alyssa, Alejandro E. Mercedes, Azamian S. Mashid, Hugo J. Bellen, Shinya Yamamoto, Michael F. Wangler, Monte Westerfield, John H. Postlethwait, Christine M. Eng, Yaping Yang, Donna M. Muzny, Patricia A. Ward, Rachel Ramoni, Alexa T. McCray, Issac S. Kohane, Ingrid A. Holm, Matthew Might, Paul Mazur, Kimberly Splinter, Cecilia Esteves, Vandana Shashi, Yong‐hui Jiang, Loren D.M. Peña, Allyn McConkie‐Rosell, Kelly Schoch, Rebecca C. Spillmann, Jennifer A. Sullivan, Sophie Nicole, David B. Goldstein, Nicholas Stong, Alan H. Beggs, Joseph Loscalzo, Calum A. MacRae, Edwin K. Silverman, Joan M. Stoler, David A. Sweetser, Richard L. Maas, Joel B. Krier, Lance H. Rodan, Chris A. Walsh, Cynthia M. Cooper, J. Carl Pallais, Laurel A. Donnell‐Fink, Elizabeth L. Krieg, Sharyn A. Lincoln, Lauren C. Briere, Howard J. Jacob, Elizabeth A. Worthey, J. J. Lazar, Kim A. Strong, Lori H. Handley, J. Scott Newberry, David Bick, Molly C. Schroeder, Donna M. Brown, Camille L. Birch, Shawn Levy, Braden Boone, Dan C. Dorset, Angela Jones, Teri A. Manolio, John J. Mulvihill, Anastasia L. Wise, Jyoti G. Dayal, David J. Eckstein, Donna M. Krasnewich, Carson R. Loomis, Laura A. Mamounas, Brenda Iglesias, Casey Martin, David M. Koeller, Thomas Metz, Euan A. Ashley, Paul G. Fisher, Jonathan A. Bernstein, Matthew T. Wheeler, Patricia A. Zornio, Daryl Waggott, Annika M. Dries, Jennefer N. Kohler, Katrina M. Dipple, Stan F. Nelson, Christina G.S. Palmer, Éric Vilain, Patrick Allard, Esteban C. Dell Angelica, Hane Lee, Janet S. Sinsheimer, Jeanette C. Papp, Naghmeh Dorrani, Matthew Herzog, Hayk Barseghyan, David R. Adams, David R. Adams, Elizabeth A. Burke, Katherine R. Chao, Mariska Davids, David D. Draper, Tyra Estwick, Trevor S. Frisby, Kate Frost, William A. Gahl, Valerie Gartner, Rena A. Godfrey, Mitchell Goheen, Gretchen Golas, Mary “Gracie” G. Gordon, Catherine Groden, Andrea Gropman, Mary E. Hackbarth, Isabel Hardee, Jean M. Johnston, Alanna E. Koehler, Lea Latham, Yvonne L. Latour, C.-Y. Lau, Paul R. Lee, Denise J. Levy, Adam P. Liebendorder, Ellen F. Macnamara, Valerie V. Maduro, May V. Malicdan, Thomas C. Markello, Alexandra J. McCarty, Jennifer L. Murphy, Michele Nehrebecky, Donna Novacic, Barbara N. Pusey, Sarah Sadozai, Katherine E. Schaffer, Prashant Sharma, Ariane Soldatos, Sara Thomas, Cynthia J. Tifft, Nathanial J. Tolman, Camilo Toro, Zaheer Valivullah, Colleen E. Wahl, Mike Warburton, Alec A. Weech, Lynne A. Wolfe, Guoyun Yu, Rizwan Hamid, John H. Newman, John A. Phillips, Joy D. Cogan,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoThe ASXL genes (ASXL1, ASXL2, and ASXL3) participate in body patterning during embryogenesis and encode proteins involved in epigenetic regulation and assembly of transcription factors to specific genomic loci. Germline de novo truncating variants in ASXL1 and ASXL3 have been respectively implicated in causing Bohring-Opitz and Bainbridge-Ropers syndromes, which result in overlapping features of severe intellectual disability and dysmorphic features. ASXL2 has not yet been associated with a human Mendelian disorder. In this study, we performed whole-exome sequencing in six unrelated probands with developmental delay, macrocephaly, and dysmorphic features. All six had de novo truncating variants in ASXL2. A careful review enabled the recognition of a specific phenotype consisting of macrocephaly, prominent eyes, arched eyebrows, hypertelorism, a glabellar nevus flammeus, neonatal feeding difficulties, hypotonia, and developmental disabilities. Although overlapping features with Bohring-Opitz and Bainbridge-Ropers syndromes exist, features that distinguish the ASXL2-associated condition from ASXL1- and ASXL3-related disorders are macrocephaly, absence of growth retardation, and more variability in the degree of intellectual disabilities. We were also able to demonstrate with mRNA studies that these variants are likely to exert a dominant-negative effect, given that both alleles are expressed in blood and the mutated ASXL2 transcripts escape nonsense-mediated decay. In conclusion, de novo truncating variants in ASXL2 underlie a neurodevelopmental syndrome with a clinically recognizable phenotype. This report expands the germline disorders that are linked to the ASXL genes. The ASXL genes (ASXL1, ASXL2, and ASXL3) participate in body patterning during embryogenesis and encode proteins involved in epigenetic regulation and assembly of transcription factors to specific genomic loci. Germline de novo truncating variants in ASXL1 and ASXL3 have been respectively implicated in causing Bohring-Opitz and Bainbridge-Ropers syndromes, which result in overlapping features of severe intellectual disability and dysmorphic features. ASXL2 has not yet been associated with a human Mendelian disorder. In this study, we performed whole-exome sequencing in six unrelated probands with developmental delay, macrocephaly, and dysmorphic features. All six had de novo truncating variants in ASXL2. A careful review enabled the recognition of a specific phenotype consisting of macrocephaly, prominent eyes, arched eyebrows, hypertelorism, a glabellar nevus flammeus, neonatal feeding difficulties, hypotonia, and developmental disabilities. Although overlapping features with Bohring-Opitz and Bainbridge-Ropers syndromes exist, features that distinguish the ASXL2-associated condition from ASXL1- and ASXL3-related disorders are macrocephaly, absence of growth retardation, and more variability in the degree of intellectual disabilities. We were also able to demonstrate with mRNA studies that these variants are likely to exert a dominant-negative effect, given that both alleles are expressed in blood and the mutated ASXL2 transcripts escape nonsense-mediated decay. In conclusion, de novo truncating variants in ASXL2 underlie a neurodevelopmental syndrome with a clinically recognizable phenotype. This report expands the germline disorders that are linked to the ASXL genes. The three additional sex-combs-like genes (ASXL1 [MIM: 612990], ASXL2 [MIM: 612991], and ASXL3 [MIM: 615115]) code for polycomb proteins that act as histone methyltransferases,1Fisher C.L. Berger J. Randazzo F. Brock H.W. A human homolog of Additional sex combs, ADDITIONAL SEX COMBS-LIKE 1, maps to chromosome 20q11.Gene. 2003; 306: 115-126Crossref PubMed Scopus (76) Google Scholar serve as epigenetic scaffolding proteins, and are involved in body patterning.2Katoh M. Functional proteomics of the epigenetic regulators ASXL1, ASXL2 and ASXL3: a convergence of proteomics and epigenetics for translational medicine.Expert Rev. Proteomics. 2015; 12: 317-328Crossref PubMed Scopus (37) Google Scholar Somatic mutations in all three ASXL genes occur across a range of malignancies.3Katoh M. Functional and cancer genomics of ASXL family members.Br. J. Cancer. 2013; 109: 299-306Crossref PubMed Scopus (102) Google Scholar Drosophila Asxl genes are involved in homeotic gene activation and silencing.4Katoh M. Katoh M. Identification and characterization of ASXL3 gene in silico.Int. J. Oncol. 2004; 24: 1617-1622PubMed Google Scholar, 5Baskind H.A. Na L. Ma Q. Patel M.P. Geenen D.L. Wang Q.T. Functional conservation of Asxl2, a murine homolog for the Drosophila enhancer of trithorax and polycomb group gene Asx.PLoS ONE. 2009; 4: e4750Crossref PubMed Scopus (50) Google Scholar In mice, Asxl2 has been reported to regulate skeletal, lipid, and glucose homeostasis and cardiac development.6Izawa T. Rohatgi N. Fukunaga T. Wang Q.T. Silva M.J. Gardner M.J. McDaniel M.L. Abumrad N.A. Semenkovich C.F. Teitelbaum S.L. Zou W. ASXL2 Regulates Glucose, Lipid, and Skeletal Homeostasis.Cell Rep. 2015; 11: 1625-1637Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 7Khan F.F. Li Y. Balyan A. Wang Q.T. WTIP interacts with ASXL2 and blocks ASXL2-mediated activation of retinoic acid signaling.Biochem. Biophys. Res. Commun. 2014; 451: 101-106Crossref PubMed Scopus (7) Google Scholar Additionally, Asxl1 and Asxl2 fine-tune adipogenesis in mice, whereby Asxl2 promotes adipogenesis and Asxl1 inhibits it.8Park U.H. Seong M.R. Kim E.J. Hur W. Kim S.W. Yoon S.K. Um S.J. Reciprocal regulation of LXRα activity by ASXL1 and ASXL2 in lipogenesis.Biochem. Biophys. Res. Commun. 2014; 443: 489-494Crossref PubMed Scopus (10) Google Scholar, 9Park U.H. Yoon S.K. Park T. Kim E.J. Um S.J. Additional sex comb-like (ASXL) proteins 1 and 2 play opposite roles in adipogenesis via reciprocal regulation of peroxisome proliferator-activated receptor gamma.J. Biol. Chem. 2011; 286: 1354-1363Crossref PubMed Scopus (59) Google Scholar Mice with homozygous Asxl2 knockout demonstrate premature death, growth retardation, impaired cardiac function, and vertebral abnormalities, indicating that this gene is required for embryonic and postnatal development.5Baskind H.A. Na L. Ma Q. Patel M.P. Geenen D.L. Wang Q.T. Functional conservation of Asxl2, a murine homolog for the Drosophila enhancer of trithorax and polycomb group gene Asx.PLoS ONE. 2009; 4: e4750Crossref PubMed Scopus (50) Google Scholar Germline mutations in ASXL1 and ASXL3 have been associated with specific genetic syndromes.10Russell B. Graham Jr., J.M. Expanding our knowledge of conditions associated with the ASXL gene family.Genome Med. 2013; 5: 16Crossref PubMed Scopus (15) Google Scholar Truncating variants in ASXL1 cause Bohring-Opitz syndrome (MIM: 605039), a severe disorder with growth retardation, microcephaly, profound intellectual disability, nevus flammeus of the face, flexion of the elbows and wrists, and ulnar deviation of the hands.11Hoischen A. van Bon B.W. Rodríguez-Santiago B. Gilissen C. Vissers L.E. de Vries P. Janssen I. van Lier B. Hastings R. Smithson S.F. et al.De novo nonsense mutations in ASXL1 cause Bohring-Opitz syndrome.Nat. Genet. 2011; 43: 729-731Crossref PubMed Scopus (199) Google Scholar, 12Bohring A. Silengo M. Lerone M. Superneau D.W. Spaich C. Braddock S.R. Poss A. Opitz J.M. Severe end of Opitz trigonocephaly (C) syndrome or new syndrome?.Am. J. Med. Genet. 1999; 85: 438-446Crossref PubMed Scopus (48) Google Scholar ASXL3 germline truncating variants are associated with Bainbridge-Ropers syndrome (MIM: 615485), characterized by severe intellectual disability, growth retardation, and clinical features overlapping those of Bohring-Opitz syndrome.13Bainbridge M.N. Hu H. Muzny D.M. Musante L. Lupski J.R. Graham B.H. Chen W. Gripp K.W. Jenny K. Wienker T.F. et al.De novo truncating mutations in ASXL3 are associated with a novel clinical phenotype with similarities to Bohring-Opitz syndrome.Genome Med. 2013; 5: 11Crossref PubMed Scopus (113) Google Scholar Variants in ASXL3 have also been reported in autism spectrum disorder.14De Rubeis S. He X. Goldberg A.P. Poultney C.S. Samocha K. Cicek A.E. Kou Y. Liu L. Fromer M. Walker S. et al.DDD StudyHomozygosity Mapping Collaborative for AutismUK10K ConsortiumSynaptic, transcriptional and chromatin genes disrupted in autism.Nature. 2014; 515: 209-215Crossref PubMed Scopus (1630) Google Scholar In contrast, ASXL2 (MIM: 612991) has thus far not been implicated in human Mendelian disease. One individual with a t(2;9) translocation resulting in a fused transcript of ASXL2 and KIAA1803 had a complex phenotype of agenesis of the corpus callosum, ocular colobomas, and periventricular heterotopias,15Ramocki M.B. Dowling J. Grinberg I. Kimonis V.E. Cardoso C. Gross A. Chung J. Martin C.L. Ledbetter D.H. Dobyns W.B. Millen K.J. Reciprocal fusion transcripts of two novel Zn-finger genes in a female with absence of the corpus callosum, ocular colobomas and a balanced translocation between chromosomes 2p24 and 9q32.Eur. J. Hum. Genet. 2003; 11: 527-534Crossref PubMed Scopus (36) Google Scholar but the relative contributions of the two genes to this individual’s phenotype are unclear. Similarly, DECIPHER lists five individuals with cytogenetic deletions encompassing ASXL2 and other genes (n = 35–141) and phenotypes including developmental delays or intellectual disabilities, among other manifestations (Table S1). Because of the contiguous deletion of several other genes, the specific contribution of the ASXL2 deletion to these phenotypes cannot be determined. Also included is an individual with a single de novo base-pair deletion causing head, neck, nervous, skeletal, skin, and respiratory system abnormalities, and potential overlapping manifestations are detailed in our individuals. In this study, we present six unrelated individuals ranging from 11 months to 31 years of age with de novo heterozygous truncating ASXL2 variants detected by whole-exome sequencing (WES). All individuals share overlapping clinical features including developmental or intellectual impairments, macrocephaly, distinct facial dysmorphisms, facial nevus flammeus, feeding difficulties in the newborn period, and hypotonia (Table 1 and Figure 1). Detailed clinical summaries are available in the Supplemental Note. Five of the individuals (individuals 1–3, 5, and 6) and their biological parents underwent trio WES, whereas individual 4 was sequenced alone, followed by Sanger sequencing of ASXL2 on the child and the biological parents. WES was performed after written informed consent was obtained through approval by institutional review boards and ethics committees. Experienced pediatricians and geneticists clinically assessed the individuals.Table 1Clinical Features of Six Individuals with ASXL2 Variants in Comparison to Those of ASXL1- and ASXL3-Associated DisordersIndividual 1Individual 2Individual 3Individual 4Individual 5Individual 6Bohring-Opitz SyndromeBainbridge-Ropers SyndromeGeneASXL2ASXL2ASXL2ASXL2ASXL2ASXL2ASXL1ASXL3MutationaMutation designations refer to transcript GenBank: NM_018263.4, NCBI Genome build GRCh37 (individuals 1, 4, and 6), and UCSC Genome Browser build hg19 (individuals 2, 3, and 5).c.2424delC (p.Thr809Profs∗32)c.2081dupG (p.Gly696Argfs∗11)c.1225_1228delCCAA (p.Pro409Asnfs∗13)c.2472delC (p.Ser825Valfs∗16)c.2971_2974delGGAG (p.Gly991Argfs∗3)c.1288G>T (p.Glu430∗)truncatingtruncatingPrenatal findingsnonenonenonenoneleft renal agenesis, cerebral ventriculomegaly, intrauterine growth restrictionnoneIUGR, polyhydramniosIUGRGrowth parameters at birthheight and weight > 97th percentile, OFC = 92nd percentileheight, weight, and OFC > 97th percentileheight = 39th percentile, length > 97th percentile, OFC = 91st percentileweight = 35th percentile (length and OFC not available)weight = 2nd percentile, length = 23rd percentile, (OFC not available)weight = 30th percentile, length = 60th percentile, OFC = 70th percentileSGASGAGrowth parameters at last examinationat 8 years: height, weight, and OFC = 100th percentile (macrocephaly)at 10 months: normal height and weight, OFC = 96th percentile (macrocephaly)at 5 years: normal height and weight, OFC > 97th percentile (macrocephaly)at 4 years: normal height and weight, OFC > 97th percentile (macrocephaly)at 7 years 10 months: height = 99th percentile, weight = 92nd percentile, OFC = 99th percentile (macrocephaly)at 12.8 years: weight = 88th percentile, height = 1st percentile, OFC = 80th percentile (relative macrocephaly)severe growth retardation, microcephalysevere growth retardation, microcephalyFeeding difficultiespresent only shortly after birthpresentpresent only in neonatal periodpresent only shortly after birthpresent for several weeks after birthpresent only shortly after birthpresent (persistent)present (severe)Hypotoniapresentpresent (persistent)present, but hypertonia in limbspresentpresentpresentpresentpresentDD and/or IDlow average cognitionmoderateseveresevereborderlinemoderateseveresevereSeizuresfebrilenonefebrile and non-febrilefebrilesuspectedpresentpresentNAFacial featureshypertelorism, arched eyebrows, long face, prominent eyes, ptosis of eyelids, epicanthal folds, broad nasal tiphypertelorism, arched eyebrows, proptosis, epicanthal folds, long eyelashes, synophrys, broad nasal tiphypertelorism, arched eyebrows, prominent eyes, ptosis of eyelids, epicanthal folds, prominent glabella, synophrys, small upper vermilion, broad nasal tiphypertelorism, arched eyebrows, long face, proptosis, small mouthhypertelorism, arched eyebrows, prominent eyes, long eyelashes, small upper vermilion, broad nasal tiphypertelorism, malar hypoplasia, low nasal bridge, short philtrum, ptosis, broad nasal tip, high narrow palatetrigonocephaly, hypertelorism, prominent forehead, long face, micrognathia, prominent eyes, upslanting palpebral fissureshigh and broad forehead, anteverted nares, hypertelorismEarslow-set, cupped, overfoldedposteriorly rotatedposteriorly rotatedposteriorly rotatedlow-set, posteriorly rotatedthick ear lobeslow-set, posteriorly rotatedlow-set, posteriorly rotatedSkinglabellar nevus flammeus; capillary malformations on trunk, neck, and behind ears; deep palmar creasesglabellar nevus flammeus, deep palmar creasesglabellar nevus flammeus, capillary malformations on back and neck, deep palmar creases, hypertrichosisglabellar nevus flammeus, normal palmar creasesglabellar nevus flammeus, fetal fingertip padsglabellar nevus flammeus, hypertrichosis, deep palmar and plantar creasesglabellar nevus flammeus, capillary malformations on philtrum and neck, deep palmar creases, hypertrichosisdeep palmar creases, hypertrichosisEpisodes of hypoglycemianone knownhas required continuous feeding since neonatal periodepisodic, starting at 2.5 years of agenone knownnone knownpresent during neonatal periodNANACongenital heart diseaseASD, PDAASD, left ventricular dysfunctionASDnone knownnone knownthickened pulmonary valveASD, VSDPDA, pulmonary artery stenosisSkeletal and/or extremity manifestationsincreased density of alveolar bone, advanced bone agekyphosisscoliosismultiple bilateral fractures, overlapping second and fourth toesnone knownadvanced bone age, thick calvarium, fusion of second and third cervical vertebrae, short metacarpals and distal phalangesscoliosis, ulnar deviation of hands, elbow and wrist flexion, scoliosisulnar deviation of hands, clenched handsBrain MRIwhite-matter volume lossincreased extra-axial cerebral space, choroid plexus papillomaincreased extra-axial cerebral spaceincreased extra-axial cerebral spacewhite-matter volume loss, ventriculomegalynormalagenesis of corpus callosum, Dandy-Walker malformationwhite-matter volume loss, Dandy-Walker malformationAbbreviations are as follows: ASD, atrial septal defect; DD, developmental delay; ID, intellectual disability; IUGR, intrauterine growth retardation; NA, not available; OFC, occipitofrontal head circumference; PDA, patent ductus arteriosus; SGA, small for gestational age; and VSD, ventricular septal defect.a Mutation designations refer to transcript GenBank: NM_018263.4, NCBI Genome build GRCh37 (individuals 1, 4, and 6), and UCSC Genome Browser build hg19 (individuals 2, 3, and 5). Open table in a new tab Abbreviations are as follows: ASD, atrial septal defect; DD, developmental delay; ID, intellectual disability; IUGR, intrauterine growth retardation; NA, not available; OFC, occipitofrontal head circumference; PDA, patent ductus arteriosus; SGA, small for gestational age; and VSD, ventricular septal defect. For all six individuals, DNA was extracted from maternal, paternal, and proband blood samples. The exome was captured with biotin-labeled VCRome 2.1 in-solution exome probes (individuals 1 and 4), the Agilent Clinical Research Exome Kit (individual 2), the Nextera Rapid Capture Exome Kit (individuals 3 and 6), or Agilent SureSelect v.4 (individual 5), and the exomes were sequenced on an Illumina HiSeq 2000 (individuals 2 and 5) or 2500 (individuals 1, 3, 4, and 6). Two paired-end 100 bp reads were used for the exome-capture sequencing. For individual 3, Trimmomatic16Bolger A.M. Lohse M. Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data.Bioinformatics. 2014; 30: 2114-2120Crossref PubMed Scopus (28414) Google Scholar was employed to remove adapters and low-quality (Phred quality score < 5) bases from the 3′ ends of sequence reads. Reads shorter than 36 bp were subsequently removed. The sequencing methodology, further processing, and variant-interpretation protocols have been described previously.17Richards S. Aziz N. Bale S. Bick D. Das S. Gastier-Foster J. Grody W.W. Hegde M. Lyon E. Spector E. et al.ACMG Laboratory Quality Assurance CommitteeStandards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet. Med. 2015; 17: 405-424Abstract Full Text Full Text PDF PubMed Scopus (14880) Google Scholar, 18Yang 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 (970) Google Scholar, 19Tanaka A.J. Cho M.T. Millan F. Juusola J. Retterer K. Joshi C. Niyazov D. Garnica A. Gratz E. Deardorff M. et al.Mutations in SPATA5 Are Associated with Microcephaly, Intellectual Disability, Seizures, and Hearing Loss.Am. J. Hum. Genet. 2015; 97: 457-464Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 20de 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 (1134) Google Scholar, 21Lelieveld S.H. Reijnders M.R. Pfundt R. Yntema H.G. Kamsteeg E.J. de Vries P. de Vries B.B. Willemsen M.H. Kleefstra T. Löhner K. et al.Meta-analysis of 2,104 trios provides support for 10 new genes for intellectual disability.Nat. Neurosci. 2016; 19: 1194-1196Crossref PubMed Scopus (271) Google Scholar Functional annotation and alteration filtering in all cases were performed against public databases (the NHLBI Exome Sequencing Project [ESP] Exome Variant Server, dbSNP138, 1000 Genomes, and Exome Aggregation Consortium [ExAC] Browser; see Web Resources). Quality metrics for all WES results are provided in Table S2. GeneMatcher, a web-based tool for researchers and clinicians working on identical genes, connected the investigators from the different institutions.22Sobreira 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 (831) Google Scholar Processing of the whole-exome data on the six unrelated individuals with overlapping clinical features (Figure 1 and Table 1) and the healthy parents of five of them took into account X-linked, autosomal-recessive, and autosomal-dominant inheritance models to identify genes with functionally relevant variants (new, clinically associated, or of low or unknown frequency) (Table S3). In the five trios, we identified one to two putatively de novo variants not present in any variant database (Table S3). De novo variants (frameshift and stop gain) in ASXL2 were detected among all individuals (Table 1): c.2424delC (p.Thr809Profs∗32) in individual 1, c.2081dupG (p.Gly696Argfs∗11) in individual 2, c.1225_1228delCCAA (p.Pro409Asnfs∗13) in individual 3, c.2472delC (p.Ser825Valfs∗16) in individual 4, c.2971_2974delGGAG (p.Gly991Argfs∗3) in individual 5, and c.1288G>T (p.Glu430∗) in individual 6 (mutation designations refer to transcript GenBank: NM_018263.4, NCBI Genome build GRCh37 [individuals 1, 4, and 6], and UCSC Genome Browser build hg19 [individuals 2, 3, and 5]). All ASXL2 variants were validated by Sanger sequencing, and the de novo origin was confirmed by parental-segregation studies (Figure S2A). Interestingly, all identified variants locate to the penultimate or last exon of ASXL2 (Figure 2). Examination of the ExAC Browser (release 0.3) for loss-of-function (LoF) variants that passed the browser’s variant-quality thresholds revealed two distinct frameshift, two distinct nonsense, and two distinct canonical splice variants, all with only a single carrier (c.2002A>T [p.Lys668∗], c.1973dupC [p.Ala658Glyfs∗49], c.1895dupA [p.His632Serfs∗17], c.1400C>A [p.Ser467∗], c.505−1G>C, and c.940−2A>G). Except for c.505−1G>C and c.940−2A>G, these variants are located in a region between the variants found in individuals 2 and 6, upstream of the variants in individuals 1, 4, and 5, and downstream of the variant found in individual 3 (Figure 2). The annotation of these LoF variants in the ExAC Browser prompted us to calculate the probability of finding ASXL2 variants by chance in exomes for neurodevelopmental phenotypes: the six ASXL2 truncating variants in our individuals occur in a collection of 12,030 subjects ascertained for neurological and neurodevelopmental disorders (GeneDx = 3,677; University Medical Center Hamburg = 123; Baylor = 6,198; Radboud University Medical Center = 1,866; and Broad Institute = 166). We used the CRAN function denovolyzeR23Ware 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: 1-15Google Scholar package with the parameters set for six truncating variants in ASXL2 and estimated that the probability of the chance occurrence of the six de novo ASXL2 mutations among 12,030 individuals is 1.47e−10. Correcting for the 18,668 protein-coding genes present in the consensus coding sequence (CCDS release 14) shows that this observation is significant genome-wide (2.744196e−6). Conversely, for the probability of finding six ASXL2 truncating variants in the ExAC Browser (60,000 samples), we found a p value of 1.79e−6, which translates to 0.03341572 when corrected for 18,668 genes. These data indicate that there are fewer than expected truncating ASXL2 variants within the control database as well. ASXL2 is also highly intolerant of LoF variants (probability of 0.99) according to the ExAC Browser pLI calculation.24Lek 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 (6602) Google Scholar The ExAC Browser endeavors not to include subjects with severe pediatric disease but is enriched with adults with heart and metabolic diseases, cancer, schizophrenia, and Tourette syndrome.24Lek 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 (6602) Google Scholar Interestingly, truncating variants in ExAC Browser control individuals have been reported for both ASXL1 and ASXL3; these overlap the disease-associated variants occurring within the last two exons of both genes.13Bainbridge M.N. Hu H. Muzny D.M. Musante L. Lupski J.R. Graham B.H. Chen W. Gripp K.W. Jenny K. Wienker T.F. et al.De novo truncating mutations in ASXL3 are associated with a novel clinical phenotype with similarities to Bohring-Opitz syndrome.Genome Med. 2013; 5: 11Crossref PubMed Scopus (113) Google Scholar, 25Ropers H.H. Wienker T. Penetrance of pathogenic mutations in haploinsufficient genes for intellectual disability and related disorders.Eur. J. Med. Genet. 2015; 58: 715-718Crossref PubMed Scopus (27) Google Scholar Similarly, other genes involved in intellectual disability, such as ARID1B, have also been found to have truncating variants in ExAC Browser control individuals.25Ropers H.H. Wienker T. Penetrance of pathogenic mutations in haploinsufficient genes for intellectual disability and related disorders.Eur. J. Med. Genet. 2015; 58: 715-718Crossref PubMed Scopus (27) Google Scholar The exact explanation for these ExAC Browser variants that overlap the disease-associated variants for the three ASXL genes remains unclear, but possibilities include somatic mosaicism, variable expressivity depending on the location of the variants in the gene, or reduced penetrance. The finding that clonal hematopoiesis in healthy subjects results in acquired somatic variants in genes (including ASXL1) that are also mutated in myeloid cancers supports the possibility that the variants in the ExAC Browser could be somatic rather than germline.24Lek 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 (6602) Google Scholar Options such as examining read counts to determine whether a variant could be postzygotic are subject to errors due to sequencing factors such as variability in capture, and so at this time, definite inferences cannot be made about the origin of these variants. Non-penetrance would be a less likely explanation, given the severity of the germline ASXL disorders, but a recent report has confirmed that incomplete penetrance of Mendelian diseases is likely to be more common than realized.26Chen R. Shi L. Hakenberg J. Naughton B. Sklar P. Zhang J. Zhou H. Tian L. Prakash O. Lemire M. et al.Analysis of 589,306 genomes identifies individuals resilient to severe Mendelian childhood diseases.Nat. Biotechnol. 2016; 34 (advance online publication): 531-538Crossref PubMed Scopus (208) Google Scholar Future studies on all three ASXL genes are necessary for better understanding the overlap between truncating variants in control and affected individuals. As for ASXL1 and ASXL3, we initially postulated that haploinsufficiency would be responsible for the ASXL2 phenotype; alternatively, a dominant-negative mechanism could cause disease. When truncating variants preferentially accumulate toward the end of a gene, the resulting transcript could escape nonsense-mediated decay (NMD) and interfere with the wild-type protein, causing an abnormal phenotype. The truncating variants in our six affected individuals are within the last two exons of ASXL2, similar to the distribution of disease-causing variants in ASXL1 and ASXL3.11Hoischen A. van Bon B.W. Rodríguez-Santiago B. Gilissen C. Vissers L.E. de Vries P. Janssen I. van Lier B. Hastings R. Smithson S.F. et al.De novo nonsense mutations in ASXL1 cause Bohring-Opitz syndrome.Nat. Genet. 2011; 43: 729-731Crossref PubMed Scopus (199) Google Scholar, 13Bainbridge M.N. Hu H. Muzny D.M. Musante L. Lupski J.R. Graham B.H. Chen W. Gripp K.W. Jenny K. Wienker T.F. et al.De novo truncating mutations in ASXL3 are associated with a novel clinical phenotype with similarities to Bohring-Opitz syndrome.Genome Med. 2013; 5: 11Crossref PubMed Scopus (113) Google Scholar, 25Ropers H.H. Wienker T. Penetrance of pathogenic mutations in haploinsufficient genes for intellectual disability and related disorders.Eur. J. Med. Genet. 2015; 58: 715-718Crossref PubMed Scopus (27) Google Scholar To get an idea of whether haploinsufficiency or a dominant-negative effect could be the underlying mechanism of disease, we performed cDNA studies. Fresh venous blood samples of individuals 1, 3, and 4 were deposited in PAXgene Blood RNA Tubes (PreAnalytiX, QIAGEN), and total RNA was extracted with the PAXgene Blood RNA Kit (QIAGEN) in accordance with the manufacturer’s instructions. 1 μg of RNA from each individual was reverse transcribed into cDNA (Superscript III, Invitrogen) with the use of random hexanucleotides (Invitrogen) according to the manufacturer’s protocol. PCR amplification for allele expression analysis of ASXL2 was carried out with the Advantage cDNA PCR Kit (Clontech, Takara Bio) and primers spanning exon-exon boundaries of ASXL2. The primers used are as follows: ASXL2_RT_11for (5′-CGACAAGAGATTGAGAAGGAG-3′) in exon 11 and ASXL2_RT_12rev (5′-CCATCAGCTGCACAATGAA-3′) in exon 12 for individuals 1 and 4 and ASXL2_RT_10for (5′-TGGAAAGAACAATTCTTTGAAAG-3′) in exon 10 and ASXL2_RT_11rev (5′-CACTCTGACTGGGAGACTACTG-3′) in exon 11 for individual 3. Three temperature “touchdown” PCRs were performed with an initial annealing temperature of 60°C, which was decreased twice by 2°C after three cycles each and then maintained at 56°C for 29 more cycles. Each cycle consisted of a denaturation step for 30 s at 94°C, an annealing step for 30 s, and a 1 min, 68°C elongation. ASXL2_RT_12seq (5′-TCATAGTG
Referência(s)