ALX 1‐ related frontonasal dysplasia results from defective neural crest cell development and migration

• DOI: 10.15252/emmm.202012013
• Publication Date: 2020-10-07T13:15:30Z
• Authors: Jonathan Pini; Janina Kueper; Yiyuan David Hu; Kenta Kawasaki; Pan Yeung; Casey Tsimbal; Baul Yoon; Nikkola Carmichael; Richard L Maas; Justin Cotney; Yevgenya Grinblat; Eric C Liao
• Keywords: General hospital

Article11 September 2020Open Access Source DataTransparent process ALX1-related frontonasal dysplasia results from defective neural crest cell development and migration Jonathan Pini Center for Regenerative Medicine, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA Shriners Hospital Children, Search more papers by this author Janina Kueper Institute Human Genetics, University Bonn, Germany Correction added on 7 July 2022, after first online publication: The affiliation has been corrected. See the associated Corrigendum at https://doi.org/10.15252/emmm.202216289 Yiyuan David Hu Kenta Kawasaki Pan Yeung Casey Tsimbal Baul Yoon Departments Integrative Biology, Neuroscience, Genetics Ph.D. Training Program, Wisconsin-Madison, Madison, WI, Nikkola Carmichael Brigham Women's Harvard Medical School, Richard L Maas Justin Cotney Genome Sciences, UConn Health, Farmington, CT, Yevgenya Grinblat Eric C Liao Corresponding Author [email protected] orcid.org/0000-0001-6385-7448 Information Pini1,2,‡, Kueper1,2,3,‡, Hu1,2, Kawasaki1,2, Yeung1,2, Tsimbal1,2, Yoon4, Carmichael5, Maas5, Cotney6, Grinblat4 *,1,2 1Center 2Shriners 3Institute 4Departments 5Department 6Genetics ‡ These authors contributed equally to work *Corresponding author. Tel: [phone]; E-mail: EMBO Mol Med (2020)12:e12013https://doi.org/10.15252/emmm.202012013 Correction(s) article migration07 2022 PDFDownload PDF text main figures. Peer ReviewDownload a summary editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract A pedigree subjects presented with (FND). sequencing analysis identified p.L165F missense variant in homeodomain transcription factor ALX1 which was imputed be pathogenic. Induced pluripotent stem cells (iPSC) were derived differentiated (NCC). NCC ALX1L165F/L165F iPSC sensitive apoptosis, showed an elevated expression several progenitor state markers, exhibited impaired compared wild-type controls. evaluated vivo using lineage tracing zebrafish model, revealed anterior stream that contributes median portion neurocranium, phenocopying clinical presentation. Analysis human culture media change level bone morphogenic proteins (BMP), low BMP2 high BMP9. Soluble BMP9 antagonist treatments able rescue phenotype. Taken together, these demonstrate mechanistic requirement migration. Synopsis Variants are implicated Frontonasal Dysplasia. This study explores role patient-derived induced zebrafish. novel mutation familial Dysplasia four children born bilateral oblique clefts ocular malformations. Neural Crest Cells harboring L165F within transcriptomic differences marker genes indicative prolonged state, dysbalance BMPs, increased rate delayed Disruption alx1 resulted perturbed Cell other alx family members. paper explained Problem causes malformations face remain poorly understood. lack understanding limited treatment counseling options, specifically families affected linked genetic cause. One such gene is called ALX1. aimed understand effect mutations genesis malformation. To do so, we reprogrammed blood into allow us retrace development. Additionally, created disruption order model malformation animal broadly. Results found crucial population exists only during time early development, termed cells. form while structures will come nervous system grow. They migrate front embryo face. patients bearing likely die when healthy donors. also show defect. Similar observed models disease same gene. Impact Understanding give tools innovate transform insufficient options currently available patients. Introduction central part contains key anatomic features sensory organs enable interact environment each other. embryologic processes midface structures, eyes, nose, upper lip, maxilla, tightly regulated (Johnston, 1966; Minoux Rijli, 2010; Rada-Iglesias et al, 2012). as centrally located prominence extends anteriorly, coalescing elements paired maxillary prominences 1966, 1975; Le Lièvre Douarin, Lièvre, 1978; Sadaghiani Thiébaud, 1987). embryonic facial distinct migrating streams cranial (NCC) conserved across vertebrates (Le Douarin 1993; Schilling 1996; Chai 2000; Olsson, 2002; Trainor Barrallo-Gimeno 2004; Wada, 2005; Dougherty differentiation highly coordinated dynamic patterns (Simoes-Costa Bronner, 2015). Key signaling pathways regulate involve BMP, Wnt, FGF, or Notch activate factors PAX3, ZIC1, TFAP2a, MSX1/2, DLX5 (Meulemans Bronner-Fraser, Khudyakov 2009; Stuhlmiller Garcia-Castro, 2012; 2013; Simoes-Costa Disruptions contribute number congenital Waardenburg syndrome (WS), velocardiofacial syndrome/DiGeorge syndrome, Hirschsprung's disease, heart conditions, craniofacial anomalies (Sedano 1970; Fox 1976; Pierpont 2007; Uz 2010) (FND) considered rare "orphan" (ORPHA250), very few cases reported literature. true prevalence FND its etiology unknown. date, six subtypes varying inheritance have described individual case reports: EFNB1 (MIM 300035) X-linked craniofrontonasal 304110); ALX3 606014) type 1 136760); ALX4 605420) 2 613451); 601527) 3 613456); ZSWIM6 615951) dominant acromelic dysostosis 603671); SPECC1L 614140) Teebi 145420; Bhoj 2015; Kayserili Smith 2014; Twigg 2004, Ullah 2016; Wieland 2004). heterogeneity phenotypes, wide range possible components, corresponds different underlying variants, environments, epigenetic modifications. examined pathogenic resulting loss function. generated investigate behavior. Cellular molecular characterizations between subject-derived control shed light developmental disrupted FND. In characterization most NCC. underscores utility complementary gain insight cellular basis Clinical consanguineous parents Amish heritage complex phenotype inherited Mendelian recessive fashion. Both parents, one unaffected sibling three (one male two females), consented enrolled (Fig 1A, subject numbers indicated red). (subjects 2) nine siblings (including 3) had normal without stigmata suggestive mild All clefts, extending either side nasal bone, involving both primary secondary palate. Among children, there some variability phenotype, where older girl (subject 4) coloboma asymmetric microphthalmia, whereas 5 6) anophthalmia, deficient lower eyelids covering shallow orbit. Subject 6 severely anophthalmia well no eyelids, leaving mucous membranes orbits exposed. Her remnant lacked lateral alar subunits surrounded nodular skin tags. Figure 1. presentation generation control, father, A. tree includes siblings, five female sibling. Subjects 1–6, red, study. 4–6 involvement. eldest right coloboma, left Tessier 4 clefts. fused orbits, open absent exposed orbital mucosa, malformed ala iPSCs samples collected 1, 5, 6. B. Whole-exome carried out (c.493 C>T) homeodomain, heterozygous (ALX1165L/165F), wild (ALX1165L/165L), homozygous (ALX1165F/165F). C. Schematic protein structure showing position substitution here (red) locations exon borders variants (purple; 2010). D. genomic sequence, variants. purple bar bottom represents FND-associated deletion previously literature (Uz 2016). E. Immunofluorescence staining markers SSEA4, OCT4, SOX2, TRA-1-60 alkaline phosphatase clones. representative clone shown genotype. Scale bar: 400 μm. F. Expression (OCT4, NANOG), endoderm (Endo., AFP, GATA4, FOXA2), ectoderm (Ecto., NESTIN, GFAP, SOX1), mesoderm (Meso., BRACH. (BRACHYURY), RUNX1, CD34) ALX1165L/165L (green), ALX1165L/165F(red), ALX1165F/165F (blue) relative undifferentiated (UND). Data represented pooled mean ± SEM experiments clones Significance: P = 0.0167 0.0005 NANOG, 0.000004 0.0082 0.0137 FOXA2, 0.00002 0.0014 SOX1, 0.0117 BRACHYURY, 0.0008 RUNX1 0.0068 CD34 comparing iPSC. 0.0013 0.0011 0.0000003 0.0003 0.0063 0.0001 0.027 0.000002 0.000009 3e−9 0.000006 ALX1165F/165L 0.0201 0.006 × 10−12 10-13 0.0031 0.0292 0.00001 10-7 0.0204 0.0009 0.000003 pooled, mathematical calculated. used determine standard error. test statistical significance, ANOVA performed. P-value < 0.05 statistically significant. 1E [emmm202012013-sup-0010-SDataFig1E.zip] Download figure PowerPoint Identification (WES) performed 1–5, corresponded sibling, children. (c.493C>T) ALX1, (ALX1165F/165F; Fig 1B). WES confirmed Sanger entire coding sequence. not connection instance nor recorded gnomAD database (preprint: Karczewski 2019; 1C D). amino acid predicted damaging causing silico (Sift, Polyphen, muttaster, fathmm) consistent autosomal pattern (Lowe, 1999; Adzhubei Schwarz Shihab 2014). Generation lines peripheral mononuclear (PBMC) obtained whole unrelated individuals father (Subjects 6; ALX1165F/165F). PBMC subsequently EV1). Overall, 22 mutant successfully isolated expanded subjects, 13 ALX1165L/165F 35 Six (3 subject), 9 controls control) fully characterized confirm their pluripotency 1E) ability generate germ layers 1F). -derived retained through reprogramming. Copy did any amplifications deletions. Click expand figure. EV1. derivation EB representation strategy Blood individual, 1), processed. Isolated infected Sendai virus, picked 21 days infection. Following expansion until passage 10, embryoid bodies formed suspension 14 days. reprogramming all underwent similar morphological changes leading formation day 21. still displayed morphology indicating self-renew. EBs. represented. iPSC-derived Given morphogenesis, protocol adapted previous (Pini 2018; 2A). indistinguishable colony immediately following 2B). 2. timeline. Maintenance Medium (MM) medium (StemFlex 1× penicillin/streptomycin), DMEM-F12, 10% fetal bovine serum, mM sodium pyruvate, penicillin/streptomycin, nonessential acids, 110 μM 2-mercaptoethanol, 10 ng/ml epidermal growth factor. Images Days 0, 14, differentiation. bars: μm (Day 0), 200 4). 2B [emmm202012013-sup-0011-SDataFig2B.zip] Overexpression plate border specifier panel center regulatory network required selected detail 3; Sauka-Spengler 2008; can broadly divided groups. group significantly differ affected, heterozygous, comprises specifiers FOXD3 P75, HAND2. second differed cells, difference heterozygote control. PAX7, MSX1, SNAI2 TWIST1 (P 2–8 subjects', DLX5; 2–14 SNAI2, TWIST1). final differentially expressed homozygous, comprised MSX2, DLX5, TFAP2A TFAP2A). Of note, overexpressed above levels putative transcriptional repressor. 3. Timeline NCC-associated differentiationGene (magenta), iPSC: FOXD3, TFAP2A, TWIST1; RT–qPCR values normalized RPLP0 GAPDH expression. Exact P-values provided Table itself 8 levels, peak reached plateaued, greatest process, around (such suggests function identifies window in-depth transcriptome future studies. Increased sensitivity apoptosis Since cycle progression predispose given importance regulating impact analyzed. Basal determined percentage Annexin V-positive (4 0.2%) (4.82 0.65%; 4A). After induction via heat shock, (87.97 2.44%) versus (24.15 0.96%). findings suggest subject's apoptosis. 4. cycle, Homozygous increase (black). data represent FACS analysis, being example experiment. Apoptosis immersion 55°C water bath min. Representative experiment condition shown. independent experiments. *: Significantly basal rate: **Significantly 0.0004). cyclins CCNA2 CCND1 (orange) passages *Significantly 0.0