TUBB 1 mutations cause thyroid dysgenesis associated with abnormal platelet physiology
2018; Springer Nature; Volume: 10; Issue: 12 Linguagem: Inglês
10.15252/emmm.201809569
ISSN1757-4684
AutoresAthanasia Stoupa, Frédéric Adam, Dulanjalee Kariyawasam, Catherine Strassel, Sanjay Gawade, Gabor Szinnai, Alexandre Kauskot, Dominique Lasne, Carsten Janke, Kathiresan Natarajan, Alain Schmitt, Christine Bôle‐Feysot, Patrick Nitschké, Juliane Léger, Fabienne Jabot‐Hanin, Frédéric Torès, Anita Michel, Arnold Münnich, Claude Besmond, Raphaël Scharfmann, François Lanza, Delphine Borgel, Michel Polak, Aurore Carré,
Tópico(s)Cellular transport and secretion
ResumoResearch Article19 November 2018Open Access Source DataTransparent process TUBB1 mutations cause thyroid dysgenesis associated with abnormal platelet physiology Athanasia Stoupa Athanasia Stoupa INSERM U1016, Faculté de Médecine, Cochin Institute, Université Paris Descartes, Sorbonne Paris Cité, Paris, France IMAGINE Institute Affiliate, Paris, France RARE Disorder Center: Centre des Maladies Endocriniennes Rares de la Croissance et du Développement, Paris, France Pediatric Endocrinology, Gynecology and Diabetology Unit, Hôpital Universitaire Necker-Enfants Malades, AP-HP, Paris, France Search for more papers by this author Frédéric Adam Frédéric Adam INSERM UMR_S1176, Paris-Sud University, Université Paris-Saclay, Le Kremlin-Bicêtre, France Search for more papers by this author Dulanjalee Kariyawasam Dulanjalee Kariyawasam RARE Disorder Center: Centre des Maladies Endocriniennes Rares de la Croissance et du Développement, Paris, France Pediatric Endocrinology, Gynecology and Diabetology Unit, Hôpital Universitaire Necker-Enfants Malades, AP-HP, Paris, France Search for more papers by this author Catherine Strassel Catherine Strassel INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS, Université de Strasbourg, Strasbourg, France Search for more papers by this author Sanjay Gawade Sanjay Gawade Department of Biomedicine, Pediatric Immunology, University of Basel, Basel, Switzerland Search for more papers by this author Gabor Szinnai Gabor Szinnai Department of Biomedicine, Pediatric Immunology, University of Basel, Basel, Switzerland Pediatric Endocrinology, University Children's Hospital Basel, University of Basel, Basel, Switzerland Search for more papers by this author Alexandre Kauskot Alexandre Kauskot INSERM UMR_S1176, Paris-Sud University, Université Paris-Saclay, Le Kremlin-Bicêtre, France Search for more papers by this author Dominique Lasne Dominique Lasne INSERM UMR_S1176, Paris-Sud University, Université Paris-Saclay, Le Kremlin-Bicêtre, France Necker Children's Hospital, Biological Hematology Service, Assistance Publique—Hôpitaux de Paris, Paris, France Search for more papers by this author Carsten Janke Carsten Janke orcid.org/0000-0001-7053-2000 Institut Curie, CNRS UMR3348, PSL Research University, Orsay, France Institut Curie, CNRS UMR3348, Université Paris Sud, Université Paris-Saclay, Orsay, France Search for more papers by this author Kathiresan Natarajan Kathiresan Natarajan Institut Curie, CNRS UMR3348, PSL Research University, Orsay, France Institut Curie, CNRS UMR3348, Université Paris Sud, Université Paris-Saclay, Orsay, France Search for more papers by this author Alain Schmitt Alain Schmitt INSERM U1016, Faculté de Médecine, Cochin Institute, Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Christine Bole-Feysot Christine Bole-Feysot Genomic Platform, INSERM UMR 1163, IMAGINE Institute, Paris Descartes University, Sorbonne Paris Cité, Paris, France Search for more papers by this author Patrick Nitschke Patrick Nitschke Bioinformatics Platform, IMAGINE Institute, Paris Descartes University, Paris, France Search for more papers by this author Juliane Léger Juliane Léger RARE Disorder Center: Centre des Maladies Endocriniennes Rares de la Croissance et du Développement, Paris, France Pediatric Endocrinology Unit, Hôpital Universitaire Robert Debré, AP-HP, Paris, France Paris Diderot University, Sorbonne Paris Cité, Paris, France INSERM UMR 1141, DHU Protect, Paris, France Search for more papers by this author Fabienne Jabot-Hanin Fabienne Jabot-Hanin Bioinformatics Platform, IMAGINE Institute, Paris Descartes University, Paris, France Search for more papers by this author Frédéric Tores Frédéric Tores Bioinformatics Platform, IMAGINE Institute, Paris Descartes University, Paris, France Search for more papers by this author Anita Michel Anita Michel INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS, Université de Strasbourg, Strasbourg, France Search for more papers by this author Arnold Munnich Arnold Munnich INSERM U1163, IMAGINE Institute, Translational Genetics, Université Paris Descartes, Sorbonne Paris Cité, Paris, France Department of Genetics, Hôpital Universitaire Necker-Enfants Malades, AP-HP, Paris, France Search for more papers by this author Claude Besmond Claude Besmond INSERM U1163, IMAGINE Institute, Translational Genetics, Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Raphaël Scharfmann Raphaël Scharfmann INSERM U1016, Faculté de Médecine, Cochin Institute, Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author François Lanza François Lanza INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS, Université de Strasbourg, Strasbourg, France Search for more papers by this author Delphine Borgel Delphine Borgel INSERM UMR_S1176, Paris-Sud University, Université Paris-Saclay, Le Kremlin-Bicêtre, France Necker Children's Hospital, Biological Hematology Service, Assistance Publique—Hôpitaux de Paris, Paris, France Search for more papers by this author Michel Polak Michel Polak INSERM U1016, Faculté de Médecine, Cochin Institute, Université Paris Descartes, Sorbonne Paris Cité, Paris, France IMAGINE Institute Affiliate, Paris, France RARE Disorder Center: Centre des Maladies Endocriniennes Rares de la Croissance et du Développement, Paris, France Pediatric Endocrinology, Gynecology and Diabetology Unit, Hôpital Universitaire Necker-Enfants Malades, AP-HP, Paris, France Fédération Parisienne pour le Dépistage et la Prévention des Handicaps de l'Enfant (FPDPHE), Paris, France Search for more papers by this author Aurore Carré Corresponding Author Aurore Carré [email protected] orcid.org/0000-0002-6795-3629 INSERM U1016, Faculté de Médecine, Cochin Institute, Université Paris Descartes, Sorbonne Paris Cité, Paris, France IMAGINE Institute Affiliate, Paris, France RARE Disorder Center: Centre des Maladies Endocriniennes Rares de la Croissance et du Développement, Paris, France Search for more papers by this author Athanasia Stoupa Athanasia Stoupa INSERM U1016, Faculté de Médecine, Cochin Institute, Université Paris Descartes, Sorbonne Paris Cité, Paris, France IMAGINE Institute Affiliate, Paris, France RARE Disorder Center: Centre des Maladies Endocriniennes Rares de la Croissance et du Développement, Paris, France Pediatric Endocrinology, Gynecology and Diabetology Unit, Hôpital Universitaire Necker-Enfants Malades, AP-HP, Paris, France Search for more papers by this author Frédéric Adam Frédéric Adam INSERM UMR_S1176, Paris-Sud University, Université Paris-Saclay, Le Kremlin-Bicêtre, France Search for more papers by this author Dulanjalee Kariyawasam Dulanjalee Kariyawasam RARE Disorder Center: Centre des Maladies Endocriniennes Rares de la Croissance et du Développement, Paris, France Pediatric Endocrinology, Gynecology and Diabetology Unit, Hôpital Universitaire Necker-Enfants Malades, AP-HP, Paris, France Search for more papers by this author Catherine Strassel Catherine Strassel INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS, Université de Strasbourg, Strasbourg, France Search for more papers by this author Sanjay Gawade Sanjay Gawade Department of Biomedicine, Pediatric Immunology, University of Basel, Basel, Switzerland Search for more papers by this author Gabor Szinnai Gabor Szinnai Department of Biomedicine, Pediatric Immunology, University of Basel, Basel, Switzerland Pediatric Endocrinology, University Children's Hospital Basel, University of Basel, Basel, Switzerland Search for more papers by this author Alexandre Kauskot Alexandre Kauskot INSERM UMR_S1176, Paris-Sud University, Université Paris-Saclay, Le Kremlin-Bicêtre, France Search for more papers by this author Dominique Lasne Dominique Lasne INSERM UMR_S1176, Paris-Sud University, Université Paris-Saclay, Le Kremlin-Bicêtre, France Necker Children's Hospital, Biological Hematology Service, Assistance Publique—Hôpitaux de Paris, Paris, France Search for more papers by this author Carsten Janke Carsten Janke orcid.org/0000-0001-7053-2000 Institut Curie, CNRS UMR3348, PSL Research University, Orsay, France Institut Curie, CNRS UMR3348, Université Paris Sud, Université Paris-Saclay, Orsay, France Search for more papers by this author Kathiresan Natarajan Kathiresan Natarajan Institut Curie, CNRS UMR3348, PSL Research University, Orsay, France Institut Curie, CNRS UMR3348, Université Paris Sud, Université Paris-Saclay, Orsay, France Search for more papers by this author Alain Schmitt Alain Schmitt INSERM U1016, Faculté de Médecine, Cochin Institute, Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Christine Bole-Feysot Christine Bole-Feysot Genomic Platform, INSERM UMR 1163, IMAGINE Institute, Paris Descartes University, Sorbonne Paris Cité, Paris, France Search for more papers by this author Patrick Nitschke Patrick Nitschke Bioinformatics Platform, IMAGINE Institute, Paris Descartes University, Paris, France Search for more papers by this author Juliane Léger Juliane Léger RARE Disorder Center: Centre des Maladies Endocriniennes Rares de la Croissance et du Développement, Paris, France Pediatric Endocrinology Unit, Hôpital Universitaire Robert Debré, AP-HP, Paris, France Paris Diderot University, Sorbonne Paris Cité, Paris, France INSERM UMR 1141, DHU Protect, Paris, France Search for more papers by this author Fabienne Jabot-Hanin Fabienne Jabot-Hanin Bioinformatics Platform, IMAGINE Institute, Paris Descartes University, Paris, France Search for more papers by this author Frédéric Tores Frédéric Tores Bioinformatics Platform, IMAGINE Institute, Paris Descartes University, Paris, France Search for more papers by this author Anita Michel Anita Michel INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS, Université de Strasbourg, Strasbourg, France Search for more papers by this author Arnold Munnich Arnold Munnich INSERM U1163, IMAGINE Institute, Translational Genetics, Université Paris Descartes, Sorbonne Paris Cité, Paris, France Department of Genetics, Hôpital Universitaire Necker-Enfants Malades, AP-HP, Paris, France Search for more papers by this author Claude Besmond Claude Besmond INSERM U1163, IMAGINE Institute, Translational Genetics, Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Raphaël Scharfmann Raphaël Scharfmann INSERM U1016, Faculté de Médecine, Cochin Institute, Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author François Lanza François Lanza INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS, Université de Strasbourg, Strasbourg, France Search for more papers by this author Delphine Borgel Delphine Borgel INSERM UMR_S1176, Paris-Sud University, Université Paris-Saclay, Le Kremlin-Bicêtre, France Necker Children's Hospital, Biological Hematology Service, Assistance Publique—Hôpitaux de Paris, Paris, France Search for more papers by this author Michel Polak Michel Polak INSERM U1016, Faculté de Médecine, Cochin Institute, Université Paris Descartes, Sorbonne Paris Cité, Paris, France IMAGINE Institute Affiliate, Paris, France RARE Disorder Center: Centre des Maladies Endocriniennes Rares de la Croissance et du Développement, Paris, France Pediatric Endocrinology, Gynecology and Diabetology Unit, Hôpital Universitaire Necker-Enfants Malades, AP-HP, Paris, France Fédération Parisienne pour le Dépistage et la Prévention des Handicaps de l'Enfant (FPDPHE), Paris, France Search for more papers by this author Aurore Carré Corresponding Author Aurore Carré [email protected] orcid.org/0000-0002-6795-3629 INSERM U1016, Faculté de Médecine, Cochin Institute, Université Paris Descartes, Sorbonne Paris Cité, Paris, France IMAGINE Institute Affiliate, Paris, France RARE Disorder Center: Centre des Maladies Endocriniennes Rares de la Croissance et du Développement, Paris, France Search for more papers by this author Author Information Athanasia Stoupa1,2,3,4, Frédéric Adam5, Dulanjalee Kariyawasam3,4, Catherine Strassel6, Sanjay Gawade7, Gabor Szinnai7,8, Alexandre Kauskot5, Dominique Lasne5,9, Carsten Janke10,11, Kathiresan Natarajan10,11,20, Alain Schmitt1, Christine Bole-Feysot12, Patrick Nitschke13, Juliane Léger3,14,15,16, Fabienne Jabot-Hanin13, Frédéric Tores13, Anita Michel6, Arnold Munnich17,18, Claude Besmond17, Raphaël Scharfmann1, François Lanza6, Delphine Borgel5,9, Michel Polak1,2,3,4,19 and Aurore Carré *,1,2,3 1INSERM U1016, Faculté de Médecine, Cochin Institute, Université Paris Descartes, Sorbonne Paris Cité, Paris, France 2IMAGINE Institute Affiliate, Paris, France 3RARE Disorder Center: Centre des Maladies Endocriniennes Rares de la Croissance et du Développement, Paris, France 4Pediatric Endocrinology, Gynecology and Diabetology Unit, Hôpital Universitaire Necker-Enfants Malades, AP-HP, Paris, France 5INSERM UMR_S1176, Paris-Sud University, Université Paris-Saclay, Le Kremlin-Bicêtre, France 6INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS, Université de Strasbourg, Strasbourg, France 7Department of Biomedicine, Pediatric Immunology, University of Basel, Basel, Switzerland 8Pediatric Endocrinology, University Children's Hospital Basel, University of Basel, Basel, Switzerland 9Necker Children's Hospital, Biological Hematology Service, Assistance Publique—Hôpitaux de Paris, Paris, France 10Institut Curie, CNRS UMR3348, PSL Research University, Orsay, France 11Institut Curie, CNRS UMR3348, Université Paris Sud, Université Paris-Saclay, Orsay, France 12Genomic Platform, INSERM UMR 1163, IMAGINE Institute, Paris Descartes University, Sorbonne Paris Cité, Paris, France 13Bioinformatics Platform, IMAGINE Institute, Paris Descartes University, Paris, France 14Pediatric Endocrinology Unit, Hôpital Universitaire Robert Debré, AP-HP, Paris, France 15Paris Diderot University, Sorbonne Paris Cité, Paris, France 16INSERM UMR 1141, DHU Protect, Paris, France 17INSERM U1163, IMAGINE Institute, Translational Genetics, Université Paris Descartes, Sorbonne Paris Cité, Paris, France 18Department of Genetics, Hôpital Universitaire Necker-Enfants Malades, AP-HP, Paris, France 19Fédération Parisienne pour le Dépistage et la Prévention des Handicaps de l'Enfant (FPDPHE), Paris, France 20Present address: Physiology and Biomedical Engineering Department, Mayo Clinic, Rochester, MN, USA *Corresponding author. Tel: +33 1 76 53 55 71; E-mail: [email protected] EMBO Mol Med (2018)10:e9569https://doi.org/10.15252/emmm.201809569 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The genetic causes of congenital hypothyroidism due to thyroid dysgenesis (TD) remain largely unknown. We identified three novel TUBB1 gene mutations that co-segregated with TD in three distinct families leading to 1.1% of TUBB1 mutations in TD study cohort. TUBB1 (Tubulin, Beta 1 Class VI) encodes for a member of the β-tubulin protein family. TUBB1 gene is expressed in the developing and adult thyroid in humans and mice. All three TUBB1 mutations lead to non-functional α/β-tubulin dimers that cannot be incorporated into microtubules. In mice, Tubb1 knock-out disrupted microtubule integrity by preventing β1-tubulin incorporation and impaired thyroid migration and thyroid hormone secretion. In addition, TUBB1 mutations caused the formation of macroplatelets and hyperaggregation of human platelets after stimulation by low doses of agonists. Our data highlight unexpected roles for β1-tubulin in thyroid development and in platelet physiology. Finally, these findings expand the spectrum of the rare paediatric diseases related to mutations in tubulin-coding genes and provide new insights into the genetic background and mechanisms involved in congenital hypothyroidism and thyroid dysgenesis. Synopsis Whole-exome sequencing in a consanguineous family with congenital hypothyroidism (CH) revealed a novel homozygous mutation in TUBB1, encoding for a member of beta-tubulins, essential for microtubule organisation. Until now, TUBB1 mutations were only known to be involved in platelet disorders. Two more mutations were identified in two distinct families with thyroid dysgenesis (TD) and abnormal platelet physiology, reinforcing the link between TUBB1 mutations and thyroid disease. TUBB1 is expressed in the developing and adult thyroid in humans and mice. Tubb1−/− mice have large platelets and show hypothyroidism with early thyroid progenitors proliferation defects, altered thyroid migration and failure of thyroid hormone synthesis. Platelet findings concerned basal activation and increased aggregation. These results expand our knowledge on genetic background of CH and TD. Introduction Thyroid dysgenesis (TD) is a feature in 65% of patients with congenital hypothyroidism (CH), the most common neonatal endocrine disorder affecting one in 2,500–3,500 newborns (Deladoëy et al, 2011; Barry et al, 2016). In France, the prevalence of CH due to TD is estimated in 1/5,000 (Barry et al, 2016). TD includes a vast spectrum of developmental thyroid anomalies encompassing athyreosis, thyroid ectopia, hypoplasia of an orthotopic gland, and hemithyroid (Barry et al, 2016; Stoupa et al, 2016). During embryogenesis, the midline thyroid anlage appears on embryonic day E.8.5 in mice and at 3 gestational weeks (GW) in humans. The midline anlage and ultimobranchial bodies migrate and fuse in the definitive pretracheal position on E13.5 in mice and at 7 GW in humans (Trueba et al, 2005; Nilsson & Fagman, 2013). The cells differentiate into thyrocytes organized in follicles or C cells (Appendix Table S1; Szinnai et al, 2007). Abnormalities at any step of thyroid development may result in TD associated with hypothyroidism or not (Maiorana et al, 2003). Previous studies of sporadic and familial TD covering a wide clinical spectrum identified mutations in nine genes: PAX8, NKX2-1, FOXE1, NKX2-5, TSHR, GLIS3, NTN1, JAG1 and BOREALIN (Dentice et al, 2006; Senée et al, 2006; Carré et al, 2009, 2014, 2017; Sura-Trueba et al, 2009; Ramos et al, 2014; Opitz et al, 2015; de Filippis et al, 2016). However, mutations in these genes are found in only 5% of all patients with TD and identification of causative mutations remains a challenging task. Our objectives were to extend the knowledge on the genetic basis of CH and TD. We performed WES (whole exome sequencing; Choi et al, 2009; Hildebrandt et al, 2009) for siblings with CH and thereby identified a mutation in TUBB1 gene. Then, we analysed 270 TD cases by targeted NGS including the TUBB1 gene. We identified two more TUBB1 mutations in patients with CH and TD. TUBB1 (Tubulin, Beta 1 Class VI) encodes for a member of the β-tubulin protein family. β-tubulins are one of two core protein families that heterodimerize to form α/β-tubulin dimers, which assemble into microtubules, one of the major cytoskeletal structures. The β1 isotype of tubulin (TUBB1) has been described as specifically expressed in platelets and megakaryocytes and involved in proplatelet formation and platelet release (Patel et al, 2015). Few mutations of TUBB1 have been identified in patients with a rare autosomal dominant disease congenital macrothrombocytopaenia, in which impaired microtubule assembly results in low platelet counts and macroplatelets (Kunishima et al, 2009; 2014; Bastida et al, 2018; Johnson et al, 2016; Burley et al, 2018). Tubb1-knock-out mice have thrombocytopaenia and spherical platelets (Schwer et al, 2001), but not known thyroid abnormalities. Our results highlight a hitherto unsuspected role for a specific tubulin isotype, Tubb1, in thyroid development and disease and extend our knowledge on genetic background of CH. Results Identification of TUBB1 mutations in a family with TD Family F1 is a consanguineous family of Algerian descent. The parents are first cousins (I.1, I.2) with five children including two females [II.1 (patient P1) and II.2 (patient P2)] with CH. Both patients were born at full term and diagnosed with CH by routine neonatal screening (Fig 1), which showed thyroid-stimulating hormone (TSH) elevation (164 and 177 μIU/ml in P1 and P2, respectively). On days 13 and 11, TSH was 67 and 202 μIU/ml in P1 and P2, respectively (normal range, N: 0.3–7 μIU/ml), and free thyroxine (T4) was 14 and 13.3 pmol in P1 and P2, respectively (N: 9.5–25 pmol; Fig 1). L-thyroxine therapy was initiated. 123I scintigraphy showed thyroid ectopia in both siblings. Another sibling (II.5, P3), aged 12 years, had thyroid hypoplasia (thyroid volume, 3.1 ml; N: 7 ± 3 ml) with a small right pyramidal lobe (17 *2 mm) and normal thyroid function tests. The parents had normal thyroid function, and two other siblings (II.3 and II.4) had normal thyroid function but were not able to undergo thyroid ultrasonography. Figure 1. Pedigrees and clinical tablePedigrees of three families with TUBB1 mutations. Family F1 has three affected individuals with homozygous mutations, family F2 has two affected individuals with heterozygous mutations, and family F3 has two affected individuals with heterozygous mutations. Thus, all seven patients (P1–P7) carry at least one mutated allele and have thyroid dysgenesis (TD) and macroplatelets. The patients are represented with filled symbols and unaffected family members with open symbols. NA, not available thyroid ultrasonography; *mild thyroid asymmetry (possibly normal) with normal thyroid function; N, not mutated; m, mutated. Download figure Download PowerPoint To look for genetic causes of CH in P1 and P2, we performed whole exome sequencing (WES) using the variant filtering and prioritization strategy described in Appendix Fig S1. Using the recessive transmission model, WES identified a novel missense homozygous TUBB1 mutation (c.479C>T, p.P160L, rs759117911) in both siblings with CH (P1 and P2) and in the sibling with thyroid hypoplasia (P3; Fig 1). Both parents and sibling II.3 were carriers. The remaining sibling (II.4) did not carry the mutation. WES identified no variants in genes known to be associated with TD or thyroid dyshormonogenesis. Search for TUBB1 mutations in a cohort with thyroid dysgenesis (TD) and congenital hypothyroidism (CH) After identification of the above-described novel TUBB1 mutation, we used targeted next-generation sequencing (NGS) to assess TUBB1 in a cohort of 270 patients with CH and TD. In a second family (F2) with a father (I.2) of Moroccan and a mother (I.1) of French descent, a female with CH and thyroid gland ectopia (P4, II.1) had a heterozygous TUBB1 mutation (c.318C>G, p.Y106X; Fig 1). CH was diagnosed upon routine neonatal screening (TSH, 250 μIU/ml) and confirmed on day 15 (TSH, 1,100 μIU/ml; free T4, 3.5 pmol/l; and free T3, 2.45 pmol/l). Thyroid scintigraphy showed an ectopic thyroid. The father carried the same heterozygous mutation; unfortunately, thyroid ultrasound was not performed, and complete phenotype was therefore not possible. In a paternal aunt (I.3, P5), an evaluation at 26 years of age for obesity and depression showed mild hypothyroidism (TSH, 6.6 μIU/ml; N: 0.1–5.5 μIU/ml; free T4, 8.7 pmol/l; N: 9.8–23.1 pmol/l). Thyroid ultrasonography and scintigraphy showed right hemithyroid. In a third family (F3), a patient (II.1, P6) with CH and an ectopic thyroid was shown by targeted NGS to have a heterozygous frameshift TUBB1 mutation (c.35delG, p.Cys12Leufs*12, rs77324804) that created a premature stop codon at amino acid 23 (Fig 1). CH was diagnosed neonatally based on TSH elevation (476 μIU/ml) and low free T4 and T3 levels (8 and 5.6 pmol/l, respectively). L-thyroxine therapy was started at 11 days of age. Both parents were of French descent. The father (I.1, P7) carried the same heterozygous mutation and had normal thyroid function with mild thyroid lobe asymmetry by ultrasonography (right lobe, 6.9 ml; left lobe, 5 ml). The other siblings and mother had normal thyroid function and morphology and did not carry the mutation. By targeted NGS, neither P4 nor P6 had any variants in genes known to cause CH (with TD or dyshormonogenesis). In the Exome Aggregation Consortium (ExAC) database, estimated allele frequencies are 0.000008 for the c.479C>T mutation (rs759117911, 20:57598961 C/T) and 0.000025 for the c35delG (rs773248042, 20:57594611 TG/T) mutation. Neither mutation has been reported in homozygous form. The c.318C>G variation has not been reported in public databases. The in silico prediction tools, PolyPhen-2, SIFT, predict that c.479C>T is probably damaging and deleterious, respectively, with a PHRED-scaled CADD score of 32 (damaging: > 15; Kircher et al, 2014). The other two mutations create a premature stop codon and have PHRED-scaled CADD score of 35. CADD is a prediction algorithm, which integrates information contained in more than 60 diverse annotations of genetic variation into a single score. The higher the CADD score, the higher the deleteriousness probability of the variant investigated. A Burden test was applied to determine whether TUBB1 was significantly enriched in rare variants in the 270 patients with CH and TD versus 406 Caucasian controls from the 1000 Genomes project. The patients in the TD cohort had athyreosis, ectopia, hemithyroid or hypoplasia. In the TD group, 14/270 patients (5.2%) exhibited at least one rare functional variant in TUBB1 compared with 8/406 controls (2%). This difference is significant from a statistical point of view (P = 0.0227). None of the patients in our cohort with TUBB1 mutations had athyreosis. Performing the same test after excluding the 77 patients with athyreosis increased the significance of the difference of TD group (n = 193) versus controls (P = 0.0095). The list of rare functional variants found in TUBB1 is provided in Table EV1. The three amino acids affected by the TUBB1 mutations are strictly conserved across species, from humans to zebrafish, and across all β-tubulins (Fig 2A). All three mutations were located in the first part of TUBB1, i.e., in the N-terminal domain needed for guanosine triphosphate (GTP) activity (Fig 2B). The c.318C>G and c.35delG mutations created a premature stop codon, thereby removing the intermediate and C-terminal domains required for microtubule-associated protein (MAP) binding (Nogales et al, 1998). Figure 2. Molecular genetics A. Alignment and conservation of residues encoded by TUBB1 orthologues and genes encoding for other β-tubulins (TUBB2A, TUBB2B, TUBB3, TUBB4A, TUBB4B, TUBB5, TUBB6 and TUBB8). Mutations are shown in green. Mutated aminoacids through species or through all TUBB are shown in red boxes. B. Location of TUBB1 mutations in the cDNA and of the corresponding changes in the protein. Exons are represented by boxes numbered from 1 to 4. The dark grey box represents the protein domain responsible for encoding guanosine triphosphate (GTP) and the light grey box the domain for microtubule-associated protein (MAP) binding. The arrows show the consequences of the three TUBB1 mutations in our patients, all of which are in the GTP domain. Mutations of this study are in bold, and published mutations associated with congenital macrothrombocytopaenia are in italic. Download figure Download PowerPoint In sum, we identified three TUBB1 mutations in three independent families of patients with CH and TD chiefly manifesting as thyroid gland ectopia. Thus, we found 1.1% of TUBB1 mutations in patients affected with CH and TD in our cohort. β1-tubulin is expressed in the developing thyroid in humans and mice β1-tubulin expression has so far been reported only in megakaryocytes and platelets (Wang et al, 1986; Lecine et al, 2000). Our finding of TUBB1 mutations in patients with TD prompted us to look for β1-tubulin expression in thyroid tissue. In human thyroid tissue, TUBB1 mRNA was expressed at 8, 10 and 12 GW and in adulthood (Fig 3A). In mouse thyroid tissue, Tubb1 was expressed at E13.5 and strongly at E15.5, E17.5 and adulthood (Fig 3B). To refine our study of Tubb1 expression, we used cells sorted from mice thyroid tissue based on well-accepted markers (Gawade et al, 2016) with stringent sorting regions, including the brightest cells for each selected marker (Pecam for endothelial cells, EpCAM for epithelial cells, Pdgfra for fibroblasts and CD45 for leucocytes). As expected, expression was strongest in platelets sorted using the specific megakaryocyte lineage marker CD41 (Appendix Fig S2A). However, Tubb1 was also expressed in EpCAM-positive epithelial-cell populations containing thyrocytes, at E17.5 and in adulthood (Fig 3C). Figure 3. β1-tubulin expression in the developing thyroid and deleterious effect of TUBB1 mutations A. TUBB1 is expressed in the developing human thyroid at 8 gestational weeks (GW), 10 GW and 12 GW and in the adult human thyroid: quantitative PCR results normalized to one of three thyroid tissues at 8 GW value (in black) and peptidylprolyl isomerase A. Experiments with four tissues per stage. Results are reported as mean ± SEM. Statistical comparisons versus 8 GW (in black) using ANOVA test showed no significant differences. B. Tubb1 is expressed in the developing mouse thyroid at E13.5, E15.5 and E17.5 and in the adult mouse thyroid: quantitative PCR results normalized to one of three thyroid tissues at E13.5 value and peptidylprolyl isomerase A. Experiments with four tissues per stage. Results are reported as mean ± SEM. Statistical comparisons versus E13.5 (black), **P < 0.01 using ANOVA test. C. Sorted mouse thyroid cells, Tubb1 ex
Referência(s)