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Licensed Unlicensed Requires Authentication Published by De Gruyter August 19, 2014

Functional characterization of the novel sequence variant p.S304R in the hinge region of TSHR in a congenital hypothyroidism patients and analogy with other formerly known mutations of this gene portion

Taise Lima Oliveira Cerqueira, Aurore Carré, Lucie Chevrier, Gabor Szinnai, Elodie Tron, Juliane Léger, Sylvie Cabrol, Chrystelle Queinnec, Nicolas De Roux, Mireille Castanet, Michel Polak and Helton Estrela Ramos ORCID logo

Abstract

Context: Thyroid dysgenesis may be associated with loss-of-function mutations in the thyrotropin receptor (TSHR) gene.

Objectives: The aim of this study was to characterize a novel TSHR gene variant found in one patient harboring congenital hypothyroidism (CH) from a cohort of patients with various types of thyroid defects.

Materials and methods: This cross-sectional cohort study involved 118 patients with CH and their family members, including 45 with familial and 73 with sporadic diseases. The thyroid gland was normal in 23 patients, 25 patients had hypoplasia, 25 hemithyroid agenesis, 21 had athyreosis, and 21 had ectopy. Genomic DNA was extracted, and 10 exons of the TSHR gene were amplified and sequenced. Mutations in other candidate genes were investigated. Ortholog alignment was performed, and TSHR functional assays were evaluated.

Results: We identified one previously unknown missense variation in the hinge region (HinR) of the TSHR gene (p.S304R) in one patient with thyroid hypoplasia. This variant is conserved in our ortholog alignment. However, the p.S304R TSHR variant presented a normal glycosylation pattern and signal transduction activity in functional analysis.

Conclusion: We report the ocurrence of a novel nonsynonymous substitution in the HinR of the large N-terminal extracellular domain of the TSHR gene in a patient with thyroid hypoplasia. In contrast with four others in whom TSHR mutations of the hinge portion were previously identified, the p.S304R TSHR variation neither affected TSH binding nor cAMP pathway activation. This TSHR gene variant was documented in a CH patient, but the current data do not support its role in the clinical phenotype.


Corresponding author: Helton Estrela Ramos, Federal University of Bahia, Health and Science Institute, Department of Biorregulation, Avenida Reitor Miguel Calmon, S/N, Sala 301, Vale do Canela, 40110-102 Salvador, Bahia, Brazil, Phone: +55 71 3283 8890, Fax: +55 71 3283 8927, E-mail: , http://orcid.org/0000-0002-2900-2099; Curso de Pós-Graduação em Biotecnologia em Saúde e Medicina Investigativa, Centro de Pesquisa Gonçalo Moniz-FIOCRUZ/BA, Salvador, Bahia, Brazil; INSERM U845, Université Paris Descartes, Sorbonne Paris Cité, Paris, France; Pediatric Endocrine Unit, Centre des Maladies Endocriniennes Rares de la Croissance, Hôpital Necker Enfants Malades, AP-HP, Paris, France; and Departamento de Biorregulação, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Bahia, Brazil

References

1. Vassart G, Pardo L, Costagliola S. A molecular dissection of the glycoprotein hormone receptors. Trends Biochem Sci 2004;29:119–26.10.1016/j.tibs.2004.01.006Search in Google Scholar

2. Duprez L, Parma J, Van Sande J, Rodien P, Sabine C, et al. Pathology of the TSH receptor. J Pediatr Endocrinol Metab 1999;12:295–302.Search in Google Scholar

3. Vassart G, Desarnaud F, Duprez L, Eggerickx D, Labbe O, et al. The G protein-coupled receptor family and one of its members, the TSH receptor. Ann N Y Acad Sci 1995;766:23–30.10.1111/j.1749-6632.1995.tb26645.xSearch in Google Scholar

4. Tonacchera M, Van Sande J, Parma J, Duprez L, Cetani F, et al. TSH receptor and disease. Clin Endocrinol (Oxf) 1996;44:621–33.10.1046/j.1365-2265.1996.746567.xSearch in Google Scholar

5. Vlaeminck-Guillem V, Ho SC, Rodien P, Vassart G, Costagliola S. Activation of the cAMP pathway by the TSH receptor involves switching of the ectodomain from a tethered inverse agonist to an agonist. Mol Endocrinol 2002;16:736–46.10.1210/mend.16.4.0816Search in Google Scholar

6. Duprez L, Parma J, Costagliola S, Hermans J, Van Sande J, et al. Constitutive activation of the TSH receptor by spontaneous mutations affecting the N-terminal extracellular domain. FEBS Lett 1997;409:469–74.10.1016/S0014-5793(97)00532-2Search in Google Scholar

7. Hamidi S, Chen CR, Mizutori-Sasai Y, McLachlan SM, Rapoport B. Relationship between thyrotropin receptor hinge region proteolytic posttranslational modification and receptor physiological function. Mol Endocrinol 2011;25:184–94.10.1210/me.2010-0401Search in Google Scholar PubMed PubMed Central

8. Jaeschke H, Schaarschmidt J, Gunther R, Mueller S. The hinge region of the TSH receptor stabilizes ligand binding and determines different signaling profiles of human and bovine TSH. Endocrinology 2011;152:3986–96.10.1210/en.2011-1389Search in Google Scholar PubMed

9. Mueller S, Jaeschke H, Gunther R, Paschke R. The hinge region: an important receptor component for GPHR function. Trends Endocrin Met 2009;21:111–22.10.1016/j.tem.2009.09.001Search in Google Scholar PubMed

10. Mueller S, Szkudlinski MW, Schaarschmidt J, Gunther R, Paschke R, et al. Identification of novel TSH interaction sites by systematic binding analysis of the TSHR hinge region. Endocrinology 2011;152:3268–78.10.1210/en.2011-0153Search in Google Scholar PubMed

11. Smits G, Govaerts C, Nubourgh I, Pardo L, Vassart G, et al. Lysine 183 and glutamic acid 157 of the TSH receptor: two interacting residues with a key role in determining specificity toward TSH and human CG. Mol Endocrinol 2002;16:722–35.10.1210/mend.16.4.0815Search in Google Scholar PubMed

12. Persani L, Calebiro D, Cordella D, Weber G, Gelmini G, et al. Genetics and phenomics of hypothyroidism due to TSH resistance. Mol Cell Endocrinol 2010;322:72–82.10.1016/j.mce.2010.01.008Search in Google Scholar

13. Sunthornthepvarakui T, Gottschalk ME, Hayashi Y, Refetoff S. Brief report: resistance to thyrotropin caused by mutations in the thyrotropin-receptor gene. N Engl J Med 1995;332:155–60.10.1056/NEJM199501193320305Search in Google Scholar

14. Calebiro D, de Filippis T, Lucchi S, Covino C, Panigone S, et al. Intracellular entrapment of wild-type TSH receptor by oligomerization with mutants linked to dominant TSH resistance. Hum Mol Genet 2005;14:2991–3002.10.1093/hmg/ddi329Search in Google Scholar

15. Calebiro D, Gelmini G, Cordella D, Bonomi M, Winkler F, et al. Frequent TSH receptor genetic alterations with variable signaling impairment in a large series of children with nonautoimmune isolated hyperthyrotropinemia. J Clin Endocrinol Metab 2012;97:E156–60.10.1210/jc.2011-1938Search in Google Scholar

16. Polak M, Sura-Trueba S, Chauty A, Szinnai G, Carre A, et al. Molecular mechanisms of thyroid dysgenesis. Horm Res 2004;62:14–21.10.1159/000080494Search in Google Scholar

17. Sunthornthepvarakul T, Hayashi Y, Refetoff S. Polymorphism of a variant human thyrotropin receptor (hTSHR) gene. Thyroid 1994;4:147–49.10.1089/thy.1994.4.147Search in Google Scholar

18. Gustavsson B, Eklof C, Westermark K, Westermark B, Heldin NE. Functional analysis of a variant of the thyrotropin receptor gene in a family with Graves’ disease. Mol Cell Endocrinol 1995;111:167–73.10.1016/0303-7207(95)03562-LSearch in Google Scholar

19. Gabriel EM, Bergert ER, Grant CS, van Heerden JA, Thompson GB, et al. Germline polymorphism of codon 727 of human thyroid-stimulating hormone receptor is associated with toxic multinodular goiter. J Clin Endocrinol Metab 1999;84:3328–35.10.1210/jc.84.9.3328Search in Google Scholar

20. Peeters RP, van Toor H, Klootwijk W, de Rijke YB, Kuiper GG, et al. Polymorphisms in thyroid hormone pathway genes are associated with plasma TSH and iodothyronine levels in healthy subjects. J Clin Endocrinol Metab 2003;88:2880–88.10.1210/jc.2002-021592Search in Google Scholar PubMed

21. Hansen PS, van der Deure WM, Peeters RP, Iachine I, Fenger M, et al. The impact of a TSH receptor gene polymorphism on thyroid-related phenotypes in a healthy Danish twin population. Clin Endocrinol (Oxf) 2007;66:827–32.10.1111/j.1365-2265.2007.02820.xSearch in Google Scholar PubMed

22. Nogueira CR, Kopp P, Arseven OK, Santos CL, Jameson JL, et al. Thyrotropin receptor mutations in hyperfunctioning thyroid adenomas from Brazil. Thyroid 1999;9:1063–68.10.1089/thy.1999.9.1063Search in Google Scholar

23. Sykiotis GP, Neumann S, Georgopoulos NA, Sgourou A, Papachatzopoulou A, et al. Functional significance of the thyrotropin receptor germline polymorphism D727E. Biochem Biophys Res Commun 2003;301:1051–56.10.1016/S0006-291X(03)00071-8Search in Google Scholar

24. Cuddihy RM, Bryant WP, Bahn RS. Normal function in vivo of a homozygotic polymorphism in the human thyrotropin receptor. Thyroid 1995;5:255–57.10.1089/thy.1995.5.255Search in Google Scholar

25. Loos U, Hagner S, Bohr UR, Bogatkewitsch GS, Jakobs KH, et al. Enhanced cAMP accumulation by the human thyrotropin receptor variant with the Pro52Thr substitution in the extracellular domain. Eur J Biochem 1995;232:62–65.10.1111/j.1432-1033.1995.tb20781.xSearch in Google Scholar

26. de Roux N, Misrahi M, Brauner R, Houang M, Carel JC, et al. Four families with loss of function mutations of the thyrotropin receptor. J Clin Endocrinol Metab 1996;81:4229–35.Search in Google Scholar

27. Karlin S, Mrazek J, Gentles AJ. Genome comparisons and analysis. Curr Opin Struct Biol 2003;13:344–52.10.1016/S0959-440X(03)00073-3Search in Google Scholar

28. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403–10.10.1016/S0022-2836(05)80360-2Search in Google Scholar

29. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22: 4673–80.10.1093/nar/22.22.4673Search in Google Scholar PubMed PubMed Central

30. Sura-Trueba S, Aumas C, Carre A, Durif S, Leger J, et al. An inactivating mutation within the first extracellular loop of the thyrotropin receptor impedes normal posttranslational maturation of the extracellular domain. Endocrinology 2009;150:1043–50.10.1210/en.2008-1145Search in Google Scholar PubMed

31. Ramos HE, Gabor Szinnai AC, Tron E, Cerqueira TL, Leger J, et al. PAX8 Mutations in congenital hypothyroidism: New evidence for phenotypic variability from normal to ectopic thyroid gland. Eur J Endocrinol 2014, in Press. DOI: http://dx.doi.org/10.1530/EJE-13-1006.10.1530/EJE-13-1006Search in Google Scholar PubMed

32. Komatsu M, Takahashi T, Takahashi I, Nakamura M, Takahashi I, et al. Thyroid dysgenesis caused by PAX8 mutation: the hypermutability with CpG dinucleotides at codon 31. J Pediatr 2001;139:597–99.10.1067/mpd.2001.117071Search in Google Scholar PubMed

33. Ramos HE, Labedan I, Carre A, Castanet M, Guemas I, et al. New cases of isolated congenital central hypothyroidism due to homozygous thyrotropin beta gene mutations: a pitfall to neonatal screening. Thyroid 2010;20:639–45.10.1089/thy.2009.0462Search in Google Scholar PubMed

34. Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, et al. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 2003;31:3497–500.10.1093/nar/gkg500Search in Google Scholar PubMed PubMed Central

35. Trueba SS, Auge J, Mattei G, Etchevers H, Martinovic J, et al. PAX8, TITF1, and FOXE1 gene expression patterns during human development: new insights into human thyroid development and thyroid dysgenesis–associated malformations. J Clin Endocrinol Metab 2005;90:455–62.10.1210/jc.2004-1358Search in Google Scholar PubMed

36. Kleinau G, Mueller S, Jaeschke H, Grzesik P, Neumann S, et al. Defining structural and functional dimensions of the extracellular thyrotropin receptor region. J Biol Chem 2011;286:22622–31.10.1074/jbc.M110.211193Search in Google Scholar PubMed PubMed Central

37. Biebermann H, Schoneberg T, Krude H, Schultz G, Gudermann T, et al. Mutations of the human thyrotropin receptor gene causing thyroid hypoplasia and persistent congenital hypothyroidism. J Clin Endocrinol Metab 1997;82:3471–80.10.1210/jc.82.10.3471Search in Google Scholar

38. Calaciura F, Motta RM, Miscio G, Fichera G, Leonardi D, et al. Subclinical hypothyroidism in early childhood: a frequent outcome of transient neonatal hyperthyrotropinemia. J Clin Endocrinol Metab 2002;87:3209–214.10.1210/jcem.87.7.8662Search in Google Scholar PubMed

39. Camilot M, Teofoli F, Gandini A, Franceschi R, Rapa A, et al. Thyrotropin receptor gene mutations and TSH resistance: variable expressivity in the heterozygotes. Clin Endocrinol (Oxf) 2005;63:146–51.10.1111/j.1365-2265.2005.02314.xSearch in Google Scholar PubMed

40. De Marco G, Agretti P, Camilot M, Teofoli F, Tato L, et al. Functional studies of new TSH receptor (TSHr) mutations identified in patients affected by hypothyroidism or isolated hyperthyrotrophinaemia. Clin Endocrinol (Oxf) 2009;70:335–38.10.1111/j.1365-2265.2008.03333.xSearch in Google Scholar PubMed

41. Nicoletti A, Bal M, De Marco G, Baldazzi L, Agretti P, et al. Thyrotropin-stimulating hormone receptor gene analysis in pediatric patients with non-autoimmune subclinical hypothyroidism. J Clin Endocrinol Metab 2009;94:4187–94.10.1210/jc.2009-0618Search in Google Scholar PubMed

42. Rapa A, Monzani A, Moia S, Vivenza D, Bellone S, et al. Subclinical hypothyroidism in children and adolescents: a wide range of clinical, biochemical, and genetic factors involved. J Clin Endocrinol Metab 2009;94:2414–20.10.1210/jc.2009-0375Search in Google Scholar PubMed

43. Tonacchera M, Agretti P, De Marco G, Perri A, Pinchera A, et al. Thyroid resistance to TSH complicated by autoimmune thyroiditis. J Clin Endocrinol Metab 2001;86:4543–46.10.1210/jcem.86.9.7791Search in Google Scholar PubMed

44. Narumi S, Muroya K, Abe Y, Yasui M, Asakura Y, et al. TSHR mutations as a cause of congenital hypothyroidism in Japan: a population-based genetic epidemiology study. J Clin Endocrinol Metab 2009;94:1317–23.10.1210/jc.2008-1767Search in Google Scholar PubMed

45. Russo D, Betterle C, Arturi F, Chiefari E, Girelli ME, et al. A novel mutation in the thyrotropin (TSH) receptor gene causing loss of TSH binding but constitutive receptor activation in a family with resistance to TSH. J Clin Endocrinol Metab 2000;85:4238–42.10.1210/jc.85.11.4238Search in Google Scholar

46. Cassio A, Nicoletti A, Rizzello A, Zazzetta E, Bal M, et al. Current loss-of-function mutations in the thyrotropin receptor gene: when to investigate, clinical effects, and treatment. J Clin Res Pediatr Endocrinol 2013;5:29–39.Search in Google Scholar

47. Tonacchera M, Di Cosmo C, De Marco G, Agretti P, Banco M, et al. Identification of TSH receptor mutations in three families with resistance to TSH. Clin Endocrinol (Oxf) 2007;67:712–18.10.1111/j.1365-2265.2007.02950.xSearch in Google Scholar PubMed

Received: 2014-5-13
Accepted: 2014-7-14
Published Online: 2014-8-19
Published in Print: 2015-7-1

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