Analysis of the PAX8 gene in 32 children with thyroid dysgenesis and functional characterization of a promoter variant

Denise Perone; Geraldo Medeiros-Neto; Célia Regina Nogueira; Antonio José Chagas; Vera Maria Alves Dias; Maria Fátima Viana; Peter Kopp

doi:10.1515/jpem-2015-0199

Article

Analysis of the PAX8 gene in 32 children with thyroid dysgenesis and functional characterization of a promoter variant

Denise Perone , Geraldo Medeiros-Neto , Célia Regina Nogueira , Antonio José Chagas , Vera Maria Alves Dias , Maria Fátima Viana and Peter Kopp

Published/Copyright: October 23, 2015

Published by

Become an author with De Gruyter Brill

Submit Manuscript Author Information Explore this Subject

From the journal Journal of Pediatric Endocrinology and Metabolism Volume 29 Issue 2

Abstract

Background: The molecular basis underlying the development of thyroid dysgenesis remains largely unknown. The objective of this study was to analyze the PAX8 gene in 32 children with congenital hypothyroidism due to thyroid dysgenesis for mutations, and to characterize the functional consequences of the mutations.

Methods: The 5′-untranslated region and the entire coding region of the PAX8 gene were analyzed in 32 children. Functional analyses with a reporter gene assay were performed in transfected PCCL3 and TSA cells.

Results: Thirty children did not have any sequence alterations. Two individuals had a previously identified monoallelic cytosine to thymine transition at position -983 in the promoter (-983C>T; mutant P. A of the ATG of the initiator codon is designated as +1), and a novel guanine to cytosine transversion in the non-coding exon 1 (-465G>C; mutant E). Functional analysis revealed that the basal transcriptional activity of the mutants is decreased compared to the wild type. Gel mobility shift assays indicated that mutant P does not interact with a transacting factor whose nature remains to be elucidated. The DNA binding property of mutant E were similar compared to the wild type.

Conclusions: These results suggest that mutations in PAX8 are most likely a very rare cause of thyroid dysgenesis. The observed sequence alterations result in diminished transcriptional activity and, in conjunction with other genetic and non-genetic modifiers, they may contribute to the pathogenesis of thyroid hypoplasia and hypothyroidism.

Keywords: congenital hypothyroidism; mutation; PAX8 gene; promoter; thyroid dysgenesis; transcription factor

Corresponding authors: Denise Perone, PhD, Department of Medicine; and Departamento de Química, Laboratório de Química Analítica e Materiais Avançados, UNESP – Universidade Estadual Paulista, Faculdade de Ciências de Bauru, Avenida Engenheiro Luiz Edmundo Coube, 14-01; Vargem Limpa, CEP: 17030-360, Bauru – SP/Brasil, E-mail: deniseperone@fc.unesp.br; and Peter Kopp, MD, Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University, Tarry 15, 303 East Chicago Avenue, Chicago, IL 60611, USA, E-mail: p-kopp@northwestern.edu

Acknowledgments

We express our gratitude to the participating families and the referring physicians.

Author contributions: DP performed the molecular analyses and functional studies, and took a lead role in writing the manuscript. GMN participated in its design and coordination and helped to draft the manuscript. CRN participated in its design and coordination and helped to draft the manuscript. AJC was involved in the molecular analyses. VMAD was involved in the clinical characterization of the patients. MFV was involved in the clinical characterization of the patients. PK oversaw the functional studies and the writing of the manuscript. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: This study was supported, in part, by the São Paulo State Research Foundation (FAPESP grant 99/03570-3), 1R01DK63024-01 from NIH/NIDDK to PK, and partial financial support from the Division of Endocrinology, Thyroid Molecular Laboratory (LIM-25), University of São Paulo Medical School.

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References

1. New England congenital hypothyroidism collaborative. Effects of neonatal screening for hypothyroidism: prevention of mental retardation by treatment before clinical manifestations. Lancet 1981;ii:1095–8.10.1016/S0140-6736(81)91287-3Search in Google Scholar

2. Toublanc JE. Comparison of epidemiological data on congenital hypothyroidism in Europe with those of other parts in the world. Horm Res 1992;38:230–5.10.1159/000182549Search in Google Scholar

3. Castanet M, Lyonnet S, Bonaiti-Pellie C, Polak M, Czernichow P, et al. Familial forms of thyroid dysgenesis among infants with congenital hypothyroidism. N Engl J Med 2000;343:441–2.10.1056/NEJM200008103430614Search in Google Scholar

4. Castanet M, Polak M, Bonaiti-Pellie C, Lyonnet S, Czernichow P, et al. Nineteen years of national screening for congenital hypothyroidism: familial cases with thyroid dysgenesis suggest the involvement of genetic factors. J Clin Endocrinol Metab 2001;86:2009–14.10.1210/jcem.86.5.7501Search in Google Scholar

5. Kopp P. Perspective: genetic defects in the etiology of congenital hypothyroidism. Endocrinology 2002;143:2019–24.10.1210/endo.143.6.8864Search in Google Scholar

6. Van Vliet G. Development of the thyroid gland: lessons from congenitally hypothyroid mice and men. Clin Genet 2003;63:445–55.10.1034/j.1399-0004.2003.00107.xSearch in Google Scholar

7. De Felice M, Di Lauro R. Thyroid development and its disorders: genetics and molecular mechanisms. Endocr Rev 2004;25: 722–46.10.1210/er.2003-0028Search in Google Scholar

8. Mansouri A, St-Onge L, Gruss P. Role of genes in endoderm-derived organs. Trends Endocrinol Metab 1999;10:164–7.10.1016/S1043-2760(98)00133-7Search in Google Scholar

9. Chi N, Epstein JA. Getting your Pax straight: Pax proteins in development and disease. Trends Genet 2002;18:41–7.10.1016/S0168-9525(01)02594-XSearch in Google Scholar

10. Damante G, Tell G, Di Lauro R. A unique combination of transcription factors controls differentiation of thyroid cells. Prog Nucleic Acid Res Mol Biol 2001;66:307–56.10.1016/S0079-6603(00)66033-6Search in Google Scholar

11. Mansouri A, Chowdhury K, Gruss P. Follicular cells of the thyroid gland require Pax8 gene function. Nat Genet 1998;19:87–90.10.1038/ng0598-87Search in Google Scholar

12. Pasca di Magliano M, Di Lauro R, Zannini M. Pax8 has a key role in thyroid cell differentiation. Proc Natl Acad Sci USA 2000;97:13144–9.10.1073/pnas.240336397Search in Google Scholar

13. Damante G, Di Lauro R. Thyroid-specific gene expression. Biochim Biophys Acta 1994;1218:255–66.10.1016/0167-4781(94)90176-7Search in Google Scholar

14. Dohan O, De la Vieja A, Paroder V, Riedel C, Artani M, et al. The sodium/iodide Symporter (NIS): characterization, regulation, and medical significance. Endocr Rev 2003;24:48–77.10.1210/er.2001-0029Search in Google Scholar PubMed

15. Macchia PE, Lapi P, Krude H, Pirro MT, Missero C, et al. PAX8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis. Nat Genet 1998;19:83–6.10.1038/ng0598-83Search in Google Scholar PubMed

16. Vilain C, Rydlewski C, Duprez L, Heinrichs C, Abramowicz M, et al. Autosomal dominant transmission of congenital thyroid hypoplasia due to loss-of-function mutation of PAX8. J Clin Endocrinol Metab 2001;86:234–8.10.1210/jc.86.1.234Search in Google Scholar

17. Congdon T, Nguyen LQ, Nogueira CR, Habiby RL, Medeiros-Neto G, et al. A novel mutation (Q40P) in PAX8 associated with congenital hypothyroidism and thyroid hypoplasia: evidence for phenotypic variability in mother and child. J Clin Endocrinol Metab 2001;86:3962–7.10.1210/jcem.86.8.7765Search in Google Scholar PubMed

18. Komatsu M, Takahashi T, Takahashi I, Nakamura M, Takada G. Thyroid dysgenesis caused by PAX8 mutation: the hypermutability with CpG dinucleotides at codon 31. J Pediatr 2001;139: 597–9.10.1067/mpd.2001.117071Search in Google Scholar PubMed

19. Meeus L, Gilbert B, Rydlewski C, Parma J, Roussie AL, et al. Characterization of a novel loss of function mutation of PAX8 in a familial case of congenital hypothyroidism with in-place, normal-sized thyroid. J Clin Endocrinol Metab 2004;89: 4285–91.10.1210/jc.2004-0166Search in Google Scholar PubMed

20. De Sanctis L, Corrias A, Romagnolo D, Di Palma T, Biava A, et al. Familial PAX8 small deletion (c.989_992delACCC) associated with extreme phenotype variability. J Clin Endocrinol Metab 2004;89:5669–74.10.1210/jc.2004-0398Search in Google Scholar PubMed

21. Grasberger H, Ringkananont U, Lefrancois P, Abramowicz M, Vassart G, et al. Thyroid transcription factor 1 rescues PAX8/p300 synergism impaired by a natural PAX8 paired domain mutation with dominant negative activity. Mol Endocrinol 2005;19:1779–91.10.1210/me.2004-0426Search in Google Scholar PubMed

22. Al Taji E, Biebermann H, Limanova Z, Hnikova O, Zikmund J, et al. Screening for mutations in transcription factors in a Czech cohort of 170 patients with congenital and early-onset hypothyroidism: identification of a novel PAX8 mutation in dominantly inherited early-onset non-autoimmune hypothyroidism. Eur J Endocrinol 2007;156:521–9.10.1530/EJE-06-0709Search in Google Scholar PubMed

23. Esperante SA, Rivolta CM, Miravalle L, Herzovich V, Iorcansky S, et al. Identification and characterization of four PAX8 rare sequence variants (p.T225M, p.L233L, p.G336S and p.A439A) in patients with congenital hypothyroidism and dysgenetic thyroid glands. Clin Endocrinol (Oxf) 2008;68:828–35.10.1111/j.1365-2265.2007.03111.xSearch in Google Scholar PubMed

24. Tonacchera M, Banco ME, Montanelli L, Di Cosmo C, Agretti P, et al. Genetic analysis of the PAX8 gene in children with congenital hypothyroidism and dysgenetic or eutopic thyroid glands: identification of a novel sequence variant. Clin Endocrinol 2007;67:34–40.10.1111/j.1365-2265.2007.02831.xSearch in Google Scholar PubMed

25. Di Palma T, Zampella E, Filippone MG, Macchia PE, Ris-Stalpers C, et al. Characterization of a novel loss of function mutation of PAX8 associated with congenital hypothyroidism. Clin Endocrinol (Oxf). 2010;73:808–14.10.1111/j.1365-2265.2010.03851.xSearch in Google Scholar PubMed

26. Narumi S, Araki S, Hori N, Muroya K, Yamamoto Y, et al. Functional characterization of four novel PAX8 mutations causing congenital hypothyroidism: new evidence for haploinsufficiency as a disease mechanism. Eur J Endocrinol 2012;167:625–32.10.1530/EJE-12-0410Search in Google Scholar PubMed

27. Hermanns P, Grasberger H, Refetoff S, Pohlenz J. Mutations in the NKX2.5 gene and the PAX8 promoter in a girl with thyroid dysgenesis. J Clin Endocrinol Metab 2011;96:E977–81.10.1210/jc.2010-2341Search in Google Scholar PubMed PubMed Central

28. Liu SG, Zhang SS, Zhang LQ, Li WJ, Zhang AQ, et al. Screening of PAX8 mutations in Chinese patients with congenital hypothyroidism. J Endocrinol invest 2012;35:889–92.Search in Google Scholar

29. Hermanns P, Grasberger H, Cohen R, Freiberg C, Dorr HG, et al. Two cases of thyroid dysgenesis caused by different novel PAX8 mutations in the DNA-binding region: in vitro studies reveal different pathogenic mechanisms. Thyroid 2013;23:791–6.10.1089/thy.2012.0141Search in Google Scholar PubMed PubMed Central

30. Carvalho A, Hermanns P, Rodrigues AL, Sousa I, Anselmo J, et al. A new PAX8 mutation causing congenital hypothyroidism in three generations of a family is associated with abnormalities in the urogenital tract. Thyroid 2013;23:1074–8.10.1089/thy.2012.0649Search in Google Scholar PubMed

31. Hermanns P, Shepherd S, Mansor M, Schulga J, Jones J, et al. A new mutation in the promoter region of the PAX8 gene causes true congenital hypothyroidism with thyroid hypoplasia in a girl with Down’s syndrome. Thyroid 2014;24:939–44.10.1089/thy.2013.0248Search in Google Scholar PubMed

32. Kumorowicz-Czoch M, Madetko-Talowska A, Dudek A, Tylek-Lemanska D. Genetic analysis of the paired box transcription factor (PAX8) gene in a cohort of Polish patients with primary congenital hypothyroidism and dysgenetic thyroid glands. J Pediatr Endocrinol Metab 2015;28:735–43.10.1515/jpem-2014-0310Search in Google Scholar

33. Kumorowicz-Czoch M, Madetko-Talowska A, Tylek-Lemanska D, Pietrzyk JJ, Starzyk J. Identification of deletions in children with congenital hypothyroidism and thyroid dysgenesis with the use of multiplex ligation-dependent probe amplification. J Pediatr Endocrinol Metab 2015;28:171–6.10.1515/jpem-2014-0040Search in Google Scholar

34. Ma D, Marion R, Punjabi NP, Pereira E, Samanich J, et al. A de novo 10.79 Mb interstitial deletion at 2q13q14.2 involving PAX8 causing hypothyroidism and mullerian agenesis: a novel case report and literature review. Mol Cytogenet 2014;7:85.10.1186/s13039-014-0085-4Search in Google Scholar

35. Ramos HE, Carre A, Chevrier L, Szinnai G, Tron E, et al. Extreme phenotypic variability of thyroid dysgenesis in six new cases of congenital hypothyroidism due to PAX8 gene loss-of-function mutations. Eur J Endocrinol 2014;171:499–507.10.1530/EJE-13-1006Search in Google Scholar

36. Vincenzi M, Camilot M, Ferrarini E, Teofoli F, Venturi G, et al. Identification of a novel pax8 gene sequence variant in four members of the same family: from congenital hypothyroidism with thyroid hypoplasia to mild subclinical hypothyroidism. BMC Endocr Disord 2014;14:69.10.1186/1472-6823-14-69Search in Google Scholar

37. Montanelli L, Tonacchera M. Genetics and phenomics of hypothyroidism and thyroid dys- and agenesis due to PAX8 and TTF1 mutations. Mol Cell Endocrinol 2010;322:64–71.10.1016/j.mce.2010.03.009Search in Google Scholar

38. Dent CL, Latchman DS. The DNA mobility shift assay. In: Latchman DS, editor. Transcription factors: a practical approach. Oxford: IRL Press, 1993:1–26.Search in Google Scholar

39. Mansouri A, Hallonet M, Gruss P. Pax genes and their roles in cell differentiation and development. Curr Opin Cell Biol 1996;8:851–7.10.1016/S0955-0674(96)80087-1Search in Google Scholar

40. Plachov D, Chowdhury K, Walther C, Simon D, Guenet JL, et al. Pax8, a murine paired box gene expressed in the developing excretory system and thyroid gland. Development 1990;110:643–51.10.1242/dev.110.2.643Search in Google Scholar PubMed

41. Zannini M, Francis-Lang H, Plachov D, Di Lauro R. Pax-8, a paired domain-containing protein, binds to a sequence overlapping the recognition site of a homeodomain and activates transcription from two thyroid-specific promoters. Mol Cell Biol 1992;12:4230–41.Search in Google Scholar

42. Tell G, Pellizzari L, Esposito G, Pucillo C, Macchia PE, et al. Structural defects of a Pax8 mutant that give rise to congenital hypothyroidism. Biochem J 1999;341(Pt 1):89–93.10.1042/bj3410089Search in Google Scholar

43. Calaciura F, Miscio G, Coco A, Leonardi D, Cisternino C, et al. Genetics of specific phenotypes of congenital hypothyroidism: a population-based approach. Thyroid 2002;12:945–51.10.1089/105072502320908277Search in Google Scholar PubMed

44. Kambe F, Nomura Y, Okamoto T, Seo H. Redox regulation of thyroid-transcription factors, Pax-8 and TTF-1, is involved in their increased DNA-binding activities by thyrotropin in rat thyroid FRTL-5 cells. Mol Endocrinol 1996;10:801–12.Search in Google Scholar

45. Amendola E, De Luca P, Macchia PE, Terracciano D, Rosica A, et al. A mouse model demonstrates a multigenic origin of congenital hypothyroidism. Endocrinology 2005;146:5038–47.10.1210/en.2005-0882Search in Google Scholar

46. Amendola E, Sanges R, Galvan A, Dathan N, Manenti G, et al. A locus on mouse chromosome 2 is involved in susceptibility to congenital hypothyroidism and contains an essential gene expressed in thyroid. Endocrinology 2010;151:1948–58.10.1210/en.2009-1240Search in Google Scholar

47. Nutt SL, Vambrie S, Steinlein P, Kozmik Z, Rolink A, et al. Independent regulation of the two Pax5 alleles during B-cell development. Nat Genet 1999;21:390–5.10.1038/7720Search in Google Scholar

48. Rhoades KL, Singh N, Simon I, Glidden B, Cedar H, et al. Allele-specific expression patterns of interleukin-2 and Pax-5 revealed by a sensitive single-cell RT-PCR analysis. Curr Biol 2000;10:789–92.10.1016/S0960-9822(00)00565-0Search in Google Scholar

Received: 2015-5-14

Accepted: 2015-8-27

Published Online: 2015-10-23

Published in Print: 2016-2-1

You are currently not able to access this content.

Articles in the same Issue

https://doi.org/10.1515/jpem-2015-0199

Keywords for this article

congenital hypothyroidism; mutation; PAX8 gene; promoter; thyroid dysgenesis; transcription factor