Nettore, Immacolata Cristina (2014) Thyroid Dysgenesis: search for mutations and functional characterizations of known and new candidate genes. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Resource language: English
Title: Thyroid Dysgenesis: search for mutations and functional characterizations of known and new candidate genes
Nettore, Immacolata
Date: 31 March 2014
Number of Pages: 114
Institution: Università degli Studi di Napoli Federico II
Department: Medicina Molecolare e Biotecnologie Mediche
Scuola di dottorato: Medicina molecolare
Dottorato: Oncologia ed endocrinologia molecolare
Ciclo di dottorato: 26
Coordinatore del Corso di dottorato:
Macchia, Paolo EmidioUNSPECIFIED
Date: 31 March 2014
Number of Pages: 114
Keywords: Thyroid dysgenesis; transcription factors
Settori scientifico-disciplinari del MIUR: Area 06 - Scienze mediche > MED/49 - Scienze tecniche dietetiche applicate
Date Deposited: 11 Apr 2014 10:23
Last Modified: 15 Jul 2015 01:02

Collection description

Background: Alterations in thyroid organogenesis occurs very frequently in humans, leading to several conditions responsible for primary congenital hypothyroidism. Such alterations, classified as “thyroid dysgenesis” (TD) include athyresis, ectopy of the gland, and thyroid hypoplasia. The molecular mechanism leading to TD are largely unknown, but, several studies linked thyroid dysgenesis to mutations in genes expressed in the developing thyroid, such as NKX2-1, NKX2-5, PAX8, FOXE1, rTSH. NKX2-1 mutations have been described in several patients with a syndromic form of primary congenital hypothyroidism associated to respiratory distress, and benign hereditary chorea (Brain–Thyroid–Lung Syndrome, BTLS). Methods: we screened 76 patients with persistent CH for mutations in known (NKX2-1, NKX2-5, PAX8) and new candidate (DNAJC17) genes by SSCP, or HRM. In addition, the NKX2-1 gene was sequenced in the members of a Brazilian family with clinical features of BTLS. Results: We found no mutations in the PAX8 or NKX2-5 genes in our cohort. The only variation observed was an already reported polymorphism (rs199939219) within the PAX8 gene that causes a change of the first nucleotide of the triplet encoding for serine at position 337 (Ser337Ala). This change, functionally tested by in vitro studies, has no effect on the PAX8 protein activity. We also looked for mutations in the DNAJC17 gene, a new candidate gene that has been demonstrated to act as phenotype modulator in mouse double heterozygous for deletion in Nkx2-1 and Pax8. In DNAJC17 we found a known polymorphism c.350A>C. Finally, we were able to identify a novel NKX2-1 mutation in the Brazilian family with the clinical findings of the BTLS. The mutation is a deletion of a cytosine at position 834 (ref. sequence NM_003317) (c.493delC) that causes a frameshift with formation of an abnormal protein from amino acid 165 and a premature stop at position 196. The last amino acid of the nuclear localization signal, the whole homeodomain, and the carboxy terminus of NKX2-1 are all missing in the mutant protein, which has a premature stop codon at position 196 (p.Arg165Glyfs*32). The p.Arg165Glyfs*32 mutant does not bind DNA, and it is unable to transactivate the thyroglobulin (Tg) and the surfactant protein-C (SP-C) promoters. Interestingly, a dose-dependent dominant negative effect of the p.Arg165Glyfs*32 was demonstrated only on the Tg promoter, but not on the SP-C promoter. This effect was also noticed when the mutation was tested in presence of PAX8 or cofactors that synergize with NKX2-1 (P300 and TAZ). The functional effect was also compared with the data present in the literature and demonstrated that, so far, it is very difficult to establish a specific correlation among NKX2-1, PAX8 and NKX2-5 mutations, their functional consequence, and the clinical phenotype of affected patients, thus suggesting that the detailed mechanisms of transcriptional regulation still remain unclear. Conclusions: We describe a novel NKX2-1 mutation in a Brazilian family and demonstrate that haploinsufficiency may not be the only explanation for BTLS. Our results indicate that NKX2-1 activity is also finely regulated in a tissue-specific manner, and additional studies are required to better understand the complexities of genotype–phenotype correlations in the NKX2-1 deficiency syndrome. No mutations have been identified in sporadic cases of TD in NKX2-5, PAX8 or DNAJC17 genes, suggesting that sporadic cases of TD are likely to be caused by epigenetic factors, rather than mutations in thyroid transcription factors or genes involved in thyroid development.


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