ICEECE2012 Oral Communications Paediatric Endocrinology (6 abstracts)
The Young Investigator Winner
I. Nettore 1 , P. Mirra 3 , A. Ferrara 1 , A. Sibilio 1 , C. Kamoi Kay 2 , P. Lorenzoni 2 , L. Werneck 2 , I. Bruck 2 , F. Bequinot 3 , P. Ungaro 3 , G. Fenzi 1 , R. Scola 2 & P. Macchia 1
1University of Naples ‘Federico II’, Naples, Italy; 2Clinical Hospital, Parana Federal University, Curitiba, Brazil; 3University ‘Federico II’, Naples, Italy.
TTF1/NKX2.1 is a transcription factor expressed in thyroid, lung and brain. Several heterozygous mutations have been described in patients with primary congenital hypothyroidism, respiratory distress and benign hereditary chorea, typical aspects of the ‘thyroid–lung–brain syndrome’.
We recently studied a family affected by some of these features, and the direct sequencing of the NKX2.1 gene demonstrated an heterozygous deletion of a cytosine at position 665 that causes a frameshift with the formation of a truncated protein.
Aim of our study was to better characterize the molecular effects determined by the C665del mutation.
The transcriptional properties of both WT and C665del NKX2.1 were investigated by cotransfection assays. As expected, the C665del mutant is unable to transactivate the Tg, the artificial C5 and the SP-C promoters. Interestingly, we demonstrated a dose-dependent dominant negative effect of the C665del on the WT NKX2.1 on Tg or C5 promoters. This dominant negative effect is absent on the SP-C promoter. To further understand the dominant-negative action, we performed cotransfection experiments with PAX8, observing that the mutant is unable to modify the PAX8 basal activity. Moreover a dominant negative effect is also present when both WT and mutant are cotransfected with PAX8. Same results can be observed in presence of known NKX2-1 coactivators such as P300 or WWTR1.
Then, we evaluated the binding of NKX2.1 to DNA in the presence of NKX2.1 C665del by electrophoretic mobility shift assay (EMSA). This binding remains unaltered but the C665del, alone, is unable to bind to DNA. These data suggest that the mutated NKX2.1 protein produces dominant-negative effects on the wt NKX2.1 in promoter-specific manner and it interferes with the activity of wt, but not with its binding to DNA.
In conclusion, these experiments allow to understand the functional relevance of the different domains in the NKX2.1 protein and show the high variability between genotype-fenotype in patients with TLB syndrome. On the basis of our data it seems reasonable to consider that the pathogenetic mechanisms underlying the effect of the mutations are very complex, and tissue-specific.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Volume 29
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Endocrine Abstracts
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