title: Identification and functional characterization of a novel mutation in the NKX2-1 gene: comparison with the data in the literature.
abstract: BACKGROUND
  NKX2-1 mutations have been described in several patients with primary congenital hypothyroidism, respiratory distress, and benign hereditary chorea, which are classical manifestations of the brain-thyroid-lung syndrome (BTLS).
  
  
  METHODS
  The NKX2-1 gene was sequenced in the members of a Brazilian family with clinical features of BTLS, and a novel monoallelic mutation was identified in the affected patients. We introduced the mutation in an expression vector for the functional characterization by transfection experiments using both thyroidal and lung-specific promoters.
  
  
  RESULTS
  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 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 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.
authors: Department of Molecular and Clinical Endocrinology and Oncology, University of Naples Federico II, Naples, Italy.
authors: Immacolata Cristina
authors: IC
authors: Nettore
authors: Paola
authors: P
authors: Mirra
authors: Alfonso Massimiliano
authors: AM
authors: Ferrara
authors: Annarita
authors: A
authors: Sibilio
authors: Valentina
authors: V
authors: Pagliara
authors: Claudia Suemi Kamoi
authors: CS
authors: Kay
authors: Paulo Josè
authors: PJ
authors: Lorenzoni
authors: Lineu Cesar
authors: LC
authors: Werneck
authors: Isac
authors: I
authors: Bruck
authors: Lucia Helena Coutinho
authors: LH
authors: Dos Santos
authors: Francesco
authors: F
authors: Beguinot
authors: Domenico
authors: D
authors: Salvatore
authors: Paola
authors: P
authors: Ungaro
authors: Gianfranco
authors: G
authors: Fenzi
authors: Rosana Herminia
authors: RH
authors: Scola
authors: Paolo Emidio
authors: PE
authors: Macchia
mesh_terms: D000293:Adolescent; D001264:Athetosis / Q000235:genetics* / Q000378:metabolism; D002819:Chorea / Q000235:genetics* / Q000378:metabolism; D018388:Codon, Terminator; D003409:Congenital Hypothyroidism / Q000235:genetics* / Q000378:metabolism; D005260:Female; D016368:Frameshift Mutation; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008297:Male; D009035:Mothers; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D009928:Organ Specificity; D011994:Recombinant Proteins / Q000378:metabolism; D012127:Respiratory Distress Syndrome, Newborn / Q000235:genetics* / Q000378:metabolism; D035781:Siblings; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D018388:Codon, Terminator; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors