Genetic analysis of the PAX8 gene in children with congenital hypothyroidism and dysgenetic or eutopic thyroid glands: identification of a novel sequence variant.
OBJECTIVE
To analyse the coding region of PAX8 in individuals with congenital (CH) or post neonatal hypothyroidism due to dysgenetic (TD) or eutopic thyroid glands.


METHODS
Forty-three children with CH and TD (13 agenesis, 23 ectopia, and seven hypoplasia), one subject with post neonatal onset of hypothyroidism and thyroid ectopia, 15 children with CH and eutopic thyroid glands and six euthyroid adults with thyroid hemiagenesis were enrolled as cases, along with 120 healthy individuals as controls.


METHODS
Exons 2-8 of the PAX8 were directly sequenced. HeLa and HEK293 cells were transfected with PAX8 wild-type (PAX8-WT), mutant PAX8, p300, thyroid transcription factor 1 (TTF-1) and thyroglobulin promoter pGL3 (TG prom-pGL3). Synergism of TTF-1 with PAX8-WT vs. mutant and activity of PAX8-WT vs. mutant in accompaniment with p300 on TG prom-pGL3 were also assessed. The luminescence produced by PAX8-WT and mutant PAX8 was measured.


RESULTS
Among patients and controls only a 15-year-old girl with thyroid ectopia showed a heterozygous transition of cytosine to thymine at position 674 in exon 6, which changed a conserved threonine at position 225 to methionine (PAX8-T225M). Her father and sister harboured PAX8-T225M without abnormal thyroid phenotypes. PAX8-T225M and PAX8-WT similarly increased luciferase activity and had a similar synergistic effect with TTF-1. At 500 ng p300, however, PAX8-T225M could not significantly increase TG promoter activity when compared to PAX8-T225M alone, while PAX8-WT +500 ng p300 induction was significantly higher than PAX8-WT alone (P < 0.001). Cotransfection of TTF-1 together with PAX8-T225M resulted in rescuing of the lack of synergism with p300.


CONCLUSIONS
PAX8 mutations in congenital hypothyroidism due to dysgenetic or orthotopic thyroid glands are rare. PAX8-T225M is probably a rare variant.
Tonacchera
Massimo
M
Dipartimento di Endocrinologia e Metabolismo, Centro di Eccellenza AmbiSEN, Università di Pisa, Pisa, Italy. mtonacchera@hotmail.com
Banco
Maria Elena
ME
Montanelli
Lucia
L
Di Cosmo
Caterina
C
Agretti
Patrizia
P
De Marco
Giuseppina
G
Ferrarini
Eleonora
E
Ordookhani
Arash
A
Perri
Anna
A
Chiovato
Luca
L
Santini
Ferruccio
F
Vitti
Paolo
P
Pinchera
Aldo
A
D000328:Adult; D002460:Cell Line; D002648:Child; D003409:Congenital Hypothyroidism / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007037:Hypothyroidism / Q000235:genetics / Q000378:metabolism; D007231:Infant, Newborn; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D016297:Mutagenesis, Site-Directed; D000072636:PAX8 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013954:Thyroglobulin / Q000235:genetics; D013960:Thyroid Function Tests; D013961:Thyroid Gland / Q000002:abnormalities* / Q000378:metabolism; D014157:Transcription Factors; D014162:Transfection / Q000379:methods
D004268:DNA-Binding Proteins; D000072636:PAX8 Transcription Factor; C083325:PAX8 protein, human; D051761:Paired Box Transcription Factors; C504003:TTF1 protein, human; D014157:Transcription Factors; D013954:Thyroglobulin; D008156:Luciferases