Molecular analysis of congenital goitres with hypothyroidism caused by defective thyroglobulin synthesis. Identification of a novel c.7006C>T [p.R2317X] mutation and expression of minigenes containing nonsense mutations in exon 7.
BACKGROUND
Thyroglobulin (TG) deficiency is an autosomal-recessive disorder that results in thyroid dyshormonogenesis. A number of distinct mutations have been identified as causing human hypothyroid goitre.


OBJECTIVE
The purpose of this study was to identify and characterize new mutations in the TG gene in an attempt to increase the understanding of the genetic mechanism responsible for this disorder. A total of six patients from four nonconsanguineous families with marked impairment of TG synthesis were studied.


METHODS
Single-strand conformation polymorphism (SSCP) analysis, sequencing of DNA, genotyping, expression of chimeric minigenes and bioinformatic analysis were performed.


RESULTS
Four different inactivating TG mutations were identified: one novel mutation (c.7006C>T [p.R2317X]) and three previously reported (c.886C>T [p.R277X], c.6701C>A [p.A2215D] and c.6725G>A [p.R2223H]). Consequently, one patient carried a compound heterozygous for p.R2223H/p.R2317X mutations; two brothers showed a homozygous p.A2215D substitution and the remaining three patients, from two families with typical phenotype, had a single p.R277X mutated allele. We also showed functional evidences that premature stop codons inserted at different positions in exon 7, which disrupt exonic splicing enhancer (ESE) sequences, do not interfere with exon definition and processing.


CONCLUSIONS
In this study, we have identified a novel nonsense mutation p.R2317X in the acetylcholinesterase homology domain of TG. We have also observed that nonsense mutations do not interfere with the pre-mRNA splicing of exon 7. The results are in accordance with previous observations confirming the genetic heterogeneity of TG defects.
Laboratorio de Biología Molecular, Cátedra de Genética y Biología Molecular, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina.
Gloria A
GA
Machiavelli
Mariela
M
Caputo
Carina M
CM
Rivolta
María C
MC
Olcese
Laura
L
Gruñeiro-Papendieck
Ana
A
Chiesa
Rogelio
R
González-Sarmiento
Héctor M
HM
Targovnik
D002675:Child, Preschool; D018389:Codon, Nonsense / Q000235:genetics / Q000502:physiology; D003409:Congenital Hypothyroidism / Q000150:complications / Q000209:etiology / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D005091:Exons; D005260:Female; D015870:Gene Expression; D005820:Genetic Testing; D006042:Goiter / Q000150:complications / Q000151:congenital / Q000209:etiology / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D020641:Polymorphism, Single Nucleotide / Q000502:physiology; D018807:Polymorphism, Single-Stranded Conformational; D017434:Protein Structure, Tertiary / Q000235:genetics; D013954:Thyroglobulin / Q000096:biosynthesis / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics*; D014162:Transfection; D019076:Transgenes / Q000235:genetics
D018389:Codon, Nonsense; D013954:Thyroglobulin