title: Novel compound heterozygous Thyroglobulin mutations c.745+1G>A/c.7036+2T>A associated with congenital goiter and hypothyroidism in a Vietnamese family. Identification of a new cryptic 5' splice site in the exon 6.
abstract: Several patients were identified with dyshormonogenesis caused by mutations in the thyroglobulin (TG) gene. These defects are inherited in an autosomal recessive manner and affected individuals are either homozygous or compound heterozygous for the mutations. The aim of the present study was to identify new TG mutations in a patient of Vietnamese origin affected by congenital hypothyroidism, goiter and low levels of serum TG. DNA sequencing identified the presence of compound heterozygous mutations in the TG gene: the maternal mutation consists of a novel c.745+1G>A (g.IVS6 + 1G>A), whereas the hypothetical paternal mutation consists of a novel c.7036+2T>A (g.IVS40 + 2T>A). The father was not available for segregation analysis. Ex-vivo splicing assays and subsequent RT-PCR analyses were performed on mRNA isolated from the eukaryotic-cells transfected with normal and mutant expression vectors. Minigene analysis of the c.745+1G>A mutant showed that the exon 6 is skipped during pre-mRNA splicing or partially included by use of a cryptic 5' splice site located to 55 nucleotides upstream of the authentic exon 6/intron 6 junction site. The functional analysis of c.7036+2T>A mutation showed a complete skipping of exon 40. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses predicted that both mutant alleles would result in the abolition of the authentic splice donor sites. The c.745+1G>A mutation originates two putative truncated proteins of 200 and 1142 amino acids, whereas c.7036+2T>A mutation results in a putative truncated protein of 2277 amino acids. In conclusion, we show that the c.745+1G>A mutation promotes the activation of a new cryptic donor splice site in the exon 6 of the TG gene. The functional consequences of these mutations could be structural changes in the protein molecule that alter the biosynthesis of thyroid hormones.
authors: Laboratorio de Genética y Biología Molecular, Instituto de Inmunología, Genética y Metabolismo (INIGEM, CONICET-UBA), Hospital de Clínicas "José de San Martín", C1120AAR Buenos Aires, Argentina; Cátedra de Genética y Biología Molecular (FFyB-UBA), C1113AAD Buenos Aires, Argentina.
authors: Cintia E
authors: CE
authors: Citterio
authors: Laboratorio de Genética y Biología Molecular, Instituto de Inmunología, Genética y Metabolismo (INIGEM, CONICET-UBA), Hospital de Clínicas "José de San Martín", C1120AAR Buenos Aires, Argentina; Cátedra de Genética y Biología Molecular (FFyB-UBA), C1113AAD Buenos Aires, Argentina.
authors: Cecilia M
authors: CM
authors: Morales
authors: Unité Endocrinologie Diabétologie Pédiatrique and Centre des Maladies Rares de la Réceptivité Hormonale, CHU-Angers, 49933 Angers CEDEX 9, France.
authors: Natacha
authors: N
authors: Bouhours-Nouet
authors: Laboratorio de Genética y Biología Molecular, Instituto de Inmunología, Genética y Metabolismo (INIGEM, CONICET-UBA), Hospital de Clínicas "José de San Martín", C1120AAR Buenos Aires, Argentina; Cátedra de Genética y Biología Molecular (FFyB-UBA), C1113AAD Buenos Aires, Argentina.
authors: Gloria A
authors: GA
authors: Machiavelli
authors: Unidad de Medicina Molecular, Departamento de Medicina, Facultad de Medicina, Universidad de Salamanca, 37007 Salamanca, España.
authors: Elena
authors: E
authors: Bueno
authors: Unité Endocrinologie Diabétologie Pédiatrique and Centre des Maladies Rares de la Réceptivité Hormonale, CHU-Angers, 49933 Angers CEDEX 9, France.
authors: Frédérique
authors: F
authors: Gatelais
authors: Unité Endocrinologie Diabétologie Pédiatrique and Centre des Maladies Rares de la Réceptivité Hormonale, CHU-Angers, 49933 Angers CEDEX 9, France.
authors: Regis
authors: R
authors: Coutant
authors: Unidad de Medicina Molecular, Departamento de Medicina, Facultad de Medicina, Universidad de Salamanca, 37007 Salamanca, España.
authors: Rogelio
authors: R
authors: González-Sarmiento
authors: Laboratorio de Genética y Biología Molecular, Instituto de Inmunología, Genética y Metabolismo (INIGEM, CONICET-UBA), Hospital de Clínicas "José de San Martín", C1120AAR Buenos Aires, Argentina; Cátedra de Genética y Biología Molecular (FFyB-UBA), C1113AAD Buenos Aires, Argentina; Unidad de Medicina Molecular, Departamento de Medicina, Facultad de Medicina, Universidad de Salamanca, 37007 Salamanca, España.
authors: Carina M
authors: CM
authors: Rivolta
authors: Laboratorio de Genética y Biología Molecular, Instituto de Inmunología, Genética y Metabolismo (INIGEM, CONICET-UBA), Hospital de Clínicas "José de San Martín", C1120AAR Buenos Aires, Argentina; Cátedra de Genética y Biología Molecular (FFyB-UBA), C1113AAD Buenos Aires, Argentina; Unidad de Medicina Molecular, Departamento de Medicina, Facultad de Medicina, Universidad de Salamanca, 37007 Salamanca, España. Electronic address: htargovnik@conicet.gov.ar.
authors: Héctor M
authors: HM
authors: Targovnik
mesh_terms: D000293:Adolescent; D000818:Animals; D044466:Asian People / Q000235:genetics*; D019556:COS Cells; D002522:Chlorocebus aethiops; D003409:Congenital Hypothyroidism / Q000235:genetics* / Q000473:pathology; D005091:Exons; D005260:Female; D006042:Goiter / Q000151:congenital* / Q000235:genetics* / Q000473:pathology; D006367:HeLa Cells; D006579:Heterozygote; D006801:Humans; D008297:Male; D010375:Pedigree; D020641:Polymorphism, Single Nucleotide; D022821:RNA Splice Sites; D017422:Sequence Analysis, DNA; D013954:Thyroglobulin / Q000097:blood / Q000235:genetics*; D014744:Vietnam
chemical_list: D022821:RNA Splice Sites; D013954:Thyroglobulin
keywords: Compound heterozygous mutations; Congenital hypothyroidism; Cryptic splice site; Mutation; Thyroglobulin gene