Compound heterozygous DUOX2 gene mutations (c.2335-1G>C/c.3264_3267delCAGC) associated with congenital hypothyroidism. Characterization of complex cryptic splice sites by minigene analysis.
Iodide Organification defects (IOD) represent 10% of cases of congenital hypothyroidism (CH) being the main genes affected that of TPO (thyroid peroxidase) and DUOX2 (dual oxidasa 2). From a patient with clinical and biochemical criteria suggestive with CH associated with IOD, TPO and DUOX2 genes were analyzed by means of PCR-Single Strand Conformation Polymorphism analysis and sequencing. A novel heterozygous compound to the mutations c.2335-1G>C (paternal mutation, intron 17) and c.3264_3267delCAGC (maternal mutation, exon 24) was identified in the DUOX2 gene. Ex-vivo splicing assays and subsequent RT-PCR and sequencing analyses were performed on mRNA isolated from the HeLa cells transfected with wild-type and mutant pSPL3 expression vectors. The wild-type and c.2335-1G>C mutant alleles result in the complete inclusion or exclusion of exon 18, or in the activation of an exonic cryptic 5' ss with the consequent deletion of 169 bp at the end of this exon. However, we observed only a band of the expected size in normal thyroid tissue by RT-PCR. Additionally, the c.2335-1G>C mutation activates an unusual cryptic donor splice site in intron 17, located at position -14 of the authentic intron 17/exon 18 junction site, with an insertion of the last 14 nucleotides of the intron 17 in mutant transcripts with complete and partial inclusion of exon 18. The theoretical consequences of splice site mutation, predicted with the bioinformatics NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses confirm that c.2335-1G>C mutant allele would result in the abolition of the authentic splice acceptor site. The results suggest the coexistence in our patient of four putative truncated proteins of 786, 805, 806 and 1105 amino acids, with conservation of peroxidase-like domain and loss of gp91(phox)/NOX2-like domain. In conclusion a novel heterozygous compound was identified being responsible of IOD. Cryptic splicing sites have been characterized in DUOX2 gene for the first time. The use of molecular biology techniques is a valuable tool for understanding the molecular pathophysiology of this type of thyroid defects.
Laboratorio de Genética Molecular Tiroidea, Instituto de Inmunología, Genética y Metabolismo (INIGEM, CONICET-UBA), Hospital de Clínicas "José de San Martín", C1120AAR Buenos Aires, Argentina; Cátedra de Genética (FFyB-UBA), C1113AAD Buenos Aires, Argentina.
Fiorella S
FS
Belforte
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 (FFyB-UBA), C1113AAD Buenos Aires, Argentina.
Cintia E
CE
Citterio
Servicio de Endocrinología, Hospital de Niños Santísima Trinidad, 5000 Córdoba, Argentina.
Graciela
G
Testa
Laboratorio de Genética Molecular Tiroidea, Instituto de Inmunología, Genética y Metabolismo (INIGEM, CONICET-UBA), Hospital de Clínicas "José de San Martín", C1120AAR Buenos Aires, Argentina; Cátedra de Genética (FFyB-UBA), C1113AAD Buenos Aires, Argentina.
María Cecilia
MC
Olcese
Servicio de Endocrinología, Hospital de Niños Santísima Trinidad, 5000 Córdoba, Argentina.
Gabriela
G
Sobrero
Servicio de Endocrinología, Hospital de Niños Santísima Trinidad, 5000 Córdoba, Argentina.
Mirta B
MB
Miras
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 (FFyB-UBA), C1113AAD Buenos Aires, Argentina.
Héctor M
HM
Targovnik
Laboratorio de Genética Molecular Tiroidea, Instituto de Inmunología, Genética y Metabolismo (INIGEM, CONICET-UBA), Hospital de Clínicas "José de San Martín", C1120AAR Buenos Aires, Argentina; Cátedra de Genética (FFyB-UBA), C1113AAD Buenos Aires, Argentina. Electronic address: cmrivolta@conicet.gov.ar.
Carina M
CM
Rivolta
D002648:Child; D003409:Congenital Hypothyroidism / Q000235:genetics* / Q000378:metabolism; D000074623:Dual Oxidases; D006367:HeLa Cells; D006579:Heterozygote; D006801:Humans; D008297:Male; D009154:Mutation; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D010375:Pedigree; D018807:Polymorphism, Single-Stranded Conformational; D022821:RNA Splice Sites; D017422:Sequence Analysis, DNA; D013961:Thyroid Gland / Q000378:metabolism
D022821:RNA Splice Sites; D000074623:Dual Oxidases; D019255:NADPH Oxidases; C431515:DUOX2 protein, human
Compound heterozygous mutations; Congenital hypothyroidism; Cryptic splice site; DUOX2 gene; Mutation