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

FIORELLA S. BELFORTE; CINTIA E. CITTERIO; GRACIELA TESTA; MARÍA C. OLCESE; GABRIELA SOBRERO; MIRTA B. MIRAS; HÉCTOR M. TARGOVNIK; CARINA M. RIVOLTA

MOLECULAR AND CELLULAR ENDOCRINOLOGY.

ELSEVIER IRELAND LTD

Lugar: Amsterdam; Año: 2016 vol. 419 p. 172 - 184

0303-7207

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) andc.3264_3267delCAGC (maternal mutation, exon 24) was identified in theDUOX2 gene. Ex-vivo splicing assays and subsequent RT-PCR and sequencinganalyses were performed on mRNA isolated from the HeLa cells transfected with wild-type and mutant pSPL3 expression vectors. The wild-type andc.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 RTPCR. 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, SPLMand MaxEntScan programs were investigated and evaluated in relation withthe experimental evidence. These analyses confirm that c.2335-1G>C mutantallele 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 gp91phox/NOX2-likedomain. In conclusion a novel heterozygous compound was identified being responsible of IOD. Cryptic splicing sites have been characterized inDUOX2 gene for the first time. The use of molecular biology techniques isa valuable tool for understanding the molecular pathophysiology of this type of thyroid defects.