title: Intronic variants of SLC26A4 gene enhance splicing efficiency in hybrid minigene assay.
abstract: The SLC26A4 genomic sequence screening in autoimmune thyroid diseases (AITD) revealed different variants types with possible pathogenic effects. Although intronic variants may have more detrimental effects than those coding, they are poorly explored. Thus, in a first assessment, our bioinformatics analysis of intronic variants predicted a pathogenic effect of c.1002-9A>C, c.1545-5T>G and c.1544+9C>T variants. Validating these variants pathogenicity may provide new clues on the AITD physiopathology. Variants were explored in a general population by PCR-RFLP. These variants effects on the mRNA processing was assessed using functional splicing assay based in DNA hybrid minigene in HeLa cell lines. The constructs splicing efficiency was investigated by real time PCR. Our results revealed that c.1002-9A>C is a rare allele (minor frequency allele (MFA)=0.007) whereas c.1545-5T>G and c.1544+9C>T are low frequency variants. The RT-PCR analysis showed that these variants did not affect the mRNA processing. However, quantifying the transcripts generated from minigene constructs proved an mRNA splicing enhancement. Our study suggests a pathogenic effect of three intronic variants on the mRNA splicing efficiency using a DNA Hybrid minigene. By quantifying these transcripts, we unveil the limit of standard RT-PCR in analyzing a splicing minigene assay.
authors.affiliation: Laboratoire Procédés de Criblage Moléculaire et Cellulaire, Centre Biotechnologie de Sfax, Tunisia.
authors.forename: Rihab
authors.initials: R
authors.lastname: Kallel-Bouattour
authors.affiliation: Laboratoire Procédés de Criblage Moléculaire et Cellulaire, Centre Biotechnologie de Sfax, Tunisia.
authors.forename: Salima
authors.initials: S
authors.lastname: Belguith-Maalej
authors.affiliation: Laboratoire Procédés de Criblage Moléculaire et Cellulaire, Centre Biotechnologie de Sfax, Tunisia.
authors.forename: Emna
authors.initials: E
authors.lastname: Zouari-Bradai
authors.affiliation: Service d'Endocrinologie, CHU, Hédi Chaker, Sfax, Tunisia.
authors.forename: Mouna
authors.initials: M
authors.lastname: Mnif
authors.affiliation: Service d'Endocrinologie, CHU, Hédi Chaker, Sfax, Tunisia.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Abid
authors.affiliation: Laboratoire Procédés de Criblage Moléculaire et Cellulaire, Centre Biotechnologie de Sfax, Tunisia; Department of Applied Biology, College of Sciences, University of Sharjah, United Arab Emirates. Electronic address: hassen.hadjkacem@cbs.rnrt.tn.
authors.forename: Hassen
authors.initials: H
authors.lastname: Hadj Kacem
mesh_terms: D000328:Adult; D000483:Alleles; D005260:Female; D006111:Graves Disease / Q000235:genetics* / Q000473:pathology; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics*; D011110:Polymorphism, Genetic; D012326:RNA Splicing; D000076988:Sulfate Transporters
chemical_list: D026901:Membrane Transport Proteins; C085609:SLC26A4 protein, human; D000076988:Sulfate Transporters
keywords: AITD; PS; SLC26A4; Splicing minigene assay