Hassen Hadj Kacem

Association of intronic repetition of SLC26A4 gene with Hashimoto thyroiditis disease.

Intronic microsatellites repeats were implicated in the pathogenic mechanisms of several diseases. SLC26A4 gene, involved in the genetic susceptibility of autoimmune thyroid disease (AITD), harbours large non-coding introns. Using the tandem repeat finder (TRF) Software, two new polymorphic microsatellite markers, rs59736472 and rs57250751, located at introns 10 and 20, respectively, were identified. A case-control design including 308 patients affected with AITD (146 GD, 90 HT and 72 PIM) and 212 unmatched healthy controls were performed for each marker (rs59736472, D7S2459 and rs57250751). Furthermore, we used PHASE 2.0 version to reconstruct haplotypes, Kolmogorov-Smirnov (KS) and the Clump analysis program for multivariate analysis. The fluorescent genotyping revealed three alleles (106,112 and 115 bp) for rs57250751 and 12 alleles for both D7S2459 and rs59736472 ranging from 134 to 156 bp and from 144 to 168 bp, respectively. The case-control analysis confirmed the positive association of D7S2459 with Hashimoto thyroiditis (HT) disease previously reported. Moreover, a significant association was found only with rs59736472 and HT disease. Haplotype-specific analysis showed that the 140-148-115 haplotype may increase the risk of HT (χ2=9.8, 1 df, P=0.0017, OR=2.07, IC [1.27-3.36]). Consequently, considering the number of repetitions of both D7S2459 and rs59736472, we found 15 alleles ranging from 45 to 59 repetitions. The case-control analysis showed a significant association of the 55 repetition with HT disease (χ2=6.32, 1 df, p c=0.012, OR=1.74, IC [1.1-2.76]). In conclusion, we suggest the association of longer alleles of intron 10 of SLC26A4 gene with HT disease.

Intronic variants of SLC26A4 gene enhance splicing efficiency in hybrid minigene assay

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.

Evaluation of alternative DNA extraction protocols for the species determination in turkey salami authentication tests

ABSTRACT Fighting food industry standard violations has become an important stake especially in developing countries. In this work, different common DNA extraction protocols, namely NaOH, Phenol Chloroform Isoamyl alcohol, CTAB, Chloroform and the commercialized DNeasy blood and tissue kit were tested and compared for turkey salami genomic authentication in mitochondrial genes species detection by Polymerase Chain Reaction (PCR). The obtained results proved that a protocol based on NaOH after an advanced grinding step was the most adequate alternative in terms of simplicity, rapidity and detection threshold even in swabs. The selected protocol encourages wide range cost-effective routine authentications.