ABSTRACT
Specific genetic variants in the mitochondrially encoded 12S ribosomal RNA gene (MT-RNR1) cause aminoglycoside-induced irreversible hearing loss. Mitochondrial DNA is usually not included in targeted sequencing experiments; however, off-target data may deliver this information. Here, we extract MT-RNR1 genetic variation, including the most relevant ototoxicity variant m.1555A>G, using the off-target reads of 473 research samples, sequenced through a capture-based, custom-targeted panel and whole exome sequencing (WES), and of 1245 diagnostic samples with clinical WES. Sanger sequencing and fluorescence-based genotyping were used for genotype validation. There was a correlation between off-target reads and mitochondrial coverage (rcustomPanel = 0.39, p = 2 × 10-13 and rWES = 0.67, p = 7 × 10-21). The median read depth of MT-RNR1 m.1555 was similar to the average mitochondrial genome coverage, with saliva and blood samples giving comparable results. The genotypes from 415 samples, including three m.1555G carriers, were concordant with fluorescence-based genotyping data. In clinical WES, median MT-RNR1 coverage was 56×, with 90% of samples having ≥20 reads at m.1555 position, and one m.1494T and three m.1555G carriers were identified with no evidence for heteroplasmy. Altogether, this study shows that obtaining MT-RNR1 genotypes through off-target reads is an efficient strategy that can impulse preemptive pharmacogenetic screening of this mitochondrial gene.
ABSTRACT
BACKGROUND: FoxE1 is a thyroid-specific forkhead transcription factor essential for thyroid gland development, as well as for the maintenance of the thyroid differentiated state in adults. FoxE1 recognizes and binds to a short DNA sequence present in thyroglobulin (Tg) and thyroperoxidase (Tpo) promoters, but FoxE1 binding to regulatory regions other than Tg and Tpo promoters remains almost unexplored. Improving knowledge of the regulatory functions of FoxE1 is necessary to clarify its role in endocrine syndromes and cancer susceptibility. METHODOLOGY/PRINCIPAL FINDING: In order to further investigate downstream FoxE1 targets, we performed a genome-wide expression screening after knocking-down FoxE1 and obtained new insights into FoxE1 transcriptional networks in thyroid follicular cells. After validation, we confirmed Adamts9, Cdh1, Duox2 and S100a4 as upregulated genes and Casp4, Creld2, Dusp5, Etv5, Hsp5a, Nr4a2 and Tm4sf1 as downregulated genes when FoxE1 was silenced. In promoter regions of putative FoxE1-regulated genes and also in the promoters of the classical thyroid genes Nis, Pax8 and Titf1, we performed an in silico search of the FoxE1 binding motif that was in close proximity to the NF1/CTF binding sequence, as previously described for other forkhead factors. Using chromatin immunoprecipitation we detected specific in vivo FoxE1 binding to novel regulatory regions in two relevant thyroid genes, Nis and Duox2. Moreover, we demonstrated simultaneous binding of FoxE1 and NF1/CTF to the Nis upstream enhancer region, as well as a clear functional activation of the Nis promoter by both transcription factors. CONCLUSIONS/SIGNIFICANCE: In search for potential downstream mediators of FoxE1 function in thyroid cells, we identified two novel direct FoxE1 target genes. To our knowledge, this is the first evidence regarding the implication of Nis and Duox2 in executing the transcriptional program triggered by FoxE1. Furthermore, this study points out the important role of FoxE1 in the regulation of a large number of genes in thyroid cells.
