Diagnostic utility of DREAM gene mRNA levels in thyroid tumours

ABSTRACT Objective The transcriptional repressor DREAM is involved in thyroid-specific gene expression, thyroid enlargement and nodular development, but its clinical utility is still uncertain. In this study we aimed to investigate whether DREAM mRNA levels differ in different thyroid tumors and how this possible difference would allow the use of DREAM gene expression as molecular marker for diagnostic and/or prognosis purpose. Materials and methods We quantified DREAM gene mRNA levels and investigated its mutational status, relating its expression and genetic changes to diagnostic and prognostic features of 200 thyroid tumors, being 101 malignant [99 papillary thyroid carcinomas (PTC) and 2 anaplastic thyroid carcinomas] and 99 benign thyroid lesions [49 goiter and 50 follicular adenomas (FA)]. Results Levels of mRNA of DREAM gene were higher in benign (0.7909 ± 0.6274 AU) than in malignant (0.3373 ± 0.6274 AU) thyroid lesions (p < 0.0001). DREAM gene expression was able to identify malignancy with 66.7% sensitivity, 85.4% specificity, 84.2% positive predictive value (PPV), 68.7% negative predictive value (NPV), and 75.3% accuracy. DREAM mRNA levels were also useful distinguishing the follicular lesions FA and FVPTC with 70.2% sensitivity, 73.5% specificity, 78.5% PPV, 64.1% NPV, and 71.6% accuracy. However, DREAM gene expression was neither associated with clinical features of tumor aggressiveness, nor with recurrence or survival. Six different genetic changes in non-coding regions of DREAM gene were also found, not related to DREAM gene expression or tumor features. Conclusion We suggest that DREAM gene expression may help diagnose thyroid nodules, identifying malignancy and characterizing follicular-patterned thyroid lesions; however, it is not useful as a prognostic marker.

Influence of the polymorphisms of the alpha-major regulatory element HS-40 on in vitro gene expression

The α-MRE is the major regulatory element responsible for the expression of human α-like globin genes. It is genetically polymorphic, and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. Most of the mutations that constitute the α-MRE haplotypes are located in flanking sequences of binding sites for nuclear factors. To our knowledge, there are no experimental studies evaluating whether such variability may influence the α-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different α-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type α-MRE) and B haplotypes were used to generate C-F haplotypes by site-directed mutagenesis. In addition, three other elements containing only the G→A polymorphism at positions +130, +199, and +209, separately, were also tested. The different α-MRE elements were amplified and cloned into a plasmid containing the luciferase reporter gene and the SV40 promoter and used to transiently transfect K562 cells. A noticeable reduction in luciferase expression was observed with all constructs compared with the A haplotype. The greatest reductions occurred with the F haplotype (+96, C→A) and the isolated polymorphism +209, both located near the SP1 protein-binding sites believed not to be active in vivo. These are the first analyses of α-MRE polymorphisms on gene expression and demonstrate that these single nucleotide polymorphisms, although outside the binding sites for nuclear factors, are able to influence in vitro gene expression.

Parsing regulatory DNA: general tasks, techniques, and the PhyloGibbs approach.

In this review, we discuss the general problem of understanding transcriptional regulation from DNA sequence and prior information. The main tasks we discuss are predicting local regions of DNA, cis-regulatory modules (CRMs) that contain binding sites for transcription factors (TFs), and predicting individual binding sites. We review various existing methods, and then describe the approach taken by PhyloGibbs, a recent motif-finding algorithm that we developed to predict TF binding sites, and PhyloGibbs-MP, an extension to PhyloGibbs that tackles other tasks in regulatory genomics, particularly prediction of CRMs.

Cloning and sequence analysis of the Antheraea pernyi nucleopolyhedrovirus gp64 gene.

Frequent outbreaks of the purulence disease of Chinese oak silkworm are reported in Middle and Northeast China. The disease is produced by the pathogen Antheraea pernyi nucleopolyhedrovirus (AnpeNPV). To obtain molecular information of the virus, the polyhedra of AnpeNPV were purified and characterized. The genomic DNA of AnpeNPV was extracted and digested with HindIII. The genome size of AnpeNPV is estimated at 128 kb. Based on the analysis of DNA fragments digested with HindIII, 23 fragments were bigger than 564 bp. A genomic library was generated using HindIII and the positive clones were sequenced and analysed. The gp64 gene, encoding the baculovirus envelope protein GP64, was found in an insert. The nucleotide sequence analysis indicated that the AnpeNPV gp64 gene consists of a 1,530 nucleotide open reading frame (ORF), encoding a protein of 509 amino acids. Of the eight gp64 homologues, the AnpeNPV gp64 ORF shared the most sequence similarity with the gp64 gene of Anticarsia gemmatalis NPV, but not Bombyx mori NPV. The upstream region of the AnpeNPV gp64 ORF encoded the conserved transcriptional elements for early and late stage of the viral infection cycle. These results indicated that AnpeNPV belongs to group I NPV and was far removed in molecular phylogeny from the BmNPV.