Impact of ultrasonographic features, cytomorphology and mutational testing on malignant and indeterminate thyroid nodules on diagnostic accuracy of fine needle cytology samples: A prospective analysis of 141 patients.

OBJECTIVE: Fine needle cytology (FNC) is the first-line diagnostic method to determine the benign or malignant nature of thyroid nodules. The gray zone of cytological classifications remains, however, a crucial and challenging area for cytopathologists. DESIGN, PATIENTS AND MEASUREMENTS: In the present study, 141 thyroid cytological samples, with ultrasonographic suspicious features, have been prospectively analysed. Molecular analyses were performed by an innovative technology using two multiplex PCRs for the amplification of BRAF, N-H-K-RAS and RET exon genes. RNA samples were studied for RET/PTC1 and RET/PTC3 rearrangements by PCR amplification, and the conditions were set-up to study, with a single experiment, both wild-type PAX8 and PAX8/PPARÉ£ rearrangements. In total, 111 samples were examined for BRAF, N-H-KRAS and RET genes. An ultrasonographic, cytological and molecular correlation was also carried out in an attempt to suggest a possible way to manage the patients with thyroid nodules. Cyto-histological correlation was available in 115 cases, and it was used to verify the global diagnostic accuracy of this combined approach. RESULTS: According to the histopathological diagnosis, FNC accuracy was 100% for TIR5 and metastases; 89% for TIR4; 84% for TIR3A and 58% for TIR3B. About 11% of the studied samples showed either RET-PTC1 or RET/PTC3 chromosomal rearrangements, and only one sample simultaneously presented RET/PTC1 and RET/PTC3 rearrangements. PAX8/PPARÉ£ rearrangement was found in 6% of the samples. CONCLUSIONS: A multidisciplinary approach to the thyroid is therefore necessary to develop innovative methods suitable for an improved diagnostic and prognostic definition of thyroid cancer.

A functional connection between dyskerin and energy metabolism.

The human DKC1 gene encodes dyskerin, an evolutionarily conserved nuclear protein whose overexpression represents a common trait of many types of aggressive sporadic cancers. As a crucial component of the nuclear H/ACA snoRNP complexes, dyskerin is involved in a variety of essential processes, including telomere maintenance, splicing efficiency, ribosome biogenesis, snoRNAs stabilization and stress response. Although multiple minor dyskerin splicing isoforms have been identified, their functions remain to be defined. Considering that low-abundance splice variants could contribute to the wide functional repertoire attributed to dyskerin, possibly having more specialized tasks or playing significant roles in changing cell status, we investigated in more detail the biological roles of a truncated dyskerin isoform that lacks the C-terminal nuclear localization signal and shows a prevalent cytoplasmic localization. Here we show that this dyskerin variant can boost energy metabolism and improve respiration, ultimately conferring a ROS adaptive response and a growth advantage to cells. These results reveal an unexpected involvement of DKC1 in energy metabolism, highlighting a previously underscored role in the regulation of metabolic cell homeostasis.

A new role for human dyskerin in vesicular trafficking.

Dyskerin is an essential, conserved, multifunctional protein found in the nucleolus, whose loss of function causes the rare genetic diseases X-linked dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome. To further investigate the wide range of dyskerin's biological roles, we set up stable cell lines able to trigger inducible protein knockdown and allow a detailed analysis of the cascade of events occurring within a short time frame. We report that dyskerin depletion quickly induces cytoskeleton remodeling and significant alterations in endocytic Ras-related protein Rab-5A/Rab11 trafficking. These effects arise in different cell lines well before the onset of telomere shortening, which is widely considered the main cause of dyskerin-related diseases. Given that vesicular trafficking affects many homeostatic and differentiative processes, these findings add novel insights into the molecular mechanisms underlining the pleiotropic manifestation of the dyskerin loss-of-function phenotype.

Intron retention: a human DKC1 gene common splicing event.

Identification of alternatively spliced transcripts produced by a gene is a crucial step in deciphering the bulk of its biological roles and the overall processes that regulate its activity. By using a combination of bioinformatic and molecular approaches we identified, cloned, and characterized 3 novel alternative splice isoforms derived from human dyskeratosis congenita 1 (hDKC1), an essential human gene causative of the X-linked dyskeratosis congenita disease and involved in multiple functions related to cell growth, proliferation, and telomere maintenance. Expression of the new isoforms, all characterized by intron retention, was confirmed by RT-PCR in a panel of diverse cell lines and normal human tissues, and despite the presence of premature termination codons, was not down-regulated by the mechanism of nonsense-mediated decay. Accumulation of these transcripts fluctuated distinctly in the diverse tissues and during in vitro differentiation of Caco2 cells, suggesting that their ratio may contribute to the gene functional diversity across different cell types. Intriguingly, the structure of one isoform leads to exonize an intronically encoded small nucleolar RNA (snoRNA), highlighting an additional layer of complexity that can contribute to overall gene regulation.