5 research outputs found
Telomerase Reverse Transcriptase Promoter Mutations in Bladder Cancer: High Frequency Across Stages, Detection in Urine, and Lack of Association with Outcome
Background: Hotspot mutations in the promoter of the gene coding for
telomerase reverse transcriptase (TERT) have been described and proposed
to activate gene expression.
Objectives: To investigate TERT mutation frequency, spectrum,
association with expression and clinical outcome, and potential for
detection of recurrences in urine in patients with urothelial bladder
cancer (UBC).
Design, setting, and participants: A set of 111 UBCs of different stages
was used to assess TERT promoter mutations by Sanger sequencing and TERT
messenger RNA (mRNA) expression by reverse transcription-quantitative
polymerase chain reaction. The two most frequent mutations were
investigated, using a SNaPshot assay, in an independent set of 184
non-muscle-invasive and 173 muscle-invasive UBC (median follow-up: 53 mo
and 21 mo, respectively). Voided urine from patients with suspicion of
incident UBC (n = 174), or under surveillance after diagnosis of
non-muscle-invasive UBC (n = 194), was tested using a SNaPshot assay.
Outcome measurements and statistical analysis: Association of mutation
status with age, sex, tobacco, stage, grade, fibroblast growth factor
receptor 3 (FGFR3) mutation, progression-free survival, disease-specific
survival, and overall survival.
Results and limitations: In the two series, 78 of 111 (70%) and 283 of
357 (79%) tumors harbored TERT mutations, C228T being the most frequent
substitution (83% for both series). TERT mutations were not associated
with clinical or pathologic parameters, but were more frequent among
FGFR3 mutant tumors (p = 0.0002). There was no association between TERT
mutations and mRNA expression (p = 0.3). Mutations were not associated
with clinical outcome. In urine, TERT mutations had 90% specificity in
subjects with hematuria but no bladder tumor, and 73% in
recurrence-free UBC patients. The sensitivity was 62% in incident and
42% in recurrent UBC. A limitation of the study is its retrospective
nature.
Conclusions: Somatic TERT promoter mutations are an early, highly
prevalent genetic event in UBC and are not associated with TERT mRNA
levels or disease outcomes. A SNaPshot assay in urine may help to detect
UBC recurrences. (C) 2013 European Association of Urology. Published by
Elsevier B. V. All rights reserved
Heterogeneous clinical phenotypes and cerebral malformations reflected by rotatin cellular dynamics
Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations has not yet been elucidated. We performed both clinical and cell biological studies, aimed at clarifying rotatin function and pathogenesis. Review of the 23 published and five unpublished clinical cases and genomic mutations, including the effect of novel deep intronic pathogenic mutations on RTTN transcripts, allowed us to extrapolate the core phenotype, consisting of intellectual disability, short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. We show that the severity of the phenotype is related to residual function of the protein, not only the level of mRNA expression. Skin fibroblasts from eight affected individuals were studied by high resolution immunomicroscopy and flow cytometry, in parallel with in vitro expression of RTTN in HEK293T cells. We demonstrate that rotatin regulates different phases of the cell cycle and is mislocalized in affected individuals. Mutant cells showed consistent and severe mitotic failure with centrosome amplification and multipolar spindle formation, leading to aneuploidy and apoptosis, which could relate to depletion of neuronal progenitors often observed in microcephaly. We confirmed the role of rotatin in functional and structural maintenance of primary cilia and determined that the protein localized not only to the basal body, but also to the axoneme, proving the functional interconnectivity between ciliogenesis and cell cycle progression. Proteomics analysis of both native and exogenous rotatin uncovered that rotatin interacts with the neuronal (non-muscle) myosin heavy chain subunits, motors of nucleokinesis during neuronal migration, and in human induced pluripotent stem cell-derived bipolar mature neurons rotatin localizes at the centrosome in the leading edge. This illustrates the role of rotatin in neuronal migration. These different functions of rotatin explain why RTTN mutations can lead to heterogeneous cerebral malformations, both related to proliferation and migration defects