Label Name: TERT
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB9172 
Last Review:  3/17/2017 10:06:57 AM
Specimen Type
Container & Volume
  Age Group   Container   Volume  
Collection Notes
  • This assay uses genomic DNA isolated from paraffin-embedded tumor tissue. Nine freshly cut (within one week) unstained slides should be submitted containing 5 mm2 to 20 mm2 of tissue. The slides should be prepared on a microtome that has been freshly wiped down with ethanol. An H&E stained slide should be prepared as well and submitted for reference purposes. The pathologist on service will circle the tumor-containing area on the H&E slide and indicate approximate percentage of tumor cells present.
  Send paraffin embedded tissue or slides at ambient temperature.
Turn Around Time -  Routine: 14 days   Stat: N/A
Reference Values


No Reference Values
  This assay uses traditional end-point PCR followed by Sanger DNA sequencing to detect mutations C250T and C228T within the promoter region of TERT. An H&E stained slide for each case is first evaluated to identify the regions of greatest tumor content. These regions are then macro-dissected from adjacent unstained formalin-fixed paraffin-embedded sections and used to prepare genomic DNA. A region of the promoter region containing C250 and C228 of the TERT gene is amplified using this DNA. The primers used in these PCR reactions contain M13 universal primer “tails” at their 5’ ends, and have 3’ ends that are complementary to their genomic target sequence. The resulting PCR products are treated with an exonuclease/ phosphatase mixture (ExoSAP-IT) to remove excess PCR primers and nucleotides. These purified DNA amplicons are then sequenced using universal M13 forward and reverse sequencing primers (M13 Forward/-20 and M13 Reverse/-27) and the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystem). The products of the completed sequencing reactions are purified with the Big Dye XTerminator Purification Kit and resolved using the ABI 3130xl Genetic Analyzer. Data is analyzed using the ABI Data Collection software v3.0, Sequencing Analysis software 5.2 and SeqScape software v2.6. Sequences are compared to the reference DNA sequence (GenBank Accession: NC_000005.9) for the TERT gene.

This test was developed and its performance characteristics determined by the DUHS Clinical Molecular Diagnostics Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.

Clinical Significance and Interpretive Data
    A recent study has identified a genetic alteration in the promoter region of TERT in malignant brain tumors. Originally, a genome-wide study examining multiple tumor types found over 70% of primary glioblastomas in adults and oligodendrogliomas positive for a mutation in the TERT promoter region.2 All TERT alterations described in the study are point mutations at one of two positions in the promoter region, described as C250T (g.-146C>T) and C228T (g.-124C>T) in the literature.3
Primary glioblastomas are the most common malignant brain tumors in adults, accounting for almost 17% of all intracranial tumors, and confer the worst survival rate.2 Patients exhibiting primary glioblastomas without a TERT mutation were observed to survive considerably longer (27 versus 14 months). While oligodendrogliomas and oligoastrocytomas also show considerably high TERT mutation rates, the clinical significance has not been studied.

    The Telomerase reverse transcriptase (TERT) gene maps to chromosome 5p15.33 and is thought to be partially responsible for the regulation of telomerase. Other studies have shown telomerase repressors to also exhibit a down-regulation of TERT.1 Somatic mutations in the TERT gene may lead to a genetic process which maintains telomere length in the absence of other epigenetic regulations.2
    • The sensitivity of DNA sequencing is high for the detection of nucleotide base changes and small deletions and insertions in the regions analyzed.
• Only the intronic region in upstream of the TERT gene (in particular mutation hot spots C228T and C250T) are examined.
• Changes in other regions of the gene are not examined, and mutations that fall in these regions will not be detected.
• Mutations in genes other than TERT will not be identified.
• Large deletions, duplications, multiple exon insertions, sequence alterations adversely affected primer binding, and complete deletion of one allele may not be identified using these methods.
• This assay may not detect an acquired mutation which is present below the 30% tumor content detection limit (i.e., mutant cell population containing at least 30% of total cells from the genomic DNA is extracted for testing).
• Clinicians should correlate results with clinical findings.

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Molecular Diagnostics Laboratory

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
 Email: michael.datto@duke.edu
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
 Email: catherine.rehder@duke.edu
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432
 Email: siby.s@duke.edu

 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

Performing Times: