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1365 Functional Testing of Variants of Uncertain Significance in TERC, TERT, and RTEL1 from Adult Patients with Telomere Biology Disorders

Program: Oral and Poster Abstracts
Session: 509. Bone Marrow Failure and Cancer Predisposition Syndromes: Congenital: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research, Bone Marrow Failure Syndromes, Inherited Marrow Failure Syndromes, Genetic Disorders, Diseases
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Rachel A Simon1*, Christy M Finke2*, Terra L. Lasho, PhD2, Christopher T. Schmitz3*, Jenna A Fernandez, PhD4, Eva M Carmona-Porquera4*, Mark E Wylam, M.D.5*, Thomas E. Witzig, MD4, Tammy M McAllister, M.A.6*, Carolyn R. Rohrer Vitek, Ed.D. / M.S.6*, Konstantinos N Lazaridis4*, Abhishek A Mangaonkar, MBBS1, Mrinal M. Patnaik, MD, MBBS1 and Alejandro Ferrer, PhD4

1Division of Hematology, Mayo Clinic, Rochester, MN
2Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN
3Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN
4Mayo Clinic, Rochester, MN
5Pulmonary and Critical Care Medicine, Mayo Clinic, Rochester, MN
6Center for Individualized Medicine, Mayo Clinic, Rochester, MN


Telomere biology disorders (TBDs) are multisystem diseases caused by accelerated telomere shortening that can present with bone marrow failure and pulmonary fibrosis, among other abnormalities. TBD diagnosis is considered when average blood telomere length (TL) measured by clinical flowFISH is <1st percentile and/or a pathogenic variant in telomere maintenance genes (most commonly TERT, TERC, RTEL1) is identified. Pathogenic variants in genes coding for the telomerase protein (TERT) and RNA component (TERC) decrease telomerase activity. Similarly, variants in RTEL1 result in defective unwinding of the t-loop at telomere ends, increasing the levels of free t-circles in cells. Variants in these genes are often classified as variants of uncertain significance (VUSs by ACMG criteria) with additional efforts needed for variant curation. To address this, we describe results of functional testing performed in TBD patients with VUSs involving TERT, TERC, and RTEL1.


TBD suspected patients were identified through the Pre-Myeloid Cancer and Bone Marrow Failure Clinic (Mayo Clinic) where they underwent clinical flowFISH and genetic testing for TBD related genes. Informed consent was obtained with approval from the Mayo Clinic Institutional Review Board. Viable peripheral blood and bone marrow samples were collected along with age matched healthy volunteers. Telomerase activity was measured in samples with TERT and TERC variants through telomeric repeat amplification protocol (TRAP) assays using the TRAPeze XL Telomerase Detection Kit (MilliporeSigma) following manufacturer instructions. HCT116 cells were used as positive controls while heat inactivated samples served as negative controls. Fluorescence was measured in a SpectraMax plate reader. Percentage of total product generated (TPG) relative to healthy age matched controls was calculated. T-circle detection assays were used on RTEL1 samples based on the protocol by Zhang et al. (2017). Final southern blotting was completed using the TeloTAGGG Telomere Length Assay Kit (Roche), and t-circle bands were visualized in a c600 GEL Imaging System (Azure Biosystems).


Ten patients (mean age = 53 years (27-71), 50% female) were included in our study. Patients 1 to 5 presented VUS in TERT (n=4) or TERC (n=1) and TL <1st percentile in lymphocytes and/or granulocytes. Patient 6 carried a known pathogenic variant in RTEL1 and was included as a positive control. Patients 7 to 9 with RTEL1 variants presented TL close to the 10th or between the 1st-10th percentile. No TL results were available for patient 10 but was tested due to the presence of a VUS in RTEL1 (Table 1). Similarly, while patient 7 carried a likely pathogenic variant, it was tested due to presenting lymphocyte TL between the 1st-10th percentile. TRAP results indicated decreased telomerase activity compared to age matched controls in all samples with TERT and TERC variants suggesting a pathogenic effect (52.%, 10.7%, 48.7%, 4.4% and 5.2% of TPG compared to age matched controls, respectively) (Table 1). On the other hand, t-circles were only seen in patient 6 (Figure 2).


We used TRAP and t-circle assays to explore the telomerase activity and presence of t-circles in patient samples with TERT/TERC and RTEL1 variants. In the case of TERT/TERC, we observed a reduction of telomerase activity in all samples. Together with blood TL below the 1st percentile, this suggests that these variants are indeed deleterious (Table 1). However, since patient 4 presented two TERT VUSs, additional testing is needed to conclude how each variant impacts telomerase activity independently. In regard to RTEL1 variants, we observed t-circles in patient 6 as expected. No t-circles could be seen on all remaining samples, including patient 7 who carried a variant classified as likely pathogenic (Figure 1). This in-frame deletion has been described once in a TBD family (Cogan et al. 2015) but was not functionally tested. This result, together with the borderline TL seen in this patient, may suggest that some compensatory mechanism exists on this sample, or that the deleterious effect of the variant is not strong enough to be detected by our assay. Further studies are currently ongoing by our group to validate these results using a cell-based transfection disease model. In summary, we show that functional testing can be a helpful tool to determine pathogenicity of VUSs in TERT/TERC and RTEL1.

Disclosures: Witzig: ADC: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; Kura Oncology: Research Funding; Salarius Pharma: Membership on an entity's Board of Directors or advisory committees. Patnaik: Epigenetix: Research Funding; Kura Oncology: Research Funding; CTI Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; StemLine: Research Funding.

*signifies non-member of ASH