Expression Levels of the TERT Gene Can be Used to Risk Stratify Aplastic Anemia Patients: Study in an Indian Cohort

Introduction: Acquired Aplastic anemia (AA) is a bone marrow failure syndrome characterized by pancytopenia and marrow hypoplasia, and is mediated by immune destruction of hematopoietic stem cells. Mutations in several genes including telomerase, a ribonucleoprotein enzyme complex, consisting of a reverse transcriptase enzyme (TERT), an RNA template (TERC), and several stabilizing proteins, and the associated shelterin complex have been found in cases of both congenital and idiopathic AA. In particular, several TERT and TER C mutations reduce telomerase activity in vitro and accelerate telomere attrition in vivo . Shortened telomeres have been observed in a third of idiopathic AA patients, but only 10% of these patients have mutations in genes of the telomerase complex. We have previously demonstrated that in addition to keeping telomeres from shortening, telomerase directly regulates transcriptional programs of developmentally relevant genes (Ghosh et al, Nat Cell Biol, 2012). We postulate that changes in expression of telomerase associated genes, specifically TERT, contribute to the etiology of aplastic anemia.

Patient cohort and Methods: In an effort to better understand the molecular and clinical correlates of this disease, 24 idiopathic AA patient blood samples were collected at a tertiary medical center in Bangalore, India. After obtaining informed consent, high throughput exome panel sequencing on an Illumina HiSeq 2500 platform of the 24 samples was performed, followed by variant calling, annotation and filtration of the generated data to reveal rare pathogenic variants. Additionally, RT-PCR analysis of harvested RNA from each patient was carried out and expression of levels of TERT measured in comparison to samples from normal individuals.

Results: Mutational profiling of 24 AA patients by exome sequencing revealed a total number of 2837 non- synonymous mutations (MAF < 0.01%). These included nonsense, frame shift, splice site, missense and in-frame mutations. Recurrent gene mutations were identified in 129 genes. However, pathway analysis of these gene mutations was uninformative with respect to disease etiology. Since DNA mutational data from the AA patients was unrevealing, and given that AA is a telomeropathy, we next chose to measure RNA expression levels of the TERT gene, an integral component of the Telomerase complex. RT-PCR of TERT expression compared to that of normal samples (n=6) revealed an 8 fold reduction in TERT expression 17/24 patients, while 7/24 patients maintained normal TERT expression.

Examination of the average number of gene mutations in the TERT-normal versus the TERT-Low patients revealed that the mutational burden was significantly higher in TERT-normal (166 mutations) compared to TERT-low (112 mutations) expressers (p=0.04). High throughput sequencing data also revealed that while TERT-normal patients had no mutations in genes associated with the telomerase/shelterin complex, TERT-low patients had mutations in TERT, TEP1, TINF2, NBN, TPP1, HSP90A and POT1 genes: all associated with the telomerase complex, most of which were predicted to be pathogenic variants. TERT-low patients also showed more evidence of clonal evolution (multiple patients with mutations in TET2, and in BCORL1, FLT-3, MLP, BRAF genes) compared to TERT-normal patients.

Clinically TERT-low patients were younger in age (average age 29) compared with the TERT-normal patients (average age 40). TERT-low patients were more likely to have severe aplastic anemia (SAA) leading to higher mortality and poorer response to therapy, with 6/17 patients dying and 4/17 not responding to ATG therapy.

Summary and Conclusions: Our observations suggest that expression of TERT can serve as a risk stratification marker in patients with AA. In spite of the higher mutational burden in TERT-normal patients (probably due to their comparative advanced age) TERT-low patients had poorer clinical outcomes which correlated with mutations in the TERT/Shelterin complex and with mutations in genes associated with clonal evolution. While these findings need to be validated in larger cohorts of patients, we suggest that evaluation of TERT expression levels in newly diagnosed AA patients could serve to risk stratify these patients and influence the choice of therapy administered.