Association of newly identified genetic variant rs2853677 of TERT with non-small cell lung cancer and leukemia in population of Jammu and Kashmir, India

Lung cancer has highest mortality rate among cancers for both males and females, whereas males are more affected than females in leukemia by the ratio 1:5:1:0 [1]. Both the cancers are aggressive in nature, and this is one of the major global health concern worldwide. Lung cancer constitutes 12.9% of all newly diagnosed cancer cases and 19.4% of all cancer associated deaths globally [2, 3] took place. Lung Cancer is the major cause of cancer related deaths in India accounting for 63,000 newly diagnosed patients each year with 52,000 deaths, and thereby contributing to 9.3% of all cancer associated deaths [4, 5]. Furthermore, lung cancer in Kashmir region (J&K) was reported to be the second most common malignancy in hospital based study. [6]. While in Jammu region (J&K), unpublished data from our research group found that lung cancer and breast cancer are most commonly occurring cancers followed by esophageal cancer.

Lung cancer and Leukemia are multifactorial in nature, where both genetic and non-genetic factors like environmental pollution, occupational exposure and smoking mainly contribute to its etiology [7]. Single nucleotide polymorphism (SNP) genotyping and Genome wide association studies (GWAS) in Japanese population have revealed that chromosome 5p15.33 is the strong candidate region associated with lung cancer [8]. Genetic variant rs2853677 of telomere maintenance gene TERT is located in an intronic region of chromosome 5p15.33 (chr5:1,253,147-1,295,069) [9]. Telomeres are actually caps located at the terminal end of the linear chromosomes, which acts as an important factor in maintaining the genomic stability. In humans, telomeres comprise of guanine rich hexa nucleotide (TTAGGG) repeats. The stability of the telomeres solely depends on the integrity of the associated telomere maintenance proteins, which always work in coordinated manner, referred to as the shelterin complex [10]. Shelterin complex comprises of different factors like Telomeric Repeat Binding Factor 1 (TRF1), Telomeric Repeat Binding Factor 2 (TRF2), TRF1-Interacting Nuclear Factor 2 (TIN2), Protection of Telomeres 1 (POT1), Tripeptidyl Peptidase 1 (TPP1) and Repressor Activator Protein 1 (RAP1) [11]. Among the binding factors TRF1 and TRF2 bind to the double stranded telomeric DNA segment as homodimers. TRF1 inhibits the action of telomerase thus acts as negative regulator of telomere length [12] and the stability of T loop is maintained by TRF2. Mutation in TRF2 results in the activation of Ataxia Telangiectasia Mutated (ATM) protein mediated DNA damage signal which leads to end-to-end telomere fusion and telomere homologous recombination, leading to typical process of telomere sister-chromatid exchange (T-SCE) [13]. However, repressor activator protein 1 (RAP1) directly binds to TRF2 protein which is crucial for efficient binding of TRF2 protein to the telomeric DNA. Another important factor POT1 protects the chromosome ends from fusion and a typical recombination and helps in the recruitment of telomerase to the telomeric DNA, thus contributing to the protection of telomeres [14]. TRF-interacting nuclear factor 2 (TIN2) protein mainly binds with other shelterin proteins like TRF1, TRF2 and TPP1, acting as a critical component of the shelterin complex and bridging all these proteins of the shelterin complex together for efficient working. [12]. Shelterin proteins possibly regulate telomere elongation by enrolling Telomerase, which is a ribonucleoprotein complex consisting of reverse transcriptase (TERT) and RNA subunit (TERC). Telomerase is inactive and suppressed in most of the normal somatic cells. During the S phase of cell cycle, it gets activated and stimulates the replication process of telomeric DNA, which is not replicated, by DNA polymerase during replication [15].

Molecular alterations in the TERT promoter lead to enhanced expression of telomerase, which maintains telomere length thereby letting the cancer cells to continuously proliferate and inhibiting the programmed cell death and apoptosis [16].

Genomic wide association studies (GWAS) and candidate gene approach (CGA) have proven to be important tools in understanding genetic complexity and heterogeneity of these multifactorial diseases through case-control association in various ethnic groups. Therefore, this study investigated the association between TERT gene polymorphism and lung cancer and leukemia risk in the less explored population of Jammu and Kashmir of North India. We observed that variant rs2853677 was strongly associated with non-small cell lung cancer and leukemia risk with odds ratio (OR) =1.8 (1.03–3.2 at 95% CI); p value (adjusted) =0.03 in NSCLC and 2.7 (1.4–5.2 at 95% CI); p value (adjusted) = 0.002 in leukemia, signifying that TERT polymorphism plays a crucial role in the pathological process of multiple cancers. Our findings are consistent with previous genome wide association studies stating the role of TERT gene in different cancers at different ethnicities [8, 18‐22]. Furthermore it was observed that the subtype adenocarcinoma (AC) and squamous cell carcinoma (SCC) of non-small cell lung cancer was found to be associated with odds ratio OR = 1.37 and 1.50 respectively whereas, the subtype undifferentiated carcinoma (UDC) was not significantly associated. The possible reason being small sample size (n = 7) However in leukemia, the subtypes Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML),Chronic Myeloid Leukemia (CML), Chronic Lymphoid Leukemia (CLL) were found to be associated with an OR = 1.75, 1.71, 1.40, 1.81 respectively. Myelodysplastic Syndrome (MDS) subtype of leukemia did not show any association with possible reason of small sample size. Additional file 1: Table S1.

Human Pulmonary neoplasms and leukemia’s are heterogeneous in nature, wherein both genetic and non-genetic factors like smoking mainly contribute to its etiology [7]. Jammu and Kashmir is Lung cancer capital of India and Tobacco sales are at highest peak. However, the genetic susceptibility to lung cancer and leukemia is still unclear; the accumulation of low risk alleles may result in inherited risk. Genetic variant rs2853677 of telomere maintenance gene TERT is located in intronic region of chromosome 5p15.33. Molecular alteration (polymorphism) in telomere maintenance genes results in destabilization of shelterin complex which could lead to constitutive activation of ribonucleoprotein complex like TERT (Fig. 2). The overexpression of TERT results in maintenance of telomere length resulting in cell immortality and allows the progression of a preneoplastic lesion towards malignant stages. So these polymorphisms might prove to be a prognostic or predictive biomarker in population group under study.

In order to determine the effect of this SNP rs2385677 on the functioning of TERT gene, the bioinformatics in silico analysis of this variant was performed by HSF (v.3.1). It showed that the variant creates broken sites that could influence the physiology of TERT gene. Moreover, the splicing effect of the variant needs to be confirmed in-vitro analysis. Although we were not able to fully evaluate the stratified analysis in different subtypes of non-small cell lung cancer and leukemia due to less sample size in each sub type. We also take it as quest to evaluate these subtypes with large sample size in future course. The present study also provides an important information of the genetic etiology of non-small cell lung cancer and leukemia and strengthens clinical findings and highlighting the role of telomere maintenance genes in leukemia and lung cancer, which may have impact to clinical and public health data in population of Jammu and Kashmir. It is also taken into account to screen other telomere maintenance genes in other Indian ethnic groups targeting different cancers [9, 19, 23, 24]. This variant has not been explored with lung cancer or leukemia in any other Indian population groups and this is the first study from the region. These findings could be potential conformations and could be used as potential predictive or prognostic markers in clinical studies of leukemia and lung cancer patients in J&K population groups.

We observed that the genetic variant rs2853677 in the TERT loci is linked with an increased non-small cell lung cancer and leukemia risk in Jammu and Kashmir (North Indian) population. Our result revealed the complex genetic regulation of telomere genetics and highlighted the critical role of telomere maintenance gene (TERT) in the pathogenesis of non-small cell lung cancer and leukemia. The limitations of the present study include, the sample size among the subtypes of non-small cell lung cancer and leukemia was relatively small. These findings may also enhance our understanding of inter-population differences in leukemia and lung cancer etiology and strengthens GWAS findings as well, although the physiological functions of these TERT SNPs need to be explored in others cancers in future study designs.