A Molecular Epidemiology Study On The Associations Between The Telomerase Polymorphisms And Genetic Susceptibility Of Childhood Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common malignant disorder inchildhood accounting for approximately three-quarters of all childhood leukemiadiagnoses and the incidence is increasing worldwide. According to the report ofsurveillence, epidemiology, and end results (SEER) in USA, each year, around3,250children are diagnosed with leukemia, of which about2,400are ALL. Approximately12,000new ALL cases were diagnosed each year in China. Despite extensive studies,little is known about the pathogenesis of ALL, although both inherited susceptibilityand specific environmental exposure (such as house painting, infection duringpregnancy, irradiation) are supposed to play a role in this process.Chromosome and gene rearrangements are the hallmark of leukemia. Abnormalchromosome extremities favor such rearrangements and are thereby involved inleukemogenesis. Telomere-driven genome instability is a widespread cause ofgenome instabiliy. Telomere is a complex DNA-protein structure located at the end ofall linear eukaryotic chromosomes. Telomerase consists of a catalytic protein subunit,human telomerase reverse transcriptase (TERT), the RNA component of thetelomerase (TERC) and the shelterin complex. The major role of human telomerase isto catalyze the addition of telomeric repeat sequences TTAGGG onto chromosomeends for stabilization of telomere length in attaining cellular immortality and maytherefore be a critical step in carcinogenesis. Moreover, telomerase is involved inDNA damage repair, chromatin regulation and genes regulation. It is known thattelomerase is active in90%of tumors, while in those cells which do not expressTERT or telomerase at all, the alternative lengthening of telomeres (ALT) pathway is assumed to preserve the length of telomeres, which are obligate for senescencebypass and for neoplastic transformation of normal cells. Recent meta-analyses havereported an association between short telomeres and increased risk of several types ofhuman cancers. Moreover, telomere length was shorter in ALL subjects than innormal bone marrow mononuclear cells. And compared to healthy individuals,telomerase activity was higher in acute leukemia children. Therefore, genetic variantsof telomerase may play important roles in the development of childhood ALL,however, there were not any studies about it.With the completion of Human Genome Projict (HGP), it is known that geneticvariantions play an important role in gene structure and function, and singlenucleotide polymorphism (SNP) is the most common type of genetic variants. In theprevious years, genome-wide association studies (GWAS) have successfullydiscovered multiple SNPs at TERT associated with risk for multiple cancers andSNPs near TERC are associated with telomere length.Moreover, in recent years, pharmacogenetics in childhood ALL has become amajor field of research and SNPs is of particular importance for drugs with a verynarrow therapeutic index, such as Methotrexate (MTX). The pharmacologic effects ofMTX are well characterized, and telomerase has emerged as one drug resistancecandidate and may be involved in drug metabolism.In the present study, we conducted a hospital-based case-control study toinvestigate the associations between genetic variants of telomerase and childhoodALL susceptibility, telomere length and the childhood ALL susceptibility and thepharmacogenetics of MTX. Our study searched for potential biomarkers forchildhood ALL susceptibility and provides clues of MTX for the childhood ALLpharmacogenetics study, which may provide a “proof-of-principle” approach forindividualized prevention, clinical decision and individualized treatment. Part1A Molecular Epidemiology Study on the Associationsbetween the TERT Polymorphisms and GeneticSusceptibility of Childhood Acute Lymphoblastic LeukemiaHuman TERT is essential for the maintenance of telomere DNA length,chromosomal stability, and cellular immortality. We hypothesized that TERTpolymorphisms are associated with risk of childhood acute lymphoblastic leukemia(ALL). In a case-control study of570cases and673cancer-free controls of Chinese,we genotyped TERT tagging single-nucleotide polymorphisms (tSNPs) to evaluatethe association and further examined the association among these polymorphisms andtelomerase activity, telomere length. We found that two TERT promoter region tSNPs(rs2735940and rs2736109) and four intron region tSNPs (rs2736100, rs2853676,rs10069690and rs4246742) were associated with risk of childhood ALL. When thesetSNPs were evaluated together by the number of putative risk alleles, a statisticallyincreased risk of ALL was associated with6-11risk alleles, compared with those with0-5risk alleles [adjusted odds ratio (OR)=1.44,95%confidence interval (95%CI)=1.13-1.84] and this association was more pronounced in B-ALL. Notably, TERTintron2polymorphisms (rs2736100and rs2853676) are associated with longertelomeres. Compared with the wild genotype, CC genotype of rs2736100increased61%and AG/AA genotype of rs2853676increased27%susceptibility of longertelomere length. However, no effect modifications were found for variant alleles andtelomerase activity. Our findings suggested that TERT genetic variants are associatedwith the susceptibility of childhood ALL. They may influence the telomere lengthhomeostasis and contributed to the development of childhood ALL. Part2A Molecular Epidemiology Study on the Associationsbetween the TERC Polymorphisms and GeneticSusceptibility of Childhood Acute Lymphoblastic LeukemiaTelomeres are involved in maintaining chromosomal stability, cellularimmortality and tumorigenesis. A recent GWAS has identified an associationbetween telomere length and two common variants (rs12696304and rs16847897) at3q26that includes TERC. We hypothesized that the two variants and relativetelomere length (RTL) would be a predictor of the risk of childhood acutelymphoblastic leukemia (ALL). A case-control study of570cases and673cancer-free controls of Chinese children was performed. We found that rs16847897CG genotype increased risk of childhood ALL compared with the CC genotype by29%. However, there was no association between this polymorphism and telomerelength. Our findings indicated that TERC variants are associated with elevated risk ofALL in Chinese children and rs16847897heterozygote may be the risk genotype ofchildhood ALL.Part3Telomere Length Contribute to Childhood AcuteLymphoblastic Leukemia RiskTelomere length is a heritable trait, and is variable among different types of cells,tissues and individuals. Telomere shortening is involved in initiation and progressionof malignancies. A series of epidemiological studies have examined the associationbetween shortened telomeres and risk of cancers, but there was no study aboutchildhood ALL in Chinese Han population. In a case-control study of570cases and673cancer-free controls of Chinese children, we examined relative telomere length. We found that there was a protective relationship between second and third quartilesof RTL and risk of ALL [adjusted OR with95%CI by quartile:0.65(0.47-0.91) and0.56(0.40-0.79)], compared with the first quartile (shortest) RTL. Our findingsindicated that extreme telomere length may be a potential predictor for future risk ofALL.Part4Telomerase Polymorphisms Are Associated withMethotrexate Elimination after High-Dose InfusionHigh-dose MTX (HDMTX) with calcium folinate (CF) rescue has been widelyused in the treatment of ALL. The high doses and therefore of a high systemicexposure to MTX was shown to significantly improve the outcome in children withALL. Nevertheless, HDMTX administration is responsible for toxic effects includingdermatitis and mucositis, which are related to serum concentrations. Inter-individualvariability in MTX disposition is well known. Genetic variants of telomerase may beinvolved in drug metabolism and are associated with the MTX metabolism.123casesof childhood ALL undergoing HDMTX chemotherapy with CF rescue were includedin the study and their genomic DNA was genotyped to identify polymorphisms attelomerase. We obtained the values of their plasma MTX concentration and liverenzymes at the same time. As a result, we found the serum MTX level was0.73±0.84μmol/L at44h after HDMTX treatment,0.21±0.27μmol/L at68h. The overallnumber with delayed MTX elimination was69(56.1%) and MTX hepatotoxicity was41(33.3%). Those cases both with delayed MTX elimination and hepatotoxicity were18(14.6%). Furthermore, TERT promoter SNP rs2735940CT/TT genotype increasedthe risk of delayed MTX elimination at44h by1.89fold compared with CC genotypeand TERT intron2SNP rs2736100AC/CC genotype increased the risk of delayedMTX elimination at44h by1.64fold compared with AA genotype. Our resultsindicated that the TERT polymorphisms may be involved in the clearance of serum MTX and act as a biological marker for the MTX treatment.