Association between TERT promoter polymorphisms and acute myeloid leukemia risk and prognosis.

Telomerase reverse transcriptase gene (TERT) promoter mutations are identified in many malignancies but not in hematological malignancies. Here we analyzed TERT and protection of telomeres 1 gene (POT1) mutations, and four different TERT SNVs in 226 acute myeloid leukemia (AML) patients and 806 healthy individuals in a case referent design, where also overall survival was assessed. A significant association for increased risk of AML was found for TERT SNVs, rs2853669 (OR = 2.45, p = 0.00015) and rs2736100 (OR = 1.5, p = 0.03). The overall survival for patients with CC genotype of rs2853669 was significantly shorter compared to those with TT or TC genotypes (p = 0.036 and 0.029 respectively). The influence of TERT rs2853669 CC on survival was confirmed in multivariable Cox regression analysis as an independent risk biomarker in addition to high risk group, higher age and treatment. No hot spot TERT promoter mutations at −228C > T or −250C > T or POT1 mutations could be identified in this AML cohort. We show that rs2853669 CC may be a risk factor for the development of AML that may also be used as a prognostic marker to identify high risk normal karyotype -AML (NK-AML) patients, for treatment guidance.


Mutations analysis TERT core promoter, TERT 1062 A > T and POT1
PCR was performed in a total volume of 20 μl, containing 5X MyTaq Reaction Buffer (comprising 1 mM dNTP and 3 mM MgCl 2 ), 1 μM for each pair primers (Supplementary Table S1), 1 U MyTaq™ DNA polymerase and 20 ng of template DNA. After an initial heat activation step at 95°C for 2 minutes, thirty five cycles of PCR were performed with the following cycling conditions: 95°C for 30 s, 61°C for 30 s, and 72°C for 30 s.

CEBPA mutations
The total reaction volume of 20 μl contained 20 ng of DNA, 1 μM of each primer, 1 U MyTaq™ DNA polymerase, 5X MyTaq Reaction Buffer, and 1 μl of 100% dimethyl sulfoxide. The following PCR conditions were used: incubation at 94°C for 3 min, 35 cycles at 94°C for 45s (denaturation), 63°C for 45s (annealing) and 72°C for 1 min (extension) followed by the final extension at 72°C for 10 min. PCR products were sequenced in both directions forward and reverse for each primer using the ABI 3500 Genetic Analyzer (Applied Biosystems, USA). NM_004364.4 CEBPA ID transcript was used to align the derived sequences.

PCR TaqMan ® SNP genotyping Genotyping
A Total volume of 10 μl per well contained 5 μl TaqMan ® Universal PCR Master Mix (2X), 0.25 μl assay mix (40X), including the two allele-specific TaqMan ® MGB probes containing distinct fluorescent dyes (FAM and VIC) and a PCR primer pair to detect specific SNV targets and 20 ng of DNA.

Pyrosequencing
The cycling conditions were 95°C for 2.5 min, followed by 35 cycles of 95°C denaturation for 30 s, 66.1°C and 58°C for annealing for rs10069690 and A1062T (rs35719940) mutation, respectively, and 30 s extension at 72°C for 30 s and with a final extension at 72°C for 5 min. Successful PCR was determined by 1.5% agarose gel electrophoresis and the genotypes were assessed on a PSQ96MD instrument (Qiagen, Sweden) as previously described [1]. In brief, the amplified and biotinylated PCR product was isolated with a Vacuum Prep Workstation (Qiagen, Sweden). The sequencing primers (Invitrogen, Paisley, UK) 5′-gggtgaggtggacaga-3′ and 5′-tgggggccaagggcg-3′for rs10069690 and rs35719940 respectively was annealed to the single-stranded DNA template for 2 min at 80°C. The plate was then transferred to the PSQ96MD instrument and sequencing was performed with the following dNTP dispensing order: cgt agc tgc and tcg atc gct for the polymorphism and the mutation respectively.

Quantitative real-time PCR
The relative mRNA expression was determined by real-time PCR with the TaqMan ® Fast Universal PCR Master Mix (Applied Biosystems) and cDNAspecific primer/probe mixes for TERT, IL-6, IL-1β and TNFα (Hs00972656_m1, amplicon length 79 bp, Hs00985639_m1, amplicon length 66 bp, Hs01555410_m1, amplicon length 91 bp and Hs01113624_g1, amplicon length 143 bp, respectively) (Applied Biosystems). The qPCR mix contained 2 μl of sample cDNA, 5 μl of TaqMan Fast Universal Master mix (2X), 0.5 μl assay mix and 2.5 μl of mili-Q water with the following cycling conditions; 20 s in 95°C and 40 cycles consisted of 3 s in 95°C, 30 s in 60°C.