Repetitive DNA sequences comprise about 45% of the mammalian genome. Long Interspersed Elements (LINE-1), a class of non-Long Terminal Repeat (LTR) retrotransposons, are amplified to more than 500,000 copies in both truncated (~0.9 kb) form or active full-length elements (~6 kb)1. A full-length LIN E-1 contains a 5′ untranslated region (UTR}, two non-overlapping open reading frames (ORF1 and ORF2}, and a 3′ UTR that ends in a poly (A) tail2. The heavily methylated LINE-1 elements are among the host cell defense mechanisms to inhibit L 1-ribonucleoprotein (L 1 RNP) formation and prevent its mobilization throughout the human genome.
Line-1 Global Methylation Analysis
Analysis Methods – Human LINE-1 methylation assay (Assay ID: ASY3201) amplifies a 146 base pair (bp) fragment located at the 5’UTR region of the LINE-1 elements (Figure 2). The LINE-1 methylation level can be quantified by Pyrosequencing, MS-HRM, and targeted bisulfite NGS (tNGBS). These three methods use the same bisulfite PCR but measure methylation levels in different ways.
- LINE-1 Pyrosequencing Assay: Sequence a 36-bp fragment to quantify methylation level of four CpG sites
- MS-H RM Methylation Assay: Provide an average methylation level of the 146-bp region using real-time PCR
- Targeted Bisulfite NGS (tNGBS): Sequence the entire 146-bp amplicon and quantify the methylation of 10 CpG sites with greater than S00x coverage
Assay Specificity – The LINE-1 Assay (ASY3201) amplifies a conserved LINE-1 element throughout the genome after sodium bisulfite modification, with a PCR amplicon size of 146 bp. It is located at the 5’UTR region of the LINE-1 elements (Figure 2). Pyrosequencing and tNGBS assays demonstrated specificity to the LINE-1 repetitive element sequence.
Assay Sensitivity – Assay sensitivity was analyzed by determining the limits of detection (LOD) and quantification (LOO). Both LOD and LOO were set at 1 ng of input DNA.
Line-1 Assay Precision & Accuracy
Assay Precision – The assay was tested for reproducibility on the same day, same operator, and same instrument
(intra-assay precision), as well as reproducibility between different instruments and operators (intermediate precision). The assay was shown to be precise down to 1 ng of input DNA.
Assay Accuracy – The accuracy of the LINE-1 assay was determined by comparing the methylation data obtained from the validation test samples to the expected values of the control DNA with known methylation levels. An
R-square value of greater than 0.9 at the LOO indicates that the assay is linear and free of PCR Bias. The average R-square for each input DNA amount was calculated and the results are shown in Figure 2. All seven input amounts, down to as low as 0.5 ng of starting DNA, show R-square values above 0.9. An example of the 5 ng input DNA amount is also shown in the figure.
A Biomarker in Cancer Research
LINE-1 hypomethylation has been found in various cancer types, such as bladder cancers4, liver cancer5, and gastrointestinal cancers6. LINE-1 methylation loss can be used as a surrogate marker for cancer diagnosis and prognosis. Genomic DNA isolated from whole blood, buffy coat, buccal cells, serum, FFPE tissue, and other biological specimens is suitable for quantifying LINE-1 methylation level using targeted bisulfite NGS, Pyrosequencing, or MS-HRM.
High-Throughput Drug Screening
Hypomethylating agents (HMAs) such as azacitidine and decitabine are FDA-approved for use in the United States in myelodysplastic syndrome and are being investigated for use in a number of tumors. The Line-1 Pyrosequencing methylation assay has been used for drug screening of compounds that influence global DNA methylation states7• Figure 2 shows the drug-induced Line-1 methylation change at different treatment time points at a selected dose.
A Surrogate Pharmacodynamic (PD) Marker
Line-1 Pyrosequencing methylation analysis has been used as a pharmacodynamic (PD) marker for several clinical trials in cancer drug development8·9• Line-1 methylation can be used as a predictor of clinical response. The time course of LINE-1 methylation following drug administration may be related to HMA concentration in blood or plasma. Simulated LINE-1 matrices can develop a PK/dose-response model which can be used to optimize the dose regimen and to evaluate the dynamic of HMAs.
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