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Methylation-specific ligation detection reaction (msLDR): a new approach for multiplex evaluation of methylation patterns

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Abstract

A new sensitive method for multiplex gene-specific methylation analysis was developed using a ligation-based approach combined with a TaqMan-based detection and readout employing universal reporter probes. The approach, termed methylation-specific Ligation Detection Reaction (msLDR), was applied to test 16 loci in 8 different colorectal cancer cells in parallel. These loci encode immune regulatory genes involved in T-cell and natural killer cell activation, whose silencing is associated with the development or progression of colorectal cancer. Parallel analysis of HLA-A, HLA-B, STAT1, B2M, LMP2, LMP7, PA28α, TAP1, TAP2, TAPBP, ULBP2 and ULBP3 by msLDR in eight colorectal cancer cell lines showed preferential methylation at the HLA-B, ULBP2 and ULBB3 loci, but not at the other loci. MsLDR was found to represent a suitable and sensitive method for the detection of distinct methylation patterns as validated by conventional bisulphite Sanger sequencing and COBRA analysis. Since gene silencing by epigenetic mechanisms plays a central role during transformation of a normal differentiated somatic cell into a cancer cell, characterization of the gene methylation status in tumours is a major topic not only in basic research, but also in clinical diagnostics. Due to a very simple workflow, msLDR is likely to be applicable to clinical samples and thus comprises a potential diagnostic tool for clinical purposes.

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Abbreviations

APM:

Antigen-presenting machinery

B2M:

Beta-2-microglobulin

COBRA:

Combined bisulphite restriction analysis

CTL:

CD8+ cytotoxic T lymphocytes

DO1:

Detector oligonucleotide 1 (“left-hand side”)—discriminatory for the unmethylated state

DO2:

Detector oligonucleotide 2 (“left-hand side”)—discriminatory for the methylated state

DOR:

Detector oligonucleotide R (“right-hand side”)

FFPE samples:

Formalin fixed paraffin embedded samples

HLA:

Human lymphocyte antigen

LMP:

Low-molecular-weight protein

MHC:

Major histocompatibility complex

msLDR:

Methylation-specific Ligation Detection Reaction

MSP:

Methylation-specific PCR

NTC:

Non-template control

Oligo:

Oligonucleotide

PA28α:

Proteasome activator subunit

TAP:

Transporter associated with antigen processing

TAPBP:

TAP binding protein

ULBP:

UL16-binding protein

STAT:

Signal transducer and transcription activator 1

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Acknowledgments

This work was supported by the Max-Planck Society and the BMBF project Bioprofile [0313349 to A.D.], by the Deutsche Krebshilfe [100708 to C.S.] and the BMBF consortium ColoNET [C.S.]

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Authors and Affiliations

  1. Institute of Pathology, University Medicine Charité, Charitéplatz1, 10117, Berlin, Germany

    Felix Bormann, Christine Sers & Stephanie Seibt

  2. Max Planck Institute of Molecular Genetics, Berlin, Germany

    Thomas Bergmann & Hans Lehrach

  3. Institute of Medical Immunology, Martin Luther University, Halle-Wittenberg, Halle, Germany

    Barbara Seliger & Diana Handke

  4. Biotechnology Center Technical University Dresden, Dresden, Germany

    Andreas Dahl

Authors
  1. Felix Bormann
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  2. Christine Sers
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  3. Barbara Seliger
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  4. Diana Handke
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  5. Thomas Bergmann
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  6. Stephanie Seibt
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  7. Hans Lehrach
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  8. Andreas Dahl
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Correspondence to Felix Bormann.

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Communicated by C. Plass.

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Bormann, F., Sers, C., Seliger, B. et al. Methylation-specific ligation detection reaction (msLDR): a new approach for multiplex evaluation of methylation patterns. Mol Genet Genomics 286, 279 (2011). https://doi.org/10.1007/s00438-011-0645-9

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