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Neue Verfahren für Einzelzellanalysen in Forschung und Diagnostik

New methods of single-cell analysis in research and diagnostics

Zusammenfassung

Die traditionelle Zytogenetik ist ein Paradebeispiel für eine Einzelzelldiagnostik, weil mit jeder gebänderten Metaphase das gesamte Genom einer Zelle – bei relativ niedriger Auflösung – untersucht wird. Dies repräsentierte über mehrere Jahrzehnte einen wichtigen Unterschied zu molekulargenetischen Untersuchungstechniken, die in der Mehrheit der Fälle auf DNA oder RNA basieren, die aus hunderten oder tausenden von Zellen extrahiert wurden. Viele Fragestellungen können jedoch nur durch Analysen auf dem Niveau einzelner oder weniger Zellen beantwortet werden. Deshalb wurden besonders in den letzten Jahren neue Einzelzelltechniken mit dem Ziel entwickelt, immer mehr Loci mit verbessertem Auflösungsvermögen simultan analysieren zu können. In dieser Übersichtsarbeit werden die diesbezüglich wichtigsten Entwicklungen der letzten Jahre zusammengefasst.

Abstract

Traditional cytogenetics is a paradigm for single-cell diagnostics; after a banding procedure, each metaphase examined represents the analysis of an entire genome of a cell, albeit at a low resolution. For several decades, this single-cell character has represented an important distinction in molecular genetics technologies, which are mostly based on DNA or RNA extracted from hundreds or thousands of cells. However, many essential questions can be addressed only by analyzing cells on the level of fewer or single cells. In the last few years, new single-cell techniques have been developed with the aim to simultaneously examine more regions with improved resolution. In this overview we summarize the most important recent developments and changes.

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Literatur

  1. 1.

    Allison M (2008) Is personalized medicine finally arriving? Nat Biotechnol 26: 509–517

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Braun S, Vogl FD, Naume B et al. (2005) A pooled analysis of bone marrow micrometastasis in breast cancer. N Engl J Med 353: 793–802

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Bruder CE, Piotrowski A, Gijsbers AA et al. (2008) Phenotypically concordant and discordant monozygotic twins display different DNA copy-number-variation profiles. Am J Hum Genet 82: 763–771

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Cremer M, Müller S, Solovei I et al. (2008) 3D-Fluoreszenz-in-situ Hybridisierung und Zellkernarchitektur. Med Genet 4

  5. 5.

    Döhner H, Stilgenbauer S, Benner A et al. (2000) Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med 343: 1910–1916

    PubMed  Article  Google Scholar 

  6. 6.

    Fiegler H, Geigl JB, Langer S et al. (2007) High resolution array-CGH analysis of single cells. Nucleic Acids Res 35: e15

    PubMed  Article  Google Scholar 

  7. 7.

    Fuhrmann C, Schmidt-Kittler O, Stoecklein NH et al. (2008) High-resolution array comparative genomic hybridization of single micrometastatic tumor cells. Nucleic Acids Res 36: e39

    PubMed  Article  Google Scholar 

  8. 8.

    Gangnus R, Langer S, Breit E et al. (2004) Genomic profiling of viable and proliferative micrometastatic cells from early-stage breast cancer patients. Clin Cancer Res 10: 3457–3464

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Geigl JB, Speicher MR (2007) Single-cell isolation from cell suspensions and whole genome amplification from single cells to provide templates for CGH analysis. Nat Protocols 2: 3173–3184

    Article  CAS  Google Scholar 

  10. 10.

    Geigl JB, Obenauf AC, Schwarzbraun T et al. (2008) Defining ‚chromosomal instability’. Trends Genet 24: 64–69

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Hu DG, Webb G, Hussey N (2004) Aneuploidy detection in single cells using DNA array-based comparative genomic hybridization. Mol Hum Reprod 10: 283–289

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Hüsemann Y, Geigl JB, Schubert F et al. (2008) Systemic spread is an early step in breast cancer. Cancer Cell 13: 58–68

    PubMed  Article  Google Scholar 

  13. 13.

    Iafrate AJ, Feuk L, Rivera MN et al. (2004) Detection of large-scale variation in the human genome. Nat Genet 36: 949–951

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Klein CA, Schmidt-Kittler O, Schardt JA (1999) Comparative genomic hybridization, loss of heterozygosity and DNA sequence analysis of single cells. Proc Natl Acad Sci USA 96: 4494–4499

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Langer S, Geigl JB, Gangnus R et al. (2005) Sequential application of interphase-FISH and CGH to single cells. Lab Invest 85: 582–592

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    Le Caignec C, Spits C, Sermon K et al. (2006) Single-cell chromosomal imbalances detection by array CGH. Nucleic Acids Res 34: e68

    Article  Google Scholar 

  17. 17.

    Mastenbroek S, Twisk M, Van Echten-Arends J et al. (2007) In vitro fertilization with preimplantation genetic screening. N Engl J Med 357: 9–17

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Pantel K, Brakenhoff RH (2004) Dissecting the metastatic cascade. Nat Rev Cancer 4: 448–456

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Piotrowski A, Bruder CE, Andersson R et al. (2008) Somatic mosaicism for copy number variation in differentiated human tissues. Hum Mutat [Epub ahead of print]

  20. 20.

    Redon R, Ishikawa S, Fitch KR et al. (2006) Global variation in copy number in the human genome. Nature 444: 444–454

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    Schardt JA, Meyer M, Hartmann CH et al. (2005) Genomic analysis of single cytokeratin-positive cells from bone marrow reveals early mutational events in breast cancer. Cancer Cell 8: 227–239

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    Schmidt-Kittler O, Ragg T, Daskalakis A et al. (2003) From latent disseminated cells to overt metastasis: genetic analysis of systemic breast cancer progression. Proc Natl Acad Sci USA 100: 7737–7742

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Sebat J, Lakshmi B, Troge J et al. (2004) Large-scale copy number polymorphism in the human genome. Science 305: 525–528

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    Sher G, Keskintepe L, Keskintepe M et al. (2007) Oocyte karyotyping by comparative genomic hybridization provides a highly reliable method for selecting „competent“ embryos, markedly improving in vitro fertilization outcome: a multiphase study. Fertil Steril 87: 1033–1040

    PubMed  Article  CAS  Google Scholar 

  25. 25.

    Speicher MR, Du Manoir S, Schröck E et al. (1993) Molecular cytogenetic analysis of formalin-fixed, paraffin-embedded solid tumors by comparative genomic hybridization after universal DNA-amplification. Hum Mol Genet 2: 1907–1914

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Stoecklein NH, Hosch SB, Bezler M et al. (2008) Direct genetic analysis of single disseminated cancer cells for prediction of outcome and therapy selection in esophageal cancer. Cancer Cell 13: 441–453

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Telenius H, Pelmear AH, Tunnacliffe A et al. (1992) Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes Chromosomes Cancer 4: 257–263

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Ullmann R (2008) Strukturelle Genomvarianten – Ausmaß, Entstehung und phänotypische Konsequenzen. Med Genet 4

  29. 29.

    Voullaire L, Wilton L, Slater H et al. (1999) Detection of aneuploidy in single cells using comparative genomic hybridization. Prenat Diagnosis 19: 846–851

    Article  CAS  Google Scholar 

  30. 30.

    Wells D, Sherlock JK, Handyside AH et al. (1999) Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genomic hybridisation. Nucleic Acids Res 27: 1214–1218

    PubMed  Article  CAS  Google Scholar 

  31. 31.

    Youssoufian H, Pyeritz RE (2002) Mechanisms and consequences of somatic mosaicism in humans. Nat Rev Genet 3: 748–758

    PubMed  Article  CAS  Google Scholar 

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Geigl, J., Speicher, M. Neue Verfahren für Einzelzellanalysen in Forschung und Diagnostik. medgen 20, 407–415 (2008). https://doi.org/10.1007/s11825-008-0138-3

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Schlüsselwörte

  • Einzelzellanalyse
  • Gleichmäßige Gesamtgenomamplifikation
  • Vergleichende genomische Hybridisierung (CGH)
  • Copy number variation (CNV)
  • Auflösungsvermögen

Keywords

  • Single-cell analysis
  • Unbiased whole-genome amplification
  • Comparative genomic hybridization (CGH)
  • Copy number variation (CNV)
  • Resolution limits