Clonal X-inactivation analysis of human tumours using the human androgen receptor gene (HUMARA) polymorphism: A non-radioactive and semiquantitative strategy applicable to fresh and archival tissue

Peter Kopp, Rolf Jaggi, Andreas Tobler, Bettina Borisch, Madeleine Oestreicher, Leah Sabacan, J. Larry Jameson, Martin F. Fey*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

39 Scopus citations

Abstract

Assessment of clonality of cellular proliferations is important in experimental and clinical cancer research. X-chromosome inactivation studies are widely used to assess clonality, but most assays require relatively large amounts of high molecular weight DNA. Two PCR-based strategies, the phosphoglycerate kinase (PGK) and the human androgen receptor (HUMARA) clonality assays allow studies of small tissue samples. The HUMARA assay was adapted to non-radioactive analysis taking advantage of an automated sequencer providing high resolution of alleles and immediate quantitation. This assay was validated by comparison with X-inactivation patterns obtained by Southern analysis with the probes M27β and PGK. Fifteen gastrointestinal carcinomas, 25 benign goiter nodules and normal peripheral leukocytes of 27 individuals (12 who were under 15 years and 15 over 80 years) were analysed. Furthermore, DNA extracted from formalin-fixed paraffin-embedded tissue (FPT) was analysed with the two PCR-based methods and compared with X-inactivation patterns determined by Southern analysis of high molecular weight (HMW) DNA. This modified HUMARA assay is reliable in most patients; as with other clonality assays, constitutive skewing in normal tissue precludes clonal analysis in some individuals. Extremely skewed X-inactivation patterns were found in normal peripheral leukocytes of 7 out of 15 old females (over 80 years) and in 1 of 12 of the young females tested (under 15 years). Comparison of results obtained with HMW and FPT DNA yielded consistent results for the HUMARA assay whereas the PGK PCR assay was much less reliable. The HUMARA assay thus permits studies of selected areas of tissue sections without significant stromal components, allowing correlation of histological and genotype findings in fresh and archival specimens.

Original languageEnglish (US)
Pages (from-to)217-228
Number of pages12
JournalMolecular and Cellular Probes
Volume11
Issue number3
DOIs
StatePublished - Jun 1997

Keywords

  • Human androgen receptor polymorphism
  • PCR
  • Tumour clonality
  • X-inactivation analysis

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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