title: Skewed X-inactivation patterns in ageing healthy and myelodysplastic haematopoiesis determined by a pyrosequencing based transcriptional clonality assay.
abstract: BACKGROUND
  Investigation of X-chromosome inactivation patterns (XCIP) by determination of differential CpG-methylation has been widely applied for investigation of female cell clonality. Using this approach the clonal origin of various tumours has been corroborated. Controversially, strong age-related increase of peripheral blood (PB) cell clonality in haematologically healthy female subjects was reported. Recently, transcriptional XCIP ratio analysis challenged these results and questioned the suitability of methylation based clonality assays.
  
  
  METHODS
  To reinvestigate XCIP-skewing in CD34, low-density mononuclear bone marrow (BM) as well as PB cells from healthy female subjects and patients with myelodysplastic syndromes (MDS), we established a transcriptional assay using pyrosequencing technique for quantification of single nucleotide polymorphism allele frequencies, representative for XCIP ratios.
  
  
  RESULTS
  Our assay provides high sensitivity for XCIP ratio assessment as determined by standard curves, reproducibility, inter-marker correlation as well as correlation with the DNA-methylation based human androgen receptor (HUMARA) assay. Notably, in agreement with most studies investigating this issue, significant age-related increase of XCIP skewing in PB cells from healthy elderly female subjects was confirmed. Moreover, XCIP ratio analysis suggests even stronger clonal manifestation in BM and CD34 cells. In MDS, XCIP skewing levels were distinctively elevated as compared with controls of similar age and higher degrees were associated with poor clinical outcome.
  
  
  CONCLUSIONS
  Transcriptional clonal profiling via pyrosequencing allows accurate assessment of XCIP ratios, confirms the validity of the DNA-methylation based HUMARA assay and reveals important insights into ageing healthy and myelodysplastic haematopoiesis.
authors: Department of Hematology and Oncology, University Hospital Mannheim, Mannheim, Germany. maximilian.mossner@medma.uni-heidelberg.de
authors: Maximilian
authors: M
authors: Mossner
authors: Florian
authors: F
authors: Nolte
authors: Gero
authors: G
authors: Hütter
authors: Jana
authors: J
authors: Reins
authors: Marion
authors: M
authors: Klaumünzer
authors: Verena
authors: V
authors: Nowak
authors: Julia
authors: J
authors: Obländer
authors: Katrin
authors: K
authors: Ackermann
authors: Silke
authors: S
authors: Will
authors: Henning
authors: H
authors: Röhl
authors: Uwe
authors: U
authors: Neumann
authors: Martin
authors: M
authors: Neumann
authors: Olaf
authors: O
authors: Hopfer
authors: Claudia D
authors: CD
authors: Baldus
authors: Wolf-Karsten
authors: WK
authors: Hofmann
authors: Daniel
authors: D
authors: Nowak
mesh_terms: D000367:Age Factors; D000368:Aged; D000369:Aged, 80 and over; D018952:Antigens, CD34 / Q000235:genetics; D001854:Bone Marrow Cells; D005260:Female; D006410:Hematopoiesis; D006801:Humans; D009190:Myelodysplastic Syndromes / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D049951:X Chromosome Inactivation / Q000235:genetics*
chemical_list: D018952:Antigens, CD34