Clonal involvement of granulocytes and monocytes, but not of T and B lymphocytes and natural killer cells in patients with myelodysplasia: analysis by X-linked restriction fragment length polymorphisms and polymerase chain reaction of the phosphoglycerate kinase gene.
To determine the clonal nature of hematopoiesis and to assess lineage involvement in patients with myelodysplastic syndromes (MDS), we used restriction fragment length polymorphisms of the X-linked genes phosphoglycerate kinase (PGK1) and hypoxanthine phosphoribosyltransferase (HPRT) and the X-linked probe M27 beta. Eleven female MDS patients heterozygous for at least one of these probes were studied: 3 with refractory anemia (RA), 2 with RA with ringed sideroblasts (RARS), 2 with chronic myelomonocytic leukemia (CMML), and 4 with RA with excess of blasts in transformation (RAEB-t). All exhibited clonal hematopoiesis as determined by Southern analysis of DNA prepared from peripheral blood (PB) and/or bone marrow (BM) cells. In three of the six patients heterozygous for the PGK1 gene, purified cell suspensions of polymorphonuclear cells (PMN), monocytes, lymphocytes, and/or T cells prepared from PB were tested. In addition, five of these patients were analyzed by a polymerase chain reaction (PCR)-based procedure as described recently. This method was slightly adapted to facilitate the analysis of cell lysates of fluorescence-activated cell sorted (FACS) monocytes, T and B lymphocytes, and natural killer (NK) cells. The outcome of Southern and PCR analysis was concordant, showing that PMN and monocytes were clonally derived, whereas circulating T and B lymphocytes and NK cells exhibited random X-chromosome inactivation compatible with a polyclonal pattern. To address the question of whether T cells are derived from unaffected progenitor cells or that their origin had antedated the onset of MDS, naive and memory T cells were analyzed separately. Both subsets showed a polyclonal pattern. However, in one patient analysis of constitutive DNA suggested a skewed methylation, and the presence of clonal lymphocytes against a background of polyclonal lymphoid cells cannot be ruled out in this patient. PCR analysis of PB and BM cells showed a nonrandom, unilateral pattern of X-inactivation, compatible with a mixture of clonally (myeloid) and polyclonally (lymphoid) derived cells. In conclusion, in some patients, MDS represents a disorder with clonal hematopoiesis restricted to cells of myeloid origin, whereas a random X-inactivation pattern is found in lymphoid cells.
Department of Hematology, University of Leiden, The Netherlands.
H
H
van Kamp
W E
WE
Fibbe
R P
RP
Jansen
M
M
van der Keur
E
E
de Graaff
R
R
Willemze
J E
JE
Landegent
D000328:Adult; D000368:Aged; D001402:B-Lymphocytes / Q000502:physiology*; D001483:Base Sequence; D015139:Blotting, Southern; D005260:Female; D017353:Gene Deletion; D006580:Genetic Carrier Screening; D006098:Granulocytes / Q000502:physiology*; D006410:Hematopoiesis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007694:Killer Cells, Natural / Q000502:physiology*; D008875:Middle Aged; D008969:Molecular Sequence Data; D009000:Monocytes / Q000502:physiology*; D009190:Myelodysplastic Syndromes / Q000201:enzymology / Q000235:genetics* / Q000276:immunology*; D009838:Oligodeoxyribonucleotides; D010735:Phosphoglycerate Kinase / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D013601:T-Lymphocytes / Q000502:physiology*; D014960:X Chromosome
D009838:Oligodeoxyribonucleotides; D007041:Hypoxanthine Phosphoribosyltransferase; D010735:Phosphoglycerate Kinase