Blood, 1 April 2003, Vol. 101, No. 7, pp. 2833-2841
RED CELLS
The spectrum of PIG-A gene mutations in aplastic
anemia/paroxysmal nocturnal hemoglobinuria (AA/PNH): a high
incidence of multiple mutations and evidence of a mutational
hot spot
Yousef Mortazavi,
Bruno Merk,
Jenny McIntosh,
Judith C. W. Marsh,
Hubert Schrezenmeier, and
Tim R. Rutherford for the
BIOMED II Pathophysiology and Treatment of Aplastic Anaemia
Study Group
From the Department of Haematology, St George's
Hospital Medical School, London, United Kingdom; Department of Medicine
III, University of Ulm, Ulm, Germany; and the Department of Pathology,
Zanjan Medical School, Zanjan, Iran.
Paroxysmal nocturnal hemoglobinuria (PNH) may arise during
long-term follow- up of aplastic anemia (AA), and many AA patients have
minor glycosylphosphatidylinositol (GPI) anchor-deficient clones, even
at presentation. PIG-A gene mutations in AA/PNH and hemolytic PNH are thought to be similar, but studies on AA/PNH have
been limited to individual cases and a few small series. We have
studied a large series of AA patients with a GPI anchor-deficient clone (AA/PNH), including patients with minor clones, to determine whether their pattern of PIG-A mutations was identical to the reported
spectrum in hemolytic PNH. AA patients with GPI anchor-deficient clones were identified by flow cytometry and minor clones were enriched
by immunomagnetic selection. A variety of methods was used to analyze
PIG-A mutations, and 57 mutations were identified in 40 patients. The majority were similar to those commonly reported, but
insertions in the range of 30 to 88 bp, due to tandem duplication of
PIG-A sequences, and deletions of more than 10 bp were also seen. In 3 patients we identified identical 5-bp deletions by conventional methods. This prompted the design of mutation-specific polymerase chain reaction (PCR) primers, which were used to
demonstrate the presence of the same mutation in an additional
12 patients, identifying this as a mutational hot spot in the
PIG-A gene. Multiple PIG-A mutations have been reported in
10% to 20% of PNH patients. Our results suggest that the large
majority of AA/PNH patients have multiple mutations. These data may
suggest a process of hypermutation in the PIG-A gene in AA
stem cells.
