Peter Hillmen

Paroxysmal nocturnal haemoglobinuria (PNH) is caused by somatic mutations in the PIG-A gene.

Paroxysmal nocturnal haemoglobinuria (PNH), an acquired clonal blood disorder, is caused by the absence of glycosyl phosphatidylinositol (GPI)‐anchored surface proteins due to a defect in a specific step of GPI‐anchor synthesis. The cDNA of the X‐linked gene, PIG‐A, which encodes a protein required for this step has recently been isolated. We have carried out a molecular and functional analysis of the PIG‐A gene in four cell lines deficient in GPI‐linked proteins, obtained by Epstein‐Barr virus (EBV) transformation of affected B‐lymphocytes from PNH patients. In all four cell lines transfection with PIG‐A cDNA restored normal expression of GPI‐linked proteins. In three of the four cell lines the primary lesion is a frameshift mutation. In two of these there is a reduction in the amount of full‐length mRNA. The fourth cell line contains a missense mutation in PIG‐A. In each case the mutation was present in the affected granulocytes from peripheral blood of the patients, but not in normal sister cell lines from the same patient. These data prove that PNH is caused in most patients by a single mutation in the PIG‐A gene. The nature of the mutation can vary and most likely occurs on the active X‐chromosome in an early haematopoietic stem cell.

Somatic mutations and cellular selection in paroxysmal nocturnal haemoglobinuria

Patients with paroxysmal nocturnal haemoglobinuria (PNH) have in their blood two red-cell populations, one normal and one deficient in proteins anchored to the membrane through a glycan phosphatidylinositol (GPI) structure. The PNH abnormality is due to a somatic mutation in the PIG-A gene, whose product is required for an early step in GPI anchor biosynthesis. We show that in two patients, two PNH clones with different mutations co-exist, and must therefore have arisen independently. This finding supports the concept that PNH develops under the pressures of a positive selection mechanism whereby GPI-anchor-deficient haemopoietic cells have a survival advantage.

Mutations in the PIG-A gene causing partial deficiency of GPI-linked surface proteins (PNH II) in patients with paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) is due to the absence or marked reduction of glycan phosphatidylinositol (GPI)‐anchored proteins on the surface of blood cells. Affected patients may have a population of red blood cells that are completely deficient (PNH III) or partially deficient (PNH II) in these proteins, or they may have both. PNH III has recently been shown to be due, in all cases examined, to a somatic mutation in the PIG‐A gene, whose product is required for an early step in GPI anchor synthesis. We now show that two patients with PNH II cells also have somatic mutations of the same gene: these produce a partial rather than a total loss of PIG‐A function.

Paroxysmal nocturnal hemoglobinuria: Correction of abnormal phenotype by somatic cell hybridization

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired blood disorder thought to result from a somatic mutation in a hemopoietic stem cell. PNH may evolve to aplastic anemia or to acute leukemia. PNH cells are deficient in proteins attached to the cell membrane via a glycosylphosphatidylinositol structure, called the GPI anchor, and the primary lesion in PNH is thought to be a defect in the biosynthesis of the GPI anchor. We have recently established permanent lymphoblastoid cell lines that have the PNH phenotype and we report now the isolation of human-human somatic cell hybrid clones obtained by fusing them with normal lymphoblastoid cells. In all of 21 hybrid clones, obtained from five different patients, the expression of three different GPI-linked proteins on the hybrid cells was normal. These findings indicate that the PNH mutant gene is recessive with respect to the normal allele and that a recessive mutation can cause a clonal preneoplastic disorder.