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.

Similar patterns of V kappa gene usage but different degrees of somatic mutation in hairy cell leukemia, prolymphocytic leukemia, Waldenstrom's macroglobulinemia, and myeloma.

To compare V kappa gene usage and the amount of somatic mutation in rearranged Ig genes from patients with lymphoproliferative disorders, we have polymerase chain reaction-amplified and sequenced a total of 26 V kappa genes from a total of 55 cases. Six sequences were obtained both from six cases of prolymphocytic leukemia (PLL) and from nine cases of hairy cell leukemia (HCL). Seven sequences were obtained both from 11 cases of Waldenström's macroglobulinemia (WM) and 29 cases of multiple myeloma (MM).

Myelodysplasia in a patient with pre-existing paroxysmal nocturnal haemoglobinuria: a clonal disease originating from within a clonal disease.

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemolytic anaemia, clonal in nature, due to somatic mutation. PNH may evolve to aplastic anaemia; more rarely to a myelodysplastic syndrome (MDS) or to acute myeloid leukaemia (AML). We have studied a patient who suffered from PNH and later developed refractory anaemia with ringed sideroblasts (RARS) associated with trisomy 8.

Genomic organization of the X-linked gene (PIG-A) that is mutated in paroxysmal nocturnal haemoglobinuria and of a related autosomal pseudogene mapped to 12q21.

The PIG-A gene, whose product is involved in one of the early steps in the synthesis of glycan phosphatidylinositol (GPI) anchors, has been recently found to be defective in all cases of paroxysmal nocturnal haemoglobinuria (PNH). By isolating genomic clones from a human phage library we now show that the PIG-A gene consists of six exons (the first of which is non-coding) spanning 17 kb of DNA, and we have mapped the gene to chromosomal position Xp22.1.

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.

Cloning of the glucose 6-phosphate dehydrogenase gene from Plasmodium falciparum.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is one of the human genetic traits that confer relative resistance against malaria caused by Plasmodium falciparum. It has been previously shown that this organism, during its intraerythrocytic development, produces its own G6PD, which has properties different from those of human G6PD. In order to investigate the role of this enzyme in parasite-host cell interactions, we have isolated the G6PD gene from Plasmodium falciparum as a set of overlapping lambda gt11 clones.

High resolution of proteins by double-inverted gradient polyacrylamide gel electrophoresis (DG-PAGE).

We have designed a new method for high resolution electrophoretic separation of proteins that have similar molecular weights. The proteins migrate first through a conventional gradient gel, in which molecular friction increases as pore size decreases. The proteins then enter an inverted sodium dodecyl sulfate (SDS) gradient gel in which friction decreases; thus, smaller molecules gradually migrate faster and achieve improved separation from larger molecules, which remain near the border between the two gels.

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.

Tissue plasminogen activator for hepatic vein thrombosis in paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder thought to arise in a multipotent haemopoietic stem cell. A distinct clinical feature is a tendency to thrombosis, with a particular predilection for the hepatic veins (Budd-Chiari syndrome). We report here on two patients with PNH who developed hepatic vein thrombosis (HVT) and who were treated with tissue plasminogen activator (t-PA).

Retroviral-mediated gene transfer of a mutant H-ras gene into normal human bone marrow alters myeloid cell proliferation and differentiation.

To investigate the effects of mutant ras expression on the growth and differentiation of normal human bone marrow, we used retrovirus-mediated gene transfer. A retrovirus (HR-1) containing a mutant ras gene (H12-ras) in addition to the selectable neo gene was transferred by cocultivation of a packaging cell line with long-term cultures of normal human bone marrow. Controls were established by cocultivating aliquots of the same bone marrow with a retrovirus (VSN-2) containing only neo.

Dyskeratosis congenita: three additional families show linkage to a locus in Xq28.

Dyskeratosis congenita (DC) is a rare inherited disorder with most families being of the X linked recessive type. We describe three families which show linkage to the marker DXS52 on Xq28. The combined maximum lod score was 2.

G6PD haplotypes spanning Xq28 from F8C to red/green color vision.

The most telomeric region of the human X chromosome within band Xq28 consists of a gene-rich region of about 3 Mb which contains the genes for coagulation factor VIIIc, glucose-6-phosphate dehydrogenase (G6PD), and red/green color vision. We have studied five polymorphic sites from this region, in a sample of normal people from the Cosenza province of Southern Italy. These sites, which span a distance of some 350 kb, are in strong linkage disequilibrium.

Specific defect in N-acetylglucosamine incorporation in the biosynthesis of the glycosylphosphatidylinositol anchor in cloned cell lines from patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal disorder arising in a multipotent hemopoietic stem cell. PNH manifests clinically with intravascular hemolysis resulting from an increased sensitivity of the red cells belonging to the PNH clone to complement-mediated lysis. Numerous studies have shown that surface proteins anchored to the membrane via a glycosylphosphatidylinositol (GPI) anchor (including proteins protecting the cell from complement) are deficient on the cells of the PNH clone, leading to the notion that GPI-anchor biosynthesis may be abnormal in these cells.

G6PD Mediterranean accounts for the high prevalence of G6PD deficiency in Kurdish Jews.

The Jews of Kurdistan are a small inbred population with a high incidence of beta-thalassaemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Recently, it was reported that the beta-thalassaemia in this population shows an unusual mutational diversity; 13 different mutations were identified, of which 4 had not previously been observed in any other population. In contrast, we now report that the G6PD deficiency, which has the highest known incidence in the world, and which affects about 70% of males, is almost entirely attributable to a single widespread mutation, G6PD Mediterranean.

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.

Genetic heterogeneity of glucose-6-phosphate dehydrogenase deficiency revealed by single-strand conformation and sequence analysis.

We have carried out a systematic study of the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency on a sample of 53 male subjects from Calabria, in southern Italy. Our sequential approach consisted of the following steps: (1) Partial biochemical characterization was used to pinpoint candidate known variants. The identity of these was then verified by restriction-enzyme or allele-specific oligonucleotide hybridization analysis of the appropriate PCR-amplified fragment.

V kappa gene segments rearranged in chronic lymphocytic leukemia are distributed over a large portion of the V kappa locus and do not show somatic mutation.

The structure of the human V kappa locus has been thoroughly investigated, but how the germ-line V kappa gene segment repertoire is actually sampled in kappa chain gene rearrangements is not known. In order to begin to answer this question we have polymerase chain reaction (PCR) amplified the rearranged V kappa genes from 26 kappa-expressing cases of chronic lymphocytic leukemia (CLL), followed by cloning and sequencing of the PCR product. All four V kappa gene families were represented amongst rearranged genes.

Variants of glucose-6-phosphate dehydrogenase are due to missense mutations spread throughout the coding region of the gene.

Glucose-6-phosphate dehydrogenase (G6PD) is remarkable for its genetic diversity in humans. Many variants of G6PD have been described with wide ranging levels of enzyme activity and associated clinical symptoms. Fifty-eight different mutations have now been identified and these account for 97 named G6PD variants.

Production and characterization of lymphoblastoid cell lines with the paroxysmal nocturnal hemoglobinuria phenotype.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic disorder caused by a somatic mutation in a hematopoietic stem cell. The fact that, in some cases, not only myeloid but also lymphoid cells are affected suggests that the mutation has occurred in a multipotent stem cell. By studying the expression of CD59 antigen (membrane inhibitor of reactive lysis) and of decay accelerating factor (DAF) on the lymphocytes of 16 patients with PNH, we found an abnormal population of lymphocytes (with absent CD59 and DAF) in 10 cases.

Dyskeratosis congenita fibroblasts are abnormal and have unbalanced chromosomal rearrangements.

Dyskeratosis congenita (DC) is a rare inherited disorder characterized by bone marrow failure, dystrophic changes in the skin and mucous membranes, and a predisposition to malignancy. The DC locus has been mapped to Xq28. The primary defect responsible for this disease remains unknown.