Genotypic and phenotypic implications in paroxysmal nocturnal hemoglobinuria (PNH): a preliminary investigation.

The genetic and biochemical defects underlying paroxysmal nocturnal hemoglobinuria (PNH) have recently been elucidated. The deficiency of the surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins caused by a somatic mutation of the PIG-A gene, an X-chromosomal gene that participates in the first step of the GPI anchor synthesis, has been shown to be responsible for PNH in all patients. The mutations of PIG-A studied to date are highly heterogeneous. They are however mainly of the frameshift type (61.5%). The characteristic abnormalities of PNH phenotypes has also been shown especially by DAF- and/or CD59-based fluorescent immunocytometry. A great degree of heterogeneity in the patterns and levels of expression of GPI-anchored proteins in various cell types was demonstrated indicating a discrepancy of lineage involvement. In this investigation, major blood cell populations, i.e erythrocytes and granulocytes were analyzed immunophenotypically, the mutations of PIG-A were identified by heteroduplex analysis and nucleotide sequencing and the consequences of PIG-A mutations were observed. All the mutations identified in 9 patients with PNH resulted in complete loss of function as clones of affected granulocytes completely negative for CD59 expression were shown in all patients. Interestingly, granulocytes in these patients contained variable proportions of affected cells varied from 50% to 100% and four of the patients had erythrocytes with diminished expression of GPI-anchored DAF and CD59 coexisting with normal and completely negative cells. Immunophenotypic analysis of reticulocytes in peripheral blood of patients with PNH demonstrated the conserved patterns of DAF and CD59 expression in circulating erythroid cells and the discrepancies between granulocytic and erythroid lineages. These findings suggested that the characteristics of abnormal phenotypes which appear to be highly variable between different hematopoietic lineages are not solely caused by mutation of PIG-A but are influenced by other factor(s).

PIG-A gene abnormalities in Thai patients with paroxysmal nocturnal hemoglobinuria.

Deficient biosynthesis of the glycosyl phosphatidyl inositol (GPI)-anchor in blood cells is implicated in the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH). Abnormal clonal cells appear in various hematopoietic cell lineages, suggesting that PNH arises as a result of somatic mutation occurred at the multipotential hematopoietic stem cell stage. We previously cloned a gene which is responsible for PNH. The gene termed PIG-A (for Phosphatidyl Inositol Glycan-class A) participates in the early step of GPI-anchor biosynthesis. Studies with cell lines and granulocytes from patients with PNH revealed that in all cases so far characterized, PIG-A is the target for the somatic mutation. In the present study, we analyzed PIG-A abnormality in granulocytes from 14 Thai-patients with PNH. PIG-A RNA was reversed transcribed and the coding region was amplified by polymerase chain reaction and cloned into plasmids. The cDNA thus obtained and genomic DNA were analyzed by mutation detection enhancement gel electrophoresis and sequencing. The assessment of function of PIG-A cDNA was based on the ability to correct the phenotype of a PIG-A deficient cell line after transfection. The result showed that all patients had PIG-A abnormality. Three patients had size abnormality of PIG-A transcripts caused by mutations at the splicing sites in the genomic DNA level. Eleven patients had PIG-A transcripts of normal sizes but had mutations in the coding region which included small deletions and insertions. Taken together with the result from Japanese and British patients, the PIG-A somatic mutations in patients with PNH are small mutations widely distributed throughout coding region and the splicing sites.

Expression cloning of genes for GPI-anchor biosynthesis

Cloning genes for glycosylphosphatydilinositol (GPI)-anchor biosynthesis is important to further understand its mechanisms and regulation. We have been using expression cloning methods in which a cDNA library was transfected into GPI-anchor-deficient mutant cells. The transfectants which restored surface expression of GPI-anchored proteins were isolated and the plasmids were rescued. In this way we previously cloned cDNAs of genes for complementation classes A and F, and named them PIG-A and PIG-F, respectively. In the present study we have cloned the gene for class B, termed PIG-B. In each case we used different methods. For cloning PIG-A cDNA we used a cDNA library made with an Epstein-Barr-virus-based vector and human class A mutant JY5 which expresses EBNA-1 protein. The EBNA-1 protein allows stable replication of oriP-containing plasmids in the episomal form. For cloning PIG-F cDNA we chose a transient expression method and cotransfected a human T-cell cDNA library made with a vector bearing an origin of replication of polyoma virus with a plasmid bearing polyoma virus large T into the class F murine thymona mutant. This cotransfection strategy was unsuccessful for cloning PIG-B due to low transfection efficiency of the class B thymoma mutant SIA-b. Thus, we first established large T-expressing SIA-b cells and then transfected them with cDNA library. PIG-B cDNA restored the surface expression of Thy-1 on SIA-b cells and also synthesis of mature type GPI-anchor precursors in these cells. The cDNA consists of 1929 bp and codes for a putative new protein of 554 amino acid residues