Defective Modification of Mannose Residues by Terminal Phosphoethanolamine Underlies Inherited GPI Deficiency.

The glycosylphosphatidylinositol (GPI) anchor is essential for expression of functionally diverse proteins at the cell surface. Disruption of the first step in GPI biosynthesis due to somatic mutation(s) of the X-linked PIG-A gene in hematopoietic cells results in paroxysmal nocturnal haemoglobinuria (PNH).

We have previously reported (

In both families the propositus presented in childhood with hepatic vein thrombosis in the absence of significant hemolysis. All three affected children since the original report have manifested neurological features which include partial seizures and cognitive delay.

Flow-cytometry revealed partial deficiency of GPI-linked proteins in blood cells and fibroblasts. Expression of GPI on the cell surface defined by FLAER staining was severely reduced. Heterogeneity was observed in the level of expression of GPI-linked proteins ranging from slightly reduced to severely deficient, probably reflecting differential affinity of the carboxyterminal anchoring signal of individual proteins for GPI.

To identify the site of block in GPI biosynthesis radiolabeling and separation of GPI intermediates by thin layer chromatography were performed. The formation of phosphatidylinositol as well as the addition and de-acetylation of N-Acetyl glucosamine were intact in deficient EBV-transformed B cell lines demonstrating that the biochemical defect in both kindreds is distinct from that of PNH. Labeling with [3H] D-mannose revealed accumulation of an identical mannose-containing intermediate in affected members of both families. This co-migrated with a moiety derived from TF cells in which there is accumulation a GPI intermediate (H6) possessing three mannose residues which lack terminal phosphoethanolamine (EtnP) modification. A defect in addition of the terminal EtnP was confirmed by alpha-mannosidase digestion.

The genes for PIG-F and PIG-O, known to regulate GPI biosynthesis at the site of block defined biochemically, were excluded as candidates by sequencing of the coding regions and targeted homozygosity mapping. Transfection of deficient B-cell lines with PIG-F or PIG-O cDNA failed to restore GPI expression. We conclude an as yet uncharacterised gene is required for terminal EtnP modification and GPI assembly. Mutation(s) of this gene is likely to be the basis for inherited GPI deficiency. The viable nature of the phenotype contrasts with that of the pig-a knock-out mouse which is embryo lethal and may be explained by the partial deficiency of GPI. The associated neurological features echo the neurodevelopmental defects observed in GPI null mice.

In summary, we describe a novel recessive genetic disorder characterised by partial GPI deficiency due to a defect of a gene required for the addition of an EtnP moiety critical for the attachment of proteins to the GPI anchor and their expression on the cell surface.