Mutations in PIGB Cause an Inherited GPI Biosynthesis Defect with an Axonal Neuropathy and Metabolic Abnormality in Severe Cases

Abstract

Proteins anchored to the cell surface via glycosylphosphatidylinositol (GPI) play various key roles in the human body, particularly in development and neurogenesis. As such, many developmental disorders are caused by mutations in genes involved in the GPI biosynthesis and remodeling pathway. We describe ten unrelated families with bi-allelic mutations in PIGB, a gene that encodes phosphatidylinositol glycan class B, which transfers the third mannose to the GPI. Ten different PIGB variants were found in these individuals. Flow cytometric analysis of blood cells and fibroblasts from the affected individuals showed decreased cell surface presence of GPI-anchored proteins. Most of the affected individuals have global developmental and/or intellectual delay, all had seizures, two had polymicrogyria, and four had a peripheral neuropathy. Eight children passed away before four years old. Two of them had a clinical diagnosis of DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures), a condition that includes sensorineural deafness, shortened terminal phalanges with small finger and toenails, intellectual disability, and seizures; this condition overlaps with the severe phenotypes associated with inherited GPI deficiency. Most individuals tested showed elevated alkaline phosphatase, which is a characteristic of the inherited GPI deficiency but not DOORS syndrome. It is notable that two severely affected individuals showed 2-oxoglutaric aciduria, which can be seen in DOORS syndrome, suggesting that severe cases of inherited GPI deficiency and DOORS syndrome might share some molecular pathway disruptions.

Keywords: DOORS syndrome; PIGB; alkaline phosphatase; epilepsy; glycosylphosphatidylinositol; inherited GPI deficiency (IGD); intellectual disability; neuropathy; seizures.

Figure 1

Phenotypes of Affected Individuals (A) The pedigrees of families with PIGB mutations. In family 5, the unaffected siblings were genotyped, and none were compound heterozygous. However, the family does not wish their carrier status to be published. (B) Photographs of affected individuals. Individual 2A at 9 years old (note a wide nasal bridge, a long and smooth philtrum, a thin upper lip, a horizontal chin crease, and upturned earlobes), 2B at 11 months old (note similar features), individual 5 at 20 years old (note a horizontal chin crease, a prominent philtrum, and slightly upturned earlobes), individual 7 at 8 months old (note a wide nasal bridge, brachytelephalangy, and nail hypoplasia), individual 8A at birth (note a wide nasal bridge, a long and smooth philtrum, and a thin upper lip), individual 10A at birth, and individual 10B at 2.5 months old (note for both hypertrichosis, a wide nasal bridge, coarse facial features, a long and smooth philtrum, a pointed chin with a horizontal crease, uplifted earlobes, and toe and nail hypoplasia). (C) EEG findings for individual 4. The top left panel depicts a sleep EEG at 9 months old, before any treatment; diffuse, low-voltage fast waves seem to be characteristic in this individual. Spindle waves are observed. High-amplitude slow waves are sometimes seen at the frontal regions predominantly. The top right panel depicts a sleep EEG at 30 months old; there are 0.5 s 12 Hz rhythmic waves at the frontal region and very small spike and high-voltage slow wave complexes. There is also a paucity of sleep markers and bilateral central spikes. The bottom left panel depicts a sleep EEG at 3 years and 10 months old. Generalized spike and wave discharge followed by voltage attenuation were seen. In the bottom right pictures, brain MRI did not demonstrate any brain anomalies such as dysplasia, atrophy, or delayed myelination. (D) Radiographs of the hands and feet of individual 10B at 2.5 months of age showing aplasia of the terminal phalange of the fifth finger bilaterally and hypoplasia or aplasia of the terminal phalanges of the toes. (E) MRI of individual 9A at 2 years and 3 months old. The left image depicts an axial T-2 sequence showing diffuse cerebral volume loss characterized by widening of the ventricles and extra CSF spaces with a prominent interhemispheric fissure anteriorly. There is suspicion of polymicrogyria along the bilateral occipital lobes, more pronounced on the right. The right image depicts an axial FLAIR sequence showing diffuse cerebral volume loss. There were signs of hypomyelination with a hyperintense signal in peri-ventricular and subcortical white matter, more pronounced at the occipital and frontal lobes bilaterally.

Figure 2

PIGB Variants (A) PIGB variants found in affected individuals. (B) Alignment of the PIGB sequence where missense mutations were found. (C) Analysis of the PIGB splicing mutation. The upper left depicts Sanger sequencing of proband 6B and the parents that used their genomic DNA showed that the c.847-10A>G mutation was homozygous in the proband and heterozygous in both parents. The lower left depicts the cDNA analysis performed with leukocytes from the proband; the analysis showed the insertion of the last nine bases of intron 7 before the canonical exon 8 as a result of the activation of an aberrant splice acceptor site. The proband did not express wild-type mRNA, which was observed in a control. At right, a graphic description of the activation of the aberrant splice acceptor site at exon 8 in PIGB occurred in the proband. Abbreviations are as follows: gDNA = genomic DNA and Ex = exon.

Figure 3

Flow Cytometry Analysis of Cell Surface GPI-APs of Granulocytes Blood samples collected from affected individuals and controls were stained with FLAER and antibodies against GPI-APs (CD16, CD24, CD55, and CD59) and were analyzed by the BD Scanto II system or the MACSQuant system. The granulocyte population was gated on the basis of cellular granularity, cell size, and CD45 level.

Figure 4

Decreased Level of GPI-AP in the Fibroblasts and Rescue by PIGB-Expressing Lentivector Skin fibroblasts derived from individuals 2A and 6B were stained with FLAER, CD73, CD87, and CD109 and were analyzed by the BD FACScanto II system and then by Cytobank software. Fibroblasts from individual 6B were transduced with PIGB-expressing-Lx304 lentivirus or empty-vector lentivirus, and these fibroblasts were stained with CD73 and CD109 as described above. The figure shows representative results from experiments done in triplicate.

Figure 5

Functional Analysis of the Mutant PIGB cDNAs Found in the Families (A) PIGB-deficient CHO cells were transiently transfected with wild-type and mutant HA-tagged PIGB cDNAs subcloned in pME (strong SRα promoter-driven vector). Restoration of the surface expression of CD59, CD55 (DAF), and uPAR was assessed two days later by flow cytometry. Black lines indicate the empty vector; green and blue lines indicate various types of mutant PIGB; the red line indicates wild-type PIGB; and light gray shadows indicate isotype controls. Various missense variants, except for I537M, driven by a strong promoter only partially rescued the expression of GPI-APs compared to wild-type PIGB. (B) Levels of the mutant PIGB. Lysates of the transfectants were subjected to SDS-PAGE and immunoblotting. For quantification, levels of PIGB were determined by dividing PIGB band intensities by GAPDH band intensities and luciferase activity to normalize for both loading and transfection efficiencies. ^∗ = truncated PIGB. Expression levels of mutant PIGB, except for the p.I5le37Met variant, were decreased to various degrees.