Targeted Molecular Therapy for Inherited Glycosylphosphatidylinositol Deficiency.

Inherited glycosylphosphatidylinositol (GPI) deficiency (IGD) is an autosomal recessive disease characterized by splanchnic vein thrombosis and epilepsy. The partial GPI deficiency in the affected children is due to disrupted addition of the first mannose residue onto the GPI intermediate PI-GlcN, a step catalyzed by the α1>4 mannosyltranferase PIGM. IGD is caused by a −270C>G substitution in the core promoter of PIGM which disrupts binding of the transcription factor (TF) Sp1 to its cognate motif resulting in markedly reduced transcription. Sp1 regulates transcription through interactions an with the basal transcriptional complex or with other TF. In some genes, Sp1 is also required for locus histone acetylation. We investigated whether histone acetylation of the PIGM locus was Sp1-dependent and whether histone deacetylase (HDAC) inhibitors such as sodium butyrate (SB) could be used to enhance PIGM transcription even in the presence of the mutated Sp1 binding motif associated with IGD. Using ChIP assays we first showed that histone 4 (H4) at the PIGM locus is constitutively acetylated in GPI+ EBV B cell lines (LBCL), consistent with the housekeeping function of PIGM. By contrast, the same H4 in GPI- LBCL from individuals with IGD was hypoacetylated indicating that efficient PIGM promoter acetylation requires an intact Sp1 core promoter motif. Upon exposure to SB, acetylation was completely restored in the affected cell lines. Consistent with this, in luciferase reporter assays performed in the presence of mithramycin, an agent that specifically inhibits Sp binding, the enhancing effect of SB on transcription driven by both wild type and mutant (−270C>G) promoter constructs was largely (but not exclusively) Sp1-dependent. The increased transcriptional activity of the mutated PIGM promoter in the presence of SB was accompanied by a more than 100-fold increase in the PIGM mRNA and almost complete restoration of surface GPI expression in the affected LBCL.

Over a period of 10 years, one of the children with IGD developed progressive, intractable and incapacitating seizures, despite a multitude of different anti-epileptic treatments including sodium valproate. At the age of 14, she was wheelchair-bound with global hypotonia, extreme drowsiness, poorly responsive, unable to feed herself and almost permanently in minor status with approximately 5 tonic-clonic seizures/day. Extensive CNS imaging showed no structural abnormalities or evidence of thrombosis. An EEG was consistent with minor status epilepticus. Guided by the effectiveness of SB in restoring PIGM transcription and GPI expression in vitro, the patient was commenced on sodium phenylbutyrate (SPB) at an oral dose of 20mg/kg tds. SPB treatment induced a progressive increase in the proportions of GPI+ granulocytes and an increase in PIGM mRNA levels in primary mononuclear blood cells from a pre-SPB level of 1.65% to 26%. A marked clinical improvement was noted after only 2 weeks of treatment, in that the patient could walk, interact with others and feed herself and became entirely seizure-free; she remains so 6 months later. No side effects were noted. These data suggest that SPB may be an effective therapeutic option for diseases caused by Sp1-dependent histone hypoacetylation and also for diseases caused by non-inactivating mutations in the coding region of genes where transcription is regulated by Sp1-dependent histone acetylation.