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Disrupted binding of the transcription factor Sp1 to the mutated promoter region of the mannosyl transferase–encoding gene PIGM causes inherited glycosylphosphatidylinositol (GPI) deficiency characterized by splanchnic vein thrombosis and epilepsy. We show that this results in histone hypoacetylation at the promoter of PIGM. The histone deacetylase inhibitor butyrate increases PIGM transcription and surface GPI expression in vitro as well as in vivo through enhanced histone acetylation in an Sp1-dependent manner. More important, the drug caused complete cessation of intractable seizures in a child with inherited GPI deficiency.
We recently described a new form of inherited GPI deficiency presenting in infancy, characterized by splanchnic vein thrombosis and seizures and inherited as an autosomal recessive trait.6 As compared with paroxysmal nocturnal hemoglobinuria, inherited GPI deficiency does not result in clinically significant hemolysis and bone marrow failure. In the families described so far, partial yet severe GPI deficiency is caused by the blocked addition of the first mannose residue onto the GPI intermediate phosphatidylinositol–glucosamine, a step catalyzed by the 
1,4-mannosyltransferase PIGM. The genetic defect in inherited GPI deficiency is a –270C
G mutation in the core promoter of PIGM, which disrupts binding of the transcription factor Sp1 to its cognate motif, resulting in markedly reduced transcription.6
Sp1 influences transcription through heterotypic interactions with the basal transcriptional machinery and with other transcription factors and by recruiting histone acetyltransferases and histone deacetylases to the promoter.7 Histone deacetylase inhibitors such as sodium butyrate enhance transcription and hold promise as therapeutic agents for a variety of diseases, including cancer.8 For a small number of genes, the hyperacetylation effect of sodium butyrate requires the presence of Sp1-binding elements or a conserved sequence to which an as-yet-unknown transcription factor binds; these promoter elements are also referred to as butyrate response elements.9 In Sp1-dependent genes containing butyrate response elements, sodium butyrate may modulate transcription through covalent modification of Sp1 as well as through histone hyperacetylation.7,9
We investigated whether the PIGM promoter contained butyrate response elements and whether modification of acetylation mediated by sodium butyrate could result in enhanced transcription of PIGM, even in the presence of the mutated Sp1-binding motif associated with inherited GPI deficiency.
Methods
Patient and Study Design
The patient was seen at King's College Hospital and Hammersmith Hospital in London. The study was approved by the respective local research ethics committees, and oral informed consent was provided in accordance with the provisions of the Declaration of Helsinki.
Cell Lines and Analyses
The generation of Epstein–Barr virus (EBV)–transformed lymphoblastoid cell lines was described previously.10 For details regarding flow cytometry, real-time polymerase-chain-reaction analysis, reporter assays, and statistical analysis, see the Supplementary Appendix, available with the full text of this article at www.nejm.org. Chromatin immunoprecipitation assays (Upstate Biotechnology) were performed according to the manufacturer's instructions except for the following modifications: immunoprecipitation was performed with antiacetylated histone 4 (Upstate Biotechnology) or rabbit IgG (Santa Cruz). After DNA-protein cross-linking reversal and proteinase K digestion, DNA was isolated with the use of a MiniElute PCR purification kit (Qiagen). Primer sequences are available from the authors on request.
Results
Clinical Report
In 1995, a 2-year-old girl (now 14 years of age) presented with hepatic vein thrombosis and the Budd–Chiari syndrome (Table 1 of the Supplementary Appendix). After one episode of hepatic decompensation owing to variceal bleeding at the age of 5 and two shunt procedures at the age of 7, portal hypertension eventually became well controlled with conservative therapy (spironolactone, propranolol, and oral anticoagulants).
Absence seizures developed at the age of 4 years and were initially well controlled with sodium valproate; lamotrigine was added when the patient was 9 years old after an episode of status epilepticus, with good control of seizures. When she was first seen in the United Kingdom at the age of 9 years, tests for thrombophilia and the results of metabolic screening were normal; the diagnosis of inherited GPI deficiency was made by flow-cytometric analysis of GPI expression on blood cells.6 During the next 5 years, the frequency of seizures increased progressively despite increases in anticonvulsant therapy. Sodium valproate was stopped and levetiracetam started at the age of 12 years; a year later, topiramate was added. When the patient was seen in the United Kingdom again at the age of 14 years, she was having multiple absence seizures and approximately five tonic–clonic seizures per day; antiepileptic treatment consisted of lamotrigine, levetiracetam, topiramate, and clobazam (Table 1 of the Supplementary Appendix).
On examination, the patient was wheelchair-bound with global hypotonia, drooling, and extreme drowsiness. She was poorly responsive and unable to feed herself, symptoms that may have reflected toxic effects of the antiepileptic therapy as well as the disease itself. Central nervous system imaging, including magnetic resonance imaging and magnetic resonance angiography and venography, showed no structural abnormalities or evidence of thrombosis. Findings on electroencephalography performed while the patient was having multiple absence episodes were grossly abnormal, with frequent multifocal and generalized epileptiform discharges and a massive photoconvulsive response, and were consistent with an electrographic diagnosis of nonconvulsive status epilepticus.
Effect of Sodium Butyrate In Vitro
Since Sp1 could be important for histone acetylation,9,11 we studied the effect of the –270CG mutation on the acetylation status of histone 4 in the promoter region of PIGM, using chromatin immunoprecipitation assays. As expected, there was no evidence of histone acetylation in the promoter of the T-cell–specific gene CD3
in lymphoblastoid B-cell lines from either the patient or her mother (Figure 1A). Consistent with the function of a housekeeping gene, histone 4 at the promoter of GAPDH was fully acetylated in both cell lines. Similarly, acetylation at PIGM, itself a housekeeping gene, was also readily detected in the maternal lymphoblastoid cell line. However, in the patient's cell line, there was no evidence of histone acetylation at the PIGM promoter, suggesting that the –270 Sp1-binding motif is crucial for histone acetylation. Histone 4 acetylation was fully restored on exposure of the patient's lymphoblastoid cell line to sodium butyrate, which suggested the presence of promoter sequences that on inhibition of histone deacetylases could substitute for the disrupted –270 motif in promoting histone hyperacetylation.
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G mutation resulted in a 55% reduction in PIGM promoter activity (Figure 1B). In the presence of sodium butyrate, transcriptional activity increased by a factor of approximately 3 with both constructs. In the presence of plicamycin (formerly called mithramycin), an agent that blocks Sp1 binding to DNA,12,13 transcriptional activity was almost entirely abolished for both constructs, suggesting that Sp1 binding is critical for efficient transcription of PIGM. Plicamycin similarly reduced but did not completely abolish sodium butyrate–enhanced transcriptional activity: 55% and 18% residual activity was observed for the wild-type and mutant constructs, respectively. The increased transcriptional activity of the PIGM promoter in the presence of sodium butyrate was accompanied by an increase by a factor of 400 in PIGM messenger RNA (mRNA) levels in the patient's cell line (Figure 1C). At the same time, expression of surface GPI, as assessed by fluorescent inactivated aerolysin (FLAER) staining, was completely restored (Figure 1D). Taken together, these findings suggested that sodium butyrate–mediated transcriptional activation could take place even in the presence of the mutated Sp1 motif, causing inherited GPI deficiency. In addition to being histone hyperacetylation–dependent, this effect is also Sp1 binding–dependent and, to a lesser extent, Sp1-independent.
Effect of Sodium Butyrate In Vivo
In view of the intractable seizures experienced by the patient and guided by the effectiveness of sodium butyrate in restoring PIGM transcription and GPI expression in vitro by the mutant as well as the wild-type promoter, we started clinical therapy with sodium phenylbutyrate at a dose of 20 mg per kilogram of body weight three times a day. At the same time, levetiracetam, clobazam, and lamotrigine were withdrawn and sodium valproate added.
The in vivo effect of sodium phenylbutyrate on PIGM RNA levels and on surface GPI was assessed prospectively in peripheral-blood cells. A progressive increase in the proportions of granulocytes staining positive for FLAER and two GPI-linked proteins was observed, as compared with levels before the administration of sodium phenylbutyrate. Assessment of the effect of this treatment on PIGM transcription showed that 3 months after therapy began, PIGM mRNA levels in primary mononuclear blood cells increased by a factor of nearly 70 from baseline (from 0.5 to 33.0%) (Figure 2C). Thus, sodium phenylbutyrate increased PIGM transcription and cell-surface GPI expression in vivo as well as in vitro.
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Discussion
We show here that binding of Sp1 to its core promoter motif is crucial for control of the acetylation status and transcriptional activity at the PIGM promoter, findings with important therapeutic implications for patients with inherited GPI deficiency. As shown in Figure 1A, the –270 motif is required for maintenance of histone acetylation in the PIGM promoter. Its disruption by the mutation responsible for inherited GPI deficiency results in significant reduction in acetylation, PIGM transcription, and synthesis and cell-surface expression of GPI. These findings are consistent with the previously described function of Sp1 in regulating transcription through recruitment to the promoter of histone acetyltransferases,14,15 histone deacetylases,7,15 or both and its function through histone acetylation.
Despite disruption of Sp1 binding to the –270 motif, sodium butyrate increased histone acetylation and transcriptional activity of the mutant PIGM promoter, thereby restoring surface GPI expression in vivo as well as in vitro. Presumably, the other three predicted Sp1-binding motifs6 were responsible for these effects. Our findings with the use of plasmid promoter constructs (Figure 1B) also suggest that inhibition of histone deacetylases by sodium butyrate can lead to enhanced PIGM transcription through binding of Sp1 (and to a lesser degree of other transcription factors) to its cognate promoter motifs in its acetyl-modified form. This hypothesis is in line with previous reports showing that inhibition of histone deacetylases, as well as histone hyperacetylation, can lead to increased transcriptional activity through acetyl modification and increased affinity binding of Sp1.16,17
Regulation of acetylation and PIGM transcription by Sp1 might be important during embryonic and fetal development, when increased acetylation is required for coordinate expression of numerous genes. In this way, maintenance of an open chromatin configuration by histone acetylation would increase PIGM transcription and expression of GPI-linked proteins critical for neural development18,19 to levels sufficient to prevent lethal neurodevelopmental defects, such as those seen in female mice with a deleted Piga gene20 but inadequate to prevent severe epilepsy. We show that manipulation of acetylation in postnatal life with sodium phenylbutyrate can have a dramatic therapeutic effect on otherwise intractable seizures. The enhancing effect of sodium butyrate on PIGM mRNA and surface GPI expression on blood cells in vivo suggest that restoration of GPI biosynthesis in the central nervous system is responsible for the drug's clinical effects. However, since direct assessment of primary neural tissue was not possible, it cannot be ruled out that the neurologic improvement was caused by alternative actions of sodium phenylbutyrate on the central nervous system. Sodium valproate is an antiepileptic agent with properties of a histone deacetylase inhibitor21 and could have contributed to seizure control, along with sodium butyrate. Indeed, in our in vitro assays using concentrations of sodium valproate similar to the in vivo therapeutic range, we observed mild restoration of GPI expression in the patient's lymphoblastoid cell line but no synergistic or additive effect with sodium butyrate (data not shown). Presumably, these levels are not sufficient for control of epilepsy in vivo.
Whether treatment with sodium phenylbutyrate eliminates or reduces the risk of thrombosis in inherited GPI deficiency is not known. Thrombosis is the only clinical feature shared by inherited GPI deficiency and paroxysmal nocturnal hemoglobinuria; in the latter, the lack of CD59, an inhibitor of membrane-attack complex of complement, is thought to lead to hypercoagulable platelets and contribute to an increased risk of thrombosis.22 In inherited GPI deficiency, 30 to 50% of platelets are CD59-deficient.6 However, sodium phenylbutyrate had no effect on the level of CD59 expression on platelets (data not shown), despite increasing granulocyte CD59 expression.
In summary, we have shown that the mutation responsible for inherited GPI deficiency disrupts an Sp1-dependent butyrate response element and is associated with hypoacetylation at the promoter of PIGM. Modification of acetylation with sodium butyrate enhances transcription of PIGM and surface GPI expression in vivo as well as in vitro and is of great therapeutic value. Therefore, sodium butyrate may be an effective therapeutic option for other diseases caused by Sp1-dependent hypoacetylation.
Dr. Almeida is a Leukaemia Research Fund Clinical Research Fellow, and Dr. Karadimitris is a Leukaemia Research Fund Bennett Senior Fellow. Drs. Murakami, Maeda, and Kinoshita are supported by grants from the Ministry of Education, Culture, Science, Sports, and Technology of Japan. Equipment was donated to the investigators by INAPA.
No potential conflict of interest relevant to this article was reported.
We thank Dr. Richard Szydlo for performing the statistical analysis.
Source Information
From the Department of Haematology, Imperial College London, Hammersmith Hospital (A.M.A., I.A.G.R., D.M.L., A.K.), and the Paediatric Liver Centre, King's College Hospital (A.B.) — both in London; and the Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Osaka (Y. Murakami, Y. Maeda, T.K.), and the Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama (T.K.) — both in Japan.
Drs. Almeida and Murakami contributed equally to this article.
Address reprint requests to Dr. Karadimitris at the Department of Haematology, Imperial College London, Hammersmith Hospital, Du Cane Rd., London W12 0NN, United Kingdom, or at a.karadimitris{at}imperial.ac.uk.
References
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