Inherited glycosylphosphatidylinositol deficiency: a review from molecular and clinical perspectives

Abstract

Glycosylphosphatidylinositol (GPI) is a highly conserved post-translational modification in eukaryotes, which is essential for anchoring various proteins to the cell surface. Dysfunction of GPI biogenesis leads to human diseases, such as inherited GPI deficiency (IGD) caused by germline mutations in GPI-related genes. With accumulating reports on individuals with IGD, there has been increasing interest and studies on disease mechanism, diagnosis, and therapy. This review outlines the biosynthetic pathway of GPI-anchored proteins (GPI-APs) and summarizes clinical IGD cases from a molecular perspective. We also review current diagnostic and therapeutic approaches for IGD. Finally, we discuss future research directions to facilitate the understanding and treatment of GPI-related disorders.

Introduction

Cell-surface proteins play crucial roles in cellular function. In eukaryotes, proteins can be anchored to the plasma membrane through two primary mechanisms: transmembrane domain or glycosylphosphatidylinositol (GPI) modification. Forming an amide bond between the protein C-terminus and the phosphoethanolamine moiety in GPI, GPI-anchored proteins (GPI-APs) are anchored to the outer leaflet of the plasma membrane, in which the glycerophospholipid tail of GPI functions analogously to a single transmembrane domain of type-I transmembrane proteins. In humans, GPI modifies more than 150 protein substrates for their correct localization to the cell surface ( Supplementary Table S1). These GPI-APs comprise various types of proteins, including enzymes, receptors, adhesion molecules, and complement regulators, which are functionally essential for a list of biological processes such as signal transduction and intercellular communication.

As a structurally complex molecule, GPI contains a phosphoethanolamine linker, a glycan core, and a glycerophospholipid tail. The biosynthetic pathway of GPI-APs is conserved across eukaryotes, consisting of two phases that involve over 20 enzymatic reaction steps and 33 proteins encoded by the phosphatidyl inositol glycan (PIG) and post-GPI attachment to proteins (PGAP) genes [1]. The first phase primarily occurs in the endoplasmic reticulum (ER), in which phosphatidylinositol (PI) undergoes stepwise modifications by glucosamine (GlcN), mannoses (Man), and ethanolamine phosphates (EtNP) to form free GPI. GPI-APs are generated by the transfer of intact GPI to protein substrates, followed by a remodeling phase involving further alterations of the lipid and glycan moieties in the ER and Golgi apparatus.

It has been known for a long time that the deficiency or dysfunction of GPI biosynthesis leads to human diseases. For example, somatic mutations of PIGA in hematopoietic cells result in paroxysmal nocturnal hemoglobinuria (PNH), a rare and acquired hematologic disorder characterized by hemolysis, thrombosis, and impaired bone marrow function [2]. Additionally, cases have been reported in recent years with GPI deficiency caused by germline mutations, referred to as inherited GPI deficiency (IGD). Currently, 24 out of 33 genes involved in the GPI biosynthetic pathway have been identified as causative factors in IGD [ 3, 4]. Although IGD shares a common pathogenesis of defective GPI structure biosynthesis, the clinical manifestations vary widely, underscoring unknown mechanisms of pathogenicity across different IGDs.

In this review, we start by outlining the biosynthetic pathway of GPI-APs. The clinical reports of IGD are then summarized from a molecular perspective. We also review approaches for the diagnosis and treatment of IGD. Finally, future directions of the field are discussed.

Structure and Biosynthetic Pathway of GPI-APs

The GPI-APs comprise a highly complex structure. The C-terminus of a GPI-AP is conjugated via an EtNP bridge to a glycan core consisting of a glycomotif, Manα2Manα6Manα4GlcNα6inositol pentasaccharide. The phospholipid tail linked to the inositol anchors GPI-APs onto the cell membrane. The glycan core undergoes various modifications, including inositol acylation, α1,2-mannosylation of the third mannose, and elongation of the first mannose by β1,4-linked N-acetylgalactosamine (GalNAc), β1,3-linked galactose, and sialic acid. Of note, a recent study showed that GPI-APs can be alternatively linked to EtNPs on the second mannose [5].

The biosynthesis of GPI-APs involves a streamlined multienzymatic cascade ( Figure 1A). Free GPI is synthesized from PI in the ER lumen in the first phase of the cascade, which takes a total of 10 steps (11 steps with an optional step). At the cytosolic side of the ER membrane, the first step begins with the transfer of N-acetylglucosamine (GlcNAc) on PI at the 6-position of inositol by the GPI N-acetylglucosamine transferase (GPI-GnT). As one of the most complex glycosyltransferases, the GPI-GnT comprises seven subunits including PIGA, PIGC, PIGH, PIGP, PIGQ, PIGY, and DPM2, with PIGA being the catalytic subunit [ 6, 7]. The core complex of PIGA, PIGC, and PIGH is stabilized by PIGQ, while DPM2 enhances the enzyme activity. PIGL, a deacetylase, catalyzes the subsequent de- N-acetylation step of GlcNAc-PI to generate glucosaminyl PI (GlcN-PI), which flips into the luminal side of the ER catalyzed by a recently discovered lipid scramblase, CLPTM1L [8]. The 2-position of inositol in GlcN-PI is then acylated (e.g., palmitoylated) by the acyltransferase PIGW, with acyl-CoA as the acyl donor. The resulting GlcN-(acyl)PI undergoes a diacyl to alkyl-acyl transition in the diacylglycerol moiety. Although the exact enzyme for this remodeling is still unclear, it requires the 1-alkyl phospholipid synthetic pathway in the peroxisome [9].

Figure 1 .

Biosynthetic pathway of GPI-Aps

(A) In the first phase, free GPI is synthesized and transferred to the proproteins. GPI-APs undergo several remodeling steps before translocating onto the plasma membrane. (B) Schematic diagram showing the genes with reported mutations causing IGD in humans. ER, endoplasmic reticulum; PM, plasma membrane; “±”, optional structures or synthetic steps; “?”, unknown enzymes. GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine.

Using dolichol-phosphate-mannose (Dol-P-Man) as the glycosyl donor, two mannose molecules (referred to as Man1 and Man2) are sequentially transferred to the GlcN-(acyl)PI to generate Manα4GlcN-(acyl)PI and Manα6Manα4GlcN-(acyl)PI. These mannosylation steps are catalyzed by two GPI-mannosyltransferases (GPI-MTs), PIGM and PIGV, which are also known as GPI-MTI and GPI-MTII, respectively [ 10, 11]. A chaperone protein PIGX stabilizes PIGM to ensure its catalytic activity [12]. In the next step, PIGN catalyzes the transfer of EtNP from phosphatidylethanolamines (PEs) to the 2-position of Man1 and generates Manα6(EtNP)2Manα4GlcN-(acyl)PI [13]. Subsequently, a third mannose (Man3) is transferred by GPI-MTIII or PIGB [14], followed by the addition of two EtNP groups to the 6-positions of Man2 and Man3 by two GPI-ethanolaminetransferases (GPI-ETs), PIGO and PIGG [ 15, 16]. PIGF stabilizes both PIGO and PIGG as a molecular chaperone [17]. The resulting EtNP6Manα2(EtNP)6Manα6(EtNP)2Manα4GlcN-(acyl)PI is the major form of GPI for synthesizing GPI-APs. GPI with a fourth mannose (Man4) linked to the 2-positon of Man3 also serves as a precursor for attachment to proteins, which is optionally synthesized by PIGZ (i.e., GPI-MTIV) [18].

The preproproteins of all GPI-APs contain an N-terminal and a C-terminal signal peptides for ER translocation and GPI attachment, respectively. The N-terminal signal guides the nascent polypeptide chain out of the ribosome into the ER, which is similar to other secretory proteins [19]. The C-terminal signal peptide usually consists of 20–30 amino acids starting from the ω+1 site, where the ω site is defined as the amino acid to which GPI is attached. Although no conserved motif is identified for the C-terminal signal peptide, the ω and ω+2 sites are found to be always small amino acids, such as Ser, Asn, Asp, Ala, Gly, Cys, and Thr [20]. Upon the cleavage of the N-terminal signal peptide, the GPI transamidase (GPI-T) recognizes the C-terminal sequence of proproteins and cleaves the amide bond between the amino acids at the ω and ω+1 sites. This process leads to the generation of a substrate-enzyme intermediate via a thioester bond between the carboxyl group of the ω amino acid and the cystine residue of PIGK, which serves as the catalytic subunit of GPI-T. The thioester is then attacked by the terminal EtNP group in GPI precursors to form nascent GPI-APs. Besides PIGK, GPI-T also contains four other subunits, GPAA1, PIGT, PIGS, and PIGU, whose functions were poorly understood until the structure of the intact GPI-T complex was recently determined [ 21– 23]. It was characterized that the PIGT and PIGU form a platform for complex assembly, PIGS stabilizes the catalytic conformation of PIGK, and the protease-like GPAA1 subunit helps recruit protein substrates instead of cleaving the C-terminal peptide.

After the attachment of GPI-AP proproteins to the GPI precursor, a series of remodeling steps take place before the mature GPI-APs are displayed on the plasma membrane. In the ER lumen, PGAP1 first removes the acyl chain on the 2-position of inositol [24]. In some cell types such as erythrocytes, this acyl chain is preserved in the mature GPI-APs, which makes GPI-APs resistant to phosphatidylinositol-specific phospholipase C (PI-PLC). Subsequently, the EtNP on Man2 is removed by PGAP5. After these two steps, the GPI-APs are transported to the Golgi apparatus by COPII-coated vesicles. In the Golgi apparatus, the fatty acid moieties of GPI-APs are further remodeled by replacing the unsaturated chains with saturated ones by PGAP3 and PGAP2, which catalyze the removal of unsaturated fatty acids and the attachment of saturated chains, respectively [25]. In addition to lipid remodeling, the glycan core can be further modified with β1,4-linked N-acetylgalactosamine (GalNAc) to Man1 by PGAP4 [26], β1,3-linked galactose (Gal) to GalNAc by B3GALT4 [27], and sialic acid by an unknown sialyltransferase.

Biological Function of GPI-APs

The GPI anchor is widely distributed across eukaryotic organisms. In vertebrates, GPI-APs are prevalent in virtually all cell and tissue types. Through interactions with specific lipids such as sphingomyelin and cholesterol, GPI-APs are enriched in lipid rafts on the plasma membrane, which facilitates their engagement in signaling pathways or their recruitment to specific membrane microdomains involved in endocytosis or cell adhesion. Cell-surface GPI-APs undergo dynamic turnover, which is regulated by a variety of cellular GPIases. For example, GPI-specific phospholipase D (GPI-PLD) specifically recognizes and cleaves the phosphodiester bond between inositol and the glycerophospholipid moiety [28]. This cleavage releases GPI-APs from the cell surface, which allows for the secretion or regulation of cell-surface levels of specific GPI-APs. Furthermore, GPI-APs play crucial roles in shaping the polarized structures of a variety of cells, such as epithelial cells and neurons. For example, certain GPI-APs are enriched on the apical surface of epithelial cells, where they participate in processes such as cell-cell adhesion, nutrient uptake, and signal transduction [29].

Among mammalian GPI-APs, representative examples include enzymes such as alkaline phosphatase (ALP), acetylcholinesterase (AChE), and 5’-nucleotidase (5’-NT). Moreover, membrane complement regulatory proteins (mCRPs), such as CD55 and CD59, which are essential in inhibiting the complement cascade, rely on GPI anchoring. In addition, GPI-APs comprise a list of adhesion proteins such as CD56 (also known as NCAM1), glypican, and members of the contactin family. Importantly, a list of GPI-anchored proteins is implicated in regulating synapse formation and neuronal activities ( Supplementary Table S1). We performed Gene Ontology (GO) analysis (using DAVID v6.8 with default parameters) on all human GPI-APs, which clearly showed enrichment in biological processes such as axon guidance and nervous system development ( Figure 2). This result highlights the potential functions of GPI-APs in the nervous system and underscores their relevance to the manifestation of neurological symptoms observed in certain cases of IGD [ 30, 31], which are discussed in the following sections.

Figure 2 .

Gene Ontology analysis of human GPI-Aps

Results highlight the enrichment of GPI-APs in pathways related to the nervous system.

Human Diseases Caused by GPI Deficiency

Given the essential functions of GPI-APs, genetic deficiencies in GPI biosynthesis manifest across a spectrum of human diseases. The first GPI deficiency with molecular mechanism unraveled is PNH [2]. Somatic mutations of PIGA in hematopoietic stem cells (HSCs) have been recognized for a long time as the singular cause of PNH. As PIGA is localized in the X chromosome, loss-of-function mutations of a single allele result in a GPI defective phenotype. Dysfunction of PIGA diminishes the GPI-GnT activity, hindering the biosynthesis of GlcNAc-PI and the subsequent production of GPI-APs. Recently, PNH cases with biallelic loss-of-function mutations of PIGT have been reported [32]. In such a case, one PIGT allele had germline mutations whereas the other PIGT was lost somatically in hematopoietic stem cells. The resulting genotype causes the inactivation of GPI-T. Another gene related to PNH is PIGB, a family with coby number-neutral loss of a germline heterozygous PIGB mutation [33]. Consequently, individuals with PNH caused by either PIGA, PIGT and PIGB variations exhibit a complete or severe loss of GPI-APs in their peripheral immune cells. Notably, among these GPI-APs, CD55 and CD59 play pivotal roles in shielding cells from complement-mediated attacks by inhibiting the complement cascade. In PNH patients, the defective GPI biosynthesis in erythrocytes reduces the levels of cell-surface CD55 and CD59, rendering them vulnerable to complement-driven cell lysis.

Conversely, germline mutations of GPI biosynthetic genes lead to IGD, a wide spectrum of diseases with more severe symptoms than PNH. Notably, various conditions, once classified by their clinical manifestations, have now been reclassified under the umbrella of IGD, such as hyperphosphatasia with mental retardation syndrome (HPMRS) and multiple congenital anomalies hypotonia seizures (MCAHS) [34]. The first case of IGD was reported in 2006, with three children from two consanguineous families identified to harbor a homozygous hypomorphic mutation in the promoter region of PIGM [35]. This mutation disrupted PIGM transcription, resulting in the impaired synthesis of Manα4GlcN-(acyl)PI and subsequently GPI-APs in specific cell types. These children exhibited symptoms such as severe seizures and thrombosis in the hepatic or portal vein, which were distinct from those in PNH patients. With the widespread adoption of whole genome sequencing in the clinic, 24 out of the 33 GPI-related genes have now been designated as causative genes for IGD ( Figure 1B; See Table 1 for a detailed summary), which are involved in core GPI biosynthesis, its transfer to proteins, and GPI-APs’ remodeling. The emerging picture is that every step in the GPI-AP biogenesis is critical.

Table 1 Summary of reported IGD cases

Genes	Reportedcases	GPI-APs 1	SerumALP 1	Seizure 2	EE 2	Hypotonia 2	DD/ID 2	Dysmorphic features 2	Hand/feet anomalies 2	Hearing impairment/loss 2	Ophthalmological anomalies 2	Cardiac anomalies 2	GU malformation 2	GI anomalies 2	CDH 2	Brain MRI/CT 3	Other manifestations
PIGA	85	↓	N/↑	+	+	+	+	+	+	+	+	+	+	+	+	①②③④	Hemochromatosis, Fryns-like phenotype
PIGQ	11	↓	N/↑	+	+	+	+	+	+	+	+	+	+	+	−	①④	Ataxia
PIGC	5	↓	N	+	−	+	+	+	+	−	+	−	−	−	−	①
PIGH	8	↓	N	+	−	+	+	+	−	−	−	−	−	+	−	①③	Scliosis, bone fracture, microcephaly
PIGP	7	↓	N	+	+	+	+	−	−	−	+	−	−	+	−	①③	Peripheral spasticity, dyskinesia
PIGY	4	N/↓	N/↑	+	+	+	+	+	+	−	−	−	−	+	−	Normal	Microcephaly
PIGL	15	↓	↑	+	−	+	+	+	+	+	+	+	−	−	+	⑤	Ichyosiform dermatosis, leukemia
PIGW	8	↓	N/↑	+	+	+	+	+	−	−	−	−	+	−	+	②③⑤⑥	Recurrent respiratory infection, inguinal hernia, umbilical hernia
PIGM	7	↓	N	+	−	+	+	−	−	−	−	−	−	−	−	②③⑦	Cerebral or portal vein thrombosis, macrocephaly
PIGX	1	NA	NA	+	−	+	+	−	−	+	+	−	−	−	−	NA
PIGV	26	↓	↑	+	−	+	+	+	+	+	+	+	+	+	−	①⑤⑧	Macrocephaly
PIGN	61	↓	N/↑	+	+	+	+	+	+	−	−	+	+	+	+	①⑤	Fryns syndrome, abdominal wall defect, cleft lip
PIGB	16	↓/N	↑	+	+	+	+	+	+	+	+	−	−	+	−	①⑤⑨	Axonal neuropathy, DOORS syndrome
PIGF	2	↓	NA	+	−	−	+	+	+	−	−	+	−	−	−	Normal	OORS syndrome
PIGO	17	↓/N	↑	+	+	+	+	+	+	−	−	+	+	+	−	②⑤	Ataxia
PIGG	30	↓/N	N	+/−	−	+/−	+	+/−	−	−	−	−	−	−	−	②	Ataxia, Emm− RBC phenotypes, microcephaly, autism spectrum disorder
PIGK	14	↓	N/↓	+/−	+	+	+	+	−	−	−	−	−	+	−	②⑤
GPAA1	18	↓	N	+/−	−	+	+	+	−	−	−	−	−	+	−	②	Ataxia, nystagmus, microcephaly and osteopenia
PIGS	13	↓/N	N/↑	+	+	+	+	+	+	+	+	−	−	+	−	②③
PIGT	51	↓/N	↓/N	+	+	+	+	+	+	+	+	+	+	+	−	②③	Osteoporosis, epileptic apnea
PIGU	5	↓/N	↓/N	+	−	+	+	+	−	−	+	−	−	−	−	①②⑤⑥	Scoliosis
PGAP1	11	N	N	+	−	−	+	+	−	−	+	−	−	−	−	①②③	Microcephaly
PGAP3	65	↓/N	↑	+	−	+	+	+	+	+	+	+	−	+	−	①②⑤⑥	Cleft palate, ataxia, microcephaly or macrocephaly, scoliosis,
PGAP2	14	↓	↑	+	−	+	+	−	−	+	−	+	−	−	−	②③⑥	Cleft palate, Microcephaly

Abbreviations: ALP: alkaline phosphatase; EE: epileptic encephalopathy; DD/ID: developmental delay/ intellectual delay; GU: genitounrinary; GI: gastrointestinal; CDH congenital diaphragmatic hernia; DOORS syndrome: deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures; OORS: onychodystrophy, osteodystrophy, impaired intellectual development, and seizures syndrome.

¹ “N”, “↓”, and “↑” are referred to as the normal, decreased, and increased level, respectively. “NA” indicates the data is not available.

² “+” and “−” indicate symptoms in the cases that have or have not been reported, respectively.

³Brain MRI/CT scanning results are abbreviated as: ① white matter immaturity/delayed myelination; ② cerebellum atrophy; ③ cerebral atrophy; ④ leukoencephalopathy; ⑤ Thin corpus callosum; ⑥ Dandy-Walker malformation; ⑦ infarction; ⑧ hydrocephalus; ⑨ polymicrogyria. “NA” indicates the data is not available.

The clinical presentation of IGD can vary widely depending on the specific genes affected and the severity of the mutations. However, there are some common clinical manifestations. For example, IGD patients usually experience neurological abnormalities, including developmental delay, intellectual disability, seizures, and hypotonia. These symptoms are often present in early life, as developmental epileptic encephalopathy has been diagnosed in IGD patients with variations of PIGA, PIGQ, PIGP, PIGY, PIGW, PIGN, PIGB, PIGO, PIGK, PIGS, and PIGT [ 36– 48]. MRI scanning of IGD patients may reveal white matter immaturity, delayed myelination, cerebral atrophy, thin corpus callosum, and cerebellar atrophy [49]. In a retrospective study covering 273 IGD cases, seizures were found to manifest as generalized, focal, or undetermined, with a predominance of motor seizures [49]. Among the focal motor seizures, myoclonic seizures (41/77) and epileptic spasms (17/77) were observed most frequently. Regarding the age of seizure onset, available data indicate a minimum onset age of 0.03 months, a maximum of 276 months, with a median onset age of 7 months.

In addition, IGD can affect multiple organ systems, leading to organ abnormalities. These may include abnormalities in the cardiovascular and gastrointestinal systems, as well as anomalies in the kidneys and other organs. Among the reported cases, it was observed that symptoms beyond those primarily affecting the neurological system frequently manifested in patients harboring mutations in genes responsible for synthesizing GPI-APs, rather than those involved in their maturations and remodeling [50]. For example, certain gastrointestinal anomaly such as Hirschsprung disease was observed in patients with variations of PIGV and PIGO. In addition, congenital diaphragmatic hernia (CDH) was observed for IGD patients with PIGA, PIGL, PIGW, and PIGN variations [ 39, 44, 51, 52]. These observations suggest functional roles of cell-surface GPI-APs in other organs beyond the brain.

Moreover, dysmorphic features are also commonly reported for IGD, such as hypertelorism, broad nasal bridge, tented upper lip, cleft palate, brachytelephalangy, and hypoplastic nails [ 4, 34]. As many receptors crucial for immune activation and response are anchored on the cell surfaces by GPI, immunological abnormalities have been described in some IGD cases. For example, recurrent respiratory tract infections, which are often fatal for individuals with IGD, have been documented in some cases [3].

Diagnosis of IGD

Although hundreds of IGD cases have been reported, there are currently no established diagnostic guidelines for this rare disease. In clinical practice, the diagnosis of IGD typically involves a combination of clinical evaluation, genetic analysis, and laboratory testing ( Figure 3). The presence of common symptoms such as intellectual disability, developmental delay, seizures, and dysmorphic features, in the absence of other evident cues, may prompt suspicion of IGD. Genetic analysis, frequently conducted through whole exome sequencing (WES) of the primary cases and their parents, facilitates the identification of variations in GPI-related genes. Most IGD-associated genes are located on autosomes, except PIGA, an X-linked gene, making the majority of IGD an autosomal recessive disorder [4]. As a result, only homozygous and compound heterozygous variations are considered pathological. Furthermore, as many variants occur within the coding regions of respective genes, the assessment of amino acid changes should follow the guidelines outlined by the American College of Medical Genetics and Genomics (ACMG) to identify potential pathogenic variations.

Figure 3 .

Diagnosis of IGD

Clinical evaluation, genetic analysis, and laboratory testing are collectively applied in the diagnosis of IGD. ALP, alkaline phosphatase; DD/ID, developmental delay/intellectual delay; GU, genitounrinary; GI, gastrointestinal.

Besides genetic analysis, follow-up laboratory testing is crucial to validate the GPI defect. Flow cytometry analysis can detect the levels of certain GPI-APs on the cell surface, which is typically performed on blood cells and skin fibroblasts. Commonly tested targets include CD55 and CD59, which are expressed in almost every cell type; and CD16, which is primarily expressed by monocytes and granulocytes. In addition, fluorescent aerolysin (FLAER), which is an inactive variant of bacterial toxin that can pan-specifically recognize the core GPI structure on all GPI-APs, is frequently used to quantify the overall level of GPI-APs [53]. Besides IGD, FLAER is also widely used in the diagnosis of PNH [2].

Of note, while most cases of IGD display a global decrease in cell-surface GPI-APs, exceptions have been observed in cases involving variations of PIGB, PIGO, PIGG, PIGS, PIGT, PIGU, PGAP1, and PGAP3 [4]. These exceptions can be partially explained by the nonessential steps, alternative GPI linkages, and potential compensatory mechanisms. For example, the loss of PIGG, which transfers an EtNP group on the Man2, appears to have minimal influence on the levels of cell-surface GPI-APs [5]. In cases carrying variations of PIGB and PIGO, although the synthesis of Man3 and Man3-EtNP is impaired, GPI-APs can be alternatively attached to the EtNP moiety on Man2, as recently discovered [42]. Moreover, it seems that the remodeling steps are not crucial for the localization of GPI-APs on the cell surface. This is exemplified by the normal level of CD59 on erythrocyte surfaces in patients with pathogenic mutations in PGAP3 [54]. Interestingly, genetic variations in some IGD cases do not uniformly affect all GPI-APs. In cases with dysfunctional PIGS, PIGT, and PIGU, there is a decrease in cell-surface CD16 on granulocytes, whereas CD55 and CD59 remain unaffected [ 55– 57]. Investigating the prioritization of GPI attachment to different GPI-APs in cells with partial GPI deficiency is an interesting future direction.

As hyperphosphatasia is frequently found in IGD patients, the serum level of alkaline phosphatase (ALP) serves as another biomarker and clinical evidence for IGD [ 4, 58]. In cells unable to synthesize free GPI, the GPI-T complex recognizes and cleaves the C-terminal hydrophobic signal peptide of a list of GPI-APs, including ALP, resulting in the secretion of these GPI-APs in soluble form [59]. However, the process does not occur in cases lacking the GPI-T activity, where ALP proproteins are subjected to ER-associated degradation (ERAD) .

Treatment of IGD

As a genetic disorder, the treatment of IGD is primarily supportive and aimed at managing the symptoms. For instance, antiepileptic drugs (AEDs) may be prescribed to control seizure activity [49]. Among the various treatments investigated, vitamin B6 therapy has shown promising efficacy in controlling neurological symptoms by augmenting intracellular pyridoxal phosphate (PLP) level [60]. Vitamin B6 contains three forms-pyridoxine, pyridoxal, and pyridoxamine-which are typically phosphorylated. The main active form, PLP, undergoes initial dephosphorylation by cell-surface alkaline phosphatase, particularly the tissue nonspecific alkaline phosphatase isozyme (TNAP), to yield free pyridoxal, which is then taken up by cells [ 60, 61]. Once inside cells, pyridoxal is re-phosphorylated into PLP, activating a myriad of PLP-dependent enzymes, with the most crucial one being γ-aminobutyric acid (GABA) synthetase. In IGD patients, the absence or reduction of cell-surface TNAP diminishes PLP uptake and subsequently affects the activities of PLP-dependent enzymes. Studies have demonstrated that vitamin B6 supplementation significantly improves outcomes in IGD by reducing seizure activity. For instance, analysis of neurotransmitter levels in cerebrospinal fluid (CSF) revealed decreased PLP and 5-methyltetrahydrofolate in a case with a PGAP2 variation [62]. Supplementation with vitamin B6 normalized these biochemical abnormalities and facilitated developmental progress. The dosage of vitamin B6 supplementation is typically 20‒30 mg/kg. In another pilot study that enrolled 9 patients diagnosed with IGDs, all patients received 20‒30 mg/kg of vitamin B6 for 1 year [63]. Seizure frequencies were markedly (>50%) and drastically (>90%) reduced in three and one patients who experienced seizures, respectively. Eight of nine patients exhibited modest improvements in development, and none of them exhibited diarrhea.

Conclusion and Perspective

The GPI modification serves as an important mechanism to anchor proteins on the cell surface. The biosynthetic pathway of GPI-APs relies on a multistep process, and any disruption at a single step may affect the entire collection of GPI-APs, i.e., GPI-APome. As a result, somatic or germline variations of genes responsible for synthesizing GPI-APs cause severe pathologies and symptoms. Unlike PNH, where the pathological mechanism is well-elucidated, the relationship between genotype and phenotype in IGD remains poorly understood. One promising future direction in this field is to develop new methods for large-scale GPI-APomics analysis, which is crucial for the identification of affected GPI-APs in IGD patients. Subsequent studies using genetically modified animal models would help elucidate which GPI-APs are causal factors for the clinical manifestations observed in IGD patients. In addition, the functions of glycan moieties on GPI-APs are currently poorly understood, which represent a potential mechanism through which dysfunctions in GPI biogenesis lead to human diseases. Future studies on the roles of glycan in GPI-APs are of great interest.

On the other hand, new diagnosis methods and therapies for IGD are urgently needed. Beyond genetic analysis, new strategies for experimentally validating the surface anchoring of GPI-APs, such as flow cytometry and chemoproteomics analysis, are promising approaches for diagnosing IGD. For therapy, one potential option is the supplementation of synthetic GPI. In vitro studies have shown that cell-surface GPI-APs in cells lacking PIGA or PIGL can be rescued by supplementing synthetic fragments of GlcNAc-PI or GlcN-PI, respectively [64]. However, supplementation with synthetic fragments of GlcN-(acyl)PI or Manα4GlcN-(acyl)PI could not rescue GPI-APs in PIGW or PIGM knockout cells, respectively, for unknown reasons. Further investigations into the underlying mechanism are needed to determine whether this approach is effective.

Similarly to other genetic disorders, gene therapy holds promise as a solution to IGD treatment. Studies using adeno-associated virus (AAV)-based therapy to express the full length of PIGO have successfully restored the expression of GPI-APs on granulocytes and rescued the growth defect in a PIGO-deficient mouse model [65]. Further explorations of gene editing technologies and expression vectors in IGD models are necessary to thoroughly assess safety and efficacy before advancing to clinical trials.

Supplementary Data

Supplementary data is available at Acta Biochimica et Biophysica Sinica online.

COMPETING INTERESTS

The authors declare that they have no conflict of interest.

Funding Statement

This work was supported by the grants from the National Natural Science Foundation of China (Nos. 22321005 and 22037001 to X.C.; Nos. 22A20339, 81601131, 82071263, and 12026606 to Y.J.), and the Beijing Natural Science Foundation (No. 5244034 to Q.T.). Q.T. is supported by the National Postdoctoral Program for Innovative Talent. X.C. is a recipient of Xplorer Prize.