The genotypic and phenotypic spectrum of PIGA deficiency

Glycosylphosphatidylinositol (GPI) is a glycolipid that is synthesized and transferred to proteins in the membrane of the endoplasmic reticulum [1]. Biogenesis of GPI anchored proteins is a conserved post-translational mechanism in eukaryotes and is important for attaching these proteins to the cell membrane, for protein sorting, trafficking, and dynamics [1], and plays an essential role in embryogenesis, neurogenesis, immune responses, and fertilization [2-6]. To date, more than 20 phosphatidylinositol glycan biosynthesis protein (PIG) subclasses have been found to be involved in GPI anchor biosynthesis and remodeling, and more than 150 proteins carry GPI anchors [1]. An increasing number of human diseases have been discovered to be due to mutations in GPI biosynthesis genes.

PIGA (MIM 311770) encodes one of the seven proteins involved in the transfer of N-acetylglucosamine (GlcNAc) from UDP-N-acetylglucosamide (UDP-GlcNAc) to phosphatidylinositol (PI) to form GlcNac-PI [1]. This is the first step of GPI anchor biosynthesis and takes place on cytoplasmic side of the endoplasmic reticulum [1]. The human PIGA gene is located on chromosome Xp22.2. It spans 162 kb and the longest transcript (NP_002632.1) encodes for a protein of 484 amino acids expressed in a wide variety of tissues including brain, liver, heart, and blood cells [7]. Somatic PIGA mutations had been well documented in PNH [MIM 300818], an acquired hemolytic disease that manifests after clonal expansion of hematopoietic cells with somatic PIGA mutations, where loss of CD55 and CD59 on erythrocytes causes complement-mediated lysis [8-12]. Unlike somatic PIGA mutations, germline mutations had not been observed until recently, and based on experiments in mice [2] and in both murine [13] and human embryonic stem cells [14] it had been proposed that germline PIGA mutations were lethal. In 2012, using an X-chromosome exome next-generation sequencing screen, Biesecker and colleagues identified a PIGA germline nonsense mutation in two siblings with an early epileptic encephalopathy with hypotonia, brain anomalies (myelination abnormalities and a thin corpus callosum), cleft palate, cardiac anomalies and early death [15]. Recently, four additional clinical reports were published on patients with germline PIGA mutations depicting a wide spectrum of phenotypes and clinical diagnoses [7,16-18], including West syndrome [18], Multiple congenital anomalies-hypotonia-seizures syndrome 2 (MCAHS2) [17,18] and Ferro-Cerebro-Cutaneous syndrome (FCCS) [16]. Here, we report a male patient with MRI brain abnormalities that resemble those of infants with maple syrup urine disease (MSUD), multi-organ involvement, therapy-responsive dyslipidemia, and reductions of mitochondrial respiratory complexes I and V on Blue Native Gel (BNG) analysis. Using WES, we identified in this patient a new missense PIGA germline mutation (g.15342986C>T, c.989G>A [NM_002641], [p.S330N]), which will be referred to as c.989G>A (p.S330N). We also review a total of 8 mutations from 9 unrelated families, summarize clinical findings, discuss genotype–phenotype correlations and identify common features that may be used to guide clinical identification of patients with germline PIGA mutations.

This study was approved as part of our TIDEX gene discovery project by the Ethics Board of the Faculty of Medicine of the University of British Columbia (UBC IRB approval H12-00067). Parents provided written informed consent for publication of this report.

To date, seven germline mutations in PIGA have been discovered using WES technologies in eight unrelated families [7,15-18]. Locations of the pathogenic variants are listed in Table 1 and highlighted on a multi-sequence alignment of PIGA proteins in Figure 2. PIGA germline mutations have been found in XLIDD male patients with a wide spectrum of clinical diagnoses (Table 1). Mothers of the affected males were confirmed to be carriers in all except for one family (Table 1) where the DNA for the mother was unavailable [18].

The phenotypic spectrum of PIGA germline mutations has shown wide variation, as reflected by the range of clinical diagnoses summarized in Table 1. However, a common set of characteristic features, shared by our patient, has emerged including infantile spasms with hypsarrhythmia on EEG (Table 1) and IDD. Generally, the phenotypic spectrum could be classified into two types (severe and less severe), as proposed by Kato et al. [18], where presence of facial dysmorphism correlates very well with the more severe spectrum of clinical manifestations (Table 1). In addition to early onset infantile spasms with hypsarrhythmia on EEG, IDD and profound developmental delay, the patients with more severe manifestations of PIGA germline mutations (patients: IV-2, IV-4 [15], III-1 [17], III-9 [16], 1, 2, and 5 [18], and our case [this report]) also present with dysmorphic facial features, multiple CNS abnormalities, such as thin corpus callosum and delayed myelination, as well as hypotonia (Table 1). Other phenotypes such as polyhydramnios, joint contractures, hyperreflexia, cardiac anomaly, severe developmental delay and elevated alkaline phosphatase are also recurrently seen in these patients, while additional phenotypes appear to be allele-specific (Table 1). Unlike patients with severe phenotypes, the less severe form of PIGA germline mutations (patients: IV-2 [7], 3 and 4 [18]) did not involve facial dysmorphism and multiple CNS abnormalities, but did present with early onset of infantile spasms with hypsarrhythmia on EEG (Table 1), and generally longer life span marked by IDD, treatable seizures and PDD (Table 1).

Early onset infantile spasms appear to be a common feature of PIGA mutations, as is seen with many other defects in GPI-anchor biosynthesis. Chiyonobu et al. [27] hypothesized that these seizures are due to intracellular pyridoxal phosphate deficiency secondary to the loss of membrane bound alkaline phosphatase, which is required to initiate pyridoxal-phosphate for transit of the plasma membrane (after which it is rephosphorylated). Given that GABA synthase requires intracellular pyridoxal-phosphate, it is the intracellular GABA deficiency which likely leads to the onset of seizures [27].

Additional features are frequently reported in patients with the severe form of PIGA germline mutations, as proposed by [18] including: hypotonia (7/8 patients with available data), elevated alkaline phosphatase (5/5 patients with available data), hyperreflexia (5/5 patients with available data), joint contractures (5/6 patients with available data), cardiac anomalies (4/4 patients with available data) and polyhydramnios (4/8 patients with available data) possibly due to dysphagia associated with hypotonia. The plagiocephaly facial dysmorphism in our and published cases is likely secondary to the hypotonia, causing facial asymmetry and distortion of proportions as well as secondary contractures. Our patient specifically appears to have a short nose with anteverted nares and a depressed nasal bridge, seen as the triangular structure at the base of the nose (Figure 1A). This feature appears to be present in the more severely affected published cases [18]. Additional features such as dental problems (e.g. microdontia, delayed eruption) [16,17], liver problems [15,16,18] visual impairment and deafness [16] appear to be recurrent as well (Table 1). On the other hand some features, such as accelerated linear growth and obesity [17], CNS iron deposition and ichthyosis [16], dyslipidemia and stomatocytosis (in the patient reported here) are reported only once. The cause of death in patients with PIGA mutations has been mainly due to cardiac arrest, pneumonia and respiratory failure (Table 1). Although the life span of the patients affected with less severe mutations is generally longer, there is a great degree of variability in life-span even in patients with the same germline mutation [15,16,18,28].

Reports of brain MRI findings in PIGA deficiency describe white matter immaturity with insufficient myelination, cerebral atrophy, thinning of the corpus callosum, and a small cerebellum [7,15,18]. Cerebral atrophy appears to progress rapidly and is associated with abnormal white matter myelination, consistent with an early neurodegenerative process [18]. Our case showed a similarly rapid progression, with an established cerebral atrophy associated with disrupted subcortical myelination at age 2.5 years. However, the most remarkable finding in the early stages of the disease was restricted diffusion in the brainstem tegmentum, superior cerebellar peduncles, subthalamus, and ventral striatum, indicative of intramyelin edema, involving selectively white matter regions that are already physiologically myelinated at birth. Kato et al. [18] also reported three cases showing restricted diffusion at the brainstem, basal ganglia, thalamus, and deep white matter. Interestingly, these findings are remarkably similar to those of the classical form of MSUD, an amino aciduria caused by deficiency of branched chain α-keto acid dehydrogenase enzyme, in which brain MRI of affected newborns or infants studied during stages of metabolic decompensation shows prominent signal changes and swelling within myelinated brain areas representing intramyelin edema, caused by a deficit of Na+/K+ ATPase activity as a result of impairment in energy production secondary to branched chain amino acids accumulation [29,30]. In particular, the findings of our case are remarkably similar to those shown in figure six in the report by Rossi and Biancheri [31], implicating that the diagnosis of PIGA deficiency could be suggested in neonates with similar brain MRI findings and unremarkable plasma amino acid and urine organic acid profiles. An explanation for the similarities remains speculative; the typical lesions in the newborn with MSUD involve preferentially those structures that are already myelinated at birth and thus with higher metabolic demands and most vulnerable to the energy deficiency and toxicity of branched-chain 2-oxo acid(s). In PIGA deficiency, although toxins have not yet been identified, we speculate that mitochondrial and thus energy deficiency could similarly play a role in the etiology of the observed intra-myelin edema. Regardless of the implicated pathophysiologic mechanism, identification of similarities in the lesion distribution and appearance on MRI represents the foundation of the pattern recognition approach, whereby neuro-imaging can guide the diagnostic process to reduce the number of unnecessary tests and time to diagnosis [32]. PIGA catalyzes the first step in GPI biosynthesis and it is anticipated that even a partial defect in activity will have a significant impact on the localization and functionality of a broad range of GPI-anchored proteins. The elevation in serum alkaline phosphatase reported here as well as in the majority of other PIGA deficient cases (likely correlated with severity of the disease) (Table 1) as well as other PIG deficiencies results from the secretion of GPI-deficient alkaline phosphatase (normally membrane-anchored) and may serve as a diagnostic clue. Normal levels of alkaline phosphatase however, do not rule out the condition, as evidenced by the mild case reported by Belet et al. [7]. The hypertriglyceridemia and serum LPL deficiency observed in our patient is also likely caused by abnormal GPI biosynthesis. LPL release from endothelial cells in response to heparin stimulation requires the activity of GPIHBP1 (GPI-anchored high-density lipoprotein binding protein 1) and primary genetic defects in this GPI-anchored protein lead to hypertriglyceridemia and non-detectable serum lipoprotein lipase [33]. The GPI biosynthesis defect resulting from PIGA mutation likely leads to a secondary GPIHBP1 deficiency, although further evaluation is required to confirm this mechanism, as a similar defect has not been described or investigated in other individuals with PIGA defects.

The germline mutational spectrum for PIGA comprises various mutation types including one nonsense mutation [15,18], one frameshift mutation that results in production of 36 amino acids shorter PIGA protein [7], one small in-frame deletion [16], and four missense mutations [17,18] (Figure 2). PIGA protein is well conserved from yeast to human (Figure 2), and all of the reported mutations, except for c.76dupT, occurred at evolutionarily conserved or semi-conserved amino acids (Figure 2). The c.76dupT frameshift mutation occurs in the non-conserved part of the protein but results in translation of the PIGA protein using a cryptic start site at amino acid position 37 producing shorter PIGA protein with the majority of the conserved amino acids intact [7]. One mutation, c.1234C>T, was recurrent (Table 1) [15,18].

Studies in mice revealed that complete disruption of the PIGA gene results in early embryonic lethality in males, while in carrier female mice late embryonic lethality is observed [3]. Based on these studies, it is believed that complete loss of PIGA function is lethal in humans. A number of studies suggest that the human mutations identified to date result in reduced, but not absent, PIGA activity and using the flow cytometry of blood granulocytes method, Kato et al. [18] showed that the phenotype severity of the PIGA germline mutations appeared to correlate with genotype and the residual functional activity of the PIGA protein [18]. Functional studies on the truncating c.1234C>T mutation (p.R412X) suggest that small amounts of full length PIGA protein were generated by the read through of the premature termination codon (PTC) [15,18]. During two stages of eukaryotic translation (elongation and termination), aminoacyl-tRNAs and termination factors compete for codon binding. When aminoacyl-tRNAs supersedes, read through of the termination codon occurs, which allows the generation of the full-length polypeptide. Depending on the amino acid inserted during the read through, the resulting protein may have normal or partial activity. Studies on base level of read through reveal 10 fold higher frequency at PTCs (<1%) [34,35] when compared to naturally occurring stop codons (<0.1%) [36,37]. In addition to c.1234C>T (p.R412X), studies on the c.328_330delCCT (p.L344Del) mutation revealed reduced GPI-anchored proteins on the patient’s granulocytes, while normal levels were observed on the red blood cells and monocytes, suggesting reduced but not absent PIGA activity [16]. Complementation assays using the c.76dupT frameshift mutation confirmed partial function of the shorter PIGA protein, which was sufficient to rescue surface expression of CD59 in a PIGA null cell line [7]. Consistent with previous findings, our patient’s mutation also led to reduced surface expression of the GPI-anchored protein CD109 on skin fibroblasts, despite normal levels of protein expression.

Patients with PIGA germline mutations share key phenotypic features with patients carrying mutations in genes encoding various PIG family members, including IDD, seizures, hypotonia, growth defects, congenital abnormalities, heart defects, and abnormal metabolic profiles. For example, inherited glycosylphophatidylinositol deficiency (MIM 610293) resulting in portal- and hepatic-vein thrombosis and absence seizures was found to be due to mutations in PIGM gene (MIM610273) [5]. Hyperphosphatasia Mental Retardation syndrome (HPMRS, MIM 239300, MIM 214749, and MIM 614207), also known as Mabry syndrome, was found to be associated with PIGV (MIM 610274), PIGO (MIM 614730),PGAP2 (MIM 615187) and PGAP3 (MIM 611801) mutations [38-42]. HPMRS is characterized by elevated alkaline phosphatase levels, IDD, seizures, hypotonia, and facial dysmorphic features [38]. CHIME syndrome (MIM 280000), also known as Zunich Neuro-Ectodermal syndrome was found to be due to mutations in PIGL (MIM 605947). Individuals with CHIME syndrome present with colobomas, congenital heart defects, early onset migratory ichthyosiform dermatosis, IDD, and ear anomalies, including conductive and sensori-neural hearing loss [43]. PIGT mutations cause congenital anomalies-seizures-hypotonia type 3, with hypophosphatasia as key feature [44,45]. The wide spectrum of human conditions associated with mutations in PIG genes reflects their role in multiple developmental processes and resembles the diversity of clinical features associated with glycosylation pathways deficiencies [43].

A variety of clinical case descriptors have been applied to individuals with germline PIGA mutations (Table 1). However, given the varied clinical spectrum reported to date, it appears that the multi-system roles of GPI-anchored proteins and private nature of most of the PIGA mutations identified will preclude the definition of a common feature set. To unify further reports on this intriguing condition, we advocate for the use of ‘PIGA deficiency’.

Careful patient phenotyping will continue to shed light on the (patho-) physiologic roles of PIGA deficiency; for example, its relation with mitochondrial structure and function. We are the first to report severely reduced amount of mitochondrial complexes (specifically I and V), but other mitochondrial defects have been reported in PIGA mutations: e.g. ‘disorganized mitochondria’ in FCC syndrome [16] and ‘abnormal ATP production’ in a patient with accelerated linear growth and obesity [17]. In fact, recent publications show that several mitochondrial membrane proteins are either modified with GPI anchor addition or associated with GPI anchored proteins, a process required for their proper function [46]. Therefore, it is of great interest to test patients with the PIGA mutations for mitochondrial defects and vice versa, to test the patients with unexplained mitochondrial phenotypes for potential PIGA mutations, especially in the presence of features described here.

Based on the patient descriptions published to date, intractable, infantile onset epilepsy with suppression burst and/or hypsarrhythmia in patients with X-linked IDD of unknown etiology should prompt clinicians to consider germline mutations in the PIGA, particularly in a patient with elevated serum alkaline phosphatase. The additional presence of dysmorphic facial features, CNS abnormalities, hypotonia, heart defects, dyslipidemia/lipoprotein lipase deficiency, or signs of intra-myelin edema on brain MRI, may increase the likelihood of mutations in PIGA specifically, and PIG family members in general. Flow cytometry of blood granulocytes has also proven a useful test for levels of GPI-anchored proteins expression in patients with PIGA mutations [7,15,16,18].