Other entities represented in this entry:
HGNC Approved Gene Symbol: PIGA
SNOMEDCT: 1963002, 774151000; ICD10CM: D59.5;
Cytogenetic location: Xp22.2 Genomic coordinates (GRCh38): X:15,319,451-15,335,554 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| Xp22.2 | Multiple congenital anomalies-hypotonia-seizures syndrome 2 | 300868 | X-linked recessive | 3 |
| Neurodevelopmental disorder with epilepsy and hemochromatosis | 301072 | X-linked recessive | 3 | |
| Paroxysmal nocturnal hemoglobinuria, somatic | 300818 | 3 |
Glycosylphosphatidylinositol (GPI) is a glycolipid that attaches dozens of different proteins to the cell surface. PIGA is 1 of several proteins required for the first step of GPI anchor biosynthesis (review by Brodsky, 2008).
For further information on the PIG gene family and GPI biosynthesis, see GENE FAMILY.
Some of the genes involved in GPI biosynthesis are represented by different complementation classes of GPI anchor-deficient mutant cells derived from human and rodent cell lines (Stevens and Raetz, 1991; Sugiyama et al., 1991; Hirose et al., 1992). By expression cloning using a GPI anchor-deficient human B-lymphoblastoid cell line belonging to complementation class A, Miyata et al. (1993) cloned PIGA. The predicted 484-amino acid PIGA protein has a single transmembrane domain.
Kawagoe et al. (1994) reported that the deduced amino acid sequence of the mouse Piga protein is 88% identical to that of the human protein. Database analysis demonstrated that a yeast gene, Spt14, is homologous and that these genes are members of a glycosyltransferase gene family.
The PIGA gene encodes 4 isoforms, 2 coding and 2 noncoding. Belet et al. (2014) found that the major isoform encodes a 484-residue protein that starts in and includes exon 2 and was expressed in all tested human tissues. The second coding isoform starts in exon 1, but skips exon 2 and produces a truncated protein of 250 residues.
PIGA Pseudogene 1
In the course of analyses of PIGA genetic alterations in patients with paroxysmal nocturnal hemoglobinuria (PNH; 300818) (see MOLECULAR GENETICS), Yu et al. (1994) amplified PIGA transcripts expressed in affected lymphocytes by RT-PCR and unexpectedly found a product differing from the authentic PIGA product by 126 nucleotide exchanges and 5 deletions in the coding region. Nagarajan et al. (1995) showed that mRNA with this sequence was coexpressed with PIGA mRNA in a wide range of cell types. Mapping of genomic DNA from human/rodent hybrids showed that the sequence derived from an intronless processed gene (PIGAP) on chromosome 12. Duplicated processed genes had been described for a number of X-linked genes, including pyruvate dehydrogenase (300502), the adenine nucleotide translocase genes (300150 and 300151), and phosphoglycerate kinase (311800). The identification of a stop codon at position 243 in the mRNA sequence of the PIGAP gene on chromosome 12 indicates that if this mRNA is translated, its protein product is probably not functional.
Iida et al. (1994) reported that the PIGA gene is at least 17 kb long and has 6 exons. They sequenced the exon-intron boundaries and described the characteristics of the 5-prime promoter region.
Using FISH, Takeda et al. (1993) mapped the PIGA gene to chromosome Xp22.1.
Ware et al. (1994) used an interspecific cross to demonstrate that the Piga gene in the mouse is also located on the X chromosome. Kawagoe et al. (1994) also mapped the mouse Piga gene to the X chromosome in a region that shows homology of synteny to Xp22.1.
GPI is synthesized in the endoplasmic reticulum (ER) and transferred to the C termini of proteins with GPI attachment signal peptides. The common core structure of GPI consists of a molecule of phosphatidylinositol (PI) and a glycan core that contains glucosamine, 3 mannoses, and an ethanolamine phosphate. Biosynthesis of GPI anchors involves at least 10 reactions and more than 20 different proteins, including various members of the PIG gene family. The first step of GPI anchor biosynthesis, the transfer of N-acetylglucosamine (GlcNAc) from uridine 5-prime-diphospho-N-acetylglucosamide (UDP-GlcNAc) to PI to yield GlcNAc-PI, is catalyzed by a 7-subunit enzymatic complex that includes PIGA, PIGC (601730), PIGH (600154), PIGP (605938), PIGQ (605754), PIGY (610662), and DPM2 (603564). The intermediate steps of GPI anchor biosynthesis, which include de-N-acetylation of GlcNAc-PI to GlcN-PI, sequential addition of 3 mannoses from dolichol-phosphate-mannose and an ethanolamine phosphate from phosphatidylethanolamine, and modification of the core with side groups during or after synthesis, involve the PIGL (605947), PIGM (610273), PIGN (606097), PIGB (604122), PIGF (600153), PIGO (614730), PIGV (610274), PIGW (610275), and PIGX (610276) proteins, as well as DPM1 (603503), DPM3 (605951), and MPDU1 (604041). The last step in GPI anchor biosynthesis is attachment of the GPI anchor to the newly synthesized proprotein via a transamidase-like reaction that involves PIGK (605087), PIGS (610271), PIGT (610272), and PIGU (608528), as well as GPAA1 (603048). During this reaction, the C-terminal GPI attachment signal is released, and the GPI-anchored protein transits the secretory pathway to reach the plasma membrane, where it resides in lipid rafts (review by Brodsky, 2008).
Using human and mouse GPI anchor-deficient cell lines, Miyata et al. (1993) showed that PIGA takes part in the synthesis of GlcNAc-PI, the first intermediate in the biosynthetic pathway of GPI anchor.
Kawagoe et al. (1994) found that transfection of the mouse Piga cDNA complemented the defects of both a Piga-deficient murine cell line and a PIGA-deficient human cell line, demonstrating that functions of the mouse and human proteins are conserved.
Watanabe et al. (1996) found that the PIGA and PIGH (600154) proteins form a protein complex and are subunits of the GPI GlcNAc transferase of the ER. They showed that PIGA is an ER transmembrane protein with a small luminal domain and a large cytoplasmic domain. The luminal domain contains information which targets the protein to the rough ER, while the cytoplasmic domain has homology to the bacterial GlcNAc transferase RfaK. Watanabe et al. (1996) concluded that the first step of GPI anchor synthesis occurs on the cytoplasmic side of the ER membrane.
Using immunoprecipitation experiments, Watanabe et al. (1998) demonstrated that PIGQ (605754) associates specifically with PIGA, PIGC (601730), and PIGH and that all 4 proteins form a complex that has GPI-GlcNAc transferase (GPI-GnT) activity in vitro.
Paroxysmal Nocturnal Hemoglobinuria
Paroxysmal nocturnal hemoglobinuria (PNH; 300818) is an acquired hematopoietic disease characterized by abnormal blood cell populations in which the biosynthesis of the GPI anchor is deficient. Deficiency of surface expressions of GPI-anchored complement inhibitors leads to complement-mediated hemolysis. Ueda et al. (1992) established affected B-lymphocyte cell lines from 2 patients with PNH, and Takahashi et al. (1993) demonstrated that the early step of GPI anchor biosynthesis was deficient in these cells. Complementation analysis by somatic cell hybridization with GPI-deficient mutant cell lines showed that these PNH cell lines belonged to complementation class A, which is known not to synthesize GlcNAc-PI. Takeda et al. (1993) found that transfection of PIGA cDNA into affected B-lymphoblastoid cell lines restored their surface expression of GPI-anchored proteins. Further analysis demonstrated that the PIGA transcript was missing or present in very small amount in cell lines established from 1 patient, but that in a cell line established from another patient, deletion of thymine in a 5-prime splice site (311770.0001) was associated with deletion of a PIGA exon located immediately 5-prime to the abnormal splice donor site. Since the PIGA gene maps to chromosome Xp22.1, and 1 of the patients studied was female, Takeda et al. (1993) concluded that the mutant PIGA gene must reside on the active X chromosome. Affected cell lines established from 5 other patients with PNH were shown to belong to complementation group class A, indicating that the target gene is the same in most, if not all, patients with PNH. This can account for the behavior of the deficiency as a dominant in hemizygous males and in females with the mutant gene on the active X chromosome in a given lymphoblastoid cell line.
Rosse (1993) indicated that all cases of PNH appear to have a defect in this gene, but the causative mutation has in all instances been unique. That many different mutations of PIGA may result in PNH may not be surprising since they arise as somatic mutations. Rosse (1993) suggested that a germline mutation resulting in defects in this biosynthetic pathway would be lethal.
Bessler et al. (1994) reviewed the evidence that PNH is caused by somatic mutations in the PIGA gene. They demonstrated a somatic point mutation in 4 cases which, with the 2 mutations reported by Takeda et al. (1993), brought to 6 the number in which formal proof of the absence of normal PIGA gene product has been shown to produce the PNH phenotype.
In granulocytes from 3 of 15 patients with PNH, Miyata et al. (1994) found size abnormalities of PIGA transcripts with different patterns, and in 1 patient a very low level of the PIGA transcript was found. Although 11 patients had transcripts of normal size, transfection assay demonstrated that in each patient some of the transcripts were nonfunctional. The percentage of nonfunctional PIGA transcripts correlated with the percentage of affected granulocytes (P = less than 0.001). Sequence analysis demonstrated somatic mutations in 2 of the patients: deletion of a T (311770.0001) and insertion of an A. The PIGA gene as the site of the defect in all patients with PNH is remarkable in light of the fact that PIGA is but 1 of at least 10 genes involved in GPI synthesis. The location of the gene on the X chromosome is probably responsible: somatic mutation in only one X chromosome is necessary to produce the mutation in a male cell or for that matter in a female cell if it occurs on the active X chromosome.
Savoia et al. (1996) found a novel mutation in the PIGA gene in each of 3 Italian patients with PNH. In each case, the mutation caused premature termination of translation of the PIGA protein.
Nafa et al. (1995) identified 15 different somatic mutations in 12 patients with PNH; 10 of them caused frameshifts. In each of 3 patients, 2 independent mutations were identified. Whereas G6PD mutations are virtually all single basepair changes that result in single amino acid replacements, most PIGA mutations are insertion-deletion mutations that cause frameshifts. The authors stated that the predominance of null mutations probably reflects the fact that the total absence of GPI-linked proteins provides a relative survival or growth advantage to the affected cells that is greater than that when the deficiency of GPI-linked proteins is only partial.
Nafa et al. (1998) described 28 previously unreported mutations. They confirmed that somatic mutations are spread throughout the entire coding region of the PIGA gene and that most frameshift mutations produce a nonfunctional PIGA protein. In addition, they found 1 total deletion of the PIGA gene, and 2 short nucleotide duplications (see 311770.0010). Although mutations are spread throughout the entire coding region, they observed more missense mutations in exon 2 than in other exons.
Luzzatto and Bessler (1996) and Luzzatto et al. (1997) reviewed the topic of PNH and gave a survey of the more than 100 somatic mutations in the PIGA gene that had been identified in patients with this disorder.
Although many of the clinical manifestations (e.g., hemolytic anemia) of PNH can be explained by a deficiency of GPI-anchored complement regulatory proteins such as CD59 (107271) and CD55 (125240), it was unclear why PNH clonal cells dominate hematopoiesis and why they are prone to evolve into acute leukemia. Brodsky et al. (1997) found that PIGA mutations confer survival advantage by making cells relatively resistant to apoptotic death. When placed in serum-free medium, granulocytes and affected CD34(+) (142230) cells from PNH patients survive longer than their normal counterparts. PNH cells were also relatively resistant to apoptosis induced by ionizing irradiation. Replacement of the normal PIGA gene in PNH cell lines reversed the cellular resistance to apoptosis. Brodsky et al. (1997) speculated that apoptosis inhibition may be the principal mechanism by which PNH cells maintain a growth advantage over normal progenitors and could play a role in the propensity of this disease to transform into more aggressive hematologic disorders. The work also suggested that GPI anchors are important in regulating apoptosis.
The clinical association between PNH and acquired aplastic anemia (AAA), and the observation that, as in AAA, PNH patients have decreased hematopoietic progenitors, may be taken to suggest a common pathogenetic process. There is strong evidence that AAA is an autoimmune disease and, as for AAA, bone marrow failure in PNH can be treated successfully with immunosuppression; thus, autoimmunity is likely to play a role in PNH as well. Specifically, it has been hypothesized that an autoimmune attack on normal stem cells targets a GPI-linked molecule and therefore preferentially spares the PNH stem cell, which thus has a growth or survival advantage (or both) in this abnormal environment. Using flow cytometric analysis of granulocytes, Araten et al. (1999) identified cells that had the PNH phenotype (lack of expression of proteins linked to the membrane by a GPI anchor) at an average frequency of 22 per million in 9 normal individuals. These rare cells were collected by flow sorting, and exons 2 and 6 of the PIGA gene were amplified by nested PCR. The authors identified PIGA mutations in 6 cases. PNH red blood cells also were identified at a frequency of 8 per million. Thus, small clones with PIGA mutations existed commonly in normal individuals, showing clearly that PIGA gene mutations are not sufficient for the development of PNH. Because PIGA encodes an enzyme essential for the expression of a host of surface proteins, the PIGA gene provides a highly sensitive system for the study of somatic mutations in hematopoietic cells. In a note added in proof, Araten et al. (1999) reported the finding of a tyr98-to-ter mutation (311770.0002) in a 61-year-old man being phlebotomized for hemochromatosis. This was confirmed in samples taken 8 weeks apart. The same mutation had been reported in a patient with PNH (Savoia et al., 1996). Thus, the same PIGA mutation that caused PNH in one person did not cause PNH in another person.
Hu et al. (2005) confirmed the finding that mutations of the PIGA gene are relatively common in normal hematopoiesis; however, they demonstrated that these mutations occur in differentiated progenitor cells rather than in hematopoietic stem cells.
Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome 2
By exome sequencing of the X chromosome in a family with multiple congenital anomalies-hypotonia-seizures syndrome-2 (MCAHS2; 300868), Johnston et al. (2012) identified a germline mutation in the PIGA gene (R412X; 311770.0011). Two affected boys carried the mutation, and 2 obligate female carriers were heterozygous for the mutation; both female carriers showed 100% skewed X inactivation. In vitro functional expression studies in PIGA-null cell lines showed that the R412X mutant protein retained some residual activity with partial restoration of GPI-anchored proteins, suggesting that it is not a null allele. The findings indicated that GPI anchors are important for normal development, particularly of the central nervous system. The patients had onset of seizures in the first weeks of life and died by 11 weeks of age. Neither patient had hemolytic anemia or clinical hemoglobinuria.
In a male patient with MCAHS2 manifest as developmental and epileptic encephalopathy-20 (DEE20; 300868), Belet et al. (2014) identified a hemizygous truncating mutation in the PIGA gene (311770.0012). The mutation, which was found by X-exome sequencing and confirmed by Sanger sequencing, was not found in 4 healthy male family members and was present in the unaffected mother of the proband, the unaffected grandmother, and a maternal aunt.
In 5 boys from 4 unrelated Japanese families with MCAHS2 manifest as DEE20 with clinical diagnoses of Ohtahara or West syndrome, Kato et al. (2014) identified a hemizygous mutation in the PIGA gene (see, e.g., 311770.0011; 311770.0013-311770.0015). The mutations were found by whole-exome sequencing. In vitro functional expression studies showed a variable loss of PIGA activity, with a correlation between severity of phenotype and degree of residual enzymatic activity.
In a boy with MCAHS2, van der Crabben et al. (2014) identified a hemizygous mutation in the PIGA gene (311770.0017).
Neurodevelopmental Disorder with Epilepsy and Hemochromatosis
In 2 affected males from a large family with neurodevelopmental disorder with epilepsy and hemochromatosis (NEDEPH; 301072), Swoboda et al. (2014) identified a hemizygous in-frame 3-bp deletion in the PIGA gene (leu110del; 311770.0016). The mutation, which was found by exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family. Flow cytometric analysis of the proband's granulocytes showed decreased cell surface levels of some GPI-anchored proteins, although CD59 (107271) expression on red blood cells was normal, suggesting that the mutant protein had some residual activity.
In 3 unrelated patients with NEDEPH, Muckenthaler et al. (2022) identified hemizygous missense mutations in the PIGA gene (R77Q, 311770.0018; L344P, 311770.0019; and S127L, 311770.0020). The mutations, which were found by exome sequencing or sequencing of a gene panel, were all inherited from an unaffected mother. A subpopulation of patient blood cells showed a slight reduction of GPI-anchored proteins, suggesting that the mutations were hypomorphic and retained some residual activity. CRISPR/Cas12a-mediated knockdown of PIGA in Hep3B liver cells eliminated the cell surface expression of GPI-anchored proteins CD59 and hemojuvelin (HJV; 608374), as well as caused decreased expression of hepcidin (606464) compared to controls. These findings indicated disruption of iron homeostasis. Transfection with wildtype PIGA rescued these defects, but expression of the L344P or R77Q mutations did not rescue hepcidin mRNA levels, consistent with a functional deficiency of PIGA. PIGA knockdown also reduced the levels of ceruloplasmin (CP), a GPI-anchored ferroxidase required for efficient cellular iron export. Reduced CP protein expression may aggravate iron overload and contribute to neurologic symptoms. The authors noted that the missense mutations had less deleterious effects than complete loss-of-function alleles, suggesting that the missense variants have residual function. These hypomorphic alleles could explain the milder neurologic phenotype, which allowed for sufficiently long survival for the iron overload phenotype to manifest.
Although a somatic PIGA mutation is responsible for deficiency of GPI-anchored proteins in PNH patients, no inherited form of GPI-anchor deficiency had been described. Piga gene inactivation in mouse embryonic stem (CES) cells followed by blastocyst injection is associated with a high rate of early embryonic lethality and low chimerism in surviving animals. Female mice heterozygous for a mutant Piga gene had never been obtained. To study the consequences of a nonfunctional Piga gene and to address the issue of a maternally inherited Piga mutation, Keller et al. (1999) generated mice carrying a Piga mutation using Cre/loxP-controlled DNA recombination, as described by Sauer and Henderson (1988). High efficiency of Piga gene recombination was obtained by targeting Piga gene inactivation directly to the preimplantation female embryo. Because of X inactivation, newborn female mice were mosaic, with cells that expressed or lacked GPI-linked proteins. To assess the importance of PIGA in different organs, Keller et al. (1999) examined the relative distribution of cells expressing or lacking GPI-linked proteins. Analysis of mosaic mice showed that in heart, lung, kidney, brain, and liver mainly wildtype Piga was active, suggesting that these tissues require GPI-linked proteins. The salient exceptions were spleen, thymus, and red blood cells, which had almost equal numbers of cells expressing the wildtype or the recombined allele, implying that GPI-linked proteins are not essential for the derivation of these tissues. PIGA(-) cells had no growth advantage, suggesting that other factors are needed for their clonal dominance in patients with paroxysmal nocturnal hemoglobinuria.
The fact that Keller et al. (1999) were able to obtain female mice that carried in virtually all cells a mutated Piga gene raised the interesting issue of whether a heritable form of paroxysmal nocturnal hemoglobinuria exists. Because of X inactivation followed by cellular selection, female mice with high levels of Piga gene recombination were born alive. A biased male/female ratio of 1.5 suggested fetal wastage of highly recombined animals not rescued by the relative growth advantage of PIGA(+) cells. An inherited Piga mutation would be expected to follow a male-lethal, female-dominant inheritance pattern, with a varied phenotype in females depending on the proportion of cells expressing the mutant Piga gene. Keller et al. (1999) found that a maternally inherited Piga mutation is embryonic lethal. In the embryo proper, X chromosome inactivation occurs at random. In contrast, in the trophoectoderm and in the primitive endoderm of the implanting embryo, the paternally derived X chromosome is preferentially inactivated. It is, therefore, conceivable that PIGA is essential in these tissues.
The experiments of Keller et al. (1999) did not exclude the possibility of sporadic mutations that, if occurring during early embryogenesis, may be found almost exclusively in females and thus mimic an X-linked dominant disease with prenatal lethality in males and a variable phenotype in females. In fact, Ogata et al. (1998) reported a female infant mosaic for an interstitial deletion within Xp22 spanning the critical region of the gene responsible for microphthalmia with linear skin defects (MLS; 309801) and the PIGA gene, as determined by microsatellite analysis.
In cells from a female patient (SS) with paroxysmal nocturnal hemoglobinuria (300818), Takeda et al. (1993) demonstrated a deletion of 207 bp from positions 982 to 1188 of the PIGA mRNA. The deletion was predicted to result in an aberrant protein with 69 amino acid residues deleted from the middle of the 484 amino acid protein. The same defect was found in a B-lymphocyte line and in the polymorphonuclear leukocytes, demonstrating that the affected cells, which were predominantly in peripheral blood, were derived from a clone of multipotential hematopoietic stem cells. Takeda et al. (1993) further demonstrated that the 207-bp deletion corresponded to a single exon and that exon skipping had resulted from a 1-bp (T) deletion in the 5-prime splice site of the intron following the skipped exon.
In an Italian patient with paroxysmal nocturnal hemoglobinuria (300818), Savoia et al. (1996) identified a C-to-A transversion at nucleotide 294 in exon 2 of the PIGA gene, resulting in a tyr98-to-ter mutation.
In a 61-year-old man who was being phlebotomized for hemochromatosis, Araten et al. (1999) identified the same mutation. Thus, the same PIGA mutation that caused PNH in one person did not cause PNH in another person. This was taken as strong support for 'dual pathogenesis of PNH' (Luzzatto and Bessler, 1996). Although a PIGA gene mutation may be necessary for the development of PNH, it is not sufficient.
In an Italian patient with paroxysmal nocturnal hemoglobinuria, Savoia et al. (1996) demonstrated an insertion of A at nucleotide 460 (460insA) of the PIGA gene, resulting in a new reading frame that was terminated by a stop codon 8 codons downstream.
In an Italian patient with paroxysmal nocturnal hemoglobinuria (300818), Savoia et al. (1996) demonstrated a deletion of 1 of the 2 cytosines at nucleotides 1114-1115 (1114delC) causing a frameshift that resulted in a termination signal 9 codons downstream.
In 1 of 4 patients developing paroxysmal nocturnal hemoglobinuria (300818) after treatment of severe aplastic anemia with antithymocyte globulin and cyclosporin, Nagarajan et al. (1995) observed a C-to-T transition of nucleotide 163 of the PIGA gene, changing codon 55 from gln to TGA (stop).
In a patient with paroxysmal nocturnal hemoglobinuria (300818), Ware et al. (1994) identified a 2-bp (GT) insertion at nucleotide position 334 of the PIGA gene leading to a premature termination codon (TGA) at nucleotide position 370. The erythrocytes and granulocytes in this patient were exclusively type III cells, indicating a complete deficiency in surface expression of glycosylphosphatidylinositol-linked proteins and causing complete loss of function.
In a patient with paroxysmal nocturnal hemoglobinuria (300818), Ware et al. (1994) identified a 1-bp deletion (C) at nucleotide position 516 of the PIGA gene leading to a premature termination codon (TAA) at nucleotide position 598. The erythrocytes and granulocytes in this patient were exclusively type III cells, indicating a complete deficiency in surface expression of glycosylphosphatidylinositol-linked proteins and causing complete loss of function.
In a patient with paroxysmal nocturnal hemoglobinuria (300818), Ware et al. (1994) identified a 2-bp (CT) deletion at nucleotide position 1408 of the PIGA gene leading to a premature termination codon (TGA) at nucleotide position 1438. The erythrocytes and granulocytes in this patient were exclusively type III cells, indicating a complete deficiency in surface expression of glycosylphosphatidylinositol-linked protein.
Maugard et al. (1997) noted that only a few cases of paroxysmal nocturnal hemoglobinuria (300818) had been described in children and adolescents. They reported the case of a male diagnosed with PNH at 12 years of age during follow-up of aplastic anemia, which had initially been diagnosed at the age of 8.5 years and was treated with cyclosporin and growth factors. Using the protein truncation test to scan for truncating mutations in PIGA mRNA reverse-transcribed and amplified from blood mononuclear cells, Maugard et al. (1997) found a donor splice site mutation, IVS5+1G-A, which had previously been described in a Japanese and a Thai adult with PNH. The recurrence in 3 unrelated patients from distinct ethnic origins suggested that this site, although not located in a CpG-type hypermutable sequence, may represent a mutation hotspot. The authors pointed out that scanning PIGA mRNA for mutations rather than genomic DNA is advantageous because it avoids the amplification of sequences from the PIGA pseudogene at 12q21.
Nafa et al. (1998) reported a detailed longitudinal study of the first patient to be treated (in 1973) for paroxysmal nocturnal hemoglobinuria (300818) with syngeneic bone marrow transplantation. The patient, a male, was 19 years old at the time of BMT. Bone marrow was derived from a monozygotic twin. The patient subsequently relapsed with PNH in 1983, and still had PNH to the time of report. Analysis of the PIGA gene in the 1990s showed an insertion-duplication in exon 6, causing a frameshift. The mutation was the insertion of 2 adenines at position 1355, followed by a duplication of the preceding 32 nucleotides (1324-1355). This introduced a frameshift at codon 452 and led to a truncated PIGA protein of only 462 amino acids. PCR amplification of the PIGA exon 6 from bone marrow slides obtained before BMT showed that this duplication was not present; instead, Nafa et al. (1998) found several single basepair substitutions in exons 2 and 6. Thus, relapse of PNH in this patient was not due to persistence of the original clones; rather, it was associated with the emergence of a new clone. These findings support the notion that the bone marrow environment may create selective conditions favoring the expansion of PNH clones. The changes found in the archival material included a 211A-G transition in exon 2, causing a thr71-to-ala substitution, and a 251C-T transition in exon 2, causing a thr84-to-ile substitution. The former change was present in 50% of clones and the latter change was present in 28% of those clones as a second mutation, suggesting that the latter mutation arose in a cell belonging to the clone that had the former mutation. A third mutation in exon 2, a 16G-T transversion causing a gly6-to-ter substitution, was present in 14% of clones. The finding of multiple mutational clones, as was the case after relapse, is not unusual in PNH.
By exome sequencing of the X chromosome in a family with multiple congenital anomalies-hypotonia-seizures syndrome-2 (MCAHS2; 300868), Johnston et al. (2012) identified a 1234C-T transition in the last exon of the PIGA gene, resulting in an arg412-to-ter (R412X) substitution and truncation of the final C-terminal 109 amino acids. The mutation was not found in multiple large control sets. In vitro functional expression studies in PIGA-null cell lines showed that the R412X mutant protein retained some residual activity with partial restoration of GPI-anchored proteins, suggesting that it is not a null allele. The findings indicated that GPI anchors are important for normal development, particularly of the central nervous system. The patients had onset of seizures associated with burst-suppression pattern on EEG in the first weeks of life; both died by 11 weeks of age. The findings were consistent with a developmental and epileptic encephalopathy (DEE).
Kato et al. (2014) identified the R412X mutation in a 6-year-old Japanese boy with MCAHS2 manifest as early infantile epileptic encephalopathy with a clinical diagnosis of Ohtahara syndrome. He had severe disability, myoclonus, and quadriplegia. In vitro functional expression studies showed that the mutant protein could partially restore GPI-anchored protein expression in PIGA-null cells, suggesting that a small amount of full-length protein was generated by read-through of the stop codon.
Fauth et al. (2016) identified a hemizygous R412X mutation (c.1234C-T, NM_002641.3) in 4 affected males from 3 unrelated families with MCAHS2. One of the families had been reported by Terespolsky et al. (1995) and originally classified as having Simpson-Golabi-Behmel syndrome type 2 (SGBS2; 300209).
In a 24-year-old man with MCAHS2 (300868) manifest as developmental and epileptic encephalopathy, Belet et al. (2014) identified a hemizygous 1-bp duplication (c.76dupT) in exon 2 of the PIGA gene, resulting in a frameshift and premature termination (Tyr26LeufsTer3). The family had previously been reported by Claes et al. (1997) as having West syndrome. The mutation, which was found by X-exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family, and was not found in the 1000 Genomes Project, dbSNP (build 135), or Exome Variant Server databases, or in an in-house control database. Patient cells showed normal PIGA expression due to the production of a normal shorter PIGA isoform that lacks exon 2. Patient cells showed normal expression of CD59 (107271), and complementation assays showed that this shorter PIGA cDNA was able to partially rescue the surface expression of CD59 in a PIGA-null cell line. Belet et al. (2014) suggested that the mutation was a hypomorph that could rescue lethality in males, but could not compensate for the MCAHS2 phenotype.
In 2 Japanese brothers with MCAHS2 (300868) manifest as developmental and epileptic encephalopathy, Kato et al. (2014) identified a hemizygous c.230G-T transversion in exon 2 of the PIGA gene, resulting in an arg77-to-leu (R77L) substitution at a highly conserved residue. The mutation, which was found by whole-exome sequencing, was not found in the Exome Variant Server database or in 573 in-house control exomes. In vitro functional expression studies showed that the mutant protein could partially restore GPI-anchored protein expression in PIGA-null cells. The patients had a slightly less severe phenotype than other patients with PIGA mutations (see, e.g., 311770.0011 and 311770.0014), which correlated with more residual PIGA enzymatic activity for the R77L protein. The patients had onset of seizures at 7 months of age.
In a Japanese boy with MCAHS2 (300868) manifest as West syndrome, Kato et al. (2014) identified a hemizygous c.6161A-T transversion in exon 2 of the PIGA gene, resulting in an ile206-to-phe (I206F) substitution at a highly conserved residue. The mutation, which was found by whole-exome sequencing, was not found in the Exome Variant Server database or in 573 in-house control exomes. In vitro functional expression studies showed that the mutant protein could partially restore GPI-anchored protein expression in PIGA-null cells. The patient had onset of seizures at 6 months of age; the phenotype was consistent with a developmental and epileptic encephalopathy (DEE).
In a 15-month-old Japanese boy with MCAHS2 (300868) manifest as West syndrome, Kato et al. (2014) identified a hemizygous c.355C-T transition in exon 2 of the PIGA gene, resulting in an arg119-to-trp (R119W) substitution at a highly conserved residue. The mutation, which was found by whole-exome sequencing, was not found in the Exome Variant Server database or in 573 in-house control exomes. The patient had onset of seizures at 3 months of age; the phenotype was consistent with a developmental and epileptic encephalopathy (DEE).
In 2 affected males from a family with neurodevelopmental disorder with epilepsy and hemochromatosis (NEDEPH; 301072), Swoboda et al. (2014) identified a hemizygous in-frame 3-bp deletion (c.328_330delCTT, NM_020473.3) in the PIGA gene, resulting in the deletion of a conserved residue (leu110del). The mutation, which was found by exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family, and was not present in the 1000 Genomes Project or Exome Variant Server databases. Flow cytometric analysis of the proband's granulocytes showed decreased cell surface levels of some GPI-anchored proteins, although CD59 (107271) expression on red blood cells was normal, suggesting that the mutant protein had some residual activity. In addition to neurologic features, the patients had cutaneous abnormalities and evidence of systemic iron overload.
In a boy with MCAHS2 (300868), van der Crabben et al. (2014) identified a hemizygous c.278C-T transition in the PIGA gene, resulting in a pro93-to-leu (P93L) substitution in a highly conserved GPI-anchored biosynthesis domain region. The mutation, which was found by X-exome sequencing and confirmed by Sanger sequencing, was not found in the dbSNP (build 137) or Exome Variant Server databases, or in 100 in-house control exomes. The mother and maternal grandmother were unaffected carriers, and the mother showed 100% skewing of the X-chromosome harboring the mutation. Functional studies of the variant were not performed.
In a 13-year-old boy (patient 1) with neurodevelopmental disorder with epilepsy and hemochromatosis (NEDEPH; 301072), Muckenthaler et al. (2022) identified a hemizygous c.230G-A transition in the PIGA gene, resulting in an arg77-to-gln (R77Q) substitution. The mutation, which was found by exome sequencing, was inherited from the unaffected mother. In vitro functional expression studies showed that the mutation resulted in a partial loss of PIGA function with decreased levels of certain GPI-anchored proteins involved in iron homeostasis.
In a 7-year-old boy (patient 2) with neurodevelopmental disorder with epilepsy and hemochromatosis (NEDEPH; 301072), Muckenthaler et al. (2022) identified a hemizygous c.1031T-C transition in the PIGA gene, resulting in a leu344-to-pro (L344P) substitution. The mutation, which was found by next-generation panel sequencing, was inherited from the unaffected mother. In vitro functional expression studies showed that the mutation resulted in a partial loss of PIGA function with decreased levels of certain GPI-anchored proteins involved in iron homeostasis.
In a 2-year-old boy (patient 3) with neurodevelopmental disorder with epilepsy and hemochromatosis (NEDEPH; 301072), Muckenthaler et al. (2022) identified a hemizygous c.380C-T transition in the PIGA gene, resulting in a ser127-to-leu (S127L) substitution. The mutation, which was found by next-generation panel sequencing, was inherited from the unaffected mother. Functional studies of the variant and studies of patient cells were not performed, but it was predicted to be a hypomorphic allele.
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