The PIGA gene plays a critical role in glycosylphosphatidylinositol (GPI) biosynthesis. PIGA variants have been linked to multiple congenital anomalies-hypotonia-seizures syndrome 2. While epilepsy is a common manifestation and patients usually show developmental delay, it is unclear whether PIGA variants are associated with pure epilepsy. Trio-based whole-exome sequencing was performed on individuals with focal epilepsy from the China Epilepsy Gene 1.0 project. The sub-molecular effect, damaging effect of the variants, and spatial-temporal expression of PIGA were analyzed to explore its role in epilepsy. Six PIGA variants were identified in seven unrelated cases with focal epilepsy. In one family, the variant co-segregated with two affected males. All the variants were inherited from the asymptomatic mothers, consistent with an X-linked recessive inheritance pattern. Five of the variants were absent in the general population and one variant had extremely low frequencies. These variants were predicted to be damaging by commonly used in silico tools. Four cases presented with developmental delay and three showed normal development. Further analysis revealed that variants associated with a severe phenotype were located within the GPI biosynthesis domain, whereas variants associated with focal epilepsy and a favorable outcome were outside functional domains, suggesting a sub-molecular effect. Patients with more severe phenotype also had variants with higher damaging scores, indicating a potential genotype-phenotype correlation. PIGA was highly expressed at the embryoid stage, decreased after birth, and then increased again during infancy and the juvenile stage, consistent with the onset age of the patients. PIGA variants are potentially associated with focal epilepsy with favorable outcome. The sub-molecular effect helps explain phenotype variations.

Phosphatidylinositol glycan class T (PIGT) is part of the glycosylphosphatidylinositol transamidase (GPI-TA) complex, crucial for various cell functions. Biallelic pathogenic variants in PIGT are associated with Multiple Congenital Anomalies-Hypotonia Seizures Syndrome 3 (MCAHS3), a rare neonatal hypotonia syndrome characterized by dysmorphic features and seizures. Diagnosing neonatal hypotonia, which has diverse congenital and acquired causes, is challenging, particularly in syndromic monogenic cases. Next-generation sequencing is essential for accurate diagnosis. This study reports a term newborn with hypotonia, dysmorphic features, seizures, and severe skeletal issues, including a humeral fracture at birth, consistent with MCAHS3. Trio whole exome sequencing (WES) analysis revealed a novel homozygous missense variant in PIGT, expanding the clinical spectrum of MCAHS3 and marking the first such case in the Thai population. The identified c.257A>G (p.His86Arg) variant manifests a severe MCAHS3 phenotype, as evidenced by reduced CD59 expression in western blot analysis, indicating impaired GPI-AP synthesis. Computational predictions suggest this mutation causes protein instability, potentially affecting GPI anchor attachment. While alkaline phosphatase (ALP), a GPI-AP crucial for skeletal mineralization, was elevated in this case, suggesting a late-stage GPI synthesis defect. The His86Arg mutation in PIGT may disrupt GPI-TA function, hindering proper protein attachment and leading to cleaved protein secretion. Further functional studies are needed to elucidate the impact of this mutation on PIGT function and MCAHS3 phenotypes.

The glycosylphosphatidylinositol (GPI) anchor is a glycolipid that anchors proteins to the eukaryotic cell surface. An anchoring process is a posttranslational modification of at least 150 molecules with various functions. Biallelic causal variants in the PIGQ gene (OMIM: * 605754) are associated with a type of disorder of glycosylphosphatidylinositol biosynthesis (PIGQ-congenital disorders of glycosylation (CDGs), also called multiple congenital anomalies-hypotonia-seizures syndrome 4 (MCAHS4, OMIM: # 618548). Only 11 patients with this condition have been reported to date. We present two novel cases of MCAHS4 with one novel and one already known variant in the PIGQ gene, detailed phenotyping, and a review of all published cases so far. We used GestaltMatcher for deep gestalt analysis and investigated its potential use in diagnosing MCAHS4 patients. In the PIGQ gene, we found one novel frameshift variant c.1092dupC, p.(Phe365LeufsTer78) and one missense c.1370T>G, p.(Leu457Arg) already listed in the ClinVar database as a variant of uncertain significance (VUS), whose pathogenicity we proved by a functional study on Chinese hamster ovarian cells. After reviewing all 13 already diagnosed MCAHS4 patients, we found that attacks of rhabdomyolysis induced by a febrile infection were documented only in our patient. Facial dysmorphism (coarse features, anteverted nares, and open mouth) seen in all analyzed MCAHS4 patients seems to be specific. Moreover, GestaltMatcher proved that MCAHS4 patients shared a similar facial phenotype. The present work expands the genotype spectrum by describing a novel causal PIGQ variant and validating the pathogenicity of an already-known VUS variant. Because of their life-threatening complications, attacks of rhabdomyolysis should be considered in MCAHS4 patients. GestaltMatcher can be an effective tool in the diagnostic setting of MCAHS4.

Phosphatidylinositol glycan anchor biosynthesis class N (PIGN) gene mutation is a rare autosomal recessive genetic disorder. PIGN is essential for the glycosylphosphatidylinositol (GPI) anchor biosynthesis pathway. These mutations are linked to multiple congenital anomalies-hypotonia-seizures syndrome 1 (MCAHS1). The affected PIGN gene leads to a significant reduction in the overall levels of GPI-anchored proteins and CD24 expression, suggesting that even partial depletion of these proteins can result in severe phenotypic manifestations. We present a case of a two-year-old boy diagnosed with spastic cerebral palsy following a hypoxic insult, who also exhibited refractory epilepsy, global developmental delay, and failure to thrive. Whole-exome sequencing confirmed a diagnosis of an autosomal recessive mutation in the PIGN gene. Given that mutations in the PIGN gene may be an underrecognized cause of epilepsy, this case report aims to highlight the importance of early diagnosis of this condition. Furthermore, our findings contribute to the expanding spectrum of PIGN gene mutations.

Germline variants in the phosphatidylinositol glycan class A (PIGA) gene, which is involved in glycosylphosphatidylinositol (GPI) biosynthesis, cause multiple congenital anomalies-hypotonia-seizures syndrome 2 (MCAHS2) with X-linked recessive inheritance. The available literature has described a pattern of almost 100% X-chromosome inactivation in mothers carrying PIGA variants. Here, we report a male infant with MCAHS2 caused by a novel PIGA variant inherited from his mother, who has a non-skewed pattern of X inactivation. Phenotypic evidence supporting the pathogenicity of the variant was obtained by flow-cytometry tests. We propose that the assessment in neutrophils of the expression of GPI-anchored proteins (GPI-APs), especially CD16, should be considered in cases with variants of unknown significance with random X-inactivation in carrier mothers in order to clarify the pathogenic role of PIGA or other gene variants linked to the synthesis of GPI-APs.