Abstract
PIGC encodes a protein essential for the biosynthesis of glycophosphatidylinositol-anchored proteins (GPI-APs). So far, three families with biallelic PIGC variants have been reported to exhibit developmental delay/intellectual disability and seizures. Our aim was to further elucidate the clinical and biomolecular characteristics of PIGC pathogenic or likely pathogenic variants. We established a cohort of 18 previously unreported probands. Clinical data were collected, and causative variants were identified though genome/exome sequencing. Variants were modelled in silico using AlphaFold2. Flow cytometry was performed to analyze the cell-surface expression of GPI-APs. The probands displayed a severe neurodevelopmental disorder characterized by developmental and cognitive impairment, early-onset and treatment-resistant seizures, and premature death affecting 10 out of 18 individuals (median age of 40 months, ranging from 40 days to 7 years). Additional features included brain imaging abnormalities (14/15), hypotonia (15/18), and skeletal anomalies (5/17). One patient exhibited mildly elevated alkaline phosphatase levels. All harbored biallelic PIGC variants, with 14 out of 18 of those being homozygous variants. Analysis of samples derived from probands and cellular models showed reduced cell surface levels of GPI-APs. This study confirms the association of PIGC biallelic variants with refractory seizures, severe developmental and cognitive impairments, and highlights their association with childhood-onset mortality. Additionally, it shows that dysfunctional PIGC results in defective biosynthesis of GPI-AP.
INTRODUCTION
Glycosylphosphatidylinositol (GPI) is a glycolipid that is synthesized and transferred to proteins in the membrane of the endoplasmic reticulum (ER) [1]. Biogenesis of GPI-anchored proteins (GPI-APs) is a conserved post-translational process in eukaryotes, which is important for the attachment of these proteins to the cell membrane as well as protein sorting, trafficking, and dynamics [1, 2]. GPI biosynthesis has been described to occur in three steps: a GPI anchor, consisting of a glycan core with a phosphatidylinositol (PI) moiety, is first formed in the ER through a series of reactions involving more than 17 proteins [3]; the anchor is then transferred, by the action of a pentameric transamidase enzyme complex, onto the carboxylic end of proteins carrying a GPI-attachment signal; the resulting immature GPI-AP undergoes different remodeling steps and modifications as it moves through the secretory pathway to reach the cytoplasmic membrane [4]. GPI synthesis and GPI-AP modification are mediated by at least 31 genes, and pathogenic variants in 24 of these genes have been associated with human disease to date [5]. The GPI biosynthesis disorders have broad clinical implications, including developmental delay (DD), intellectual disability (ID), seizures, and diverse congenital anomalies.
Phosphatidylinositol glycan class C, PIGC (MIM# 601730) encodes an ER protein, a member of the GPI N-acetylglucosaminyltransferase complex (GPI-Gnt), that catalyzes the transfer of N-acetylglucosamine (GlcNAc) to PI at position six to initiate the GPI-AP biosynthesis pathway. This leads to the biosynthesis of the N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI) intermediate [6]. PIGC was first reported as a candidate gene for embryonic lethality [7]. Only five individual probands from three unrelated families have been reported so far, with moderate-severe DD/ID, and seizures (Glycosylphosphatidylinositol biosynthesis defect, MIM# 617816) [8, 9] due to rare homozygous or compound heterozygous variants in PIGC. Both missense and protein-truncating pathogenic variants have been previously described [8, 9]. The reports of these probands simply refer to seizures and limited data regarding the epileptology is provided [8, 9]. Thus, the epileptology of the PIGC-related encephalopathy, including evolution and treatment response, remains poorly understood.
Flow cytometry analysis is a useful tool to screen GPI-AP disorders. A GPI attachment signal is present at the C terminus of every GPI-AP precursor, but these signals vary in strength among different proteins. When the biosynthesis of GPI is disrupted by pathogenic variants, GPI-AP with weak attachment signals is preferentially altered. Hence, the extent of the decrease is different among various GPI-APs.
In this study, we describe clinical findings of 18 previously unreported probands with PIGC-associated encephalopathy from ten unrelated families, with a particular focus on epileptology. We also explore the effect of PIGC pathogenic variants on GPI-AP biosynthesis using proband-derived cells as well as a transfected cellular model.
MATERIALS AND METHODS
Probands
Probands with genotypes of interest were identified through an international network of Epilepsy and Genetics departments. Clinical details from affected individuals, including data regarding magnetic resonance imaging (MRIs) and electroencephalograms (EEG), were collected from their clinicians. The experimental protocol was approved by the institutional review boards of the Centre Hospitalier Universitaire (CHU) Saint-Justine Research Center. All probands or, in the case of minors, their parents or legal guardians gave informed consent. The clinical information was collected by interviewing families and/or from hospital records of the probands and their family members.
Genetic analyses
Whole genome sequencing was performed in two probands P11 and P13 as part of 100,000 Genome Projects (100KGP [10]; Cambridge South REC: 14/EE/1112). Primary analysis based on multiple panels was negative for P11 and P13. Whole exome sequencing was performed in the remainder of probands. Genomic data was then interrogated using an in-house panel-agnostic pipeline as previously described [11–14]. The predicted causal variants validation in all probands and segregation analysis in parents were confirmed by Sanger sequencing. Bioinformatics tools like EIGEN [15], FATHMM-MKL [16], LRT [17], M-CAP [18], MutationTaster [19], PROVEAN [20], and SIFT [21] were used to predict the pathogenicity of the variants. The frequency of the identified variants in PIGC (NM_153747.2) in the control population was determined using the Genome Aggregation Database (gnomAD v4.1.0). The American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) guidelines were used for variant interpretation and classification [10, 22].
Fluorescence-activated cell sorting analyses
Cell surface GPI-AP expression in fibroblasts and granulocytes from the probands was assessed by flow cytometry analysis using various GPI-AP markers: FLAER (Cedarlane, Burlington, NC, USA), CD16 (BioLegend, San Diego, CA, USA), CD59 (BioLegend, San Diego, CA, USA), CD73 (BioLegend, San Diego, CA, USA) and CD109 (BioLegend, San Diego, CA, USA). FLAER binds directly to the GPI anchor and thus serves as a marker of total GPI-APs, whereas the rest of the markers are GPI-AP specific. Blood samples from probands (P12, P13 and P14) and healthy controls were stained with Alexa488-conjugated inactivated aerolysin (FLAER; Protox Biotech, Victoria, BC, Canada), PE-conjugated anti-human CD16 (BioLegend, San Diego, CA, USA), FITC-conjugated anti-human CD24 and CD55 (BD Bioscience, San Jose, CA, USA) for 1 h on ice in 0.5% BSA incubation buffer. The red blood cells were lysed with fluorescence-activated cell sorting (FACS) lysing solution (BD Bioscience, San Jose, CA, USA) for 15 min and the white blood cells were pelleted and resuspended in 1ml PBS; GPI-AP expression on the surface of granulocytes was measured by a BD FACSCanto II system (BD Biosciences) and analyzed by Cytobank software.
Fibroblasts from P9 were collected and harvested at 80%-90% confluency and then stained with FLAER-Alexa 448, FITC-conjugated mouse anti-human CD73 (BioLegend, San Diego, CA, USA) or PE-conjugated mouse anti-human CD109 (BioLegend, San Diego, CA, USA) for 1 h on ice in the incubation buffer. The cells were then fixed in 4% formaldehyde. Non-specific binding was washed off before the cells were analyzed by a BD FACSCanto II system (BD Biosciences) and either Cytobank or FlowJo software.
Probands were investigated at different institutions using different markers and therefore the histograms are not standardized across probands.
Cellular model analyses
The impact of variants found in family 8 was determined by using a Pigc-deficient cell model to assess if they lead to a loss-of-function. cDNA of PIGC with variants p.(C265Ffs*11) and p.(G96R) were cloned into a pME expression vector. This vector is driven by a strong SRα promoter. By electroporation, plasmids were transfected into T1M1-Thy1(−)c (TIMI) cells [23]; these are Pigc-defective murine lymphoma cells. Transfection efficiency was monitored by co-transfecting luciferase-expression vector. Pathogenicity of the two variants was studied by assessing the restoration of the surface expression of GPI-APs by flow cytometry using mouse CD90 (PharMingen, San Diego, CA, USA), FLAER (Cedarlane, Burlington, NC, USA) and mouse CD48 (BioLegend, San Diego, CA, USA).
RESULTS
We ascertained 18 probands from 10 unrelated families, each with biallelic pathogenic variants in PIGC (Fig. 1A). The phenotypic details are summarized in Table 1, while genotypic details, including variant interpretation, are summarized in Table 2.
Fig. 1. Pedigrees and clinical images of patients with PIGC-related encephalopathy.
Panel A displays the ten pedigrees of the cases reported in this study, providing a visual representation of the familial relationships and inheritance patterns associated with the PIGC-related encephalopathy. Panels B–F show facial images of probands with notable dysmorphic features: P6 (panel B) exhibits long face, malar hypoplasia, short philtrum, pointed chin, mild retrognatia, and low-set large ears; P9 (panel C), a long face with a high forehead, sparse eyebrows, wide palpebral fissures, and a prominent nose; P10 (panel D), upturned earlobes, mild retrognatia, and mild enophtalmia; P11 (panel E), strabismus, wide and depressed root of the nose, upturned earlobes; P12 (panel F), strabismus, low-set ears, wide and depressed root of the nose, macrostomia, tented upper lip, gum hypertrophy, and myopathic facies.
Table 1.
Phenotypes identified in patients from present and previous study with biallelic pathogenic variants in PIGC.
| Family | Family 1 | Family 2 | Family 3 | Family 4 | Family 5 | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Patient | P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 | P9 | P10 | P11 | P12 |
| ID number in original paper | - | - | - | - | - | - | - | - | - | - | - | - |
| Variant (NM_153747.2) | c.437_445del, p.(T146_T148del) (hom) | c.437_445del, p.(T146_T148del) (hom) | c.437_445del, p.(T146_T148del) (hom) | c.437_445del, p.(T146_T148del) (hom) | c.437_445del, p.(T146_T148del) (hom) | c.437_445del, p.(T146_T148del) (hom) | c.437_445del, p.(T146_T148del) (hom) | c.437_445del, p.(T146_T148del) (hom) | c.437_445del, p.(T146_T148del) (hom) | c.437_445del, p.(T146_T148del) (hom) | c.437_445del, p.(T146_T148del) (hom) | c.859G>T; p.(E287T*) (hom) |
| Age | 4y (died) | 40 days (died) | 3y6mo (died) | 2y (died) | 7y (died) | 4y (died) | 8mo (died) | 3y (died) | 1.6y (died) | 6y (died) | 2y7mo | 3y |
| Sex | Female | Female | Female | Female | Male | Male | Male | Female | Male | Male | Male | Female |
| Age at last follow-up | 3y | 1mo | 3y | 18mo | 5y | 3y | 6mo | 2y8mo | 12mo | 6y | 2y7mo | Birth |
| Head circumference at last follow-up | 43 cm (−3.5 SD) | 36 cm (−0.71 SD) | 43 cm (−3.5 SD) | 41.7 cm (−4.3 SD) | 42.5 cm (−6.25 SD) | 43 cm (−3.91 SD) | 39 cm (−3.44. SD) | 42 cm (−3.57 SD) | 42 cm (−2.8 SD) | 45 cm (−4.8 SD) | 46.5 (−3.5 SD) | 35 cm (0.1 SD) |
| Height at last follow-up | 81 cm (−3.50 SD) | 52 cm (−0.63 SD) | 79 cm (−4.01 SD) | 72 cm (−3 SD) | 92 cm (−3.5 SD) | 92 cm (−1.00 SD) | 69 cm (0.75 SD) | 81 cm (−0.29 SD) | 70 cm (−2.4 SD) | 97 cm (−3.6 SD) | 88cm (−1.14 SD) | 47 cm (−2 SD) |
| Weight at last follow-up | 7 kg (−5.10 SD) | 3 kg (−3 SD) | 6.5 kg (−5.47 SD) | 7 kg (−3.8 SD) | 11 kg (−3 SD) | 7 kg (−5.36 SD) | 5 kg (−3.44 SD) | 7 kg (−4.19 SD) | 6.8 kg (−3.3 SD) | 7 kg (−6.06 SD) | 9kg (−3.53 SD) | 3.15 kg (−0.52 SD) |
| Degree of global developmental impairment | Profound | Unable to evaluate due to age | Profound | Profound | Profound | Profound | Profound | Profound | Profound | Profound | Profound | Profound |
| Motor development | Severely hypotonic, limited movements | Severely hypotonic, limited movements | Severely hypotonic, limited movements | Severely hypotonic, limited movements | Severely hypotonic. Unable to sit. Briefly holds own head | Severely hypotonic, no achieved milestones | Severely hypotonic. Unable to sit or hold own head | Severely hypotonic, no achieved milestones | Severely hypotonic, limited movements | Severely hypotonic limited movements | Severely hypotonic limited movements | Severely hypotonic, limited movements |
| Verbal development | Nonverbal | - | Nonverbal | Nonverbal | Nonverbal | Nonverbal | Nonverbal | Nonverbal | Nonverbal | Nonverbal | Nonverbal | NA |
| Seizures | Yes | No | Yes | Yes | Yes | Yes | Yes | Yes | Yes | Yes | Yes | Yes |
| Age at onset of seizures | 6mo | Not relevant | 6mo | 3mo | 1mo | 1y6mo | 1mo | 7mo | 8mo | 11mo | 11mo | NA |
| Seizure types | FTS | Not relevant | FTS | FTS, atonic, My | FTS, FBTCS, atonic, My | FTS, My | FTS, My | FS | FTS, atonic, My | FTS | FTS | NA |
| Epilepsy classification | Focal | - | Focal | Combined focal and generalized | Combined focal and generalized | Combined focal and generalized | Focal | Focal | Combined focal and generalized | Focal | Focal | NA |
| Treatment resistant seizures | Yes | Not relevant | Yes | Yes | Yes | Yes | Yes | Yes | Yes | Yes | Yes | Yes |
| Ictal EEG findings | Focal discharge | Not relevant | Multifocal discharges | Multifocal discharges, GSW, PSW | Multifocal discharges, GSW, PSW | Multifocal discharges, GSW, PSW | Multifocal discharges | Multifocal discharges | Multifocal discharges, GSW, PSW | Multifocal discharges | Multifocal discharges | NA |
| Hearing loss | No | NA | No | No | No | Respond to high sounds | Respond to high sounds | NA, but no respond to sounds | No | No | No | NA |
| Ophtalmology | Hypopig-mented fundus | No | Strabis-mus | Strabismus, hypopigmen-ted fundus | Strabismus | No | No | Strabismus | Strabismus | No | Strabismus | No |
| Dysplastic nail | No | No | No | Yes | No | No | No | No | Yes | No | No | NA |
| Skeletal anomalies | No | No | No | Clinodactyly | Scoliosis, pectus excavatum, arachnodactyly | No | No | No | Arachno dactyly | No | No | NA |
| MRI findings | CH | NA | CH, abnormal CC, CVL, enlarged LV | CH, abnormal CC, CVL, SCVL, WMI, enlarge LV and TV | CH, abnormal CC, CVL and SCVL | CH, CVL, SCVL | CVL | CH, CVL | CH, abnormal CC, WMI | CH, abnormal CC, CVL, enlarged LV, WMI | CH, abnormal CC, CVL, enlarged LV, WMI | NA |
| Serum AP | NA | No | No | No | Normal | No | No | No | No | NA | NA | Normal |
| Other | Possible SUDEP, MC | Died of possible pneumonia. | Possible SUDEP, MC | Possible SUDEP, MC, GER, severe constipation, dermatitis | Possible SUDEP, MC, GER cryptorchidism,severe constipation | Possible SUDEP, MC | Possible SUDEP, MC | Possible SUDEP, MC hepatomegaly | Possible SUDEP, MC, HT | Hepato-megaly and jaundice detected two weeks before death | Head nodding | NA |
| Family | Family 6 | Family 7 | Family 8 | Family 9 | Family 10 | Family 11 | Family 12 | Family 13 | |||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Patient | P13 | P14 | P15 | P16 | P17 | P18 | P19 | P20 | P21 | P21 | P22 |
| ID number in original paper | - | - | - | - | - | - | A-II-2 dvardson et al., 20178 | A-II-4 Edvardson et al., 20178 | B-II-1 Edvardson et al., 20178 | 1 Pons et al., 2020 9 | 2 Pons et al., 2020 9 |
| Variant (NM_153747.2) | c.77A>G; p.(D26G) (hom) | c.422C>T, c.138C>A; p.(T141I), p.(Y46*) | c.794_796delinsT, c.286G>A; p.(C265Ffs*11), p.(G96R) | c.794_796delinsT, c.286G>A; p.(C265Ffs*11), p.(G96R) | c.77A>T, c.286G>A; p. (D26V), p.(G96R) | c.3G>A, p.(M1?) (hom) | c.566T>G: p.(L189W) (hom) | c.566T>G: p.(L189W) (hom) | c.61C>T, c.635T>C; p.(R21*), p.(L212P) | c.12_13insTTGTGACTAACA; p.(Q4_P5insL*) (hom) | c.12_13insTTGTGACTAACA; p.(Q4_P5insL*) (hom) |
| Age | 3y | 26y | 4y (died) | 2y | 2.5y | 5y | 9y | 6y | 3y | 5y | 20mo |
| Gender | Female | Female | Female | Female | Male | Female | Male | Female | Male | Male | Female |
| Age at last follow-up | 18mo | 26y | 2.5y | Birth | 27mo | 5y | NA | NA | NA | 5y | 20mo |
| Head circumference | 48.5 cm (1.5 SD) | 56.7 cm (2.1 SD) | 43 cm (−3.38 SD) | 34.5 cm (0.2 SD) | 52 cm (1.90 SD) | 46.5 cm (−2.75 SD) | NA | NA | NA | 39 (3 SD) | 34 (1 SD) |
| Height | 81.6 cm (0.4 SD) | 170 cm (1 SD) | 79 cm (−3 SD) | NA | 95 cm (0.91 SD) | 102 cm (−1.24 SD) | NA | NA | NA | 47 (−1 SD) | 45 (1 SD) |
| Weight | 11.45 kg (−0.3 SD) | 70 kg (0.7 SD) | 10.2 kg (−2.25 SD) | 3.25 kg (0.4 SD) | 14.5 kg (0.63 SD) | 17 kg (−0.48 SD) | NA | NA | NA | 3.14 (−0.5 SD) | 2.69 (1 SD) |
| Degree of global developmental impairment | Severe | Mild | Severe | Severe | Moderate | Profound | Severe | Moderate | Severe | Severe | Severe |
| Motor development | Severely hypotonic | Walked at 17 m of life | Severely hypotonic. Limited movements | NA | Hypotonic, sits, crawls | Profound delay | Walked at 6 yrs of life | Walked at 2 yrs of life | Severely hypotonic | Severely hypotonic, walked at 3 yrs of life | Severely hypotonic |
| Verbal development | Nonverbal | Few sentences | Nonverbal | NA | Nonverbal | Nonverbal | Few words | Short sentences | Nonverbal | Nonverbal | Nonverbal |
| Seizures | Yes | Yes | Yes | No | Yes | Yes | Yes | Yes | Yes | Yes | Yes |
| Age at onset of seizures | 8mo | 6mo | 2mo | Not relevant | 8mo | NA | < 1y | < 1y | < 1y | 12mo | 7mo |
| Seizure types | FTS, unknown onset TCS | Atypical AS, GTCS | ES | Not relevant | FTS | Mainly generalized tonic | Unknown onset TCS | Unknown onset TCS | Unknown | My and unknown onset CS | FS of unknown type |
| Epilepsy classification | Focal | Generalized | Focal | Not relevant | Focal | Generalized | Unclassified | Unclassified | Unclassified | Generalized and also unclassified | Focal |
| Treatment resistant seizures | Yes | Yes | Yes | Not relevant | Yes | Yes | NA | NA | Yes | Yes | NA |
| Ictal EEG findings | Multifocal discharges | GSW, PSW | Hypsarythmia, multifocal discharges | Not relevant | NA | NA | NA | NA | NA | NA | NA |
| Hearing loss | No | No | Yes | NA | No | No | NA | NA | NA | NA | NA |
| Ophtalmology | Strabismus, hypopigmented fundus | Hyperopia | Abnormal but not further specified | Abnormal but not further specified | Nystagmus | NA | NA | NA | NA | High myopia | Oculocephalic deviation |
| Cardiomyopathy | No | DCM | No | No | No | Tetralogy of Fallot | NA | NA | NA | NA | NA |
| Dysplastic nail | NA | Yes | No | No | No | No | NA | NA | NA | NA | NA |
| Skeletal anomalies | No | No | Scoliosis, preaxial polydactyly | No | Kyphosis | No | NA | NA | NA | Pectus excavatum, short limbs | Pectus carinatum, short limbs |
| MRI | Hyperintensity in central tegmental tracts | CH | CH, hyperintensity in globus pallidi and central tegmental tracts | NA | Normal (15mo) | Widening of TH, HCC, hypomyelination, narrowing of SP | Normal | Normal | Normal | Normal at 3mo and 2y of life | Normal at 4mo of life |
| Serum AP | Dyskinesia | NA | Elevated (538u/L) | NA | Normal | Elevated (650u/L) | Normal | Normal | Normal | NA | NA |
| Other | Scoliosis | Chronic constipation, mild sleep apnea | Died of possible pneumonia. Thrombocy topenia. RAI. Hydronephrosis Anteriorly placed anus. | Anteriorly placed anus, accessory nipples, hydro nephrosis | Sleep disturbance, chorea, opisthotonus | NA | NA | NA | NA | Hepatosplenomegaly. Recurrent airway infections. Limb hypertonia | Recurrent airway infections. Obstructive sleep apnoea. HT, Hypertonia |
+: present; −: absent; AP alkaline phosphatase, AtypAS atypical absences, CC corpus callosum, CH cerebellar hypoplasia, Comp het compound heterozygous, CS clonic seizure, CVL cortical volume loss, DCM dilated cardiomyopathy, ES epileptic spasms, FBTCS focal to bilateral tonic-clonic seizures, FS focal seizure, GE gastroesophageal reflux; GSW generalized spike and slow wave, GTCS generalized tonic clonic seizures, hom homozygous, HT Hypothyroidism, HCC hypoplastic CC, LV lateral ventricles, mo month(s), MC microcephaly, My myoclonic seizure, NA not available, PSW polyspike and slow wave, RAI recurrent airway infections, SCVL subcortical volume loss, SP superior peduncles, SUDEP sudden unexpected death in epilepsy, TH temporal horns, TV third ventricle, WMI white matter immaturity, y years.
Table 2.
Summary of PIGC variant classification according to 2015 ACMG/AMP based criteria and in silico pathogenicity prediction.
| Patient ID | Variants in PIGC (NM_153747.2) | Chromosomal position (hg38) | Interpretation (ACMG/AMP criteria) | In silico predictions | Allele frequency according to gnomAD browser v4.1.0 |
|---|---|---|---|---|---|
| P1-11 | c.437_445del, p.(T146_T148del) | chr1-172442178-TGGTGTCAG | Pathogenic (PS3, PM2, PM4, PP1, PP3) | Predicted pathogenic by SIFT and PROVEAN | Variant not reported |
| P12 | c.859G>T, p.(E287T*) | chr1-172441764-C-A | Pathogenic (PVS1, PS3, PM2, PP3) | Predicted pathogenic by EIGEN, FATHMM-MKL, LRT, MutationTaster | 0.000001274 |
| P13 | c.77A>G, p.(D26G) | chr1-172442546-T-C | Likely pathogenic (PS3, PM2, PP3) | Predicted pathogenic by EIGEN, FATHMM-MKL, LRT, M-CAP, MutationTaster, PROVEAN, SIFT | 0.000001239 |
| P14 | c.422C>T, p.(T141I), | chr1-172442201-G-A | Likely pathogenic (PS3, PM2, PP3) | Predicted pathogenic by EIGEN, FATHMM-MKL, LRT, M-CAP, MutationTaster, PROVEAN, SIFT | 0.000004957 |
| c.138C>A, p.(Y46*) | chr1-172442485-G-T | Pathogenic (PVS1, PS3, PM2, PP3) | Predicted pathogenic by EIGEN, FATHMM-MKL, LRT, MutationTaster | Variant not reported | |
| P15, P16 | c.794_796delinsT, p.(C265Ffs*11) | chr1-172441827-GAC-A | Pathogenic (PVS1, PS3, PM2, PP3) | Predicted pathogenic by EIGEN, MutationTaster | Variant not reported |
| c.286G>A, p.(G96R) | chr1-172442337-C-T | Likely pathogenic (PS3, PM2, PP3) | Predicted pathogenic by EIGEN, FATHMM-MKL, LRT, M-CAP, MutationTaster, PROVEAN, SIFT | 0.00001922 | |
| P17 | c.77A>T, p.(D26V) | chr1-172442546-T-A | Likely pathogenic (PS3, PM2, PP3) | Predicted pathogenic by EIGEN, FATHMM-MKL, LRT, M-CAP, MutationTaster, PROVEAN, SIFT | 0.000007434 |
| c.286G>A, p.(G96R) | chr1-172442337-C-T | Likely pathogenic (PS3, PM2, PP3) | Predicted pathogenic by EIGEN, FATHMM-MKL, LRT, M-CAP, MutationTaster, PROVEAN, SIFT | 0.00001922 | |
| P18 | c.3G>A, p.(M1I) | chr1-172442620-C-T | Pathogenic (PVS1, PS3, PM2, PP1) | Predicted pathogenic by SIFT | Variant not reported |
Genotypic findings
Exome sequencing identified eight novel variants in PIGC, illustrated in Fig. 2: homozygous c.437_445del, p.(T146_T148del) in P1-11, c.859 G > T, p.(E287*) in P12, c.77 A > G, p.(D26G) in P13, compound heterozygous variants c.422 C > T, p.(T141I) and c.138 C > A, p.(Y46*) in P14, compound heterozygous variants c.794_796delinsT, p.(C265Ffs*11), and c.286 G > A, p.(G96R) in P15-16, compound heterozygous variants c.77 A > T, p.(D26V) and c.286 G > A, p(G96R) in P17, and homozygous, potential start-loss variant c.3 G > A, p.(M1?) in P18. Sanger sequencing confirmed these variants in the probands, and parental testing revealed heterozygosity.
Fig. 2. Schematic representations of PIGC protein and variants.
Panel A: Visualization of the PIGC protein using Protter (https://wlab.ethz.ch/protter/start/). This schematic highlights the topology of the PIGC protein, with previously reported variants shown in blue and novel variants identified in the current study marked in red. This representation helps in understanding the spatial distribution of different variants within the protein structure and their potential impact on function. Panel B: This schematic further illustrates the PIGC protein using ProteinPaint (https://proteinpaint.stjude.org/) and its associated variants, offering a user-friendly view of the genetic landscape. As in Panel A, previously reported variants are depicted in blue, while novel variants from this study are indicated in red, providing a consolidated view for easy cross-reference and assessment.
All variants are rare in frequency databases (allele frequency ranging from 0 to 1.922 × 10−5 in gnomAD v4.1.0); none have been reported in homozygous state in healthy probands. In silico analysis consistently predicted these variants as pathogenic (Table 2).
Variant computational modeling
Due to the absence of experimental structural data, we used a predicted AlphaFold2 model to analyze disease-associated mutations in PIGC. The predicted structure reveals eight transmembrane helices (TMHs), with both N- and C-termini located in the cytosol. Apart from unstructured N- and C-terminal regions, PIGC is largely devoid of loop domains and is thus embedded in the membrane (Fig. 3A).
Fig. 3. Structural Interpretation of PIGC-related encephalopathy variants.
Overview (panel A) and close-ups (panels B–D) of mutated regions. Nonsense (gray), frameshift (gray), and deletion (black) mutations are displayed as Cα spheres. Point mutations (magenta) are depicted as Cα spheres or side-chain sticks, with surrounding residues shown as stick (panels B, D) or sphere (panel C) representations.
Nonsense mutations, including those at Tyr46 and Arg21, as well as a frameshift mutation at Cys265, are expected to result in truncated, non-functional proteins. However, the nonsense mutation at Glu287 produces a protein with a relatively short truncation of only 10 residues, which is unlikely to disrupt overall folding. Thus, this residue might play a role in mediating PIGC’s interactions with other subunits of the GPI-GnT complex. Similarly, the D26G/D26V mutations could also impact subunit binding.
The deletion mutation Δ146-148 likely disrupts protein folding, as these residues are positioned at the start of TMH4. The short preceding loop connecting TMH3 and TMH4 is likely insufficient to compensate for this deletion, impairing structural integrity.
Mutations within transmembrane regions likely induce steric clashes, leading to destabilization of PIGC. Gly96 on TMH2, which packs against TMH6, if replaced by bulky, positively charged arginine, could cause steric clashes with adjacent residues, such as Leu216 and Trp213 (Fig. 3B). Thr141 on TMH3 packs closely with TMH5 and TMH6 at the membrane periphery; replacement with isoleucine may introduce clashes with nearby residues including Cys188, Val187, and Ser191 (Fig. 3C). Leu189 on TMH5 is in proximity to aromatic residues Trp72 on TMH1 and Tyr150 on TMH4. Replacement with tryptophan at this position could cause significant steric clashes (Fig. 1D). Finally, Leu212, located in the middle of TMH6, when mutated to proline, is expected to disrupt α-helical structure and cause misfolding.
Phenotypic findings
Neurodevelopmental impairment.
The majority (16/18, 89%) of probands had profound ID/DD, being nonverbal and unable to walk or sit unsupported. Proper head control, social smiling, and fixing-and-following were frequently absent. For proband P2, clinical assessment was limited due to her passing at 1 month of age, possibly secondary to pneumonia. However, as the vast majority of our cohort, she was severely underweight (12/18, 67%) and hypotonic (15/18, 83%). Only two probands, P14 and P17, had ID/DD described as mild or moderate. Proband P14 had mild ID and the ability to communicate using few sentences by the age 26 years; she was able to sit by 6 months and walk by 17 months. P14 and P17 represented 2/4 of probands with compound heterozygous variants, while all probands with homozygous variants had severe to profound ID/DD. These findings suggest that the PIGC-related encephalopathy is characterized by severe neurodevelopmental impairment, but moderate or even mild phenotypes are also possible.
Brain imaging anomalies.
Developmental anomalies were seen in all proband but one proband (P17), who underwent cerebral MRI (14/15). These included cerebellar atrophy (11/14), prominent cortical and/or subcortical volume loss (8/14), abnormal corpus callosum (7/14), hypomyelination (4/14), and enlarged ventricles (4/14). An abnormal signal in the central tegmental tracts was detected in P13 and P15. Additionally, proband P18, exhibited widening of temporal horns and narrowing of superior peduncles. These findings suggest that the PIGC-related encephalopathy is tightly linked to abnormal findings in cerebral MRI.
Neuromotor impairment.
Movement disorders were present in our cohort, although not previously described. Dystonia was noted in P4 and P9, while P13 exhibited dyskinetic movements in both upper and lower limbs. Spasticity was not noted in any of our cases, but some probands had brisk reflexes. Proband P15 had upper limb hypertonia and displayed dystonic movements of the arms. P4 also suffered from breath-holding spells, while P15 had recurrent respiratory tract infections and breathing difficulties. These findings suggest that the PIGC-related encephalopathy can present with additional neuromotor anomalies.
Epileptic activity.
Seizures were reported in all probands who survived beyond the first month of age, except for P16. Seizures began at a mean age of 7 months (range 1 month to 18 months) (Table 1). Nine probands presented with focal epilepsy, two probands with generalized seizures, and the rest had either combined or unclassified epilepsy. Only 2/17 probands had isolated focal seizures, while 14/17 had focal to bilateral or generalized tonic-clonic seizures. Atonic and myoclonic seizures were noted in 3/17 and 6/17 probands, respectively. Proband P14 had atypical absences, and P5 had isolated epileptic spasms. Over the followup period, seven probands developed additional seizure types.
Focal tonic seizures could be provoked by fever, typically lasting 3–10 min and occurred several times weekly. Atonic seizures had an onset around 6-8 months of age and typically lasted a few seconds; they occurred several times daily without known triggers. The myoclonic jerks had an onset between 4 and 12 months of age, occurring several times daily, with frequency increasing due to fever. Convulsive status epilepticus occurred in at least 9/16 probands, often in the first 3 years of life.
EEG recordings were available for 13/16 probands with seizures, revealing interictal EEG patterns from an early age. They typically showed focal or diffuse background slowing with interictal multifocal epileptic discharges, particularly in the frontotemporal or posterior regions. While multifocal discharges were seen in 11/13 probands, P1 had unifocal epileptiform discharges. In those classified with generalized or combined focal and generalized epilepsy, generalized ictal spike-wave activity was recorded during seizures. In P15, EEG showed hypsarrhythmia, which improved after treatment but remained encephalopathic.
All probands with epilepsy experienced drug-resistant and ongoing seizures, contrasting with previously published cases where 2/5 (A-II-2 and A-II-4) were responsive to antiseizure medications (ASM) (Table 1). No single ASM proved consistently effective across multiple probands. While some ASMs appeared beneficial for certain probands, they were ineffective or exacerbated seizures in others. Levetiracetam, vigabatrin and topiramate were reported to reduce seizure frequency by at least 50% for a period of at least 6 months. None of the probands were placed on a ketogenic diet or given pyridoxine. Proband P15 developed infantile spasms at 2 months old and was treated with prednisolone and vigabatrin. Vigabatrin was stopped due to drowsiness and bradycardia. She was initially started on levetiracetam, which was eventually replaced with valproate and topiramate. Clobazam proved most helpful in controlling her seizures and she remained on a low-maintenance dose (5mg twice a day). These findings suggest that epileptic activity in the PIGC-related encephalopathy tends to be severe and complex to treat.
Childhood-onset mortality.
Out of the 18 probands, 10 were deceased. The age at death ranged from 40 days to 7 years of life (median age of 40 months). Nine probands died within the first four years of life (Table 1). The causes of death were due either to respiratory failure (2/10) or possible sudden unexpected death in epilepsy (SUDEP) (8/10). These findings suggest that high and early mortality should be a concern among patients with PIGC-related encephalopathy.
Other phenotypic findings.
Dysmorphic features were noted in nearly all probands (Fig. 1B–F). These included prominent/high forehead, deep-set eyes, large and low-set ears, wide mouth with full lips, full cheeks and pointed chin. Skeletal anomalies were also common, such as scoliosis (P5, P13 and P15), pectus excavatum (P5), joint laxity (P6, P9 and P12), clinodactyly (P4), arachnodactyly (P5 and P9), and pre-axial polydactyly (P15). Additionally, three probands presented with dysplastic fingernails (P4, P9 and P14). Anteriorly placed anus and hydronephrosis were noted in probands P15-16. These findings suggest that dysmorphisms are predominant in the PIGC-related encephalopathy.
Detection of secondary GPI-AP reduction by flow cytometry
Flow cytometry analysis revealed a 53% and 42% decrease in FLAER and CD16 levels in granulocytes of proband P12 (Fig. 4A). Although P12 had a nonsense variant, p.E287*, the premature stop codon was at the end of the last exon, likely allowing the transcript to escape NMD and produce a truncated protein, resulting in reduced surface expression of GPI-APs.
Fig. 4. Flow cytometry analysis of granulocytes derived from PIGC-encephalopathy patients.
Panels A–C: Flow cytometry histograms granulocytes from probands P10 A, P11 B, and P12 C compared to healthy controls. These histograms represent at least two independent experiments using three different control samples. Due to investigations being carried out at different institutions with various markers and using either Cytobank or FlowJo software, histograms were not standardized across probands, reflecting the real-world complexity and variability in lab conditions. Panel D: Analysis of Pigc-deficient TIMI cells, a mouse lymphoma cell line, which were transfected with either wild-type or mutant PIGC-expressing plasmids (pME) under the control of a strong promoter. Two days post-transfection, the restored expression of various GPI-anchored proteins (GPI-APs) was assessed through flow cytometry. Results revealed that the C265Ffs*11 mutant exhibited no activity, whereas the G96R mutant demonstrated decreased activity compared to the wild-type PIGC, indicating variant-specific functional impairments. Panel E: Expression levels of affected proteins were comparable to those in wild-type cells, as confirmed by western blot analysis. Band intensities were normalized to GAPDH levels, serving as the loading control, to ensure accurate quantification of protein expression across samples.
In proband P13 granulocytes, we showed a decrease of 63% in FLAER levels and 68% in both CD55 and CD16 (Fig. 4B). Proband P14 granulocytes showed reductions in FLAER, CD24 and CD16 to 56%, 42%, and 49% compared to controls, respectively (Fig. 4C). In fibroblasts from proband P11, levels of FLAER, CD73 and CD109 were decreased to 51%, 80%, and 45% compared to healthy probands (data not shown).
Functional analysis of variants in knockout cells is an alternative option to study variant consequences, offering insights when proband cells are unavailable. We performed functional analysis on two variants using Pigc-deficient TIMI cells, focusing on a newly reported missense variant, and a frameshift variant found in family 8. The experiments demonstrated that the mutants p.(C265Ffs*11) showed no activity, while the p.(G96R) mutant had decreased activity compared to the wildtype protein (Fig. 4D, E). Overall, these findings suggest that variants leading to PIGC-related encephalopathy are linked to decreased GPI cellular signal.
DISCUSSION
We report the largest cohort so far of individuals affected with PIGC-related encephalopathy and further characterize the related neurological features. Inherited GPI-anchoring disorders are a relatively newly defined group of clinical conditions. Their identification can be particularly challenging due to overlapping features with many other neurodevelopmental disorders, such as ID/DD, abnormal movements, and epilepsy. Our study significantly expands the current understanding by more than tripling the number of probands with PIGC-related encephalopathy, allowing for a deeper exploration of the phenotypical spectrum, neurological features, and seizure pattern associated with this condition. We expand the known clinical range to include mild and moderate ID/DD, although severe ID/DD remains predominant. We also expand the spectrum of seizure types, to include focal tonic seizures, absence and atypical absence seizures, focal to bilateral or generalized tonic-clonic seizures, and epileptic spasms, in addition to the previously reported myoclonic and tonic-clonic seizures. Despite earlier reports suggesting that epilepsy in PIGC-related encephalopathy might respond well to antiseizure medications (ASMs), epilepsy is generally treatment-resistant within our cohort. Additionally, our study underscores a concerning trend toward a high early mortality, due to possible SUDEP or respiratory failure related to airway infections. Our observation classifies PIGC-related encephalopathy as one of the most severe GPI disorders with very poor prognosis. These findings should increase awareness among pediatricians, child neurologists, epileptologists, and geneticists to enhance healthcare management and life expectancy in PIGC-related encephalopathy.
Pathogenic variants in PIGC were first identified in an embryonic lethal case by autozygosity mapping [7]. Later, three probands from two unrelated families were reported to have generalized bilateral tonic-clonic seizures and severe ID/DD [8]. Two of these subjects were ambulant and capable of spoking either a few words or short simple sentences. Additionally, two siblings from one of the families were reported to have multisystem disorders alongside ID/DD and seizures [9].
In contrast, most of our probands exhibit profound ID/DD. Notably, only probands P14 and P17 had mild or moderate ID/DD. These two cases represented half of the probands with compound heterozygous variants (Fig. 3), whereas all probands with homozygous variants had severe to profound ID/DD. This observation may imply that in compound heterozygotes, the presence of one less severe variant could allow for a milder phenotype, as exemplified by the missense variant seen in P17. However, a larger cohort of cases is needed to reinforce our findings and to further explore into the natural history of this disorder.
Fourteen out of 18 probands had homozygous variants (Fig. 3). Among these, the stop-gain variant, p.(E287*), is in the only coding exon, which is also the last exon. This suggests that the associated transcript might escape the NMD pathway [24]. In our study, we did not observe solid genotype-phenotype correlations. Clinical presentations ranged from isolated ID/DD and seizure to additional multisystem involvement and even embryonic lethality. Additional research with a larger cohort and more comprehensive data are needed to explore whether specific variant locations correlate with particular clinical outcomes.
PIGC has been shown to restore the biosynthesis of GPI anchors and the subsequent cell surface expression of GPI-APs when transfected into mammalian cells deficient for PIGC function [23]. PIGC has been reported to form a complex with PIGA, PIGH, PIGQ, PIGP, PIGY, and DPM2 collectively constituting the GPI N-acetylglucosaminyl transferase (GPI-GnT). This complex plays a crucial role in the transfer of N-acetylglucosamine (GlcNAc) to phosphatidylinositol (PI) using UDP-GlcNAc [6, 23, 25–29]. Flow cytometry analysis of granulocytes from probands with PIGC variants showed a striking decrease in CD16 levels and a moderate reduction in CD59 and FLAER levels [8]. Our flow cytometry results align with these previous findings and provide further evidence that dysfunctional PIGC results in defective GPI biosynthesis.
In conclusion, we report 18 new probands with PIGC-related encephalopathy, expanding the known phenotypical spectrum, and identifying a high risk of early mortality. We also initiate delineation efforts for genotype-phenotype correlations. Using proband-derived cells as well as a transfected Pigc-deficient cellular model, we demonstrated that PIGC pathogenic variants lead to a decrease in GPI-AP signal in flow cytometry. This further corroborates the essential role of PIGC in neurodevelopment.
ACKNOWLEDGEMENTS
The authors are grateful to the families for cooperating in this study. This research was made possible through access to data in the National Genomic Research Library, which is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The National Genomic Research Library holds data provided by patients and collected by the NHS as part of their care and data collected as part of their participation in research. The National Genomic Research Library is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure.
FUNDING
AB is funded by a BRIDGE - Translational Excellence Programme grant funded by the Novo Nordisk Foundation, grant agreement number: NNF20SA0064340. PMC is supported by awards from the CIHR and the FRQS. MZ is supported by Science and Technology Development Fund (STDF) grant 33650. MIUR project “Dipartimenti di Eccellenza 2023-2027” to the Department of Neurosciences “Rita Levi Montalcini” (University of Turin); Italian Ministry for Education, University and Research (Ministero dell’Istruzione, dell’Università e della Ricerca - MIUR) PRIN2020 code 20203P8C3X. The whole-exome sequencing was performed as part of the Autism Sequencing Consortium and was supported by the NIMH (MH111661). This research was supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC) Programme and the Wellcome Trust (203141/Z/16/Z). This study has been delivered through the NIHR Manchester BRC (NIHR203308). The views expressed are those of the author(s) and not necessarily those of the, the NIHR or the Department of Health and Social Care. Dr Adam Jackson is supported by Solve-RD. The Solve-RD project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 779257.
Footnotes
COMPETING INTERESTS
We declare that the authors do not have any conflict of interest, and all have read and approved the final manuscript.
ETHICS DECLARATION
The study protocol was approved by CHU Sainte-Justine Research Ethics Board (#MP-21-2016-962). Samples were collected from probands and families after written informed consent was obtained from the parents or legal guardians, and the data was de-identified. A written medical photography consent was obtained from the parents of P6, P9, P10, P11, and P12.
DATA AVAILABILITY
All clinical data generated or analyzed during this study are included in this published article.
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Associated Data
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Data Availability Statement
All clinical data generated or analyzed during this study are included in this published article.