Bi-allelic Variants in the GPI Transamidase Subunit PIGK Cause a Neurodevelopmental Syndrome with Hypotonia, Cerebellar Atrophy, and Epilepsy

Abstract

Glycosylphosphatidylinositol (GPI)-anchored proteins are critical for embryogenesis, neurogenesis, and cell signaling. Variants in several genes participating in GPI biosynthesis and processing lead to decreased cell surface presence of GPI-anchored proteins (GPI-APs) and cause inherited GPI deficiency disorders (IGDs). In this report, we describe 12 individuals from nine unrelated families with 10 different bi-allelic PIGK variants. PIGK encodes a component of the GPI transamidase complex, which attaches the GPI anchor to proteins. Clinical features found in most individuals include global developmental delay and/or intellectual disability, hypotonia, cerebellar ataxia, cerebellar atrophy, and facial dysmorphisms. The majority of the individuals have epilepsy. Two individuals have slightly decreased levels of serum alkaline phosphatase, while eight do not. Flow cytometric analysis of blood and fibroblasts from affected individuals showed decreased cell surface presence of GPI-APs. The overexpression of wild-type (WT) PIGK in fibroblasts rescued the levels of cell surface GPI-APs. In a knockout cell line, transfection with WT PIGK also rescued the GPI-AP levels, but transfection with the two tested mutant variants did not. Our study not only expands the clinical and known genetic spectrum of IGDs, but it also expands the genetic differential diagnosis for cerebellar atrophy. Given the fact that cerebellar atrophy is seen in other IGDs, flow cytometry for GPI-APs should be considered in the work-ups of individuals presenting this feature.

Keywords: GPI8; PIGK; glycosylphosphatidylinositol (GPI); inherited GPI deficiency disorders (IGDs); transamidase.

Figure 1

Pedigrees of Families with Bi-allelic PIGK Variants

Figure 2

Facial and MRI Features of Subjects with PIGK Variants Clinical features in two siblings of Family 2. Facial dysmorphisms: long face, sparse hair, high anterior hairline, prominent forehead, broad and laterally sparse eyebrows, thin upper lip, antihelix shelf, prominent antitragus, dental crowding, abnormally shaped teeth, and brachydactyly in hands and feet. Magnetic resonance imaging (MRI) findings: cerebellar atrophy in Individuals 2A, 2B, 3, 5, 6, and 8.

Figure 3

PIGK Variants and Conservation of Affected Residues (A) Location of the variants on the PIGK gene and corresponding protein. Introns not drawn to scale. (B) Multiple alignment of phosphatidylinositol glycan (PIG) anchor biosynthesis class K (PIGK) orthologs showing conservation of the affected residues in vertebrates.

Figure 4

Cell Surface GPI-AP Levels in Blood Cells of Affected Individuals Blood samples from the individuals in Families 1, 2, and 3 and control cells were stained with FLAER and CD16. Figure shows representative analysis of cell surface GPI-AP levels of granulocytes from triplicate experiments.

Figure 5

Rescue Assays in Fibroblasts Skin fibroblasts derived from Individuals 1A (upper panels) and 7 (lower panels) were transduced with PIGK-expressing-Lv105 lentivirus or empty Lv105-lentivirus, and non-transduced cells were stained with FLAER, CD73, and CD109. The figure shows representative results from experiments done in triplicate.

Figure 6

In Vitro Functional Studies for PIGK Variants Identified in Individuals 1A and 1B (A) Fluorescence-activated cell sorting (FACS) analysis of phosphatidylinositol glycan (PIG) anchor biosynthesis class K (PIGK)-deficient CHO cells transfected with mutant or wild-type (WT) PIGK cDNA driven by a strong promoter, pME vector (upper panels), or a weak promoter, pTK vector (lower panels). (B) Expression of WT and mutant PIGK-GST protein in PIGK deficient CHO cells transfected with mutant or WT PIGK cDNA driven by a strong promoter, pME vector. GAPDH is a loading control.