PGAP2 mutations, affecting the GPI-anchor-synthesis pathway, cause hyperphosphatasia with mental retardation syndrome

Abstract

Recently, mutations in genes involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor have been identified in a new subclass of congenital disorders of glycosylation (CDGs) with a distinct spectrum of clinical features. To date, mutations have been identified in six genes (PIGA, PIGL, PIGM, PIGN, PIGO, and PIGV) encoding proteins in the GPI-anchor-synthesis pathway in individuals with severe neurological features, including seizures, muscular hypotonia, and intellectual disability. We developed a diagnostic gene panel for targeting all known genes encoding proteins in the GPI-anchor-synthesis pathway to screen individuals matching these features, and we detected three missense mutations in PGAP2, c.46C>T, c.380T>C, and c.479C>T, in two unrelated individuals with hyperphosphatasia with mental retardation syndrome (HPMRS). The mutations cosegregated in the investigated families. PGAP2 is involved in fatty-acid GPI-anchor remodeling, which occurs in the Golgi apparatus and is required for stable association between GPI-anchored proteins and the cell-surface membrane rafts. Transfection of the altered protein constructs, p.Arg16Trp (NP_001243169.1), p.Leu127Ser, and p.Thr160Ile, into PGAP2-null cells showed only partial restoration of GPI-anchored marker proteins, CD55 and CD59, on the cell surface. In this work, we show that an impairment of GPI-anchor remodeling also causes HPMRS and conclude that targeted sequencing of the genes encoding proteins in the GPI-anchor-synthesis pathway is an effective diagnostic approach for this subclass of CDGs.

Figure 1

Phenotypic Features of HPMRS Associated with Mutations in PGAP2 (A and B) Face of individual A from family A at the ages of 3 (A) and 28 years (B). (C) Normal-appearing fingernails of the affected individual in family A. (D and E) Facial dysmorphism of the affected individual in family B at the age of 2 years includes wide palpebral fissures, a short nose with a broad nasal bridge, a tented upper lip, and a small jaw. (F) Distal tapering of fingers and mild nail hypoplasia of the fifth digit of the affected individual in family B.

Figure 2

Identification and Segregation of the PGAP2 Mutations Pedigrees showing segregation of the HPMRS phenotype with deleterious variants in PGAP2 in families A (A) and B (B). Circles represent females, squares represent males, filled symbols represent affected individuals, and dots within the symbols represent heterozygotes. Sequence reads show the mutation in short read alignments visualized in integrative genome viewer.

Figure 3

Reduced Activity of Altered Forms of PGAP2 in Restoring Surface Expression of GPI-Anchored Proteins after Transfection into PGAP2-Null Cell Lines PGAP2-deficient CHO cells were transiently transfected with wild-type or altered forms (p.Arg16Trp, p.Thr160Ile [family A], and p.Leu127Ser [family B]) of pTA Flag-PGAP2 isoform 8 driven by a weak promoter. Restoration of the surface expression was assessed 2 days later by flow cytometry. p.Arg16Trp and p.Thr160Ile detected in family A and p.Leu127Ser detected in family B did not restore the surface expression of CD59 and CD55 as efficiently as the wild-type PGAP2. The reduction of surface protein levels associated with p.Arg16Trp was less severe. This correlates with a lower sequence conservation of this position and a milder phenotype in individual II-1of family A.