Hypomorphic mutations in PGAP2, encoding a GPI-anchor-remodeling protein, cause autosomal-recessive intellectual disability

Abstract

PGAP2 encodes a protein involved in remodeling the glycosylphosphatidylinositol (GPI) anchor in the Golgi apparatus. After synthesis in the endoplasmic reticulum (ER), GPI anchors are transferred to the proteins and are remodeled while transported through the Golgi to the cell membrane. Germline mutations in six genes (PIGA, PIGL, PIGM, PIGV, PIGN, and PIGO) in the ER-located part of the GPI-anchor-biosynthesis pathway have been reported, and all are associated with phenotypes extending from malformation and lethality to severe intellectual disability, epilepsy, minor dysmorphisms, and elevated alkaline phosphatase (ALP). We performed autozygosity mapping and ultra-deep sequencing followed by stringent filtering and identified two homozygous PGAP2 alterations, p.Tyr99Cys and p.Arg177Pro, in seven offspring with nonspecific autosomal-recessive intellectual disability from two consanguineous families. Rescue experiments with the altered proteins in PGAP2-deficient Chinese hamster ovary cell lines showed less expression of cell-surface GPI-anchored proteins DAF and CD59 than of the wild-type protein, substantiating the pathogenicity of the identified alterations. Furthermore, we observed a full rescue when we used strong promoters before the mutant cDNAs, suggesting a hypomorphic effect of the mutations. We report on alterations in the Golgi-located part of the GPI-anchor-biosynthesis pathway and extend the phenotypic spectrum of the GPI-anchor deficiencies to isolated intellectual disability with elevated ALP. GPI-anchor deficiencies can be interpreted within the concept of a disease family, and we propose that the severity of the phenotype is dependent on the location of the altered protein in the biosynthesis chain.

Figure 1

Affected Children of Family MR043 The three affected individuals with PGAP2 mutations; there are no major dysmorphisms or syndromic gestalt.

Figure 2

PGAP2 Exon-Intron Structure and PGAP2 Sequence and Structure (A) The gene structure for the PGAP2 transcript variants 1 and 12. Isoform 1 (transcript variant 1) has a 61 aa insertion (Ala56 to Gly116) corresponding to an ectopic exon 3. It seems that transcript 12 (isoform 8) is the active one (Y. Murakami, unpublished data). Arrows denote the mutations. (B) The alignment of the human PGAP2 isoform 8 shows homology to mammalian, zebrafish, and frog orthologous proteins. The TM1-5 alpha-helix regions (predicted by TMHMM²²) are marked by rectangles, and the altered amino acids are denoted (RefSeq accession numbers NP_001243169.1 [Homo sapiens], NP_001092581.1 [Bos Taurus], NP_446347.1 [Rattus norvegicus], NP_663558.1 [Mus musculus], NP_001233740.1 [Cricetulus griseus], NP_001106477.1 [Xenopus tropicalis], and NP_001013562.1 [Danio rerio]). (C) PGAP2 is a transmembrane protein, and the p.Tyr99Cys and p.Arg177Pro alterations are located in the Golgi lumen.

Figure 3

Results of Functional Analyses in CHO Cells cDNAs encoding PGAP2 isoform 8 were subcloned into the vectors under promoters of different strengths and were expressed in PGAP2-deficient CHO cells. (A) Immunoblot of cell lysates isolated with the strong SRα promoter after 2 days of expression showed similar levels of wild-type (lane 1) and altered (lane 2) PGAP2 proteins; an empty-vector construct did not express the protein (lane 3). The protein levels were normalized (levels are shown underneath the blot) with the intensities of GAPDH expression shown at the left part of the blots. (B) Expression of the DAF and CD59 cell-surface proteins after transient transfection was monitored by FACS as a measurement of PGAP2 activities and is shown for the various promoter constructs. The left panel shows the p.Tyr99Cys alteration, and the right panel shows the p.Arg177Pro alteration. Under the strong SRα promoter, both altered proteins restore the cell-surface expression of DAF and CD59, suggesting a hypomorphic alteration. Expression under the weak promoters TK and TATA-box shows reduced activity of altered PGAP2 (see Results and Discussion for details).