Case report: Functional characterization of a de novo c.145G>A p.Val49Met pathogenic variant in a case of PIGA-CDG with megacolon

Abstract

A subgroup of congenital disorders of glycosylation (CDGs) includes inherited GPI-anchor deficiencies (IGDs) that affect the biosynthesis of glycosylphosphatidylinositol (GPI) anchors, including the first reaction catalyzed by the X-linked PIGA. Here, we show the first PIGA-CDG case reported in Mexico in a male child with a moderate-to-severe phenotype characterized by neurological and gastrointestinal symptoms, including megacolon. Exome sequencing identified the hemizygous variant PIGA c.145G>A (p.Val49Met), confirmed by Sanger sequencing and characterized as de novo. The pathogenicity of this variant was characterized by flow cytometry and complementation assays in PIGA knockout (KO) cells.

Keywords: CD59 antigen; CDG (congenital disorder of glycosylation); GPI (glycosylphosphatidylinositol); PIGA; exome.

FIGURE 1

Barium enema. (A). Anteroposterior view showing chronic megarectum. (B). Oblique view showing severe dilatation of the sigmoid and rectum with loss of haustral markings.

FIGURE 2

Sanger sequence chromatograms of gDNA showing the PIGA variant NM_002641.4(PIGA):c.145G>A (p.Val49Met). The patient shows the mutation at codon position 49 of exon 2. Both mother and father are non-carriers.

FIGURE 3

CD16 expression in granulocytes and rescue analysis in the PIGA-KO HEK293 cells. (A) Histogram depicting median fluorescence intensity (MFI) of CD16-PE in granulocytes from the patient (blue histogram) and healthy control (red histogram) (healthy control MFI = 5,130 vs. patient MFI = 4,450). (B) Rescue of CD59 and DAF expression on the PIGA-KO HEK293 cells using a weak TATA box only promoter (pTA). The histograms represent the MFI for the rescue of CD59 and DAF expression by the wild type and Val49Met (V49M) variant using the pTA promoter. Values for three independent experiments and their statistical significance were graphed. CD59: wild type MFI = 943 vs. variant MFI = 584. DAF: wild type MFI = 1,074 vs. variant MFI = 785. Representative Western blotting of the mutant (Val49Met) and wild type (Wild) PIGA protein expression showing that the PIGA mutant protein was more expressed than the wild-type protein. (normalized with the intensities of GAPDH, the loading control, and luciferase activities used for evaluating transfection efficiencies). PIGA protein was detected by anti-His mAb (arrow), and loading control was revealed with anti-GAPDH. Empty vector = vector without insert gene. Histograms in (A) y axis show cell counts; the x axis shows fluorescence intensity.