PMC9619068_01
Barium enema. (A). Anteroposterior view showing chronic megarectum. (B). Oblique view showing severe dilatation of the sigmoid and rectum with loss of haustral markings.
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F1
He still suffers from severe chronic constipation that has caused a megacolon (Figures 1A,B), requiring treatment with polyethylene glycol 3350 and/or sennosides and glycerine enemas.
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.
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F2
Sanger sequencing data identified that the variant is a de novo mutation in view that sequencing of parental gDNA showed that the mother was a non-carrier (Figure 2).
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.
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F3
A 13% reduction in CD16 was observed in the patient compared to the healthy control (Figure 3A). Rescue of DAF expression was also found to be significantly deficient (73% of wild type; p < 0.001) (Figure 3B). Western blot from cell lysates showed similar expression of both wild type and mutant protein (Figure 3B).