PMC6859795_01
Pedigree and clinical phenotype of proband. (A) Pedigree, with affected individual (the proband) in black and unaffected individuals in white. PIK3R1 genotyping of the pedigree identified a de novo mutation in the proband; WT, wild type. (B) Peripheral vasculitis of digits and heel. (C) Facial gestalt reminiscent of SHORT syndrome (short stature, hyperextensibility of joints and/or inguinal herniae, ocular depression, rieger anomaly of the eye, and teething problems): ocular depression, nasal deviation, and prominent mandible present in the proband (see main text and Supplemental Table 1). (D) Sanger sequencing confirmed a heterozygous splice site G/A mutation (black/green overlapping line) at position c.1425 + 1 of PIK3R1 gene in family member II-2 only. This mutation is absent (single black line corresponding to wild type "G" allele) in the other family members (I-1, I-2, II-1, and II-3).
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F1
The pedigree is summarized in Figure 1A. She had spontaneously developed vasculitic lesions of the fingers, toes, and heels (Figure 1B) at the age of 6 years, with no obvious infectious, cold-exposure, or other trigger. Her past medical history was remarkable, with intrauterine growth retardation and short stature: age 17 years; height 152 cm (second percentile); weight 38.6 Kg (<<0.4th centile); facial dysmorphia with deep-set eyes, astigmatism prominent jaw, and unilateral choanal atresia (Figure 1C). Supplemental Table 5), which also confirmed that neither parent had the mutation. Sanger sequencing was also then used to further (and definitively) confirm this as a de novo mutation in the proband and also confirm its absence in the parents and the two unaffected siblings (Figure 1D).
Immunofluorescence microscopy of intracellular C1q in M2 macrophages derived from the proband and a healthy control. (A) Immunofluorescent microscopy images taken, using confocal microscopy (original magnification 63x; Supplemental Methods), of unstimulated and stimulated permeabilized macrophages (72 h with 10 muM of dexamethasone and 200 ng/ml of gamma interferon, IFN-gamma) from the proband and a healthy control stained with C1q (green) and DAPI (blue). (B) Flow cytometry (see Supplemental Methods) demonstrated comparable intracellular and surface C1q staining from a healthy control and the proband monocytes following stimulation with dexamethasone and IFN-gamma for 72 h; FSC-H, forward scatter height. (C) C1q levels in the supernatant of monocyte derived macrophages (MDM) from the proband and healthy controls before and after stimulation with dexamethasone and IFN-gamma for 72 h (as described above). Cultured macrophages from the proband released comparable amounts of C1q into supernatants as healthy controls. Bar chart indicates mean of duplicate measurements in proband and three healthy controls; error bars indicate standard error of mean.
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F2
IFN-gamma and 10 muM dexamethasone for 72 h (see Supplemental Methods). Intracellular C1q staining of macrophages was clearly demonstrated in the proband and controls at baseline and following stimulation (Figure 2A). This observation was confirmed using flow cytometry (Supplemental Methods), which also demonstrated comparable intracellular and cell surface C1q staining (Figure 2B) of monocytes in the proband compared with healthy control. Lastly, cultured macrophages from the proband released comparable amounts of C1q into supernatants as healthy controls (Figure 2C).