Figure 1.
Patient clinical presentation and aberrant activation of stimulator of interferon genes (STING) and NF-κB pathway by G348R recombinant RFC (rRFC). (A) Oral lesions (i) and dark skin discoloration of the knee (ii) and arm (iii) of P1. (B) Immunologic characteristics prior to folate supplementation. Phytohemagglutinin stimulation index reflects values during supplementation. (C) Serum and red blood cell (RBC) folate levels in P1 and P2 during supplementation (ages 8 and 5 years, respectively) demonstrating low RBC folate levels (below the fifth percentile) despite normal serum levels. The solid line represents median and dotted lines represent 5th and 95th percentile concentrations of RBC folate in healthy controls.10 (D) Schematic diagram of the folate and CDN transporter RFC, showing 12 TMDs and the intracellular N-terminus and C-terminus. The G348R mutation affects the ninth transmembrane domain. (E) HEK293T cells expressing wild-type (WT) rRFC, G348R rRFC (G348R), or plasmid vector control (Vec) alongside STING were coincubated in the presence/absence of the cyclic dinucleotide 2’3’-cGAMP (10 μg/mL, 2 hours). Phosphorylation of STING (p-STING) was assessed by Western blotting. The G348R variant induced less STING phosphorylation. The G348R variant induced less STING phosphorylation (bottom), as quantified by densitometry (mean ± standard deviation of 4 independent experiments; 2-tailed Mann-Whitney test). (F) Induction of p65 phosphorylation (p-p65) was impaired following 2’3’-cGAMP incubation in G348R compared with WT rRFC expressing HEK293T cells, as quantified by densitometry (mean ± standard deviation of 3 independent experiments; 2-tailed Mann-Whitney test). (G) Downstream induction of IFN-β, assessed by quantitative polymerase chain reaction, revealed impaired messenger RNA (mRNA) gene expression in G348R expressing HEK293T cells compared with WT (replicates n = 3). (H) Incubation of patient lymphocytes (P1, P2) with 2’3’-cGAMP (40 μg/mL) resulted in reduced STING phosphorylation compared with the healthy controls (Ctrl). Results are normalized against total STING protein levels (bottom). IgG, immunoglobulin G; PHA, phytohemagglutinin; WBC, white blood cell.

Patient clinical presentation and aberrant activation of stimulator of interferon genes (STING) and NF-κB pathway by G348R recombinant RFC (rRFC). (A) Oral lesions (i) and dark skin discoloration of the knee (ii) and arm (iii) of P1. (B) Immunologic characteristics prior to folate supplementation. Phytohemagglutinin stimulation index reflects values during supplementation. (C) Serum and red blood cell (RBC) folate levels in P1 and P2 during supplementation (ages 8 and 5 years, respectively) demonstrating low RBC folate levels (below the fifth percentile) despite normal serum levels. The solid line represents median and dotted lines represent 5th and 95th percentile concentrations of RBC folate in healthy controls.10 (D) Schematic diagram of the folate and CDN transporter RFC, showing 12 TMDs and the intracellular N-terminus and C-terminus. The G348R mutation affects the ninth transmembrane domain. (E) HEK293T cells expressing wild-type (WT) rRFC, G348R rRFC (G348R), or plasmid vector control (Vec) alongside STING were coincubated in the presence/absence of the cyclic dinucleotide 2’3’-cGAMP (10 μg/mL, 2 hours). Phosphorylation of STING (p-STING) was assessed by Western blotting. The G348R variant induced less STING phosphorylation. The G348R variant induced less STING phosphorylation (bottom), as quantified by densitometry (mean ± standard deviation of 4 independent experiments; 2-tailed Mann-Whitney test). (F) Induction of p65 phosphorylation (p-p65) was impaired following 2’3’-cGAMP incubation in G348R compared with WT rRFC expressing HEK293T cells, as quantified by densitometry (mean ± standard deviation of 3 independent experiments; 2-tailed Mann-Whitney test). (G) Downstream induction of IFN-β, assessed by quantitative polymerase chain reaction, revealed impaired messenger RNA (mRNA) gene expression in G348R expressing HEK293T cells compared with WT (replicates n = 3). (H) Incubation of patient lymphocytes (P1, P2) with 2’3’-cGAMP (40 μg/mL) resulted in reduced STING phosphorylation compared with the healthy controls (Ctrl). Results are normalized against total STING protein levels (bottom). IgG, immunoglobulin G; PHA, phytohemagglutinin; WBC, white blood cell.

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