PMC9196877_01
In silico analysis of c.649G>A (p.Gly217Arg) and c.706A>G (p.Arg236Gly) mutations on the ORC1 protein structure and function. (A) Partial electropherograms showing the detected mutations. Multiple alignment of the modified ORC1 aminoacid residues with the protein sequence from different species using the Clustal W2 software. (B)
In silico validation of the two SLC25A15 gene mutations identified. (C) Modeling of the tridimensional protein structural changes introduced by the two identified mutations using the human ORC1 homology model. Normal residues are marked in blue (top) and the changes introduced by mutations in red (bottom).
fimmu-13-861516-g001.jpg
PMC9196877_02_fimmu-13-861516-g001.jpg
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As shown in Figure 1A, multiple sequence alignment analysis revealed that G217R and R236G are both non-conservative substitutions in amino acid residues evolutionarily conserved among species, which suggests they are crucial for protein structure and function. Moreover, different bioinformatics prediction tools such as Clustal W, SIFT, Polyphen, CADD, REVEL and Metal R indicated that both mutations are highly detrimental for the secondary structure and function of the protein since both substituted amino acids differ in polarity, charge, size, and other biochemical properties (Figure 1B). Conversely, the residue Gly217 belongs to the Pro-Gly level 1 immediately above the substrate binding region, playing the crucial hinge/kink function, which allows conformational changes and the access of the substrate to the ORC1 binding site (Figure 1C).
PMC9196877_02
In silico analysis of c.649G>A (p.Gly217Arg) and c.706A>G (p.Arg236Gly) mutations on the ORC1 protein structure and function. (A) Partial electropherograms showing the detected mutations. Multiple alignment of the modified ORC1 aminoacid residues with the protein sequence from different species using the Clustal W2 software. (B)
In silico validation of the two SLC25A15 gene mutations identified. (C) Modeling of the tridimensional protein structural changes introduced by the two identified mutations using the human ORC1 homology model. Normal residues are marked in blue (top) and the changes introduced by mutations in red (bottom).
fimmu-13-861516-g001.jpg
PMC9196877_02_fimmu-13-861516-g001.jpg
f1
As shown in Figure 1A, multiple sequence alignment analysis revealed that G217R and R236G are both non-conservative substitutions in amino acid residues evolutionarily conserved among species, which suggests they are crucial for protein structure and function. Moreover, different bioinformatics prediction tools such as Clustal W, SIFT, Polyphen, CADD, REVEL and Metal R indicated that both mutations are highly detrimental for the secondary structure and function of the protein since both substituted amino acids differ in polarity, charge, size, and other biochemical properties (Figure 1B). Conversely, the residue Gly217 belongs to the Pro-Gly level 1 immediately above the substrate binding region, playing the crucial hinge/kink function, which allows conformational changes and the access of the substrate to the ORC1 binding site (Figure 1C).
PMC9196877_03
PMC9196877_04
Functional analysis of cellular adaptive immune compartment. (A) Lymphoproliferative responses of peripheral blood mononuclear cells (PBMC) from the patient (P) and control (C) individuals to different mitogenic stimuli [concanavalin A (Con A), phytohemagglutinin (PHA), Pokeweed (PKW), candidin, and antigens of Mycobacterium tuberculosis or Trychophyton spp.] or medium alone (mock). Cells were cultured (3.0x105/well) in quadruplicate for each condition and incubated for 96 h at 37 C/5% CO2 as previously described. During the last 18 h of culture, wells were pulsed with 1 microCi of [methyl-3H] thymidine in fresh medium. Cells were harvested onto glass fiber filters and labeled material was counted in a beta scintillation counter. Results were expressed as counts per minute (cpm). (B) IFNgamma, IL-17A, TNF-alpha and IL-10 concentrations in PBMCs culture supernatants determined by sandwich ELISA and results expressed as pg/ml. Experiments were performed at least in quadruplicate and repeated twice with similar results. Data are shown as mean +- SEM, the patient and sex and age-matched control individuals (n=6 healthy females, aged 3-7 years old). Red bars correspond to the average values of the patient and white bars correspond to the average values of the controls analyzed. Mann-Whitney test; *p< 0.05, **p< 0.01 and ***p< 0.005.
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A (Con A), phytohemagglutinin (PHA), Pokeweed (PKW), candidin, and antigens of Mycobacterium tuberculosis or Trychophyton spp. were assessed. As shown in Figure 2A, significantly reduced proliferation rates in response to most of the mitogens assayed were observed in the patient with respect to controls. When assaying the production of inflammatory cytokines, significantly reduced levels of IFNgamma, IL-17A and TNFalpha secretion were detected in culture supernatants of PBMC from the patient compared with controls (Figure 2B). IL-10 were detected in culture supernatants from the patient PBMC stimulated with Con A, PKW, and candidin with respect to controls (Figure 2B). In parallel, we quantified the levels of CD4+ and CD8+ T cells, B cells and NK cells in the patient peripheral blood by flow cytometry.
Phenotypic analysis of T and B cells. (A) Flow cytometry gating strategy for the assessment of phenotypic cell markers in live CD4 and CD8 T cells, and B cells from PBMC after red blood cell depletion by lysing buffer treatment. (B, C) Frequencies of naive T cells, central memory (CM), effector memory (EM), and terminally differentiated effector memory cells (TEMRA) within the CD4+ (B) or CD8+ (C) T cell populations from the patient (P) under study and controls (C). (D) Frequencies of mature, immature and memory B cells, and plasmablasts in peripheral blood from the patient (P) under study and controls (C). Representative flow cytometry dot plots. Experiments were performed in triplicates. Data are shown as mean +- SEM, the patient and sex and age-matched control individuals (n=6 healthy females, aged 3-7 years old). Red bars correspond to the average values of the patient and white bars correspond to the average values of the controls analyzed. Mann-Whitney test; *p< 0.05 and **p< 0.01. The following fluorescent-labeled anti-human antibodies (BioLegend) were used: CD3 (PerCP), CD4 (APC), CD19 (APC), CD45RA (PE-Cy7), CCR7 (PE), CD24 (FITC) and CD38 (PE). Dead cells were excluded using LIVE-DEAD  fixable (Invitrogen). Data were collected on FACS-CANTO II flow cytometer (BD Biosciences) and analyzed using FlowJo software (version 7.6.2). Proper compensation using Fluorescence Minus One (FMO) controls were used.
fimmu-13-861516-g003.jpg
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Although total B cell counts were within reference values, significantly reduced frequencies of plasmablasts, immature and memory B cells were found in the patient with respect to sex and age-matched healthy controls (Figure 3D). Conversely, the patient revealed significantly higher frequencies of mature B cells than control individuals (Figure 3D).
PMC9196877_05
Phenotypic analysis of T and B cells. (A) Flow cytometry gating strategy for the assessment of phenotypic cell markers in live CD4 and CD8 T cells, and B cells from PBMC after red blood cell depletion by lysing buffer treatment. (B, C) Frequencies of naive T cells, central memory (CM), effector memory (EM), and terminally differentiated effector memory cells (TEMRA) within the CD4+ (B) or CD8+ (C) T cell populations from the patient (P) under study and controls (C). (D) Frequencies of mature, immature and memory B cells, and plasmablasts in peripheral blood from the patient (P) under study and controls (C). Representative flow cytometry dot plots. Experiments were performed in triplicates. Data are shown as mean +- SEM, the patient and sex and age-matched control individuals (n=6 healthy females, aged 3-7 years old). Red bars correspond to the average values of the patient and white bars correspond to the average values of the controls analyzed. Mann-Whitney test; *p< 0.05 and **p< 0.01. The following fluorescent-labeled anti-human antibodies (BioLegend) were used: CD3 (PerCP), CD4 (APC), CD19 (APC), CD45RA (PE-Cy7), CCR7 (PE), CD24 (FITC) and CD38 (PE). Dead cells were excluded using LIVE-DEAD  fixable (Invitrogen). Data were collected on FACS-CANTO II flow cytometer (BD Biosciences) and analyzed using FlowJo software (version 7.6.2). Proper compensation using Fluorescence Minus One (FMO) controls were used.
fimmu-13-861516-g003.jpg
PMC9196877_05_fimmu-13-861516-g003.jpg
f3
Although total B cell counts were within reference values, significantly reduced frequencies of plasmablasts, immature and memory B cells were found in the patient with respect to sex and age-matched healthy controls (Figure 3D). Conversely, the patient revealed significantly higher frequencies of mature B cells than control individuals (Figure 3D).