PMC8185152_01
Confirmation of novel IL-10RA variants. (A) c.395T>G variant (red arrow) confirmed by Sanger sequencing. (B) Validation of the ex.1del (p.?) variant by quantitative PCR. Genomic DNA was extracted from peripheral blood from the patient, her parents and healthy controls. Reactions were set up using one set of primers upstream and downstream of exon 1 in triplicate. The beta-actin gene served as a reference gene. The 2-DeltaDeltaCT method was used to calculate the copy number. Samples with normalized ratios (NRs) <0.1 denote individuals with homozygous deletion, samples with NRs of about 0.5 denote individuals with heterozygous deletion, sample with NRs of ~1 denote healthy individuals (two copies), and samples with NRs of ~1.5 or more denote individuals with copy number gain.
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F1
Furthermore, two IL-10RA variants were confirmed by Sanger sequencing (Figure 1A) or quantitative PCR (Figure 1B).
Western blot showing the patient had normal expression of p-STAT3 in peripheral blood mononuclear cells (PBMCs) in response to IL-6 (positive control) stimulation but showed defective STAT3 phosphorylation under IL-10 stimulation. -, untreated; +, treated.
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F2
Both IL-10 and IL-6 were able to induce the phosphorylation of STAT3 in PBMCs from healthy control and the patient's father and mother (Figure 2).