Figure 1
Novel STAT1 mutation associated with abnormal mRNA splicing and protein expression. (A) Family pedigree. Subjects I.1 and I.2 are first cousins. The proband is indicated with an arrow. Healthy persons are shown in white; deceased siblings are indicated by line crossing. (B) Genomic sequence analysis of exon 3 showing a homozygous nucleotide substitution at position 372 (G > C) affecting the donor splicing site. Both parents are heterozygous for the same mutation. (C) Schematic diagram of the first 5 exons of the STAT1 mRNA patient. Coding exons are indicated with Roman numerals and delineated by vertical bars. The upper band corresponds to the wild-type form. The lower band was observed for the cells of the patient and corresponds to the form of STAT1 mRNA lacking exon 3. (D) RT-PCR of STAT1 fragment spanning from exon 1 to exon 5 shows a lower size fragment in the patient. (E) Schematic representation of STAT1 protein structure, including the position of the 33 amino acid residue deletion (Δ91-124) in the N-terminal domain as a result of the G372C mutation affecting the donor splicing site of exon 3. Other domains included the coiled-coil (CC), DNA-binding (DNA-B), linker (L), Src-homology 2 (SH2), tail segment (TS), and transactivation (TA) domains. The position of Tyr701 (Y) is shown. (F) Expression of STAT1 and STAT1Δ3 mRNAs in NK cells from control and patient. Target gene expression was normalized against the housekeeping gene (GAPDH) and presented as n-fold increase over the expression in the healthy control. The experiments shown are representative of 4 independent experiments. Data are mean ± SE. *Significant difference in the response between the patient and control subject (P < .05). (G) Flow cytometry analysis of STAT1 expression in T cells (CD3+ cells), as measured by intracellular staining with anti-STAT1 mAb, was performed as described in “Cytokine, ELISA assay, and flow cytometry.” Mean fluorescence intensity is indicated. The results are representative of 4 independent experiments. (H) Immunoprecipitation and Western blot analysis of STAT1α and STAT1β were performed using an antibody against STAT1 on PBMCs (30 μg of total lysates) from the patient and a control subject. Analysis shows that the 2 bands corresponding to STAT1α and STAT1β were less abundant and at a reduced molecular weight.

Novel STAT1 mutation associated with abnormal mRNA splicing and protein expression. (A) Family pedigree. Subjects I.1 and I.2 are first cousins. The proband is indicated with an arrow. Healthy persons are shown in white; deceased siblings are indicated by line crossing. (B) Genomic sequence analysis of exon 3 showing a homozygous nucleotide substitution at position 372 (G > C) affecting the donor splicing site. Both parents are heterozygous for the same mutation. (C) Schematic diagram of the first 5 exons of the STAT1 mRNA patient. Coding exons are indicated with Roman numerals and delineated by vertical bars. The upper band corresponds to the wild-type form. The lower band was observed for the cells of the patient and corresponds to the form of STAT1 mRNA lacking exon 3. (D) RT-PCR of STAT1 fragment spanning from exon 1 to exon 5 shows a lower size fragment in the patient. (E) Schematic representation of STAT1 protein structure, including the position of the 33 amino acid residue deletion (Δ91-124) in the N-terminal domain as a result of the G372C mutation affecting the donor splicing site of exon 3. Other domains included the coiled-coil (CC), DNA-binding (DNA-B), linker (L), Src-homology 2 (SH2), tail segment (TS), and transactivation (TA) domains. The position of Tyr701 (Y) is shown. (F) Expression of STAT1 and STAT1Δ3 mRNAs in NK cells from control and patient. Target gene expression was normalized against the housekeeping gene (GAPDH) and presented as n-fold increase over the expression in the healthy control. The experiments shown are representative of 4 independent experiments. Data are mean ± SE. *Significant difference in the response between the patient and control subject (P < .05). (G) Flow cytometry analysis of STAT1 expression in T cells (CD3+ cells), as measured by intracellular staining with anti-STAT1 mAb, was performed as described in “Cytokine, ELISA assay, and flow cytometry.” Mean fluorescence intensity is indicated. The results are representative of 4 independent experiments. (H) Immunoprecipitation and Western blot analysis of STAT1α and STAT1β were performed using an antibody against STAT1 on PBMCs (30 μg of total lysates) from the patient and a control subject. Analysis shows that the 2 bands corresponding to STAT1α and STAT1β were less abundant and at a reduced molecular weight.

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