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New and recurrent gain-of-function STAT1 mutations in patients with chronic mucocutaneous candidiasis from Eastern and Central Europe.

Soltész B, Tóth B, Shabashova N, Bondarenko A, Okada S, Cypowyj S, Abhyankar A, Csorba G, Taskó S, Sarkadi AK, Méhes L, Rozsíval P, Neumann D, Chernyshova L, Tulassay Z, Puel A, Casanova JL, Sediva A, Litzman J, Maródi L - J. Med. Genet. (2013)

Bottom Line: The c.1154C>T (T385M) mutation affecting the DNA-binding domain (DBD) resulted in a gain of STAT1 phosphorylation in a Ukrainian patient.Impaired Candida-induced IL-17A and IL-22 secretion by leucocytes and lower levels of intracellular IL-17 and IL-22 production by T cells were found in several patients.Haplotype studies indicated that the c.820C>T (R274W) mutation was recurrent due to a hotspot rather than a founder effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious and Pediatric Immunology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary, EU.

ABSTRACT

Background: Chronic mucocutaneous candidiasis disease (CMCD) may result from various inborn errors of interleukin (IL)-17-mediated immunity. Twelve of the 13 causal mutations described to date affect the coiled-coil domain (CCD) of STAT1. Several mutations, including R274W in particular, are recurrent, but the underlying mechanism is unclear.

Objective: To investigate and describe nine patients with CMCD in Eastern and Central Europe, to assess the biochemical impact of STAT1 mutations, to determine cytokines in supernatants of Candida-exposed blood cells, to determine IL-17-producing T cell subsets and to determine STAT1 haplotypes in a family with the c.820C>T (R274W) mutation.

Results: The novel c.537C>A (N179K) STAT1 mutation was gain-of-function (GOF) for γ-activated factor (GAF)-dependent cellular responses. In a Russian patient, the cause of CMCD was the newly identified c.854 A>G (Q285R) STAT1 mutation, which was also GOF for GAF-dependent responses. The c.1154C>T (T385M) mutation affecting the DNA-binding domain (DBD) resulted in a gain of STAT1 phosphorylation in a Ukrainian patient. Impaired Candida-induced IL-17A and IL-22 secretion by leucocytes and lower levels of intracellular IL-17 and IL-22 production by T cells were found in several patients. Haplotype studies indicated that the c.820C>T (R274W) mutation was recurrent due to a hotspot rather than a founder effect. Severe clinical phenotypes, including intracranial aneurysm, are presented.

Conclusions: The c.537C>A and c.854A>G mutations affecting the CCD and the c.1154C>T mutation affecting the DBD of STAT1 are GOF. The c.820C>T mutation of STAT1 in patients with CMCD is recurrent due to a hotspot. Patients carrying GOF mutations of STAT1 may develop multiple intracranial aneurysms by hitherto unknown mechanisms.

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STAT1 dephosphorylation in nuclear extracts. Western blotting was performed on nuclear extracts of EBV-transformed lymphocytes from P8 and P4, carrying the c.1154C>T (T385) and c.820C>T (R274W) heterozygous missense mutations of STAT1, respectively (table 1). (A) Before nuclear extracts were prepared, cells were treated with 2000 U/ml IFN-γ, 2000 U/ml IFN-α or 50 μg/ml IL-27 for 30 min. The data shown are representative of three independent experiments. The T385M (P8) and R274W (P4 and P5) STAT1 proteins are more strongly phosphorlylated than the WT protein in control cells. (B) Effect of the tyrosine kinase inhibitor staurosporine on STAT1 dephosphorylation in nuclear extracts of cells from healthy controls (left), P8 (middle) and P4 (right) with CMCD. EBV-transformed lymphocytes were treated with 2000 U/ml IFN-γ for 30 min and then with staurosporine (concentration, 8 ng/ml) for 15 and 30 min. Staurosporine induced the dephosphorylation of nuclear STAT1 in control cells but not in cells from P8 and P4. C, control; EBV, Epstein–Barr Virus; IFN, interferon; P, patient; STAT, signal transducer and activator of transcription; WB, western blot.
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JMEDGENET2013101570F2: STAT1 dephosphorylation in nuclear extracts. Western blotting was performed on nuclear extracts of EBV-transformed lymphocytes from P8 and P4, carrying the c.1154C>T (T385) and c.820C>T (R274W) heterozygous missense mutations of STAT1, respectively (table 1). (A) Before nuclear extracts were prepared, cells were treated with 2000 U/ml IFN-γ, 2000 U/ml IFN-α or 50 μg/ml IL-27 for 30 min. The data shown are representative of three independent experiments. The T385M (P8) and R274W (P4 and P5) STAT1 proteins are more strongly phosphorlylated than the WT protein in control cells. (B) Effect of the tyrosine kinase inhibitor staurosporine on STAT1 dephosphorylation in nuclear extracts of cells from healthy controls (left), P8 (middle) and P4 (right) with CMCD. EBV-transformed lymphocytes were treated with 2000 U/ml IFN-γ for 30 min and then with staurosporine (concentration, 8 ng/ml) for 15 and 30 min. Staurosporine induced the dephosphorylation of nuclear STAT1 in control cells but not in cells from P8 and P4. C, control; EBV, Epstein–Barr Virus; IFN, interferon; P, patient; STAT, signal transducer and activator of transcription; WB, western blot.

Mentions: It was initially suggested that the STAT1 mutations in CMCD patients affected the STAT1 CCD only, resulting in a GOF because of a gain of phosphorylation.9 We detected a recently reported mutation, c.1154C>T (T385M), affecting the STAT1 DBD, in one of the Ukrainian CMCD patients (table 1; patient P8).13 EBV-B cells from P4 and P8, heterozygous for the R274W and T385M mutations, respectively, were treated with IFN-γ, IFN-α and IL-27, all of which predominantly activate STAT1.9 Western blotting showed that treatment with these cytokines resulted in stronger STAT1 phosphorylation in the nuclear extracts of cells from both patients than in those from controls (figure 2A), demonstrating that the heterozygous alleles were associated with a dominant phenotype of gain of STAT1 phosphorylation. Incubation with the TYK inhibitor staurosporine and comparison with the control lymphoblasts showed that the dephosphorylation of IFN-γ-treated EBV-B cells heterozygous for the STAT1 T385M or R274W alleles was impaired (figure 2B). By contrast, after pervanadate treatment, the phosphorylation of T385M STAT1 and R274W STAT1 was similar to that of WT STAT1 (see online supplementary figure S2). These data confirm that the T385M and R274W amino acid substitutions in STAT1 result in a gain of STAT1 phosphorylation due to a loss of dephosphorylation.913


New and recurrent gain-of-function STAT1 mutations in patients with chronic mucocutaneous candidiasis from Eastern and Central Europe.

Soltész B, Tóth B, Shabashova N, Bondarenko A, Okada S, Cypowyj S, Abhyankar A, Csorba G, Taskó S, Sarkadi AK, Méhes L, Rozsíval P, Neumann D, Chernyshova L, Tulassay Z, Puel A, Casanova JL, Sediva A, Litzman J, Maródi L - J. Med. Genet. (2013)

STAT1 dephosphorylation in nuclear extracts. Western blotting was performed on nuclear extracts of EBV-transformed lymphocytes from P8 and P4, carrying the c.1154C>T (T385) and c.820C>T (R274W) heterozygous missense mutations of STAT1, respectively (table 1). (A) Before nuclear extracts were prepared, cells were treated with 2000 U/ml IFN-γ, 2000 U/ml IFN-α or 50 μg/ml IL-27 for 30 min. The data shown are representative of three independent experiments. The T385M (P8) and R274W (P4 and P5) STAT1 proteins are more strongly phosphorlylated than the WT protein in control cells. (B) Effect of the tyrosine kinase inhibitor staurosporine on STAT1 dephosphorylation in nuclear extracts of cells from healthy controls (left), P8 (middle) and P4 (right) with CMCD. EBV-transformed lymphocytes were treated with 2000 U/ml IFN-γ for 30 min and then with staurosporine (concentration, 8 ng/ml) for 15 and 30 min. Staurosporine induced the dephosphorylation of nuclear STAT1 in control cells but not in cells from P8 and P4. C, control; EBV, Epstein–Barr Virus; IFN, interferon; P, patient; STAT, signal transducer and activator of transcription; WB, western blot.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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JMEDGENET2013101570F2: STAT1 dephosphorylation in nuclear extracts. Western blotting was performed on nuclear extracts of EBV-transformed lymphocytes from P8 and P4, carrying the c.1154C>T (T385) and c.820C>T (R274W) heterozygous missense mutations of STAT1, respectively (table 1). (A) Before nuclear extracts were prepared, cells were treated with 2000 U/ml IFN-γ, 2000 U/ml IFN-α or 50 μg/ml IL-27 for 30 min. The data shown are representative of three independent experiments. The T385M (P8) and R274W (P4 and P5) STAT1 proteins are more strongly phosphorlylated than the WT protein in control cells. (B) Effect of the tyrosine kinase inhibitor staurosporine on STAT1 dephosphorylation in nuclear extracts of cells from healthy controls (left), P8 (middle) and P4 (right) with CMCD. EBV-transformed lymphocytes were treated with 2000 U/ml IFN-γ for 30 min and then with staurosporine (concentration, 8 ng/ml) for 15 and 30 min. Staurosporine induced the dephosphorylation of nuclear STAT1 in control cells but not in cells from P8 and P4. C, control; EBV, Epstein–Barr Virus; IFN, interferon; P, patient; STAT, signal transducer and activator of transcription; WB, western blot.
Mentions: It was initially suggested that the STAT1 mutations in CMCD patients affected the STAT1 CCD only, resulting in a GOF because of a gain of phosphorylation.9 We detected a recently reported mutation, c.1154C>T (T385M), affecting the STAT1 DBD, in one of the Ukrainian CMCD patients (table 1; patient P8).13 EBV-B cells from P4 and P8, heterozygous for the R274W and T385M mutations, respectively, were treated with IFN-γ, IFN-α and IL-27, all of which predominantly activate STAT1.9 Western blotting showed that treatment with these cytokines resulted in stronger STAT1 phosphorylation in the nuclear extracts of cells from both patients than in those from controls (figure 2A), demonstrating that the heterozygous alleles were associated with a dominant phenotype of gain of STAT1 phosphorylation. Incubation with the TYK inhibitor staurosporine and comparison with the control lymphoblasts showed that the dephosphorylation of IFN-γ-treated EBV-B cells heterozygous for the STAT1 T385M or R274W alleles was impaired (figure 2B). By contrast, after pervanadate treatment, the phosphorylation of T385M STAT1 and R274W STAT1 was similar to that of WT STAT1 (see online supplementary figure S2). These data confirm that the T385M and R274W amino acid substitutions in STAT1 result in a gain of STAT1 phosphorylation due to a loss of dephosphorylation.913

Bottom Line: The c.1154C>T (T385M) mutation affecting the DNA-binding domain (DBD) resulted in a gain of STAT1 phosphorylation in a Ukrainian patient.Impaired Candida-induced IL-17A and IL-22 secretion by leucocytes and lower levels of intracellular IL-17 and IL-22 production by T cells were found in several patients.Haplotype studies indicated that the c.820C>T (R274W) mutation was recurrent due to a hotspot rather than a founder effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious and Pediatric Immunology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary, EU.

ABSTRACT

Background: Chronic mucocutaneous candidiasis disease (CMCD) may result from various inborn errors of interleukin (IL)-17-mediated immunity. Twelve of the 13 causal mutations described to date affect the coiled-coil domain (CCD) of STAT1. Several mutations, including R274W in particular, are recurrent, but the underlying mechanism is unclear.

Objective: To investigate and describe nine patients with CMCD in Eastern and Central Europe, to assess the biochemical impact of STAT1 mutations, to determine cytokines in supernatants of Candida-exposed blood cells, to determine IL-17-producing T cell subsets and to determine STAT1 haplotypes in a family with the c.820C>T (R274W) mutation.

Results: The novel c.537C>A (N179K) STAT1 mutation was gain-of-function (GOF) for γ-activated factor (GAF)-dependent cellular responses. In a Russian patient, the cause of CMCD was the newly identified c.854 A>G (Q285R) STAT1 mutation, which was also GOF for GAF-dependent responses. The c.1154C>T (T385M) mutation affecting the DNA-binding domain (DBD) resulted in a gain of STAT1 phosphorylation in a Ukrainian patient. Impaired Candida-induced IL-17A and IL-22 secretion by leucocytes and lower levels of intracellular IL-17 and IL-22 production by T cells were found in several patients. Haplotype studies indicated that the c.820C>T (R274W) mutation was recurrent due to a hotspot rather than a founder effect. Severe clinical phenotypes, including intracranial aneurysm, are presented.

Conclusions: The c.537C>A and c.854A>G mutations affecting the CCD and the c.1154C>T mutation affecting the DBD of STAT1 are GOF. The c.820C>T mutation of STAT1 in patients with CMCD is recurrent due to a hotspot. Patients carrying GOF mutations of STAT1 may develop multiple intracranial aneurysms by hitherto unknown mechanisms.

Show MeSH
Related in: MedlinePlus