Translational and clinical immunology
Alanine-scanning mutagenesis of human signal transducer and activator of transcription 1 to estimate loss- or gain-of-function variants

https://doi.org/10.1016/j.jaci.2016.09.035Get rights and content

Background

Germline heterozygous mutations in human signal transducer and activator of transcription 1 (STAT1) can cause loss of function (LOF), as in patients with Mendelian susceptibility to mycobacterial diseases, or gain of function (GOF), as in patients with chronic mucocutaneous candidiasis. LOF and GOF mutations are equally rare and can affect the same domains of STAT1, especially the coiled-coil domain (CCD) and DNA-binding domain (DBD). Moreover, 6% of patients with chronic mucocutaneous candidiasis with a GOF STAT1 mutation have mycobacterial disease, obscuring the functional significance of the identified STAT1 mutations. Current computational approaches, such as combined annotation-dependent depletion, do not distinguish LOF and GOF variants.

Objective

We estimated variations in the CCD/DBD of STAT1.

Methods

We mutagenized 342 individual wild-type amino acids in the CCD/DBD (45.6% of full-length STAT1) to alanine and tested the mutants for STAT1 transcriptional activity.

Results

Of these 342 mutants, 201 were neutral, 30 were LOF, and 111 were GOF mutations in a luciferase assay. This assay system correctly estimated all previously reported LOF mutations (100%) and slightly fewer GOF mutations (78.1%) in the CCD/DBD of STAT1. We found that GOF alanine mutants occurred at the interface of the antiparallel STAT1 dimer, suggesting that they destabilize this dimer. This assay also precisely predicted the effect of 2 hypomorphic and dominant negative mutations, E157K and G250E, in the CCD of STAT1 that we found in 2 unrelated patients with Mendelian susceptibility to mycobacterial diseases.

Conclusion

The systematic alanine-scanning assay is a useful tool to estimate the GOF or LOF status and the effect of heterozygous missense mutations in STAT1 identified in patients with severe infectious diseases, including mycobacterial and fungal diseases.

Section snippets

Functional assay based on systematic alanine-scanning mutagenesis

A vector from which to express HaloTag STAT1 was obtained from the Kazusa cDNA/ORF clone collection (FHC013013). The codons of the vector-encoding residues from L136 to F487 of STAT1, except the 10 alanines (A188, A230, A246, A254, A267, A401, A402, A415, A469, and A479), were individually substituted with GCC, the codon most frequently encoding alanine in human subjects, by using site-directed mutagenesis. The activities of the mutants were measured with a luciferase reporter assay with the

Functional assay based on systematic alanine-scanning mutagenesis

Alanine-scanning mutagenesis is a widely used technique in the determination of the catalytic or functional role of protein residues. We systematically investigated the effects of alanine substitutions in the CCD/DBD of STAT1 with a GAS reporter assay after IFN-γ stimulation. We generated 176 alanine mutants in the CCD and 166 alanine mutants in the DBD. We defined the individual alanine mutants as LOF or GOF if they showed less than 0.3 times or greater than 1.6 times the GAS transcriptional

Discussion

We systematically evaluated the effect of amino acid substitutions (342 alanine mutants) in the CCD/DBD of STAT1 using a GAS reporter assay. The GAS reporter assay is an accurate and practical method with which to assess LOF and GOF STAT1 mutations.11, 12, 15, 19, 20, 21 This assay allowed us to explain 100% of known LOF mutations and 78.1% (86.7% in the CCD and 64.3% in the DBD) of known GOF mutations in the CCD/DBD of STAT1 by using alanine substituents of the same residues. It showed that

References (61)

  • X. Wang et al.

    Exome sequencing reveals a signal transducer and activator of transcription 1 (STAT1) mutation in a child with recalcitrant cutaneous fusariosis

    J Allergy Clin Immunol

    (2013)
  • P.P. Lee et al.

    Penicillium marneffei infection and impaired IFN-gamma immunity in humans with autosomal-dominant gain-of-phosphorylation STAT1 mutations

    J Allergy Clin Immunol

    (2014)
  • S. Kataoka et al.

    Extrapulmonary tuberculosis mimicking Mendelian susceptibility to mycobacterial disease in a patient with signal transducer and activator of transcription 1 (STAT1) gain-of-function mutation

    J Allergy Clin Immunol

    (2016)
  • L. Martinez-Martinez et al.

    A novel gain-of-function STAT1 mutation resulting in basal phosphorylation of STAT1 and increased distal IFN-gamma-mediated responses in chronic mucocutaneous candidiasis

    Mol Immunol

    (2015)
  • M. Tanimura et al.

    Recurrent inflammatory aortic aneurysms in chronic mucocutaneous candidiasis with a gain-of-function STAT1 mutation

    Int J Cardiol

    (2015)
  • J. Toubiana et al.

    Heterozygous STAT1 gain-of-function mutations underlie an unexpectedly broad clinical phenotype

    Blood

    (2016)
  • L. Dotta et al.

    Clinical heterogeneity of dominant chronic mucocutaneous candidiasis disease: presenting as treatment-resistant candidiasis and chronic lung disease

    Clin Immunol

    (2016)
  • M. Al Rushood et al.

    Autosomal dominant cases of chronic mucocutaneous candidiasis segregates with mutations of signal transducer and activator of transcription 1, but not of Toll-like receptor 3

    J Pediatr

    (2013)
  • X. Mao et al.

    Structural bases of unphosphorylated STAT1 association and receptor binding

    Mol Cell

    (2005)
  • G.R. Stark et al.

    How cells respond to interferons

    Annu Rev Biochem

    (1998)
  • C. Mertens et al.

    Dephosphorylation of phosphotyrosine on STAT1 dimers requires extensive spatial reorientation of the monomers facilitated by the N-terminal domain

    Genes Dev

    (2006)
  • S. Dupuis et al.

    Impaired response to interferon-alpha/beta and lethal viral disease in human STAT1 deficiency

    Nat Genet

    (2003)
  • A. Chapgier et al.

    A partial form of recessive STAT1 deficiency in humans

    J Clin Invest

    (2009)
  • I.A. Kristensen et al.

    Novel STAT1 alleles in a patient with impaired resistance to mycobacteria

    J Clin Immunol

    (2011)
  • S. Dupuis et al.

    Impairment of mycobacterial but not viral immunity by a germline human STAT1 mutation

    Science

    (2001)
  • A. Chapgier et al.

    Novel STAT1 alleles in otherwise healthy patients with mycobacterial disease

    PLoS Genet

    (2006)
  • O. Hirata et al.

    Heterozygosity for the Y701C STAT1 mutation in a multiplex kindred with multifocal osteomyelitis

    Haematologica

    (2013)
  • M. Tsumura et al.

    Dominant-negative STAT1 SH2 domain mutations in unrelated patients with Mendelian susceptibility to mycobacterial disease

    Hum Mutat

    (2012)
  • E.P. Sampaio et al.

    A novel STAT1 mutation associated with disseminated mycobacterial disease

    J Clin Immunol

    (2012)
  • L. Liu et al.

    Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis

    J Exp Med

    (2011)
  • Cited by (0)

    Supported in part by Grants in Aid for Scientific Research from the Japan Society for the Promotion of Science (22591161 to M.K. and 25713039, 16H05355, and 16K15528 to S.O.) and was supported in part by the Practical Research Project for Rare/Intractable Diseases from Japan Agency for Medical Research and development (AMED). This study was also supported in part by Research on Measures for Intractable Diseases funding from the Japanese Ministry of Health, Labour and Welfare (H22-Nanchi-ippan-078 to M.K.), GSK Japan Research Grant 2014, and the Kurozumi Medical Foundation. The Laboratory of Human Genetics of Infectious Diseases is supported by institutional grants from INSERM, University Paris Descartes, the Rockefeller University, the St Giles Foundation, the US National Institute of Allergy and Infectious Diseases (grant no. R37AI095983 and U01AI109697), and grants from the French National Research Agency (ANR) under the “Investments for the future” program (grant no. ANR-10-IAHU-01). The sequence analysis was supported by the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University.

    Disclosure of potential conflict of interest: J.-L. Casanova serves on the ADMA Scientific Advisory Board and serves as a consultant for ADMA, Nimbus, and Vitae Pharmaceuticals. O. Ohara received grant support from the Japan Agency for Medical Research and Development. S. Okada receives grant support from the Japan Agency for Medical Research and Development, Grants in Aid for Scientific Research from the Japan Society, GlaxoSmithKline, and Kurozumi Medical Foundation. The rest of the authors declare that they have no relevant conflicts of interest.

    These authors contributed equally to this work.

    View full text