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

Reiko Kagawa, Ryoji Fujiki, Miyuki Tsumura, Sonoko Sakata, Shiho Nishimura, Yuval Itan, Xiao Fei Kong, Zenichiro Kato, Hidenori Ohnishi, Osamu Hirata, Satoshi Saito, Maiko Ikeda, Jamila El Baghdadi, Aziz Bousfiha, Kaori Fujiwara, Matias Oleastro, Judith Yancoski, Laura Perez, Silvia Danielian, Fatima AilalHidetoshi Takada, Toshiro Hara, Anne Puel, Stéphanie Boisson-Dupuis, Jacinta Bustamante, Jean Laurent Casanova, Osamu Ohara, Satoshi Okada, Masao Kobayashi

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10 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)232-241
Number of pages10
JournalJournal of Allergy and Clinical Immunology
Issue number1
Publication statusPublished - Jul 2017


All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Immunology

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