title: Alanine-scanning mutagenesis of human signal transducer and activator of transcription 1 to estimate loss- or gain-of-function variants.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pediatrics, Hiroshima University Graduate School of Biomedical & Health Sciences, Hiroshima, Japan.
authors.forename: Reiko
authors.initials: R
authors.lastname: Kagawa
authors.affiliation: Department of Technology Development, Kazusa DNA Research Institute, Chiba, Japan.
authors.forename: Ryoji
authors.initials: R
authors.lastname: Fujiki
authors.affiliation: Department of Pediatrics, Hiroshima University Graduate School of Biomedical & Health Sciences, Hiroshima, Japan.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Tsumura
authors.affiliation: Department of Pediatrics, Hiroshima University Graduate School of Biomedical & Health Sciences, Hiroshima, Japan.
authors.forename: Sonoko
authors.initials: S
authors.lastname: Sakata
authors.affiliation: Department of Pediatrics, Hiroshima University Graduate School of Biomedical & Health Sciences, Hiroshima, Japan.
authors.forename: Shiho
authors.initials: S
authors.lastname: Nishimura
authors.affiliation: St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY.
authors.forename: Yuval
authors.initials: Y
authors.lastname: Itan
authors.affiliation: St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY.
authors.forename: Xiao-Fei
authors.initials: XF
authors.lastname: Kong
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan; Structural Medicine, United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan.
authors.forename: Zenichiro
authors.initials: Z
authors.lastname: Kato
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Ohnishi
authors.affiliation: Department of Pediatrics, Hiroshima University Graduate School of Biomedical & Health Sciences, Hiroshima, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Hirata
authors.affiliation: Department of Pediatrics, Hiroshima University Graduate School of Biomedical & Health Sciences, Hiroshima, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Saito
authors.affiliation: Department of Pediatrics, Okazaki City Hospital, Aichi, Japan.
authors.forename: Maiko
authors.initials: M
authors.lastname: Ikeda
authors.affiliation: Genetics Unit, Military Hospital Mohamed V, Hay Riad, Rabat, Morocco.
authors.forename: Jamila
authors.initials: J
authors.lastname: El Baghdadi
authors.affiliation: Laboratory of Clinical Immunology, Inflammation and Allergy, Faculty of Medicine and Pharmacy, Hassan II University of Casablanca, Casablanca, Morocco; Clinical Immunology Unit, Department of Pediatric Infectious Diseases, Averroes University Hospital, Casablanca, Morocco.
authors.forename: Aziz
authors.initials: A
authors.lastname: Bousfiha
authors.affiliation: Department of Pediatrics, National Hospital Organization Fukuyama Medical Center, Hiroshima, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Fujiwara
authors.affiliation: Department of Immunology, "Juan Pedro Garrahan" National Hospital of Pediatrics, Buenos Aires, Argentina.
authors.forename: Matias
authors.initials: M
authors.lastname: Oleastro
authors.affiliation: Department of Immunology, "Juan Pedro Garrahan" National Hospital of Pediatrics, Buenos Aires, Argentina.
authors.forename: Judith
authors.initials: J
authors.lastname: Yancoski
authors.affiliation: Department of Immunology, "Juan Pedro Garrahan" National Hospital of Pediatrics, Buenos Aires, Argentina.
authors.forename: Laura
authors.initials: L
authors.lastname: Perez
authors.affiliation: Department of Immunology, "Juan Pedro Garrahan" National Hospital of Pediatrics, Buenos Aires, Argentina.
authors.forename: Silvia
authors.initials: S
authors.lastname: Danielian
authors.affiliation: Laboratory of Clinical Immunology, Inflammation and Allergy, Faculty of Medicine and Pharmacy, Hassan II University of Casablanca, Casablanca, Morocco; Clinical Immunology Unit, Department of Pediatric Infectious Diseases, Averroes University Hospital, Casablanca, Morocco.
authors.forename: Fatima
authors.initials: F
authors.lastname: Ailal
authors.affiliation: Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Takada
authors.affiliation: Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Toshiro
authors.initials: T
authors.lastname: Hara
authors.affiliation: St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY; Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Paris, France; Paris Descartes University, Sorbonne Paris Cité, Imagine Institute, Paris, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Puel
authors.affiliation: St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY; Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Paris, France; Paris Descartes University, Sorbonne Paris Cité, Imagine Institute, Paris, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Boisson-Dupuis
authors.affiliation: St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY; Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Paris, France; Paris Descartes University, Sorbonne Paris Cité, Imagine Institute, Paris, France; Center for the Study of Primary Immunodeficiencies, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors.forename: Jacinta
authors.initials: J
authors.lastname: Bustamante
authors.affiliation: St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY; Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Paris, France; Paris Descartes University, Sorbonne Paris Cité, Imagine Institute, Paris, France; Pediatric Hematology-Immunology Unit, Assistance Publique-Hôpitaux de Paris, Necker Hospital for Sick Children, Paris, France; Howard Hughes Medical Institute, New York, NY.
authors.forename: Jean-Laurent
authors.initials: JL
authors.lastname: Casanova
authors.affiliation: Department of Technology Development, Kazusa DNA Research Institute, Chiba, Japan; Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Ohara
authors.affiliation: Department of Pediatrics, Hiroshima University Graduate School of Biomedical & Health Sciences, Hiroshima, Japan; St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY. Electronic address: saok969@gmail.com.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Okada
authors.affiliation: Department of Pediatrics, Hiroshima University Graduate School of Biomedical & Health Sciences, Hiroshima, Japan.
authors.forename: Masao
authors.initials: M
authors.lastname: Kobayashi
mesh_terms: D000409:Alanine / Q000235:genetics*; D001681:Biological Assay; D005260:Female; D020022:Genetic Predisposition to Disease; D006801:Humans; D008297:Male; D016296:Mutagenesis; D009154:Mutation; D009164:Mycobacterium Infections / Q000235:genetics*; D000072417:Protein Domains; D050794:STAT1 Transcription Factor / Q000235:genetics*
chemical_list: D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D000409:Alanine
keywords: Mendelian susceptibility to mycobacterial disease; Signal transduction and activator of transcription 1; alanine scanning; chronic mucocutaneous candidiasis; reference database