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 Ailal; Hidetoshi Takada; Toshiro Hara; Anne Puel; Stéphanie Boisson-Dupuis; Jacinta Bustamante; Jean-Laurent Casanova; Osamu Ohara; Satoshi Okada; Masao Kobayashi

doi:10.1016/j.jaci.2016.09.035

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

J Allergy Clin Immunol. 2017 Jul;140(1):232-241. doi: 10.1016/j.jaci.2016.09.035. Epub 2016 Dec 20.

Authors

Reiko Kagawa¹, Ryoji Fujiki², Miyuki Tsumura¹, Sonoko Sakata¹, Shiho Nishimura¹, Yuval Itan³, Xiao-Fei Kong³, Zenichiro Kato⁴, Hidenori Ohnishi⁵, Osamu Hirata¹, Satoshi Saito¹, Maiko Ikeda⁶, Jamila El Baghdadi⁷, Aziz Bousfiha⁸, Kaori Fujiwara⁹, Matias Oleastro¹⁰, Judith Yancoski¹⁰, Laura Perez¹⁰, Silvia Danielian¹⁰, Fatima Ailal⁸, Hidetoshi Takada¹¹, Toshiro Hara¹¹, Anne Puel¹², Stéphanie Boisson-Dupuis¹², Jacinta Bustamante¹³, Jean-Laurent Casanova¹⁴, Osamu Ohara¹⁵, Satoshi Okada¹⁶, Masao Kobayashi¹

Affiliations

¹ Department of Pediatrics, Hiroshima University Graduate School of Biomedical & Health Sciences, Hiroshima, Japan.
² Department of Technology Development, Kazusa DNA Research Institute, Chiba, Japan.
³ St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY.
⁴ 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.
⁵ Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan.
⁶ Department of Pediatrics, Okazaki City Hospital, Aichi, Japan.
⁷ Genetics Unit, Military Hospital Mohamed V, Hay Riad, Rabat, Morocco.
⁸ 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.
⁹ Department of Pediatrics, National Hospital Organization Fukuyama Medical Center, Hiroshima, Japan.
¹⁰ Department of Immunology, "Juan Pedro Garrahan" National Hospital of Pediatrics, Buenos Aires, Argentina.
¹¹ Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
¹² 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.
¹³ 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.
¹⁴ 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.
¹⁵ Department of Technology Development, Kazusa DNA Research Institute, Chiba, Japan; Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.
¹⁶ 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: [email protected].

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.

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.

Keywords: Mendelian susceptibility to mycobacterial disease; Signal transduction and activator of transcription 1; alanine scanning; chronic mucocutaneous candidiasis; reference database.

MeSH terms

Alanine / genetics*
Biological Assay
Female
Genetic Predisposition to Disease
Humans
Male
Mutagenesis
Mutation
Mycobacterium Infections / genetics*
Protein Domains
STAT1 Transcription Factor / genetics*

Substances

STAT1 Transcription Factor
STAT1 protein, human
Alanine

Abstract

MeSH terms

Substances

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