Kagawa Reiko, Fujiki Ryoji, Tsumura Miyuki, Sakata Sonoko, Nishimura Shiho, Itan Yuval, Kong Xiao-Fei, Kato Zenichiro, Ohnishi Hidenori, Hirata Osamu, Saito Satoshi, Ikeda Maiko, El Baghdadi Jamila, Bousfiha Aziz, Fujiwara Kaori, Oleastro Matias, Yancoski Judith, Perez Laura, Danielian Silvia, Ailal Fatima, Takada Hidetoshi, Hara Toshiro, Puel Anne, Boisson-Dupuis Stéphanie, Bustamante Jacinta, Casanova Jean-Laurent, Ohara Osamu, Okada Satoshi, Kobayashi Masao
Department of Pediatrics, Hiroshima University Graduate School of Biomedical & Health Sciences, Hiroshima, Japan.
Department of Technology Development, Kazusa DNA Research Institute, Chiba, Japan.
J Allergy Clin Immunol. 2017 Jul;140(1):232-241. doi: 10.1016/j.jaci.2016.09.035. Epub 2016 Dec 20.
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.
We estimated variations in the CCD/DBD of STAT1.
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.
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.
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.
人类信号转导与转录激活因子1（STAT1）的种系杂合突变可导致功能丧失（LOF），如孟德尔遗传性分枝杆菌病患者；或功能获得（GOF），如慢性黏膜皮肤念珠菌病患者。LOF和GOF突变同样罕见，且可影响STAT1的相同结构域，尤其是卷曲螺旋结构域（CCD）和DNA结合结构域（DBD）。此外，6%携带GOF STAT1突变的慢性黏膜皮肤念珠菌病患者患有分枝杆菌病，这使得已鉴定的STAT1突变的功能意义变得模糊。目前的计算方法，如联合注释依赖缺失法，无法区分LOF和GOF变异。
我们评估了STAT1的CCD/DBD中的变异情况。
我们将CCD/DBD（占全长STAT1的45.6%）中的342个野生型氨基酸逐个突变为丙氨酸，并检测这些突变体的STAT1转录活性。
在荧光素酶测定中，这342个突变体中，201个为中性，30个为LOF突变，111个为GOF突变。该测定系统正确估计了所有先前报道的STAT1的CCD/DBD中的LOF突变（100%），而GOF突变的估计略少（78.1%）。我们发现GOF丙氨酸突变体出现在反平行STAT1二聚体的界面处，这表明它们会使该二聚体不稳定。该测定还精确预测了我们在2例无关的孟德尔遗传性分枝杆菌病患者中发现的STAT1的CCD中的2个低表达和显性负性突变E157K和G250E的作用。
系统性丙氨酸扫描测定是一种有用的工具，可用于评估严重传染病（包括分枝杆菌病和真菌病）患者中鉴定出的STAT1杂合错义突变的GOF或LOF状态及影响。
