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Loss of Function Mutation in ELF4 Causes Autoinflammatory and Immunodeficiency Disease in Human

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Abstract

Monogenic autoinflammatory diseases (mAIDs) are a heterogeneous group of diseases affecting primarily innate immunity, with various genetic causes. Genetic diagnosis of mAIDs can assist in the patient’s management and therapy. However, a large number of sporadic and familial cases remain genetically uncharacterized. Deficiency in ELF4, X-linked (DEX) is recently identified as a novel mAID. Here, we described a pediatric patient suffering from recurrent viral and bacterial respiratory infection, refractory oral ulcer, constipation, and arthritis. Whole-exome sequencing found a hemizygous variant in ELF4 (chrX:129205133 A > G, c.691 T > C, p.W231R). Using cells from patient and point mutation mice, we showed mutant cells failed to restrict viral replication effectively and produced more pro-inflammatory cytokines. RNA-seq identified several potential critical antiviral and anti-inflammation genes with decreased expression, and ChIP-qPCR assay suggested mutant ELF4 failed to bind to the promoters of these genes. Thus, we presented the second report of DEX.

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Data Availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Code Availability

Not applicable.

Abbreviations

mAIDs:

Monogenic autoinflammatory diseases

ChIP-qPCR:

Chromatin immunoprecipitation-quantitative real-time PCR IEI

IEI:

Inborn errors of immunity

PIDs:

Primary immunodeficiency diseases

WES:

Whole-exome sequencing

NF-kB:

Nuclear factor-kB

PBMC:

Peripheral blood mononuclear cells

BMDM:

Bone marrow-derived macrophage

VSV:

Vesicular stomatitis virus

HSV-1:

Herpes simplex virus 1

URT:

Upper respiratory tract

WBC:

White blood cell

IVIG:

Intravenous immune globulin

NSAIDs:

Non-steroidal anti-inflammatory drugs

ANCA:

Anti-neutrophil cytoplasmic antibody

BCG:

Bacille Calmette-Guérin

DPT:

Diphtheria-pertussis-tetanus

WT:

Wild type

MUT:

Mutant

DEGs:

Differentially expressed genes

DEX:

Deficiency in ELF4, X-linked

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Acknowledgements

We thank the patient and his family for their cooperation, especially for the patient’s mother whose perseverance in caring for her child gives us strength to pursue a definitive genetic diagnosis and next a more effective personalized treatment. We also thank all other members of You lab (Peking University Health Science Center) for insightful discussion of the results and practical technical tips.

Funding

This study is funded by Sanming Project of Medicine in Shenzhen (SZSM201812002), Livelihood Project of Chongqing, China (cstc2018jscxmsybX0005), National Natural Science Foundation of China (81974255), National Natural Science Foundation of China (81900136), Postdoc Fund at Chongqing Medical University, and Ministry of Education Key Laboratory Foundation at Children’s Hospital of Chongqing Medical University.

Author information

Authors and Affiliations

  1. National Clinical Research Center for Child Health and Disorders (Chongqing), Children’s Hospital of Chongqing Medical University, Chongqing, China

    Gan Sun, Luyao Qiu, Lang Yu, Yunfei An, Yuan Ding, Lina Zhou, Junfeng Wu, Xi Yang, Zhiyong Zhang, Xuemei Tang, Xiaodong Zhao & Hongqiang Du

  2. Ministry of Education Key Laboratory of Child Development and Disorders, Children’s Hospital of Chongqing Medical University, Chongqing, China

    Gan Sun, Luyao Qiu, Lang Yu, Yunfei An, Yuan Ding, Lina Zhou, Junfeng Wu, Xi Yang, Zhiyong Zhang, Xuemei Tang, Xiaodong Zhao & Hongqiang Du

  3. Chongqing Key Laboratory of Child Infection and Immunity, Children’s Hospital of Chongqing Medical University, Chongqing, China

    Gan Sun, Luyao Qiu, Lang Yu, Yunfei An, Yuan Ding, Lina Zhou, Junfeng Wu, Xi Yang, Zhiyong Zhang, Xuemei Tang, Xiaodong Zhao & Hongqiang Du

  4. Department of Rheumatology & Immunology, Children’s Hospital of Chongqing Medical University, Chongqing, China

    Yunfei An, Junfeng Wu, Xi Yang, Zhiyong Zhang, Xuemei Tang & Hongqiang Du

  5. Beijing Key Laboratory of Tumor Systems Biology, Department of Immunology, School of Basic Medical Sciences, Institute of Systems Biomedicine, Peking University Health Science Center, Beijing, 100000, China

    Huawei Xia, Lili Cao & Fuping You

  6. The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400014, China

    Xiaodong Zhao

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  1. Gan Sun
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  2. Luyao Qiu
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  3. Lang Yu
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Contributions

HQ. D and XD. Z conceived and supervised the project, reviewed and revised the manuscript; HQ. D designed the experiments, analyzed data, and wrote the manuscript. G. S performed the experiments, analyzed data, and followed the patient. FP. Y provided plasmids, viruses, experimental expertise, and scientific sights in designing the project and revising the manuscript. HW. X constructed ELF4 mutant plasmids. LL. C performed ChIP-qPCR experiment. Others provided essential help for clinical management and follow-up of the patient and valuable clinical expertise in reviewing the manuscript.

Corresponding authors

Correspondence to Xiaodong Zhao or Hongqiang Du.

Ethics declarations

Ethics Approval and Consent to Participate

The ethics committee of Children’s Hospital of Chongqing Medical University approved the study. Blood samples collection were performed in accordance with the Declaration of Helsinki. Written informed consent for participation in the study were obtained from patients’ parents.

Consent for Publication

Written informed consent for publication of the study was obtained from patients’ parents.

Conflict of Interest

The authors declare no competing interests.

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Supplementary Information

Below is the link to the electronic supplementary material.

10875_2022_1243_MOESM1_ESM.jpg

Supplementary Fig.1 Mutant cells failed to restrain viral replication effectively. A Plaque-formation assays of culturesupernatant of peripheral monocytes from healthy control (HC) and patient (P)after 12 h infection with VSV or HSV-1-GFP. B mRNA quantification of VSV in peripheral monocytes from healthycontrol (HC) and patient (P) after 12 h infection with VSV. C Microscopy analysis (left images,magnification, 5×) of HeLa cells transfected with human WT ELF4 (hWT ELF4),human W231R mutant ELF4 (hMut ELF4), mouse WT ELF4 (mWT ELF4) or mouse W231Rmutant ELF4 (mMut ELF4) after 12 h infection with HSV-1-GFP. Right graph,quantification of HSV-1-GFP infected cells indicated by GFP expression, 2-3random fields were counted. DMicroscopy analysis (left images, magnification, 5×) of BMDM from WT mice (WT)and point mutation mice (Mut) after 6h and 12 h infection with HSV-1-GFP. Rightgraph, quantification of HSV-1-GFP infected cells indicated by GFP expression,2-3 random fields were counted. Data were pooled from at least 3 independent experimentsand represented as mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Unpaired two-sided Student’s t test (JPG 854 KB)

Supplementary Fig.2 Generation summary of ELF4 point mutation mice (JPG 105 KB)

10875_2022_1243_MOESM3_ESM.jpg

Supplementary Fig. 3Loss of function of ELF4 leads to increased virus-induced inflammation. A mRNA quantification of indicatedinterferons of peripheral monocytes from healthy control (HC) and patient (P)after 12 h infection with VSV or HSV-1-GFP. B IFN-b mRNA quantification of HeLa cells transfected with human WTELF4 (hWT ELF4), human W231R mutant ELF4 (hMut ELF4), mouse WT ELF4 (mWT ELF4)or mouse W231R mutant ELF4 (mMut ELF4) after 12 h infection with HSV-1-GFP. C mRNA quantification of indicatedinterferons of BMDM from WT mice (WT) and point mutation mice (Mut) after 6 hinfection with VSV or HSV-1-GFP. DmRNA quantification of indicated cytokines of peripheral monocytes from healthycontrol (HC) and patient (P) after 12 h infection with VSV or HSV-1-GFP. E ELISA quantification of indicatedcytokines in peripheral monocytes isolated from healthy control (HC) andpatient (P) after 12 h infection with VSV or HSV-1-GFP. F mRNA quantification of indicated cytokines and ELISAquantification of IL-6 in peritoneal macrophage from WT mice (WT) and pointmutation mice (Mut) after 6 h infection with VSV or HSV-1-GFP. Data were pooledfrom at least 3 independent experiments and represented as mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Unpaired two-sided Student’s t test (JPG 1540 KB)

10875_2022_1243_MOESM4_ESM.jpg

Supplementary Fig. 4 A Representative image of flow cytometryanalysis of IL-17A producing CD4+T cells, gated on CD3+CD4+T cells. B Flow cytometry analysis showing percentageof IL-17A producing CD4+T cells differentiated from naïve CD4+ Th17 in vitrounder non-pathogenic conditions (TGFβ + IL-6), cells were restimulated with PMAand Ionomycin. WT n = 3, Mut n = 3. CMouse IL-17A ELISA analysis of Th17 culture supernatant after restimulation. WTn = 3, Mut n = 3. Data were represented as mean±SD.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Unpairedtwo-sided Student’s t test (JPG 449 KB)

10875_2022_1243_MOESM5_ESM.jpg

Supplementary Fig. 5 MutantELF4 fails to activate potential antiviral or anti-inflammatory genes. A mRNA quantification of indicatedgenes in BMDM from WT mice (WT) and point mutation mice (Mut) after 6 hinfection with VSV or HSV-1-GFP. B mRNAquantification of indicated genes in peripheral monocytes from healthy control (HC)and patient (P) after 6 h infection with VSV or HSV-1-GFP. C ChIP-qPCR experiments showing Flag-ELF4 binding to promoters ofindicated genes in HEK293T cells transfected with empty vector, WT ELF4, W231Rmutant ELF4 or W251A mutant ELF4. Data were pooled from at least 3 independent experimentsand represented as mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Unpaired two-sided Student’s t test (JPG 515 KB)

10875_2022_1243_MOESM6_ESM.jpg

Supplementary Fig. 6Cistrome database mining. Elf4 antibody binding peaks in the promoters ofindicated genes. Raw data were derived from Cistrome database (JPG 1821 KB)

Supplementary data-R2 (DOCX 36 KB)

SupplementaryTable 4 DEGs between uninfected WT and mutant BMDM cells (XLS 27 KB)

SupplementaryTable 5 DEGs between VSV-infected WT and mutant BMDM cells (XLS 48 KB)

SupplementaryTable 6 DEGs between HSV-1-infected WT and mutant BMDM cells (XLS 31 KB)

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Sun, G., Qiu, L., Yu, L. et al. Loss of Function Mutation in ELF4 Causes Autoinflammatory and Immunodeficiency Disease in Human. J Clin Immunol 42, 798–810 (2022). https://doi.org/10.1007/s10875-022-01243-3

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  • DOI: https://doi.org/10.1007/s10875-022-01243-3

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