The Effect of Nudcd3 Deficiency on V(D)J Recombination

In the study of congenital immunodeficiency, especially in the field of T cell developmental abnormalities, identifying key genes and pathways is of great significance. Researches on Severe Combined Immunodeficiency (SCID) and Omenn Syndrome (OS) have highlighted the crucial roles of T cell receptor (TCR) rearrangement and signaling, as well as the survival signals delivered by interleukin 7 receptor (IL-7R) during the development of T lymphocytes. However, the genetic mechanisms of these diseases are still not completely clear, and many unknown genes may be involved. Therefore, Chen et al. conducted an in-depth study to clarify the role of a new gene, Nudc domain-containing 3 (Nudcd3), in SCID and OS.

Research Background and Objectives

SCID and OS are two severe, life-threatening immunodeficiency diseases, where patients have high susceptibility to infections. SCID is characterized by a complete lack of T and B cells, while OS presents with limited oligoclonal T cell expansion upon partial developmental blockade, causing inflammation of the skin and viscera as well as SCID-like susceptibility to infection. In recent years, many countries have begun screening newborns for T-cell immunodeficiencies to facilitate early precise treatments such as enzyme replacement, gene therapy, or hematopoietic stem cell transplantation (HSCT). However, molecular diagnosis of these diseases still faces unprecedented challenges. Known SCID-related genes include RAG1 and RAG2 recombination enzymes and their corresponding components in DNA repair mechanisms, but these are apparently not enough to explain all cases. Thus, exploring new pathogenic genes is significant for improving precision medicine.

Research Source

This study was completed in cooperation by researchers from Newcastle University, Wellcome Sanger Institute, Erasmus University Medical Center, University of Birmingham, University of Sussex, and other scientific research institutions. The research paper was published in the journal “Science Immunology” on May 24, 2024, with Sophie Hambleton serving as the corresponding author.

Research Process

The research team discovered a homozygous deletion mutation in the Nudcd3 gene in 11 children from four consanguineous families with familial cases, by using whole-exome sequencing (WES) and integrative analysis. The impact of the Nudcd3 mutation was further detailed through the following steps:

1. Identification of Mutation Sites and Protein Expression: A missense mutation in the Nudcd3 gene (c.155G>A) was found, replacing glycine with aspartic acid, which was confirmed as highly deleterious by multiple prediction programs such as CADD and PolyPhen2. The protein expression of Nudcd3 was significantly reduced in the patient’s cells, whereas mRNA expression was normal, indicating that the variant is a low-expressor phenotype.

2. Protein Interaction and Functional Analysis: Immunoprecipitation and mass spectrometry analysis showed that the self-dimerization ability of the Nudcd3G52D variant protein was significantly weakened, but its interaction with known protein-binding partners (such as HSP family proteins) showed no significant change. In addition, significant aberrant distributions of T lymphocyte subsets were revealed by single-cell RNA sequencing (scRNA-seq).

3. V(D)J Recombination Assay: Experiments in vitro and in a mouse model demonstrated that the Nudcd3 variant significantly affected the recombinase activity during V(D)J recombination, particularly playing a key regulatory role in the redistribution of RAG1 protein from the nucleolus to the nucleoplasm.

4. Mouse Model Validation: Using CRISPR-Cas9 technology, the research team constructed a mouse model carrying the same G52D mutation and found that these mice exhibited immunodeficiency and small body phenotypes. However, these effects were relatively milder in mice compared to humans. Retrospective transplantation experiments also proved that Nudcd3-deficient cells faced developmental difficulties of T and B cells in the in vivo environment.

Research Findings

1. Impact of Mutation on V(D)J Recombination: The study showed that the loss of Nudcd3 mainly hindered V(D)J recombination by affecting the residence and redistribution of RAG1 protein in the nucleolus. In patient cells, RAG1 predominantly remained in the nucleolus, whereas it should be guided by RAG2 to distribute throughout the nucleus under normal circumstances.

2. Abnormal Distribution of Immune Cells: Using single-cell RNA sequencing, it was found that the T cell subsets of patients with Nudcd3 defects, especially CD4^+, CD8^+, and γδ T cells, showed significant phenotypic abnormalities. Moreover, monoclonal T cells predominated, and the usage of TCR genes was significantly reduced, exhibiting an extreme monoclonal profile.

3. Phenotypic Features of the Mouse Model: By reintroducing the G52D mutation in mice, it was confirmed that this led to significant immunodeficiencies in mice as well, but their other bodily organ functions were mostly normal.

Conclusions and Implications

After multi-level and multi-angle experimental verifications, the research team confirmed the absolute necessity of Nudcd3 in V(D)J recombination and lymphocyte development. This discovery not only reveals a new genetic mechanism but may also provide important clues for future diagnosis and treatment of SCID and OS. Some patients have been cured using therapies such as HSCT, which also demonstrates the importance of early diagnosis and treatment.

Highlights of the Study

1. Identification of Nudcd3 Mutation: A clear association between the Nudcd3 mutation and severe immunodeficiency diseases was identified for the first time.
2. Multi-level Verification: The research underwent comprehensive verification from gene sequencing and protein interactions to mouse models.
3. Guidelines for Precision Medicine: The pathogenic mechanism of the Nudcd3 gene was elucidated, providing new targets for the precision medicine of SCID and OS.

Future Prospects

Future research is expected to further decipher the interaction mechanisms between Nudcd3 and other molecular partners and explore if there are other similar auxiliary proteins. This will help our understanding of V(D)J recombination and related diseases and may lead to the development of new intervention strategies.

This study not only provides new insights into the molecular mechanisms of immunodeficiency disorders but also opens a new direction for the development of precision medicine. By deeply understanding the mechanisms of Nudcd3’s action, we may develop more effective treatment protocols to save more patients suffering from immunodeficiency diseases.