A systematic analysis of recombination activity and genotype-phenotype correlation in human recombination-activating gene 1 deficiency

Abstract

Background The recombination-activating gene (RAG) 1/2 proteins play a critical role in the development of T and B cells by initiating the VDJ recombination process that leads to generation of a broad T-cell receptor (TCR) and B-cell receptor repertoire. Pathogenic mutations in the RAG1/2 genes result in various forms of primary immunodeficiency, ranging from T−B− severe combined immune deficiency to delayed-onset disease with granuloma formation, autoimmunity, or both. It is not clear what contributes to such heterogeneity of phenotypes. Objective We sought to investigate the molecular basis for phenotypic diversity presented in patients with various RAG1 mutations. Methods We have developed a flow cytometry–based assay that allows analysis of RAG recombination activity based on green fluorescent protein expression and have assessed the induction of the Ighc locus rearrangements in mouse Rag1−/− pro-B cells reconstituted with wild-type or mutant human RAG1 (hRAG1) using deep sequencing technology. Results Here we demonstrate correlation between defective recombination activity of hRAG1 mutant proteins and severity of the clinical and immunologic phenotype and provide insights on the molecular mechanisms accounting for such phenotypic diversity. Conclusions Using a sensitive assay to measure the RAG1 activity level of 79 mutations in a physiologic setting, we demonstrate correlation between recombination activity of RAG1 mutants and the severity of clinical presentation and show that RAG1 mutants can induce specific abnormalities of the VDJ recombination process.

Modeling altered T-cell development with induced pluripotent stem cells from patients with RAG1-dependent immune deficiencies

“…5 Using an in vitro cellular platform in which RAG activity can be measured by analyzing recombination at an inverted green fluorescent protein (GFP) cassette flanked by RSS, we have shown that the phenotypic diversity of human RAG deficiency correlates with the residual function of the mutant RAG protein. 10 We found that mutations associated with OS have residual, yet markedly decreased, recombination activity. The observation that OS and T 2 B 2 NK 1 SCID may occur in affected members of the same family suggests that RAG mutations associated with these phenotypes can only support, at best, limited repertoire diversity.…”

Programmed DNA breaks in lymphoid cells: repair mechanisms and consequences in human disease

“…Moreover, it was recently shown that mutations affecting the recombination activity of RAG1 correlate with clinical outcome. Near complete loss of the enzymatic activity leads to SCID but, where enzymatic activity remains, phenotypes are less severe 33. These phenotypes include not only Omenn syndrome but also leaky SCID, SCID with expansion of γδ T cells (often associated with cytomegalovirus infections), combined immune deficiency with granuloma and/or autoimmunity and idiopathic CD4 + T‐cell lymphopenia.…”

Three faces of recombination activating gene 1 (RAG1) mutations

“…Maybe we need further investigations to detect second mutation in the presented patient #3 as said Pico-Knijnenburg et al [18]. Atypical SCID result from RAG genes mutations shows varying numbers of T and B cells and varied clinic manifestations such combined immune defi ciency with granuloma and/or autoimmunity, γδ T lymphocytes expansion which is often associated with cytomegalovirus infection, idiopathic CD4 + T cell lymphopenia which presenting with extensive chickenpox and recurrent pneumonia, and early onset-autoimmunity [15]. The pleomorphic manifestations of RAG defi ciency result from hypomorphic mutations are explained by residual RAG protein activity [7].…”

“…Second classic SCID patient also died at the age of 9 months while unrelated donor screening. Hypomorphic mutations in the RAG1/2 genes have been associated with different clinical and immunologic phenotypes that include OS and atypical SCID [15]. OS presents erythroderma, eosinophilia, lymphadenopathy, and increased serum IgE levels as presented in patient #3.…”

“…The pleomorphic manifestations of RAG defi ciency result from hypomorphic mutations are explained by residual RAG protein activity [7]. To date, at least 76 distinct mutations have been described RAG1 gene [15]. Sometimes epigenetic factors including compound genetic defects, environmental factors, and infections effect on the clinical manifestation of RAG1 gene mutations and may alter phenotypical characteristics of the mutations of RAG1 gene.…”