Similar recombination-activating gene (RAG) mutations result in similar immunobiological effects but in different clinical phenotypes

Abstract

Background: V(D)J recombination takes place during lymphocyte development to generate a large repertoire of T- and B-cell receptors. Mutations in recombination-activating gene 1 (RAG1) and RAG2 result in loss or reduction of V(D)J recombination. It is known that different mutations in RAG genes vary in residual recombinase activity and give rise to a broad spectrum of clinical phenotypes.

Objective: We sought to study the immunologic mechanisms causing the clinical spectrum of RAG deficiency.

Methods: We included 22 patients with similar RAG1 mutations (c.519delT or c.368_369delAA) resulting in N-terminal truncated RAG1 protein with residual recombination activity but presenting with different clinical phenotypes. We studied precursor B-cell development, immunoglobulin and T-cell receptor repertoire formation, receptor editing, and B- and T-cell numbers.

Results: Clinically, patients were divided into 3 main categories: T(-)B(-) severe combined immunodeficiency, Omenn syndrome, and combined immunodeficiency. All patients showed a block in the precursor B-cell development, low B- and T-cell numbers, normal immunoglobulin gene use, limited B- and T-cell repertoires, and slightly impaired receptor editing.

Conclusion: This study demonstrates that similar RAG mutations can result in similar immunobiological effects but different clinical phenotypes, indicating that the level of residual recombinase activity is not the only determinant for clinical outcome. We postulate a model in which the type and moment of antigenic pressure affect the clinical phenotypes of these patients.

Keywords: B- and T-cell receptor repertoire; RAG deficiency; V(D)J recombination; autoimmunity; immune repertoire analysis; next generation sequencing; receptor editing.

Fig 1

RAG expression and precursor B-cell compartment. A, Recombination activity of the c.519delT (delT), c.delA368/A369 (delAA), p.P874GX82, p.R559S, and p.R759C RAG1 mutations was compared with wild-type (WT) RAG1. Only the delT and the delAA RAG1 mutations result in low levels of residual recombination activity. B, Composition of the precursor B-cell compartment in control subjects (n = 9), 3 patients with the “classical” SCID phenotype, 2 patients with OS, and 6 patients with CID. C, Relative RAG1 expression levels correlated to RAG2 expression in all the analyzed RAG patients, as determined by using RQ-PCR.

Fig 2

Heat maps of the different combinations of immunoglobulin DH-JH (A) and VH-JH (B), as determined in the unique junctions (defined by the unique combination of VH, DH, JH, and nucleotide sequences of CDR3).

Fig 3

Functional characteristics of IGH junctions. A, Functional characteristics of the IGH junctions were determined in 3 patients with RAGD in PB or BM. Distribution of CDR3 length frequencies in BM and PB was similar in control subjects and patients with RAGD; however, P18 had increased numbers of junctions, with a CDR3 length of 16 and 23 amino acids. B, Sequence logo showed no similarity of the 16-amino-acid CDR3s of P23 but high similarity of CDR3s of 16 amino acids using the IGHV6-1 gene and the 22-amino-acid CDR3s. C, The frequency of long CDR3s (≥15 amino acids) was decreased in P15 and P16. D,IGHV4-34 use was increased in P16 and P18. E and F, The percentage of IGKV and IGKJ genes was determined in 6 control subjects, 5 patients with OS, and 4 patients with CID. IGKV use was normal (Fig 3, E), but hardly any IGKJ5 gene was used (Fig 3, F).

Fig 4

TRB repertoire. TR spectratyping profiles of TRB gene rearrangements using the BIOMED-2 TRB tube B. The upper 2 panels show the monoclonal and the polyclonal controls. The patient panels SCID (P2 and P4), OS (P8), and CID (P21) show a restricted TRB repertoire.

Fig 5

Model for development of clinical phenotype in patients with RAGD. RAGD results in reduced V(D)J recombination, leading to fewer B and T cells with a limited repertoire. In an attempt to compensate for low numbers, B and T cells start to proliferate, but the repertoire remains limited and imbalanced, so that selection and immune regulation are impaired. Most likely the type of antigenic stimulation together with the incomplete and imbalanced repertoire that has been developed will affect the eventual clinical phenotype with immune dysregulation problems.

Fig E1

Immunoglobulin heavy chain gene usage. Frequency of IGHV(A), IGHD(B), and IGHJ(C) gene use in control BM and PB and in P16 BM and PB, P15 PB, and P18 PB.