Hypomorphic Rag mutations can cause destructive midline granulomatous disease

Abstract

Destructive midline granulomatous disease characterized by necrotizing granulomas of the head and neck is most commonly caused by Wegener granulomatosis, natural killer/T-cell lymphomas, cocaine abuse, or infections. An adolescent patient with myasthenia gravis treated with thymectomy subsequently developed extensive granulomatous destruction of midface structures, palate, nasal septum, airways, and epiglottis. His lymphocyte numbers, total immunoglobulin G level, and T-cell receptor (TCR) repertoire appeared normal. Sequencing of Recombination activating gene-1 (Rag1) showed compound heterozygous Rag1 mutations; a novel deletion with no recombinase activity and a missense mutation resulting in 50% Rag activity. His thymus was dysplastic and, although not depleted of T cells, showed a notable absence of autoimmune regulator (AIRE) and Foxp3(+) regulatory T cells. This distinct Rag-deficient phenotype characterized by immune dysregulation with granulomatous hyperinflammation and autoimmunity, with relatively normal T and B lymphocyte numbers and a diverse TCR repertoire expands the spectrum of presentation in Rag deficiency. This study was registered at www.clinicaltrials.gov as #NCT00128973.

Figure 1

Granulomatous inflammation and immunophenotype on Subject P. (A) Photographs of palatal fistula at initial presentation (left), nasal fistulous tract with collapse of left alar rim (middle), and inflamed epiglottis (right). (B) Imaging studies with the use of computed tomography show the marked subglottic stenosis characteristically seen in WG (i) and megacolon (ii). Extensive inflammation in the nasopharynx (iii) and colon (iv) was evident on positron emission tomographic scans. (C) A summary of lymphocyte, immunoglobulin, and antibody levels at chronologic time points from initial diagnostic work up. *Patient on γ globulin supplement. The normal range referenced the values within 95% confidence interval, n = 40. (D) A time line depicting patient P's major clinical events chronologically (the time line is not to scale) and the progression of provisional diagnoses.

Figure 2

Diversity of T-cell receptor repertoire. (A) A diverse distribution of CDR3 lengths in most of the Vβ families was observed in the spectratype of patient P (red) compared with a healthy control (black), whereas a partially corrected γc-deficient severe combined immunodeficient patient (blue) displayed an oligoclonal repertoire. (B) Quantitative analysis of Vβ distribution in CD4⁺ and CD8⁺T (C) cells showed some under-presentation in a few Vβ families and over-represented in others, particularly in CD8⁺ T cells. The shaded bars represent the patient's cells. The error bars indicate ± 1 SD.

Figure 3

Immunohistopathology of lymph node, skin, and thymus. (A) Lymph node biopsy from patient P shows preserved nodal architecture with well-formed secondary B follicles and multiple epithelioid granulomas with multinucleated giant cells in the paracortical area. In contrast, lymph node at autopsy from patient's sister showed no secondary B follicles and marked polyclonal plasmacytosis (B). (C) Skin biopsy (heel) shows dense granulomatous inflammation involving mid dermis around vessels, clusters of epithelioid cells, and multinucleated giant cells. (D) Thymic biopsy shows abnormal thymic architecture with loss of corticomedullary demarcation (CMD), fat replacement and absence of Hassal bodies (HBs); moderate number of thymocytes, mostly of a blastic immature phenotype, and are admixed with TECs (top left). Immunostains highlight a diffuse epithelial network mostly composed of cytokeratin-5⁺cytokeratin-8⁻ (CK5⁺CK8⁻) double-positive immature TECs and no expression of Cld4 and AIRE (middle left). In contrast, normal thymus shows defined CMD and presence of HBs (top right and inset) with normal distribution of CK5⁺CK8⁻ and CK5⁻CK8⁺ single-positive cortical (c) and medullary (m) TECs, with expression of claudine-4 (Cld4) and AIRE (middle right). Severe depletion of thymic S-100⁺ DCs and Foxp3⁺ regulatory T cells (Tregs) are seen in patient (bottom left), whereas normal distribution of medullary S-100⁺ DCs and Foxp3⁺ Tregs were present in control thymus (bottom right; Foxp3⁺ cells are indicated by arrows). Hematoxylin and eosin (H&E) staining, 4×, 40× (inset), and 60× original magnification; immunostains for CK5, CK8, S-100, and Foxp3, 20× magnification; immunostains for Cld4 and AIRE, 40× magnification.

Figure 4

In vitro genetic and functional evaluation of the Rag 1 defect from Subject P. (A) Sequence analysis of the patient (middle) and his father (top) and mother (bottom), showing the novel mutation 1621delC and a previously reported 1566G > T, missense mutation (A). Both of these mutations are located within the catalytic core of RAG1 (B). (B) The effect of patient mutations on Rag1 protein. Both mutations are predicted to disrupt the catalytic domain of Rag1 with a missense W522C and the novel deletion L541CfsX30 with the resulting truncated open reading frame as indicated. (C) Mouse pro-B Abelson line deficient for Rag1 was infected with inverted substrate. (D) Subclones 1, 2, and 3 with single copy stable integrants were transduced with wild-type, G1566T, or 1621delC, then treated with STI-571 (an abl kinase inhibitor) to promote cell differentiation and induction of Rag activity. The level of GFP expression (x-axis) indicated the recombinase activity level, and only minimal GFP level was detected in the absence of STI-571.

Figure 5

Stimulated cytokine responses from PBMCs. PBMCs from patient P (triangles) were challenged with indicated stimuli in vitro on multiple occasions over 3 years, and the cytokine production compared with PBMCs from healthy persons (circles) (n = 50, not age or sex matched). (A) IL-1β production was significantly increased after stimulation with pan-T cell and viral mimic (PHA and poly I:C). (B) IL-8 production in response to PHA, phorbol 12-myristate 13-acetate (PMA), or ssRNA was elevated compared with controls. (C) Production of tumor necrosis factor-α (TNFα) is also markedly elevated (P < .001) in response to PHA. Other cytokines measured were either not significantly different or were reduced compared with the healthy controls. LPS indicates lipopolysaccharide; ION, ionomycin; and SAC, Staphylococcus aureus Cowan I.

Figure 6

A model depicting key immunologic phenotypes and characteristic features in the spectrum of Rag-deficient genotypes.