Approaches to patients with variants in RAG genes: from diagnosis to timely treatment

Abstract

Introduction:

Patients with primary immunodeficiency (PID) secondary to abnormal recombinase activating genes (RAG) can present with broad clinical phenotypes ranging from early severe infections to autoimmune complications and inflammation. Immunological phenotype may also vary from T^-B^- severe combined immunodeficiency (SCID) to combined immunodeficiency (CID) or antibody deficiencies with near normal T and B cell counts and even preserved specific antibody response to pathogens. It is not uncommon that RAG variants of uncertain significance (VUS) are identified by serendipity during a broad genetic screening process and pathogenic RAG variants are increasingly recognized among all age groups, including adults. Establishing the pathogenicity and clinical relevance of novel RAG variants can be challenging since RAG genes are highly polymorphic. This review paper aims to summarize clinical phenotypes of RAG deficiencies and provide practical guidance for confirming the direct link between specific RAG variants and clinical disease. Lastly, we will review current understanding of treatment option for patients with varying severity of RAG deficiencies.

Area covered:

This review discusses the different phenotypes and immunological aspects of RAG deficiencies, the diagnosis dilemma facing clinicians and an overview for current and advancement in treatments.

Expert opinion:

A careful analysis of immunological and clinical data and their correlation with genetic findings help to determine the significance of the genetic polymorphism. Advances in functional assays, as well as anti-cytokines antibodies, make it easier to resolve the diagnostic dilemma.

1. Introduction

The adaptive immune system is unique due to the capability to recognize millions of antigens in a pre-immune state before environmental exposure. This diversity stems from a process where T and B lymphocytes break their own DNA in the regions coding the antigen receptor genes, reshuffles the segments (recombination) and create a unique construct of variable (V), diversity (D) and joining (J) gene to form the antigen-binding site of the variable region of T and B cell receptors (TCR, BCR) (1, 2). This process, termed V(D)J recombination, generates a highly diverse BCR (in the bone marrow) and TCR (in the thymus) that can recognize millions of antigens (3) Each gene segment of V, D, and J is flanked by recombination signal sequences (RSSs) (1, 4). As a first step in the V(D)J recombination process, a DNA breakage occurs in a synaptic complex just before RSS by a heterotetramer endonuclease complex with recombination-activating gene 1 (RAG1) and 2 (RAG2). Specifically, RAG2 reads the histone codes of active DNA, RAG1 recognizes the RSS and RAG2 directs RAG1 to nick DNA (5). Together they create a DNA break and secure the blunt end to recruit the non-homologous end joining (NHEJ) complex for repair and further diversification of junctional regions(6). The RAG enzyme complex is restricted to lymphocytes and only expressed during early T and B cell development whereas NHEJ is ubiquitous. RAG enzyme expression is high during the G0/G1 phase, during which V(D)J rearrangement largely occurs, and is low during the rest of the cell cycle (7).

Homozygous or compound heterozygous pathogenic variants in RAG often cause immunodeficiency, and the clinical severity partly depends on the level of impairment of recombinase activity (8). In fact, relative recombinase activity level and genotype-phenotype correlation have been reported in large patient cohorts (9, 10). Of note, environmental factors may modify the clinical phenotype further as underscored by variability of clinical presentation within the same family (11, 12).

Pathogenic variants of RAG genes may result in zero recombination activity, therefore leading to highly restricted T and B cell repertoire with near absence of T and B cells coined as T^-B^-NK⁺ severe combined immunodeficiency (SCID) (13). When RAG recombinase activity is severely reduced but not absent (<5%), patient may develop leaky SCID (LS) or its inflammatory variant, Omenn syndrome (OS) with expansion of oligoclonal Th2-skewed cells that promote an atopic phenotype with dermatitis, colitis and highly elevated immunoglobulin E (IgE) (14–16). Patients with SCID, LS or OS often present with severe infections and/or systemic inflammations early in life and are fatal without curative therapy such as a hematopoietic stem cell transplant (HSCT).

The spectrum of clinical phenotypes of partial RAG deficiencies further broadened in recent years with the realization that infections are complemented with autoimmunity and granulomas. Immunological evaluations point toward late onset combined or primary antibody deficiency or naïve CD4+ T-cell lymphopenia (TCL) (17–23). Below, we will discuss variants of RAG deficiencies pertaining to their variable phenotypic and immunologic characteristics (see Table 1), defects in tolerance mechanisms, and diagnostic approaches to confirm novel variant immunophenotypes via functional analysis. Lastly, we will review treatment options for non-infectious complications and curative therapy.

Table 1.

Diverse clinical and immunological phenotypes of RAG deficiency. The RAG complex recombination activity and the T and B cell counts generally increase when moving left to right among the variants of RAG deficiency.

Variant of RAG Deficiency	SCID	OS	AS/γδ LS	CID-G/AI	PAD	TCL
T cells	Absent No TRECs	Oligoclonal expansion with CD45RO predominance	↓ αβ T cells with CD45RO predominance oligoclonal expansion of γδ T cells in γδ LS	Moderately restricted TCR repertoire with CD45RO predominance	Variable (low to normal) with CD45RO predominance	CD4+ lymphopenia with CD45RO predominance
B cells	Absent	Absent in blood, may be expanding in secondary lymphoid organs	Variable (mainly low)	Progressive decline, skewed BCR repertoire, expansion of unswitched memory B cells	Variable (low to normal)	Variable with expansion of unswitched memory B cells
NK cells	Normal number, ↑ cytotoxicity	Normal number with skewed maturation	Normal number with skewed maturation	Normal number with skewed maturation, mildly ↑ cytotoxicity	N.A.	N.A.
Other Blood Test Results	↓ IgG	↑ eosinophil ↑ IgE ↓ IgG	- Variable (low to normal) IgG, IgM, IgA - Anticytokine antibodies (e.g. IL-α, IL-ω, IL-12)	- Variable (low to normal) IgG - Anticytokine antibodies - Autoantibodies related to autoimmune disease	Hypogamm. Low vaccine titers, low to normal IgG	Normal IgG
Clinical Phenotype	Early severe opportunistic infections, diarrhea and failure to thrive	Diffuse erythroderma, chronic diarrhea with failure to thrive, lymphadenopathy & hepatosplenomegaly	Disseminated CMV, and VZV, cytopenias and ↑ risk of lymphoproliferative disease	Late onset infections, varied autoimmunity and granulomas	Recurrent sinopulmonary infections, cytopenias, GLILD	Mild to moderate viral infections (e.g. VZV) or skin rash with eosinophilia

SCID: T^-B^-NK⁺ SCID; OS: Omenn syndrome; AS/γδ LS: atypical or leaky SCID with γδ T cells; CID-G/AI: combined immunodeficiency with granulomas and/or autoimmunity; PAD: primary antibody deficiency; TCL: CD4⁺ T-cell lymphopenia; GLILD: granulomatous lymphocytic interstitial lung disease

2. Autoimmunity associated with RAG deficiencies

Among patients with RAG deficiencies, as the percent of RAG protein activity increases, the vulnerability to infections decreases, however, the susceptibility to autoimmunity increases (Figure. 1). SCID (T^-B^-NK⁺) patients have severe infections and little or no autoimmunity since they are devoid of T and B cells secondary to absent RAG activity, whereas CID patients with granulomas and/or autoimmunity (CID-G/AI) have milder infections but frequent autoimmune complications due to dysregulated T and/or B cells subsequent to partial RAG activity. Within a cohort with diverse variants of RAG deficiencies (n = 22), the prevalence of serum autoantibodies was 64% in CID-AI/G, TCL and LS combined, 25% in OS, 20% in SCID and <1% in healthy controls (calculated from Table 1 and Fig. 2 in reference 23).

Figure 1. RAG recombination activity positively correlates with T and B cell numbers and repertoire diversity, and affects clinical and immunological phenotypes.

Environmental factors, such as viral infections, as well as other genetic causes can further influence the diversity of the T and B cell receptor (TCR, BCR) repertoires, and clinical/immunological phenotypes.

Note: it is original figure

Figure 2. Infant between 1–2 months old with Omenn syndrome.

Note the generalized dermatitis with erythroderma and desquamation. The blue color is due to an antiseptic application.

Although the etiology for autoimmunity in RAG deficiencies is not fully understood, several central and peripheral tolerances checkpoints are impaired and therefore, may contribute to the survival of autoreactive T and B cells (Figure 1). Regarding, central tolerance checkpoints for cell mediated immunity, partial RAG activity deficiency reduces the TCR repertoire (i.e. junctional) diversity, complementarity-determining regions 3 (CDR3) length and T cell numbers. It also disrupts thymus structure and T cell development (8, 24–26). Disrupted thymic T cell development hinders T cell-stromal cell communication necessary for maturation of thymic epithelial cells and their expression of the autoimmune regulator (AIRE). Decreased AIRE expression limits negative selection of autoreactive T cells centrally or their transformation to T regulatory (reg) cells peripherally. (27, 28) Decreased numbers and/or function of T reg cells impairs peripheral tolerance as it allows survival of autoreactive T cells. Interchangeably, lack of a diversified TCR repertoire may hinder control of autoreactive T cells despite appropriate numbers of T reg cells (29). Concerning central tolerance checkpoints for humoral immunity, partial RAG deficiency decreases VDJ recombination and receptor editing in the bone marrow resulting in reduced BCR diversity and B cell numbers, and survival of autoreactive clones despite the low B cell count. Further, B cell lymphopenia with inflammatory milieu can result in elevated level of B-cell–activating factor (BAFF), which is associated with impaired peripheral tolerance (8). The survival and proliferation of autoreactive T and B cell clones likely contribute to broad spectrum autoantibodies including those targeting cytokines (23, 26, 30).

Another mechanism of autoimmunity in RAG deficiencies is due to failure to control acute infections which leads to persistent inflammation. This in turn results in autoimmune-induced tissue damage due to over secretion of cytokines and vasoactive molecules (31, 32).

3. Variable phenotypes of RAG deficiencies

The RAG1 and RAG2 genes are highly polymorphic. Over 200 distinct RAG1 and RAG2 pathogenic variants have been reported (33). As mentioned above, patients with these variants may present with distinct clinical phenotypes that range from severe infections to autoimmunity and inflammation. Epigenetic modifiers such as viral infections may also contribute to this phenotypic diversity (8) (Figure 1). Following is a description of the major phenotypes of RAG deficiency.

3.1. T^-B^-NK⁺ SCID

SCID is a heterogeneous group of disorders that primarily affects T cell immunity and leads to severe, often fatal infections (34). Affected patients will not survive beyond their second year of life unless diagnosed in time and receive HSCT to reconstitute the immune system (35). With the implementation of newborn screening (NBS) for SCID, diagnosis can be made very early in life, prior to any symptoms. Patients additionally have B cell deficiency with very low antibody production that results in pan-hypogammaglobulinemia. B cell dysfunction is either secondary to abnormal T cell help and/or intrinsic (primary) B cell defect when both early T and B cell development is compromised. A classic example of the latter one is disruption in the process of V(D)J recombination including RAG defects.

Pathogenic null variants in both alleles of RAG1 or RAG2 genes are associated with complete block in V(D)J recombination and fully absent or highly restricted TCR and BCR repertoires. Since TCR and BCR signaling is essential in T and B cell development, ultimately these compartments contract and result in severe lymphopenia. Initially, patients are often asymptomatic after birth, under the protection of placentally-transferred maternal antibodies. Around 4–6 months of age, gradual decline of maternal antibodies leads to opportunistic sinopulmonary and gastrointestinal (GI) infections, chronic diarrhea and failure to thrive. Infective agents include opportunistic bacterial infections (Pseudomonas), fungi (Pneumocystis jirovecii, Candida and Aspergillus species) and viruses [respiratory syncytial virus, cytomegalovirus (CMV), herpes simplex virus (HSV), varicella zoster virus (VZV), parainfluenza, and adenovirus]. Breastfeeding by CMV positive mother can be an important and avoidable source of infection. Live vaccines, such as Bacille Calmette-Guerin (BCG) or rotavirus can escalate this process.

In the era of newborn screening for SCID, the face of this disorder is changing dramatically. Once patients are diligently identified in the first month of life, the initiation of antimicrobial prophylaxis, immunoglobulin replacement therapy and recommendations on isolation and cessation of breastfeeding by CMV positive mothers have kept many of SCID infants less vulnerable and in most cases asymptomatic (36). In these cases, the immunological definition of typical SCID, and not the clinical history of infections, confirms the need for HSCT. The definition of typical SCID identified by NBS include <300 cell/μl (autologous) CD3+ T cells, <10% of lower limit of normal proliferation to mitogen phytohemagglutinin and a proven deleterious defect in a known SCID gene (37, 38). TCR repertoire studies by spectratyping or deep sequencing show oligclonality. Relative recombination activity of pathogenic variants, measured in vitro, average <1% for RAG1 and <10% for RAG2 variants in SCID cases (9, 10).

3.2. Omenn Syndrome (OS)

OS is an inflammatory variant of SCID characterized by early life diffuse erythroderma, chronic diarrhea with failure to thrive, lymphoproliferation (lymphadenopathy, hepatosplenomegaly) and viral or fungal pneumonitis (Figure 2) (16, 39). TCR repertoire studies by spectratyping or deep sequencing show oligoclonality. In OS cases, relative recombination activity of pathogenic variants in vitro average 8% for RAG1 and 10% for RAG2 variants (9, 10).

The inflammatory changes in OS primarily contribute to the expansion of poorly functional non-maternal oligoclonal TCRαβ+ T cell population that is Th2 skewed and secretes IL-4 and IL-5. This milieu promotes increased eosinophil count and markedly elevated IgE level (14–16, 39). Curiously, circulating mature B cells are undetectable in peripheral blood, however data from rag1 and rag2 hypomorphic mouse models suggest the accumulation of immunoglobulin secreting B cells with broad specificity in end organs, such as the spleen (24, 26).

Although RAG1 and RAG2 hypomorphic variants were the first reported cause of OS; there are several other SCID genes linked to OS including defects in metabolic pathways (AK2, RMRP, ADA), T cell signaling (IL7RA, IL2RG, ZAP70), V(D)J recombination [DCLRE1C (Artemis)], LIG4 (DNA ligase IV) (25, 40–44), partial thymus defect (partial DiGeorge syndrome with p22q11 deletion or TBX1 mutation) and even coloboma, heart defects, atresia choanae, growth retardation, genital abnormalities, and ear abnormalities syndrome, i.e. CHARGE syndrome (45). Similar to SCID, patients with OS lack antigen-specific immunity which result in extreme susceptibility to infections, from early onset severe sinopulmonary and GI infections along with other cutaneous and extracutaneous symptoms. In many cases, viral infection triggers the transition from leaky SCID to OS phenotype (25, 44–46). Although systemic steroid and cyclosporine may alleviate inflammation, OS is fatal in the first couple years of life unless treated with definitive therapy such as HSCT.

3.3. RAG deficiencies with leaky SCID with γδ T cells (γδT-LS)

Another form of hypomorphic RAG deficiency is characterized by oligoclonal expansion of T cells in the setting of viral infections (similar to Omenn syndrome) but with the following differences: (1) non-specific expansion of γδ TCR positive T cells with restricted repertoire; (2) low to near normal immunoglobulin level and specific antibodies to infectious agents or vaccines (mainly herpesviruses but less to tetanus vaccine); (3) production of autoantibodies including those reactive to red blood cells; (3) history of herpesvirus infections (CMV, VZV, HSV) with cytopenia and (4) Epstein–Barr virus (EBV) associated proliferation. The patients are mostly healthy in the first few months of life before herpes virus infections occur. The most prominent features are cytopenias after infections with disseminated CMV and VZV, and higher risk of lymphoproliferative disease induced by EBV (47, 48). TCR repertoire studies by spectratyping or deep sequencing show oligoclonality. Relative recombination activity of pathogenic variant in vitro is <3% for RAG1 in γδT-LS cases (9, 10). Aggressive management of infections with appropriate therapy may delay autoimmune complications but would not prevent it. Therefore, HSCT is an essential and curative option in this subset of patients.

3.4. Combined immunodeficiency with granulomas and/or autoimmunity (CID-G/AI)

In the past few years, increasing evidence showed the role of RAG deficiencies in immune dysregulation in the form of autoimmunity or granulomatous diseases (18, 23, 49). It was found that hypomorphic RAG genes variants with more preserved T and B cell receptor repertoire secondary to less impaired recombination activity (>10–15%) when compared to OS are the culprit for this phenotype (21, 50). Patients mostly present later in life with autoimmunity, predominantly autoimmune cytopenias (AIC), as an initial presentation (22). In some cases, patient may be recognized early, around 1–2 years of age in association with scheduled live vaccination (51) or acute viral infection (52). The clinical presentation includes the combination of autoimmunity and inflammation such as autoimmune hemolytic anemia (AIHA), idiopathic thrombocytopenic purpura (ITP), autoimmune neutropenia (AN), vitiligo, psoriasis, Guillain-Barré syndrome (30, 53, 54), vasculitis, inflammatory myopathy, and hepatitis (22, 30). Another form of immune dysregulation is the granulomatous disease that can affect the skin, mucosal tissue and lungs (18, 22, 49, 54) (Figure 3).

Figure 3. Granuloma within the dermis of a combined immunodeficiency patient with partial RAG deficiency.

A. Several granulomas (green arrow) with central necrosis and granulomatous inflammation with surrounding areas of necrosis (blue stars) is visible. There is also prominent lymphohistiocytic inflammation (light gray arrow) (magnification 10X). B. It consists of a lymphohistiocytic wall surrounding a central area of necrosis indicated by the blue star (Maginifcation 40x). C. Vacuolation of keratinocytes in basal layer and intercellular edema (brown arrows) and apoptotic keratinocytes (orange arrows)(magnification 10X). Patient is described in “Sharapova Hum Immunol 2013 Late onset combined immune deficiency associated to skin granuloma due to heterozygous compound mutations in RAG1 gene in a 14 years old male”. Sample is courtesy of Dr. Svetlana Sharapova (Minsk, Belarus). Interpretation is by Dr. Ignacio Gonzalez-Gomez (Molecular Pathology, Johns Hopkins All Children’s Hospital, St. Petersburg, Florida).

Specifically, a recent case series of 85 patients with RAG deficiencies and immune dysregulation highlighted that molecular diagnosis was delayed to a median of 5 years from the first clinical signs of immune dysregulation in this group (22). The majority of patients presented with more than one complication indicative of immune dysregulation, with the most common etiologies being autoimmune cytopenias (AIC) (84.1%), granulomas 157 (23.8%), and inflammatory skin disorders (19.0%). Infections, including live viral vaccinations, were an initiating trigger for autoimmunity in 36.5% of cases. Similar findings were recently reported in a review that included 68 patients with CID G/AI in which autoimmune cytopenia was the most common manifestation (53%) followed by other organ-specific autoimmunity (55). Granulomas were identified in about 35% of cases, and lung and skin were commonly affected organs. Of note, these two cohorts had shared patients as part of the data was based on previously published cases.

Lastly, recently several adult patients with RAG deficiencies have been reported with progressive inflammatory lung disease (21).

The immune phenotype of these patients is notable for low normal to preserved immunoglobulin levels and B cell count, even with hypergammaglobulinemia in selected cases (22). However, both B cell count and immunoglobulin levels may decline with age (56). IgE level is mostly normal, unlike in OS. High fraction of activated T cells with memory phenotype (CD45RO+) and low number of naïve T cells is a key immunological feature along with the presence of broad-spectrum antibodies, including those targeting cytokines interferon alpha (IFN-α), IFN-ω and IL-12 (23). If left untreated, immune dysregulation in CID-G/AI, can lead to many complications including lymphomas (18).

In some cases of CID-G/AI and other variants of RAG deficiencies that have higher RAG activity, the total diversity of the TCR and BCR repertoires may be normal, however, the repertoires are skewed. TCR repertoire studies by spectratyping or deep sequencing show skewing and BCR repertoire studies are notable for less usage of distal elements (12, 57). Relative recombination activity of pathogenic variant. measured by an in vitro assay average >10% for RAG1 and >20% for RAG2 variants in CID-G/AI phenotype. It is not uncommon that patients carry two different variants in separate alleles (compound heterozygous). In this case, relative recombination activity is best assessed in a bi-cistronic system with both variants (21, 58).

Overall, our experience with patients with CID-G/AI phenotype highlights a late-onset disease with autoimmunity, such as early onset AIC, and hypergammaglobulinemia that may confound the diagnosis of RAG deficiencies and emphasize the importance of T and B cell immunophenotyping, TCR/BCR repertoire studies and measurement of anti-cytokine antibodies, as a useful diagnostic tool.

3.5. Partial RAG deficiencies presenting initially as primary antibody deficiency

The increasing use of whole exome sequencing among a broad selection of patients with PID allow RAG deficiencies patients to be discovered in groups other than SCID and CID variants. Indeed, partial RAG deficiencies is now reported with initial presentation as primary antibody deficiency, including common variable immunodeficiency (CVID), selective antibodies deficiency with lack of polysaccharide-specific antibody responses, and IgA deficiency (sIgA) (19, 20, 59). The presence of autoimmune features is variable. A late presentation with sinopulmonary infections, hypogammaglobulinemia and inadequate response to vaccinations that fit the International consensus document (ICON) for CVID has been described in two patients with hypomorphic compound heterozygous RAG variants (19). Interestingly, in both patients, opportunistic infections and immune dysregulation in the form of AIC and granulomatous-lymphocytic interstitial lung disease (GLILD) developed despite immunoglobulin replacement therapy (IgRT). Similarly, CVID was diagnosed in two other patients with granulomatous disease in the liver, autoimmune neutropenia, mucosa-associated lymphoid tissue lymphoma (MALToma) and the patients died despite proper IgRT (60). We also identified several patients in a large cohort study from the United Kingdom and Austria with antibody deficiency syndromes (21). Immune phenotyping in these studies are notable for low fraction of naïve T cells, variable lymphocyte proliferation, and variable B cells count and immunoglobulin levels, which suggest a more appropriate designation of CID-G/AI phenotype.

Polysaccharide antibody deficiency was diagnosed in early adulthood in two patients who presented with sinopulmonary infections and vasculitis (in one only) who were found to have hypomorphic RAG deficiencies (20). As an unusual example, milder presentation in the form sIgA deficiency with absence of opportunistic infections, and autoimmune and/or granulomatous disease was reported in one patient (59).

Laboratory clues that may lead to identification of hypomorphic RAG deficient patients in the wide cohort of antibody deficient PID cases are a low fraction and low absolute count of naïve T cell. In fact, Kamae et al. have proposed the use of T cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) assays to distinguish CID cases from CVID (61).

3.6. RAG deficiencies with CD4+ T-cell lymphopenia (TCL)

A much less severe and late-onset form of hypomorphic RAG deficiencies that causes isolated CD4+ T cell lymphopenia has been described in two reports. One case with compound heterozygous RAG1 variants presented in childhood with chickenpox and VZV pneumonia that responded to medical treatment and only required prophylactic antibiotics to prevent infections (17). The other patient presented in his 30s with pruritic skin rash, eosinophilia and skin biopsy but no granulomatous lesions. Sequencing revealed heterozygous RAG1 variant. The patient was treated with prophylactic antibiotics accompanied by skin care hygiene protocol used in patients with atopic dermatitis (62). Both patients had CD4+ lymphopenia with skewness toward activated memory CD45RO phenotype, normal B cell with a low fraction of class-switched memory (IgD-CD27+) subsets, normal immunoglobulin levels, and intact humoral immunity. Although, autoantibodies against self-antigens (cytokines) were detected in the first case, the patient was clinically asymptomatic with no evidence of autoimmunity (17, 23).

4. Incidence of RAG deficiencies

Based on NBS results in eleven states of the United States (US), the incidence of typical and atypical SCID is 1 in 58,000 newborns (37). RAG genes are the second or third most common cause of SCID in the US with a predicted incidence of 1 in 336,000 newborns, depending on the study (37, 63). Further, it is noted that RAG is a dominant genetic defect among leaky SCID patients (41%) in the era of newborn screening (prospective protocol 6901, 2010–2018, NCT01186913, n=68) (63). This is a dramatic change form 11% in a retrospective study (6902, 1968–2012, n=81). In addition, we estimated that there are about twice as many RAG1/2 hypomorphic patients (1:181,000) than patients with RAG1/2-deficient SCID, LS, and OS based on a query of the ExAC database (ExAC.v3; http://exac.broadinstitute.org)) with whole exome data from more than 60,000 unrelated individuals (64).

Additionally, in countries with high rate of consanguinity, SCID (1 in 7,500) in general, and pathogenic RAG variants are even more frequently identified (in 20% of cases) (65, 66). Similarly, frequent accumulation of RAG variants is also described in Amish communities (67).

Lastly, a large cohort study from the United Kingdom and Austria has identified partial RAG deficiencies in 1:500 adult patients with antibody deficiency syndromes highlighting the importance of genetic testing among adults in PID (21).

5. Epigenetic modification of phenotype

The fact that the same mutation in RAG can manifest in distinct phenotypes give evidence of epigenetic factors modifying genetic factors affecting disease expression. For instance, among three patients from the same extended family who inherited the same homozygous mutation of the RAG2 gene, two patients continued to manifest T^-B^-SCID whereas the third patient changed from T^-B^-SCID phenotype to that of OS following parainfluenza virus 3 infection (46). Another example is the dramatic difference in disease penetrance reported in two brothers with identical homozygous mutations in RAG1 resulting in 10% RAG activity. The younger sibling (9 years old) was severely affected with recurrent infectious and several inflammatory/autoimmune disorders whereas his older brother (15 years old) merely had vitiligo, did not have recurrent infections, and was in good health (11). Further, the younger brother had low B cells (CD19), IgA deficiency, and low T cells (CD4, and CD8), especially low naïve T cells (CD4/CD45RA), whereas these values were normal in his older brother. Lawless et al. reported a similar example of discordant phenotypes among two patients with identical hypomorphic RAG mutations (patient 12 and 13 in supplementary data tables) although it is not indicated whether the two patients were genetically related (21). Although it is possible that the discordance in immune phenotypes observed in patients with identical hypomorphic RAG mutations could be explained by additional unknown or undiscovered mutations, it is more likely that epigenetic factors, such as history of viral infections, accounts for the phenotypic differences.

Viral infections in RAG deficiencies can lead to complications of lymphoproliferation, malignancy, granulomas and inflammation, and autoimmunity as illustrated in the following examples: (1) three young girls with immunodeficiency due to compound heterozygous mutations in RAG experienced complications following viral infections including cutaneous and/or visceral granulomas and B-cell lymphoma (18); (2) two patients with hypomorphic RAG mutations developed cutaneous and/or visceral granulomas following live rubella vaccination (68); (3) a large retrospective study of patients with RAG deficiency and autoimmune/inflammatory complications (n = 63) found that viral infections occurred just before autoimmune manifestations in 29% of cases (22); and (4) a prospective study of RAG deficiency patients with variable phenotypes revealed most autoantibodies were directed against cytokines and most patients with anti-cytokine antibodies suffered severe viral infections previously (23).

Animal experiments give evidence that viral infections may induce production of autoantibodies. A mouse model of LS due to hypomorphic rag mutation produced autoantibodies in response to repeated stimulation with toll-like receptor agonists mimicking viral infection. The autoantibodies had broad specificity for single and double-stranded DNA, and nuclear proteins. The fact that the specificity of autoantibodies in RAG deficiency patients differed from those in the mouse model of LS may reflect the fact that most of the patients with autoantibodies had CID-AI/G; this variant has greater RAG activity than LS. The variability of phenotypes, which are subject to viral infections and other epigenetic modifiers, presents a challenge for diagnosing RAG deficiency.

6. Diagnostic dilemmas in RAG deficiencies

The correlation between genetic, immunological and clinical data has to be carefully examined and interpreted to correctly diagnose patients with RAG deficiency variants. These patients have a wide variety of infections, autoimmunity and inflammation in different degrees. Beyond infections linked to SCID, early herpes viral infections predominate including EBV, CMV, and varicella. Patients also can have infection from adenovirus or from live vaccines such as BCG (23, 47, 48, 52).

Autoimmunity may affect multiple organs. The most dominant autoimmune manifestation is AIC, especially AIHA (22, 55). Patients with both a PID, such as RAG deficiency, and AIC are often refractory to treatment, which is typically multilinear. The worst prognoses have been reported in cases with vasculitis (22) and rheumatological complications such as chronic recurrent multifocal osteomyelitis (CRMO) (54). Other inflammatory complications include development of chronic granulomas in several organs (18, 19, 49), chronic skin and intestinal inflammation (16, 32), and EBV-induced lymphoproliferation (18).

Regarding their immunological diagnosis, RAG deficiency patients have progressive decline of naïve CD4 T cell counts. Many cases may be diagnosed by newborn screening with low TRECs secondary to abnormal T cell development in the thymus. The T cell repertoire may be skewed to variable degrees; similarly, lymphocyte proliferations with mitogens and antigens are also variable. In cases with more preserved RAG activity, B cells can present normally in number but may decline with age. Immunoglobulin levels can range from absent to near normal levels based on the clinical phenotype. The serological hallmark of the disease is broad-spectrum autoantibodies with some targeting specific cytokines: IFN-α, IFN-ω, ανδIL-12. Severe skewing of CD4 T cells towards memory phenotype is a common key finding. Testing for broad spectrum antibodies, including those targeting cytokine antibodies, may be useful (22, 23, 52).

The genetic diagnosis of RAG deficient patients can involve missense or nonsense variants in homozygous or compound heterozygous form. Since RAG genes are highly polymorphic, it is not uncommon that a variant of uncertain significance (VUS) is identified by genetic testing. Allele frequency of the variant in the healthy population is informative (gnomAD: https://gnomad.broadinstitute.org (former ExAc). High frequency variants (>0.01% of population) are more suggestive of benign mutation. The effect of the mutation on the function or the structure of the protein and may give additional insight into these variants and can be estimated with prediction programs, SIFT (https://sift.bii.a-star.edu.sg) and PolyPhen (http://genetics.bwh.harvard.edu/pph2/).

Overall, ultimately direct or indirect readout of recombinase activity is needed for confirmation of the pathogenicity of novel RAG variants and its association with the clinical and immunological phenotypes. This approach may include TCR/BCR repertoire studies (indirect assay, in vivo) and/or in vitro recombination assays (direct assay). In case of RAG deficiencies, the BCR repertoire is skewed towards decreased distal V, D and especially J (J5 and J6) BCR families (12), and TCRα repertoire is lacking Vα7.2 secondary to absent distant TCR rearrangement (57). The skewing of TCR and BCR repertoire reflects well the level of underlying RAG activity.(12, 21, 50, 59). However, similar repertoire changes may occur in other V(D)J recombination defects such as Artemis (DCLRE1C) deficiency.(57) Therefore, to make a distinction, several direct in vitro measurements of RAG enzyme recombination activity have been described. There are two most recently used approaches. In the first method, Notarangelo et al., transfected a retroviral vector construct for RAG1 to rag1 deficient Abelson murine leukemia virus transformed pro-B cell line that contains an inverted intrachromosomic green florescent protein (GFP) gene flanked by RSS. Relative recombinase activity of RAG1 is measured by flipping the GFP gene permitting its expression, which is measured by flow cytometry; the proportion of cells expressing GFP (mutant /wild type) corresponds to relative recombinase activity of the RAG variant (9).

The second recently used method, described by Thwaites et al., is a polymerase chain reaction (PCR) based assay (69). In this approach, increasing amount of mouse wild type or mutant RAG1 or RAG2 plasmid is co-transfected with target plasmid pJH299 into NIH3T3 cells. Relative recombinase activity is determined by the level of recombined plasmid by PCR.(69) The advantage of the second system is that relative recombinase activity can be measured in a bi-cistronic system (21, 56).

In case of compound heterozygous variants, the bi-cistronic in vitro recombination assay system is essential. Note that in vitro assays are a crude approximation of what occurs in vivo. It is still unclear in what combination the mutant alleles are used in the RAG heterotetramer, and the assay is based on a construct with only one variant of RSS. Therefore, TCR and BCR repertoire studies may be important to reflect the “real-life” RAG recombinase activity (50, 57). The need for such studies is supported by the fact, that patients from families with the exact same variants may present with distinct clinical and immunological phenotypes.

7. Prevention and management of infectious complications in RAG deficiencies

Early detection of children with SCID in NBS will hopefully prevent most early opportunistic infections including candidiasis, Pneumocystis jiroveci, gastrointestinal and/or disseminated viral infections; and severe local or systemic bacterial infections such as sinopulmonary infections, mastoiditis or meningitis. Patient management in a protected environment with high-efficiency particulate air filtration (HEPA) or laminar air flow (LAF) is recommended to lower the incidence of fungal infections, especially with Aspergillus and Pneumocystis jirovecii. Prophylactic antibiotics and antifungal medications also help reduce the risk of further infections and are important in asymptomatic neonates awaiting HSCT (Table 2). Evaluation for CMV carrier state in breastfeeding mothers is crucial and breastfeeding should be on hold until full clearance. As humoral immunity is also affected, intravenous immunoglobulin (IVIG) is required. It is crucial to avoid live vaccines including BCG since they can cause fatal disseminated disease. All blood products should be irradiated because of the risk of graft-vs-host disease (GvHD) from donor T cells. Despite all prophylactic measurements, the risk for severe viral infections will only be minimized with restoration of the immune system by timely HSCT.

Table 2.

Prophylactic treatment options in RAG deficiency to prevent infectious complications.

Condition	Medication	Dose
		Pediatric	Adult
Prophylactic treatment for infectious complications	Trimethoprim-sulfamethoxazole	5 mg/kg per day of trimethoprim as a single dose or twice daily	160 mg of trimethoprim daily or twice daily
	Azithromycin∗§	• <25kg: 30mg/kg per week (may be given in divided doses three times weekly or as a single weekly dose) • 25–40kg: 250mg/dose three times weekly • >40kg: 500mg/dose three times weekly	500 mg weekly or 250 mg every other day
	Acyclovir	80 mg/kg 4 times daily
	Fluconazole	6 mg/kg once daily
	IVIG	400–600 mg/kg every 4 weeks

^∗to prevent chronic rhinosinusitis, otitis media or bacterial infection in patient with bronchiectasis

^§not part of standard prophylaxis for infants

IVIG: intravenous immunoglobulin

On the contrary, clinical management for patients with idiopathic CD4 T cell lymphopenia (ICL) and pathogenic RAG variants is less clear. Immune cell function and repertoire studies may provide insight into how advanced the patient’s disease is. In the first case report of ICL by Kujipers et al, watchful waiting with prophylactic antibiotics was used for a few years. The patient eventually underwent HSCT as a young adult when frequency of infections increased, did well post-HSCT, but eventually died secondary to infections (personal communication T. Kuijpers) (17). Along the same line, a patient with partial RAG and IgA deficiencies received HSCT early in life, which may have prevented progressive decline of immune function and infectious complications (59).

8. CID-G/AI phenotype: management of autoimmune and hyperinflammatory complications

The presentation of autoimmune and/or inflammatory complications varies across individuals with hypomorphic RAG deficiencies. Autoimmune cytopenias are the most common, followed by granulomas, and skin disorders including vitiligo, psoriasis, and alopecia. Usually, patients present with various combinations of infection susceptibility and autoimmune or inflammatory complications.

In case of AIC, first line therapy may include corticosteroids for AIHA, high dose IVIG and corticosteroids for ITP, and granulocyte-colony stimulating factor (G-CSF) for AN. In a large case series, AIC were dominated by AIHA, followed by ITP and AN, respectively. Autoimmune cytopenias were refractory to intravenous immunoglobulin, steroids, and rituximab in the majority of cases (64.7%, 73.7%, and 71.4% for AIHA, ITP, and AN, respectively). Occurrence of Evans syndrome was associated with lack of response to first-line therapies (22). Onset at older age, single-lineage involvement and less severe cytopenia may predict favorable response to treatment (22). Other autoimmune disorders and single or multiorgan granulomas have been managed with high dose IVIG and corticosteroids with limited response. Anti-TNF, e.g. infliximab, has shown a moderate effect in granulomatous disease (Table 3). Other second-line therapies such as cyclophosphamide, cyclosporine, anti-CD52 (alemtuzumab), adalimumab (TNFα inhibitor), rituximab (anti-CD20) and/or sirolimus have been tried and failed to achieve a sustained response in most patients.

Table 3.

Mainstay treatment options used to treat autoimmunity and hyperinflammatory complications associated with partial RAG deficiency

Condition	Medication	Dose
Autoimmune cytopenias (AIC)	Corticosteroids	1–1.5 mg/kg of prednisone or its equivalent daily
	IVIG (high dose)	1–2 g/kg/day
	infliximab	IV 3–5 mg/kg at 0, 2, and 6 weeks, followed by 3–5 mg/kg every 8 weeks
	adalimumab	40 (rheumatological) to 80 (IBD) mg every other week
	rituximab	375 mg/m2 weekly for a median of 4 weeks OR 100 mg weekly for 4 weeks
	HSCT	Per protocol
	Granulocyte-colony stimulating factor (G-CSF)	5 mcg/kg once daily; adjust the dose based on ANC and clinical response
Rheumatologic disease	Corticosteroids	1–1.5 mg/kg of prednisone or its equivalent daily
	IVIG (high dose)	1–2 g/kg/day
	rituximab	375 mg/m2 weekly for a median of 4 weeks OR 100 mg weekly for 4 weeks
Granulomas	Corticosteroids	1–1.5 mg/kg of prednisone or its equivalent daily
	IVIG (high dose)	1–2 g/kg/day

IVIG: intravenous immunoglobulin

Since autoimmune and/or inflammatory complications accompanied by susceptibility to infections may manifest years before molecular detection of RAG deficiencies, some patients may have undergone experimental treatment with HSCT for CID prior to molecular diagnosis. In our case series of RAG deficient patients, treatment-refractory immune dysregulation prompted HSCT in 20 patients (25%) (22).

Inflammatory lung disease in partial RAG deficiencies may be progressive. Based on a cohort of 15 patients, lung disease was mostly treated as an infectious process, and anti-inflammatory treatment with non-steroid sparing agent was only attempted in 2 of 15 cases (13%). Similarly, treatment of vasculitis can be a challenge in partial RAG deficiencies. In our cohort of seven patients, only skin manifestation responded well to first-line steroid treatment, whereas systemic vasculitis with severe end organ complications was refractory to first- or second-line therapy, including steroids and immunosuppression and required HSCT for curative management (70).

9. HSCT in RAG deficiencies

The cornerstone treatment of RAG deficiencies is HSCT, and there is unanimity in children with null mutations T-B-NK+ SCID, and LS variants including OS and RAG deficiencies with expanding γδ T cells. Selection of donor, source of hematopoietic stem cells (HSC) and conditioning regimens are important determinants of favorable outcome (36).

HSCT is highly successful with up to 90% survival without conditioning when donors are HLA-identical matched siblings or family donors (MSD/MFD) using unmanipulated HSCs as based on results of a 3-center study analyzing 145 patients treated between 1985 and 2009 (71). While earlier data had shown worse outcomes in OS phenotype compared to classical SCID (72, 73), more recent data from PIDTC showed no difference in survival, immune reconstitution or GvHD between patients with OS and with typical SCID patients (74). Interestingly, outcomes were not different in patients with null RAG versus hypomorphic mutations (74). In the absence of a MSD/MFD, HSCT may be performed from either a matched unrelated donor (MUD) or a mismatched related/haploidentical donor, usually a parent. Both donor sources generally require use of conditioning regimens to ensure engraftment and both T and B cell reconstitution. The intensity of myelosuppressive treatment required remains controversial. Donor T cells need to be removed from haploidentical transplants either by graft manipulation (positive or negative selection) or post-transplant cyclophosphamide to prevent GvHD. A high rate of graft failure was noted following haploidentical HSCT without myeloablative conditioning (71) and long-term T and B cell reconstitution is observed primarily in patients following conditioning (75), The presence of double negative CD3+ T cells in thymic niches has been postulated to hinder engraftment (76). Moreover, natural killer (NK) cells may influence the engraftment process in RAG deficiencies. Rarely measured clinically, RAG deficiencies prime NK cells for increased cytotoxicity in human and animal studies (77, 78), suggesting that NK cells may contribute to graft rejection in non-conditioned HSCT.

Patients with CID-G/AI phenotype are not severely lymphopenic since residual recombinase activity allows development of lymphocytes with a limited TCR and BCR repertoire. In this context, “leaky” T and B cells survive in early developmental stages and occupy the bone marrow and thymic niches. However, along with NK cells, this may create a milieu favoring inflammatory complications triggered by infections. For patients with CID-G/AI phenotype, data comparing conditioning regimens, graft manipulation and outcome data is limited (22, 23, 79, 80).

With the introduction of NBS for SCID via TRECs that is offered in the US and other countries, early detection of SCID became more prevalent. Several studies have highlighted that HSCT at early age (<3.5 months) and prior infections exposure has the greatest success rate (35, 74). There is an ongoing discussion whether and which conditioning regimen should be used for these asymptomatic patients. If a matched family donor is available, HSCT can be performed without graft manipulation and conditioning. This approach circumvents toxicity, but usually does not result in B-cell engraftment. With alternative donors (MUD, or haploidentical), conditioning is highly recommended to avoid graft failure and ensure full immune reconstitution including B cell immunity and long-lasting T cell production. Toxicity in this very young group of SCID patients includes both acute (e.g. hepatic veno-occlusive disease) as well as long-term toxicities.

Most published hypomorphic RAG deficient patients with late-onset were considered for HSCT only after immune dysregulation progressed despite therapeutic immune modulation. It is at present an open discussion, when a RAG deficient patient with variants resulting in a more preserved recombinase activity should be transplanted, especially when diagnosed by NBS. However, since most hypomorphic RAG deficient patients will present at some point in their life with infectious and/or autoimmune complications which increases risks of HSCT, early HSCT needs to be considered in particular when a HLA-matched family donor is available.

10. Gene therapy for RAG deficiencies

Gene therapy (GT) has been proposed to overcome problems of allogeneic HSCT, including donor selection, need for conditioning and GvHD. So far, it has been tested in murine models of RAG deficiencies and a phase I trial is pending review in Europe (Recomb Clinical trial for RAG1-SCID). Both retrovirus and lentivirus strategies for corrected gene transduction have been tried in mice with variable results (81–85). GT in SCID (T^-B^-NK⁺) individuals risks autoimmune complications as in Omenn-like syndrome if restoration of RAG expression is low. Such was the outcome in a murine lentiviral GT trial in RAG1 knockout (KO) mice (81). However, a previous study using the same lentiviral vectors in RAG1 KO mice of the same strain had much higher RAG expression levels and did not report OS-like features. Thus, the level of RAG1 expression in transduced stem cells and after transplantation and reconstitution in lymphocytes is a crucial determinant of outcome and may be responsible for the observed variable results of GT (86). To prevent possible genotoxicity with increased gene expression, such as malignancy, the minimum effective vector copy number and cell dose should be established (84).

The most recent GT study was in an irradiated mouse model of OS using a lentivirus vector of human genes. The study resulted in partial reconstitution of T and B cells (including naïve T cells), normal B and T cell function (i.e. immunoglobulin levels and response to mitogens, respectively), polyclonal TCR repertoire, normal vaccine responses, and decreased autoimmune manifestations (85). These encouraging findings in an animal model demonstrate that GT can benefit SCID due to RAG deficiencies with autoimmune/inflammatory pathology.

Gene editing with CRISPR-Cas9 has recently been used to create mouse model for new variants of RAG deficiencies (87). However, no mouse or clinical trials are currently published for correcting RAG deficiencies with gene editing.

11. Conclusion

RAG deficiencies can present with different phenotypes reflecting variable recombination activity levels which are crucial for both T and B cells development. In the era of a revolution of genetic screening, there is a need for confirmatory testing methods for pathogenic RAG variants. Tools may include repertoire studies, immune cell characterization and serological biomarkers, as well as in vitro functional studies. Despite the advances in early detection of null variants via newborn screen for SCID, there is exigent demand to describe the clinical path and response to treatment specifically for patients with CID-G/AI. Lastly, while HSCT has improved over the years, in particular with better understanding and usage of conditioning, there is still room to explore alternative approaches to overcome risks of graft rejection and poor immune reconstitution. Finally, gene therapy with more precise transgene or gene editing has a promising future.

12. Expert Opinion

In the era of genetic screening, atypical presentations of primary immune diseases with well-known genetic defects often occur. An excellent example of this is RAG defects, which may present with broad range phenotypes starting from severe combined immunodeficiency to conditions dominated by CID-G/AI. The age range of patients may also vary. Beyond infancy, many RAG-deficient patients are identified in late childhood or even during adulthood with milder forms of antibody or combined immunodeficiencies.

Resultant of national NBS efforts, we can now identify RAG deficiencies at birth at the asymptomatic stage. Beyond classical and atypical variants of SCID, we may also be able to identify patients who are currently in the asymptomatic stage but could evolve to CID-G/AI phenotype with permissive RAG variants (>10–15% predicted relative recombinase activity). As within the family with the same RAG mutations, patients may develop very different clinical phenotypes; it is still unclear what will be the natural history of these patients. We currently lack specific biomarkers to identify who will be at risk for autoimmune and inflammatory phenotypes, and which patients should receive HSCT in the asymptomatic stage from these asymptomatic group of patients with permissive RAG variants.

Autoimmune and inflammatory complication, such as multilineal autoimmune cytopenias, granulomas, alopecia, vasculitis, are commonly seen in partial RAG deficiencies and may be the first sign of the underlying immunodeficiency. Therefore, physicians need to become more vigilant and include RAG genes testing within the genetic screening of patient presenting with multiple autoimmune diseases and/or hyperinflammation, especially among those who are refractory to first and second lines of therapies.

Immunological evaluation can be misleading as many patients have immunoglobulin levels and B cell compartments preserved in varying degrees. OS with high IgE and TH2 skewing is only one face of partial RAG deficiencies. Patients with CID-G/AI phenotype more likely present with normal IgE.

Genetic testing is the first affirmative step for RAG deficiencies. However, a cautious approach needs to be practiced when interpreting genetic results. It is known that RAG genes are highly polymorphic and many times, variants of uncertain significance (VUS) are being identified. Therefore, it is recommended that the novel mutant RAG gene is functionally tested for relative recombinase activity. In vitro assays are available in single allele and bi-cistronic systems (for compound heterozygous mutations) on a research basis. In vivo assessment of RAG function can be drawn from T and B cell receptor (TCR, BCR) repertoire studies. Some of these studies are extensive and can be accomplished on the grounds of research collaboration with laboratories that focus on V(D)J recombination. However, recently simplified tools are emerging that may be used for in vivo validation of novel hypomorphic RAG variants. These may include flow cytometry or PCR-based detection of certain TCR-Vα segments in immune cells. Curiously, antibodies targeting cytokines (IFN-α, IFN-ω and IL-12) have been detected in a high fraction of the patients and may be used as possible biomarkers.

Over the few coming years, it will be essential to address how NBS impacts the identification of patients with a broad spectrum of immunological phenotypes of RAG deficiencies at the asymptomatic stage. As the next step after early diagnosis, most patients will be referred for consideration of HSCT. It will be crucial to assess the decision-making patterns for moving forward with HSCT and how donor source and conditioning regimens are selected, and how it impacts outcomes. Gene therapy is currently awaiting clinical trial approval and may become available. It is unclear how well it can achieve immune reconstitution and what late effects we can expect with gene therapy for RAG deficiencies.

In the next decade, we will continue identifying patients with partial RAG deficiencies in all ages as newborn screening for SCID is still not available globally and non-SCID patients with low TRECs may not get sequenced for RAG genes. It is of utmost importance that we work closely with our colleagues in Hematology (autoimmune cytopenias, lymphoproliferation), Rheumatology (vasculitis, arthritis, chronic recurrent multifocal osteomyelitis) and Dermatology (granulomas, alopecia, vitiligo, psoriasis), Neurology (neuropathy, myopathy, Guillen-Barre syndrome) and Pulmonology (granulomas, progressive interstitial lung disease) to identify partial RAG-deficient cases, hence, expedite their targeted therapy and transition to evaluation for definitive treatments such as bone marrow transplant or GT.

In summary, a careful analysis of immunological and clinical data and their correlation with genetic findings help to determine the significance of the genetic polymorphism. Advances in functional assays, as well as anti-cytokines antibodies, make it easier to resolve the diagnostic dilemma. NBS for SCID will continue to identify RAG deficiency at the asymptomatic stage, which requires a clear consensus on how to approach it with HSCT. GT is currently under investigation and may play a role in treatment.

Article Highlights.

1. RAG deficiency is an autosomal recessive disease that can present with wide variety of clinical phenotypes (infections, autoimmunity) partly secondary to variability in remnant recombinase activity (phenotype-genotype correlation) and influence of epigenetic modifiers (such as infections).

2. RAG genes are highly polymorphic and variants of uncertain significance (VUS) may be discovered among patients during broad genetic screening.

3. The causative link between the VUS in RAG genes and clinical phenotype should be established by a rigorous diagnostic approach.

4. Functional evaluation of RAG activity among patients with VUS in RAG genes may include in vitro recombinase assays and/or B and T cell receptor repertoire studies.

5. Patients with homozygous or compound heterozygous pathogenic RAG variants and clinical features of autoimmunity may fail conventional treatment approach and can be eligible for hematopoietic stem cell transplant.