Heterogeneity in RAG1 and RAG2 deficiency: 35 cases from a single-centre

Abstract Recombination activating genes (RAG)1 and RAG2 deficiency leads to combined T/B-cell deficiency with varying clinical presentations. This study aimed to define the clinical/laboratory spectrum of RAG1 and RAG2 deficiency. We retrospectively reviewed the clinical/laboratory data of 35 patients, grouped them as severe combined immunodeficiency (SCID), Omenn syndrome (OS), and delayed-onset combined immunodeficiency (CID) and reported nine novel mutations. The male/female ratio was 23/12. Median age of clinical manifestations was 1 months (mo) (0.5–2), 2 mo (1.25–5), and 14 mo (3.63–27), age at diagnosis was 4 mo (3–6), 4.5 mo (2.5–9.75), and 27 mo (14.5–70) in SCID (n = 25; 71.4%), OS (n = 5; 14.3%), and CID (n = 5; 14.3%) patients, respectively. Common clinical manifestations were recurrent sinopulmonary infections 82.9%, oral moniliasis 62.9%, diarrhea 51.4%, and eczema/dermatitis 42.9%. Autoimmune features were present in 31.4% of the patients; 80% were in CID patients. Lymphopenia was present in 92% of SCID, 80% of OS, and 80% of CID patients. All SCID and CID patients had low T (CD3, CD4, and CD8), low B, and increased NK cell numbers. Twenty-eight patients underwent hematopoietic stem cell transplantation (HSCT), whereas seven patients died before HSCT. Median age at HSCT was 7 mo (4–13.5). Survival differed in groups; maximum in SCID patients who had an HLA-matched family donor, minimum in OS. Totally 19 (54.3%) patients survived. Early molecular genetic studies will give both individualized therapy options, and a survival advantage because of timely diagnosis and treatment. Further improvement in therapeutic outcomes will be possible if clinicians gain time for HSCT.


Introduction
The recombination activating genes (RAG) 1 and 2 have essential roles in the early stage of V(D)J [variable (diversity) joining segments] recombination, which provides the plasticity of the adaptive immune system to give reaction to diverse antigens.Therefore, defect in the V(D)J recombination process leads to a restricted antigen receptor repertoire in the adaptive immune system [1].
Schwarz et al. [2] first described RAG gene mutations in patients with T-negative (T-), B-negative (B-), and natural killer cell-positive (NK+) severe combined immunodeficiency (SCID) in 1996.Further studies showed that human RAG gene mutations have a broad spectrum of clinical and immunological phenotypes other than classical SCID [3,4].In SCID patients, clinical findings usually begin in the first year of life, generally soon after birth.Life-threatening opportunistic viral and fungal infections are common.Patients experience recurrent sinopulmonary infections, interstitial pneumonitis, protracted diarrhea, and failure to thrive.Lymphopenia and severe hypogammaglobulinemia are frequent findings.Hematopoietic stem cell transplantation (HSCT) should be planned just after the diagnosis of SCID because T-and B-cell reconstitution is curative for SCID [5,6].
A rare clinical presentation of RAG deficiency is Omenn syndrome (OS).RAG genes have a partial V(D)J recombination activity in OS [1,7].Omenn syndrome may result when certain hypomorphic RAG½ gene mutations result in partial V(D)J recombination activity, and leads to an activated oligoclonal T cell proliferation and infiltration in several organs, especially in skin, gut, and liver.The findings of OS are generalized erythroderma, lymphadenopathy, hepatosplenomegaly, eosinophilia, hypogammaglobulinemia, and high immunoglobulin (Ig) E levels.Clinical follow-up and treatment are similar to SCID [7,8].
If hypomorphic RAG gene mutations are present, residual RAG protein activity is possible which will cause delayed-onset disease forms and mimic common variable immunodeficiency (CVID) or combined immunodeficiency (CID).In delayed-onset RAG deficiency patients, autoimmune cytopenia (AIC), vasculitis, nephritis, and granulomatous lesions in various tissues and organs are common in addition to recurrent sinopulmonary infections [9,10].Idiopathic CD4+ T-cell lymphopenia [11], IgA deficiency [12], selective deficiency of polysaccharide-specific antibody responses [13], and hyper-IgM syndrome [14] are other delayed-onset and atypical presentations.Different clinical phenotypes with the same RAG defect even in the same family may support the role of epigenetic factors on the phenotype [1].
Herein, we aimed to elucidate the clinical features, molecular diagnosis, and outcomes of RAG½ deficient patients followed by a tertiary pediatric immunology department over a twenty-year period by describing the variable clinical presentation.

Patients and study design
This study enrolled 35 RAGD patients from 30 families diagnosed in a twenty-year period (1999-2019) at Hacettepe University, Ihsan Dogramaci Children's Hospital, Division of Pediatric Immunology.We retrospectively noted the clinical and laboratory data from medical records.We recruited the patients into three groups according to the clinical presentations/immunological findings; typical T(-)B(-)NK(+) SCID, OS, and delayed-onset CID (leaky SCID and atypical SCID) [9].Hacettepe University Institutional Review Board approved the study, and the parents of the patients signed the informed consent.
SCID patients were diagnosed by using the European Society of Immunodeficiency Disorders (ESID) Criteria [15] and International Union of Immunological Societies (IUIS) guidelines [16].OS criteria included the presence of erythroderma or atopic/seborrheic dermatitis in the absence of maternal engraftment [7,17].Delayed-onset patients with RAG½ mutations were diagnosed with delayed-onset CID, depending on the clinical symptoms and laboratory data [9].
The demographic characteristics of the patients (age of manifestation, age at diagnosis, gender, family history, etc.), clinical and laboratory findings, genetic mutations, HSCT outcomes, and survival were evaluated.RAG deficient patients those with high CD3 count, and high CD45RO value were assessed in terms of maternal engraftment, and karyotype and chimerism analyses were performed.

Sanger sequencing
DNA was isolated from peripheral blood mononuclear cells after separation using Ficoll-Paque (GE Healthcare, Little Chalfont, UK) according to the manufacturer's instructions.Sequence analysis of RAG½ was performed following PCR amplification of the coding regions with TaqGoldTM (Life Technologies), followed by direct sequencing on an ABI Prism 3130 XL fluorescent sequencer (Applied Biosystems, Bleiswijk, the Netherlands).

Targeted primary immunodeficiency panel screening
The molecular analyses of the patients were performed in the Hacettepe Pediatric Immunology Laboratory [18], Erasmus Center, and CeMM Research Center by using next-generation sequencing (NGS) for primary immune deficiency (PID) [19] and the Sanger Technique.

Statistical analysis
Statistical analysis was performed by using SPSS ® version 22.0 for Windows (IBM SPSS, Chicago, IL, USA).Quantitative parameters were reported as means and SD, or as medians with 25th and 75th percentile values in case of skewed distribution.Categorical variables were described using absolute frequencies and proportions with a 95% CI.A P-value of <0.05 was considered statistically significant.Kaplan-Meier test was used for survival analysis.

Patient characteristics
Thirty-five RAG-deficient patients (65.7% male) were included in the study.Eighty percent of cases had parental consanguinity, and 57.1% of the cases had a history of immunodeficiency in siblings or other family members.We subdivided the patients into three groups considering the clinical presentations and immunological findings: typical SCID (patients P1-25); OS (P26-30), and delayed-onset CID (P31-35) [9,15,16].

RAG ½ mutations and affected domains
Twenty-five patients had RAG1, and 10 patients had RAG2 deficiency.The RAG½ mutations, and affected RAG½ domains are shown in Table 1 and Fig. 1A and B. Mutations were mostly found in the core region for RAG1 and RAG2 genes.P26 and P27 were cousins, and had novel RAG2 mutations affecting the C-terminal non-core domain.
All patients with RAG1 and RAG2 deficiency had homozygous mutations, except three patients (RAG 1 deficient P19 and P33 and RAG2 deficient P9) had compound heterozygous mutations.Among the thirty-five patients included in this study, three in the RAG1 gene and six in the RAG2 gene, a totally of nine novel mutations were reported and depicted in Table 1 and Fig. 1A and B. P33 and P34 were previously reported [25,27].
Almost all autoimmune findings were generally associated with the CID group, albeit a patient with vitiligo was in the SCID group and patients with alopecia were in the OS group.(Table 2).Inflammatory disorders including hepatomegaly and/or splenomegaly, lymphadenopathy and several forms of dermatitis were quite common in all groups (Table 2/Fig.2).

Infectious diseases
CMV infection developed in 8/35 patients (SCID = 6, CID = 1, and OS = 1), and in two SCID patients (P11 and P20) retinitis developed as a complication.Immune thrombocytopenic purpura associated with CMV infection developed in P11 at the age of 1.5 mo [2].Foscarnet and ganciclovir were given.After referral, she was diagnosed with SCID and treated with HSCT successfully.The other SCID patient developed CMV retinitis during the disease course and underwent HSCT.Despite ganciclovir and CMV hyperimmunoglobulin, blindness developed.
Warts occurred in two siblings in the CID group (P31 and P32); the lesions were resistant to cryotherapy and laser in one.They had a previously reported RAG1 mutation (R561H; c.1682 G > A) [7].
Bacillus-Calmette-Guérin (BCG) is a live-attenuated vaccine and is contraindicated in SCID patients.Unfortunately, it is administered soon after birth since tuberculosis is still a public health problem in some countries [28].BCG is in the national vaccination schedule, and applied at the age of 2 mo in Turkey.As the median (IQR) age at diagnosis was 5 (3-10) mo in our cohort, 23 out of 35 patients received BCG vaccine before the diagnosis of PID.All BCG-vaccinated patients received isoniazid (INH) and rifampicin (RIF) for tuberculosis prophylaxis.Four SCID patients (P5, P7, P9, and P21) were diagnosed with BCGitis after HSCT and treated with additional anti-mycobacterial drugs.
Fifty-two percent of SCID patients, 80% of OS patients, and 20% of CID patients had low IgA, IgG, and IgM on admission.Normal/high IgG levels in some of the SCID patients were attributed to partially transplacental IgG transfer from their mothers.Most of the patients especially in the OS group had profound hypogammaglobulinemia on the first visit.Laboratory findings of RAG-deficient patients are summarized in Table 3.

Classification of patients with RAG½ deficiency
Typical severe CID patients Twenty-five patients, 19 males and 6 females were diagnosed with typical T(-) B(-) NK(+) SCID (patients [P] 1-25).The median age of clinical manifestations was 1 (0.5-2) mo and the age at diagnosis was 4 (3-6) mo.The parental consanguinity ratios were 15/17 and 5/8 in patients with RAG1 and RAG2 deficiency, respectively.Early onset of life-threatening infections and lymphopenia were common findings in SCID patients.Almost all patients except P1 and P11 [2] had lymphopenia [29].Eczema and diaper dermatitis were also common.Clinical and laboratory characteristics are given in Tables 2 and 3.

OS patients
Five female patients were diagnosed with OS (P26-P30).P26 and P27 were cousins and had novel RAG2 mutations.The median age of clinical manifestations was 2 (1.25-5) mo, and the age at diagnosis was 4.5 (2.5-9.75)mo.All except one OS patient were born to consanguineous parents (P27's parents were from the same village).Dermatitis was a common finding in all OS patients.Diffuse erythroderma, exfoliative dermatitis, and diffuse seborrheic dermatitis were present sometimes with alopecia and nail dystrophy.They had very low B-cell counts.Eosinophilia was present in 3/5.Only one patient P28 had elevated IgE [17].

Survival and outcome
Twenty-eight patients (80%) (SCID; 22, OS; 2, and CID; 4) underwent HSCT.Nineteen had an HLA-matched family donor, five had haploidentical (parent) donors, and four patients had a matched unrelated donor (MUD) (Table 4).Eleven out of 28 patients received pre-transplant conditioning before HSCT, those who did not receive were in the SCID group, and one in the OS group (Table 4).The median (IQR) age at HSCT was 7 (4-13.5)mo, and the success of HSCT was 67.9% (19/28).There was a significant difference in the median (IQR) age at HSCT among the clinical groups (P = 0.002).Median age at HSCT was 6 (3.5-9.9)mo in the SCID group, and it was 90.3 (51.4-115.3)months in the CID group.All except P24 who did not receive pretransplant conditioning are alive and well after HSCT (16/17).Twelve patients (SCID; 11, OS; 1) received immunosuppressive treatments (methylprednisolone and cyclosporine) for GVHD (Table 4).P21 with a previously reported homozygous RAG1 mutation (Y589*; c.1879C > G) [17] underwent HLA-identical HSCT from his mother at 4 mo of age.A liver biopsy for persistent transaminase elevation revealed GVHD.MPZ and cyclosporine (CYC) were given.Skin exfoliation, thickening, excoriation, and pancytopenia suggest bone marrow failure developed despite the treatment.Afterwards, he was diagnosed with chronic GVHD.He is under tacrolimus and mycophenolate mofetil (MMF) treatments for chronic GVHD.P24 had a novel RAG1 mutation and was diagnosed with isolated liver GVHD and treated with CYC, MMF, and etoposide.Liver functions deteriorated and progressive liver failure developed despite plasmapheresis and mesenchymal stem cell transplantation.All other patients with acute GVHD were treated with MPZ and/or CYC (Table 4).Autoimmune cytopenia developed in two SCID patients after HSCT.P8 developed idiopathic thrombocytopenic purpura (ITP) 6 years after HSCT [17], and he was successfully treated with intravenous immunoglobulin (IVIG) therapy.Autoimmune hemolytic anemia developed 6 mo after HSCT in P13 with a novel RAG2 mutation.Unfortunately, despite the treatment [IVIG, pulse steroids, plasmapheresis (three times), CYC, cyclophosphamide, rituximab, and MMF] and supportive care for persistent AIHA, the patient died before the second HSCT planned from another HLA-matched sibling donor.
In total, 16 patients died during the disease course, including nine patients who underwent HSCT (SCID; 6, OS; 1, and CID; 2).Nineteen patients (54.3%) are alive, and well after HSCT.The 10-year-survival analysis is shown in Fig. 3A for distinctive clinical groups, and in Fig. 3B according to the type of RAG deficiency.Survival differed in the groups; it was maximum in the SCID patients (64%) who mostly had an HLA-matched family donor, and minimum in the OS patients (20%) only P29 survived after a successful HSCT with the full-matched family donor.(Table 4).There was no difference between RAG1 and RAG2 deficient SCID patients in terms of HSCT outcomes, autoimmunity, and survival (P > 0.05).

Discussion
Here, we present a large cohort of RAG ½ deficient patients (25 patients with RAG1, 10 with RAG2 deficiency) during a 20-year period from Turkish origin with nine novel mutations.In our study patients were classified as SCID, OS, and CID, and it was also depicted that identical mutations can cause distinct clinical presentations.All novel mutations except one caused SCID phenotype.We believe that the identified novel variants in this study can contribute to the literature, and help to understand the nature of the RAG deficiency.
RAG deficiency was described in various studies from all over the world with different clinical pictures from the first cases until today with raising awareness.RAG mutations were particularly reported from highly consanguineous populations for instance Middle East region [30][31][32]and Turkey [33,34], whereas there were also large case series from the Slavic countries [35], Italy [36] and Latin America [37] in which consanguineous marriages seen relatively less common.Noteworthy, similar mutations have been reported from different ethnic origins requiring more research.
Due to the high rate of consanguinity in our population [38,39], we had a higher rate of homozygous mutations compared to other European nations [36].RAG½ deficiency is the predominant genetic reason for SCID phenotype in Turkey, and the reported frequency among studies varies between 15.4% and 26% [6,40].Furthermore, in the present study, most of the patients had SCID phenotype similar to the studies from the Middle East region [30,32], and in contrast to Slavic [35] and Italian [36] cohorts in which OS was more prevalent.
The RAG½ gene mutations have a broad spectrum of phenotypes, ranging from SCID, OS, and delayed-onset CID/ AS.The RAG deficient patients with SCID and OS generally present with opportunistic infections in early infancy.Diagnosis of delayed-onset CID due to hypomorphic RAG½ deficiency is more challenging due to clinical variation.In some patients, the diagnosis may not be possible [9,41].

Table 2. Continued
A single mutation may result in a variety of clinical manifestations [7,13,16].Patients with the same RAG mutation may have different phenotypes even in the same family [42], possibly due to epigenetic factors including gene modifiers, environmental factors, infections, and iatrogenic factors [43].Furthermore, researchers showed that similar mutations in the N-terminal truncation of the RAG1 protein cause different RAG residual protein activity, which leads to distinct clinical phenotypes [44].
The published studies regarding RAG1 and RAG2 deficiencies indicated that more than 60 RAG1 and RAG2 mutations are located in the core regions of the RAG proteins and they affect DNA binding, catalytic activity, or protein stabilization [45].The core region mutations in our study also comprised the majority of the identified variants.In addition, two patients in our study with OS had a non-core region variant like in the articles of Grazzini et al. [46] and Matthews et al. [47].These OS patients had severe ichthyosis-like skin lesions and alopecia, and unfortunately deceased before HSCT.
We observed an overall distribution of the causative variants including different types of monoallelic or biallelic variations located in different regions of the RAG1 and RAG2 genes.In addition, we did not detect a founder variant like in the study reported from Slavic countries [35].Although the consanguinity rate is high among our patients, we think that they are coming from different regions of the country.
In our study group, most of the patients had RAG1 mutations in line with the literature [35,36].Interestingly, the majority of the novel mutations were RAG2 mutations presenting with SCID phenotype.P13 and P14 in the SCID group had the same novel homozygous missense mutation in the RAG2 protein core region, which is proposed to disturb the interaction with RAG1 and recombination signal sequence (RSS) and leads to RAG2 c.2152G > T mutation causing p.Trp317Cys.The tryptophan at this position is essential for interaction with RAG1 and cleavage of the DNA and the RSS [48,49].
In the present study, recruiting some of the patients (P1, P33, and P35) to a clinical group according to the ESID criteria was challenging.Other parameters and clinical characteristics were indicative in grouping.The estimated prevalence of RAG½ mutations, leading to partial enzyme activity and a later presentation varies between 1% and 1.9% in adult PID cohorts [50].An important finding of this cohort is that the ratio of hypomorphic defects was shown to be 5/25 (20%) for RAG1 deficiency.
Treatment-resistant severe vasculitis was present in P34 and complicated with digital necrosis [26,27].He had a relatively delayed-onset CID caused by a homozygous RAG1 mutation (c.2095C > T; R699W).Similarly, in our study vasculitis was reported in RAG deficient patients.Henderson et   .Partial RAG deficiency with vasculitis was reported in another study in six patients [53].More than half of our patients (SCID; n = 13, OS; n = 4 and CID; n = 1) had a history of intractable diarrhea, a common symptom in SCID patients.It may present with IBDlike disease, autoimmune enteropathy, duodenitis, or severe noninfectious diarrhea.Detected infective agents are pneumocystis jirovecii, Candida species, and viral infections, such as cytomegalovirus (CMV) and adenovirus [54].
Viral infections are an important cause of morbidity and mortality in the course of RAG deficiency and are challenging for patients.Varicella infections, complicating with subsequent pneumonitis and ITP were reported in RAG-deficient patients [55,56].Another accompanying viral infection is CMV, which may progress to retinitis in PID patients.Early suspicion and effective treatment are crucial to prevent visual morbidity and loss in CMV retinitis [57,58].Two siblings (P31 and P32) diagnosed with CID presented with widespread warts in our cohort.Efficient cellular and cytotoxic immunity provided by T and NK cells is necessary to cope with HPV infections [59].
A wide range of autoantibodies, anti-cytokine antibodies, and neutralizing antibodies against interferon-α and interferon-ω, may develop in RAG-deficient patients following viral infections [56,60].A meta-analysis showed that autoimmunity and inflammatory diseases developed in 67.1% of 134 RAG deficiency.Autoimmune and inflammatory diseases have been reported in delayed-onset CID patients, whereas they were rare in OS and SCID patients [41].Autoimmune cytopenia, granuloma, skin cancer, vasculitis, neuropathy, interstitial lung disease, and myopathy were detected in 76.2% of patients with RAG1, and 23.8% of the patients with RAG2 deficiency [41].
In our study, AIC developed after the HSCT was performed without a conditioning regimen (P8 and P13).Autoimmune cytopenia following HSCT, especially AIHA, was considered a serious post-HSCT complication with a poor prognosis [61].Viral infections usually precede the onset of AIC [41].The ratio of AIC was 2/35 (6%) in RAG½ deficiency in this cohort.
In the present study, 80% of all RAG½ patients who underwent HSCT had a survival rate of 54.3%.The median age at HSCT was 7 (4-13.5)mo, and the HSCT success in the RAG½ deficiency SCID group was 72.7% (16/22), a higher outcome than the general SCID-HSCT outcome (65.7% survival rate over 20 years) in Turkey [6].Severe pneumonia was the leading cause of death in patients after HSCT.All RAGdeficient patients were diagnosed with SCID in a recently published study from Israel and the HSCT success rate was 68% [32].The lack of newborn screening has a negative impact on the survival of our study patients, because, it causes a delay in both the PID diagnosis and timely HSCT.
In conclusion, we evaluated a considerable number of RAGD patients and identified certain novel mutations.A high proportion of patients presented with classical SCID phenotype.Early diagnosis, which will be accomplished after national neonatal screening, could improve clinical outcomes and survival.Patients with the lowest survival ratio, the delayed onset/CID patients, were the patients with the most frequent ratio of autoimmune/inflammatory findings.Thus, patients with autoimmunity and inflammation, including vasculitis, should be referred to immunology clinics and evaluated for delayed onset/CID.Early molecular diagnosis may also help in timely management.Definite and individualized therapeutic interventions which could only be possible after early diagnosis will provide a survival advantage, especially for delayed onset/CID patients until HSCT.

Table 1 .
The analysis of the RAG mutations in our cohort