Keywords

3.1 Introduction

The phenocopies of primary immunodeficiency diseases have been characterized during the last decades and manifest as a clinical phenocopy to patients with genomic mutations affecting the same biological pathway. In this chapter we will provide an updated description of the clinical manifestations, diagnosis, and treatment.

3.2 Phenocopies of PID Caused by Somatic Mutations

The phenocopies of primary immunodeficiency diseases manifest as a clinical phenocopy to patients with genomic mutations affecting the same biological pathway (Fig. 3.1) [1].

Fig. 3.1
figure 1

Classification of phenocopies of primary immunodeficiency diseases. Phenocopies of PID are a group of diseases caused by somatic mutations or autoantibodies against various cytokines. Clinical manifestations can mimic those of other PIDs. The diseases belonging to each group are shown in the figure

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The traditional definition of a mosaic is any pattern or image made from multiple pieces; its individual elements can be recognized just by close inspection. In biological organisms, mosaicism denotes an individual with more than one genetically distinct cell population [2]. It might be imperceptible unless closely analyzed. If it takes place during embryonic development, germline and somatic cells will be affected. Otherwise, only somatic cells will be affected. Mosaicism can be caused by DNA mutations, epigenetic factors, and chromosomal abnormalities [3].

Somatic variants require high-throughput sequencing techniques to be detected. During data analysis specific algorithms are fundamental, as these mutations have very low allele frequencies in the population. High deep reads are recommended in order to increase the accuracy [4]. Single-cell sequencing has enabled us to detect somatic mutations as heterozygous variants that occur in a subset of cells [5].

Below, we describe several conditions characterized by somatic mutations and mimicking primary immunodeficiency diseases (PID) (Table 3.1).

Table 3.1 Phenocopies of PID. Immunophenotype and clinical characteristics associated with their similar genetic counterpart
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3.2.1 Autoimmune Lymphoproliferative Syndrome (ALPS) Caused by Somatic Mutation in TNFRSF6 Gene

Autoimmune lymphoproliferative syndrome is a condition of impaired lymphocyte homeostasis, resulting from mutations in genes involved in the Fas pathway. Clinical manifestations include lymphadenopathy, splenomegaly and autoimmune cytopenias. Patients have a predisposition to malignancy, especially lymphomas [6, 7].

FAS (CD95/Apo1) is a cell receptor that belongs to the tumor necrosis factor receptor (TNFR) superfamily. It is codified by the gene TNFRSF6. Upon binding to its ligand (Fas ligand), Fas starts a series of events leading to apoptosis to maintain lymphocyte homeostasis [8]. Its role was initially identified in mouse models with a germline mutation in TNFRSF6 that manifest with autoimmunity [9]. In humans, patients develop a syndrome known as autoimmune lymphoproliferative syndrome (ALPS) [10,11,12].

If the mutation is clear, ALPS can be categorized as ALPS type Ia (FAS/TNFRSF6), ALPS type Ib (FAS ligand), and ALPS type II (caspase 8 or 10 genes). ALPS type III is caused by somatic mutations, and it is the second most common type of ALPS. Somatic mutations have been described in patients without germline mutations but a clinical phenotype similar to other types of ALPS: lymphadenopathy, splenomegaly, hepatomegaly, autoimmunity, elevated DNT cells, increased serum FAS ligand, and elevated levels of IL-10 and vitamin B12 [13,14,15,16]. The number of reported cases due to somatic mutations has increased over the last years.

Patients with ALPS type III have a later onset, and the symptoms remain mild for a long period and hence lead to diagnostic delay [17]. The clinical phenotype can suggest a somatic mutation, but this is not enough to make the diagnosis. In vitro studies in cells of these patients have shown Fas-mediated apoptosis, with a higher degree compared to patients with ALPS type Ia [14].

Diagnosis is challenging; all patients with ALPS phenotype, elevated serum biomarkers, and no germline mutation should be evaluated for somatic mutations. The identification of somatic mutations is established by sequencing FAS on double-negative T cells (DNT) [13]. DNT cells seem to be originated from activated peripheral single-positive T cells that received a death-inducing signal but cannot go to apoptosis as they harbor a Fas defect [18].

Treatment is similar to ALPS patients with germline mutations. It focuses on treatment of disease manifestations such as lymphoproliferation and autoimmune cytopenias [19]. Patients require steroid therapy and more than 50% immunosuppressive drugs to control autoimmunity. Malignancy can be treated with conventional protocols. As secondary options, intravenous gammaglobulin, plasmapheresis, and bortezomib should be considered [20]. Hematopoietic stem cell transplantation (HSCT) has been used for refractory patients [21].

3.2.2 RALD: Ras-Associated Autoimmune Leukoproliferative Disease (ALPS like)

Ras-associated autoimmune leukoproliferative disorder (RALD) is characterized by autoimmune manifestations, persistent monocytosis, leukocytosis, and non-malignant lymphoproliferation. Clinical and laboratory features overlap with those of juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML) [22]. The somatic mutations affect genes of the Ras family, KRAS, NRAS, and RAS, involved in myeloid and lymphoid lineages [23]. Mutations found in RALD patients are also reported in around 25% of JMML patients, suggesting a shared molecular etiology [24]. The presence of autoimmunity supports RALD diagnosis, but these patients can have malignant cell transformation and evolve to JMML [25].

RAS (named for their role in forming rat sarcomas) encodes for GTPases important in cell division, cell differentiation, and apoptosis. Opposite to ALPS, DNT cells or serum vitamin B12 levels are not always increased, and there is no defect in Fas-mediated apoptosis. A key feature of RALD is persistent absolute or relative monocytosis [23, 26]. The autoimmune manifestations can mimic lupus with low complement levels and elevated autoantibodies (dsDNA) [27]. Patients with mutations in NRAS may have DNT cells elevation [28]. Restricted clonal expansion of TCR and BCR in one patient has been reported; this might explain the reduce lymphocyte repertoire and immunodeficient state in this disease [29].

There are some reported cases with cutaneous involvement known as RALD cutis. Patients present with panniculitis-like erythematous plaques and sweet syndrome. Usually, they have a benign course [30, 31].

Management is based on corticosteroid therapy and other immunomodulatory agents for the autoimmunity. Rituximab has been published as an effective option in patients with refractory cytopenias [32].

3.2.3 Cryopyrinopathies

NLRP3 auto-inflammatory disorders (NLRP3-AIDs) were previously known as cryopyrin-associated periodic syndromes (CAPSs), including overlapping entities with increasing severity: familial cold auto-inflammatory syndrome (FCAS); Muckle-Wells syndrome (MWS); chronic infantile neurological, cutaneous, and articular syndrome (CINCA); and neonatal-onset multisystem inflammatory disease (NOMID) [33].

NLRP3-AIDs are autosomal dominant disorders caused by germline mutations in NLRP3. The gene encodes for cryopyrin, which leads to hyperactivation of IL-1β [33]. Somatic mutations have been described. Patients present a late onset of the disease and milder symptoms [34,35,36,37,38,39,40]. Clinical manifestations include fever, joint involvement, and skin rash. Laboratory workup reveals neutrophilic leukocytosis, elevated C-reactive protein, and erythrocyte sedimentation rate.

A prompt molecular diagnosis is critical; it requires high-deep next-generation sequencing techniques and specific pipelines [41].

Treatment targets IL-1β, and anti-IL1 (anakinra, rilonacept, and canakinumab) are generally effective [42].

A somatic mutation in NLRC4, the caspase recruitment domain-containing 4 gene, was found in a Japanese male child with auto-inflammatory symptoms compatible with neonatal-onset multisystem inflammatory disease. The patient had complete response to anakinra [43].

3.2.4 Hypereosinophilic Syndrome Due to Somatic Mutations in STAT5b Gene (STAT5b Gain-of-Function Mutation)

Somatic mutations in STAT5b have been described in hematologic malignancies [44,45,46]. Recently, a somatic mutation in STAT5b was found in two patients with eosinophilia, atopic dermatitis, and urticarial rash. The first one, a 3-year-old girl, presented autoimmunity manifestations (alopecia totalis). She had history of one event of pneumonia and measles-like illness 10 days after MMR vaccination. The other patient had a severe clinical presentation with recurrent events of bronchiolitis, worsening eosinophilia, failure to thrive, and delayed speech. Gut biopsy revealed eosinophilic infiltrates. She underwent umbilical cord stem cell transplant but died later. Functional tests in CD3-CD4+ T cells showed increase STAT5B responsiveness [47]. Management was based on steroid therapy.

3.3 Phenocopies of PIDs Caused by Autoantibodies against Various Cytokines

Autoantibodies can be found in healthy individuals; they are mainly IgM and have moderate affinity for self-antigens contributing to the homeostasis of the immune system [48]. In contrast, high-affinity and high-titer autoantibodies reflect the loss of balance in effector functions of the immune system. Clinical presentation is correlated with the affected cytokine pathway. These diseases present as a clinical phenocopy of patients with germline mutations in the same associated pathway [49]. Here, we review current knowledge focusing on diseases with increased susceptibility to infections.

3.3.1 Autoantibodies against IL-17 and/or IL-22

Chronic mucocutaneous candidiasis (CMC) is a disorder characterized by recurrent or persistent candida infections involving the skin, nails, and mucous membrane [50]. When the disease is associated with autoimmune hypoparathyroidism and primary adrenocortical insufficiency is named APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy). APECED is caused by a loss-of-function mutation in AIRE, an essential gene in central immune tolerance [51]. These conditions occur in association with impaired IL-17 and IL-22 immunity [50, 52].

Several genetic mutations lead to impair production of IL-17 and IL-22: IL17F, IL17RA, IL-17RC, and TRAF3IP2/ACT1 [53,54,55]. Autoantibodies against IL-17F, IL-17A, and IL-22 have been found in patients with CMC [56]. In one study, high titers of autoantibodies against IL-17A, IL-17F, and/or IL-22 were found in 33 APECED patients; from those 29/33 developed CMC [57]. Along with these findings, autoantibodies against IL-17A (41%), IL-17F (75%), and/ or IL-22 (91%) were detected in more than 150 APECED patients, mainly among the CMC group. Remarkably, in this study, autoantibodies were also found in patients with thymoma who later developed CMC [51].

Diagnosis is based on the detection of autoantibodies by techniques such as western blotting and enzyme-linked immunosorbent assay (ELISA). Management includes antifungal therapy and treatment of associated endocrine and infectious manifestations. Members of the azole family are usually effective for the treatment of CMC [58, 59].

3.3.2 Autoantibodies Against IL12p70

IL-12p70 is a heterodimeric molecule consisting of IL-12p35 and IL-12p40 subunits; it signals through a heterodimeric receptor complex of IL-12Rβ1 and IL-12Rβ2 [60]. The signaling pathway IL-12p40-STAT4-IFNγ is involved in protection against intracellular pathogens, such as mycobacterium [61].

There is just one case of anti-IL-12p70 autoantibodies detected in one Cambodian patient. She presented with severe recurrent Burkholderia gladioli lymphadenitis and was demonstrated to have isolated neutralizing anti-IL-12p70 autoantibodies as the only immune defect [62]. Interestingly, patients with myasthenia gravis or thymoma have high titers of autoantibodies against IL12p40, but they do not develop infections [63].

3.3.3 Autoantibodies to Interferon-α (IFN-α)

Type I IFNs include IFN-α, IFN-β, and IFN-ω. IFN-α is involved in the transcription of type 1 interferon genes and acts via phosphorylation of STAT1/2 [64, 65]. Autoantibodies to type I IFNs have been detected in healthy donors [66], autoimmune diseases [67,68,69], malignancy [70], APECED [71], and thymoma [72, 73]. Infections have been reported in a patient with dermatomal varicella zoster reactivation. IFN-α was given as treatment with good response [74].

3.3.4 Autoantibodies to IFN-ƴ and Susceptibility to Intracellular Pathogens

Interferon-gamma (IFN-ƴ) is produced by type 1 helper T lymphocytes and NK cells and is crucial for immunity against intracellular pathogens. The IFN-ƴ receptor is composed of two subunits IFNƴR1 and IFNƴR2, which combine in duplicate, to form a tetramer, and bind IFN-ƴ. IFN-ƴ downstream signaling is mainly through the JAK-STAT1 pathway [52, 75]. Autoantibodies against IFN-ƴ may impair the production of IL-12. Mendelian susceptibility to mycobacterial disease (MSMD) is a condition that predisposes to infections by low pathogenicity mycobacteria such as nontuberculous mycobacteria (NTM) or bacille Calmette-Guérin (BCG) [76]. Patients are also susceptible to Salmonella, Candida, and Mycobacterium tuberculosis. Genetic defects along these pathways confer similar infection susceptibility [77].

The first cases reporting an association between anti-IFN-ƴ autoantibodies with severe atypical NMT infection were published in 2004 [78, 79]. Supporting these data, several cases are published, including one study in which 85 patients were enrolled [78,79,80,81,82,83,84,85,86,87,88]. Other opportunistic infections have been reported, including Salmonella, Burkholderia, Penicillium, Histoplasma, Cryptococcus, and viruses, in particular varicella zoster virus (VZV) [89]; these infections resemble those observed in patients with germline mutations in the IFN-ƴ-IL12 axis.

A high prevalence rate among patients from South East Asia was observed; this was later explained by the discovery of a strong HLA association: HLA-DQB1*05:01/05:02 and DRB1*15:02/16:02. In addition, a major epitope, P12-131, located at the C-terminus of IFN-ƴ was identified [90, 91].

Patients with opportunistic infections and neutrophilic dermatosis (Sweet syndrome) were reported to have anti-IFN-ƴ autoantibodies [92]. Lymph nodes are the main site of involvement [93], and 80% of patients have skin manifestations such as reactive dermatoses, erythema nodosum, pustular psoriasis, and exanthematous pustulosis [89, 94].

Laboratory workup reveals features of chronic inflammation including anemia, leukocytosis, elevated erythrocyte sedimentation rate, polyclonal hypergammaglobulinemia, and elevated C-reactive protein (CRP) and/or β2-microglobulin. Other immunological parameters are normal [89]. Undetectable levels or low levels of IFN-ƴ suggest the presence of autoantibodies. Autoantibodies can be measured using particle-based technology or ELISA [95, 96]. For screening, QuantiFERON-TB Gold In-Tube (QFT-GIT) test can be useful [97].

Management is based on antimicrobial therapy. NTM are usually refractory to first-line therapy and often require second-line drugs for months to years. If the response is poor, immunomodulatory agents can help to decrease autoantibody production. Rituximab has been used in four cases; all patients had a decrease in anti-IFN-γ autoantibody levels. Use of rituximab was reported in a series of four cases, all of which responded clinically, with commensurate decrease in neutralizing capacity [98]. Plasmapheresis and cyclophosphamide were used in one patient [99].

3.3.5 Autoantibodies Against Granulocyte Macrophage Colony Stimulation Factor (GM-CSF)

Granulocyte macrophage colony stimulation factor (GM-CSF) is a growth factor which promotes the immune activation, proliferation, and differentiation of neutrophils, dendritic cells, erythrocyte progenitors, macrophages, and megakaryocytes [100]. In the lung, it is essential for function and differentiation of alveolar macrophages. GM-CSF induces phosphorylation of STAT5, nuclear translocation, and induction of transcription factor PU.1. Together, GM-CSF and PU.1 are essential for surfactant catabolism in the pulmonary alveoli [101,102,103,104].

High titers of neutralizing autoantibodies against GM-CSF are associated with pulmonary alveolar proteinosis (PAP) [105]. PAP is a disease linked to congenital or acquired defects in the GM-CSF signaling pathway, causing the impairment of GM-CSF-dependent catabolism of surfactant and leading to accumulation in pulmonary alveoli [106]. PAP is classified in different types according to the underlying pathogenesis: primary PAP characterized by the disruption of GM-CSF signaling which can be autoimmune [107] or hereditary (mutations in CSF2RA or CSF2RB) [108, 109], secondary PAP in patients on immunosuppressive therapy or malignancies [110], and congenital PAP caused by mutations in genes involved in surfactant production [86,87,88]. Histopathological findings are alveolar filling with acellular periodic acid-Schiff (PAS)-positive proteinaceous material [111].

Autoimmune PAP is the most common, representing approximately 90% of cases [112]. Autoimmune PAP can cause respiratory failure, and it presents between 20 and 50 years of age. The presentation is heterogeneous; it can range from asymptomatic to progressive respiratory failure. Autoantibodies can be detected in the bronchoalveolar lavage (BAL) fluid [113]. It has been suggested its levels may correlate with disease severity and predict the need for additional treatment.

Patients with autoimmune PAP can present defects in neutrophil functions, manifesting as infections by Nocardia [114, 115], nontuberculous mycobacteria (NMT) [116], Histoplasma [117], and Cryptococcus [118]. Pulmonary and extrapulmonary infections do not always develop in the same patient. To date, it remains unknown why some patients have just PAP and others just infections.

Useful tools for the diagnostic are pulmonary function tests, which may reveal a restrictive pattern [119]; high-resolution computed tomography (HRCT) of the lungs, which could show a “crazy paving” pattern [120]; and levels of autoantibodies in BAL lavage [121].

The first-line treatment in PAP are whole-lung lavage to remove the proteinaceous material contained in the alveoli and long-term antimicrobial agents for patients with infections [122]. Inhaled and subcutaneous GM-CSF were effective in some studies [123,124,125]. Rituximab has been used in a small number of patients [126, 127].

3.3.6 Antibodies to Interleukin-6

IL-6 is a cytokine involved in the acute-phase response and in chronic inflammation. It is produced by B and T lymphocytes, macrophages, endothelial cells, hepatocytes, and synovial cells. It regulates the acute phase response in the liver with induction of serum C-reactive protein (CPR) and elevated erythrocyte sedimentation rate [128,129,130].

Autoantibodies to IL-6 have been found in healthy controls [131, 132] and in four patients associated with severe bacterial infections. The first patient was a 4-year-old boy with a history of recurrent staphylococcal cellulitis and abscesses [133]. The second case was detected in a 20-month-old female with severe septic shock [134]. The third was a 67-year-old man with fatal thoracic empyema by Escherichia coli and Streptococcus intermedius, and the fourth was a 56-year-old woman with multiple abscesses by Staphylococcus aureus [135]. Management included supportive care and antibiotic treatment.

All patients had undetectable levels of CRP despite severity of infections, suggesting impaired IL-6 activity. Functional assays with plasma of patients showed block of activity of IL-6 in vitro. However, IL-6 production from peripheral blood monocytes was normal. Hence, patients with autoantibodies against IL-6 have increased susceptibility to staphylococcal infections; a hint toward the diagnosis is low levels of CRP, despite severity of infection.

3.3.7 Autoantibodies in Good Syndrome

Good syndrome is defined as the triad of thymoma, immunodeficiency, and hypogammaglobulinemia [136]. Clinical manifestations are increased susceptibility to bacterial infections with encapsulated organisms and opportunistic viral and fungal infections. Patients have combined B and T cell immunodeficiency [137, 138]. Anti-cytokine autoantibodies have been identified in these patients and are a potential cause of immunodeficiency [139, 140]. This disorder should be treated by resection of the thymoma and immunoglobulin replacement to maintain adequate trough IgG values. Anti-cytokine autoantibodies have been also associated with infection in patients with thymoma [63]. These need to be further studied.