**1. Introduction**

Inborn errors of immunity (IEI), formerly known as primary immunodeficiency disorders (PIDs), are inherited disorders caused by damaging germline variants in single genes, resulting not only in increased susceptibility to infections but also in allergic, autoimmune, autoinflammatory, nonmalignant lymphoproliferative, and malignant manifestations. According to the most recent report by the International Union of Immunological Societies (IUIS), the identified IEI were classified in 10 tables with subtables segregating groups of disorders into overlapping phenotypes: (1) immunodeficiencies affecting cellular and humoral immunity (combined immunodeficiencies); (2) combined immunodeficiencies with associated or syndromic features; (3) predominantly antibody deficiencies; (4) diseases of immune dysregulation; (5) congenital defects of phagocyte number or function; (6) defects in intrinsic and innate immunity; (7) autoinflammatory diseases; (8) complement deficiencies; (9) bone marrow failure disorders; and (10) phenocopies of IEI. The 55 novel monogenic gene defects positioned in the last IEI update enhanced the total number of IEI to 485 [1,2].

The COVID-19 pandemic had an impact on various fields of medicine. In the context of clinical immunology and IEI, it has uncovered several new IEI [1]. Each time, the appearance of new pathogens is a potential challenge for the general population and also healthcare systems because of the lack of significant pre-existing immune memory. Similarly, in the case of pathogens learned about so far, patients with specific germline genetic variants (causing known and unknown IEI) may be more exposed to severe disease than the general population. Research on the COVID-19 pandemic course led to the detection of genes and mechanisms necessary for anti-SARS-CoV-2 immunity. About 2–3% of cases of severe SARS-CoV2 infection resulted from germline LOF/LOE variants in the

**Citation:** Pieniawska-Smiech, K.; ´ Pasternak, G.; Lewandowicz-Uszy ´nska, A.; Jutel, M. Diagnostic Challenges in Patients with Inborn Errors of Immunity with Different Manifestations of Immune Dysregulation. *J. Clin. Med.* **2022**, *11*, 4220. https://doi.org/10.3390/ jcm11144220

Academic Editors: Rita Consolini and Giorgio Costagliola

Received: 24 June 2022 Accepted: 18 July 2022 Published: 20 July 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

type 1 IFN signaling pathway: *TLR3*, *UNC93B1*, *TICAM1*, *TBK1*, *IRF3*, *IRF7*, *IFNAR1*, and *IFNAR2* [1]. According to Asano et al., X-linked recessive TLR7 deficiency is a highly penetrant genetic etiology of severe COVID-19 among 1.8% of males below the age of 60 years [3].

The defects of the number or the function of immune system elements determine the clinical presentation of an IEI. Family history, as well as personal and clinical data, are considered a core element of patient initial management. Extensive anamnesis and clinical evaluation are the main tools for a suspected diagnosis of IEI [4]. The early diagnosis of IEI can be life-saving but remains challenging due to the low prevalence of these pathologies. This can result in the delay of diagnosis and consequently in a worse prognosis [5].

Disease manifestation appearance (i.e., Nijmegen breakage syndrome (NBS), Shwachman-Diamond syndrome, and DiGeorge syndrome), as well as subject growth during both in utero life and later, may suggest the diagnosis of IEI and provide an important diagnostic clue [6]. Severe and/or recurrent infections, consanguinity, or an unexplained death in one's family are well-known signs of IEI; however, more attention should be paid to signs of immune dysregulation. Immune dysregulation is defined as a breakdown or malfunction of molecular control of immune system processes, and it is used to characterize an array of autoimmune and inflammatory conditions [7]. According to IUIS classification, there are 10 IEI categories based on their underlying molecular defect. One of them is called 'diseases of immune dysregulation'. Moreover, it has been established that other patients with humoral, cellular, or innate immune system deficiencies are also at risk of autoimmune or inflammatory conditions [8]. Currently, signs of immune dysregulation are of great importance in defining IEI, as well as an increased tendency to infection.

The modern diagnostics of IEI include various diagnostic measures, such as a simple blood count with particular attention paid to the total absolute lymphocyte count, the serum immunoglobulin levels, and the complete sequencing of the exome or genome [9]. However, during the clinical evaluation of a patient with suspected or confirmed IEI, we should be aware of the possible problems and finer points that may restrict diagnosis in patients with IEI. The aim of this review is to summarize these diagnostic challenges, in particular, in the context of immune dysregulation in IEI patients.

#### **2. Allergic Disease**

Allergy develops on account of disturbed function of the immune system. The immune system depends on a complex balance of activation, to defend against invasive, foreign pathogens, and control, to differentiate between self and foreign matter. Allergic reactions are exaggerated immune responses against specific allergens [10,11]. The comorbidity of IEI and allergy appears because of the impairment of the immune system, leading to infectious susceptibility; however, it is still able to trigger an allergic response [8]. The mechanisms underlying the relationship between atopy and immunodeficiency are better recognized, thanks to the discovery and characterization of genetic variants, often showing "a new face of old disorders" [8]. Several studies indicated the potential mechanisms leading to such dysregulation, which include the failure of central thymic tolerance, an imbalance between the effector and regulatory T-cell function, a failure in the production of counter regulating interferon-gamma (IFN-γ), disturbed cytokine production, and possible differences in microbial colonization and infection patterns [8,12,13].

Thanks to growing interest in the coexistence of allergy and IEI, the topic has been investigated in a number of studies. However, the results are still inconsistent. For example, in one Iranian study atopic dermatitis (AD) was present in 52% of patients with selective IgA deficiency (sIgAD) [14], while among Brazilian patients with sIgAD, AD was found in 2.3% [8,15]. In the USIDENT study, AD was most commonly reported in patients with a deficiency of the nuclear factor κB (NFkB) essential modulator (62.5%), the Wiskott–Aldrich syndrome (WAS: 41.5%), combined immunodeficiency (CID: 33.3%), selective IgM deficiency (33.3%), and autosomal-dominant hyper-IgE syndrome (AD-HIES; 25%) [8,16]. A cohort study of patients with early onset severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) demonstrated that atopy was present in 56% of the patients, including mild AD in 11.1%. Severe AD was not a common feature [17]. A possible explanation of the diverse results are ethnic and geographical diversity and differences in methodological approaches.

Potential diagnostic difficulties may start even at the beginning in diagnosing IEI. An underlying, sometimes severe immune deficiency can manifest as common allergic symptoms, and IEI may masquerade allergic atopic patients [10]. In clinical practice, there are few warning signs of an underlying IEI among atopic phenotypes, and these include severe atopic disease, usually with a poor response to standard therapies, early-onset of the disease, a positive family history for IEI and/or severe familial atopy, and immunological abnormalities [11].

The standard screening tests for antibody deficiency include the measurement of immunoglobulin, IgG, IgA, and IgM levels in serum and the interpretation according to age-related reference values [18]. The routine measurement of serum IgE is not obligatory in the management of patients with suspected antibody deficiency and a history of recurrent infections. Previously, the level of total IgE was considered as a marker to catch allergic patients, but because it is nonspecific, it cannot confirm the allergy status of a patient [19,20]. Non-immunodeficient patients have variable IgE concentrations associated with atopic disease such as allergic rhinitis (AR), asthma, food allergy (FA), and AD, as well as other conditions, including parasitic disease [21]. However, in the context of PID, IgE measurement plays a role, especially in patients with concomitant eczema. Elevated IgE is common in a number of IEI, such as HIES, WAS, Netherton syndrome, immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, and Omenn syndrome [22]. One phenotype of complete DiGeorge syndrome, which is known as atypical complete DiGeorge syndrome, has oligoclonal T cell expansion with elevated IgE levels with concomitant generalized rash and lymphadenopathy [23]. The pathophysiological role of increased IgE in these disorders was not clearly characterized; however, there are few hypotheses [13]. Increased IgE production is associated not only with well-defined genetic syndromes but also with humoral, cellular, innate, and combined immunodeficiency disorders [5]. However, a high IgE (>180 IU/mL) is very rare in common variable immunodeficiency (CVID) (0.3% of patients) [21].

There are particular PIDs associated with atopy, especially eczema and elevated serum IgE, which can be confirmed by genetic tests and the identification of specific mutations. Mutations in the *WAS* gene on the X chromosome, which encodes the WAS protein (WASP), are a cause of Wiskott–Aldrich syndrome, characterized by recurrent infections, thrombocytopenia with small platelets, and eczema [8]. The mechanism for atopy in WAS is not fully described; however, impairment of regulatory T-cell (Treg) function is a possible contributor [8,24–26]. In total, 33% of patients with WAS and 20% of patients with X-linked thrombocytopenia (XLT) had positive food allergen-specific IgE (sIgE), in a study conducted by Lexmond et al. [8,27]. Food sensitization was generally detected with greater sensitivity using sIgE testing than by skin prick testing (SPT).

A dominant-negative heterozygous mutation in signal transduction and the activator of transcription 3 (STAT3) leads to autosomal-dominant hyper-IgE syndrome (AD-HIES), previously known as Job syndrome, with characteristic features such as chronic eczema, recurrent staphylococcal skin infections, pneumonia, increased serum IgE, and eosinophilia [10]. Skin findings distinguishing it from AD include a distinctive thickened texture of the facial skin, retroauricular fissures, and severe folliculitis of the axillae and groin [5]. Serum IgE levels are often >2000 IU/mL, and eosinophilia levels are often >700 cells/mL (eosinophilia does not correlate with the elevation in IgE), but patients usually do not suffer from symptomatic allergic disease such as AR, FA, or anaphylaxis [10,28]. Disturbances in the inflammatory process, and associated immune regulatory defects, are present. In clinical practice, a lower limit of 2000 IU/mL is often considered as a cutoff for AD-HIES. However, patients with HIES with lower IgE levels and STAT3 pathogenic variants have been reported [29]. Moreover, the serum IgE level does not correlate with

the severity and activity of the disease, and paradoxically patients with STAT3 loss-offunction (LOF) mutations are rather protected from severe allergic reactions. A potential explanation of this protection is disturbed mast cell degranulation, as well as vascular reaction to histamine caused by the STAT3 mutation itself [8,30–32]. SPT results and clinical symptoms of allergy are consistent with the specific IgE (sIgE) results in AD-HIES. Both skin and blood test results are comparable between patients with AD-HIES and healthy controls [32]. Defective neutrophil chemotaxis has been described among AD-HIES patients, and variable specific antibody production is seen [5,33]. Patients may require immunoglobulin replacement.

At the end of 20th century, the National Institutes of Health HIES scoring system was originally presented where a score of 30 has a sensitivity of 87.5 percent and a specificity of 80.6 percent [34]. It is noteworthy that some patients (e.g., some young children), may not meet the scoring criteria. Thereupon, in cases of positive family history of HIES and some distinctive features, according to experts, molecular screening should still be performed even if the score is below 30. Other diagnostic guidelines takes into account five cardinal clinical features (recurrent pneumonia, newborn rash, pathologic bone fractures, characteristic facies, and high palate) with total IgE level and Th17 cell count [35]. Molecular genetic testing is crucial to establish the diagnosis of the AD-HIES.

Autosomal-recessive-HIES (AR-HIES) is characterized by highly elevated serum levels of IgE, eczema, recurrent staphylococcal abscesses, and hypereosinophilia. In contrast to AD-HIES, where patients are usually free from allergic manifestations, 50% to 70% of patients with AR-HIES suffer from severe allergies, i.e., eczema, anaphylaxis to food, and environmental allergies, and 30% have asthma [10,32]. Pulmonary disease is usually asthma-related as compared with AD-HIES, with pneumatocele and lung damage due to prior infections [10].

Some patients with DOCK8 or TYK2 deficiency were previously classified as AR-HIES with harmful allergic symptoms [36]. Now, we better recognize the differences in the clinical features. DOCK8 deficiency is a combined immunodeficiency characterized by allergic inflammation, severe atopy, high IgE, susceptibility towards cutaneous viral infections, and malignancy [37]. TYK2 deficiency is also a combined immunodeficiency with recurrent skin viral infections, while eczema and elevated IgE are variably found. A study conducted by Boos et al. revealed that total serum IgE levels similarly increased in STAT3-HIES, DOCK8 deficiency, and AD patients. The ratio of aeroallergen-specific IgE to total IgE was the highest in AD, whereas patients with DOCK8 deficiency showed the highest specific serum IgE against food allergens. Th2-cell numbers were significantly increased in DOCK8 deficiency and AD patients compared to STAT3-HIES patients and controls. The study showed that hyper-IgE syndromes and atopic dermatitis patients showed a different sensitization pattern of serum IgE corresponding to the allergic disease manifestations and Th-cell subset data, suggesting a key role of DOCK8 in the development of FA [32]. Moreover, according to Wilkie et al., defective Treg function may contribute to the increased skin inflammation and the eczema in DOCK8 deficient patients [38]. IEI with elevated IgE are summarized in Table 1.


**Table 1.** Inborn errors of immunity with elevated IgE.


**Table 1.** *Cont.*



On the other hand, low levels of IgE interest immunologists. Selective IgE deficiency (defined as a significant decrease in the levels of IgE (<2.5 IU/mL) in patients whose other immunoglobulin levels, including IgG, IgG subclasses, and IgA levels, are normal) has not been included in international classification systems for IEI [1]. Low serum levels of IgE can be associated with some well-defined IEI: common variable immunodeficiency (CVID), IgG subclass deficiencies, sIgAD, ataxia-telangiectasia (A-T), and agammaglobulinemia [39–41]. According to studies, an undetectable serum IgE (<2 IU/mL) occurs in only 3.3% of the general population [21]. In contrast, Lawrence et al. found that an undetectable IgE occurs in 75.6% of patients with CVID [21]. Another finding was a significant correlation between serum IgE with serum IgG, suggesting that lower IgE occurs in patients with more severe hypogammaglobulinemia. Moreover, false-negative results may appear using traditional methods of sIgE measurement, and allergen sIgE was not detectable in 96.5% of patients with CVID. Many patients with CVID report symptoms of rhinitis, wheeze, or adverse reactions to antibiotics, but it is difficult to detect allergic sensitization among them, especially using SPT or serum sIgE [41–43]. In these cases, sensitization should be confirmed using different methods, for example, an oral provocation challenge, and bronchial provocation tests with allergens [41]. The interpretation of food-specific IgE values and their usefulness in predicting symptomatic food allergies in the context of IEI patients is a potential field for further studies.

While diagnostics among PID patients during Ig replacement therapy (IRT) are often challenging, in the context of allergy, studies suggest that current Ig products are not a significant source of IgE [21].
