The chromosome 22q11.2 deletion syndrome (22q11.2 DS), also known as DiGeorge syndrome (DGS) or velocardiofacial syndrome (VCFS), is a genetic condition resulting from the impaired development of structures originating from the third and fourth pharyngeal pouches in the germinal stage. The clinical features of the syndrome include hypoparathyroidism and hypocalcemia, thymic hypoplasia, conotruncal heart defects, facial dysmorphism, and palatoschisis. The complex phenotype of children affected with 22q11.2 DS may show considerable intersubject variability, and the expanded clinical manifestations comprise craniofacial, neurological, cognitive, behavioral, ocular, speech and hearing, musculoskeletal, and internal organs, as well as airway, gastrointestinal or renal abnormalities [1]. The phenotypes may vary considerably among patients with immunodeficiency and immune dysregulation, including autoimmunity, allergy, and lymphoproliferative sequelae.

The incidence of 22q11.2 DS has been estimated to range from 1:3000 [2] to 1:4000 live births [1], placing it among frequent syndromic diseases. However, the rate of its clinical suspicion is still challenging, and the process of establishing the definitive diagnosis is an odyssey [3] due to the remarkable heterogeneity of clinical phenotypic expressions and overlapping manifestations with other categories of syndromic disorders [4,5,6]. The disease entities, such as, but not limited to, coloboma-heart defects-choanal atresia-retardation of growth and development-ear anomalies (CHARGE) syndrome [7,8], cardio-facio-cutaneous (CFC) syndrome [9,10], and Takenouchi-Kosaki syndrome (TKS) [11,12], require special pediatrician awareness and multidisciplinary care, as their distinctive phenotypes are associated with immunodeficiencies. Furthermore, a common denominator of 22q11.2 DS and other syndromic disorders is an increased susceptibility to recurrent infections, which may not result only from inborn errors of immunity; a multiplicity of developmental anatomical malformations and organ dysfunctions are also important contributing factors. These extra-immune phenotypes in 22q11.2 DS, with neurological, psychomotor, hormonal, circulatory, and respiratory pathophysiological mechanisms, are substantially underpinning the definitive clinical manifestations.

Immunodeficiency is a key feature of 22q11.2 DS and is secondary to thymic aplasia or hypoplasia with subsequent impaired thymocyte development. The third and fourth pharyngeal pouches are a common embryonic precursor for the thymus, parathyroid glands, and conotruncal regions of the heart. In 22q11.2 DS, maldevelopment of these organs is due to impaired migration of the neural crest cell into pouch ectoderm [85]. In the setting of abnormal thymic migration, but with the preservation of residual microscopic nests of thymic epithelial cells, mild to moderate reductions in T cell numbers accompanied by only a mild deficit in T cell function occur in most affected children. However, even in these patients, the T-cell thymic output of recent thymic emigrants, assessed by T-cell receptor excision circles (TRECs) analysis, is very low and decreases with age [86,87]. Full thymic aplasia appears occasionally, in approximately 1% of cases with 22q11.2 DS [86,88]. Interestingly, beyond the TBX1 hemizygosity, other genes, such as CRKL in the affected 22q11.2 region, may also have a gene dosing, modifying effect on the phenotypic expression of the syndrome. CRKL is expressed in neural crest-derived tissues and involves thymic development. The effect of compound heterozygosity for TBX1 and CRKL deletion on clinical features and thymus development is additive [89].

Consequently, a wide spectrum of T-cell alterations is seen in 22q11.2 DS, ranging from near normal to near completely immunodeficient. Mild T cell immunodeficiency may be found in children with apparently hypoplastic thymus because ectopic retropharyngeal thymic tissue may be preserved [90,91]. Furthermore, dynamic changes in immunodeficiency are observable over time, and the direct effect of thymic hypoplasia on T cell counts is most apparent in early infancy [86,87]. They tend to normalize by adulthood in most patients, due to the increased secretion of interleukin (IL)-7 that stimulates the thymic output and peripheral proliferation of T cells [92]. The most common deficits include low total CD3+ T cell percentage and absolute count, as well as low numbers of naive CD4+ T helper and CD8+ T cytotoxic/suppressor cells. The naive T cell compartment shows a progressive decline with patients’ age, leading to a predominantly memory phenotype of T cells at the periphery [93].

Referring to anatomical maldevelopment and dysfunction in generating T cells, the humoral immunodeficiency and B cell abnormalities in children with 22q11.2 DS are secondary to T cell deficits [94]. Low immunoglobulin production, most frequently affecting IgM and occasionally IgG and rendering the need for immunoglobulin replacement therapy, as well as the defective immune response to polysaccharide antigens, have been reported in children with the syndrome [95]. Among the B cell subsets, low switched memory B cells expanding by adulthood, accompanied by decreased somatic hypermutation despite increased follicular T helper cells, have been shown, reflecting a dysregulated B cell compartment and compromised T cell help [96,97,98]. Recurrent respiratory infections, such as adenotonsillitis, otitis media, bronchitis, pneumonia, as well as sepsis [99,100], also occur.

Immune dysregulation in 22q11.2 DS has been foremostly ascribed to T-cell lymphopenia and deficiency in CD3+CD4+CD25++, FOXP3+ regulatory T cells, which play a crucial role in maintaining immune homeostasis and self-tolerance. Reduced thymic output related to an increased naive T cell subset, and subsequent T regulatory cell activation, control the expansion of the T cell compartment. A reduced number of regulatory T cells, with their activated phenotype and loss of suppressive capacity in children with 22q11.2 DS, are key features of deregulated T cell homeostasis [101]. Beyond the T cell compartment, immunophenotype anomalies also encompass peculiar B cell developmental disorders with increased naive B cells and deficit in switched memory B cells [102] which are biomarkers of immune dysregulation in 22q11.2 DS. It has been postulated that the individual patient’s immunophenotype may be influenced by genetic modifiers outside the microdeletion locus which regulatethe expression of TBX1. Rare DNA variants in transcriptional regulators involved in retinoic acid signaling, NCOR2 and EP300, were found to be associated with parameters of the immune functions, such as immunoglobulin levels, lymphocyte response to antigens and mitogens, and flow cytometric lymphocyte compartment. Retinoic acid plays an important role in maintaining immune homeostasis by enhancing the differentiation of regulatory T cells, modulating epithelial and mucosal immune responses, and regulating proinflammatory cytokine activity. Hence, genetic modifiers contributing to the individual’s genetic background and modulating variable penetrance may influence the immune response in 22q11.2 DS [103,104].

Autoimmune disorders have been described in as many as 23% of pediatric patients with the syndrome [105], manifesting as autoimmune thyroid disease, juvenile idiopathic arthritis, autoimmune cytopenia (thrombocytopenia, hemolytic anemia, neutropenia), celiac disease, psoriasis, vitiligo, autoimmune hepatitis, and inflammatory bowel disease [100,105,106,107]. It has also been hypothesized that psychotic disorders, developmental regression, and cognitive impairment in children with 22q11.2 DS may have a causal relationship with autoimmune encephalitis [108].

The peripheral homeostatic expansion of T cells driven by low thymic output and T cell lymphopenia may contribute to Th2-skewed lymphocyte phenotype and atopic manifestations, such as eczema and asthma [109]. In these children with 22q.11.2 DS, the overall frequency of atopic diseases has been estimated to reach 70% and the frequency of asthma to as many as 50% [110]. Coexisting gastroesophageal reflux and sinopulmonary infections may have an impact on its clinical course [111].

The thymus dysfunction and immunophenotypic abnormalities within the T cell compartments in 22q11.2 DS make affected children susceptible to cancerogenic viruses, such as Epstein-Barr virus (EBV) and human papilloma virus (HPV), that might be linked to an increased risk of malignant transformation. It has also been postulated that chronic immune activation of peripherally expanded T cell population in dysfunctional cellular immunity may underpin the predisposition to developing lymphoproliferative disorders and lymphomagenesis [112], as T cell and B cell lymphomas [113,114,115], as well as acute lymphoblastic leukemia [116], have been reported in children with 22q11.2 DS. The spectrum of malignancies in affected pediatric patients also includes solid tumors—namely Wilms tumor—hepatoblastoma, neuroblastoma, thyroid carcinoma [116], pineoblastoma [112], and xanthoastrocytoma [117].

The chromosomal 22q11.2 DS is a clinically highly variable microdeletion syndrome with differently expressed phenotypes, with wide interfamilial and intrafamilial variability in patients sharing the same genetic underpinnings [118]. This is due to both the remarkable complexity of the 22q11.2 region with LCR blocks and the high susceptibility of this region to meiotic errors, as well as the epigenomic and environmental factors influencing the phenotypic variability [119]. Based on functional genomic assessments, it has also been hypothesized that theories on single-gene haploinsufficiency in 22q11.2 DS cannot be supported. In the setting of diminished 22q11.2 gene dosage, shared molecular functions, convergence on cellular processes, and related consequences on the genetic level point to the matrix or multigenic interactions that translate into the multiplicity of phenotypes [120]. All these genetic factors, together with age-related developing symptomatology, contribute to diagnostic challenges in 22q11.2 DS pediatric patients on the diverse individual, family, social, and population levels. The summary of modifying variants influencing the TBX1 penetrance and related phenotypic expression is shown in Table 1 [17,18,19,52,53,103,104,118,120,121,122,123,124].

The diagnosis of 22q11.2 DS has been traditionally based on the recognition of clinical features and cytogenetic testing using the fluorescence in situ hybridization (FISH) technique. FISH is perceived as the golden standard genetic testing method to confirm the diagnosis of microdeletion syndromes. However, poor clinical accuracy, the low confirmatory rate in the screening of suspected microdeletion syndromes, and failure to detect other than the targeted microdeletion are the major drawbacks of this method. An important limitation of this method is a failure to identify atypical and nested deletions because it can recognize deletions in the proximal part of the critical region, including the typical LCR22A-D deletion. FISH is admittedly inexpensive, yet still a highly labor-intensive and time-consuming procedure [125]. Importantly, due to a marked clinical variability from minimal to full manifestation in patients with 22q11.2 DS, precision in defining clinical criteria is important for referring patients to undergo FISH analysis. Since FISH alone cannot provide a reliable diagnosis of 22q11.2 DS, other diagnostic methods have been developed, such as comparative genomic hybridization (CGH), multiplex ligation-dependent probe amplification (MLPA), multiplex quantitative real-time polymerase chain reaction (qPCR), and high-resolution single-nucleotide polymorphism (SNP) microarray analysis [126]. Although the FISH method is still routinely used in laboratories, the MLPA assay has been perceived as an alternative that is superior to the FISH technique as it is less costly, less time-consuming, and laborious, and does not require cell cultures. It has been proposed that a locus-specific approach, using FISH or MLPA assays, could be offered to children strongly meeting clinical and dysmorphology criteria for 22q11.2 DS [126,127]. Even though FISH is a state-of-the-art procedure for patients with the clinical suspicion of 22q11.2 DS, at present, patients are usually diagnosed by indirect whole genome studies [128]. Referring to the regulatory role of the TBX1 gene during development of the heart, thymus, and parathyroid glands, as well as during formation of the palate, teeth, and craniofacial features, there has been growing evidence that TBX1 is a candidate gene for 22q11.2 DS [129,130]. Therefore, in patients with clinically evident disease in whom a deletion of 22q11.2 has not been identified by FISH or microarray tests, TBX1 gene testing is recommended. Genotype first approach has therefore been proposed, and whole genome sequencing as the first-line method in the not-too-distant future, with FISH to be used as a confirmation of patient and family screening results [125].

Newborn screening (NBS) for severe combined immunodeficiency (SCID) is identifying a subset of infants with 22q11.2 DS due to T cell lymphopenia and low TREC numbers, and, hence, it is not a universally reliable detecting method. Therefore, direct NBS for 22q11.2 DS to recognize affected neonates using the genomic approach with multiplex qPCR assay, targeted to the TBX1 gene within the LCR22A-B region and CRKL within the LCR22C-D region, has been elaborated [131].

Whereas postnatal phenotypes have been widely characterized and categorized, prenatal diagnosis of 22q11.2 DS remains challenging due to a low rate of inheritance (10% of cases) and mild unrecognized parental features. Fetal ultrasound imaging may provide information on findings characteristic for 22q11.2 DS, such as polyhydramnios, hypoplasia or aplasia of the fetal thymus, central nervous system anomalies, such as asymmetric ventriculomegaly and dilated cavum septum pellucidum, and, rarely, skeletal anomalies, among others, such as bilateral talipes and anomalous vertebrae [132].

The golden standard method for detecting 22q11.2 microdeletions remains first trimester screening by chromosomal microarray (CMA), which is performed in invasively obtained prenatal samples, such as chorionic villi and amniotic fluid. An important technical advance in prenatal noninvasive screening for 22q11.2 is cell-free DNA testing [133]. Cell-free DNA fragments present in maternal plasma, which derive from both the mother and the embryo as a result of apoptosis of the cytotrophoblast. An external layer of the placenta is used for qualitative and quantitative assays. The fetal fraction is screened for common trisomy and 22q11.2 deletion, as well as for other rare trisomies and microdeletions. Targeted technologies, such as single-nucleotide polymorphism (SNP)-based, digital analysis of selected regions (DANSR), and targeted capture enrichment assay (TCEA) technologies, as well as the genome-wide methodology massively parallel shotgun sequencing (MPSS), are advanced techniques used in cell-free DNA analysis [133,134,135,136,137].

The marked phenotypic variability of 22q11.2 DS is accompanied by a wide scope of immune deficits, ranging from mild to moderate T cell lymphopenia in the partial form of DiGeorge syndrome (pDGS) to profound combined T and B cell immunodeficiency in the complete form (cDGS). In the first case, hypoplastic ectopic thymus or microscopic thymic rests are found and successful spontaneous immunocorrection has been reported, whereas the latter case is characterized by complete athymia [138]. In those most severely immunocompromised children, immune reconstitution may be achieved by thymus transplantation, providing the ability to produce naive T cells showing a broad T cell receptor repertoire. To facilitate the optimal establishment of thymic allograft, stability of comorbidities, such as attaining cardiopulmonary function, upper airway stabilization, appropriate weight gain, and metabolic compensation, are essential to avoid graft failure. The management of concurrent disorders plays a fundamental role in the timing of thymus transplantation, which requires optimal planning and sequencing [139]. Another approach to cDGS is T cell-replete hematopoietic stem cell transplantation; however, due to the absence of the thymus, engraftment of post-thymic T cells may result in poor quality of immune reconstitution [94,140,141]. Although 22q11.2 DS has been perceived as a T cell deficiency, disorders of B cell maturation and reduced numbers and functions in naive, unswitched, and switched memory B cells have also been reported. Humoral immunodeficiency in children with 22q11.2 DS may considerably vary from hypogammaglobulinemia with low all immunoglobulin isotypes and the need to receive immunoglobulin replacement therapy (approximately 6% and 3% of them, respectively) [95] to low serum IgA or IgM and impaired antigen-specific vaccine response in sporadic cases [142,143]. Despite the vast majority of children with 22q11.2 DS having normal serum immunoglobulin levels, due to the variable degree of cellular immunity impairment, they are susceptible to acute and recurrent infections, among others such as sinusitis, otitis media, mastoiditis, pneumonia, urinary tract infections, and viral infections [144,145]. Important questions are then raised about indications for preventive measures against infections in those children who do not qualify for immunoglobulin replacement therapy but present with T-cell lymphopenia. Antibiotic prophylaxis is indicated for children with 22q11.2 DS, first of all in those with low IgA serum levels and panhypogammaglobulinemia presenting with recurrent respiratory tract infections during epidemic season. The prophylactic regimens include daily or alternate-daily use of amoxicillin or azithromycin and co-trimoxazole in children with advanced T-cell lymphopenia posing the risk of Pneumocystis jiroveci infection [136,146,147].

Active immunization in children with 22q11.2 DS requires optimizing to provide vaccination coverage against vaccine-preventable infections. Live vaccine practices with Bacille Calmette-Guerin (BCG), and vaccines against measles-mumps-rubella (MMR), varicella (VAR), and an intranasal live attenuated influenza vaccine (LAIV), are contraindicated in children with this syndrome as T cell lymphopenia makes them susceptible to adverse effects following live immunization (AEFLI). Furthermore, in those children who, due to hypogammaglobulinemia, receive immunoglobulin replacement therapy either intravenously or subcutaneously, live vaccines are inactivated by administered antibodies and thereby are contraindicated. Inactivated vaccines can be safely administered to immunodeficient patients as they do not pose the risk of an uncontrolled spreading of vaccine microorganisms in the patient’s body, and they are not inactivated by supplemented immunoglobulins. However, the immune response to vaccines with antigen-specific antibodies and memory B cell generation may be significantly reduced [148].

However, many individuals with 22q11.2 DS with mild to moderate immunosuppression receive live viral MMR and varicella vaccines despite the known diagnosis and tolerate them well, without serious adverse effects [149,150]. Given the risk of natural infection, the benefits of protection following immunizations with live vaccines outweigh the risks of potential AEFLI. To assess the safety of live attenuated vaccines and evaluate the ability to generate an effective immune response in children with 22q11.2 DS, immunological investigations prior to the administration of live vaccines have been proposed [151]. The recommended immunology workup practices include lymphocyte immunophenotyping with the evaluation of total CD3+ T cells, CD4+ T helper cells, CD8+ T cytotoxic cells, and CD3+CD4+CD45RA+CD31+ recent thymic emigrants, as well as response to mitogen phytohemagglutinin (PHA). Live vaccines can be safely administered in children showing a total T cell count above 0.5 × 10⁹/L, a cytotoxic T cell count above 0.2 × 10⁹/L, and a normal response to mitogen [151].