Primary immunodeficiencies and secondary immunodeficiencies

Abstract

Introduction

The first primary immunodeficiency (PID) was reported in the 1950s by Colonel Ogden Bruton, who described a male patient without the gamma globulin fraction (immunoglobulin) in blood who was inherently susceptible to multiple and severe infections. It took approximately another four decades to identify the molecular basis of this X-linked recessive immune disorder, which is now called X-linked agammaglobulinemia (XLA) caused by pathogenic variants in the Bruton’s tyrosine kinase ( BTK ) gene. Similarly, two physicians on two different continents approximately 15 years apart described male patients with a triad of bleeding diathesis, eczema, and diarrhea, which also appeared to be X-linked recessive in inheritance. ^, The disease was named Wiskott-Aldrich syndrome after the two physicians, and again it took almost four decades to identify the specific gene associated with this defect, now called Wiskott-Aldrich syndrome ( WAS ) gene. Today in the 21st century we are aware of more than 400 single gene (monogenic) defects associated with various inborn errors of immunity, which includes primary immunodeficiencies (PIDs) and primary immune dysregulatory disorders (PIRDs). We have come a long way in our understanding of human immunity, and our ability to diagnose, treat, and manage these conditions, but we still have a long way to go.

The cellular components of the immune system include both adaptive (T cells and B cells) and innate immune cells (natural killer [NK] cells, granulocytes, monocytes, dendritic cells). Additionally, there are other specialized cells including various types of innate lymphoid cells (ILCs) that along with a variety of cells and tissues play a key role in the modulation of the immune response. The immune system also produces and is regulated by several soluble biomarkers, including cytokines, chemokines, and other biologically active molecules that drive and modify the immune response. This chapter will not delve into the details of the development of the immune system or these cellular subsets, or the immune response and its regulation.

Key serum proteins of the immune system include antibodies or immunoglobulins produced by terminally differentiated B cells, plasma cells. A brief discussion of the role of antibodies is provided here. Antibodies are a critical component of the host defense mechanism able to neutralize toxins or viruses, or bind to bacteria or fungi and prepare them for killing by phagocytes and the complement system proteins. There are five major isotypes of immunoglobulin heavy chains—IgG, IgA, IgM, IgD, and IgE. Each of these has distinct functions within the humoral immune response, and pair with either kappa or lambda light chains. Within the IgG family of immunoglobulins, there are four subclasses (IgG1, G2, G3, and G4), while IgA has two subclasses (IgA1 and IgA2). The immunoglobulins either are secreted into plasma or present on the surface of B cells as membrane-bound immunoglobulin. Immunoglobulin allotypes are reflective of genetic differences in the constant region segments of antibody molecules and are either classified as Gm, Am, Em, or Km, depending on the alleles associated with each isotype. Idiotypes, on the other hand, are unique epitopes within the variable (V) regions of antibodies and allow discrimination of one antibody from another. IgG is the most abundant immunoglobulin in human serum and has a half-life of 23 days while IgM, IgA, IgD, and IgE have half-lives of 5, 7, 2.8, and 2.3 days, respectively. It is important to bear in mind that the half-life of IgG is not invariant but rather is related to the concentration of the immunoglobulin in circulation. Therefore for patients with low concentrations of IgG (hypogammaglobulinemia), the half-life may be as long as 35 days, while in patients with hypergammaglobulinemia, it can be as short as 10 days. The regulation of IgG half-life is mediated by the neonatal Fc receptor (FcRn) and permits recycling of the IgG molecule. Deficiency of immunoglobulins is the most common of all inborn errors of immunity and falls into three broad categories, based on the degree of antibody deficiency, presence or absence of B cells, and whether there are other syndromic manifestations. Immunoglobulin is transferred through the placenta, especially IgG, while IgA is present in secretions, such as tears, breast milk, and saliva and is a critical component of mucosal immunity. Selective IgA deficiency is probably the most common immunodeficiency to be described, and most patients are clinically asymptomatic while some may have an increased incidence of infections. Maternal IgG is present in the first 6 months of life and gradually wanes allowing endogenous immunoglobulin production to take over. If the physiologic hypogammaglobulinemia of infancy persists beyond 6 months, it is often referred to as transient hypogammaglobulinemia of infancy (THI). Patients with THI may be either asymptomatic and normalize over time, or have recurrent infections. Replacement with external immunoglobulin is commonly not recommended except if there is life-threatening infections. It may take several months to years to normalize immunoglobulin levels but typically most patients in this category would have normalized IgG levels by 4 years of age.

IgG subclass deficiencies, on the other hand, are conditions in which total IgG (and subclass IgG1, which is the most abundant subclass) is normal but one or more other subclasses are decreased due to reduction in production of one or more isotypes. As with THI, measurement of subclasses and treatment with replacement therapy is only recommended in the context of aberrant functional antibody responses and significant infections.

As previously alluded to in the abstract, inborn errors in immunity (IEIs), often referred to as primary immunodeficiencies (PIDs), are heterogeneous disorders affecting all components of the immune system, and occurring either as the dominant phenotype or in conjunction with other anomalies affecting one or more organ systems (syndromic immunodeficiencies). The spectrum of IEI can present with a clinical phenotype of susceptibility to infection, autoimmunity, predisposition to malignancy or atopy, lymphoproliferation, or combinations of these. While autoimmunity and immunodeficiency may appear mutually exclusive, studies of IEI reveal that monogenic defects can cause immune dysregulation, which not only predisposes to autoimmunity, but may also be associated with increased susceptibility to infections. With the advent of next generation sequencing (discussed later in this chapter), identification of molecular defects associated with aberrant immunity has become increasingly facile, and allowed for various permutations within the same gene associated with distinct phenotypes (e.g., gain-of-function [GOF] and loss-of-function [LOF] variants ^, ) to be revealed. In addition, identification of specific molecular defects has opened the door to personalized therapies and targeted treatments.

The IUIS classification of IEI ^, has grouped genetic defects associated with immunologic diseases into nine broad categories, based on the immune defect or predominant clinical phenotype ( Fig. 100.1 ). A tenth category includes phenocopies of immunodeficiencies, which will be briefly described in this chapter. The nine categories include: (1) combined (T and B cell) immunodeficiencies, (2) combined immunodeficiencies (CIDs) with syndromic or associated features, (3) humoral or antibody deficiencies as the major phenotype, (4) defects of immune dysregulation, (5) congenital defects of phagocytes, (6) defects of innate/intrinsic immunity, (7) autoinflammatory conditions, (8) complement deficiencies, and (9) bone marrow failure syndromes. Representative examples of these monogenic disorders and phenocopies of PIDs are provided in this chapter along with an overview of treatment and management of IEI. The final section of this chapter is focused on secondary immunodeficiencies.

Group 1. Combined immunodeficiencies affecting cellular and humoral immunity

This category includes a variety of genetic defects associated with diverse clinical phenotypes but unified by a defect in both arms of adaptive immunity—T and B cells. Severe combined immunodeficiencies (SCIDs) are a prototype of this group of disorders.

Severe combined immunodeficiencies

SCID is characterized by defect in T-cell numbers and/or function. In addition, other components of the adaptive (B cells) and innate immune system (NK cells) may be affected. The disease manifests at a median of 4 to 6 months and most infants are asymptomatic at birth. Typical infections in these patients include recurrent viral, bacterial, and fungal opportunistic infections including pneumonias, diarrhea, and failure to thrive (FTT). Due to compromised cellular immunity, live vaccines are contraindicated. The number of genetic defects associated with SCID has rapidly multiplied in recent years. ^, Most of the SCID defects present with an autosomal recessive inheritance except for a few, which demonstrate X-linked inheritance, including the prototypic SCID defect, X-linked SCID caused by pathogenic variants in the IL2RG gene, encoding the common gamma chain of the interleukin (IL)-2 receptor. SCID-associated conditions can be categorized based on whether B and NK cells are affected, in addition to T cells. The four main categories of SCID based on T-, B-, and NK-cell quantitation include T−B−NK−, T−B−NK+, T−B+NK−, and T−B+NK+ defects ( Table 100.1 ). The clinical spectrum ranges from the classic or typical manifestations to leaky SCID, caused by partial loss-of-function (LOF) or hypomorphic gene defects in the same genes that cause the typical phenotype. Also, if the leaky SCID presentation occurs in conjunction with other features of erythroderma, elevated IgE, eosinophilia, organomegaly, or oligoclonal T cell expansion, then the phenotype is consistent with Omenn syndrome. ^, Most forms of SCID are associated with the absence of a thymic shadow on a chest X-ray, which ought to trigger suspicion for a primary immunodeficiency. However, there are certain forms of SCID, such as Coronin-1A and CD3 delta subunit deficiencies, which have a visible thymus.

TABLE 100.1

Examples of Classification of Severe Combined Immunodeficiencies Defects Based on Lymphocyte Subset Phenotyping

Category	Gene Defect
T−B+NK− SCID
IL-2 receptor common gamma chain (X-linked)	IL2RG
Janus Kinase 3 (JAK3) deficiency	JAK3
T−B+NK+ SCID
IL7 receptor chain α	IL7RA
CD45	PTPRC
CD3 δ	CD3D
CD3 ε	CD3E
CD3 ζ	CD3Z
Coronin 1A	CORO1
Winged helix deficiency	FOXN1
PAX1 deficiency	PAX1
T−B−NK+SCID
Recombinase activating genes 1 and 2	RAG1 and RAG2
DNA crosslink repair enzymes 1C (Artemis)	DCLRE1C
DNA-dependent protein kinase catalytic subunit (DNA-PKcs)	PRKDC
DNA ligase IV deficiency	LIG4
CERNUNNOS/XLF deficiency	NHEJ1
T−B−NK− SCID
Adenylate kinase 2 (reticular dysgenesis)	AK2
Adenosine deaminase 1 (ADA) deficiency	ADA1 /ADA

NK, Natural killer; SCID, severe combined immunodeficiencies.

The diagnosis of SCID includes a combination of clinical features with laboratory testing. A simple complete blood count (CBC) can be informative as most patients with the classic forms of SCID have significantly decreased absolute lymphocyte counts (ALC < 2500 cells/μL; CD3+ T cells < 300 cells/μL). However, in some cases of leaky SCID or Omenn syndrome there may be either maternal engraftment or oligoclonal expansion of T cells, which can raise the ALC. Maternal engraftment (ME) can be ruled out by performing a karyotype analysis on peripheral blood of male infants, while for female infants, a short tandem repeat assay (STR) would be necessary to differentiate between maternal and infant T cells. The STR assay may also be used for ME in male infants and is likely less expensive than karyotyping. Flow cytometry for activated T cells expressing HLA DR and distribution of memory and naïve T cells can be informative, though these cannot discriminate between maternal engraftment and autologous oligoclonal expansion of T cells.

Most flow cytometry assays use a panel of basic markers for discriminating between naïve and memory T cells utilizing CD45RA and CD45RO, respectively, along with CD4 and CD8 to identify the main subsets in blood. However, additional markers such as CCR7 and CD62L are useful in ensuring the naïve T cells identified are truly naïve, and not antigen-experienced T cells that have re-expressed CD45RA (T-cell effector memory cells expressing CD45RA, TEMRA). More recently, a few studies have documented reference intervals for various lymphocyte and T-cell subsets in premature infants, neonates, healthy children, and adults. It has also become common to assess recent thymic emigrants by flow cytometry using a combination of CD4, CD45RA, and CD31. ^, While immunophenotyping is the mainstay of the early laboratory diagnosis of SCID, functional assessment of T cells is also incorporated into most diagnostic algorithms. The most basic T-cell functional assay is measurement of T-cell proliferation after stimulation of whole blood or peripheral blood mononuclear cells with a polyclonal lectin stimulant, a mitogen such as phytohemagglutinin (PHA), or Pokeweed mitogen (PWM). The readout can utilize more traditional methods, such as radioactive thymidine (3H-t) or flow cytometry. More complex T-cell proliferation assays can be performed depending on the specific clinical context (e.g., antigen-specific proliferation, stimulation with anti-CD3/anti-CD28 or anti-CD/IL-2), the latter of which causes T-cell proliferation in response to CD3 receptor and other costimulatory molecule crosslinking. These assays are more useful at an advanced diagnostic stage of evaluation rather than as a first-tier diagnostic test.

In addition to these laboratory criteria, the clinical phenotype and family history is very useful in identification of patients with asymptomatic SCID. Patients often have a failure to thrive and serious adverse reactions to live vaccines, such as the Bacillus Calmette Guerin (BCG) and rotavirus.

Specific forms of SCID, which causes loss of enzyme function, such as adenosine deaminase (ADA) or recombinase activating gene (RAG) deficiencies, can also be confirmed by direct measurement of ADA enzyme activity and associated accumulation of toxic metabolites, or measurement of recombinase activity in T cells, respectively, in addition to the aforementioned laboratory tests. Hypomorphic (partial loss-of-function) forms of RAG1 and RAG2 deficiencies are often associated with a combined immunodeficiency along with autoimmunity and inflammatory manifestations, such as granulomatous disease ^, and can present at ages beyond infancy, including childhood and early adulthood.

Some forms of SCID are associated with defects in DNA repair and these tend to cluster within the T−B−NK+ grouping. These forms of SCID are frequently referred to as radiosensitive (rs)-SCID, and include genetic defects, such as DNA-PKcs ( PRKDC ), Artemis ( DCLRE1C ), Cernunnos ( NHEJ1 ), and DNA Ligase IV ( LIG4 ), among others. These radiosensitive forms of SCID along with other forms of syndromic combined immunodeficiency, such as ataxia telangiectasia (AT), can be rapidly diagnosed using a flow cytometry assay that measures the nonhomologous end-joining (NHEJ) pathway of DNA repair after induction of DNA double-strand breaks (DSBs) ( Fig. 100.2 ). ^,

Newborn screening for severe combined immunodeficiencies

The early diagnosis of SCID has been revolutionized with the inclusion of this condition in the recommended uniform screening panel (RUSP) for newborn screening (NBS) in the United States 2010. Several other countries also perform NBS for SCID though they may not have a RUSP. SCID met all the criteria required for a public health initiative, such as NBS: (1) a lethal condition, which is asymptomatic in the newborn period, (2) availability of a biomarker, which can be used on dried blood spots (DBS), and (3) a curative treatment. The use of T-cell receptor excision circles (TREC), a by-product of T-cell receptor rearrangement, as a biomarker for T-cell production by the thymus revolutionized large-scale screening for SCID ( Fig. 100.3 ), and though this condition was intended to be the primary target of NBS, it soon became apparent that other conditions associated with T-cell lymphopenia (TCL) could also be identified by NBS for SCID ^, as secondary targets. An early family-based study revealed that diagnosis by NBS SCID reduced the mortality associated with SCID, by allowing life-saving interventions, such as hematopoietic cell transplantation (HCT), to be instituted early, ^, improving the long-term outcomes. Currently, all 50 states in the United States perform TREC-based NBS SCID, and several other countries or regions within countries have either implemented routine NBS SCID or are performing pilot studies. While TREC-based screening facilitates detection of severe early-onset TCL, there are PIDs with late-onset TCL, which are not identified by NBS and are identified by other tests at ages beyond the neonatal period. The Clinical Laboratory Standards Institute (CLSI) produced a guidelines document on TREC-NBS-SCID (Clinical Laboratory Standards Institute, 2013, Newborn blood spot screening for Severe Combined Immunodeficiency by measurement of T-cell receptor excision circles; Approved Guideline) several years ago, and a current revision is underway, which will be published at the end of 2021.

NBS SCID has enabled assessment of the true population prevalence for SCID, and in an early study in the United States, it was estimated to be 1:58,000; more recently, from data in California, the incidence was estimated at 1:65,000 (95% confidence interval, 1:51,000 to 1:90,000). However, certain ethnic groups have founder mutations for specific genes and thus have a higher incidence of those specific genetic forms of SCID, including the Navajo Nation (Artemis ( DCLRE1C ) SCID), Somali (ADA-SCID), and Amish and Mennonite ( RAG1, RAG2, IL7RA ).

Beyond the well-described genetic defects associated with the more classic SCID conditions, which are treated by HCT, NBS SCID has allowed for diagnosis of newer SCID defects, which are specific for thymic defects, and can only be treated with thymus transplantation (e.g., FOXN1 and PAX1 deficiencies). ^, Diagnosis of SCID caused by these defects can enable selection of the appropriate treatment reducing morbidity and mortality.

Measurement of TREC in blood can not only be used for population-based screening but also in the diagnostic laboratory for evaluation of TCL in various contexts and for monitoring recovery of thymic function post-HCT. Since TREC is an extra-chromosomal product derived from VDJ gene rearrangement during production of the T-cell receptor, it is diluted by cell division in the periphery. Therefore infants have the highest levels of TREC, which steadily decreases with age, and this is particularly so after puberty and in adults ( Fig. 100.4 ). Besides TREC measurements, diversity of the T-cell repertoire (TCR Vβ) can be analyzed by different methods including flow cytometry, fragment length analysis (spectratyping), and next-generation sequencing. These assays can offer advanced diagnostic assessment of patients, especially with complicated phenotypes who have evidence of TCL or oligoclonal expansion of T cells.

As with any other clinical condition, there are several other immune defects that can present with overlapping phenotypes with SCID, and these may be either other primary immunodeficiencies or even secondary immunodeficiencies, including HIV or loss of T cells due to other conditions, such as chylothorax, gastroschisis, gestational diabetes, or prenatal exposure to maternal immunosuppression among many others. There are many genetic defects associated with CIDs, which can present with failure to thrive and opportunistic infections reminiscent of SCID, and while these cannot be addressed in detail in this chapter, beyond representative examples, they are described elsewhere in the literature. ^,

Molecular diagnosis of SCID, as with other PIDs and PIRDs is most frequently performed by next-generation sequencing (NGS) methods, which is discussed elsewhere in this chapter.

The definitive form of treatment for SCID and related conditions is hematopoietic cell transplantation (HCT) aimed at achieving immune reconstitution and correction of the underlying defect. ^, HCT has been shown to dramatically improve survival and outcomes in SCID patients. For certain forms of SCID, such as ADA-SCID and X-linked SCID ( IL2RG ), gene therapy is available, either clinically or under a clinical trial, which has shown effective immune recovery. For ADA-SCID, enzyme replacement therapy (ERT) with recombinant enzyme has proven effective at bridging therapies until a more definitive form of treatment is available. In addition to these longer-term treatment options, supportive therapies, such as prophylactic antibiotics and replacement immunoglobulin, has proved efficacious in reducing infectious complications prior to transplant. If the patient has viral infections, such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, or Varicella, virus-specific cellular therapies can be administered if antivirals have not proven effective. Many of these supportive therapies are not restricted to SCID, and can be used for any of the PIDs and PIRDs, with significant complications and mortality.

Group 2. Combined immunodeficiencies with syndromic or associated features—DiGeorge syndrome

Angelo DiGeorge first recognized DiGeorge syndrome (DGS; OMIM #188400) in 1965 when he described a series of infants with hypoparathyroidism associated with thymic aplasia. Defective remodeling of the pharyngeal region during embryogenesis causes this syndrome. The classic triad of thymic hypoplasia/aplasia, congenital heart defects, and hypoparathyroidism with associated facial characteristics is recognized in most patients with DGS. ^, The majority (90%) of DGS patients have a microdeletion, either 3 or 1.5 Mb, on chromosome 22q11.2 (22q11.2del), which typically results in heterozygosity of 36 and 56 genes with the smaller and larger deletion, respectively. Chromosome 22q11.2 deletion syndrome is the most common microdeletion syndrome reported in humans, reported in 1:4000 live births. This complex multiorgan disorder encompasses previously described overlapping genetic syndromes including velocardiofacial syndrome (VCFS; Shprintzen syndrome), the conotruncal anomaly face syndrome (CTAF; Takao syndrome), autosomal dominant Opitz G/BBB syndrome, Sedlackova syndrome, and Cayler cardiofacial syndrome.

In addition to 22q11.2del, the DiGeorge phenotype may be seen with other chromosomal abnormalities and genetic defects. Congenital features overlapping with 22q11.2del/DiGeorge phenotype are also observed in offspring of mothers with gestational diabetes or diabetic embryopathy, isotretinoin teratogenicity, and fetal alcohol syndromes.

Thymic aplasia or hypoplasia results in absence or decreased levels of circulating T cells, especially naïve T cells in infants and children. Approximately 20% of DGS infants may be identified through NBS SCID due to decreased TREC copies. DGS infants with completely absent T cells are classified as complete DiGeorge syndrome (cDGS) and represent approximately 1% of DGS patients. These patients are phenotypically no different from classic forms of SCID and require immediate therapeutic intervention for survival. Atypical forms of cDGS resembling Omenn syndrome have been reported. The other DGS patients who do not have cDGS are classified as having partial DiGeorge syndrome (pDGS).

The clinical phenotype of 22q11.2del/DGS is extremely variable, differing from patient to patient, even within the same family. The congenital malformations frequently involve the heart (conotruncal malformations, tetralogy of Fallot, aortic arch abnormalities, truncus arteriosus, ventricular septal defects, and vascular rings), abnormalities of the palate (clefts and velopharyngeal incompetence), facial dysmorphism, renal and/or skeletal anomalies, hypoparathyroidism with hypocalcemia, and immunodeficiency. Developmental delay, autism and autism spectrum, attention deficit disorders, and psychiatric illnesses may become apparent with time. ^,

The clinical phenotype of DGS is extremely diverse. Recurrent upper respiratory tract infections (URI) are a common feature and occur in 35 to 40% of children. These infections do not necessarily correlate with T-cell numbers, and may be related to a combination of factors including anatomic anomalies. Opportunistic infections are rare and usually seen only in the cDGS phenotype. Autoimmunity has an overall frequency of 8 to 10% in DGS, with autoimmune cytopenias being the most common. DGS patients with autoimmune cytopenias have decreased naïve T cells and class-switched memory B cells.

While cytogenetic analysis for the chr.22q11.2del by fluorescence in situ hybridization (FISH) was commonly used, more recently, use of single-nucleotide polymorphisms (SNP) or comparative genomic hybridization (CGH) arrays and NGS for the TBX1 gene have gained traction, though single gene haploinsufficiency is unlikely to account for all DGS phenotypes. Other diagnostic immunology laboratory tests include a complete blood count (CBC), flow cytometry for lymphocyte subsets (T, B, and NK cells), and, if TCL is present, evaluation for naïve and memory T cells, and immunoglobulin levels, vaccine antibody responses, especially to inactivated vaccines. More complex immune studies, such as assessment of thymic function, B-cell differentiation subsets, T-cell receptor repertoire diversity, and T-cell proliferation to mitogens and antigens may also be performed but might require the use of a reference diagnostic immunology laboratory. Besides the immunologic studies, cardiac and renal evaluation, measurement of serum calcium and phosphorus, as well as a chest X-ray to assess for the presence of a thymic shadow should be obtained.

The degree of TCL in the majority of pDGS patients is variable, but patients with autoimmunity appear to have overall lower total T cells, CD4+ T cells, and naïve CD4+ T cells, and there is an accelerated loss of naïve T cells and constrained T cell receptor repertoire diversity in older pDGS patients. Defects in switched memory B cells have also been reported in DGS patients. T-cell function, especially proliferation to mitogens and antigens, is also variable, and though most pDGS patients may respond to mitogens, such as PHA, the response to specific antigens, for example Candida and Tetanus toxoid, is less likely to be normal.

Treatment of DGS patients requires a multidisciplinary approach to address the varied clinical needs unique to each patient. Inactivated vaccines can be administered to all pDGS patients. However, the use of live vaccines demonstrates a lack of consistency in the immune parameters used to determine competence for these vaccines. Some criteria use a CD4+ T-cell count greater than 400 cells/μL with CD8+ T cells greater than 250 cells/μL and normal proliferation to mitogens; others do not use any T-cell proliferation criteria, and yet others do not perform any immunologic assessment.

Patients with pDGS are typically managed conservatively with antibiotics, when needed, and use of immunoglobulin replacement if there is hypogammaglobulinemia and deficits in class-switched memory B cells. Complete DGS patients, on the other hand, typically require thymus transplantation, which is performed in very few centers, one in North America and the United Kingdom. ^, While HCT has been attempted in cDGS and anecdotally reported as successful, it is unlikely to serve the purpose for most patients as there is no appropriate environment for maturation of T-cell precursors differentiated from the hematopoietic stem cells. Therefore thymus transplantation would be the treatment of choice for these patients, though newer therapies with thymic organoids may supplant or be used alongside thymus transplantation.

Group 2. Combined immunodeficiencies with syndromic or associated features—Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS; OMIM #301000; gene WAS ), caused by pathogenic variants in the WAS gene on chromosome Xp11.23, is a PID first described in 1937 by Wiskott, and demonstrated as an inherited X-linked recessive disorder by Aldrich in 1954. ^{, ,} The estimated prevalence is reported between 1:100 to 250,000 live births. The protein encoded by WAS , WASp, is primarily expressed in the cytoplasm of hematopoietic cells and belongs to a family of proteins involved in the formation of actin filaments. Absence of WASp affects many immunologic processes including effective immunologic synapse formation, chemotaxis, migration and adhesion, NK cytotoxicity, peripheral regulatory T-cell (Treg) homeostasis, B-cell homeostasis, and Fas-mediated apoptosis. A small amount of WASp has been found in the nucleus and a role in the prevention of DNA double strand breaks has been demonstrated in vitro . ^,

Clinical manifestations can be variable with all patients having microthrombocytopenia and risk for a bleeding diathesis. Beyond this, the spectrum of disease includes varying degrees of eczema, infections, diarrhea, autoimmunity (particularly autoimmune cytopenias), and cancer predisposition. Those patients with isolated thrombocytopenia with/without mild-to-moderate eczema and infrequent infections are classified as having X-linked thrombocytopenia (XLT), while those with the full spectrum of disease are classified as having “classic” WAS. The overall life expectancy of patients with XLT is reported similar to males in the general population, but many experience high morbidity related to disease-related complications. For “classic” WAS, long-term survival is limited if not treated with HCT, though gene therapy trials show promise. The clinical phenotype can be highly variable and is related to the amount of WASp expressed, which in turn is dependent on the specific pathogenic variant. WAS is a gene that shows several allelic variants, including the loss-of-function (LOF) phenotype associated with XLT, while a gain-of-function (GOF) phenotype is associated with X-linked myelodysplasia and X-linked neutropenia. WAS is also associated with a high degree of somatic reversion mosaicism though this does not appear to influence the clinical phenotype.

WAS is diagnosed in the laboratory by platelet measurement—size and number. Small platelet size is a hallmark of the disease, but this requires laboratory expertise to ensure the measurements are accurate. A complete blood count (CBC) will provide a platelet count along with absolute lymphocyte count among other parameters. Serum IgG in WAS patients can be low or normal, but serum IgA is often increased, and IgA nephropathy can be seen as a complication in these patients. IgE levels are variable but can frequently be elevated, and this pattern of serum immunoglobulins may not always be apparent in the youngest patients. Other immunologic tests include measurement of T-cell and B-cell function, as well as immunophenotyping. These results can be variable, depending on the patient, and cannot be used independently for a diagnosis. Frequently, spontaneous NK-cell cytotoxicity is decreased resulting in increased viral infections in these patients. One of the most useful tests for the definitive diagnosis of WAS, besides gene sequencing, is intracellular flow cytometry in lymphocytes, monocytes, and granulocytes for WASp. Completely absent protein expression would support a more severe clinical phenotype while partially preserved protein expression is likely to reflect a milder clinical phenotype, though this may evolve over time. Carrier females can also be identified by flow cytometry due to the presence of two populations for WASp, and occasionally somatic revertants can also be identified by this testing method.

The treatment for WAS is not unlike that for SCID with a variety of supportive therapies, including antibiotics and replacement immunoglobulin (and this is particularly essential if the patient has had a splenectomy for the management of autoimmune cytopenias). As with other combined immunodeficiencies on the more severe end of the spectrum, live vaccines should be avoided to prevent risk of vaccine-associated infection. Patients with autoimmune manifestations would require modulation with immunosuppression prior to HCT. For “classic” WAS patients and XLT patients at higher risk for developing malignancies, HCT remains a curative treatment. ^, Gene therapy trials for WAS show promise in early clinical trials.

Group 3. Antibody deficiencies—common variable immunodeficiency

Common variable immunodeficiency (CVID), first described in 1954, ^, encompasses a heterogeneous group of antibody deficiency disorders defined by specific laboratory criteria associated with a range of clinical manifestations, which encompass susceptibility to infection, autoimmunity, and lymphoproliferation. The term “CVID” appears a misnomer given the heterogeneity in clinical phenotypes and molecular defects, but nonetheless it persists, and the most recent IUIS classification has redefined it as Common Variable Immunodeficiency Disorders. Not all the clinical phenotypes associated with this group of disorders appear to be monogenic and some may be oligogenic or polygenic.

CVID is the most commonly diagnosed primary immunodeficiency in adults with an estimated prevalence of 1:25,000 to 1:50,000, affecting both genders equally. Most patients are diagnosed between the second and fourth decade of life though clinical manifestations may occur earlier, suggesting a bimodal distribution. Delay in diagnoses ranges between 4 and 7 years among adult patients. While most cases of CVID occur sporadically, in 5 to 25% of patients a positive family history is encountered.

The noninfectious clinical manifestations of CVID appear to be associated with the highest mortality. ^, Approximately 94% of CVID patients present with infections indicating that not all patients present with this manifestation, and in some patients, the presenting feature may be autoimmunity and/or lymphoproliferation. ^, As exemplified by humoral immunodeficiencies, sinopulmonary infections are the most common, along with gastrointestinal (GI) infections with norovirus, Giardia, etc. among the most pernicious. Chronic lung disease due to infections and interstitial lung disease with or without noncaseating granulomas is not uncommon, though granulomas may be seen in other organs as well, including the central nervous system. ^, Other noninfectious complications are recognized in at least one-third of CVID patients, if not more, and include multiple target organs. In particular, autoimmune cytopenias and GI complications have been recognized in this entity, ^, and may present before other clinical and laboratory features of the disease. Another significant complication is nodular regenerative hyperplasia of the liver, occurring in approximately 5% of these patients, as well as lymphoid nodular hyperplasia, in other organs, including the gut. Malignancy in CVID is not rare, and largely tends to be hematopoietic neoplasias or adenocarcinomas of the gut or other organs. ^{, , , ,}

The laboratory diagnosis of CVID mandates primary hypogammaglobulinemia involving IgG and at least one or more of the other isotypes, along with abnormalities in functional antibody responses to vaccines (e.g., protein vaccines—Tetanus and Diphtheria toxoid, pneumococcal polysaccharide, Salmonella polysaccharide), B-cell differentiation (low switched memory B cells and other B-cell subset defects). ^{, ,} Most patients with CVID have normal numbers of circulating B cells, and 1% of patients may have decreased to absent B cells. This is relevant because some patients with X-linked agammaglobulinemia may be diagnosed in adulthood and incorrectly classified as “CVID.”

Though not all diagnostic criteria recommend detailed B-cell subset immunophenotyping by flow cytometry, practical value in the evaluation of patients has been well established and is widely used. The term “primary” hypogammaglobulinemia implies that other causes of hypogammaglobulinemia have been eliminated including infections, drugs, malignancies, and secondary losses due to lymphatic alterations. The challenges in interpretation of vaccine antibody responses in primary immunodeficiencies is a topic in itself, though several guidelines exist. ^,

Some patients diagnosed with CVID also appear to have defects in T cells—number and function. This has been referred to as late-onset combined immunodeficiency (LOCID) and these patients may require different management strategies, therefore patients with a CVID diagnosis who have defects in total T cells or naïve T cells should be assessed more carefully for an underlying combined immunodeficiency.

While the current diagnostic criteria for CVID do not mandate genetic testing, it is part of accepted clinical practice to perform genetic testing by next-generation sequencing (NGS) methodologies, ^, as identifying the specific molecular defect has impact on prognosis and therapeutic management of disease. Even if only a small proportion of CVID patients (approximately 30%) ^, have monogenic defects identified by NGS, the value of knowing the specific molecular etiology has an immeasurable impact on clinical practice, patient care, and genetic counseling.

There are currently 14 different genetic defects with CVID phenotypes annotated in OMIM (Online Mendelian Inheritance in Man) ( Table 100.2 ). The genetic defects in CVID may be inherited as autosomal dominant or autosomal recessive conditions, causing either LOF) GOF, or haploinsufficiency. There are other humoral immunodeficiencies with distinct clinical phenotypes, such as X-linked lymphoproliferative disease I (XLP1) caused by pathogenic variants in the SH2D1A gene, which may be mistakenly diagnosed as CVID, and therefore rare X-linked recessive conditions can also be included in the differential diagnosis of CVID. Other gene defects associated with antibody deficiencies described in the literature include TNFSF12 (TWEAK deficiency), IL21R (IL-21 receptor deficiency), PLCG2, PTEN, TRNT1, RAC2, VAV1, ATP6AP1 (X-linked), ARHGEF1, SH3KBP1 (CIN85; X-linked), SEC61A1, ITPKB (haploinsufficiency due to microdeletion of chromosome 1q42.1–q42.3), and mannosyl-oligosaccharide glucosidase deficiency. ^,

Several of the above noted gene defects have unique phenotypes, which has resulted in their reclassification from “classic CVID” diagnosis to distinct inborn errors of immunity classified by gene name. For example, patients with defects in ICOS, or dominant negative pathogenic variants in Ikaros ( IKZF1), or IL21/IL21R gene defects, are now categorized among the combined immunodeficiency disorders. In addition, pathogenic variants within the same gene may cause either LOF or GOF consequences, further segregating them out as different diseases. For example, autosomal recessive deficiency of PIK3CD and PIK3R1 causes a profound decrease or absence of B cells with agammaglobulinemia, while GOF mutations in PIK3CD causes an activation of p110δ and the resultant activated p110Delta syndrome (APDS1). An autosomal dominant defect in PIK3R1 causes APDS2 (SHORT syndrome), and both these conditions are categorized under the CVID umbrella, in the genetics lexicon, while in immunology they are recognized and classified as distinct entities. Other genetic defects grouped under “CVID” but with distinct immunologic and clinical phenotypes have also been re-classified based on the gene defect and its impact on immune function (e.g., LRBA, CTLA4 associated with LATAIE and CHAI disorders, which affect Treg function) or CVID-like diseases associated with EBV driven lymphoproliferation (autosomal recessive defects in TNFRSF7 causing CD27 deficiency).

Treatment of CVID consists of passive replacement of immunoglobulin every 3 to 4 weeks by intravenous (IVIg) or subcutaneous (subcu Ig, SCIg) routes, antimicrobial therapy, and management of noninfectious manifestations with immunomodulatory therapies. A meta-analysis showed that a trough level of 1000 mg/dL of Ig replacement was effective in reducing pulmonary complications and demonstrated a significant inverse correlation between annual infection rate and serum IgG concentration. Usually, higher doses result in higher IgG trough levels and are given to patients with end-organ damage including those with chronic lung disease, bronchiectasis, and enteropathy. Hematopoietic cell transplantation (HCT) is typically not used for the management of “unspecified CVID” and is only considered in context of severe disease. However, targeted therapies, such as abatacept for LRBA deficiency or leniolisib for APDS1, based on a molecular diagnosis has shown success along with other supportive therapies.

X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA; OMIM #300755; gene BTK ) is a rare humoral immunodeficiency affecting 1:100-379,000 live births and caused by pathogenic variants in the Bruton’s tyrosine kinase gene ( BTK ). The protein encoded by this gene, also called Btk, is essential for B-cell development and maturation. Consequently, patients with XLA lack or may have very few B cells in peripheral blood, have absent or hypoplastic secondary lymphoid organs, such as tonsils and adenoids, lymph nodes have a distorted architecture, plasma cells are not generated, serum concentrations of immunoglobulins are low or absent, and adaptive immunity is impaired. At least half of XLA patients are identified in the first year of life when maternal immunoglobulin starts to wane, and approximately 85% are identified in the first five years of life. Some patients with hypomorphic variants in BTK may have a progressive loss of B cells and hypogammaglobulinemia and may be started on treatment with replacement immunoglobulin without a diagnosis, or may be classified as CVID if a young adult. These patients have sinopulmonary infections, gastrointestinal infections, meningitis, and encephalitis, which could be either bacterial or viral in origin. Noninfectious complications include inflammatory bowel disease, neutropenia, and arthritis, sometimes with rare pathogens, such as Ureaplasma urealyticum . Long-term outcome data suggest that despite IgG replacement therapy, patients with XLA continue to have sinopulmonary infections resulting in chronic lung disease, an important contributor to mortality. From a large series of 783 XLA patients from 40 centers in 32 countries, it was shown that complications including enteroviral meningoencephalitis, inflammatory bowel disease, and arthritis contribute to morbidity. While individually these complications may be uncommon, collectively these problems are seen in 20% of patients with XLA. Survival beyond 20 years depends on geographic location (lowest survival seen in Asia and Africa), with 62% of centers that followed adult patients reporting greater than 75% survival beyond 20 years of age.

The laboratory diagnosis includes a complete blood count (CBC), serum immunoglobulins (IgG, IgA, and IgM), and flow cytometry for lymphocyte subsets (T, B, and NK cells). If B cells are absent or significantly decreased and serum immunoglobulins are low, intracellular flow cytometry for the Btk protein is performed in monocytes, as Btk is typically expressed in B cells, monocytes, and platelets. Since B cells are absent or very low in XLA, an alternative cell subset like monocytes is used for the specific protein analysis ( Fig. 100.5 ). Depending on the detection antibody used in the flow cytometry assay, and the location of the variant in the BTK gene, protein may be present, decreased, or absent. Presence of protein does not negate a diagnosis of XLA, and in such cases, genetic testing of the BTK gene is required to confirm a diagnosis ( Fig. 100.6 ). T-cell numbers and function are intact in patients with XLA.

Treatment of XLA consists of effective antimicrobial therapy for management of infections and therapeutic use of pooled human IgG immunoglobulin preparations (replacement immunoglobulin), similar to the treatment of CVID. Prophylactic antibiotics for those patients with chronic lung disease was found to be beneficial in a recent double blind, placebo-controlled, randomized trial of low-dose azithromycin prophylaxis. Live vaccines should not be administered to XLA patients, particularly oral polio vaccine. Given the selective advantage of functional Btk for normal B-lymphocyte lineage commitment, HCT can provide a cure. Despite advancement in transplant practices, HCT is rarely offered to patients, especially in western countries, when medical management is readily available because the transplant-related morbidities, which include conditioning chemotherapy, graft versus host disease (GVHD), and infections while awaiting engraftment, may outweigh the potential benefit.

For the last two decades, knowledge of gene therapy strategies and genome editing methodologies has advanced such that ex vivo cellular engineering, utilizing viral vectors to treat monogenic disorders of immunity, and clinical trials are underway. ^, XLA is a unique disorder for gene correction because circulating levels of immunoglobulins have no effect on B-cell development or peripheral B-cell maturation, and human bone marrow provides an environment which permits B-cell reconstitution, independent of age. Preclinical work has demonstrated the rescue of Btk-dependent B-cell development, albeit incomplete, in the double knock-out mouse model for Btk- and Tec (Btk/Tec−/−) that phenocopies human XLA. ^, This provides proof-of-concept and a first step toward the development of gene-corrected autologous transplantation for XLA as an alternative therapeutic approach to conventional management and HCT.

Group 4. Diseases of immune dysregulation—autoimmune lymphoproliferative syndrome

Autoimmune lymphoproliferative syndrome (ALPS) (also known as Canale-Smith syndrome) encompasses a group of rare genetic disorders caused by defects in the extrinsic apoptotic pathway (FAS-mediated), resulting in dysregulated lymphocyte homeostasis. The key characteristics include chronic non-malignant, noninfectious proliferation with lymphadenopathy, hepatosplenomegaly, polyclonal hypergammaglobulinemia, and autoimmune cytopenias along with elevation of T cells lacking CD4 and CD8, “double-negative T cells” (DNT) (>1.5% of total lymphocytes or >2.5% of CD3+ lymphocytes). ^, Flow cytometry for DNT cells expressing the alpha-beta T-cell receptor (TCRαβ+) is one of the diagnostic laboratory tests ( Fig. 100.7 ) and is used in conjunction with other immunophenotyping assays along with evaluation of the B220 marker, which is aberrantly expressed on the above-described DNT cells. Measurement of other biomarkers, including soluble Fas ligand (sFasL), vitamin B12, and IL-10 are useful in establishing the diagnosis of ALPS in patients who meet the Revised NIH diagnostic criteria. ^, Genetic testing is useful in confirming the diagnosis, and while most cases of ALPS are associated with germline variants, somatic variants in DNT cells can also cause a form of ALPS, which requires sorting of these specific cells and subsequent genetic analysis. Assessment of apoptosis defects in vitro can also provide confirmation of a Fas-pathway defect. However, if patients have been previously treated, especially with steroids, the assay is nondiagnostic. A list of ALPS genetic defects and ALPS-like disorders is provided in Tables 100.3 and 100.4 , respectively. Patients with an overlapping phenotype of ALPS and CVID have also been described but these patients usually have hypogammaglobulinemia and not elevated levels of immunoglobulins.

The treatment of ALPS primarily focuses on reduction of lymphoproliferation and associated complications, as well as control of the autoimmune cytopenias. Steroids and sirolimus are frequently used for management with success. ^{, ,} HCT, though curative, is restricted to patients who are refractory to immunosuppression. Patients with ALPS-like diseases are often treated with various immunomodulatory agents, depending on the underlying molecular defect, and aimed at reducing the morbidity associated with the various clinical features. ALPS patients require life-long surveillance due to the increased risk of developing lymphomas.

Group 5. Phagocytic defects—chronic granulomatous disease

Chronic granulomatous disease (CGD) is a primary immunodeficiency largely affecting the innate immune system and caused by LOF variants in any of the five genes encoding the subunits of the phagocytic activity enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, present mainly in phagocytes. The five subunits of the NADPH oxidase complex include the two membrane-bound proteins, gp91phox ( CYBB gene; X-linked) and p22phox ( CYBA gene; autosomal recessive (AR)), and the three cytosolic components, p47phox ( NCF1 gene; AR), p67phox ( NCF2 gene; AR), and p40phox ( NCF4 gene; AR) ( Fig. 100.8 ). Very recently, a new gene defect associated with CGD has been described due to pathogenic variants in the CYBC1 gene encoding the EROS protein. ^, When phagocytes are stimulated, activated enzyme oxidase transfers electrons from the NADPH substrate to molecular oxygen resulting in the formation of reactive oxygen species (ROS), including superoxide (SO ₂ ⁻ ). NADPH oxidase deficiency results in defective production of ROS resulting in impaired microbial killing and excessive inflammation. CGD is characterized by severe recurrent bacterial and fungal infections and may be associated with hyperinflammatory complications, including inflammatory bowel disease.

The incidence of CGD varies based on population and the incidence in the West is approximately 1:200,000. ^, Infections in CGD patients are most frequently caused by catalase-positive microbes such as Staphylococcus aureus , Burkholderia cepacia, Klebsiella , Serratia marcescens, Salmonella , Aspergillus, and Candida species, though infections with catalase-negative organisms may also be seen. ^,

The laboratory diagnosis of CGD involves measurement of NADPH oxidase activity using the nonfluorescent dye, dihydrorhodamine (DHR) 123, which on stimulation of neutrophils is oxidized to rhodamine 123, which is fluorescent and can be detected by flow cytometry. The Nitroblue tetrazolium (NBT) test was widely used in the past but has largely been replaced by the flow cytometry-based DHR test, which is more sensitive and reliable and less subjective. A variety of stimulants can be used to activate neutrophils; however, phorbol myristate acetate (PMA) is the most common in the clinical laboratory. Rac2 deficiency, which can partially overlap in phenotype with CGD, ^, shows a normal result in the DHR assay when stimulated with PMA but provides an abnormal result when stimulated with a synthetic bacterial peptide, N -formylmethionyl-leucyl-phenylalanine (fMLP). Rac2 deficiency has a mixed phenotype and can overlap with CVID, but also has been identified by NBS SCID with TCL. ^, In the DHR test ( Fig. 100.9 ), comparison is made to an unstimulated sample, and the median fluorescence intensity (MFI) data is gathered for both the stimulated and unstimulated samples. The stimulation index (ratio of stimulated MFI to unstimulated MFI) is the most reliable measure of NADPH oxidase activity and can be used for serial measurement, both for diagnosis of disease and monitoring post-treatment. While the flow cytometry pattern can offer some correlation with the genotype, it is not always completely reliable, and therefore has to be confirmed independently, either by flow cytometric assessment of NADPH oxidase specific proteins in neutrophils and monocytes, which can be useful in identification of atypical forms of CGD, or genetic testing. Additionally, NADPH oxidase specific proteins can also be detected in B cells, while T cells can be used as a negative control. Confirmation of NCF1 defects (p47phox deficiency) by genetic analysis is challenging due to the presence of two pseudogenes. Therefore the p47phox flow assay is very useful for a rapid confirmation. The phenotype of p40phox is somewhat different from the other CGD defects, ^, and has a different pattern by flow cytometry. Complete myeloperoxidase deficiency (cMPO) can cause a false-positive (abnormal) result on the DHR flow assay, and therefore this has to be excluded before confirming a diagnosis of CGD, though this can often be triggered by a specific pattern on the DHR flow assay (see Fig. 100.9 ). The DHR flow assay can also be used for identification of carrier females for X-linked CGD. Carrier females for CYBB variants (X-linked CGD) show skewed lyonization, and can develop clinical symptoms, especially with age (see Fig. 100.9 ). ^, The DHR flow assay can also be used to monitor disease-specific chimerism post-hematopoietic cell transplant, and is useful for monitoring and prognosis. The DHR assay is particularly sensitive to sample quality, and poor transport conditions can adversely affect the interpretation of the assay (see Fig. 100.9 ). Residual NADPH oxidase activity and production of ROS can be assessed by the ferricytochrome c assay, which is very specific and sensitive ^, ; however, the assay requires fresh neutrophils, which makes it challenging for most clinical laboratories.

Treatment of CGD consists of life-long antifungal and antibacterial prophylaxis. ^, Recombinant IFN-gamma may be used along with antimicrobial drugs as an adjunctive therapy. The only curative therapy that has gained widespread acceptance is HCT. ^, Pioglitazone, a drug used most commonly for the management of diabetes has been postulated to be useful in CGD as it can improve mitochondrial ROS formation and reduce inflammation by reversing impaired efferocytosis, and is currently in a clinical trial. Gene therapy trials for X-linked CGD are available, and may be an alternative to HCT for some CGD patients.

Group 6. Defects of innate immunity

The compartmentalization of the immune system, in an effort to simplify its complexity, has led to the binary organization of adaptive and innate immunity. The adaptive immune system, comprised mainly of T and B cells, requires generation of antigen-specific receptors, and thus can take a few days (∼96 hours) to actively initiate participation in the immune response. Several of the preceding sections describe varied monogenic defects of the adaptive immune system. On the other hand, the innate immune system exists in the germline state and can immediately mount an immune response. They include not only cellular components, but also mucosal and epithelial barriers, naturally produced anti-microbial compounds, and receptors, called pathogen-recognition receptors, which recognize pathogen-associated molecular patterns (PAMPs) stimulating the secretion of bioactive substances, such as cytokines. The cells of the innate immune system include neutrophils, monocytes and macrophages, dendritic cells (DCs), NK cells, NKT cells, and other ILCs. There are several monogenic defects that affect either number or function of innate immune cells, or cytokines produced by these cells, which are critical for protection against infections with a variety of pathogens. ^{, ,} One example of an innate immune deficiency was described in the immediately preceding section, CGD. This section will briefly focus on Toll-like receptor (TLR) defects, as an example of innate immune defects. TLRs are capable of recognizing specific microbial and host-derived molecules, and enabling rapid and early detection by the host of infection or other injurious signals. A variety of cells of the immune system express these TLRs, including T cells, B cells, DCs, macrophages, and epithelial cells among others. Signaling through the TLRs enables initiation of host defense mechanisms, which results in cytokine production or secretion of other bioactive moieties with antimicrobial functions. Study of TLR defects enables our understanding of the critical role of the innate immune system and, in particular, these receptors in the host immune response. Human TLRs include both extracellular and intracellular receptors, and are ten in number. Each of these recognize different PAMPs, and danger-associated molecular patterns (DAMPs). A variety of ligands, microbial and endogenous ( Table 100.5 ), stimulate these TLRs and mediate downstream signals and induce cellular functions. With few exceptions, TLRs utilize the MyD88 and IRAK (IL-1 receptor-associated kinase) complex to transduce intracellular signals. One of the most important effects of TLR signaling is the activation of the nuclear factor κB (NFκB) pathway. The NFκB family of proteins consist of five members, which in the inactive state are maintained in the cytosol, bound to inhibitory κB (IκB) proteins, or tethered by p100, in the canonical and noncanonical pathways respectively. The five NFκB proteins include NFκB1 (p50), NFκB2 (p52), RelA (p65), RelB, and c-Rel, which are transcription factors that function as hetero- or homodimers. While some of the gene defects affecting the NFκB pathway of signaling cause either a combined immunodeficiency (CID) or a humoral immunodeficiency, with a CVID-like phenotype, other defects affect individual TLRs, or MyD88, IRAK-4 or other components of the innate immune system, or cause an autoinflammatory phenotype. As a prototype of innate immune defects, IRAK-4 (interleukin-1 receptor-associated kinase-4) deficiency causes recurrent infections, mainly by pyogenic, largely Gram-positive bacteria, like Streptococcus pneumoniae , and results in a poor inflammatory response with low-grade fever. Another common infectious organism is Staphylococcus aureus . A few patients have had invasive infections caused by Gram-negative organisms. The most noteworthy feature of IRAK-4 deficiency is improvement in clinical phenotype with age, with most patients having infections in childhood prior to adolescence. This suggests an age-related compensation of immune function. Similar to phagocyte defects such as leukocyte adhesion deficiency (LAD) type 1, some patients with IRAK-4 deficiency can demonstrate delayed separation of the umbilical cord. Innate immune defects, such as IRAK-4 deficiency, are most often diagnosed in the laboratory by assessing cytokine production in response to TLR stimulation using a variety of ligands, and IRAK4 gene sequencing. Flow cytometric detection of membrane-bound L-selectin on neutrophils has also been described as a rapid method for assessment of TLR defects. Since this disease can be fatal in childhood, prophylactic antibiotics and replacement immunoglobulin therapy along with vaccination for encapsulated pathogens are mandatory. In general, though, the treatment of innate immune defects depends on the specific genetic defect and associated clinical phenotypes, and thus can be quite variable.

Group 7. Autoinflammatory conditions

There is considerable overlap in the phenotypes and pathways affected by monogenic defects of the immune system, as evidenced by the IUIS categories and the examples provided above. Nonetheless, single gene defects associated with an autoinflammatory phenotype are categorized as a distinct entity. The concept of autoinflammation was proposed to explain the periodic fevers and systemic inflammation involving cells of the myeloid compartment but lacking features of classic autoimmune diseases, such as presence of autoantibodies or self-reactive T cells. Now it is recognized that autoinflammation involves aberrant activation and stimulation of the innate immune system. Inflammasomes, an integral part of the innate immune system, regulate the immune response to both extrinsic and intrinsic factors. Two groups of inflammasomes are recognized and include the NOD-like receptor (NLR) proteins—NLRP1, NLRP3, NLRP6, NLRP12, NLRC4 (IPAF), and the ALR-AIM2-like receptors, AIM2. ^, NLRP3, often regarded as the prototypic inflammasome, consists of a PRR (pathogen recognition receptor), a caspase-recruitment domain (ASC), which is an adaptor protein, and an enzyme, caspase 1, which promotes maturation of the pro-inflammatory cytokines, IL-1β and Il-18. The inflammasome is activated by triggering of the PRRs by PAMPs or DAMPs, and this in turn interacts with the ASC, leading to caspase 1 activation, which directs cleavage of the inactive precursors of IL-1β and IL-18 to their active forms. The periodic fever syndromes, a prototypic inflammasomopathy, includes the cryopyrin-associated periodic syndrome (CAPS) due to GOF variants in the NLRP3 gene, encoding the protein cryopyrin. The other most common form of periodic fevers is Familial Mediterranean Fever (FMF) due to pathogenic variants in the MEFV gene, encoding the pyrin protein, which is part of the inflammasome complex. Patients with FMF tend to have recurrent fever episodes, elevated acute phase reactants (APR) with inflammatory arthritis, and can also have inflammatory bowel disease. This condition is most typically seen in non-Ashkenazi Jews and other Eastern Mediterranean peoples. The list of autoinflammatory diseases continues to expand with identification of new monogenic defects, and these are not exhaustively covered in this section. NLRC4 GOF defects are associated with a severe form of infantile enterocolitis (AIFEC—autoinflammation with enterocolitis) but can also be associated with a relatively milder phenotype, familial cold autoinflammatory syndrome type 4 (FCAS4). Besides germline GOF variants in NLRC4 , somatic variants have been reported in one patient who presented with an AIFEC phenotype, while another patient presented with a NLRP3-type phenotype of neonatal-onset multisystem disease (NOMID). Treatment of most inflammasomopathies is with targeted therapies directed against IL-1β or IL-18.

Beyond the inflammasomopathies, genetic disorders associated with defective regulation of type I interferons, type I interferonopathies, are included under the umbrella of autoinflammatory conditions. ^, While the original designation of type I interferonopathies was confined to a few genetic disorders, such as Aicardi-Goutières syndrome (AGS), monogenic systemic lupus erythematosus (SLE), and spondyloenchondrodysplasia (SPENCD), now several others have been added to this group, including proteasome-associated autoinflammatory syndromes (PRAAS), Interferon (IFN)-stimulated gene 15 (ISG15) deficiency, Singleton-Merten syndrome and its atypical forms (SMS), and stimulator of IFN genes (STING)-associated vasculopathy with onset in infancy (SAVI). Type 1 interferons are a key component of the host anti-viral and in some cases, anti-bacterial response. The viral and bacterial pathogens that stimulate a Type I IFN response are sensed by various PRRs, including the TLRs, RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and other sensors, including AIM2 (inflammasome) among others, in the cytoplasm or endosomes of infected cells. In addition to the family of type 1 IFNs, which includes 13 IFN-α and a single IFN-β, there are IFN regulatory factors (IRF), which translocate to the nucleus and induce transcription of the type I IFNs. Type I IFNs perform two key functions, antiviral activity and anti-proliferative activity. All type I IFNs can mediate antiviral activity at very low concentrations, in most cells, while the anti-proliferative function is very cell specific and depends on the expression of the type I IFN and its cellular receptors, and affinity of binding to the receptor. Genetic defects in many of these type I IFN-associated diseases results in susceptibility to severe viral infections, herpes, and influenza.

Laboratory assessment of type I interferonopathies with enhanced type I IFN signaling is based on measuring increased expression of type I IFN. However, currently there is no such test available in a clinical laboratory. An assay has been developed and validated in the research setting, which measures expression of six interferon-stimulated genes (ISGs) as a surrogate for induction of type I IFN signaling. These six ISGs include IFI27, IFI44L, IFIT1, ISG15, RSAD2, and SIGLEC1. While these have been shown to be of value in the diagnosis of specific interferonopathies, they have not been tested in all these disorders or indeed all autoinflammatory conditions. In addition, increased expression of these six ISGs may be seen in other conditions with aberrant activation of innate immunity, but not classically considered as a type I interferonopathy (e.g., ADA2 deficiency, PRKDC defects among others). Increased Type I IFN signaling may also be seen in the context of cytokine blockade (IL-1β and tumor necrosis factor α [TNFα]) for other autoinflammatory diseases, which has clinical implications. An alternative scoring system was developed for IFN-response genes using 28 genes in the NanoString methodology, and used for the evaluation of a subset of patients with autoinflammatory diseases. The value of such assays extends beyond diagnosis and could be used for monitoring disease activity, response to therapy, and long-term outcomes.

Group 8. Complement deficiencies

Complement comprises several soluble and membrane-bound proteins and their associated receptors, which play a critical role in innate immunity and regulation of the immune response. The prevalence of complement deficiencies has not been accurately ascertained in all populations, but several national or global registries consider it to account for 1 to 10% of all PIDs, while another more recent review indicates an estimated prevalence of 0.03% in the general population. This chapter will not discuss the complement system in detail, as it is covered elsewhere in this book. However, a very brief discussion on complement deficiencies will be provided here. Monogenic defects of the complement proteins can be organized into (1) susceptibility to infections, especially with encapsulated pathogens, (2) predisposition to autoimmunity, specifically SLE, (3) and dysregulation resulting in specific diseases, such as atypical hemolytic uremic syndrome (aHUS) and thrombotic microangiopathy (TMA). Fig. 100.10 provides an algorithm for the diagnostic evaluation, based on the European Society for Immunodeficiencies (ESID) review on complement defects, and Table 100.6 is an overview of the known complement deficiencies, adapted from the European Society of Immunodeficiencies (ESID) review.

Group 9. Bone marrow failure syndromes—dyskeratosis congenita

The bone marrow failure syndromes (BMFSs) are a heterogeneous group of disorders, caused by either intrinsic (germline) or acquired defects resulting in ineffective hematopoiesis and cytopenias, affecting one or more lineages. ^, Inherited BMFS (iBMFS) are either autosomal recessive or X-linked conditions, many of them with high penetrance which usually, but not always, present in infancy. However, some of these have a broad clinical spectrum, and partial loss-of-function variants can result in a later onset. Other marrow-associated conditions, such as GATA2 deficiency or some of the short telomere syndromes (telomeropathies) have variable onset, are usually autosomal dominant, and manifest in adolescence or adulthood. Other iBMFS include dyskeratosis congenita, Hoyeraal-Hreidarsson syndrome, Fanconi anemia, Shwachman-Diamond syndrome, aplastic anemia, severe congenital neutropenia, and Diamond-Blackfan anemia, among others. Most of these conditions are identified by clinical phenotype and next-generation sequencing, though in some cases a molecular defect may not be clearly identified. As our understanding of the pathology of these disorders expands, other “classic” syndromic immunodeficiencies, such as Cartilage Hair Hypoplasia (CHH), may also be included in the overlap of iBMFS. Dyskeratosis congenita (DKC; Zinsser-Engman-Cole syndrome) is a prototypic iBMFS associated with a spectrum of immunodeficiency and telomere dysfunction. ^, Hoyeraal-Hreidarsson syndrome is at the severe end of the spectrum of DKC and presents with a severe combined immunodeficiency phenotype along with other syndromic features (microcephaly, cerebellar hypoplasia) but milder forms may present with aplastic anemia. The clinical features of the classic form includes the triad of skin pigmentation anomalies, leucoplakia, and nail dystrophy. However, it is important to remember that not all DKC patients may present with this classic phenotype. In addition to these, there may be varying degrees of bone marrow failure, immunodeficiency, susceptibility to malignancy, and pulmonary fibrosis. DKC is also a prototype of telomere defects, as it is caused by pathogenic variants in the genes DKC1 (X-linked), TERC (autosomal recessive or dominant), and RTEL1 (autosomal recessive or dominant), all involved in maintenance of the telomere complex and its function. The autosomal recessive and dominant forms may present with different clinical features and age of onset. DKC is associated with a very high risk for developing malignancy and includes both hematopoietic and non-hematopoietic tumors. Telomeres are “caps” on the end of chromosomes comprised of repetitive units of (TTAGGG) _n , and critical to cell survival and chromosome integrity. They shorten with cell division, and several components of the telomere complex, including telomerase (includes TERT, the reverse transcriptase, and TERC, the RNA component) and dyskerin (encoded by DKC1 ) with others counteract this physiologic attrition. Short telomeres are associated with premature cellular senescence, and may result in abnormal organ function due to tissue ageing, even of a subset of cells within that tissue. Patients with DKC who have bone marrow failure with associated immunodeficiency require HCT to rescue the hematopoietic/immunologic defect; however, outcomes are not very robust and require ongoing follow-up and improved approaches. One of the key diagnostic tests to identify abnormally short telomeres in patients with relevant clinical phenotypes is to use a combination of flow cytometry and fluorescence in situ hybridization (FISH), called flow-FISH ( Fig. 100.11 ), in various peripheral blood subsets. This test is sensitive for measurement of telomere length, especially if less than the first percentile compared to healthy controls, though some patients may show evidence of short telomeres before the onset of clinical symptoms. While telomere length may be measured in many peripheral leukocyte subsets, the most meaningful information may be obtained from analysis in lymphocyte and granulocyte subsets. Telomere length measurement is available in the clinical diagnostic setting in only two laboratories in North America, and therefore it is a highly specialized test. It has proven utility in clinical management of at least a subset of patients with short telomeres and associated clinical phenotypes, including selection of conditioning regimen for HCT.

Group 10. Phenocopies of primary immunodeficiencies

While inborn errors of immunity refers to genetic disorders of the immune system, within the IUIS classification, a category was included for disorders with no germline genetic defect, but rather caused by autoantibodies or somatic variants in genes associated with an immunodeficiency. These disease phenotypes mimicked those of germline immunodeficiencies and were thus called “phenocopies of inborn errors of immunity.” As would be expected with nongermline genetic defects, these diseases do not follow a Mendelian order of inheritance. This section focuses on autoantibody-associated phenocopies of errors of immunity, though somatic variants can result in phenocopies, and some examples have been provided in the Group 4 section (somatic FAS, NRAS, and KRAS defects associated with ALPS). Most phenocopies of PIDs are associated with anti-cytokine autoantibodies ( Table 100.7 ), and these are usually high-titer, neutralizing IgG antibodies associated with a specific phenotype. Diagnosis of autoantibody-mediated immunodeficiency requires measurement of the autoantibodies using immunoserology assays, either ELISA or multiplex bead-based assays, which detect ligand binding and offer assessment of the titer. However, since not all autoantibodies which bind ligand mediate neutralizing function, it is useful to have alternate methods that assess biological impact and clinical significance. Flow cytometry has been used to detect the functional consequences of certain anti-cytokine autoantibodies, such as those to interferon-γ (IFNγ) and GM-CSF. Consideration of a phenocopy of PID in the differential diagnosis depends on the clinical phenotype, age of the patient, and presence of negative genetic testing results (see Table 100.7 ).

Treatment of anti-cytokine autoantibodies is focused on management of clinical symptoms, removal of the autoantibody or halting its production. Therefore removal of proteinaceous material from lungs of patients with pulmonary alveolar proteinosis (PAP) caused by autoantibodies to GM-CSF by therapeutic bronchoalveolar lavage (BAL) is one example. Others include plasmapheresis, use of B cell–depleting therapies such as anti-CD20 antibodies, use of other immunomodulatory therapies, or treating the clinical consequences caused by autoantibodies.