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JMD 2004, Vol. 6, No. 2
Copyright © 2004 American Society for Investigative Pathology & Association for Molecular Pathology

The Molecular Pathology of Primary Immunodeficiencies

From the Division of Anatomic Pathology, Department of Pathology, University of Utah; and Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology, Salt Lake City, Utah

	Introduction

Top Introduction Classification Epidemiology Approach to Diagnosis Laboratory Assessment Diagnostic Criteria B Cell Immunodeficiencies... T Cell Immunodeficiencies CD3{epsilon} and CD3{gamma}... Severe Combined... T-B-SCID T-B+ SCID Other SCIDs Treatment of SCIDs Defects of Phagocytic Cells Other Immunodeficiencies Immunodeficiency with Albinism Conclusions References

 
Primary immunodeficiencies are a heterogeneous group of disorders, which affect cellular and humoral immunity or non-specific host defense mechanisms mediated by complement proteins, and cells such as phagocytes and natural killer (NK) cells.^{1, 2, 3} These disorders of the immune system cause increased susceptibility to infection, autoimmune disease, and malignancy. There are over 80 primary immunodeficiencies, many of which are very rare, and in most cases associated with inherited genetic defects. One in 500 individuals, in the United States, is born with a defect in some component of the immune system.⁴ In the majority of cases, the primary immunodeficiencies manifest in the first year of life.⁴ They can, however, present at any age, including adulthood.⁵ The advent of molecular genetic analyses now allows for the detection and confirmation of immunodeficiencies that were otherwise not severe enough during childhood to have led to a specific diagnosis. In addition, effective treatment for many disorders has led to increased survival of many children with primary immunodeficiency into adult life.

Recent advances in molecular techniques have led to the identification and characterization of more than 25 newly recognized immunological disease genes since 1997.^{6, 7} The identification of many genes responsible for primary immunodeficiencies has provided insights regarding the spectrum of clinical severity seen in a particular disorder and the phenotypic overlap resulting from mutations of different genes.

This review focuses on the molecular genetic features of primary immunodeficiencies with emphasis on the molecular pathophysiology of the diseases. In large part, the molecular detection of gene mutations leading to these diseases is carried out in research laboratories. Thus, information regarding comparison of analytical methods and prioritization of specific targets for study is largely lacking. Diagnostic perspectives for disease entities for which this information exists are included in their respective sections. We have organized the review into categories of B cell, T cell, severe combined immunodeficiencies, and defects of phagocytes and other miscellaneous immunodeficiencies. The clinico-pathological and immunological aspects are beyond the scope of this review and are available elsewhere. Various websites containing pertinent databases including those organized by the Pan-American Group for Immunodeficiencies (PAGID) http://www.clinimmsoc.org/pagid/, mutation registries, and the large European Society for Immunodeficiencies (ESID) registry which contains clinical data for over 7000 patients from 24 countries are listed in Table 1 .⁸ In addition, the website for GeneTests-GeneClinics which provide information regarding specific laboratories and the nature of the molecular assays is also shown in Table 1 .

	Classification

Top Introduction Classification Epidemiology Approach to Diagnosis Laboratory Assessment Diagnostic Criteria B Cell Immunodeficiencies... T Cell Immunodeficiencies CD3{epsilon} and CD3{gamma}... Severe Combined... T-B-SCID T-B+ SCID Other SCIDs Treatment of SCIDs Defects of Phagocytic Cells Other Immunodeficiencies Immunodeficiency with Albinism Conclusions References

	Epidemiology

Top Introduction Classification Epidemiology Approach to Diagnosis Laboratory Assessment Diagnostic Criteria B Cell Immunodeficiencies... T Cell Immunodeficiencies CD3{epsilon} and CD3{gamma}... Severe Combined... T-B-SCID T-B+ SCID Other SCIDs Treatment of SCIDs Defects of Phagocytic Cells Other Immunodeficiencies Immunodeficiency with Albinism Conclusions References

	Approach to Diagnosis

Top Introduction Classification Epidemiology Approach to Diagnosis Laboratory Assessment Diagnostic Criteria B Cell Immunodeficiencies... T Cell Immunodeficiencies CD3{epsilon} and CD3{gamma}... Severe Combined... T-B-SCID T-B+ SCID Other SCIDs Treatment of SCIDs Defects of Phagocytic Cells Other Immunodeficiencies Immunodeficiency with Albinism Conclusions References

The nature of the pathogens and sites of infections can provide insight as to the underlying immunodeficiency. Defects involving B cell function result in recurrent sinopulmonary infections, often with bacterial septicemia. The lack of antibody production may also increase susceptibility to invasive disease with enteroviruses, resulting in chronic viral meningitis, and giardiasis. T cells are essential for the control of viral and fungal disease, however they also provide helper function to B cells for effective antibody responses. Thus, T cell disorders present as combined T and B cell immunodeficiency with susceptibility to both bacterial and chronic, invasive viral, and fungal pathogens. Patients with disorders of granulocytes are susceptible to staphylococcal diseases and gram-negative infections.

The primary immunodeficiencies are commonly inherited disorders, thus a family history is one of the best diagnostic clues. Unfortunately, because these diseases are rare with low carrier frequencies, a negative family history does not rule out a primary immunodeficiency. Furthermore, occurrence of new mutations, especially for X-linked disorders, is so high that the majority of patients with proven X-linked immunodeficiency mutations have no history of affected male relatives.

	Laboratory Assessment

Top Introduction Classification Epidemiology Approach to Diagnosis Laboratory Assessment Diagnostic Criteria B Cell Immunodeficiencies... T Cell Immunodeficiencies CD3{epsilon} and CD3{gamma}... Severe Combined... T-B-SCID T-B+ SCID Other SCIDs Treatment of SCIDs Defects of Phagocytic Cells Other Immunodeficiencies Immunodeficiency with Albinism Conclusions References

 
Initial laboratory evaluation for immunodeficiency should include a minimum of tests that can be performed reliably by any laboratory.⁹ The initial screening should include a complete blood count and quantitation of serum IgG, IgM, and IgA levels. Laboratories should provide age-matched normal values for cell counts, immunoglobulin measurements and proper controls for functional studies. Other readily available tests are: 1) Quantification of blood mononuclear cell populations: T cells (CD3, CD4, CD8, TCR ß, TCR ); B cells (CD19, CD20, CD21, Ig); NK cells (CD16/CD56); monocytes (CD15); activation markers (HLA-DR, CD25, CD80 for B cells), CD154 for T cells. 2) T cell functional evaluation: delayed hypersensitivity skin tests (PPD, Candida, histoplasmin, and tetanus toxoid); proliferative response to mitogens (anti-CD3 antibody, phytohemagglutinin, concanavalin A) and allogeneic cells (mixed lymphocyte response); cytokine production. 3) B cell functional evaluation: natural or commonly acquired antibodies; response to immunization proteins and carbohydrate antigens; and quantitative IgG subclass determination. 4). Phagocyte function: reduction of nitroblue tetrazolium; chemotaxis assays, and bactericidal activity.

	Diagnostic Criteria

Top Introduction Classification Epidemiology Approach to Diagnosis Laboratory Assessment Diagnostic Criteria B Cell Immunodeficiencies... T Cell Immunodeficiencies CD3{epsilon} and CD3{gamma}... Severe Combined... T-B-SCID T-B+ SCID Other SCIDs Treatment of SCIDs Defects of Phagocytic Cells Other Immunodeficiencies Immunodeficiency with Albinism Conclusions References

 
The PAGID and ESID¹⁰ have established diagnostic criteria for immunodeficiencies which are divided into three categories: definitive, probable, and possible. These criteria provide objective guidelines that ensure that the same definitions are being used universally for diagnosis and clinical research studies. Patients with a definitive diagnosis are assumed to have a greater than 98% probability that in 20 years they would still be given the same diagnosis. Detection of a gene mutation is the most reliable method of making a diagnosis. In some disorders, the absence of the specific transcript or protein is diagnostic, although in others this may be hampered by transient or low levels of expression. The clinical and laboratory findings in several of the X-linked immunodeficiencies are sufficiently distinctive that if coupled with a family history that is specific to X-linked inheritance, a definitive diagnosis can frequently be made. In families with a known mutation in a particular gene, pre- or peri-natal testing can be used to establish a definitive diagnosis in a newborn or fetus. Table 4 highlights the major genetic abnormalities required for definitive diagnosis of primary immunodeficiency disorders.

View larger version (39K): [in this window] [in a new window]   Figure 1. Algorithm for evaluation and diagnosis of a patient with suspected severe combined immunodeficiency.

	B Cell Immunodeficiencies-Predominantly Antibody Defects

Top Introduction Classification Epidemiology Approach to Diagnosis Laboratory Assessment Diagnostic Criteria B Cell Immunodeficiencies... T Cell Immunodeficiencies CD3{epsilon} and CD3{gamma}... Severe Combined... T-B-SCID T-B+ SCID Other SCIDs Treatment of SCIDs Defects of Phagocytic Cells Other Immunodeficiencies Immunodeficiency with Albinism Conclusions References

Molecular and Cellular Defects
X-linked agammaglobulinemia was the first immunodeficiency to be characterized at the genetic level.¹³ XLA is caused by a block in B cell differentiation due to mutations involving the Bruton kinase gene, Btk, which encodes a tyrosine kinase that regulates the activity of signaling pathways by phosphorylation.^{14, 15} BTK is activated by immunoglobulins and other membrane receptors that in turn activate phospholipase C and calcium influx. Although the specific defect in the signaling pathway that impairs B-cell development is unknown, it is proposed that BTK is important in mediation of survival signals.¹⁶ The phenotypic heterogeneity seen in patients does not reflect the consequences of different mutations as most of the mutations identified thus far lead to complete absence of BTK protein. Modifier genes and environmental factors have been postulated to play a role in influencing the expression pattern of the B cell deficiency. Flow cytometric methods to detect expression of BTK protein expression¹⁷ and molecular diagnostic tests allow the detection of the carrier state and enable prenatal diagnosis of the disorder.¹⁸ However, since approximately one third to one half of XLA cases are sporadic, alternative mechanisms of diagnosis are necessary. Furthermore, deleterious mutations of the µ heavy chain gene,¹⁹ lambda 5/14–1 surrogate light chain gene,²⁰ Blnk gene which encodes an adaptor protein that has a critical role in pro-B to pre-B cell progression,²¹ and the CD79a gene²² have been identified in agammaglobulinemia patients.

Hyper-IgM Syndrome
Hyper-IgM syndrome (HIM) represents a group of distinct entities characterized by defective normal or elevated IgM in the presence of diminished IgG and IgA levels.²³ Seventy per cent of the cases are X-linked in inheritance,²⁴ and others are autosomal recessive.²⁵ Male patients with X-linked hyper-IgM have a history of recurrent pyogenic infections, and are particularly susceptible to Pneumocystis carinii. They are also prone to profound neutropenia, autoimmune hemolytic anemia, and thrombocytopenic purpura. Liver disease including sclerosing cholangitis, viral hepatitis as well as hepatic lymphoma are common and their frequencies increase with age.²⁶ The long-term survival rate for patients with XHIM is poor despite regular use of intravenous immunoglobulins. Less than 30% of the patients are alive at 25 years of age. Major causes of death include Pneumocystis carinii pneumonia early in life, liver disease, and malignancies in later life.²⁷ Allogeneic bone marrow transplantation²⁸ or non-myeloablative bone marrow transplantation from matched, unrelated donors²⁹ have been successful in the treatment of hyper-IgM syndrome.

Molecular and Cellular Defects
The genetic anomaly in X-linked hyper-IgM syndrome has been mapped to Xq26, and resides in mutations of the CD40 ligand gene now known as CD154.³⁰ CD40L is a member of the tumor necrosis factor family that binds to its receptor (CD40) expressed in B cells. Interactions between the CD40 ligand present on activated T cells and CD40 on B cells is required for productive isotype switching in B cells.³¹ The defect in X-linked hyper-IgM syndrome is a failure of isotype switch. Failure of this switch results in defective formation of germinal centers and immunoglobulin switching. Studies of CD40L (CD154) knockout mice that are also susuceptible to Pneumocystis carinii infections show that the defect of CD40L expression prevents CD40-mediated up-regulation of CD80/CD86 expression in B cells and other antigen presenting cells, ultimately resulting in poor T cell priming and defective type 1 immune response.³²

Another form of X-linked hyper-IgM is associated with ectodermal dysplasia (XHM-ED). Interestingly, the expression of CD40 and CD40L are normal but a specific missense mutation in the putative zinc-finger domain of the gene that encodes nuclear factor B (NF-B) essential modulator (NEMO, also known as IKK) have been described in these patients.³³ The mutations of NEMO prevent CD40L-mediated degradation of inhibitor of NF-B (IB-) resulting in defects in immunoglobulin class-switching, inability of antigen-presenting cells to synthesize the NF-B-regulated cytokines such as IL-12 and TNF- when stimulated with CD40L.³³

A minority of patients show an autosomal recessive inheritance pattern named hyper IgM-2. A mutation of the activation-induced cytidine deaminase (AID) gene, the product of which is selectively expressed in B cells from germinal centers³⁴ has been reported. There is a similar defect in immunoglobulin switching, however significant differences exist in that they have lymph nodes with hyperplastic germinal centers and patients do not suffer from opportunistic infections. The B cells characteristically exhibit few somatic mutations of the variable part of the rearranged immunoglobulin genes. The exact mechanism of how a member of a RNA editing enzyme family leads to the defect is unknown.

More recently, homozygous mutations of the CD40 gene leading to lack of surface expression of CD40 have been reported in another form of autosomal recessive hyper IgM. The clinical and immunological features are indistinguishable from those of the X-linked form.³⁵

Common Variable Immunodeficiency
Common variable immunodeficiency (CVID) refers to a heterogeneous group of disorders which are characterized by defective antibody formation.⁴ CVID is the most frequent of the primary immunodeficiency diseases among populations of European descent and affects both sexes equally.³⁶ The feature common to all patients is hypogammaglobulinemia, generally affecting all antibody classes but sometimes only IgG. Several modes of inheritance (autosomal recessive, autosomal dominant, and X-linked) have been reported, however sporadic cases are most common. The usual age at presentation is in the second or third decade of life. CVID is the most common clinically significant primary immunodeficiency disease that can present initially in adult life.⁵

The clinical manifestations of CVID include manifestations of antibody deficiency, ie, recurrent pyogenic sinopulmonary infections. Recurrent attacks of herpes simplex are common, and herpes zoster develops in about 20% of patients.³ Some patients may have unusual enteroviral infections with a chronic meningoencephalitis and a dermatomyositis-like illness. In addition, CVID patients are also prone to persistent episodes of diarrhea caused by Giardia lamblia. A high frequency of autoimmune diseases including rheumatoid arthritis, pernicious anemia, hemolytic anemia, thrombocytopenia, and neutropenia, is also seen.³ A syndrome resembling sarcoidosis can also affect some patients with CVID. The granulomas tend to involve the lung, liver, spleen, and conjunctivae. They are also at risk for inflammatory bowel diseases such as Crohn’s disease, celiac disease, and nodular lymphoid hyperplasia. A few patients present with opportunistic infections such as Pneumocystis carinii, mycobacteria, or fungal infections.

Molecular and Cellular Defects
The primary cause of CVID is not known. In part because CVID comprises a heterogeneous group of disorders, non-random recurrent cytogenetic abnormalities unique to CVID have not been identified. Current evidence suggests that the humoral defect in CVID patients is as a result of insufficient in vivo stimulus for B cell activation rather than an intrinsic inability of the B lymphocytes to undergo terminal differentiation into plasma cells.³⁷ Furthermore, circulating B cells from CVID patients failed to undergo somatic hypermutation in immunoglobulin-variable region genes, similar to cord blood B cells. They were also unable to produce IgA on engagement of the Ig receptor suggesting the presence of severe deficiency of switched memory B cells (CD27+ IgM–IgD–) in CVID patients.^{38, 39}

Abnormalities in T cell signaling and thus defective T cell to B cell interactions, are thought to underlie the diminished in vivo stimulation of B cell activation and differentiation into immunoglobulin secreting plasma cells.^{3, 7} Experimental evidence indicates that the molecular basis of abnormal B cell differentiation is varied. Some CVID patients have mutations interfering with the regulation of the expression of immunoglobulin genes.^{40, 41} Others have functional abnormalities of CD4⁺ (helper) cells or CD8+ (suppressor) T cells, as well as defective B cells⁴² and apoptosis.⁴³ An aberrantly spliced lck transcript, a key molecule in T cell receptor-mediated signaling, lacking the entire exon 7 associated with decreased expression of LCK protein has been described in a patient with CVID.⁴⁴ Low levels of serum interleukin 2 (IL-2) and interferon gamma (IFN-)⁴⁵ due to defects in these cells can contribute to hypogammaglobulinemia.

Recent studies have also implicated genes within the HLA complex predisposing to CVID; many patients have deletions of the C4A gene or possess rare alleles of the C2 gene. Both CVID and isolated IgA deficiency may affect different individuals of the same family, suggesting that they may be related disorders with a common genetic defect.⁴¹ A subset of male patients with CVID have mutations in the X-linked lymphoproliferative disease gene SH2D1A/SAP⁴⁶ suggesting a link between defects in the SAP gene to abnormal B cell development. The diagnosis is based on the exclusion of other known causes of humoral immune defects.

IgA Deficiency
Selective IgA deficiency is the most common form of immunodeficiency in the Western world, affecting approximately 1 in 600 individuals.⁴⁷ Only about one third of the patients are particularly prone to infections. Most patients have IgA levels below 5 mg/dl. The serum concentrations of the other immunoglobulins are usually normal, but patients have a high incidence of autoantibodies, many with allergies including food reactions, allergic conjunctivitis, rhinitis, urticaria, atopic eczema, and bronchial asthma.⁴⁸ In about two thirds of the cases, the deficiency does not lead to an increased occurrence of infections, whereas the remaining patients suffer from bacterial infections in both the upper and lower respiratory tract.

Molecular and Cellular Defects
The major role of IgA is to facilitate presentation of antigen to mucosal T cells. The pathogenesis of IgA deficiency involves a block in B cell differentiation that occurs due to defective interaction between T and B cells. This is demonstrated by the observation that IL-12 treatment can overcome IgA deficiency by providing adequate T cell priming in mice.⁴⁹ The pathogenesis of IgA deficiency is associated with genes within the major histocompatability complex such as HLA-B8, SC01, and DR3.⁵⁰ Other studies have implicated genomic polymorphisms in the tumor necrosis factor gene⁵¹ as a protective factor in IgA deficiency. The defect is manifested at the stem cell level as transfer of bone marrow from an IgA-deficient donor to a normal recipient results in IgA deficiency in the recipient.⁵²

Selective IgG Subclass Deficiencies
Selective deficiencies of IgG subclasses, with or without IgA deficiency, are caused by defects in several genes. IgG2 deficiency is most common in children, whereas adults more often have low levels of IgG3.⁴ Hyper-IgE syndrome is a rare immunodeficiency state characterized by recurrent skin and pulmonary abscesses and extremely elevated serum IgE levels. It is a single-locus disease with an autosomal dominant pattern of transmission with variable expressivity.⁵³ Genotypic analysis of 19 kindreds with multiple cases of hyper-IgE syndrome with polymorphic markers in a candidate region on human chromosome 4 suggests the proximal 4q region as a candidate locus for the disease.⁵⁴ Single-strand conformation polymorphism analysis followed by DNA sequencing revealed mutations in the subunit of the interleukin-4 receptor ⁵⁵ in 3 of 3 patients with hyper-IgE. The R576 is a novel IL-4 receptor allele causing a change from glutamine to arginine at position 576 (R576) in the cytoplasmic domain of the IL-4 receptor protein. However, this allele is also commonly found in those patients with atopic dermatitis suggesting that the mutation may predispose persons to allergic disease rather than a primary genetic cause of the disease.

	T Cell Immunodeficiencies

Top Introduction Classification Epidemiology Approach to Diagnosis Laboratory Assessment Diagnostic Criteria B Cell Immunodeficiencies... T Cell Immunodeficiencies CD3{epsilon} and CD3{gamma}... Severe Combined... T-B-SCID T-B+ SCID Other SCIDs Treatment of SCIDs Defects of Phagocytic Cells Other Immunodeficiencies Immunodeficiency with Albinism Conclusions References

 
Purine Nucleoside Phosphorylase Deficiency
The ubiquitous purine nucleoside phosphorylase (PNP) plays a key role in the purine salvage pathway.⁵⁶ PNP deficiency is a lethal, autosomal recessive disease that causes profound T cell deficiency with variable deficiencies in the humoral system. Patients with PNP deficiency experience recurrent bacterial, viral, and fungal infections usually beginning early in life. Immunodeficiency is accompanied by a neurological disorders and developmental retardation.⁵⁷

Molecular and Cellular Defects
The PNP protein reversibly catalyzes the degradation of the purine nucleosides inosine and deoxyinosine to hypoxanthine and that of guanosine and deoxyguanosine to guanine. Deficiency of nucleoside phosphorylase leads to a dysfunction of the purine salvage pathway and accumulation of deoxyguanosine triphosphate (dGTP) which is preferentially toxic to T cells compared to B cells.⁵⁸ Mutations result in truncated proteins leading to variable levels of enzymatic activity.⁵⁹ Recent mouse models of purine nucleoside phosphorylase deficiency suggest that accumulation of dGTP in the mitochondria result in impaired mitochondrial DNA repair with enhanced sensitivity of T cells to spontaneous DNA damage leading to T cell apoptosis.⁶⁰

Zap-70 Deficiency
ZAP-70 deficiency is inherited in an autosomal recessive manner. Recurrent and opportunistic infections occur within the first year of life.⁴ There is lymphopenia involving CD8+ T cells, with normal numbers of non-functional CD4⁺ T cells and severe combined immunodeficiency.⁶¹ Zap-70 (-associated polypeptide of 70 kd) is a tyrosine kinase that binds to the TCR’s phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) sequences. Recruitment of ZAP-70 to the TCR and its subsequent phosphorylation and activation, largely by Lck, is essential for downstream signaling events.⁶² In Zap-70 deficiency, a mutation within the kinase domain of ZAP-70 results in abolished protein expression. Signaling through TCR is defective, influencing T cell development with a selective block in positive selection of CD8+ cells. There is failure of peripheral CD4⁺ T cell proliferative response to mitogens or anti-CD3 antibody. By contrast, the activity of natural killer cells, B cells, and serum immunoglobulin levels are normal. The severe combined immunodeficiency associated with ZAP-70 deficiency is fatal unless treated by allogeneic bone marrow transplantation.

	CD3 and CD3 Deficiencies

Top Introduction Classification Epidemiology Approach to Diagnosis Laboratory Assessment Diagnostic Criteria B Cell Immunodeficiencies... T Cell Immunodeficiencies CD3{epsilon} and CD3{gamma}... Severe Combined... T-B-SCID T-B+ SCID Other SCIDs Treatment of SCIDs Defects of Phagocytic Cells Other Immunodeficiencies Immunodeficiency with Albinism Conclusions References

 
Rare congenital immunodeficiencies are caused by mutations in the ⁶³ and ⁶⁴ subunits of CD3. They are inherited in an autosomal recessive (11q23) manner and result in moderate to severe immunodeficiency due to decreased circulating CD3+ T cells, and poor responses to T cell mitogens. They show decreased lymphocyte membrane expression of TCR/CD3.

	Severe Combined Immunodeficiencies (SCIDs)

Top Introduction Classification Epidemiology Approach to Diagnosis Laboratory Assessment Diagnostic Criteria B Cell Immunodeficiencies... T Cell Immunodeficiencies CD3{epsilon} and CD3{gamma}... Severe Combined... T-B-SCID T-B+ SCID Other SCIDs Treatment of SCIDs Defects of Phagocytic Cells Other Immunodeficiencies Immunodeficiency with Albinism Conclusions References

 
The severe combined immunodeficiencies (SCIDs) syndrome is characterized by gross impairment of both the humoral and cell-mediated immunity and by susceptibility to overwhelming fungal, bacterial, and viral infections. The syndrome comprises a heterogeneous group of primary immunodeficiencies associated with various defects of the immune system involving T, B, and sometimes natural killer cells.

	T-B-SCID

Top Introduction Classification Epidemiology Approach to Diagnosis Laboratory Assessment Diagnostic Criteria B Cell Immunodeficiencies... T Cell Immunodeficiencies CD3{epsilon} and CD3{gamma}... Severe Combined... T-B-SCID T-B+ SCID Other SCIDs Treatment of SCIDs Defects of Phagocytic Cells Other Immunodeficiencies Immunodeficiency with Albinism Conclusions References

 
RAG1 Deficiency, RAG2 Deficiency, and Omenn Syndrome
Two related deficiencies of recombination activating genes, RAG1 and RAG2 result in a spectrum of severe combined immunodeficiencies called RAG1/RAG2 deficiency and Omenn syndrome. Both conditions are inherited as autosomal recessive diseases. RAGs are crucial proteins that play a role in activating V(D)J recombination in the B cell and T cell receptor genes required for generation of the diversity of the recognition sites. Absence or defective V(D)J recombination results in the arrest of B and T cell development such that most of the circulating lymphocytes in affected patients are natural killer cells. Mutations that lead to total absence of RAG1 or RAG2 gene product (null mutations) are known to lead to severe combined immune deficiency without mature lymphoid cells,⁶⁵ whereas mutations that result in partial V(D)J recombinase activity due to missense mutation on at least one allele lead to Omenn syndrome.⁶⁶ A clinical phenotype similar to Omenn syndrome has been also seen as a result of engraftment of maternal T cells as a complication of a transplacental transfusion.⁶⁷ Thus, analysis of the RAG genes by direct sequencing may be an effective way to provide accurate diagnosis of RAG-deficient as opposed to RAG-independent V(D)J recombination defects.

Omenn syndrome is an immunodeficiency disease with autoimmune features resembling graft-versus-host disease.⁶⁸ It is characterized by the absence of circulating B cells and infiltration of many organs by activated oligoclonal T cells. Most present with infantile diffuse erythrodermia, alopecia, protracted diarrhea, lymphadenopathy, hepatosplenomegaly, fever, hypereosinophilia, and elevated serum IgE levels leading to failure to thrive and ultimately death. Protein loss due to diarrhea and exudative erythrodermia often leads to generalized edema. The condition is fatal unless it is corrected by bone marrow transplantation.

Molecular and Cellular Defects
Molecular studies of patients with Omenn syndrome⁶⁶ have revealed missense mutations of the RAG1 and RAG2 genes which result in impaired but not absent rearrangement of the B cell receptor and T cell receptor genes. These findings indicated that the immunodeficiency manifested in patients with Omenn syndrome arises from mutations of RAG1 and RAG2 genes that decrease the efficiency of V(D)J recombination. More recent studies have shown that some cases of Omenn syndrome are characterized by mutations identical to those seen in T-B-SCID patients suggesting the role of additional factors that may play a part in the development of Omenn syndrome.⁶⁹ An analysis of TCR repertoire demonstrates exquisite restriction of TCR clonotypes indicating antigen-driven proliferation of T cells. The TCR from some patients lacked N- or P-nucleotide insertions and used proximal variable and joining gene segments, suggesting abnormal intrathymic T cell development. Abnormal assembly of gene segments and truncated rearrangements within non-productive alleles also suggest abnormalities in the TCR rearrangement mechanism.⁷⁰

Adenosine Deaminase Deficiency
Adenosine deaminase (ADA) deficiency accounts for about half of the autosomal recessive forms of SCIDs. It is one of the most severe immunodeficiencies and is associated with severe depletion of B cells, T cells, and NK cells. It is the second-most prevalent form of SCID accounting for approximately 20% of the group. Affected individuals die from overwhelming opportunistic infections within the first few months of life if untreated. ADA follows purine nucleoside phosphorylase in purine nucleoside catabolism, but deficiency in this enzyme causes even more severe symptoms than PNP deficiency that is largely limited to T cells. In addition to immunological defect, most patients with ADA deficiency also have skeletal abnormalities.

Molecular and Cellular Defects
ADA, an enzyme of the purine salvage pathway, catalyzes the conversion of adenosine and 2'-deoxyadenosine to inosine and 2'-deoxyinosine, respectively. ADA deficiency results in accumulation of the toxic metabolites, adenosine and deoxyadenosine, which accumulate in the cells of affected patients. Deficiency of adenosine deaminase results in a profound decrease in the maturation of lymphocyte precursors.⁷¹ Defective enzymatic activity in lymphocyte precursors results in selective accumulation of dGTP that inhibits cellular division. Typical patients are diagnosed by age 6 months and rarely survive beyond 1 to 2 years unless immune function is restored by stem cell transplation or enzyme replacement therapy. Partial ADA deficiency has been found in less severely affected patients with delayed or late/adult onset of immune deficiency^{72, 73} and autoimmunity.⁷¹ About 70 known mutations, the majority missense, span the 32-kb, 12 exon ADA gene on chromosome 20q.⁷⁴ Expression studies demonstrate that missense mutations appear more deleterious yielding 0.005% to 0.6% of the ADA activity from wild-type cDNA whereas splicing mutations are associated with mild severity.⁷⁴ Studies of ADA-deficient mice show multiple defects of T cells including increased thymic apoptosis as well as defective T cell receptor signaling.⁷⁵ Treatment of ADA deficiency by gene transfer has also been attempted.⁷⁶ Flow cytometric analysis of ADA expression can be used to assess the expression of ADA in follow-up of patients treated in clinical gene transfer protocols.⁷⁷

	T-B+ SCID

Top Introduction Classification Epidemiology Approach to Diagnosis Laboratory Assessment Diagnostic Criteria B Cell Immunodeficiencies... T Cell Immunodeficiencies CD3{epsilon} and CD3{gamma}... Severe Combined... T-B-SCID T-B+ SCID Other SCIDs Treatment of SCIDs Defects of Phagocytic Cells Other Immunodeficiencies Immunodeficiency with Albinism Conclusions References

 
SCIDs with lack of circulating T cells but a normal number of B cells accounts for 30 to 50% of all cases of human SCIDs. The X-linked form is the most common form that provides rationale for the prevalence of SCID being three times as common in boys as in girls. The second most common variant is autosomal recessive and due to mutations of the JAK3 gene.

X-Linked SCID
This group of immunodeficiency diseases appears to be as a result of several genetic causes. Affected infants are susceptible to recurrent severe infections caused by a wide range of pathogens including, Candida albicans, Pneumocystis carinii, Pseudomonas, cytomegalovirus, and varicella. Death from varicella, herpes, adenovirus, or cytomegalovirus may occur very soon after infection. Infants with severe combined imunodeficiency invariably have profound lymphopenia.³ The number of natural killer cells may be normal or high. In contrast to the autosomal recessive forms of SCID in which both T and B cells are profoundly deficient, the X-linked form of SCID is characterized by the presence of normal number of peripheral blood B-cells.

Molecular and Cellular Defects
Approximately 50% of patients with SCID have an X-linked recessive pattern of inheritance. It has been mapped to Xq13.⁷⁸ These patients have a mutation in the common cytokine receptor gamma chain gene that encodes a shared, essential component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15.⁷⁹ The mutation results in the expression of the gamma chain which exhibits reduced binding to JAK3.⁸⁰ Thus, the early lymphoid progenitor cells lacking intact interleukin receptors, fail to be stimulated by these growth factors that are vital to the normal development of T and B cells. Affected newborns are profoundly deficient in thymic size and there is progressive T cell deficiency resulting from ineffective rescue from apoptosis or replication senescence.⁸¹ Prenatal mutational screening by single-strand conformational polymorphism, heteroduplex analysis and dideoxy fingerprinting (ddF) followed by direct sequencing for mutations in the gamma chain reveal that ddF is the most sensitive method for detection of heterozygous mutations.⁸² Allogeneic stem cell transplantation even in utero, as well as ex vivo gene therapy can correct the immunodeficiency in these patients.⁸³

JAK3 Deficiency
Mutations of the JAK3 gene lead to a form of non-X-linked autosomal recessive form of SCID. Clinically, they resemble infants with X-linked SCID with elevated levels of B cells and very low levels of T cells and natural killer cells in the blood.⁷⁹ The JAK3 gene maps to chromosome 19p12–13.1 and encodes the JAK3 protein, an intracellular tyrosine kinase, which is crucial for signal-transmission of cytokine receptors to signal transducers and activators of transcription (STATs). Once recruited into the cytokine receptor complex, STATs are phosphorylated and then translocated into the nucleus to regulate transcription at multiple sites. As a result, there is almost complete absence of JAK3 kinase activity with impairment of IL-2 and IL-4 signaling.^{84, 85} Due to the multiple cytokines using this signaling pathway, an early and severe block in T and natural killer cell development combined with impaired B cell function is observed. The identification of the genomic organization of the human JAK3 gene into 23 exons⁸⁶ has made rapid mutation detection and prenatal diagnosis feasible.⁸⁷ Twenty-seven unique mutations have been identified that affect all seven structural Jak homology (JH) functional domains. The ability to test the function of a specific and expressed mutant in a phosphorylation assay using physiological substrates (JAK3 and STAT5) has enabled the verification of the consequences of the observed mutations. While some mutations result in absence of protein activity, others such as the C759R substitution results in constitutive phosphorylation of JAK3 which cannot be up-regulated by cytokine stimulation and thus block signal transduction.⁸⁸ Thus, demonstration of JAK3 protein expression by Western blot does not rule out functional JAK3 deficiency until cytokine-induced phosphorylation of JAK3 itself and/or STAT5 is excluded. JAK3 deficiency is a candidate for gene therapy. In vitro biochemical correction of JAK3-deficient human B-cell derived cell lines has been accomplished.⁸⁹

	Other SCIDs

Top Introduction Classification Epidemiology Approach to Diagnosis Laboratory Assessment Diagnostic Criteria B Cell Immunodeficiencies... T Cell Immunodeficiencies CD3{epsilon} and CD3{gamma}... Severe Combined... T-B-SCID T-B+ SCID Other SCIDs Treatment of SCIDs Defects of Phagocytic Cells Other Immunodeficiencies Immunodeficiency with Albinism Conclusions References

 
Bare Lymphocyte Syndrome Type I (TAP1 and TAP2 Deficiency)
Bare lymphocyte syndrome is characterized by a severe decrease of HLA class I and/or class II molecules. Patients show reduced numbers of CD8+ T cells and lack natural killer cell activity. HLA class I expression depends on the formation of a peptide-loading complex composed of class I heavy chain, ß₂-microglobulin, the transporter associated with antigen processing (TAP) and tapasin which links TAP to the heavy chain. Type I bare lymphocyte syndrome is caused by a deficiency in the TAP proteins encoded by genes within the major histocompatibility complex and plays a role in presentation of antigenic peptides to T cells. TAP transports peptides from the cytoplasm into the inner lumen of the endoplasmic reticulum and thus defects in TAP induce poor peptide loading on class I heavy chains. The TAP complex is a composed of TAP1 and TAP2 which, via the ATP-binding cassette transporter, translocates peptides from the cytosol to the waiting MHC class I molecules in the endoplasmic reticulum. TAP1 and TAP2 deficiency have identical clinical presentations that manifest within the first 6 years of life with recurrent bacterial infections of the upper respiratory tract.

Molecular and Cellular Defects
Mutations of both TAP1 and TAP2 genes result in deficient expression of class I HLA proteins on the cell surface with defects in natural killer cell cytotoxicity.^{90, 91} A novel genetic cause of type I bare lymphocyte syndrome has been identified within the Tapasin gene resulting from a deletion of 4 exons by Alu-mediated recombination.⁹²

Bare Lymphocyte Syndrome Type II (MHC Class II Deficiency)
The cell surface glycoproteins of the MHC class II are crucial players in the immune response. Defective expression of major histocompatibility complex class II molecules account for 5% of SCID.⁹³ Children with autosomal recessive form of hereditary MHC class II deficiency, or bare lymphocyte syndrome type II are extremely susceptible to bacterial, viral, and fungal infections, beginning in the first year of life. Mortality rate is high, with most children dying from overwhelming infections by the age of 4 years.⁹⁴

Molecular and Cellular Defects
The genetic lesions responsible for this syndrome do not lie within the MHC-II locus itself, but reside instead in genes encoding transcription factors controlling MHC-II expression. Different subtypes of human MHC II molecules are transcribed from TATA-less promoters that contain conserved S, X, and Y boxes. Protein complexes that bind to these proximal promoter elements attract the class II transactivator (CIITA) by an unknown mechanism. S and X boxes bind a tripartite regulatory factor X (RFX) complex, while the Y box binds the nuclear factor Y (NFY) complex. While the genes for MHC-II determinants remain intact, different mutations have been found in four trans-acting factors, RFX5, RFXAP, RFXANK(B), and CIITA.⁹⁵ MHC II molecules expressed on the surface of B cells present processed peptide fragments to the TCR of CD4 + T helper cells, triggering the antigen-specific T cell response. The regulatory factor (RF) X, a complex binding to the X-box of MHC-II promoters in the nucleus, is mutated in one type.

CD4 +T cells are decreased in all forms, although circulating lymphocyte numbers are normal and immunoglobulin numbers can also be decreased. Mutant forms of CIITA and all of the known subunits of RFX have been found among patients with the bare lymphocyte syndrome because all mutations cause absolute deficiency of MHC class II proteins.^{96, 97}

CD45 Deficiency
A molecular defect of CD45 is a rare cause of severe combined immunodeficiency with an autosomal recessive inheritance.^{98, 99} CD45 is an abundant transmembrane tyrosine phosphatase expressed on all leukocytes and is required for efficient lymphocyte signaling, integrin-mediated adhesion, and migration of immune cells. Mutations leading to loss of a component of the extracellular domain of CD45 result in very low circulating T cells but a normal number of B cells. The T cells are unresponsive to mitogens and serum immunoglobulin levels usually decrease with age. A homozygous 6-bp deletion in the CD45 gene resulting in a loss of glutamic acid 339 and tyrosine 340 in the first fibronectin type II module of the extracellular domain of CD45 has been reported.⁹⁸ A male patient with a deficiency in CD45 due to a large deletion at one allele and a point mutation at the other resulting in aberrant splice site⁹⁹ has also been reported.

	Treatment of SCIDs

Top Introduction Classification Epidemiology Approach to Diagnosis Laboratory Assessment Diagnostic Criteria B Cell Immunodeficiencies... T Cell Immunodeficiencies CD3{epsilon} and CD3{gamma}... Severe Combined... T-B-SCID T-B+ SCID Other SCIDs Treatment of SCIDs Defects of Phagocytic Cells Other Immunodeficiencies Immunodeficiency with Albinism Conclusions References

 
SCIDs have been among the first diseases to be cured by bone marrow stem cell transplantation, and there is no need for pretransplant immunosuppression. Preliminary studies show that they are also good candidates for gene therapy. Retroviral reconstitution of the gene defect into autologous marrow hematopoeitic cells have led to full correction of X-linked SCIDs.¹⁰⁰

	Defects of Phagocytic Cells

Top Introduction Classification Epidemiology Approach to Diagnosis Laboratory Assessment Diagnostic Criteria B Cell Immunodeficiencies... T Cell Immunodeficiencies CD3{epsilon} and CD3{gamma}... Severe Combined... T-B-SCID T-B+ SCID Other SCIDs Treatment of SCIDs Defects of Phagocytic Cells Other Immunodeficiencies Immunodeficiency with Albinism Conclusions References

 
Most congenital phagocytic disorders are diagnosed in the first year of life, but leukocyte adhesion deficiencies and chronic granulomatous disease may not be diagnosed until adulthood. Chronic granulomatous disease tends to exhibit increased susceptibility to catalase-positive organisms, whereas defects of the interferon--interleukin 12-pathway are characteristically associated with mycobacteria and other intracellular pathogens. Myeloperoxidase deficient individuals will generally not be susceptible to infections except for patients with diabetes who develop severe candidiasis.

Cyclic Neutropenia
Cyclic neutropenia occurs sporadically and by an autosomal-dominant inheritance pattern. The typical presentation is that of recurrent, severe neutropenia (an absolute neutrophil count of less than 200 cells per cubic millimeter) lasting 3 to 6 days of every 21-day period. Diagnosis depends on serial measurements of absolute neutrophil counts over a period of several weeks.¹⁰¹ Patients are usually asymptomatic, however, during periods of severe neutropenia, they develop recurring episodes of fever, aphthous ulcers, gingivitis, and cellulitis. Deep tissue infections and bacteremia from Clostridium species are the most serious complications.

Molecular and Cellular Defects
Although neutropenia in these disorders has been attributed to impaired or ineffective neutrophil production, the molecular and cellular basis for these diseases has remained largely unknown. Positional cloning studies have led to the mapping of candidate genes to chromosome 19p13.3, a region containing the genes for three neutrophil proteases: azurcidin, proteinase 3, and neutrophil elastase. Sequence analysis of the PCR-amplified genomic DNA revealed that all affected members in 13 families and one sporadic case of cyclic neutropenia harbored a mutation within the neurophil elastase gene (ELA2), with most of the mutations occurring at the junction of exons 4 and 5.¹⁰² Neutrophil elastase is a serine protease chiefly synthesized by promyelocytes. The mutations affect the catalytic site of the enzyme, resulting in inactive elastase.¹⁰³ The link between inactive elastase and the cyclic changes in the level of neutrophils remains to be determined. However, defects in the granulocyte colony-stimulating factor signaling pathway is thought to destabilize normal steady-state conditions and increase the number of circulating lymphocytes, reticulocytes, and platelets during neutropenia.¹⁰⁴

Subsequent studies also showed that 90% of patients with classic congenital neutropenia also had mutations in ELA2 gene, however a greater diversity of mutations was found.¹⁰⁵ Those mutations associated with cyclic neutropenia occur in proximity to the active site and the binding pocket for the enzyme’s substrate whereas the mutations responsible for congenital neutropenia would be predicted to change the molecular folding of the protein possibly affecting the storage of the enzyme in the primary granules.¹⁰⁶

Severe Congenital Neutropenia
Severe congenital neutropenia is a heterogeneous disorder related to cyclic neutropenia. There is severe neutropenia with an absolute neutrophil count of less than 500 cells per cubic milliter, recurrent bacterial infections, and absence of myeloid maturation with arrest at the promyelocyte stage.¹⁰⁷ The disease manifests in the first months of life with infectious complications from Staphylococcus aureus and Burkholderia aeruginosa resulting in cellulitis, perirectal abscess, meningitis, and stomatitis.¹⁰⁸ The disease is more severe than cyclic neutropenia and is three to four times more common.¹⁰⁹ The disease was initially described as an autosomal recessive disease, however it can occur sporadically and as an autosomal dominant disorder.

Molecular and Cellular Defects
The underlying genetic abnormality in severe congenital neutropenia is largely unknown. In about 10% of the patients, a heterozygous mutation of the granulocyte colony-stimulating factor receptor is identified. Germline mutations of the gene encoding neutrophil elastase (ELA2) have been observed in a large percentage of (22 of 25) patients studied according to a recent report.¹⁰⁵ This gene mutation has been found to play a role in other neutropenic disorders including cyclic neutropenia, but not in Shwachman-Diamond syndrome patients. Accelerated apoptosis of neutrophil precursors is a common feature of both cyclic and severe congenital neutropenia. Nevertheless, the mechanism by which mutations of the ELA2 gene cause the premature death of these cells is unclear.¹¹⁰ The enzyme neutrophil elastase is synthesized in neutrophil precursors early in the process of primary granule formation. It is currently presumed that the mutant elastase functions aberrantly within the cells to accelerate apoptosis of the precursors resulting in ineffective and oscillatory production. Although mutations in ELA2 may be necessary for the phenotype of congenital neutropenia, it may not be sufficient.¹¹¹ In most patients, treatment with G-CSF diminishes the number of infections.^{107, 112} ELA2 mutations may provide a background for the G-CSF receptor mutations that occur in the transformation to acute myeloid leukemia. More recently, a novel mutation affecting the the GTPase binding domain of the Wiskott-Aldrich syndrome protein (WASP) has been described in an X-linked form of severe congenital neutropenia.¹¹³

Shwachman-Diamond Syndrome
Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency, skeletal abnormalities, bone marrow dysfunction, and recurrent infections.¹⁰⁸ Neutropenia, either cyclic or intermittent, with or without pancytopenia can occur.¹¹⁴ Recurrent infections involving sinuses, lungs, bones, skin, and urinary tract begin during the first year of life.¹⁰⁸ Patients have increased risk of bone marrow aplasia, myelodysplasia, and myeloid leukemia.¹¹⁵

A variety of immune defects have been observed including decreased in vitro B lymphocyte proliferation, lack of specific antibody production, low numbers of CD4⁺ T cells and decreased numbers of natural killer cells.¹¹⁶ Additionally, there is hyperactivation of the Fas-mediated apoptotic pathway resulting in higher tendency of bone marrow mononuclear cells to undergo apoptosis.¹¹⁷ The significant immune dysfunction seen in these patients suggests that Shwachman-Diamond syndrome may involve a marrow defect accounting for aberrant function of hematopoietic and lymphopoietic lineages. The mapping of the SDS locus to the centromeric region of chomosome 7 (7p10–7q11)¹¹⁸ is interesting as both myelodysplastic syndrome and AML frequently exhibit monosomy 7, isochromosome 7, and deletion of 7q.¹¹⁹

More recently, mutations involving a previously uncharacterized gene, SBDS, have been identified in patients with Shwachman-Diamond syndrome.¹²⁰ The mutations are located in the interval of 1.9 cM at 7q11 resulting from gene conversion involving exon 2 in 89% of unrelated individuals with Shwachman-Diamond syndrome (141 of 158 families) with 60% (95 of 158 families) carrying two converted alleles. SDBS encodes a predicted protein of 250 amino acids and thought to be involved in RNA processing. The complex and pleiotropic phenotype associated with this syndrome suggests however, the presence of several mutations responsible for the disease.

Leukocyte Adhesion Defect
Leukocyte adhesion deficiency is a rare, autosomal recessive genetic disorder in which neutrophils fail to mobilize and migrate to sites of injury. There is delayed separation of the umbilical cord in infancy, followed by severe, scarring skin infections, gingivitis, and systemic bacterial infections. Because of abnormal aggregation and migration, even when there is no infection, the patients have twice the normal number of neutrophils in the peripheral blood.¹²¹

Molecular and Cellular Defects
The gene defects associated with this disorder involve CD18 and CD11c, both of which are components of surface integrin complexes that are essential for neutrophil aggregation and attachment to endothelial surfaces.¹²² Leukocyte adhesion deficiency type 1 is an autosomal recessive disorder resulting from a lack of ß₂ integrin adhesion molecules on neutrophils.¹²³

The second type of leukocyte adhesion deficiency is a defect of carbohydrate fucosylation and is associated with growth retardation, dysmorphic features, and neurological deficits.¹²⁴ These patients lack CD15s, sialyl-Lewis^X, a ligand for the selectin family. In these patients, there is no fucosylation of other glycoconjugates that are required for interactions with P-selectins and E-selectins on endothelial cells.¹²⁵ The genetic defect has not been determined, however treatment with oral fucose has reduced the frequency of infections and fevers.¹²⁵

Rac2 Deficiency
Dominant-negative mutations resulting in deficiency of ras-related C3 botulinum toxin substrate