Genes that Matter…™
Reviews in Immunology





Severe Combined Immunodeficiency (SCID)
including Omenn Syndrome and Zap-70 Deficiency

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Severe Combined Immunodeficiency (SCID)
including Omenn Syndrome (SCID/OS) and Zap-70 deficiency


Frequently used abbreviations: ADA - adenosine deaminase; Ado - adenosine; D - diversity; dAdo - 2'-
deoxyadenosine; dATP - deoxyadenosine triphosphate; GVHD - graft-versus-host disease; HSCT - hematopoietic
stem cell transplantation; IL - interleukin; IL-7R - interleukin-7 receptor; J - joining; Jak - Janus kinase; NHEJ -
non-homologous end-joining; NK - natural killer cell; OS -Omenn Syndrome RAG1/2 - recombination-activating
proteins 1/2; RSS - recombination signal sequences; RS-SCID - radiation-sensitive SCID; SCID - Severe Combined
Immunodeficiency; STAT - signal transducer and activator of transcription proteins; TCR - T-cell receptor; V -
variable; XSCID - X-linked SCID; Zap-70 - ζ-chain associated protein of 70 kDa


Introduction

Severe Combined Immunodeficiency (SCID) is characterized by severe lymphopenia and lack of adaptive immunity
and, if untreated, leads to death through infection. SCID occurs with an estimated incidence of 1 in 75,000 births (1)
and is considered a pediatric emergency because of the potentially lethal outcome of recurrent or persistent
infections suffered by SCID patients. Several monogenic causes with different modes of inheritance have been
identified for SCID (reviewed in 2).

Genetic testing for SCID can allow distinction between the various forms of this syndrome. Knowledge of the
defective gene may have implications for treatment and prognosis. This knowledge may also enable more
effective genetic counseling, in addition to facilitating identification of asymptomatic carriers and timely
initiation of treatment in affected descendants of carriers.


Types and Causes of SCID

Depending on the underlying genetic defect, four different primary phenotypes associated with SCID have been
characterized (for comprehensive reviews, refer to references 1-3). Categorization is based on the classes of
lymphocytes that are absent or severely reduced. T-cell lymphopenia is generally common to all forms of SCID, but
levels of B and natural killer (NK) cells vary depending on the genetic defect. For certain rare subtypes of SCID, T
cells may be present, but their function is impaired.

Table 1
Lymphocyte
Associated Mode of
Gene
Gene Product
Phenotype
Inheritance
IL2RG
Common γ chain of IL receptors (γc)
X-linked recessive
T− B+ NK−
JAK3
Janus kinase 3 (Jak3)
autosomal recessive
ADA
Adenosine deaminase (ADA)
T− B− NK−
autosomal recessive
RAG1
Recombination-activating protein 1 (RAG1)
autosomal recessive
RAG2
Recombination-activating protein 2 (RAG2)
T− B− NK+
autosomal recessive
DCLRE1C
DNA-cross-link repair protein 1C (Artemis)
autosomal recessive
IL7R
IL-7 receptor α chain (IL-R α, CD127)
autosomal recessive
CD3D
CD3 δ chain
T− B+ NK+
autosomal recessive
CD3E
CD3 ε chain
autosomal recessive
ZAP70
ζ-associated protein of 70 kDa (Zap-70)
T+(CD4+CD8−)B+NK+
autosomal recessive

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T−B+NK− SCID
T−B+NK− SCID is the most common type of SCID. It is most often caused by X-linked recessive mutations in IL2RG
(XSCID), which encodes the γ chain (γc) common to several cytokine receptors such as IL-2R, IL-4R, IL-7R, IL-9R,
IL-15R, and IL-21R (reviewed in 4). T−B+NK− SCID has also been associated with autosomal recessive mutations in
JAK3, which codes for the γc-associated tyrosine kinase known as Janus kinase 3 (Jak3) (reviewed in 5). Mutations
in IL2RG or JAK3 lead to defective signaling through the γc receptors, resulting in an absence of both T cells and NK
cells. B cells are present at normal levels, but have impaired function.

Ligand binding to cytokine receptors containing the γc subunit results in activation of Jak3. Activated Jak3
phosphorylates the cytoplasmic domain of the cytokine receptor, creating a binding site for the signal transducer and
activator of transcription (STAT) proteins. Upon binding, STAT proteins are also phosphorylated by Jak3, resulting in
their dimerization and translocation to the nucleus where they activate transcription of various genes necessary for
the growth and differentiation of lymphocytes (reviewed in 4, 6). Mutations in either γc or Jak3 disrupt signaling of all
γc-dependent cytokines. Defective IL-7 signaling is thought to be primarily responsible for the T-cell deficiency
observed in patients with T−B+NK− SCID (7), while failed IL-15 signaling leads to the NK-cell deficiency (8, 9).
Although B cells are present at normal or elevated levels, B-cell activation, maturation, and class switch
recombination are impaired, probably due to the combined effects of defective IL-4 and IL-21 signaling as well as
failure of T-cell help (10). The T-cell lymphopenia associated with T−B+NK− SCID allows infection by opportunistic
organisms, while the lack of NK cells causes susceptibility to pathogens such as viruses. The defect in B-cell function
further contributes to susceptibility to recurrent infections.

T−B−NK− SCID
T−B−NK− SCID results from a purine metabolism defect due to autosomal recessive mutations in ADA, the gene
encoding adenosine deaminase. Defects in adenosine deaminase allow accumulation of cytotoxic adenosine
derivatives in lymphoid organs, resulting in the death of lymphocyte precursors and a lack of T cells, B cells, and NK
cells (reviewed in 11).

Adenosine deaminase (ADA) catabolizes adenosine (Ado) and 2’-deoxyadenosine (dAdo) generated by apoptotic
cells (reviewed in 11, 12). The large number of apoptotic cells in the lymph nodes, thymus, and bone marrow
continually generate high concentrations of Ado and dAdo. Breakdown of dAdo, in particular, is important because its
accumulation is highly toxic to lymphoid cells. Excess dAdo causes an increase in levels of deoxyadenosine
triphosphate (dATP). Increased dATP levels block DNA synthesis by inhibiting ribonucleotide reductase (13) and
trigger lymphocyte apoptosis by facilitating apoptosome formation (14, 15). It is thought that the pro-apoptotic effects
of elevated levels of dAdo and dATP are the primary cause of T-, B-, and NK-cell deficiencies observed in ADA-
related SCID patients. The resulting combined B-cell, T-cell, and NK-cell lymphopenia allows severe, persistent, or
recurrent infections with all types of pathogens, including pyogenic and opportunistic bacteria, as well as viruses.

T−B−NK+ SCID
T−B−NK+ SCID is caused by autosomal recessive mutations in at least three genes necessary for antigen receptor
rearrangement, RAG1, RAG2, and DCLRE1C (ARTEMIS). Defects in these genes lead to impaired development of
both B and T cells, while NK-cell development is normal (reviewed in 1). Notably, mutations in RAG1, RAG2, or
DCLRE1C allowing limited production of B and T cells result in a condition that is clinically distinct from SCID,
commonly known as Omenn Syndrome (OS) (16, 17) (see below).

RAG1 and RAG2 encode the recombination-activating proteins RAG1 and RAG2, respectively. These proteins play a
fundamental role in the rearrangement of the antigen-binding domain of B- and T-cell receptors by initiating V(D)J
recombination (18, 19). Sites of recombination are specified by recombination signal sequences (RSS) that flank
each variable (V), diversity (D), and joining (J) gene segment. RAG1 recognizes and binds to the RSS, and then
recruits RAG2 to form a stable complex on the DNA (20, 21). Within this complex, the RAG proteins introduce a DNA
double-strand break at the junction of the RSS and the coding sequence. DNA cleavage by the Rag1/Rag2 complex
generates covalently sealed hairpins at the ends of the V, D, and J coding sequences that require further processing.
The DCLRE1C gene product, DNA cross-link repair protein 1C (commonly known as Artemis), is recruited to the DNA
hairpin structures by components of the non-homologous end joining (NHEJ) DNA repair machinery. Upon joining the
NHEJ complex, Artemis cleaves the DNA hairpin structures (22), allowing other NHEJ components to repair the
damaged DNA, thus completing the recombination process. Defects in either RAG1, RAG2, or Artemis prevent
productive rearrangement of both the B- and T-cell receptors, which is a prerequisite for B and T cell maturation,
thereby blocking development of B and T cells at very early stages (23, 24). The resulting T- and B-cell lymphopenia
leads to increased susceptibility to a wide range of infections, including opportunistic pathogens. NK-cell levels are
normal or elevated. Defects in Artemis also cause cellular sensitivity to ionizing radiation, likely due to a role for
Artemis in the general DNA double-strand break repair pathway (25). DCLRE1C-related SCID is therefore also
known as radiation-sensitive SCID (RS-SCID).


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T−B+NK+ SCID
T−B+NK+ SCID is often associated with autosomal recessive loss-of-function mutations in IL7R, which encodes the α
subunit of the IL-7 receptor (reviewed in 6). Additional causes of this type of SCID include autosomal recessive loss-
of-function mutations in CD3D and CD3E, which code for proteins necessary for signaling through the pre-T-cell
receptor (pre-TCR) or the TCR (reviewed in 1). Autosomal recessive loss-of-function mutations in ZAP70, which
encodes a signaling protein that associates with the CD3 complex, give rise to a rare subtype of SCID characterized
by the selective absence of CD8+ T cells (reviewed in 26, 27).

IL-7R α, the IL7R gene product, is a component of the interleukin-7 receptor (IL-7R) (reviewed in 28, 29). Interaction
of IL-7 with IL-7R leads to recruitment of the intracellular signaling molecules Jak1 and Jak3. Phosphorylation of IL-
7R α by Jak proteins activates multiple downstream signaling pathways which are important for transcriptional
activation of genes involved in T-cell differentiation (30), T-cell survival and maturation (31), and TCR rearrangement
(32). Disruption of IL-7 signaling arrests T-cell development at the double negative (CD4−CD8−) stage, preventing
productive TCR rearrangement (33) and leading to T-cell lymphopenia.

The CD3D and CD3E gene products, CD3 δ and CD3 ε, respectively, are components of the invariant CD3 protein
complex that pairs with the variable antigen-recognition subunits to form both the pre-TCR and the TCR. The CD3
complex is comprised of the CD3 γ, δ, ε, and ζ transmembrane protein subunits; each subunit contains at least one
immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic tail (reviewed in 34). Upon ligand binding to
the pre-TCR or the TCR, these ITAMs are phosphorylated, initiating a signaling cascade that results in transcriptional
activation of genes involved in T-cell differentiation and proliferation. CD3 association with the pre-TCR is required for
the transition from double negative to double positive T cells during thymocyte maturation and for rearrangement of
the TCRα chain (35). Surface expression of and signaling by the α/βTCR/CD3 complex is necessary for thymocyte
maturation from double positive to single positive CD4 or CD8 T cells (reviewed in 36). Defects in any of the CD3
subunits can result in impaired TCR surface expression and/or signaling, thereby hindering thymocyte maturation.
Defects in CD3 δ or CD3 ε lead to an arrest of thymocyte development at the double negative (CD4−CD8−) stage,
causing a severe deficiency of both α/β and γ/δ T cells (36, 37).

Loss-of-function mutations in IL7R, CD3D, or CD3E result in severe T-cell lymphopenia. NK and B cells are present
at normal levels, but B-cell activation and class-switch recombination are impaired, probably due to the failure of T-
cell help. The T-cell lymphopenia associated with IL7R-, CD3D-, and CD3E-related SCID leads to recurrent
opportunistic infections, while the defect in B-cell function further contributes to susceptibility to severe or recurrent
infections.

Zap-70 is a Syk family protein tyrosine kinase expressed exclusively in T cells and NK cells. Zap-70 is recruited to the
ζ chain of the invariant CD3 protein complex of the TCR in response to TCR engagement (38, 39). Once associated
with the TCR/CD3 complex, Zap-70 is phosphorylated and activated. Activation of Zap-70 leads to recruitment of
additional downstream signaling molecules, inducing a signaling cascade that results in transcriptional activation of
genes required for T-cell activation, differentiation, proliferation, and function (reviewed in 40, 41). Loss-of-function
mutations in ZAP-70 disrupt T-cell development at the transition from double positive (CD4+CD8+) to single positive T
cells, leading to a selective absence of peripheral CD8+ T cells, likely due to a defect in the positive selection of these
cells (42). CD4+ T cells are present at normal or elevated levels, but do not function properly, indicating that Zap-70 is
not essential for CD4+ T-cel development (43), but required for their activation and proliferation. As in T−B+NK+ SCID,
NK and B cells are present at normal levels, but in most cases, B-cell function is impaired (26).

Omenn Syndrome
Mutations in RAG1 or RAG2 that allow residual enzyme activity (so called hypomorphic mutations) result in impaired
V(D)J recombination and limited TCR diversity and can give rise to a distinct SCID phenotype commonly known as
Omenn Syndrome (OS), which is distinguished from classic SCID by the presence of oligoclonal T cells, elevated
levels of serum IgE, and eosinophilia (reviewed in 17).

Although hypomorphic mutations in RAG1 or RAG2 are the best characterized cause of OS, they do not account for
all cases of OS (44). Hypomorphic mutations in IL7R (45) and DCLRE1C (ARTEMIS) (16) have also been implicated
in OS, and the identification of additional causes is anticipated.


Clinical Presentation of SCID

Symptoms of SCID usually appear within the first few months after birth, following a brief asymptomatic period due to
the presence of maternal Igs. Infants with SCID typically suffer from severe and persistent infections, intractable
diarrhea, and failure to thrive (2). Infections are characteristically caused by opportunistic organisms such as

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Pseudomonas, Salmonella, Candida albicans, or Pneumocystis carinii or viruses such as respiratory syncytial virus,
adenovirus, or cytomegalovirus. Lymph nodes and tonsils are absent. X-ray imaging will usually reveal a small
thymus lacking thymocytes; however, in rare cases, a nearly normal-sized thymus may also be observed (36). SCID-
associated deficiency in endogenous T cells can be initially masked by the temporary presence of maternal
lymphocytes that have crossed the placenta during gestation (46). Engraftment of such maternal T cells or T cells
received through postnatal blood transfusions can lead to graft-versus-host disease (GVHD) (47). The defect in B-cell
function results in hypogammaglobulinemia and failure to mount a specific antibody response to immunization (48).

ADA-related SCID (T−B−NK− SCID) is further characterized by the presence of skeletal and neurological
abnormalities and/or excretion of large amounts of dAdo in the urine (12). The level of clinical severity and immune
dysfunction associated with mutations in ADA varies substantially, with mutations that allow some ADA activity giving
rise to milder disease or later onset of symptoms (12).

DCLRE1C-related SCID (T−B−NK+ SCID or RS-SCID) is also associated with increased sensitivity of both bone
marrow and skin fibroblasts to ionizing radiation (49, 50), B-cell lymphomas (51), and oral and genital ulcers (52).

Zap-70 deficiency is clinical y similar to classic SCID, but is distinguished by the selective absence of CD8+ T cells in
the presence of normal or elevated CD4+ T cells levels. Although CD4+ T cells are present, proliferative responses to
antigens are severely reduced. In most cases of Zap-70 deficiency, lymph nodes and tonsils are palpable and X-ray
imaging will show a normal-sized thymus (26). Thymic biopsy will reveal the presence of double positive (CD4+CD8+)
thymocytes in the cortex, while CD4+, but not CD8+ single positive cells are present in the medulla (42).

Omenn Sydrome can be distinguished from SCID by the presence normal or elevated T-cell levels with a restricted,
oligoclonal T-cell repertoire, elevated levels of serum IgE, and eosinophilia. In rare cases, B cells are also present;
however both B cells and T cells are oligoclonal and non-functional (53). Lymph nodes, liver, and spleen are enlarged
(17). Patients with OS also suffer from exudative erythrodermatitis and chronic diarrhea due to infiltration of the skin
and intestines by activated T cells, resulting in symptoms that resemble graft-versus-host disease (GVHD) (25).


Diagnosis of SCID

Patients with SCID and related SCID subtypes are usually diagnosed during the first few months after birth. SCID is
suspected in infants presenting with recurrent, severe, or unusual infections and lymphopenia. Diagnosis currently
relies on detection of a reduction in total lymphocyte counts, detection of diminished T-cell levels by flow cytometry,
and lymphocyte functional tests, and is supported by a family history of death in infancy due to infection or a family
history of SCID. Biochemical testing may be considered to identify ADA deficiency (12).

Diagnosis of SCID and its subtypes can also be achieved through genetic testing. Importantly, genetic testing allows
detection of carriers, allowing more effective genetic counseling. Genetic testing of newborns known to have a
genetic predisposition for SCID can al ow diagnosis and treatment of SCID before any life-threatening infections
develop. Such early treatment has been correlated to increased survival (47).


Treatment of SCID

Hematopoietic stem cell transfer (HSCT) has proven successful in controlling the defect in T-cell mediated immunity
(2), although continued intravenous immunoglobulin therapy is often necessary if B cells do not engraft. HSCT is also
the recommended treatment for OS (17) and Zap-70 deficiency (26). When performed before serious infection
develops, HSCT can result in a survival rate as high as 97% (3), making early diagnosis critical.

ADA-related SCID patients who are unable to receive HSCT may benefit from enzyme replacement with polyethylene
glycol-conjugated ADA (PEG-ADA), which has been shown to increase T-lymphocyte levels and improve cellular
immune function.

Recent clinical trials suggest that gene therapy will be another promising treatment option for both ADA-related SCID
and IL2RG-related XSCID; however, clinical trials are currently on hold due to development of a form of leukemia in
two IL2RG gene-therapy recipients (2, 47). Research into the development of gene therapy-based replacement of
both Jak3 (5) and Zap-70 (54, 55) is also ongoing, but has not yet yielded viable treatment options.

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Genetics of SCID

IL2RG-related XSCID is an X-linked recessive disorder, exclusively affecting males. Of note, mutations in IL2RG
resulting in a milder form of XSCID, distinguished by the presence of circulating T cells and delayed onset of
infections, have also been characterized (56-59).

Loss-of-function mutations in CD3E that allow residual expression of CD3 ε are associated with milder
immunodeficiency characterized by recurrent bacterial and viral infections (36, 60).

Except for IL2RG-related XSCID, all other known types of SCID are inherited in an autosomal recessive manner,
affecting both males and females.

Although very rare in the general population, DCLRE1C-related SCID occurs with an incidence of 1 in 2000 live births
within the Athabascan-speaking Navajo and Apache Native American population (often referred to as Athabascan
SCID or SCIDA) due to a founder mutation in DCLRE1C (52).


Table 2
Lymphocyte
Relative
Affected Gene
Affects
Phenotype
Frequency (3)
IL2RG
46% males
only
T− B+ NK−
JAK3
6.9%
males and females
T− B− NK−
ADA
16.1%
males and females
RAG1
3.4%
T− B− NK+
RAG2
males and females
DCLRE1C
1.1%
IL7R
10.3%
T− B+ NK+
CD3D
0.6%
males and females
CD3E
0.6%
T+ (CD4+CD8−) B+ NK+
ZAP70
n/a
males and females

unknown
~15%



Omenn Syndrome
Loss-of-function mutations in RAG1, RAG2, DCLRE1C, or IL7R can result in either SCID or OS (16, 17, 45). Both
SCID and OS are inherited in an autosomal recessive manner, that is, both copies of the affected gene must harbor a
loss-of-function mutation for a disease phenotype to be expressed. If both gene copies contain a mutation that leads
to complete loss of enzyme function, the resulting phenotype is SCID. If at least one copy of the gene contains a
mutation that allows residual enzyme activity, the phenotype will be OS. Thus, a patient affected by OS could also be
a carrier of a SCID-associated mutation. With only one exception (a frameshift mutation in RAG1), all known OS-
associated mutations in RAG1 and RAG2 are missense mutations (reviewed in 17, 53).

Of note, hypomorphic mutations in both copies of DCLRE1C can give rise to milder disease characterized by residual
V(D)J recombination activity in vitro and partial T- and B- lymphocyte deficiency. These patients also exhibit
increased frequency of B-cell lymphomas (51).

OS and SCID have been reported within the same family (49), implying that the identical genetic defect can cause
both phenotypes. However, GVHD in SCID patients may also lead to OS-like symptoms (17).


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Testing for SCID

Genetic testing can confirm a diagnosis of SCID or establish a diagnosis before infections develop. Such early
diagnosis is important, since timely treatment has been shown to increase survival. Genetic testing can also
distinguish between the different molecular causes of SCID, with important implications for genetic counseling.
Importantly, genetic testing al ows detection of asymptomatic carriers of SCID-associated mutations. This is critical in
cases of X-linked diseases such as XSCID, since female carriers are asymptomatic, while their sons are at a 50%
risk of being affected.


How is Genetic Testing for SCID Performed?
DNA for sequencing is obtained from leukocytes present in a small blood sample. The coding sequences of the
genes in question are amplified in a highly specific manner through a polymerase chain reaction (PCR), and all
PCR products are fully sequenced. Sequencing results are interpreted, and a detailed result report is sent to
the patient's physician.



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Document Outline

• SCID title page_vr0040607.doc
• SCID_Rvw_061306.doc

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