Arch Dis Child 2001;84:169–173 169 Arch Dis Child: first published as 10.1136/adc.84.2.169 on 1 February 2001. Downloaded from CURRENT TOPIC

Severe combined immunodeficiency—molecular pathogenesis and diagnosis

H B Gaspar, K C Gilmour, A M Jones

Severe combined immunodeficiencies (SCID) has been defined. In some cases pre- are a heterogeneous group of inherited disorders natal diagnosis of an aVected fetus may not characterised by profound abnormalities in T,B, lead to termination of pregnancy, but can allow and natural killer cell development and func- preparation for BMT early in the neonatal tion.1 They arise from a variety of molecular period, or even in utero in selected cases.2 defects, and the deficits in both cell mediated Thirdly, knowledge of the genetic defect has and humoral immunity lead to similar presenta- allowed a greater understanding of the molecu- tions in all the defined conditions. Children lar pathogenesis of the disease with the characteristically present with failure to thrive, possibility of designing more rational therapies recurrent , and increased susceptibility in some cases, and for developing strategies for to opportunistic . The age of presenta- somatic .34 tion is variable but occurs typically between 3 Even before a molecular defect is identified, and 6 months when the protective eVect of analysis of the immunological profile can maternally transmitted immunoglobulin has provide an indication of the nature of the diminished, although atypical and late presenta- underlying genetic abnormality. Nearly all tions are well described. cases of SCID have very low or absent numbers Over the past 10 years there have been enor- of T . Patients are then grouped mous advances in the understanding of the into those who have B lymphocytes (T−B+ molecular basis of the diVerent forms of SCID SCID) and those who do not (T−B− SCID). (see table 1). These have led to several Further subclassification can then be made improvements in diagnosis and management. according to the presence or absence of natural Firstly, unambiguous assignment of a molecu- killer (NK) cells. For instance SCID caused by lar diagnosis is now possible in many cases. gamma chain (ãc) or JAK3 deficiency has a http://adc.bmj.com/ This is particularly important in children who characteristic T−B+NK− profile (see fig 1). In have evidence of combined (cellular and all cases, the absolute count may humoral) immunodeficiency, but with be useful in making the diagnosis of SCID. “milder” clinical phenotypes than infants with Very low or absent counts (less than 2.7 × 109/l) classical SCID. Some of these children are may be seen in the T−B− forms and in T−B+ Molecular found to have identical molecular defects to SCID, the lymphoycte count is usually below those causing SCID, and in these cases the long

Immunology Unit, the lower end of the age related range. on September 30, 2021 by guest. Protected copyright. Institute of Child term outlook is now known to be poor enough Health, 30 Guilford to justify bone marrow transplantation (BMT) SCID caused by abnormalities in the Street, London at an early stage. Secondly, accurate carrier common gamma chain or JAK3 WC1N 1EH, UK H B Gaspar detection and first trimester prenatal diagnosis Of the diVerent molecular defects that result in are possible in any family where the precise SCID, the most common is the X linked form Clinical Immunology Table 1 Major types of SCID and their genetic defect Laboratory, Camelia Botnar Laboratories, Disorder (year of definition of molecular Diagnostic tests other than direct mutation Great Ormond Street basis) Chromosomal location Gene analysis Hospital for Children NHS Trust, London, X linked severe combined Xq13 Common ã chain (ãc) ãc expression by FACS analysis UK immunodeficiency (1993) K C Gilmour Adenosine deaminase (ADA) 20q12–13 Adenosine deaminase Red cell ADA levels and metabolites deficiency (1983) Purine nucleoside phosphorylase 14q11 Purine nucleoside Red cell PNP levels and metabolites Department of (PNP) deficiency (1987) phosphorylase Immunology, Great Recombinase activating gene (RAG 11p13 RAG1 and RAG2 Ormond Street 1&2) deficiency (1996), Omenn’s Hospital for Children syndrome (1998) NHS Trust receptor deficiencies (1987) 11q23 CD3ã/CD3å A M Jones Zap70 deficiency (1994) 2q12 ZAP-70 ZAP-70 expression JAK3 deficiency (1995) 19p13 JAK3 JAK3 expression/signalling IL-7 receptor deficiency (1998) 5p13 IL-7 receptor á IL-7 receptor á expression Correspondence to: MHC class II deficiency Dr Gaspar (1993) 16p13 CIITA HLA-DR expression [email protected] (1998) 19p12 RFX-B (1995) 1q21 RFX5 Accepted 27 September (1997) 13q13 RFXAP 2000

www.archdischild.com 170 Gaspar, Gilmour, Jones Arch Dis Child: first published as 10.1136/adc.84.2.169 on 1 February 2001. Downloaded from NK– γc/JAK3 SCID explain the immunological profile of XSCID and JAK3 SCID. Data from in vitro studies and B+ from “knockout” mice models have shown that functional IL-7/IL-7R and IL-15/IL-15R me- NK+ IL-7Rα deficiency diated signalling pathways are essential for normal T and NK cell development respec- tively.11 12 Abnormalities in IL-2 and IL-4 T– signalling may further explain the functional defects. Prior to the identification of the genetic NK– ADA SCID defects, diagnosis was based on family history and clinical and immunological profile. Link- B– age analysis and examination of X inactivation pattern in T cells of female relatives (a NK+ RAG1/2 deficiency unilateral pattern is seen in female carriers) was ADA SCID used to guide diagnosis and carrier status but Figure 1 Immunophenotypes in SCID. could only oVer a degree of probability. These techniques have largely been replaced by direct 13 14 (X-SCID) which arises from defects in ãc.5 analysis of the ãc and JAK3 genes. If a This molecular defect results in the absence of mutation is identified, carrier assessment for T and NK cell development but normal B cell female relatives can be made with absolute cer- numbers, although recent evidence suggests tainty and accurate prenatal diagnosis can be there are intrinsic ãc mediated defects of B cell oVered. More rapid tests based on the expres- function.6 A similar, though much rarer, sion patterns and function of the mutant ãcor clinical and immunological phenotype arises JAK3 protein are also now available for from an autosomal recessive defect in the gene diagnosis of aVected infants. Approximately encoding the tyrosine kinase JAK3 (Janus 65–90% of children with X-SCID have abnor- associated kinase 3).78 c was initially identi- mal expression of ãc on the surface of ã 15 fied as a component of the high aYnity mononuclear cells (our own unpublished data interleukin 2 receptor (IL-2R), but is now support this), allowing confirmation of the known to be an essential component of the molecular diagnosis by flow cytometric analysis IL-4, -7, -9, and -15 cytokine receptor of peripheral blood mononuclear cells (fig 3). complexes.9 Stimulation of the receptor com- In infants aVected by T−B+NK SCID who plex by cytokine results in the heterodimerisa- have normal ãc expression, further dissection tion of the receptor subunits and phosphoryla- of the signalling pathway can now be under- tion of the JAK3 molecule which binds taken. IL-2 stimulation of mononuclear cells specifically to the ã chain subunit. Tyrosine results in tyrosine phosphorylation of JAK3 at phosphorylated JAK3 in turn phosphorylates specific tyrosine based motifs. A monoclonal

one of the STAT (signal tranducers and activa- directed against phosphotyrosine http://adc.bmj.com/ tors of ) family of transcription residues can be used to show JAK3 activation, factors which then dimerises and translocates so abnormalities in this signalling pathway can to the nucleus where it binds to specific sites to be detected at a protein level prior to genetic initiate transcriptional events10 (fig 2). The analysis. The variability in clinical presentation specific function of the diVerent cytokines can in these forms of SCID and especially in JAK3 deficiency again underlines the need to identify the molecular defect so that earlier referral for

IL-2 bone marrow transplantation can be made. on September 30, 2021 by guest. Protected copyright. With the development of successful somatic gene therapy protocols for X-SCID it is again essential that a rapid molecular diagnosis is α β γ made.3 JAK3 SCID caused by abnormalities in purine metabolism JAK1 Approximately 20% of all cases of SCID arise STAT5 from an autosomal recessive deficiency of adenosine deaminase (ADA), a ubiquitously P expressed “housekeeping” enzyme that is STAT5 P required for the degradation of adenosine and STAT5 deoxyadenosine (dAdo) following DNA break- down. ADA SCID has a T−B− profile while NK cell numbers are variable. The abnormali- P P ties in T and B cell development have been attributed to a number of diVerent mecha- STAT5 16 STAT5 nisms. Deficiency of ADA results in the accu- mulation of dAdo and deoxyadenosine triphos- phate (dATP). Raised dAdo directly inactivates the enzyme S-adenosylhomocysteine hydrolase (SAH hydrolase)17 which is required for normal Figure 2 ãc/JAK3 signalling pathway. methylation reactions; dAdo has also been

www.archdischild.com Severe combined immunodeficiency 171 Arch Dis Child: first published as 10.1136/adc.84.2.169 on 1 February 2001. Downloaded from A patient erythrocytes is less than 1% of normal and is also significantly reduced in amniocytes or fibroblasts cultured from chorionic villus biopsy samples, thus allowing this assay to be used for prenatal diagnosis. Genetic analysis can also be used if a mutation has been identi- fied.21 A rarer disorder of purine metabolism is purine nucleoside phosphorylase (PNP) defi- ciency.22 This enzyme, like ADA, is expressed in all tissues and is required to maintain the balance between production of dephosphor- B ylated purines, detoxification to uric acid, and salvage back to the nucleotide concentration. Lack of PNP results in accumulation of a number of purine substrates, the most impor- tant being deoxyguanosine, which is phosphor- ylated to deoxyguanosine triphosphate (dGTP). Similar to dATP in ADA deficiency, dGTP exerts a lymphotoxic eVect by inhibition of ribonucleotide reductase.23 The immuno- logical defects in PNP deficiency are variable C but T cell function is most severely aVected. Neurological defects are common and are mainly related to motor dysfunction. Cerebral palsy, spastic paresis, and ataxic diplegia have all been described.24 25 Diagnosis is based on analysis of enzyme activity and measurement of intracellular dGTP concentrations.

SCID caused by abnormalities in V(D)J Common γ chain PE recombination Figure 3 Flow cytometric analysis of ãc expression in The assembly of functional B and T cell recep- X-SCID patients. Peripheral blood mononuclear cells from tor complexes is essential for the normal devel- a control sample (A) and two patients with T−B+ SCID (B, C) were stained with an antibody to common ã chain. opment of B and T lymphocytes. Both An increase in fluorescence activity from isotype control is receptors consist of immunoglobulin or immu- seen in (A), indicating normal ã chain expression. In (B) noglobulin like molecules, the diversity of and (C) no ã chain expression is seen, thus confirming the diagnosis of X linked SCID. which is generated through the process of

V(D)J recombination. During this carefully http://adc.bmj.com/ reported to induce chromosome breakage and regulated process, combinations of V (vari- apoptosis in immature thymic lymphocytes. In able), D (diversity), and J (joining) genes are addition raised dATP inhibits ribonucleotide assembled to create unique sequences that reductase which is necessary for normal DNA code for specific receptor chains. The initial synthesis. Because of the high turnover of lym- step in this process is the introduction of a phocytes, the highest amount of ADA expres- DNA double strand break (dsb) at specific sion is found in lymphoid tissue; this may sequences that flank each receptor gene

explain the severe lymphotoxic eVects of ADA segment. This event is mediated by the action on September 30, 2021 by guest. Protected copyright. deficiency. of two recombination activating genes, RAG1 Approximately 85–90% of ADA deficient and RAG2.26 Subsequently, the double strand patients present within the first year of life with break is processed and modifed gene segments severe clinical manifestations. However, there are joined together. This latter process requires is considerable heterogeneity in the clinical and the action of a number of other molecules immunological phenotype with 15–20% of essential to the DNA dsb repair in all cells patients being diagnosed at 1–8 years of age.18 types: XRCC4, DNA ligase IV, Ku 70, Ku80, In some of these cases the pattern of infection and the catalytic subunit of DNA dependent is less severe, and this correlates with a less protein kinase (DNA-PKcs).27 Murine models profound lymphocyte abnormality and degree show that defects in any one of these molecules of metabolic derangement. A few adults who can give rise to a T−B− SCID phenotype.28–32 were investigated for recurrent infections have In human SCID, a subgroup of T−B−NK+ also been shown to have a delayed onset ADA patients have been shown to have defects in the deficiency.19 Analysis of the ADA gene for RAG1 or RAG2 genes.33 In an in vitro model, has shown a correlation between the the mutant RAG proteins arising from these mutation, amount of residual ADA activity of mutations were shown to be practically devoid the mutant protein, and the clinical pheno- of all V(D)J recombination activity, suggesting type.20 Diagnosis of ADA SCID can be made that in these patients recombination events rapidly by analysis of dATP concentrations and cannot be initiated, and T and B cell receptor ADA activity in washed red cells. In red cells of complexes cannot be assembled. normal individuals, dATP concentrations are Mutations in the RAG genes have also been undetectable but are notably raised in patients shown to give rise to Omenn’s syndrome, a with ADA deficiency. ADA catalytic activity in form of SCID which has a characteristic clini-

www.archdischild.com 172 Gaspar, Gilmour, Jones

cal and immunological phenotype. Patients three complementation groups, RFX5 in group Arch Dis Child: first published as 10.1136/adc.84.2.169 on 1 February 2001. Downloaded from present with the characteristic infective compli- C,40 RFXAP in group D,41 and most recently, cations of SCID, but in addition develop an RFX-B in group B.42 Group A patients do not erythrodermic rash with lymphadenopathy and display this defect and mutations have been hepatosplenomegaly (this clinical presentation found in another protein, CIITA, which acts as can also be seen in SCID patients with engraft- an MHC transactivator and is essential for the ment of maternal lymphocytes). There is constitutive and inducible expression of all usually an associated eosinophilia and raised MHC class II genes.43 Diagnosis is made by IgE but otherwise variable immunoglobulin flow cytometric analysis of peripheral blood concentrations. Circulating B lymphocytes are lymphocytes for the expression of MHC class usually low or absent but T cells are detectable II molecules. More detailed analysis of the and display an activated phenotype. Detailed genetic defect can then be undertaken. studies have shown that these T cells are oligo- 34 clonal and display restricted TCRVâ usage. Other genetic defects leading to SCID In some families, children with both Omenn’s The genetic abnormalities that lead to SCID in syndrome and classical T−B− SCID have the majority of aVected patients have been out- occurred, suggesting that the same gene defect 35 lined. However, a number of other much rarer can give rise to the diVerent phenotypes. Fur- defects have been identified in a few patients. ther analysis of Omenn’s syndrome patients Abnormalities in components of the TCR revealed that RAG gene mutations were 36 receptor complex, CD3å and CD3ã have been responsible. Mutant RAG proteins from described in four patients to date.44 45 The phe- Omenn’s patients display residual V(D)J re- notype in these patients was less severe than in combination activity, suggesting that assembly classical SCID and an indication of the of some TCR complexes is possible and may diagnosis can be gained from the mean fluores- explain why oligoclonal T cell populations are cence intensity of the TCR–CD3 complex on detected. At present definitive molecular diag- flow cytometric analysis. Other defects in TCR nosis of T−B− SCID and Omenn’s syndrome complex signalling can cause an SCID pheno- is dependent on the detection of RAG gene type. Mutations in the ZAP-70 protein (which mutations. Routine protein expression analysis binds to the æ chain of the CD3 complex) result is not possible since the RAG genes are not in a selective CD8 lymphopenia.46 It is thought expressed in mature cells found in peripheral that this molecule is essential for positive selec- blood, and lymphocyte precursors are in insuf- tion of CD8+ T cells during thymic matura- ficient numbers in bone marrow. However, tion. A severe form of CD4+ lymphopenia has analysis of clonality in T and B cells in the been shown to result from defective expression peripheral circulation may give an indication of of the protein tyrosine kinase Lck which is a defect in the V(D)J recombination process. involved in proximal TCR signalling.47 Other RAG defects are found in the majority of indvidual SCID patients for whom novel patients with T−B−NK+ SCID, but a signifi- molecular defects has been identified have also cant number of T−B−NK+ SCID patients been reported.48 49 http://adc.bmj.com/ remain in whom no genetic defect has been identified. Some of these patients show in- creased sensitivity to ionising radiation, sug- Strategies for diagnosis gesting a defect in dsb repair mechanisms.37 38 Early clinical suspicion of susceptibility to However, analysis of the RAG genes and genes infection is the first important step in making a known to be involved in dsb repair has not diagnosis. Some reports have suggested that detected any abnormalities, suggesting that the absolute lymphocyte count found on most

automated full blood count analyses may be a on September 30, 2021 by guest. Protected copyright. other, as yet unidentified, gene defects may be 50 responsible. simple way to diagnose SCID. However, while this may be useful in T−B− forms, it may MHC class II deficiency (type 2 bare be misleading in X-SCID and JAK-3 SCID lymphocyte syndrome) where the lymphocyte count may be normal. In Major histocompatibility complex (MHC) these and all other forms of SCID, analysis of class II deficiency is an autosomal recessive lymphocyte subsets is necessary in order to immunodeficiency syndrome resulting from detect the abnormalities in development of defects in trans-acting factors essential for specific subpopulations. Following the aloga- transcription of MHC class II genes; it is char- rithm in fig 1 can provide a useful guide to the acterised by the presence of normal amounts of molecular abnormality. Patients should then be dysfunctional T and B cells (T+B+ SCID). All investigated at specialist laboratories where the bone marrow derived cells in aVected individu- appropriate functional and genetic assays can als fail to express MHC class II antigens (DR, be performed. DP, and DQ) and HLA-DM. In vitro studies have shown a specific defect in the binding of a Summary protein complex, RFX, to the highly conserved As we enter the post genome era, the genes X box of the MHC class II promoter in some of responsible for many diVerent types of SCID these patients.39 Further studies in patients have been identified and it is very likely that have revealed the presence of four genetic many more will follow. The challenge remains to complementation groups (A, B, C, and D), use this information to expand our understand- reflecting the existence of four MHC class II ing of the pathogenesis of the disease and to regulators. Mutations have been found in continue to develop improved methods of diag- subunits of the RFX complex in patients of nosis and treatment for aVected individuals.

www.archdischild.com Severe combined immunodeficiency 173

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