review

Autoimmune lymphoproliferative syndrome: molecular basis of disease and clinical phenotype Austen Worth,1,2 Adrian J. Thrasher1,2 and H. Bobby Gaspar1,2 1

Department of Clinical Immunology, Great Ormond Street Hospital NHS Trust, London, UK, and 2Molecular Immunology Unit, Institute of Child Health, University College London, London, UK

Summary Autoimmune lymphoproliferative syndrome (ALPS) is a variable clinical condition manifest by lymphoproliferative disease, autoimmune cytopenias and susceptibility to malignancy. Central to the cellular pathogenesis is defective FASinduced apoptosis, which in turn leads to dysregulation of lymphocyte homeostasis. The majority of patients have heterozygous mutations in the FAS (TNFRSF6) gene, but the condition is genetically heterogeneous and mutations in FAS ligand and caspase-8 and caspase-10, all of which are involved in Fas mediated signalling, have also been identified. This review provides a detailed insight into the pathophysiology of lymphocyte apoptosis and how this relates to the variable and complex clinical manifestations of ALPS. Keywords: Autoimmune lymphoproliferative syndrome, apoptosis, lymphocyte homeostasis, FAS, autoimmunity. In 1967, Canale and Smith described five patients with lymphadenopathy, splenomegaly and autoimmune cytopenias that mimicked malignant lymphoma (Canale & Smith, 1967). Investigation of two similar patients with progressive lymphoproliferative disease and autoimmunity by Sneller et al (1992) revealed an increase in a normally rare population of T cells characterised by the surface phenotype T-cell receptor (TcR)ab+ CD4) CD8) or double negative T cells (DNTs). They proposed that these patients may exhibit the human equivalent of a murine disease caused by the lpr and gld mutations, in which mice have a raised DNT subset, autoimmunity and lymphoproliferation and which had been studied as a model for systemic lupus erthythematosis. In the same year, Watanabe-Fukunaga et al (1992) reported that lpr mice lacked cell surface expression of Fas (CD95, Apo-1) a receptor which causes apoptosis of activated lymphocytes.

Correspondence: Dr H. Bobby Gaspar, Molecular Immunology Unit, Institute of Child Health, University College London, 30 Guilford

This link was proved in 1995 with the demonstration of lymphocyte apoptosis defects and Fas mutations in two groups of patients with the above clinical syndrome (Fisher et al, 1995; Rieux-Laucat et al, 1995; Le Deist et al, 1996), with a single mutated allele sufficient to cause disease. A similar mutation was then identified by Drappa et al (1996) in some of the patients described by Canale and Smith (1967). The clinical syndrome was termed autoimmune lymphoproliferative syndrome (ALPS) and subsequently many other cases have been described (Rieux-Laucat et al, 1995; Drappa et al, 1996; Bettinardi et al, 1997; Dianzani et al, 1997; Pensati et al, 1997; Sneller et al, 1997; Haas et al, 1998; Kasahara et al, 1998; Sleight et al, 1998; van der Werff ten Bosch et al, 1998; Jackson et al, 1999; Rieux-Laucat et al, 1999; Vaishnaw et al, 1999; van der Burg et al, 2000; Bleesing et al, 2001; Oren et al, 2002; Alvarado et al, 2004; Deutsch et al, 2004; Teachey et al, 2005). A disease definition was proposed by Straus et al (1999) (Table I), based on the experience of the largest cohort of recognised ALPS patients. Clinically, ALPS patients have the triad of lymphoproliferative disease, autoimmune cytopenias and susceptibility to malignancy. Several patients with a clinical syndrome of ALPS have been found to have a normal FAS [also termed TNFRSF6 (tumour necrosis family receptor super family 6)] gene, and in rare patients, mutations in other intermediates in the Fas signalling pathway have been discovered. This has led to a more recent classification of ALPS based on molecular pathology (RieuxLaucat et al, 2003a; Sneller et al, 2003) (Table II). The vast majority of patients have ALPS Ia, and the inheritance of this condition is usually autosomal dominant with variable clinical penetrance. Autoimmune lymphoproliferative syndrome is the first human disease whose aetiology has been attributed to a primary defect in apoptosis, or programmed cell death (PCD), and has given us a unique insight into the control of PCD, lymphocyte homeostasis and the termination of an immune response. This review explores the details of death signalling that the study of ALPS has helped to elucidate; how these defects cause the clinical phenotype of ALPS and the mechanisms controlling lymphocyte function and disease.

Street, London WC1N 1EH, UK. E-mail: [email protected]

doi:10.1111/j.1365-2141.2006.05993.x

ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 133, 124–140

Review Table I. Disease definition of ALPS. Required feature Chronic non-malignant lymphadenopathy ± splenomegaly Raised (>1%) circulating DNT cell Defective antigen induced apoptosis in cultured activated lymphocytes in vitro Supportive features Autoimmune disease Positive family history of ALPS Characteristic lymph node or splenic histology Mutation in gene coding for Fas

Table II. ALPS classification. Ia Ib II III

TNFRSF6 mutation Fas ligand gene mutation Caspase 8 or 10 gene mutation Unknown genetic cause

Where identification of FasL, caspase 8 and caspase 10 mutations is not available. ALPS is more practically classified as type Ia or type non-Ia.

et al, 1992). This is sequentially followed by cell and organelle shrinkage, acidification of cell cytoplasm, chromatin condensation, nuclear fragmentation and zeiosis (or ‘blebbing’) of the plasma membrane (Majno & Joris, 1995). The blebbing progresses to budding and fragmentation of the cell into apoptotic bodies. Phagocytic cells have receptors for phosphatidylserine, allowing apoptotic cells and apoptotic bodies to be phagocytosed. This process occurs whilst the cell is still viable, and as a result the proinflammatory cytoplasmic cell contents are excluded from the extracellular space and an inflammatory response is avoided (Savill & Fadok, 2000). As a result, there is no bystander cell damage and the apoptotic cell is removed with minimal disruption to the surrounding tissue. This pattern of PCD is common to all cell types and conserved between species, suggesting a common effector machinery. First dissected in primitive organisms (Horvitz, 2003; Hay et al, 2004), it has been found that several key genes that instigate PCD are highly conserved throughout evolution (Boyce et al, 2004). Even in simple organisms the control of these effector mechanisms is extremely complex with an ongoing balance between pro-apoptotic and anti-apoptotic signals.

Normal lymphocyte apoptosis Death receptor apoptosis signalling pathway Apoptosis and lymphocyte homeostasis The adaptive immune response effectively responds to a diverse range of pathogens by maintaining small numbers of a broad repertoire of antigen-specific lymphocytes. Upon antigen recognition the specific lymphocyte pool undergoes rapid and prolific expansion with a doubling time for activated lymphocytes being as little as 4Æ5 h (Kurts et al, 1997). This pathogen-initiated proliferation poses a serious risk to the host organism due to damage of healthy cells as a result of nonspecific effector mechanisms and bystander damage, due to cross-reactivity between pathogen-specific antigen receptors and self-antigens, and from the risk of malignant transformation in chronically activated lymphocytes undergoing high rates of proliferation. To remain safe and effective the immune system must eliminate these cells just as efficiently. Mechanisms must exist to prevent uncontrolled expansion of potentially harmful self-reactive lymphocytes. A balance must exist between too few lymphocytes, which may lead to ineffective and delayed pathogen clearance, and an overactive lymphocyte population, which may lead to autoimmunity and impaired immune surveillance. This immune system homeostasis is not confined to lymphocytes, but also involves the expansion and elimination of other effector and antigen presenting cells. Programmed cell death is the mechanism by which this balance is achieved, and apoptosis is the morphological description of the characteristic cellular changes in the best described form of PCD. Once a cell is committed to apoptosis the first indication is the ‘cell membrane flip’ where phosphatidylserine is externalised on the cell membrane (Fadok

Activated T cells are the key to a specific immune response and as such are particularly susceptible to apoptosis. T cells are eliminated if exposed to too much stimulation (active apoptosis) or too little (passive apoptosis) (Lenardo et al, 1999). Following initial antigen recognition and TcR signalling, T lymphocytes proliferate and their function matures in response to cytokines, especially interleukin 2 (IL-2). Active apoptosis is mediated by death receptors (Fas and TNFR1 and 2) and their ligands [FAS ligand (FasL) and tumour necrosis factor (TNF)] both of which are upregulated by TcR ligation in the context of IL-2. Once ligated, the death receptors activate the caspase cascade, causing cell death in a transcription independent pathway (Itoh et al, 1991). The caspases (cysteine proteases which cleave at an aspartate residue) are a group of 12 highly conserved zymogens expressed in all mammals (Boyce et al, 2004) and play a central role in the apoptosis of cells (Cohen, 1997; Salvesen & Dixit, 1997; Thornberry & Lazebnik, 1998). They are synthesised as inactive procaspases that are activated by cleavage into large and small subunits which then undergo conformational changes, facilitating the formation of active tetramers of two small and two large subunits (Salvesen & Dixit, 1999). This activating cleavage is performed by previously activated caspases (autoprocessing) and is dependent on initial procaspase dimerisation (Fig 1). Once active, caspases have highly specific endoprotease activity, selectively cleaving between domains usually only at one site per protein, causing either activation or inactivation of the target protein. Functionally, caspases can be divided into different groups. Caspases 2, 8, 9 and 10 are initiator caspases, which transduce

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Review

Fas L

Fas

FADD DISC Procaspase 8 or 10 Activation Caspase 8 or 10

Procaspase 3 or 7

Autocatalysis Proteolysis Positive feedback

Effector caspases

Target proteins

Autocatalysis

Apoptosis Fig 1. Death-inducing signalling complex (DISC) formation following Death Receptor activation. Cross-linked Fas L binding to Fas results in a conformational change in the death domain of Fas molecules, facilitating the actin-mediated recruitment and binding of Fas-associated death domain (FADD) via homologous death domains. FADD then binds procaspases 8 or 10 via homologous death effector domains (DEDs) found on both the carboxyl terminal of FADD and the caspase recruitment domain (CARD) of procaspase 8 or 10 (Kischkel et al, 1995; Boldin et al, 1996; Muzio et al, 1996; Kischkel et al, 2001; Wang et al, 2001) to form the DISC. Formation of the DISC facilitates the autoprocessing and activation of caspase 8 or 10. Caspase 8 and 10 then process effector caspases 3 and 7 resulting in apoptotic cell death. Caspase 8 and 10 activation is inhibited by c-FLIP (and vFLIP, a homologous viral protein), DED-containing adaptor proteins that are competitive antagonists for FADD binding (Garvey et al, 2002; Peter & Krammer, 2003).

apoptosis signals (e.g. death receptor ligation) into caspase cascade activation. Once active, their main substrates are effector caspases or other intermediaries in the apoptosis signalling network. For activation, initator procaspases must bind to a polyprotein complex [death-inducing signalling complex (DISC), apoptosome or pidosome] which, by binding several procaspases in close proximity and favourable allosteric arrangement, allows activating proteolysis (Salvesen & Dixit, 1999). Caspases 3, 6 and 7 are the effector caspases that are directly activated either by initiator caspases or by previously activated effector caspases. This step acts as a major amplification step in the response. Inactivation targets for effector caspases are widespread, but include proteins responsible for 126

cell shape (actin, cytokeratin -18, paxillin), transcription factors (STAT1 and GATA-1), DNA repair proteins (PARP), kinases (PKC, PAK2 and MST1), antiapoptotic factors (Bcl-2 and Bcl-x) and caspase inhibitors (IAPs). Substrates that are activated include proteins involved in cell membrane blebbing (e.g. Gelosin, ROCK-1 and PAK-2), DNA degradation (cleavage of ICAD and releasing caspase activated DNase), nuclear shrinkage (lamin A, B1, B2 and NUMA) and proapoptotic proteins (Bid and Bad) (Shi, 2002). Because caspase activation generates a positive feedback loop, a group of caspase inhibitors called inhibitory apoptosis proteins (IAPs) are present in the cytosol and prevent low grade caspase activation from inducing cell death (Fig 1).

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Review

Death receptor signalling The death receptors are a subset of the tumour necrosis factor receptor superfamily (TNFRSF), which, in addition to death receptors, contains nerve growth factor receptors and costimulatory receptors, such as CD40 and CD27. These receptors consist of between one and four extracellular cysteine rich domains (CRDs), and the death receptors have a functionally important 60–80 amino acid intracellular signalling domain called the death domain (DD) (Itoh & Nagata, 1993; Tartaglia et al, 1993) (Fig 2). Despite only 25% sequence homology among death receptor DDs there is a high degree of structural conservation, each having an antiparallel six helix bundle and a hydrophobic core (Nagata & Golstein, 1995; Huang et al, 1996; Locksley et al, 2001; Collette et al, 2003). Ligands for these receptors are members of the tumour necrosis factor superfamily, and structurally these share an extracellular TNF homology domain of approximately 150 amino acids length (Collette et al, 2003; Muppidi & Siegel, 2004). Members of both receptor and ligand families can exist in a soluble form due to alternative splicing or metalloproteinase cleavage (e.g. TNF, FasL and lymphotoxin) (Tanaka et al, 1995). Death receptors and their ligands are shown in Table III. Fas is the best understood death receptor, and it is expressed on immune system cells, liver, cardiac, ovary and lung tissue

(van der Burg et al, 2000). It contains three CRD domains and CRD2 and CRD3 are the major contact sites for FasL (Orlinick et al, 1997; Starling et al, 1997). Studies of truncation mutants and have shown that CRD1 contains a ‘preligand assembly domain’ (PLAD) which induces Fas trimerisation at the cell surface prior to FasL binding (Chan et al, 2000; Siegel et al, 2000a)(Fig 2). FasL also trimerises both on the cell surface and in solution (Tanaka et al, 1995) and upon binding the Fas trimer, has the capability of cross-linking receptor complexes. Within seconds of binding trimerised FasL or anti Fas monoclonal antibody, Fas complexes form on the cell surface, and evolve into clusters visible by immunofluorescence after 10 min. These clusters then progress to receptor capping (30 min) and finally internalisation of the Fas complexes by endocytosis (1 h) (Algeciras-Schimnich et al, 2002; Siegel et al, 2004). It has been proposed that FasL binding induces changes in the Fas DD, facilitating homophilic association between the individual fas DDs (Holler et al, 2003). The nature and mechanism of this change is at present unknown. Death domain modification induces recruitment and binding of an adaptor protein, FADD (Fas-associated DD), via a DD at its amino terminal (Chinnaiyan et al, 1996) and procaspase 8 and/or procaspase 10 then bind. This polyprotein complex, DISC, forms within minutes of Fas ligation (Gajate et al, 2000; Algeciras-Schimnich et al, 2002) and is dependent

(A) Gene Structure

Extracellular cDNA Exons

225 1

391

529

3

2

PLAD CRD 1

Intracellular

4

639

700

763

846

5

6

7

8

871 9

Death domain

CRD 2 CRD 3 (B) Protein Structure

Signal peptide

PLAD

Cysteine-rich domain Transmembrane domain

CRD 2

Death domain

CRD 3

3¢ untranslated

Death domain

Monomeric Fas

Trimeric Fas

Fig 2. Fas gene and protein structure.

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Review Table III. Death receptors. Death receptor

Synonym

Ligand

Fas TNF-R1 TNF-R2 DR3 Trail-R1 Trail-R2 DR6 NGFR

TNFRSF6, APO-1, CD95 and APT1 TNFRSF1a, CD120a and p55-R TNFRSF1b, CD120b and p75 TNFRSF12, APO-3, TRAMP, WSL-1 and LARD TNFRSF10a, DR4 and APO-2 TNFRSF10b, DR5, TRICK2a and KILLER TNFRSF21 TNFRSF16

FasL (TNFSF6) TNF (TNFSF2) TNF (TNFSF2) APO3L (TNFSF12 and TWEAK) TRAIL (TNFSF10 and APO2L) TRAIL (TNFSF10 and APO2L) Unknown NGF

TNF, tumour necrosis factor; TNFRSF, tumour necrosis factor receptor superfamily; TNFSF, tumour necrosis factor superfamily; DR, death receptor; FasL, Fas ligand; NGF, nerve growth factor; NGFR, nerve growth factor receptor.

on actin polymerisation for FADD recruitment (Refaeli et al, 1998; Algeciras-Schimnich et al, 2002). The DISC facilitates autoprocessing of caspase 8 and caspase 10 and activation of the caspase cascade as described above (Martin et al, 1998; Salvesen & Dixit, 1999), but is competitively antagonised by the adaptor molecule cFLIP. The key to successful apoptotic signalling via the Fas pathway appears to be the cooperativity of homotypic protein interaction causing superclustering of signalling molecules on the outside of the cell and of DISCs at the inner membrane. This allows activated caspase 8 to be locally concentrated and overcome constitutive caspase inhibitors, driving forward the positive feedback loop of the caspase cascade. This model explains the different sensitivity to apoptosis seen between Fas stimulation by multivalent ligands with the capacity to cross-link (e.g. anti-Fas IgM or oligomerised cell surface FasL) or divalent (anti-Fas IgG) ligands (Siegel et al, 2000a; Holler et al, 2003), and the fact that metalloproteinase-cleaved soluble monomeric FasL is actually antiapoptotic to many cell lines (Suda et al, 1997; Schneider et al, 1998; Tanaka et al, 1998). Upregulation of Fas and FasL as a result of TcR ligation, leads to Fas-induced apoptosis, but the exact nature of the Fas– FasL interaction in vivo is not known. Interaction between membrane–bound Fas and membrane–bound FasL on the same cell (suicide), membrane–bound interactions between neighbouring T-cells (fratricide), autocrine (suicide) and paracrine (fratricide) FasL secretion are all possible mechanisms and combinations of some or all of these may occur in vivo. The interaction between membrane bound receptor is, however, likely to give a far more powerful death signal due to greater capacity for cross linkage (Yokota et al, 2005).

Molecular mechanisms of lymphocyte homeostasis Commitment to apoptosis is determined by a balance of proand anti-apoptotic signals, which are processed in the context of the cell’s state of maturation or activation. In activated T-cells IL-2 seems to be the central external factor that influences this balance. The control IL-2 exerts has been called ‘proprioceptive’ or feedback control (Lenardo et al, 1999), 128

with IL-2 levels sensed by the cell and an appropriate response, either apoptosis or survival, being executed by the mechanism described above. Resting and memory cells have low levels of Fas expression and the need for an IL-2 survival signal is overcome by an upregulation of anti-apoptotic factors, such as Bcl-2, induced by the developmental stage of the cell (Itoh et al, 1991). Once activated T cells are proliferating (in response to IL-2), receptivity to pro-apoptotic signals increases with the number of cell cycles they have undergone and they become particularly sensitive to apoptosis during late G1/S phase (Boehme & Lenardo, 1993; Fournel et al, 1996; Radvanyi et al, 1996). During this time, signals that would have caused resting T cells to be activated and proliferate (such as TcR ligation and cross linking) are likely to induce apoptosis. By having a ‘narrow window of survival’ for activated T cells, the immune response can be controlled, with passive apoptosis removing active cells once antigen has been eliminated and active apoptosis removing cells specific to chronic antigens and self-reactive T cells. The result is a dynamic T-cell response with individual cells having short lifespans, and ongoing immune responses being maintained by recruitment and proliferation from the naı¨ve cell pool. This high turnover of lymphocytes is a vital self-protective mechanism, but this type of homeostasis would be unsuitable for other less potentially dangerous cell types, such as neurons. It also suggests that the role of Fas in lymphocyte homeostasis is primarily in the elimination of ageing, chronically activated or autoreactive T cells, and that the elimination of the lymphocytes following an acute infection is mediated by the cytokine withdrawal, intrinsic apoptotic pathway (Strasser & Pellegrini, 2004). This is confirmed, both by the finding that in ALPS patients lymphocytes actually reduce rather than accumulate following an acute infection (see below), and the fact that lpr mice develop lymphadenopathy even if raised in a germ-free environment and fed on antigen-free food (Maldonado et al, 1999). Although Fas appears to be the principal death receptor involved in T-cell apoptosis, other death receptors undoubtedly contribute pro-apoptotic signals. The TNF–TNFR2 interaction in addition to Fas signalling appears to be critical for induction of apoptosis in CD8+ cells (Sarin et al, 1995;

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Review Zheng et al, 1995). The rare mature T cell that manages to satisfy both passive and active survival criteria matures to enter the memory pool, a process which is likely to be mediated by intermediaries of the apoptosis signalling pathways (Wu et al, 2004). Activated T cells, by expressing FasL, also play an important role in controlling immune responses by inducing apoptosis in other immune cells expressing Fas (Rathmell et al, 1996; Kiener et al, 1997; Koppi et al, 1997), but again, other death receptors appear to play important roles as well. Dendritic cells, for example, are far more sensitive to apoptosis from TNF-related apoptosis-inducing ligand (TRAIL) ligation than Fas ligation (Kiener et al, 1997). FasL ligation is a potent mechanism of cytotoxicity for T cells and natural killer (NK) cells, important in killing of virally infected and transformed cells. Expression of FasL by cells in immune privileged tissues has been implicated in the mechanism of exclusion of immune cells in these tissues. Fas, however, has no role in the central clonal deletion of autoreactive thymocytes cells during T-cell development.

Autoimmune lymphoproliferative syndrome Ia - Fas deficiency Clinical features Lymphoproliferation is the key clinical finding in ALPS and combined lymphadenopathy and massive splenomegaly is the most common presentation. The median age of first presentation is 24 months of age although with increasing awareness of the condition adults with autoimmune complications are now more frequently being diagnosed (Deutsch et al, 2004). Lymphadenopathy ranges from the upper limits of normal for age, to massive anatomically distorting swellings, associated with thoracic and intra-abdominal lymphoid masses (Avila et al, 1999). Seventy-five per cent of patients have hepatomegaly in addition, but occasional patients present with only one of lymphadenopathy or splenomegaly (Bleesing et al, 2000; Rieux-Laucat et al, 2003b). The lymphoid hyperplasia is chronic and consistent in nature, although reductions are often seen in association with bacterial and viral infection, as was described in the original Canale–Smith Syndrome patients (Canale & Smith, 1967). It is not usually associated with systemic symptoms, such as fevers, rigours or night sweats, which would suggest alternative diagnoses. Lymphadenopathy is usually more pronounced in infancy, often regresses during adolescence and, in selected patients, spontaneous resolution has been seen (Infante et al, 1998; Rieux-Laucat et al, 1999), although late presentation with lymphadenopathy has also been described (Bleesing et al, 2000). Autoimmune disease is less consistent and only develops in 50–70% of patients (Kwon et al, 2003; Rieux-Laucat et al, 2003a,b). Blood cytopenias are most frequently seen with autoimmune haemolytic anaemia developing in 29–38%, immune mediated thrombocytopenia in 23–34% and autoimmune neutropenia in 19–27%. These cytopenias are usually

severe, difficult to treat and can be life-threatening, with autoantibody-mediated blood cell destruction being exacerbated by hypersplenism. Recently, 12 patients with a diagnosis of Evans Syndrome (a chronic relapsing condition of unknown aetiology where patients have two or more autoimmune blood cytopenias) were investigated for ALPS. Half were found to have raised DNTs and defective Fas-induced apoptosis (Teachey et al, 2005). Rarer autoimmune phenomena seen include glomerulonephritis, optic neuritis, Guillian–Barre´ syndrome, primary billiary cirrhosis/autoimmune hepatitis, arthritis, vasculitis, childhood linear IgA disease and factor VIII coagulopathy (Pensati et al, 1997; Sneller et al, 1997; Sleight et al, 1998; Rieux-Laucat et al, 1999; Fang et al, 2000; van den Berg et al, 2003; Wong et al, 2004). Skin rashes of probable autoimmune origin and urticaria are both common (Bleesing et al, 2000; Auricchio et al, 2005). Several other conditions, including seizure disorder, ovarian failure and autism, have been suggested to have an autoimmune aetiology (Shenoy et al, 2000). The risk of developing autoimmunity is life-long and, in contrast to lymphoproliferation, tends to be less prominent during infancy and becomes more severe with age. Established autoimmune conditions follow an intermittent, remitting and resolving pattern. Autoantibodies are more common than overt clinical disease and present in up to 92% of patients (Carter et al, 2000; Stroncek et al, 2001; Kwon et al, 2003; Rieux-Laucat et al, 2003b). Anticardiolipin and a positive direct antigen test are seen most frequently (in over 50%), and anti-platelet antibodies, anti-neutrophil antibodies, anti-nuclear antibodies and rheumatoid factor are all commonly positive (Carter et al, 2000; Kwon et al, 2003; Rieux-Laucat et al, 2003b). Interestingly, the most common autoantibodies associated with thrombocytopenia and neutropenia are the same as those seen in idiopathic thrombocytopenia purpura and autoimmune neutropenia in patients without ALPS (Grodzicky et al, 2002; Kwon et al, 2003). Despite this, there is poor correlation between presence of an autoantibody and presence of the appropriate cytopenia. The number and titre of autoantibodies appears to be correlated to the severity of lymphadenopathy and circulating DNT cell count (Kwon et al, 2003), which is counterintuitive considering the clinical observation that lymphadenopathy and autoimmunity have quite different clinical courses. Increased susceptibility to malignancy, particularly haematological malignancy is the most worrying clinical phenomenon associated with ALPS. Current data suggests lymphoma develops in about 10% of ALPS patients and the relative risks of developing Hodgkin and non-Hodgkin lymphoma are 51· and 14·, respectively (Straus et al, 2001). Interestingly two of the cases of Hodgkin disease had the rare histological subtype of nodular lymphocyte predominent (NLP), suggesting a specific link between ALPS and this unusual histological subgroup of Hodgkin disease (Straus et al, 2001; van den Berg et al, 2002). A wide range of other malignancies have also been described in ALPS patients (Drappa et al, 1996), although

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Review unlike other immunodysregulatory conditions, these malignancies show no evidence of being virally driven. Fas mutations are commonly found in clonal malignant cells from patients without germline Fas mutations. 10% of plasmacytomas (43 · 23), 11% of non-Hodgkin lymphomas (43 · 36) and 28% of bladder carcinomas (43 · 38) have Fas gene mutation in malignant cells.

Diagnosis Diagnosis is based on the disease definition shown in Table I. The identification of DNTs on fluorescent-activated cell sorting has been cited as a useful screening tool for centres with limited experience of lymphocyte apoptosis assays (Oren et al, 2002; Teachey et al, 2005), and if combined with appropriate clinical features, peripheral lymphocytosis, circulating autoantibodies and polyclonal hypergammaglobulinaemia, is extremely suggestive of ALPS. The upper limit of normal for DNTs (1%) has been questioned, with a normal threshold of 2Æ6% identified among normal controls in one group (Teachey et al, 2005). Assessment of Fas-mediated apoptosis is achieved by culturing Ficol-separated peripheral blood lymphocytes activated by phytohaemoglutanin (PHA) or anti-CD3 monocloncal antibody (or both), in IL-2-containing medium for 1 week and then exposing them to anti- Fas monoclonal antibody for 12 h (Fisher et al, 1995; Rieux-Laucat et al, 1995; Drappa et al,

Normal control

CD4 / CD8

(A) Flow cytometry

Treatment Lymphoproliferative disease in ALPS does respond to corticosteroids and other immunosuppressants, but symptoms recur upon dose reduction, and long-term side-effects outweigh benefits unless lymphoproliferation is causing critical obstructive disease. Advice concerning splenic rupture and avoidance of contact sports is recommended. Autoimmune cytopathies also respond well to corticosteroids and short courses of high dose treatment have been more effective at controlling these conditions. Immune thrombocytopenia is less sensitive to intravenous immunoglobulin

(B)ApoptosisAssay

n

CD4 / CD8

TcR

ALPS Patient

1996; Sneller et al, 1997; Kasahara et al, 1998). Apoptotic cell death has been assessed by a variety of methods, including hypodiploid nucleic quantification, terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) method, propidium iodide surface staining, annexin surface staining and dye exclusion (Nicoletti et al, 1991; Gavrieli et al, 1992; Ashany et al, 1995; Rieux-Laucat et al, 1995). ALPS patients show wide variation in the degree of impairment to Fasmediated apoptosis but it is consistently reduced compared with normal controls. By culturing cells in activating medium prior to performing the apoptosis assays, the cells tested in this in vitro assay are a very different population from those freshly isolated from the patient. Typical DNT subset and apoptosis assay findings from an ALPS patient are shown in Fig 3.

n

Annexin FITC

n

TcR

Annexin FITC

n

Annexin FITC

Unstimulated T cells

Annexin FITC

Stimulated T cells

Fig 3. Double-negative cells and apoptosis assays (a) fluorescence-activated cell sorted plots for freshly isolated peripheral lymphocytes, sorted by TcRab against CD4 or CD8. (b) Freshly isolated lymphocytes were cultured for 1 week with IL-2 and phytohaemaglutanin, and then a further 12 h with anti-Fas monoclonal antibody. Apoptosis was detected by Annexin staining, which binds cell surface phosphatidyl serine.

130

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Review therapy than conventional idiopathic thrombocytopenia purpura (Rieux-Laucat et al, 2003b). In resistant cases splenectomy has proved to be effective in controlling thrombocytopenia and anaemia, although the abnormalities do remain and the consequences of postsplenectomy pneumococcal sepsis seem to be worse in ALPS patients than in non-ALPS patients (Dianzani et al, 1997; Infante et al, 1998). Postsplenectomy antibiotic prophylaxis until late adolescence and into adulthood for patients with previous pneumococcal sepsis has been advocated (Rieux-Laucat et al, 2003b). Mycophenolate mofetil (Rao et al, 2005) and rituximab with vincristine (Heelan et al, 2002) have been used with success for severe refractory immune thrombocytopenia, and regular granulocyte colonystimulating factor injections have been of benefit in treating neutropenia with recurrent infections (Rieux-Laucat et al, 2003b). When blood transfusion is necessary it has been recommended that blood should be ABO and D compatible, negative to any antigen to which the patient has autoantibodies and matched for C, E and K antigens to prevent antibody formation to these immunogenic antigens (Carter et al, 2000). The anti-malarial drug pyrimethamine and sulphadoxine (Fansidar
; Roche, NJ, USA) has induced a reduction in lymphadenopathy and improvement in autoimmune cytopathies in a case series of seven ALPS patients (van der Werff ten Bosch et al, 1998, 2002), although only two of these patients had ALPS type Ia. Two of these patients have maintained a remission of their symptoms for >1 year after stopping the drug, but only one patient showed a reduction in DNT cell counts. Bone marrow transplant has been used successfully in two cases (one of which was homozygous) with a severe, worsening clinical phenotype (Benkerrou et al, 1997; Sleight et al, 1998).

Prognosis Because ALPS has only recently been identified and classified as a distinct disease, accurate long-term follow-up data does not yet exist (Rieux-Laucat et al, 2003b); however, medical histories of patients identified at an older age and detailed family histories of patients give an indication of the expected long-term outcome. ALPS-related death and serious morbidity usually arise from development of haematological malignancy, complication of severe autoimmune disease or postsplenectomy sepsis.

Genetic penetration The human Fas gene is located on chromosome 10q24.1 and consists of six exons (Inazawa et al, 1992; Behrmann et al, 1994; Cheng et al, 1995) (Fig 2). The majority of patients with ALPS Ia are heterozygous for a mutation in this gene. Over 70 different Fas gene mutations have now been described in ALPS patients, two-thirds of them in the intracellular domain (ICD) (most involving the death domain), and one-third in the extracellular domains (ECD). The vast majority of these are

insertions, substitutions or deletions of one or two base pairs in exons or splice sites of the gene. Approximately, half of the mutations result in modification of amino acid sequence, and half cause premature truncation of the protein. Investigation of relatives of index cases of ALPS has revealed an autosomal dominant inheritance pattern with incomplete penetrance (Infante et al, 1998; Jackson et al, 1999; RieuxLaucat et al, 1999) in the majority of cases and a recessive inheritance pattern in occasional cases (Le Deist et al, 1996; Bettinardi et al, 1997; Pensati et al, 1997; van der Burg et al, 2000). Even patients with ALPS within a family, caused by the same mutation can have very different clinical phenotypes (age of presentation, severity of lymphadenopathy or autoimmunity). Despite not expressing florid disease many of the asymptomatic individuals with one mutated Fas gene do have minor signs of lymphoproliferation and autoimmunity on close examination (Fisher et al, 1995; Drappa et al, 1996; Le Deist et al, 1996; Bettinardi et al, 1997; Infante et al, 1998; van der Burg et al, 2000). Clinical penetration is highest with ICD mutations, with missense mutations causing clinical symptoms in 90% and truncation mutations causing symptoms in 70%. By contrast, ECD mutations have a clinical penetration of 30% (Jackson et al, 1999; Rieux-Laucat et al, 1999; Vaishnaw et al, 1999). This is the only consistent correlation between genotype and clinical phenotype found in ALPS (Bettinardi et al, 1997; van der Burg et al, 2000). Four patients (Le Deist et al, 1996; Kasahara et al, 1998; van der Burg et al, 2000; Sneller et al, 2003) have been described with homozygous Fas mutations and minimal Fas expression on activated T cells (also known as ALPS0; Rieux-Laucat et al, 2003a). These patients have demonstrated a more severe phenotype, with antenatal onset of lymphoproliferation, hydrop foetalis, lymphocytic pulmonary infiltrates and severe blood cytopenias from birth. Although the clinical penetration is varied, there is a 100% penetration in functional assays with all carriers of Fas mutations having defective Fas-induced T-cell apoptosis. The degree of inhibition of apoptosis is again varied, even for the same mutation in different individuals. In the homozygous patients, there is an absence of Fas expression at the cell surface and as a result the disease is caused by lack of Fas function or haploinsufficiency. Two of the described homozygous patients had DD mutations (Rieux-Laucat et al, 1995; Kasahara et al, 1998), whereas one patient had an ECD mutation predicted to lead to a severely truncated Fas molecule (van der Burg et al, 2000). The latter case demonstrated normal levels of mRNA transcripts of the mutant Fas gene. Patients with heterozygous ICD mutations show normal levels of Fas expression, but those with ECD mutations have either a normal or slightly reduced expression of Fas at the cell surface. Both groups show equal mRNA transcription of mutant and wild type Fas genes (Vaishnaw et al, 1999). Patients with reduced cell surface Fas expression, like the homozygous patients, have disease caused by haploinsufficiency, although in heterozygous patients the resultant clinical defect is much less profound (Fig 4). For these mutations, the mutant protein is either rapidly degraded

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WT

Haploinsufficient mutant

Dominant Negative mutant

Weak activation

MUTANT

Strong activation

MUTANT

MUTANT

DISC

No activation

Fig 4. Cross-linked WT Fas facilitates cooperative binding of FADD and procaspases. Cross-linked WT Fas facilitates cooperative binding of FADD and procaspases, facilitating efficient caspase processing, high concentrations of activated caspases and a strong apoptotic signal. Mutations that cause haploinsufficiency (e.g. ECF mutations with reduced surface Fas expression) process the procaspases, but less efficiently due to less available Fas at the cell surface. Dominant negative mutations result in Fas trimers containing mutant Fas, which inhibits the caspase processing capacity of the whole trimer by impairing cooperative binding of DISC components.

in the cytosol, retained in the endoplasmic reticulum due to interruption of targeting signals or is unable to anchor to the cell membrane and as is released as soluble protein. For most ICD mutations and some ECD mutations the mutant protein is expressed and is stable at the cell surface, and causes disease by interfering with DD signalling or inhibiting FasL binding, respectively. This disruption of function has been demonstrated by the assessment of Fas function in wild type and mutant co-transfection studies (Jackson et al, 1999; Martin et al, 1999; Vaishnaw et al, 1999). Demonstration of Fas receptor preassociation at the cell surface via the PLAD domain (Chan et al, 2000; Siegel et al, 2000a) provides an explanation for this dominant negative effect, because Fas trimers will form on the cell surface with seven of eight complexes containing at least one mutant protein. A single mutant protein within the trimer will be sufficient to inhibit the structural change in Fas that allows the cooperative recruitment of FADD to form the DISC (intracellular mutations), or reduce the affinity of trimerised FasL complex binding (extracellular mutations) (Fig 4). Some ECD mutations that result in reduced levels of Fas at the cell surface still cause dominant negative inhibition of wild type Fas-induced apoptosis in transfection studies, although the level of impairment was significantly less than that seen with ICD mutations (Rieux-Laucat et al, 1999; Vaishnaw et al, 1999). A possible explanation is suggested by the finding that even very

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small amounts of mutant Fas, relative to wild type Fas, produce a dominant negative effect and inhibition of apoptosis (Yokota et al, 2005), and therefore even transient, low expression of ECD mutant Fas could bind wild-type PLAD domains and have a significant impact on apoptosis signalling. Interestingly, this effect is exaggerated when T-cell-mediated Fas-induced apoptosis (a more physiological stimulus) was studied. This model explains how the majority of ICD mutations and some ECD mutations show dominant inheritance, whereas occasional ECD mutations demonstrate recessive inheritance. The striking finding that family members with both ICD or ECD mutations can be disease-free despite having the same Fas mutation as a severely affected close relative, cannot be explained by the above model. This finding can only be explained by a ‘second signal’ required for the full development of clinical ALPS. This is similar to the lprcg mouse model where autoimmunity only develops in certain strain genetic backgrounds. This second signal could be genetic or environmental, and may impair apoptosis signalling in a Fas independent manner. With 100% clinical penetration in some family lines (Jackson et al, 1999), another unanswered question is whether a second signal is always required for ALPS disease, or whether some mutations are capable of inhibiting Fas-induced apoptosis so profoundly that a single defect is sufficient. Dissecting the molecular mechanisms underlying the

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Review penetration of Fas mutations will lead to a greater understanding of the molecular interplay that underlies apoptosis signalling.

Immunobiology Lymphocytosis is a consistent finding in ALPS, and this is due to an expansion of the following lymphocyte subsets; CD8+ T cells, ab+ DNTs. Many of these abnormalities are also seen in asymptomatic individuals with a Fas gene mutation although the expansion of HLA-DR+ T cells and CD57+ T cells appears to be predictive of ALPS disease (Bleesing et al, 2001). Expansion of ab+ DNTs is the most prominent and consistent immunological difference between ALPS patients and healthy controls. The level of DNTs appears to correlate to the degree of lymphoproliferation seen in the patient, but once again there is enormous variation in the degree of this abnormality (from 1% to 75% DNTs) (Fisher et al, 1995; Dianzani et al, 1997; Sneller et al, 1997; Infante et al, 1998; Jackson et al, 1999; Rieux-Laucat et al, 1999). Asymptomatic individuals with Fas mutations show significantly increased absolute DNT counts compared with healthy controls, although this increase is much smaller on average than the levels seen with ALPS patients, and the proportion of DNT remains 75%) on DNTs supports the theory that these cells are post-terminally differentiated CTLs. Against a CD8+ cell origin for DNTs are studies of mice with deficient CD8 T-cell development and CD8a deficient patients who demonstrate that DNTs can develop in the absence of CD8 (Mixter et al, 1995; Ohteki et al, 1995; Calle-Martin et al, 2001). As CD8+ T cells develop cytotoxic activity they revert back from CD45RO subtype expression to CD45RA. In addition, CTLs downregulate CD27 and CD28 costimulatory molecules (Hamann et al, 1999), but in human ALPS DNTs these markers are upregulated suggesting ongoing stimulation. In summary, the origin of ALPS DNTs remains unproven, but it is likely they represent an

arrested T-cell developmental stage not seen in healthy patients, which are chronically activated in the specific in vivo environment. Although evidence suggests their accumulation is responsible for the lymphoproliferative disease in ALPS, it is unclear if they have any role in the pathogenesis of autoimmune disease or malignancy (Goldman et al, 2002).

Histopathology Lymph node biopsies of patients with ALPS reveal retention of lymph node architectural features with marked paracortical Tzone expansion. The pleomorphic lymphocytes responsible for this expansion range from small cells to lymphoblasts (Lim et al, 1998; Maric et al, 2005). Immunohistochemistry revealed that the majority of these cells are DNT cells (upto 70% of T cells) with an identical phenotype to the circulating abDNTs. These cells show evidence of active proliferation but very little apoptosis is seen. Among cells in the expanded paracortex that are not DNTs, the ratio of CD4 to CD8 T cells is markedly lower than in normal patients, and among these cells polyclonal plasma cell expansion is seen. There are also changes in the reactive germinal centres, including marked follicular hyperplasia and plasmocytosis. Interestingly, a normal pattern of apoptosis is seen in these areas, there is a normal proportion of CD4+ cells, and the majority of T cells are CD45RO+. A recent study has identified a histiocyte infiltration characteristic of Rosai–Dorfman disease (or sinus histiocytosis with massive lymphadenopathy) in 44% of ALPS lymph nodes, suggesting a link between the two diseases (Maric et al, 2005). Splenic biopsies have revealed follicular hyperplasia in an expanded white pulp, and a massively expanded red pulp containing cells phenotypically identical to those seen in the lymph node paracortical regions. No histology assessed has detected a difference between ALPS patients with a Fas mutation and those with normal Fas gene (Lim et al, 1998).

Basis of autoimmunity Animal models of autoimmunity have given conflicting evidence of the role of Fas in disease pathogenesis. In nonobese diabetic (NOD) mice Fas-mediated apoptosis plays an important role in the destruction of islet cells (Kim et al, 2000), yet in experimental autoimmune encephalomyelitis (EAE) Fas-mediated T-cell apoptosis is important in the induction of remissions (Sabelko-Downes et al, 1999; Suvannavejh et al, 2000). Similar contradictory findings have been found in human multiple sclerosis and type 1 diabetes (Dowling et al, 1996; Zipp et al, 1998; Comi et al, 2000; Huang et al, 2000; Pouly et al, 2000; Dianzani et al, 2003; Deutsch et al, 2004; Wong et al, 2004). The pattern of autoimmunity seen in ALPS differs from that in the lpr/gld mice models, with multi-organ disease and glomerulonephritis being typical in mice, whereas blood cytopenias are far more common in ALPS. The dysregulation of Fas function in autoimmunity is

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Review N o r ma l F a s f unc tio n Impaired f as function

U n c h e ck e d expansion of autoreactive iymphocytes

Antibody and T cell mediated autoimmunity

Overactive fas function Normal l ymphocyte homeostasis

Peripheral t olerance to autoreactive l ymphocytes maintained

Excessive Fas mediated k illing in t arget tissues

Cytotoxic cell mediated autoimmunity

Fig 5. Autoimmunity and Fas dysregulation.

explored in Fig 5 (Siegel et al, 2000b; Grodzicky & Elkon, 2002; Dianzani et al, 2003). The role of Fas sensitivity may be more important as a determinant of severity of autoimmune disease once the disease has been established as a result of an independent aetiology. In ALPS, autoimmunity is invariably only seen in association with appropriate autoantibodies, suggesting that the autoimmunity is primarily B-cell mediated. Although lymphocytes expanded following infection will be eliminated in ALPS (via cytokine withdrawal apoptosis), autoreactive lymphocytes will continue to receive antigenic stimulation, and therefore cytokine-based survival signals. Autoreactive B-cells resistant to FasL ligation on the surface of T cells may then survive and mature in the T-cell zones of lymph nodes, and produce autoantibodies (Rathmell et al, 1995). This production will be enhanced by the TH2 predominance of the cytokine response. The differential penetration of lymphadenopathy and autoimmunity in ALPS has led to the hypothesis that a third signal or genetic defect is required for the development of autoimmunity in ALPS patients (Rieux-Laucat et al, 2003a). This fits with a multiple-defect model for autoimmunity, where resistance to Fas-mediated apoptosis is one of several factors which determine type and severity of autoimmune disease (Dianzani et al, 2003).

function. Perhaps, it is surprising that patients with ALPS express as little malignancy as they do, and the predominance of lymphoid malignancy suggests the involvement of a common specific mechanism. Co-transfection studies and detection of nuclear factor (NF)-jB in activated peripheral blood lymphocytes of ALPS patients showed that, unlike the apoptosis signalling via Fas, the activation of NF-jB by Fas did not express dominant negative inhibition in the presence of heterozygous Fas mutations. As a result, lymphocytes in heterozygote ALPS patients are susceptible to unopposed NF-jB activation (Legembre et al, 2004a), which could contribute to the growth, survival and ultimately malignant transformation of these cells (Lin & Karin, 2003; Ruland & Mak, 2003). In addition, Fas stimulation of apoptosis resistant tumour cell lines by FasL has recently been shown to increase the mobility and invasiveness of the tumour cells via a NF-jB pathway (Barnhart et al, 2004; Legembre et al, 2004b,c). The association of malignancy with Fas death domain mutations but not with extracellular mutations also supports a hypothesis whereby low threshold Fas signalling is required but apoptosis signalling is impaired. Mutations causing haploinsufficiency will have reduced amounts of normal signalling and therefore not be exposed to unopposed NF-jB activation.

Basis of susceptibility to malignancy

ALPS Ib – FasL deficiency

The role of FAS as an oncogene is well demonstrated by the number of malignancies which have developed Fas mutations during their evolution (43 · 23, 43 · 36, 43 · 38), and the role of such a mutation in avoiding immune surveillance can be easily understood. Lymphocytes, the cells most profoundly affected by impaired apoptosis in ALPS, are particularly susceptible to malignant transformation because of high rates of cell division, and in B cells because of DNA recombination events that are central to their maturation and effector

A single case of FasL deficiency has been described (Wu et al, 1996), which was detected as a result of screening patients with system lupus erthythematosus (SLE). The patient presented at 52 years of age with a clinical syndrome typical of SLE, but with marked lymphadenopathy, no splenomegaly and no evidence of cytopenias. He showed defective activationinduced apoptosis, but increased T-cell proliferative responses to mitogens. Fas-mediated apoptosis was normal and there was no rise in DNTs. The patient was heterozygous for the

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Review mutation suggesting a dominant mutation (as would be predicted from the experience with Fas mutation), but it has not been possible to demonstrate inheritance. Although this case has been labelled as ALPS, the clinical and immunological phenotype is quite different from classical ALPS.

ALPS II – caspase 8 and 10 deficiency A small number of patients have now been identified with a typical ALPS clinical condition, normal Fas and FasL genes, but a mutation in the caspase 10 gene. Of the two patients described by Wang et al (1999) one was heterozygous for a caspase 10 defect that was inherited from the patient’s asymptomatic mother, but the other patient had a homozygous caspase 10 defect with one mutant allele inherited from each asymptomatic parent. Both patients demonstrated lymphoproliferation and autoimmune complications from the first year of life, and had DNT levels that were high, even for ALPS patients. The mutation in the homozygous case (V410I) has been described as a common polymorphism in the Danish population with an incidence of 6Æ8% in healthy controls, but no asymptomatic homozygous case has been described (Gronbaek et al, 2000). In addition to having TcR activation and Fas-mediated apoptosis defects, these patients also showed apoptotic defects in response to TRAIL stimulation, and co-transfection studies of the heterozygous mutation demonstrated inhibition of death signalling via TNFR1, DR3, TRAILR1 and TRAILR2. Apoptosis induced by cytokine withdrawal and staurosporin was normal. The immunological impact of this additional defect is a dendritic cell apoptosis lesion, leading to dendritic cell accumulation in the T areas of lymph node, which is not seen in ALPS Ia. The dominant negative effect of a caspase 10 mutation is important because it demonstrates the central role this enzyme plays in apoptosis signalling, which cannot be overcome by caspase 8. In fact, mutant caspase 10 must not only inhibit autoactivation, but also the activation of caspase 8. Investigation of these patients and their mutations has been important in elucidating the formation and signalling of the DISC, as described above (Kischkel et al, 2001; Wang et al, 2001). Chun et al (2002) described two siblings with a homozygous caspase 8 defect who, in addition to having lymphoproliferation, had an immunodeficiency characterised by recurrent herpes simplex virus infection, recurrent sinopulmonary infections and impaired vaccine responses but no autoimmunity. They showed defective Fas-induced apoptosis, impaired IL-2 production in response to TcR stimulation, defective induction of surface activation markers on lymphocytes following stimulation, defective NK cell activation and impaired B-cell antibody production. These patients were able to form a DISC, but it was non-functional and unable to induce caspase 3 activation. Carriers of the mutation were asymptomatic. This case demonstrates the importance of caspase 8 activation in the signalling of naı¨ve T-cell activation, in addition to its role in apoptosis signalling (Chun et al, 2002).

With so few of these signalling intermediary mutations described, and the complexity of the signalling network, it is difficult to know how much of their clinical condition is attributable to the single defect identified, and how much is dependent on other genetic conditions. Caspase 8 deficiency in mice is lethal in the early embryonic period, and perhaps the very rare mutations identified represent patients who have highly favourable polymorphisms of associated signalling molecules, allowing some residual signalling, and therefore viability of the individual. Alternatively, the converse may be true, and many other individuals do possess these and similar mutations, but their symptoms are not severe enough to have been identified for further investigation.

ALPS III – unknown defects Even among the first descriptions of ALPS there were patients with no identifiable mutation in the Fas signalling cascade (Sneller et al, 1997), but the clinical features, histology and immunophenotypes of the lymphocyte subset in these patients are indistinguishable from ALPS Ia. Dianzani et al (1997) described a series of patients with a clinical syndrome resembling ALPS, with defective Fas mediated apoptosis, but normal levels of DNT cells and absent Fas and FasL mutations. These patients also demonstrated defective ceramide-induced apoptosis but normal apoptosis in response to methylprednisolone. These findings suggest a defect downstream from the DISC formation. There is considerable heterogeneity of functional defects among these patients and, like classical ALPS, these patients show an inheritable component with variable penetration, again suggesting a multiple gene involvement (Ramenghi et al, 2000; Dianzani et al, 2003). Recently, another subgroup of patients has been identified with an ALPS-like clinical phenotype and raised DNTs but normal Fas-induced apoptosis. These patients had somatic Fas mutations identified from DNT cells and were mosaic for Fas expression with wild type Fas expressed in other cell types and tissues. The apoptosis resistance of the DNTs resulted in ALPS disease, but the normal lymphocytes present in peripheral blood expanded during the apoptosis assay to give a normal assay result (Holzelova et al, 2004). As our knowledge of ALPS increases it is becoming more obvious that the ‘ALPS-like clinical phenotype’ is broad, and is likely to include conditions which have already been independently classified (e.g. Evans syndrome, Rosai–Dorfman disease, NLP Hodgkin disease). It is also apparent that resistance to Fas-induced apoptosis is important in a wide range of diseases.

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