Abstract
New forms of Primary Immunodeficiencies (ID) are characterised by a clinical picture, which at times does not clearly suggest an immunological disease. This can make the diagnosis difficult. Very often most of these IDs belong to a complex syndromic picture, which can involve many systems. Today technological progress has enabled the identification of new disease processes, thus ensuring better characterisation of clinical pictures and especially recognition of patients with genetic immunodeficiencies who have yet to be diagnosed. In the framework of new IDs a particularly interesting group comprises cytolitic activity defects. Besides forms in which excessive apoptotic activity gives rise to pathological pictures, there are clinical conditions in which reduced cytolitic activity in the framework either of cell growth regulating processes or of mechanisms issuing from the immune response causes unusual clinical pictures of immunodeficiency, which can be misdiagnosed for this very reason. Alterations can involve two pathways: one involved in target cell cytolisis caused by the exocytosis of intracytoplasmic granules containing granzymes and perforin and the Fas/Fas ligand system. The first category numbers the following diseases: Griscelli’s syndrome, Chediak-Higashi’s syndrome and lymphohistiocytosis. Unlike perforin-mediated cytolitic activity defects, Fas pathway defects are characterised by intense hyperimmune symptoms. This second category numbers the following syndromes: ALPS, APECED and IPEX; paradoxically autoimmunity is the first introductory sign of immunodeficiencies.

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Primary Immunodeficiencies are a relevant health problem in the national territory. The incidence of serious primary forms is low, counting around 1 case every 10,000 born. However, despite the relative rarity of this paediatric disease group, it has been calculated that 5-10% of the paediatric population presents symptoms that can suggest a diagnosis of immunodeficiency. Besides, the identification of new forms of primary immunodeficiencies, whose clinical picture is neither very evident nor clearly suggests an immunological disease, indicates that many cases may not be correctly diagnosed in the appropriate time. Hence the real incidence of IDs cannot be currently defined. Everything can be blamed on the availability of technological resources in the framework of diagnostic procedures and thus suggests the need to create an integrated network of diagnostic centres evenly distributed throughout the country to enable physicians to speedily rule out possible immunodeficiencies. The recent and constant transfer of knowledge from basic subjects to clinical problems and the application of innovative technologies have considerably improved the levels of knowledge of these diseases, especially enabling the identification of new disease processes, a better pathogenetic characterisation of clinical pictures and speedy recognition of emerging problems, but they have especially enabled to mark out patients who can receive a complete diagnostic definition today and who had so far not been identified as individuals suffering form immunodeficiencies.
IDs discovered so far comprise over 100 forms with different symptom gravity and pathogenetic processes. Along with the typical forms in which cell, humeral or combined immunodeficiency appears under a clinical profile with an adequately clear picture, there are atypical cases in which the signs of the disease’s onset suggest a basic immunodeficiency to a lesser degree. Very often most immunodeficiencies belong to a complex syndromic picture, which hence involves many systems, thus configuring complex clinical pictures in which the immune involvement can be obscured by another system’s prevalent involvement.
Recent proof has also enabled experts to understand how an autoreactive response can paradoxically be a clinical sign of immunodeficiency since an autoreaction can result from alterations in the immune system’s homeostasis. Lymphocytes, which expand and proliferate in vivo after contact with the antigen, will later undergo apoptosis to maintain a constant number of cells. Programmed cell death or apoptosis is a physiological process that is characterised by alterations in cell morphology and DNA fragmentation into oligonucleotides.
Many intracellular signalling molecules participate in this process. In a broad sense cell lysis occurs either through the activation of the Fas/Fas ligand system pathway or through pathways, which by means of exocytosis of intracytoplasmic granules containing granzymes and perforin, cause target cell cytolysis (Fig. 1).
Recent proof indicates that these processes’ dysregulation is the pathogenetic process of certain diseases in man. In particular, alterations in processes that regulate programmed cell death play a relevant role in giving rise to virus-induced anergy conditions such as the measles virus or the chickenpox virus.
Besides an inappropriate apoptosis has been documented both at a morphological
and molecular level in combined hereditary immunodeficiency conditions. Hence this apoptosis is the process that causes lymphopenia, which is not present at the onset, in the advanced phases of these forms of congenital T immunodeficiency.
Besides forms in which excessive apoptotic activity gives rise to pathological pictures, there are however clinical conditions in which reduced cytolitic activity in the framework both of cell growth regulating processes and of processes issuing from the immune response causes immunodeficiency pictures with unusual clinical symptoms that can be misdiagnosed for this very reason.
As an example, we wish to present clinical cases, which show the clinical progress of these new immunological disorders to better define the clinical picture’s eleipofibrinogements, its progress and the molecular process involved.

Fig. 1

CASE 1
Alessia was admitted at 12 years in poor general conditions due to persistent fever, hepatosplenomegaly, cytopenia and enlarged lymph nodes. Her clinical history prior to admission reported normal clinical progress of common viral childhood infections, which made an immunodeficiency syndrome unlikely. However early biochemical investigations detected a reduction in the number of red and white blood cells, hypertriglyceridemia, hypofibrinogemia, hypoalbuminaemia, and hyperferritinemia. Serological data indicated the possibility of an infectious cause (Epstein Barr or Herpes Virus) for the fever. The functional analysis of Natural Killer cell activity detected the virtual absence of their activity, while their absolute number was normal. During the detailed diagnostic study, the analysis of the perforin gene, a key molecule in cytolitic processes, highlighted the presence of a mutation. Later the histological evaluation on a liver biopsy sample showed the presence of intense haemophagocytosis, thus confirming the suspicion of a genetic
Natural Killer activity deficiency with haemophagocytic lymphohistiocytosis. Unfortunately Alessia’s general conditions worsened drastically and she could not survive the acute episode for long.

CASE 2
Gianmarco was apparently well till he was 1 year old, when he presented widespread rash, erythema and urticaria unrelated to any apparent cause. Later at the age of 4 he presented a low platelet count, hepatosplenomegaly and lymphadenopathy.
Laboratory tests highlighted an increase in muscle enzymes, circulating immunocomplexes and the presence of autoantibodies. Besides, the functional analysis of Fas-induced apoptosis on lymphocytes in the peripheral blood was pathological. On the basis of a family tendency for autoimmune diseases, such as thyrosis and connectivopathy, and of the functional pathological test, he was diagnosed with Autoimmune Lymphoproliferative Syndrome (ALPS). This diagnosis was confirmed at a molecular level by the identification of a Fas gene mutation. Gianmarco currently lives a normal life for his age.
These cases, which differ greatly both in clinical presentation and progress, can however both be traced back to a defect in cytolitic activity.
Immunodeficiencies resulting from defective cytolitic activity In Alessia’s case the defect concerned processes, which caused cytolitic activity related to the role performed by T cells and Natural Killer cells in antitumoural and anti-viral immunity. In fact the cytolitic granules present in them contain molecules, which cause citotoxicity and this process is mediated by the exocytosis of granules and followed by the release of granzymes and perforin. In particular, in the process of target cell cytolisis caused by the exocytosis of intracytoplasmic granules, the molecular alteration can be caused by a perforin defect or by the inability to secrete cyolitic granules during exocytosis. As shown in Table 1, this category numbers many diseases, whose common factor is a defect in cytolitic activity, which generally ensures adequate defence from infections and especially from viruses. The main forms are: Griscelli’s syndrome, Chediak-Higashi’s syndrome and lymphohistiocytosis in the family.

Tab. 1

Griscelli’s syndrome is caused by a defect in the gene RAB27A, which encodes a “GTP binding protein” that is associated with cytolitic granules. Patients present many episodes of fever and lymphocyte organ infiltrations. Often there is hepatosplenomegaly, lymphadenopathy, pancytopenia and hyperpigmentation of the skin.
There is often sudden neurological deterioration with epilepsy, neuromotor deficiency and finally coma. These neurological alterations caused by white matter degeneration are especially present in frontoparietal areas. Progress is fatal in untreated cases. The death rate depends on an early diagnosis.
Chediak-Higashi’s syndrome is a rare multiorgan autosomic recessive transmission disorder that is characterised by oculocutaneous albinism, frequent infections caused by pyogenes, a tendency for bleeding and neurological disorders. It is associated with a defect in LYST, which regulates lysosomial traffic. The pathogenetic process is characterised by massive lysosomial inclusions, which cause relevant cytoplasmic damage and phagocytosis.
At the onset there can be neutropenia followed later by anaemia and a low platelet count. In the advanced phase of the disease many individuals develop lymphohistiocyte deposits in the liver, spleen, lymph nodes and bone marrow with these organs’ subsequent enlargement and malfunction. Since the clinical presentation is quite heterogeneous and therapeutic decisions must be personalised to suit each case, many patients die before they are 10 years old. A therapeutic possibility is hallogenic bone marrow transplant from an identical HLA brother or, failing a donor relation, from compatible bone marrow or umbilical cord.
Haemophagocytic lymphohistiocytosis (HLH) is a disease, which is characterised by a massive activation of the immune system, especially of hystiocytes.
This picture is generally associated with fever, hepatosplenomegaly, pancytopenia, hypertriglyceridaemia and hypofibrinogemia and it sets in early in life though its onset can occur at a later date. From a functional perspective experts have observed Natural Killer cells and cytotoxic T lymphocytes’ reduced or absent lytic capacity. Its hereditary forms are transmitted in a recessive autosomic manner. Only recently have these cells been identified in patients with HLH mutations in the gene, which encodes perforin, a molecule that performs an important lytic activity. Generally the evident disease is triggered by a common viral infection, whose progress is usually benign in healthy individuals. On the basis of current knowledge, it seems that HLH can appear after a symptom-free period that can be even very long and that there are no predictive functional
and/or biochemical parameters of evolution during the disease’s asymptomatic phase. These observations back the theory that there are unrecognised cases of HLH and hence that the disease is underdiagnosed. Hence we recommend following a diagnostic track for all individuals who present a particularly serious progress of a viral infection. In 40% of cases individuals with HLH show a mutation in the perforin gene. Many mutations have been described so far and some of them are well characterised, while for many others we have still to find a clear correlation between genotype and phenotype.
Immunodeficiency and Hyperimmunity Unlike cytolytic activity defects mediated by perforin, which lack these autoimmune symptoms, Fas pathway defects are characterised by intense hyperimmune symptoms. As illustrated in Table 2, the second category numbers the following syndromes: autoimmune polyendocrinopathycandidiasis- ectodermal distrophy (APECED); immunodysregulation - polyendocrinopathyenteropathy - Xlinked syndrome (IPEX), autoimmune - lymphoproliferative - syndrome (ALPS) and immunodeficiency with hyperimmunity caused by a lack of caspase. All these syndromes are characterised by the presence of autoimmune diseases, which can involve any system. These diseases are the only model of autoimmune disease associated with a single genetic defect. Besides, the identification of these new immunodeficiency syndromes, whose clinical picture is characterised by “clustering” of distinct autoimmune diseases in one individual, has considerably contributed towards further comprehension of autoimmune diseases’ genetic aspects.
These new IDs have been identified in man thanks to pilot studies conducted on animal models (mouse Lpr and Gld), which have enabled to understand how there can be a monogenic cause behind these hyperimmune syndromes. Even if a genetic tendency is recognised for a certain number of diseases, as witnessed by the association between autoimmune symptoms and some specific HLA system features, this association would not suffice alone to explain these hyperimmune phenotypes’ pathogenesis on a hereditary basis.
In a high percentage of cases functional studies have highlighted a Fas-induced apoptosis defect, which is the cause of the clinical phenotype. Experts have recently also identified molecular alterations associated with these pictures. In particular, APECED is associated with alterations in the “autoimmunity regulating” gene (AIRE), while the IPEX form is associated to alterations in the transcription factor FOX P3. Besides, mutations of 2 caspases, 8 and 10, have been identified in patients with hyperimmunity and, in the case of caspase 8, hyperimmunity associated with functional
immunodeficiency. In the case of ALPS instead 4 forms are currently known, as illustrated in Table 2.

Tab. 2

ALPs is a disorder that is characterised by chronic lymphocyte expansion and subsequent benign lymphocyte infiltration of various organs. Suspicious signs are lymphadenopathy (generally cervical and axillary) and hepatosplenomegaly. As illustrated in Table 3, the main clinical symptoms are: haemolytic anaemia, thrombocytopenia, neutropenia, urticaria, glomerulonephritis, autoimmune hepatitis, biliary cirrhosis, Guillan Barré’s syndrome, rheumatoid arthritis in youth, vitiligo and alopecia. They are rarely associated with: hydrops fetalis, autism, dyserythropoiesis, hypereosinophilia and high incidence of tumours. Though it is a benign disorder, long term complications require careful observation in time and appropriate treatment, such as the use of cortisone treatment, chemotherapy, antimalaria drugs, growth factors and surgery (splenectomy due to very large splenomegaly associated with hypersplenism and hepatomegaly). The most serious of these 4 forms is form 0, which lacks the Fas molecule.

Tab. 3

APECED is an autoimmune multiglandular syndrome caused by a defect in a single gene called AIRE (autoimmune regulator). The gene’s product is expressed in certain organs such as foetal thymus, lymph nodes, liver and spleen. It is rare but relatively very frequent in some ethnic groups: Finland, Sardinia and Iran. The disorder is autosomic recessive with a high degree of penetration.
Symptoms usually appear in early infancy but some patients develop the disease during the following decades. The phenotype is variable even in the same family. Besides immunodeficiency, it comprises both endocrine and non endocrine disorders: gonad diseases (primary hypogonadism), ectodermal dystrophy (onychodystrophy, anomalies in hair follicles, tooth enamel dystrophy, baldness), vitiligo, keratoconjunctivitis, hypothyroidism subsequent to thyroiditis, Sjogren’s syndrome, insulin dependent diabetes mellitus (IDDM), pernicious anaemia, atrophic gastritis and also chronic active hepatitis, cholelithiasis and malabsorption. Tab 4

Tab. 4

IPEX is an immunodeficiency, which is associated with autoimmune symptoms such as enteropathy and polyendocrinopathies.
It has an early onset with signs and symptoms already in the neonatal period or in early infancy. Enteropathy is characterised by: diarrhoea, gastrointestinal bleeding and ileum disorders.
Polyendocrinopathies comprise insulindependent diabetes mellitus and autoimmune thyroiditis. Tab. 5 illustrates the main clinical signs: atopical dermatitis, autoimmune thrombocytopenia, neutropenia, anaemia, leucocytosis, lymphadenopathy, respiratory distress, vascular anomalies, sepsis and at times serious infections (peritonitis, pneumonia, arthritis caused by Enterococci and Staphylococci) subsequent to immunodeficiency.
Cases of ulcerative colitis, glomerulonephritis, interstitial nephritis and muscular atrophy are reported less frequently.
The prognosis is serious: many patients die early due either to cachexia or to the immunosuppressive treatment required.

Tab. 5

Conclusions
New forms of ID have the special feature of being complex syndromes with a heterogeneous clinical presentation in which it is not always easy to recognise the immunodeficiency.
Besides, the knowledge transfer from basic subjects to clinical problems and the application of innovative technologies have already caused a significant improvement in the level of knowledge on individual diseases with evident positive effects on public health. Paradoxically at times the immunodeficiency can appear with a picture of autoimmunity. The failure to recognise immunodeficiency at an early stage as a pathogenetic mechanism behind a clinical phenotype greatly increases the ranks of these hospitalised patients, who are often labelled with diagnoses that identify the predominant organ disease (i.e. unexplained encephalopathy fulminans, hepatosplenomegaly, cluster of autoimmune diseases, etc.) with a considerable increase in public expense following poor understanding of the disease.
Doubtless multispecialist paediatric facilities can considerably contribute towards the recognition of these primary IDs through an integrated multidisciplinary approach. The presence of appropriate infrastructures, which are especially distributed homogenously throughout the country, is the tool required for the early identification of new cases and to reduce patients’ undue migration to other facilities either in the national territory or abroad. Concluding, we believe that the recognition of some warning signs can be useful to recognise these new immunodeficiencies, which deserve an appropriate detailed diagnostic study.
Warning signs of new immunodeficiencies resulting from disorders in apoptosis-regulating cytolitic activities or processes.
A viral infection’s particularly negative clinical progress, despite a long symptomfree period; Unexplained lymphadenopathy; “Clustering” of autoimmune diseases and/or immunodeficiencies; Positive family tendency to develop immunological and/or autoimmune disorders; Considerable lymphocyte organ infiltration.

Claudio Pignata, Pasqualina Ferri
Department of Paediatrics,
“Federico II” University, Naples, Italy