Severe combined immunodeficiency (SCID) is the most severe forms of inborn immunodeficiencies, which are characterized in most cases by complete absence of T-cell-mediated immunity and by impaired B-cell-function [121, 123, 220]. The over-all incidence is about 1:50,000 to 1:100,000 newborns, possibly there may be a higher incidence due to early lethality in undiagnosed cases in the case of patients who succumb in the course of overwhelming infections before the diagnosis of immunodeficiency is made. The differential diagnostic to “pure” cellular immunodeficiencies might be difficult in some conditions.

T-B+ SCID (OMIM*600802) are characterized by impaired development of mature T-cells while B-cells are present but non-functional. This form presents the most frequently observed SCID phenotype and can be observed in 30–50 % of all cases [88, 627]. T-B+ SCID can be further distinguished according to the presence or absence of NK-cells.

In the case of γc-deficiency and JAK3 deficiency, NK-cells are virtually absent (T-B + NK- SCID), whereas NK cell development is intact in SCID patients with T-B + NK+ phenotype. While NK cells are present in normal number in the IL-7 receptor α deficiency and in defects of the different subunits of the TCR, the CD3 γ, δ, ε and ξ-chain, NK cells are reduced in number in the CD45 deficiency.

The gene IL2RG covers 4,5kB of genomic DNA in Xq13.1 and contains a coding sequence of 1124 nucleotides distributed into eight exons. It is constitutively expressed in lymphoid cells including both T-, B- and NK-cell-lineages [378] and encodes the gamma c chain of the interleukin-2 receptor. The gamma c is a type I transmembrane protein which is transported to the cell membrane after cleavage of a signal peptide.

Defective production of interleukin-2 had been observed in an immunodeficient patient who had detectable circulating T-cells [698]. The observation that the knock-out mouse for IL-2 shows disturbed peripheral T-cell homeostasis and autoimmunity, but does not display a SCID phenotype [590], suggested already that XL-SCID is not caused exclusively by impaired IL-2 mediated signaling. This hypothesis has further been confirmed by the identification of mutations in the IL-2RA gene encoding the interleukin-2 receptor alpha chain (CD25), a subunit of the tripartite high-affinity receptor for interleukin, in a patient who showed decreased numbers of peripheral T cells and abnormal T-cell proliferation but normal B cell development and autoimmune features [597] like the murine IL-2 knock-out. The complex XL-SCID phenotype can be explained by the fact, that the interleukin-2 receptor gamma chain is not only part of the interleukin-2 receptor, but also of the IL-4, IL-7, IL-9, IL-15 and IL-21 receptors [406] [185] and has been therefore also designated “common gamma chain” [378]. Multiple cytokine mediated pathways are thus abrogated in the gamma C deficiency giving rise to the pronounced defect in T-cell maturation. Exceptionally patients with gamma c deficiency may develop some autologous T-cells which may be associated to a milder clinical phenotype [174, 436, 568].

IL-7 provides survival and proliferative signals through the IL-7 receptor and plays thus a critical role in early T-cell development. SCID with T-B + NK+ phenotype in humans due to mutations in the Interleukin-7 receptor alpha gene was first described by Puel et al. in 1998 in two patients with failure to thrive, diarrhea, recurrent otitis, viral infections and candidiasis [525]. Other patients with defect in the IL-7Rα have subsequently been described [85, 524, 554].

Up to now few cases of CD45 deficiency have been identified. A 2-month-old infant with mutations in the CD45 tyrosine phosphatase gene was described by Kung et al. [356]. This patient presented with low CD4 numbers while B-cell counts were normal and NK cells were found albeit in reduced number (T-B + NK+ SCID). The TCR αβ T cells were lacking, but γδ-cells were present. More recently a second case was reported by Tchilian in 2001 [648]. CD45 deficiency has thus to be examined in T-B + NK+ SCID phenotype when the more common etiologies have been ruled out.

CD3γ deficiency has been described in Turkish and Spanish patients [32, 509]. A defect of the δ chain has been found in a Canadian patient [144]. A French patient presented a CD3ε deficiency [616, 651]. Complete CD3δ and γ-deficient patients who present with SCID-symptoms have been described [161, 643]. A 4-month-old boy with primary immunodeficiency was reported to have a homozygous germ-line mutation of the gene encoding the CD3ξ subunit of the T-cell receptor-CD3 complex [542]. Interestingly, the CD3ξ-deficiency was partially corrected by somatic mutations resulting in a milder phenotype and in decreased numbers of circulating T cells. A second patient with complete CD3ξ deficiency resulting in T-B + NK+ SCID was described recently [547].

Despite the huge heterogeneity on the molecular level, the clinical manifestations of the different SCID forms are comparable, as shown by the observations in large cohorts of SCID patients in Europe and the United States of America which has revealed that the clinical presentation with regard to the infectious events is quite similar [88, 627]. The onset of manifestations is characteristically early, often already before the third month of life. Despite the protection through maternal antibodies, SCID patients develop recurrent infections with protracted course and unexpected complications. Before the age of 6 months the SCID patients develop chronic diarrhea, interstitial pneumonia and/or therapy-resistant mucocutaneous candidiasis. Infections with opportunistic germs like Pneumocystis jiroveci (beforehand Pneumocystis carinii) or Cryptosporidium are currently present. But also intracellular microorganisms like Listeria, Salmonella typhi, Toxoplasma and Mycobacteria can be found. Other manifestations are due to infections due to Aspergillus sp or viral infections like Adenovirus, Respiratory Syncytial Virus (RSV), CMV, Herpesvirus or EBV. The suspicion of SCID is always to be considered as a “pediatric emergency” with the risk of a rapidly fatal evolution if the immunodeficiency remains undetermined.

The clinical alarm signs in an infant which should direct our attention to a possible immunodeficiency or failure to thrive or loss of weight (often observed between the 3rd and the 6th month of age), chronic diarrhea, atypical eczematous skin manifestations, absence of adequate response to current antibiotics, recurrent candidiasis and persistent respiratory symptoms (chronic cough, chronic respiratory obstruction, progressive tachypnea or dyspnea). The clinical examination of a “classical” SCID patient reveals a hypoplasia of the lymphatic tissues (lymph-nodes, tonsils), there is no thymic shadow in the chest radiography. Consanguineous setting is in favor of an inborn error of the immune system as many deficiencies follow an autosomal recessive inheritance-pattern and are thus more frequently observed in consanguineous families. Lymphopenia and hypogammaglobulinemia are additional factors that should lead to further immunological investigations.

Vaccination with live vaccines is contraindicated in SCID patients. BCG vaccination in SCID patients causes disseminated infections that may be fatal. Infiltrating and ulcerating lesions at the impact of the vaccination and in the regional lymph nodes, but also systemic propagation with papular cutaneous lesions, osteolytic lesions and organ impairment of liver, spleen, lymph-node and lung may occur. As the BCG vaccination is no longer generally recommended in many countries, it should be checked if a patient has been exposed to BCG vaccination, and if so, adequate antibiotic treatment should be initiated even in the absence of any clinical manifestation. In the case of oral live polio vaccine or upon contact to recently vaccinated persons, central nervous poliomyelitis-infections and carditis may occur.

Other SCID manifestations concern in rare cases chronic hepatitis or sclerosing cholangitis. Cutaneous manifestations interests consist in recurrent warts, Molluscum contagiosum, atypical eczematous skin lesions, alopecia, seborrhoic skin manifestations as well as cellulitis.

The maternal alloreactive T-cells may lead to the clinical picture of “Graft versus host disease” (GVHD). Habitually asymptomatic, the so-called “materno-fetal” may touch different organs. Frequently exist maculopapular rush and hypereosinophilia, more rarely found are liver involvement with disturbed liver enzymes, profuse diarrhea or pancytopenia. Transfusion of non-irradiated blood-products can generate a fatal GVHD, thus only irradiated products should be used.

Anamnesis is a central element in the establishment of diagnosis and allows the identification of those children for whom immediate immunological explorations are indicated. As in most cases SCID follows autosomal recessive or X-linked inheritance, it is very important to perform an exact inquiry of family history and to analyze the genealogical background of the patient. Attention has to be paid to any other family member presenting infectious susceptibility, auto-immune manifestation or tumor-disease. Cases of unidentified infant death have to be reported. Obviously, autosomal recessive inborn errors are more frequent in a consanguine setting.

Basic investigations should contain a complete white blood count. Eosinophilia can be frequently observed in SCID patients. Absolute lymphocyte counts are often less than 1000/μl, but normal lymphocyte counts do not exclude SCID, as some forms of SCID present with absolute lymphocyte counts which may be within normal range. This may be the case on one hand in SCID-forms in which T-cell maturation is only impaired in a limited way (e.g. PNP deficiency), on the other hand in patients with “leaky” or atypical SCID who present hypomorphic mutations, which allow a residual function of the defective protein.

A special situation is the persistence of maternal T-cells after transplacental materno-fetal transfusion. In these cases, the presence of maternal T-cells should be eliminated through chimerism analysis: in male patients by in situ XX/XY hybridization of the CD3 positive cells, in girls by molecular biological methods (HLA or VNTR analysis of CD3 positive cells). In some cases, skin-, liver- or intestinal biopsies may be necessary to rule out a materno-fetal GVHD. HIV-infection should be ruled out systematically in all cases of suspected SCID.

Analysis of humoral immunity should be performed by dosage of immunoglobulins IgG, IgA and IgM. Antibody production in SCID patients is deeply reduced or completely abolished. In the first months of life a normal IgG-level may be observed due to the transmission of maternal antibodies during pregnancy, whereas a reduced IgM level is more significant. A detailed exploration of humoral immunity through analysis of specific antibody-levels following vaccination, allohemagglutinins or IgG-subclass is not useful before the second year of life, but should be done in older infants with suspected immunodeficiency. In case of enteropathy it is important to determine values for albumin in order to rule out an exudative enteropathy that may generate a “secondary” hypogammaglobulinemia through enteral protein loss. Sometimes intestinal biopsies may be justified, as lymphopenia may be observed in the context of lymphangiectasia.

In order to perform precise immunological diagnostic, a center for pediatric immunology should be contacted promptly. The characterization of the lymphocyte subpopulations can be achieved by flow cytometry and allows in most cases a first diagnostic classification of the SCID type with regard to the presence or absence of the different lymphocyte-populations (CD4+ and CD8+ T lymphocytes, CD19+ B-cells and CD3-CD16/56+ Natural Killer cells). It is important to determine in the same time the absolute lymphocyte count. Normal range of the different lymphocyte subpopulations are age dependent. For age-related normal values see [131, 165, 601].

The T-cell function can be assessed in specific laboratory assays in vitro by testing the lymphocyte proliferation upon stimulation through so-called mitogens or through specific antigens, the latter is only meaningful after vaccination (e.g. tetanus, tuberculin) or after infection (e.g. Candida, CMV or VZV). T cell receptor excision circles or TREC are episomal DNA circles that are generated during V(D)J recombination by endjoining of the removed genomic DNA segments, they attest continuing thymic output. These TREC can be analyzed by polymerase chain reaction [180], patients with impaired T-cell maturation lack TREC.

Depending on the characterization of the specific immunophenotype of the patient, different diagnostic hypothesis can be formulated. A molecular diagnosis should be achieved based on the identification of the underlying gene defect, but in no case should the adequate treatment be postponed because the definitive diagnosis is pending. Enzymatic determination of ADA and PNP should always be performed in distance to eventual blood-transfusions.

Ultrasound of the thymus or chest radiography allows the evaluation of the size of the thymus which is generally reduced in the SCID patients. In the case of ADA-SCID patients, an alteration of the anterior rips may be observed. Additional imaging may be necessary in the context of infectious complications. In all cases a detailed microbiological work-up should be performed. Direct identification through culture or with the help of polymerase chain reactions (PCR) should be privileged as serological analysis is not significant in immunodeficient patients with abolished antibody production. Bronchoalveolar lavage or digestive endoscopy with biopsies may be necessary in order to attempt microbiological documentation.

Theoretically, gamma c deficiency could be present exceptionally in females in the case of Turner syndrome (45X0), and in the very rare females with constitutionally unbalanced X-chromosome inactivation. Diagnosis should be confirmed by genetic analysis of the IL2RG. IL2RG mutations have been reported in different ethnical groups. IL2Rgbase [521], a database of identified mutations, is available on the web http://​www.​genome.​gov/​DIR/​GMBB/​SCID. The majority of mutation concerns single nucleotide changes leading to nonsense and missense-mutations, but there are also insertions, deletions and splice mutations. Mutations are not evenly distributed within the gene. There exist recurrent mutations at several positions, so-called “hot-spots”, most mutations concern the exon 5, followed by exon 3 and 4 [521]. Prenatal diagnosis at 11 weeks of gestational age is possible once the mutation is identified in a given family.

Female carriers remain healthy, they show non-random X-inactivation in T-, B- and NK-cells with the non-mutated X-chromosome being the active X-chromosome in their lymphocytes [133, 523], whereas myeloid cells show random inactivation. This underlines the important function of the common gamma c for the development of the lymphoid cell-lineages. This non-random X-inactivation in lymphoid cells has been used for the diagnosis of the carrier status.

At the slightest suspicion of SCID, adequate prophylaxis and treatment has to be initiated immediately, with the aim to treat acute infections and to prevent their recurrence. It is essential to isolate any suspected SCID patient in a sterile environment and to apply drastic hygienic measures. Suspicion of SCID is always a “pediatric-immunological emergency”, as exclusively a rapid and adequate treatment in specialized centers allows the initiation of a curative therapy. The preparations for hematopoietic stem cell transplantation (HSCT) should be launched immediately at diagnosis of SCID, a specialized center should be contacted and the patient should be transferred promptly. HLA-typing of the patient, his eventual siblings and his parents has to be performed as soon as possible. The guideline written by the Primary Immune Deficiency Treatment Consortium (PIDTC) is a useful protocol, which could be considered in treatment of SCID [600].

Exclusively irradiated blood-products should be transfused; CMV negative patients should receive only CMV negative blood-products.

At diagnosis, SCID patients are often in poor a nutritional condition and present chronic intestinal infection and inflammation which lead to impaired intestinal absorption. A high caloric parenteral nutrition is justified to cover the energetic requirement especially as due to infections energy requirement is higher in SCID patients than in age matched controls. The parenteral nutrition and anti-infectious intravenous therapy requires a central venous line. During central venous line placement tracheal secretions for additional microbiological analysis should be obtained in children with respiratory symptoms. In some cases a fibroblast biopsy for further genetic or functional investigations with regard to the underlying immunodeficiency can be justified.

Except for infants with complete Di George syndrome who lack an HLA identical donor and who need a cultured allogenic thymic transplantation, all children with inborn immunodeficiencies may be cured by allogenic HSCT, which is actually the treatment of choice for severe combined immunodeficiencies.

Up to now, only a few patients were treated by somatic gene therapy in clinical studies. For ADA-SCID enzyme replacement therapy is available. The first successful bone marrow transplantations were performed in 1968 [41, 237] shortly after the description of the „major human histocompatibility system“[24]. Since then more than 1300 patients with primary immunodeficiency have been transplanted worldwide. In the beginning, only unfractionated HSCT with HLA identical donors could be performed. Only about 20 % of the patients dispose of an HLA identical sibling. The development of T-cell depletion techniques starting in the beginning of the 1980’s [532] allowed the transplantation from haploidentical parental donors. Bone marrow, peripheral blood stem cells (PBSC) harvested by cytapheresis or cord blood can be used as source for HSCT.

Best results with regard to survival and immune reconstitution can be observed when using HLA-identical sibling-donors. In some cases the search for an HLA identical unrelated donor can be justified, if the patient’s HLA-type allows the identification of an HLA-matched unrelated donor in a reasonable time span. In clinically critic situations or in the case of a rare HLA-type in the patient, no time should be wasted with an unrelated donor search and haploidentical HSCT with one of the parents should be prepared.

Considerable progress has been observed with regard to survival rates: the first report in 1977 on the outcome of SCID patients showed survival with functional graft in only 14 out of the 69 transplanted patients [73]. In 2004, Buckley et al. report survival rates of 84 % in the case of HLA identical siblings, 71 % in HLA-matched unrelated donors and 63 % in haploidentical donors [82, 83]. The most frequent reasons of death concern infectious complications, veno-occlusive disease and graft versus host disease. In isolated cases in utero transplantation has been reported, but there seems to be no advantage in comparison to HSCT performed early after birth.

The first successful treatment by gene therapy was observed in the case of X-linked SCID, this was the proof of principle that gene therapeutic correction of the hematopoietic stem cell is feasible [106] and results in sustained immune reconstitution [278]. However, the occurrence of severe adverse effects has been observed subsequently [279, 280] with the appearance of leukemic transformation in actually 4 patients out of 10 in the French patient group and one patient treated at the Great Ormond Street Hospital. Occurrence of genotoxicity with retroviral vectors led to development of new generations of safer and efficient vectors such as self-inactivating gammaretroviral or lentiviral vectors as well as major advances in integrome knowledge [107, 218].

Allogenic HSCT is a curative treatment for XL-SCID patients and shows good success with regard to survival [30, 82]. The best results are achieved with an HLA identical related donor. In the case of haploidentical donors the immune reconstitution with regard to humoral immunity might be mediocre as patients often present only partial chimerism after HSCT with persistence of autologous B-lymphocytes, so that immunoglobulin-substitution has to be continued after HSCT [30]. Two isolated cases have been reported of successful in utero bone marrow transplantation, in which fetuses between 17 and 20 weeks of gestation received haploidentical T-depleted BMT via intraperitoneal infusion [221, 702]. In the follow-up, both patients showed adequate immune reconstitution and independence from immunoglobulin substitution [50, 53].

The observation in a single patient that spontaneous reversion of the genetic defect may occur in vivo, probably within a T-cell progenitor, and can generate functional T-cells [628], and a stable T-cell repertoire [76], was a powerful argument for the selective advantage of the corrected cell and opened the way for the development of gene therapy, an innovative therapy option for inborn immunodeficiencies. In 1999 a first clinical gene therapy trial was initiated in the Necker Hospital in Paris with inclusion of XL-SCID patients who lacked HLA-identical donor. The XL-SCID was the first disease in humans which was treated successfully by gene therapy. It could be demonstrated that the retroviral-mediated gene transfer of the gamma-c gene allowed sustained restoration of the patients’ immune function [106, 278]. This was the proof of principle that gene-transfer in hematopoietic stem cells can restore the development of the immune system. The appearance of severe adverse events due to insertional oncogenesis with development of uncontrolled T-cell proliferation were first observed in two patients [279, 280], at the time of this writing in total 4 patients have been identified with leukemic transformation which appeared after gene therapy.

Additional gene therapy trials for XL-SCID were launched by Thrasher et al. at the Great Ormond Street Hospital [236]. Until recently, no severe adverse events have been documented in this trial in which a similar protocol to the French one is used; the differences regard essentially the culture conditions and the vector design. However, Thrasher et al. reported a case of leukemia caused by the gene therapy in December 2007. Chinen et al. reported also on gene therapy for XL-SCID [119]. Such unfortunate adverse events led to extensive investigations to define the retrovirus integration profiles, which led to development and implementation of new generations of safer vectors [107].

As it has been explained in the 2.2 section, SCID is a heterogeneous group of diseases that affect cellular and humoral immune function. Twenty to thirty percent of all SCID patients have a phenotype where circulating T cells and B cells are almost entirely absent but natural killer (NK) cells are present (T-B-NK+ SCID, OMIM*601457) [216]. This particular form of SCID has an autosomal recessive pattern of inheritance and is most commonly caused by a defect in the Recombination Activating Genes (RAG1, OMIM*179615; RAG2, OMIM*179616) [238, 480]. There are also some types of T-B-NK+ SCID with sensitivity to ionizing radiation (OMIM*602450), which are caused by mutation in the gene encoding Artemis (DCLRE1C, OMIM*605988), CERNUNNOS (OMIM*611290), LIG4 (OMIM*601837), and PRKDC (OMIM*600899). Moreover such phenotype in addition to microcephaly and growth retardation (OMIM*611291) is due to mutations in the NHEJ1 gene (OMIM*611290). DNA ligase IV deficiency (OMIM*606593) is another form of T-B- SCID, which is characterized by a profound but not complete defect in the development of T and B lymphocytes (T-B-NK+ SCID) associated with various degrees of microcephaly, developmental defects and growth delay. There is a high heterogeneity with level of immunodeficiency in DNA Ligase IV deficiency, ranging from no immunodeficiency to profound SCID phenotypes. Patients with Cernunnos deficiency are characterized by severe T lymphopenia, progressive B lymphopenia and microcephaly [80].

The immune system encounters a vast array of foreign antigens, the recognition of which is facilitated by antigen-specific immunoglobulins (Ig)/B cell receptors (BCR), or T cell receptors (TCR). Immunoglobulins and B cell receptors control humoral immunity, recognizing soluble antigens, while T cell receptors are responsible for binding and reacting against antigens presented via cells using the human leukocyte antigen molecule. The diversity in the variable region of antigen receptors is created through random somatic recombination of genetic elements, forming a contiguous coding segment for a functional unit. This receptor also serves as a checkpoint in lymphocyte development; lack of it causes T cells to be blocked at the CD4, CD8 double negative stage and B cells do not mature past the pro B compartment [706]. T-cells lacking receptors cannot undergo selection in the thymus to become CD4⁺ or CD8⁺ immunocompetent cells, and IgM⁺ B cells are not exported from the bone marrow, resulting in T-B- SCID.

The principle genes that control the mechanism responsible for recombination of the antigen receptors are called Recombination Activating Genes 1 and 2 (RAG1 and RAG2). The RAG genes are convergently expressed specifically in lymphocytes and the RAG proteins that are produced act as a heterodimer, targeting the variable (V), diversity (D) and joining (J) components of T cell receptors (TCR) and immunoglobulins (Igs) which are then randomly selected from pre-existing gene segments and joined together through a process of recombination.

There are seven antigen receptor loci in mammals; TCR α, β, γ and δ loci along with Immunoglobulin receptors H, k and λ loci. The N-terminal variable part of TCR β and δ, and Ig heavy chain (H) are assembled through V, D and J recombination, while TCR α and γ and the Ig light chains are produced from V and J segments only. These gene fragments are recombined together and then joined, through RNA splicing, to a constant (C) region to produce a functional receptor. Because each locus comprises numerous copies of each V, D or J segment, random joining of these different regions of DNA can produce in excess of 10¹⁴ possible receptor combinations which are capable or recognizing the array of antigens encountered.

Each V, D and J gene are flanked by a recombination signal sequence (RSS) which is recognized by the RAG complex. Each RSS comprises a conserved palindromic seven base pairs (bp), followed by an AT-rich nine base pair motif, separated by either 12 or 23 bp of weakly conserved DNA. The length of the spacer is vital for producing functional receptors because recombination occurs only between RSS with 12 and 23 bp spacers [658]. Hence, V and J regions are flanked by RSS with different spacers so that V-J recombination occurs in preference to a non functional V-V or J-J arrangement. If the D segment is involved, such as for the IgH antigen receptor loci, appropriate spacers flank it to ensure the regions are joined in the correct order.

As demonstrated by experiments in vitro [425], RSS with unlike spacers are joined when the RAG complex produces a double strand break at the border of the palindromic heptamer motif, leaving a 3’ hydroxyl group that is then covalently joined to the same nucleotide position on the opposite strand. This results in DNA with a conserved coding sequence and a hairpin structure on the coding terminus. This action also excises the DNA between the recognition sites to produce a blunt 5’ phosphorylated signal terminus on the section that is looped out. The RAG proteins remain associated with all the cleaved ends of DNA [7]. The blunt signal ends are then ligated, typically without any modification [385], to form an excision circle with an exact signal joint (Fig. 2.1) [669]. These DNA circles are generally lost from the genome through dilution during cell division.

The second stage of V(D)J recombination requires the resolution of the hairpin ends to form a functional, rearranged reading frame. The ligation of the coding joint is imprecise compared to that of the signal ends with the loss or addition of approximately 15 nucleotides. This adds further variation to the receptor domain, although it does carry the risk of producing non-functional genes through frameshift mutations or introduction of premature stop codons. The addition or loss of nucleotides arises firstly by the random opening of the hairpin within the coding region, rather than exactly at the covalently closed terminus [562, 733]. If the hairpin is opened asymmetrically, the overhang can be filled in by the addition of short palindromic (P) repeat nucleotides upon resolution of the structure [381]. RAG1/2 can mediate hydrolysis of hairpins in vitro [58, 605] but while their presence appears to be required [323, 562, 721], Artemis (DCLRE1C) is the most likely candidate to open the RAG-generated coding hairpin [450]. This protein is phosphorylated by the DNA protein kinase catalytic subunit (DNA-PKcs) activating an endonuclease capable of cleaving hairpin DNA [183, 399]. Coding ends are also modified through template-independent addition of random N (GC rich) nucleotides by terminal deoxynucleotidyl transferase (TdT) [251, 347, 561]. Joining of homologous regions or truncation of random nucleotides at the ends of the free DNA are further mechanisms implicated in producing additional junctional diversity [563].

The lose ends of the modified coding signal are joined by ubiquitous proteins involved in the non-homologous end joining (NHEJ) pathway. DNA-dependent protein kinase (DNA-PK) recognizes open DNA ends, mediated by the DNA-binding subunits KU70 and KU80 and catalytic subunit DNA-PKcs. The final joining of the double strand breaks of RAG-associated cleavages is probably due to a complex of several factors [266]: a novel protein, XRCC4 [384] associates with DNA ligase IV [139, 266] and the protein Cernunnos or XLF [9, 80, 96] to ligate double strand breaks. Mutations of these NHEJ factors can lead to immunodeficiency [80, 372].

The observation of patients presenting a T-B-NK+ phenotype with increased sensitivity to ionizing radiation without mutations in the known factors involved in non homologous end joining (NHEJ) in mammals (Ku70, Ku80, DNA-dependent protein kinase catalytic subunit, XRCC4, DNA ligase IV, or Artemis) [145] indicated that there were still other NHEJ-repair-factors to be discovered. Recently a new factor was identified through the study of five human SCID patients with severe progressive T and B cell lymphopenia and increased sensitivity to ionizing radiation: CERNUNNOS or XRCC4-like factor (XLF), was cloned contemporarily via a complementation strategy in Cernunnos deficient patients’ fibroblasts [80] and via its capacity to interact with XRCC4 [9], respectively. Cernunnos is located on the long arm of chromosome 2 (2q35) and its cDNA comprises 2063 nucleotides giving rise to a protein of 299 amino acids. Cernunnos shows homology to XRCC4 [96] and forms a complex with XRCC4 and DNA-ligase IV, its precise molecular function remains to be elucidated, but it can be considered as a “new” factor of the NHEJ pathway. With regard to V(D)J recombination, the fidelity of signal joints is impaired in Cernunnos deficiency with various length of nucleotide deletions [80, 145].

RAG1 and RAG2 are located on chromosome 11p13, 8 kb apart. The proteins are the only lymphoid specific factors required for recombination of RSS sites. When the genes are artificially expressed in non-lymphoid cells where rearrangement does not normally occur, a test substrate is recombined [491, 587], suggesting that the remaining required factors are available in all cell lineages. Equally, lack of either RAG 1 or RAG 2 in humans or mice [444, 602] leads to an absence of mature T and B cells with no other defects, implying that RAG genes function only in lymphoid cells.

As homozygous or compound heterozygous mutation cause disease, this form of SCID follows an autosomal recessive pattern of transmission. RAG1 and RAG2 are arranged in an unusual tail-to-tail configuration, sharing a 3’ untranslated region and both lacking introns [6]. There is no homology between the genes, but they are highly conserved in animals, emphasising their importance. In addition to this genomic configuration, the close arrangement of the genes suggests the genes may have appeared at the same time in early vertebrates through an insertion of a mobile genetic element [56, 655].

In addition to mutations of RAG1 or RAG2, T-,B- SCID in humans has been caused by aberrant expression of Artemis [450], Ligase IV [56, 81, 489, 536] and Cernunnos/XLF [80]. Because these genes are also involved in DNA double strand break repair, SCID caused by their disruption is also associated with radiosensitivity [615].

Out of 174 cases of SCID examined at one American Medical Center, 3.4 % were due to RAG mutations, 1.1 % due to Artemis and 16.1 % caused by ADA deficiency [87] although worldwide, RAG mutations account for approximately 50 % of T-B- SCID [86].

Mutations of a given gene can generate a multitude of clinical phenotypes depending on the type of mutation and additional somatic mutations, environmental and regulatory factors. Hypomorphic mutations in the genes RAG1 or RAG2 have been shown to generate an oligoclonal T-cell repertoire which in the case of Omenn syndrome will expand and display self-reactivity [159, 679]. The observation, that identical mutations in RAG1 or RAG2 can be observed in Omenn syndrome but also in typical and atypical SCID patients [478, 592, 679], sometimes in the same kindred [134], suggests the involvement of one or more modifying factors.

An interesting phenotype of hypomorphic RAG1 mutations was described in several patients with TCRαβ T cell lymphopenia, severe cytomegalovirus (CMV) infection and autoimmunity [163, 191]. T cells have been shown to be of autologous origin. De Villartay et al. describe four unrelated patients from consanguineous families who present hypomorphic mutations in Rag1, three of the four identified mutations have already been described in patients with Omenn syndrome (del T631, del 368-369 and R841W). The missense mutation Q981P found in the forth patient involves amino acids within the minimal core of RAG1 leading to a protein with residual RAG1 activity [163]. The remaining patient developed EBV-associated lymphoproliferation and presented an R561H RAG1 mutation [191] which had also been described previously in Omenn syndrome patients. It can be speculated that in these patients due to hypomorphic mutations in Rag1, a limited T cell repertoire is generated. The early occurrence of CMV infection may then induce a huge expansion of oligoclonal non γδ T cell clones.

Patients with attenuated forms of T-B-NK + SCID have been described, for example a patient who survived for 6 years without HSCT carrying mutations in RAG1, an R559S substitution on one allele and an R897X substitution on the second allele [354]. This patient presented maternal derived T cells and autologous peripheral B cells which were shown to be functional as specific anti HSV antibodies were observed. In fact, it has become obvious that the clinical spectrum for Rag1/Rag2 defects comprises not only complete abolition of V(D)J recombination leading to typical T-B-NK + SCID patients and hypomorphic mutations giving rise to Omenn Syndrome: more and more “atypical” SCID forms are identified [680, 681]. Thus genetic analysis of the Rag1 and Rag2 genes should be considered also in atypical clinical presentations.

Hypomorphic mutations in the Artemis gene may be found in patients that show clinical and immunological features that are indistinguishable from Omenn syndrome due to mutations in Rag1 or Rag2 [190].

In our patient group, four patients of two different kindreds showed a combined immunodeficiency with profound B- and T-lymphopenia and severe hypogammaglobulinemia generated by mutations in the last exon leading to truncation of the Artemis-protein and thus leaving intact the metallo-beta-lactamase domain [451, 454]. These “hypomorphic” mutations display a partial V(D)J recombination activity as assessed in the functional V(D)J assays in patients’ fibroblasts and have an incomplete complementation of the sensitivity to ionizing radiation compared with a cell line fully deficient in Artemis. The patients present polyclonal T and B lymphocyte populations albeit in low number. Interestingly, two out of the four patients developed EBV-associated B-cell lymphoma; in three of the four patients a general genomic instability was found. It has thus been hypothesized that Artemis may play an important role in genome stability. According to the hypothesis of Kinzler and Vogelstein [338], Artemis may be considered as genomic “caretaker” involved in the repair of genomic lesions and thus guaranteeing genomic stability. This hypothesis was emphasized by the observations of chromosomal fragments, fusion and detached centromers in different cell lines of Artemis knock-out mice [559] indicating genomic instability in these mice [557]. Artemis/p53-deficient mice succumb to progenitor B cell tumors [558]. Furthermore, it has recently been described that tumorigenesis in several tissues is accelerated in Artemis deficient mice in a Trp 53 heterozygous setting, emphasizing the tumor suppression role for nonhomologous end-joining in lymphoid and non lymphoid cells [490, 714]. These findings suggest that Artemis deficient patients may be at risk for the development of lymphoid and non-lymphoid malignancies.

DNA ligase IV (LIG4) which is located on chromosome 13.q22-q34: the cDNA encoding a polypeptide of 844 amino acids [697] is essential for embryonic development and its complete deficiency causes early lethality accompanied by defective lymphogenesis and defective neurogenesis in knock-out mice [226, 235]. DNA ligase IV is a component of the non homologous end-joining and participates thus in the repair of DNA double strand breaks (dsb) that arise during DNA damage induced by ionizing radiation but also in the context of endogenously induced DNA dsb during V(D)J recombination. As detailed in the Sect. 2.3, V(D)J recombination is initiated by the lymphoid specific proteins RAG1 and RAG2 that introduce a DNA dsb between a coding segment (V, J or D) and the specific recombination signal sequence (RSS). This generates four different extremities: two blunt signal ends and two hairpin sealed coding ends, which are then resolved by the NHEJ-DNA repair pathway composed of at least six factors: DNA-PKcs, Ku70, Ku80, Artemis, DNA-ligase IV and XRCC4. Whereas the signal ends can be directly ligated by the complex formed by DNA-ligase IV with XRCC4 [139, 265] giving rise to a precise signal joint, the coding ends have to be processed prior to their ligation which generates an imprecise coding joint. V(D)J recombination in patients’ fibroblasts shows only moderate impairment with an almost normal recombination frequency of coding- and signal joint formation, but the fidelity of the signal joint formation in DNA ligase IV deficient patients is highly compromised [81].

The Lig4 ^Y288C mouse strain presents hypomorphic mutations in the DNA ligase IV gene and is characterized by growth retardation and immunodeficiency. The diminished DNA double-strand break repair in Lig4 ^Y288C mice causes a progressive loss of hematopoietic stem cells and bone marrow cellularity during ageing [476], thus it can be speculated that DNA ligase IV may be required beyond V(D)J recombination for lymphoid homeostasis explaining why DNA ligase IV deficiency can cause profound immunodeficiency despite the fact that there is only moderate in vitro impairment of V(D)J recombination in DNA ligase IV deficient patients.

Hypomorphic mutations of DNA ligase IV have been described in humans, first in a 14 year old leukemia patient who overresponded to radiotherapy [536, 537]. The observed increased cellular sensitivity to ionizing radiation was the clue to the diagnosis of DNA ligase IV deficiency. Interestingly, this patient did not display developmental or immunological abnormalities before the onset of leukemia.

Van der Burg et al. identified a homozygous missense mutation in the PRKDC (DNA-PKcs) gene in a girl with T-B- SCID and increased cellular sensitivity to radiation [666]. Few years later, Woodbine et al. showed compound heterozygous mutations of that gene in a case with severe neurologic abnormalities. Functional studies revealed a loss of function, resulting in decreased protein expression, loss of kinase activity, and impaired NHEJ and DSB repair [716].

Symptoms of T-B- SCID are similar to all other SCID and are generally manifested as early opportunistic infections with impaired growth by the second or third month after birth. Patients often present with candidiasis, chronic persistent infections of the airways, and local or systemic bacterial infections. These most commonly cause rhinitis, otitis, mastoiditis, abscesses, conjunctivitis and meningitis. Chronic diarrhea associated with gram-negative enteric bacterial sepsis causes a failure to thrive.

Maternal T cells are engrafted in half of all patients and Natural killer cells are present in this form of SCID. After decline of maternal immunoglobulins, no antibodies circulate in the peripheral blood and the lack of mature B and T cells is often accompanied by an absence of a thymus, tonsils and cervical lymph nodes.

DNA ligase IV deficient patients with a varying degree of T and B immunodeficiency, microcephaly, facial dysmorphy, growth retardation and developmental delay have been described [56, 81, 199, 489]. Some patients present exclusively a T-B-NK+ SCID phenotype without any growth or developmental defects [667]. After the first leukemia patient, who had been reported to have a mutation in DNA Ligase IV, several other patients have been identified with DNA ligase IV deficiency and lymphoproliferation or lymphoid malignancy: EBV associated B cell lymphoproliferation in two patients [81, 657], and acute T-cell leukemia in another patient [56].

Cernunnos deficient patients present recurrent bacterial, viral and/or parasitic infections like those observed in other SCID patients. Developmental defects, microcephaly, bone and urogenital malformations, and a “bird like face” could be other features of Cernunnos deficiency.

To diagnose T-B- SCID, a full lymphocyte count and flow cytometry should be performed on peripheral blood, including markers for B, T and NK cells. RAG and Artemis deficient patients will generally lack T cells and B cells with NK cells present.

For full T-B- SCID, patients generally lack a thymus on X-ray or ultrasound imaging. Once an initial diagnosis has been determined based on physical examination, further investigation can be performed to establish the molecular basis of disease. DNA sequencing can reveal the mutation responsible and if parental mutation has been previously determined, prenatal diagnosis can be offered [642, 678].

The immunophenotype of DNA ligase IV deficient patients may be very heterogeneous, ranging from an almost complete T-B-NK+ SCID phenotype to milder presentation with various degrees of lymphopenia and hypogammaglobulinemia [241, 489]. Radiosensitivity and microcephaly are important clues to diagnosis, but there may be rare cases without the typical microcephaly. Other characteristic features that should lead to the suspicion of DNA ligase IV deficiency are developmental retardation and growth delay.

The diagnosis can be confirmed by sequencing of the genes.

Upon presentation, management of life-threatening infection is the immediate concern and is treated with antibiotics and antifungal drugs specific for the pathogen (often Candida spp, Pneumocystis jiroveci or Staphylococcus aureus). Antiviral agents should be used only if necessary.

Isolation of the patient with meticulous skin and mucosal hygienic practice is essential to prevent further infection. Prophylactic antibiotics, antifungal agents and intravenous immunoglobulins are usually required. Parenteral or enteral nutrition is an option when patients have severe diarrhea and are failing to thrive through malnutrition.

Patients should not be immunized with live viral vaccines, as they can cause fatal symptoms.

If left untreated, all forms of T-B- SCID are fatal. Bone marrow or stem cell transplantation is the only curative therapy available, although the mortality rate with this treatment is higher when compared to other types of SCID [30]. However, HSCT outcome in DNA ligase IV deficiency may be limited by complications due to increased sensitivity to conditioning regimens, even if “reduced intensity” conditioning is chosen, and more severe GVHD due to the DNA repair deficiency. It can be speculated that long-term outcome may be compromised by occurrence of secondary malignancies; nevertheless the observation period after the few performed HSCT for DNA ligase IV deficiency is still too short to draw final conclusions.

Gene therapy is a possibly future option for treating this disease. Advances treating other types of SCID have been made [10, 106, 236, 278, 494], using retroviruses to deliver functional copies of the affected gene to patients’ stem cells ex vivo. The treated cells can then be re-implanted and give rise to an effective immune system. Gene therapy vectors to treat T-B- SCID are currently being tested [365, 455, 722] and may soon provide an alternative treatment in situations when bone marrow donors are unavailable. Occurrence of genotoxicity with retroviral vectors led to development of retroviral vectors devoid of its enhancer element, which showed safety and efficacy of this method [218]. The guideline written by the Primary Immune Deficiency Treatment Consortium (PIDTC) is a useful protocol, which could be considered in treatment of SCID [600]

Omenn syndrome (OS, OMIM*603554) is a related disease first described by Gilbert Omenn in 1965 after observing a consanguineous family with an unusual skin disorder [493].

Omenn syndrome is caused primarily by missense mutations in RAG1 or RAG2, which do not entirely abrogate V(D)J recombination [679, 685]. Partial activity of the recombination activating genes allows some T cell clones develop and survive, but because of the oligoclonal nature of the population, patients remain immunodeficient. The severity of disease is variable and can be partially attributable to genotype although there are exceptions: identical mutations in RAG genes have been discovered in both T-B- SCID and OS patients [134, 240]. As OS describes a heterogeneous range of symptoms and is not a molecular definition, the disease can be the result of mutations in genes other than the RAGs [243], such as Artemis [190] or IL-7Rα [238, 480].

Symptoms are similar to other SCID but also characterized by lymphadenopathy and hepatosplenomegaly which are problems unusual in other types of SCID. Patients also suffer from alopecia and an exudative erythrodermia that is associated with episodes of Staphylococcus aureus sepsis. This skin condition becomes apparent as pachydermia which progresses to desquamation, resulting in protein loss through the skin which, in conjunction with diarrhea, causes hypoproteinaemia and edema. Normal to elevated levels of T cells can be present but these cells have a skewed T-helper-2 (Th2) profile [118] and due to their highly oligoclonal nature [159, 287], are poorly functional. Th2 cells produce elevated levels of interleukins 4 and 5 which lead to hypereosinophilia and despite the absence of B cells, increased serum levels of IgE.

To diagnose OS, lymphocyte count and flow cytometry should be performed on peripheral blood. An initial misdiagnosis of atopic dermatitis or a food allergy is possible in Omenn syndrome. Engraftment of maternal T cells in utero can cause a skin condition with a similar appearance to graft-versus-host type illness, but OS can be differentiated by lack of T cell chimerism and eosinophilia, where lymphadenopathy and hepatosplenomegaly are also hallmarks of the syndrome. In OS, B cells are absent but an oligoclonal population of T cells is present with an activated antigen stimulated Th2 cell profile, as shown by presence of CD30 of the T cell surface with a CD45RO positive phenotype. These cells are responsible for the increased IL-4 and IL5 levels in serum.

Immunoglobulins A and M are absent whilst levels of IgE and maternal IgG will be elevated. OS T cell lymphocyte stimulation assays against concanavalin A (conA), pokeweed mitogen (PWM) and phytohemagglutanin (PHA) are absent or greatly decreased. Lymphocytes will however show some response to stimulation with anti-CD3, superantigens and phorbol myristate acetate (PMA).

Patients with OS generally lack a thymus on X-ray or ultrasound imaging. DNA sequencing can reveal the mutation responsible and if parental mutation has been previously determined, prenatal diagnosis can be offered [642, 678].

Therapeutic procedures are the same as for other forms of SCID. Dermatitis can be treated with immunosuppression and topical steroids. Immunosuppression of the patients’ oligoclonal T cells has decreased incidence of graft versus host disease [281]. The guideline written by the Primary Immune Deficiency Treatment Consortium (PIDTC) is a useful protocol, which could be considered in treatment of SCID [600]

Purine nucleoside phosphorylase is a key enzyme in the purine salvage pathway. PNP catalyzes the phosphorylation of inosine, deoxyinosine, guanosine and deoxyguanosine to yield guanine or hypoxanthine and ribose -1- phosphate or 2’-deoxyribose 1-phosphate. These ubiquitous purine metabolic pathways are responsible for the proper balance between the production of dephosphorylated purines, detoxification by further degradation to uric acid, and salvage by metabolism back to the nucleotide level. PNP is also responsible for catalyzing guanosine and deoxyguanosine back into the GTP pool. Maintenance of low and balanced intracellular deoxynucleoside triphosphate pools is critical for the fidelity of DNA synthesis and repair [128, 197, 437, 531].

The metabolic consequences of the PNP deficiency is the accumulation of all four PNP substrates; inosine, deoxyinosine, guanosine and deoxyguanosine [127]. Because PNP activity is obligatory to purine degradation, no uric acid is produced [127]. Of the four metabolites only deoxyguanosine can be phosphorylated further in mammalian cells [201, 690]. As a result, cells from patients with PNP deficiency accumulate abnormally high levels of intracellular dGTP [127]. The high concentration of dGTP is believed to cause lymph toxicity in patients with PNP deficiency.

Much of these metabolic effects on the immune system were learned from animal models. Three mutant mice lines were generated with a single amino acid substitution and partial PNP enzymatic activity (1–5 % of wild-type) [614]. The PNP mutant mice developed partial immune deficiency after 2–3 months consistent with the partial reduction in PNP enzymatic activity. The total number of thymocytes was reduced with a decrease in the number of CD4 + CD8+ double positive cells and an increase in immature CD4-CD8- double-negative cells. In parallel spleen, T cells were reduced by 50 % and their response to T-cell mitogen was impaired partially. The overall conclusion of this study was that the progressive T-cell defect is similar to the human disorder. It is likely that the partial nature of the mutations in the PNP may hinder direct comparison with the human disease and further insight into the mechanism of the immune deficiency.

The authors’ group [33] generated a PNP-deficient mouse by gene targeting resulting in a complete absence of PNP enzymatic activity. The PNP-deficient mice develop severe immune deficiency at an early age characterized by abnormal intrathymic T-cell differentiation, progressively reduced peripheral T cell with impaired immune function, and minimal abnormalities of B lymphocytes or other tissues. The observed immune phenotype of the PNP-deficient mice is similar to clinical observations in patients with PNP deficiency.

The following observations of the immune phenotype of PNP-deficient mice shed light on the mechanism by which PNP deficiency may cause immune deficiency: (1) The development of T cells in PNP-deficient mice is affected at the CD4 + CD8+ double-positive intrathymic stage of differentiation; (2) in PNP-/- mice, the double-positive thymocytes undergo enhanced apoptosis in vivo markedly increased rates of activation induced apoptosis in vitro; and (3) apoptosis of double-positive thymocytes can be induced by inhibition of PNP in the presence of deoxyguanosine. The deoxyguanosine-induced apoptosis of double-positive thymocytes is inhibited by over expression of Bcl-2 or by inhibition of caspase activity.

Together, the experimental evidence supports the following hypothesis explaining the mechanisms of the immune deficiency caused by PNP deficiency:

It should also be added that mutations in the ADA gene [3], which normally breaks down toxic products of the purine scavenging pathway, cause apoptosis in lymphocytes. As such this also results in a T-B- SCID, but patients also lack natural killer cells [250].

PNP deficiency is a rare disease with an estimated frequency of 4 % among patients with SCID [95]. Patients with PNP deficiency typically have a triad of symptoms including neurologic abnormalities, autoimmune phenomena, and recurrent and unusual infections.

Similar to children with other types of severe immune deficiency, PNP deficiency may come to medical attention during the first year of life because of prolonged diarrhea, oral thrush, or respiratory infections [146, 295]. Other infections include meningitis, recurrent otitis, sinusitis, mastoiditis, pharyngistis, pneumonia, and skin infection [128, 146, 222]. Patients are extremely susceptible to viral infections such as varicella, cytomegalovirus, Epstein-Barr virus, parainfluenza [95], and the polyoma JC virus [504]. There is a considerable heterogeneity both in age of presentation and severity of symptoms. In some cases significant infections are delayed until later in life [128, 146, 189, 222, 504] or have only mild symptoms, which may be credited to residual PNP activity [283].

Neurologic abnormalities are common in PNP deficiency [146, 283], and more than 20 % of cases seek medical consultation due to neurologic symptoms that can not be explained by infections or preceding signs of immune deficiency [617]. The majority of neurologic manifestations are related to the motor system dysfunction, such as non-progressive cerebral palsy, spastic paresis, or tonus abnormalities. Disequilibrium characterized by hypotonia, pronounced difficulty in maintaining posture and upright position, associated with spastic diplegia and ataxia [283] or spastic paraplegia have also been described [496, 641]. Other neurologic findings include tremor, developmental delay, hyperactivity, behavioral problems, and varying levels of mental retardation, some of which may be related to recurrent brain infarcts.

One third of the patients manifest autoimmune phenomena, which may be the presenting feature [95, 222]. These include autoimmune hemolytic anemia (associated with autoantibodies to erythrocytes) [146], idiopathic thrombocytopenic purpura, autoimmune neutropenia, arthritis, pericarditis, and systemic lupus erythematosus [84]. Patients with autoimmune disorders may test positive for rheumatic factors and antinuclear antigens [100].

PNP deficiency is an autosomal recessive disorder. The gene that encodes PNP is localized on chromosome 14Q13.1 [708]; and several disease-causing mutations have been identified [28, 38, 146, 441, 499, 585, 663]. Different mutations in the PNP gene produce proteins with variable degrees of enzymatic activity that correlate with accumulation of nucleoside substrates and with the clinical course. Retention of partial enzyme activity may lead in some patients to less severe metabolic abnormalities, delayed presentation, milder clinical symptoms, and immune dysfunction [128].

All patients with PNP deficiency have purine nucleoside abnormalities (elevated inosine and deoxyinosine, and also guanosine and deoxyguanosine in blood and urine). Uric acid blood level is typically below 2.0 mg. Normal or slightly decreased uric acid levels are found in few patients with partial enzyme activity [59, 283]. Low serum uric acid levels also may be caused by proximal renal tubular diseases (e.g., Fanconi syndrome) or xanthinuria, in which blood and urine levels are extremely low [301]. Other metabolic abnormalities found in patients with PNP deficiency include elevated dGTP, undetectable in normal individuals, and depletion of GTP in erythrocytes to about 10 % of normal levels [295].

PNP activity can be determined by measuring the rate of conversion of radioactivity labeled inosine to hypoxanthine [95] or by spectrophotometry in which the coupled conversion of inosine to uric acid in the presence of xanthine oxidase is tested [544]. Normal PNP activity varies in different human cell and tissues extracts; the diagnosis of PNP deficiency is based commonly on enzyme activity in hemolysate [249]. Undetectable or lower than 1 % activity is usually found in patients with PNP deficiency [100], but activity as high as 4.8 % of normal control was associated with immune deficiency, although with a mild course and delayed presentation [574]. Determination of PNP activity could be affected by recent erythrocyte transfusion [189]. It is advised in these instances to measure inosine, guanosine and their deoxy analogue concentrations in the urine, or PNP activity in mononuclear cells or peripheral blood T cells [283, 295].

Prenatal exclusion of PNP deficiency can be performed by measuring the enzyme activity in fetal red blood cells [222] and amniocytes or by determining the purine profile in amniotic fluid. The advantage of the latter is that purine levels are available within a short time after amniocentesis [100]. Assessing PNP activity in chorionic villi is an effective alternative that can be performed early in the course of pregnancy [100].

The thymus of patients with PNP deficiency is small; however, unlike most other types of SCID, occasional poorly formed Hassall’s corpuscles can be demonstrated [95]. Lymph nodes seem depleted and lack paracortical fields. In most patients there is a low absolute lymphocyte count (frequently less than 500 cells/mL). T-cell function assessed by responses to mitogens and by skin test for Candida and other delayed hypersensitivity immunogens are reduced or absent [249, 295]. Decreased total lymphocytes and T-cells numbers were reported in PNP deficiency. In some patients, T-cell numbers and function fluctuate with time [222, 544], whereas in those with delayed presentation, mitogenic responses may be moderately reduced to normal [574]. Humoral immunity as assessed by B-cell number, immunoglobulin levels, and specific antibody formation are normal in most cases with PNP deficiency [128]. In a small group of patients, humoral aberrations including low levels of immunoglobulins, poor specific antibody production, reduced isohemagglutinins [617] or monoclonal gammopathy were documented [538]. The number of NK cells varies among patients [283].

The differential diagnosis of PNP deficiency should particularly consider disorders that combine significant immune deficiencies and neurologic abnormalities, including A-T, zinc deficiency, and biotin-dependent carboxylase deficiency. Because a dysplastic marrow and anemia may be an early symptom of PNP deficiency [182], congenital hypoplastic anemia (Diamond Blackfan syndrome), transcobalamine 2 deficiency, and type I hereditary orotic aciduria, which may be associated with immunodeficiency, also should be considered in the differential diagnosis.

The only available cure for patients with PNP deficiency is HSCT. There are a few reports of successful restoration of immune function in patients with PNP after HLA-matched sibling HSCT [43, 99]. Myeloablative conditioning is required in order to reduce the risk of rejection caused by residual immune function frequently documented in these patients. Conditioning regimens included cyclophosphamide and busulfan, without [43], or with ATG [167], or alternatively busulfan and fludarabine [124]. In the absence of a matched related donor, cord blood has been recently used successfully in a patient with PNP deficiency [459]. Whether these patients can benefit from matched unrelated donor marrow or cord blood transplants remains to be determined in a larger group of patients. In addition, HSCT may not reverse neurological manifestation as previously observed [43].

Regarding ADA deficiency, although HSCT is the treatment of choice, several patients benefit from enzyme replacement with PEG-ADA [42, 466].

When bone marrow transplant is unavailable, enzyme replacement using PEG-PNP could provide temporary remedy similar to the treatment of patients with ADA deficiency [295]. Its efficiency has been recently tested, demonstrating complete immune reconstruction of PNP-/- mice unfortunately, PEG-PNP is not commercially available [33]. Other future therapies such as enzyme replacement with TAT-PNP [659] or gene therapy are now undergoing pre-clinical studies.

In the past, several other modalities of therapy were proposed for PNP deficiency. Erythrocyte transfusions used as enzyme replacement were originally encouraging, but subsequently proved inefficient [621]. Other treatment including deoxycytidine and tetrahydrouridine [630, 695], guanine [695], adenine, uridine, and hypoxanthine [128, 621] showed no benefit. Attempts to restore immune function in patients with PNP deficiency with thymus transplant, or with thymosine fraction 5 were also unsuccessful.

Supportive treatment is warranted in patients with PNP deficiency, as in all immune deficiency states [222]. Immunoglobulin therapy should be considered in cases who have antibody deficiency or autoimmune manifestation [617].

The life expectancy of individuals with PNP deficiency has been poor. Most of the patients who did not receive a bone marrow transplant died during early childhood. The oldest reported patient reached the second decade of life [645]. Death has occurred from overwhelming infections, such as generalized chickenpox complicated by pneumonia and carditis, or pneumonia and chronic pulmonary disease. A high frequency of malignancy was also noted, including pharyngeal tumors, lymphoma, and lymphosarcoma [128, 429, 585].

Reticular dysgenesis (OMIM*267500) or AK2 deficiency is the most severe form of combined immunodeficiency, characterized by congenital agranulocytosis, lymphopenia. The disease was first described by de Vall and Seyneheve in 1959 [164]. The patients also suffer from lymphoid and thymic hypoplasia with absent cellular and humoral immunity functions.

Reticular dysgenesis is due to mutations in the AK2 gene (OMIM*103020), which is an adenylate kinase, a mitochondrial enzyme [364, 498].

In addition to severe infections that can be seen in other combined immunodeficiencies, affected newborns with AK2 deficiency have bilateral sensorineural deafness [364].

Severe neutropenia as well as severe T- and NK- cell lymphopenia are characteristics for AK2 deficiency, while the B cell lineage could variably be affected [364].

HSCT is the treatment of choice for those with AK2 deficiency. A recent study suggested potential use of antioxidants as a supportive therapeutic modality for these patients as well [545].

DOCK2 deficiency (OMIM*616433) is a very recently described autosomal recessive combined immunodeficiency, affecting T-cell number and function, with variable defects in B- and NK- cell function.

DOCK2 deficiency (OMIM*616433) is due to homozygous or compound heterozygous mutations in the DOCK2 gene (OMIM*603122). Five patients with DOCK2 deficiency have already been reported [175].

Patients with DOCK2 deficiency suffer from early onset severe invasive bacterial and viral infections.

Three reported patients experienced invasive bacterial and viral infections, associated with T-cell lymphopenia and reduced in vitro T-cell proliferation, while remaining two patients also had B-cell lymphopenia, and poor antibody responses [175].

HSCT is the treatment of choice in patients with DOCK2 deficiency. Two patients with DOCK2 deficiency died, while three who underwent HSCT, which was successful [175].

Hyper IgM (HIGM) syndrome, originally termed “dysgammaglobulinemia” is immunodeficiency conditions, characterized by defective production of Ig requiring a switch process, i.e. IgG, IgA and IgE, whereas the IgM concentration is either normal or increased. Although rare cases of HIGM with autosomal recessive inheritance have been reported recently, most cases are inherited as an X-linked recessive trait and are due to a mutation in the CD40 ligand encoding gene [21, 35, 171, 231, 296, 348]. The gene responsible for some autosomal recessive forms was identified as CD40 [206]. The clinical and biological characteristics of both HIGM syndromes associated with a defect in the CD40 ligand-CD40 interaction are very similar and point to the importance of this interaction in the immune response. These characteristics distinguish them from other HIGM with Ig CSR deficiencies (see Chap. 3 for more details) [203, 207, 484].

The X-linked form of HIGM (XHIGM or HIGM1) syndrome (OMIM*308230) is due to a mutation in CD40 ligand (CD40L also called CD154). The CD40L gene (OMIM*300386), also called tumor necrosis factor superfamily 5, TNFS5) maps on the X chromosome region q26 and is organized in five exons and four introns. CD40L is a type II transmembrane glycoprotein 261 amino acids long that is mainly expressed on activated CD4 T lymphocytes as a trimer. The crystal structure of the extracellular part of CD40L shows that hydrophobic and hydrophilic residues are crucial for CD40 binding [327]. Different mutations of the gene have been described in a large number of patients, including missense mutations, deletion, insertions, non-sense mutations and splice site mutation [375, 486, 594]. Although the mutations described involve all parts of the gene, most of them are located in exon 5, affecting regions that are conserved in sequence analogy with tumor necrosis factor [486]. The majority of missense mutations described affect the folding and stability of the molecule rather than the CD40-binding site directly [327, 486]. There is no a clear phenotype–genotype correlation, however, some mutations allowing a residual binding of CD40 are associated with a less severe phenotype [148, 594]. Some rare cases of XHIGM have been described in girls secondary to a skewed X inactivation chromosome [162, 307].

In 2001, Ferrari et al. [206] identified CD40 gene (OMIM*109535) mutation in three patients from two unrelated families with autosomal recessive HIGM syndrome (HIGM3)(OMIM:606843). It is a rare situation and less than 20 patients are reported [15, 326, 358, 395, 424]. So far, all patients described had homozygous mutations. CD40 is a type I transmembrane protein 277 amino acids long and is included in the TNF-R superfamily. CD40 is constitutively expressed on B cells, monocytes, macrophages, dendritic cells and non-hematopoietic cells. The CD40 gene displays 9 exons. CD40 mutations affect splice sites or consist of amino acid substitution or deletion. In most cases, CD40 is not expressed at the membrane level. However, recently a homozygous CD40 deletion was described, including the stop codon, resulting presumably to a longer non-functional protein, which is detected at the membrane level.

The CD40-CD40L interaction plays a major role in the cross talk between immune cells. Engagement by CD40L induces CD40 signal transduction in B and dendritic cells. CD40 could already be trimerized independently of CD40L engagement by its pre-ligand-associated domain (PLAD) identified in the extracellular regions of TNFR members [113].I). The CD40L-CD40 interaction plays a crucial role in T cell-dependent B cell proliferation and differentiation in the presence of a second signal (such as IL-4 or IL10). It is consequently critical for germinal center formation and for the generation of a secondary antibody repertoire. The latter results from two main processes. First, there is class switch recombination that leads to the expression of different immunoglobulin isotypes. The second process consists of the somatic hypermutations characterized by a high rate accumulation of point mutations in the V regions of Ig genes and allows the selection of B cells bearing a high affinity antigen specific BCR. Altogether, these processes lead to high affinity antibody production and to the generation of memory B cells and of long-life plasma cells. Although rare somatic mutations can be detected in IgM-bearing B lymphocytes [700], the main consequence of a defect in CD40/CD40L interaction is the absence of generation of a secondary antibody repertoire. However, several sources of evidence indicate that XIGM1 and HIGM3 are not solely a humoral immunodeficiency. CD40 triggering also plays a central role in T cell mediated activation of monocytes-dendritic cells [18, 104, 223, 315]. Engagement of CD40 on dendritic cells leads to their maturation and the secretion of IL-12 a cytokine with a major role in TH1 immunity. Failure to produce IL-12 and thereby interferon γ [108] is a likely event in the T cell immunity defect observed in HIGM affecting CD40-CD40L interaction.

This section summarizes the clinical manifestations observed in HIGM1 patients; the disorder has been recognized since 1993 and has been the object of many reports [375, 380, 710]. However, the clinical manifestations observed in the patients with HIGM3 are very similar [15, 206, 326, 395, 424].

In most cases, age at the time of diagnosis is between 3 months and 2 years and the clinical presentation evokes a combined immunodeficiency. However, it seems that variability in susceptibility to opportunistic infection in HIGM1-deficient patients could exist since some patients develop such infection early in life while others do not, at least not until adulthood.

The most common clinical manifestations observed in HIGM-1 patients are infections, especially infections involving the respiratory tract. First of all, the pneumonias that occur in more than 80 % of patients, and pneumocystis jiroveci, accounts for most of the cases in infancy. It is noticeable that this infection is the first manifestation of the disease in over one-third of patients. The occurrence of such an infection in a young patient has to evoke this diagnosis, especially if hypogammaglobulinemia is associated. Lung infections can also be due to viruses including CMV, adenovirus, herpes simplex or bacteria such as pseudomonas or staphylococcus. Finally, mycobacteria including bacillus Calmette-Guerin (BCG) and fungi such as Histoplasmosis and Cryptococcus can be responsible for lower respiratory tract infections. Upper respiratory tract infections including sinusitis and otitis are also common and affect more than 40 % of patients.

Gastrointestinal problems also affect over 50 % of patients. These problems are often of infectious origin especially due to Cryptosporidium. Diarrhea associated with Gardia lamblia, Salmonella or Entamoeba histolytica have been reported [380]. Inflammatory bowel disease and intestinal hyperplasia may cause chronic diarrhea in some patients. The intestinal problems follow a chronic course leading to failure to thrive, and parenteral nutrition is required. The liver is often affected. The common lesion is sclerosing cholangitis that is most often related to Cryptosporidium infection and that may require liver transplantation. Hepatitis has been reported either with or without a proven viral etiology. As with other immunodeficiencies, the risk of neoplasm, especially lymphoma, is increased. But in HIGM1 the risk of neoplasm also includes carcinomas affecting the liver, pancreas, biliary tree [293, 380, 462]. These observations suggest that physiological CD40 expression on regenerating or inflamed bile duct epithelium could play a role in triggering local immune response. [293].

The most typical hematological abnormality is neutropenia that is observed in over 60 % of patients. It is usually chronic and can be exacerbated by infectious episodes and be associated with oral ulcers and gingivitis. Chronic infections can lead to anemia, but some of them are related to Parvovirus B19 infection [61].

Neurologic problems including meningitis and encephalitis have also been reported. Despite the frequent absence of identification, several organisms are involved such as Toxoplasma, Cryptococcus and Mycobacteria [380]. Moreover, viruses including enterovirus and JC virus are responsible for some neurological features [284, 639].

Some cases of arthritis, nephritis and hyperparathyroidism have been reported. The osteopenia observed in some patients suggests a regulatory role for CD40L in bone mineralization [393].

The characteristic serum Ig profile observed in HIGM1 and HIGM3 consists in markedly decreased serum IgG, IgA and IgE and normal to increased IgM levels. Indeed, a normal IgM level is observed at the time of diagnosis in around 50 % of the patients, especially in young patients [380]. However, nearly 70 % of patients will present a Hyper IgM during their lifetime. In some cases, the level of IgG, which is generally very low, can reach normal values. In the same way, some patients present normal or high IgA level as well as IgE. These near normal immunoglobulin profiles, sometimes associated with an antibody response to T cell-dependent antigens, could be associated with a milder phenotype [61, 148].

In both HIGM1 and HIGM3, T-cell counts were generally normal, although a low proportion of CD45R0 memory T cells is frequently observed [315]. Whereas total B cell count is normal in most cases, the B cell population is characterized by the lack of B cells that do not express IgD and that express CD27, which correlates with the failure of class switch recombination and of somatic hypermutation processes [5, 326, 395].

The screening assay for diagnosis of HIGM1 is based on the absence of CD40 binding on the patient’s activated T cells. Usually, T-cell activation is driven by the association of phorbol ester and ionomycin, and the expression of a functional CD40 ligand is revealed by binding fluorescent chimeric CD40-Ig molecules assessed in flow cytometry. Some monoclonal fluorescent anti-CD40L antibodies which recognize the binding site of CD40 can be used cautiously for the diagnosis [375]. However, some CD40L mutations associated with milder phenotypes allow a residual CD40 binding and the level of fluorescent intensity has to be taken into consideration for a suitable interpretation. Moreover, when a defect of CD40 binding is detected, it is important to rule out a T cell activation defect which could lead to an absence of CD40Ligand expression without intrinsic defect in this molecule. The final diagnosis requires CD40L molecular analysis. Carrier detection in females has to be performed by direct sequencing when the searched-for mutation is known. Therefore, prenatal diagnosis can be offered by using a chorionic Villi biopsy taken at week 8–10 of pregnancy. Direct mutation identification, if known in the family at risk, or an intragenic polymorphic marker can be used [172].

The screening assay for the diagnosis of HIGM3 was founded on the absence of CD40 expression assessed by immunofluorescence. However, some mutant proteins can be expressed and recognized by monoclonal antibodies. Then, the diagnosis of CD40 deficiency requires genetic analysis.

The treatment included immunoglobulin substitution that resulted in a marked decrease of upper and lower respiratory tract bacterial infections. In some cases, immunoglobulin replacement therapy also led to the resolution of lymphoid hyperplasia when it existed before treatment. Under immunoglobulin treatment, IgM level often drops to normal value. The neutropenia is also frequently corrected by this substitution. However, in some patients presenting severe and symptomatic neutropenia, treatment by granulocyte-colony-stimulating factor has been given, successfully in most cases. Depending on the frequency and the severity of opportunistic infection, especially by Pneumocystis jiroveci, a prophylactic antibiotherapy using trimethopim-sulfamethoxasazole is recommended, especially when the patient had presented a previous episode of opportunistic infection. In spite of these preventive measures, the survival rate is still poor, although variable from one series to another. An important cause of death is still opportunistic infections, including Pneumocystis jiroveci, CMV and mycobacteria. But it is noticeable that severe liver disease is responsible for many deaths, particularly in the European cohort. Indeed, in the US registry, these complications seem to be less frequent. This could reflect a lower incidence of Cryptosporidium infection. Neoplasm complications are also an important element in the prognosis. Consequently, more aggressive treatment such as HSCT has to be considered. Indeed, HSCT using either bone marrow from familial HLA identical [69, 314, 652] or matched unrelated donors [25, 244, 336, 380] or cord blood [735] has been performed in patients with HIGM1 with an overall cure rate of 58 %. Recently, a haploidentical T-cell depleted peripheral blood stem transplantation has been performed successfully in a patient. Injection of donor T lymphocytes reverted a mix chimerism characterized by an increasing proportion of autologous cells [317]. The absence of preexisting liver or lung disease and an HSCT from HLA-matched sibling or closely mated unrelated donor may increase the success rate. [528, 535]. A careful follow-up of the lung and liver functions, with regular screening for Cryptosporidium infection and the monitoring of the neutropenia could allow proposing HSCT to at-risk patients before complications that constitute a pejorative factor especially when matched related donor is not available. According the CD40 expression on non-hematopoietic cells, stem cell transplantation as treatment in HIGM3 patients is more uncertain. However, three out of four patients with HIGM3 who received HSCT has been cured [15, 357, 424].

Recently, patients received therapeutics targeting the CD40 using either recombinant CD40 ligand or agonist anti-CD40 antibody [204, 316]. In the three patients treated by recombinant CD40 ligand, whereas the capability of T lymphocytes to synthesize IFN-γ and TNF-α was improved, the specific antibody response was not corrected. However, the architecture and size of lymph nodes changed, with an expansion of follicular dendritic cells, but no germinal center was observed. The decrease of the Cryptosporidium burden detected in two patients treated by agonist anti-CD40 antibody could be related to the improvement of the production of TNF-α and IFN-γ by T-cells. Perhaps, these treatments would open a new avenue allowing limitation of complications due to infections and consequently to perform HSCT in better conditions.

Di George syndrome (DGS, OMIM*188400) is a developmental disturbance of neural crest occurring during the embryogenesis and is attributed to the haploinsufficiency of one or more of the genes located on the chromosomal region 22q11.2 [2, 153, 334]. This condition was first described by Angelo DiGeorge in 1965 as the association of immunodeficiency and congenital absence of thymus gland which had been noted early in the twentieth century [132]. The syndrome is classically defined as a congenital T-cell immunodeficiency secondary to aplasia or hypoplasia of the thymus gland associated with congenital heart defects and hypocalcaemia, due to small or absent parathyroid glands. The most common cause of the syndrome is a hemizygous deletion of 22q11.2, seen in approximately 90 % of DGS patients and may occur as frequently as once in 4000–6000 live births, affecting both sex equally [169]. It is one of the most frequent genetic diseases, considering that it may be underestimated because of the rate of perinatal deaths observed in many cases with a severe congenital heart defect.

DGS is characterized by malformations attributed to abnormal development of the pharyngeal arches and pouches. The common threat among all the organs involved in DGS is that their development is dependent on migration of neural crest cells to the region of pharyngeal pouches. Lammer and Opitz described DGS as a field defect in which a group of tissues, that are interdependent on each other for normal growth, develop in an abnormal fashion [64, 339]. Although DGS has traditionally been described as abnormal development of the third and fourth pharyngeal pouches, defects involving the first to sixth pouches are also known to occur. Animal studies have shown that acute ethanol exposure in mice at a time when neural crest cells are migrating results in a craniofacial phenotype similar to DGS [696]. Exposure to teratogens during pregnancy, including alcohol, retinoids, bisdiamine, can result in similar phenotypic syndromes [264, 696]. Thus, it is postulated that any intrauterine insult to the facial neural crest can result in similar features of DGS.

A 3-Mb deletion within 22q11.2 is present in majority of cases, with a smaller 1.5-Mb deletion found in less than 10 % and some unique smaller deletions in a few number of cases [198, 595]. Most deletions are de novo, with 10 % or less inherited from an affected parent. At least 40 genes have been identified within this region. In spite of efforts to identify candidate gene(s), no single gene deletion has been shown to be sufficient for the development of DGS. Consequently, it is possible that more than one gene could contribute to the phenotype since DGS patients with different type of deletions have similar phenotypes.

Among the most investigated genes, TUPLE1 (TUP-like enhancer of split gene-1) (OMIM*600237), reported by Halford et al. [282], is an attractive candidate for the central features of the syndrome. It shows evidence of expression during the critical period of development of the outflow tract of heart, and of the neural crest derived aspects of face and upper thorax.

Moreover, TBX1 (OMIM*602054), encodes for a “T box” transcription factor, is involved in the regulation of developmental processes, and is mostly affected in the majority of DGS patients [45, 153]. Yagi et al. identified 3 mutations within TBX1 in unrelated patients with 22q11.2 syndrome phenotype, but no detectable deletion in 22q11.2 [719]. One mutation was found in a case of sporadic velocardiofacial syndrome/conotruncal anomaly face, and a second in a sporadic case of Di George syndrome. The third mutation was shown in 3 patients from a family with velocardiofacial syndrome/conotruncal anomaly face. These findings indicated that TBX1 mutations were responsible for five major phenotypes of the 22q11.2 syndrome, namely, abnormal facies (conotruncal anomaly face), cardiac defects, thymic hypoplasia, velopharyngeal insufficiency of the cleft palate, and parathyroid dysfunction with hypocalcaemia. These mutations did not appear to be responsible for typical mental retardation that is commonly seen in patients with the deletion form of 22q11.2 syndrome.

Other implicated genes include Crkl and COMT genes. Crkl encodes an adaptor protein, which is highly expressed in neural crest derived tissue during development. Crkl ^-/- mice die in uterus, whereas heterozygous ones survive [275, 387]. Catechol-O-methyltranferase (COMT), also located within the commonly deleted region [270], is involved in the metabolism of catecholamines. The V158M polymorphism (COMT158^met) seems to result in decreased enzyme activity and to be associated with the development of psychiatric disease in patients with chromosome 22q11.2 deletion syndrome [264, 362]. In contrast, some studies have shown that patients carrying the met allele have a better cognition performance and that COMT V158M polymorphism affects minimally the executive function in 22q11.2 deletion syndrome [254, 598]. Deletions on the short arm of chromosome 10 p13-14 are also associated with a DGS-like phenotype, but are much less common than 22q11.2 deletions with an estimated frequency of 1 in 200,000 live births. Other chromosomal abnormalities that have been found in patients with presumed DGS include deletions on chromosomes 17p13, and 18q21 [267].

Although many reports have greatly contributed to the understanding of the clinical features and the pathophysiology of the disease, the DGS phenotype is much more variable and extensive than initially recognized, and several aspects still need to be clarified [49, 75, 132, 391, 456, 570, 671].

DGS has commonly been characterized as a triad of clinical features: congenital cardiac defects, immunodeficiency and hypocalcemia. A variety of cardiac malformations are seen, in particular affecting the outflow tract. These include tetralogy of Fallot, type B interrupted aortic arch, truncus arteriosus, right aortic arch and aberrant right subclavian artery.

Moreover, newborns and infants with DGS may have dysmorphic facial features. Ears are typically low set and deficient in the vertical diameter with abnormal folding of the pinna. Telecanthus with short palpebral fissures is seen. Both upward and downward slanting eyes have been described. The philtrum is short and the mouth relatively small. In older children the features overlap velocardiofacial (Shprintzen) syndrome with a rather bulbous nose, square nasal tip and hypernasal speech associated with submucous or overt palatal clefting.

Neonatal hypocalcaemia, due to hypoplasia of the parathyroid glands, is characteristic and may be sufficiently severe to present as tetany or seizures. However, it could be intermittent and resolve during the first year of life as the parathyroid glands hypertrophy. Latent hypoparathyroidism may occur in both children and adults [141].

Feeding difficulties and gastroesophageal reflux are also described. Renal abnormalities such us single kidney, multicystic dysplasic kidney, horseshoe kidney, and duplicated collecting system occur in approximately one-third of DGS patients. Short stature and variable mild to moderate learning difficulties are common. Other clinical features seen more rarely include hypothyroidism and deafness. Cases presenting later, tend to have a milder spectrum of cardiac defect with ventricular septal defect being common.

Various psychiatric disorders have been also described both in children and adults [248, 640].Different behavioral, psychiatric, and communication disorders include attention deficit-hyperactivity disorder (ADHD), anxiety, language and speech delays, and affective disorders. An estimated 25 % of children with 22q11 deletion syndrome develop schizophrenia in late adolescence or adulthood. A recent study on 112 individuals aged 8–45 years revealed diagnoses of psychosis in 11 % of cases with a peak occurrence of psychosis risk during adolescence [646]. Neurological abnormalities consist of structural brain anomalies (small vermis, small posterior fossa and small cysts adjacent to the anterior horns) and increased risk of developing seizures, in a minority polymicrogyria and periventricular nodular heterotopia have been observed [27, 439].

Thymic hypoplasia or aplasia leading to defective T-cell function is the hallmark of DGS. Patients with the chromosome 22q11.2 deletion have a broad range of T-cell counts and proliferative responses. Complete absence of thymus (‘complete’ DGS) accounts for less than 0.5 % of patients and exhibit a severe T-cell immunodeficiency, resembling a SCID phenotype. In ‘complete’ DGS few T cells are detectable in peripheral blood (1–2 %) and there is no response to T cell mitogenes. T-cell receptor excision circles (TRECs), as a measure of newly emigrated thymic cells, are reduced [292].A recent report has described two patients with absent T cells and DGS associated with 22q.11 deletion and carrying pathogenic mutations in the DCLRE1C (Artemis) gene [294]. Since TRECs are absent or low in complete DGS, newborn screening using TREC detection is useful for early diagnosis of the disease and for the prevention of infections [359, 564].

In contrast, the majority of patients with 22q11.2 deletion syndrome and immune defects exhibit mild to moderate deficits in T cell numbers (so-called ‘partial’ DGS). Immunodeficiency in these patients is not caused by the absence of thymus, but due to abnormal thymic migration. Many patients have microscopic nests of thymic epithelial cells that account for their ability to produce T cells. A normal-sized thymus is not necessary for normal T cell development, and patients with a very small thymus, even in an ectopic location, may have a T cell response to mitogens that ranges from below normal to normal. As such, total T cell numbers may not accurately reflect immune [411]. The majority of ‘partial’ DGS patients have normal T cell proliferations, although some patients show low mitogen responses. Therefore, mitogen responsiveness should be considered the most important parameter to assess T cell function and to better discriminate DGS as ‘partial’ or ‘complete’.

Most DGS patients have normal antibody levels, function and avidity. The aberrant regulation of B cells by the deficient T cells might also result in hypergammaglobulinemia. On the other hand, hypogammaglobulinemia, IgA deficiency, delayed acquisition of appropriate anti-tetanus and anti-diphtheria antibody titers have been described as well. In a cohort, 55 % of patients showed impaired specific antibody responses to pneumococcal polysaccharide antigen [242]. Impaired T–B cell interaction is likely to explain the defective T-dependent antibody responses. In another study 43 % of patients exhibited evidence of antibody deficiency (IgA deficiency, IgM deficiency, IgG subclass deficiency or specific antibody deficiency) and a significant correlation between the presence of recurrent infections and humoral abnormalities (P < 0.01) was found. CD27⁺ memory B cell subsets were reduced in patients with defective humoral immunity [215]. A recent study performed on over 1000 patients of partial DGS with a median age of 3 year, showed that 2.7 % were under immunoglobulin replacement. In the over 3 years age group, 6.2 % had IgG levels below 5 g/l. Amongst patients over 3 years of age, around 0.7 % had complete and 1 % partial IgA deficiency, while 23 % had low levels of IgM [506]. There was not association between low T cells counts and Immunoglobulin levels in any of the isotypes. Unfortunately this study did not evaluate the B cell numbers, although previous studies reported to be normal or sometimes low but normalizing during life [321]. The repertoire of IgH usage is normal; however, further studies are needed to clarify whether abnormalities in somatic hypermutation might occur.

Patients with DGS who present with infections as the first manifestation are unusual because cardiac malformations and hypocalcaemia are so severe that they usually manifest in the neonatal period. In fact, most of the early deaths are due to cardiac defects. However, recurrent infections are a major problem and an important cause of later mortality. Increased susceptibility to infections, caused by organisms typically associated with T-cell dysfunction, is observed. These include systemic fungal infections, Pneumocystis jiroveci infection, and disseminated viral infections [410, 581]. Moreover, the combination of impaired immune response and abnormal palatal anatomy may be associated with high frequency of upper respiratory tract infectious.

Immunodeficiencies are frequently associated with autoimmunity, and the incidence of autoimmune disorders is increased in Di George syndrome as well [318]. In one study of 20 patients with 22q11.2 deletion syndrome, 10 % had evidences of autoimmune disease [242]. In particular, autoimmune cytopenias [150, 379], juvenile rheumatoid arthritis–like polyarthritis [637] and autoimmune endocrinopathy [151] have been described. A number of immune defects may predispose to the development of autoimmunity in these patients including increased infection, persistent antigen stimulation. However, in partial DGS autoimmunity is not predominantly found in those with the most severe or frequent infections [433]. It is more likely that defective central tolerance or impaired development of natural CD4⁺CD25⁺ T-regulatory cells may have a role in predisposition to autoimmunity. Controversial data are reported in literature on peripheral tolerance. Indeed, one study performed on partial DGS patients demonstrated a significant decrease in the percentage of CD4⁺CD25⁺ T cells when compared to normal control, which was most marked in infancy. Another study reported CD4⁺ CD25⁺ cells in patients with pDGS. However, no difference was observed in the percentage of CD4⁺CD25⁺ T cells in 22q11.2 deletion syndrome patients with and without evidence of autoimmune disease [636]. Abnormal thymic development in DGS may thus result in impaired expression of autoimmune regulator gene (AIRE) and potentially of other transcription factors that regulate expression of organ-specific antigens in the thymus, resulting in defective central tolerance [105, 150]. However, so far any report indicates defect in AIRE expression in thymic tissue from partial DGS cases and indeed, since autoimmune disease is limited to one or two organs in patients with partial DGS, it is likely that negative selection most occur to most antigens [150].

There is a wide range of phenotypic variability associated with the 22q11.2 deletion syndrome as conotruncal anomaly face (Takao syndrome), and isolated outflow tract defects of the heart. While some patients present with classic findings of DGS, others have relatively slight features such as minor dysmorphic facial traits or mild cognitive impairment. Consequently, none of the phenotypic features is considered pathognomonic for the 22q11.2 deletion. Furthermore, the deletion does not predict the organ effects or disease severity and the phenotypic expression does not seem to be related to the deletion size, to date. In addition, there are many published examples of affected kindreds demonstrating that the clinical presentation can be broadly different even within a single family [330, 373].

The dysmorphic facial appearance, in an individual with a major outflow tract defect of the heart or a history of recurrent infection, should raise suspicion. In infancy, hypocalcaemia, a characteristic feature, is usually evident with low parathyroid hormone (PTH) levels. Chest radiography may detect an absent thymic shadow, although this finding does not always correlate with immune function. Newborns should be evaluated for T cell production and function. A complete blood count (CBC) and the measurement of the CD4+ subset of T cells can assess the presence and severity of lymphopenia. Meanwhile it is important to evaluate T cell proliferative responses and not merely the number of T cells. In vitro studies of T cell function offer the most reliable estimate of the extent of immunodeficiency. Evaluation of humoral immunity reveals variable immunoglobulin levels and depends on the degree of T cell deficiency. Patients with partial DGS generate good antibody response to protein vaccines [40].

The investigation of choice is a standard karyotype to exclude major rearrangements, and fluorescence in situ hybridization (FISH) using probes within the deletion segment, preferably those close to the translocation breakpoint site. A 10p13-14 FISH study should also be considered if there is clinical evidence for DGS, but negative 22q11 FISH study. A positive FISH test for chromosome 22q11.2 deletion or a 10p deletion ascertains the diagnosis. For patients without the deletion diagnosis is based on the clinical phenotype, although precise diagnostic criteria are difficult to establish [75, 428, 570, 671] (Table 2.1). Parents should be screened for carrier status.

Several approaches have been attempted over time to achieve an immune reconstitution. Implantation of whole thymus was first described by Cleveland et al. in 1968. Later, several other trials of fetal thymic tissue implantation were performed [423, 656]. Recently success has been reported using allogeneic, partially HLA-matched postnatal thymus tissue to transplant infants with the complete DGS [411, 412].Thymic tissue is obtained from cardiac surgery and kept in culture for 2–3 weeks prior to transplantation that is performed into the quadriceps muscle of the patient [150].Two trails are currently ongoing and so far, of 60 patients treated the survival was 72 %. Death after transplant is caused by systemic viral infections such as cytomegalovirus and chronic lung disease. Transplanted thymi show a normal morphology and in patients with successful transplantation, patients develop host derived naïve T cells with normal T cell repertoire, normal mitogen responses and a normalization of the TCR repertoire in circulating regulatory T cells [120]. However, there are other disappointing reports for thymus transplantation. In particular, development of autoimmunity represents the main problem, mainly hypothyroidism and immune-cytopenias [413] and importantly autoimmune signs mimick the spectrum of autoimmunity observed in partial DGS.

In complete DGS, bone marrow and peripheral blood T-cell transplantation from HLA-matched sibling donor has been also efficacious [57, 74, 259, 421]. Long term survival has been reported but with low rate (41–48 %), as compared with survival after HSCT [245]. Mortality is referred to graft versus host disease or viral infections.

The prognosis of DGS patients varies significantly according to the degree of involvement of the cardiac and immune system. Heart problems are the major cause of deaths early in childhood and opportunistic infections are the second most fatal complication. In most children who survive, the number of T cells rises spontaneously as they mature. Children who were successfully bone marrow or peripheral blood transplanted as well as those who received thymus transplant and achieved a good immune reconstitution, remained free of infections long time after. Survivors are likely to be mentally retarded and to have other developmental and neurologic difficulties in later life.

CHARGE syndrome (OMIM*214800) is association of Coloboma, Heart anomaly, choanal Atresia, Retardation, Genital and Ear anomalies.

De novo heterozygous mutation in the CHD7 gene (OMIM*608892), on chromosome 8q12, resulting in haploinsufficiency has been reported in CHARGE syndrome [166, 582]. There is also a report showing that heterozygous in the SEMA3E gene (OMIM*608166), on chromosome 7q21, could cause this syndrome [366].

Coloboma of eye, heart anomaly, choanal atresia, mental retardation, microphallus, and abnormalities of ear are the main features of CHARGE syndrome. Some other anomalies such as facial palsy, cleft palate, and dysphagia are also common.

Distinctive clinical phenotype could help in making the diagnosis. Four major signs of diagnostic criteria are coloboma, choanal atresia, characteristic ear anomalies, and cranial nerve involvement [62, 675]. Various defects of thymus and associated T cell abnormalities have been reported in cases of with CHARGE syndrome [713].

A combination of medical and surgical care is needed in patients with CHARGE syndrome [302].

The Combined Immunodeficiency with Alopecia Totalis due to FOXN1 deficiency (OMIM*601705) constitutes the human counterpart of the nude mouse.

In 1994, the genetic basis of the well-known, “nude” mouse, associating hairlessness and congenital athymia, was reported for the first time. It involves a new gene, Winged Helix – Nude whn (also called Foxn1), and consists in a single base deletion in exon 3. This frameshift mutation leads to a predicted aberrant protein.

The protein FOXN1 is a member of the forkhead/winged-helix transcription factor family. It is mainly expressed in thymus epithelia and in skin [468] and plays a crucial role in the differentiation of thymic epithelial cells (TEC) [632] as well skin epithelial cells [434]. FOXN1 is involved in the morphogenesis of the three dimensional thymic structure and the development of cortical and medullary TEC is FOXN1-dependent in fetal life [555]. The expression of FOXN1 could be upregulated by wingless (wnt) proteins which play an important role in cell-fate specification [44, 555, 556, 662]. The mutation observed in nude mice leads to a protein deprived of the DNA binding domain.

Five years later, in 1999, J. Franck et al. identified a homozygous mutation of the human gene FOXN1 (OMIM*600838), localized on the chromosome 17, in two siblings. This mutation, R255X, is a nonsense mutation and predicts complete absence of functional protein [225]. The two patients were born from consanguineous parents in a small community in southern Italy. It was secondarily shown that this mutation is present in 6.52 % of this population, and is related to a single ancestral origin [4]. More recently, this mutation has been found in another patient born from consanguineous Portuguese parents. Another FOXN1 homozygous mutation has been found in deficient patient born from mixed French/African parent. This missense mutation, C987T (R320W) alters the DNA binding site of the proteins [17, 414]. The study of a FOXN1 deficient fetal thymus confirm that FOXN1 mutation abrogate prenatal T-development [677]. A novel FOXN1 mutation has also been reported, resulting in SCID phenotype [122].

In the patients reported, alopecia affecting the scalp, the eyebrows and the eyelashes associated with nail dystrophy was noted at birth, as well as bilateral epicantal fold in two patients [414, 516].

Subsequently, between 2 and 4 months of age, they developed immunodeficiency symptoms. The first one had with a clinical picture mimicking Omenn’s Syndrome, including erythrodermia, diarrhea and hepatosplenomegaly, and died at 12 months of age following recurrent infections and severe failure to thrive. Two patients developed erythrodermia probably related to the presence of circulating T lymphocytes. One out four patients reported had BCG invasion after vaccination and another one, a HHV6 infection associated with anemia and neutropenia. These two patients received thymus transplantation.

It is noticeable that in a FOXN1 deficient fetus, anencephaly and spina bifida were found to be associated with the absence of thymus [23].

Only one patient had a total absence of T lymphocyte [414]. The others displayed a T-cell lymphopenia affecting mainly the CD4 population. However, in one patient who had a moderate lymphopenia, the non-maternal circulating T-lymphocytes, predominantly double negative CD4-CD8- displayed a restricted repertoire, and no TREC was detected consistent with the absence of naïve CD45RA T-lymphocytes like in atypical SCID or atypical DiGeorge syndrome. B and NK cell populations are present at normal or high level. Proliferations induced by PHA or anti-CD3 monoclonal antibody are variable from one patient to another.

One out of the two patients received non depleted HLA identical bone marrow transplantation from her healthy heterozygous brother, with successful engraftment [517]. CD4 and CD8 T lymphocytes increased promptly and are stable 6 years later. However, the CD4 T population displays only a memory phenotype CD45RO. This suggests that, as expected, CD4 recovery mainly results from the expansion of graft T lymphocytes.

Moreover, the Vβ repertoire of CD4 lymphocytes is similar in the donor and the engrafted patient. Conversely, the prompt recovery of naïve CD45RA CD8 population suggests extrathymic lymphopoiesis. However, CD8 compartment reconstitution is poor as judged by restricted TCR-Vβ diversity. T cell proliferation restored early after transplantation has further decreased to reach 20 % of the normal value. In spite of this incomplete immune T reconstitution, humoral immunity is restored as judged by the production of specific antibodies after immunization, especially with antigen unknown by donor. However, the patient is free of infections at 6-year follow-up.

Taking advantage of the experience of thymus transplantation in DiGeorge syndrome, two patients with FOXN1 mutation received this treatment at 14 and 9 months of age. The first patient, who had an atypical picture, received treatment with cyclosporin, steroids, rabbit anti-thymocyte globulin and daclizumab before the procedure. The second patient who had no circulating T-lymphocyte did not receive any immunosuppression treatment before transplant [414]. In both patients, the immune reconstitution evaluated at 5 and 2.9 years, respectively, after transplantation is characterized by the presence of naïve T-lymphocytes, diversified TCR repertoire, normal T-cell proliferative response, normal immunoglobulin levels and normal specific antibody response. As observed in some DiGeorge patients after thymus transplantation, one patient developed an autoimmune thyroid disease at 1.6 years after transplantation [412].

Immuno-osseous disorders are a heterogeneous group of disorders, characterized by combined abnormalities in immune and skeletal systems. These disorders are manifest at birth mainly because of skeletal abnormalities; however, there are variants that may present later in life (Table 2.2).

Schimke syndrome or Schimke Immuno-Osseous Dysplasia (SIOD, OMIM*242900) was first classified as a new lysosomal storage disease by Schimke in 1974 [588]. He described a 6-year old girl with spondyloepiphyseal dysplasia, progressive renal failure, lymphopenia and signs of defective cellular immunity. The increased amounts of urinary chondroitin 6-sulphate, led him to speculate the condition as a new presentation of mucopolysaccharidosis, which was not confirmed in later studies [67, 619]. SIOD is an autosomal recessive multisystem disorder with invariant defining features of spondyloepiphyseal dysplasia, progressive proteinuria leading to renal dysfunction [66, 67, 584, 588]. T-cell immunodeficiency is frequently observed, associated with opportunistic infections, autoimmune diseases and non-Hodgkin’s lymphoma [48, 66, 734].There are some other features, which are variable among patients, including hypothyroidism, bone marrow failure, numerous cutaneous lentigines, early-onset cerebral ischemic attacks, migraine type headaches, and peculiar faces [66, 67, 170, 196, 337, 584, 619].

Metaphyseal Chondrodysplasia, McKusick type, also known as Cartilage Hair Hypoplasia (CHH, OMIM*250250), was first described 1965 in Amish families [432] and later identified in multiple ethnic groups and particularly among the Amish and the Finns [540]. This condition is an autosomal recessive disorder that results in short-limb dwarfism. It is predominantly associated with cell-mediated immunodeficiency. Other associated conditions are chondrodysplasia, fine and sparse hair, Hirschprung disease, skin hypopigmentation, increased risk of malignancy, defective hematopoiesis [401, 404, 635].

Several studies had postulated various pathogenesis for SIOD, such as autoimmunity [322, 619] or metabolic defects [65, 126, 396, 588], that could not explain all the features of SIOD. For example scientists noticed that the disease does not recur in the transplanted tissues, neither tissue transplantation protects other tissues from disease process [196, 512]. In 2002, Boerkoel showed that mutations in SMARCAL1 gene (OMIM*606622), (SW1/SNF matrix-associated actin-dependent regulator of chromatin, subfamiliy a-like 1), encoding a DNA stress response enzyme, are the causative molecular defect of SIOD [67].However, the role of this gene in the pathogenesis was not recognized at that time. Using a murine model, it was later shown that SMARCAL1 was expressed throughout development and is involved in all affected tissues [196].

The molecular defect of CHH has been identified in the gene for RNAase, RMRP (OMIM*157660), mapped to 9p21-p12. RMRP is a ribonucleoprotein present in the nucleus and mitochondria [541, 635]. RNase MRP has two functions: cleavage of RNA during the mitochondrial DNA synthesis and nuclear cleaving of pre-rRNA. Mutations in RMRP affect cell growth by impairing ribosomal assembly and altering cyclin-dependent cell-cycle regulation [649]. Four distinct skeletal disorders have found to be associated with RMRP mutations: CHH, metaphyseal dysplasia without hypertrichosis (MDWH; MIM 250460), kyphomelic dysplasia (MIM211350) and anauxetic dysplasia (MIM607095) [352, 650]. Furthermore, it has been shown that RMRP mutations are responsible for a variable spectrum of immunodeficiencies and should be considered even in patients without skeletal dysplasias.

Schimke immuno-osseous affects both sexes equally [66, 619]. Facial features in SIOD patients are characteristic with a broad-low nasal bridge and bulbous nasal tip. Spondyloepiphyseal dysplasia is a constant feature, which manifests as truncal short stature. Vertebrae are usually flattened and ovoid. Increased lumbar lordosis is invariant and leads to a protuberant abdomen. Thoracic kyphosis, short neck, skull and rib abnormalities have also been mentioned. Epiphyseal changes are most consistently observed in the proximal femurs. Capital femoral epiphyses are small and laterally displaced with hypoplastic iliac wings, and shallow dysplastic acetabular fossae [66, 360, 619].

Most of these children have multiple hyperpigmented macules, measuring a few millimetres, mainly on the trunk with extension to the extremities and face. These cutaneous lentigines usually progress with age [66, 584], but there is a report of regression during adolescence [66].

SIOD patients present with growth retardation and normal or nearly normal developmental milestones. There is invariable evidence of intrauterine growth retardation in them and maximum height of adult patients rarely reaches more than 150 cm; however, the bone age does not suggest hormonal deficiency. Growth hormone studies are normal in most cases and they do not respond to hormone supplementation. Up to 50 % of the patients may have high TSH levels with normal T4 and free T3 levels; however, L-Thyroxin supplementation improves TSH levels without any effect on the course of disease [66].

The other constant feature of SIOD is renal failure, which usually starts with proteinuria and progresses to an end-stage disease within 1–11 years. The renal failure is refractory to treatment with glucocorticoids, cyclosporin A and cyclophosphamide [66]. Histopathology specimens usually show focal, segmental glomerulosclerosis (FSGS) and interestingly, there is no report of recurrence of FSGS in transplanted kidney; neither of improvement in other organ systems after renal transplantation [66]. Hypertension is relatively common [66, 584].

Immune dysfunction in SIOD usually presents with lymphopenia and/or T-cell dysfunction. Lymphocytopenia might be episodic in some patients, but all the patients show evidence of T-cell dysfunction. The CD3⁺/CD4⁺ lymphocyte counts are reduced; whereas, the CD3⁺/CD8⁺ lymphocyte counts can be either low or normal [66, 584]. All SIOD patients show a reduced response to at least one T-cell specific mitogen [66]. Similarly they respond poorly to T-cell dependent B-cell mitogens (pokeweed mitogen), but normally to B-cell specific mitogen [66]. Absolute B-cell counts are normal in most patients while immunoglobulin levels might be reduced in some [584]. Delayed hypersensitivity skin tests were negative in one patient [584]. Adverse effects to vaccinations have not been reported yet. Interestingly, a high frequency of non-Hodgkin’s lymphoma has been observed in patients and Smarcal 1-deficient are more sensitive to several genotoxic agents [46].

Arteriosclerosis is a common complication, leading to cerebral vascular accidents. Older patients frequently develop migraine-type headaches [337].The vascular disease is progressive and is not halted by renal transplantation, anticoagulants, or antimigraine medications. Large arteries including the aorta and carotids might be affected as well which is much in advance of their chronological age [125].

Recurrent fungal (oral thrush, candidal dermatitis), viral (Herpes simplex), or bacterial infections (gingivitis, sinusitis, pneumonia, septicemia) are seen in almost 50 % of the patients [66, 584, 619]. The onset of infections usually follows growth failure and is preceded by ischemic events [66]. Opportunistic infections, including Pneumocystis jiroveci, fulminant viral infections (Cytomegalovirus and Epstein-Barr virus), and atypical mycobacterial infections have also been reported [66, 581, 583]. Recurrent infections are not associated with milder juvenile form of the disease [66].

Other findings in SIOD include microdontia with absence of dental pulp, microdontia, hypodontia, or malformed deciduous and permanent molars. Immunohistochemical analyses showed expression of SMARCAL1 in all developing teeth, raising the possibility that the malformations are cell-autonomous consequences of SMARCAL1 deficiency [143, 394, 448].

Eye refraction difficulties and optical neuropathy, testicular hypoplasia with azospermia, fatty infiltration of cardiac wall, pulmonary emphysema, and high pitch voice [125, 620].

Cartilage-hair Hypoplasia is equally distributed in both sexes and has been seen throughout the world [431]. The predominant feature in CHH is short-limb dwarfism which is evident at birth, metaphyseal flaring and irregularities. Globular epiphyses at the knees and ankles are also the typical radiographic signs. Other skeletal features can be variable which include incomplete extension at the elbow, anterolateral chest deformity with flaring of the ribs at the costochondral junction, Harrison grooves, genu varum, and excessively long fibula distally relative to the tibia [432]. Skeletal age can be reduced in some patients. Mild scoliosis has been observed in 25 % of the patients [403]. Bonafe et al. suggested that a diagnostic feature of CHH is cone-shaped epiphyses in the phalanges [68]. Anterior angulation of the entire sternum in CHH was described by Glass [255]. The mean adult height is 131.1 and 122.5 cm in males and females, respectively [403].

Skin manifestations of CHH are also variable. Most of these patients have hypopigmented skin. Finger nails are foreshortened and dysplastic. The hair is fine, sparse and light-colored. Under light microscopy, hair looks abnormally small caliber and hypoplastic with lack of the central pigmented column [401].

Involvement of the immune system in CHH was noted by the time it was first described as an unusual susceptibility to varicella infections [432]. Patient could be affected by severe herpes labialis. Markedly impaired function of T cells as well as lymphopenia and neutropenia have been described in CHH [401]. In spite of decreased CD4⁺ cells, B lymphocyte count is usually normal while natural killer cell population is normal only in 40 % of patients. Lymphocyte stimulation studies with mitogens were subnormal in most patients. Humoral immunity may also be affected, with deficiencies in immunoglobulin A and G subclasses [402, 487]. Buckley et al. have considered a case of CHH in their series of 108 patients with SCID; however, CHH is not a common cause for SCID. Moreover, a generalized hematopoietic impairment has been described which involve all myeloid lineages in patients with CHH. Severe anemia and defective erythrogenesis requiring transfusion affect up to 79 % of patients, but can undergo spontaneous and permanent remission before adulthood in the majority of the patients [707]. Moreover, patients showing skeletal changes typical of (CHH) associated with some Omenn-like clinical signs, such as infections, erythroderma, lymphoadenopathy and hepatosplenomegaly have been reported to carry mutations in the ribonuclease mitochondrial RNA-processing [40, 551]. There is a statistically significant increased risk of cancer among CHH patients which is mainly attributable to non-Hodgkin lymphoma and basal cell carcinoma [401, 487]. The latter can be partly related to skin hypopigmentation. The prognosis of these patients after development of malignancies is poor.

Hirschprung disease was described in some of these patients, which may lead to aganglionic megacolon [635]. Splenomegaly with portal hypertension, dental abnormalities and defective spermatogenesis are other less known features.

Skeletal abnormalities are manifest at birth and most of these children are born with evidence of intrauterine growth retardation and a short stature with a mean relative length of −3.0 SD [403]. Immunology tests could be impaired as discussed before. Other diagnostic features depend on the presentation and complications. Imaging studies can be diagnostic in some cases, but genetic testing is needed for confirmation. Thanks to recent advances, these conditions can be suspected and diagnosed in prenatal clinics [66, 394, 584].

Severely affected SIOD patients usually present with growth failure in the neonatal period and die within the first decade of life. On the other hand, the milder juvenile form of the disease usually presents with growth failure and renal dysfunction between 8 and 13 years of age and progresses to renal failure over the next 6–12 years into adulthood. Patients with severe phenotype have at least one null allele. However, the severity and age of onset do not invariably predict survival [125, 288, 394].

There is no proven treatment for SIOD or CHH. Medical and supportive care may prolong survival of severely affected patients [394]. Combined renal and HSCT may treat the renal failure, bone marrow failure and immunodeficiency in SIOD, but not arteriosclerotic changes [512]. A study performed on five transplanted patients [47] showed a poor outcome likely due increased sensitivity to genotoxic agents. Of note, SIOD patients are prone to restrictive lung disease due to skeletal dysplasia [448] and conditioning regimens containing busulfan and cyclophosphamide associate with increased risk to develop pulmonary side effects. On this basis, reduced conditioning regimens should be considered although high incidence of acute GVHD is observed [46]. Patients usually die within the first two decades of life from infections (23 %), stroke (17 %), renal failure (15 %), complications of organ transplantation and lymphoproliferative disease (9 %), gastrointestinal bleeding, (6 %) bone marrow failure and unspecified lung diseases [46].Prophylactic and early administration of antibiotics reduces the severity and frequency of infections [66] and prolongs survival of early-onset patients [394]. Dislocated hips may warrant surgical treatment.

For CHH patients, management would similarly include treatment of complications. Acyclovir can be used for the treatment of severe varicella infections [187, 715]. These patients should not receive live attenuated vaccines like, however, varicella vaccine would be worthy of consideration [286, 289]. For CHH associated with severe immunodeficiency and or autoimmunity, HSCT should be considered before the development of severe infections and severe complications such as malignancies or organ damage that can influence the outcome of the disease [71, 707].

The association of hereditary multiple intestinal atresias (HMIA) and profound immunodeficiency has first been described in 1990. Recently, using whole exome sequencing, two teams of researchers identified TTC7A (OMIM*609332) as the causative gene of this disease (OMIM*243150) [116, 579].

Human TTC7A protein contains nine tetratricopeptide repeat (TPR) domains [149]. The TPR domains are degenerate 34-amino-acid repeat motifs that are found in many diverse proteins in all organisms and are thought to mediate protein–protein interaction, although in the vast majority of cases, the identity of a particular ligand has not yet been identified. TPR-containing proteins are involved in numerous cellular processes such as transcription, cell cycle, protein translocation, protein degradation and even host defense against invading pathogens. Spontaneously arising mutations in the mouse TTC7A ortholog, Ttc7, are known [703]. Between them, the spontaneous autosomal recessive mutation, the flaky skin (fsn) mutation, causes anemia, skin disorders (psoriasis) and gastric hyperplasia. It was mentioned that thymic histology of 8-week old fsn^–/fsn^– mice show a markedly reduced cortex cellularity (although data were not shown), and both neonate and adult fsn^–/fsn^– mice show a significant reduction on number of lymphocyte [52]. Papillomas in the stomach and increase apoptosis of cecal enterocyte were observed. Other Ttc7 mutation in mouse, the autosomal recessive hea mutation, results in a lethal severe anemia with lymphopenia, for both CD4+ and CD8+ T cells [328]. The TTC7A is abundantly expressed in human thymus especially in thymic epithelial cells and in Hassall corpuscle. As the thymus in hea/hea mutant mice, TTC7A deficient patient thymus display lymphoid depletion without a clear corticomedullary demarcation. These observations suggest that Ttc7 and TTC7A play a crucial role in the thymocyte differentiation in mice as in human.

As intestinal atresia is the most common etiology of congenital small bowel obstruction accurate treatment needs to differentiate HMIA which is the rarest form of these diseases. The intestinal manifestations are present in fetal life since bowel distensions are often seen on fetal ultrasound performed as early as 17 weeks of gestation, which can also detect hydramnios and intraluminal calcifications, which seems to be specific of this condition [39, 77]. Prematurity and hypotrophy at birth with intrauterine growth retardation are often reported [60]. HMIA affects the entire gastrointestinal tract especially the small intestine and the colon, which differentiates it from non-hereditary MIA. Others anomalies are associated in sporadic cases as malrotation, septal ventricular defect, omphalocele and choanal atresias [60, 208].

The particularity of these atresias is to recur after surgical intervention which is consequently ineffective in restoring intestinal transit and which leads to short bowel syndrome.

The main infectious complications are peritonitis and bacterial sepsis, which are the cause of death, in most cases. The bacteria involved are often enteric bacteria as Streptococcus faecalis, Enterococcus faecalis and Enterobacter cloacae. However, one patient died from Pneumocystis jirovicii related pneumonia at 2 months of age.

The sieve-like appearance of the atretic bowel section and the diffuse inflammation characterize the histological lesions of the bowel.

The sieve-like appearance consists of multiple small cysts with common muscularis, propria and submucosa but with own mucosa and muscularis mucosa. The inflammatory lesions are diffuse, of variable stages, involve the mucosa and are associated with ulcerations. The lumen of the bowel contains mixed inflammatory cells and fibroblasts. Dense submucosal fibrosis is often noted as intraluminal and intramural calcifications [19, 368].

The immunodeficiency associated with the HMIA remained unidentified for a long time and consequently, only few immunological investigations are reported. This is probably due the death very early in the life. This association was first described in 1990. One patient reported here had a SCID phenotype with complete absence of T lymphocyte [446]. In contrast, B lymphocytes were detectable. The occurrence of a post-transfusion GVHd in two patients [446, 687] and of fatal Pneumocystis jirovicii pneumonia in another one [51] confirm that immunodeficiency is frequently associated, though often ignored. However, some reports mention hypogammaglobulinemia associated in some cases with lymphopenia. Recently, this lymphopenia has been more often characterized [19, 51, 130, 252, 445]. Severe hypogammaglobulinemia seems to be a constant feature. In contrast, T-cell lymphopenia is variable from one patient to another patient. Some patients had a profound lymphopenia similar to the one observed in SCID patients and others profound CD8 T lymphopenia whereas CD4 lymphocytes are normally present with a normal proportion of CD45RA + CD31+ naïve cells [51, 116] and personal data. In conclusion, the CD8 lymphopenia is probably a feature shared by most of the patients. The mitogen-induced proliferation is variable. B cell lymphopenia is also often observed.

Surgery gives only poor results because the recurrence of atresias and the consequent short bowel syndrome justify the exclusive parenteral nutrition that constitutes a non-curative treatment, not without adverse events, especially liver alterations.

So, long as the precise function of TTC7A is not elucidated, it is difficult to propose a rational curative treatment. Indeed, two non-exclusive approaches can be proposed, i.e., HSCT and bowel transplantation.

HSCT was performed in at least three patients. Two of these patients died after HSCT, one of infection [116] and the other due to complications of the intestinal disease [579]. The only remaining patient after HSCT received a familial well-matched HSCT without any conditioning regimen. Twenty-two months after HSCT, the patient displays a nearly full chimerism, and a good immune reconstitution of T- and B-cell compartments with presence of naïve T-lymphocytes [116]. However, the patient is always dependent on parenteral nutrition because of a short bowel syndrome.

Bowel transplantation has been reported in one HMIA patient [252]. The patient had liver disease secondary to the parenteral nutrition and consequently received a 1 of 6 HLA matched liver-small bowel transplantation at 16 months of age. Two years after, liver and intestinal functions are normal without evidence of allograft rejection.

Surprisingly, an engraftment of T- and B-lymphocytes from donor was observed in this patient. The T-lymphocytes display a phenotype consistent with an intestine origin (CD3 + CD4-CD8- TCRγ/δ and CD3 + CD4-CD8αα + TCRγ/δ+). IgM and IgG levels were improved but without specific antibody production after immunization. However, after CMV and parainfluenza-3 infections, virus specific antibodies were produced.

Now, we know the causative gene of this complex disease and we can expect that the elucidation of the TTC7A function will allow an accurate curative treatment of patients in a short delay.

MHC class II deficiency is a rare immunodeficiency (OMIM*209920) in autosomal recessive transmission. Most patients are of North African origin (Tunisia, Morocco, Algeria). However, patients of various origins, including Europe, United States, and Middle-East have been described. This syndrome is also called «Bare lymphocyte syndrome». It is characterized by the absence of expression of HLA class II molecules. This absence of expression is the result of a mutation in the genes encoding one of the 4 trans-acting elements that regulate the expression of HLA class II molecules.

This immunodeficiency was initially subdivided into four functional complementation groups: A, B, C, D. These four complementation groups were confirmed when the 4 genes involved were identified, that is, the genes encoding the Class II transactivator (CTIIA in group A, OMIM*600005) [623], the regulatory factor X associated protein containing ankyrin repeat (RFXANK also called RFX-B in group B, OMIM*603200) [416, 463], the fifth member of the regulatory factor X family (RFX5 in group C, OMIM*601863) [622] and the regulatory factor associated protein (RFXAP in group D, OMIM*601861) [188]. Identification of the molecular origin of this immunodeficiency contributed to the clarification of the respective roles of these factors in the regulation of the transcription of HLA Class II molecules. HLA Class II molecules DR, DP, DQ are α/β heterodimers. In humans, the genes encoding these different chains are located on chromosome 6. The molecules are expressed constitutively by thymic epithelial cells, by the antigen presenting cells (B lymphocytes, dendritic cells and monocytes/macrophages) and by activated T lymphocytes. Aside from this constitutive expression, the expression of HLA class II molecules can be induced specifically by interferon γ. HLA Class II molecule expression is regulated by a proximal region promoter called S-Y comprised of 4 cis-acting DNA elements called the S, X, X2 and Y boxes [351, 533, 682]. The RFX ubiquitous complex composed of RFX5, RFXANK and RFXAP binds box X. CREB binds box X2, and NF-Y binds box Y. The totality of factors that bind the S-Y module constitute a complex called “enhanceosome”. In case of a mutation in the gene encoding one of the components of RFX observed in patients presenting a MHC class II deficiency belonging to groups B C and D, the S-Y site is unoccupied [260, 324], proving that each of these components is indispensable for binding the enhanceosome on the S-Y site. Binding of the enhanceosome on the S-Y module is necessary for the transcription of molecule MHC class II genes, but it is not sufficient (Fig. 2.2). In fact, recruitment of the inducible CIITA coactivator, whose gene is mutated in patients with an MHC class II deficiency of group A, is indispensable.

In most patients (environ 60 %), the affected gene is RFXANK (group B) and mutations modify the Ankyrin repeat region, a region whose integrity is required for RFXANK function. The RFXANK mutation 752del G-25, linked with a founding effect, is observed in almost all North African patients [460, 495, 711]. Mutations in the RFXAP gene (group D) account for about 20 % of patients. These mutations result in synthesis of truncated proteins or the absence of transcription because a homozygous 75 bp insertion in the 5′-UTR, which impaired the activity of the RFXAP promoter.[668].The mutations observed in group A patients (about 15 %) involve the CIITA gene [416]. These mutations are diverse: missense mutations, non-sense mutations and splice site mutations. In the remaining patients (group C), mutations in the RFX5 gene generally lead to synthesis of truncated proteins [417]. Punctual mutations in RFX 5 or CIITA are associated with milder phenotypes [470, 712].

Despite the heterogeneity of molecular origins responsible for the different groups of patients presenting MHC Class II deficiency, clinical manifestations are similar [54, 195, 343, 575]. However, mild forms associated with certain mutations have been described [181, 290, 712].

Patients present recurrent infections characteristic of combined immunodeficiency. Susceptibility to bacteria, viruses and fungi testifies to the severity of this immunodeficiency. The first infection occurs in infancy, at an average age of 4 months, and exceptionally after the age of 1 year. These recurrent infections essentially involve the gastrointestinal tract, the lungs, the upper respiratory tract and the urinary tract.

Digestive problems are common. They take the form of diarrhea starting most often during the first year of life, becoming chronic and associated with malabsorption leading to delayed height–weight development. Histology findings commonly include villous atrophy associated with intraepithelial infiltration by lymphocytes and macrophages. These types of diarrhea are very often associated with Candida, Giardia lamblia and cryptosporidium infections. However, viruses (enterovirus species or adenoviruses), gram-negative bacteria (E. coli, Salmonella species, Shigella, Pseudomonas) and gram-positive bacteria (Staphylococcus and enterococcus) are also frequently involved.

Hepatic abnormalities take multiple forms. Sclerosing cholangitis secondary to chronic infection due to Cryptosporidium develops secondarily in over half the patients and constitutes a major factor in prognosis. Hepatitis cases are most often of viral origin. Cholangitis cases of bacterial origin (pseudomonas, Enterococcus and streptococcus) have also been observed.

Meningitis and meningoencephalitis of viral origin can cause death in some cases. Enteroviruses including the polioviruses, the Herpes simplex virus, the coxsackievirus and the adenovirus have been reported. Infectious pyelonephritis and septicemias can also occur. Autoimmune cytopenias, particularly hemolytic anemias and neutropenias are described in about 10 % of patients.

The immunological consequences of lack of MHC class II expression orient the diagnosis. These features can be accounted for by the lack of MHC Class II expression on Antigen Presenting Cells [195, 343]. The first characteristic is the inability to develop antigen specific humoral and cellular responses. Delayed–type hypersensitivy skin tests and in vitro Antigeng specific stimulation are negative in all patients. By contrast, responses to mitogens are normal. Humoral immunity is also always impaired. Hypogammaglobulinemia is variable from one patient to another, from agammaglobulinemia to a slight decrease in one immunoglobulin isotype (mainly IgA and IgG2). In all cases, specific antibody production is impaired. Patients display normal T cell count. However, most of them present CD4 lymphopenia. By analogy with MHC Class II -/- mice, the latter could reflect the abnormal selection and maturation of CD4 T lymphocytes in the absence of MHC class II expression on the thymus [271]. However, some MHC class II expression has been detected on medullary thymus cells from dead children and from aborted fetuses [269]. This finding suggests leakiness of the defect or the presence of an alternative regulation pattern of MHC class II gene transcription in thymic cells, that can account for partially preserved CD4 T cell differentiation and their normal repertoire building assessed by Vβ and Vα usage [367, 543].

The final diagnosis requires mutation detection. The existence of the 4 different genes involved makes molecular analysis difficult. Different strategies can be proposed to direct the molecular analysis. First, in case of consanguinity, the study of polymorphic markers flanking the four genes involved can be useful. Second, according to the frequency of the mutation 752delG-25 in patients of North Afriqua origin, it is judicious to search for this mutation first in this population. In other cases, a functional identification of the gene affected could be helpful. Recently a functional test based on direct correction of the genetic defect by transduction of cells from patients with lentiviral vectors encoding CIITA, RFXANK, RFX5 or RFXAP has been proposed as a valuable tool for the diagnosis and classification of new MHCII-deficiency patients [419]. Molecular characterization is a crucial step for proposing an appropriate prenatal diagnosis at 8–10 weeks of gestation in at-risk families.

MHC class II deficiency has a very poor prognosis. Supportive care associating symptomatic and prophylactic treatment of infection can reduce the frequency and the severity of clinical problems. Intravenous immunoglobulin injections are a part of this care. In some cases, parenteral nutrition is need. However, except in some patients who may survive for relatively long periods, this supportive care, as complete as possible, does not prevent progressive organ failure and death that occurs in most cases before 20 years of age [495].

The only radical treatment that can be proposed is HSCT for which some successful outcomes have been reported [30, 342, 575]. However, it appears that HSCT in MHC class II deficiency is associated with a lower survival rate than other immunodeficiencies because graft rejection, aGVHD and opportunistic infections. Indeed, the survival rates vary from 40 to 80 % in HLA matched situation and is <50 % in HLA mismatched one [14, 30, 219, 342, 495, 514, 534, 575, 607, 611]. In addition, in case of successful engraftment, the immune reconstitution is poor [14, 495] and the patients remain susceptible to infection [611]. The occurrence of aGVHd and the occurrence of lethal infection after transplantation are associated with viral infection status before stem cell transplantation [534]. These observations suggest that stem cell transplantation could be improved by performing the transplantation at the time of diagnosis that would minimize the risk of viral infection.

MHC class I deficiency (OMIM*604571), is characterized by low expression of the MHC class I molecules. This is true whatever the molecular basis. In no case, a complete absence of MHC class I molecule expression has never been described. To date, less than 20 patients with elucidated MHC class I deficiency have been reported and only one presented tapasin deficiency [718] and two presented a β2- microglobulin deficiency. Others display a deficiency of either TAP 1 or TAP2 [155, 158, 232, 418, 442, 705, 718]. However, some asymptomatic subjects present non-elucidated low expression of MHC class I molecule [507]. Only elucidated MHC class I deficiency will be discussed in this section.

MHC class I molecules are expressed ubiquitously and present endogenous peptides to CD8+ T cell. Consequently, MHC class I molecules are designated as the central agents of anti-viral immune response. The peptides, usually eight or nine amino-acids in length, and binding MHC class I molecules result from the degradation of newly synthesized protein carried out by the proteasome. They are further translocated in the endoplasmic reticulum by the two transporters associated with antigen processing proteins (TAP1 and TAP2), where they are loaded onto the MHC class I heavy chain/β2-microglobulin heterodimer. This loading is dependent on the peptide –loading complex that contains the heterodimer TAP1/TAP2, the thiooxido-reductase ERp57 and the glycoprotein chaperone calreticuline and tapasin (Fig. 2.3) [346, 717]. The role of tapasin seems to be multiple and complex. However, it is clear that tapasin stabilizes the TAP1/TAP2 complex, links it to MHC class I molecules and facilitates loading of peptides with progressively higher affinity [94, 717]. The peptide-loaded MHC class I molecules are further transported to the cell membrane where expression takes place. Membrane expression of MHC class I molecules is dependent on their association with high affinity peptides. MHC class I molecules that do not bind high affinity peptides do not travel through the Golgi apparatus and the empty MHC class I molecules expressed at the membrane level are unstable. Consequently, a defect in either TAP1/TAP2 complex or in tapasin leads to low MHC class I expression.

TAP1 and TAP2 molecules include a core domain, 10 and 9 transmembrane domains respectively and a catalytic nucleotide-binding domain. The genes encoding these two proteins, TAP1 (OMIM*170260) and TAP2 (OMIM*170261), are located in the HLA class II region [110, 369, 736]. So far, 12 families presenting a defect in TAP1/TAP2 complex have been reported. Homozygous TAP1 and TAP2 mutations have been found in seven and five families respectively [155, 158, 232, 418, 442, 705]. All theses mutations lead a premature stop codon and consequently to a truncated non functional protein.

Only one patient presenting a tapasin (TAPBP, OMIM*601962) mutation has been described [718]. The tapasin molecule contains a short cytoplasmic tail, a transmembran region and an N terminal intraluminal region. The mutation described consists in a large deletion of 7.4 kb leading to a putative frame shifted and truncated protein that is not detectable.

Homozygous mutation of Beta-2 microglobulin encoding gene (B2M; OMIM*109700) has been found associated with a familial hypercatabolic hypoproteinemia in two siblings born from consanguineous parents [691]. This mutation leads to a substitution of a very conserved alanine to a proline which would affect the secondary structure of the protein and consequently its entry in the endoplasmic reticulum.

The clinical consequences of TAP1/TAP2 deficiency are variable from one subject to another. Some patients are asymptomatic [157, 500]. In most cases, symptoms, when they exist, occur late in childhood, at about 4–7 years of age. Despite the few patients described, no difference in clinical manifestation can be detected between TAP1 and TAP2 deficiency. Two typical features have been reported [110, 154, 233, 736]. The first consists in chronic infections affecting the respiratory tract and the second in skin granulomatous lesions.

In most cases, the respiratory tract is involved. Chronic infections of the upper respiratory tract are often the first manifestation and are responsible for purulent rhinitis, pansinusitis and otitis media. Frequent association with nasal polyposis has to be noted. Secondly, the infections extend to the lower respiratory tract and a to a chronic inflammatory lung disease that progressively degrade the lung tissues, including bronchiolitis, bronchiectasis, and emphysema. These lesions inevitably evolve into a respiratory insufficiency. Death may be secondary to this degradation but may also occur during an acute infection. The pathogen most often involved in respiratory alteration is Haemophilus influenza, but others can be detected such as Streptococcus pneumonia, Klebsiella, Pseudomonas aeruginosa and Toxoplasma gondii. Altogether, respiratory manifestations can mimic cystic fibrosis.

Skin lesions are present in half the patients and can be the only manifestation in patients without respiratory involvement [442]. They start with local inflammation that progressively extends, ulcerates, and evolves into chronic necrotizing granulomatous lesions mimicking Wegener disease [442, 518, 705]. In most cases, they are localized on the legs. However, some such lesions have been described on the face, around the mouth and the nose, and in some cases are very mutilating, associated with perforation and destruction of the nasal cartilage. In some cases, these granulomatous lesions are related to vasculitis [442, 518, 705] associated with infiltration by NK cells and, to a lesser extent, TCRγ/δ T lymphocytes [442]. More recently, such skin lesions have been reported in association with Toxoplasma gondii infection [176]. Moreover, such granulamotous lesions can involve the upper respiratory tract, but have never been found in patient lung biopsies.

Recently, necrotizing retinocchoroiditis related to Toxoplasma gondii has been reported as the only clinical manifestation in a 14 year-old patient [500].

In spite of the role of the MHC class I in the peptide presentation to CD8 T lymphocytes, it is noticeable that no patient presents severe viral infection and there is no evidence of a higher incidence of neoplasm in these subjects. This observation suggests that either other effectors such as NK cells and TCR γ/δ T lymphocytes could be efficient enough to eliminate virus infected cells in this situation, or independent TAP peptide presentation is sufficient to trigger TCR α/β CD8 lymphocytes. NK cells and TCR γ/δ T lymphocytes, beneficial in virus clearance, could however generate granulomatous and epithelial lesions, the lack of MHC class I dependent inhibition of their cytotoxic activity allowing the killing of uninfected cells [233, 739]. Epithelial lesions could favor bacterial colonization. Moreover, the TAP dependent MHC class I presentation of exogenous peptides of bacterial origin could play a more important role in the antibacterial defense than previously thought [154, 736].

Clearly, there is no correlation between mutation and clinical severity. The environmental context and/or genetic background could constitute determinant factors in the development of clinical manifestations.

The only patient presenting tapasin deficiency suffered from primary chronic glomerulonephritis for 10 years at time of diagnosis. This 54 year-old woman does not present any manifestation that can be related to an immunodeficiency, except Herpes Zoster virus infection [718].

The two siblings with β2-microglobulin deficiency presented with forearm deformity including shortened ulna and bowed radius. Except these symptoms, the patients were healthy until adulthood. After a miscarriage in the 7th month of pregnancy at 21 years of age, the first patient developed skin ulcerations on the legs related to granulomatous lesions and subsequently severe idiopathic thrombocytopenic purpura. Her affected younger brother did not present any clinical manifestations. However, the chest x-ray detected a granulomatous lesion in the lung [686].

With the exception of two patients who present T cell lymphopenia, most TAP1/2 deficient patients have normal T cell count. However, most of them present a slight CD8 TCRα/β lymphopenia in contrast with the TAP-/- mouse model [670]. However, it seems that a more severe CD8 TCRα/β lymphopenia could exist early in life and be partially corrected later [154]. CD8 T lymphocytes display a diversified α/β repertoire [154] and cytotoxic activity, at least against EBV [156, 157]. In most patients, TCRγ/δ T lymphocyte count is increased, especially T lymphocytes bearing Vδ1 chain, and these lymphocytes can kill autologous cells [157, 442]. NK cells, that are present in the normal range show poor spontaneous cytotoxic activity against MHC class I deficient targets, that is corrected after cytokine-mediated activation. Moreover, activated NK cells can kill autologous cells [418, 683, 737, 738]. The killing of autologous cells by TCRγ/δ T lymphocytes and activated NK cells could play a role in the pathogenesis of epithelial lesions.

In most cases, hypergammaglobulinemia involving different isotypes is observed. However, some patients present a hypogammagloblinemia involving one or more isotypes [418, 518]. Antibodies to common viruses are present even in case of hypogammaglobulinemia, and often at high titer [177].

In contrast, the two β2-microglobulin deficient patients presented a hypo IgG contrasting with normal levels of IgA and IgM. The association with a low level of albumin is characteristic of a hypercatabolic hypoproteinemia due to the lack of neonatal Fc Receptor (nFcR). Indeed, nFcR is a heterodimer composed of a β2-microglobulin and a non-classical MHC class I α-chain and it protects its ligands i.e IgG and albumin from the degradation [26].

The diagnosis is based on low MHC class I expression assessed by immunofluorescence. In case of TAP1/2 or TAPBP deficiency, residual expression is 30–100 fold less than in controls [155, 158, 442]. The consequence of β2-microglobulin on the MHC Class I expression has not been directly studied, but the transfection of the mutant cDNA did not restore the MHC Class I expression of the β2-microglobulin deficient cell line Daudi [31, 691]. Final diagnosis requires mutation detection. The involvement of TAP1/TAP2 or tapasin can be assessed by HLA typing in consanguineous families that confirms the linkage to the chromosome 6. In contrast, B2M gene is on the chromosome 15. A functional test based on direct correction of the genetic defect by infection of patient cell line with recombinant vaccinia virus expressing TAP1, TAP2 or both subunits could assist genetic diagnosis [154, 567].

Chronic lung colonization evolves to respiratory failure which may lead to the patient’s death. Based on the similarity of these respiratory manifestations with those observed in cystic fibrosis, it is legitimate to propose to symptomatic patients with TAP deficiency management analogous with that recommended in cystic fibrosis, including prophylactic antibiotherapy in association with physiotherapy [233]. In spite of the absence of humoral immunodeficiency, treatment using intravenous immunoglobulin has been reported useful in patients with severe pneumonia.

The lesions of the upper respiratory tract may require local medical treatment (local washing and topical steroids) or surgical (polypectomy) treatment. However, surgery has to be carefully considered because, in one patient, surgical intervention for chronic sinusitis has been reported to accelerate the nasal disease [233].

Treatment of skin granulomatous lesions is based only on optimal antiseptic topical care [233]. Immunosuppressive treatment including steroids in combination with either cyclophosphamide, methotrexate, azathioprine or cyclosporin, has worsened skin lesions as well as lung manifestations and has to be avoided. In the same way, immunomodulatory intervention based on the use of Interferon α or γ is also disappointing, since it is associated with lesion progression [705].

A curative treatment has not been reported so far. Lung transplantation could be considered if the hypothesis concerning the role played by NK and TCR γ/δ cells in lesion pathogenesis is confirmed. The immunoglobulin substitution in the β2-microglobulin deficient patients has not been proposed but we can speculate that it would be challenging as in other pathologies associated with IgG loss as nephrotic syndrome and protein-losing enteropathy. The rationale of HSCT that would provide MHC class I positive hematopoietic cells could be debated.

Two immunodeficiencies characterized by the isolated absence of CD8+ T cells have been identified, caused by a defect in either ZAP70 (OMIM*269840) [112, 193] or CD8α chain (OMIM*608957) [152]. In spite of this shared feature, the clinical and biological consequences are very different. The ZAP-70 deficiency constitutes a SCID, while the CD8 α defect is considered non–severe and compatible with life. Both are inherited as an autosomal recessive trait.

The differentiation and activation of T lymphocytes require TCR-dependent signal transduction including tyrosine phosphorylation of many substrates. The tyrosine kinase ZAP70 (Zeta associated protein-70) (OMIM*176947), belonging to the tyrosine kinase Syk family, plays a major role in this biochemical pathway. The antigen recognition is assured by the TCR, while the CD3 complex consisting of the γ, δ, ε₂, ζ₂ chains transmits an intracytoplasmic signal by recruiting tyrosine kinases from the Src and Syk families. The CD3 complex contains, in its intracytoplasmic portion, a total of ten ITAM motifs (Immunoreceptor Tyrosine-based Activation Motif), targeted for phosphorylation. Three of these motifs are carried by CD3ζ, and one by each of the other chains, CD3γ, CD3δ and CD3ε. Phosphorylation of these motifs by protein kinases of the Src family leads to recruitment by the ζ chain of ZAP-70, which is then phosphorylated and activated by the tyrosine kinase p56lck [111, 699]. ZAP-70 phosphorylates different substrates and consequently induces a calcium signal and MAPK activation leading to immune response [256] (Fig. 2.4). The normal thymic differentiation of CD4 positive T lymphocytes in ZAP-70 deficient patients proves that in humans, in contrast with the ZAP-/- mouse model, CD4 differentiation can occur in the absence of this tyrosine kinase [34, 467, 549, 552]. Syk, highly expressed in patient thymocytes, may compensate for the loss of ZAP-70 in CD4, but not in CD8 thymic selection [239, 479]. ZAP-70 is also expressed in NK cells.

To date, around twelve different ZAP70 mutations have been reported in the literature, but we can suppose that some ZAP-70 deficient patients are not reported. Most patients are born of related parents and present a homozygous mutation [34, 112, 192–194, 239, 325, 420, 435, 473, 479, 513, 549, 660, 664]. The mutations described include missense mutations, splice site mutations and deletions. Most mutations involve the catalytic domain but in fact affect protein stability. Two missense mutations found in a compound heterozygote patient, one affecting the first SH2 domain and the other affecting the kinase domain, are associated with a temperature-dependent instability of ZAP-70 [420]. A hypomorphic mutation, a single G-to-A substitution in a non-coding intron, which allows residual expression of normal protein, was observed in a patient with a moderate clinical and immunological phenotype [513].

CD8 molecules are expressed on the cell surface either as a αα homodimer in NK cells and TCRγ/δ T lymphocytes, or as an αβ heterodimer in TCRα/β T lymphocytes. However, surface expression of CD8β is dependent on expression of CD8α because CD8β polypeptides are otherwise retained in the endoplasmic reticulum and degraded. CD8 constitutes a coreceptor for TCR recognition of MHC class I–binding peptides and is necessary for the maturation, positive selection and activation of class I MHC restricted cytotoxic T lymphocytes. To date, two cases of CD8α deficiency (CD8A, OMIM*186910) have been reported in two families [152, 407]. Both are Spanish gypsy patients and present the same homozygous mutation. It is a missense mutation, Gly111Ser, affecting a very conserved position. This mutation is restricted to the Spanish Gypsy population and a study of microsatellite markers has shown that it is derived from a common founder and that it is detected at a 0.4 % rate in this population [407].

Patients with ZAP-70 deficiency present infections indistinguishable from these observed in other severe combined immunodeficiencies. They occur in most cases within the first year of life and involve bacterial, viral and fungal pathogens. In some cases, opportunistic infections such as Pneumocystis jiroveci related pneumonia or a CMV uncontrolled infection are the first manifestations of the disease. Frequently, Candida is responsible for cutaneous and oral infections and even for septicemia. Other infections due to various virus including varicella zoster virus, Rotavirus and parainfluenza have been reported, as well as lower and upper respiratory tract bacterial infections. These infections are often associated with a failure to thrive. Moreover, the patient presenting mutations associated with a thermo- sensitive ZAP-70 was affected by infiltrative erythematous skin lesions on his face and extremities [420]. In contrast with other SCID patients, some ZAP70 deficient patients display palpable lymph node and a normal sized thymus detected by chest radiology. Some patients presented with erythrodermia, eosinophilia and increased IgE level as observed in Omenn’s syndrome [664]. However, in the case of a partial ZAP-70 deficiency due to the hypomorphic mutation, the patient displayed an attenuated and late onset form of the disease without autoimmunity [513].

The severity of this later immunodeficiency contrasts with the late onset of clinical manifestations in both CD8α deficient patients described so far. The age at diagnosis in the latter is 25 and 16 years [152, 407]. However, both patients suffered from recurrent respiratory infections very close to those observed in TAP deficiency since the childhood. In the first patient described the pulmonary lesions led to death at 33 years of age. The main pathogens reported are Pseudomonas aeruginosa and Haemophilus influenza. Similarities with TAP deficiency are numerous. Some subjects who present the same CD8 deficiency are as healthy as the siblings of the first case described, and patients do not present high incidence of viral infection.

ZAP-70 and CD8α deficiencies share a common feature: the lack of blood CD8 T lymphocytes. However, other biological findings are very different and are going to be described sequentially.

ZAP-70 deficient patients have a normal or high blood lymphocytes count. Except for the absence of CD8 TCR α/β T lymphocytes (in most cases, less than 3 % of blood lymphocytes), other lymphocyte populations, including CD4 T lymphocytes, and TCRγ/δ T lymphocytes, are normally present. NK cell count usually normal cells are reported slightly decreased in one case [325]. CD4 lymphocytes display a normal Vβ repertoire [552], suggesting that ZAP-70 is not indispensable for CD4 lymphocyte selection. However, peripheral CD4 lymphocytes function poorly. In vitro proliferation assays are useful to orient the final diagnosis. The proliferative as well as the IL-2 secretive responses to PHA and anti-CD3 antibody are absent and restored in part by exogenous IL2. Antigen-induced proliferations are also poor. In contrast, the association of a phorbol ester (PMA) with a calcium ionophore (ionomycin) that bypasses proximal TCR/CD3 signaling induces normal T cell proliferation. The lack of calcium mobilization and poor protein tyrosine phosphorylation after CD3 triggering confirm a defect in a proximal signal step [34, 239]. These functional abnormalities were also observed in the case of the partial deficiency whereas the patient displayed a T cell lymphopenia including CD4 lymphocytes [513].

Humoral immunity is variably altered. Hypogammaglobulinemia involving all isotypes associated with a complete absence of specific antibodies observed in most patients contrasts with the normal or high level of immunoglobulins reported in others [596]. Some of these patients display normal antibody response after tetanus immunization. In any case, the hypogammaglobulinemia does not constitute an absolute diagnostic criterion.

Blood T lymphocyte phenotype is characteristic of patients with CD8α deficiency. The patients present normal TCRα/β CD3, TCRγ/δ CD3 and CD4 T lymphocyte counts. Surprisingly, the lack of CD8 T cells is associated with an increased T cell population that expresses CD3 and TCRα/β, but expresses neither CD4 nor CD8 [152, 407]. This population is polyclonal and displays a normal Vβ repertoire. It probably represents a population with CD8 cytotoxic T lymphocytes, since it expresses a phenotype associated with effector cytotoxic T lymphocytes (CD11b+, CD57+ and CD28-) and transcripts for CD8α and CD8β [152].