Immunodeficiency associated with DNA repair defects

Cellular DNA is subjected to an onslaught of damaging insults which threaten cellular control and replication. It is therefore unsurprising that a plethora of damage response mechanisms operate to maintain genomic stability. These include processes that recognize and repair the damage, cell cycle checkpoints that prevent cell cycle progression in the presence of damage, and mechanisms, such as apoptosis, that remove damaged cells. In contrast, the development of effective immune responses is totally dependent on the generation of some 10¹² genetically diverse cells, each bearing a unique receptor capable of recognizing a unique antigen/MHC combination. Only in this way can the immune system recognize the vast array of antigens that may be encountered. Higher organisms create this huge number of genetically diverse cells by breaking, randomly re-sorting and then joining the DNA sequences coding for antigen receptors by adapting the DNA repair mechanisms normally utilized to maintain genome stability. Individuals with defective DNA repair mechanisms have pleiotropic phenotypes including a predisposition to cancer, neurodegeneration and developmental abnormalities. Increasingly, immunodeficiency is recognized as a feature of some of these syndromes. Further evidence for the overlap between DNA repair and immune development is the finding that some individuals with poorly understood immunodeficiencies exhibit cellular ionizing radiation sensitivity, probably as a consequence of defective repair of radiation-induced DNA damage. This review discusses the common molecular defects identified in DNA repair-defective syndromes associated with immunodeficiency, as well as detailing the clinical features seen in affected individuals.

Mechanisms of dna repair

DNA damage can result from exposure to exogenous agents, such as radiation and chemicals in the environment, as well as from endogenous DNA-damaging agents that arise as by-products of cellular metabolism. Damage can also occur during DNA replication, and from such spontaneous events as de-purination and de-amination of nucleotides. The range of lesions arising in DNA is large, but they can be subdivided into four distinct classes: strand breaks, damage or modification to a single base, dimer formation or cross-links between adjacent or opposing bases, and damage to the phosphodiester backbone. A classification of the repair mechanisms that operate to deal with these lesions together with details of human syndromes known to be defective in these processes is given in Fig. 1 andTable 1. Cell lines from immunodeficient patients are most frequently associated with sensitivity to ionizing radiation (IR). Although IR induces an especially broad spectrum of lesions, double strand breaks (DSBs) represent the most significant lethal lesion. We will therefore focus here on damage response mechanisms that operate to repair DNA DSBs.

Fig. 1

Repair mechanisms

Table 1

Genes involved in common DNA repair pathways and identified hereditary disorders with identified defects in these pathways

Repair pathway	Genes involved	Hereditary disorders
Base excision repair	Glycosylases; HAP1; DNA polβ; XRCC1,	None yet identified
	DNA ligase III, PARP
Nucleotide excision repair	XPA-G; DNA polε; PCNA, RFC; RPA,	Xeroderma pigmentosum
	transcription complex. CSA, CSB
Mismatch repair	HMsh2, 3 and 6; Mlh1; hPms2	Hereditary colon cancer
Homologous recombination	HRad51; hRad52, hRad54; XRCC2 and	None yet identified*
	XRCC3
Non-homologous end joining	DNA-PK (Ku70, Ku80 and DNA-PKcs);	Possibly rare cases of severe
	XRCC4; DNA ligase IV	combined immunodeficiency.

*

BRCA1 and BRCA2 may also operate in homologous recombination (HR), but this has not yet been definitively proven.

In lower organisms the majority of DSBs are repaired by homologous recombination, a process whereby a homologous chromosome or sister chromatid acts as a template to repair the break. This high-fidelity repair process is particularly beneficial for the repair of complex breaks where bases may be lost or damaged at the break site. Homologous recombination has been well characterized in lower eukaryotes, and proteins that operate in the process, including Rad50–58, have been identified [1]. Homologues of many of these proteins have been identified in higher organisms but their contribution to the repair of breaks induced by IR is unclear [2–4]. In contrast, the major mechanism for the repair of DNA DSBs in mammalian cells is non-homologous end-joining (NHEJ), a process that rejoins breaks with the use of little or no homology. Five proteins that operate in NHEJ have been identified, namely Ku70, Ku80 and DNA-PKcs, three components that comprise the DNA–PK complex, as well as XRCC4 and DNA ligase IV (reviewed in [5]). Significantly, rodent cell lines defective in these proteins are impaired in their ability to carry out V(D)J gene recombination which is critical to the formation of a diverse repertoire of T and B cell antigen receptors, and knock-out mice, when viable, display severe combined immunodeficiency (SCID) phenotypes [6–10]. These studies demonstrate that the NHEJ machinery is recruited to effect rearrangements during V(D)J recombination and provided the first evidence for a direct overlap between the cellular machinery that repairs radiation-induced DNA damage and that which ensures the development of a diverse repertoire of T and B cells.

In addition to DNA repair, checkpoints operate at critical points in the cell cycle, including the G₁/S and G₂/M boundaries and during S phase [11,12]. In lower organisms, such as yeast, checkpoint control enhances survival after DNA damage by preventing cell cycle progression in the presence of DNA damage, or before critical processes, such as replication or mitosis, have been completed. However, it is not clear whether checkpoints serve the same function in mammalian cells, and there are to date no examples where unusual sensitivity to DNA-damaging agents can be attributed directly to lack of checkpoint control. Nevertheless, two syndromes associated with immunodeficiency have defective cell cycle checkpoints (see below).

RELATIONSHIP BETWEEN IMMUNE DEVELOPMENT and DNA REPAIR

The immune response depends upon the ability of the organism to recognize a wide array of foreign antigens. During development, T and B lymphocyte receptors use similar mechanisms during V(D)J gene recombination or immunoglobulin isotype class switching in order to generate a diverse repertoire using a limited set of gene segments. The best understood of the recombination mechanisms is the rearrangement of the variable (V), diversity (D) and joining (J) gene segments that together make up the TCR or BCR, a process that is termed V(D)J recombination [13]. This process is initiated by the introduction of a site-specific DSB at the segments to be rearranged by two recombination-activating gene proteins (RAG1 and RAG2), and the NHEJ machinery is then employed to rejoin and rearrange the segments (reviewed in [14]). Sequence changes, including deletions, additions and mutations, are frequently formed at the junctions and serve to enhance the diversity of the process. Several mechanisms operate to effect these sequence changes, including the use of terminal deoxynucleotidyl transferase [15]. The mechanism of class switching is less well understood and it is not clear whether it involves homologous recombination (HR), NHEJ or a distinct mechanism. Finally the mechanism of somatic hypermutation introduces mutations in the structure of the BCR, resulting in minor conformational changes. This generates stronger BCR/antigen avidity for some cells which undergo clonal expansion (reviewed in [16]). It is likely that a specific ‘hypermutable’ polymerase controls this process. The mechanisms creating variability are likely to require helicases, polymerases and DNA ligases, all proteins that operate during DNA repair processes. Thus potentially, defects in a range of proteins could confer overlapping deficiencies in DNA repair and immune responsiveness. A number of immunodeficiencies with associated repair defects have been well characterized, and increasingly we are recognizing poorly defined immunodeficiencies with apparent DNA repair defects, in which the molecular defect remains elusive. Below we discuss syndromes, as well as a few individual patients, in which immunodeficiency is associated with DNA repair defects.

Ataxia-telangiectasia

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by diverse clinical features including immunodeficiency, progressive cerebellar ataxia, oculocutaneous telangiectasies, clinical radiosensitivity, chromosome instability and elevated risk of developing lymphoid malignancies [17–19]. The immunodeficiency is characterized by both cellular and humoral impairment, but clinical manifestations are extremely variable, ranging from normal to profoundly reduced responses to bacterial antigens [20]. Recurrent sinopulmonary infection is common, occasionally leading to finger clubbing and bronchiectasis, and is associated with hypogammaglobulinaemia [21]. Low or absent IgA, IgE or IgG, particularly IgG2 subclass, are frequently found and are due to a defect in B cell maturation, rather than B cell lymphopenia [20]. Autoantibodies have also been documented in some A-T patients [22]. Cellular immunodeficiency is characterized by defective thymic development, with macroscopic absence of the thymus at post mortem examination. Lymphopenia has been described with associated impairment of in vitro proliferation to mitogens [20]. A-T patients are proficient in V(D)J recombination but significantly, translocations involving the immunoglobulin and TCR loci are found at elevated frequency in A-T lymphocytes [23–25]. The wide range of clinical characteristics of A-T is coupled with a pleiotropic phenotype at the cellular level [17,19,26]. A-T cell lines display γ-radiation sensitivity, defects in cell cycle checkpoint control including an inability to arrest at the G₁/S and S phase checkpoints and, for cells in G₂ at the time of irradiation, an impaired G₂/M arrest [27–30]. Furthermore, p53, a key protein involved in signal transduction and required for G₁/S arrest, fails to be stabilized following radiation exposure [31,32]. However, the radiosensitivity of A-T cells appears to be distinct from the checkpoint defects and there is circumstantial evidence that A-T cells also have a DNA repair defect (see [33] for further discussion of this point). A-T cells rejoin DNA DSBs relatively efficiently, suggesting that the NHEJ pathway functions normally and homologous recombination can also take place in A-T cells [34,35]. The defective protein in A-T cells is a member of the phosphoinositol 3-kinase (PI 3-K) family of kinases, has serine threonine protein kinase activity [36] and is implicated in the phosphorylation of a range of proteins involved in damage response mechanisms, including p53 and Chk1 [37,38]. Taken together, these and additional findings suggest that this protein acts as an early sensor of DNA damage, and activates by phosphorylation, a number of damage response mechanisms including a p53-dependent pathway that controls apoptosis and G₁/S arrest. Some of the clinical heterogeneity may be explained by the nature of the ATM mutation. Whilst the majority of ‘classical’ A-T patients have frameshift mutations that are likely to inactivate gene function and express no (or undetectable quantities of) protein, a subset of milder ‘AT variants’ have missense mutations which allow the production of some normal protein [36,39–42].

Nijmegen breakage syndrome

Nijmegen breakage syndrome (NBS) is another rare autosomal recessive disorder also associated with diverse clinical features overlapping with, but distinct from those of A-T [25]. In common with A-T, NBS patients manifest humoral and cellular immune dysfunction, clinical radiosensitivity, chromosome instability and a predisposition to cancer [43–45]. In contrast to A-T, they display a characteristic ‘bird-like’ facial appearance, microcephaly and growth retardation. Whilst the clinical phenotype is variable, most affected children have recurrent bacterial sinopulmonary infection which may lead to bronchiectasis. Hypogammaglobulinaemia is common, particularly IgA and IgG subclass deficiency, and impaired antibody responses to antigens are reported [46]. Mild to moderate lymphopenia is present with impaired in vitro lymphocyte proliferative responses to mitogens [44]. Autoimmune phenomena have occasionally been reported. At the cellular level, A-T and NBS cells also show overlapping but distinct phenotypes. In addition to radiosensitivity, NBS cells also display some cell cycle checkpoint defects [47–51]. The protein defective in NBS, NBS1, nibrin or p95, has been identified and shows functional homology but only limited sequence homology to Xrs2, a protein that participates in the DNA repair response mechanism in yeast. p95 interacts with HMre11 and Hrad 50, homologues of two proteins with which Xrs2 interacts in yeast [52,53]. Yeast mutants defective in any of these components are impaired in NHEJ as well as HR [54,55]. However, NBS1 cells are proficient in DSB repair and thus do not appear to be defective in the NHEJ process in higher organisms [56]. NBS1 appears to be recruited to the site of DNA DSBs and thus may function together with ATM to sense DNA DSBs [57]. A number of NBS variant patients have also been described. Significantly, at least a subset of these do not have mutations in NBS1, showing that defects in a second protein can confer NBS-like characteristics ([58,59]; our unpublished observations).

Bloom's syndrome

Bloom's syndrome (BS) is another rare autosomal recessive disorder associated with immunodeficiency, genomic instability and a predisposition to cancer. Additional characteristics include growth deficiency, sun-sensitive erythema of the face, and impaired fertility [60]. The clinical phenotype is variable, with respiratory infection leading to chronic lung disease being the most common manifestation of immunodeficiency. Prolonged low levels of IgM have been reported [61] and sinopulmonary infection is associated with hypogammaglobulinaemia [62]. Cells from BS patients characteristically show an increased frequency of chromatid gaps, breaks and rearrangements, most particularly quadriradials, and increased sister chromatid exchanges (SCEs) [63]. The latter two features are indicative of aberrant HR events. Recently, the protein defective in BS has been identified and belongs to the RecQ helicase family, named after a homologous Escherichia coli protein [64]. Homologues in yeast have also been identified and additional related proteins in human cells include RECQC and WRN, the protein defective in the ageing disorder Werner's syndrome [65]. Current evidence, including the presence of abnormal replication intermediates and retarded replication fork progress in BS cells, suggests that the Bloom's protein may function either during replication or in a post-replication recombination process that resolves aberrant structures generated during replication [63]. However, BS cells do not display any appreciable sensitivity to either UV or IR.

Defects in dna ligases

Multiple DNA ligases have been identified in higher organisms, of which DNA ligase I is the major ligase utilized during replication; DNA ligase III functions to rejoin single strand breaks during base excision repair (BER), and DNA ligase IV, which unlike the other ligases can rejoin DNA DSBs, functions in NHEJ [66]. A point mutational change in DNA ligase I which severely compromised, but did not abolish DNA ligase I activity, was identified in a unique immunodeficient individual who suffered recurrent sinopulmonary infection leading to bronchiectasis. IgA and IgG subclass deficiency were present, with selective anti-polysaccharide deficiency [67,68]. Although initially lymphocyte numbers were normal, there was impaired in vitro proliferation to mitogens, and the patient became lymphopenic with absent in vitro lymphocyte proliferation, and clinical cellular immunodeficiency. The cell lines derived from this patient showed only minor sensitivity to DNA-damaging agents [67,69].

A non-inactivating mutational change in DNA ligase IV has also been identified in a leukaemia patient, who was dramatically over-sensitive to radiotherapy [70,71]. Perhaps surprisingly, the patient did not display any overt immunodeficiency, but immunological parameters were not formally examined. It is suggested that the residual DNA ligase IV function is sufficient to handle the few breaks that arise during B and T cell development but is unable to repair the large number of breaks arising following radiotherapy. This is the first description of a patient with a defect in a component of the NHEJ machinery and demonstrates that such a defect is compatible with life. Interestingly, ligase IV deficiency is embryonically lethal in mice [72,73].

Immunodeficiency in xeroderma pigmentosum patients

Xeroderma pigmentosum (XP) is a rare clinical disorder associated with sun sensitivity and a high risk of cutaneous malignancy in sun-exposed areas [74]. Defects in any of seven proteins, designated XPA to XPG, can confer an XP phenotype and these proteins jointly operate in nucleotide excision repair (NER), a repair mechanism that handles the damage induced by UV exposure [75,76]. Consistent with this, XP cells are highly sensitive to UV irradiation. Immunodeficiency has been reported in some but not all XP patients [77]. Impaired natural killer cell cytotoxicity in particular has been described [78], and whilst defective interferon production has been suggested as the mechanism, the basis is currently unknown [77]. UV damage is known to suppress the immune response in normal cells and the presence of elevated unrepaired DNA damage in XP patients is likely to enhance this immunosuppression. Thus it appears that at least some of the immunosuppression in XP develops as a consequence of the repair defect rather than as a direct result of the abnormal XP proteins impairing immune development [79]. In support of this, defective post-UV immunity has been observed in XP-A mice [80].

Non-characterized immunodeficiency associated with radiosensitivity

The finding that mice defective in NHEJ showed features of SCID prompted an examination of cell lines from unusual immunodeficiency patients for radiosensitivity. Defects in RAG1 and RAG2, the proteins required for the introduction of the site-specific DSBs during V(D)J recombination, have been identified in several patients with T-B-autosomal recessive SCID, but as anticipated such defects do not confer radiosensitivity, since the subsequent repair steps operate normally. However, a subgroup of patients with T-B-SCID with normal RAG1 and RAG2 expression do show increased cellular radiosensitivity, one of which is a patient displaying Ommen's syndrome [81–84]. Contrary to expectation, the majority of these are proficient in DNA DSB rejoining and are not defective in the Ku80-DNA-PK complex. One cell line however, with a defect in DNA DSB rejoining, has been identified (personal observations). Some, but not all, have defects in V(D)J recombination using a cellular assay to monitor the process [83].

A number of poorly characterized immunodeficiency patients display radiosensitivity, although the basis underlying the sensitivity is unclear. Two siblings had lymphopenia, absent lymph nodes and thymus, hypogammaglobulinaemia and evidence of autoimmunity. Cells from the younger sibling showed radiosensitivity, not associated with a cell cycle checkpoint defect or V(D)J recombination defect, suggesting a defect in an as yet unidentified damage response complex [85].

Increased chromosomal radiosensitivity has also been observed in patients with common variable immunodeficiency (CVID), a poorly characterized, heterogeneous group of immunodeficiencies, with associated hypogammaglobulinaemia, decreased antibody response following antigen challenge, autoimmunity, lymphopenia and in vivo and in vitro cellular dysfunction [86]. Interestingly, these patients also have an increased incidence of malignancies, particularly lymphoid tumours [87]. Irradiated lymphocytes show higher chromosomal aberrations and lower mitotic indices than control lymphocytes, indicating an increase in chromosomal radiosensitivity in at least some of these patients [88,89]. Specific defects in cell cycle check points or DSB machinery have not been examined.

Conclusion

Our knowledge of immunodeficiency associated with DNA repair defects is far from complete. Some conditions are now well characterized, and our understanding of some of the mechanisms that cells use to repair damaged DNA has helped to reveal the basis for immunodeficiency in diseases such as A-T and NBS. Some repair pathways are incompletely understood, and others remain unrecognized as yet. Although patients who display features both of DNA repair-defective syndromes and immunodeficiency are rare, the elucidation of disease mechanisms in these patients enables us to correlate genotype with phenotype. This allows prognostic assessment and the evolution of better treatment plans, in particular in determining which patients will benefit from bone marrow transplantation. Perhaps one day, many of these diseases will be amenable to gene therapy. More immediately, the study of these patients has the potential to provide insight into both damage response mechanisms and the process of immune development. In some cases, the patients represent the human homologue of ‘knock-out’ mice, providing a unique insight into human rather than murine DNA repair mechanisms; in other cases, where the affected gene is essential, they provide valuable information that cannot be easily ascertained from knock-out mouse systems. Ultimately, such studies will reveal the curious way by which mammalian cells have subtly changed the mechanisms designed to maintain genomic stability into a process that creates genetic diversity.