Update on DNA-Double Strand Break Repair Defects in Combined Primary Immunodeficiency

The meiotic recombination 11 homologue 1 (MRE11), RAD50 and Nijmegen breakage syndrome protein 1 (NBS1) proteins play a pivotal role in sensing DNA-dsb and coordinating the response to initiate cell cycle checkpoint arrest and commence DNA repair or initiate apoptosis. This compound (the MRN complex), which exhibits dual single strand DNA endonuclease and double strand DNA exonuclease activity, comes together as a heterodimer complex to execute three indispensable functions in DNA-dsb repair:

Human disease has been described due to mutations in MRE11 (Ataxia-Telangiectasia-like disorder, OMIM #604391) [4‐6], RAD50 (Nijmegen Breakage Syndrome-like disorder) [7•,8] and NBN, and although the somatic phenotype shows some common features, significant immunodeficiencies are confined to patients with NBN mutations giving rise to Nijmegen Breakage syndrome (NBS) (OMIM #251260).

The activated MRN complex initiates the cell cycle checkpoint response by promoting the localized activation of ataxia-telangiectasia mutated (ATM) protein, which is a central component of the signal transduction pathway through a variety of cellular signalling pathways in response to DNA damage, including cell cycle control, apoptosis, senescence, transcription, chromatin structure alteration and DNA repair. Activated ATM phosphorylates the MRN complex, resulting in cascade of phosphorylation of hundreds of ATM substrates [9] (Figure 1ii). Whilst the majority (~80%) of irradiation-induced DNA-dsbs are repaired by the NHEJ pathway independently of ATM, a minority are repaired by a pathway requiring ATM and Artemis [10]. Defects in ATM give rise to ataxia-telangiectasia (AT) (OMIM #208900).

The NHEJ repair pathway for DNA-dsbs has three aims:

A series of eight proteins have been identified as the critical NHEJ components, which are involved in the ligation of DNA-dsb [11]. The DNA-binding subunits Ku70 and Ku80 together form a ring shaped heterodimer that acts as an anchor protein binding the DNA ends and protecting them from exonucleolytic activity. A single Ku heterodimer binds to each DNA end, and interacts with DNA protein kinase catalytic subunit (DNA-PKcs) to form a holoenzyme, DNA-PK. DNA-PK acts as a bridge between two Ku heterodimer-bound DNA ends, acting to stabilize the local DNA structure to enable end-processing and DNA ligation. The Ku enzymes have not yet been implicated in human disease. Mutations in PRKDC, which encodes the DNA protein kinase catalytic subunit lead to CID (OMIM #615966).

Artemis, encoded by DCLRE1C, is phosphorylated by activated DNA-PKcs, which initiates the endonuclease activities of Artemis allowing resolution of complex DNA ends including the heterologous loop and stem-loop DNA structures that contain single-stranded DNA adjacent to double-stranded DNA (Figure 1iii). Defects in DCLRE1C lead to a number of immunodeficiencies including SCID and CID (OMIM #602450). DNA ligase 4, XRCC4 and Cernunnos-XRCC4-like factor (XLF) act as link proteins, bridging the DNA ends – DNA ligase 4 is required for the ligation reaction that rejoins the DNA-dsbs. DNA ligase 4 (OMIM #606593) and cernunnos-XLF (OMIM #611291) have been implicated in human immunodeficiency. Patients with XRCC4 deficiency are described (OMIM #616541), but although short stature and microcephaly are features, immunodeficiency has not been described. Most of the NHEJ process functions separately from ATM signalling, although a fraction contingent upon Artemis requires ATM activity, demonstrating some relationship between the signalling and repair machinery.

The most recent factor involved in NHEJ to be described is Paralog of XRCC4 and XLF (PAXX), which has a similar structure to XRCC4, and interacts and binds with Ku, stabilizing the NHEJ protein assembly [12••, 13••, 14••] (Figure 1iv). To date, no human disease has been described involving PAXX, and it is not clear whether defects have any impact on adaptive immunity.

Artemis is critical for VDJ recombination, demonstrated by null mutations in DCLRE1C leading to a T-B-NK+ SCID phenotype with absent immunoglobulins [15], which was initially described in Athabascan-speaking native Americans [16]. Patients present as with any other form of SCID, classically in early infancy with viral or pneumocystis pneumonitis, persistent viral diarrhoea and growth failure [17]. There is a systemic increased cellular sensitivity to ionizing radiation. Although SCID is the most common presentation, other clinical phenotypes are described including atypical SCID with hyper IgM [18•], Omenn syndrome [19], progressive CID presenting from later infancy and typified by recurrent infection of the gastrointestinal or sino-respiratory tracts, T- and B-lymphocytopaenia, hypogammaglobulinaemia and autoimmune cytopaenias, some with EBV-associated B lymphomas [20, 21], and antibody deficiency with a common variable immunodeficiency phenoytype [22]. Autoimmunity is commonly described in the non-SCID presentations [23]. The severity of the clinical phenotype correlates with the levels of recombination and DNA repair activity conferred by the protein, determined by the type and genetic locus of the mutation [24]. Microcephaly is not a feature of DCLRE1C mutations.

The outcome of HSCT in patients with SCID is significantly better in those transplanted without pre-existing infection [25, 26]. This observation has directed the institution of newborn screening programs for SCID in many states in the USA [27], by identification of DNA remnants present in lymphocytes, and left over following successful VDJ recombination, revealing successful T lymphocyte receptor re-arrangement (T lymphocyte receptor excision circles – TRECs). T lymphocyte neo-genesis, successful thymopoiesis and durable T lymphocyte engraftment, as well as probability of B lymphocyte function requires haematopoietic stem cell engraftment, most likely achieved by use of preparative chemotherapy [28, 29]. The use of alkylating-containing conditioning regimens confers longterm immune-reconstitution in patients with DCLRE1C mutations [30], but leads to significant long-term sequalae [31], and possibly an increased risk of early conditioning-related mortality [32•]. These factors have driven the quest for safer alternatives to achieve functioning immunity. Two promising developments which have entered clinical trials are lentiviral-vector gene-addition therapy [33••, 34••] and the use of chemotherapy free antibody-based conditioning [35]. Longterm results are awaited, but preliminary results are encouraging.

AT is a rare systemic disorder, inherited in an autosomal recessive fashion, and caused by mutations in ATM [36]. Systemic clinical features include progressive cerebellar ataxia, oculocutaneous telangiectasia (Fig. 2), gonadal sterility, and growth retardation. Patients experience a high frequency of lymphoid tumours. Immunodeficiency may lead to frequent sinopulmonary infections that in conjunction with recurrent aspiration, lead to chronic lung disease [37]. Interstitial lung disease, including lymphoid interstitial pneumonitis is also reported. Patients with AT have a variable incidence of infections;- some experience no more than unaffected siblings, whereas others manifest progressive respiratory infection because of humoral and cellular immunodeficiency. Immune responses, especially to bacterial polysaccharide antigens, are generally reduced [38]. Mutations in ATM do not lead to a significant block in lymphocyte development. Constancy of VDJ recombination rather than completion of VDJ recombination may be affected in the absence of ATM, which may be important to monitor recombination intermediate products. IgA, IgE and IgG subtypes are reduced or absent in AT patients and, in some, IgM may be raised, which may represent a more severe immunological defect with worse prognosis [39‐41]. Heterozygote carriers do not display the classical manifestations of disease, but may have a higher incidence of solid tumours, particularly breast cancer in female carriers [42]. Ataxia is normally the earliest clinical manifestation of AT [43]. Most infants appear to be normal during the first year of age - walking develops normally, although some demonstrate ataxia in infancy with a delay in walking. Abnormal eye movements generally develop from the second year of life onwards [44, 45] and oculocutaneous telangiectasias appear on the bulbar conjunctivae and exposed areas of the skin after the neurological deficit has manifest. Café-au-lait patches are also common.

Patients with AT have a significantly increased risk of developing malignancy - from 10 years of age, the risk of developing a tumour is around 1% annually. Around 25% will develop malignancy, of which lymphoma and leukaemia predominate [46•]. Malignancy may be difficult to treat – the tumours are often aggressive, and because of the systemic nature of the disease, significant morbidity and mortality to chemotherapy and radiotherapy may be experienced. Currently there are no curative therapies for patients with AT, median age of death is 25 years [47], and older patients have significant multi-system co-morbidities [48••].

There is a tentative suggestion that chronic inflammatory processes, driven in part by persistent genotoxic stress, participate in the pathogenesis in AT [49, 50]. Furthermore, in addition to the intrinsic genomic instability, impaired immune surveillance may contribute to tumorogenesis and development. Murine Atm-deficient T-lymphocytes demonstrate impaired proliferative capacity because of replication stress [51]. These observations raise the possibility that modification of the immune system may alleviate some of the disease sequelae. Haematopoietic stem cell transplantation has been considered for AT patients. Use of fully myelo-ablative conditioning is associated with significant morbidity and mortality [52••]. The adoption of a reduced intensity conditioning regimen gives better survival and enables donor engraftment and immune reconstitution [53‐55•]. The long-term outcome with respect to systemic damage to tissues has yet to be determined, and there may be an increase in secondary malignancies, as there is for Fanconi anaemia patients who have been transplanted [56]. Furthermore, the efficacy of HSCT in terms of ameliorating damage secondary to inflammation, and malignancy due to enhanced tumour surveillance are unproven to date. Adoption of HSCT for patients with HSCT would best be undertaken in the context of clinical trials, with careful long-term follow up.

Newborn screening for SCID detects TRECs, the by-product of successful T lymphocyte development. Significant T-lymphocytopenia will therefore be detected by TREC screening, and non-SCID conditions may be picked up incidentally, including patients with AT [57•, 58•]. This presents an ethical dilemma as:

The first two points can be resolved by ensuring that a genetic diagnosis is available prior to embarking upon a stem cell procedure. The final points require sensitive counselling before further testing is performed. Many parents would prefer to have information regarding an early diagnosis of AT, although a significant minority would prefer not to know [59••,60].

Nijmegen breakage syndrome (NBS) is typified by a characteristic dysmorphic facial appearance, which becomes exaggerated with advancing age. Intrauterine growth retardation is usually present and patients exhibit profound microcephaly at birth. They display a receding forehead, receding mandible and prominent midface [61]. Other features include short stature, congenital skeletal (clinodactyly, syndactyly) and renal abnormalities, and mild non-progressive mental retardation. Premature ovarian insufficiency is reported in females. Although reported worldwide, there is a particularly high prevalence among Central and Eastern European Slavic populations due to a founder mutation effect (a homozygous deletion of five nucleotides (657_661del5)) [62]. Occasional patients may have normal head size, although display other features typical of NBS [63]. Sino-pulmonary infection is common and patients are susceptible to malignancies, particularly B lymphocyte-lineage lymphomas (Fig. 3), and autoimmune manifestations, including pulmonary granulomata and interstitial lymphocytic lung disease [64, 65]. Cellular and humoral immune deficiency are widely reported, but with a spectrum of clinical expression ranging from clinically-silent laboratory abnormalities (reduced CD4+, CD8+ T-lymphocytes, thymic emigrants, low percentage of naïve T-lymphocytes, increased memory T-lymphocytes, reduced TCRαβ/ TCRγδ ratio in peripheral blood) to clinically relevant immunodeficiency, particularly hypogammaglobulinaemia, which presents with recurrent, chronic respiratory tract infections. This may cause development of bronchiectasis, and many patients require immunoglobulin substitution therapy. Opportunistic infections are rare and there is generally no correlation between the degree of cellular deficiency and infection severity of infections [66]. Some patients with Nijmegen breakage syndrome may be identified on newborn screening for SCID with very low levels of TRECs [67•].

Malignancy remains the most significant risk for patients with NBS, with most tumours arising from the lympho-reticular system. By 20 years of age, over 40% of patients have acquired a malignancy, and the median age at which malignancy develops is 10 years [68]. Because the repair defect is systemic, patients often fail to tolerate treatment, and have a higher frequency of chemotherapy-related toxicity, and severe or fatal infectious complications during chemotherapy than observed in otherwise healthy children receiving chemotherapy. The adverse response to treatment results in a high rate of treatment failure and increased risk of developing secondary malignancies.

Given these issues, the arguments for considering HSCT to treat patients with NBS are perhaps more compelling than for patients with AT – the neurological problems associated with NBS are not progressive, and many early deaths may be prevented by altering immune surveillance. Regimens using reduced intensity conditioning or Fanconi-anaemia type protocols have resulted in successful outcomes [52••, 62, 64, 69]. The role of pre-emptive HSCT has yet to be formalized, but could be contemplated before malignancy develops, particularly in patients with clinical immunodeficiency, recurrent or chronic infection despite immunoglobulin therapy, latent viral infection (especially EBV) or severe autoimmunity. However, careful long-term follow up is required to determine whether the incidence of secondary malignancy might be lower than that recorded in non-transplanted individuals. One report documents a loss of donor myeloid chimerism in patients receiving reduced intensity conditioning, but improved chimerism when treosulfan was added to the regimen [70]. Follow up was short, however, at only 3 years and longer follow up is required to determine whether the risk of secondary malignancy is increased with the higher intensity regimen.

A few patients have been described with hypomorphic mutations in LIG4. Most have microcephaly, although individuals with a normal head circumference are described [71]. The spectrum of presentation in individuals with LIG4 deficiency is wide with cases of SCID or atypical SCID [72, 73], Omenn syndrome [74], CID [75, 76], asymptomatic CD4+ T-lymphocytopenia [77], predisposition to malignancy [71], marrow hypoplasia and even asymptomatic individuals [78••], some of whom display microcephaly and growth failure [79]. As well as immunodeficiency, patients are at significant risk of developing malignancies, predominantly lymphomas or leukaemias, often, but not always associated with EBV [71, 75, 80]. Whilst microcephaly and growth failure are characteristic, they are not universally present, and the clinico-immunological phenotype is indistinguishable from that of patients with NBS. T-lymphocytopenia is often present, and peripheral B-lymphocytes may be almost absent. A high IgM and low IgA and IgG may be seen, due to the role of LIG4 in class switch recombination. Treatment is expectant, with antimicrobial prophylaxis and immunoglobulin replacement as required. Treatment of malignancy is difficult, as tumours are often aggressive and the systemic distribution of the defect means that patients are poorly tolerant of chemotherapy and radiotherapy [71]. The role of HSCT is unclear. A number of patients have successfully been treated, particularly when reduced intensity conditioning regimens are employed [52••], but the optimal patient selection and timing have yet to be determined. Whilst HSCT will correct the immunodeficiency and associated marrow hypoplasia, it is unclear whether successful treatment will prevent development of malignancy or whether late-onset conditioning-related secondary malignancies will develop.

Cernunnos-XLF interacts closely with DNA ligase 4. To date, only a few patients with hypomorphic mutations in NHEJ1 have been described. The phenotypic features are similar to those of patients with NBS and LIG4 deficiency, namely profound microcephaly with variable degrees of learning difficulty, growth failure, SCID or CID [81‐83]. Lymphoma has also been described in one patient [84•]. The immunological phenotype is similar to that in patients with LIG4, namely T-lymphocytopenia with profound peripheral B-lymphocytopenia, high IgM and low IgA and IgG. A few patients have successfully been transplanted using reduced intensity conditioning regimens [52••].

DNA-PKcs acts early in the repair of DNA-dsb, and interacts with artemis. Only six patients with defects in PRKDC have been described to date, too few to confidently ascribe a typical phenotype. The first patient had a SCID phenotype, not associated with microcephaly [85]. Microcephaly has been described in one patient with SCID [86]:- other presentations have described autoimmunity with granulomata [87•, 88•]. Stem cell transplantation has been successfully attempted, but there are too few data to make any clear recommendations. It could be predicted that patients will react more like those with artemis deficiency than deficiencies of other core NHEJ proteins.

Ataxia telangiectasia-like disorder is caused by mutations in MRE11, part of the MRN complex which associates with NBS1, and is extremely rare, with few patients reported worldwide [92, 93•]. Clinical features mimic those of patients with AT although telangiectasia are absent and progressive cerebellar ataxia occurs later and progresses more slowly. Immunoglobulin levels are normal, but deficiency in pneumococcal polysaccharide antigen antibodies has been reported, particularly to pneumococcal polysaccharide antigen [94]. Some patients are reported to exhibit microcephaly.

Only two patients have been described with NBS-like features, in whom compound heterozygous or homozygous mutations in RAD50, the third component of the MRN complex, were found [7•,8]. The clinical features more resemble NBS than ATLD, with intrauterine growth retardation, microcephaly, and poor postnatal growth. There was no history of excessive infections and immunoglobulin levels were normal.

Numerous individuals have been described with mutations in XRCC4 [95‐99]. Whilst the clinical phenotype is quite severe, with microcephaly, growth retardation and developmental delay, and there is severe radiosensitivity with a DNS-dsb defect, VDJ recombination appears preserved, with no significant immunodeficiency.

Whilst these proteins are represented in NHEJ and presumably VDJ recombination, to date, no human disease has been described attributable to defects in these genes.