DNA Ligase IV deficiency syndrome is a rare autosomal recessive disorder caused by hypomorphic mutations in the DNA ligase IV gene (LIG4) 1. The gene product of the LIG4 gene functions in nonhomologous end-joining (NHEJ), a major repair pathway for DNA double-strandbreaks in mammalian cells that is activated following DNA damage, but also during class switch 2 and during V(DJ) recombination 3. The clinical phenotype shows overlap with a number of other rare syndromes, including Seckel syndrome, Nijmegen breakage syndrome, and Fanconi anemia. As a result, the clinical diagnosis is often delayed and established by exclusion. LIG4-deficient patients are characterized by microcephaly, growth retardation starting in utero, distinctive facial appearance ("bird-like face"), developmental delay, immunodeficiency, pancytopenia, and pronounced clinical and cellular radiosensitivity 145. According to their radiosensitive cellular phenotype, LIG4-deficient patients belong to the group of human radiosensitivity syndromes which include ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), Rad 50 and Mre 11 deficiency, thrombocytopenia absent radii syndrome (TAR), Artemis syndrome, and Cernunnos-XLF syndrome 15678. LIG4-deficient patients share features with the genetic instability syndrome Fanconi anemia (FA), including growth failure, bone marrow failure and increased risk of leukemia 5. FA patients are successfully treated by hematopoietic stem cell transplantation (HSCT), preferably from matched sibling donors 910111213, whereas HSCT has only rarely been applied in patients with radiosensitivity syndromes 11. We present the clinical course of a LIG4-deficient patient who is in good condition five years after a matched sibling donor bone marrow transplantation (BMT).

The patient is the second of three children of healthy, non-consanguineous parents. There was no history of hereditary disorders in the family. During the 22^nd week of pregnancy sonography suggested microcephaly and severe growth retardation. Spontaneous uncomplicated delivery occured at the 35^th week. The baby was small for gestational age (42 cm), and birth weight was 1500 g. Head circumference was not recorded but described as severely reduced. Developmental milestones were somewhat delayed: The girl started walking at the age of 15 months and began to use single words at the age of 18 months. Because of her pronounced microcephaly, short stature, psychomotoric delay and her distinctive facial appearance ("bird-like face"; cf. figure 1) she received the tentative diagnosis of Seckel syndrome. Her growth velocity remained below the third percentile (figure 2). Starting at age five the child developed thrombocytopenia and anemia followed by leukocytopenia (figure 3). A combination of pancytopenia with features like pre- and postnatal growth retardation, telecanthus, epicanthal folds, ptosis, and broadening of the bridge and tip of the nose similar to our patient had been previously described in two children with Dubowitz syndrome 1415 leading to consideration of this diagnosis. Our patient increasingly suffered from recurrent infections of the inner ear and respiratory tract with low immunoglobulin levels, requiring frequent hospital admissions. Bone marrow histology (at age 8) showed hypoplastic marrow with cellular dysplasia. This finding was remarkable since cytopenia in LIG4-deficient patients has previously been attributed to autoimmunity rather than to a primary bone marrow defect 16. Standard chromosome analysis revealed a female karyotype (46, XX) without evidence for numerical aberrations. There were no signs of monosomy 7 or trisomy 8 in bone marrow preparations, but chromosome breakage studies of peripheral blood mononuclear cells showed increased spontaneous breakage. Bleomycin-treatments of 72 h peripheral blood mononuclear cell cultures yielded strongly increased breakage rates (cf. table 1), suggesting a radiosensitive cellular phenotype and the diagnosis of Nijmegen breakage syndrome. However, no mutations were found in the nibrin gene. In order to confirm the cellular radiosensivity phenotype, cell cycle analysis using bivariate BrdU-Hoechst ethidium bromide flow cytometry was performed. As illustrated in figure 4, following irradiation with 1.5 Gy and 72 h in vitro culture, the patients' mononuclear blood cells proved as radiosensitive as cells from patients with ataxia telangiectasia. Given a normal serum alpha-fetoprotein (AFP) level and the absence of clinical features of AT, the diagnosis of AT was considered unlikely, but diagnostic considerations were extended to the AT-like syndromes which include the DNA ligase IV deficiency syndrome. As reported previously 1 mutation analysis of the LIG4 gene revealed the mutations G469E (maternal) and R814X (paternal), confirming the diagnosis of DNA ligase IV deficiency syndrome.

The clinical history of our patient illustrates the difficulty of establishing the correct diagnosis in rare growth failure and dysmorphy syndromes. The key observations that finally lead to the correct diagnosis were increased cellular sensitivity towards the radiomimetic bleomcycin (in terms of chromosome breakage), and increased cellular sensititvity to ionizing radiation (in terms of G2 phase cell cycle blockage). After exclusion of the Nijmegen breakage syndrome by mutation analysis and exclusion of AT on clinical grounds (supported by normal AFP levels), sequencing of the LIG4 gene established the diagnosis of DNA ligase IV deficiency syndrome. Due to the extensive phenotypic overlap it is conceivable that patients with features of the Dubowitz syndrome who in addition suffer from aplastic anemia 1415 might eventually turn out to be affected by the DNA ligase IV deficiency syndrome.

As offspring of a non-consanguinous couple, our patient was diagnosed as compound heterozygous for an amino acid change (glycine replaced by glutamate) on the maternal allele and a stop codon mutation on the paternal allele 1. Analysis of the impact of the R814X ligase IV mutation in vivo (hamster cell experiments) revealed an impaired interaction between ligase IV and XRCC4 (X-ray repair complementing defective repair in Chinese hamster cells 4 protein), and an impaired DNA ligase IV adenylate complex formation with reduced ligation activities 19. When the G469E ligase IV mutation was analyzed in several experiments, a normal interaction with XRCC4 was observed, but adenylation and ligation activity were severely reduced, with very little residual ligation activity 19. These results suggest that, at least in hamster cells, both mutations are pathogenic. Since LIG4 null-mutant mice are embryonic lethal 20 and biallelic null mutations have not been described to date in other LIG4-deficient patients 2122, viability of the DNA ligase IV deficiency syndrome appears to require at least one allele with a hypomorphic mutation. A preserved pattern of TCR-alpha and beta junctions in the T cells and only mild to moderate impairment of class switch and V(D)J recombination suggests the presence of residual LIG4 activity in DNA ligase IV deficient patients 2122. The relatively mild degree of immunodeficiency observed in our patient agrees with the notion of residual LIG4 function as prerequisite for viability. However, at least some LIG4 deficient patients exhibit a severely compromised immune system, including complete absence of B lymphocytes and severely reduced numbers of T cells (SCID phenotype; 2122). This suggests that DNA ligase IV, beyond its well established role in V(D)J recombination, may have additional functions in the lymphocytic cell lineage 22.

Together with the XRCC4 protein, DNA ligase IV functions in non-homologous end joining (NHEJ), the major repair process of double strand breaks in mammalian cells 23. DNA repair via NHEJ takes place during G0, G1, and early S phase of the cell cycle and requires Ku (a Ku70/Ku80 heterodimer), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and the XRCC4/LIG4 complex proteins 242526. It is assumed that Ku or Rag binds to the DSB first, then recruits DNA-PKcs and Artemis, a nuclease that trims DNA ends allowing the LIG4/XRCC4 complex to perform religation 242526. In addition, NHEJ is essential for the generation of T cell receptor and immunoglobulin diversity. Impairment of class switch and of V(D)J recombination therefore explain the immunodeficiency and proneness to multiple myeloma and lymphoma observed in LIG4-deficient patients 1726. It does not, however, explain the propensity to bone marrow failure. In analogy to the bone marrow failure syndromes Fanconi anemia and Dyskeratosis congenita which are characterized by instability of chromosomes or telomeres 2728, genetic instability due to LIG4 deficiency might explain increased radiosensitivity and bone marrow failure in LIG4-deficient patients.

It has previously been shown in mice that ligase IV is essential for neuronal cell development 29: Disruption of NHEJ due to ligase IV deficiency leads to impaired prenatal differentiation of neuronal cells. This may explain the mild to moderate mental retardation observed in our patient.

Like in Fanconi anemia and a single previously reported patient with the Nijmegen breakage syndrome, our patient illustrates that HSCT can be an effective treatment of LIG4-deficiency. Since our patient had a matched sibling donor available, a six-year history of progressive bone marrow failure requiring increasing rates of blood product substitution, and since she showed signs of developing myelodysplastic syndrome (MDS), HSCT was the most promising therapeutic option. It should be pointed out, however, that HSCT in our LIG4-patient was prompted by bone marrow hypo-and dysplasia rather than by a SCID phenotype as in other patients with LIG4 syndrome 2216.

To our knowledge, this is the first detailed description of a modified conditioning regimen for BMT and of the clinical course after transplantation in a Ligase IV deficiency patient. In other publications 14 BMT is briefly mentioned, but neither the procedure nor the outcome have been described in detail in any of the previous patients. Due to our patient's cellular radiosensitivity, she most likely would not have tolerated a conditioning regimen using total body irradiation and high dose cyclophosphamide. In addition, such aggressive conditioning would have increased her risk of secondary malignancies 30. Our protocol therefore used a mild conditioning regimen without any irradiation, a reduced dose of cyclophosphamide, and no methotrexate for GvHD prophylaxis. This reduced toxicity lead to rapid engraftment without severe side effects, complete and stable chimerism at 4 months, and good clinical condition at four years after transplant.

HSCT using mild conditioning without irradiation qualifies as treatment of choice in LIG4-deficient patients who have a matched sibling donor. In addition, the development of bone marrow failure in our patient suggests that at least some forms of Ligase IV deficiency belong to the inherited bone marrow failure syndromes.

BrdU = bromodesoxyuridine; GvHD = graft versus host disease; FSH = follicle stimulation hormone; LH = luteinizing hormone; AFP = alpha-fetoprotein; TCR = T-cell receptor; SCID = severe combined immunodeficiency disease; MDS = myelodysplastic syndrome

The author(s) declare that they have no competing interests.

BG was the attending physician performing BMT. FZ and JS were responsible for patient care and follow up. The Berlin group did cytogenetic and Bleomycin testing as well as the mutation analysis. The Wurzburg group carried out the flow cytometric testing. AB and DS were responsible for writing the manuscript.