Allogeneic hematopoietic stem cell transplantation in two brothers with DNA ligase IV deficiency: a case report and review of the literature

Three children were born to Caucasian, non-consanguineous parents, who were in good health. There was no history of hereditary disorders in the family. The oldest child, a girl, was healthy and developed according to age.

In June 2007, at the age of 2 years and 7 months, the older brother was referred to the hemato-oncological department of the University Children’s Hospital Tuebingen. He presented with microcephaly and remarkable growth retardation (weight 10.3 kg (<3rd percentile), length 83 cm (<3rd percentile), head circumference 44 cm (<3rd percentile)). His skin appeared scaly, dry and rather pale; his hair was dry, brittle and scarce. Furthermore, the patient suffered from chronic bloody diarrhea, due to a persistent infection with norovirus. No neurological deficits were detected. The laboratory findings revealed leucopenia (white blood cells 2900/μl), anemia (hemoglobin 6.8 g/dl) and mild thrombocytopenia (thrombocytes 168,000/μl). The number of lymphocytes (484/μl) was reduced, with CD3+ T cells on the lower limit (282/μl), no B cells (CD19+ 1/μl), sufficient CD4+ T cells (214/μl), remarkably few CD8+ T cells (34/μl) and reduced CD16/56+ NK cells (81/μl). The immunoglobulin levels were within the normal range (IgG 924 mg/dl, IgA 112 mg/dl, IgM 63 mg/dl). The subclass analysis of IgG showed very low levels of IgG2 (IgG1 705 mg/dl, IgG2 43 mg/dl, IgG3 24 mg/dl, IgG4 < 7 mg/dl). Conspicuously, the boy did not show adequate antibody titers after having received the standard vaccinations for his age. The first year of the boy’s life had been unremarkable. But during his second year of life the patient suffered many viral and bacterial infections, mainly affecting the respiratory and the urinary tract. Cytomegalovirus (CMV) was detected several times in the urine and norovirus in the stool. There were several episodes of unclear fever. The boy had to be hospitalized multiple times due to severe infections and was treated with antibiotics eight times in 1 year. Furthermore the failure to thrive became obvious as well as a mild hepatomegaly. Several differential diagnoses such as cystic fibrosis, coeliac disease, gastrointestinal diseases or severe underlying infections such as hepatitis were ruled out in the previously treating hospital. As an immunodeficiency syndrome was suspected, he was treated with antibiotic chemoprophylaxis and intravenous immunoglobulins, starting when he first presented to the Children’s Hospital Tuebingen. In order to identify the type of immunodeficiency syndrome a genetic analysis was performed in June 2007. At the age of 32 months the genetic findings revealed a DNA ligase IV deficiency, associated with SCID, with a compound heterozygous mutation in exon two of DNA LIG4 gene ((NM_002312) exon 2: c.613delT p.S205LfsX29 heterozygous and exon 2: c.845A > T p.H282L heterozygous). Both parents carry a heterozygous mutation (mother: ligase IV exon 2: c.845A > T p.H282L, heterozygous; father: ligase IV exon 2: c.613delT p.S205LfsX29 heterozygous). The sequencing of the RAG-1/2, Artemis, XLF and TCF3 genes did not show any abnormalities. In the chromosomal analysis a balanced translocation (46, XY, t (1;19) (q21; p13)) was found. The same karyotype could be detected in the healthy father. Thus, this chromosomal defect is considered an incidental finding.

In August 2007, at the age of 33 months, HSCT was performed due to the severity of the immunodeficiency. Conditioning was myeloablative with fludarabine (4 × 40 mg/m²), thiotepa (1 × 10 mg/kg BW), melphalan (2 × 70 mg/m²) and anti-thymocyte globulin (10 mg/kg BW, divided 1 × 1 mg/kg BW, 3 × 3 mg/kg BW). The boy suffered from severe mucositis during aplasia. He received 8.77 × 10⁶/kg BW CD34+ hematopoietic progenitor cells, 1.524 × 10⁷/kg BW CD3+ cells derived from the bone marrow of an HLA-identical (10/10 match), non-related female donor. The graft-versus-host disease (GvHD) prophylaxis was carried out with CSA (cyclosporine A 3 mg/kg BW, target level 150–200 ng/ml, day − 1 until death on day + 142), and methotrexate 10 mg/m² (day + 1, + 3, + 6). Leukocyte and granulocyte take was observed at day + 15. In the following days, the child developed acute GvHD of the skin (II°-III°, day + 17) which was initially treated locally with tacrolimus and glucocorticoids and then systemically with prednisolone (5 mg/kg BW). After an initial improvement, the GvHD of the skin relapsed and was then treated with methylprednisolone (5 mg/kg BW). A slow recovery was observed. Further complications, due to HSCT, were a foscavir-associated ulcer of the penis, CSA-associated hypertension, a reactivation of Epstein-Barr virus (EBV) and CMV, as well as a possible candidemia (positive serology for Candida antigen in the peripheral blood three times) without any clinical symptoms. Treatment was changed from amphotericin B to caspofungin. Complete donor chimerism was observed 1 month after HSCT. On day + 32, 5690/μl WBC, 1414/μl lymphocytes, (960/μl CD3+, 16/μl CD19+, 189/μl CD4+, 752/μl CD8+, 291/μl CD16/56+) were detected in the peripheral blood (Fig. 1).

A veno-occlusive disease (VOD) of the liver was diagnosed on day + 58 and on day + 74 the boy developed severe acute intestinal GvHD stadium IV, with bloody and watery diarrhea (stool volume > 1000 ml/m² body surface area per day). The symptoms stabilized for a short time with high-dose methylprednisolone (10 mg/kg BW per day, for 3 days), but then relapsed again. Further treatment consisted of somatostatin-infusions and symptomatic substitution of thrombocytes, erythrocytes, fresh frozen plasma (FFP) and coagulation factors. A causal immunosuppressive combination therapy, consisting of tacrolimus, steroids, CSA and infliximab was unable to stop the intestinal bleeding sufficiently. Furthermore the boy received 3.7 × 10⁶/kg BW mesenchymal stem cells from his father as GvHD treatment. A colonoscopy showed necrosis and ulcers of the colonic mucosa with diffuse bleeding. A probable pulmonary aspergillosis was detected on day + 105. Aspergillus-galactomannan-antigen was positive (1.9 EIA, rising quickly to 4.8 EIA) and the computed tomography (CT) scan showed intrapulmonary noduli suspected to be aspergillomas (Fig. 2a). Therefore, amphotericin B was added to the antifungal treatment with caspofungin. On day + 135, a severe intestinal bleeding, associated with intestinal GvHD, occurred (hemoglobin decreased from 14 g/dl to 5 g/dl) and the patient was transferred to the intensive care unit (ICU) for massive transfusions of blood, thrombocytes, FFP and coagulation factors. He suffered from acute renal failure and transfusion-related acute lung injury. In combination with the aspergillosis, this led to an acute pulmonary hemorrhage and the need for mechanical ventilation.

Moreover, while being transferred to the ICU, the boy was already suffering from hemiparesis and somnolence. These neurological symptoms worsened and a magnetic resonance imaging (MRI) was conducted. The scan showed severe brain damage that could be associated with a cerebral aspergillosis (Fig. 2b) although Aspergillus antigen was not detected in the cerebrospinal fluid. On day + 143 after HSCT, the patient succumbed to multiple organ failure. The autopsy report confirmed the suspected, invasive, cerebral aspergillosis (Fig. 2c, d).

Another boy was born into the family in November 2006. Although this boy, 2 years younger than his brother, was hypotrophic at birth (weight 2.610 kg (<3rd percentile), length 49 cm (7th percentile), head circumference 33 cm (<3rd percentile)), the first 3 months of his life were uneventful. Subsequently, numerous pulmonary infections and chronic bronchitis with cough, pulmonary obstruction and secretion occurred. As with his brother, the laboratory findings revealed leucopenia (WBC 2400/μl) anemia (hemoglobin 7.9 g/dl) and mild thrombocytopenia (207,000/μl). The number of lymphocytes was reduced (245/μl) with extremely reduced counts of B, T, and NK cells (CD3+ 70/μl, CD19+ 2/μl, CD4+ 51/μl, CD8+ 13/μl, CD16/56+ 11/μl). With the exception of IgA (< 6 mg/dl), the immunoglobulin levels were within the normal range (IgG 395 mg/dl, IgM 46 mg/dl). The subclass analysis of IgG was normal (IgG1 279 mg/dl, IgG2 63 mg/dl, IgG3 30 mg/dl, IgG4 < 7 mg/dl). Similar to his older brother, the boy was treated with antibiotic, antifungal, and antiviral chemoprophylaxis and intravenous immunoglobulins. At the age of 8 months, the diagnosis of DNA ligase IV deficiency was confirmed genetically, at the same time as his brother was diagnosed. He carries the same compound heterozygous mutation in exon two of LIG4 gene (exon 2: c.613delT p.S205LfsX29 heterozygous and exon 2: c.845A > T p.H282L heterozygous) and the same karyotype (46, XY, t (1; 19) (q21; p13)).

At the age of 18 months, chronic, infiltrative changes in the lung structure were identified in the CT scan. Furthermore, there was a progressive bone marrow failure. Based on those findings, HSCT was performed. The treatment protocol was adapted because his brother had suffered from severe GvHD. This time, allogeneic HSCT was performed with a combination of dosage-reduced, myeloablative conditioning and T cell-depletion (CD3/CD19 depletion) as well as CD34-selection. The chemotherapy consisted of fludarabine (4 × 40 mg/m²), melphalan (2 × 60 mg/m²) and anti-thymocyte globulin (10 mg/kg BW, divided 1 × 1 mg/kg BW, 3 × 3 mg/kg BW). Apart from a skin reaction with depigmentation, chemotherapy was tolerated well. There was no mucositis. The patient received 40 × 10⁶/kg BW CD34+ hematopoietic progenitor cells and 1.62 × 10⁴/kg BW CD3+ cells from a HLA-identical (10/10 matched), unrelated male donor. Besides the T cell-depletion no other GvHD prophylaxis was performed. Leukocyte and granulocyte take was observed at day + 10. On day + 29, complete chimerism and 1960/μl white blood cells (0/μl CD3+, 11/μl CD19+, 0/μl CD4+, 0/μl CD8+, 189/μl CD16/56+) were observed in the peripheral blood (Fig. 1c, d). There were no severe infections or GvHD post-transplant. About 6 months after the HSCT, the patient suffered from hemolytic uremic syndrome with acute renal failure, hypertension and edemas. The child developed a dilated cardiomyopathy due to hypertension. These symptoms were treated and disappeared during the following years. The cell count for B and T cells has normalized during the past couple of years (Fig. 1c, d), and the current T cell receptor repertoire, analyzed via Vα spectratype, shows a polyclonal diversity of T cell receptors reflecting a healthy immune competence (Fig. 3b). Today, the boy is 12 years old and is living a normal life. He attends a regular high school, plays sports and enjoys a good quality of life. At the 10-year follow up, he presented with extreme dwarfism and dystrophy (weight 14.1 kg (14.5 kg <3rd percentile), length 110.8 cm (27 cm <3rd percentile) head circumference 45.1 cm (<3rd percentile)) (Fig. 3a). The bone age, calculated according to Greulich and Pyle [21], was in accordance with the chronological age. His maximum final adult height was calculated to be 135 cm. The laboratory findings showed normal blood counts with hemoglobin 13.4 g/dl, thrombocytes 277,000/μl and WBC 9900/μl with 4059/μl lymphocytes (2030/μl CD3+, 755/μl CD19+, 1023/μl CD4+, 398/μl CD8+, 321/μl CD16/56+). Without any substitution the immunoglobulin levels were generally low, but for IgA and IgM within the normal range; only IgG remained below the standard value (IgG 411 mg/dl, IgA 87 mg/dl, IgM 63 mg/dl) at the 10-year follow up. Complete donor chimerism was observed. The endocrinological findings revealed a hypergonadotropic hypogonadism (luteinizing hormone 6.4 IU/l (normal range: 1–3.5 IU/l), follicle-stimulating hormone 64.4 IU/l (normal range: < 0.2–7.5 IU/l)). The echocardiographic examination showed further improvement with very mild residuals of the dilated cardiomyopathy.

Due to the rarity of DNA ligase IV deficiency and the limited number of patients undergoing HSCT, there is no general recommendation as to which conditioning protocol should be followed. Because of the severe radiosensitivity, irradiation should not be part of the conditioning regimen [11, 19]. Patients with defects in NHEJ suffer from an increased sensitivity to alkylating agents. For best survival and minimal late effects, it is suggested to minimize the exposure to alkylating agents. In accordance with our report, myeloablative conditioning does not seem to be necessary for performing HSCT successfully. On the contrary, myeloablative conditioning regimens are associated with a higher mortality and morbidity compared to reduced intensity conditioning regimens [29, 30]. In the future, transplantation of gene-corrected autologous CD34+ cells might simplify the conditioning regimen [31].

Another major difference between the two cases is the GvHD prophylaxis. In the first case, the prophylaxis was carried out with CSA (cyclosporine A, day − 1 until death) and methotrexate 10 mg/m² (day + 1, + 3, + 6). In contrast, there was no GvHD prophylaxis apart from CD3/CD19 depletion and anti-thymocyte globulin in the second case. In an experimental in vitro study, it was shown that CSA can induce double-strand breaks in cells with DNA ligase IV deficiency. Furthermore, double-strand breaks particularly occurred in busulfan and fludarabine pre-treated DNA LIG4 deficient cells [32]. Fludarabine was part of the chemotherapy in the first case. Moreover the dosage of CSA (3 mg/kg) was rather low in successfully transplanted cases [10, 19]. The role of CSA in HSCT in patients with DNA LIG4 deficiency has still to be determined in further studies, but it might have a negative impact on the outcome in this selected group of patients inducing double-strand breaks.

In the 10-year follow up, the younger brother was in good shape, except for the ligase IV deficiency-related dwarfism and dystrophy. A fully recovered immune system, was confirmed by Vα spectratype (Fig. 3b). Hypergonadotropic hypogonadism was an unexpected finding. To the authors’ knowledge, there is no proven association between ligase VI deficiency and hypergonadotropic hypogonadism. But there are four descriptions of hypogonadism in LIG4 deficiency patients [3, 9, 11, 19]. Further investigation is needed to clarify if there are some endocrinological disorders associated with LIG4 deficiency. Thus, the cause of this endocrinological problem in the boy remains unknown. The laboratory findings indicate a primary testicular damage. One might speculate that it could also be related to HSCT’s toxicity, or to the late orchidopexy, which was performed at the age of 4 years.

The data presented in this case report does not allow any generalization concerning treatment recommendations or cause-effect relationships as this is a retrospective description of two patients with a rare genetic disease. The strength of this case can be found in the fact that two brothers sharing the same genetic background and who are suffering from an identical mutation are treated with different HSCT regimen resulting in vastly different outcomes with one brother dying and the other one - receiving a milder regimen - fully recovering. Therefore further studies to carefully evaluate the efficacy of HSCT as a treatment option, as well as the conditioning regimen and GvHD prophylaxes in patients with DNA ligase IV deficiency are required.