Patients with adenosine deaminase deficiency surviving after hematopoietic stem cell transplantation are at high risk of CNS complications

One cause of autosomal recessive severe combined immunodeficiency (SCID) is a deficiency of the enzyme adenosine deaminase (ADA).^1,2  ADA is an ubiquitously expressed enzyme involved in the degradation of adenosine and deoxyadenosine. In ADA-deficient patients these nucleosides and their phosphorylated metabolites accumulate, which, by mechanisms not yet completely clarified, induce a profound impairment in particular of the lymphoid system.^2,3  Affected infants usually present early in life with life-threatening infections and the prognosis is extremely poor unless the immunodeficiency is treated. Less severe forms of ADA deficiency have been described and are characterized by a delayed onset of symptoms and initially less profound immunologic abnormalities.² 

Beside the immunodeficiency, other clinical findings may be encountered in ADA-deficient patients, including skeletal abnormalities and rarely also neurologic complications. In an analysis of 117 SCID patients, developmental delay was reported in 6 of 16 ADA-deficient patients as a disease-specific finding.⁴  In another report, 3 of 23 ADA-deficient patients suffered from cortical blindness and pyramidal and extrapyramidal motor dysfunction.⁵  In a further report, 2 siblings with ADA deficiency were found to have sensorineural hearing deficit.⁶  Because complications from infections usually predominate in the clinical presentation of infants with ADA deficiency, the full spectrum of nonimmunologic manifestations of the disorder and their natural course may be obscured.

As in patients with nonmetabolic forms of SCID, hematopoietic stem cell transplantation (HSCT) is able to correct the immunodeficiency in ADA deficiency. After HLA-identical HSCT, excellent results have been reported with survival rates approaching 100%.^4,7,8  Exploration of HLA-mismatched HSCT in ADA deficiency has provided less favorable results compared to those obtained in other variants of SCID, mainly due to a high rate of graft failures.^4,9-11  By the use of cytoreductive conditioning, this difficulty was overcome.^8,12  Therapeutic alternatives in ADA deficiency are long-term enzyme replacement therapy with polyethylene glycol-ADA (PEG-ADA)^13,14  as well as more recently introduced successful gene therapy.^15-17 

We analyzed cumulative experience of HSCT in 15 ADA-deficient patients treated at 2 centers and report a high rate of marked neurologic abnormalities in long-term surviving patients.

Approval was obtained from the Institutional Review Board of the University of Ulm for these studies. Informed consent was obtained in accordance with the Declaration of Helsinki.

Within a group of 170 patients with SCID diagnosed at our institutions since 1982, 15 patients suffered from ADA deficiency and were treated by HSCT (13 at Children's Hospital in Ulm, 2 at Children's Hospital in Munich). These patients were of different origin (5 German, 4 Turkish, 3 Swiss, 1 Afghan, 1 Albanian, 1 Austrian). Five patients were born to consanguineous parents (Table 4). Two patients were identical twins. The diagnosis of ADA deficiency was based on enzyme activity in erythrocytes, which was undetectable in 14 and markedly reduced in one case.

In 7 cases HLA-matched family donors (MFDs) were available. These were either siblings (n = 5) or HLA-genotypically identical parents (n = 2). In 6 cases, donors were HLA-haploidentical parents (mismatched family donors, MMFDs) and in 2 cases matched unrelated donors (MUDs). Two patients had received prior enzyme replacement therapy with PEG-ADA (UPN U322 for 3 weeks, U457 for 9 months), which was discontinued several weeks before transplantation (Table 2). Patients received bone marrow except for 3 patients who received peripheral-blood stem cells (PBSCs) harvested after G-CSF treatment of donors (Table 2). Transplants from MMFD and from one MUD (UPN U442) were depleted of T cells to prevent graft versus host disease (GvHD), using soybean lectin agglutination and E-rosette formation or positive selection of CD34⁺ cells for PBSCs. Recipients of T-cell–depleted transplants received no further GvHD prophylaxis. Regimens used in the other patients are listed in Table 2. Eight patients underwent pretransplantation conditioning, which consisted of a combination of busulfan and cyclophosphamide with some variation in the dosage of busulfan (Table 2 lists details).

Prior to treatment, parents gave written informed consent for the treatment procedures and for anonymous scientific evaluation.

Lymphocyte phenotyping was performed by fluorescence-activated cell sorting (FACS) analysis using commercially available monoclonal antibodies. T-cell function was determined by standard proliferative assays as previously described.¹⁸  Chimerism of white blood cells was analyzed by HLA typing, fluorescence in situ hybridization (FISH), or short tandem repeat (STR) analysis. Mixed chimerism was analyzed in more detail. For this, T cells (CD3⁺), B cells (CD19⁺), and monocytes (CD14⁺) were isolated by FACS (FACSAria, Becton Dickinson, Heidelberg, Germany) resulting in more than 98% purity of cell populations. Erythrocyte chimerism was assessed by blood group determination. After normalization of immune functions, patients were discharged home and were usually re-evaluated once yearly at our centers.

Posttransplantation ADA activity in red blood cells was determined by a radiochemical thin-layer chromatographic method (conversion of [¹⁴C]adenosine to inosine) at Duke University Medical Center (Durham, NC) and in 3 patients at the Children's Hospital (Zurich, Switzerland). Intracellular levels of deoxyadenosine-X-phosphate (dAXP) and adenosine-X-phosphate (AXP) were determined by high-pressure liquid chromatography from samples of packed red cells collected at the most recent presentation of the patients.^19,20 

Coding sequences and the exon/intron boundaries of the ADA gene were amplified using the Taq polymerase system (Hot Start, Qiagen, Hilden, Germany) from DNA originating from 13 individuals from 13 families. Primers used for polymerase chain reaction (PCR) and sequencing are available on request.

Most patients (14 of 15) presented within the first 4 months of life with respiratory tract infections of viral or bacterial origin, with intractable diarrhea and failure to thrive (Table 1). Diagnosis in 2 infants was made immediately after birth because of positive family histories. During the first months of life, none of the patients had unusual neurologic findings. One patient developed bacterial meningitis at 6 months of age, which was treated successfully. One patient (UPN U457) had a less severe form of ADA deficiency with only minor infections during the first year of life. With the exception of this patient, all other patients had absent or extremely low numbers of T, B and natural killer (NK) cells and thus findings characteristic of severe ADA deficiency. If detectable, T cells were autologous and not of maternal origin as demonstrated by HLA-typing or XX/XY-FISH analysis. HSCT was performed within the first 6 months of life except in 3 children, of whom 2 were older than 1 year.

After HLA-identical family donor transplantation all 7 patients survive after a mean follow-up time of 9.5 years (range, 3.9-13.5 years). These patients continue to show complete immune reconstitution, with T-cell counts ranging from 1000 to 2300/μL and with regular in vitro T-cell proliferative responses. B-cell numbers are low in 5 of 7 patients; however, immunoglobulin serum levels are normal without substitution and all patients developed positive antibody responses after vaccination (Table 3). Chimerism analysis shows T cells to be exclusively of donor origin. Analysis of B cells and monocytes reveals mixed chimerism with variable proportions of donor and host cells (Table 4). In 4 of 7 cases donors and recipients had different blood groups, and in all 4 cases mixed red-cell chimerism was observed.

Six patients received transplants from MMFDs and 2 patients from MUDs. In these cases we used cytoreductive conditioning prior to HSCT with the exception of 2 cases, who initially underwent transplantation without conditioning: UPN U26 from his haploidentical father (this case has been previously reported¹⁸ ) and U457 from a MUD. Transplanted cells in both cases failed to engraft. Subsequent transplants following conditioning were successful. Causes of death in 3 patients were early infections, associated with acute GvHD in one case. A second patient (UPN U51) developed acute and chronic GvHD, which resolved completely.

The overall survival rate in the 8 patients is 63% (MMFD 4 of 6; MUD 1 of 2), and after a mean follow-up of 14.6 years (range, 4.6-22.2 years), all 5 long-term surviving patients show stable and complete immune reconstitution. Chimerism analysis reveals all blood cells including red cells to be of donor origin, except in one patient who had a small proportion of autologous red cells.

Molecular analysis of the ADA gene was performed in 13 cases and revealed 2 deletions, 1 nonsense mutation, and 8 missense mutations (Table 4). The missense mutations have all been described previously^22-24  and all except one have been tested for residual ADA activity in Escherichia coli.²²  For the homozygous missense mutation found in patient U457 with delayed onset of disease, some residual ADA activity would be predicted (class II according to Arredondo-Vega et al²² ). All other missense mutations would be classified as class I (minimal or no residual ADA activity). The genotype, therefore, is in high concordance with the clinical presentation with early onset of disease in all but one patient.

ADA activity in red cells after HSCT was highest in conditioned patients who developed complete donor red-cell chimerism (Table 4). After HSCT without conditioning, 4 of 6 patients showed detectable levels of ADA activity, which, however, were much lower as compared to conditioned patients. Red-cell dAXP levels were variable, ranging from 2.5- to 17-fold of the upper normal limit (2 nmol/mL packed RBCs; Table 4). These are markedly lower than dAXP levels in red cells of untreated ADA-deficient SCID patients which may approach 2000 nmol/mL.²⁵ 

Six of the 12 long-term survivors are in excellent health and have no clinical complications, whereas 6 patients suffer from significant neurologic and cognitive deficits, as summarized in Table 5. These patients have learning disabilities, precluding attendance of regular (pre-) school, and require continuous special support. In addition, 4 patients show persistently abnormal gait and 5 patients exhibit a sensorineural hearing deficit. Furthermore, in 4 cases hyperactivity is a prominent problem. Common to all 6 patients was a marked delay in reaching developmental milestones, which usually became obvious during the second year of life. Remarkably, in none of the patients did we observe progressive deterioration or loss of acquired neurologic abilities during later childhood and adolescence.

Most patients had cranial ultrasound studies, magnetic resonance imaging (MRI) or computed tomography (CT) scans before transplantation and at follow-up evaluations. With the exception of one patient these were normal (Table 5). In the exceptional case, who had suffered from bacterial meningitis prior to successful transplantation, nonspecific bilateral frontal and parietal calcifications were noted.

Of particular interest was our experience in the identical twins (UPN M1052 and M1053). They underwent transplantation simultaneously from a healthy HLA-identical sister without conditioning. There were no major differences in medication or infections in the pretransplantation and posttransplantation period. One twin shows a distinctly more severe learning disability and is suffering from epilepsy with only partial response to anticonvulsive therapy. Her sister is less severely affected and has never experienced seizures.

We analyzed various parameters in the 12 surviving patients to recognize possible correlations with neurologic outcome. All infants had an uneventful perinatal and neonatal history. CNS infections were neither diagnosed nor suspected, except in the one case mentioned with bacterial meningitis, from which he recovered. All survivors had severe forms of ADA deficiency with early onset of infections and complete absence of enzyme activity in red cells. No evidence was found for a correlation of neurologic outcome with parameters such as genotype of the disease, age at transplantation, use of conditioning prior to transplantation, donor type or donor-cell chimerism, posttransplantation dAXP, levels and serious complications such as mechanical ventilation for respiratory failure (Tables 1 and 6).

In ADA deficiency, transplantation of hematopoietic stem cells serves 2 purposes. One is the establishment of a functioning immune system by donor lymphocytes, similar as in other SCID variants. The other is the improvement of metabolic abnormalities by the enzymatic activity provided by donor cells. The latter mechanism may be important, in particular, for the control of nonimmunologic manifestations associated with this systemic metabolic disease.

In the study presented here we evaluated the outcome of HSCT in 15 SCID patients with ADA deficiency. Twelve patients are alive with complete reconstitution of immune functions after a mean follow-up period of 12 years (range, 4-22 years). Following HLA-identical family donor transplantation the survival rate is 100%, confirming similar good results reported previously.^4,7,8  In these patients, who underwent the procedure without conditioning, donor-cell engraftment is incomplete and restricted mainly to T cells, whereas B cells and other blood cells remain predominantly of host type. This is in contrast to patients receiving a transplant after conditioning, which always resulted in complete donor-cell chimerism. It is interesting to note that humoral immunity is normal in the former group of patients, despite mixed B-cell chimerism and lower absolute numbers of B cells compared to conditioned patients (Table 3). The contribution of autologous B cells to humoral immune reconstitution after HSCT in ADA deficiency remains an open question.

As also reported by others, transplantation of T-cell–depleted, HLA-haploidentical stem cells in ADA-deficient patients carries a substantial risk for complete graft failure. This is in contrast to the experience in nonmetabolic variants of SCID where donor T cells commonly develop in the absence of conditioning. The basis for this discrepancy remains unclear. One hypothesis is that ADA enzyme activity provided by the transplant enhances the potential of residual host lymphocytes to resist engraftment of allogeneic donor cells. This hypothesis is supported by the successful engraftment after conditioning observed in 2 of our patients who formerly had rejected their grafts. In our series 4 of 6 patients are alive with normal immunity after HLA haploidentical, parental donor transplantation. Nevertheless, the need for conditioning represents a major limitation to this approach due to the risk of developing toxic complications. An alternative treatment in ADA deficiency represents long-term enzyme replacement therapy with PEG-ADA by regular intramuscular injections.^2,26,27  This therapy has been effective in restoring cellular and humoral immunity in the majority of treated patients. Significant disadvantages are high costs and the necessity for lifelong application of this therapy. Moreover, recent studies indicate that immunologic functions may decrease after several years.^28,29  Recently, gene therapy has been introduced with success in the treatment of ADA deficiency.^15-17  Results are promising but experience is confined to a very limited number of patients and longer follow-up is required to evaluate the full potential as well as possible risks of this new treatment.

A major finding in our study is a high rate of neurologic abnormalities that we observed in 6 of 12 long-term surviving patients. These patients became symptomatic during the first 2 years of life and suffer from significant learning disabilities as well as other neurologic abnormalities including sensorineural hearing deficit, muscular hypertonia, and problems with coordination and expressive speech. Based on our experience in approximately 100 long-term surviving patients undergoing transplantation for nonmetabolic variants of SCID, we have no evidence that the transplantation procedure as applied in this disorder carries a significant risk to induce CNS complications. There are only 2 other patients, both suffering from SCID of unknown etiology, who show unexplained mental retardation after HSCT. We previously reported sensorineural hearing defects in patients with SCID and reticular dysgenesis as an isolated finding not associated with mental retardation or other neurologic findings.³⁰ 

In the present study we obtained no evidence for a correlation of neurologic abnormalities with complications before or after HSCT or with variables of the transplantation procedure such as the use of conditioning, donor type, or length of hospitalization. Our findings thus would indicate that CNS abnormalities observed in ADA-deficient patients most likely are caused by metabolic abnormalities of the underlying disorder rather than representing complications of the treatment procedure. One other previous study addressed neurologic outcome after HSCT in ADA-deficient patients.³¹  In this study, which included 16 patients as well as a control group given transplants for nonmetabolic SCID, the authors reported striking behavioral abnormalities in ADA-deficient patients. In contrast to our findings an evaluation of motor function did not reveal abnormalities. Interestingly, in ADA-deficient patients the authors observed an inverse correlation of cognitive function with toxin levels before transplantation. In our patients no correlation to genotype or posttransplantation dAXP levels could be demonstrated. Unfortunately, no samples were available for determination of dAXP levels before transplantation. However, indirect parameters such as early age at clinical presentation, pretransplantation red-cell ADA activity, and severe genotypes indicate a homogeneous composition of our group in this regard.

CNS abnormalities including motor dysfunction have been described in several ADA-deficient patients at diagnosis and before the initiation of any therapy.^5,32,33  Hirschhorn et al postulated that these abnormalities may reflect interactions of high concentrations of adenosine with known adenosine A1 receptors in nervous tissue.⁵  A metabolic basis of CNS manifestations was strongly supported by findings in 2 clinical studies, where improvements of neurologic symptoms were noticed during enzyme replacement therapy using multiple partial-exchange transfusions, which resulted in a rapid reduction in concentrations of accumulated metabolites.^5,33 

Although none of the patients in our study revealed remarkable neurologic abnormalities at diagnosis, our finding clearly indicates that there is a substantial risk of developing such abnormalities during further follow-up after HSCT, raising the important question as to why treatment by HSCT apparently fails to prevent this complication in a substantial proportion of patients. Similar to transfused red cells used in the studies mentioned, engrafted donor blood cells after transplantation are able to provide “enzyme replacement.” This was demonstrated in previous studies where marked improvement but no normalization of metabolic abnormalities was observed after HSCT.^34,35  The variable neurologic outcome after HSCT cannot be explained by differences in the degree of metabolic correction as shown in this study. This perplexing variability is also demonstrated by our experience in the identical twins, where one is distinctly more severely affected neurologically than the other, although both received transplants simultaneously from the same donor. It should be pointed out that similar neurologic problems have so far not been reported in patients receiving long-term enzyme replacement with PEG-ADA. This therapy, which has been established since 1987 and has been used in more than 150 ADA-deficient patients, was shown to induce rapid and complete normalization of metabolic abnormalities.^2,14,25  In fact, plasma ADA activity in patients on maintenance replacement therapy with PEG-ADA usually exceeds by several fold the total blood ADA activity in healthy individuals.²⁵  The question thus must be raised whether this treatment approach may be more effective compared to HSCT with regard to protection of the CNS.

In summary, we report an unexpected high rate of neurologic complications in ADA-deficient patients after HSCT. We were unable to identify transplantation-related parameters correlating with this complication. It remains an important issue whether other treatment options such as regular enzyme replacement with PEG-ADA may offer advantages to prevent neurologic damage.

Contribution: M.H., M.-H.A., A.S., and W.F. designed and performed the study and prepared the initial draft of the manuscript; M.S.-S., C.S., B.B., T.G., and H.B. assisted in patient data collection; M.S.H., M.T.R., U.P., D.L., and K.S. provided laboratory data; all authors contributed to the discussion and the preparation of the final draft.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Wilhelm Friedrich, Universitätsklinik für Kinder- und Jugendmedizin, Eythstrasse 24, 89075 Ulm, Germany; e-mail: wilhelm.friedrich@uniklinik-ulm.de.

We thank Tatjana Kersten and Gabi Keller for their technical assistance.