Neurologic Status of Patients with Purine Nucleoside Phosphorylase Deficiency Before and After Hematopoetic Stem Cell Transplantation

Purine nucleoside phosphorylase (PNP) deficiency is a rare autosomal recessive combined immunodeficiency. The phenotype is profound T cell deficiency with variable B and NK cell functions and results in recurrent and persistent infections that typically begin in the first year of life. Neurologic findings occur in approximately two-thirds of patients. The mechanism of neurologic abnormalities is unclear. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for PNP deficiency. We report here six patients from five unrelated families with PNP deficiency treated in two centers in Turkey. We evaluated the neurological status of patients and compared to post-transplantation period if available. Then, we performed PubMed, Google Scholar, and Researchgate searches using the terms “PNP” and “hematopoietic stem cell transplantation” to find all reported cases of PNP transplantation and compared to our cohort. Six patients were treated in two centers in Turkey. One patient died from post-transplant complications. The other four patients underwent successful HSCT with good immune reconstitution after transplantation (follow-up 21–48 months) and good neurological outcomes. The other patient with a new mutation is still waiting for a matching HLA donor. In PNP deficiency, clinical manifestations are variable, and this disease should be considered in the presence of many different clinical findings. Despite the comorbidities that occurred before transplantation, HSCT currently appears to be the only treatment option for this disease. HSCT not only cures immunologic disorders, but probably also improves or at least stabilizes the neurologic status of patients.


Introduction
Purine nucleoside phosphorylase (PNP) is an enzyme of the purine salvage pathway.In purine metabolism, PNP catalyzes the phosphorylation of inosine, guanosine, and deoxy nucleosides to hypoxanthine and guanine, respectively.The metabolites are detoxified or converted back to the nucleotide.In different tissues, the PNP enzyme is expressed to varying degrees; the highest expression is found in lymphoid tissue.The accumulation of toxic metabolites (especially deoxyguanosine to deoxyguanosine triphosphate (dGTP)) is toxic to T cells in lymphoid tissue.Accumulation of dGTP leads to inhibition of ribonucleotide reductase and blocks DNA synthesis, proliferation, and repair mechanisms [1,2].In addition, double-positive thymocytes are more susceptible to activation-induced apoptosis because these accumulations disrupt mitochondrial membrane potential [3].
PNP deficiency is a rare autosomal recessive combined immunodeficiency.The phenotype is profound T cell deficiency with variable B and NK cell functions and results in recurrent and persistent infections that typically begin in the first year of life.Patients account for approximately 4% of all patients with severe combined immunodeficiency (SCID) [4].Patients present with autoimmune manifestations, neutropenia, neurologic abnormalities, or infections resembling those of SCID or combined immunodeficiency [5][6][7][8][9][10][11][12][13].Neurologic manifestations may precede infectious and autoimmune complications [14].Approximately twothirds of patients present with neurologic findings; in addition, one-third of patients have autoimmune manifestations.
The mechanism of neurologic abnormalities such as ataxia, hyper-/hypotonia, tremors, spasticity, and varying degrees of developmental delay is unclear.Although toxic purines impair neurological functions, neurotoxicity cannot explain every abnormality.GTP is essential for neurotransmission in the central nervous system; low GTP levels may be the main reason for the neurologic findings [14,15].
Low plasma uric acid level has been suggested as a marker for diagnosis, but Walker et al. found normal uric acid levels in five patients.Therefore, this marker is deceptive [19].Diagnosis can be made by measuring PNP enzyme activity or by genetic analysis of the PNP gene.
We report here six patients from five unrelated families with PNP deficiency treated in two centers in Turkey.

Patient Data
We reviewed the medical records of five patients with PNP deficiency and prospectively screened them using the Denver Developmental Screening Test (DDST), which suggests age-appropriate milestones.Patient data included medical history, transplant history, posttransplant clinical course, and immunologic parameters collected at baseline until the patients' last review.

Literature Review
We performed PubMed, Google Scholar, and Researchgate searches using the terms "PNP" and "hematopoietic stem cell transplantation" to find all reported cases of PNP transplantation.

Patients
We performed clinical, laboratory, and genetic evaluation in a cohort of 6 patients (patients 1-6) with PNP deficiency (Tables 1 and 2) (see Case Descriptions section in the supplemental material).Three patients (P1, P2, P3) had severe viral or bacterial infections (e.g., recurrent LRTI, CMV viremia, tuberculosis), two patients (P4 and P6) suffered from cough and mild wheezing, and one patient (P5) had failure to thrive.P1, P2, and P3 had severe neurologic abnormalities and developmental delays.Although P4 and P3 were siblings and had the same mutations, P4 had a milder phenotype.It is likely that the early diagnosis and early HSCT were an advantage for him.P5 and P6 also had a mild phenotype.
The extended laboratory evaluation of the immune system in all patients is summarized in Table 1.In this cohort, all patients had lymphopenia, and two of them (P1 and P5) also had neutropenia.Immunophenotype analysis revealed a T-B-NK + phenotype.Low uric acid levels were found in all patients.PNP enzyme levels were low in all 3 patients in whom PNP enzyme levels could be measured.
In all but 1 of the 6 patients, mutations had been described in previous reports.Genetic analysis of P6 revealed a novel mutation.P5 was also compound heterozygous, one of which is a novel mutation (Table 3).
Five patients underwent HSCT, and one (P3) died after 14 days.One (P6) is awaiting HSCT because there is no suitable HLA donor.Survival rate was 80% in our cohort (4/5), P3 died on day 14 after HSCT due to respiratory infection.P5 was found to have a steroid-responsive acute GIS GVHD.Post-transplant neurological status was assessed by DDST, and the assessment revealed that all surviving patients in this cohort had clearly reached new developmental milestones.Comparing P3 and P4, who were from the same family and had the same mutations, we found that P3 had worse neurodevelopmental features at the same age than his sibling (P4) who received HSCT.

Literature Review
In the literature review, the 20 case reports with HSCT for PNP deficiency were described in Table 3.A total of 3/20 patients had neutropenia, malignancy and autoimmunity, especially autoimmune cytopenia.The median age at transplantation was 40 months (range 6.5-168 months).Survival was 85%, with two patients dying immediately after transplantation from infectious complications and the other 14 days after HSCT from veno-occlusive disease.All surviving patients are doing well; median follow-up after transplantation is 25 months (8-108 months).Two patients still required IVIG treatment [4,7].Most patients have chimerism of 90% or greater, and one patient with mixed chimerism was 60% clinically and immunologically adequate [10].Neurologic sequelae are relatively mild; two patients were reported to have no improvement in development [16,22], whereas in the others, it was reported that "assessment at this age concluded that the patient had reached new developmental milestones".Therefore, neurologic improvement in development was noted in most patients after transplantation.

Discussion
We described six patients with PNP deficiency from four different families in Turkey.The mutations found in our five patients were described in previous reports.P5 was compound heterozygous; one is novel mutation (c.389 T > G (p.Met130Arg).P6 was homozygous for a novel mutation in the PNP gene (c.59_60delACinsCT(p.His20Pro)), and her phenotype was mild; without severe infections and neurological development was almost normal (Fig. 1).Despite    their identical genotype, P3 and P4 had different presentations; P3 presented later and was diagnosed at 5 years of age.At the time of diagnosis, he showed recurrent respiratory infections, including M. tuberculosis, gross motor developmental delay, and was dystonic.Unfortunately, he died on day 14 after transplantation due to respiratory infection.P4 presented earlier and was diagnosed after his brother's diagnosis.Probably, the early diagnosis was an advantage for him.He underwent HSCT after 12 months.Despite the decline in donor chimerism, the patient is infection-free and has developed new developmental skills.The two cases presented here may provide evidence that neurologic involvement may increase over the years and that early HSCT may prevent this.Although there are no systematic studies of genotype/phenotype correlation in PNP deficiency, we hypothesize that the clinical spectrum of PNP deficiency is variable and not related to genotype.Clinical variability ranges from SCID phenotype to the presence of neurologic symptoms only.Typically, patients with PNP deficiency have severe T cell defects causing bacterial, viral, and fungal infections, but also variable B and NK defects with/without decreased serum immunoglobulin levels [17].In our cohort, three patients (P4, P5, and P6) were diagnosed in the first year of life and were infection-free or had mild infections.The others had severe bacterial, viral, and fungal infections, such as CMV viremia, recurrent respiratory infections, and oral candidiasis in later life.However, immunological examination revealed T and B cell deficiency with variable serum immunoglobulin levels in all our patients.Although immunoglobulin levels could be normal, in three patients both B and T cells were affected by similar dysfunction as in previous studies [14].Purine metabolism is essential for all cells, and the cellular function can be ensured by a proper functioning purine-salvage pathway.PNP is a ubiquitous enzyme that provides purine homeostasis [1,2].In addition to immunological dysfunction, the occurrence of diseases of other tissues in PNP deficiency is not surprising.Besides recurrent infections autoimmune manifestations or malignancy (especially EBV-related malignancy) may be seen [13][14][15][16][17][18].Autoimmune cytopenia such as AIHA and ITP are the most common autoimmune manifestations [4,13,14,16].Autoimmune mechanisms can also be a cause of neutropenia [6].In our cohort, only one patient (P1) had autoimmune manifestations (AIHA, ITP), and neutropenia was documented in P1 and P6.
Neurologic dysfunction, which occurs in two-thirds of patients (66%), may be helpful in diagnosing the disease [26].The mechanism of neurologic disease in PNP deficiency is not well understood; PNP deficiency results in an accumulation of deoxyguanosine.This accumulation is catalyzed by deoxyguanosine kinase (mitochondrial enzyme) or deoxycytidine kinase (nuclear enzyme) to dGTP.dGTP inhibits DNA synthesis, proliferation, and repair mechanisms [15].In addition, this molecule induces apoptosis and causes cellular toxicity.27Even if immunologic disorders are explained by lymphotoxicity, neurotoxicity cannot explain all neurologic abnormalities.GTP is essential for neurotransmission in the central nervous system, and low GTP levels may be the main reason for the neurologic findings [14,15].Moreover, de novo synthesis of GTP in the brain is limited, supporting this mechanism [20].In a previous study, PNP-deficient mice were found to have a small cerebellum, corpus callosum, and thalamus [28].Following this, the recent studies reported abnormal MRI results; one patient had significant neurologic impairment with atrophy of the cerebellum and cerebral cortex with loss of white matter myelination [29], and two other patients had brain atrophy [5,30].However, Torun et al. described two patients with normal MRI images [14].In all our patients, neurologic symptoms were mild or marked.In particular, late presentation (P1, P2, P3) was associated with more advanced neurological dysfunction.In addition, P1 suffered a cardiopulmonary arrest requiring resuscitation during HSCT, and a cranial CT examination revealed cerebrocerebellar atrophy and minimal ventriculomegaly.Cranial MRI examination of P4 after transplantation revealed normal findings.A low plasma uric acid level has been suggested as a marker for diagnosis, but Walker et al. found normal uric acid levels in five patients; therefore, this marker is deceptive [19,26].PNP enzyme activity or genetic analysis of the PNP gene is the gold standard diagnostic tests [4].
In PNP deficiency, the neonatal screening program fails in early diagnosis because TREC and KREC levels may be normal in infancy.Immunologic injury is caused by the gradual accumulation of toxic metabolites.La Marca et al. described a spectrometric method for early diagnosis of dried blood spots to identify PNP metabolites [31].
Almost all patients with PNP deficiency died in the second decade of life from infectious complications.Only one patient reported surviving without HSCT at the age of 21 years.The baby of the PNP carrier was born after her pregnancy under intensive observation [32].Despite the comorbidities that occurred before transplantation, HSCT currently appears to be the only treatment option for this disease.Moreover, the success of HSCT was reported to be 85% in this study.Another disease is deficiency of another enzyme in the purine rescue pathway, adenosine deaminase, and the survival rate is 80% after transplantation [33].In our cohort, P3 died after HSCT on day 14 because of respiratory infection.The other four patients survived after transplantation and were free of infection despite posttransplant complications.
Neurological improvement after transplantation is difficult to assess.Because new developmental milestones are usually achieved over time and patients are relatively young at the time of transplantation, it is difficult to determine what neurologic progress they would have made if they had not been transplanted [14,20].It is unclear whether HSCT is the cause of neurologic improvement; however, it appears that most patients do not have new neurologic impairments after transplantation.After transplantation, neurologic status may improve in some patients, whereas in others, further neurologic impairment may be arrested.Carpenter et al. and Hallet et al. reported that "no new developmental milestones were achieved" [16,22].However, our review found that 19 of the posttransplant patients who reported their neurologic status after HSCT achieved new developmental milestones with good immune reconstitution (Table 3).A previous study also reported that Purkinje cell layer can be corrected by early treatment in mouse models [28].
In the review, some researchers reported that early treatment better prevents neurological complications.Early diagnosis and treatment play a critical role in preventing neurologic damage and other complications [14,28].In our cohort, late-diagnosed patients had severe neurologic findings, whereas early-diagnosed patients had mild findings.Nevertheless, some neurologic deficits remain after both groups reached new developmental milestones after HSCT.In addition, some studies reported that patients' neurological status did not improve in the first year after transplantation, but they clearly reached new developmental milestones at repeated follow-up [13,21].Unfortunately, this study and previous studies failed to provide functional or biochemical evidence of changes in neurons and neurologic outcome after transplantation.Ideally, patients could be divided into two groups: the transplanted group and the nontransplanted group, and the neurological outcomes of the two groups could be compared.However, HSCT is essential for these patients.Nevertheless, in this cohort, two siblings of the same age had different phenotypic features depending on whether they had undergone HSCT or not, and the patient with HSCT had milder neurological abnormalities.Consequently, HSCT stabilizes the neurological status of patients.Moreover, this review shows that HSCT is more likely to improve neurological status.Further studies are needed to find functional or biochemical evidence of the neurological effects of HSCT in PNP deficiency.

Fig. 1
Fig. 1 The schematic representation of patients variants.* Shown in 2 patients.**Compound heterozygous.Novel mutations are shown with red color

Table 1
Pre-transplantation and Post-transplantation Immune Work-up

Table 3
Patients diagnosed with PNP deficiency who underwent HSCT in the literature