Patients
Patient 1 (P1) was born to consanguineous parents. He had a sister who died of a seizure of unknown cause at the age of 3 years. His medical history showed treatment-resistant oral candidiasis, recurrent upper respiratory tract infections, lymphopenia, neutropenia, thrombocytopenia, and autoimmune hemolytic anemia (AIHA), which was treated with intravenous immunoglobulin (IVIG) and systemic steroids. After his first year of life, he required multiple hospitalizations for sepsis and upper respiratory tract infections. The thorax computer tomography (CT) showed a mosaic pattern and ground-glass infiltrates. Bone marrow biopsy revealed erythroid hyperplasia, normoblastic maturation, and a relative decrease in myeloid series. At 18 months of age, he underwent immunologic evaluation, which revealed failure to thrive and global developmental delays. Computed tomography of the skull (CT) showed only a cavum septum pellucidum and a vergae variant. On examination, growth parameters were below the third percentile. He had hepatosplenomegaly and lymphadenopathy. A neurological examination revealed global developmental delay and mild truncal hypotonia. On immunologic examination, immunoglobulin levels were normal except for a low IgA level (but the patient had been treated with IVIG several times); his lymphocyte subset analyses revealed a T-B-NK + phenotype, and uric acid levels were very low (1.3 mg/dl), suggesting PNP deficiency (Table 1). Autoantibodies, including ANA, anti- GAD, and direct Coombs were positive. Homozygous mutation c.349G > A (p.Ala117Thr) was detected by genetic sequencing of the PNP gene. While awaiting a suitable HLA donor, the patient received prophylactic trimethoprim-sulfamethoxazole (TMP-SMZ), fluconazole, acyclovir, isoniazid (due to BCG vaccination before diagnosis), and immunoglobulin replacement therapy. During the follow-up period, the patient developed vision loss in both eyes. CMV retinitis was diagnosed, and the left eye was treated with intravitreal ganciclovir injection; unfortunately, treatment could not be given because of complete vision loss in the right eye. CMV viremia also developed, which was treated with systemic ganciclovir. Liver biopsy was performed to explain the elevated liver function test, and the result was normal. At the time of HSCT, he was 38 months old and could not yet speak or walk but could sit without support. The patient underwent HSCT from a matched family donor (healthy sister). His conditioning regimen consisted of treosulfan, fludarabine. Mesenchymal stem cells were given at day − 1 to provide engraftment. The patient was transplanted with an unmanipulated bone marrow graft. For prophylaxis of graft-versus-host disease (GVHD), he received cyclosporine A and mycophenolate mofetil (MMF). Neutrophil engrafment was achieved at day + 15 and thrombocyte engraftment was achieved at day + 34. At the first, sixth, and twelfth months after HSCT, he had complete donor chimerism; he was also free of GVHD. After HSCT, cranial examination CT showed cerebrocerebellar atrophy and minimal ventriculomegaly. Currently, he is 6 years and 2 months old and has low IgM and normal IgG levels without infections; leukopenia and anemia have also improved (Table 1). Neurological assessment with DDST is delayed, but improvement is noted (Table 2).
Table 1
Pre-transplantation and Post-transplantation Immune Work-up
|
P1
|
P2
|
P3
|
P4
|
P5
|
P6
|
|
Pre
HSCT
|
Post
HSCT
|
Pre
HSCT
|
Post
HSCT
|
Pre
HSCT
|
Pre
HSCT
|
Post
HSCT
|
Pre
HSCT
|
Post
HSCT
|
Pre
HSCT
|
Age(months)
|
22
|
71
|
14
|
69
|
43
|
6
|
35
|
6
|
40
|
12
|
Neutrophils/mm3
|
500
|
2620
|
2390
|
2210
|
2900
|
2560
|
2500
|
1910
|
2700
|
1500
|
Lymphocytes/mm3
|
600
|
3330
|
330
|
5090
|
1100
|
1400
|
4470
|
600
|
2800
|
500
|
% CD3+
|
N/A
|
83
|
23,7
|
75,12
|
23
|
41,9
|
83.04
|
54
|
70
|
45,8
|
% CD3 + CD4+
|
N/A
|
40,1
|
7,5
|
44,21
|
58
|
11,6
|
34.83
|
78
|
36
|
34,3
|
% CD3 + CD8+
|
N/A
|
42,8
|
16
|
49,14
|
23
|
21,8
|
58.55
|
10
|
21
|
10,4
|
% CD19+
|
N/A
|
11,4
|
6,2
|
12,66
|
47
|
30,5
|
14.63
|
6
|
12,2
|
16,8
|
% CD3-CD16 + 56+
|
N/A
|
N/A
|
70,1
|
32,62
|
21
|
N/A
|
4.68
|
N/A
|
16
|
36,2
|
Recent Thymic Emigrant (%)
|
N/A
|
N/A
|
N/A
|
59,3
|
46
|
N/A
|
64.15
|
53
|
58,2
|
1
|
IgM (mg/dl)
|
101
|
47
|
105
|
98
|
N/A
|
48
|
< 0,01
|
34
|
53
|
48,1
|
IgA (mg/dl)
|
44,4
|
55
|
313
|
124
|
155,4
|
17
|
9
|
14
|
116
|
15,4
|
IgG (mg/dl)
|
492
|
748
|
1869
|
766
|
N/A
|
438
|
742
|
340
|
985
|
346
|
Total IgE (kU/L)
|
2.67
|
< 0,2
|
1,78
|
N/A
|
< 16,5
|
375,6
|
2,35
|
6
|
379
|
6,09
|
Uric Acid (mg/dL)
|
1.3
|
3,6
|
2,4
|
5,1
|
1,7
|
1,7
|
3,09
|
0.1
|
4
|
0,1
|
PNP analyze
|
N/A
|
N/A
|
N/A
|
14,8
|
|
58,5
|
N/A
|
6,9
|
Autoantibody Status
|
ANA(+)
D. Coombs(+)
AntiGAD (+)
|
Anti-dsDNA(-)
Anti TTG(-)
D.Coombs(-)
|
N/A
|
D.Coombs(-)
|
???
|
|
D.Coombs(-)
|
Table 2
Pretransplantation and Posttransplantation Neurological Development
|
Neurological Development
|
|
Pretransplantation
|
Posttransplantation
|
P1
|
18 months:
• Global developmental delay
• mild truncal hypotonia
38 months:
• unable to speak
• unable to walk
• sit without support
|
74 months:
• speak with articulation problems
• walk and run with mild ataxia.
DDST:
• Social and Emotional: 36 months
• Language/Communication:51 months
• Gross motor skills: 33 months
• Minor motor skills: 51 months
|
P2
|
12 months:
• gross motor delay (only hold head)
• gross delay in communication and language (no speech)
|
73 months:
• walk with mild ataxia from 30 months
• speech is appropriate 45 months of age.
DDST:
• Social and Emotional: 30 months
• Language/Communication: 45 months
• Gross motor skills: 18 months
• Minor motor skills: 51 months
|
P4
|
6 months:
• mild hypotonia
• hold head
• sit with support
|
37 months
• Walk without support
• Speak several single words
DDST
• Social and Emotional: 24 months
• Language/Communication: 24 months
• Gross motor skills: 18 months
• Minor motor skills: 22 months
|
P5
|
3 months:
• hold head steady, unsupported
6 months:
• roll over from tummy to back
|
60 months
• Walk without support
• Speak several single words
DDST
• Social and Emotional: 36 months
• Language/Communication: 15 months
• Gross motor skills: 27 months
• Minor motor skills: 16 months
|
Patient 2 (P2) was born to consanguineous parents The family history was significant for another sibling who had died of sepsis at 18 months of age. He presented at 12 months of age with severe gastroenteritis requiring hospitalization and was referred to immunology for treatment of resistant diarrhea. Neurologically, he had significant motor delay and delayed speech development. Immunologic examination revealed normal immunoglobulin levels, positive vaccine-specific antibodies, and T- and B-cell lymphopenia (T- B- NK + phenotype). Uric acid levels were low (2.5 mg/dl). Autoantibodies included antibodies to gliadin and tissue transglutaminase, and the direct Coombs test was negative. Clinical exome sequencing (CES) revealed a homozygous mutation of the PNP gene (deletion of exon 3–5). Before HSCT, he received prophylaxis with TMP-SMZ, fluconazole, isoniazid, and immunoglobulin replacement therapy. CMV retinitis developed and was treated with systemic ganciclovir. The patient underwent HSCT from a HLA full matched sibling donor at 18 months of age using treosulfan, fludarabine. Mesenchymal stem cells were given at day − 1 to provide engraftment. Cyclosporine A and MMF were used for GVHD prophylaxis. Neutrophil engrafment was achieved at day + 13 and thrombocyte engraftment was achieved at day + 27. Immediately after transplantation, the patient suffered from ileus, which was successfully treated. The patient's chimerism on days 40, 42, 61, and 65 showed mixed chimerism (72% at day 40 and 53% at day 65). On day 79, the patient received an infusion of donor lymphocytes. On day 99, he had complete donor chimerism. After HSCT, he underwent bone osteotomy and fixation for a slipped femoral epiphysis. Today, 6 years and 1 month after transplantation, he is doing well, has complete donor chimerism, is free of infection, and has normal immunoglobulin levels (Table 1). In terms of development, he has not been able to catch up with his delay but began to walk unassisted at 30 months of age, although he has mild ataxia. His speech is at the developmental level of 3 years and 9 months. However, his development improves after HSCT (Table 2).
Patient 3 (P3) was born of consanguineous parents. The patient presented with recurrent respiratory infections including tuberculosis and severe developmental delays in all areas at the age of 4 years. Neurological examination revealed severe mental retardation, ataxia, dysarthria, dystonia, and gross motor delay. Head holding, sitting without support, and walking without support were delayed at 6, 12, and 24 months, respectively. Cranial examination CT was normal. Thoracic examination CT revealed bilateral acino-nodular infiltration and lymphadenopathy. On immunologic examination, lymphocyte subset analysis showed T- and B-cell lymphopenia. Genetic analysis revealed a homozygous missense mutation of the PNP gene. The patient underwent bone marrow transplantation with a full matched unrelated donor at the age of 60 months but unfortunately died on day 14 due to respiratory infection.
Patient 4 (P4) was born to consanguineous parents with meconium aspiration syndrome that required hospitalization for the first 7 days. The patient's brother (P3) had died after a diagnosis of PNP deficiency. At 6 months of age, he presented with upper extremity cramps, wheezing, eczema, food allergy, and vomiting. Physical examination was normal except for mild hypotension. Immunologic examination revealed normal IgG, IgM, and IgA levels, high IgE, T-cell, and B-cell lymphopenia, and a low uric acid level (1.7 mg/dl). Skin prick tests with cow's milk, egg white, and egg yolk were also positive (Table 1). PNP enzyme assay revealed low PNP levels (14.8 nmol/h/mg). Genetic testing revealed a homozygous missense mutation of the PNP gene (p.R67P(c.200G > C)). Prophylaxis was started with TMP-SMZ, fluconazole, ganciclovir, and immunoglobulin replacement therapy. The patient underwent bone marrow transplantation at 16 months of age with a fully matched male unrelated donor. He received a conditioning regimen of treosulfan and fludarabine. GVHD prophylaxis consisted of cyclosporine A and MMF. Neutrophil engrafment was achieved at day + 13 and thrombocyte engraftment was achieved at day + 21. Chimerism analysis revealed 100% donor chimerism in all cells (at days 28 and 99). 5 months after HSCT, the patient developed cervical and mesenteric multiple lymphadenopathies and EBV viremia (copy number: 68521). A lymph node biopsy was consistent with posttransplant lymphoproliferative disease. The patient was treated successfully with acyclovir and rituximab (3 doses every week). During follow-up, chimerism had decreased to 32% 12 months after HSCT. The PNP enzyme test was re-evaluated, and PNP activity was less than 10% of the mean of controls, although the patient was infection-free. In addition, immunologic examination revealed a normal IgG level, low IgA and IgM levels, and a normal lymphocyte count despite an impaired CD4/CD8 ratio (Table 1). His neurologic development showed no further deterioration. At 37 months of age, his gross and fine motor skills were equivalent to what would be expected at 18 and 22 months, respectively. His cognitive, social-emotional, and language abilities were similar to those of a 2-year-old child. The assessment concluded that he had clearly reached new developmental milestones.
Patient 5 (P5) was born to non-consanguineous parents with a normal perinatal period. At 4 months of age, she presented with failure to thrive, mild neutropenia, and severe lymphopenia without infection. Neuromotor development was largely normal (holding her head without support at 3 months and rolling from her abdomen to her back). Because leukopenia persisted, a bone marrow aspiration was performed, which was normal. At 9 months of age, a very low uric acid level was noted (0.1 mg/dl), and a PNP enzyme test was performed; enzyme levels were low and dAXP was not detected. Genetic analysis revealed that the patient was compound heterozygous for a novel mutation and a described mutation in the PNP gene (c.389T > G (p.Met130Arg) and c.265G > A (p.Glu89Lys)). While she was waiting for a suitable HLA donor, DDST results at 8 months of age were adequate for her age, except for gross motor skills, which were at 6 months. At 12 months of age, she received a bone marrow transplant with 5/10 HLA loci from an haploidentical related donor. GVHD prophylaxis consisted of cyclosporine A. At day 28, chimerism was 100%. Within a few months, low donor chimerism (63%) and low PNP activity were detected. At month 6 after transplantation, the patient received donor lymphocyte infusion (DLI) three times due to low chimerism and PNP activity. Following DLI, diarrhea and elevated liver enzymes were evaluated as GVHD and treated with steroids. at 18 months after HSCT, chimerism was 96%. Today, she is 5 years old and has a normal immunologic workup, including immunoglobulin and lymphocyte counts, subgroup analyses, and proliferation (Table 1). Neurologic assessment with DDST is delayed, and her gross and fine motor skills are what would be expected at 27 and 16 months, respectively. Their cognitive, social, and emotional abilities are equivalent to those of a 3-year-old child. Language skills are appropriately identified at 15 months. The assessment concluded that she has clearly reached new milestones in her development.
Patient 6 (P6) was born to consanguineous parents. She was healthy until she was 12 months old, when she was referred to a pediatric unit for cough and severe lymphopenia and was found to have decreased IgG levels. Neurologically, she had significant motor delay but normal language development: at 10 months, she was sitting with support, and at 1 year, she was speaking two words. The immunological examination revealed T-B-NK + lymphopenia, low IgG, and low uric acid levels (0.1 mg/dl). PNP enzyme assay was evaluated, and PNP enzyme levels were low, ADA activity was normal, and dAXP was undetectable (Table 1). Genetic analysis revealed that the patient was homozygous for a novel mutation in the PNP gene ((c.59_60delACinsCT (p.His20Pro). The disruption of this protein causing PNP deficiency has been reported previously.11 Unfortunately, there was no suitable HLA donor. While waiting for HSCT, the patient received prophylactic TMP-SMZ, fluconazole, ganciclovir, and immunoglobulin replacement therapy. She is currently 21 months old and waiting for a matching HLA donor. On neurological examination, she speaks two-word sentences, and her motor skills are developing in accordance with her age, although she does not walk without assistance.