Hematopoietic stem cell transplantation rescues the immunologic phenotype and prevents vasculopathy in patients with adenosine deaminase 2 deficiency

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Letter to the Editor
Hematopoietic stem cell transplantation res-
cues the immunologic phenotype and prevents
vasculopathy in patients with adenosine
deaminase 2 deficiency
To the Editor:
Recently, recessively inherited loss-of-function mutations in
CECR1 (cat eye syndrome chromosome region, candidate 1),
which encodes adenosine deaminase 2 (ADA2), were identified
in patients with a complex immunologic and vascular pheno-
type.
1,2
Possible mechanisms for this disorder are proinflamma-
tory polarization and disturbed endothelial integrity.
1,2
Zhou
et al
1
reported that aggressive systemic immunosuppressive
treatment was not effective in controlling inflammation but
hypothesized that hematopoietic stem cell transplantation
(HSCT) might be curative given that bone marrow–derived mono-
cytes and macrophages are the main source of secreted ADA2.
Here we report on 2 related patients with homozygous
p.Arg169Gln missense mutations in ADA2 located within the
putative receptor-binding domain.
3
Our observations in these sib-
lings demonstrate the clinical heterogeneity associated with
ADA2 deficiency and show that HSCT can be an effective
therapy. In the index patient the clinical course was dominated
by autoimmunity and lymphoproliferation with a combined
immunodeficiency–like phenotype, which prompted HSCT
from a healthy sibling. Despite early complications, transplanta-
tion was successful both in rescuing the immunologic phenotype
and in preventing vascular disease; at 5 years after HSCT, the
patient remains off treatment.
The index patient (P1) was the second child of a father of
Moroccan descent and a white mother. He was first admitted at
age 6 months for complicated human respiratory syncytial virus
infection. At this time, hypogammaglobulinemia was noted (see
Table E1 in this article’s Online Repository at www.jacionline.
org). At age 12 months, P1 presented with fever, lymphadenitis,
generalized lymphadenopathy, and hepatosplenomegaly. Staphy-
lococcus aureus was cultured from the lymph nodes, and fever
resolved within 24 hours of starting amoxicillin–clavulanic
acid treatment. Pancytopenia, hypogammaglobulinemia, and the
absence of specific antibodies were found (see Table E1). Results
of blood PCRs for EBV, cytomegalovirus, human herpesvirus
(HHV) 6, HHV-8, and adenovirus were negative. However,
adenovirus and norovirus were detected in the stool. Computed
tomographic scans confirmed generalized lymphoproliferation
with mediastinal and intra-abdominal lymphadenopathy and
splenomegaly. Lymphoma was suspected, but the results of lymph
node biopsy and bone marrow examination were normal. Macro-
phage activation syndrome as the cause of the pancytopenia and
lymphoproliferation was excluded based on serum markers
(including soluble IL-2 receptor) and the absence of hemo-
phagocytosis on bone marrow examination. A primary immune
deficiency (PID) with predominant lymphoproliferation and
autoimmunity was suspected, and known genetic causes
were excluded. Prednisone (2 mg/kg) led to resolution of the
thrombocytopenia and splenomegaly. However, attempts to taper
led to a relapse of thrombocytopenia. Despite the addition of
mycophenolate mofetil, sirolimus, tacrolimus, cyclosporine,
and mercaptopurine, the cytopenia and lymphoproliferation
persisted.
Because of growth failure secondary to chronic corticosteroid
treatment, HSCT was considered at the age of 3 years. The
patient’s HLA-identical healthy elder brother was chosen as the
donor. After conditioning with oral busulfan and cyclophospha-
mide, 7.5 310
6
CD34
1
bone marrow–derived hematopoietic
stem cells per kilogram were infused. Anti–graft-versus-host-
disease (GvHD) prophylaxis consisted of cyclosporine, whereas
steroids were slowly tapered. Antiviral prophylaxis consisting
of acyclovir and intravenous immunoglobulin (IVIG) administra-
tion and antifungal prophylaxis with fluconazole was added.
The transplantation was complicated by late engraftment of
neutrophils (day 26 <1.5 310
9
/L) and persistent severe
thrombocytopenia (<10 310
9
/L) refractory to transfusion,
although at day 28, whole blood chimerism was greater than
95%. At day 36, magnetic resonance imaging (MRI) of the brain,
which was performed because of severe sudden-onset headache,
identified a pineal gland hemorrhage (see Fig E1,A,in
this article’s Online Repository at www.jacionline.org). The
thrombocyte level was 2 310
9
/L but increased to greater
than 50 310
9
/L at day 40 after 2 infusions of rituximab.
Veno-occlusive disease (VOD) was diagnosed according to the
Seattle criteria at day 60 and was accompanied by a relapse of
thrombocytopenia. VOD responded well to fluid restriction.
Platelet levels of greater than 100 310
9
/L were reached at
day 111. Adenovirus reactivation was found at day 40, with
accompanying intestinal GvHD grade III, which responded to
corticosteroids. Cyclosporine was stopped at day 150. IVIG was
discontinued at day 180. Immunoreconstitution at day 360 was
excellent, including normal antibody levels, normal numbers
of B- and T-lymphocytes, and normal T-cell proliferation in
response to PHA. Moreover, response to polysaccharide vaccine
was normal (data not shown).
Five years after transplantation, P1 is clinically well and off all
medication. No more lymphoproliferation has occurred, and the
most recent MRI of the brain 5 years after HSCT did not show any
signs of acute or chronic small infarcts.
Two years after transplantation of P1, his younger brother (P2)
presented at age 5 months with profound Coombs (2) anemia
(hemoglobin, 2 g/dL), which was attributed to PCR-verified
HHV-6–associated erythroblastopenia. At this time, immuno-
logic analysis of P2 was normal. Several episodes of PCR-verified
facial herpes simplex virus infection followed. At age 23 months,
P2 was admitted with abdominal pain and ileus refractory to
conservative treatment. He had generalized lymphadenopathy
and hepatosplenomegaly, as well as hypogammaglobulinemia
and intermittent lymphopenia and neutropenia (see Table E1).
Results of blood polyomavirus PCR were positive. Bone marrow
examination was normal. Partial enterectomy was performed;
biopsy showed an atypical ulcerative bowel disease devoid of
plasma cells (see Fig E1,B), as can be seen in patients with
common variable immunodeficiency.
4
No cytomegalovirus,
Ó2014 The Authors. Published by Elsevier Inc. on behalf of the American Academy of
Allergy, Asthma & Immunology. This is an open access article under the CC BY-NC-
ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
1

EBV, herpes simplex virus, HHV-6, polyomavirus, or adenovirus
could be detected in the biopsy specimen, and no signs of vascu-
litis could be observed in the entire surgical specimen.
Obstruction persisted despite aggressive systemic immunosup-
pressive treatment and was only relieved after treatment with
sirolimus. At this time, IVIG was started, and sirolimus was slowly
tapered without clinical relapse. Subsequently, P2 did not receive
any immunosuppressive treatment for a period of 13 months but
was solely treated with IVIG. At 3.5 years of age, P2 had
neurologic manifestations in the form of 2 episodes of acute loss
of balance in the absence of fever or signs of systemic
inflammation on blood analysis, Repeated MRI of the brain did
not reveal any lesions compatible with ischemic or hemorrhagic
stroke. A transient ischemic attack (TIA) was suspected, and
treatment with sirolimus was restarted.
Whole-exome sequencing was performed on the untreated
patient (P2), the parents, and the healthy sibling (for details, see
the Methods section in this article’s Online Repository at www.
jacionline.org). We hypothesized a recessive model of inheri-
tance. After filtering out common polymorphisms, we identified
a homozygous c.G506A variant in CECR1, resulting in a
p.Arg169Gln missense mutation in ADA2. Sanger sequencing
on DNA obtained from the cheek swab of the patient who under-
went transplantation confirmed that he was also homozygous for
this variant. Both parents were carriers, whereas the sibling donor
was homozygous for the wild-type form of CECR1 (see Fig E2 in
this article’s Online Repository at www.jacionline.org).
ADA2 enzyme activity in plasma (Table I) was essentially
absent in P2, the patient who did not undergo transplantation,
whereas in post-HSCT plasma from P1, ADA2 activity was
comparable with that of his healthy donor and in the range for
healthy control subjects. Both parents have intermediate plasma
ADA2 activity. Of note, neither adenosine nor deoxyadenosine
levels were increased (<0.4 mmol/L) in plasma of P2 (these levels
have not been measured in previous patients). Both P1 and P2 had
normal ADA1 activity in dried blood spots, and deoxyadenosine
nucleotides were undetectable.
Although it has been speculated that the clinical consequences
of ADA2 deficiency might be due to increased extracellular
adenosine, our findings suggest this is not the case and that ADA2
actually has a minimal role compared with ADA1 in adenosine
metabolism in vivo, which is consistent with the very different
substrate affinities of the 2 ADA enzymes (see the Methods
section in this article’s Online Repository).
Because of the observed immunodeficiency, we performed
extensive profiling of peripheral immune cells of P2 (for details,
see the Methods section in this article’s Online Repository). Of
the major mononuclear leukocyte cell types surveyed, CD4
1
T-cell numbers were increased and CD8
1
T-cell numbers were
reduced in P2 compared with those in healthy age-matched con-
trol subjects. B-cell, natural killer cell, and dendritic cell numbers
were within 1 SD of the mean of the healthy control subjects
(Fig 1,A). Within the T-lymphocyte population, we found
defective T-cell activation, with increased naive and low effector
and memory subsets (Fig 1,Band D). Within the T
H
cell
population, numbers of regulatory T cells were increased,
whereas T
H
1, T
H
2, and follicular helper CD4
1
T-cell numbers
were low (Fig 1,C). T-cell proliferation in response to
Candida species, tetanus, and PHA was normal (data not shown).
Within the B-lymphocyte population, naive B-cell numbers were
increased at the expense of memory and plasmablasts (Fig 1,E),
which is suggestive of a defect in B-lymphocyte differentiation or
T-cell provision of help. Limited immunoprofiling performed
before HSCT showed similar findings in P1 (see Table E2 in
this article’s Online Repository at www.jacionline.org).
Because ofthe presence of severe inflammation inP1, serum IL-6
levels were measured from initial evaluation to last follow-up
(Fig 1,F). IL-6 levels were persistently high before HSCT and
before engraftment, but after HSCT IL-6 levels slowly decreased
and were undetectable at 3 years post-HSCT. In P2 serum IL-6
levels were extremely increased, despite the absence of clinical
signs of inflammation, with levels peaking at the time of bowel
obstruction and at the time of the suspected TIAs. IL-6 was unde-
tectable inthe healthy sibling and in healthy control subjects. More-
over, TNF-awas not detectable in the serum of P1 andP2 at the time
of the highest IL-6 levels. The immune profile of the other family
members wasnormal (data not shown). Together, these data demon-
strate a profound defect in T cell–dependent antibody-mediated
responses and a failure to regulate normal inflammatory cytokine
production in ADA2-deficient patients, adding to the previously
identified function of ADA2 in in vitro stimulation of T
H
cells.
5
PIDs with autoimmunity and lymphoproliferation dominated
the clinical image in our patients. The index patient P1 presented
with persistent autoimmune pancytopenia and lymphoprolifera-
tion, whereas P2 had an episode of lymphoproliferation, bowel
involvement, and 2 possible TIAs. Both patients only had fever
during infectious episodes, and unlike previously reported
patients, neither showed skin involvement or clear signs of
vasculitis. P1 had a stroke as an apparent early complication of
HSCT in the context of prolonged and severe thrombocytopenia.
Only 3 years after initial presentation, P2 presented with 2
potential TIAs, although transient labyrinthitis caused by a viral
infection could not be excluded. Therefore in retrospect
vasculitis and inflammation might have been present at
a subclinical level in both patients, but vasculopathy and
inflammation did not dominate the clinical presentation, as is
the case in the patients reported by Zhou et al
1
and Elkan et al.
2
Interestingly, serum IL-6 levels were increased in both patients
in the absence of clinical and (routine) biochemical signs of
inflammation. This suggests that ADA2 deficiency might lead
to a subclinical state of inflammation. This phenotypic
discrepancy cannot be explained entirely by CECR1 genotype
because the p.Arg169Gln mutation was previously observed in
hemizygous and homozygous form.
1,2
The ADA2-deficient
patients previously described had decreased serum immuno-
globulin levels and enhanced B-cell apoptosis in vitro.
1
By
TABLE I. Plasma ADA2 activity in the affected pedigree
Sample Age (y)
Plasma ADA2 activity
(mU/mL)
Patient 1 after HSCT 8 22.07
Patient 2 3 0.11
Healthy sibling (5HSCT donor) 10 19.14
Father 43 7.20
Mother 40 2.91
Reference values for plasma ADA2 activity (mU/mL), mean 6SD
(min-max)
ADA2 deficient (n 54) 1.0 60.4 (0.6-1.4)
ADA2 carriers (n 54) 4.9 60.3 (4.6-5.3)
Control subjects (n 551pooled human plasma) 14.0 66.1 (4.8-21.3)
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2LETTER TO THE EDITOR

FIG 1. Serum IL-6 levels and immunoprofiling in ADA2-deficient patients. A, Major blood leukocyte subsets.
B, CD4
1
T-lymphocyte subsets. C, T
H
cell lineages. D, CD8
1
T-lymphocyte subsets. E, B-cell subsets. P2’s
values are shown as filled circles, and values of healthy age-matched control subjects are indicated by
open circles. Means and SDs (error bars) shown exclude values for the patient. F, IL-6 levels in sera of P1
and P2. The vertical line indicates the moment of HSCT followed by pineal stroke in P1. The gray shading
indicates the periods in which P2 was treated with sirolimus. DC, Dendritic cell; mDC, myeloid dendritic
cell; NK, natural killer cell; NKT, natural killer T cell; pDC, plasmacytoid dendritic cell; RTE, recent thymic
emigrant; TCM, central memory T cell; TEM, effector memory T cell; TEMRA, CD45RA-expressing effector
memory T cell; Tfh, follicular T cell; Th17, IL-17–expressing helper T cell; Treg, regulatory T cell.
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LETTER TO THE EDITOR 3

contrast, our patients had abnormalities suggesting an in vivo
defect in T-cell activation and proliferation, corresponding to
their increased susceptibility to viral infections and combined
immunodeficiency–like phenotype. Taken together, these
observations suggest that ADA2 deficiency has a more varied
clinical phenotype than initially reported and that the diagnosis
should be considered in cases of undiagnosed PID characterized
by lymphoproliferation and autoimmunity, even in the absence
of overt vasculopathy or inflammation.
As reported by Zhou et al,
1
we found that treatment with a
variety of immunosuppressive medications resulted in poor
disease control in P1. However, both at the time of bowel
obstruction and at the time of potential TIA, P2 seemed to
respond well to sirolimus treatment. Sirolimus reduces M1
macrophage differentiation and IL-6 production.
6
Because
ADA2 deficiency drives macrophages toward a more
proinflammatory M1 profile,
1
we present sirolimus as a
potential therapeutic option to at least temporarily control
inflammatory complications in ADA2-deficient patients. TNF-a
was undetectable in the serum of our patients. However, this
finding does not at all exclude a role for this cytokine in disease
pathogenesis. Indeed, etanercept led to a significant response in
all patients reported by Elkan et al
2
and should therefore be
considered as a potential treatment.
In the index patient P1 we successfully performed an
allogeneic HSCT. At 5 years after HSCT, consecutive clinical
and biochemical investigations in P1 have shown no signs of
immunologic disorder and no additional strokes. This result
supports the potential of HSCT as a long-term treatment strategy
for ADA2 deficiency. However, caution is warranted because the
HSCT procedure in P1 was characterized by severe early
complications. Indeed, ADA2-deficient patients might present
as high-risk candidates for HSCT. First, the inflammatory
response associated with conditioning is superimposed on the
inflammatory state intrinsic to ADA2 deficiency, which might
negatively affect engraftment. Second, the compromised
endothelial integrity observed in patients with ADA2 deficiency
could predispose to development of VOD, a potentially fatal
complication of HSCT. This combination of inflammation
and endothelial injury might further increase the risk of
stroke in the pre-engraftment and early postengraftment phases,
7
as observed in P1. It is reasonable to hypothesize that
ADA2-deficient patients might benefit from VOD prophylaxis
with defibrotide, as well as from pretreatment with anti–IL-6
mAbs, rituximab, or both. Moreover, treatment with etanercept
peri-HSCT could be considered in the context of ADA2
deficiency, especially given its usefulness in preventing and
treating acute GvHD. However, given the underlying
immunodeficiency, the risk of infection needs to be carefully
balanced when using anti–IL-6 and anti–TNF-amAbs.
Allogeneic HSCT restored normal plasma ADA2 activity in P1,
which is consistent with bone marrow–derived monocytes and
macrophages being the main sources of secreted ADA2. Whether
ADA2 plays a role in other tissues and the effect of this on
long-term prognosis remains unclear. A recent report on HSCT in a
patient with ADA2 deficiency with a 9-year follow-up is promising
and supports our findings.
8
However, it is plausible that the
benefit from HSCT to our patient is entirely due to restoration
of normal plasma ADA2 levels. If true, future treatment with
exogenous ADA2 might provide an alternative therapy
for ADA2 deficiency in patients in whom allogeneic HSCT is
contraindicated.
Lien Van Eyck, Jr, MD
a
Michael S. Hershfield, MD
b
Diana Pombal, MSc
a
Susan J. Kelly, PhD
b
Nancy J. Ganson, PhD
b
Leen Moens, PhD
c
Glynis Frans, MPharm
c
Heidi Schaballie, MD
d
Gert De Hertogh, MD, PhD
e
James Dooley, MSc
a
Xavier Bossuyt, MD, PhD
c
Carine Wouters, MD, PhD
d
Adrian Liston, PhD
a
*
Isabelle Meyts, MD, PhD
d
*
From
a
the Department of Immunology and Microbiology, Autoimmune Genetics Labo-
ratory, VIB and University of Leuven, Leuven, Belgium;
b
Duke University Medical
Center, Durham, NC;
c
the Department of Immunology and Microbiology, Experi-
mental Laboratory Immunology, University of Leuven, Leuven, Belgium;
d
the
Department of Immunology and Microbiology, Childhood Immunology, Department
of Pediatrics, University Hospitals Leuven and University of Leuven, Leuven,
Belgium; and
e
the Department of Pathology, University of Leuven, Leuven, Belgium.
E-mail: Isabelle.Meyts@uzleuven.be.
*These authors equally contributed to this work as senior authors.
Supported by the Research Foundation Flanders (FWO), the VIB, and the European
Research Council grant IMMUNO. I.M. is supported by a KOF mandate of the KU
Leuven, Belgium, and by the Jeffrey Modell Foundation.
Disclosure of potential conflict of interest: L. Van Eyck has received research support
from Research Foundation Flanders (FWO) and is employed by Un iversity Hospital
Leuven. M. S. Hershfield has consultant arrangements with and has received research
support from Sigma-Tau Pharmaceuticals and has a patent with and receives royalties
from Creata Pharmaceuticals. D. Pombal is employed by VIB. G. Frans has received a
GOA grant from the Catholic University of Leuven, Belgium. H. Schaballie has
received research support from Research Foundation Flanders (FWO). J. Dooley
has received research support from the European Research Council. X. Bossuyt has
received research support from the Research Council of Catholic University Leuven.
A. Liston has received research support from the European Research Council,
Research Foundation Flanders (FWO), and VIB. The rest of the authors declare
that they have no relevant conflicts of interest.
REFERENCES
1. Zhou Q, Yang D, Ombrello AK, Zavialov AV, Toro C, Zavialov AV, et al.
Early-Onset Stroke and Vasculopathy Associated with Mutations in ADA2.
N Engl J Med 2014;370:911-20.
2. Elkan PN, Pierce SB, Segel R, Walsh T, Barash J, Padeh S, et al. Mutant adenosine
deaminase 2 in a polyarteritis nodosa vasculopathy. N Engl J Med 2014;370:921-31.
3. Zavialov AV, Yu X, Spillmann D, Lauvau G, Zavialov AV. Structural basis for the
growth factor activity of human adenosine deaminase ADA2. J Biol Chem 2010;
285:12367-77.
4. Malamut G, Verkarre V, Suarez F, Viallard JF, Lascaux AS, Cosnes J, et al. The
enteropathy associated with common variable immunodeficiency: the delineated
frontiers with celiac disease. Am J Gastroenterol 2010;105:2262-75.
5. Zavialov AV, Gracia E, Glaichenhaus N, Franco R, Zavialov AV, Lauvau G.
Human adenosine deaminase 2 induces differentiation of monocytes into
macrophages and stimulates proliferation of T helper cells and macrophages.
J Leukoc Biol 2010;88:279-90.
6. Mercalli A, Calavita I, Dugnani E, Citro A, Cantarelli E, Nano R, et al. Rapamycin
unbalances the polarization of human macrophages to M1. Immunology 2013;140:
179-90.
7. DiCarlo J, Agarwal-Hashmi R, Shah A, Kim P, Craveiro L, Killen R, et al.
Cytokine and chemokine patterns across 100 days after hematopoietic stem cell
transplantation in children. Biol Blood Marrow Transplant 2014;20:361-9.
8. Van Montfrans J, Zavialov A, Zhou Q. Mutant ADA2 in vasculopathies. N Engl J
Med 2014;371:481.
http://dx.doi.org/10.1016/j.jaci.2014.10.010
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4LETTER TO THE EDITOR

METHODS
The study was performed in accordance with the modified version of the
Declaration of Helsinki. The study was approved by the Ethics Committee of
UZ Leuven. Written informed consent was obtained before DNA isolation
from blood of all family members and from cheek epithelium of the
transplanted patient.
Functional assays
PBMCs were isolated from heparinized blood of patients, family members,
and control subjects and analyzed by using flow cytometry, as previously
described.
E1
Serum IL-6 levels were measured by means of ELISA, according
to the manufacturer’s instructions (BD Bioscience, San Jose, Calif).
Whole-exome sequencing
We performed whole-exome sequencing on the untreated patient and on the
unaffected parents and sibling. Genomic DNA samples for whole-exome
sequencing were prepared from heparinized peripheral blood by using the
QIAamp DNA Blood Midi Kit (QIAGEN, Hilden, Germany). Exome
sequence libraries were prepared with a SeqCap EZ Human Exome Library
v3.0 kit (Roche NimbleGen, Madison, Wis). Paired-end sequencing was
performed on the Illumina HiSeq2000 (Genomics Core Facility, University of
Leuven, Leuven, Belgium). BWA software was used to align the sequence
reads to the Human Reference Genome Build hg19. The GATK Unified
Genotyper was used to identify single nucleotide variants and insertions/
deletions. ANNOVAR was used for annotation.
Sanger sequencing
A somatic DNA sample of the patient undergoing transplantation was
obtained from a cheek swab by using the GenElute Mammalian Genomic
DNA Miniprep Kit (Sigma-Aldrich, St Louis, Mo). The region of interest in
exon 2 of CECR1 was sequenced with the primers 59-GTTTGTACCAAGG-
GAGACACCTACC-39and 59-CTGGCTGGTGAGGAATGTCAC-39. Sanger
sequencing was performed on an ABI 3730 XL Genetic Analyzer (Applied
Biosystems, Foster City, Calif) at the LGC Genomics Facility in Berlin, Ger-
many. Sequencing data were analyzed by using DNADynamo (Blue Tractor
Software, Llanfairfechan, United Kingdom).
Flow cytometry
PBMCs were isolated from heparinized blood of patients and control
subjects by using lymphocyte separation medium (MP Biomedicals, Solon,
Ohio) and frozen in 10% dimethyl sulfoxide (Sigma). Thawed cells were
stained with antibodies (from eBioscience [San Diego, Calif], unless stated
otherwise) against CD11c (3.9), CD3 (SK7), CD4 (RPA-T4), CD8a
(RPA-T8), CD19 (HIB19), CD45RA (HI100), CD56 (MEM188), HLA-DR
(LN3), forkhead box protein 3 (FOXP3; 206D; BioLegend, San Diego,
Calif), IFN-g(4S.B3 IL-17, eBio64DEC17), IL-2 (MQ1-17H12), CXCR5
(IgG23; R&D Systems, Minn eapolis, Minn), CD31 (WM-59), CCR7 (3D12),
IgM (MHM-88, BioLegend), CD27 (O323), IgE (IgE21), CD24 (eBioSN3,
SN3 A5-2 H10), CD38 (HIT2), gd T-cell receptor (B1.1), CD56 (MEM188),
CD14 (61D3), CD123 (6H6) , and IL-4 (8D4-8). For cytokine staining, T cells
were stimulated ex vivo for 5 hours in 50 ng/mL phorbol 12-myristate
13-acetate (Sigma) and 500 ng/mL ionomycin (Sigma) in the presence of
GolgiStop (BD Biosciences) before staining. Before intracellular staining,
cells were first surface stained as described, fixed, and permeabilized with
fixation/permeabilization buffer (eBioscience) for forkhead box protein 3
staining or Cytofix/Cytoperm (BD) for other intracellular stainings. All
data were acquired on BD FACSCanto II and analyzed with FlowJo (Tree
Star, Ashland, Ore).
ELISA for measurement of IL-6 levels in serum
An in-house validated ELISA was used based on a commercially available
antibody pair (BD Biosciences).
Measurements of ADA1 and ADA2 activity in
plasma
ADA2 activity in plasma was measured by using the HPLC method
described by Zhou et al.
E2
ADA1 activity and concentrations of total adeno-
sine and deoxyadenosine nucleotides in extracts of dried blood spots were
measured, as previously described.
E3,E4
The concentrations of adenosine
and deoxyadenosine in plasma were determined by means of HPLC analysis
of a neutralized perchloric acid extract of plasma. In brief, 200 mL of plasma
was acidified with 40 mL of 5 N perchloric acid and centrifuged, and the su-
pernatant was neutralized with 3 N KOH and 1 M KHCO
3
. After centrifuga-
tion, 100 mL of the supernatant was analyzed on a C18 mBondapak column
(Waters Corporation, Milford, Mass) by using 0.05 mol/L NH
4
H
2
PO
4
,8%
methanol, and 1% acetonitrile (pH 5.2; flow rate, 0.5 mL/min) as the mobile
phase and monitoring absorbance at 260 and 280 nm with a diode array detec-
tor. The lower limit of quantitation for adenosine and deoxyadenosine in this
assay was 0.8 mmol/L; the lower limit of detection was taken as half the lower
limit of quantitation or 0.4 mmol/L.
RESULTS
X-linked lymphoproliferative disease type I and II, Wiskott-
Aldrich syndrome, autoimmune lymphoproliferative syndrome,
ADA1 deficiency, purine nucleoside phosphorylase deficiency,
and immune dysregulation–polyendocrinopathy–enteropathy–X-
linked syndrome were excluded by means of functional and
genetic analyses.
REFERENCES
E1. Danso-Abeam D, Zhang J, Dooley J, Staats KA, Van Eyck L, Van Brussel T, et al.
Olmsted syndrome: exploration of the immunological phenotype. Orphanet J
Rare Dis 2013;8:79.
E2. Zhou Q, Yang D, Ombrello AK, Zavialov AV, Toro C, Zavialov AV, et al. Early-
Onset Stroke and Vasculopathy Associated with Mutations in ADA2. N Engl J
Med 2014;370:911-20.
E3. Hershfield MS, Fetter JE, Small WC, Small WC, Bagnara AS, Williams SR, et al.
Effects of mutational loss of adenosine kinase and deoxycytidine kinase on deox-
yATP accumulation and deoxyadenosine toxicity in cultured CEM human T-lym-
phoblastoid cells. J Biol Chem 1982;257:6380-6.
E4. Arredondo-Vega FX, Santisteban I, Richard E, Bali P, Koleilat M, Loubser M,
et al. Adenosine deaminase deficiency with mosaicism for a ‘‘second-site sup-
pressor’’ of a splicing mutation: decline in revertant T lymphocytes during
enzyme replacement therapy. Blood 2002;99:1005-13.
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LETTER TO THE EDITOR 4.e1

FIG E1. Va sculopathology and immunopathology in patients with ADA2 deficiency. A, Sagittal T1-weighted
MRI of P1 showing pineal gland hemorrhage (arrow).B, Hematoxylin and eosin staining of jejunal ulcera-
tion in P2 showing chronic ulcer with predominant eosinophils (arrows), some neutrophils and lympho-
cytes, and very few plasma cells. Plasma cells stained by means of CD138 staining are indicated by
arrows in the inset.
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4.e2 LETTER TO THE EDITOR

FIG E2. Familial inheritance of CECR1 mutation. The region of interest in
exon 2 of CECR1 was sequenced by means of Sanger sequencing. A-E,
Sequence reads for the father (Fig E2, A), mother (Fig E2, B), healthy sibling
(HSCT donor; Fig E2, C), patient 1 after HSCT (chimerism accounts for pres-
ence of a minor G peak; Fig E2, D), and patient 2 (Fig E2, E). F, Family tree of
the affected pedigree, indicating affected patients and CECR1 genotype.
J ALLERGY CLIN IMMUNOL
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LETTER TO THE EDITOR 4.e3

TABLE E1. Clinical presentation, laboratory values, and therapeutic history of ADA2-deficient patients
Patient 1 Patient 2
Clinical phenotype
Clinical presentation Hypogammaglobulinemia, pancytopenia,
lymphoproliferation
Hypogammaglobulinemia, (intermittent)
lymphopenia and neutropenia,
lymphoproliferation
Viral infections confirmed by means of PCR RSV, adenovirus, norovirus HHV-6, HSV, polyomavirus
Stroke Hemorrhage in the pineal gland None
Laboratory values*
White blood cell count (kU/mL) 2.02 9.08
Neutrophil count (kU/mL) 0.3 6.4
Lymphocyte count (kU/mL) 1.0 1.7
Hemoglobin (g/dL) 8.8 9.7
Thrombocytes (kU/mL) 25 300
ALT (5-38 U/L) 44 15
AST (0-41 U/L) 64 6
IgG (3.02-9.85 g/L) <1.00 2.77
IgA (0.13-1.08 g/L) <0.07 0.11
IgM (0.26-1.60 g/L) 0.09 0.27
IgE (0-91 IU/mL) <230
IgD (<10 U/mL) 0 0
ANA Negative Not determined
ANCA Negative Not determined
Thrombocyte autoantibodies Anti-gpIIB-IIIa antibody present Not determined
Erythrocyte autoantibodies Anti-MNS1 antibody present Not determined
Lymphocyte count (kU/mL) [at moment of
immunophenotyping]
0.39 3.0
Therapeutic history
Immunosuppressive medication Corticosteroids, sirolimus, mycophenolate mofetil,
tacrolimus, cyclosporine, mercaptopurine
Corticosteroids, azathioprine, sirolimus
Immunoglobulin substitution Yes (before HSCT) Yes
Allogeneic HSCT Yes No
ALT, Alanine aminotransferase; ANA, antinuclear antibody; ANCA, antineutrophil cytoplasmic antibody; AST, aspartate aminotransferase; HSV, herpes simplex virus; RSV,
respiratory syncytial virus.
*Values were obtained at initial clinical presentation unless stated otherwise.
J ALLERGY CLIN IMMUNOL
nnn 2014
4.e4 LETTER TO THE EDITOR

TABLE E2. Relative frequencies of peripheral blood leukocyte populations in P1 before HSCT compared with those in healthy
age-matched control subjects
Subset Defining surface markers Patients (%)
Healthy volunteers (%)
Range (minimum-maximum)
T cells CD3
1
80.6 52.9-65.2
CD4
1
T cells CD4
1
CD8
2
63.8 29.4-65.2
/Treg CD25
1
Foxp3
1
10.0
CD8
1
T cells CD4
2
CD8
1
0.83 17.6-23.2
B cells CD19
1
5.56 11.8-30.4
/Transitional CD38
high
CD24
high
0.2
/Naive CD27
2
IgD
1
97.8
/Immature CD27
1
IgD
1
0.1
/Switched memory CD27
1
IgD
2
0.3
CD3
1
, CD4
1
, and CD8
1
T cells and CD19
1
B cells are shown as percentages of total lymphocytes. Regulatory T (Treg) cells are shown as percentages of CD4
1
T cells. B-cell
subsets are shown as percentages of CD19
1
B cells.
Foxp3, Forkhead box protein 3.
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LETTER TO THE EDITOR 4.e5