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A single-center experience in 20 patients with infantile malignant osteopetrosis.

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Research Article
A single-center experience in 20 patients with infantile
malignant osteopetrosis
Evelina Mazzolari,1* Concetta Forino,1 Alessia Razza,1 Fulvio Porta,1 Anna Villa,2,3 and
Luigi Daniele Notarangelo4
Infantile malignant osteopetrosis (IMO) includes various genetic disorders that affect osteoclast development and/or function. Genotype–phenotype correlation studies in IMO have been hampered by the rarity
and heterogeneity of the disease and by the severity of the clinical course, which often leads to death early
in life. We report on the clinical and molecular findings and treatment in 20 consecutive patients (11 males,
nine females) with IMO, diagnosed at a single center in the period 1991–2008. Mean age at diagnosis was
3.9 months, and mean follow-up was 66.75 months. Mutations in TCIRG1, OSTM1, ClCN7, and TNFRSF11A
genes were detected in nine, three, one, and one patients, respectively. Six patients remain genetically
undefined. OSTM1 and ClCN7 mutations were associated with poor neurologic outcome. Among nine
patients with TCIRG1 defects, six presented with hypogammaglobulinemia, and one showed primary pulmonary hypertension. Fourteen patients received hematopoietic cell transplantation; of these, nine are alive
and eight of them have evidence of osteoclast function. These data may provide a basis for informed deciC 2009 Wiley-Liss, Inc.
sions regarding the care of patients with IMO. Am. J. Hematol. 84:473–479, 2009. V
Introduction
Autosomal recessive infantile malignant osteopetrosis
(IMO, OMIM 259700), is an uncommon but severe disorder
of osteoclast development and/or function with an average
incidence of 1:200,000 to 1:300,000 [1–4]. The condition is
most commonly diagnosed soon after birth or within the
first years of life with severe symptoms of abnormal bone
remodeling, including significant hematologic abnormalities
with bone marrow failure and extramedullary hematopoiesis, resulting in hepatosplenomegaly, a characteristic
macrocephaly with frontal bossing, exophtalmus, bone fractures, and failure to thrive. Bony encroachment can cause
cranial nerve entrapment, leading to visual impairment,
optic atrophy, and hearing loss, and less frequently also to
facial palsy or difficulties with swallowing and feeding.
Radiologic abnormalities include diffuse sclerosis of the
bones with a typical ‘‘bone-in-bone’’ appearance, signedu-mask (Batman sign) and metaphyseal changes. In
addition, some patients present severe and progressive
neurologic impairment and psychomotor retardation, which
can hardly be linked to the bone resorption defect [5]. A
variety of gene defects that affect osteoclast development
and/or function have been shown to account for IMO.
At present, allogeneic hematopoietic cell transplantation
(HCT) represents the only curative treatment for IMO
[6–12]. For patients who are not considered eligible for
HCT, calcitriol and interferon gamma have been used to
slow the hematologic progression of the disease, but with
only modest clinical results [13,14].
The purpose of this single-center study is to review the
clinical, molecular, and therapeutic findings in a cohort of
20 patients with IMO, with the aim of linking genotype with
clinical phenotype and response to treatment. These data
may provide a basis for informed decisions regarding the
care of patients with IMO.
parental informed consent and approval of the study by the Institutional
Review Board of the Spedali Civili, Brescia, the following data were collected: clinical phenotype, growth, fractures, infections, hematologic
status and transfusion dependency, evidence of cranial nerves compression, and neurologic and psychomotor development according to
milestones and Griffith’s scale.
To define the degree of hematopoietic deficiency at diagnosis, the
number of red cell and platelet transfusions per month up to the time of
diagnosis was recorded. Patients were given a score of ‘‘0’’ if they
received no transfusions and a score of ‘‘1’’ or ‘‘2’’ if one or more red
cell transfusions per month were used, respectively. A score of ‘‘3’’ indicated that both platelet and red cell transfusions were required. Serum
levels of calcium, alkaline phosphatase, lactate dehydrogenase, and
parathyroid hormone were used to identify signs of abnormal bone
remodeling. Bone biopsies at diagnosis were performed in four patients
(P9, P11, P14, and P18). Immunoglobulin serum levels were determined in 18 patients. Radiologic studies of bone, electroencephalography (EEG), and brain neuroimaging by computerized tomography (CT),
and/or magnetic resonance were recorded when available. In particular,
vision and hearing loss were documented by ophthalmoscopy, audiometry, and evoked responses. Visual impairment was defined as
moderate in patients with partial optical atrophy; reduced vision evoked
potential responses and presence of eye fixation. Severe visual impairment was defined by total optical atrophy, absence of evoked potential
responses, and roving eye movements. When possible, visual acuity
was compared with neuroimaging of the optic foramina. Echocardiography was used to screen for pulmonary hypertension.
Patients and Methods
Received for publication 22 February 2009; Revised 20 April 2009; Accepted
27 April 2009
Clinical and laboratory features at diagnosis. Between 1991 and
2008, 20 patients (11 males, 9 females) with a diagnosis of IMO were
followed at the Department of Pediatrics of the University of Brescia,
Italy. Median follow-up was 66.75 months (range 9–194 months). The
patients’ clinical features at diagnosis are summarized in Table I. On
1
Department of Pediatrics, University of Brescia, Brescia, Italy; 2Institute of
Biomedical Technologies, Consiglio Nazionale delle Ricerche, Milan, Italy;
3
Istituto Clinico Humanitas, IRCCS Rozzano, Milano, Italy; 4The Manton
Center for Orphan Disease Research and Division of Immunology, Children’s
Hospital, Boston, Massachusetts
Conflict of interest: Nothing to report.
Contract grant sponsor: N.O.B.E.L. Program from Fondazione Cariplo (to
LDN and AV) and Manton Foundation (to LDN).
*Correspondence to: Evelina Mazzolari, MD, Department of Pediatrics,
University of Brescia, P.le Spedali Civili,1, 25123 Brescia, Italy.
E-mail: mazzolar@med.unibs.it
Am. J. Hematol. 84:473–479, 2009.
Published online 14 May 2009 in Wiley InterScience (www.interscience.wiley.
com).
DOI: 10.1002/ajh.21447
C 2009 Wiley-Liss, Inc.
V
American Journal of Hematology
473
http://www3.interscience.wiley.com/cgi-bin/jhome/35105
research article
TABLE I. Molecular and Clinical Features at Diagnosis in 20 Patients With Infantile Malignant Osteopetrosis
Patient
1
2
3
4
5
6
7
8
9
Gene
TCIRG1
ClCN7
TCIRG1
TCIRG1
TCIRG1
10
11
TNFRSF11A
12
TCIRG1
13
TCIRG1
14
15
TCIRG1
16
OSTM1
17
18
19
20
OSTM1
OSTM1
TCIRG1
TCIRG1
Mutation
Allele 1: G4680T(IVS7,11)
Allele 2: 11647–11650del
Allele 1: T23373C
Allele 2: 1485–1565del
Allele 1: T4525C(IVS6,12)
Allele 2: A2418T(IVS2,14)
Not identified
Not identified
Allele 1: T11622C
Allele 2: not identified
Not identified
Not identified
Allele 1: C8980A
Allele 2: 8280–9560del
Not identified
Allele 1: c1301 G>A
Allele 2: c1301 G>A
Allele 1: C4391A(IVS5,26)
Allele 2: C4391A(IVS5,26)
Allele 1: C10311A
Allele 2: C10311A
Not identified
Allele 1: G11279A(IVS18,11)
Allele 2: G11279A(IVS18,11)
Allele 1: G105801A(IVS5,15)
Allele 2: G105801A(IVS5,15)
Allele 1:105688-9insG
Allele 2: not identified
Allele 1: G105801A(IVS5,15)
Allele 2: G105801A(IVS5,15)
Allele 1: G11651C
Allele 2: G8521A
Allele 1: 8521delG
Allele 2: not identified
Effect
Splice-site defect
frameshift
L641P
frameshift
Splice-site defect
Splice-site defect
Y512X
frameshift
W434X
W434X
Splice-site defect
Splice-site defect
Y625X
Y625X
Splice-site
Splice-site
Splice-site
Splice-site
frameshift
defect
defect
defect
defect
Splice-site defect
Splice-site defect
G792R
G410R
frameshift
Age at diagnosis
(months)
1
3
Length
(percentile)
3
rd
th
10
th
Visual
impairment
Transfusional
support score
IgG (mg/dl)
Moderate
1
180
Severe
1
393
No
0
0.5
50
7
2
4.5
th
10 –25
<3rd
<3rd
Moderate
Moderate
Severe
1
3
1
811
75 at 2 yr
464
1000
422
5.5
2
6.5
3rd
3rd–10th
3rd
Severe
Moderate
No
1
1
0
511
n.a.
265
th
5
7
75th
<3rd
Severe
Severe
0
0
415
480
4
<3rd
No
0
101
4
<3
Moderate
1
100
2
2
rd
<3
3rd
Severe
Severe
1
3
70
120
9.5
<3rd
Severe
1
435
1
rd
3
Severe
2
n.a.
0.5
rd
<3
Severe
3
209
Severe
1
165
Moderate
0
77
rd
th
th
6
5 –10
5
<3
rd
n.a.: not available. Low serum levels of IgG are indicated in Italics. Normal values: lactate deydrogenase 125–300 U/L; phosphatase, alkaline <270 U/L; calcium, total
8.8–10.8 mg/dl; serum IgG : 1 mo: 251–906 mg/dl, 2–4 mo: 176–601 mg/dl, 5–12 mo: 172–1,069 mg/dl.
Mutation analyses were performed as described for the following
genes: TCIRG1, ClCN7, OSTM1, TNFRSF11A (RANK), and TNFSF11
(RANKL) [15–23]. The molecular findings were correlated to clinical
phenotype and outcome.
Treatment and outcome evaluation. Overall, 14 patients received a
total of 18 HCTs (Table II). Of these, five were from a matched sibling
donor (MSD), nine from a matched unrelated donor (MUD), and four
from a mismatched related donor (MMRD). HLA compatibility was
defined by A, B, DRB1 serotyping and, since 2001, by high-resolution
molecular HLA typing.
All patients received myeloablative conditioning with regimens
according to protocols of the Inborn Errors Working Party of the European Group for Blood and Marrow Transplantation. Briefly, most
patients received busulfan 4–5 mg/kg/day (adjusted to 800 ng/ml
steady-state blood level) for 4 days orally, plus cyclophosphamide 50
mg/kg/day for 4 days intravenously (i.v.). To improve engraftment in
recipients of MUD- or MMRD-HCT, additional cytotoxic drugs were
used: Arabinoside-C (3 g/m2/dose every 12 hr i.v. for up to four doses),
thiotepa (10 mg/kg/day i.v. for 1 day), etoposide (300 mg/m2/day i.v. for
3 days), and fludarabine (40 mg/m2/day i.v. for 4 days). Total body irradiation (TBI) (12 Gy) was used in two patients (P3 and P4) and total
lymphoid irradiation was used in patients P1 and P8 before performing
a second transplant. To achieve immunosuppression, rabbit antithymocyte globulin (ATG, 2 mg/kg/day i.v for 4 days), alemtuzumab (0.2 mg/
kg/day i.v. for 4 days), or anti-CD2 monoclonal antibody (mAb) in combination with anti-LFA1 mAb (0.2 mg/kg/day i.v.) were used. The MSDand MUD-HCTs were performed using unseparated marrow grafts, and
a median number of 4.43 3 108/kg nucleated cells (range 3–7.5) and
11.07 3 106/kg CD341 cells (range 4–24.47) were infused. The
MMRD-HCTs were performed following in vitro bone marrow T-cell
depletion with Campath-1M plus complement, and a mean of 7.5 3
108/kg nucleated cells (range 5–10) were infused. For GvHD prophylaxis, all patients received cyclosporine (5 mg/kg/day per os), starting
on day 1; dosage was adjusted to a 100 ng/ml target blood level.
GvHD was graded according to Glucksberg’s criteria. All patients were
transplanted in single laminar airflow rooms and received prophylactic
broad-spectrum antibiotics, acyclovir, and intravenous immunoglobulins.
474
In addition, they were screened weekly for individual flora and CMV,
Epstein-Barr virus, and adenovirus reactivation. Chimerism was
assessed on peripheral blood mononuclear cells, granulocytes, and
CD31 and CD191 cells by microsatellite analysis at the DQ alpha,
D1S80, and ApoB loci.
Time to myeloid engraftment was defined as the first day after HCT
in which the absolute neutrophil count was greater than 500/lL for
three consecutive days. Hematopoietic reconstitution was defined also
based on the last day of platelet and erythrocyte transfusion. Indirect
evidence of osteoclast function was monitored by evaluating calcium
serum levels, clearing of the bones as assessed by X-rays or bone biopsy (in patient P4).
Six patients were not transplanted and received medical support.
Five of these patients (P15, P16, P17, P18, and P19) had very severe
clinical conditions and were considered not eligible for HCT. One
patient (P14) had no MSD or MUD available, and his parents refused
the option of MMRD-HCT.
Transplanted and untransplanted patients were monitored for growth,
gross motor, fine motor and psychomotor development, tooth development, spleen and liver size, infections, and hematologic parameters.
Statistical analysis. Overall and event-free survival after HCT were
calculated according to Kaplan–Meier’s method [24]. Death, severe
infections, graft failure, graft loss, acute GvHD of grade 3, and
chronic GvHD were considered in the analysis of event-free survival.
Results
Molecular characterization and clinical features
at diagnosis
Twenty patients had a diagnosis of IMO at the mean age
of 3.9 months (range 15 days to 9.5 months). In 14 of 20
patients (Table I), IMO-causing gene mutations were found.
In particular, mutations of the TCIRG1 and the OSTM1
gene were identified in nine patients (45%) and in three
patients (15%), respectively. One patient (P2) was mutated
in the ClCN7 gene, and another patient (P11) carried muta-
American Journal of Hematology
research article
TABLE II. Characteristics and Outcome of Hematopoietic Cell Transplantation
Patient
1
2
3
Age at
HCT
(months)
6
8
4
2
Donor
Nucleated
cells
(3108/kg)
MMRD
MMRD
MSD
MMRD
5
6.5
4.3
10
Myeloid
engraftment
(day)
Chimerism
GvHD
FU (mo.)
Outcome
ARA-C, BU, CY
TLI
BU, CY
VP-16, BU,
CY, rATG
TBI, THIOTEPA,
FLUDA, rATG
BU, CY,
MabCAMPATH
TBI, THIOTEPA,
FLUDA, rATG
No
No
135
128
n.a.
n.a.
D
D
No
No
No
No
2
1
46
90.5
133
D
110
D
112
D
No
98
Second HCT
Deceased at day 127
Deceased at 14.2 yr
Late failure 128 mo,
second HCT
Deceased at
118.5 mo
Late failure 120 mo,
second HCT
Alive, hypogonadotropic,
hypogonadism,
hypothyroidism
Acute rejection at
day135, alive with
disease
Alive
Alive
Second HCT
Conditioning
regimen
92
MUD
3
15
MUD
6.2
81
MUD
3
5
18
MMRD
8.5
VP-16, BU, CY,
antiCD2, antiLFA1
114
mixed
No
176
6
7
8
6.5
9.5
14
MSD
MSD
MUD
7.5
6
5
125
124
No
D
D
n.a.
Acute, Grade II
No
No
163
145
1.5
9
15.5
20.5
MUD
MUD
3.5
3
114
116
D
D
No
Acute, Grade II
1
92
Deceased at day128
Alive
10
11
12
6
12
10
MSD
MSD
MUD
3.7
3.6
3
132
111
130
D
D
D
Acute, Grade I
Acute, Grade II
Acute, grade II
66
51
40
13
10.5
MUD
4
112
D
Acute, Grade II
1
Alive
Alive
Alive, hemolytic anemia,
aplastic anemia
Deceased at day134
20
10
MUD
6.2
BU, THIOTEPA, CY
BU, CY
BU, THIOTEPA,
CY, rATG
TLI
BU, THIOTEPA,
CY, rATG
BU, CY
BU, THIOTEPA, CY
BU,THIOTEPA,
CY, CAMPATH
BU, THIOTEPA,
CY, rATG
BU, CY, rATG
127
D
Acute, Grade I
17
4
Acute, Grade
III chronic
No
18.5
81
Cause of death
Respiratory failure
Neuro-degeneration
Extensive cGvHD
IP CMV
IP CMV
Alive
HCT, hematopoietic cell transplantation; MMRD, mismatched related donor; MSD, matched sibling donor; MUD, matched unrelated donor; VP-16, etoposide; ARA-C,
cytarabine; BU, busulfan; CY, cyclophosphamide; FLUDA, fludarabine; TLI, total lymphoid irradiation; TBI, total body irradiation; rATG, rabbit antithymocyte globulin; Campath,
alemtuzumab; n.a., not applicable; D, donor; GvHD, graft versus host disease; FU, follow-up; IP, interstitial pneumonitis; cGvHD, chronic graft versus host disease.
tions in the TNFRSF11A gene [16]. No mutations in known
IMO-causing genes were identified in six (30%) of the 20
patients.
At diagnosis, 17 patients (85%) had typical craniofacial
features of IMO. However, macrocephaly was absent in all
three patients (P16, P17, and P18) with OSTM1 gene
defects (Table III), and two of them (P16 and P18) were even
microcephalic. Most patients had narrowing of the nasal passage with a high and narrow hard palate, causing significant
obstruction to airflow and chronic rhinorrea. Five patients
(P7, P9, P13, P14, and P19) presented restrictive chest wall
associated with recurrent hypoventilation and dyspnea. A
similar clinical picture was observed in a 3-month-old infant
(P15) with TCIRG1 mutation, who was admitted to the pediatric intensive care unit with the diagnosis of interstitial pneumonia and hypoxemia. In this patient, heart ultrasound
revealed severe pulmonary hypertension (arterial pulmonary
pressure, 90–95 mmHg) with low cardiac output.
Length-for-age was below the third percentile in 14
patients (70%). Three of the six patients whose length was
at or above the third percentile did not carry mutations in
any of the known IMO-causing genes.
With the exception of patient P3, all the remaining 19
patients (95%) showed generalized hypotonia, motor developmental delay, poor social interaction, and cognitive
impairment at diagnosis (95%). Among 17 patients who
were evaluated by CT and/or MRI of the brain, no abnormalities of gray or white matter were detected, and modest
cerebral atrophy was detected in patient P16. Clinical and
neuroradiologic signs of hydrocephalus were present in 11
patients (55%); however, only two of them (P6 and P13)
required extracranial shunt.
At diagnosis, 11 patients (55%) had severe and six
patients (30%) had moderate visual impairment, whereas
normal vision was present in three subjects. All of the
patients with OSTM1 mutations and the single patients with
American Journal of Hematology
ClCN7 and TNFRSF11A defects presented with severe visual problems, whereas TCIRG1 mutations were variably
associated with visual abnormalities (Table III). Only four of
the 11 patients with severe visual impairment had stenosis of
the optic foramina. No correlation was found between age at
diagnosis and severity of visual problems (data not shown).
None of the patients showed evidence of hearing loss.
At diagnosis, all patients had diagnostic signs of abnormal bone remodeling, with diffuse bone sclerosis. A bone
biopsy was performed in four patients (P9, P11, P14, and
P18). The number of osteoclasts was severely reduced in
patient P11; the remaining patients shared a severe defect
of bone resorption, despite the presence of osteoclasts.
Fractures were observed at diagnosis in 20% of the
patients (P6, P14, P17, and P18) and were more common
in patients with OSTM1 mutations (of whom, two of three
had more than one fracture) than in patients with TCIRG1
defect, of whom only one of nine showed a single fracture
at diagnosis (Table III).
Overall, serum levels of both lactate dehydrogenase
(mean: 1037 U/L; range: 125–1227 U/L; normal values
125–300 U/L) and alkaline phosphatase (mean: 921 U/L;
range: 156–2583 U/L; normal values: <270 U/L) were
markedly increased, and they were within the normal range
only in four subjects (two with unidentified gene defect, and
one each with TCIRG1 and TNFRSF11A mutations).
Twelve patients (60%) had low and unstable levels of
serum calcium (range: 4.8–8.2 mg/dl), which caused
neonatal seizures in two of them (P1 and P3). Notably,
hypocalcemia was more common in patients with TCIRG1
mutations (Table III).
All patients had signs of compensatory extramedullary
hematopoiesis; spleen and liver were palpable at least 4
cm below the costal edge in 18 of them (90%). Signs of
hematopoietic deficiency, with need for transfusional
support, were documented only in 14 of 20 (70%) patients
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research article
TABLE III. Clinical and Laboratory Features by Genotype in 20 Patients With IMO at Diagnosis
Gene
defect
Number
of patients
Macrocephaly
TCIRG1
OSTM1
ClCN7
TNFRSF11A
Unknown
9
3
1
1
6
9
0
1
1
6
Hydrocephalus
Bone
fractures
Short
stature
Need for
transfusional
support
Developmental
delay
Severe
visual
abnormalities
Hypocalcemia
Low
serum IgG
6
0
0
1
4
1
2
0
0
1
7
3
0
1
3
5
3
1
0
5
8
3
1
1
6
7
3
0
1
3
7
1
1
1
2
6
1
0
0
1
For each genotype, the number of patients with the indicated clinical or laboratory feature is shown.
(Table I). Four of the six patients who did not require transfusional support carried TCIRG1 mutations.
Eight patients (40%) had low IgG serum levels (range:
77–209 mg/dl) at diagnosis (Table I). In particular, hypogammaglobulinemia was observed in six of nine patients
with mutations in the TCIRG1 gene (Table III).
Hematopoietic cell transplantation
We performed a total of 18 HCTs in 14 patients (Table
II). The mean age at the first transplant was 10.3 months
(range 2–20.5 months) and the mean time from diagnosis
to HCT was 6.3 months (range 1–16 months). No significant early drug-related toxicity was observed with the conditioning regimens reported in Table II. Donor engraftment
after the first transplant was documented in 12 of 14
patients (85.7%). In particular, full chimerism was initially
observed in all five recipients of MSD-HCT, one of three
patients treated by MMRD-HCT, and in five of six recipients
of MUD-HCT. The mean time to myeloid engraftment was
day 122 (range: day 110 to 135), and independence from
platelet and erythrocyte transfusion was achieved at day
148 (range: day 124 to 1110) and day 140 (range: day
121 to 160), respectively.
Acute graft rejection was documented at day 135 in patient
P5, who had initially achieved mixed chimerism after MMRDHCT. This patient is currently alive with disease, following
parental refusal to perform a second transplant. Among the
four patients who received a second transplant, sustained
engraftment was documented in patients P3 and P4.
Acute GvHD Grade I-II was observed in 7 of 14 patients
(50%). One patient (P3) experienced acute GvHD Grade III,
followed by extensive and progressive chronic GvHD after
the second transplant (from a MUD-HCT), and died because
of this complication. Other significant adverse events
included severe hemolytic anemia, followed by aplastic anemia, in P12, 2 years after MUD-HCT. Elevated calcium levels
(>13.5 mg/dl) were observed in two patients, at 15 days
(P11) and at 9 months after HCT (P9). Patient P4 shows
central hypogonadism and hypothyroidism, likely the result
of the TBI used in the preparative regimen for HCT.
Long-term follow-up and survival
Overall survival in the whole cohort is 40.9% (9 of
20 patients), with a mean follow-up of 66.75 months (range
9–194 months) (Fig. 1).
All the six patients who did not receive HCT died, at a
mean age of 17.1 months (range: 10.5–31 months). The
causes of death in this group included severe neurologic
deterioration in three patients (P16, P17, and P18; all
OSTM1 mutated), septic shock in two (P14 and P19) and
pulmonary hypertension followed by sudden death at 10
months of age in one patient (P15). Interestingly, although
the three patients with OSTM1 deficiency were transfusion
dependent early in life, they showed progressive and spontaneous improvement of hematologic parameters over time,
476
Figure 1. Overall survival in 20 patients with infantile malignant osteopetrosis.
Survival for the entire population of 20 patients (——), for the 14 patients who
have received hematopoietic stem cell transplantation (), and for the six
patients who did not receive transplant (- - - - -) is shown.
and they did not require transfusional support after the first
month of life.
Among the 14 recipients of HCT, nine patients are
currently alive (64%) and eight patients (57%) had evidence
of osteoclast function at the last evaluation. One patient
(P5) is alive with disease at 16.2 years of age. When the
18 different transplants were considered, event-free survival
was 80% after MSD-HCT, 27.8% after MUD-HCT, and 0%
after MMRD-HCT (Fig. 2).
Five patients died after HCT, at a mean age of 39.6
months (range: 9–110.5 months). Causes of death in these
patients included interstitial pneumonia in three patients
(P1, P8, and P13), progressive neurodegeneration (P2),
and chronic GvHD (P3).
Eleven of the 14 patients who received HCT survived at
least 1 year after HCT (mean: 98.5 months; range: 17–179
months) and could be evaluated for possible clinical
improvement. Normalization of calcium serum levels and
clearing of the bones were demonstrated in all the eight
patients who achieved stable donor cell engraftment.
However, length at the time of last evaluation remained more
than 2 s.d. below the normal range (mean: 23.35 s.d.;
range: 21.39 to 27.36) in 8 of the 11 patients who survived
at least 1 year after HCT, despite full donor chimerism.
In addition to the six patients with TCIRG1 defects who presented with low serum IgG, one additional TCIRG1-mutated
patient (P3) developed profound hypogammaglobulinemia
(IgG: <100 mg/dl) after late graft failure following HCT.
Five of the 11 patients who survived at least 1 year after
HCT had severe visual impairment before transplantation,
and no changes of visual ability have been observed during
follow-up. Three patients had moderate visual impairment
before HCT, and two of them (P4 and P5) have developed
progressive disease. Of these, patient P4 has achieved full
donor chimerism and osteoclast function after two trans-
American Journal of Hematology
research article
plants, whereas patient P5 remains with persistence of disease after graft reject and parental refusal to attempt a second transplant. Three patients with TCIRG1 mutations (P3,
P9, and P12) did not have evidence of visual impairment
before HCT and have remained with good visual function
until the last follow-up visit (P9 and P12) or until death due
to chronic GvHD 19 months after the second HCT (P3).
None of the patients have developed hearing loss, as
assessed by evoked potentials.
All eight survivors with donor stem cell engraftment show
normal tooth development. In contrast, abscesses associated with maxillary and mandibular osteitis were documented in patient P5 with autologous reconstitution. Similar
problems were observed in P3, following late graft failure.
As mentioned earlier, 19 of 20 patients had evidence of
developmental delay at diagnosis. Three of the 20 patients
(P1, P8, and P13) died very early after HCT and therefore
could not be further evaluated. Evolution of neurologic and
behavioral status in the remaining 17 patients is shown in
Table IV. Among these, the three infants with OSTM1 defects
(P16, P17, and P18) developed severe and progressive
encephalopathy and myoclonic epilepsy early in life, and
they died at 19, 11, and 31 months of age, respectively, without receiving HCT. In two of these patients (P17 and P18),
Figure 2. Event-free survival after HCT for IMO. For each transplant, survival
was considered from the date of transplantation. Events included death, graft failure, loss of engraftment, severe infections, acute GvHD of grade 3, and chronic
GvHD. Data are shown for transplants from matched sibling donors (n 5 5;——),
mismatched related donors (n 5 4;- - - - -), and from matched unrelated donors (n
5 9;).
epileptogenic activity was documented by EEG before the
appearance of clinical evidence of epilepsy. Despite full donor chimerism after MSD-HCT, patient P2 with ClCN7 mutations died at 50 months of age with progressive neurodegenerative disease, which became evident at 1 year of age.
Patients P14, P15, and P19 showed progression of developmental and psychomotor delay until they died. Three
patients (P6, P7, and P11) with different genetic defects
have developed autistic spectrum disorder and are alive but
require special education and family support. Two patients
(P12 and P20) are alive with expressive language delay after
HCT. In contrast, cognitive and neuromotor development at
the time of last follow-up was normal in the remaining four
patients (P3, P4, P5, and P9), despite the fact that P5 has
persistence of disease and P4 has severe visual disability.
Discussion
IMO is an uncommon but severe congenital disease
caused by defects in osteoclast development and/or function that result in impaired bone resorption [25–27]. In addition, some of the gene defects also interfere with function
of other cell types. Despite recent advances in the molecular characterization of IMO [28,29], genotype–phenotype
correlation studies have been hampered by the rarity and
heterogeneity of the disease and the severity of the clinical
course that often leads to death early in life, thus, precluding long-term analysis of bone-extrinsic manifestations.
Furthermore, different mutations in the same gene may
result in variability of the clinical phenotype, further complicating genotype–phenotype correlation.
Despite these problems, this single-center report suggests that the frequency of certain clinical and laboratory
features of IMO may vary depending on the underlying
genetic defect (Table III). In particular, as previously
reported [30,31], none of the patients with OSTM1 mutations in our series showed macrocephaly or hydrocephalus.
On the other hand, patients with TCIRG1 mutations had a
higher frequency of hypocalcemia but showed lower tendency to develop bone fractures.
Although neurodevelopmental problems were very common, regardless of the underlying genetic defect, ClCN7
and OSTM1 mutations were associated with severe neurologic progression, as previously reported [17,30–32].
Recently, studies in clcn72/2 mice have shown a widespread degeneration of the central nervous system (CNS)
that includes retinal degeneration [33,34]. Furthermore, rescue of the osteopetrotic phenotype by inducing selective
TABLE IV. Neurological Features During Follow-up
Patient
Mutated gene
HCT
Motor
delay
Cognitive
delay
Language problems
Epilepsy
Encephalopathy
Behavioral
disorders
Age at last
FU (months)
2
3
4
5
6
7
9
10
11
12
14
15
16
17
18
19
20
ClCN7
TCIRG1
Unknown
Unknown
TCIRG1
Unknown
TCIRG1
Unknown
TNFRSF11A
TCIRG1
Unknown
TCIRG1
OSTM1
OSTM1
OSTM1
TCIRG1
TCIRG1
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
No
Yes
Yes
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
No
No
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes, receptive
No
No
No
Yes, unusual
Yes, unusual
No
No
Yes, expressive and receptive
Yes, expressive
Yes
Yes, receptive and expressive
Yes
Yes
Yes
Yes
Yes, expressive
No
No
No
No
Yes
No
No
No
Yes
No
No
No
Yes
Yes
Yes
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
No
No
No
No
No
No
Yes, autism
Yes, autism
No
No
Yes, autism
No
No
No
No
No
No
No
No
50a
111a
194
194
169
152
113
72
63
50
18a
10.5a
19a
11.5a
31a
13a
27
a
These patients have died.
HCT, hematopoietic cell transplantation; FU, follow-up
American Journal of Hematology
477
research article
expression of clcn7 in osteoclasts showed that the neurologic defects of clcn72/2 mice are not secondary to poor
osteoclast function, but are rather specific to lack of clcn7
expression in the CNS. Mutation analysis is important to identify ClCN7-mutated patients who are unlikely to benefit from
HCT because of severe progressive neurodegeneration.
The pathophysiology of the early-onset, severe neurologic
phenotype associated with OSTM1 mutations remains
poorly defined [18,19]. Studies in mice have shown that the
ostm1 and clcn7 proteins colocalize in late endosomes and
lysosomes of various tissues, and that levels of clcn7 protein
are markedly reduced in the gray-lethal (gl) mice (mutated in
ostm1), suggesting that ostm1–clcn7 interaction may be important for protein stability [35]. Signs of neurodegenerative
storage disorder, detected by MRI, have been reported in
some, but not all, OSTM1-deficient patients [31,32]. Recent
data have indicated that ostm1 may be important for Wnt/bcatenin signaling and that ostm1 mutations may impair the
transcription of Lef1-dependent genes [36]. Finally, defects
of myelination have been observed in gl mice, and abnormalities of the white matter have been reported in some
patients with OSTM1 mutations [30]. In agreement with
these observations, a low content of sphingomyelin, sulfatide, and galactosylceramide has been reported in the brain
of gl mice [37]. However, no signs of demyelination were
identified by MRI and CT in the three OSTM1-deficient
patients from our series. The observation that patients with
OSTM1 mutations develop intractable epilepsy in the first
months of life suggests interference with cortical brain function and possibly a primary neuronal involvement. Because
of the progressive and irreversible neurologic deterioration
associated with OSTM1 mutations, HCT is not indicated in
patients with this variant of IMO.
Three patients of various genotype in our series have
developed autistic disorder after successful HCT. To our
knowledge, this association has not been previously
reported. Because these patients do not share a common
genetic defect, it is unlikely that autism is a component of
specific molecular subgroups of IMO.
Reports from the literature have indicated that HCT
(especially if performed beyond 3 months of age) is usually
ineffective in preserving vision in patients with IMO [12].
Our data support this notion. Of 11 patients with severe visual impairment at diagnosis, only four had evidence of
restriction of optic foramina. We have not observed
improvement of vision in any of the patients who have
achieved donor chimerism after HCT. Importantly, in our
series, seven of the eight patients who were diagnosed
early in life (<3 months of age) had already developed
moderate (n 5 3) or severe (n 5 4) visual impairment.
These data indicate that visual defects may occur very
early (potentially, even during prenatal life) in IMO, thus,
reducing the efficacy of HCT (even when performed in the
first months of life) on visual function.
The occurrence of PPH after HCT has been previously
reported [38,39], but the reasons for this association have
remained obscure. We have identified one patient with
TCIRG1 mutations who has developed severe primary pulmonary hypertension (PPH) in the absence of HCT, suggesting that PPH in patients with IMO does not simply reflect a
drug-related adverse event. Furthermore, the fact that several other TCIRG1-mutated patients did not present with
PPH argues against the hypothesis that the association of
IMO and PPH may represent a novel and specific disease
entity [39]. It has been hypothesized that development of
PPH may depend on hypoxemia, which, in patients with
IMO, is often determined by nasal airways and chest wall
restriction, leading to obstructive sleep apnea [39]. Alternatively, abnormalities of osteoprotegerin (OPG) [40] and pos-
478
sibly of granulocyte-macrophage colony stimulating factor
levels may also play a key role [41]. In particular, it has been
recently shown that OPG stimulates proliferation and migration of pulmonary artery smooth cells, and serum levels of
OPG are increased in patients with pulmonary hypertension
[42]. In our patient, PPH had originally been misdiagnosed
as interstitial pneumonia, and a similar diagnostic error has
been previously reported in other cases of IMO with PPH
[38,39]. Evaluation of pulmonary arterial flow should be
included in the assessment of each patient with IMO to permit early diagnosis of PPH and to attempt treatment.
We have identified previously unrecognized immunologic
abnormalities in patients with IMO. In particular, of nine
patients with TCIRG1 mutations, seven developed hypogammaglobulinemia during the course of the disease, in the
absence of signs of protein loss or hypercatabolism. Disruption of the tcirg1 gene in oc/oc osteopetrotic mice leads to a
block in B-cell development at the pro-B stage and to
impaired T-cell activation [43]. Interestingly, the TCIRG1
gene encodes both for the a3 subunit of the vacuolar proton
pump required for bone resorption and for the protein
TIRC7, which is important for T-cell activation [44–46]. However, TIRC7 is not expressed in B lymphocytes. Studies in
larger cohorts of patients may allow to assess whether
defects in T-cell activation and function may account for
hypogammaglobulinemia in patients with TCIRG1 mutations.
Alternatively, data in oc/oc mice suggest that abnormalities
in the bone marrow microenvironment (including reduced IL7 secretion) may also play a role [47,48].
Immunologic defects have been recently reported also in
IMO associated with mutations in the TNFRSF11A (RANK)
gene [16]. Mutations of this gene and of TNFSF11
(encoding for RANK ligand, RANKL) account for osteopetrosis associated with severe immunologic problems in
mice. In particular, rank-deficient mouse thymic epithelium
is unable to support negative selection of autoreactive T
lymphocytes, leading to catastrophic autoimmunity [49].
This defect cannot be corrected by HCT. We have not
observed any evidence of autoimmune manifestations in
our patient with IMO due to TNFRSF11A mutations. This
could reflect significant differences in the requirement for
RANK expression in the thymus to delete autoreactive T
cells in mice and humans. Alternatively, autoimmunity may
develop only after prolonged follow-up in patients with
RANK deficiency who have been successfully engrafted
with donor stem cells.
Defects of insulin secretion have been reported in oc/oc
mice, reflecting expression of tcirg1 in endocrine tissues
[50,51]. However, we did not detect abnormalities of glucose levels in our series of TCIRG1-mutated patients.
Since 1977, HCT represents the only curative therapy for
IMO [8–12]. The results presented in this study are similar
to what has been reported in the literature, with better
outcome for patients who have received HCT from a MSD.
We have not observed any significant early toxicity or venoocclusive disease, in particular, that had been frequently
observed after HCT for IMO [52]. On the other hand, in
agreement with previous reports [12], we have found that
growth retardation and vision impairment usually do not
improve after HCT, even in patients who achieve full donor
engraftment. Earlier intervention may be required to prevent
these complications. Recently, transplantation of bone marrow and fetal liver cells in utero [53,54] or gene therapy at
neonatal age [55] have been shown to cure osteopetrosis
in oc/oc mice; however, these options would not be available to most patients with IMO.
Genotype–phenotype correlation studies may help refine
eligibility criteria for HCT. This study has shown that the frequency and severity of certain clinical and laboratory abnor-
American Journal of Hematology
research article
malities in patients with IMO may vary, depending on the
underlying genetic defect. However, larger series of cases
will be required to substantiate a genotype–phenotype
correlation that might have prognostic and therapeutic
implications.
Acknowledgments
We thank the patients’ families for their support and the
nurses of the bone marrow transplantation unit for their
dedication in the care of these patients. We also thank Mrs.
Lisa Giles for careful editing of the manuscript.
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