OMIM 259700) is an autosomal recessive disease
manifesting with anaemia,
hepatosplenomegaly, visual impairment due to optic
atrophy and deafness. Most of the children die during
infancy or early childhood without curative treatment
Infantile malignant osteopetrosis (arOP; ARO;
Novel mutations in Indian patients with autosomal recessive infantile
Shubha R. Phadke, Bjoern Fischer*, Neerja Gupta**, Prajnya Ranganath, Madhulika Kabra** & Uwe Kornak*,+
Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India
*Institute for Medical Genetics, Charité Universtaetsmedizin, Berlin, Germany, ** Department of Pediatrics
All India Institute of Medical Sciences, New Delhi, India & +Max Planck Institute for Molecular Genetics
Received April 23, 2009
Background & objectives: Although clinical reports have described infantile malignant autosomal
recessive osteopetrosis (ARO) in Indian patients, no published data are available about the genetic causes
of ARO in this population. We investigated the main genetic causes of ARO in eight Indian patients with
early postnatal onset and the typical severe clinical course including visual impairment and anaemia.
Methods: Mutation screening in the genes CLCN7 and TCIRG1 was done on genomic DNA from 8
affected individuals (diagnosed on the basis of clinical and haematological parameters and characteristic
radiological changes of increased bone density) and their parents. In one family, after detection of both
mutations in the proband, targeted mutation analysis was also done in chorionic villus samples for
Results: Six patients had mutations in TCIRG1 and two patients harboured mutations in CLCN7 gene.
Three of the five different TCIRG1 mutations identified and both CLCN7 mutations were novel mutations.
Except for the already known mutation p.Ile720del, all TCIRG1 mutations disrupt conserved splice
consensus sequences or lead to premature stop codons. In contrast, both CLCN7 mutations only lead to
missense changes of conserved amino acids. In a foetus harbouring TCIRG1 mutations osteopetrosis was
visible radiologically at 23 wk of gestation.
Interpretation & conclusions: That the CLCN7 mutations provoke a phenotype as severe as the one
caused by TCIRG1 loss of function suggests the affected residues to be crucial for the function of the
ClC-7 chloride channel or chloride/proton-exchanger. Our data also show that ARO can manifest as
early as in the second trimester of pregnancy.
Key words Autosomal recessive - CLCN7 - India - infantile - malignant osteopetrosis - mutations - TCIRG1
by bone marrow transplantation. Though there are
no data available about prevalence, it is not a very
rare disease in India1,2. Osteopetrosis is caused by a
defect in osteoclast function. The degradation of the
mineralized extracellular matrix of the bone requires
acid secretion by the osteoclast ruffled membrane. This
Indian J Med Res 131, April 2010, pp 508-514
proton transport is driven by a vacuolar (v-) type H+-
ATPase that is anchored to the ruffled membrane by the
a3 subunit. Mutations in the gene TCIRG1 (ATP6V0A3)
encoding the a3 subunit were found to cause infantile
malignant osteopetrosis3,4. Other genes mutated in
ARO are CLCN7 and OSTM1, that together form a
chloride channel or chloride/proton-exchanger which
also resides in the ruffled membrane and facilitates
acidification5,6. Mutations in the PLEKHM1 gene cause
milder forms of autosomal recessive osteopetrosis7,8. A
mild form of osteopetrosis associated with renal tubular
acidosis is caused by carbonic anhydrase II gene9. In
contrast to all ARO forms mentioned before, a minority
of patients has strongly reduced osteoclast numbers.
In some of these patients mutations in TNFSF11,
encoding the osteoclast differentiation factor RANKL,
were identified10. Mutations in the RANK receptor also
cause osteoclast-poor osteopetrosis with additional
immunological abnormalities11. Here, we report the
mutation spectrum in Indian patients with autosomal
recessive malignant osteopetrosis.
Material & Methods
Patients: Eight patients diagnosed to have infantile
malignant autosomal recessive osteopetrosis (ARO)
in the Medical Genetics department of the Sanjay
Gandhi Postgraduate Institute of Medical Sciences
(SGPGIMS), Lucknow, Uttar Pradesh and the Genetics
unit of the Pediatrics department of the All India
Institute of Medical Sciences, New Delhi, during 2003
to 2008, were chosen for the study. The diagnosis of
ARO was based on typical clinical and haematological
parameters and characteristic radiological changes of
increased bone density.
Sample collection: This study was conducted as part of
a large project ongoing in the Department and ethical
clearance was obtained from the Ethic committee of
the Institute (SGPGIMS, Lucknow). Informed consent
was obtained from the parents of all affected children.
Up to 5 ml EDTA blood was collected through
venipuncture. DNA was extracted from 1 ml of this
venous EDTA blood using the QIAamp DNA mini kit
(Qiagen, Hilden, Gernmay).
Mutation analysis: Patient DNAs were investigated
for mutations in the genes TCIRG1 (ATP6V0A3) and
CLCN7 by amplifying all exons and flanking intronic
regions by PcR using genomic DNA as a template.
PcR conditions and primers have been described
previously4,6,10,12. Dye terminator sequencing was
performed using Big Dye (Applied Biosystems,
Fosterville, USA) sequencing mix and an AB 3730
capillary sequencer (Applied Biosystems, Fosterville).
Sequences were compared with the reference
sequences using the software DNASTAR (DNASTAR,
Madison, USA). Reference cDNA sequences were: 1.
CLCN7: NM_001287, 2. TCIRG1: NM_006019. In
one family, after detection of both the mutations in the
proband, prenatal diagnosis was done three times by
mutation testing in DNA from chorionic villi samples.
The molecular diagnostic tests were performed in an
accredited laboratory (No.: DAP-ML-3869.00 (ISO
15189:2003 and ISO/IEc 17025:2005).
Molecular modeling: The 3D structure of the bacterial
cLc chloride channel Ecclc was reconstructed by
loading the PDB file 1KPK (download under http://www.
the published structure into PyMOL (version pre-1.0)
(DeLano Scientific, Palo Alto, CA, USA)13. Residue
Val297 corresponds to Ecclc residue Leu186.
screening of the patients are given in Table I and Table
II, respectively. Three of the patients were Hindu, three
were Muslims and two were Sikhs. In two families
consanguinity was known. The age at diagnosis was
between 3 and 18 months and the mean life expectancy
without curative bone marrow transplantation was 3.7
yr. Optic atrophy and visual impairment were major
features reported in six patients. In contrast, seizures
only appeared in one patient. Blood analysis revealed
severe anaemia in all affected children.
The clinical data and the results of the mutation
and showed a severe generalized osteosclerosis of the
long bones, the spine and the skull base (Fig. 1). The
long bones were abnormally modeled, had no bone
marrow cavity and frequently displayed a bone-within-
bone appearance (Fig. 1).
Radiographic data were available for all patients
CLCN7 were detected in all eight cases. Six patients
harboured disease-causing mutations in TCIRG1
(Table II, Fig. 2). Patients 4, 5 and 6 were homozygous
for a mutation: c.2236+1G>T, c.1554+2T>A and
c.2160_2162del (Ile721del). DNA was not available
for patient 2, but both the parents were found to
be heterozygous for the mutation c.2160_2162del
(1721del) (Fig. 2). Patients 1 and 3 were compound
heterozygous. While patient 1 displayed the mutations
c.1554+2T>A and c.2160_2162del (Ile721del) already
Mutations on both alleles of the genes TCIRG1 or
PHADKE et al: INDIAN PATIENTS WITH AR INFANTILE OSTEOPETROSIS 509
Table I. clinical details of the patients
Presenting complaintOther features
1 ARM No /
Increasing head sizeObstructive
Optic atrophy2 MI6M ? /
0.7Failure to thrive, distension of
Anaemia, distension of abdomen3 RT 12M 7 Optic atrophy
4 AF18M NA Noisy breathing, prominent eyes,
Inability to focus, anaemia
5 KH6F 1 Optic atrophy
6 PR3M NA Anaemia, previous sib died of
7 AM18M 5.6Hypocalcemic seizures,
8 HA13F 1.6
Table II. Results of laboratory investigations and mutation analysis
(X 103/ cmm)
(N: 5 - 15)cmm)
(N:1.5 - 4)
Mutations in TCIRG1
1 AR6.1 38.0 0.845%No 386 Het
2 MI* 7.4 58.00.78 35%Myelocytes,
3 RT**7.0 1.041.1+1316 c.1684c>T
Mutations in CLCN7
Hom 22.7 0.59
7 AM6.7 12.9 0.99-- 2280Hom
8 HA 8.211.1 0.8214-- Hom
*Proband’s sample was not available. Both parents were heterozygous for the mutation. **Heterozygosity in the parents was confirmed
N, normal range; TLc, total leucocyte count; ALP, serum alkaline phospha tase
510 INDIAN J MED RES, APRIL 2010
found in the other probands, patient 3 harboured the
two distinct mutations c.1684c>T (Gln562X) and
c.1653_1654insGTGG (Val551fsX670) (Table II).
In the two remaining cases we found novel
homozygous mutations in the second most common
ARO gene, CLCN7: c.889G>A (p.Val297Met) in
patient 7 and c.1856c>T (p.Pro619Leu) in patient 8.
The possibility of these two novel mutations being
polymorphisms was ruled out by demonstrating their
absence in 80 healthy Indian controls (equivalent to
160 alleles). Sequence alignments revealed that both
residues are highly conserved in clc-7 orthologs from
different vertebrate species (Fig. 3). Furthermore,
Val297 is also conserved in the muscle-specific
clc-1 chloride channel or chloride/proton exchanger
indicating a functional importance. Indeed, molecular
modeling of the corresponding residue Leu186 in the
Escherichia coli Ecclc indicates that the longer non-
polar side chain introduced by a mutation to Met could
have an influence on the selectivity filter of the ClC
chloride/proton exchanger (Fig. 3).
older siblings who had died in infancy and also in the
family of patient 6 the next sibling born was affected
with osteopetrosis. The mother of patient 3 underwent
prenatal diagnosis by chorionic villus sampling in her
next three pregnancies. The first prenatal diagnosis
showed absence of mutations in the prenatal sample
and the pregnancy resulted in the birth of a normal girl.
In the second prenatal diagnosis the presence of both
TCIRG1 mutations (p.Gln562X and p.Val551fsX670)
in chorionic villus DNA was detected and the
pregnancy was terminated at 23 wk of gestation. The
radiograph of the foetus showed mildly increased
bone density and marrow cavity obstuction (Fig. 4).
The third prenatal mutation screening showed normal
results; but in the early third trimester the foetus showed
Patients 1 and 4 had a history of two affected
Fig. 1. Radiological phenotype of patient 8 with a CLCN7 mutation. (a) The skull shows the typical sclerosis of the skull base. (b) Sclerosis
of the vertebral bodies and iliac wings is pronounced. (c) The bone-within-bone appearance, especially visible in the metacarpal bones, and
the sclerosis and undermodelling of the distal radius and ulna is evident.
Fig. 2. Mutations in TCIRG1 (ATP6V0A3) identified in Indian
osteopetrosis patients. (a) The position of splice site mutations
is given by arrows in a schematic representation of the genomic
organization of the TCIRG1 gene. The number of every fifth exon
is given. (b) The position of all mutations not affecting slice sites
are indicated in a model of the transmembrane topology of the
PHADKE et al: INDIAN PATIENTS WITH AR INFANTILE OSTEOPETROSIS 511
Fig. 3. Mutations identified in the ClC-7 chloride-proton antiporter. (a) Multiple alignments of the clc-7 protein sequences from human (h),
mouse (m), chick (c) and zebrafish (d) and of human ClC-1 (hClC-1) around the position of the mutations Val297Met and Pro619Leu. Note
complete conservation of the affected residues in all clc-7 orthologs. While Val297 is also conserved in hclc-1 the region containing Pro619
is very divergent between clc-7 and clc-1. (b) Position of the two missense mutations in the clc topology model. While Val297Met resides
in helix G Pro619Leu is located after the regulatory helix R. (c) 3D modeling of the position of residue Leu186 which corresponds to Val297
in the clc protein from E. coli (Ecclc). Although Leu186 is not directly involved in binding of chloride and the formation of the selectivity
filter of the chloride/proton exchanger, it lies in proximity to relevant residues. (d) closer modeling of the chloride binding site including
residues Ser107, Phe357 and Tyr445. Leu186 is near the chloride ion (represented as black ball, scaling not correct). A further protrusion of
the side chain due to mutation of Leu186 to Met could sterically affect the binding site.
Fig. 4. Post-termination radiograph of an TCIRG1 mutation-positive (a) and an unaffected fetus (b) at 23 wk of gestation. (a) Note mild
increase in general bone density. Obliteration of the bone marrow cavity and mild bone-within-bone appearance is seen in femora. (b) Note
that there is a clear separation of cortical bone and medullary cavity in the normal foetus.
512 INDIAN J MED RES, APRIL 2010
significant growth retardation. The karyotype of the
foetus was normal and ultrasonography did not show
any malformation. The outcome of this last pregnancy
is not known as the family was lost to follow up.
(ARO; arOP) is a serious lethal disorder usually leading
to death in infancy and childhood. The only curative
treatment is haematopoietic stem cell transplantation14.
The clinical course of the patients reported here was
similar to the commonly seen presentation of ARO15.
All patients presented with severe anaemia due to
constriction of the bone marrow cavity. Only two
patients did not show evidence of optic atrophy due
to optic nerve encroachment. As was already outlined
by Susani et al16, the phenotype caused by TCIRG1
mutations is relatively uniform. It is, however, striking
that the life expectancy differs very much between
the individual cases. In our patients, one (patient 2)
died at 0.7 yr whereas case 3 survived for 7 yr. The
seizures in patient 7 were found to be associated
with hypocalcaemia. Since CLCN7 mutations were
identified in this case, it is also possible that the seizures
were a sign of neuronopathic changes, which are more
frequent if CLCN7 mutations are present17.
Autosomal recessive malignant osteopetrosis
cause for ARO and are found in approximately 50
per cent of the cases18. A total of about 44 different
TCIRG1 mutations have been published16. In this study
TCIRG1 mutations were identified in six of the eight
patients. Of the five TCIRG1 mutations identified,
c.2160_2162del (Ile721del) and c.1554+2T>A have
been reported previously16. The splice site mutation
in one of the patients, c.2236+1G>T has not been
described; but at the same location a G>A mutation has
been previously reported19. The other two mutations
c.1684c>T (Gln562X) and c.1653_1654insGTGG
(Val551fsX670) are novel. Since both mutations lead
to a loss of several transmembrane helices of the v-type
ATPase subunit a3 these clearly entail a loss of function
like most of the mutations described so far. Population-
specific common mutations for TCIRG1 gene have been
observed in other studies. All nine costa Rican patients
reported by Sobacchi et al19 had either or both of two
missense mutations (G405R and R444L). In a study
of 55 patients of autosomal recessive osteopetrosis,
Susani et al16 reported two mutations namely; c.1674-
1G>A (aberrant splicing: r.1674_1884del) and
c.2005c>T (protein variation: p.Arg669X), in 17 and
16 alleles, respectively. These two alleles constituted
Mutations in TCIRG1 gene are the most frequent
30 per cent of all TCIRG1 abnormalities. About 40 per
cent of all TCIRG1 mutations are splice site mutations.
This proportion was also found in our patients. Given
the high numbers of splice site mutations attempts
have been undertaken to prevent abnormal splicing by
addition of mutated U1 snRNAs16.
mutations in the gene CLCN718. CLCN7 encodes
the chloride channel or chloride/proton exchanger
clc-7 that co-operates with the gene product of
TCIRG1, the a3 subunit of the v-type H+-ATPase20.
Both are necessary for resorptive activity of the
osteoclast. On an average 15 per cent of all ARO
cases are due to CLCN7 mutations. In our eight
patients we found two CLCN7 mutations suggesting
a higher frequency. Both CLCN7 mutations are
missense mutations of highly conserved residues
and have not been previously reported. According
to the topology of clc-7 p.Val297Met resides in
helix G and p.Pro619Leu is located after helix R in
the c-terminus13. Although helix G is not directly
involved in chloride ion binding, the side chain of
Val297 points towards the ion binding site and it
is reasonable to speculate that the substitution by
an amino acid like methionine with a longer side
chain is able to disturb the architecture of the ion
binding site and thus alter the function. Indeed,
when the corresponding amino acid Val275 in clc-1
was mutated to the larger residue Trp the chloride
channel function was still measurable, but had
The second most common cause of ARO are
already develops in the second trimester and can in
principle be visualized radiologically by an absence of
a marrow cavity in the long bones. However, in earlier
stages the diagnosis by radiology or ultrasonography
can be difficult22,23. Therefore, mutation screening is the
best means to provide a reliable prenatal diagnosis.
Our study demonstrates that the ARO phenotype
autosomal recessive malignant osteopetrosis in India,
similar to the data for other populations available in
the literature. However, the mutational spectrum seems
to be different from that in other populations as two
of the five TCIRG1 mutations detected are novel. The
previously reported c.1554+2T>A and c.2160_2162del
(I721del) appear to be common mutations in Indian
patients of infantile osteopetrosis. Data on more
patients will be useful in deciding on the strategy for
mutation detection in Indian patients.
TCIRG1 appears to be an important gene for
PHADKE et al: INDIAN PATIENTS WITH AR INFANTILE OSTEOPETROSIS 513
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Rae Bareilly Road, Lucknow 226 014, India
acknowledge the OSTEOPETR E-Rare grant from the BMBF (to
UK) and the Indian council of Medical Research, New Delhi for
Authors thank claire Schlack for technical assistance, and
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