The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 3 1047-1051
Copyright © 1999 by The Endocrine Society
Locus Heterogeneity of Autosomal Dominant Osteopetrosis (ADO)1
Kenneth E. White,
Daniel L. Koller,
Istvan Takacs,
Kenneth A. Buckwalter,
Tatiana Foroud and
Michael J. Econs
Departments of Medicine (K.E.W., I.T., M.J.E.), Medical and
Molecular Genetics (D.L.K., T.F., M.J.E.), and Radiology (K.A.B.),
Indiana University School of Medicine, Indianapolis, Indiana 46202
Address correspondence and requests for reprints to: Michael J. Econs, M.D., Indiana University School of Medicine, 975 W. Walnut St. IB445, Indianapolis, Indiana 46202. E-mail:mecons{at}iupui.edu
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Abstract
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Autosomal dominant osteopetrosis (ADO), is a heritable disorder that
results from a failure of osteoclast-mediated bone resorption. The
etiology of the disorder is unknown. A previous linkage study of one
Danish family mapped an ADO locus to chromosome 1p21. We have studied
two families from Indiana with ADO. The present study sought to
determine if the ADO gene in these families was also linked to
chromosome 1p21. We used six microsatellite repeat markers, which
demonstrated linkage to the 1p21 ADO locus in the Danish study, to
perform linkage analysis in the new kindreds. Multipoint analysis
excluded linkage of ADO to chromosome 1p21 (logarithm of the odds
score < -7.00) in both families. In addition, no haplotype
segregated with the disorder in either family. In summary, the present
investigation ruled out linkage of ADO to chromosome 1p21 in two
families from Indiana. Our results demonstrate that there is locus
heterogeneity of this disorder; therefore, mutations in at least two
different genes can give rise to the ADO phenotype.
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Introduction
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AUTOSOMAL dominant osteopetrosis (ADO),
also known as Albers-Schönberg disease, is an inherited,
osteosclerotic disorder that results from inadequate
osteoclast-mediated skeletal resorption (1, 2). ADO has variable
penetrance (3, 4) and occurs with an estimated prevalence between 1 in
20,000 (4) and 1 in 100,000 (3). The disorder is referred to as the
"benign" form of osteopetrosis, to distinguish it from the
autosomal recessive "malignant" forms of the disease, which are
typically fatal if untreated (3). Although some individuals with ADO
are asymptomatic, patients may present with dense but brittle bones
that are prone to fracture, bone pain, osteomyelitis (particularly of
the mandible), and nerve entrapment syndromes (3). Anemia with
extramedullary hematopoiesis may also develop as a result of
insufficient marrow space (3).
Individuals affected with ADO display several radiographic and
biochemical hallmarks. Upon X-ray analysis, patients frequently have
osteosclerosis; endobone ("bone within bone") formation at the
hips, spine, and extremities; as well as vertebral end-plate sclerosis
(Rugger-Jersey spine) (5, 6). Biochemically, serum levels of the
brain-specific isoform of creatine kinase (CK-BB) (7, 8) and
tartrate-resistant alkaline phosphatase (TRAP) (3) may be elevated in
affected individuals. Interestingly, obligate carriers of a defective
ADO gene have been identified in multiple families and show no
detectable radiographic or biochemical manifestations of the disorder
(3, 4, 9).
Although the disorder results from insufficient osteoclast
mediated bone resorption, the etiology of ADO is presently unknown and
may not be understood until the gene is identified. In an effort to
determine the chromosomal location of the disease gene, a linkage study
was undertaken by Van Hul and colleagues (10) in a single Danish ADO
family. Several candidate regions were analyzed, and linkage to 1p21
was identified (10). The ADO locus mapped to an 8.5 cM region between
the markers D1S486 and D1S2792 [logarithm of the odds (LOD) > 4.00
]. Because only a single family was used in this linkage analysis, it
is unknown whether all ADO cases result from mutations in the same
gene, or if the disease can result from mutations in more than one gene
(locus heterogeneity). Therefore, the goal of the present study was to
determine if the ADO gene locus in two ADO families of non-Danish
descent also linked to chromosome 1p21.
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Materials and Methods
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Patients
Family number EOP1 was previously reported by Johnston et
al. (3), and family number EOP3 was referred to us by the
Department of Medical and Molecular Genetics at Indiana University
School of Medicine. We obtained blood samples from 8 members of family
EOP1 and 19 members of family EOP3. Phenotypic evaluation of family
members continues to proceed as medical records and radiographs
become available. Only individuals with radiographic evidence of
vertebral end-plate sclerosis or endobones were considered affected. If
radiographic evidence of ADO was not present in an individual, but they
had an affected parent or sibling and an affected child, they were
considered an obligate gene carrier. Radiographs were read by a
radiologist specializing in metabolic bone disorders (K.A.B.), who was
blinded to all other phenotypic and genotypic information. Radiographs
from normal spouses were included as negative controls, when available,
to assure consistency of film evaluation. The study was approved by the
Indiana University School of Medicine Institutional Review Board, and
all patients gave written, informed consent before participating.
PCR amplification of microsatellite repeat markers
DNA extraction from whole blood was performed as described
previously (11, 12, 13, 14, 15). PCR primer pairs for the microsatellite repeat
markers (Research Genetics, Inc.; Huntsville, AL) were
used to amplify 30 ng of genomic DNA. The PCR products were
32P radiolabeled by 5' labeling of the forward primer with
T4 polynucleotide kinase (Gibco BRL; Gaithersburg, MD) and
[32P]ATP (DuPont NEN; Boston, MA). The
PCR products were resolved by electrophoresis on standard acrylamide
sequencing gels and visualized by autoradiography. The sequences for
all primers are available from the Centre d’Etude du
Polymorphisme Humain (CEPH) marker database
(http://www.cephb.fr/cephdb/). Paternity was verified by
examining an additional 19 highly polymorphic microsatellite markers on
other chromosomes, in addition to the 6 markers evaluated within the
ADO region on chromosome 1.
Linkage analysis
In accordance with the previously published linkage study (10),
an autosomal dominant disease model with 60% penetrance was used for
all linkage analyses with a population disease allele frequency of 1
per 100,000 chromosomes. Maximum likelihood estimates of the population
marker allele frequencies were obtained from the observed pedigree data
with the USERM13 subroutine of the MENDEL (Ann Arbor, MI) computer
package (14). USERM13 was also used to calculate polymorphic
information content (PIC) for each marker. Two-point LOD scores were
computed using the program MLINK from the FASTLINK implementation of
the LINKAGE (New York, NY) package of programs (15, 16).
Order and distances for all markers except AMY2B were taken from the
Marshfield Center for Medical Genetics sex-averaged map of chromosome 1
(http://www.marshmed.org/genetics) (17). The interval
flanked by the markers D1S486 and D1S221 included the entire 1p21
region from a previous linkage study (10), according the Marshfield
Center map. Genetic mapping data from the Genetic Location Database
(http://cedar.genetics.soton.ac.uk/public html)
(18) placed marker AMY2B in the interval between D1S495 and D1S239; for
lack of additional information, AMY2B was assumed to lie halfway
between these two markers. No recombination was observed between D1S239
and D1S248 in the CEPH families used for mapping by Marshfield;
therefore, a short arbitrary distance between them (0.1 cM) was
assumed. The final chromosome 1p21 genetic map was tel-D1S486 —2.68
—D1S495 —1.07 —AMY2B —1.07 —D1S239 —0.10 —D1S248 —3.22
—D1S221-cen (distances in cM). The computer program VITESSE
(Pittsburgh, PA) (19) was used for multipoint linkage calculations.
Multipoint LOD scores were computed at each marker position and at five
equally-spaced points in each interval, using information from all six
markers for each calculation.
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Results
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Radiographic evidence of ADO in the Indiana families
Radiographs from members of family EOP1 displayed typical
features of ADO (9). Representative radiographs from the proband of
family EOP3, an 18-yr-old female, are shown in Fig. 1
, A, B, and C. Upon examination of a
lateral chest X-ray, vertebral end-plate sclerosis as well as endobone
formation in the spine was readily apparent at age 15 yr (Fig. 1A). In
addition, marked endobone formation was evident in her sternum (Fig. 1A). Of note, lateral chest X-rays taken at 3 yr of age showed slight
vertebral sclerosis that was not as obvious as in the film taken at age
15 (Fig. 1B). Left foot x-rays taken at 12 yr showed an onion-skin like
appearance of endobone formation in the heel (Fig. 1C).
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Figure 1. Radiographs of an ADO patient. A, Lateral
chest X-ray of an affected female at 15 yr of age from family EOP3
showing the characteristic vertebral end-plate sclerosis
(arrow on left side of panel A), as well as endobone
formation in the sternum (arrow on right side of panel
A). B, Lateral chest x-ray of the same individual at 3 yr of age, in
which signs of ADO in the spine and sternum are far less evident. C,
Radiograph of the left foot of the same patient at 12 yr of age,
displaying endobone formation in the heel.
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Linkage analysis with 1p21 microsatellite repeat markers
To test our ADO families for linkage to chromosome 1p21, we used 6
dinucleotide microsatellite repeat markers (see Materials and
Methods) shown to localize to the 8.5 cM ADO region defined in the
Danish kindred (10). Two-point LOD scores did not support linkage with
any of the 6 chromosome 1p21 markers (Table 1). More importantly, multipoint linkage
analysis of the region, defined by the telomeric marker D1S486 and by
the centromeric marker D1S221, produced LOD scores of less than -2.0
(at 
= 0) for the individual families and, when combined, yielded
LOD scores below -7.0 throughout the entire region (Fig. 2). These LOD scores surpass the
traditional criteria for exclusion of linkage (LOD of < -2.0, or
100:1 odds against linkage) by a factor of 105. In
addition, the scores calculated were stable across order-of-magnitude
changes in the disease allele frequency estimate, as well as changes in
the penetrance function (not shown). These results demonstrated that
the two Indiana ADO families do not link to the previously described
ADO region on chromosome 1p21.
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Figure 2. Multipoint LOD score analysis of the ADO
region. LOD score is shown on the Y-axis, map position on the X-axis
(in cM). The results for individual families EOP1 (dashed
line) and EOP3 (hairline), as well as the
combined multipoint analyses (heavy line), are shown.
The standard LOD of -2.0 used to exclude linkage is present as a
dotted line.
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ADO haplotype analysis
Chromosome 1 haplotypes were determined across the ADO interval
and are shown in Fig. 3. Visual
inspection of the markers revealed no haplotype segregating with the
disorder in either family. Of note, sisters II:1 and II:3 in family
EOP1 each carry a different 1p21 haplotype from the
disease-transmitting parent (I:1) (Fig. 3A). Furthermore, two brothers
from family EOP3, individuals II:1 and II:3, who both must carry the
ADO haplotype because they each have affected children, received
completely different 1p21 chromosomal regions from their parents,
individuals I:1 and I:2 (Fig. 3B). The origin of the ADO mutation,
however, is unknown in this family as both I:1 and I:2 have
insufficient radiographic evidence for diagnosis, and neither has a
family history of ADO. These haplotypes provide further evidence that
the ADO gene in these families is not localized between markers D1S495
and D1S221. In sum, our results demonstrate that ADO displays locus
heterogeneity; therefore mutations in at least two different genes, one
at the 1p21 locus and one at another unmapped locus, can give rise to
clinically indistinguishable ADO phenotypes.
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Figure 3. ADO pedigrees with haplotypes. Filled
symbols indicate affected individuals; open
symbols, unaffected or unknown. Circles
represent females; squares, males.
"C" within a symbol indicates an obligate carrier.
Marker haplotypes are listed below each individual. Question
marks (?) indicate an unknown genotype, and
brackets indicate an inferred genotype. A, Family EOP1.
B, Family EOP3. Phenotypic data for individual II:3 (*) is incomplete
to definitively conclude whether he is a carrier or is mildly
affected.
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Discussion
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Identifying genes that are responsible for osteosclerotic
disorders provides an opportunity to discover novel proteins that
regulate bone structure and function (20). The first step in a
positional cloning strategy is to identify a region of the genome that
contains the disease gene locus, and the second step is to identify
potential candidate genes within the linked region. The
characterization of natural or targeted gene knockout animals has led
to a large number of models for the recessive forms of osteopetrosis
(21, 22, 23, 24). These animal models indicate that the resorptive capabilities
of osteoclasts are regulated through multiple pathways and support the
idea that aberrations in several genes could give rise to
osteo-petrosis.
The linkage results and haplotype analysis (Table 1
, Figs. 2 and 3)
exclude mutations in the ADO gene locus on chromosome 1p21 as a cause
of ADO in our families. Therefore our results indicate that ADO is
caused by mutations in at least two different genes. The phenotype in
the Indiana and Danish families is remarkably similar. Although
speculative, the clinical similarity between the families supports the
possibility that the ADO gene on chromosome 1p21 and the as yet
unmapped ADO gene locus responsible for the disease in our kindreds may
interact as part of a complex or may be part of the same biochemical
pathway. Alternatively, it is plausible that one gene could code for a
circulating hormone or factor and the other gene code for its receptor.
Mutations in either locus may lead to decreased effective circulating
concentration of the hormone or may cause insufficient target receptor
number and thereby result in ADO. Another dominant disorder, autosomal
dominant polycystic kidney disease (ADPKD) is caused by mutations in
several distinct but homologous genes (25). By analogy, it is possible
that the two ADO genes possess similar functions that regulate the
resorptive activities of osteoclasts. If the two genes are related
through function or primary structure, then isolation of the ADO gene
at one locus could potentially allow rapid identification of the second
ADO gene.
The variable penetrance of ADO (4) remains an interesting facet in the
genetic and clinical investigation of the disorder. There are numerous
examples of both asymptomatic gene carriers and severely affected
individuals within the same family (3, 4, 9). The determination that
multiple genes for ADO exist may help to shed light on the issue of
clinical variability among family members who presumably carry the same
disease allele. It is possible that if the ADO genes at both loci
interact, functional polymorphisms at the nonmutated locus may
influence the presence and/or severity of disease in individuals who
have a mutant allele at the other locus. Although speculative at the
present time, this idea may be addressed after the isolation of the ADO
genes from both loci.
In summary, we demonstrate in this report that locus heterogeneity
exists for ADO; therefore, alterations in at least two genes can give
rise to the same phenotype. Elucidation of both ADO genes will provide
important insight into the pathogenesis of ADO and will improve our
understanding of the regulation of the homeostatic mechanisms involved
in osteoclast function and skeletal resorption. Cloning the genes may
also reveal potential therapeutic targets and thus help to improve the
treatment of ADO patients.
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Footnotes
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1 This research was supported by a grant from the Charles E. Culpeper
Foundation, by NIH Grants AR42228 and AG05793, and by a Department of
Medical and Molecular Genetics PHS Training Grant T32-HD07373
(D.L.K.). 
Received October 16, 1998.
Revised December 30, 1998.
Accepted January 5, 1999.
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Abstract
Introduction
