Vitamin D Receptor Gene Polymorphisms Predict Acquired Resistance to Clodronate Treatment in Patients with Paget’s Disease of Bone

Abstract

Bisphosphonates are first-choice drugs for treatment of Paget’s disease of bone (PDB); nevertheless, acquired resistance to
bisphosphonate therapy has been described in PDB patients. The 1,25(OH)2D3/vitamin D receptor (VDR) system influences the effectiveness of antiresorptive treatments in metabolic bone disorders. This
study evaluated the relationship between acquired resistance to clodronate treatment and BsmI, TaqI, and FokI VDR polymorphisms in Caucasian patients with polyostotic PDB (n=84). We also evaluated the influence of mutations in exons 7 and 8 of the sequestosome 1 (SQSTM1) gene on the occurrence of this phenomenon. All patients were treated from diagnosis for several cycles with intravenous clodronate
infusion (1500mg/cycle). Acquired resistance to clodronate treatment was defined as the failure of total alkaline phosphatase
serum levels to be suppressed to at least 50% of the patient’s previous highest levels during a subsequent treatment course
with the same compound, which produced a>50% response after the first exposure. During an observation period of 10.6±2.7years,
31 PDB patients (36.9%) showed acquired resistance to clodronate. It was observed that the bb and TT
VDR genotypes as well as a lower persistence of the biochemical response to the first treatment course were significantly and
independently associated with the risk of developing resistance to clodronate treatment. SQSTM1 gene mutations, considered altogether, did not influence the occurrence of this phenomenon. Our results indicate that 3′VDR allelic variants and duration of biochemical response to the first treatment course are independent predictors of acquired
resistance to clodronate treatment in patients with polyostotic PDB.

Full text

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Vitamin D Receptor Gene Polymorphisms Predict Acquired
Resistance to Clodronate Treatment in Patients with Paget’s
Disease of Bone
Giuseppe Mossetti Æ Æ Luigi Gennari Æ Æ Domenico Rendina Æ Æ
Gianpaolo De Filippo Æ Æ Daniela Merlotti Æ Æ Vincenzo De Paola Æ Æ
Pina Fusco Æ Æ Teresa Esposito Æ Æ Fernando Gianfrancesco Æ Æ
Giuseppe Martini Æ Æ Ranuccio Nuti Æ Æ Pasquale Strazzullo
Received: 29 April 2008/Accepted: 29 October 2008/Published online: 20 November 2008
? Springer Science+Business Media, LLC 2008
Abstract
treatment of Paget’s disease of bone (PDB); nevertheless,
acquired resistance to bisphosphonate therapy has been
described in PDB patients. The 1,25(OH)2D3/vitamin D
receptor (VDR) system influences the effectiveness of anti-
resorptivetreatmentsinmetabolicbonedisorders.Thisstudy
evaluated the relationship between acquired resistance to
clodronate treatment and BsmI, TaqI, and FokI VDR poly-
morphisms in Caucasian patients with polyostotic PDB
(n = 84). We also evaluated the influence of mutations in
exons 7 and 8 of the sequestosome 1 (SQSTM1) gene on the
occurrence of this phenomenon. All patients were treated
from diagnosis for several cycles with intravenous clodro-
nate infusion (1500 mg/cycle). Acquired resistance to
clodronate treatment was defined as the failure of total
alkalinephosphataseserumlevelstobesuppressedtoatleast
50% of the patient’s previous highest levels during a sub-
sequent treatment course with the same compound, which
produced a[50% response after the first exposure. During
an observation period of 10.6 ± 2.7 years, 31 PDB patients
Bisphosphonates are first-choice drugs for
(36.9%) showed acquired resistance to clodronate. It was
observedthatthebbandTTVDRgenotypesaswellasalower
persistenceofthe biochemicalresponsetothefirst treatment
course were significantly and independently associated with
the risk of developing resistance to clodronate treatment.
SQSTM1 gene mutations, considered altogether, did not
influence the occurrence of this phenomenon. Our results
indicate that 30VDR allelic variants and duration of bio-
chemical response to the first treatment course are
independent predictors of acquired resistance to clodronate
treatment in patients with polyostotic PDB.
Keywords
Sequestosome 1 ? Genetic variability ? Clodronate ?
Acquired resistance
Paget’s bone disease ? Vitamin D receptor ?
Paget’s disease of bone (PDB; OMIM 602080) is a dis-
order of unknown aetiology characterized by an anarchic
bone turnover which affects one or more sites throughout
the skeleton. This disorder is more likely sustained by
osteoclasts (OCL), which show several morphological
and physiological abnormalities [1]. In particular, the
early OCL precursors from PDB patients appear to be
hyperresponsive to ligand of receptor activator of nuclear
factor (NF)-(B (RANKL) and to 1,25(OH)2D3 [2, 3].
Kurihara and coworkers have demonstrated that hyper-
responsivity to 1,25(OH)2D3 is dependent on increased
expression of TATA box-associated factor II (TAFII)-17,
a vitamin D receptor (VDR) coactivator, and that the
TEI-9647(25-[23S]dehydro-1a-hydroxyvitamin
26,23-lactone), an antagonist of the VDR-mediated dif-
ferentiation of human leukemia (HL-60) cells, inhibits
OCL formation induced by 1,25(OH)2D3 from pagetic
bone marrow cells [4, 5]. Based on this experimental
D(3)-
G. Mossetti (&) ? D. Rendina ? P. Strazzullo
Department of Clinical and Experimental Medicine, Federico II
University Medical School, Via S. Pansini 5, Naples 80131, Italy
e-mail: giumosse@unina.it
L. Gennari ? D. Merlotti ? V. De Paola ? G. Martini ? R. Nuti
Department of Internal Medicine, Endocrine-Metabolic Sciences
and Biochemistry, University of Siena, Siena, Italy
G. De Filippo
Pediatric Endocrinology Unit, Gaetano Rummo Hospital,
Benevento, Italy
P. Fusco ? T. Esposito ? F. Gianfrancesco
Institute of Genetics and Biophysics ‘‘Adriano Buzzati-
Traverso’’, Italian National Research Council, Naples, Italy
123
Calcif Tissue Int (2008) 83:414–424
DOI 10.1007/s00223-008-9193-7

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evidence, they suggested that PDB should be considered
a VDR coactivator disease [6].
Other than its possible role in PDB pathogenesis, the
1,25(OH)2D3/VDR system plays a major role in bone
homeostasis [7]. Genetic variability of the VDR gene may
influence several aspects of bone metabolism, including the
responses to calcium intake, to calcium and vitamin D
supplementation, to hormone replacement therapy, and to
bisphosphonate treatment, in normal and osteoporotic
subjects [7–17]. Bisphosphonates are the treatment of
choice for patients with PDB. They suppress the abnor-
mally increased rate of bone remodeling by decreasing
OCL activity, and induce clinical and biochemical remis-
sion in the majority of PDB patients [1, 18, 19]. Although
disease remission can be maintained for a long time after
discontinuation of treatment, PDB recurrences occur fre-
quently. These recurrences can adequately respond to a
new bisphosphonate course, but several studies have
described the phenomenon of impaired response to con-
secutive bisphosphonate
resistance to bisphosphonate [20–25].
In view of the biological properties of the 1,25(OH)2D3/
VDR system and the described hypersensitivity of pagetic
OCLs to 1,25(OH)2D3, we investigated whether VDR
genetic variability plays a role in the phenomenon of
acquired resistance to bisphosphonate treatment in patients
with polyostotic PDB treated since diagnosis with several
cycles of intravenous clodronate infusion. Moreover, con-
sidering the role of mutations in the gene sequestosome 1
(SQSTM1; also known as p62; OMIM 601530) in PDB
pathogenesis, we also performed sequence analysis of
exons 7 and 8 of the SQSTM1 gene. All PDB-related
SQSTM1 mutations are located in these regions; all of them
cause an amino acid substitution in the SQSTM1 ubiquitin-
binding domain or generate proteins with a partially or
entirely truncated ubiquitin-binding domain. Functional
studies have demonstrated that loss of the capacity for
ubiquitination is common to all PDB-related SQSTM1
mutations when the biological properties of the SQSTM1
protein are analyzed at physiological temperature [26].
treatments,i.e.,acquired
Patients and Methods
Patients
We investigated 84 Caucasian patients (48 males and 36
females; mean age, 60.2 ± 9.1 years) with polyostotic
PDB, all born in Campania, a region of southern Italy
characterized by an elevated clinical severity of disease
[27]. The entire cohort belongs to a group of pagetic out-
patients referred to the Department of Clinical and
Experimental Medicine, Federico II University of Naples.
The diagnosis was based on unequivocal evidence of the
radiological and scintigraphic features of PDB [18]. Dis-
ease extension, expressed as the number of affected sites,
was evaluated according to the criteria proposed by Davie
et al. [28].
Before and 16 weeks after each clodronate therapeutic
cycle, a fasting venous blood sample was drawn and serum
ionized calcium, magnesium, and phosphate, total alkaline
phosphatase (ALP), intact parathyroid hormone, 25OHD3,
and 1,25(OH)2D3 were determined. Twenty-four-hour
urine samples were also collected and analyzed for creat-
inine and hydroxyproline values. The determination of
these serum and urinary parameters were performed as
previously described [29]. To evaluate the prevalence of
VDR allelic variants in the general population of southern
Italy, 387 unrelated age- and sex-matched control subjects,
without a known personal or family history of PDB and
without biochemical and clinical evidence of PDB, were
randomly recruited among members of the Postgraduate
School of Internal Medicine family reference panel (one
per family) and among patients’ spouses.
Assessment of Clodronate Efficacy
All enrolled patients have been treated since diagnosis with
several cycles of intravenous clodronate infusion (300 mg/
day in 500 ml of saline solution infused over 3 h for
5 days) according to Yates and coworkers [30]. The
sequential changes in serum levels of total ALP (normal
range, 98–275 U/L) were used to assess the long-term
pharmacological response to clodronate treatments [18].
The acquired resistance to clodronate treatment was
defined as the failure of total ALP to be suppressed to at
least 50% of the patient’s previous highest levels (usually
the pre-any-treatment level) during a subsequent treatment
course with the same compound (clodronate), which pro-
duced a[50% response after the first exposure [20].
Response to treatment was defined as a reduction of at least
75% in total ALP excess (the difference from the midpoint
of the reference range) [31]. A calcium dietary intake of
1000 mg/day and a vitamin D supplementation (800 IU
cholecalciferol per day) were assured during the entire
clinical follow-up.
Exclusion Criteria
Patients with abnormal serum transaminase levels (which
might impair the interpretation of total serum ALP) and/or
neoplastic bone disorders (either related or not related to
PDB) were excluded from the study. Moreover, given the
reported high prevalence of familial PDB cases in southern
Italy [27], only one member of each PBD family was
admitted to the study, based on the date of enrollment, to
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avoid a clustering effect on results. The diagnosis of
familial disease was excluded or confirmed after an eval-
uation of all first-degree relatives of each enrolled patient,
as described previously [27].
The procedures used in the study were in accordance
with the guidelines of the Helsinki Declaration on human
experimentation. The protocol was approved by the local
Ethics Committee of Federico II University of Naples. The
purpose of the research and the procedures included in the
protocol were explained to all patients and written
informed consent was obtained before they entered the
study.
VDR Genotyping
According to results from previous functional studies by
Whitfield and coworkers, we decided to perform a con-
textual evaluation of genetic polymorphisms located in
both the 30and the 50regions of the VDR gene [32]. In fact,
the 30VDR polymorphisms (BsmI and TaqI) may alter
mRNA stability or, more likely, the efficiency of its
translation into VDR protein, whereas FokI 50VDR poly-
morphism affects the interaction between VDR and TAFII
[32]. DNA extraction and genotypic analysis of BsmI,
TaqI, and FokI polymorphisms were performed by
restriction endonuclease analysis as described previously
[33]. All PCR products were verified by sequence analysis.
Genotypes were defined as B, T, and F (in the absence of
restriction sites) or b, t, and f (in the presence of restriction
sites). All patients were genotyped for common 30- and 50-
VDR
polymorphisms.
BsmI
(rs731236) polymorphisms are located, in linkage dis-
equilibrium, in the 30region of the VDR gene. FokI
polymorphism (rs10735810), located in the 50region of the
VDR gene, is not linked to the 30polymorphisms and alters
the transcription initiation site, resulting in a protein three
amino acids shorter in length with higher activity as a
transcription factor [8, 9].
(rs1544410)and
TaqI
SQSTM1 Genotyping
Exons 7 and 8 of the SQSTM1 gene were amplified using,
respectively, two pairs of primers located in the flanking
introns: 50-CATGCGTGCTCCCCGACTGT-30/50-GCCCT
GCAGTGGAGAACATC-30for exon 7 and 50-CTCTGG
GCAGGCTCGGACAC-30/50-CTTGCACCCTAACCCCT
GAT-30for exon 8. We performed PCRs (25 ll) using Taq
DNA polymerase (1 U; Fermentas), 1 9 buffer, dNTPs
(0.2 mM; Amersham), primers (0.5 lM), and DNA
(50 ng). PCR conditions were as follows: initial denatur-
ation at 94?C for 3 min, followed by 35 cycles at 94?C for
30 s and 62?C for 30 s, extension at 72?C for 45 s, and a
final extension for 10 min at 72?C. Samples were then
ExoSap digested (Amersham) and sequenced using the Big
Dye Terminator Ready Reaction Kit (Applied Biosystems).
Sequencing reactions were performed on a 9700 Thermal
Cycler (Applied Biosystems) for 25 cycles at 95?C for
10 s, 60?C for 5 s, and 60?C for 2 min. After the
sequencing, each reaction was column-purified (Amer-
sham) to remove excess dye terminators. Sequencing of the
products was performed on an ABI prism 3710 Genetic
Analyser (Applied Biosystems).
Statistical Analysis
Statistical analysis was performed using the SPSS (SPSS
Inc., Chicago, IL, USA) statistical package (version 11.5).
Data in tables are presented as mean ± standard deviation
for continuous variables and as absolute and percentage
values for discrete variables. Patients who developed
acquired resistance to clodronate treatment were compared
to those who did not. The unpaired Student’s t-test was
used for continuous variables to evaluate differences
between groups. For comparison of discrete variables, the
chi-square test or Fisher’s exact test was used. Logistic
regression analysis was used to evaluate the independent
role of clinical and laboratory variables related to the
occurrence of acquired resistance to clodronate treatment.
Moreover, Cox analysis was performed to assess the vari-
ables related tothe time-dependent
occurrence of acquired resistance to clodronate treatment.
To estimate VDR haplotype frequencies, the computer
program Estimated Haplotype (EH; Linkage Program
version 5.1) was used according to criteria proposed by Ott
[34]. All statistical tests were two-sided. A p-value\0.05
was considered statistically significant. The p-value of this
test is asymptotically equal to the p-value obtained in Ar-
mitage’s trend test (ATT). The ATT takes into account
genotypes rather than alleles, avoiding a possible bias
caused by doubling the sample size. It assumes an additive
(or codominant) disease model where all disease alleles
have an equal and independent contribution to disease risk.
The ATT is valid and powerful under a broad range of
disease mechanisms. Allele and genotype frequencies,
dominant and recessive genetic models, and odds ratios
were calculated to characterize the distribution of distinct
genotypes in different phenotypic subgroups of the popu-
lation(using theFINETTI
http://ihg.ghs.de/cgi-bin/hw/hwa1.pl).
Assuming a resistance rate of 30–40% [23] and an
allelic frequency approaching 50% (as observed in the
overall pagetic cohort and in controls for BsmI and TaqI
polymorphisms), our sample had 70–80% power to detect a
25% difference in allelic distribution between responders
and nonresponders.
likelihood of
program,availableat:
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Results
Response to Pharmacological Treatment
The clinical characteristics of PDB patients with or without
acquired resistance to clodronate therapy are reported in
Tables 1 and 2. Gender, age at enrollment, age at PDB
diagnosis, extension of disease, place of birth and resi-
dence, occurrence of familial disease, time of observation,
and serum levels of relevant biochemical parameters at
PDB diagnosis were not different between the study
groups.
Overall, during an observation period of 10.6 ±
2.7 years, 53 PDB patients (64.1%) continued to respond to
clodronate, while 31 (36.9%) developed acquired resis-
tance to the treatment after an average treatment period. As
reported in Table 2, the pharmacological response to
clodronate treatment was achieved in all PDB patients after
the first course, and levels of serum total ALP or other
biochemical variables were not significantly different at
this time between the groups of patients that subsequently
developed resistance or continued to respond to treatment
(p[0.05, ANOVA). In particular, 16 weeks after the first
clodronatecourse,a47.8 ± 10.1
decrease in total ALP was observed in PDB responder and
nonresponder groups, respectively (p\0.01 vs. baseline,
t-test for paired samples). However, the durations of
pharmacological response between the first and the second
clodronate treatment, expressed as months, were signifi-
cantly lower in resistant compared to responder PDB
patients (p\0.01 for both; Table 1).
and 54.3 ± 9.1%
As reported in Table 2, after the second clodronate
course, serum levels of total ALP before and after treat-
ment were significantly higher in resistant PDB patients
compared to responders (p\0.01, ANOVA). After the
third clodronate course, serum levels of total ALP after
treatment were significantly decreased compared to those
observed before treatment in responder PDB patients
(p\0.01, t-test for paired samples) but not in resistant
PDB patients.
Genetic Analysis
Four different mutations in the SQSTM1 gene were
detected in 15 PDB patients (17.8%; 6 females, 4 with
familial disease): the mutations A427D and P387L were
detected everyone in one patient, while the mutations
G425E and P392L were detected in 3 and 10 PDB patients,
respectively [35, 36]. To maximize the power of the sta-
tistical analyses, and considering the common mechanism
by which SQSTM1 mutations were related to PDB patho-
genesis [26], we considered these mutations altogether as
‘‘SQSTM1 mutations.’’ The prevalence of SQSTM1 muta-
tions was similar in PDB patients with versus without
acquired resistance to clodronate treatment (Table 1).
The prevalence of different VDR genotypes and the
respective allelic frequencies are reported in Table 3. In
both PDB patients and controls the VDR allelic frequencies
and estimated VDR haplotype frequencies were similar and
comparable to previously published data from Caucasian
unrelated subjects [37] as well as to data previously
observed in healthy subjects from the same geographic area
Table 1 Clinical characteristics of PDB patients with (resistant) or without (responders) acquired resistance to clodronate therapy: mean ± SD
Resistant (n = 31) Responders (n = 53)
M/F17:1431:22
Mean age at enrollment (year)
Paget’s involvement (no. of sites)a
58.2 ± 8.961.1 ± 9.4
6.5 ± 2.6 5.4 ± 2.7
Place of birth (n)
Rural districts 15 (48.4%)22 (41.5%)
Urban districts16 (51.6%)31 (58.5%)
Mean age at diagnosis (year)48.1 ± 7.9 50.2 ± 8.5
SQSTM1 mutations 5 (16.1%)10 (18.9%)
Familial disease (%) 5 (16.1%)
6.2 ± 1.8b
4.9 ± 2.1b
8 (15.1%)
Duration of response to first treatment (months)
8.6 ± 2.8
Duration of response to second treatment (months)
8.5 ± 3.0
Note: All patients were treated since diagnosis with several cycles of intravenous clodronate infusion (300 mg/day in 500 ml 0.9% NaCl infused
over 3 h for 5 days; total dose of clodronate administered in each cycle, 1.5 g) [30]. Acquired resistance to clodronate treatment was defined as
failure of the total ALP to be suppressed to at least 50% of the patient’s previous highest level (usually the pre-any-treatment level) during a
subsequent treatment course with the same compound (clodronate), which produced a[50% response after the first exposure [20]
aThe number of sites involved by PDB was evaluated according to criteria proposed by Davie et al. [28]
bSignificantly different compared to responder PDB patients (p\0.001, ANOVA)
G. Mossetti et al.: Pharmacogenomics in Paget’s Bone Disease417
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Table 2 Biochemical parameters in PDB patients with (resistant) or without (responders) acquired resistance to clodronate therapy before and after each therapeutic cycle: mean ± SD
Parameter (control value)
PDB patients
Clodronate treatment (300 mg/day i.v. for 5 days; total dose in each cycle, 1.5 g)
1st cycle
2nd cycle
3rd cycle
Last cycle
Day 0
Day 120
Day 0
Day 120
Day 0
Day 120
Day 0
Day 120
tALP (198 ± 57 U/L)
Resistant
2074 ± 600
1083 ± 241a
1680 ± 487b
1239 ± 221a,b
1750 ± 411b
1440 ± 280b
2017 ± 357b
2004 ± 387b
Responder
1914 ± 855
876 ± 271a
1007 ± 245
594 ± 185a
624 ± 205
371 ± 125a
471 ± 105
265 ± 95a
25(OH)D3(79.6 ± 3.1 nmol/L)
Resistant
77.1 ± 11.9
81.4 ± 10.4
85.6 ± 14.1
79.8 ± 12.5
72.8 ± 10.5
80.7 ± 12.3
69.1 ± 12.8
76.4 ± 9.9
Responder
74.7 ± 2.1
78.5 ± 9.5
80.6 ± 11.3
82.1 ± 12.6
71.8 ± 10.6
74.5 ± 10.9
65.7 ± 9.9
71.5 ± 10.2
1,25(OH)2D3(91.1 ± 11.3 pmol/L)
Resistant
87.2 ± 10.6
87.3 ± 10.9
85.6 ± 12.1
84.9 ± 13.2
77.1 ± 13.8
81.5 ± 12.5
71.9 ± 14.0
73.9 ± 13.5
Responder
84.7 ± 11.7
91.1 ± 11.3
87.2 ± 10.6
85.1 ± 12.1
79.5 ± 13.8
82.2 ± 4.1
75.6 ± 12.9
80.0 ± 12.6
PTH (2.5 ± 0.9 pmol/L)
Resistant
2.7 ± 0.7
2.8 ± 0.3
2.5 ± 0.4
2.6 ± 0.4
2.7 ± 0.4
2.5 ± 0.4
2.7 ± 0.4
2.5 ± 0.4
Responder
2.7 ± 0.3
2.8 ± 0.3
2.5 ± 0.4
2.5 ± 0.4
2.7 ± 0.4
2.5 ± 0.4
2.7 ± 0.3
2.4 ± 0.4
Calcium (1.21 ± 0.01 nmol/L)
Resistant
1.21 ± 0.02
1.19 ± 0.02
1.20 ± 0.02
1.18 ± 0.03
1.18 ± 0.02
1.21 ± 0.02
1.19 ± 0.03
1.20 ± 0.03
Responder
1.22 ± 0.01
1.20 ± 0.02
1.21 ± 0.02
1.19 ± 0.02
1.18 ± 0.03
1.22 ± 0.02
1.20 ± 0.02
1.22 ± 0.02
Magnesium (0.97 ± 0.08 mmol/L)
Resistant
0.89 ± 0.09
0.85 ± 0.10
0.87 ± 0.09
0.86 ± 0.08
0.85 ± 0.12
0.91 ± 0.11
0.87 ± 0.10
0.90 ± 0.09
Responder
0.91 ± 0.07
0.88 ± 0.08
0.90 ± 0.09
0.89 ± 0.08
0.88 ± 0.11
0.92 ± 0.09
0.91 ± 0.08
0.92 ± 0.09
Phosphate (1.16 ± 0.25 mmol/L)
Resistant
1.14 ± 0.19
1.13 ± 0.17
1.18 ± 0.20
1.15 ± 0.18
1.19 ± 0.14
1.22 ± 0.15
1.15 ± 0.21
1.19 ± 0.18
Responder
1.13 ± 0.21
1.13 ± 0.18
1.17 ± 0.19
1.14 ± 0.17
1.16 ± 0.18
1.18 ± 0.19
1.21 ± 0.20
1.14 ± 0.18
Urinary Hp/Cr (24.6 ± 5.1 lmol/mmol)
Resistant
58.2 ± 3.1
40.1 ± 3.5a
47.5 ± 3.6
38.7 ± 3.8a
49.5 ± 3.6§
45.8 ± 4.1§
60.3 ± 3.4§
61.4 ± 4.7§
Responder
57.5 ± 3.2
39.8 ± 3.4a
46.0 ± 4.2
36.2 ± 2.9a
40.8 ± 3.8
31.9 ± 2.7a
34.5 ± 3.0
27.1 ± 2.5a
Note: tALP, total alkaline phosphatase serum levels; PTH, parathyroid hormone; Hp/Cr, urinary hydrossiproline/creatinine ratio. Control values were determined in 387 age- and sex-matched
control subjects recruited among members of the Postgraduate School of Internal Medicine family reference panel (one per family) and among patients’ spouses
aSignificantly different compared to pretreatment values (p\0.05, t-test for paired samples)
bSignificantly different compared to responder PDB patients (p\0.05, ANOVA)
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[12–14, 33]. The prevalence of BsmI genotypes was also
comparable to that reported by Donath et al. in a cohort of
69 Caucasian PDB patients [38]. The frequencies of VDR
genotypes met the Hardy-Weinberg equilibrium in both
PDB patients (p = 0.088, 0.129, and 0.696, for BsmI, TaqI,
and FokI genotypes, respectively) and healthy controls
(p = 0.487, 0.246, and 0.337 for BsmI, TaqI, and FokI
genotypes, respectively). Interestingly, the prevalences of
TT and bb VDR genotypes and of T and b VDR alleles
among pagetic patients who developed acquired resistance
to clodronate treatment were significantly higher compared
to those observed among PDB patients without acquired
resistance to clodronate treatment (p\0.023 in all cases,
(v2test). Conversely, FokI VDR genotypes and alleles
showed a similar prevalence between the study groups
(Table 3). When the clinical characteristics of PDB
patients (summarized in Tables 1, 2) were analyzed
according to different VDR genotypes, we did not find any
significant difference between responder and nonresponder
groups. Moreover, the prevalence of SQSTM1 mutations
was similar within the 30- and 50-VDR genotypes examined.
Allelic and genotype p-values and odds ratio (ORs) as
well as dominant and recessive genetic models were cal-
culated for each SNP using FINETTI software. The results
are reported in Table 4. The BsmI b allele and bb genotype
frequencies in PDB-resistant patients were significantly
higher than in PDB responder patients (B vs. b allele fre-
quencies, p = 0.003; BB vs. bb genotype frequencies,
p = 0.006). This analysis was suggestive of a recessive
model of transmission for BsmI. No significant differences
were found for either the BsmI allele or the genotype fre-
quency comparing PDB-resistant patients vs. controls.
Similar results were obtained analyzing TaqI VDR poly-
morphisms. Both T allele and TT genotype frequencies
were significantly higher in PDB-resistant patients com-
pared to PDB-responder patients (t vs. T allele frequencies,
p = 0.003; tt vs. Tt ? TT genotype frequencies, p =
0.006). This analysis was suggestive of a dominant model
of transmission for TaqI. No significant differences were
found for either the TaqI allele or the genotype frequency
comparing PDB-resistant patients vs. controls. A strong
statistical significance (p = 0.006) was also obtained using
the ATT for both BsmI and TaqI.
Consideringthelinkage
between BsmI and TaqI VDR polymorphisms, we used two
different models of multinomial logistic regression analysis
to evaluate the relative impact of each VDR polymorphism
on the risk of developing acquired resistance to clodronate
disequilibriumoccurring
Table 3 Prevalence of BsmI, TaqI, and FokI VDR polymorphisms and respective allelic frequencies in PDB patients with (resistant) or without
(responders) acquired resistance to clodronate therapy and in healthy control subjects
VDR genotype & allelePatients with PDBControls (n = 387)
v2p-value
Responders (n = 53) Resistant (n = 31)All (n = 84)
BsmI
BB
23 (43.4%)5 (16.1%) 28 (33.3%)112 (28.9%)
Bb
20 (37.7%) 14 (42.2%)
12 (38.7%)a
34 (40.5%) 186 (48.1%)0.450
bb
10 (18.9%)
22 (26.2%)89 (23.0%)
B
62.338.7
61.3a
53.653.0
b
37.7
46.447.0
TaqI
TT
11 (20.7%) 12 (38.7%)a
23 (27.4%) 98 (25.3%)
Tt
20 (37.7%)15 (48.4%) 35 (41.7%) 182 (47.1%)0.667
tt
22 (41.5%)4 (12.9%)
62.9a
26 (30.9%)107 (27.6%)
T
39.6
48.248.8
t
60.4 37.1 51.8 51.2
FokI
FF
21 (39.6%)11 (35.5%) 32 (38.1%)146 (37.7%)
Ff
23 (43.4%)15 (48.4%)38 (45.2%) 176 (45.5%)0.998
ff
9 (17.0%)5 (16.1%)14 (16.7%) 65 (16.8%)
F
61.359.760.760.5
f
39.640.339.339.5
Note: Cross-genotyping in the 50-end and the 30-end VDR loci showed a similar prevalence of FokI polymorphisms across BsmI and TaqI VDR
sites. The frequencies of VDR genotypes met the Hardy-Weinberg equilibrium in both PDB patients (p = 0.088, 0.129, and 0.696 for BsmI, TaqI,
and FokI genotypes, respectively) and healthy controls (p = 0.487, 0.246, and 0.337 for BsmI, TaqI, and FokI genotypes, respectively). v2p-
values for comparison between all PDB patients and healthy controls
aSignificantly higher compared to PDB without acquired resistance to clodronate
G. Mossetti et al.: Pharmacogenomics in Paget’s Bone Disease419
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therapy after the first course, accounting for duration of
biochemical response to first clodronate treatment, gender,
age at enrollment, age at PDB diagnosis, extension of
disease, place of birth and residence, occurrence of familial
disease and SQSTM1 mutations, time of observation,
duration of pharmacological response to second clodronate
treatment, and serum levels of relevant biochemical
parameters at diagnosis. The results indicate that both the
TaqI and the BsmI VDR polymorphisms, as well as the
duration of biochemical response to the first treatment
course, but not the Fok1 VDR polymorphisms, were inde-
pendent predictors of acquired resistance to clodronate
treatment (Table 5).
To evaluate the time-dependent likelihood of the
occurrence of acquired resistance to clodronate treatment,
we decided to perform a Cox regression analysis. Using
this mathematical model, the likelihood was corrected for
gender, age at enrollment, age at PDB diagnosis, extension
of disease, place of birth and residence, occurrence of
familial disease, and SQSTM1 mutations. As shown in
Fig. 1, TT and bb VDR genotypes as well as a shorter (\6-
month) persistence of the biochemical response to the first
treatment course were the variables related to a significant
risk of developing acquired resistance to clodronate treat-
ment over time.
Discussion
The most important and intriguing finding of the present
study is the observation that TaqI and BsmI polymorphisms
in the VDR gene predicted the occurrence of acquired
Table 4 Association results of BsmI, TaqI, and FokI VDR polymorphisms in resistant PDB patients (group 1), responder PDB patients (group 2),
and healthy controls (group 3)
Allele B vs. b
Genotype BB vs. Bb
Genotype BB vs. bb
Genotype BB vs. Bb ? bb
Armitage’s test
OR95% CI
p
OR 95% CI
p
OR95% CI
p
OR 95% CI
p
OR
p
BsmI
Group 1 vs. 22.61.3–4.90.003 3.20.9–10.5 0.04 5.51.5–19.8 0.0063.9 1.3–11.90.012.3 0.006
Group 1 vs. 3 0.9 0.7–1.30.8 0.70.4–1.2 0.20.9 0.5–1.8 0.90.8 0.5–1.3 0.40.9 0.9
Allele t vs. T
Genotype tt vs. Tt
Genotype tt vs. TT
Genotype tt vs. Tt ? TT
OR95% CI
p
OR95% CI
p
OR95% CI
p
OR95% CI
p
TaqI
Group 1 vs. 22.51.3–4.9 0.0034.1 1.1–14.5 0.026 1.5–22.90.006 4.71.4–15.6 0.006 2.40.006
Group 1 vs. 3 0.9 0.7–1.30.8 0.70.4–1.3 0.40.9 0.5–1.80.9 0.80.5–1.40.50.9 0.8
Allele F vs. f
Genotype FF vs. Ff
Genotype FF vs. ff
Genotype FF vs. Ff ? ff
OR95% CI
p
OR95% CI
p
OR95% CI
p
OR95% CI
p
FokI
Group 1 vs. 2 1.00.5–2.01.01.20.4–3.30.61.0 0.2–3.90.91.20.4–2.9 0.71.00.8
Group 1 vs. 3 0.90.7–1.40.9 0.90.5–1.6 0.90.90.5–1.9 0.90.9 0.6–1.6 0.90.9 0.9
Note: p-values for allele and genotype distributions are shown. To assess the dosage effect of possessing zero, one, or two copies of the risk
alleles (b, T, f; i.e., an additive effect), Armitage’s test for linear trend in proportions was performed on the genotype frequency data. Genetic risk
magnitudes (effect size) were estimated by calculating odds ratios (ORs) with 95% confidence intervals (CI). All statistical tests were two-tailed.
Statistical significance was defined as a p-value\0.05. Hardy-Weinberg equilibrium and dominant and recessive genetic models (using the
FINETTI program, available at: http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl) were analyzed. BsmI was suggestive of a recessive
model of transmission; TaqI, of a dominant model
Table 5 Multiple logistic regression of variables univariately associated with the occurrence of acquired resistance to clodronate treatment
Variable
b
SE
p
Adjusted HR (95%CI)
bb VDR genotype2.6590.8670.002 14.28 (2.61–78.02)
TT VDR genotype1.931 0.8790.028
\0.001
6.89 (1.23–38.67)
Duration of biochemical response to first clodronate treatment (months) –0.635 0.1750.53 (0.38–0.75)
Note: Data were corrected for gender, age at enrollment, age at PDB diagnosis, occurrence of familial disease and SQSTM1 gene mutations,
extension of disease, place of birth and residence, time of observation, and levels of biochemical parameters reported in Table 2
420 G. Mossetti et al.: Pharmacogenomics in Paget’s Bone Disease
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resistance to clodronate treatment, independently of age,
gender, age at diagnosis, extension of disease, place of
birth and residence, occurrence of familial disease, and
SQSTM1 mutations, in PDB patients who had a satisfactory
biochemical response to the first treatment course. A
shorter persistence of the biochemical response to the first
treatment course was also associated with the risk of
developing resistance over time. To the best of our
knowledge, this is the first description of biochemical and
genetic factors associated with the occurrence of acquired
resistance to bisphosphonate, other than pamidronate [25].
Conversely, we did not find any difference in the response
to clodronate treatment between patients with and those
without SQSTM1 mutation, even though only 15 mutations
were detected in our sample. Further studies in larger
samples will be necessary to address this issue fully.
Moreover, given the limited sample size, our study had a
reduced power to detect significant differences in VDR
haplotype distributions (associating 30and 50polymor-
phisms) between responder and nonresponder patients.
We previously reported that about a third of patients
treated with clodronate showed the phenomenon of
acquired resistance to treatment [23]. In a previous study,
Gutteridge and coworkers evaluated the occurrence of
acquired pharmacological resistance to bisphosphonate
treatment in pagetic patients [20]. They demonstrated that
when these patients were treated with the same amin-
obisphosphonate for two courses, some of them showed
similar nadir values of bone turnover markers, but with a
shorter remission time, after the second course. The nadirs
were significantly higher in a subgroup of these patients
after a third course of therapy. On the other hand, PDB
patients showing an acquired partial resistance to one
aminobisphosphonate (usually after two or more previous
courses) were still capable of remission after exposure to
another compound of the same class [20, 22, 23].
The mechanisms of acquired resistance to bisphospho-
nate treatment in PDB are actually poorly understood.
Lyles proposes two potential explanations for this phe-
nomenon [21]. First, bisphosphonate therapy may select a
subgroup of osteoclasts that become resistant to the apop-
totic effect of the drug. Second, continued therapy with a
bisphosphonate may induce a number of enzymes which
confer resistance to a subset of osteoclasts or their
Fig. 1 Variables related to the time-dependent likelihood of the occurrence of acquired resistance to clodronate treatment (Cox analysis)
G. Mossetti et al.: Pharmacogenomics in Paget’s Bone Disease421
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precursor cells. An alternative explanation has been sug-
gested by Papapoulos and colleagues. They propose that
the resistance to action of bisphosphonate in PDB is caused
by disease-related factors rather than decreased respon-
siveness of the bisphosphonate molecular target [25]. This
observation is supported by the findings that the response
to pamidronate could be impaired exclusively in patients
with extensive PDB [25].
Given the importance of the 1,25(OH)2D3/VDR system
in regulating bone homeostasis and the activity of pagetic
OCLs [2, 7–9], all the above mechanisms could be sig-
nificantly influenced by genetic variation at the VDR locus.
In fact, particular VDR variants may either indirectly or
directly induce the development of pagetic OCL that are
resistant to current bisphosphonate treatment. Further in
vitro and in vivo studies are required to test this hypothesis.
However, despite the high number of studies on population
samples, the influence of these polymorphisms on VDR
protein function and signaling remains largely unknown.
Polymorphisms at the 30end of the VDR gene do not
produce any change in the amino acid sequence of the
protein, making the hypothesis of functional differences in
VDR activity unlikely. An alternative hypothesis is that
these variants affect VDR gene transcription and/or mRNA
stability [39]. Interestingly, a tight linkage between BsmI or
TaqI variants (which are in strict linkage disequilibrium)
and differences in the length of a microsatellite poly(A)
repeat at the 30-UTR of the VDR gene, *1 kb upstream of
the poly(A) tail, a region which is important for mRNA
stability, has been described [10, 40]. Alleles b and T are
highly associated with a long length of poly(A) repeats,
while alleles B and t are linked to a short poly(A) length.
Experimental observations in human fibroblast lines evi-
denced increased activity of the long hVDR allele [41].
Theoretically, the exposed findings might also be indicative
of linkage disequilibrium between BsmI or TaqI polymor-
phisms and another functional variant within the VDR
locus or another nearby gene.
The strength of our study resides in our ability to eval-
uate a genetically homogeneous cohort living in a
relatively restricted geographical region, treated over time
with the same drug, and followed for more than 10 years,
ideal conditions to evaluate the impact of genetic variation
on the response to a pharmacological treatment. However,
the fact that the study focused on only one drug could also
represent a limitation. Clodronate is a non-nitrogen-con-
taining bisphosphonate, metabolically incorporated into the
nonhydrolyzable analogue of ATP (AppCCl2p) [42–44].
The intracellular accumulation of this metabolite inside
OCL inhibits ATP-dependent intracellular enzymes [42–
44]. On the contrary, aminobisphosphonates (such as
pamidronate, alendronate, risedronate, and ibandronate) are
able to inhibit the farnesyl diphosphonate synthase, an
enzyme in the mevalonate pathway [45–48]. Despite these
different biological properties, all bisphosphonates are able
to induce cellular apoptosis by caspase activation, and this
biological property is essential for inhibition of bone
resorption mediated by clodronate [49, 50]. In experi-
mental models, caspase-mediated apoptosis is dependent
on the 1,25(OH)2D3/VDR system [51]. To date, the pos-
sibilitythatour observations
clodronate therapy may be generalized to treatments with
other bisphosphonates remains to be determined. Another
limitation of the study could be identified in the restricted
size of our study cohort, which, however, includes only
PDB patients with poliostotic disease. Moreover, only one
member of each PBD family was admitted to the study, to
avoid a clustering effect on results, and we excluded
patients with PDB-related neoplasms. We also analyzed
VDR genotypes as independent variables, to maximize the
power of the statistical analyses. The results of these sep-
arate analyses were concordant: TT and bb VDR genotypes
and T and b VDR alleles, which are in strong linkage dis-
equilibrium, were able to predict the occurrence of
acquired resistance to clodronate treatment in PDB
patients. Since there are many SNPs across the VDR gene
locus and we only analyzed the most studied 30and 50
variants, the present study was able to capture only a small
amount of the variation across the gene. It is likely that
combination of haplotype blocks at the VDR gene in larger
samples, rather than single polymorphisms, could provide
more information in this context. Indeed, recent evidence
from population-based studies suggested intragenic inter-
action between polymorphisms in different VDR haplotype
blocks in protein transcription and activity [52, 53].
An exact comparison between our findings and those
provided by studies indicating an influence of VDR geno-
types on the effectiveness of antiresorptive treatments for
osteoporosis [12–16, 54] is difficult because drugs were
administered by different routes, at different dosages, and
in patients with different disorders. Moreover, previous
studies evaluating the pharmacological response to bis-
phosphonate treatment were performed using alendronate,
an aminobisphosphonate showing pharmacodynamic and
pharmacokinetic properties different from those of clodr-
onate. Despite these limitations, our results were in
agreement with those reported by Marc and coworkers,
who analyzed the influence of BsmI VDR genotypes on the
pharmacological response to oral etidronate, a non-nitro-
gen-containing bisphosphonate with pharmacodynamic and
pharmacokinetic properties similar to those of clodronate,
in osteoporotic patients [55].
In the expectation that pharmacogenomics will, hope-
fully soon, help in the planning of individualized drug
therapy in medical conditions having a genetic background
[56], a challenging perspective of our results is that the
in patientsreceiving
422G. Mossetti et al.: Pharmacogenomics in Paget’s Bone Disease
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evaluation of genetic variation at the VDR locus, integrated
with information from a diverse set of biomarkers, may be
used to better define therapeutic strategies in PDB and
other skeletal disorders [57].
Acknowledgments
Rendina for their statistical advice.
We are indebted to Pasquale and Italiacornelia
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