Article

Case-parent analysis of variation in pubertal hormone genes and pediatric osteosarcoma: a Children's Oncology Group (COG) study

Division of Pediatric Epidemiology and Clinical Research, University of Minnesota Minneapolis MN 55455.
International Journal of Molecular Epidemiology and Genetics (Impact Factor: 1.3). 12/2012; 3(4):286-93.
Source: PubMed
ABSTRACT
Osteosarcoma (OS) is a rare malignant bone tumor with an overall incidence rate of 4.6 cases per million children aged 0-19 years in the United States. While the etiology of OS is largely unknown, its distinctive age-incidence pattern suggests that growth and development is crucial in genesis. Prior studies have suggested that variants in genes in the estrogen metabolism (ESTR) and insulin-like growth factor/growth hormone (IGF/GH) pathways are associated with OS. We examined 798 single nucleotide polymorphisms (SNPs) in 42 genes from these pathways in a case-parent study (229 complete triads and 56 dyads) using buccal cell samples. Relative risks (RR) and 95% confidence intervals (CI) associated with transmitting one or two copies of the variant were estimated using log-linear models. After Bonferroni correction, 1 SNP within the ESTR pathway (rs1415270: RR = 0.50 and 8.37 for 1 and 2 vs. 0 copies, respectively; p = 0.010), and two SNPs in the IGF/GH pathway (rs1003737: RR = 0.91 and 0.0001 for 1 and 2 vs. 0 copies, respectively; p <0.0001 and rs2575352: RR = 2.62 and 0.22 for 1 and 2 vs. 0 copies; p < 0.0001) were significantly associated with OS incidence. These results confirm previous findings that variation in the estrogen metabolism and bone growth pathways influence OS risk and further support a biologically and epidemiologically plausible role in OS development.

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Int J Mol Epidemiol Genet 2012;3(4):286-293
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Original Article
Case-parent analysis of variation in pubertal hormone
genes and pediatric osteosarcoma: a Children’s
Oncology Group (COG) study
Jessica RB Musselman
1
, Tracy L Bergemann
2
, Julie A Ross
1,3
, Charles Sklar
4
, Kevin AT Silverstein
5
, Erica K
Langer
1,3
, Sharon A Savage
6
, Rajaram Nagarajan
7
, Mark Krailo
8
, David Malkin
9
, Logan G Spector
1,3
1
Division of Pediatric Epidemiology and Clinical Research, University of Minnesota, Minneapolis MN 55455;
2
Medtronic, Mounds View, MN 55112;
3
University of Minnesota Masonic Cancer Center, Minneapolis MN 55455;
4
Memorial Sloan-Kettering Cancer Center, New York City, New York 10065;
5
Supercomputing Institute for Ad-
vanced Computational Research University of Minnesota, 55455;
6
Clinical Genetics Branch, Division of Cancer
Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, MD 20852;
7
Divi-
sion of Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati OH 45229;
8
Department of Preventive
Medicine, University of Southern California, Los Angeles CA 90089;
9
Division of Hematology/Oncology, Depart-
ment of Pediatrics, The Hospital for Sick Children, University of Toronto, Toronto CA M5G 1X8
Received August 21, 2012; Accepted October 19, 2012; Epub November 15, 2012; Published November 30,
2012
Abstract: Osteosarcoma (OS) is a rare malignant bone tumor with an overall incidence rate of 4.6 cases per mil-
lion children aged 0-19 years in the United States. While the etiology of OS is largely unknown, its distinctive age-
incidence pattern suggests that growth and development is crucial in genesis. Prior studies have suggested that
variants in genes in the estrogen metabolism (ESTR) and insulin-like growth factor/growth hormone (IGF/GH) path-
ways are associated with OS. We examined 798 single nucleotide polymorphisms (SNPs) in 42 genes from these
pathways in a case-parent study (229 complete triads and 56 dyads) using buccal cell samples. Relative risks (RR)
and 95% condence intervals (CI) associated with transmitting one or two copies of the variant were estimated us-
ing log-linear models. After Bonferroni correction, 1 SNP within the ESTR pathway (rs1415270: RR = 0.50 and 8.37
for 1 and 2 vs. 0 copies, respectively; p = 0.010), and two SNPs in the IGF/GH pathway (rs1003737: RR = 0.91 and
0.0001 for 1 and 2 vs. 0 copies, respectively; p <0.0001 and rs2575352: RR = 2.62 and 0.22 for 1 and 2 vs. 0
copies; p < 0.0001) were signicantly associated with OS incidence. These results conrm previous ndings that
variation in the estrogen metabolism and bone growth pathways inuence OS risk and further support a biologically
and epidemiologically plausible role in OS development.
Keywords: Osteosarcoma, case-parent study, growth and development, insulin-like growth factor pathway, estro-
gen metabolism pathway
Introduction
Osteosarcoma (OS) is the most commonly
occurring bone cancer in children <20 years of
age, with about 440 new cases diagnosed
annually in the United States and Canada [1, 2].
Several lines of evidence link OS etiology to fac-
tors related to pubertal development and bone
growth. First, incidence of OS is distributed bi-
modally, with the primary peak occurring during
adolescence, corresponding to the pubertal
growth spurt and its accompanying increase in
endogenous sex and growth hormone levels
[3-5]. OS incidence peaks earlier in females,
which corresponds to the earlier onset of puber-
ty in females [6]; however, overall incidence is
higher in males, who are generally taller than
females. The association of OS incidence with
growth and puberty corroborates ndings of
associations among greater height [5, 7-12],
rate of growth [12], and birth weight [13, 14]
and increased risk of OS in both humans and
canines, although not all studies have found
such associations [15, 16]. Furthermore, the
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Pubertal hormone gene variants and osteosarcoma
287 Int J Mol Epidemiol Genet 2012;3(4):286-293
most common sites of OS are areas of rapid
bone growth during puberty [6, 17, 18].
There are few known causes of OS, and these
account for only a small proportion of cases.
The only widely accepted exogenous risk fac-
tors are high-dose ionizing radiation [19, 20]
and prior chemotherapy with alkylating agents
[19, 20]. In addition, there are several associ-
ated syndromes [21-23] including Li-Fraumeni
Syndrome [24-26], hereditary retinoblastoma
[27-29], Werner [30], Bloom [31], and
Rothmund-Thomson Syndromes [32, 33].
Because growth and puberty are heritable
traits, it is possible that other forms of genetic
variation may explain sporadic OS incidence.
In particular, evidence suggests that genes
underlying pubertal growth might be key in OS
etiology. Linear bone growth is under the inu-
ence of several genes, including those from the
sex steroid hormone control pathway (e.g.,
estrogen metabolism (ESTR)) and insulin-like
growth factor/growth hormone (IGF/GH) path-
way, which regulate rates of bone growth [34-
39] through release of estrogen, IGF1, and
growth hormones. The timing and extent of
adolescent bone growth is also under substan-
tial genetic control, with heritability estimates
ranging from 50 to 80% for the time of onset of
puberty and peak bone mineral density [40,
41]. Hence, variation in genes in the IGF/GH
and ESTR pathways are reasonable candidates
for exploration in OS etiology. A candidate gene
study by Mirabello et al. identied 12 SNPs in
the DNA repair gene and growth/hormone
pathways as being associated with OS in a
study of ~5000 tag SNPs in 255 candidate
genes [3]. In the same population, Savage et al.
identied two polymorphisms of the IGF2
receptor gene that were signicantly more fre-
quent in OS cases [42, 43]. That study included
adult and pediatric cases and a similar
approach has not been taken in a study com-
prised solely of pediatric OS cases.
In order to assess the association of OS with
genes putatively related to adolescent bone
growth, we conducted a case-parent study con-
sisting of nearly 300 cases of pediatric OS
recruited through the Children’s Oncology
Group and one or both of their biological par-
ents. Using these triads/dyads, we employed
log-linear regression to assess 798 SNPs
across 42 candidate genes in biologically plau-
sible pathways for an association with OS.
Materials and methods
Study population
OS cases were identied through the Childhood
Cancer Research Network (CCRN)--the registry
component of the Children’s Oncology Group
(COG) [44]. As part of registering all pediatric
patients with the COG data center, the CCRN
consent process allows for informed consent
for the release of personal identiers on
patients and parents along with permission to
contact in the future. Eligible cases were
patients with a primary diagnosis of OS (ICCC
9180-9200; OMIM #259500) at age <20 years
made at a North American COG institution
between December 24, 2007 and March 31,
2010, who had at least one living biological par-
ent willing to participate and understand
English or Spanish. Initial contact was through
an introductory letter followed by a telephone
call during which study staff summarized the
goals of the study and provided respondents
with an opportunity to ask questions and
assess eligibility. If consent over the telephone
was given, assent forms (if appropriate), buccal
cell collection kits (mouthwash or cytobrushes),
a paper questionnaire, and return mailers were
sent. The study sample consisted of the 290
cases who returned a buccal cell kit for them-
selves and at least one biological parent (mean
age at diagnosis 12.9 years). Thus, this study
population represents one of the largest and
highly powered studies for assessing genetic
variants associated with OS in a pediatric
population.
SNP selection
Tag SNPs were selected as follows: rst, tran-
scripts and gene boundaries for gene symbols
were obtained from Ensembl release 50. SNPs
were then found by uploading the list of gene
names to Ensembl’s BioMart, and then addi-
tional SNPs were obtained directly at hapmap.
org [45] that were 20,000 bp up and down-
stream of gene boundaries using the data from
the International HapMap Project Data Rel
23a/phase II Mar08 data le where tag SNP
data was downloaded from the CEU population
using the “tagger pairwise method” with an
R-square cutoff of 0.8 and a minor allele fre-
quency (MAF) cutoff of 0.0. The nal set of
SNPs were then chosen if they were annotated
as veried in the dbSNP or SNP500 databases
[46] or were coding SNPs predicted to be dele-
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Pubertal hormone gene variants and osteosarcoma
288 Int J Mol Epidemiol Genet 2012;3(4):286-293
terious [47, 48] or SNPs selected from the
Phase I HapMap data using the Tagger algo-
rithm with r
2
>0.8.
Genotyping assays
Buccal cell samples were stored at 4°C and
batched for DNA extraction. Samples were cen-
trifuged, cells pelleted and washed, and DNA
was extracted using the Puregene kit (Gentra
Systems, Minneapolis, MN). DNA was quanti-
ed, aliquoted and stored in hydration solution
at -20°C. The Sequenom platform iPLEX Gold
method was used for genotyping. Multiplexed
PCR was performed in 5μl reactions on a 384-
well plate. Reactions contain 0.5 U HotStar Taq
polymerase (Qiagen), 100 nM primers, 1.25X
HotStar Taq buffer, 1.625 mM MgC1
2
2, and
500 μM dNTPs. Enzyme activation was per-
formed at 94°C for 15 minutes, followed by
DNA amplication with 45 cycles at 94°C for 20
seconds, 56°C for 30 seconds, and 72°C for 1
minute, followed by a three minute extension at
72°C. Unincorporated dNTPs were removed
using shrimp alkaline phosphatase (0.3 U,
Sequenom, San Diego). SBE primers at concen-
trations from 0.625 μM to 1.25 μM and iPLEX
enzyme and buffers in 9 μl reactions were used
to carry out single base extension. Reactions
were desalted and SBE products were mea-
sured using the MassARRAY system. TYPER
software (Sequenom) was used to analyze
mass spectra in order to generate genotype
calls and allele frequencies.
DNA was quantied and normalized to a house-
keeping gene. A total of 866 SNPs were select-
ed and genotyped following our SNP selection
procedure. SNPs were excluded if they had less
than a 90% genotyping rate in our sample, were
non-variable, or had a sample MAF of < 1%.
Individuals were excluded if >10% genotypes
were missing. If the individual excluded was a
case, the entire triad/dyad was excluded. In
addition, an algorithm was created to assess
for discrepancy between the genotypes of the
parents and their associated case child.
Individuals were excluded whose genotypes
demonstrated >5% inconsistency between the
parents and the associated case child (e.g.
paternal genotype contains two copies of the
variant and the child’s genotype contains no
copies). A total of 798 tag SNPs, 186 complete
triads and 72 dyads met the above quality con-
trol criteria and were included for analysis. All
original candidate genes had at least one SNP
included in the nal analysis except for IGFBP6.
Statistical analysis
This study uses the case-parent design, which
involved enrolling and genotyping OS cases and
their parents [49, 50] and testing whether the
distribution of candidate susceptibility alleles
in cases signicantly deviates from what would
be expected by Mendelian inheritance. We
used a log-linear regression model to assess
for signicant over- or undertransmission of a
variant allele among OS cases and their biologi-
cal parents [51, 52]. Briey, the log-linear model
estimates relative risks (RR) of possessing 0, 1,
or 2 copies of the variant by assessing the joint
likelihood of the case, maternal, and paternal
genotypes where the statistical model is condi-
tioned on the presence of disease in the off-
spring. A likelihood ratio test (LRT) determines
the statistical signicance for each SNP vari-
ant. The least common allele among cases was
selected to serve as the variant for each SNP.
Families were informative if at least one biologi-
cal parent was available for genotyping.
Estimation and inference were performed in
the presence of missing parental data using a
likelihood-based algorithm [51]. Bonferroni cor-
rections were performed within each of two
pathways--the ESTR pathway (29 genes, 519
SNPs after quality control) and the IGF/GH
pathway (13 genes, 279 SNPs after quality
control)--to correct for multiple statistical com-
parisons. Statistical analyses were performed
using custom programs in R (http://www.r-pro-
ject.org).
Results
A summary of case identication, enrollment,
and DNA collection is provided in Figure 1.
During the eligibility period, a total of 660 OS
cases were identied at COG institutions. Of
those, families of 602 (91%) cases consented
to participation in the CCRN and potential
future contact for study participation. Twenty of
those who consented to CCRN were considered
ineligible due to lack of an available biological
parent (n=17) or lack of a parent who spoke
either English or Spanish (n=3). Of those who
were contacted and eligible (n=582), 445 fami-
lies (76% contacted) gave consent over the
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Pubertal hormone gene variants and osteosarcoma
289 Int J Mol Epidemiol Genet 2012;3(4):286-293
phone and were sent a DNA kit, of which 290
(65% sent) were returned.
Demographics and clinico-pathologic charac-
teristics of the cases are presented in Table 1.
A majority (95%) of the families came from the
United States. Cases were also primarily white
(86%). Tumors were most often located in the
long bones (92%). For the log-linear analysis,
six families were not included because there
was either a diagnosis of or a family history
consistent with Li-Fraumeni Syndrome. An
additional 30 triads were excluded due to
excessive missing genotyping data from the
case, and an additional 42 parents were exclud-
ed for the same reason. A further four fathers
were excluded due to discordance above our
5% cutoff between paternal and progeny geno-
types. A total of 68 SNPs were excluded, leav-
ing a total of 798 SNPs for analysis, 519 on the
ESTR pathway and 279 on the IGF/GH pathway.
After Bonferroni correction (for 519 SNPs with-
in ESTR and 279 within IGF/GH), two SNPs on
the IGF/GH pathway remained signicant (Table
the minor allele (un-corr p<0.0001; corr-p
=0.02).
Discussion
We employed a candidate gene based approach
in a case-parent study to assess the involve-
ment of genes in biologically plausible path-
ways for a possible association with pediatric
OS. The IGF/GH and ESTR pathways are rea-
sonable candidates for analysis due to previ-
ously established associations of growth and
puberty with OS. The heritability of growth and
hormone regulation provides additional ratio-
nale for examining genes in these pathways
[3-7, 10, 11, 40, 41].
After Bonferroni correction for multiple statisti-
cal tests, we identied one SNP in each of three
genes of interest as signicant. In the IGF/GH
pathway, the IGF2R gene (on chromosome 6)
plays a role in activating growth factor beta and
degrading IGF2, while IGFALS (on chromosome
16) increases the half life of IGF. SNPs in the
IGF2R gene were previously reported to be
Figure 1. Flow chart describing the identication and enrollment of the cases
that make up the study population from the initial population of childhood
osteosarcoma cases treated at Children’s Oncology Group institutions dur-
ing the eligibility period. Patient Identication, Contact, Consent, Enrollment,
and Participation in a case-parent study of childhood osteosarcoma; April 1,
2007-March 31, 2010.
2, Figure 2). The rst SNP,
rs1003737 is located in the
intronic region of the IGF2R
gene on chromosome 6, and
was associated with a RR of
0.91 and 0.0001 for one
and two copies versus no
copies, respectively, of the
minor allele (un-corr p<0.00-
01; corr-p <0.0001). The
second SNP, rs2575352, is
upstream of the IGFALS
gene on chromosome 16
and was associated with a
RR of 2.62 and 0.22 for one
and two copies versus no
copies of the minor allele,
respectively (un-corr p<0.00-
01; corr-p <0.0001). An
additional SNP from the
ESTR pathway also main-
tained signicance after
Bonferroni correction: rs141-
5270 downstream of the
androgen receptor (AR) gene
of the X chromosome was
associated with a RR of
0.53 and 8.26 for one and
two copies, respectively,
compared to no copies of
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Pubertal hormone gene variants and osteosarcoma
290 Int J Mol Epidemiol Genet 2012;3(4):286-293
associated with increased risk of OS. However,
the SNP found in our study, rs1003737, was in
low LD with the previously reported SNPs
(r-squared 0.10) [42]. The third SNP is located
on the X chromosome about 20 kb downstream
of the AR gene in a region that plays a role in
the N-terminal, the DNA-binding, and the andro-
gen-binding domains [42, 43, 45]. To our knowl-
edge, neither the particular function of these
specic SNPs nor their involvement in other
malignancies has been reported.
Previous studies used a case-control approach,
which is limited by the need to identify an
appropriate control group and the possibility of
population stratication in genetic analyses. In
contrast, for the case-parent approach, no con-
trols are needed and population stratication is
inherently corrected for. The statistical model
employed in a case-parent design also allows
for the estimation of RRs rather than odds
ratios-- the standard measure of association in
case-control studies. Further, our focus on
pediatric malignancies is a strength given the
availability and willingness of biological parents
to participate in research, particularly when
compared to the underwhelming participation
rates for population based controls. Including
solely pediatric OS cases could also improve
our understanding of etiology should it differ
from that of older patients; this is especially the
case in light of our large sample size, given the
rarity of the tumor. We appear to have taken the
rst comprehensive look at genetic variation in
the ESTR pathway as an OS risk factor. Our
identication of a signicant SNP associated
with the AR gene suggest that this pathway
might also be of biological relevance. It should
also be noted that while Bonferroni corrections
were used in both our analyses and that of pre-
vious studies, we elected to correct at the path-
way level and thus our criteria for statistical
signicance was considerably more stringent.
This could partially explain both the reduction
Table 1. Demographic Characteristics from a Case-Parent Study of Childhood Osteosarcoma, Chil-
dren’s Oncology Group, United States and Canada, 2007-2010 (n=290)
Case Trait Category Count %
Gender
Male 170 58.6
Female 120 41.4
Age at diagnosis (years)
0-4 2 0.7
5-9 51 17. 6
10-14 130 44.8
15-19 107 36.9
Race
a
White 237 85.6
Black 21 7.6
Other 19 6.9
Ethnicity
b
Non-Hispanic 239 86.0
Hispanic 39 14.0
Country of Residence
United States 274 94.5
Canada 16 5.5
Tumor Location
c
Lower Limb 221 77. 5
Upper Limb 42 14.7
Chest 6 2.1
Hip 9 3.2
Spine 2 0.1
Head 5 1.8
Histology
Chondroblastic 47 20.2
Osteoblastic 68 29.2
Fibroblastic 16 6.9
Telangiectatic 15 6.4
High grade 74 31.8
Low grade 5 2.1
Periosteal 4 1.7
Mixed 4 1.7
a
13 unknown;
b
12 unknown;
c
5 missing.
Page 5
Pubertal hormone gene variants and osteosarcoma
291 Int J Mol Epidemiol Genet 2012;3(4):286-293
in number of signicant SNPs compared to the
ndings by Mirabello et al. as well as our failure
to conrm signicant SNPs from that study.
While we did identify statistically signicant
SNPs in our proposed pathways, we recognize
that interpretation of our results must be con-
sidered in the context of the caveats associat-
ed with a candidate gene based approach, pri-
marily the reliance on current knowledge
regarding the biology of OS and the limitation in
the number of SNPs examined compared to a
genome-wide approach. Despite these short-
comings, we selected a biologically plausible
set of genes based on previous research indi-
cating the primacy of the IGF/GH and ESTR
pathways in promoting linear bone growth [34]
and on preliminary data implicating the IGF/GH
axis in adult and pediatric OS [42], which made
candidate genes in the IGF/GH and ESTR path-
ways obvious choices for a study in children.
Conrmation of these results in future studies
would lend additional support to the associa-
tion of bone growth pathways in pediatric OS.
Acknowledgements
This research was supported by National
Institutes of Health grants U01CA122371, T32
CA099936-S1, K05 CA157439, U01CA98543,
U10CA13539, the Childrens Cancer Research
Fund, Minneapolis, MN, grant 97834 from the
Canadian Institutes for Heath Research (CIHR),
and the intramural research program of the
Division of Cancer Epidemiology and Genetics,
National Cancer Institute.
Address correspondence to: Logan G Spector,
Division of Pediatric Epidemiology and Clinical
Research, University of Minnesota Cancer Center,
Table 2. Signicant SNPs from the IGF/GH and ESTR pathways and associated relative risks after cor-
rection for multiple testing from a study of childhood osteosarcoma
Pathway Gene SNP id Chromosome Region MAF
a
RR1
b
RR2
c
Un-corr-p Corr-p
d
IGF/GH IGF2R rs1003737 6 intronic 0.30 0.91 0.0001 <0.0001 <0.0001
IGFALS rs2575352 16 upstream 0.35 2.62 0.22 <0.0001 <0.0001
ESTR AR rs1415270 X downstream 0.22 0.53 8.26 <0.0001 0.02
a
: minor allele frequency;
b
: relative risk for one vs. no copies;
c
: relative risk for two vs. no copies;
d
: Bonferroni corrected within
pathway (519 SNPs for ESTR and 279 SNPs for IGF/GH).
Figure 2. This gure depicts both the uncorrected p-values and the p-values after correction for multiple testing for
each of the SNPs analyzed in the two pathways. We present the p-values (corrected and uncorrected) on the -log
scale and provide a horizontal line so that the reader can easily see the threshold necessary for signicance in this
analysis. P-values for each SNP by pathway before and after Bonferroni correction
a
α = 0.05. Inset box indicates
pathway symbol, results for 798 SNPs in 42 candidate genes (29 ESTR and 13 IGF/GH). Dashed line represents
signicance threshold for α.
Page 6
Pubertal hormone gene variants and osteosarcoma
292 Int J Mol Epidemiol Genet 2012;3(4):286-293
420 Delaware Street SE, MMC 715, Minneapolis,
MN 55455. Phone: (612)624-3912; Fax: (612) 624-
7147; E-mail: spector@umn.edu
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