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Regular Article
CLINICAL TRIALS AND OBSERVATIONS
Genetic risk factors for the development of osteonecrosis in children
under age 10 treated for acute lymphoblastic leukemia
Seth E. Karol,1 Leonard A. Mattano Jr,2 Wenjian Yang,3 Kelly W. Maloney,4 Colton Smith,3 ChengCheng Liu,3
Laura B. Ramsey,3 Christian A. Fernandez,3 Tamara Y. Chang,1 Geoffrey Neale,5 Cheng Cheng,6 Elaine Mardis,7
Robert Fulton,7 Paul Scheet,8 F. Anthony San Lucas,8 Eric C. Larsen,9 Mignon L. Loh,10 Elizabeth A. Raetz,11
Stephen P. Hunger,12 Meenakshi Devidas,13 and Mary V. Relling3
1
Department of Oncology, St. Jude Children’s Research Hospital, Memphis, TN; 2HARP Pharma Consulting, Mystic, CT; 3Department of Pharmaceutical
Sciences, St. Jude Children’s Research Hospital, Memphis, TN; 4Center for Cancer and Blood Disorders, Children’s Hospital Colorado, Denver, CO;
5
Hartwell Center for Bioinformatics and Biotechnology, St. Jude Children’s Research Hospital, Memphis, TN; 6Department of Biostatistics, St. Jude
Children’s Research Hospital, Memphis, TN; 7McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO; 8Department
of Epidemiology, University of Texas MD Anderson Cancer Center, Houston, TX; 9Department of Pediatrics, Maine Medical Center, Portland, ME;
10
Department of Pediatrics, University of California School of Medicine, San Francisco, CA; 11Department of Pediatrics, University of Utah, Salt Lake City,
UT; 12Department of Pediatrics, Children’s Hospital of Philadelphia and the University of Pennsylvania Perelman School of Medicine, Philadelphia, PA;
and 13Department of Biostatistics, Colleges of Medicine, Public Health and Health Professions, University of Florida, Gainesville, FL
Osteonecrosis is a dose-limiting toxicity in the treatment of pediatric acute lymphoblastic
leukemia (ALL). Prior studies on the genetics of osteonecrosis have focused on patients ‡10
• Variants in genes important for years of age, leaving the genetic risk factors for the larger group of children <10 years
incompletely understood. Here, we perform the first evaluation of genetic risk factors for
mesenchymal stem cell
osteonecrosis in children <10 years. The discovery cohort comprised 82 cases of
differentiation influence the
osteonecrosis and 287 controls treated on Children’s Oncology Group (COG) standard-risk
risk of osteonecrosis in
ALL protocol AALL0331 (NCT00103285, https://clinicaltrials.gov/ct2/show/NCT00103285),
children with ALL under 10
with results tested for replication in 817 children <10 years treated on COG protocol
years old.
AALL0232 (NCT00075725, https://clinicaltrials.gov/ct2/show/NCT00075725). The top repli• Variants in genes in the
cated single nucleotide polymorphisms (SNPs) were near bone morphogenic protein 7
glutamate signaling pathway
[BMP7: rs75161997, P 5 5.34 3 1028 (odds ratio [OR] 15.0) and P 5 .0498 (OR 8.44) in the
influence osteonecrosis in
discovery and replication cohorts, respectively] and PROX1-antisense RNA1 (PROX1-AS1:
children with ALL regardless
rs1891059, P 5 2.28 3 1027 [OR 6.48] and P 5 .0077 [OR 3.78] for the discovery and replication
of age.
cohorts, respectively). The top replicated nonsynonymous SNP, rs34144324, was in a
glutamate receptor gene (GRID2, P 5 8.65 3 1026 [OR 3.46] and P 5 .0136 [OR 10.8] in the
discovery and replication cohorts, respectively). In a meta-analysis, the BMP7 and PROX1-AS1 variants (rs75161997 and rs1891059,
respectively) met the significance threshold of <5 3 1028. Top replicated SNPs were enriched in enhancers active in mesenchymal stem
cells, and analysis of annotated genes demonstrated enrichment in glutamate receptor and adipogenesis pathways. These data may provide
new insights into the pathophysiology of osteonecrosis. (Blood. 2016;127(5):558-564)
Key Points
Introduction
Progressive intensification of multiagent chemotherapy has improved
outcomes for children with acute lymphoblastic leukemia (ALL), with
cure rates exceeding 90%.1-6 Results have been particularly favorable
for patients ,10 years of age with National Cancer Institute (NCI)
standard-risk (SR) ALL.7,8 Unfortunately, such intensified therapy has
also been associated with significant increases in the occurrence of
therapy-related osteonecrosis.9-12
Prior studies have identified multiple clinical risk factors for the
development of osteonecrosis, including female sex, European ancestry, the administration of 3 weeks of continuous rather than alternateweek dexamethasone during delayed intensification, and intensive vs
standard therapy.9-11 However, age remains the strongest and most
consistently identified factor, with patients 10 to 20 years old at greatest
risk.10,11,13-15
Given this age-related risk, the majority of children in prior investigations of genetic predisposition to osteonecrosis were .10 years.10,15
However, because ALL is so common in young children, up to 40% of
osteonecrosis cases develop in children ,10 years of age.10
In this study, we identified genetic factors associated with the development of osteonecrosis in the largest cohort of SR ALL patients
evaluated to date and validated these findings in a cohort of NCI highrisk ALL patients ,10 years of age.
Submitted October 7, 2015; accepted November 10, 2015. Prepublished
online as Blood First Edition paper, November 20, 2015; DOI 10.1182/blood2015-10-673848.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2016 by The American Society of Hematology
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BLOOD, 4 FEBRUARY 2016 x VOLUME 127, NUMBER 5
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BLOOD, 4 FEBRUARY 2016 x VOLUME 127, NUMBER 5
GENETIC RISKS OF OSTEONECROSIS IN YOUNG CHILDREN
559
Figure 1. Consort diagram of 369 patients in discovery
cohort treated on AALL0331.
Methods
osteonecrosis; cases had moderate (grade 2), severe (grade 3), or disabling
(grade 4) osteonecrosis.
Patients
Genotyping
The discovery cohort consisted of children with newly diagnosed SR
B-precursor ALL treated on the Children’s Oncology Group (COG)
AALL0331 (NCT00103285) protocol with germline DNA available;
of these, all patients with osteonecrosis as of June 30, 2012 were submitted
for genotyping as cases. Controls were taken from a previously genotyped
subcohort of AALL0331 (supplemental Methods, available on the Blood
Web site). Of 111 cases who were evaluable after induction and developed
grade 2 to 4 osteonecrosis, genotyping was completed for 96. Patients
(N 5 34, 14 cases and 20 controls) who received alternate-week dexamethasone after protocol amendment 2C (see supplemental Methods for
details) were excluded from the analysis (Figure 1). Controls were excluded
if ,1000 days of follow-up data were available, a time when the majority of
osteonecrosis was detected. Following these steps, the discovery cohort
included 82 osteonecrosis cases and 287 controls (Figure 1).
The replication cohort consisted of children ,10 years of age at diagnosis treated for newly diagnosed high-risk B-precursor ALL on the COG
AALL0232 (NCT00137111) protocol. The characteristics of this cohort
have been previously described.15 The analysis was limited to the 817 children
(20 cases, 797 controls) with genotype and phenotype data available (supplemental Figure 1).
Informed consent was obtained from patients $18 years of age and from
parents or guardians of patients ,18 years of age in accordance with the
Declaration of Helsinki. The COG AALL0331 and AALL0232 protocols
were approved by the NCI and the institutional review boards of participating institutions.
Patients on both protocols were prospectively monitored for clinical signs
and symptoms of osteonecrosis, and the diagnosis was confirmed by imaging
according to institutional preference. Osteonecrosis was considered a reportable
event and was graded using the NCI Common Terminology Criteria for Adverse
Events Version 4.0; controls had absent (grade 0) or asymptomatic (grade 1)
Genotyping of single nucleotide polymorphisms (SNPs) for AALL0331 and
AALL0232 was performed using the Illumina Human Exome BeadChip v1.1
and the Affymetrix Gene Chip Human Mapping Array 6.0. For SNPs not interrogated by direct genotyping, genotypes were imputed using MaCH-Admix
software (University of North Carolina), using phase 1 release v3 of the 1000
Genomes Project (http://www.1000genomes.org) as the reference genomes.16
For a subset of 15 cases of osteonecrosis, whole exome sequencing was
performed. Library generation was performed using NimbleGen SeqCap
EZ Exome Enrichment Kit v2.0, and sequencing was performed using
Illumina HiSequation 2000. Raw reads were aligned against human genome
hg19 using Burrow-Wheeler Aligner.17 Raw reads were converted to genotypes
using the Genome Analysis Toolkit version 3.2.18
Definition of genetic ancestry
Genetic ancestry for all patients was defined using STRUCTURE v2.2.3
as previously described.19 When ancestry was assessed as a categorical
variable, individuals were classified as white, black, or Hispanic based on
percentage inferred genetic ancestry as follows: .90% Northern European
(CEU) were classified as white; .70% West African (YRI) classified as
black; those with Native American ancestry .10% and greater Native
American than West African ancestry were classified as Hispanic. Patients
not falling into these groups were categorized as Other.
Quality control
All SNPs with a call rate of ,95% or poor clustering were excluded. SNPs
were excluded if they deviated from Hardy-Weinberg equilibrium in whites
(P , 1 3 1024). SNPs with a minor allele frequency (MAF) .5% and SNPs
with a MAF .0.5% with the minor allele as the risk allele for the development of osteonecrosis were included.
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BLOOD, 4 FEBRUARY 2016 x VOLUME 127, NUMBER 5
KAROL et al
Statistical analysis
AALL0331 treatment arms were clustered into therapy groups based on premaintenance therapy duration, dexamethasone and asparaginase dosing, and
previously reported therapy groups at increased risk for the development of
osteonecrosis (supplemental Methods; supplemental Table 1).20 Within the
discovery cohort, SNP genotypes were compared between cases and controls
while controlling for age, genetic ancestry as continuous variables, sex, and
therapy group. Evaluation used a time-to-event proportional hazard regression analysis with patients censored at the time of diagnosis of osteonecrosis
or at last follow-up. Relapse and off-protocol therapy were treated as competing events. Analysis was performed using the R survival package (v2.38-1)
implemented in PLINK (v1.07; http://pngu.mgh.harvard.edu/purcell/plink/)
using Rserve.21,22 Additional data analysis was performed using R (v3.1.1;
www.r-project.org).23
To test for validation, a parallel genome-wide association study (GWAS)
was performed in the replication cohort. SNP alleles identified in the discovery cohort were considered validated if the P values in both cohorts were ,.05
(supplemental Figure 2). A meta-analysis was performed for all SNPs passing
quality control in both the discovery and replication cohorts using Stouffer’s
Z-score method weighted by the GWAS standard error of the SNP in each
cohort.24
Enhancer enrichment analysis was performed using HaploReg (v2).25
Validated SNPs with a meta-analysis P , .001 were evaluated for enhancer
enrichment across available Roadmap samples. Associations were sorted according to their enrichment for enhancers and strongest enhancers.26
SNPs validated in both cohorts with a meta-analysis P , .001 were annotated
to genes, with the closest gene selected for intergenic SNPs. Pathway analysis
was performed using QIAGEN Ingenuity Pathway Analysis (www.qiagen.com/
ingenuity) to identify canonical biological pathways enriched within top SNPs.
Only pathways with $5 genes associated with osteonecrosis were considered.
Figure 2. Cumulative incidence of osteonecrosis in the discovery cohort based
on genotype for top BMP7 variants. All 5 patients in the discovery cohort (82 cases,
287 controls) carrying variant alleles of 2 SNPs in high LD (rs77556622 [T.C], rs76599360
[C.T]; P 5 1.13 3 1029) located 39 to BMP7 developed osteonecrosis. Patient age,
ancestry, sex, and therapy group are shown next to each case of osteonecrosis in
the variant allele group. Therapy groups are defined in the supplemental Materials,
with group 1 receiving the most intense therapy and group 4 receiving the least intense.
respectively) are predicted to increase glucocorticoid receptor binding
based on binding site motif alterations.
Meta-analysis
Results
GWAS for osteonecrosis
We identified covariates to include in the GWAS for osteonecrosis
in the discovery cohort (supplemental Table 2), which included age
(P 5 1.88 3 10-10), therapy group (P 5 6.8 3 1024 for the difference
across all 4 groups), sex (P 5 .008), and genetic ancestry (P 5 .02 for
European ancestry).
A GWAS of the discovery cohort adjusting for the covariates
of age, sex, therapy group, and ancestry (supplemental Table 2)
identified 16 SNPs in 6 genes associated with osteonecrosis at a
P value threshold of ,5 3 1028 (supplemental Table 3). Two SNPs,
rs77556622 and rs76599360 (both at P 5 1.13 3 1029), were
located ;11 and 20.7 kb, respectively, 39 of BMP7 (bone morphogenic protein 7). The risk alleles (C and T, respectively) for both
SNPs were present in 5 of 82 cases and 0 of the 287 controls and
conferred a 22-fold increase in osteonecrosis risk (95% confidence interval [CI] 8.15-59.6). Patients with these SNPs developed
osteonecrosis despite clinical characteristics placing them at low
risk, including 3 patients in the lowest risk treatment group, 2
males, and 1 patient with black ancestry (Figure 2).
In the replication cohort, 10 of the top 100 SNPs from the discovery
cohort were validated, including 2 SNPs (rs79085477, rs75161997)
in BMP7 in full linkage disequilibrium (LD) with one another, with
P 5 5.34 3 1028 (hazard ratio [HR] 15.0, 95% CI 5.64-39.7) in the
discovery cohort and P 5 .0498 (HR 8.43, 95% CI 1.002-71.1) in
the replication cohort. The top replicated SNPs represented 5 loci across
4 genes including BMP7 (2 SNPs), PROX1-AS1 (6 SNPs), LINC00251
(1 SNP), and DOK5 (1 SNP) (Table 1). Interestingly, the top validated
SNPs in both BMP7 and PROX1-AS1 (rs7516997 and rs1891059,
Meta-analysis of both cohorts identified 5 loci with genome-wide
significant associations with osteonecrosis (P , 5 3 1028), 3 of which
were significant in both cohorts, including loci near PROX1-AS1 and
BMP7 (Table 1; Figure 3). The top SNP from the meta-analysis
(rs9632544 near ERV3-1) was significant in the discovery but not the
replication cohort (P 5 2.36 3 1029 and P 5 .052, respectively).
In the meta-analysis, the top replicated coding SNP was rs34144324,
a missense variant in GRID2 (glutamate receptor, ionotropic, d2)
with a meta-analysis P 5 7.07 3 1027 (HR 3.78, 95% CI 2.23-6.39;
P 5 8.65 3 1026 and P 5 .014 in the discovery and replication cohorts,
respectively). In the 15 patients with whole exome sequencing available, 12 nonsynonymous variants were sequenced with a P , .001 in
the meta-analysis. There was 99.4% agreement in variant calls between
the array-based genotype calls and the sequencing genotype calls,
including 100% agreement for the GRID2 SNP.
Enhancer enrichment analysis
SNPs validated in both cohorts with a meta-analysis P , .001 (N 5 3271)
were evaluated for enhancer enrichment using HaploReg. We focused
on tissues in which SNPs were enriched in both all enhancers and the
strongest enhancers: colon smooth muscle, adipose-derived mesenchymal stem cells (MSCs), bone marrow–derived MSCs, and duodenal smooth muscle. When restricting the analysis to validated
SNPs with a meta-analysis P , 1 3 1025 (N 5 140; supplemental
Table 4), enhancer enrichment was observed only in bone marrow–
derived MSCs (2-fold enrichment for all enhancers [P 5 .0148] and
4.3-fold enrichment for the strongest enhancers [P 5 2.81 3 1024]).
Adipose-derived MSCs remained enriched for SNPs in all enhancers
(1.8-fold, P 5 .011), with marginal enrichment in the strongest enhancers (1.9-fold, P 5 .068; Table 2).
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561
Table 1. Top SNPs from the discovery cohort validated in replication cohort
RSID
Gene
Risk allele
Discovery MAF
cases
Discovery MAF
controls
Discovery cohort P*
Discovery cohort
HR (95% CI)
Replication cohort P†
rs79085477
BMP7
T
0.03
0
5.34 3 1028
15.0 (5.64-39.7)
.0498
0.061
0.017
2.28 3 1027
6.48 (3.19-13.2)
.008
rs75161997
rs1891059
T
PROX1-AS1
rs115602884
A
T
rs74533616
T
rs141059755
LINC00251
G
0.018
0.002
8.48 3 1027
23.3 (6.65-81.5)
.003
rs80223967
PROX1-AS1
G
0.061
0.019
6.63 3 1027
6.1 (2.99-12.4)
.01
rs17021408
PROX1-AS1
C
0.061
0.023
1.01 3 1026
5.89 (2.89-12.0)
.01
0.018
0.0017
1.71 3 1026
21.0 (6.03-72.9)
.003
rs61818937
rs117532069
A
DOK5
A
*P value for association of SNP genotype with osteonecrosis risk in discovery cohort (N 5 369).
†P value for association of SNP genotype with osteonecrosis in the replication cohort (N 5 817).
Pathway analysis
There were 3271 SNPs with meta-analysis P , .001 and significant
associations with osteonecrosis (P , .05) in both the AALL0331 and
AALL0232 cohorts (the top 140 with P , 1 3 1025 are shown in
supplemental Table 4). The 459 genes to which these SNPs annotated
were significantly enriched within 8 canonical pathways. Of these,
7 contained overlapping glutamate receptor genes, with the glutamate
receptor signaling pathway most overrepresented (P 5 9.92 3 1024,
10.5% of genes in pathway associated with osteonecrosis). The
adipogenesis pathway was the only overrepresented pathway (P 5 .0148,
5.5% of genes in pathway associated with osteonecrosis) whose genes
did not overlap with glutamate receptor signaling (Table 3; supplemental Table 5).
Discussion
Osteonecrosis has been recognized in recent years as a major toxicity
of pediatric ALL therapy. Children ,10 years of age constitute ;75%
of pediatric ALL cases.27 Thus, although osteonecrosis incidence is
low in this age group, those ,10 constitute up to 40% of cases of
osteonecrosis.10 It is hypothesized that inherited risk factors may be
more penetrant in young rather than older patients. Better understanding
of the individual risk factors for the development of osteonecrosis
in younger children and the underlying biology in this population and
in older patients is needed to improve prediction and management
of this often debilitating complication. These patients who developed
osteonecrosis despite being at low risk based on age and treatment
Figure 3. Manhattan plot of meta-analysis for osteonecrosis in patients 1 to 10 years of age. SNPs (N 5 9157) with associations with osteonecrosis (P , .05) in both the
discovery and replication cohorts are shown in red or orange. The top coding SNP (rs34144324 in GRID2, meta-analysis P 5 7.07 3 1027) and loci reaching genome-wide
significance (PROX1-AS1, rs1891059, P 5 8.72 3 1029; LINC00251, rs141059755, P 5 1.30 3 1028; BMP7, rs79085477 and rs75161997, P 5 8.29 3 1029) are noted.
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BLOOD, 4 FEBRUARY 2016 x VOLUME 127, NUMBER 5
KAROL et al
Table 2. Top validated SNPs (meta-analysis P < 1 3 1025) are
enriched in enhancers active in MSCs
All enhancers
Tissue type
Bone marrow–derived
Fold enrichment
Strongest enhancers*
P
Fold enrichment
P
2
.0148
4.3
2.81 3 1024
1.8
.011
1.9
.068
MSC cultured cells
Adipose-derived MSC
cultured cells
*Strongest enhancer chromatin state as previously defined.26
factors may provide unique insights into the genetic variations that influence development of osteonecrosis.
The occurrence of osteonecrosis is highly dependent on the therapeutic regimen that patients receive. Thus, the identification of genetic
variants that increase osteonecrosis risk in patients ,10 years of age
treated on both SR (AALL0331) and high-risk therapy (AALL0232)
protocols suggests the identified variants may be important to
osteonecrosis risk in this age group in the context of any modern ALL
protocol (using both minimal residual disease–directed intensification of
therapy, pegylated-asparaginase, and a delayed intensification phase).
We performed the first GWAS for osteonecrosis risk in patients
exclusively ,10 years of age and identified novel risk variants near
BMP7 and PROX1-AS1 (Table 1). Studies have shown that BMP7 is
released in response to bone damage in osteoarthritis and spondyloarthritis, and its release is increased by mechanical stress.28-31 Bone
morphogenic proteins can induce mesenchymal precursor cells to differentiate into osteoblasts and inhibit the formation of osteoclasts,32,33
and thus variants affecting this gene could contribute to osteonecrosis
by altering bone metabolism and formation both prior to and during
ALL therapy. Moreover, BMP7 is known to be toxic to vascular
smooth muscle.34 The prominent role of local arteriopathy, at least in
part due to vascular smooth muscle damage, in the development of
osteonecrosis35 supports the possibility that BMP7 could contribute
to osteonecrosis via direct toxic effects on local bone vasculature.
PROX1 has been shown to control the differentiation of lymphatic
endothelial cells from vascular endothelial cells.36 PROX1 was also
noted to be downregulated in familial combined hyperlipidemia, and it
has been argued that this results in reduced clearance of plasma lipids in
this disease.37 It is possible that the identified variants in PROX1-AS1
increase the risk of osteonecrosis by altering lipid trafficking either
within the compartment of the bone marrow or by increasing plasma
lipids, a previously identified risk factor.10
The importance of the identified SNPs in osteonecrosis is further
supported by the findings of the enhancer enrichment analysis. The top
92 validated SNPs were enriched for locations within enhancers active
in tissues closely related to osteonecrosis, specifically mesenchymal
progenitors (Table 2). These cells, under the influence of cytokines
including BMP738 and environmental signals including glucocorticoids,39 vitamin D, and mechanical load29 can differentiate into either
osteoblasts or adipocytes.40,41 SNPs significantly associated with
osteonecrosis (P , .001) were linked to 7 genes in the adipogenesis
pathway (Table 3), supporting the importance of genes affecting
mesenchymal differentiation in osteonecrosis. These findings are
additionally supported by the findings in prior GWAS linking variants
in fat and cholesterol metabolism genes (including ACP1) with
osteonecrosis.10
Pathway analysis demonstrated that variation in glutamate receptor
signaling contributes to osteonecrosis in children ,10 years of age.
Variants in 6 genes in the glutamate receptor pathway were associated
with the development of osteonecrosis in this cohort, including the top
validated nonsynonymous variant (rs34144324 in GRID2; Table 3).
Interestingly, the glutamate receptor signaling pathway was the top
pathway represented by genetic variants in a cohort of HR patients of all
ages.15 Although glutamate receptor signaling appears to play a role in
osteonecrosis across the age spectrum, the association appears stronger
in older patients than in young patients, as both the top SNP and top
pathway in a cohort dominated by older patients were related to
glutamate signaling.15 In contrast, in young patients, genetic variation
in genes expressed in mesenchymal and adipose tissues appear more
important as supported by single SNP, enhancer, and pathway analyses
(Tables 1-3). It is also possible that the apparent stronger association of
glutamate receptor signaling in the prior study reflects the effects of
high-risk therapy in addition to differences in age. However, prior
findings in the Vanderbilt BioVU cohort, in which most patients
were .10 years old and who developed osteonecrosis after glucocorticoids outside the treatment of ALL,15 suggests this association may be
more due to age than therapy intensity.
Prior genome-wide studies10,15 failed to identify an association
between osteonecrosis and BMP7. This is likely due to the greater
number of older patients in prior genome-wide studies. The reasons for
the specificity of BMP7 to osteonecrosis risk in younger patients but
not older patients are not clear. This may be explained by interactions
between BMP7 and vitamin D, as vitamin D levels vary across the
pediatric age range.42,43 BMP7 expression in the absence of vitamin D
induces osteoblast differentiation and mineralization, but this effect is
reversed in the presence of 1-25(OH)2 vitamin D3.44 Thus, it is possible
that variation in BMP7 is important for osteonecrosis risk in younger
but not older children due to age related differences in vitamin D levels.
Alternatively, because BMP7 can induce angiogenesis,45,46 it is possible variants in BMP7 have a smaller impact on osteonecrosis in older
children and adolescents when bony growth is complete and the bony
vasculature is already established.15 The BMP7 and PROX1-AS1 variants replicated in AALL0232 patients ,10 years of age (P 5 .0498 and
P 5 .008, respectively) but not in patients $10 years of age (P 5 .73
and P 5 .74, respectively), which also suggests the effects of the
variants are age rather than treatment specific. Future evaluation of
these findings in additional SR and high-risk patient populations under
age 10 will allow further understanding of the interaction of younger
age and treatment intensity in the development of osteonecrosis.
Although less common in children ,10 years of age, osteonecrosis
remains a major therapeutic toxicity in this age group. The findings of
this GWAS provide new insight into the genetic features associated
with the development of osteonecrosis in younger patients while
Table 3. Top nonoverlapping pathways enriched in SNPs
associated with osteonecrosis (meta-analysis P < .001) in combined
discovery and replication cohorts (1186 total patients)
Gene
Top SNP(s) tagged
to gene*
Meta-analysis
P value of
top SNP(s)
GRID2
rs34144324
7.07 3 1027
signaling
GRM5
rs308894
1.09 3 1024
(P 5 9.91 3 1024)
GRM7
rs112943394, rs112050403
1.22 3 1024
GRIN2B
rs1805491
4.21 3 1024
Pathway
(P for enrichment)
Glutamate receptor
GNG7
rs71339112
9.74 3 1024
SLC17A6
rs7127241
4.79 3 1024
Adipogenesis
BMP7
rs79085477, rs75161997
8.28 3 1029
(P 5 .015)
EBF1
rs71593317
1.26 3 1026
NR1D2
rs62253386, rs62253388
4.07 3 1025
CTBP1
rs2306475
3.27 3 1024
SOX9
rs17224938
7.39 3 1024
FZD10
rs10773748
1.82 3 1025
BMP2
rs117896134
3.16 3 1024
*Multiple SNPs in full LD are shown if all have equal associations with osteonecrosis.
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BLOOD, 4 FEBRUARY 2016 x VOLUME 127, NUMBER 5
verifying that glutamate receptor genetic variation may be important
across all age groups.15 Our findings support the notion that some genetic
risk factors for osteonecrosis are age specific, whereas others are not.
Acknowledgments
This study was supported by the National Institutes of Health,
National Institute of General Medical Sciences (grants GM92666,
GM115279, and GM97119), National Cancer Institute (grants
CA21765, CA36401, CA142665, CA156449, CA98543 [COG
Chair’s grant], CA98413 [COG Statistical Center], and CA114766
[COG Specimen Banking]), Leukemia & Lymphoma Society (grant
6168), and the American Lebanese Syrian Associated Charities.
GENETIC RISKS OF OSTEONECROSIS IN YOUNG CHILDREN
563
Authorship
Contribution: E.A.R. and M.V.R. conceived and designed the study;
S.E.K., L.A.M., W.Y., K.W.M., C.S., C.L., T.Y.C., M.L.L., S.P.H.,
M.D., and M.V.R. collected and assembled the data; S.E.K., W.Y.,
C.S., T.Y.C., C.C., and M.V.R. analyzed and interpreted the data;
and all authors contributed to the writing of the manuscript.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: Mary V. Relling, Pharmaceutical Department,
St. Jude Children’s Research Hospital, 262 Danny Thomas Place,
Room I-5112, Memphis, TN 38105; e-mail: mary.relling@stjude.
org.
References
1. Pui CH, Campana D, Pei D, et al. Treating
childhood acute lymphoblastic leukemia without
cranial irradiation. N Engl J Med. 2009;360(26):
2730-2741.
2. Hunger SP, Lu X, Devidas M, et al. Improved
survival for children and adolescents with acute
lymphoblastic leukemia between 1990 and 2005:
a report from the children’s oncology group. J Clin
Oncol. 2012;30(14):1663-1669.
3. Schrappe M, Moricke A, Reiter A, et al.
Key treatment questions in childhood acute
lymphoblastic leukemia: results in 5 consecutive
trials performed by the all-bfm study group from
1981 to 2000. Klin Padiatr. 2013;225(Suppl 1):
S62-S72.
4. Vora A, Goulden N, Mitchell C, et al. Augmented
post-remission therapy for a minimal residual
disease-defined high-risk subgroup of children
and young people with clinical standard-risk and
intermediate-risk acute lymphoblastic leukaemia
(UKALL 2003): a randomised controlled trial.
Lancet Oncol. 2014;15(8):809-818.
5. Schmiegelow K, Forestier E, Hellebostad M, et al;
Nordic Society of Paediatric Haematology and
Oncology. Long-term results of NOPHO ALL-92
and ALL-2000 studies of childhood acute
lymphoblastic leukemia. Leukemia. 2010;24(2):
345-354.
6. Conter V, Bartram CR, Valsecchi MG, et al.
Molecular response to treatment redefines all
prognostic factors in children and adolescents
with B-cell precursor acute lymphoblastic
leukemia: results in 3184 patients of the AIEOPBFM ALL 2000 study. Blood. 2010;115(16):
3206-3214.
7. Vora A, Goulden N, Wade R, et al. Treatment
reduction for children and young adults with
low-risk acute lymphoblastic leukaemia defined
by minimal residual disease (UKALL 2003): a
randomised controlled trial. Lancet Oncol.
2013;14(3):199-209.
8. Matloub Y, Bostrom BC, Hunger SP, et al.
Escalating intravenous methotrexate improves
event-free survival in children with standard-risk
acute lymphoblastic leukemia: a report from the
Children’s Oncology Group. Blood. 2011;118(2):
243-251.
9. Mattano LA Jr, Sather HN, Trigg ME, Nachman
JB. Osteonecrosis as a complication of treating
acute lymphoblastic leukemia in children: a report
from the Children’s Cancer Group. J Clin Oncol.
2000;18(18):3262-3272.
10. Kawedia JD, Kaste SC, Pei D, et al.
Pharmacokinetic, pharmacodynamic, and
pharmacogenetic determinants of osteonecrosis
in children with acute lymphoblastic leukemia.
Blood. 2011;117(8):2340-2347, quiz 2556.
11. Mattano LA Jr, Devidas M, Nachman JB, et al;
Children’s Oncology Group. Effect of alternateweek versus continuous dexamethasone
scheduling on the risk of osteonecrosis in
paediatric patients with acute lymphoblastic
leukaemia: results from the CCG-1961
randomised cohort trial. Lancet Oncol. 2012;
13(9):906-915.
12. Hyakuna N, Shimomura Y, Watanabe A, et al;
Japanese Childhood Cancer and Leukemia Study
Group (JCCLSG). Assessment of corticosteroidinduced osteonecrosis in children undergoing
chemotherapy for acute lymphoblastic leukemia:
a report from the Japanese Childhood Cancer and
Leukemia Study Group. J Pediatr Hematol Oncol.
2014;36(1):22-29.
13. Patel B, Richards SM, Rowe JM, Goldstone AH,
Fielding AK. High incidence of avascular necrosis
in adolescents with acute lymphoblastic
leukaemia: a UKALL XII analysis. Leukemia.
2008;22(2):308-312.
14. Toft N, Birgens H, Abrahamsson J, et al. Toxicity
profile and treatment delays in NOPHO ALL2008comparing adults and children with Philadelphia
chromosome-negative acute lymphoblastic
leukemia [published online ahead of print April 13,
2015]. Eur J Haematol. doi:10.1111/ejh.12562.
15. Karol SE, Yang W, Van Driest SL, et al. Genetics
of glucocorticoid-associated osteonecrosis in
children with acute lymphoblastic leukemia.
Blood. 2015;126(15):1770-1776.
16. Liu EY, Li M, Wang W, Li Y. MaCH-admix:
genotype imputation for admixed populations.
Genet Epidemiol. 2013;37(1):25-37.
17. Li H, Durbin R. Fast and accurate short read
alignment with Burrows-Wheeler transform.
Bioinformatics. 2009;25(14):1754-1760.
18. DePristo MA, Banks E, Poplin R, et al. A
framework for variation discovery and genotyping
using next-generation DNA sequencing data. Nat
Genet. 2011;43(5):491-498.
19. Pritchard JK, Stephens M, Donnelly P. Inference
of population structure using multilocus genotype
data. Genetics. 2000;155(2):945-959.
20. Mattano LA, Devidas M, Friedmann AM, et al.
Effect of dexamethasone (dex) dose modification
on osteonecrosis (on) risk associated with
intensified therapies for standard risk acute
lymphoblastic leukemia (sr-all): a report from the
children’s oncology group (cog) study aall0331.
J Clin Oncol. 2013;31(15 suppl). Abstract 10002.
21. Purcell S, Neale B, Todd-Brown K, et al. PLINK:
a tool set for whole-genome association and
population-based linkage analyses. Am J Hum
Genet. 2007;81(3):559-575.
22. Urbanek S. Rserve: a fast way to provide r
functionality to applications. In: Proceedings of
the 3rd International Workshop on Distributed
Statistical Computing; March 20-22, 2003;
Vienna, Austria.
23. Team RDCR. A Language and Environment
for Statistical Computing. Vienna, Austria: R
Foundation for Statistical Computing; 2006.
24. Willer CJ, Li Y, Abecasis GR. METAL: fast
and efficient meta-analysis of genomewide
association scans. Bioinformatics. 2010;26(17):
2190-2191.
25. Ward LD, Kellis M. HaploReg: a resource for
exploring chromatin states, conservation, and
regulatory motif alterations within sets of
genetically linked variants. Nucleic Acids Res.
2012;40(Database issue):D930-D934.
26. Ernst J, Kheradpour P, Mikkelsen TS, et al.
Mapping and analysis of chromatin state
dynamics in nine human cell types. Nature.
2011;473(7345):43-49.
27. McNeil DE, Coté TR, Clegg L, Mauer A. SEER
update of incidence and trends in pediatric
malignancies: acute lymphoblastic leukemia. Med
Pediatr Oncol. 2002;39(6):554-557, discussion
552-553.
28. Itoh F, Asao H, Sugamura K, Heldin CH, ten Dijke
P, Itoh S. Promoting bone morphogenetic protein
signaling through negative regulation of inhibitory
Smads. EMBO J. 2001;20(15):4132-4142.
29. Santos A, Bakker AD, Willems HM, Bravenboer
N, Bronckers AL, Klein-Nulend J. Mechanical
loading stimulates BMP7, but not BMP2,
production by osteocytes. Calcif Tissue Int.
2011;89(4):318-326.
30. Honsawek S, Chayanupatkul M, Tanavalee A,
et al. Relationship of plasma and synovial fluid
BMP-7 with disease severity in knee osteoarthritis
patients: a pilot study. Int Orthop. 2009;33(4):
1171-1175.
31. Park MC, Park YB, Lee SK. Relationship of bone
morphogenetic proteins to disease activity and
radiographic damage in patients with ankylosing
spondylitis. Scand J Rheumatol. 2008;37(3):
200-204.
32. Maurer T, Zimmermann G, Maurer S, Stegmaier
S, Wagner C, Hänsch GM. Inhibition of osteoclast
generation: a novel function of the bone
morphogenetic protein 7/osteogenic protein 1.
Mediators Inflamm. 2012;2012:171209.
33. Bi W, Gu Z, Zheng Y, Wang L, Guo J, Wu G.
Antagonistic and synergistic effects of bone
morphogenetic protein 2/7 and all-trans retinoic
acid on the osteogenic differentiation of rat bone
marrow stromal cells. Dev Growth Differ. 2013;
55(9):744-754.
34. Zhang S, Fantozzi I, Tigno DD, et al. Bone
morphogenetic proteins induce apoptosis in
human pulmonary vascular smooth muscle cells.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
564
BLOOD, 4 FEBRUARY 2016 x VOLUME 127, NUMBER 5
KAROL et al
Am J Physiol Lung Cell Mol Physiol. 2003;285(3):
L740-L754.
35. Janke LJ, Liu C, Vogel P, et al. Primary
epiphyseal arteriopathy in a mouse model of
steroid-induced osteonecrosis. Am J Pathol.
2013;183(1):19-25.
39. Smith E, Frenkel B. Glucocorticoids inhibit
the transcriptional activity of LEF/TCF in
differentiating osteoblasts in a glycogen
synthase kinase-3beta-dependent and
-independent manner. J Biol Chem. 2005;
280(3):2388-2394.
43. Modan-Moses D, Pinhas-Hamiel O, MunitzShenkar D, Temam V, Kanety H, Toren A. Vitamin
D status in pediatric patients with a history of
malignancy. Pediatr Res. 2012;72(6):620-624.
44. Eichner A, Brock J, Heldin CH, Souchelnytskyi S.
Bone morphogenetic protein-7 (OP1) and
transforming growth factor-beta1 modulate
1,25(OH)2-vitamin D3-induced differentiation of
human osteoblasts. Exp Cell Res. 2002;275(1):
132-142.
36. Johnson NC, Dillard ME, Baluk P, et al. Lymphatic
endothelial cell identity is reversible and its
maintenance requires Prox1 activity. Genes Dev.
2008;22(23):3282-3291.
40. Neumann K, Endres M, Ringe J, et al. BMP7
promotes adipogenic but not osteo-/chondrogenic
differentiation of adult human bone marrowderived stem cells in high-density micro-mass
culture. J Cell Biochem. 2007;102(3):626-637.
37. Horra A, Salazar J, Ferré R, et al. Prox-1
and FOXC2 gene expression in adipose tissue:
A potential contributory role of the lymphatic
system to familial combined hyperlipidaemia.
Atherosclerosis. 2009;206(2):343-345.
41. Knippenberg M, Helder MN, Zandieh Doulabi B,
Wuisman PI, Klein-Nulend J. Osteogenesis
versus chondrogenesis by BMP-2 and BMP-7
in adipose stem cells. Biochem Biophys Res
Commun. 2006;342(3):902-908.
45. Ramoshebi LN, Ripamonti U. Osteogenic
protein-1, a bone morphogenetic protein,
induces angiogenesis in the chick chorioallantoic
membrane and synergizes with basic fibroblast
growth factor and transforming growth factorbeta1. Anat Rec. 2000;259(1):97-107.
38. Luu HH, Song WX, Luo X, et al. Distinct roles
of bone morphogenetic proteins in osteogenic
differentiation of mesenchymal stem cells.
J Orthop Res. 2007;25(5):665-677.
42. Choudhary A, Chou J, Heller G, Sklar C.
Prevalence of vitamin D insufficiency in survivors
of childhood cancer. Pediatr Blood Cancer. 2013;
60(7):1237-1239.
46. Yeh LCC, Lee JC. Osteogenic protein-1 increases
gene expression of vascular endothelial growth
factor in primary cultures of fetal rat calvaria cells.
Mol Cell Endocrinol. 1999;153(1-2):113-124.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2016 127: 558-564
doi:10.1182/blood-2015-10-673848 originally published
online November 20, 2015
Genetic risk factors for the development of osteonecrosis in children
under age 10 treated for acute lymphoblastic leukemia
Seth E. Karol, Leonard A. Mattano Jr, Wenjian Yang, Kelly W. Maloney, Colton Smith, ChengCheng
Liu, Laura B. Ramsey, Christian A. Fernandez, Tamara Y. Chang, Geoffrey Neale, Cheng Cheng,
Elaine Mardis, Robert Fulton, Paul Scheet, F. Anthony San Lucas, Eric C. Larsen, Mignon L. Loh,
Elizabeth A. Raetz, Stephen P. Hunger, Meenakshi Devidas and Mary V. Relling
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