Atypical femoral fracture is a well-documented adverse reaction to bisphosphonates. It is strongly related to duration of bisphosphonate use, and the risk declines rapidly after drug withdrawal. The mechanism behind bisphosphonate-associated atypical femoral fracture is unclear, but a genetic predisposition has been suggested. With the aim to identify common genetic variants that could be used for preemptive genetic testing, we performed a genome-wide association study. Cases were recruited mainly through reports of adverse drug reactions sent to the Swedish Medical Products Agency on a nation-wide basis. We compared atypical femoral fracture cases (n = 51) with population-based controls (n = 4891), and to reduce the possibility of confounding by indication, we also compared with bisphosphonate-treated controls without a current diagnosis of cancer (n = 324). The total number of single-nucleotide polymorphisms after imputation was 7,585,874. A genome-wide significance threshold of p < 5 × 10−8 was used to correct for multiple testing. In addition, we performed candidate gene analyses for a panel of 29 genes previously implicated in atypical femoral fractures (significance threshold of p < 5.7 × 10−6). Compared with population controls, bisphosphonate-associated atypical femoral fracture was associated with four isolated, uncommon single-nucleotide polymorphisms. When cases were compared with bisphosphonate-treated controls, no statistically significant genome-wide association remained. We conclude that the detected associations were either false positives or related to the underlying disease, i.e., treatment indication. Furthermore, there was no significant association with single-nucleotide polymorphisms in the 29 candidate genes. In conclusion, this study found no evidence of a common genetic predisposition for bisphosphonate-associated atypical femoral fracture. Further studies of larger sample size to identify possible weakly associated genetic traits, as well as whole exome or whole-genome sequencing studies to identify possible rare genetic variation conferring a risk are warranted.
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Introduction
For over a decade, atypical fracture of the femoral bone (AFF) has been a well-documented adverse drug reaction (ADR) associated with long-term bisphosphonate use [1]. AFF is normally preceded by weeks or months of thigh pain and is in contrast to ordinary fragility fractures related to no or minimal trauma [2]. The term ‘atypical’ refers to the deviant transverse pattern of the fracture-line revealed on plain radiographs of the affected femur [2]. Although not all AFFs occur after bisphosphonate exposure, there is a strong correlation with duration of bisphosphonate use. A more than 100-fold increase in risk is seen after 4–5 years of bisphosphonate use, and the risk declines rapidly after cessation of treatment [3‐5].
By now, clinicians, the scientific community and patients have come to realize the many challenges associated with AFFs. Over the last decade, a 50% decrease in prescriptions of bisphosphonates for primary and secondary prevention of fragility fractures has been seen [6]. This significant decline in preventive medication is believed to be due to fear of ADRs.
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A major challenge in the prevention of AFF is the overall lack of knowledge about the mechanism behind this fracture type. Theories highlight long-term buildup of micro-cracks in the bone due to an over-suppression of bone remodeling that eventually leads up to failing skeletal integrity and stress fractures [7]. Predisposing risk factors are long-term use of bisphosphonates [3], female sex [3, 8], Asian ethnicity [9], and bowing of the femur [10]. Since only a minority of bisphosphonate users develop AFF, pathophysiological theories include a predisposing genetic trait, altered collagen cross-linking, accumulation of microdamage, increased mineralization, reduced heterogeneity of mineralization, variation in rates of bone turnover, and reduced vascularity [2].
A recent systematic review found six published studies that investigated the role of genetics on AFF in a total of 44 patients [11]. The review also identified 23 cases of AFF associated with seven different monogenetic bone disorders, of which seven cases had been exposed to a bisphosphonate. There is thus some evidence of rare genetic susceptibility loci for bisphosphonate-associated AFF. If common risk variants, i.e., genetic variants occurring among at least 1%, also exist, as has been shown for many rare adverse drug reactions [12], it might be feasible to predict patients at risk through preemptive genotyping. We performed the largest case–control GWAS to date, to determine whether common genetic variants contribute to risk of bisphosphonate-associated AFF. We also performed candidate gene analyses of 29 genes that have been implicated in AFF [11].
Materials and Methods
Sample Description
The basis for case recruitment was through nation-wide spontaneous ADR reports sent from healthcare professionals to the Swedish Medical Products Agency between the years 2006 and 2015. Each patient should be at least 18 years of age and able to give informed consent. Case definition for AFF was according to the American Society for Bone and Mineral Research [2].
We collected clinical data (demographics, medical history, drug treatment history, X-ray images, and ancestry) through interviews using a standardized questionnaire, and by obtaining and reviewing medical records. Prior to genetic analysis, each case including radiographs was evaluated by at least one senior consultant in orthopedics.
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Overall, 71 reported cases were available. Of these, 18 cases were not possible to include (five were deceased, five could not be reached, four declined to participate, two were not suitable to be contacted according to the reporting physician, one was not able to perform the interview, and in one case the reporting physician could not be reached). Of the remaining 53 cases, two did not pass radiograph adjudication (ordinary fragility fractures) and therefore 51 cases, all with complete fractures, were included in the study. We compared the cases with two sets of controls. In the main analysis, we utilized 4891 population controls from the Swedish Twin Registry [13], all non-related individuals. The proportion of women in this population was 46%, and birth years ranged from 1911 to 1958 (1911–1919, 0.78%; 1920–1929, 10.3%; 1930–1939, 27.7%; 1940–1949, 45.7%; 1950–1958, 15.5%). Information on diseases and drug treatments for controls was available by linkage to individual data from the Swedish National Patient Register and the Swedish Prescribed Drug Register. Complete linkage is enabled by use of the individual personal registration number provided to all Swedish citizens. To determine whether any positive GWAS findings might be due to confounding by indication, we also defined a matched control group, consisting of patients who had collected at least one prescription of a bisphosphonate and who did not have a current cancer diagnosis. This gave a total of 324 controls that had been prescribed bisphosphonates and thus resembling the same source population of individuals as the cases, i.e., bisphosphonate users. Four out of five matched controls were women, which corresponds well with the overall proportion of women/men prescribed bisphosphonates in Sweden according to the Swedish Prescribed Drug Register. None of the cases with AFF had a current diagnosis of cancer.
Genome-Wide Array Data and Analyses
DNA was extracted from peripheral venous blood. Cases were genotyped with the Illumina Infinium OmniExpressExome 1 M array, and controls were genotyped with the Illumina HumanOmniExpress 700 K array. Genotype calls were generated using the Genome Studio software from Illumina and the Genome Reference Consortium human assembly GRCh37.
Genotyping quality control (QC) and data management was performed using PLINK v1.9 [14]. The resulting merged data included 604,238 SNPs post QC. Imputation was performed using the Sanger imputation server [15]. The pipeline with Eagle2 (v2.0.5) prephasing [16] and PBWT imputation [17] was used with the haplotype reference consortium panel as reference (v1.1) [15]. The total number of SNPs after imputation and QC was 7,585,874. All cases and controls were within the European cluster according to genetic principal component analysis (PCA), except for one case of Chilenean origin (Supplemental Fig. 1). Additional details on QC, PCA and imputation can be found in the Supplement.
Logistic regression on a genome-wide level was performed using PLINK v1.9 [14]. All genome-wide analyses were adjusted for the first four principal components. SNP effects were modeled only as additive and the conventional genome-wide significance threshold p < 5 × 10−8 was used to correct for multiple testing [18]. Results are presented as Manhattan plots. QQ-plots are presented in Supplemental Figs. 2 and 3.
Candidate Gene Analyses
In addition to genome-wide analyses, we performed candidate gene analyses in the imputed data set for a panel of 29 genes that have been implicated in AFF (Table 1) [11]. We examined a panel consisting of 8709 SNPs distributed in these genes. We both tested all 51 cases vs all 4891 controls and all 51 cases vs the 324 matched controls. Adjustment for multiple testing was done with Bonferroni correction (0.05/8709 ≈ 5.74 × 10−6).
Table 1
Candidate genes tested in the study
Gene
Chromosome
Start position
End position
ACKR3 (CXCR7)
2
237476430
237491001
ACOXL
2
111490150
111875799
ALPL
1
21835858
21904905
CCDC147
10
106113522
106214848
CNGB1
16
57917503
58005020
COL1A2
7
94023873
94060544
CRYBB2
22
25615489
25627836
CTSK
1
150768684
150780799
CYP1A1
15
75011883
75017951
DOCK2
5
169064251
169510386
EDC3
15
74922899
74988633
FN1
2
216225163
216300895
FOXK2
17
80477589
80602538
GGA3
17
73232694
73258444
GGPS1
1
235490665
235507847
HHAT
1
210501596
210849638
LIPN
10
90521163
90537999
MVD
16
88718343
88729569
NAT8B
2
73927636
73928467
NGEF
2
233743396
233877982
OR2L13
1
248100493
248264224
OR51T1
11
4903049
4904113
PCK2
14
24563262
24579807
PPEF2
4
76781020
76823724
SF3B3
16
70557691
70608820
SLC15A5
12
16341419
16430619
SLC2A6
9
136336217
136344259
SYDE2
1
85622556
85666729
SYTL2
11
85405267
85522184
Genes implicated in atypical femoral fractures [11]
Power Calculation
Given a genome-wide significance level of p < 5 × 10−8 and using an additive genetic model, our sample size was powered to detect common genetic variants with effect sizes of clinical utility [19]. We had approximately 80% power to detect an odds ratio (OR) of 3–4 for variants with a minor allele frequency (MAF) of 40%, and 80% power to detect an OR of 4–5 for variants with a MAF of 20% (Supplemental Figs. 4 and 5). Given the significance level of p < 5.74 × 10−6 in the candidate gene analyses, we had 80% power to detect an OR of about 3 for variants with a MAF of 40%, and 80% power to detect an OR of about 4 for variants with a MAF of 20% (Supplemental Figs. 6 and 7).
Results
Characteristics of the 51 cases (48 women and 3 men) of bisphosphonate-associated AFF and the 324 matched controls are shown in Table 2. Most of the cases were of Swedish ethnicity (n = 47), while one each was of Finnish, Norwegian, British or Chilean origin.
Table 2
Characteristics of cases of bisphosphonate-associated atypical femoral fractures and matched controls
AFF (n = 51)
Matched controls (n = 324)
Gender (n female, [proportion female])
48 [0.94]
257 [0.79]
Agea (mean, years [range])
70.7 [47-86]
71.5 [52-93]
PPI (n, [proportion])
17 [0.33]
100 [0.31]
Systemic corticosteroids (n, [proportion])
17 [0.33]
123 [0.38]
Alendronic acid (n, [proportion])
47 [0.92]
264 [0.81]
Zoledronic acid (n, [proportion])
2 [0.039]
4 [0.012]
Risedronic acid (n, [proportion])
4 [0.078]
51 [0.16]
Etidronic acid (n, [proportion])
0 [0]
7 [0.022]
Ibandronic acid (proportion)
0 [0]
1 [0.0031]
Clodronate (proportion)
0 [0]
0 [0]
Oral administration (proportion)
49 [0.96]
320 [0.99]
Indication for treatment with bisphosphonate
Unknown
Osteoporosis (n)
45
Prophylaxis due to corticosteroid treatment (n)
2
Unknown (n)
4
Fracture location
N/A
Femur (n)
51
Matched controls were individuals who had collected at least one prescription of a bisphosphonate. We excluded as matched controls those individuals who had a diagnosis of cancer (any type) 12 months prior to or following first collection of a prescription of a bisphosphonate. Note that some patients have received more than one bisphosphonate
aAge at time of onset of AFF for cases, and time of first recorded collection of a prescription of a bisphosphonate for controls
AFF atypical femoral fractures
Genome-Wide Association Analyses—Cases Versus All Population Controls
Bisphosphonate-associated AFF was significantly associated with four isolated single nucleotide polymorphisms (SNP) (Fig. 1a; Table 3). The first SNP was rs7729897, which is located in an intergenic region upstream of the NR3C1 gene (nuclear receptor subfamily 3 group C member 1) on chromosome 5, OR 10.27 [95% confidence interval (CI) 4.95, 21.31] p = 4.00 × 10−10. The NR3C1 gene encodes a glucocorticoid receptor, which functions as a transcription factor that activates glucocorticoid responsive genes, and as a regulator of other transcription factors [20]. Variants of this gene have been associated with decreased bone mineral density in patients with endogenous hypercortisolism [21, 22].
Fig. 1
a Manhattan plot of the genome-wide association analysis—cases vs all controls. b Manhattan plot of the genome-wide association analysis—cases vs matched controls. Analyses of 51 cases of bisphosphonate-associated atypical femoral fractures versus a all 4891 population controls, and b 324 matched controls. There were 7,585,874 SNPs after imputation, and adjustment was made for genetic principal components 1–4. The red line shows the threshold for genome-wide significance of 5 × 10−8. a Four SNPs were statistically significant when cases were compared with all 4891 controls. The top SNP was rs7729897, located in an intergenic region upstream of the NR3C1 gene (nuclear receptor subfamily 3 group C member 1) on chromosome 5, odds ratio (OR) 10.27 [95% confidence interval (CI) 4.95, 21.31] p = 4.00 × 10−10. There was also a significant association with rs11465606 positioned in an intronic region within the IL18R1 gene (interleukin 18 receptor 1) on chromosome 2, OR 6.15 [95% CI 3.32, 11.37], p = 7.13 × 10−9. A third significant association was with rs145787127, which is located in an intron region of the NTN1 (netrin 1) gene on chromosome 17, OR 7.37 [95% CI 3.63, 14.93], p = 3.08 × 10−8. The fourth significant association was with rs144094653, located close to the pseudogene TUBB8P5 (tubulin beta 8 class VIII pseudogene 5 on chromosome 12, OR 7.68 [95% CI 3.70, 15.91], p = 4.20 × 10−8. SNP single nucleotide polymorphism. b There were no statistically significant findings when cases were compared with matched controls. SNP single nucleotide polymorphism
Table 3
Top genome-wide associations with bisphosphonate-associated atypical femoral fractures
CHR
SNP
BP
Minor allele
N
OR
L95
U95
p
GTPS
MAF cases
MAF controls
Gene
5
rs7729897
142970862
G
4942
10.27
4.949
21.31
4.000 × 10−10
G/C
0.098
0.01
2
rs11465606
102988300
A
4942
6.149
3.324
11.37
7.131 × 10−9
A/C
0.128
0.024
IL18R1
17
rs145787127
9142414
A
4942
7.366
3.633
14.93
3.076 × 10−8
A/G
0.098
0.016
NTN1
12
rs144094653
38593619
A
4942
7.675
3.704
15.91
4.201 × 10−8
A/G
0.088
0.014
3
rs73111385
63645410
G
4942
5.042
2.811
9.045
5.755 × 10−8
G/A
0.137
0.031
SNTN
1
rs113093597
165017843
A
4942
6.144
3.137
12.03
1.205 × 10−7
A/G
0.098
0.017
4
rs191328328
174611710
C
4942
8.951
3.917
20.46
2.013 × 10−7
C/T
0.069
0.009
9
rs12336042
108538200
A
4942
6.731
3.252
13.93
2.774 × 10−7
A/T
0.088
0.015
TMEM38B
3
rs76646538
2694727
C
4942
3.933
2.317
6.676
3.950 × 10−7
C/T
0.157
0.04
CNTN4
3
rs6768500
2693258
C
4942
3.932
2.316
6.675
3.962 × 10−7
C/G
0.157
0.04
CNTN4
12
rs147502517
103265420
T
4942
7.191
3.354
15.42
3.972 × 10−7
T/G
0.078
0.012
PAH
14
rs72698961
96278663
G
4942
5.128
2.701
9.734
5.762 × 10−7
G/A
0.118
0.027
2
rs74476239
182754649
C
4942
6.925
3.232
14.84
6.477 × 10−7
C/T
0.078
0.012
2
rs78658531
182741934
G
4942
6.925
3.232
14.84
6.477 × 10−7
G/A
0.078
0.012
2
rs78797265
182736267
T
4942
6.925
3.232
14.84
6.477 × 10−7
T/G
0.078
0.012
2
rs78890965
182734044
T
4942
6.925
3.232
14.84
6.477 × 10−7
T/C
0.078
0.012
10
rs112889159
899303
T
4942
8.161
3.564
18.69
6.807 × 10−7
T/A
0.069
0.01
LARP4B
8
8:2410672
2410672
T
4942
7.257
3.318
15.88
6.952 × 10−7
T/C
0.078
0.013
12
rs116973965
34352942
A
4942
8.121
3.542
18.62
7.524 × 10−7
A/G
0.069
0.011
2
rs56272862
32379663
G
4942
3.995
2.307
6.917
7.610 × 10−7
G/A
0.157
0.045
SPAST
17
17:77861401
77861401
T
4942
7.375
3.328
16.34
8.604 × 10−7
T/G
0.069
0.01
2
rs72796871
32393157
A
4942
3.971
2.292
6.878
8.662 × 10−7
A/G
0.157
0.046
SLC30A6
6
rs1773013
2560712
A
4942
3.277
2.041
5.261
9.011 × 10−7
A/G
0.245
0.092
2
rs2303553
182783653
C
4942
6.723
3.141
14.39
9.218 × 10−7
C/T
0.078
0.012
SSFA2
2
rs77278954
182793839
A
4942
6.723
3.141
14.39
9.218 × 10−7
A/G
0.078
0.012
SSFA2
2
rs78774163
182780126
A
4942
6.723
3.141
14.39
9.218 × 10−7
A/G
0.078
0.012
SSFA2
8
rs74463341
9228334
C
4942
7.027
3.219
15.34
9.852 × 10−7
C/G
0.078
0.014
2
rs145475960
103130361
A
4942
5.499
2.778
10.89
9.995 × 10−7
A/T
0.098
0.02
SLC9A4
2
2:102820009
102820009
G
4942
5.092
2.652
9.779
1.014 × 10−6
G/C
0.108
0.024
IL1RL2
2
rs13419200
182758257
C
4942
6.666
3.115
14.27
1.026 × 10−6
C/A
0.078
0.012
SSFA2
8
rs74382792
62356700
G
4942
6.941
3.181
15.15
1.134 × 10−6
G/A
0.078
0.014
CLVS1
17
rs57769213
77879893
G
4942
7.199
3.251
15.94
1.138 × 10−6
G/C
0.069
0.011
20
rs140824800
12541106
A
4942
7.289
3.265
16.28
1.254 × 10−6
A/G
0.069
0.011
12
rs146647050
38191129
T
4942
5.931
2.871
12.25
1.514 × 10−6
T/G
0.088
0.019
7
rs142711375
46602409
G
4942
6.503
3.028
13.97
1.581 × 10−6
G/A
0.078
0.014
5
rs79287094
142892785
G
4942
8.409
3.522
20.08
1.624 × 10−6
G/A
0.069
0.01
9
rs150057407
3276207
G
4942
4.92
2.563
9.445
1.672 × 10−6
G/T
0.108
0.024
RFX3
12
rs143302148
39100013
T
4942
7.507
3.286
17.15
1.739 × 10−6
T/C
0.069
0.011
CPNE8
20
rs76232775
60768910
A
4942
4.44
2.408
8.186
1.789 × 10−6
A/G
0.118
0.03
MTG2
23
rs149305693
27808447
C
4942
8.11
3.435
19.15
1.799 × 10−6
C/T
0.069
0.012
9
rs148123055
100176616
G
4942
6.626
3.044
14.42
1.886 × 10−6
G/A
0.078
0.014
TDRD7
23
rs1433806
27812073
A
4942
8.08
3.421
19.08
1.887 × 10−6
A/G
0.069
0.011
12
rs150862851
38793434
G
4942
7.435
3.255
16.98
1.928 × 10−6
G/A
0.069
0.012
23
rs36115712
27825140
A
4942
8.066
3.415
19.05
1.931 × 10−6
A/G
0.069
0.012
23
rs146644158
27819452
T
4942
8.047
3.408
19
1.969 × 10−6
T/C
0.069
0.012
23
rs4829082
27805106
T
4942
8.047
3.408
19
1.969 × 10−6
T/C
0.069
0.012
23
rs6630571
27814160
A
4942
8.047
3.408
19
1.969 × 10−6
A/G
0.069
0.012
20
rs149264569
49715107
G
4942
6.037
2.878
12.66
1.971 × 10−6
G/C
0.088
0.019
6
rs9386997
111414038
A
4942
7.389
3.237
16.87
2.038 × 10−6
A/T
0.069
0.011
SLC16A10
15
rs62026663
45485831
C
4942
3.221
1.987
5.221
2.060 × 10−6
C/T
0.235
0.096
SHF
9
rs187960516
36238454
A
4942
7.132
3.164
16.07
2.155 × 10−6
A/G
0.069
0.01
CLTA-GNE
23
rs140339686
27830115
T
4942
7.963
3.372
18.81
2.226 × 10−6
T/C
0.069
0.012
23
rs4829084
27827112
A
4942
7.963
3.372
18.81
2.226 × 10−6
A/G
0.069
0.012
6
rs73010912
155067310
A
4942
6.174
2.903
13.13
2.265 × 10−6
A/G
0.088
0.015
SCAF8
6
rs6921109
111448767
T
4942
7.309
3.203
16.68
2.297 × 10−6
T/A
0.069
0.011
SLC16A10
6
rs7760668
111446502
C
4942
7.309
3.203
16.68
2.297 × 10−6
C/A
0.069
0.011
SLC16A10
23
rs139460593
27817042
C
4942
7.925
3.357
18.71
2.319 × 10−6
C/T
0.069
0.012
6
rs72993420
155087077
G
4942
6.129
2.882
13.03
2.475 × 10−6
G/A
0.088
0.015
SCAF8
15
rs62026667
45491136
G
4942
3.174
1.963
5.133
2.475 × 10−6
G/C
0.235
0.097
SHF
15
rs142484525
95512720
T
4942
6.364
2.944
13.76
2.535 × 10−6
T/A
0.069
0.011
Top GWAS results based on 7,585,874 SNPs after imputation in 51 cases versus all 4891 population controls. All results were adjusted for genetic principal components 1–4. The threshold for statistical significance was p < 5 × 10−8
GWAS genome-wide association study, CHR chromosome, SNP single nucleotide polymorphism, BP base pair, N number, GTPS Guanosine-5′-triphosphates, MAF minor allele frequency, OR [95% CI] odds ratio with 95% confidence interval, p p value
×
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The second SNP was rs11465606 positioned in an intron within the IL18R1 gene (interleukin 18 receptor 1) on chromosome 2, OR 6.15 [95% CI 3.32, 11.37], p = 7.13 × 10−9. The third SNP was rs145787127, which is located in an intron of the NTN1 (netrin 1) gene on chromosome 17, OR 7.37 [95% CI 3.63, 14.93], p = 3.08 × 10−8. Genetic variation within NTN1 has been linked to osteoporosis [23]. The last SNP was rs144094653, located close to the pseudogene TUBB8P5 (tubulin beta 8 class VIII pseudogene 5 on chromosome 12, OR 7.68 [95% CI 3.70, 15.91], p = 4.20 × 10−8.
Genome-Wide Association Analyses—Cases Versus Controls with Bisphosphonate Use
No statistically significant association with gene status was revealed when cases of bisphosphonate-associated AFF were compared with matched controls (Fig. 1b; Table 4).
Table 4
Top genome-wide associations with bisphosphonate-associated atypical femoral fractures—cases vs matched controls
CHR
SNP
BP
Minor allele
N
OR
L95
U95
p
GTPS
MAF case
MAF control
Gene
16
rs7188484
88918607
T
375
3.576
2.153
5.94
8.605 × 10−7
T/G
0.431
0.196
GALNS
3
rs6768500
2693258
C
375
7.634
3.379
17.25
1.021 × 10−6
C/G
0.157
0.023
CNTN4
3
rs76646538
2694727
C
375
7.634
3.379
17.25
1.021 × 10−6
C/T
0.157
0.023
CNTN4
1
rs1913592
18550837
C
375
3.346
2.06
5.435
1.055 × 10−6
C/T
0.529
0.279
IGSF21
12
rs4765913
2419896
A
375
3.114
1.942
4.995
2.454 × 10−6
A/T
0.412
0.188
CACNA1C
16
rs12444242
88911043
T
375
3.269
1.987
5.38
3.125 × 10−6
T/C
0.402
0.182
GALNS
16
rs12447646
88910824
A
375
3.269
1.987
5.38
3.125 × 10−6
A/G
0.402
0.182
GALNS
16
rs12449164
88909788
T
375
3.269
1.987
5.38
3.125 × 10−6
T/C
0.402
0.182
GALNS
16
rs8054592
88912039
T
375
3.269
1.987
5.38
3.125 × 10−6
T/C
0.402
0.182
GALNS
16
rs12932521
88914235
T
375
3.242
1.97
5.335
3.679 × 10−6
T/C
0.402
0.184
GALNS
16
rs34858110
88914598
C
375
3.242
1.97
5.335
3.679 × 10−6
C/A
0.402
0.184
GALNS
16
rs71395332
88909028
T
375
3.243
1.97
5.336
3.683 × 10−6
T/C
0.402
0.184
GALNS
16
rs12598981
88916036
T
375
3.217
1.955
5.293
4.278 × 10−6
T/G
0.402
0.185
GALNS
16
rs11076726
88912899
T
375
3.219
1.953
5.306
4.503 × 10−6
T/G
0.422
0.201
GALNS
8
rs17063092
3104832
C
375
2.958
1.86
4.703
4.614 × 10−6
C/T
0.461
0.238
CSMD1
7
rs12538221
24123003
T
375
5.237
2.575
10.65
4.867 × 10−6
T/C
0.167
0.045
7
rs71526045
24118952
A
375
5.237
2.575
10.65
4.867 × 10−6
A/G
0.167
0.045
17
rs61753147
8809025
A
375
5.265
2.58
10.74
4.995 × 10−6
A/G
0.167
0.035
PIK3R5
16
rs34495980
88906555
A
375
3.177
1.93
5.232
5.544 × 10−6
A/C
0.402
0.188
GALNS
15
rs4776851
67180920
A
375
6.06
2.776
13.23
6.075 × 10−6
A/G
0.137
0.031
16
16:88906780
88906780
G
375
3.152
1.914
5.191
6.427 × 10−6
G/A
0.402
0.19
GALNS
16
rs13337256
88907043
G
375
3.152
1.914
5.191
6.427 × 10−6
G/A
0.402
0.19
GALNS
16
rs3784881
88905888
T
375
3.152
1.914
5.191
6.427 × 10−6
T/C
0.402
0.19
GALNS
18
rs116941264
75460371
A
375
10.78
3.833
30.33
6.609 × 10−6
A/G
0.098
0.011
7
rs2727797
36628761
T
375
3.142
1.909
5.169
6.613 × 10−6
T/C
0.676
0.44
AOAH
10
rs7082862
134341963
G
375
3.851
2.139
6.93
6.916 × 10−6
G/C
0.226
0.071
2
rs11465606
102988300
A
375
6.86
2.958
15.91
7.252 × 10−6
A/C
0.128
0.022
IL18R1
8
rs17319624
3105800
A
375
3.161
1.906
5.242
8.180 × 10−6
A/G
0.363
0.176
CSMD1
10
rs36009580
73627786
G
375
2.965
1.839
4.78
8.200 × 10−6
G/C
0.412
0.194
3
rs2717296
182456980
C
375
2.864
1.802
4.552
8.603 × 10−6
C/T
0.686
0.426
8
rs17319596
3104594
C
375
2.846
1.795
4.513
8.681 × 10−6
C/T
0.461
0.245
CSMD1
8
rs17319617
3105038
A
375
3.103
1.878
5.126
9.811 × 10−6
A/C
0.363
0.176
CSMD1
8
rs34162586
3105087
C
375
3.103
1.878
5.126
9.811 × 10−6
C/G
0.363
0.176
CSMD1
8
rs35729878
3104896
G
375
3.103
1.878
5.126
9.811 × 10−6
G/C
0.363
0.176
CSMD1
15
rs62026663
45485831
C
375
3.643
2.054
6.463
9.814 × 10−6
C/T
0.235
0.083
SHF
8
8:3104001
3104001
T
375
3.578
2.032
6.3
1.007 × 10−5
T/G
0.245
0.096
CSMD1
8
rs117459261
3103995
T
375
3.578
2.032
6.3
1.007 × 10−5
T/A
0.245
0.096
CSMD1
8
rs73185574
3106144
C
375
3.099
1.874
5.124
1.041 × 10−5
C/T
0.363
0.179
CSMD1
8
rs142418205
3097543
G
375
3.218
1.912
5.418
1.093 × 10−5
G/A
0.343
0.167
CSMD1
16
rs8062286
88917502
A
375
3.102
1.873
5.138
1.101 × 10−5
A/G
0.402
0.198
GALNS
7
rs3801298
36569019
T
375
3.098
1.87
5.131
1.123 × 10−5
T/C
0.716
0.486
AOAH
18
rs3016811
589690
T
375
2.609
1.7
4.002
1.126 × 10−5
T/C
0.628
0.381
18
rs518302
589635
G
375
2.609
1.7
4.002
1.126 × 10−5
G/A
0.628
0.381
2
rs6723676
22414978
A
375
2.821
1.774
4.484
1.159 × 10−5
A/C
0.559
0.327
20
rs149264569
49715107
G
375
10.89
3.744
31.68
1.170 × 10−5
G/C
0.088
0.011
4
rs116838635
112534842
A
375
5.704
2.617
12.43
1.187 × 10−5
A/G
0.137
0.034
17
rs111859148
32210110
C
375
3.174
1.893
5.323
1.191 × 10−5
C/T
0.304
0.13
ASIC2
17
rs2348157
32210243
G
375
3.174
1.893
5.323
1.191 × 10−5
G/C
0.304
0.13
ASIC2
17
rs56174865
32214269
A
375
3.174
1.893
5.323
1.191 × 10−5
A/G
0.304
0.13
ASIC2
17
rs66923090
32215593
A
375
3.174
1.893
5.323
1.191 × 10−5
A/G
0.304
0.13
ASIC2
17
rs67026511
32215830
G
375
3.174
1.893
5.323
1.191 × 10−5
G/A
0.304
0.13
ASIC2
17
rs67236820
32215903
A
375
3.174
1.893
5.323
1.191 × 10−5
A/G
0.304
0.13
ASIC2
17
rs67809660
32215544
C
375
3.174
1.893
5.323
1.191 × 10−5
C/T
0.304
0.13
ASIC2
17
rs68033423
32215432
C
375
3.174
1.893
5.323
1.191 × 10−5
C/T
0.304
0.13
ASIC2
17
rs68085213
32215389
C
375
3.174
1.893
5.323
1.191 × 10−5
C/T
0.304
0.13
ASIC2
17
rs72818938
32215882
C
375
3.174
1.893
5.323
1.191 × 10−5
C/T
0.304
0.13
ASIC2
17
rs8069564
32215953
T
375
3.174
1.893
5.323
1.191 × 10−5
T/C
0.304
0.13
ASIC2
17
rs8070346
32212347
C
375
3.174
1.893
5.323
1.191 × 10−5
C/G
0.304
0.13
ASIC2
17
rs8074055
32215922
C
375
3.174
1.893
5.323
1.191 × 10−5
C/T
0.304
0.13
ASIC2
17
rs8076707
32212839
C
375
3.174
1.893
5.323
1.191 × 10−5
C/T
0.304
0.13
ASIC2
Top GWAS results based on 7,585,874 SNPs after imputation in 51 cases versus 324 matched controls. All results were adjusted for genetic principal components 1–4. The threshold for statistical significance was p < 5 × 10−8
GWAS genome-wide association study, CHR chromosome, SNP single nucleotide polymorphism, BP base pair, N number, GTPS Guanosine-5′-triphosphates, MAF minor allele frequency, OR [95% CI] odds ratio with 95% confidence interval, p p value
Candidate Gene Analyses—Cases Versus All Population Controls
When cases of bisphosphonate-associated AFF were compared with all population controls, there were no statistically significant associations (Fig. 2a; Table 5; Supplemental Table 1).
Fig. 2
a Manhattan plot of the candidate gene analyses—cases vs all 4891 controls. b Manhattan plot of the candidate gene analyses—cases vs matched controls. Analyses of 51 cases of bisphosphonate-associated atypical femoral fractures versus a all 4891 controls, and b 324 matched controls. Adjustment was made for genetic principal components 1–4. The red line shows the threshold for statistical significance of 5.74 × 10−6. There were no statistically significant associations in either analysis
Table 5
Top candidate gene associations with bisphosphonate-associated atypical femoral fractures
CHR
SNP
BP
Minor allele
N
OR
L95
U95
p
GTPS
MAF cases
MAF controls
Gene
2
rs181660819
111578634
G
4942
5.42
2.284
12.87
1.271 × 10−4
G/A
0.059
0.013
ACOXL
5
rs116741837
169450719
T
4942
5.474
2.28
13.14
1.425 × 10−4
T/C
0.059
0.013
DOCK2
16
rs17821406
57919041
T
4942
2.713
1.612
4.566
1.721 × 10−4
T/C
0.167
0.068
CNGB1
2
rs138252364
111483994
C
4942
3.573
1.756
7.272
4.435 × 10−4
C/G
0.088
0.027
16
rs12446558
57915370
A
4942
2.533
1.484
4.325
6.607 × 10−4
A/T
0.157
0.068
10
rs116907192
106148123
T
4942
4.48
1.811
11.08
1.172 × 10−3
T/C
0.049
0.012
CCDC147
2
rs140272071
111510669
A
4942
3.247
1.592
6.62
1.197 × 10−3
A/G
0.088
0.03
ACOXL
2
rs3789117
111712123
C
4942
2.15
1.342
3.443
1.447 × 10−3
C/T
0.235
0.128
ACOXL
16
rs116919349
57911019
G
4942
2.323
1.361
3.966
1.998 × 10−3
G/A
0.157
0.074
16
rs17240952
57910443
C
4942
2.322
1.36
3.964
2.013 × 10−3
C/T
0.157
0.074
5
rs10063658
169131347
T
4942
3.151
1.52
6.531
2.035 × 10−3
T/C
0.088
0.026
DOCK2
5
rs111717777
169128756
G
4942
3.103
1.497
6.435
2.336 × 10−3
G/A
0.088
0.027
DOCK2
16
rs79806773
57917473
C
4942
2.283
1.34
3.891
2.396 × 10−3
C/G
0.157
0.075
9
rs76038546
136345878
C
4942
2.614
1.401
4.877
2.543 × 10−3
C/A
0.118
0.052
9
9:136352590
136352590
T
4942
2.567
1.38
4.775
2.903 × 10−3
T/C
0.118
0.052
1
rs114420253
248103804
A
4942
2.896
1.432
5.856
3.071 × 10−3
A/G
0.088
0.034
OR2L13
5
rs262864
169200927
A
4942
1.999
1.263
3.163
3.094 × 10−3
A/G
0.255
0.142
DOCK2-
9
9:136338187
136338187
C
4942
0.218
0.0786
0.602
3.312 × 10−3
C/A
0.039
0.148
SLC2A6
10
rs117846723
106186205
A
4942
3.646
1.53
8.691
3.514 × 10−3
A/C
0.059
0.018
CCDC147
2
rs55739979
216234981
C
4942
3.244
1.469
7.162
3.593 × 10−3
C/G
0.069
0.022
FN1
5
rs116213385
169457689
T
4942
3.561
1.515
8.372
3.595 × 10−3
T/A
0.059
0.018
DOCK2
2
rs3827546
111718499
C
4942
1.988
1.242
3.183
4.219 × 10−3
C/G
0.235
0.136
ACOXL
2
rs3789119
111707405
T
4942
1.81
1.205
2.719
4.283 × 10−3
T/C
0.372
0.25
ACOXL
5
rs114254961
169213503
A
4942
3.425
1.455
8.059
4.813 × 10−3
A/G
0.059
0.019
DOCK2
10
rs117402638
106199934
G
4942
3.428
1.449
8.111
5.04 × 10−3
G/T
0.059
0.02
CCDC147
7
7:94036547
94036547
T
4942
1.728
1.172
2.548
5.772 × 10−3
T/C
0.461
0.326
COL1A2
2
2:111621582
111621582
G
4942
2.274
1.265
4.09
6.061 × 10−3
G/T
0.137
0.067
ACOXL
5
rs76019338
169229582
A
4942
1.878
1.197
2.946
6.108 × 10−3
A/G
0.265
0.155
DOCK2
15
rs116916068
74920220
A
4942
2.311
1.265
4.222
6.427 × 10−3
A/G
0.128
0.061
CLK3
5
rs12520941
169218189
T
4942
1.867
1.19
2.93
6.606 × 10−3
T/G
0.265
0.156
DOCK2
2
rs74791643
111823562
G
4942
4.237
1.493
12.03
6.68 × 10−3
G/A
0.039
0.011
ACOXL
5
rs76621262
169356148
C
4942
4.081
1.477
11.28
6.686 × 10−3
C/G
0.039
0.011
DOCK2-FAM196B
2
rs2670632
111586327
T
4942
1.708
1.157
2.521
7.042 × 10−3
T/G
0.471
0.334
ACOXL
1
rs72763242
248187347
A
4942
3.607
1.408
9.236
7.502 × 10−3
A/G
0.049
0.015
OR2L13
2
rs3789100
111731713
C
4942
1.887
1.18
3.017
8.03 × 10−3
C/T
0.226
0.135
ACOXL
2
rs7564385
111734779
T
4942
1.887
1.18
3.017
8.03 × 10−3
T/C
0.226
0.135
ACOXL
1
rs4654971
21897903
C
4942
2.261
1.235
4.141
8.226 × 10−3
C/T
0.118
0.055
ALPL
1
rs3738098
21894785
T
4942
2.256
1.232
4.133
8.432 × 10−3
T/G
0.118
0.055
ALPL
2
rs11687442
216246210
G
4942
1.726
1.148
2.595
8.653 × 10−3
G/T
0.392
0.272
FN1
1
1:21903180
21903180
T
4942
2.242
1.223
4.108
8.987 × 10−3
T/C
0.118
0.055
ALPL
2
rs3789101
111729489
C
4942
1.868
1.168
2.989
9.099 × 10−3
C/G
0.226
0.136
ACOXL
2
rs12694363
216254032
A
4942
1.694
1.139
2.519
9.227 × 10−3
A/G
0.441
0.316
FN1
16
rs117529794
58005931
T
4942
3.933
1.387
11.15
0.01001
T/C
0.039
0.011
5
rs10462993
169497539
A
4942
1.757
1.143
2.701
0.01015
A/G
0.284
0.183
DOCK2
1
rs2242421
21904574
G
4942
2.15
1.199
3.856
0.01022
G/A
0.137
0.067
ALPL
1
rs7533989
210801954
G
4942
1.693
1.132
2.53
0.01029
G/C
0.412
0.296
HHAT
7
rs3750109
94042814
C
4942
1.907
1.163
3.126
0.01052
C/T
0.206
0.115
COL1A2
5
rs112139518
169198357
A
4942
2.562
1.245
5.272
0.01059
A/G
0.088
0.032
DOCK2
17
rs76141655
80570428
A
4942
2.777
1.264
6.101
0.01099
A/G
0.069
0.028
FOXK2
16
rs79070935
70578817
A
4942
3.157
1.296
7.689
0.01137
A/C
0.049
0.015
SF3B3
5
rs10462992
169497534
T
4942
1.738
1.13
2.672
0.01189
T/C
0.284
0.185
DOCK2
16
rs411657
57941094
T
4942
0.586
0.387
0.889
0.01198
T/C
0.333
0.461
CNGB1
10
rs11202848
90532166
A
4942
2.174
1.185
3.989
0.01211
A/C
0.118
0.057
LIPN
10
rs11202852
90544073
A
4942
2.174
1.185
3.989
0.01211
A/G
0.118
0.057
10
rs12572022
90545882
A
4942
2.174
1.185
3.989
0.01211
A/C
0.118
0.057
RCBTB2P1
10
rs17112679
90527569
C
4942
2.174
1.185
3.989
0.01211
C/T
0.118
0.057
LIPN
10
rs11202853
90545416
A
4942
2.17
1.183
3.982
0.01234
A/G
0.118
0.057
RCBTB2P1
5
rs264838
169134768
T
4942
2.496
1.216
5.124
0.01264
T/C
0.088
0.033
DOCK2
16
rs17240980
57933771
C
4942
2.094
1.171
3.745
0.01268
C/T
0.137
0.071
CNGB1
5
rs73318247
169155152
T
4942
2.492
1.214
5.116
0.01284
T/G
0.088
0.033
DOCK2
Top results after imputation in 51 cases versus all 4891 controls. All results were adjusted for genetic principal components 1–4. The threshold for statistical significance was p < 5.74 × 10−6
GWAS genome-wide association study, CHR chromosome, SNP single nucleotide polymorphism, BP base pair, N number, GTPS Guanosine-5′-triphosphates, MAF minor allele frequency, OR [95% CI] odds ratio with 95% confidence interval, p p value
×
Candidate Gene Analyses—Cases Versus Matched Controls
When cases of bisphosphonate-associated AFF were compared with matched controls, no statistically significant associations were revealed (Fig. 2b; Table 6; Supplemental Table 2).
Table 6
Top candidate gene associations with bisphosphonate-associated atypical femoral fractures
CHR
SNP
BP
Minor allele
N
OR
L95
U95
p
GTPS
MAF cases
MAF controls
Gene
9
9:136352590
136352590
T
375
5.239
2.305
11.91
7.72 × 10−5
T/C
0.118
0.029
9
rs76038546
136345878
C
375
4.854
2.165
10.88
1.254 × 10−4
C/A
0.118
0.031
2
rs138252364
111483994
C
375
5.002
1.983
12.61
6.47 × 10−4
C/G
0.088
0.02
2
rs140272071
111510669
A
375
4.58
1.838
11.41
1.09 × 10−3
A/G
0.088
0.022
ACOXL
5
rs262864
169200927
A
375
2.337
1.395
3.915
1.269 × 10−3
A/G
0.255
0.117
DOCK2-
2
2:111621582
111621582
G
375
3.334
1.599
6.951
1.319 × 10−3
G/T
0.137
0.049
ACOXL
10
rs116907192
106148123
T
375
8.746
2.298
33.28
1.47 × 10−3
T/C
0.049
0.008
CCDC147
2
rs181660819
111578634
G
375
6.45
2.037
20.42
1.524 × 10−3
G/A
0.059
0.011
ACOXL
5
rs114254961
169213503
A
375
6.187
2
19.14
1.56 × 10−3
A/G
0.059
0.015
DOCK2
1
1:21877265
21877265
C
375
3.204
1.525
6.731
2.115 × 10−3
C/G
0.118
0.045
ALPL
1
rs113561139
21909239
C
375
3.158
1.507
6.619
2.316 × 10−3
C/G
0.118
0.046
11
rs78214094
4909900
C
375
13.59
2.376
77.71
3.364 × 10−3
C/T
0.039
0.003
MMP26
2
rs3789106
111720884
G
375
1.871
1.229
2.847
3.453 × 10−3
G/T
0.51
0.363
ACOXL
2
rs13003263
111710045
T
375
0.5213
0.3336
0.8147
4.244 × 10−3
T/C
0.382
0.532
ACOXL
2
rs3789115
111712251
A
375
0.5213
0.3336
0.8147
4.244 × 10−3
A/G
0.382
0.532
ACOXL
2
rs4577288
111713046
T
375
0.5213
0.3336
0.8147
4.244 × 10−3
T/G
0.382
0.532
ACOXL
2
rs6750439
111711536
T
375
0.5213
0.3336
0.8147
4.244 × 10−3
T/C
0.382
0.532
ACOXL
1
rs114420253
248103804
A
375
3.541
1.487
8.432
4.281 × 10−3
A/G
0.088
0.031
OR2L13
2
rs1877655
111712703
C
375
0.5211
0.3331
0.8152
4.308 × 10−3
C/T
0.372
0.523
ACOXL
2
rs2341914
111713724
T
375
0.5211
0.3331
0.8152
4.308 × 10−3
T/C
0.372
0.523
ACOXL
2
rs2341915
111713661
T
375
0.5211
0.3331
0.8152
4.308 × 10−3
T/C
0.372
0.523
ACOXL
2
rs2880190
111713595
T
375
0.5211
0.3331
0.8152
4.308 × 10−3
T/A
0.372
0.523
ACOXL
2
rs4619626
111713057
T
375
0.5211
0.3331
0.8152
4.308 × 10−3
T/C
0.372
0.523
ACOXL
9
9:136338187
136338187
C
375
0.2256
0.08075
0.6302
4.498 × 10−3
C/A
0.039
0.156
SLC2A6
1
rs116121521
21876957
C
375
2.892
1.388
6.027
4.585 × 10−3
C/T
0.118
0.049
ALPL
2
rs11687442
216246210
G
375
1.926
1.223
3.034
4.69 × 10−3
G/T
0.392
0.258
FN1
5
rs111913365
169447265
G
375
6.491
1.769
23.82
4.807 × 10−3
G/A
0.049
0.009
DOCK2
5
rs76469325
169447222
T
375
6.491
1.769
23.82
4.807 × 10−3
T/G
0.049
0.009
DOCK2
5
rs116741837
169450719
T
375
4.669
1.595
13.66
4.918 × 10−3
T/C
0.059
0.019
DOCK2
2
rs112273617
233841768
C
375
9.255
1.96
43.71
4.966 × 10−3
C/T
0.039
0.005
NGEF
2
rs149536245
111709828
G
375
9.066
1.939
42.4
5.095 × 10−3
G/A
0.039
0.005
ACOXL
1
rs141276685
21888425
A
375
2.836
1.363
5.904
5.313 × 10−3
A/G
0.118
0.051
ALPL
15
rs116916068
74920220
A
375
2.647
1.32
5.31
6.104 × 10−3
A/G
0.128
0.052
CLK3
2
rs3789119
111707405
T
375
1.862
1.189
2.918
6.643 × 10−3
T/C
0.372
0.253
ACOXL
10
rs10887854
90540941
G
375
2.884
1.342
6.2
6.677 × 10−3
G/A
0.118
0.051
10
rs10887855
90541206
T
375
2.884
1.342
6.2
6.677 × 10−3
T/C
0.118
0.051
10
rs11202848
90532166
A
375
2.884
1.342
6.2
6.677 × 10−3
A/C
0.118
0.051
LIPN
10
rs11202850
90535654
G
375
2.884
1.342
6.2
6.677 × 10−3
G/T
0.118
0.051
LIPN
10
rs11202851
90537942
T
375
2.884
1.342
6.2
6.677 × 10−3
T/C
0.118
0.051
LIPN
10
rs11202852
90544073
A
375
2.884
1.342
6.2
6.677 × 10−3
A/G
0.118
0.051
10
rs11202855
90547504
A
375
2.884
1.342
6.2
6.677 × 10−3
A/G
0.118
0.051
10
rs12572022
90545882
A
375
2.884
1.342
6.2
6.677 × 10−3
A/C
0.118
0.051
RCBTB2P1
10
rs17112679
90527569
C
375
2.884
1.342
6.2
6.677 × 10−3
C/T
0.118
0.051
LIPN
10
rs17112704
90529566
T
375
2.884
1.342
6.2
6.677 × 10−3
T/A
0.118
0.051
LIPN
2
rs71431135
111809400
G
375
0.1615
0.04269
0.6107
7.222 × 10−3
G/A
0.029
0.119
ACOXL
7
7:94036547
94036547
T
375
1.779
1.166
2.716
7.547 × 10−3
T/C
0.461
0.31
COL1A2
2
rs13024581
111823835
C
375
0.1635
0.0432
0.6186
7.648 × 10−3
C/T
0.029
0.119
ACOXL
2
rs2118908
111824592
G
375
0.1635
0.0432
0.6186
7.648 × 10−3
G/A
0.029
0.119
ACOXL
2
rs71431138
111818383
C
375
0.1635
0.0432
0.6186
7.648 × 10−3
C/T
0.029
0.119
ACOXL
2
rs13034863
111810020
G
375
0.1646
0.04355
0.6224
7.842 × 10−3
G/C
0.029
0.117
ACOXL
2
rs34121532
111810633
G
375
0.1646
0.04355
0.6224
7.842 × 10−3
G/A
0.029
0.117
ACOXL
2
rs35875858
111811106
G
375
0.1646
0.04355
0.6224
7.842 × 10−3
G/C
0.029
0.117
ACOXL
2
rs36091399
111810844
T
375
0.1646
0.04355
0.6224
7.842 × 10−3
T/G
0.029
0.117
ACOXL
2
rs71431134
111808175
G
375
0.1646
0.04355
0.6224
7.842 × 10−3
G/C
0.029
0.117
ACOXL
2
rs71431136
111809837
C
375
0.1646
0.04355
0.6224
7.842 × 10−3
C/T
0.029
0.117
ACOXL
2
rs78210391
216273212
A
375
6.618
1.64
26.71
7.943 × 10−3
A/C
0.039
0.008
FN1
2
rs17483962
111826292
C
375
0.1672
0.04419
0.6328
8.441 × 10−3
C/G
0.029
0.117
ACOXL
2
rs17549841
111826389
T
375
0.1672
0.04419
0.6328
8.441 × 10−3
T/C
0.029
0.117
ACOXL
2
rs35812219
111826286
T
375
0.1672
0.04419
0.6328
8.441 × 10−3
T/C
0.029
0.117
ACOXL
2
rs74848138
111825521
G
375
0.1672
0.04419
0.6328
8.441 × 10−3
G/A
0.029
0.117
ACOXL
Top results after imputation in 51 cases versus 324 matched controls. All results were adjusted for genetic principal components 1–4. The threshold for statistical significance was p < 5.74 × 10−6
GWAS genome-wide association study, CHR chromosome, SNP single-nucleotide polymorphism, BP base pair, N number, GTPS Guanosine-5′-triphosphates, MAF minor allele frequency, OR [95% CI] odds ratio with 95% confidence interval, p p value
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Discussion
We were hoping to find a strong common genetic susceptibility trait for AFF to predict patients at high risk of this ADR. Our results indicate that there is no common genetic variant that can be used for this purpose. The only significant finding on a genome-wide level was with four SNPs when cases were compared with population controls, but these were uncommon SNPs, all of which were single hits, meaning that these associations are likely false positives [24, 25], although two may theoretically be related to the treatment indication (NR3C1 and NTN1). None of these specific SNPs have, however, previously been implicated in AFF or osteoporosis [11, 26‐28]. After reducing the risk of confounding by indication with the use of a comparison to bisphosphonate-treated controls, no statistically significant association remained.
At this time we are therefore left to models based on pharmacological and clinical considerations to minimize the risk of AFF. The prevailing pathophysiological theory of AFF is that bisphosphonates lead to over-suppression of bone remodeling [29]. Because bisphosphonates preferentially suppress the targeted repair mechanism, increased numbers of micro-cracks and reduced heterogeneity of the bone can be seen in bone tissue from animals and humans [7, 30‐32]. The combination of these can lead to accumulation of micro-cracks during normal loading and propagation to larger cracks, eventually resulting in complete AFF. Studies have shown that the risk of developing an AFF is on average 50-fold greater for a bisphosphonate user compared to a nonuser, and more than 100-fold greater after 4–5 years of treatment [3, 5, 33]. In contrast, discontinuation of the drug will lead to a steep decline in the risk for developing an AFF [3]. In addition, different bisphosphonates might vary in terms of risk [3, 5, 34]. Hence, treatment duration and choice of bisphosphonate could be subject to manipulation in order to gain maximum treatment benefit while reducing the risk of AFF.
Many attempts have been made to identify risk factors that may predispose bisphosphonate users to AFF. A potential genetic influence has been suggested as a possible explanation to why only a minority of bisphosphonate users develop AFF. For instance, studies have revealed that polymorphisms in the gene encoding farnesyl diphosphate synthase (FDPS) may affect bone mineral density and bone turnover following bisphosphonate treatment in some patients, while not in others [35‐38]. A possible genetic cause is also supported by studies that have demonstrated a difference in risk of AFF based on ethnicity, with Asians being at higher risk. A recent study by Lo et al. revealed a hazard ratio of 6.6 for females of Asian ethnicity compared with Caucasian women [9]. In addition, theories of a possible genetic trait have been long existing for other bisphosphonate ADRs that manifest in the skeleton [39].
There are several limitations to this study. First, matching of controls was done using bisphosphonate exposure as a proxy for osteoporosis as the Swedish Patient Register mainly includes information on diagnoses from hospital care. We were thus unable to identify controls who were prescribed a bisphosphonate for osteoporosis prevention. Secondly, although this is the largest genetic study of bisphosphonate-associated AFF to date, the number of included cases is still low. This means that the power to detect weakly associated common variants and strongly associated rare variants is low. It is also possible that several variants, inherited independently of one another, are required to infer a risk of AFF, in which case they will go undetected. To elucidate this would require a larger study and whole genome or exome sequencing, which was beyond the scope of this study. Lastly, there are suggestions that the association between bisphosphonate use and AFF is mainly driven by a genetic predisposition [11]. However, since 4–5 years of bisphosphonate use in Swedish women is associated with a 125-fold increase in risk of AFF [3], the potential underlying causal genetic risk allele(-s) should have a firm relation with both AFF and bisphosphonate use to entirely extenuate the exponential increase in risk with duration of bisphosphonate use. Noteworthily, a more moderately strong effect modification between bisphosphonates and genetic predisposition might still exist, but the current study is too small to disentangle such genetic modifying effects.
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That several genetic loci, perhaps varying between individuals, might explain at least some cases of bisphosphonate-associated AFF has been proposed by some studies, although methodological issues and other limitations makes it difficult to conclude whether the findings are of relevance for a larger population of individuals with bisphosphonate-associated AFF. In the study by Pérez-Núñez et al. that compared 13 women with AFF and 268 female controls, 21 loci were more frequent in the fracture group [40]. Most patients accumulated two or more allelic variants, and the number of variants was different between patients with fractures and the controls, suggesting that several genes may be involved. The study was, however, limited by the fact that the controls were a mix of normal and osteoporotic women, and that only 12 of the 13 cases had been exposed to bisphosphonates. In another study, Roca-Ayats et al. performed whole-exome sequencing in three sisters who had all developed AFF following bisphosphonate treatment, and compared with three unrelated patients with bisphosphonate-associated AFF [41]. They detected 37 rare nonsynonymous mutations in 34 genes, but the results are questionable due to lack of validation and a small sample size. In a further study, Funck-Brentano et al. performed sequencing of four genes amongst two patients with bisphosphonate-associated AFF and found genetic variants in one, a rare heterozygous mutation in COL1A2 (c.213G > A; p.Arg708GIn) [42]. Limitations of this study include the small sample size. While these findings suggest a polygenic model in which an accumulation of susceptibility variants may lead to a predisposition to bisphosphonate-associated AFF, larger studies are required to provide solid evidence.
Conclusion
With this genome-wide association and candidate gene study, we were unable to find evidence of common genetic traits predisposition for bisphosphonate-associated AFF. This does not rule out the possibility of weakly associated genetic traits or the presence of rare genetic variants that confer a risk. Further studies of larger sample size as well as whole-exome or whole-genome sequencing studies are warranted.
Acknowledgements
This work was supported by the Swedish Research Council (Medicine 521-2011-2440, 521-2014-3370 and 2015-03527); Swedish Heart and Lung Foundation (20120557, 20140291 and 20170711); Selander’s foundation; Thuréus’ foundation; the Swedish Medical Products Agency; the Clinical Research Support (ALF) at Uppsala University; and Östergötland County Council (LIO-698411). We thank research nurses Ulrica Ramqvist, Elisabeth Stjernberg, Charlotta Haglund and Elisabeth Balcom, and research assistants Sofie Collin, Eva Prado Lopez, Agnes Kataja Knight, Agnes Wadelius, and Martha Wadelius, Department of Medical Sciences, Clinical Pharmacology, Uppsala University, Uppsala, Sweden, for recruiting and interviewing cases and for database administration. We are grateful to Tomas Axelsson for SNP array genotyping at the Department of Medical Sciences, SNP&SEQ Technology Platform, which is funded by the Science for Life Laboratory, Swedish Research Council, and Uppsala University. Computations were performed on resources provided by SNIC through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX). We acknowledge Patrik Magnusson and Barbro Sandin at the Department of Medical Epidemiology and Biostatistics, Karolinska Institutet for access to data from the Swedish Twin Registry, which is managed by Karolinska Institutet and receives funding through the Research Council Swedish under Grant No. 2017-00641.
Compliance with Ethical Standards
Conflict of interest
Mohammad Kharazmi, Karl Michaëlsson, Jörg Schilcher, Niclas Eriksson, Håkan Melhus, Mia Wadelius, and Pär Hallberg declare that they have no conflict of interest.
Ethical Approval
The study was approved by the regional ethical review boards in Uppsala and Stockholm (2010/231 in Uppsala; 2007/644-31 and 2011/463-32 in Stockholm).
Informed Consent
Written informed consent was obtained from all participants.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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