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A Genome-Wide Association Study of Bisphosphonate-Associated Atypical Femoral Fracture

  • Mohammad KharazmiEmail author
  • Karl Michaëlsson
  • Jörg Schilcher
  • Niclas Eriksson
  • Håkan Melhus
  • Mia Wadelius
  • Pär Hallberg
Open Access
Original Research

Abstract

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.

Keywords

Genome-wide association study Atypical fractures Bisphosphonate Drug-related side effects and adverse reactions Pharmacogenetics 

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, 4, 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.

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.

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

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

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, 27, 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, 31, 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, 36, 37, 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.

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.

Notes

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.

Author Contributions

Study design: PH and MW. Data collection: MK, MW, KM, JS and PH. Data analysis: NE. Data interpretation: MK, PH, MW, KM, JS, HM and NE. Drafting manuscript: MK and PH. Revising manuscript content: MK, PH, MW, KM, JS, HM and NE. Approving final version of manuscript: MK, PH, MW, KM, JS, HM and NE.

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.

Supplementary material

223_2019_546_MOESM1_ESM.docx (618 kb)
Supplementary material 1 (DOCX 617 kb)

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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.

Authors and Affiliations

  1. 1.Department of Surgical SciencesUppsala UniversityUppsalaSweden
  2. 2.Department of Clinical and Experimental Medicine, Faculty of Health SciencesLinköping UniversityLinköpingSweden
  3. 3.Department of Medical SciencesUppsala UniversityUppsalaSweden
  4. 4.Uppsala Clinical Research CenterUppsala UniversityUppsalaSweden

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