Osteoporosis Genes Identified by Genome-wide Association Studies

C H A P T E R

16 Osteoporosis Genes Identified by Genome-wide Association Studies Fernando Rivadeneira and André G. Uitterlinden Genetic Laboratory Ee5-79, Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands

I. INTRODUCTION Without doubt the field of osteoporosis (just as most of all other medical fields) was revolutionized by the advent of the Human Genome Project1 at the end of the 1980s. An exponential increase in the number of publications in the field was observed shortly after its launch with the trend continuing to its completion in the year 2001, and even more so thereafter (Figure 16.1). This impressive wave of studies concerning the genetics of osteoporosis in humans has now begun a new era of discoveries with the advent of so-called genome-wide association studies (GWAS),2–4 whose design has been comprehensively described in “Genome Wide Associations Studies”. Before the GWAS era, the literature about the genetics of osteoporosis and fracture had been confined to a very large number of “genome-wide linkage” and “candidate gene association” studies. With few exceptions, the majority were small, inadequately-powered studies generating controversial and frequently non-reproducible reports5 on variants in about 150 candidate gene regions for osteoporosis (HUgeNet website http://www. hugenavigator.net/). Some shortcomings were tackled in the GENOMOS consortium where large-scaled evidence (n = 20 000–45 000 which, by current standards, is still substantial) was produced on a limited number of “the usual suspects” in genetics of osteoporosis including the ESR1,6 VDR,7 COLIA1,8 TGFB19 and LRP5/610 genes. These results are summarized in Table 16.1. Although a few polymorphisms were indeed identified as being associated with either bone mineral density (BMD) or fracture (such as for LRP5 very significantly associated at p < 5 × 10−8, the current standard for declaring genomewide significance), this effort was restricted to known Genetics of Bone Biology and Skeletal Disease DOI: http://dx.doi.org/10.1016/B978-0-12-387829-8.00016-0

polymorphisms and did not interrogate the genetic contribution to osteoporosis at a genome-wide level as is now possible in GWAS. As reviewed in Chapter 7, it is expected that, for most complex traits and common diseases,11–13 hundreds (if not thousands) of variants with weak (but real) effects14 will be underlying the genetic architecture of the trait and disease. From this perspective, only wellpowered studies based on several independent populations (for replication), with a well-defined selection of polymorphisms and gene regions and a robust control for multiple hypothesis-testing in the analysis, will be suited to identify genuine genetic effects.15 While the GWAS approach (with some few exceptions) incorporates in its design several of these properties, the number of loci identified depends on the total sample size obtained in a given study: more samples will lead to more GWAS “hits”. Therefore, to the present, only a small fraction of all the variants expected to be associated with a complex trait have been detected so far. In addition, a very limited number of these loci discovered at a genome-wide significant level (i.e. 1 in 20 or p = 0.05 after the Bonferroni correction) may still represent false-positive reports. Nevertheless, after replication in tens to hundreds of thousands of individuals, it is very likely that genome-wide significant signals associated at a stringent p < 5 × 10−8 will contain associated variants which represent true underlying biological mechanisms influencing the trait in question. Another important aspect for the interpretation of the GWAS discoveries is that, with few exceptions, most of the genes claimed as “underlying the GWAS signals” have been labeled as such without robust functional evidence supporting or demonstrating its candidacy.

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© 2012 2013 Elsevier Inc. All rights reserved.

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16.  Osteoporosis Genes Identified by Genome-wide Association Studies

Usually, allocating a gene to a genetic signal is based on physical distance and current knowledge of biology, neither of which suffices to assign unequivocally the gene to the single nucleotide polymorphism (SNP) effect. Therefore, even though the circumstantial evidence for biologic candidacy may be strong, such assignments should be interpreted with caution until conclusive evidence becomes available. This will also vary from region to region depending on the relative location of the associated SNPs, the linkage disequilibrium properties between the markers, the patterns of recombination rate, and the number of genes underlying the signal. All of these parameters must be considered while evaluating the candidacy of the gene(s) underlying the GWAS signals. From this perspective, the likelihood of a gene being responsible for a GWAS signal will be a function of the properties of the signal and the candidacy characteristics (known biological function) of the gene (Table 16.2).

Nevertheless, just as for any candidate gene evaluation, these assessments will be based on previous knowledge about the gene, which by itself constitutes a liability considering that the GWAS approach is a hypothesis-free screen of the genome. This implicates that new and not (properly) annotated areas of the genome can be found in a given GWAS, leaving much new biology to be discovered. This way, not scoring high on the candidacy dimension may just reflect the lack of knowledge, and does not exclude any gene from being truly involved in the biologic mechanisms leading to trait variation.

II.  GENOME-WIDE ASSOCIATION STUDIES OF OSTEOPOROSIS We define the set of candidate osteoporosis genes included in this chapter as those proposed to have been TABLE 16.2  Likelihood of a Gene being Responsible for a GWAS Signal A. Properties of the GWAS Signal Arising from a SNP (or a Variant in High LD) 1. Location in relation to the gene (exonic, intronic, promoter, 3’UTR or intergenic) 2. Functionality (promoter/regulatory region, non-synonymous, synonymous, transcription factor binding or splice regulation) 3. Physical distance from the gene 4. Co-location in the same linkage disequilibrium block of the gene B. Candidacy Characteristics by which a Gene is Weighted Based on Presence of 1. SNP (or a variant in high LD) constituting an eQTL associated with gene expression 2. KO model organism (mouse or zebra fish) with a skeletal phenotype* 3. Monogenic human syndrome (OMIM) with a skeletal phenotype* 4. Involvement in a bone-active pathway*

FIGURE 16.1  Historical trend of the number of publications in the field of “genetics of osteoporosis”.

*Candidacy in relation to osteoporosis traits.

TABLE 16.1  Pre-GWAS Results of the GENOMOS Consortium BMD

Fracture

Gene

SNPs (n)

Sample (n)

Femoral Neck

Lumbar Spine

NonVertebral Vertebral

ESR1

3

18 917

–

–

20–30%

10–20%

6

VDR

5

26 242

–

–

10%

–

7

COL1A1

1

20 786

0.15 SD

0.15 SD

10%

–

8

TGFB1

5

28 924

–

–

–

–

9

LRP5

2

37 760

0.15 SD

0.15 SD

12–26%

6–14%

10

LRP6

1

37 760

–

–

–

–

10

III.  DISORDERS OF BONE AND JOINT

Reference

III.  Genes Identified by Genome-Wide Association Studies on Bone Mineral Density

discovered by the GWAS approach within adequately powered settings (n >10 000 subjects) in relation to BMD, the most accessible and up to now, the most prolific of the osteoporosis traits. An overview of GWAS studies on osteoporosis is presented in Table 16.3. The first GWAS report concerning osteoporosis was published in 2007 by Kiel and colleagues.16 The effort failed to identify any associated loci at a genome-wide significant level as a reflection of the limited sample size (n = 1141) and sparse SNP content (100  K) of the study. In 2008, in Richards et  al,17 variants in LRP5 and TNFRSF11B (OPG) were reported as associated with lumbar spine and femoral neck BMD in 8557 UK and Dutch individuals. Almost simultaneously, Styrkarsdottir et  al published a report on 5861 Icelandic individuals, with replication in an additional 7925 European individuals,18 which identified variants mapping also to TNFRSF11B (OPG), together with additional ones mapping to the TNFSF11 (RANKL), ESR1, ZBTB40 and the major histocompatibility complex (MHC) loci. A subsequent report from this group published in early 2009 was based on an extended set including 6865 Icelandic individuals, with replication in another 8510 European individuals.19 They identified variants mapping to TNFRSF11A (RANK), SOST, MARK3 and SP7 (osterix). Shortly afterwards, variants in the osterix gene were also identified by Timpson et  al in an effort based on 1518 UK children, followed by replication in adults, including an “extremes truncate selection” of 132 Australian individuals with high or low BMD, and in 3692 individuals of European descent.20 During mid-2009, Cho et al published a study examining ultrasound of the radius, tibia and the heel in 8842 Korean individuals (with replication in additional 7861 individuals) postulating FAM3C and SFRP4 as new BMD loci.21 Though Speed of Sound (SOS) ultrasound does not directly measure BMD, it

is associated with fracture risk. At the end of 2009, the GEFOS consortium reported a large leap in the discoveries described in Rivadeneira et al.22 Within the setting of the Genetic Factors of Osteoporosis (GEFOS) consortium (), variants were identified in 13 additional loci in a study of 19 195 individuals of Northern European origin. These 13 loci reached genome-wide significance for the first time and included WLS (former GPR177), SPTBN1, CTNNB1 (β-catenin), MEPE/SPP1/IBSP, MEF2C, STARD3NL, SHFM1, LRP4, SOX6, DCDC5, FOXC2, CRGR1 and HDAC5. After that, two different studies of BMD, using extreme-ascertainment designs based on 800 Chinese and on 1955 Australian individuals, provided evidence for additional loci after replication in nearly 20 000 individuals (18 898 and 20 898, respectively). In the first study, Kung et  al23 identified variants in JAG1 associated at a genome-wide significant level (GWSL) of p  <  5  ×  10−8. In the second study, Duncan et  al24 identified variants mapping to GALNT3 and RSPO3 associated at a GWSL, while variants in SOX4, LTBP3, and CLNC7 were suggestive of association. With the exception of variants that mapped to MHC and LBT3, all other loci described in this chapter have been found associated at GWSL by the recently published second effort on BMD of the GEFOS consortium,25 which has now brought the number of identified BMD loci to 56.

III.  GENES IDENTIFIED BY GENOMEWIDE ASSOCIATION STUDIES ON BONE MINERAL DENSITY Table 16.4 lists the 39 genes from 27 loci with variants consistently associated with BMD at a GWSL of p < 5 × 10−8.

TABLE 16.3  GWAS of BMD GWAS Populations

Discovery

Total

Reported Hits

Replicated Hits

Reference

Framingham Osteoporosis Study

1141

1141

0

0

16

Twins UK & Rotterdam Study

2094

8557

2

2

17

deCODE 1

5861

13 786

5

4

18

ALSPAC

1518

5474

1

1

20

deCODE 2

6865

15 375

4

4

19

KARE GEFOS 1 Hong Kong Osteoporosis Study AOGC GEFOS 2

245

8842

16 703

2

2

21

19 125

19 125

20

20

22

800

18 898

1

1

23

1955

20 898

2

2

24

32 961

83 894

56

56

25

III.  DISORDERS OF BONE AND JOINT

TABLE 16.4  List of BMD Loci with their Candidate Genes and Functional Biological Annotations Chromosomal Location

Gene Name

Description

Size (kb)

MIM MIM Gene Morbid Accession Accession MIM Morbid Description

1p31.3

WLS

wntless homolog (Drosophila)

135

6 11 514

1p36.12

ZBTB40

zinc finger and BTB domain containing 40

79.3

6 12 106

WNT4

wingless-type MMTV integration site family, member 4

24.0

6 03 490

2p16.2

SPTBN1

spectrin, beta, non-erythrocytic 1

213.4

1 82 790

2q24.3

GALNT3

UDP-N-acetyl-alpha-Dgalactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3)

47.1

3p22.1

CTNNB1

catenin (cadherin-associated protein), beta 1, 88 kDa

4q22.1

IBSP

eQTL

MGI KO mouse with Skeletal Phenotype

Pathway Member

MGI:1915401

Wnt

NM_014870 6 11 812

46,XX sex reversal with dysgenesis of kidneys, adrenals, and lungs

6 01 756

2 11 900

Tumoral calcinosis, hyperphosphatemic, familial; HFTC

MGI:894695

65.3

1 16 806

1 81 030

Salivary gland adenoma, pleomorphic

MGI:88276

integrin-binding sialoprotein

12.3

1 47 563

MGI:96389

MEPE

matrix extracellular phosphoglycoprotein

25.4

6 05 912

MGI:2137384

SPP1

secreted phosphoprotein 1

7.8

1 66 490

5q14.3

MEF2C

myocyte enhancer factor 2C

185.9

6 00 662

6p22.3

SOX4

SRY (sex determining region Y)-box 4

4.9

1 84 430

Reference 46

6q22.33

RSPO3

R-spondin 3

79.2

6 10  574

MGI:1920030

6q25.1

ESR1

estrogen receptor 1

472.9

1 33 430

7p14.1

SFRP4

secreted frizzled-related protein 4

119.8

6 06 570

STARD3NL

STARD3 N-terminal like

52.4

6 11 759

7q21.3

SHFM1

split hand/foot malformation (ectrodactyly) type 1

228.3

6 01 285

7q31.31

FAM3C

family with sequence similarity 3, member C

47.5

6 08 618

WNT16

wingless-type MMTV integration site family, member 16

15.7

6 06 267

TNFRSF11B

tumor necrosis factor receptor superfamily, member 11b

28.6

6 02 643

8q24.12

NM_030761

NM_000582 6 13 443

6 08 446

Wnt

MGI:98389

Mental retardation, stereotypic movements, epilepsy, and/or cerebral

Myocardial infarction, susceptibility to, 1

Wnt

Endochondral Ossification Endochondral Ossification

Wnt

MGI:1352467 Wnt

1 83 600

2 39 000

TABLE 16.4  List of BMD Loci with their Candidate Genes and Functional Biological Annotations

Split-hand/foot malformation 1; SHFM1

Paget disease, juvenile

Lexicon Pharmaceuticals

Wnt

MGI:109587

OPG/RANK/ RANKL

Size (kb)

MIM MIM Gene Morbid Accession Accession MIM Morbid Description

low density lipoprotein receptorrelated protein 4

61.8

6 04 270

ARHGAP1

Rho GTPase activating protein 1

23.5

6 02 732

F2

coagulation factor II (thrombin)

20.3

1 76 930

DCDC1

doublecortin domain containing 1

297.3

6 08 062

DCDC5

doublecortin domain containing 5

426.6

6 12 321

11p15.1

SOX6

SRY (sex determining region Y)box 6

773.1

6 07 257

11q13.2

LRP5

low density lipoprotein receptorrelated protein 5

136.7

6 03 506

Chromosomal Location

Gene Name

Description

11p11.2

LRP4

11p13

2 12 780

Cenani–Lenz syndactyly syndrome; CLSS

eQTL

MGI KO mouse with Skeletal Phenotype

Pathway Member

AB011540

MGI:2442252

Wnt

MGI:3606252 6 13 679

Prothrombin deficiency, congenital

MGI:98368

Endochondral Ossification

6 07 636

Van Buchem disease, type 2

MGI:1278315

Wnt

1 44 750

Hyperostosis corticalis generalisata, benign form of worth, with torus palatinus

MGI:1278315

Wnt

2 59 770

Osteoporosis-pseudoglioma syndrome (OPPG)

MGI:1278315

Wnt

6 08 084

Bone mineral quantitative trait locus 1, high bone mass included

MGI:1278315

Wnt

12q13.13

SP7

Sp7 transcription factor

18.7

6 06 633

6 13 849

Osteogenesis imperfecta, type XI

MGI:2153568

13q14.11

TNFSF11

tumor necrosis factor (ligand) superfamily, member 11

45.3

6 02 642

2 59 710

Osteopetrosis, autosomal recessive 2; OPTB2

MGI:1100089

14q32.32

MARK3

MAP/microtubule affinityregulating kinase 3

118.5

6 02 678

16p13.3

CLCN7

chloride channel 7

30.6

6 02 727

16q24.1

FOXL1

forkhead box L1

3.2

6 03 252

FOXC2

forkhead box C2 (MFH-1, mesenchyme forkhead 1)

1.7

6 02 402

1 53 400

Lymphedema-distichiasis syndrome

17q12-22

CRHR1

corticotropin-releasing factor receptor 1

213.9

17q21.31

SOST

sclerostin

5.1

6 05 740

2 69 500

Sclerosteosis; SOST

HDAC5

histone deacetylase 5

46.9

6 05 315

18q21.33

TNFRSF11A

tumor necrosis factor receptor superfamily, member 11a, NFKB activator

62.4

6 03 499

6 12 301

Osteopetrosis, autosomal recessive 7; OPTB7

20p12.2

JAG1

jagged 1

36.3

6 01 920

6 01 920

Jagged 1; JAG1

OPG/RANK/ RANKL

NM_002376 6 11 490

Osteopetrosis, autosomal recessive 4; OPTB4

MGI:1347048 MGI:1347481

TGF-beta Wnt

NM_003839

MGI:1921749

Wnt

MGI:1314891

OPG/RANK/ RANKL Notch

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16.  Osteoporosis Genes Identified by Genome-wide Association Studies

1p31.3 WLS The (Drosophila) wntless homolog is a 135  kD gene formerly known as G protein-coupled receptor 177 (GPR177) gene. WLS is a novel factor part of the highly evolutionary conserved Wnt signaling pathway involved in bone cell differentiation and development. The reported variants, showing association with lumbar spine and femoral neck BMD,22 map to a narrow linkage disequilibrium (LD) block within an intronic region of the gene. WLS has been shown to be required for cell surface expression of Wnt3a proteins by HEK cells, and capable of activating nuclear factor-kappa B (NF-κB) when expressed in HEK cells.26 In addition, Wls has been shown to be a Wnt trafficking regulator in mouse embryogenesis.27–29

1p36.12 ZBTB40 and WNT4 ZBTB40 is a 79 kb gene located in a well-recognized linkage region. GWAS signals in ZBTB40 were first reported by Styrkarsdottir et al18 as associated with hip and spine BMD, and further replicated in the subsequent GWAS meta-analyses of the GEFOS consortium.22 In the latter effort, there was evidence for an independent signal mapping to the 5’ region of WNT4, a smaller gene of size 24 kb located about 310 kb upstream from ZBTB40. ZBTB40 encodes a protein of unknown function but which is expressed in bone. Given its zinc finger and BT domains, it is likely the protein has DNA binding properties and may be involved in protein– protein interactions. Wnt4 belongs to the Wnt family of signaling proteins and is involved in several developmental processes including regulation of cell fate and patterning during embryogenesis30 by inhibition of β-catenin. Wnt4 is involved in the control of female development and the prevention of testes formation, where mutations in the gene have shown to produce sex reversal syndromes.31 During hematopoietic stem cell differentiation, WNT4 acts as a non-canonical activator of Wnt signaling,32 though its role on bone remains unknown.

2p16.2 SPTBN1 SPTBN1 is a 213 kb gene that encodes a subform of β-spectrin, which is a major cytoskeletal scaffold protein. This locus was first reported by Styrkarsdottir et al19 as associated with lumbar spine BMD with subsequent replication at GWSL in the first GEFOS metaanalysis.22 The functional role of this gene in bone remains unclear although, in mice, targeted inactivation of Elf (mouse homolog) results in disruption of transforming growth factor-beta (TGFβ) signaling with severe phenotypic alterations across multiple systems

similar to that observed in double knockouts (KOs) of SMAD3 and SMAD4 proteins.33

2q24.3 GALNT3 GALNT3 is a gene of 47 kb encoding for an enzyme involved in biosynthesis of oligosaccharides and has high homology to other members of the GalNAc-transferases family members. Variants in and around GALNT3 were first reported as associated at GWSL with both hip and lumbar spine BMD in Australian and Northern European populations.24 Mutations in the GALNT3 gene cause autosomal recessive hyperostosis–hyperphosphatemia syndrome.34

3p22.1 CTNNB1 This 65 kb gene encodes β-catenin, a key transcription factor of the Wnt signaling involved in osteoblast differentiation from mesenchymal stem cells35,36 and regulation of osteoclast activation.37 Beta-catenin dependent (so-called canonical) Wnt signaling is a major regulator of chondrogenesis, osteoblastogenesis, and osteoclastogenesis. Deletion of the gene in osteoblasts results in osteopenia while stabilization results in high bone mass.38 Common variants located in the 5’ region of the gene were found associated for the first time at genomewide significant level with femoral neck BMD in the first GEFOS meta-analysis.22

4q22.1 IBSP, MEPE and SPP1 This locus clusters a series of relatively small phylogenetically-related genes encoding for matricellular phosphoglycoproteins important for bone formation and mineralization39 including MEPE, IBSP (integrin binding sialoprotein) and SPP1 (osteopontin). All three genes are expressed in bone and exhibit a skeletal phenotype when deleted. MEPE spans 25 kb and encodes one of these phosphoglycoproteins. Targeted inactivation of Mepe in mice results in increased BMD.40 Even though the first GEFOS meta-analysis22 showed that the variants achieving GWSL for association with lumbar spine BMD mapped close to MEPE, the involvement of the other genes in the region cannot be ignored. In a previous report, Styrkarsdottir et al19 reported variants mapping close to IBSP as suggestive of association with hip BMD. IBSP is a 12 kb gene located about 42 kb away from MEPE, encoding a major structural protein of the bone matrix that binds tightly to hydroxyapatite and appears to form an integral part of the mineralized matrix. In addition, Koller et  al41 showed that the effect on IBSP was also present in premenopausal women. Ibsp KO mice have impaired bone growth and mineralization, concomitant with dramatically reduced bone formation

III.  DISORDERS OF BONE AND JOINT

III.  Genes Identified by Genome-Wide Association Studies on Bone Mineral Density

with thinner cortices, despite greater trabecular bone volume than wild-type mice, and low rates of skeletal turnover,42 indicating impairments of both new bone formation and osteoclast activity. SPP1 is a small gene spanning 7 kb (located about 122 kb away from MEPE) that codes for osteopontin (OPN). Mice with deletion of the OPN gene are resistant to ovariectomy–as well as unloading–induced bone loss.42,43

5q14.3 MEF2C MEF2C is a large 186 kb gene encoding a member of the MADS box transcription enhancer factor 2 (MEF2) family of proteins. MEF2C has been primarily implicated in muscle function, although it is known to play a role in endochondral ossification44 and is a potential regulator of the SOST gene by interaction with a conserved enhancer, which is deleted in van Buchem’s disease.45 The first report implicating variants in MEF2C as associated with BMD variation in Northern Europeans came from the first GEFOS meta-analysis22 which has subsequently been replicated.25 In addition, variants in MEF2C have also been associated with human stature.12

6p22.3 SOX4 SOX4 is a small intronless gene of about 5 kb encoding a member of the SOX (SRY-related HMG-box) family of transcription factors. Sox4 homozygous KO mice die, but heterozygous mice display osteopenia and reduced bone strength.46 Further, in rat osteoblasts, Sox4 has been shown to be involved in cellular proliferation and differentiation, functioning upstream of Osx (Osterix) but independently of Runx2. Variants in and around SOX4 presented with suggestive evidence for association with both hip and lumbar spine BMD in Australian and Northern European populations.24 This was replicated at a GWSL in the second GEFOS metaanalysis on lumbar spine and femoral neck BMD,25 with variants mapping in between CDKAL1 and SOX4.

6q22.33 RSPO3 RSpo proteins are general regulators of canonical Wnt signaling, acting through a common biochemical mechanism involving competition with DKK1 and reduction of the internalization of LRP6 by the cell.47 Homozygous RSPO3 KO mice display embryonic lethality, and no mutations in humans have been reported to date. Variants upstream from RSPO3, a gene spanning 79 kb, were first reported as associated at genome-wide significant level with BMD of the femoral neck and at nominal level with lumbar spine BMD in Australian and Northern European populations.24

249

6q25.1 ESR1 The estrogen receptor type 1 gene (ESR1) is a very large gene, spanning 473 kb, which historically has been a strong candidate for genetic regulation of bone mass. Three main polymorphisms have been thoroughly studied in many candidate gene studies, including a TA microsatellite repeat in the promoter region, the PvuII and the XbaI restriction fragment length polymorphisms. None of these polymorphisms is associated with BMD variation, as reported by the prospective large-scale individual level meta-analysis from the GENOMOS study involving 18 917 individuals, but did show a significant association with fracture that was independent of BMD.48 The first GWAS report postulating variants in the ESR1 locus as associated at genome-wide significant level with BMD variation in humans was performed in Icelandic individuals with replication on other Northwestern European populations.18 This study showed signals mapping to C6orf97, while this and other independent variants (not in LD) mapping within ESR1 were subsequently replicated in larger GWAS.19,22 Interestingly, this ESR1 locus not only displays multiple signals (allelic heterogeneity) for BMD, but also for other phenotypes also assessed by GWAS, including breast cancer risk and body height.12,49

7p14.1 SFRP4 and STARD3NL Variants in this locus were first reported associated at genome-wide significant level with BMD derived from forearm, heel and finger ultrasound in Korean populations21 and with lumbar spine BMD in the first GEFOS meta-analysis in European individuals22 mapping in between the STARD3NL and SFRP4 genes. STARD3NL is a 52 kb long gene which encodes a cholesterol endosomal transporter but its potential role on bone metabolism remains unclear. In contrast, SFRP4 is known to inhibit Wnt signaling and, as shown in SAMP6 mice, Sfrp4 negatively regulates bone formation and decreases BMD.50 This inhibition of canonical Wnt signaling is suggested to be mediated by lower responsiveness for the Wnt3A ligand in osteoblasts derived from the SAMP6 strains. Attenuation of canonical Wnt signaling by overexpression of SFRP4 in osteoblasts results in reduction of trabecular bone mass;51 a finding consistent with lower BMD and ultrasound properties of the trabecular-rich bone content of the lumbar spine and heel skeletal sites.

7q21.3 SHFM1 and FLJ42280 This locus was first described in the first GEFOS BMD meta-analysis as holding several variants associated at genome-wide significant level with both lumbar

III.  DISORDERS OF BONE AND JOINT

250

16.  Osteoporosis Genes Identified by Genome-wide Association Studies

spine and femoral neck.22 These variants map close to FLJ42280, a gene encoding a hypothetical protein of unknown function. The large linkage disequilibrium block harboring the GWAS signal also includes the split hand and foot malformation or SHFM1 gene, also known as deleted in split-hand/split-foot 1 region or DSS1 gene. More than a single gene, the region is characterized by genomic rearrangements leading to deletion of DSS1 and the distalless-related homeogenes DLX5 and DLX6. The latter two genes code for members of the Wnt signaling pathway, and cause ectrodactyly in mice and humans52 when both are deleted or mutated. This may also be as a consequence of “functional haploinsufficiency” when the chromosomal rearrangement physically separates the genes from their control elements. The ectrodactyly syndrome is a genetically heterogeneous limb developmental defect characterized by the absence of digital rays and syndactyly of the remaining digits.53

7q31.31 FAM3C and WNT16 Genetic variants in the region mapping close to FAM3C gene have been previously reported in the literature as associated with derived BMD and speed of sound as measured by quantitative ultrasound in the radius and calcaneous in a Korean population.21 This chromosome 7 region harbors two genes (among others) and an open reading frame sequence including WNT16, FAM3C and C7orf58. In the second GEFOS BMD meta-analysis, variants mapping to WNT16 were found associated at GWSL with femoral neck and lumbar spine BMD, and also with increased fracture risk.25

8q24.12 TNFRSF11B (OPG) Large-scale confirmation that variants in TNFRSF11B are associated at genome-wide significant level with lumbar spine and femoral neck BMD were first reported in British and Dutch populations (i.e. the Twins UK and the Rotterdam Study)17 together with other efforts involving Icelandic and Northern European populations (deCODE Genetics).18 This finding was subsequently replicated by several additional efforts22,24,25,54,55 including Asian populations.55 The tumor necrosis factor receptor superfamily member 11b is a gene that spans 28.6 kb which encodes the osteoprotegerin (OPG) protein, an endogenous inhibitor of bone resorption. OPG is integral to the OPG/RANK/RANKL pathway whose relevance in the regulation of bone resorption has been well established.56 In addition, mutations in this gene have been identified in juvenile Paget’s disease57 as described in “Paget’s Disease”. The functional mechanisms by which TNFRSF11B alleles predispose to osteoporosis

are incompletely understood but, in the TwinsUK/ Rotterdam GWAS, expression of the BMD lowering allele was associated with reduced expression of TNFRSF11B in lymphoblasts.17

11p11.2 LRP4 and ARGHAP1 and F2 The low lipoprotein receptor related protein 4 (LRP4) gene spans 62 kb and was proposed as a candidate for regulation of total hip BMD by a GWAS meta-analysis based on Icelandic (deCODE Genetics) and Northern European populations19 which identified variants mapping close to the gene associated at suggestive level. A subsequent effort in five Northern European populations found variants in the locus associated at genomewide significant level.22 Nevertheless, the GWAS signals map within a region of high linkage disequilibrium containing several genes including the Rho GTPase activating protein 1 (ARHGAP1) gene and the coagulation factor II (F2) gene. At the moment it cannot be determined which of these genes is responsible for the reported associations. Regarding the candidacy of ARGHAP1, it is known that small GPTases such as Rho play an important role regulating bone cell activity while LRP4 is homologous to LRP5 which is known to regulate BMD (see below). The involvement of F2 cannot be completely discarded despite the lack of knowledge of relevant biology. Further work will be required to define the functional mechanisms underlying the associations that have been reported in this genomic region.

11p13 DCDC5 and DCDC1 The doublecortin domain containing 5 (DCDC5) and 1 (DCDC1) are big genes spanning 297 and 427 kb respectively and have emerged as possible candidates for regulation of lumbar spine BMD by the first GEFOS GWAS meta-analysis on BMD.22 This showed an association with the rs16921914 located 62  kb downstream of the doublecortin domain containing 1 (DCDC1) and 73 kb upstream of the DCDC5 gene. Doublecortin domains are found in a wide variety of genes and are involved in mediating protein–protein interactions.58 Alternatively spliced transcript variants encoding distinct isoforms of DCDC5 have been found, but the full-length nature of such variants is not determined. Genes that contain these domains are highly expressed in the central nervous system and mutations in some members of this gene family have been associated with neurological disorders. DCDC1 has been shown to be expressed in the testis. However, neither gene appears to be highly expressed in bone and the mechanisms by which these genes might regulate BMD remain unclear.

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III.  Genes Identified by Genome-Wide Association Studies on Bone Mineral Density

11p15.1 SOX6 The SRY (sex determining region Y)-box 6 (SOX6), which is a large gene spanning 773 kb, emerged as a candidate for regulation for BMD in the first GEFOS GWAS meta-analysis on populations of European descent, showing a genome-wide significant association signal with femoral neck BMD, situated 297 kb upstream from SOX6.22 Both SOX5 and SOX6 encode transcription factors which play essential roles in chondrocyte differentiation and endochondral ossification, are needed for growth plate multilayered establishment and for proper and timely development of endochondral bones.59 Therefore, genetic variation in this candidate gene may well be mediating skeletal development and influence BMD variation at the population level.

11q13.2 LRP5 The low density lipoprotein receptor-related protein 5 (LRP5) gene spans 137 kb and has for decades already been known to play an important role in bone biology. Its role as a critical regulator of bone mass was first established by linkage studies60–62 identifying mutations in the gene as producing the osteoporosis–pseudoglioma syndrome60,63,64 and several forms of high bone mass syndromes65–68 including autosomal dominant forms of type 2 van Buchem disease67 and osteosclerosis/endosteal hyperostosis.68 Genetic variants in the LRP5 locus have been identified as significant determinants of BMD by several of the GWAS to date17,22,69 but, interestingly, not in those associations arising from Icelandic populations.18,19,55 Even though several early candidate gene association studies showed that common variants in LRP5 underlie variation of BMD in the general population, it was only within (the largest study run to date from) the GENOMOS consortium including ≈45 000 individuals, that two non-synonymous coding variants were robustly associated with BMD, at a level of significance surpassing the current stringent standards of genome-wide level. An association with risk of fracture was also observed in that study and has been recently confirmed in the latest effort of the GEFOS consortium.25 Many common LRP5 variants have been studied in association studies, but the most likely functional candidates are a valine to methionine variant in exon 9 at codon 667 (V667M) and an alanine to valine substitution at position 1330 (A1330V) in exon 18 which are also precisely tagged by the GWAS signal.

12q13.13 SP7 SP7 is a small gene spanning 19 kb which encodes Osterix, a transcription factor exerting an essential role in osteoblast differentiation.70,71 Variation in the gene

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was first reported to influence BMD variation in an Icelandic population19 followed by a report showing its effect on peak bone mass accrual in children from the UK.20 Since then, several GWAS have shown association with variants mapping in the SP7 region.22,24,25 Additional studies are required to investigate the mechanisms underlying these associations with BMD. Noteworthy is the fact that a homozygous single basepair deletion in SP7 has been shown to cause Type IX osteogenesis imperfecta.

13q14.11 TNFSF11 (RANKL) The tumor necrosis factor (ligand) superfamily member 11 gene spans 45 kb and encodes RANKL, a member of the TNF superfamily which stimulates bone resorption by activating RANK signaling. Variants in RANKL have been consistently associated with lumbar spine BMD19,22,24,25 since the first report arising from Icelandic populations (deCODE Genetics).18 In a recent report based on peripheral quantitative computed tomography, variants in this locus distinct from those associated with DXA BMD (i.e. not in linkage disequilibrium), were associated at genome-wide significant level with volumetric and cortical bone density in 999 UK adolescents and 935 young Swedish adults.72 These findings imply that allelic heterogeneity may be governing the associations with areal and volumetric BMD seen in this locus. Even though the involvement of the OPG–RANK–RANKL pathway in bone metabolism is well established,56 the exact functional mechanisms by which genetic variation in these genes regulate BMD remains to be investigated.

14q32.32 MARK3 This gene has a size of 119 kb and encodes MAP/ microtubule affinity-regulating kinase 3, a member of the AMP kinase superfamily of proteins.73 Variants in this gene were first reported as associated at genomewide significant level with total hip BMD in an Icelandic population after replication in other European populations.19 In the first GEFOS meta-analysis on femoral neck and lumbar spine, variants in MARK3 were significant but not at genome-wide significant level.22 Nonetheless, variants in this locus did achieve a GWSL in the most recent effort involving an expanded set of populations of European descent and including East-Asian cohorts.25 Mechanisms, by which variations in this gene might affect BMD and/or bone physiology, are yet to be described.

16p13.3 CLCN7 The chloride channel 7 is a gene spanning 31 kb and coding for ClC-7. CIC-7 is particularly important for

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regulating pH and needed for the adequate resorptive function of osteoclasts through lysosomal acidification. CLCN7 mutations cause (infantile malignant) type 4 autosomal recessive osteopetrosis (OPTB4), and (Albers– Schonberg) type 2 autosomal dominant osteopetrosis (OPTA2), also called “marble bone disease”. Variants in CLCN7 were first reported as suggestive of association with total hip, femoral neck and lumbar spine BMD in Australian populations,24 while variants in this locus have now reached genome-wide significance in the second GEFOS meta-analysis of GWAS.25

16q24.1 FOXL1 and FOXC2 This locus was first identified by the first GEFOS meta-analysis in European populations as containing variants associated with lumbar spine BMD.22 The region contains a cluster of small genes of the “forkhead” (or winged helix) FOX gene family that are part of the TGFβ pathway and are involved in organogenesis. The GWAS signal maps close to the forkhead box L1 (FOXL1) gene of size 3 kb and the forkhead box C2 (FOXC2) gene spanning 2 kb. In contrast to FOXL1 of which little is known except that mutations in FOXC2 have been reported as causing the lymphedema-distichaisis syndrome,74,75 a disorder characterized by lymphedema of the limbs coupled with various other features but no evident skeletal phenotype. Nevertheless, Foxc2-deficient mice exhibit aortic arch anomalies and defects of skeletogenesis in the craniofacial bones and vertebral column,76 while inactivating mutations affecting the FOX gene cluster involving FOXC2 can cause severe malformations of the VACTERL (vertebral, anal, cardiac, tracheoesophageal, renal and limb) type in humans, which include vertebral malformations.77 Further, FOXC2 expression is regulated by bone morphogenetic proteins78 and can effect osteoblast differentiation of mesenchymal cells through activation of canonical Wnt-β-catenin signals79 and via upregulation of integrin beta 1.80

17q12-22 CRHR1 This locus was identified by the first GEFOS GWAS meta-analyses on populations of European descent proposing corticotropin-releasing factor receptor 1 (CRHR1) as the closest candidate gene to be influencing BMD variation at the population level.22 CRHR1 is a gene that spans 214 kb coding for a member of the corticotropin-releasing factor family of G-protein-coupled receptors that binds neuropeptides involved in regulation of the hypothalamic–pituitary–adrenal pathway. Nevertheless, the involvement of CRHR1 on this BMD signal cannot be unequivocally determined considering that the GWAS signals lies in a region of very high linkage disequilibrium (LD) containing many genes. The

high LD in the region arises from a common inversion polymorphism spanning about 900 kb, which has been subject to positive selection and which has been highly preserved for more than 3 million years in humans of Northern European descent.81

17q21.31 SOST and HDAC5 The first GWAS report identifying variants mapping to this locus was based on a discovery setting based on Icelandic individuals.19 In that study, variants mapped close to an open reading frame sequence (C17orf53) and to a 5 kb gene encoding sclerostin. A non-synonymous variant in C17orf53 was associated with hip BMD but not at genome-wide significant (GWS) level, while the marker mapping in the vicinity of SOST did achieve GWS. Sclerostin is a protein produced by osteocytes which inhibits bone formation by blocking the binding of members of the Wnt family to the LRP5 receptor.82 This way, SOST arises as an excellent candidate for genetic regulation of BMD considering that inactivating mutations of SOST cause high bone mass syndromes of the type seen in sclerosteosis and van Buchem’s disease.83,84 Variants mapping to SOST did not achieve genomewide significance in the first GEFOS meta-analysis with lumbar spine or femoral neck BMD.22 Nevertheless, a meta-analysis using a candidate gene approach examining 150 genes on the same exact datasets did identify variants mapping to SOST as significantly associated with BMD,85 while the second GEFOS meta-analysis has now identified variants mapping to SOST as associated at genome-wide significant level with BMD.25 Further, the first GEFOS meta-analysis also identified variants as associated with FN-BMD at GWS level in this locus, but mapping in an intronic region of the histone deacetylase 5 (HDAC5) gene22 on what appears to be an independent signal from that mapping to SOST and the same one arising from C17orf53. At this moment, it is still not possible to determine from which gene is this signal arising as the function of C17orf53 is unknown. On the other hand, HDAC5 is a 50 kb gene which is ubiquitously expressed and involved in transcriptional regulation through MEF2 inhibition86 and involved in muscle differentiation.87

18q21.33 TNFRSF11A The tumor necrosis factor receptor superfamily member 11a (TNFRSF11A) is a 62 kb gene that encodes the receptor activator of the NF-κB or RANK protein. The RANK receptor is expressed in osteoclasts and plays a critical role in regulating osteoclast differentiation and function, constituting a key element for the stimulation of bone resorption. Mutations in the RANK gene are responsible for the osteoclast-poor form of autosomal recessive osteopetrosis associated with

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V.  Conclusions and Perspective

hypogammaglobulinemia (OPTB7).88 The first study to identify common variants in the gene associated at genome-wide significant level was on Icelandic populations 19 and subsequently consistently replicated.22,25

20p12 JAG1 The first report identifying variants mapping close to JAG1 as associated with lumbar spine BMD came from Asian populations23 but has been replicated in the second large scale meta-analysis of the GEFOS consortium, including populations of both European and Asian descent.25 Jagged-1 is a ligand of the Notch receptor which, after binding, triggers the release of the intracellular unit of the Notch receptor from the membrane. This translocates to the nucleus and activates transcription factors key for cell differentiation and morphogenesis processes.89 In 2008, two reports in mice showed that Notch signaling stimulates early proliferation of osteoblastic lineages and that when knocked down results in an osteoporosis-like phenotype.90,91 In addition, JAG1 may be mediating some anabolic responses considering that PTH stimulation of osteoblast results in expression of JAG1.92

IV.  GWAS IN OTHER ETHNIC GROUPS AND FOR OTHER OSTEOPOROSIS PHENOTYPES It is now clear that BMD is the trait producing the largest yield of identified loci probably due to the combination of favorable properties including its high heritability, measurement precision and widespread use (allowing collections of samples of large size). Several other genes

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like IL21R,93 PTH,93 ALDH7A1,94 TBC1D8,54 OSBPL1A,54 RAP1A,54 PLCL1,95 RTP3,96 ADAMTS1897 and TGFBR397 have been postulated as involved in diverse osteoporosis traits including BMD,93,97 bone geometry,95,96 fracture94 and derived phenotype combinations.54 Nevertheless, several of these reported associations are under or just over the genome-wide significance threshold, are based on small and heterogeneous groups of individuals or have not been replicated to date in well-powered settings summing several tens of thousands of individuals.25 Even though populationspecific findings are a possibility, it has recently been shown that the vast majority of GWAS associations replicate across populations of different ethnic backgrounds.55 Additional replication is needed to consolidate these findings as robust candidate genes of osteoporosis.

V.  CONCLUSIONS AND PERSPECTIVE To date at least 56 loci have shown robust association with BMD containing an even larger number of candidate genes potentially underlying these GWAS signals. Here, we have reviewed a subset of them, mainly those that have been reported in the literature associated at genome-wide significant level and been replicated. Interestingly, many of these variants map in the vicinity of genes of unknown function (representing cutting-edge new biology), while several other factors cluster within critical biological pathways relevant for bone biology like Wnt signaling, OPG–RANK–RANKL and mesenchymal cell differentiation. Thus far, these genes identified by GWAS incorporate variants which together explain 3–4% of the variation in BMD (Figure 16.2). This suggests that FIGURE 16.2  Genetic architecture underlying BMD variation.

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both common and rare variation underlies the genetic architecture of bone mineral density and many more genetic determinants are yet to be identified. One of the expectations from genetic studies of common diseases like osteoporosis is the possibility to be able to improve risk prediction (i.e. as compared to what is currently possible with the use of clinical factors and/ or biomarkers like BMD). Nevertheless, this goal is not yet feasible to achieve since the small fraction of phenotypic variance explained still provides little added value in terms of prediction; hence, limiting at the moment any such derived clinical utility. From a distinct clinical perspective (i.e. translation to therapies), GWAS have pinpointed many factors in critical molecular pathways (e.g. Wnt signaling) which withhold factor candidates for therapeutic applications. Such potential is highlighted by the identification (among others) of genes encoding proteins that are currently subject to mediate novel bone medications. This is the case for denosumab, a human monoclonal antibody against RANKL, which is a protein inhibiting bone resorption. Yet even more interesting is the identification of several factors which can constitute targets for true bone-building drugs, exemplified by the identification of the sclerostin gene for which an antisclerostin antibody is expected soon to be available in the market. Hence, from the perspective of their therapeutic potential, translation of the GWAS discoveries into clinical applications is an upcoming reality.

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III.  DISORDERS OF BONE AND JOINT