Genetic of osteoporosis in Asia

Takayuki Hosoi, M.D., Ph.D.Department of Advanced Medicine, National Center for Geriatrics and

Gerontology

osteoporosis

normal Definition predisposition to fragile fractures due to deteriorated bone strength

Prevalent fractures spine, radius, humerus, hip

Epidemiology patients: 10 million in Japan hip fractures: 　 120 thousands/year

Diagnosis bone mineral density X-ray of spine bone turnover markers differential diagnosis

Therapy to prevent fractures bisphosphonates, SERM, vitamin D, vitamin K, calcitonin

Osteoporosis

cases/year

0

20,000

40,000

60,000

80,000

100,000

1987 1992 1997 2002

malefemale

13,500

39,700

19,00020,800

58,000

71,600

25,300

92,600

Orimo et al 2004

Incidence of hip fractures

Risk factors for fragility fractures considered at the pharmacological intervention of osteoporosis

　　　● low bone mineral density　　　● previous fragility fractures　　　● age　　 ● excessive alcohol consumption (more than 2 units/day)　　　● smoking　　　● parent’s hip fracture

　

Guideline for the prevention and treatment of osteoporosis 2006

Risk factors for fragility fractures considered at the pharmacological intervention of osteoporosis

　　　● low bone mineral density　　　● previous fragility fractures　　　● age　　 ● excessive alcohol consumption (more than 2 units/day)　　　● smoking　　　● parent’s hip fracture

　

Guideline for the prevention and treatment of osteoporosis 2006

WHO Fracture Risk Assessment Tool (FRAX TM)

agesex

heightweight

fracture after 50-year oldparent’s history of hip fracture

current smokinguse of steroid

alcohol more than 2 units/daydisease causing secondary osteoporosisBMD of femoral neck or total hip, or BMI

10-year

absolute risk

for fractur

e

Kanis et al.

WHO Fracture Risk Assessment Tool (FRAX TM) age

sexheightweight

fracture after 50-year oldparent’s history of hip fracture

current smokinguse of steroid

alcohol more than 2 units/daydisease causing secondary osteoporosisBMD of femoral neck or total hip, or BMI

10-year

absolute risk

for fractur

e

Kanis et al.

Heritability of bone mineral density

lumbar spine 92 % femoral neck 73 % Ward’s triangle 85 % trochanter 57 %

Pocock NA et al. J Clin Invest 1987; 80:706-710

Genes for osteoporosis ?

genome-wide approach

candidate gene approach

The determinants of statistical power

Sample size

Linkage disequilibrium pattern

Allele frequency difference

Population stratification

Phenotype difference

Quality control

Multiple testing

Complex etiology

Shen H et al. JBMR 2005

Major statistical issues in association studies

Coincidence of SNPs between the racial groups

Carlson CS et al.Nature Genetics 33:518-521, 2003

Coincidence of SNPs between the racial groups

Carlson CS et al.Nature Genetics 33:518-521, 2003

Only 50 % of SNPs are sharedby the two groups.

Genes for osteoporosis ?

genome-wide approach

candidate gene approach

Genes for osteoporosis ?

genome-wide approach

candidate gene approach

Ionannidis JPA, et al. J Bone Miner Res 2007; 22: 173-183.Meta-Analysis of Genome-Wide Scans Provides Evidence for Sex- and Site-Specific Regulation of Bone Mass.

cytogenic location observed rank p value

1p13.3-q23.3 93.34 0.004 3p25.3-p22.1 95.52 　　 0.006 11p12-q13.3 89.02 0.014 18p11-q12.3 88.72 0.014 18pter-p11 85.51 0.028 1q32-q42.3 84.88 0.030 12q24.31-qter 84.88 0.030 12p24.31-qter 84.65 0.032 11q13.3-q22.1 84.03 0.036 　　　　

Results of weighted analsyses for lumbar spine BMD

Ionannidis JPA, et al. J Bone Miner Res 2007; 22: 173-183.Meta-Analysis of Genome-Wide Scans Provides Evidence for Sex- and Site-Specific Regulation of Bone Mass.

cytogenic location observed rank p value

9p31.1-q33.3 101.33 0.002 14q13.1-q24.1 99.88 0.003 17p12-q21.33 95.27 0.010 14q23.3-q32.12 91.20 0.022 9q21.32-q31.1 91.20 0.020 5q14.3-q23.2 88.01 0.038

Results of weighted analsyses for femoral neck BMD

intron

exon

promotor

gene gene

rSNP iSNP

cSNP

sSNP

gSNP

rSNP: regulatory SNPiSNP: intronic SNPcSNP: coding SNPsSNP: silent SNPgSNP: genome SNP

Single nucleotide polymorphisms (SNPs) for genome-wide association study

variation in the quality of gene products

variation in the quantity of gene products

Systematic use of genome wide SNPs

SNPs registered in J-SNP : 170,000 SNP ｓ

Emi and Hosoi et al.

Systematic use of genome wide SNPs

SNPs registered in J-SNP : 170,000 SNP ｓ

Missense polymorphisms in the exons and polymorphisms in the promotor regions2,000 SNP ｓ

Emi and Hosoi et al.

Systematic use of genome wide SNPs

SNPs registered in J-SNP : 170,000 SNP ｓ

Missense polymorphisms in the exons and polymorphisms in the promotor regions2,000 SNP ｓ

First screenig for the association with bonemineral density 　　　　　　　　　　　　　 50 SNP ｓ

Emi and Hosoi et al.

Systematic use of genome wide SNPs

SNPs registered in J-SNP : 170,000 SNP ｓ

Missense polymorphisms in the exons and polymorphisms in the promotor regions2,000 SNP ｓ

First screenig for the association with bonemineral density 　　　　　　　　　　　　　 50 SNP ｓ

Interleukin-1-receptor-associated kinase (IRAK1)brain natriuretic peptidegonadotropin releasing hormoneLDL receptor-related protein 5 (LRP5)

Emi and Hosoi et al.

Systematic use of genome wide SNPs

SNPs registered in J-SNP : 170,000 SNP ｓ

Missense polymorphisms in the exons and polymorphisms in the promotor regions2,000 SNP ｓ

First screenig for the association with bonemineral density 　　　　　　　　　　　　　 50 SNP ｓ

Interleukin-1-receptor-associated kinase (IRAK1)brain natriuretic peptidegonadotropin releasing hormoneLDL receptor-related protein 5 (LRP5)

Emi and Hosoi et al.

Ｗｎｔ

ＬＲＰ５

Ｆｒｉｚｚｌｅｄ

Dkk

Kremen

Dsh

P

GSK3β

β-catP

Axin

APC

β-cat P

β-catTCFDNA

porteasome

transcription

sFRPs

c-mycc-junfra-1cyclin D1PPAR δmatrilysinetc.

other pathways

JB Richards et al. Bone mineral density, osteoporosis, andosteoporotic fractures; a genome-wide association study.Lancet 371; 1505-1512, 2008

rs4355801 on chromosome 8 close to TNFRSF11B(osteoprotegerin) gene

rs3736228 on chromosome 11 in the LRP5 (low density lopoprotein-receptor-related protein) gene non-synonymous base-pair change (Ala1330Val)

Genes for osteoporosis ?

genome-wide approach

candidate gene approach

tissue non-specific alkaline phosphatase (TNSALP) (JBMR 2005)

γ-glutamyltransferase (GGCX) 　　 (Bone 2007)

Our attempts to search the functional SNPs related to osteoporosis and nutrition

Hosoi 2007

tissue non-specific alkaline phosphatase (TNSALP) (JBMR 2005)

γ-glutamyltransferase (GGCX) 　　 (Bone 2007)

Our attempts to search the functional SNPs related to osteoporosis and nutrition

Hosoi 2007

● tissue non-specific ALP (TNSALP) bone liver● intestinal ALP: small intestine

● placental ALP: placenta

● germ cell ALP: testis, thymus

ALP- １

ALP-2

ALP-3

ALP-4

ALP-5

isozymes

NTP-PPi-ase 5’-NT

TNSALP

ATP PPi

AMP

Pi

AdenosinePi

PEA PLP

EA

PL

NTP-PPi-ase : nucleoside triphosphate pyrophosphataseTNSALP : tissue non-specific alkaline phosphatasePEA : phosphoethanolaminePLP : pyridoxal phosphateEA : ethanolaminePL : pyridoxal

1p34-p36.1

Exon 1B 1L 2 3 4 5 6 7 8 9 10 11 12

chromosome 1

Structure of human TNSALP gene and mRNAs

787 T>C (Tyr246His)876 A>G (Pro 275 Pro)

D’ ＝ 1.0000, r2 = 1.0000, χ2 = 1002.00, p < 0.001

　

TTn = 128

TCn = 248

CCn = 128

Distribution of 787 C/T genotype of TNSALP gene

elderly women in an area of Japan

TNSALP 787T ( Tyr 246) : TNSALP 787T>C (His246)

A

:

B

Location of the investigated SNPs (787T, 787T>C) in the three-dimensional modeled structures of human TNSALPs.

787T (Tyr246) 787T>C (His246)

PLAP Arg241

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

Z score

787T (homozygotes, TT)

Heterozygotes (TC)

787T >C (homozygotes, CC)

＞ 74 yrs. (n = 207)

Ｐ＝ 0.002

　　　　　　　 TNSALPs n Km (mM)

787T (Tyr246Tyr) 15 0.505 + 0.064787T>C (Tyr246His) 15 0.290 + 0.002 **

** p < 0.01

[ 基質 ]

sameVmax

different Km

787T

787T>C

JBMR 2005

V

Hypothesis for TNSALP 787 T>C (Tyr246His)

lower Km = higher affinity for the substrate

higher efficiency in providing Pi to the site of calcification

　　　　　　　 higher BMD

＜ aging＞

excessive phosphate intake

excessive phosphate intake

PTH synthesis and secretion ↑

excessive phosphate intake

PTH synthesis and secretion ↑

excessive phosphate intake as a possible risk factor for

osteoporosis

excessive phosphate intake

PTH synthesis and secretion ↑

excessive phosphate intake as a possible risk factor for

osteoporosis

TNSALP polymorphism 　?

0

20

40

60

80

0 500 1000 1500 2000 2500

P intake (mg/ day)

inta

ct

PT

H (

pg/m

l)

787T

Heterozygotes

787T >C

r =0.302p =0.019*

Association of phosphate intake and serum PTH (young male)

Goseki and Hosoi et al. in press

Association of phosphate intake and serum PTH (young male)

0

20

40

60

80

0 500 1000 1500 2000 2500

P intake (mg/day)

inta

ct

PTH

(p

g/m

l)

r =0.604p =0.006**

TNSALP ７８７ T>C

Goseki and Hosoi et al. in press

excessive phosphate intake

PTH synthesis and secretion ↑

excessive phosphate intake as a possible risk factor for

osteoporosis

TNASLP polymorphism?

minor genes

major genes

lifestyle factors

disease

nutritionphysical activityhabits

minor genes

major genes

lifestyle factors

disease

nutritionphysical activityhabits

tissue non-specific alkaline phosphatase (TNALP) (JBMR 2005)

γ-glutamyltransferase (GGCX) 　　 (Bone 2007)

Our attempts to search the functional SNPs related to osteoporosis and nutrition

Hosoi 2007

OH

OH

R

O

O

R

O

O

O

R

CH3

H

n

R :

lCH2 lHCH lCOOH

lCH2 lHC - COOH lCOOH

O2 CO2

NAD(P)+

NAD(P)H

SH SH l l~~~

S S l l~~~

S S l l~~~

SH SH l l~~~

vitamin K epoxide

vitamin K epoxide reductase (#)

γ-glutamyl calboxylase

vitamin Knaphthoquinone

vitamin K hydro-naphthoquinone

#

##

## NADH or NADPH- linked quinone reductases

SNPs in GGCX gene

SNP 　 codon SNP freq. 　 a.a. exon8 (Arg/Gln) 8762 G/A 0.66: 0.34 Arg/Gln

325 exon 9-1 9167 C/T 0.66: 0.34 none

406 exon 9-2 9191 C/T 0.94: 0.06 none

414

exon 9-1 Human GGCX exon8(Arg/Gln) exon 9-2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

SNP1 SNP2 D D’ r2　 　 exon 8 exon 9-1 0.2232 1 1　 　 exon 8 exon 9-2 -0.0208 0.9991 0.00334　 　 exon 9-1 exon 9-2 -0.0208 0.9991 0.00334

Membrane topology of GGCX and the locationd of a functional SNP

SNP （Arg325Gln） catalytic center (343,345)

Ｃ Ｃ terminterminalal

Ｎ Ｎ terminterminalal

Microsome membrane

Hosoi 2005

0

20

40

60

80

100

120

140

160

180

200

0 200 400 600 800 1000

Vitamin KH2 (mM)

0

50

100

150

200

250

300

0 1 2 3 4

FLEEL (mM)

14C

O2

inco

rpor

atio

n(p

mol

/30m

in/m

g pr

otei

n)

14C

O2

inco

rpor

atio

n(p

mol

/30m

in/m

g pr

otei

n)325G ｌｎ325G ｌｎ

325Arg

325Arg

Gln/Gln(11)

Gln/Arg(54)

Arg/Arg(48)

0.2.4.6.81

1.2

0

.5

1

1.5

2

2.5

Gln/Gln(11)

Gln/Arg (54)

Arg/Arg(48)

BMD（ｆｅｍｏｒａｌ　ｔｏｔａｌ , ｚ ｓｃｏｒｅ ） P=0.03

adjusted endplate sclerosis score P=0.04

Mean±S.D Kruskal Wallis test

Osteocalcin molecule and three carboxylation sites

Under-carboxylation of osteocalcin can be used as a marker of vitamin K deficiency in bone metabolism.

0

1

2

3

0 1 10 100MK- 7 (ng/ ml)

ucO

C/ inta

ct

OC r = - 0.572

p = 0.003

.1

　　 Arg/Arg 　　　　　　　　　 A ｒｇ /Gln 　　　　　　　　

0

1

2

3

0 1 10 100MK- 7 (ng/ ml)

r = - 0.260p = 0.166

.1

Goseki and Hosoi J Vitaminol Nutr Sci 2007

Serum MK-7 and uc OC/intact OC correlated only in the GGCX genotye Arg/Arg.

ucOC as an indicator for vitamin K deficiency

Persons with GG (Arg/Arg) genotype of GGCX maybe affected by vitamin K deficiency more than those with other genotypes.

or

Vitamin K intervention may be more effective in persons with GG (Arg/Arg) than in those with other genotypes.

Explanation of the results

minor genes

major genes

lifestyle factors

disease

nutritionphysical activity habits

Effects of lifestyle factors may be modulated by genetic variations.

Nutrition factors and genetic polymorphisms

nutrient 　　　　　　　 genes

Folate MTHFRvitamin B12 MTRvtamin D VDRFat APOEAlcohol ADHLactate LDOxidative stress 　 PON １

Modern Nutrition in Health and Disease 2006

Nutrition factors and genetic polymorphisms

nutrient 　　　　　　　 genes

Folate MTHFRvitamin B12 MTRvtamin D VDRFat APOEAlcohol ADHLactate LDOxidative stress PON １

phosphate TNSALP vitamin K GGCX

? ?

0.5

0

1.0

0

1.0

0.5

low 　　　　　　　 intake of nutrient 　　　　　　 high

risk by deficiency risk by excess

Dietary goal for preventing diseases

dietary goal

0.5

0

1.0variation bygenotypes ?

0

1.0

0.5

low 　　　　　　　 intake of nutrient 　　　　　　 high

risk by deficiency risk by excess

Dietary goal for preventing diseases may be modulated by the genotypes.

Thank you for your attention!