GENETIC SUSCEPTIBILITY

FACTORS FOR
OSTEOPOROSIS!

Gene 2! Gene 3!

Gene 1! Gene 4!

Complex Disease!

Environmental!
factor 1! Environmental!
Environmental! Environmental! factor 4!
factor 2! factor 3!
 How do we know that a disease
is a genetic disease?

Higher concordance than in:!

in:!

monozygous twins ! dizygous twins!

Hereditary component of coronary

artery disease

Twin Concord. Concord.

studies MZ DZ

Danish Men 39 % 26 %
(n = 352)
Women 44 % 14 %
Swedish Men 40,5 % 32,8 %
(n = 2810)
Women 32,6 % 16,3 %
How do we know that a disease
is a genetic disease?

Higher risk for:! than for:!

?! ?!

a relative of an! a relative of healthy!

affected individual! individuals!

GENETICS of OSTEOPOROSIS!

HEREDABILITY OF BONE MASS :!

!!50-70 % INTERGENERATIONAL STUDIES !

!!80-90 % TWIN STUDIES !

! 46-62 % AFTER ADJUSTMENT OF BONE MINERAL

DENSITY BY AGE, WEIGHT AND LIFE STYLE!
 Genetic bases of
diseases
GENE WITH A! SUSCEPTIBILITY!
MAJOR EFFECT! GENES!
•! Large effect •! Mild effect

•! Rare mutations •! Common mutations

(polymorphisms)
•! It explains only a small
part of the population •! They explain most of the
variability population variability

Genetic bases of
diseases
GENE WITH A! SUSCEPTIBILITY!
MAJOR EFFECT! GENES!
•! Large effect •! Mild effect

•! Rare mutations •! Common mutations

(polymorphisms)
•! It explains only a small
part of the population •! They explain most of the
variability population variability
 Type of Inheritance

GENE WITH A ! SUSCEPTIBILITY!

MAJOR EFFECT! GENES!
•! Complex inheritance
•! Mendelian •! Many genes
•! Many enviromental
factors
•! Gene-gene and gene-
environment interactions

Type of Inheritance

GENE WITH A ! SUSCEPTIBILITY!

MAJOR EFFECT! GENES!
•! Complex inheritance
•! Mendelian •! Many genes
•! Many enviromental
factors
•! Gene-gene and gene-
environment interactions
 MUTATION and
POLYMORPHISMS!
In human genetics:!
Mutation! Pathogenic changes!

Polymorphisms! Neutral changes!

(markers)!

Susceptibility!

POLYMORPHISMS!

Microsatellites! i.e.: (CA)n!

SNPs! i.e.: C G!

DIPs! i.e.: 7A 8A!

Transcription

Transcriptionn Factors
 Possible effect of a
polymorphism in a regulatory
region

Individual 1 Individual 2

Polymorphisms/environment
interaction
EF!
I A

Individual 1! Individual 2!
A! EF!
EF!
A

I! I
 Strategies to find genes
responsible for complex diseases

Linkage!
Anonymous loci !

Sib pairs!

Association!
Candidate genes!

Sib pairs!

* Replaced now by the

positional candidate search
 Strategies to find genes
responsible for complex diseases

Linkage!
Anonymous loci!

Sib pairs!

Association!
Candidate genes!

Sib pairs!

SIB PAIRS

General! Dominant model! Recessive model!

AB! CD! AB! CD! AB! CD!

AC! AC!(1/4)! AC! AC! (1/2)! AC! AC! (1/1)!

AD! AD!
BC!
BD!
Los loci identificados en
diferentes screenings
NO coinciden!

Devoto et al. , 98!

Mitchell et al. , 98!

Duncan et al. , 99!

Karasik et al. , 02!

DeCode, 03!
Modificado de Uitterlinden en European Calcified Tissue Society 1999
http://www.ectsoc.org/reviews/005_uitt.htm

Strategies to find genes

responsible for complex diseases

Linkage!
Anonymous loci!

Sib pairs!

Association!
Candidate genes!

Sib pairs!
 ASSOCIATION
C T
Candidate!
gene! 1! 2! 3! 1! 2! 3!

Phenotype! Phenotype!

(pathology risk)! (pathology risk)!

Linkage disequilibrium!
b! B!

(1)!

X1! X2!
b! B!

(2)!

X1! X2!

B - b : detected polymorphism!
X1 - X2 : change responsible for the disease!
 Individuos necesarios para los
distintos estudios

LINKAGE! SIB PAIRS!

ASOCIACION!

POLYMORPHISMS IN THE
COLIA1 GENE PROMOTER
AND OSTEOPOROSIS
Osteoporosis is characterized by:!

!Reduced bone mineral density !

(enhanced bone loss)!
Bone tissue micro-!
architecture deterioration!
Enhanced risk of!
fracture.!

The fracture risk depends both on the bone mass pick

and on the bone loss rate.!

Pick Quick loser

Young loser
Osteopenic

Fracture treshold Fracture treshold

Age
Age
NO GENETIC FACTORS !

TABACCO!

SEDENTARY LIFE!

GASTROINTESTINAL AND HEPATIC

DISEASES!

CORTICOID THERAPY!

ALCOHOL, WHICH ENHANCES CALCIUM

ELIMINATION!

GENETIC!

MULTIFACTORIAL!

INCIDENCE IN DEVELOPPED COUNTRIES!

40% WOMEN AND 12% MEN!

BONE MASS HEREDABILITY:!

! 50-70 % INTERGENERATIONAL STUDIES!
! 80-90 % TWIN STUDIES!
! 46-62 % BMD ADJUSTED BY AGE, WEIGHT AND
LIFE STYLE.!
 CANDIDATE GENES !

VITAMIN D RECEPTOR !

ESTROGEN RECEPTOR !

IL-6!

TGF!!
TYPE I COLLAGEN!

Controversial results have been obtained for these genes in

different association studies .!

TYPE I COLLAGEN IS THE MAIN PROTEIN IN BONE!

MUTATIONS IN ITS CODING REGION CAUSE

OSTEOGENESIS IMPERFECTA!

EXCEPTIONALLY, MILD MUTATIONS IN ITS

CODING REGION CAN CAUSE SEVERE
OSTEOPOROSIS !
 PROMOTER OF THE " 1 TYPE I COLLAGEN GENE!
F1!
F3! F2!

-2.3 Kb! -2.0 Kb! -1.8 Kb! -1.67 Kb! -1.50 Kb!

SPECIFIC REGULATORY REGION IN OSTEOBLASTS IN VIVO !

SSCP F3 ! ! SSCP F2 ! ! SSCP F1!

PCOL2 PCOL1

-1997 G/T -1663indelT

 ANALYSIS OF VARIANCE (ANOVA)!

FONT! ! !dof ! !F ! !Sig.!

AGE ! ! !1 ! !4.986 ! !0.026!

MENOPAUSE !1 ! !9.312 ! !0.003!
WEIGHT ! !1 ! !10.092! !0.002!
PCOL2 ! !2 ! !4.324 ! !0.014!
PCOL1 ! !2 ! !1.441 ! !0.239!
PCOL1*PCOL2 !2 ! !4.011 ! !0.019!

DEPENDENT VARIABLE : BMD!

PCOL1!
PCOL2!

8T!
GRE!
Sp1!

8A!
8T!
Extract - + + + + + + + !!
8A!
7T!
7A!

8T!
*!
8A! *!
 G!
Competitor:!
PCOL2! C!

GRE!
Sp1!
G!
C! Extract ! - + + + + + + +!

T!
A!

Labelled oligo!
G!
*
C! *

competitors!
PCOL2! C! A!
G!
C! Extract -! +! +! + + + + +

T!
A!

Labelled oligo!

C! *
García-Giralt et al. (2002) J.Bone Min.Res. 17: 384-393"
 Luc+
SP
IR
IR ***
*
LP ***
-1997 G/T -1663indelT
IRD

0 1 2 3
Relative Luciferase Activity (LUC/BGAL)

COL1A1 gene promoter haplotypes

4

* * * *
2 LP

* * IRD
1

0
G/8T T/8T G/7T T/7T SP IR G/8T T/8T G/7T T/7T
 COL1A1 promoter:independent alleles
4

3 G T 8T 7T G T 8T 7T LP

2
* IRD
1

0
G

-1

-1

-1

-1

-1

-1

-1

-1
/8

/8
99

99

66

66

99

99

66

66
T

T
7

3i

3i

3i

3i
nd

nd

nd

nd
G

T
el

el

el

el
T

T
8T

7T

8T

7T

Identification of
Transcription Factors
that bind
PCOL1 and PCOL2
 Comparisons with COL1A1 promoters in animal models!

•! Bidwell and his group (Indiana, USA) have been

performing functional studies on the rat COL1A1 promoter
for many years !

•! By gel-shift analyses they defined two polypirimidinic

sites (A and B) bound by a nuclear matrix protein (NP/
NMP4)!

•! NP/NMP4 is supposed to be a architectural transcription

factor with capacities to bend the DNA!

•! **PCOL1 lies within the human homolog for site B**!

PCOL1 binds a protein from the CIZ/NMP4 family

AB - NMP4 NFAT - NMP4 NFAT
NE - + + + - + + +

Supershift analysis
using polyclonal
antibodies (AB)
against NMP4 and
NFAT.

PROBE PCOL1 PCOL2

 How are the studies of the
polymorphisms in the COL1A1 gene
promoter supposed to follow ?

Identification of other
Transcription Factors which bind
PCOL1 and PCOL2
 AFFINITY CHROMATOGRAPHY
Cell culture and
Biotinylated Oligonucleotides extraction of nuclear
proteins

Streptavidin-coated Incubation Incubation

magnetic beads

Obtention of the proteins bound to the oligonucleotide

separation elution separation

EMSA /Southwestern

2-D Electrophoresis

MALDI-TOF
 GENOMOS Project n = 20.000!

1.Rotterdam

GENE POLYMORPHISMS

Vitamin D
BsmI ApaI TaqI Cdx FokI
Receptor
3502 / 7081 3502 / 7081 3502 / 7081 3402 / 7081 2589 / 7081

Estrogen
PvuII XbaI (TA)n VNTR
Receptor -!
4746 / 7081 4746 / 7081 3100 / 7081

G-800A C-509T Leu10Pro Arg25Pro Thr263Ile

TGF-"
3070 / 7081 3059 / 7081 3063/ 7081 3054 / 7081 3069 / 7081

COLIA1 Sp1

3055/7081

ESR1

6q25.1, 140 kb, 7 promoters (osteoblasts: promoter B)

XbaI!
(TA)n! PvuII!
 XX vs. Xx and xx
Aarhus F
Aberdeen F
Barcelona F
Cambridge F
Cambridge M
DOPS F
Florence F
Oxagen F
Oxagen M
Rotterdam F
Rotterdam M
TOTAL-FE
TOTAL-RE
WOMEN-FE
WOMEN-RE

.1 .2 .4 .6 .8 1 2 4 6

Odds ratio for vertebral fractures (95% CI)

 Natàlia Garcia-Giralt! Anna Enjuanes!
Mariona Bustamante! Susana Jurado!
Lídia Águeda!
Leo Mellibovsky!
Susana Balcells! Xavier Nogués!
Daniel Grinberg! Adolfo Díez!
DEPT. GENÈTICA! URFOA!
FAC. DE BIOLOGIA! HOSPITAL DEL MAR-IMIM!
UNIVERSITAT DE BARCELONA! INSTITUT MUNICIPAL!
D’ASSISTÈNCIA SANITÀRIA!

+
++ IMAS
+
 Daniel Grinberg Susana Balcells

Mariona Bustamante Susana Jurado

Anke Wesselius
Lídia Águeda

Grupo Osteoporosis - UB