Introduction
The Genetics of
Osteoporosis
Mr. Christopher Vidal B.Sc.(Hons.)
Supervisor: Prof. Angela Xuereb Ph.D.
(Co-supervisor: Prof. Alex Felice Ph.D.)
„ Osteoporosis is a complex metabolic bone disease
characterised by a decrease in bone mineral density
and deterioration of the microarchitecture of bone
leading to an increased risk of fractures.
„ The pathogenesis of osteoporosis depends upon
peak bone mass reached early in life and the rate of
bone
loss
in
later
years,
especially
in
postmenopausal women.
„ An individual’s increased risk of disease depends
upon both environmental and genetic factors.
Genetics of Osteoporosis
Genes associated with osteoporosis
„ Osteoporosis is a polygenic disease where
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Vitamin D Receptor (VDR)
Oestrogen Receptor (ER1)
Osteoprotegerin (TNFRSF11B)
Collagen Type 1 (COL1A1,COL1A2)
Lipoprotein Related Protein-5 (LRP5)
Methylenetetrahydrofolate reductase (MTHFR)
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Transforming growth factor-β (TGFβ)
Bone Morphogenic Protein 2 (BMP2)
Androgen receptor (AR)
Bone Gla Protein (osteocalcin)
Sclerostin (SOST)
Tumour Necrosis Factor Receptor 2 (TNFR2)
Lysyl Hydroxylase (PLOD1)
Interleukin 6 (IL-6)
Insulin-like Growth factor 1 (IGF-1)
T-Cell Immune Regulator (TCIRG1)
multiple gene variants each having a small
effect contribute to an individual’s increased
susceptibility to the disease.
„ A number of chromosomal loci and single
nucleotide polymorphisms (SNPs) found
within candidate genes have been associated
with BMD and/or fracture risk.
[Ref.1]
[Ref.2]
[Ref.3]
[Ref.4]
[Ref.5]
[Ref.6]
[Ref.7]
[Ref.8]
[Ref.9]
[Ref.10]
[Ref.11]
[Ref.12]
[Ref.13]
[Ref.14]
[Ref.15]
[Ref.16]
Genetic Studies
Aims of Study
„ Linkage Studies (gene mapping)
„ To evaluate the role of candidate loci in
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The search for the co-inheritance of phenotype and genotype in
families with multiple affected individuals
Analysis for co-inheritance with disease of highly polymorphic markers
(STRs) spread at regular intervals in the genome.
The LOD score is the probability expressed as a log base 10 of the
likelihood ratio of linkage between marker and disease.
Whole genome scan
Candidate gene approach
„ Association Studies (case-control)
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The search for association of a marker allele with disease by
comparing its frequency in affected and unaffected individuals in a
population.
Analysis of single nucleotide polymorphisms (SNPs) that can be
functional or non-functional.
If association is found then determine whether the SNP itself is causing
the disease or whether it is in linkage disequilibrium with a nearby
mutation.
families with multiple affected individuals
„ To confirm the role of candidate loci indicated
by previous linkage studies in Maltese
families
„ To localise and confirm susceptibility alleles
for osteoporosis in the Maltese population
1
Study Approach
Patient Recruitment
„ Linkage
„ 125 postmenopausal women were recruited from new subjects
study in two extended Maltese
families using selected markers at eight
candidate regions where known genes for
osteoporosis are found (candidate gene
approach).
„ Association study in a random population of
postmenopausal women.
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Pedigree 1
Methodology - STR genotyping
„ DNA was extracted from peripheral blood leucocytes by salting
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out
DNA concentration was measured by spectrofluorometry using
PicoGreen (Molecular Probes Inc, USA). DNA concentrations
were adjusted to a final concentration of 10ng/ul.
39 STRs located on 8 chromosomal regions were analysed in
27 family members from 2 pedigrees.
STRs had an average heterozygosity of 0.76 with an average
spacing of 8.6cM.
PCR was performed using fluorescently labelled primers and
fragment analysis was performed by denaturing capillary
electrophoresis technique using a 3730xl ABI Genetic Analyser.
This work was performed in a commercial laboratory (McGill
University and Genome Quebec Innovation Centre, Canada)
referred to the Bone Density Unit, St. Luke’s Hospital.
Menopause was defined as amenorrhoea of at least six months
duration.
Two families were recruited from probands selected from
individuals that participated in the association study and were
observed to have severe osteoporosis as defined by WHO
criteria (BMD t-score <-2.5) and low z-score at the LS and/or
FN. These individuals were also relatively young in age.
All participants from these families answered a detailed
questionnaire concerning medical conditions, use of
medications, lifestyle and dietary habits as well as family history
of osteoporosis.
BMD at the LS and FN was measured by DEXA using a Norland
385 densitometer.
Peripheral blood was collected for DNA analysis and
biochemical tests to exclude secondary osteoporosis.
Pedigree 2
Table 1. Chromosomal Regions
Analysed in this study
Chromosome
Locus
Genes
1
1p36
MTHFR, TNFR2, PLOD1,
ALP, Chloride channel
8
8q24
TNFRSF11B (OPG)
11
11q12-13
LRP5, TCIRG1
12
12q13.1-q13.2
VDR, COL2A1, BMP11,
PTH-like hormone
17
17q21.31-q22
COL1A1, SOST
18
18q22.1
RANK
20
20p12
BMP-2
X
Xq11.2-q12
AR
2
Methodology – SNP genotyping
Statistical Analysis
„ DNA was extracted by salting out from peripheral blood
„ Linkage and Association analyses were performed
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leucocytes.
Target sequences of 17 SNPs found within 6 candidate genes
(VDR, ER1, TNFRSF11B, COL1A1, LRP5, MTHFR) were
amplified by PCR using appropriate primers.
PCR products were then digested using specific restriction
enzymes to the polymorphic region. An SNP either introduces or
abolishes a cut site for an endonuclease.
Digested products were then analysed by agarose or
polyacrylamide gel electrophoresis.
Serum procollagen type I (Prolagen C) and urinary
deoxypyridinoline crosslinks (DpD) were used as biochemical
markers of bone formation and resorption.
Genotyping was performed at the DNA Laboratory, Medical
School, G’Mangia, Malta.
Results – Linkage Analysis
„ The pedigrees were analysed separately using both parametric
and non-parametric analyses.
„ For the mild phenotype, affected individuals were defined as
those having an age and sex matched z-scores of <-1.0.
„ A more severe phenotype was also used, where affected
individuals in the first generation were defined according to
WHO criteria as osteoporotic (t-score <-2.5), while affected
individuals in the second generation (pre-menopausal women
and men) were defined by a z-score of <-2.0 (as recommended
by the International Society of Clinical Densitometry).
„ Four different penetrances were used in the analysis ranging
from a complete dominant to a recessive mode of inheritance.
Results – Family 1
using statistical software:
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Cyrillic v2.1 to construct the pedigrees and input STR
genotypes
MLINK
GENEHUNTER v1.2 to compute LOD and NPL scores
Linkage Disequilibrium Analyser (LDA) used to test for
LD between different SNPs
EH software used to calculate LD between haplotypes
and disease by comparing cases and controls
SPSS student version 9.0 used to test for correlation
between BMD and different genotypes
Table 2. Clinical Characteristics
Variable
Affected
Unaffected
N (M/F)
17 (5/12)
11 (8/3)
Age (years)
50.0
41.8
Weight (Kg)
63.3
67.8
Height (cm)
155.6
162.8
BMI (kg/m2)
26.0
25.7
Obesity (Normal/Obese)
15/2
9/2
Fractures (no/yes)
14/3
10/1
Ionised Calcium (mmol/l)
1.28
1.29
Smokers/non-smokers
3/14
3/8
LS BMD (g/cm2)
0.785
1.000
FN BMD (g/cm2)
0.741
0.879
Fig.1 Inheritance of STRs on
Chromosome 8
„ No evidence of linkage was observed with loci 1p36,
18q22.1, 20p12 and Xq11.2-q12 in this family.
„ An NPL score of 3.69 (p=0.009) and LOD of 1.34
were observed to marker D8S1128 for a dominant
mode of inheritance of a low BMD phenotype. When
a severe phenotype was tested both NPL and LOD
increased to 4.17 and 2.28 (Fig.1 – 3).
„ An NPL of 3.61 (p=0.03) was observed to marker
D17S1293 with a LOD score of 1.93 for a dominant
mode of inheritance with 90% penetrance (Fig. 4 –
6).
„ Marker D11S2002 gave an NPL score of 2.65
(p=0.04) when a severe phenotype was analysed.
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Fig. 2 NPL Plot of Chromosome 8
Fig.3 LOD of Chromosome 8
Fig.4 Inheritance of STRs on
Chromosome 17
Fig.5 NPL Plot of Chromosome 17
Fig.6 LOD Plot of Chromosome 17
Results – Family 2
„ No evidence of linkage was observed for markers on
chromosomes 1, 8, 11, 12 and X in this family.
„ Highest NPL of 6.11 (p=0.002) was observed to
marker D17S1865 with an LOD of 2.04 on
chromosome 17, for a low BMD phenotype (Fig. 7 –
10).
„ Suggestive linkage was observed with a severe
phenotype and marker D18S484 with an NPL score
of 3.24 (p=0.04) and a LOD of 1.47 for a dominant
mode of inheritance with penetrance 0.90.
„ When added, the highest NPL score between the two
families was of 7.08 for marker D17S1865 on
chromosome 17.
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Fig.7 Inheritance of STRs on
Chromosome 17
Fig.8 NPL Score for Chromosome 17
Fig.9 LOD Score for Chromosome 17
Fig.10 Informative Markers on
Chromosome 17
Results – Association Study
Fig. 11 TNFRSF11B (OPG) T950C Genotype Distribution
Between Unaffected and Affected (Osteopenic +
Osteoporotic) Individuals
„ All polymorphisms studied in the Maltese population
100
80
83
60
59
40
Percentage (%)
were found to be in Hardy-Weinberg equilibrium.
„ No statistical significant difference (p>0.05) was
reached for LS and FN BMD between individuals of
different genotypes, for all SNPs studied.
„ Statistical significance difference was observed in the
distribution of genotypes between affected and
unaffected individuals for the TNFRSF11B T950C
SNP (χ2 = 9.7; p = 0.01; df = 2) with the TT genotype
found more frequently in the affected group (Fig. 11).
„ A significant odds ratio of 2.11 (95% CI: 1.26 – 3.55)
was obtained when assuming the T allele of the
same polymorphism as a risk for low BMD.
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43
41
OPG T950C (HpaI)
TT
20
17
TC
0
CC
Normal
Low BMD
Bone status LS and FN
chi=9.7; df=2; p=0.01
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Table 3 VDR Gene Haplotype Frequencies in Affected
and Unaffected Individuals
Linkage Disequilibrium
„ Strong LD was observed between a number of SNPs within the
VDR, ER1, TNFRSF11B, COL1A1 and LRP5 genes.
„ Haplotype frequencies within these genes were then compared
between affected and unaffected individuals using the EH
software.
„ No association was observed between VDR, ER1 and LRP5
haplotypes with an increased risk of osteoporosis (Table 3)
„ Significant association was observed between haplotypes in the
TNFRSF11B (Table 4) and COL1A1 genes with an increased
risk osteoporosis.
„ The A-T-G haplotype in the promoter region of the TNFRSF11B
was more frequently found in affected individuals (51.7%) when
compared to normal controls (34.1%) (Table 4).
Haplotype
G-T-T (B-A-T)
Unaffected
0.011
Affected
0.009
G-G-T (B-a-T)
G-T-C (B-A-t)
G-G-C (B-a-t)
A-T-T (b-A-T)
0.011
0.378
0.000
0.156
0.000
0.438
0.000
0.170
A-G-T (b-a-T)
A-T-C (b-A-t)
A-G-C (b-a-t)
0.444
0.000
0.000
0.384
0.000
0.000
χ2 = 9.18; p = 0.24; df = 7
The Role of OPG in Osteoclast
Differentiation and Activation
Table 4 TNFRSF11B Gene Haplotype Frequencies in
Affected and Unaffected Individuals
Haplotype
A-T-G
Affected
0.517
Unaffected
0.341
A-T-C
A-C-G
A-C-C
G-T-G
0.000
0.052
0.347
0.000
0.015
0.012
0.496
0.080
G-T-C
G-C-G
G-C-C
0.051
0.024
0.090
0.023
0.105
0.000
χ2 = 24.66; p < 0.001, df = 7
Conclusions
„ In this study evidence of linkage was observed to a marker
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found in locus 17q21.31-q22 indicating that a gene responsible
for osteoporosis might be present in this region.
In a family with high incidence of severe osteoporosis and
hypertension, suggestive linkage was observed to other loci
where candidate genes for both diseases are known to be
found. This might help in the identification of a gene that links
osteoporosis with hypertension and atherosclerosis (Table 5).
Differences in loci identified between the two families is an
evidence of genetic heterogeneity in the disease.
Association of the A-T-G haplotype with increased risk for low
BMD indicates the possible role of the TNFRSF11B gene
variants in postmenopausal bone loss.
Also an increased frequency in affected individuals of the
haplotype in the COL1A1 gene promoter, shows the importance
of this structural gene in bone mass.
Table 5. Candidate Genes Indicated by
Linkage
Locus
Osteoporosis
Hypertension
17q21.31-q22
SOST, COL1A1, NOG
Angiotensin 1 converting
enzyme (ACE) *
8q24
TNFRSF11B*
Angiopoietin 1, Squalene
epoxidase
11q12-13
LRP5
Angiotensinase C (PCP) **
•*Genes already associated with both osteoporosis and atherosclerosis
•**Essential Hypertension Susceptibility Locus
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Future Studies
Acknowledgments
„ Increasing the number of STRs on chromosome 17
„ Prof. Angela Xuereb for her constant supervision and
where strong evidence of linkage was observed. This
helps in narrowing the interval and so increasing the
chance to localise the gene (high resolution scan).
„ Sequencing of interesting genes to identify possible
mutations that might be responsible for osteoporosis.
„ Gene expression studies to identify the biological
effects of polymorphisms found in the promoter
region of the TNFRSF11B.
support throughout the study.
„ Prof. Alex Felice for his supervision and use of
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facilities at the Laboratory of Molecular Genetics,
University of Malta.
Dr. Raymond Galea for his assistance in patient
recruitment.
Prof. Mark Brincat for his support.
Dr. Marios Kambouris for his informative discussions
and linkage software.
This work was supported by the Research Fund
Committee, University of Malta
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