Note: Descriptions are shown in the official language in which they were submitted.
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POLYMORPHISM IN A TGF-SS GENE CORRELATED TO OSTEOPORPSIS
This invention relates to diagnostic method and apparatus based upon
polymorphism of a TGF-f3 Gene. More specifically, this invention relates to
a method for diagnosis of pre-disposition to disease by screening for the
presence of a polymorphism. The invention also relates to apparatus for
screening for the potymorphism. The invention further relates to TGF-t3
genes containing a polymorphism and to a probe therefor.
Osteoporosis is a common age-related condition characterised by reduced
bone mineral density (BMD), deterioration in skeletal microarchitecture and
an increased risk of fragility fracture. One in three Caucasian women will
experience an osteoporotic fracture during their lifetime and it is estimated
that the annual health cost of such fractures amounts to around USS14
billion in the USA alone.
Hormone replacement therapy is an established treatment for osteoporosis
and has proved successful in halting further decline in bone density that is
characteristic in women suffering from this disease. Hormone replacement
therapy is generally not, however, able to bring about a reversal of
osteoporosis, that is to say it is not capable of inducing an increase in the
bone density of sufferers. The same criticism is made of other known
treatments for osteoporosis.
It would, accordingly, be of particular advantage to be able to identify with
increased accuracy those individuals having a predisposition or increased
susceptibility to osteoporosis. Suitable therapy could then be put into
place before the effects of osteoporosis set in.
Genetic factors play an important role in determining bone mineral density
SUBSTIME SHEET (RULE 26)
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(BMD) in later life, with the genetic influence mediated through effects on
both peak mass and on age- and menopause- related bone loss. At the
menopause there is an increase in the production and activity of various
cytokines and growth factors within the bone microenvironment.
Bone mineral density (BMD) in later life is a strong predictor of subsequent
osteoporotic fracture and is determined by both the peak value achieved
during skeletal growth and by age- and menopause- related bone loss.
Family and twin studies suggest a strong genetic component to the
determination of peak bone mass, with 50-85% of the population variance
in BMD being attributable to genetic factors. Twin studies in
postmenopausal and elderly women also support a persistent and
significant genetic influence on bone mass in later life. This rnay represent
either a strong residual effect from the genetic contribution to peak bone
mass or an independent genetic effect on the regulation of bone loss.
Indirect assessment of bone turnover through biochemical markers
suggests a genetic regulation of bone metabolism that may translate into
differing effects on bone loss although to date only two twin studies have
directly attempted to explore the genetic contribution to age- and
menopause- related bone loss with conflicting and uncertain results.
Osteoporosis is a complex disease that is likely to have a polygenic
aetiology, and candidate gene analysis has demonstrated that
polymorphisms of the vitamin D receptor (uDR) locus the oestrogen
receptor (ER) locus and the type I collagen alpha 1 (COL1 A1 ) locus are all
potential genetic markers for bone mass and bone loss. WO-A-97/28280
describes two polymorphisms in the promoter region of the TGF-~B1 gene
that have been shown to influence the amount of TGF-~1 protein in the
blood and correlates these polymorphisms with predisposition to a number
of disease states including hypertension, cancer and osteoporosis.
Y
AMENDED SHEET
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Langdahl et al. (Bone Vo1.20, No.3, 1997: 289-294) describes a sequence
variation in an intron of a TGF-Beta 1 gene, the 713-SdeIC variation. The
sequence variation is a single base deletion eight bases upstream of the
start of intron 5. The 713-BdeIC variation is correlated with an increase in
bone turnover but in both the non-osteoporotic normal control individuals
and the total group of osteoporotic patients bone mass was unaffected by
the presence of this variation.
Yamada et al. (J. Bone. Miner. Res. Vol. 13, No.10, 1998:1569-1676)
relates to a T-~C transition at nucleotide 29 in the signal sequence region
of the TGF-Beta 1 gene. Yamada et al. describe the correlation between
this variation and susceptibility to osteoporosis in postmenopausal
Japanese women.
The search for further genetic markers for use in diagnosis of disease,
AMENDED SHEET
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including diagnosis of osteoporosis and predisposition thereto, nevertheless
continues.
It is an object of this invention to provide method and apparatus for
detecting individuals having a predisposition or susceptibility to
osteoporosis. It is a further object of the invention to identify individuals
having such a predisposition or susceptibility by identifying those
individuals on the basis of genotype. It is another object of the invention
to provide a therapy for those individuals. Still further objects of the
invention are to provide an isolated gene comprising a polymorphism
indicative of predisposition to osteoporosis and probe therefor.
Accordingly, a first aspect of the invention provides a method of diagnosis
comprising determining genotype of an intron of a TGF-f3 gene.
The method of the invention typically comprises determining whether an
individual is homozygous or heterozygous for the gene and a particular
polymorphism thereof and thereby determining if the individual possesses
a risk polymorphism, the risk polymorphism being correlated with
osteoporosis or disposition thereto. The method is conveniently used to
screen for an individual at risk of a condition or disease correlated with a
polymorphism of this gene. In a preferred embodiment of the invention the
TGF-~B gene is a TGF-,B1 gene and the polymorphism is a T-~C
polymorphism located in intron 5 of said TGF-,81 gene. However, it is
envisaged that other polymorphisms that are located within the same or a
different intron may be so correlated with the presence of the intron 5
polymorphism of the invention that the two polymorphisms are in linkage
disequilibrium. Thus, diagnosis of disease by determining genotype of the
further polymorphism may lead to a similarly reliable diagnosis of
osteoporosis or predisposition thereto.
The method of the invention determines whether the individual being tested
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has a TGF-f3 gene which is identical with the published sequence or
whether that individual has a gene which differs from the published
sequence, i.e. is a polymorphism of the published sequence. In carrying
out the invention, an individual's TGF-f3 gene genotype is generally
determined by analysis of a section of the gene, rather than by analysis of
the entire gene. If the sequence of that section is found to be the same as
the corresponding section in the wild type sequence, then that individual
is classified as having the wild type gene.
In use of an embodiment of the invention to be described below in further
detail, an individual is screened to determine whether he or she possess a
TGF-f3 gene which is the published sequence or is a polymorphism thereof
in which there is a polymorphism in one or more of its introns. In this
specific embodiment, the' presence of a homozygous polymorphism in an
intron of the gene correlates with a predisposition to osteoporosis.
In an example of the invention, described in more detail below, the
presence of a BstUl restriction site in intron 5 of a TGF-,81 gene is
correlated with increased risk of osteoporosis or predisposition to
osteoporosis as well as increased bone turnover and reduced BMD. This
novel restriction site polymorphism is produced as a result of a T->C
substitution introducing the 5'-CG ,~ CG-3' recognition site for BstUl.
Screening is carried out, for example, using PCR primers adapted to amplify
a portion of gene in the region of and including the site of the
polymorphism It is preferred that the PCR primers are selected so as to
amplify a region of the gene that surrounds the region and includes at least
six nucleotides on either side. PCR techniques are well known in the art
and it would be within the ambit of a person of ordinary skill in this art to
identify primers for amplifying a suitable section of the gene. PCR
techniques are described for example in EP-A-0200362 and EP-A-0201 i 84.
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A second aspect of the invention provides for an isolated DNA fragment
comprising all or a part of the sequence of SEQ ID N0:1. The sequence in
SEQ ID N0:1 is the last fifty bases of intron 5 of the human TGF-~1 gene
including the T-~C substitution at position 31 of that sequence. Such a
DNA fragment may suitably be inserted into a vector for transformation into
prokaryotic or eukaryotic cells. Alternatively, the fragment may serve as a
template for production of a nucleic acid probe sequence for use in
applications such as northern or southern blotting or RNase protection.
Thus the invention provides DNA probes capable of distinguishing between
a wild type gene, to which the probe does not bind, and a polymorphism
thereof, such as one containing a polymorphism in intron 5 to which the
probe does bind.
The invention is of advantage in that by screening for the presence of the
polymorphism it is possible to identify individuals likely to have a genetic
predisposition to this disease.
A third aspect of the invention provides diagnostic means comprising PCR
primers adapted to amplify a region of a TGF-f3 gene, preferably a DNA
segment comprising intron 5.
Suitable primers are recited in the example below and correspond to SEQ
ID NOS: 2 and 3. The invention further provides a diagnostic kit comprising
PCR primers for use in amplification of an intron in a TGF ~ gene and
reference means to enable determination of the genotype, optionally within
a container. Such reference means typically include control reactions, a
written manual or diagrammatic representations indicating positive and
negative results.
The kit of the invention may also comprise an amount of the restriction
enzyme BstUl enabling PCR amplimers of the intron 5 region of the TGF-~B1
gene to be analysed for the presence of the novel T->C polymorphism of
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the invention.
Accordingly, a fourth aspect of the invention provides a method of therapy
comprising screening an individual for a predisposition to osteoporosis and,
if a genetic predisposition is identified, treating that individual to delay
or
reduce or prevent the osteoporosis, wherein a predisposition to
osteoporosis is correlated with a polymorphism in an intron of a TGF ~B1
gene.
A suitable treatment to prevent or reduce or delay osteoporosis is hormone
replacement therapy. The use of this therapy is well known in the art.
According to the invention, hormone replacement therapy can thus be
commenced in individuals likely to have a predisposition to osteoporosis but
in whom osteoporosis has not yet begun to any significant extent.
It is believed that the use of hormone replacement therapy carries with it
a concomitant increased risk of breast cancer. The invention offers the
advantage that the increased risk of breast cancer associated with hormone
replacement therapy can be accepted only by those women who are known
to have a likelihood of predisposition to osteoporosis. In an embodiment
of this aspect of the invention, the predisposition of an individual to
osteoporosis is assessed by determining whether that individual is
homozygous for the wild type TGF-f3 gene, is heterozygous for the wild
type and the, or is homozygous for the polymorphism - indicating risk of
predisposition to osteoporosis.
According to the invention, an individual who is homozygous for the risk
polymorphism is classified as being at highest risk.
Another suitable treatment is use of bisphosphonates. Two specific
treatments involve using xanthine oxidase inhibitors or substituted
benzodiazepines and are described in US-A-5436258 and US-A-54419fi4,
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the contents of which are incorporated herein by reference. Still further
treatments will be known to a person of skill in the art. Potential
treatments are described, for example, in JP-A-09030977, WO-A-
97/06254, JP-A-09025293, WO-A-97/04799, WO-A-97/03060 and JP-A-
09012592, the contents of which are incorporated herein by reference.
Currently authorised treatments for osteoporosis include the use of
oestrogens, with and without progestogen, the use of selective oestrogen
receptor modulators, the use of anabolic steroids such as nandrolone, the
use of the bisphosphonates alendronate and disodium etidronate. Further
treatments suitably include the use of salcatonin, administration of calcium
supplements and use of isoflavones or other plant derived steroids.
In pharmaceutical treatment of osteoporosis, all routes of administration are
suitable and include but'' are not limited to oral, injection intravenously,
intraperitoneally,intramuscularlyandsubcutaneously,intranasalandtopical
administration. Typical dosages and durations of treatment are as
described in clinician's textbooks such as British National Formulary,
incorporated herein by reference.
Currently, none of the osteoporosis medications that have been approved
by the Food and Drug Administration (FDA) for postmenopausal women
have been approved for men.
Testosterone replacement therapy may be prescribed for a man with a low
testosterone level.
Calcitonin is a medication that slows or stops bone loss and may relieve
the pain of fractures in some patients. Calcitonin is approved by the FDA
for the treatment of osteoporosis in postmenopausal women. While its
effect in men has not been studied, evidence suggests that it may work the
same in men as in women. Calcitonin is available as an injection and as a
nasal spray. Its use is described in US-A-5440012, incorporated herein by
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reference.
Bisphosphonates are a class of drugs that have been shown to help
preserve and increase bone density by slowing or stopping bone loss. The
FDA has approved the bisphosphonate known as alendronate for the
treatment of postmenopausal osteoporosis in women; it is currently being
studied for treatment of osteoporosis in men. There are other
bisphosphonates under development - and in fact etidronate has been
approved, though only outside the USA.
Sodium fluoride has recently been recommended for approval by an FDA
committee. Parathyroid hormone, calcitriol, and others are investigational
drugs. It will be some time before research: findings are available on these
preparations.
Decrease in bone mineral density can also be slowed by taken calcium
supplements, and some suggested levels are 1,000 mg of calcium a day for
women on oestrogen replacement therapy and 1,500 mg of calcium daily
for women not receiving oestrogen therapy.
Thus, a range of treatments, for those suffering or predisposed to
osteoporosis are known and all are believed suitable for use in combination
with its diagnosis according to the present invention.
Optionally, the assessment of an individual's risk factor is calculated by
reference both to the presence of a TGF-f3 gene polymorphism and also to
other known genetic or physiological or dietary or other indications. The
invention in this way provides further information on which measurement
of an individual's risk can be based.
The invention thus also provides a method of identifying, and optionally
treating, an individual predisposed or susceptible to osteoporosis, said
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method comprising determining genotype of a first gene in said individual,
wherein genotype of said first gene is correlated with genotype of a TGF-(3
gene in said individual.
The invention further again provides a method of predicting response to
osteoporosis therapy, comprising diagnosing genotype of a TGF-f3 gene, in
accordance with the first aspect of the invention.
This latter aspect of the invention thus enables informed choice of therapy,
including choice of type of therapy and choice of amount or strength of
therapeutic agent, to be made for a given individual predisposed to
osteoporosis. Moreover, for a given individual already suffering from
osteoporosis, the invention enables an assessment of whether the currently
f
prescribed therapy is likely to be effective in treating the disease or if an
alternative therapy regime will be more successful: In a specific
embodiment of the invention, diagnosis of a risk polymorphism in a TGF-f3
gene indicates that hormone replacement therapy, or an equivalent, is likely
to be effective. More specifically, possessing two copies of the risk
polymorphism indicates an increased level of therapy is likely to be
appropriate.
Bone mineral density (BMD) in later life is a major determinant of
osteoporotic fracture risk and has been shown to be under strong genetic
influence. Segregation analysis within families and data from twin studies
has suggested that this genetic effect on BMD is probably mediated by a
number of genes each having small individual effects. Transforming
growth factor 13 (TGF-f3) is an important regulatory cytokine and is found
in high concentrations in the bone matrix. TGF-f3 is therefore a plausible
candidate for the genetic regulation of BMD. In total 91 1 DZ pairs and 386
MZ pairs (age range 18-76 years) were studied, with measurements of
BMD using DXA and calcaneal ultrasound. In accordance with the present
invention, a novel T-~C polymorphism was identified in intron 5 with an
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allele frequency of 0.25 within the DZ subjects. Comparison of the
variance in femoral neck BMD between the MZ and DZ twins showed a
heritability of 62% at this site. BMD at the femoral neck was 5% lower in
subjects homozygous for the presence of the TGF-f3 polymorphism when
compared to the other two genotype groups. No effect was seen at the
lumbar spine, ultradistal radius, or with ultrasound measurements. Results
were unaffected after adjustment for potential confounders. Linkage
analysis within the DZ twin pairs confirmed the significance of this
polymorphism on hip BMD.
BMD is one of the strongest predictors of fracture risk (Cummings et al.
(1993)) and a large number of twin and family studies have suggested a
strong genetic influence on this trait, with up to 85% of the population
variance in BMD being attributable to genetic factors (Seeman et al. ( 1989);
Arden et al. (1996); Jouanny et al. (1995).
Normal skeletal morphogenesis is dependent on a complex interaction
between osteoblasts, osteoclasts, and local growth factors. During growth
and development the processes of osteoblastic bone formation and
osteoclastic bone resorption appear coupled, thereby maintaining skeletal
integrity and preserving bone mass and shape. Non-invasive measures of
bone turnover have shown that after the menopause there is an increase
in bone resorption, leading to a loss of bone and subsequent development
of osteoporosis. Several studies have also shown that in women with
osteoporotic fracture the remodelling balance is more negative when
compared to age-matched women with no fracture history. It is believed
that this postmenopausal increase in bone resorption in the oestrogen
deficient state is mediated in part by cytokines (interleukins 1 and fi,
tumour necrosis factor and various growth factors (transforming growth
factor, insulin-like growth factors). Twin studies have also suggested that
there is a genetic influence on the general process of bone turnover in pre-
and post-menopausal women, with higher correlations for biochemical
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markers of both bone formation and resorption seen in identical compared
to non-identical twins (Kelly et al. ( 1991 ); Garnero et al. ( 1996)1.
Transforming growth factor f3 (TGF-f3) is synthesised by osteoblasts and
osteoclasts in vivo and has three isoforms. High concentrations of all these
isoforms can be extracted from the mineralised bone matrix, and although
TGF-(3 is found in a variety of tissues, the concentration of TGF-f3 appears
to be highest in the bone matrix. Osteoblasts produce TGF-f3 largely as a
matrix-bound latent complex composed of 390 amino acids. It is only
released during bone resorption, with subsequent activation in the acidic
environment below the ruffled border of the resorbing osteoclast. This
suggests that TGF-f3 may play a central regulatory role in the coupling that
exists between bone formation and resorption. Active TGF-f3 is formed of
two identical disulphide-linked polypeptide chains consisting of the 112
amino acids from the C-terminal part of the precursor protein. The TGF-(3
gene maps to chromosome 19q 13 and contains 7 exons. The active
component of TGF-f3 is encoded by part of exon 5, exon 6, and part of
axon 7. The TGF-f3 gene may therefore be an important candidate gene for
the development of osteoporosis, with variation at this locus being
associated with differences in BMD and therefore risk of fracture.
A further aspect of the invention provides for a method of diagnosing
osteoarthritis and predisposition to osteoarthritis in an individual
comprising
determining the genotype of intron 5 in a TGF-~1 gene, wherein absence
of a BstUl restriction site is correlated with risk of osteoarthritis.
It is known that predisposition to osteoarthritis and osteoporosis may be
linked in that the process of bone deposition seen in osteoarthritis is under
the control of similar signalling factors to those that control the process of
bone resorption, as seen in osteoporosis. The activities of osteoblasts
(bone forming cells) and osteoclasts (bone resorbing cells) are to an extent
controlled by the same group cytokines including TGF-~3. Accordingly, a
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further aspect of the present invention provides for a method of diagnosing
osteoarthritis and predisposition to osteoarthritis in an individual
comprising
determining the genotype of intron 5 in a TGF-~1 gene. The risk of disease
or predisposition thereto is inversely related to risk of or predisposition to
osteoporosis, thus for example absence of a BstUl restriction site is
correlated with risk of osteoarthritis, and mutatis mutandis for all other
embodiments of the invention.
The present invention is now illustrated by way of the following example.
Example
Methods
Subjects
The subjects studied were Caucasian, female monozygous (MZ) and
dizygous (DZ) twins (age range 18-76 years) recruited after national media
campaigns. All subjects were healthy and did not suffer from diseases
specifically affecting bone, and were broadly representative of the normal
United Kingdom population as previously described. Twins completed a
nurse-administered questionnaire detailing medical, obstetric and
gynaecological histories, full drug histories, dietary calcium assessment,
exercise levels, smoking status, and alcohol intake. Twin zygosity was
determined by questionnaire and in doubtful cases this was confirmed with
multiplex DNA fingerprinting.
Measurements
BMD was measured at the lumbar spine (L1-4), non-dominant hip (femoral
neck, total hip), and non-dominant ultradistal radius using DXA on a
Hologic QDR-2000. Reproducibility as assessed by the coefficient of
variation (CV%) from duplicate measures in healthy volunteers was
between 0.8% and 1.6% at the skeletal sites measured. Subjects were
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classified as having osteoporosis according to the World Health
Organisation diagnostic criteria if their BMD measurement was 2.5 standard
deviations (SD) below the mean peak young adult value (i.e. T -score < -
2.5).
Calcaneal ultrasound was measured using a McHue Cuba Clinical scanner.
This produced two output variables: broadband ultrasound attenuation
(BUA) and velocity of sound (VOS). Reproducibility as assessed by the
CV% in duplicate measures on 30 subjects was 2.5% (BUA) and 0.44%
(VOS).
Polvmorahism identification
DNA was prepared for each subject from peripheral blood leucocytes using
a standard phenol extraction method. Common single nucleotide
polymorphisms (SNPs) in the TGF-f3 gene were detected by sequence
analysis of 24 unrelated dizygotic (DZ) individuals and comparison made
with the published sequence (Accession no. Y00112). This strategy was
chosen to identify polymorphisms with an allele frequency of at least 0.1
within the study group. Oligonucleotide primer pairs were designed to
cover the TGF-f31 coding regions, and the promoter and 5'-untranslated
region up to position 1363. Following amplification by the polymerase
chain reaction (PCR) the products were purified with the Advanced Genetic
Technologies Corp 96 well PCR purification system and sequenced using
PE Applied Biosystems dRhodamine Terminator cycle sequencing kit. The
sequencing reactions were analyzed on an ABI 377 DNA sequencer.
Restriction enzyme digests
Polymorphism screening within the DZ group was performed using PCR-
restriction fragment length polymorphism (RFLP) based methods with
restriction enzyme digest.
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BstUl PCR-RFLP analysis of the intron 5 SNP
PCR amplification was performed using the ~PCR primers SEQ ID NOS: 2
and 3. Reactions were performed in 25,u1 with the following composition:
0.3NM primers, 0.2mM dNTPs, 1 mM MgCL2, 1 X Taqgold buffer, 1.25
units Taqgold (P.E. Applied Biosystems) and 50ng genomic DNA.
Thermocycling was performed on a MJ Research DNA Engine Tetrad PTC-
225 thermal cycler using the following conditions: 95°C for 14 minutes,
35 cycles of 94°C for 15 seconds, 60°C for 15 seconds,
72°C for 30
seconds, followed by final extension of 72°C for 10 minutes. The 230 by
PCR product was digested with 3 units of BstUl (New England Biolabs) at
60°C for 2 hours producing polymorphic fragments of 202 and 28 bp.
Products were analysed by agarose gel electrophoresis with size
determination after transillumination under ultra-violet light. Alleles were
coded as A 1 = presence of restriction site, and AO = absence of site.
Statistical analysis
Differences in the mean value in each variable for the three TGF-f3 intron
5 genotypes (AOAO, AOA1 and A1A1) were tested using generalised
estimating equations (GEE)(Zeger et al. (1986)). This method takes into
account the dependence of measurements within twin pairs in estimating
the significance of the differences. The GEE models were extended to
include potential confounding variables (age, menopausal status).
Significant GEE results were followed up using a quantitative genetic
modelling approach in which additional phenotypic data on MZ twins were
used. A model was specified which provided estimates for the genetic and
environmental variance components and estimates for the means of each
possible genotype (AOAO, AOA1 and A1A1). This enabled us to estimate
the percentage of the genetic variation in the phenotype that could be
attributed to the TGF-f3 polymorphism. Model fitting was performed using
Mx. Parameters were estimated by normal-theory maximum-likelihood,
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where the model were fitted to the raw data. Linkage analysis within the
DZ twins was also analysed using the MAPMAKER/SIBS programme
lversion 1.0). This calculates the maximum likelihood sharing probabilities
for each DZ pair and as parental information was not available this utilised
estimated allele frequencies from within the total DZ group. Evidence for
linkage of the polymorphism to the trait was taken as a nominal lod score
>_ 1.00 or a nominal P value S 0.05.
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Results
Sequence analysis of 24 unrelated individuals identified a novel
polymorphism in the TGF-f3 gene. The novel polymorphism was a T-~C
substitution in intron 5, 20bp upstream of exon 6. This substitution in
intron 5 introduces a BstUl site (recognition sequence 5'-CG 1 CG-3'), and
subsequent screening for this within the whole DZ cohort using the BstUl
PCR-RFLP showed the allele frequencies to be 0.75 and 0.25, with the
genotype distributions being in Hardy Weinberg equilibrium.
In total, data was available on 911 DZ and 386 MZ twin pairs. Intron 5
genotype results were available on 1664 of the DZ subjects. Reasons for
absence of genetic results included inadequate DNA extraction or failure of
the PCR-RFLP assay. No 'significant differences were observed, however,
between subjects with and without TGF-f3 genotype results.
Characteristics of the study population are shown in Table 1. In
comparison with the DZ twins, the MZ twins were on average slightly older
by 2 years and had a higher percentage of women who were
postmenopausal. Within the DZ group there were no differences in age,
height, weight, smoking status or hormone replacement (HRT) use between
the three TGF-f3 intron 5 genotype groups. There were however, small
differences in the proportion of postmenopausal women (Table 1 ).
The TGF-f3 intron 5 genotype A1A1 was associated with hip BMD when
compared to the other 2 genotypes, with a 5% reduction in femoral neck
BMD and a 3.8% reduction in total hip BMD (Table 2). These findings
were unaltered after adjustment for the small difference observed in
menopausal status. These results at the hip, suggest a recessive pattern
of risk associated with carriage of the rarer A1 TGF-f3 intron 5 allele. No
genotypic association was seen at the lumbar spine, ultradistal radius or in
the calcaneal ultrasound parameters of BUA and VOS. The prevalence of
clinically defined osteoporosis (T-score <-2.5) at the femoral neck in the
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genotype group A1A1 was 18% compared to 10% in the AOAO and AOA1
groups. This demonstrates a 70% increased risk for a subject with the
A1A1 genotype having femoral neck osteoporosis when compared to the
other 2 genotypes, with an odds ratio (95% confidence interval) of 1.71
(0.95, 3.07), P = 0.07.
Having demonstrated an association between TGF-f3 intron 5 genotype and
hip BMD, twin modelling and variance component analysis was utilised to
estimate the proportion of the genetic variance explained by this
polymorphism. This analysis confirmed previous studies showing high
heritabilities for BMD and ultrasound parameters at the various skeletal
sites. At the femoral neck the estimated heritability was 0.62, with the
proportion of the population genetic variance explained by the intron 5
TGF-f3 polymorphism at this site being 0.60%.
The results of the single point linkage analysis are shown in Table 3. These
demonstrate linkage between the intron 5 polymorphism and femoral neck
BMD. For the measurement of BUA the nominal P value approaches 0.05
although the lod score does not exceed 1.00.
Thus, in accordance with the present invention, a novel T-~C polymorphism
in a TGF-f3 intron, specifically intron 5 in an embodiment of the invention,
of the human TGF-f3 gene. Our data demonstrate both association and
linkage between this polymorphism and hip BMD in a large group of
unselected, normal female twins. The polymorphic C allele was present at
a relatively high allelic frequency in the female population (0.25), and there
appeared to be a recessive pattern of risk associated with this allelic
variant. Hip BMD was 3-5% lower in women who were homozygous for
the carriage of the polymorphic allele, when compared to women who were
either heterozygous or homozygous for the commoner allele. Women who
were homozygous for the polymorphism also had a 70% increased risk of
having osteoporosis at the femoral neck in comparison to the other two
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genotypes.
These cross-sectional data indicate that subjects who are homozygous for
an intronic polymorphism of the TGF-f3 gene have reduced BMD at the
femoral neck and total hip. The observation that these findings were
confined to the hip (and predominantly at the femoral neck), rather than
being seen at the spine and distal radius suggest a site-specific association.
The absence of any genotype association with bone ultrasound measures
also suggests that the TGF-f3 intron 5 polymorphism has a predominant
effect on BMD rather than on bone quality or structure. Although the
proportion of population variance in BMD attributable to this polymorphism
appears low, this reflects the recessive risk associated with this locus as
only approximately 6% of the population would be expected to be of the
genotype A1A1. Our data indicate, however that these subjects have a
70% increased risk of having osteoporosis and therefore face significantly
increased risk of fracture. TGF-f3 genotype therefore identifies an at risk
sub-group of women who would benefit from targeted intervention. This
is also illustrated by recent findings from a large population study
examining the relationship between a polymorphic variant in the promoter
region of the type I collagen 1 a gene and osteoporosis. In this study the
polymorphic allele was associated with a 2-fold increased risk of fracture
despite only explaining 0.3 to 0.4% of the population variance in BMD.
There is currently a large amount of data suggesting that TGF-f3 may play
a central role in the regulation of BMD and bone turnover. In vivo studies
have shown that local injection of TGF-f3 under the periosteum stimulates
cartilage and bone formation (Noda et al. (1989)) and that systemic
injection of TGF-f32 also leads to a generalised increase in osteoblastic
activity. In vitro, TGF-f3 induces extracellular matrix secretion by
osteoblasts, inhibits matrix mineralisation, and modulates osteoprogenitor
cell proliferation. The rate of bone formation is altered in the TGF-(3
knockout mouse (Geiser et al. ( 1996)) and administration of TGF-13 corrects
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the bone density deficiency in elderly mice with osteoporosis. Oestrogen
action on bone also appears to be mediated via effects on TGF-f3 and
induction of osteoclast apoptosis. Allelic variation at the TGF-f3 locus may
therefore be important in determining the therapeutic response of the
oestrogen and selective oestrogen receptor modulators on bone and other
tissues.
The functional significance of our findings on TGF-f3 activity is uncertain,
although as the polymorphism is 20 by upstream of exon 6 it could have
some influence on the active component of the TGF-f3 protein. The lack of
coding sequence variation identified in this study and others suggests that
the amino acid sequence of the active form of TGF-f3 is highly conserved
with strong selective pressures against variant proteins. It has been
reported that up to 15% of human diseases are caused by point-sequence
variation in splice regions resulting in either exon skipping or cryptic
splicing and although the intron 5 polymorphism is not located in a splice
donor or acceptor site it could affect a branch point. The intronic
polymorphic sequence change may also affect messenger RNA stability and
further studies will be required to examine these possibilities. If this
polymorphism is not functional, then these findings may suggest that the
TGF-f3 polymorphism is actually in linkage disequilibrium with a novel
disease locus mapping to this chromosomal region. The finding of a
positive linkage result at this locus with hip BMD would also implicate this
chromosomal region and multipoint linkage analysis will be required to
refine the location of the putative disease locus.
The invention thus provides method and apparatus for diagnosis of
osteoporosis or predisposition thereto.
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Table 1
Mean ( t SD) characteristics of female twin subjects
variable MZ total DZ totalDZ subjects
724) ~" ~ according
~ ~58~ to TGF-~1
intron
5 genotype
HVHU 1 "j--
(n = 848) (n = 609) (n =
Age (yrs) 50.1 47.5 47.6 47.5 g0)
49.1
( 13.4) ( 11.3) ( 11.0) ( 1 1.9) (9.6)
Height (cm) 162 (6) 163 (6) 162 (6) 163 (6) 162 (6)
Weight (kg) 63.9 65.9 66.0 65.4 65.4
(10.7j (12.3) (12.1) (12.1) (12.7)
S a b j a c t s 67 54 51 56 62
postmenopausal (%)
Subjects ever smoking44 48 45 49 51
(%)
Subjects ever use 30 30 28 31 31
of
HRT (%)
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Table 2
Mean ( t SD) BMD at lumbar spine, hip and forearm, and calcaneal ultrasound
measurements in Dt subjects according to their TGF ~f39 intron 5 genotype
Variable T F X81 Intron
5 Genotype
0 n = A n = n =
848)
L a m b a r s 0.998 (0.147)1.011 (0.149) 1.005 (0.146)
p i n a
BMD (g/cm2)
Femoral neck 0.812 (0.131)0.810 (0.130) 0.770 (0.112)*'
BMD (g/cm2)
Total hip BMD 0.924 (0.132)0.925 (0.131 0.889 (0.117)
) ~
(g/cm2) '
Ultradistal radius0.455 (0.068)0.458 (0.069) 0.451 (0.068)
BMD (g/cmz)
VOS (m/sec) 1659 (52) 1660 (52) 1661 (51 )
BUA (dB/MHz/cm) 78 ( 19) 78 ( 19) 76 ( 17)
* AlAl vs AOAO, P = 0.005; AlAl vs AOA1, P = 0.04
$ AlAl vs AOA1, P = 0.05
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Table 3
Single point lod scores for TGF-~1 intron 5 genotype and BMD/IJltrasound
measures
in DZ twin pairs.
Variable TGF-,81 Intron 5
Lod score Nominal P-va ue
Lumbar spine 0.365 0.195
BMD (g/cm2)
Femoral neck 1.091 0.025
BMD (g/cm2)
Total hip BMD 0.479 0.138
(9/cmz
Ultradistal radius 0.570 0.105
BMD (g/cm2)
VOS (m/sec) 0.159 0.392
BUA (dB/MHz/cm) 0.785 0.057
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References:
Arden NK, Baker J, Hogg C, Baan K, Spector TD 1996. The heritability of bone
mineral density, ultrasound of the calcaneus and hip axis length: a study of
postmenopausal twins. J Bone Miner Res 11: 530-534.
Cummings SR, Black DM, Nevitt MC, Browner W, Cauley J, ~nsrud K, Genant HK,
Palermo L, Scott J, Vogt TM 1993. Bone density at various sites for prediction
of hip fractures. The study of osteoporotic fractures research group. Lancet
341:
72-75.
Garnero P, Arden NK, Griffiths GO, Delmas PD, Spector TD 1996 Genetic
influences on bone turnover in postmenopausal tv~ins. J Clin Endocrinol Metab
81: 140-146.
Geiser AG, Helvering LM, Zeng QQ, Sato M 1996 Bone growth in the TGF ~1
knockout mouse. J Bone Miner Res 11: S378. (Abstr)
Jouanny P, Guillemin F, Kuntz C, Jeandel C, Pourel J 1995. Environmental and
genetic factors affecting bone mass: similarity of bone density among members
of healthy families. Arthritis Rheum 38: 61-67.
Kelly PJ, Hopper JL, Macaskill GT, Pocock NA, Sambrook PN, Eisman JA 1991
Genetic factors in bone turnover. J Clin Endocrinol Metab 72: 808-813.
Noda M, Camilliere JJ 1989 In vivo stimulation of bone formation by
transforming
growth factor,8. Endocrinology 124: 2991-2994.
Seeman E, Hopper JL, Bach L, Cooper ME, Parkinson E, McKay J, Jerums G
1989. Reduced bone mass in daughters of women with osteoporosis. N Engl J
Med 320: 554-558.
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Zeger SL, Liang KY 1986 Longitudinal data analysis for discrete and continuous
outcomes. Biometrics 42: 121-30
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MOLLOY, HELEN R
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