Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-08-15
2004-07-13
Johannsen, Diana B. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023500, C536S024310, C536S025320
Reexamination Certificate
active
06762023
ABSTRACT:
BACKGROUND OF THE INVENTION
Osteoporosis is a disabling condition characterized by low bone density and fragile bones. The term “osteoporosis”, encompasses diseases of many different etiologies that all result in a reduction in the mass of bone per unit volume. Osteoporosis can be seen without association with other disease (idiopathic) and also in conjunction with other diseases (e.g., rheumatoid arthritis, diabetes, calcium deficiency). The decreased bone mass characteristic of the disease is not due to a decrease in the ratio of mineral to organic phase of bone, nor to any known abnormality in the mineral or organic matter of bone. Histological changes seen include a decrease in cortical thickness and a decrease in the number and size of the trabeculae of cancellous bone. Ostcoporosis is the most common of all metabolic bone diseases and is seen commonly in the elderly.
Since bone mass is decreased in osteoporosis, the rate of bone resorption exceeds that of bone formation. There is a normal age-related loss of bone mass in adults. However, this age-related loss of bone begins earlier and proceeds more rapidly in women. The reasons for the age-related bone loss are not known, although risk factors identified have included race (white women being at greatest risk), sex (females), sedentary lifestyle, and less muscular development. Other factors associated with risk of developing osteoporosis include early menopause, smoking, and excessive alcohol consumption.
Diagnosis of osteoporosis is most often done in conjunction with a study of bone density by radiography. Although clinical laboratory tests such as levels of calcium and phosphorus in blood can be examined, these measures are usually normal in osteoporotic patients. Only about 20% of postmenopausal women with osteoporosis exhibit hypercalciuria, or increased excretion of calcium in urine. Therefore, such laboratory findings are not indicative of the presence of disease, and clearly would not be indicative of risk of developing disease. To date, the prediction of risk of developing disease relies on family history of the disease. However, no genetic test is currently available to screen individuals.
The ethnic differences shown to exist in the propensity to develop osteoporosis have led researchers to believe that genetic factors play a dominant role in the etiology of this disease. Several genes have been shown to be associated with low bone density and research has focused on identifying those genes that may act as markers of disease. Common allelic variations of the vitamin D receptor gene have been found to be associated with decreased bone density in certain populations, including premenopausal women and young girls (Wood, R. J. and Fleet, J. C.
Ann. Rev. Nutrit
. 1998 18:233-258). Bone mineral density has also been associated with genetic variation in the estrogen receptor gene, both by itself and in conjunction with variations in the vitamin D receptor gene (Willing et al.
J. Bone Min. Res
. 1998 13:695-705). In Japanese women, the HLA-A*24-B*07-DRB*01halotype has been linked to low peak bone mass (Tsuji et al.
Hum. Immunol
. 1998 59:243-249). A variant of the gene encoding transforming growth factor-beta 1 has also been associated with low bone mass in osteoporotic women and with low bone mass and increased bone turnover in normal women (Langdahl et al.
Bone
1997 20:289-294). A polymorphism of the COLIAL gene has been identified as a potential marker for low bone mass and vertebral fracture in women (Grant et al.
Nat. Genet
. 1996 14:203-205). However, none of these studies has examined the relationship between tumor necrosis factor alpha receptor 2 gene and osteoporosis or bone mass in women.
Devoto et al. (
Eur. J. Hum. Genet
. 1998 6:151-157) determined that there was a gene or genes on chromosome 1 of humans that was linked to low bone density. However, there are many potential genes on this chromosome that could be involved in the etiology of low bone density.
It has now been found that individuals at risk of developing osteoporosis can be identified by assessing the genotype of the tumor necrosis factor alpha receptor gene in a sample of DNA obtained form the individual. A polymorphism in the gene on chromosome 1 for tumor necrosis factor alpha receptor 2 has now been shown to be associated with low bone density.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method of screening individuals for risk of developing osteoporosis. This method is based on assessment of the genotype of the tumor necrosis factor alpha gene in a DNA sample obtained from the individual. The presence of polymorphisms at nucleotides 593, 598 and 620 in exon 10 of the tumor necrosis factor alpha 2 receptor gene is associated with low bone density.
Another object of the present invention is to provide a kit for screening individuals for risk of developing osteoporosis which comprises a means for assessing the genotype of the tumor necrosis factor alpha 2 receptor gene in an individual.
DETAILED DESCRIPTION OF THE INVENTION
In the present invention a method is provided for screening individuals for the presence of a polymorphism in the tumor necrosis factor alpha receptor 2 (TNFR2) gene. The presence of this genotype in individuals has now been shown to be associated with low bone density and the risk of developing osteoporosis. The association between TNFR2 gene and low bone density was based on the observation that there is a polymorphism in this gene at a particular region of the gene sequence. This polymorphism was identified by Kaufman et al. (
Hum. Genet
. 1994 94:418-422), however, no characterization of its nature or examination of the relationship with bone density was disclosed. In the present invention, a relationship of this polymorphism with bone density has now been identified.
In these experiments, two polymorphic sites within the TNFR-2 gene, including a microsatellite repeat within intron 4 and a three nucleotide variation in the 3′untranslated region of the gene were assayed in three groups of individuals. Test group 1 was comprised of individuals from families used for linkage analysis of the low BMD (mean bone density) trait. Test group 2 was made up of unrelated individuals from an osteoporosis clinic. The third group was made up of unrelated individuals from a general clinic population with no testing for or knowledge of their bone status. This third group was used as reference for allele frequency comparisons. The composition and mean bone density Z-scores for test groups 1 and 2 are shown in Table 1.
TABLE 1
Results of ANOVA among the males and females
comprising the two test groups for age, body mass
index and bone density
GROUP 1
GROUP 2
P
F ratio
Male
Female
Male
Female
Value
(df)
Number
22
44
12
81
Age
56.2
57.3
43.7
52.6
0.011
3.846
(yrs ± SE)
(2.5)
(1.9)
(3.8)
(1.5)
(3,153)
Body Mass
28.4
25.9
25.0
24.8
0.084
2.259
Index (±SE)
(1.3)
(0.9)
(1.7)
(0.6)
(3,149)
Mean BMD Z-score (±SE)
L2-L4
−0.56
−1.17
−1.02
−0.99
0.442
0.901
(0.38)
(0.21)
(0.49)
(0.14)
(3,155)
Femoral neck
−0.50
−0.74
−0.73
−0.74
0.790
0.348
(0.27)
(0.13)
(0.39)
(0.09)
(3,150)
Trochanter
0.04
−0.64
−0.84
−0.59
0.054
2.611
(0.31)
(0.16)
(0.30)
(0.11)
(3,146)
Ward's
−0.21
−0.76
−0.61
−0.70
0.232
1.446
triangle
(0.22)
(0.16)
(0.45)
(0.11)
(3,146)
Only age was significantly different among the groups (p=0.011), due to the younger mean age of the males in the second group. However, ANOVA of age as the dependent variable with genotype for the two polymorphisms as class variable indicated that age was unaffected by genotype. Therefore the results of genotype testing in the two groups were pooled for subsequent analyses.
The microsatellite repeat within intron 4 of the TNFR2 gene has been reported previously to have 5 alleles (Beltinger et al.
Genomics
1996 35:94-100). In these test groups, 6 alleles were observed with a statistically significant difference in the frequency of distributio
Drexel University
Johannsen Diana B.
Licata & Tyrrell P.C.
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