Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-10
2004-10-12
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023500, C536S024310, C536S024330
Reexamination Certificate
active
06803197
ABSTRACT:
The present invention relates to a prognostic method and prognostic means based on polymorphisms in the vitamin D receptor and collagen I&agr;1 genes. In particular, the present invention relates to a method for determining susceptibility to bone damage by screening for polymorphisms in vitamin D receptor or collagen I&agr;1 genes.
Osteoporosis is a common disease characterized by reduced bone mineral density (BMD), deterioration of bone micro-architecture and increased risk of bone damage, such as fracture.
1
It is a major public health problem which affects quality of life and increases costs to health care providers. In European populations, one in three women and one in twelve men over the age of fifty is at risk. The disease effects 25 million people in the USA, where the incidence of disease is 25% higher than it is in the UK, and a further 50 million people in Japan and Europe combined. It is estimated that by the middle of the next century the number of osteoporosis sufferers will double in the West, but may increase six-fold in Asia and South-America. Fracture is the most serious endpoint of osteoporosis, particularly fracture of the hip which affects up to 1.7 million people worldwide each year. It is estimated that by the year 2050, the number of hip fractures worldwide will increase to over 6 million, as life expectancy and age of the population increase.
Treatment of osteoporosis is unsatisfactory. In particular, once bone damage has occurred as a result of osteoporosis, there is little a physician can do other than let the bone heal. In the elderly, this may be a slow and painful process. Diagnosis of those at risk of developing osteoporosis allows more effective preventative measures. Strategies for the prevention of this disease include development of bone density in early adulthood, and minimisation of bone loss in later life. Changes in lifestyle, nutrition and hormonal factors have been shown to affect bone loss.
7-14
Osteoporosis can be considered a complex genetic trait with variants of several genes underlying the genetic determination of the variability of the phenotype. Low bone mineral density (BMD) is an important risk factor for fractures, the clinically most relevant feature of osteoporosis. Segregation analysis in families has shown that BMD is under polygenic control
12,13
while, in addition, biochemical markers of bone turnover have also been shown to have strong genetic components
14-16
. Several candidate genes have been analysed in relation to BMD but the most widely studied gene in this respect, the vitamin D receptor (VDR) gene, explains only a small part of the genetic effect on BMD
8
. Numerous studies, focussing on the BsmI allele of the vitamin D receptor gene have concluded that absence of the restriction site correlates with low bone mineral density. Most genetic analyses have focussed on BMD as a determinant of fracture risk and not so much on fractures themselves as an endpoint in the analysis. Recently, an SpI polymorphism in the COLIA1 gene encoding the most abundant bone matrix protein, was found to be associated with reduced BMD and, more importantly, also with increased risk of osteoporotic fracture
9,10
. An emerging theme from these studies seems to be the association of the absence of the BsmI restriction site with reduced bone mineral density, which in turn signifies increased risk of bone damage such as fracture.
Few studies have addressed genetic association with the clinically most important endpoint of osteoporosis, namely bone damage, in particular fracture. Accordingly, it is an object of the present invention to improve the prognosis of predisposition or susceptibility to bone damage.
In a first aspect of the present invention, there is provided a method of determining susceptibility to bone damage in a subject, said method comprising analyzing genetic material of a subject to determine which allele(s) of the vitamin D receptor gene is/are present.
Thus, the present invention satisfies the pressing need for identification of those individuals susceptible to bone damage, thus facilitating the development of preventative measures. For example, those at risk may avoid damage by modifying their lifestyle and implementing bone strengthening measures, such as regular exercise and a healthy diet. Typically, the method of the present invention comprises analysis of polymorphisms in the VDR gene to determine susceptibility to bone damage. The method may include determining whether one or more particular alleles are present, or which combination of those alleles (i.e. the haplotype) is present. The method may further comprise determining whether subjects are homozygous or heterozygous for alleles or haplotypes of the vitamin D receptor gene.
Vitamin D is a potent regulator of bone and calcium homeostasis, as well as of cellular differentiation and replication in many tissues, and mediates its effects through the vitamin D receptor (VDR). Cloning of the vitamin D receptor has shown it to be a member of the ligand-activated superfamily, which are natural regulators of a number of physiological and developmental processes. Evidence suggests that the vitamin D receptor activates expression of the osteocalcin gene through interaction with a palindromic sequence in the promoter of the gene.
27
The osteocalcin gene product is a marker of bone turnover in normal and disease states, and inter-individual variation in its circulating levels have been associated with polymorphisms in the vitamin D receptor gene.
The vitamin D receptor gene (12q12) comprises inherited polymorphisms between exon 7 and the 3′ UTR of the VDR gene, as shown in FIG.
1
. These alleles are denoted B/b, A/a and T/t for restriction enzyme sites BsmI, ApaI and TaqI respectively (or enzymatic or chemical procedures having similar specificity), where a lower case letter denotes the presence of a wild type restriction site which is capable of being cleaved, and a capital letter denotes the presence of a mutant restriction enzyme site which is not capable of being cleaved by the relevant restriction enzyme. For the purposes of the present invention, determination of which alleles are present in a particular gene may be referred to as determining the genotype of a subject for a particular gene. It is apparent from the above that each copy of the vitamin D receptor gene will comprise a specific combination of the three alleles, this combination being referred to as the haplotype of the gene. For example, the haplotype may be baT, indicating the presence of cleavable BsmI and ApaI sites, and a non-cleavable TaqI site. Direct haplotyping of the VDR gene has allowed five different haplotypes to be determined, of which three are common.
11
The present invention is based upon the surprising observation of a correlation between the presence of the b allele of the vitamin D receptor and susceptibility to/or risk of (where the terms are used interchangeably) bone damage, such as fracture. The invention goes further to show that presence of the a and/or T alleles, and in particular the haplotype baT is/are associated with increased risk of bone damage. A subject having the baT haplotype may show a higher risk of fracture compared to a subject having the bAt, or BAt haplotype which confers the lowest risk of fracture. The results are unexpected, as previous studies have shown the b allele, and particularly the baT haplotype, to be associated with high bone density, which itself is not associated with fracture risk. Thus, in contrast to previous results, the present inventions have shown that susceptibility to bone damage is independent of bone mineral density in a subject. By screening for the presence of alleles of the vitamin D receptor gene, susceptibility to bone damage may be assessed without the need for analysis of bone mineral density.
Preferably, the method of the first aspect of the present invention further comprises determining whether the alleles present are associated with risk of bone damage. This may be performed by comparing the alleles present in a sub
Pols Huibert Adriaan Pieter
Uitterlinden Andreas Gerardus
Van Leeuwen Johannes Petrus Thomas Maria
Christensen O'Connor Johnson & Kindness PLLC
Erasmus Universiteit Rotterdam
Myers Carla J.
Sakelaris Sally
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