Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-30
2004-10-26
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S023500, C536S024310, C536S024330, C536S024100
Reexamination Certificate
active
06808881
ABSTRACT:
The present invention relates to a prognostic method and prognostic means based on polymorphisms in the vitamin D receptor gene. In particular, the present invention relates to a method for determining susceptibility to heart disease by screening for polymorphisms in the vitamin D receptor gene.
Heart disease such as myocardial infarction is a complex phenotype caused by interaction of a number of genetic and environmental factors.
1
Advances in molecular genetics have led to the identification of a number of genetic risk factors for heart disease, such as gene variants involved in thrombosis
2,3
and lipid metabolism
4
. However, another important risk factor for heart disease such as myocardial infarction is a deregulated calcium homeostasis which is required for maintenance of neuromuscular activity
5
. Disturbances of the calcium balance have been implicated in hypertension, ventricular hypertrophy, aortic calcification and arrythmnias all of which are important risk factors associated with myocardial infarction.
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. The major physiological function of the active metabolite of vitamin D, i.e. 1,25(OH)
2
D
3
, is to maintain serum calcium levels in the normal range. This is accomplished by stimulating the small intestine to increase it's efficiency of absorbing calcium from the diet and to mobilize calcium stores from bone. The action of the hormone is mediated by the vitamin D receptor (VDR), a steroid transcription factor that mediates hormone response by initiating gene transcription and mRNA translation
6
. Involvement of the vitamin D endocrine system in the etiology of heart disease was suggested by epidemiological studies which found serum levels of 25-hydroxyvitamin D
3
to be reduced in myocardial infarcton patients compared with control individuals
7
. Furthermore, congestive hean failure associated with vitamin D deficiency has been described in a three and a half month old child
8
. The involvement of the vitamin D endocrine system in cardiac arrythria was suggested by case reports involving the observation of atrial flutter in a foetus with X-linked vitamin D resistant rickets
9
and sick sinus syndrome that was cured by administration of vitamin D to a 77-year old caucasian woman
10
. Further studies in the rat have also demonstrated an important role of the vitamin D endocrine system in cardiovascular function, wherein vitamin D3-deficiency can lead to large increases in the contracile function of the heart
17-20
.
The above studies establish a link between the via D endocrine system and risk of heart disease in an individual. Further studies (Carling et al, JCE&M, 82 (6) 1772-1775 (1997); Carling et al, Nature Medicine, 1 (12) 13099-1310 (1995) and Carling et al, JCE&M 83 (7) 2255-2259 (1998) show an association between the b, a and T alleles of the vitamin D receptor gene and hyperparathyroidism. Hypertension is considered to be a feature of hyperparathyroidism (Boucher, B. J, of Nutr., 79 315-327 (1998). However, the genetic component of heart disease such as myocarlial infarction or cardiac arrythmia is poorly defined. As a result, methods of diagnosing risk of heart disease, or treating those suffering from heart disease, are restricted.
Thus, in a first aspect of the present invention, there is provided a method of determining susceptibility to heart disease in a subject, said method comprising analysing genetic material of a subject to determine which allele(s) of the vitamin D receptor is/are present.
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 with similar specificities), 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.
16
The present invention is based on the discovery of a genetic component of heart disease. It has now been shown that presence of the b, a or T alleles, and in particular the baT haplotype of the vitamin D receptor is/are associated with increased risk of heart disease. Thus, the presence of such alleles of the vitamin D receptor gene may be used to determine susceptibility to heart disease.
Preferably, the method of the first aspect of the present invention comprises the additional step of determining whether the alleles present are associated with risk of heart disease. This may be performed by comparing the alleles present in the genetic material of the subject with genotypes of the vitamin D receptor having known degrees of risk of heart disease. For example, a visual aid detailing alleles and their relative risk of heart disease may be used to determine whether the genotype of the subject is associated with a high or low risk of heart disease.
The first aspect of the present invention may also comprise the additional step of determining aspects of calcium metabolism, such as calcium levels, in a subject. Preferably, the daily calcium intake is measured. This feature of the first aspect is based on the observation that the correlation between vitamin D receptor genotype and heart disease may be dependent upon dietary calcium intake.
The method of the first aspect may be performed in vitro. Preferably, the method is performed on tissue or fluid removed from the body of the subject. Thus, the present invention relates to a non-invasive method, the results of which provide an indication of susceptibility to heart disease but do not lead to a diagnosis upon which an immediate medical decision regarding treatment has to be made.
The present invention may be performed on any subject for whom it is desirable to determine risk of heart disease. Preferably, the subject may be a mammal. Most preferably, the method of the first aspect is performed on humans, preferably male.
Heart disease may include atrial or ventricular hypertrophy, aortic calcification, arrythmina, hypertension, and most importantly, myocardial infarction.
In a preferred feature of the first aspect of the present invention, there is provided a method of determining susceptibility to heart disease, said method comprising analysing genetic material of a subject to determine which of the B/b, A/a or T/t alleles of the vitamin D receptor gene is/are present. The method may comprise determining whether one or more of the above alleles is present. The subject may further be classified as being heterozygous or homozygous for one or more alleles. Preferably, the method comprises the additional step of determining whether the allele(s) present is/are associated with risk of heart disease, wherein presence of the b, a or T alleles is associated with increased risk of heart disease. Homozygosity for the a, b or T allele may further increase the susceptibility to heart disease in a subject.
In a preferred feature of the first
Pols Huibert Adriaan Pieter
Uitterlinden Andreas Gerardus
Van Leeuwen Johannes Petrus Thomas Maria
Erasmus Universiteit Rotterdam
Johnson Kindness PLLC
Myers Carla J.
O'Connor Christensen
Sakelaris Sally
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