DNA assay for the prediction of autoimmune diabetes

Details DNA assay for the prediction of autoimmune diabetes DNA assay for the prediction of autoimmune diabetes

2001-07-23

2003-03-18

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C530S350000, C536S022100

Reexamination Certificate

active

06534272

ABSTRACT:

BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to a novel DNA assay for the diagnosis and/or prediction of autoimmune diabetes.
(b) Description of Prior Art
Diabetes is a major cause of morbidity and mortality in industrialized societies. It has been estimated that one of every seven health-care dollars goes to treating diabetes and its complications. Type 1 diabetes (also called insulin-dependent or juvenile diabetes, henceforth referred to in this document as “diabetes”) is due to the autoimmune destruction of the insulin-producing pancreatic &bgr;-cells. Type 1 diabetes is less common than type 2, accounting for only 10-20% of cases in Caucasians. However, because it is much more severe and starts much earlier in life, it accounts for a large proportion of diabetes-related morbidity and mortality.
Type 1 diabetes involves autoimmune destruction of the insulin-producing pancreatic &bgr;-cells. Insulin, an autoantigen in this process, is expressed in human thymus at levels dependent on alleles at the upstream INS VNTR, to which the IDDM2 susceptibility locus has been mapped. Chromosomes carrying the dominantly protective (Bennett S T et al., 1995
, Nat. Genet
. 9(3):284-292), long INS VNTR alleles (class III) produce 2-3 times higher levels of insulin gene (INS) mRNA than those with predisposing, short class I alleles (Vafiadis P et al., 1997, Nat Genet. 15(3):289-292; Pugliese A et al., 1997, Nat. Genet. 15(3):293-297).
Prevention of Diabetes
It is estimated that by the time symptoms of diabetes appear, more than 95% of the &bgr;-cell mass has been destroyed. Given the irreversibility of this destruction, the most promising approach to the disease is prevention. This will require an intervention, at some time before symptoms appear, aimed at modulating the immune system to prevent the antigen-specific autoimmune reaction. Although the specific causative autoantigen(s) in diabetes is (are) not known, insulin, the main product of the &bgr;-cell, appears to be an autoantigen of major importance.
Based on this, the DPT (diabetes prevention trial) a large study, is now underway in the United States. Oral insulin administration is used in the hope of helping individuals at risk for diabetes acquire immune tolerance to insulin. The results of this study will not be known for a number of years. It is possible that it will be superseded by other studies, which will be based on more precise scientific rationale provided by current spectacular advances in immunology. There is a tangible likelihood that in the coming decade a safe and effective method of preventing or reversing the diabetes autoimmune process will have been found.
Identification of Individuals at Risk
An effective intervention to prevent diabetes is very unlikely to be inexpensive, safe and convenient enough to be applied to the general population (i.e. in the fashion of infectious disease vaccines). The intervention will most likely need to be targeted to individuals that can be identified as being at a substantial risk for diabetes.
Autoantibody Testing
The DPT and similar trials have focused on first-degree relatives of diabetics that are positive for autoantibodies known to predict diabetes. These autoantibodies against protein components of the &bgr;-cell become positive at least a year or two before the onset of clinical diabetes. However, even if diabetes is prevented with 100% efficacy in all first-degree relatives of diabetics, this will only abolish less than 10% of new cases of diabetes, as the majority of individuals with diabetes do not have a previously affected first-degree relative. To be meaningful, an effective prevention will have to be applicable to the general population. Screening the general population for antibodies has been shown to be feasible in practice, and predictive of diabetes. However, since antibodies only become positive at a finite time point before the onset of diabetes, most people destined to become diabetic at some future point will be negative when tested early in life. Therefore, antibody screening of the general population will need to be repeated at intervals of 1-2 years, a totally impractical proposition.
Genetic Testing
An alternative way of predicting high risk for diabetes is through DNA testing. It is known that genetic predisposition plays a major role in diabetes. Identical twins of individuals with diabetes have a risk that ranges from 30 to >60% in various studies, as opposed to fraternal twins, that are concordant only in 5-10% of the cases (Bennett S T et al., 1996
, Ann. Rev. Genet
. 30:343-370). The predisposition is not inherited in a Mendelian fashion, which means that this complex phenotype requires predisposing genetic material involving more than a single gene. Linkage studies have identified as many as 18 different genetic loci that are potentially linked to diabetes (in genetics, the term “locus” is used instead of “gene”, when only a location in the genome is known, but the gene[s] involved remain[s] to be identified). Although many of these loci will probably turn out to be statistical artifacts, it is clear that in order to define the “diabetes genotype”, markers at more than one loci will have to be typed. A good definition of the “diabetes genotype” would be: a combination of alleles that correctly predicts diabetes with a probability that approaches that predicted from being the identical twin of a diabetic. Obviously, a risk of at least 30% would justify preventive intervention.
So far only two of the several loci have been defined precisely enough to be used to this end. They are termed IDDM1 and IDDM2, and can be used already to obtain a certain degree of risk estimation (Bennett S T et al., 1996
, Ann. Rev. Genet
. 30:343-370). Using similar approaches several more loci can be likewise defined, that together predict diabetes with specificity that approaches that of the idealized diabetes genotype. Specificity in a diagnostic test is the percentage of positive tests that turn out to be true. The twin studies show that 30-60% is the best specificity that can be achieved with DNA testing, but even a specificity of 10% or less can be very useful depending on how safe, simple and inexpensive future preventive interventions turn out to be.
Once defined, the loci could be genotyped on a few drops of blood obtained at birth as part of neonatal screening programs currently in place for other diseases. Those individuals who exceed a certain threshold of risk can then be followed with antibody testing or treated prior to the appearance of autoantibodies, depending on what the optimal strategy will be determined to be.
Conventional Testing at the IDDM2 locus
It involves determination of the VNTR by Southern blotting, a cumbersome technique that is not easily amenable to miniaturization and automation (Bennett S T et al., 1995
, Nat. Genet
. 9(3):284-292). Polymerase chain reaction (PCR) has been used to identify class I alleles, but there is no published report of successful amplification of class III alleles.
More importantly, the conventional Southern blotting approach only allows classification of individuals as having no class III allele (genotype: I/I) or having at least one class III allele (genotype I/III or III/III). Individuals in the latter category will be assigned a risk that is approximately 4-fold less (Bennett S T et al., 1996
, Ann. Rev. Genet
. 30:343-370), and this estimate will be entered in a formula along with information from other IDDM loci, in order to calculate risk. The risk assignment can be erroneous in individuals carrying the specific alleles S1 or S2.
Given the foregoing, it would be desirable to develop a novel DNA assay for the diagnosis and/or prediction of autoimmune diabetes which overcomes the drawbacks of the prior art. More particularly, an assay by which alleles predisposing for diabetes could be distinguished from protective alleles would be desirable.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide a PCR-based method to distinguish not only

Affiliated with

Also associated with

Rating

DNA assay for the prediction of autoimmune diabetes does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with DNA assay for the prediction of autoimmune diabetes, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and DNA assay for the prediction of autoimmune diabetes will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3069230