BACKGROUND OF THE INVENTION
The invention relates to a method for determining the prognosis of a patient with a neurological disease.
Neurological diseases include Alzheimer's disease (AD), Creutzfeldt-Jakob disease, Huntington's disease, Lewy body disease, Pick's disease, Parkinson's disease (PD), amyotrophic lateral sclerosis, multiple sclerosis (MS), neurofibromatosis, and diseases without a necessary genetic component such as brain injury, stroke and multi-infarct dementia (MID). Most of these diseases are typified by onset during the middle adult years and lead to rapid degeneration of specific subsets of neurons within the neural system, ultimately resulting in premature death. There are no known cures and few therapies that slow the progression of these diseases.
Parkinson's disease (PD) is a common neurodegenerative disorder which first appears in mid- to late-life. Familial and sporadic cases occur, although familial cases account for only 1-2 percent of the observed cases. The neurological changes which cause this disease are somewhat variable and not fully understood. The disorder generally develops asymmetrically with tremors in one hand or leg and progresses into symmetrical loss of voluntary movement. Eventually, the patient becomes incapacitated by rigidity and tremors. In the advanced stages the disease is frequently accompanied by dementia.
Diagnosis of both familial and sporadic cases of Parkinson's disease can only be made after the onset of the disease symptoms. Anticholinergic compounds, propranolol, primidone and levadopa are frequently administered to modify neural transmissions and thereby suppress the symptoms of the disease, though there is no known therapy which halts or slows the underlying progression.
Multiple Sclerosis (MS) is a neurodegenerative disease of the brain and spinal cord in which a breakdown occurs in the myelin sheathing of the nerve fibers. MS is currently incurable and treatments are few and usually result in only temporary improvements of the disease symptoms.
Stroke is the sudden death of a portion of the brain cells due to a lack of oxygen. A stroke occurs when blood flow to the brain is impaired resulting in abnormal brain function. Brain blood flow can be impaired by blockage or rupture of an artery to the brain.
In the United States, about 400,000 people a year will suffer from a stroke, and up to 40% of these strokes may be fatal. The cost of strokes is not just measured in the billions of dollars lost in work, hospitalization, and the care of survivors in nursing homes. The major cost of a stroke is the loss of independence that occurs in 30% of the survivors. What was a self-sustaining and enjoyable life style may lose most of it's quality after a stroke and family members can often find themselves in a new role as caregivers.
Other cerebral vascular diseases that present similar sequelae to stroke are multi-infarct dementia (MID), vascular dementia (VaD), and cardiovascular injury or accident. In addition, diseases such as AIDS can often have vascular dementia as a complication. As with the above diseases, there are no known cures for these diseases and most therapies only aid rehabilitation or lower the risk of having another vascular incident.
Apolipoprotein E (apoE) functions as a ligand in the process of receptor mediated internalization of lipid-rich lipoproteins. ApoE is probably also involved in reverse lipid transport. In the central nervous system (CNS), apoE plays a central role in the mobilization and redistribution of cholesterol and phospholipid during membrane remodeling associated with synaptic plasticity. The importance of apoE in the brain is further underscored by the absence of other key plasma apolipoproteins such as apoA1 and apoB in the brain.
The apoE gene on chromosome 19 has three common alleles (E2, E3, E4), which encode three major apoE isoforms. The frequency of the apoE4 allele has been shown to be markedly increased in sporadic Alzheimer's Disease (AD) and late onset familial Alzheimer's disease (AD). This gene dosage effect was observed in both sporadic and familial cases (i.e., as age of onset increases, E4 allele copy number decreases). Women, who are generally at a greater risk of developing Alzheimer's disease, show increased apoE4 allele frequency when compared to age matched men.
The cholinergic hypothesis of geriatric memory dysfunction has raised some fundamental questions regarding the heterogeneity of responses toward different cholinomimetics in AD. The absence of clear beneficial effects of choline and lecithin on geriatric patients with and without AD is still perplexing. Furthermore, multiple clinical studies using esterases inhibitors such as physostigmine and tacrine have shown that contrary to results found in young subjects, the optimal acute dose necessary to facilitate performance on memory tasks varied considerably among individual aged subjects.
Neurological diseases provide a unique series of complications for the clinicians, patients, and care givers; the diseases often progress rapidly and disrupt a vast number of major life functions. The progressive nature of these diseases makes the passage of time a crucial issue in the choice and administration of different treatment options. It would be desirable to know the severity of the prognosis for patients diagnosed with various neurological diseases.
SUMMARY OF THE INVENTION
We have discovered a method for determining the prognosis of patients with a non-AD neurological disease such as Parkinson's disease, Multiple Sclerosis, or stroke. Our prognostic methods provide a prognosis for the patient, including a prediction of the relative outcome of the patient in terms of rate of progression, severity of disease symptoms, and longevity. The prognostic methods allow clinicians, patients, and family members to make informed choices about therapeutic regimes. This method will also provide for more rapid and cost effective treatment by determining the relative appropriateness of various therapeutic and palliative choices. Even where drug therapy is inappropriate, the prognostic method will provide patients, and their family members, a more informed and realistic expectation of patient outcome including an insight into the most effective rehabilitation strategy, and a forecast of the patient's risk for future disease.
In the first aspect, the invention provides a method of determining the prognosis for a patient diagnosed with a non-AD neurological disease. The method includes: a) identifying a patient already diagnosed with a non-AD disease; b) determining the apoE genotype or phenotype of a patient; and c) converting the data obtained in step b) into a prognosis determination. The prognosis may include a prediction of drug efficacy, patient outcome, and patient risk for future disease events. In preferred embodiments, the method of the invention may further include the steps of determining the BChE genotype or phenotype of a patient, obtaining a patient profile, which may, preferably, include the patient's sex, age, and/or genotype (e.g., presenilin, apolipoprotein E, or BChE genotype).
In other preferred embodiments of the prognostic method, the patient is diagnosed with a disease selected from the group consisting of: Parkinson's disease (PD), multiple sclerosis (MS), and stroke which shall also include multi-infarct dementia (MID), vascular dementia (VaD), and cardiovascular injury or accident, for example, as a complication of AIDS.
In a second aspect, the invention provides a method for determining the prognosis of future risks of disease in a asymptomatic mammal. In preferred embodiments the mammal is a human and the method further involves a determination of the mammals BChE genotype or phenotype, obtaining a patient profile, which may, preferably, include the mammal's sex, age, and/or genotype (e.g., presenilin, apolipoprotein E, or BChE genotype).
In a related aspect, the invention provides a kit for performing the prognosis. The kit includes a means for converting the patient profile into a prognosis. In a preferred embodiment, the kit contains a means for performing the steps of the conversion. In another preferred embodiment, the kit contains a means for compiling the data for said patient profile and for formatting said patient profile. In another preferred embodiment, the kit contains a computer software program to perform the data analysis.
It should be noted that the therapies suggested by the prognostic method may be used alone, or in combination with other known therapies that are not otherwise contraindicated for the patient.
For the purpose of the present invention the following terms are defined below.
“Non-AD Neurological disease” means any disease other than Alzheimer's disease, which involves the neuronal cells of the nervous system. Specifically included are: prion diseases (e.g., Creutzfeldt-Jakob disease); pathologies of the developing brain (e.g., congenital defects in amino acid metabolism, such as argininosuccinicaciduria, cystathioninuria, histidinemia, homocystinuria, hyperammonemia, phenylketonuria, and tyrosinemia, and fragile X syndrome); pathologies of the mature brain (e.g., neurofibromatosis, Huntington's disease, depression, amyotrophic lateral sclerosis, multiple sclerosis, and stroke); conditions that strike in adulthood (e.g. Creutzfeldt-Jakob disease, Huntington's disease, Lewy body disease, Parkinson's disease, Pick's disease, amyotrophic lateral sclerosis, multiple sclerosis, neurofibromatosis), brain injury, stroke, multi-infarct dementia (MID), vascular dementia (VaD), pathologies of the brain (e.g., brain mishaps, brain injury, coma, infections by various agents, and dietary deficiencies) and, cardiovascular injury or accident, for example, as a complication of AIDS.
“Drug efficacy” means the a determination of an appropriate drug, drug dosage, administration schedule, and prediction of therapeutic utility.
“Already diagnosed” means already diagnosed as having a neurological disease or having a genetic predisposition or risk for acquiring a neurological disease.
“Patient profile” means data pertaining to the patient for whom the prognostic analysis is being performed. Data may include information on the patient's diagnosis, age, sex, and genotype. The patient's profile may also include materials from the patient such as blood, serum protein samples, cerebrospinal fluid, or purified RNA or DNA.
“Asymptomatic” means that the mammal or human subject has no clinical symptoms of a disease but nonetheless may be a “silent” carrier of a genotype determined by the method of the invention to result later or be likely to result later in the onset of disease symptoms.
“ApoE genotyping” means determination of the type and number of apoE alleles present in the patient, whether determined by nucleic acid sequencing, PCR or RT-PCR amplification, examination of apoE protein, or by other methods available to those skilled in the art.
“Allele load” means the relative ratio of apoE2, 3, and 4 alleles in the patient's chromosomal DNA. The allele load may be determined by comparing the relative numbers of the patient's already known apoE allele types.
“PCR or RT-PCR amplification” means subjecting a DNA sample to a Polymerase Chain Reaction step or an RN-A sample to a Reverse Transcriptase-Polymerase Chain Reaction step, such that, in the presence of appropriately designed primers, a DNA fragment is synthesized or fails to be synthesized and thereby reveals the allele status of a patient.
“BChE genotype” means a determination of the patient's alleles that encode the butyrylcholinesterase gene product. This may be accomplished by nucleic acid sequencing, RT-PCR, PCR, examination of the BChE protein, a determination of the BChE enzyme activity, or by other methods available to those skilled in the art.
“BCHE-K allele” means the polymorphism of the butyrylcholinesterase (BCHE) gene which has a point mutation at nucleotide 1615 that changes amino acid residue 539 from alanine to threonine and can result in an enzyme with reduced catalytic activity. Other polymorphisms of this locus exist (e.g., deletions (BCHBE*FS4), missense mutations (BCHE*24 M, * 1005, *250P, *267R, *330I, *365R, *418S, *515C, *539T), and nonsense mutations (BCHE*119STOP, *465STOP)) and are included within the scope of the invention.
“Prognosis” means a method whereby diagnostic data, including the patient's neurological diagnosis and genetic data, such as the patient's apoE and BChE genotype, are processed to provide therapeutic options and prognoses. Processing may include, but not be limited to, the collection of a patients medical history including age and sex, apoE and BChE genotyping using appropriately designed primers and using a RT-PCR or PCR amplification step, apoE and BChE phenotyping using an antibody-mediated method or enzymatic test, and statistical analysis step that converts this raw data into a prognosis. The prognosis may include a prediction of a patient's response to drug therapy, recovery time, age of disease onset, treatment efficacy, rehabilitation time, symptomology of attacks, and risk for future disease. For example, a high apoE4 allele load could be used as a positive predictor for stroke patients that respond well under drug therapy and as negative predictor of PD and MS patient response to drug therapy. A prognosis may also be determined for asymptomatic and healthy subjects in order to forecast future disease risks an determine what preventive therapies should be considered or invoked in order to lessen these disease risks. The prognosis may include the use of a computer software program to analyze patient data and run statistical cross-checks against relational databases that are constantly being updated.
DETAILED DESCRIPTION OF THE INVENTION
Here we show that a correlation of age, sex, apoE genotype, and BChE genotype, may be used to formulate a prognosis for a given patient with a neurological disease. The prognosis can include a prediction of both relative age of onset, rate of disease progression, and risk for future disease.
We have investigated the relationship between the apoE4 genotype and cholinergic deficits, and we observed that the greater the number of apoE4 alleles the lower the apoE level. Furthermore, reduction in ChAT activity in the hippocampus and temporal cortex of AD cases is inversely proportional to the apoE4 allele copy number (i.e. where the apoE4 allele copy number is increased the ChAT activity is decreased). In addition, we found that another presynaptic marker of cholinergic projection, the nicotinic receptor, was markedly reduced in apoE4 AD subjects. Conversely, we have found that a typical post-synaptic marker, M1-muscarinic receptor, is unaltered in AD versus non-AD subjects, irrespective of whether apoE4 is present or not. The M2-muscarinic receptor, a composite pre- and post-synaptic marker, is also unaffected by the apoE4 allele gene dosage. We have also observed that the presence of the apoE4 allele lowers the age of onset of neurological disease and worsens the prognosis.
The above findings clearly indicate the existence of distinct genetic entities in neurological disease which correlate with differential degrees of alterations of cholinergic innervation. In turn, the innervation level correlates with the prognosis, including the ability to respond to cholinomimetic drugs.
We believe the correlation between apoE4 allele load and reductions in ChAT activity and nicotinic receptors may be explained by at least two distinct phenomena. First, phospholipids such phosphatidylcholine (PC) and phosphatidylethanolamine (PE), that can serve precursors to choline in the synthesis of acetylcholine (Ach), could be transported into neurons via the classical apoE-LDL receptor pathway. An isoform-dependent impaired regulation of the transport of phospholipids in the brain of apoE4 carriers could explain the reduced levels of PC, PE and choline reported in AD (Pettegrew J. W., 1989, Ann. NY Acad. Sci., 568:5-28; Nitch R M et al., 1992, Proc. Natl. Acad. Sci., 89:1671-1675). This, in turn, may lead to decreased Ach synthetic capacities. This hypothesis is consistent with membrane defects reported in AD subjects such as changes in membrane fluidity in the hippocampus and in the platelets of AD patients. The loss of cholesterol reported in AD and the effect of apoE4 on nicotinic binding activity are consistent with the apoE4/impaired lipid homeostasis hypothesis.
In addition to the above, the reduction in neuronal ChAT activities and choline levels in both AD and non-AD patients could parallel the loss of cholinergic neurons. The analysis of the number of acetylcholinesterase-positive neurons in the nucleus basalis of Meynert (NBM) and the diagonal band of Broca (DBB) in AD patients revealed marked losses of cholinergic neurons in apoE4 carriers versus apoE3 homozygous AD cases.
Although these observations were initially made in AD patients, we have discovered that our observations regarding apoE allele load and drug therapies can be generalized to non-AD neurological diseases because the underlying mechanism altered by the apoE allele load is not AD-specific. Our discovery indicates that the apoE4 allele load, taken together with patient profile parameters, can predict individual variations in a patients response to drug treatment, rehabilitation, and risk for future disease. The method of the invention provides useful predictions for patients with diseases such as stroke, Parkinson's disease, and Multiple Sclerosis as discussed in the Examples herein. Prospective-retrospective analyses of patients that are either good or poor responders to drugs designed to ameliorate the conditions of stroke, Parkinson's disease, and Multiple Sclerosis, (e.g., aspirin, antithrombotics, ticlopidine (Ticlid™), levodopa-carbidopa, (Sinernet™) and interferon β-1B (Betaseron™)) are presented in the Examples. We further propose that the method of the invention also can be used to generate prognostic protocols for other cerebral vascular injuries that involve similar disease mechanisms to stroke. Such diseases would include multi-infarct dementia (MID), cardiovascular injury, brain injury, or cerebral vascular pathologies as a complication AIDS.
The prognostics method is useful for allowing the physician to select the most appropriate drug, drug dosage, administration or palliative therapy for a patient. The invention also provides a method for forecasting future patient disease risk. This forecast method allows the patient and clinician to consider and invoke preventive therapeutic regimens before disease strikes or to at least lower the risk of future disease events.
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.