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  Methods for detecting neurological disordersUSPTO Application #: 20060040315Title: Methods for detecting neurological disorders Abstract: In one aspect, the present invention features methods for detecting at least one neurological disorder in a patient, the method comprising obtaining a biological sample from the patient; and detecting at least one aberrant human glutamate transporter 2 (EAAT 2) mRNA in the sample as being indicative of the neurological disorder in the patient. In a particular aspect, the invention is useful for detecting amyotrophic lateral sclerosis (ALS) in the patient. (end of abstract) Agent: Drinker Biddle & Reath Attn: Intellectual Property Group - Philadelphia, PA, US Inventors: Jeffrey D. Rothstein, Chieng-Liang Glenn Lin, Lynn A. Bristol USPTO Applicaton #: 20060040315 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20060040315. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0001] 1. Field of the Invention [0002] The present invention features methods for detecting at least one specified neurological disorder in a subject. In one aspect, the invention relates to novel polynucleotides for detecting the neurological disorder. In a related aspect, the invention provides methods for identifying, analyzing, and using the polynucleotides. Further provided are screening methods for detecting therapeutic compounds with capacity to treat the neurological disorder. The present invention has a variety of uses including detecting a specified motor neuron disorder in a patient. [0003] 2. Background [0004] Neurological disorders can significantly impact the central nervous system (CNS) and motor neuron units. For example, certain neurological disorders of the CNS are known to adversely affect the brain and associated structures. Neurological disorders affecting motor neuron units have been grouped into motor neuron diseases and peripheral neuropathies. See generally Kandel, E. R. et al; (1991) in Principles of Neuroscience, Appleton & Lange, Norwalk, Conn.; and Rowland, L. P. (ed.) (1982) in Human Motor Neuron Diseases. New York. Raven Press. [0005] An illustrative motor neuron disease is amyotrophic lateral sclerosis (ALS). ALS has been reported to be a chronic neuromuscular disorder having recognized clinical manifestations. For example, it has been suggested that degeneration of cortical and spinal/bulbar motor neurons may play a key role in the disorder. ALS is nearly always fatal. About 95% of all ALS cases are sporadic, with many of the remaining cases showing autosomal dominant inheritance. See e.g., Kunc R. W. et al., (1992) Motor Neuron Diseases In Diseases of the Nervous System, Asbury et al. eds. (Philadelphia W. B. Saunders) pp. 1179-1208; Brown, R. H., (1996) Amer. Neurol. 30:145; Siddique, T. and Deng., H. X. (1996) Hum. Mol. Genetics 5:1465). [0006] Specific CNS disorders have been also described. In particular, some have been attributed to cholinergic, dopaminergic, adrenergic, serotonergic deficiencies or combinations thereof. CNS disorders of severe impact include pre-senile dementia (sometimes referred to as Alzheimer's disease (AD) or early-onset Alzheimer's disease), senile dementia (dementia of the Alzheimer's type), Parkinson's disease (PD), and Huntington's disease (HD, sometimes referenced as Huntington's chorea). Such CNS disorders are well-represented in the human population. See generally; Gusella, J. F. et al. (1983) Nature 306: 234; Borlauer. W. and Jprmuloewoca. P. (eds.) (1976); Adv. in Parkinsonism: Biochemistry, Physiology, Treatment. Fifth International Symposium on Parkinson's Disease (Vienna) Basel: Roche; and references cited therein. [0007] Significant attention has been directed towards understanding the etiology of motor neuron diseases. For example, abnormal levels of certain excitotoxic neurotransmitters have been reported to adversely contribute to many motor neuron diseases. In particular, glutamate-mediated excitotoxicity is recognized to have a critical role in ALS. See e.g., Rothstein J. D. et al., (1990) Ann. Neurol. 28: 18. ; Rothstein J. D. et al. (1992) N. Engl. Med. 326: 1464; Rothstein J. D. et al. (1993) PNAS (USA) 90: 6591; and Lacomblez, L. et al., (1996) Lancet 347: 1179. [0008] There has been substantial efforts towards understanding mechanisms for reducing glutamate levels in the nervous system. For example, high-affinity, sodium-dependent glutamate transport is one reported means of inactivating glutamate. In particular, astrocytic excitatory amino acid transporter 2 (EAAT 2) proteins are believed to have substantial functions in that inactivation. See e.g., Rothstein J. D. et al. (1994) Neuron 28: 18; Rothstein J. D. et al., (1995) Ann. Neurol. 38: 78. and references cited therein. [0009] In particular, investigations have suggested that EAAT 2 is a predominant glutamate transporter. More particularly, certain antisense knockdown studies have been reported to demonstrate that EAAT 2 loss can lead to excitotoxic neuronal degeneration and progressive motor impairment. Studies of ALS and other neurodegenerative disorders have related impaired glutamate transport to loss of the EAAT 2 protein. In particular, up to 60% to 70% of the sporadic ALS patients examined have a 30% to 95% loss of the EAAT 2 protein. See e.g., Haugeto et al., supra; Rothstein J. D., et al., (1996) Neuron 16: 675; Bristol, L. A. and Rothstein, J. D. (1996) Ann. Neurol. 39: 676. [0010] There have been attempts to treat or prevent neurological disorders of the CNS and the motor neuron units. However, most existing therapies do not always stem the development or severity of the disorders in afflicted patients. See e.g., Rowell, (1987) Adv. Behav. Biol. 31: 191; Rinne, et al. Brain Res. (1991) 54: 167; U.S. Pat. No. 5,210,076 to Berliner; Yurek, D. M. (1990) Ann. Rev. Neurosci. 13: 415, and Rowland et al. supra. [0011] Substantial research effort has focussed on developing effective methods for detecting neurological disorders in patients. However, many existing methods are not always effective or reliable. For example, some methods are optimized to analyze post-mortem samples. Such methods provide little benefit for the afflicted patient. Other methods rely on testing living patients for specific cognitive or motor skills. However, such tests can be difficult to perform or interpret in some settings. [0012] Accordingly, there is a need in the field for effective and reliable methods for detecting neurological disorders in a living patient. There is general recognition that such methods would positively impact many existing therapies. It would be particularly desirable to have methods for detecting specific neurological disorders in a living patient before disease onset or at an early stage of disease progression. SUMMARY OF THE INVENTION [0013] The present invention features methods for detecting at least one specified neurological disorder in a subject. In one aspect, the methods include obtaining a biological sample from the subject and detecting at least one type of aberrant human glutamate transporter 2 mRNA in the sample. Presence of the aberrant mRNA is indicative of the neurological disorder in the subject. The invention also relates to novel polynucleotides that can be used to detect the neurological disorder. Further provided are methods for isolating a variety of aberrant human glutamate transporter 2 polynucleotides. The invention also provides screening methods for detecting compounds useful in the diagnosis or treatment of specified neurological disorders. The present invention has a variety of uses including monitoring efficacy of a therapy for treating the neurological disorder. [0014] In general, we have discovered aberrant human glutamate transporter 2 mRNAs in patients suffering from or suspected of suffering from a specific neurological disorder. It was found that incidence of the aberrant mRNAs substantially increased in affected nervous system regions. For example, incidence of the aberrant mRNAs could be detected in patients afflicted with a specific motor neuron disease. Significantly, the present invention provides sensitive and reliable methods for detecting specific neurological disorders in living patients with minimal impact to the nervous system. [0015] The term "human glutamate transporter 2" is sometimes abbreviated herein as "EAAT 2". The term "EAAT 2" will be particularly used to refer to the human astroglial glutamate transporter 2 gene as well as normal or aberrant polynucleotides derived from that gene. [0016] As will be discussed more fully below, aberrant EAAT 2 polynucleotides of this invention are novel molecular markers that can be used to detect specific neurological disorders. In particular, aberrant EAAT 2 mRNAs of this invention are intron-retention or exon-skipping variants of normal EAAT 2 mRNA. The aberrant EAAT 2 mRNAs were found in a majority of patients that were known to have or were suspected of having a specified neurological disorder. However, the aberrant EAAT 2 mRNAs were not found in control samples obtained from apparently healthy and non-affected donors. Accordingly, detection of at least one type of aberrant EAAT 2 mRNA in the patient is taken to be indicative of the neurological disorder in that patient. [0017] The neurological disorders that can be detected in accord with the present invention include specific disorders that have been reported to be associated with excitotoxicity. Particularly included are specified neurological disorders affecting motor neuron function. Specifically included are neurological disorders impacting the CNS or motor neuron units such as amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Parkinson's disease (PD), and Alzheimer's disease (AD). As will be pointed out below, in selected disorders, at least one type of aberrant EAAT 2 mRNA has been detected in a majority of patients that have or were suspected of having the disorder. As noted, those aberrant mRNAs were not detected in apparently healthy and non-affected donors. [0018] The present invention has a number of important advantages. For example, the invention can be used to detect a specified neurological disorder in a living patient with minimal impact to the nervous system. For example, one embodiment of the present invention features a method for detecting at least one type of aberrant EAAT 2 mRNA in a replaceable nervous system fluid that can be readily obtained from the patient. In this embodiment, presence of at least one type of aberrant EAAT 2 mRNA in the fluid is indicative of the neurological disorder in that patient. [0019] In some instances, the patient to be tested may not yet manifest overt signs of any neurological disorder. In this instance, the methods of the invention can serve as an indicator of predisposition for or susceptibility to the disorder. In contrast, many prior methods for detecting neurological disorders rely on difficult cognitive or motor tests. Other more definitive tests are typically formatted to characterize post-mortem nervous system tissue. Unlike these prior methods, the present invention provides reliable and sensitive methods for detecting neurological disorders while the patient is living. Significantly, opportunities for early medical intervention are increased by practice of the invention. [0020] In one aspect, the present invention provides an assay-method for detecting at least one specified neurological disorder in a patient who has or is suspected of having that neurological disorder. The method generally involves obtaining a biological sample from the patient and detecting at least one type of aberrant EAAT 2 mRNA in the sample as indicative of the neurological disorder. In one embodiment of the method, detection of the aberrant EAAT 2 mRNA is achieved by amplifying the sample in a polymerase chain reaction (PCR) or suitable related method. Typically, the amplification method selected will be sufficient to make complementary polynucleotides, typically complementary DNA (cDNA), from the sample. That is, the PCR or related method is used to make nucleic acid copies of the mRNA which copies are usually cDNA copies but can be at least partially RNA copies (cRNA) in some instances. A preferred amplification method is a reverse transcriptase-PCR (RT-PCR) method using at least two oligonucleotide primers. In a particular embodiment, amplified cDNA made from aberrant EAAT 2 mRNA (if present in the sample) is sequenced and the resulting DNA sequence is compared to the sequence of the normal EAAT 2 cDNA (FIGS. 1A-C and SEQ ID NO: 1). The comparison allows determination of the aberrant EAAT 2 mRNA structure in most instances. In many cases, a suitable control sample is included to provide suitable co-amplification of normal EAAT 2 mRNA. Preferred control samples generally provide a baseline for any basal expression of the normal EAAT 2 mRNA. In another particular embodiment, DNA sequence from the cDNA is substantially homologous or identical to specific aberrant EAAT 2 cDNA sequences disclosed below. [0021] In another aspect of the present invention, assay methods are provided for detecting the neurological disorder in the patient in which aberrant EAAT 2 mRNA is detected by making a polynucleotide library from the biological sample. In one embodiment, the polynucleotide library is a cDNA library and the methods include detecting in the library at least one nucleic acid that includes a sequence substantially homologous or identical to specified aberrant EAAT 2 cDNA sequences disclosed below. [0022] Further provided by the present invention are methods for detecting at least one specified neurological disorder in the patient in which the methods involve obtaining a first biological sample from a suitable control donor and a second biological sample from the patient, amplifying nucleic acid in the samples independently, and detecting in the second sample any amplified nucleic acid as indicative of the neurological disorder in the patient. In one embodiment of the method, mRNA in the first and second samples is independently amplified under conditions capable of producing cDNA from at least one type of aberrant EAAT 2 mRNA (if present) in the sample. In this embodiment, the aberrant mRNA includes intron sequence from the EAAT 2 gene. The method is especially useful for detecting specific aberrant EAAT 2 mRNAs which retain at least one intron or intron fragment from the EAAT 2 gene. Presence of a suitable amplification product in the second sample, when compared to the first sample (control), is an indication of the neurological disorder in the patient. Continue reading... Full patent description for Methods for detecting neurological disorders Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods for detecting neurological disorders patent application. Patent Applications in related categories: 20080113379 - Method for the detection of cytosine methylations in immobilized dna samples - A method is described for the analysis of cytosine methylation patterns in genomic DNA samples. In the first method step, the genomic DNA is isolated from cells or other accompanying materials and bound essentially irreversibly to a surface. 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