Patent Description:
<CIT> discloses a method for diagnosing Alzheimer's Disease using PKC-elicited gene expression profiles.

This invention provides a method for determining whether a human subject is afflicted with AD or non-ADD when the subject is suspected of being afflicted with AD or non-ADD, comprising the steps of.

whereby (i) the subject is afflicted with AD if the expression level measured in step (b) is consistent with that gene's expression level in corresponding synchronized cells derived from AD patients, and (ii) the subject is afflicted with non-ADD if the expression level measured in step (b) is consistent with that gene's expression level in corresponding synchronized cells derived from non-ADD patients.

This invention also provides a method for determining whether a human subject is afflicted with AD or non-ADD when the subject is suspected of being afflicted with AD or non-ADD, comprising the steps of.

whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with the genes' expression levels in corresponding synchronized cells derived from AD patients, and (ii) the subject is afflicted with non-ADD if the expression levels measured in step (b) are consistent with the genes' expression levels in corresponding synchronized cells derived from non-ADD patients.

This invention further provides a method for determining whether a human subject is afflicted with AD or non-ADD when the subject is suspected of being afflicted with AD or non-ADD, comprising the steps of.

In this application, certain terms are used which shall have the meanings set forth as follows.

As used herein, "administer", with respect to an agent, means to deliver the agent to a subject's body via any known method. Specific modes of administration include, without limitation, intravenous, oral, sublingual, transdermal, subcutaneous, intraperitoneal and intrathecal administration.

In addition, the various agents can be formulated using one or more routinely used pharmaceutically acceptable carriers. Such carriers are well known to those skilled in the art. For example, oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc). Injectable drug delivery systems include, for example, solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA's). Implantable systems include rods and discs and can contain excipients such as PLGA and polycaprylactone.

As used herein, "Alzheimer's disease" means a concurrent affliction with the following three symptoms: (i) dementia; (ii) amyloid plaques; and (iii) neurofibrillary tangles. Dementia can be diagnosed during life. Cerebral amyloid plaques and neurofibrillary tangles can, for example, be diagnosed during autopsy. This definition of Alzheimer's disease is the one provided by the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH), and is known as the "gold standard. " All subjects from whom samples were taken and studied, and for which data are presented herein, are autopsy-confirmed AD and non-ADD patients.

As used herein, a gene's expression level is "consistent" with that gene's expression level in corresponding synchronized cells derived from AD patients if it is the same as, or close to, that expression level. For example, assume that gene X's TPM measure in synchronized cells derived from AD patients is <NUM> and its TPM measure is <NUM> in the same type of cells derived from non-ADD patients that are synchronized in the same way. A subject's gene X expression level would be consistent with gene X's AD expression level if it were, for example, below <NUM>, below <NUM>, below <NUM>, below <NUM> or, ideally, <NUM> or lower.

As used herein, "culturing" lymphocytes is achieved, for example, by conducting the culturing at a temperature and in a growth factor milieu permissive of cell growth. In another embodiment, "culturing" lymphocytes is performed under conditions (e.g., those described herein for proliferation) that preserve lymphocyte viability. In one embodiment, the temperature, salinity and protein milieu permissive of cell growth is <NUM>, RPMI <NUM> Medium with <NUM>% fetal bovine serum ("FBS") and <NUM>% penicillin ("PS"). In one embodiment of this invention, the lymphocyte-culturing step is performed for more than three hours. Preferably, the lymphocyte-culturing step is performed for more than six hours (e.g., overnight). B-lymphocyte can be cultured to over-confluence, i.e., high density/µl. The high density is determined as the plateau that is typically more then <NUM>% in the growth curve. Then, the lymphocytes are starved overnight.

Methods for obtaining lymphocytes from a subject's blood are known, and include, for example, flow cytometry, Ficoll (a hydrophilic polysaccharide that separates layers of blood), and gradient centrifugation. Additionally, in the subject methods, the lymphocytes (e.g., B lymphocytes) can be used in immortalized or primary (i.e., non-immortalized) form. Methods for immortalizing lymphocytes (e.g., B lymphocytes) are known, and include, for example, treating the lymphocytes with Epstein-Barr virus ("EBV").

As used herein, "culturing" skin fibroblasts is achieved, for example, by conducting the culturing at a temperature and in a growth factor milieu permissive of cell growth. In another embodiment, "culturing" skin fibroblasts is performed under conditions (e.g., those described below for proliferation) that preserve skin fibroblasts viability. In one embodiment, the temperature, humidity and protein milieu permissive of cell growth is <NUM>, DMEM Medium with <NUM>% fetal bovine serum ("FBS") and <NUM>% penicillin ("PS"). In one embodiment of this invention, the skin fibroblast-culturing step is performed for more than three hours. Preferably, the skin fibroblast-culturing step is performed for more than six hours (e.g., overnight).

Methods for obtaining skin fibroblasts from a subject's blood are known, and include, for example, skin punch biopsy, and growing cells out of explants. When cell confluence reaches <NUM>%, cells are passaged. Typically after two passages, fibroblasts are purified in a proportion greater than <NUM>%.

As used herein, cells "derived" from a subject are cells that arise through culturing and/or other physical manipulation performed on cells directly removed from the subject. For example, cultured skin fibroblasts derived from a subject are those skin fibroblasts that arise through culturing a sample of skin cells (e.g., contained in a punch biopsy) directly removed from the subject.

As used herein, "diagnosing Alzheimer's disease", with respect to a symptomatic human subject, means determining that there is greater than <NUM>% likelihood that the subject is afflicted with Alzheimer's disease. Preferably, "diagnosing Alzheimer's disease" means determining that there is greater than <NUM>%, <NUM>%, <NUM>% or <NUM>% likelihood that the subject is afflicted with Alzheimer's disease. As used herein, the phrase "determining whether the subject is afflicted with Alzheimer's disease" is synonymous with the phrase "diagnosing Alzheimer's disease.

As used herein, "diagnosing non-ADD", with respect to a symptomatic human subject, means determining that there is greater than <NUM>% likelihood that the subject is afflicted with non-ADD. Preferably, "diagnosing non-ADD" means determining that there is greater than <NUM>%, <NUM>%, <NUM>% or <NUM>% likelihood that the subject is afflicted with non-ADD. As used herein, the phrase "determining whether the subject is afflicted with non-ADD" is synonymous with the phrase "diagnosing non-ADD.

As used herein, a gene is "differentially expressed between corresponding synchronized cells derived from AD patients and those derived from non-ADD patients" if, for example, the gene's TPM measure in synchronized cells derived from AD patients is different than in the same type of cells derived from non-ADD patients that are synchronized in the same way. For example, gene X would be differentially expressed between corresponding synchronized cells derived from AD patients and those derived from non-ADD patients if its TPM measure in synchronized cells derived from AD patients were <NUM> and its TPM measure were <NUM> in the same type of cells derived from non-ADD patients that are synchronized in the same way.

As used herein, an agent "favorably" affects the expression level of a gene whose expression level correlates with AD if it either decreases or increases that expression toward a level correlative with a non-AD state. For example, if the expression level of gene X is lower in an AD patient than in a non-afflicted patient, an agent favorably affecting the expression level of that gene would increase its expression level. Similarly, if the expression level of gene X is higher in an AD patient than in a non-afflicted patient, an agent favorably affecting the expression level of that gene would decrease its expression level.

As used herein, "measuring" the expression level of a gene means quantitatively determining the expression level via any means for doing so (e.g., Total RNA Sequencing, (<NUM> million reads, 2x75bp PE)). Preferably, measuring the expression level of a gene is accomplished by measuring the number of RNA transcripts for that gene per million total RNA transcripts (i.e., "TPM" via FastQ data, and FPKM estimation per sample) present in the cell-derived RNA population being studied. For example, measuring the expression level of gene X in a synchronized cell population might yield a result of <NUM> TPM.

As used herein, a subject afflicted with "non-Alzheimer's dementia" means a subject showing dementia such as, for example, that which characterizes Parkinson's disease, Huntington's disease and frontotemporal dementia.

As used herein, a "population" of cells includes any number of cells permitting the manipulation and study required to assess gene expression. In one embodiment, the population of cells includes at least <NUM>,<NUM>,<NUM> cells. In another embodiment, the population of cells includes between <NUM>,<NUM> cells and <NUM>,<NUM>,<NUM> cells, between <NUM>,<NUM> cells and <NUM>,<NUM> cells, between <NUM>,<NUM> cells and <NUM>,<NUM> cells, between <NUM> cells and <NUM>,<NUM> cells, between <NUM> cells and <NUM> cells, and fewer than <NUM> cells (e.g., one cell).

As used herein, the term "subject" includes, without limitation, a mammal such as a human, a non-human primate, a dog, a cat, a horse, a sheep, a goat, a cow, a rabbit, a pig, a rat and a mouse. Where the subject is human, the subject can be of any age. For example, the subject can be <NUM> years or older, <NUM> years or older, <NUM> years or older, <NUM> or older, <NUM> or older, <NUM> or older, <NUM> or older, <NUM> or older, or <NUM> or older. The instant methods are envisioned for all subjects, preferably humans (and preferably symptomatic).

As used herein, a human subject who is "suspected of being afflicted with AD or non-ADD" is a subject displaying at least one symptom consistent with both AD and non-ADD, e.g., dementia.

As used herein, "synchronizing" a population of cells means placing at least a majority of cells in that population in the same cell cycle stage (namely, in the G1, S, G2 or M stage, and preferably in the G1, S or G2 stage). In one embodiment, synchronizing a population of cells means placing at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>% or preferably at least <NUM>% of cells in that population in the same cell cycle stage. In another embodiment, synchronizing a population of cells means placing the cells in that population in the same cell cycle stage that they would be in if cultured to over-confluence and then starved. Cell confluence followed by serum starvation typically arrests the cells in the G0/G1 stage [<NUM>-<NUM>].

Doses, i.e., "therapeutically effective amounts", include, for example, a single administration, and two or more administrations (i.e., fractions). In one embodiment, the therapeutically effective amount of a drug approved for a non-Alzheimer's indication is the dose and dosing regimen approved for that non-Alzheimer's indication.

As used herein, "treating" a subject afflicted with a disorder shall include, without limitation, (i) slowing, stopping or reversing the disorder's progression, (ii) slowing, stopping or reversing the progression of the disorder's symptoms, (iii) reducing the likelihood of the disorder's recurrence, and/or (iv) reducing the likelihood that the disorder's symptoms will recur. In the preferred embodiment, treating a subject afflicted with a disorder means (i) reversing the disorder's progression, ideally to the point of eliminating the disorder, and/or (ii) reversing the progression of the disorder's symptoms, ideally to the point of eliminating the symptoms.

The treatment of AD can be measured according to a number of clinical endpoints. These include, without limitation, (a) lowering, stabilizing or slowing progression of (i) dementia, (ii) synaptic loss, (iii) amyloid plaques and/or (iv) neurofibrillary tangles, and/or (b) favorably affecting the expression level of a gene whose expression level correlates with AD.

This invention provides accurate gene-based methods for determining whether a human subject is afflicted with AD or non-ADD when the subject is suspected of being afflicted with AD or non-ADD. The subject methods are based, at least in part, on the surprising discovery that synchronizing a patient's suitable cell population and then measuring the expression levels of genes that are differentially expressed between AD and non-ADD cells permits accurately diagnosing the patient as having either AD or non-ADD. Certain gene expression-altering agents for the use in treating AD are also described.

Specifically, this invention provides a method for determining whether a human subject is afflicted with AD or non-ADD when the subject is suspected of being afflicted with AD or non-ADD, comprising the steps of.

In one embodiment of the subject method, the suitable cells derived from the subject are cultured skin cell fibroblasts. In another embodiment, the suitable cells derived from the subject are cultured B lymphocytes (preferably immortalized B lymphocytes).

Methods for synchronizing cell populations are known in the art. In one embodiment of the subject method, synchronizing the population of suitable cells comprises culturing the cells to over-confluence and then starving the resulting over-confluent cells.

Ideally in the subject method, the gene is known to be differentially expressed by a significant margin. In one embodiment, the gene is known to be differentially expressed by at least <NUM>% between corresponding synchronized cells derived from AD patients and those derived from non-ADD patients. Preferably, the gene is known to be differentially expressed by at least <NUM>% between corresponding synchronized cells derived from AD patients and those derived from non-ADD patients. Another way of expressing the degree of differential expression is "% change" or "%Ch", which is equal to [ADexpression - Non-ADDexpression / Non-ADDexpression].

In one embodiment, the gene expression levels set forth in Table <NUM>, including PHBP8 and, one, two or more, three or more, four or more, are indicative of AD. In a preferred embodiment, the gene expression levels set forth in Table <NUM>, including PHBP8 and, one, two or more, three or more, four or more, are indicative of AD. For example, as shown in Table <NUM>, a PSMB9 expression level greater than <NUM> TPM is indicative of AD. In yet another embodiment, AD-indicative expression levels for each other gene disclosed herein are readily determined based on the data presented.

In a further preferred embodiment of the subject method, step (b) comprises measuring the expression levels of a plurality of genes including PHBP8, each gene being known to be differentially expressed between corresponding synchronized cells derived from AD patients and those derived from non-ADD patients. The plurality of genes can be of any suitable size, such as at least two genes, at least five genes, at least <NUM> genes, at least <NUM> genes, and at least <NUM>,<NUM> genes including PHBP8. Preferably, each gene of the plurality of genes is known to be differentially expressed by at least <NUM>% (and more preferably by at least <NUM>%) between corresponding synchronized cells derived from AD patients and those derived from non-ADD patients. In yet another preferred embodiment of the subject method, the plurality of genes comprises PHBP8 and one or more genes selected from the group consisting of AC004057. <NUM>, AC092651. <NUM>, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70.

in the subject method where the expression levels of a plurality of genes are measured, the expression levels measured in step (b) are "consistent" with those in corresponding synchronized cells derived from AD patients if, for example, for at least a majority of gene expression levels measured, each such level is independently consistent with that gene's expression level in corresponding synchronized cells derived from AD patients.

In the subject method, measuring the expression level of a gene can be accomplished by any suitable method known in the art. In the preferred embodiment, measuring the expression level of a gene comprises measuring the number of that gene's RNA transcripts per number of total transcripts.

In a preferred embodiment, the subject invention provides a method for determining whether a human subject is afflicted with AD or non-ADD when the subject is suspected of being afflicted with AD or non-ADD, comprising the steps of.

In another preferred embodiment, the subject invention provides a method for determining whether a human subject is afflicted with AD or non-ADD when the subject is suspected of being afflicted with AD or non-ADD, comprising the steps of.

A method for determining whether a human subject is afflicted with AD, non-ADD or a disorder which is neither ("NDS") when the subject is suspected of being afflicted with AD or non-ADD is described, comprising the steps of.

whereby (i) the subject is afflicted with AD if the expression level measured in step (b) is consistent with that gene's expression level in corresponding synchronized cells derived from AD patients, (ii) the subject is afflicted with non-ADD if the expression level measured in step (b) is consistent with that gene's expression level in corresponding synchronized cells derived from non-ADD patients, and (iii) the subject is afflicted with neither AD not non-ADD if the expression level measured in step (b) is consistent with that gene's expression level in corresponding synchronized cells derived from NDS subjects. The various embodiments of the diagnostic methods above for determining whether a human subject is afflicted with AD or non-ADD apply, mutatis mutandis, to this method.

An agent known to favorably affect the expression level of one or more genes whose expression levels correlate with Alzheimer's disease for the use in treating a human subject afflicted with Alzheimer's disease by administering to the subject a therapeutically effective amount of the agent is described. Preferably, the genes are selected from the group consisting of AC004057. <NUM>, AC092651. <NUM>, ACP6, ADAM20, ASXL2, C2CD5, CARNS1, FAM149B1, GLIS3-AS1, IL18R1, LINC01393, LZIC, MAP1LC3B2, NHLH1, NORAD, NPPA-AS1_3, OSMR-AS1, PAN3, PHBP8, PSMB9, RAB3IP, RDH16, RFESDP1, RPL5, SCG2, SDHD, SHISA5, SLC45A3, SNHG14, TTC26, URB2, USMG5, WASF2, ZCWPW2, ZNF444, and ZNF70. In one embodiment, the genes are selected from the group consisting of IL18R1, PSMB9, TTC26, WASF2, ACP6, CARNS1, NPPA-AS1_3, SCG2 and SDHD. In another embodiment, the gene is IL18R1, PSMB9, TTC26, WASF2, ACP6, CARNS1, NPPA-AS1_3, SCG2 or SDHD.

An agent selected from the group consisting of carfilzomib (Kyprolis®, Onyx Pharmaceuticals), bortezomib (Velcade®, Takeda Oncology), bumetanide (Bumex®, Hoffman-La Roche), furosemide (Lasix®), torsemide (Demadex®), flavin mononucleotide, phosphoric acid, riboflavin, gamma-aminobutyric acid, adenosine monophosphate, histidine, L-arginine, cisplatin, clozapine, cyclosporin A, dexamethasone, etanercept, ethanol, filgrastim, glucose, haloperidol, heparin, infliximab, leflunomide, nitric oxide, oxygen, polyethylene glycol, prednisolone, progesterone, tacrolimus, thalidomide, zinc, calcitriol, calcium, serine, acetylcholine, capsaicin, dopamine, histamine, lithium, norepinephrine, succinic acid, formic acid, tromethamine, citric acid, 10Z-hymenialdisine (Tocris), JIB <NUM> (Tocris), CRT <NUM> (Tocris), celastrol, dihydroeponemycin, noradrenaline bitartrate (Tocris), or any other drug listed in Table <NUM> for the use in treating a human subject afflicted with Alzheimer's disease by administering to the subject a therapeutically effective amount of the agent, is described. In a preferred embodiment, the agent is carfilzomib which, in one embodiment, is administered in the manner stated on the FDA-approved label for one of its approved indications (e.g., in the manner approved for treating multiple myeloma, wherein the formulation is injectable and is administered at a dose of <NUM> or <NUM>). In another preferred embodiment, the agent is bortezomib which, in one embodiment, is administered in the manner stated on the FDA-approved label for one of its approved indications (e.g., in the manner approved for treating multiple myeloma, wherein the formulation is injectable and is administered at a dose of <NUM>, or <NUM>/m<NUM>). In another preferred embodiment, the agent is bumetanide which, in one embodiment, is administered in the manner stated on the FDA-approved label for one of its approved indications (e.g., in the manner approved for treating edema, wherein the formulation is oral and is administered at a dose of <NUM>, <NUM> or <NUM> daily, every other day, or daily for <NUM>-<NUM> days followed by a <NUM>-<NUM>-day rest period). In another preferred embodiment, the agent is furosemide which, in one embodiment, is administered in the manner stated on the FDA-approved label for one of its approved indications (e.g., in the manner approved for treating edema or hypertension, wherein the formulation is oral and is administered at a dose of <NUM>, <NUM>, <NUM> or <NUM> per day (e.g., <NUM> 2x daily)). In another preferred embodiment, the agent is torsemide which, in one embodiment, is administered in the manner stated on the FDA-approved label for one of its approved indications (e.g., in the manner approved for treating edema or hypertension, wherein the formulation is oral and is administered at a dose of <NUM>, <NUM>, <NUM> or <NUM> per day).

In another preferred embodiment, the agent is any of cisplatin, clozapine, cyclosporin A, dexamethasone, etanercept, filgrastim, haloperidol, heparin, infliximab, leflunomide, prednisolone, progesterone, tacrolimus, thalidomide or calcitriol which, in one embodiment, is administered in the manner stated on the FDA-approved label for one of its approved indications.

As for each of 10Z-hymenialdisine, JIB <NUM>, CRT <NUM>, celastrol, dihydroeponemycin, noradrenaline bitartrate, and other non-FDA-approved drugs, the preferred route of administration is oral, and the preferred dosage is from <NUM>/kg to <NUM>/kg, from <NUM>/kg to <NUM>/kg, from <NUM>/kg to <NUM>/kg, from <NUM>/kg to <NUM>/kg, or from <NUM>/kg to <NUM>/kg.

This invention will be better understood by reference to the examples which follow, but those skilled in the art will readily appreciate that the specific examples detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

The initial findings of the gene differential expression in synchronized skin fibroblasts, between the Alzheimer's Disease patients (AD; n=<NUM>) and the Non-Alzheimer's Disease Demented patients (Non-ADD; n=<NUM>), were cross-correlated with the second batch of samples (AD; n=<NUM>; Non-AD n=<NUM>). For the purpose of separating the two batches of samples, we called the first set of samples the "Training Set" and the second set of samples the "Validation Set.

The genes were ranked in decreasing statistical significance order, i.e., with the highest statistical significance first (examples in Tables <NUM> and <NUM>). The ranking is based on the t-test (two tailed, unequal variance) for the two groups of samples AD and Non-ADD. The comparison of the two lists of genes was made as described below.

The number of statistically significant genes is similar in the training and validation sets (<FIG>), with smaller differences for lower statistical significance (P<<NUM>) and larger differences for higher statistical significance (P<<NUM>). The larger difference for the higher statistical significance (P<<NUM>) could be due not only to the different number of samples in the validation set (<NUM>) when compared to the training set (<NUM>), but also to the different types of Non-ADD samples in the two sets. This difference suggests a high diversity of dysregulated pathways.

The majority of the genes (n=<NUM>) presented in Tables <NUM> and <NUM> are under highest statistical significance (P<<NUM>), and all of them are under high statistical significance (P<<NUM>). The presence of the first <NUM> genes from the training set (Table <NUM>) was checked in the list of <NUM>,<NUM> genes from the validation set (P<<NUM>; <FIG>). Similarly, the presence of the first <NUM> genes from the validation set (Table <NUM>) was checked in the list of <NUM>,<NUM> genes from the training set (P<<NUM>; <FIG>). The first <NUM> genes from Tables <NUM> and <NUM> are under highest statistical significance therefore it is very likely to have the highest impact in Alzheimer's disease detection, treatment, and pathways dysregulation. The cross-correlation of the first <NUM> genes in each set was made with a larger pool of genes from the opposite set (P<<NUM>) to accommodate the diversity in Non-ADD samples as well as to compensate for different numbers of samples in the validation (<NUM>) and training sets (<NUM>). However, in the end only the genes with similar statistical significance are considered as representing the core of dysregulation for AD.

The results of these initial findings in the highest statistically significant <NUM> genes suggests that about <NUM>% of the genes which are dysregulated the training set are also dysregulated in the validation set. However, only about <NUM>% of these genes show the same statistical significance in both training and validation set (Table <NUM>).

Those genes showing the same statistical significance in the training and validation sets are at the core of the dysregulated pathways and will be very likely at the core of the genetic biomarkers for AD and at the core of the therapeutic targets for AD.

The average and standard deviations were calculated for the Transcripts Per Million (TPM) values for each of the two groups - Alzheimer's disease (AD) and Non-Alzheimer's Disease Demented (Non-ADD) for each gene. The reference intervals were then calculated according to Horn and Pesce (Reference <NPL>) as the average plus minus two standard deviations. The reference intervals calculated in this way assure that <NUM>% of all the possible values in each population (AD or non-ADD) are considered.

If there is no overlap between the reference intervals of AD and Non-ADD, there is a gap between the two bell-shaped curves and that indicates unequivocal diagnosis.

If there is an overlap in the reference intervals for AD and Non-ADD (light grey in Table <NUM>), then there is a possibility of having a false positive or a false negative in the diagnosis. The genes that show overlap in the reference intervals, i.e., no gap (light grey) were eliminated from the final vector diagnosis. The genes that show an average of zero in one of the groups, either in the AD group or in the Non-ADD group, were also eliminated.

The cut-offs for each of the remaining <NUM> genes (Table <NUM>) was determined as the middle of the gap in the reference intervals.

The AD diagnosis is based on the <NUM> components/genes of the vector. For each one of the components, the greater than (>) or smaller than (<) the cut-off value is indicated for each gene, in the last column.

Claim 1:
A method for determining whether a human subject is afflicted with Alzheimer's disease ("AD") or non-Alzheimer's dementia ("non-ADD") when the subject is suspected of being afflicted with AD or non-ADD, comprising the steps of
(a) synchronizing a population of suitable cells derived from the subject; and
(b) in the resulting synchronized cell population, measuring the expression level of a gene known to be differentially expressed between corresponding synchronized cells derived from AD patients and those derived from non-ADD patients, wherein the gene is PHBP8,
whereby (i) the subject is afflicted with AD if the expression level measured in step (b) is consistent with that gene's expression level in corresponding synchronized cells derived from AD patients, and (ii) the subject is afflicted with non-ADD if the expression level measured in step (b) is consistent with that gene's expression level in corresponding synchronized cells derived from non-ADD patients.