Gene expression-based tests for alzheimer's disease

Abstract

This invention provides 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, wherein the suitable cells are cultured skin cell fibroblasts or cultured B lymphocytes; and (b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1, SHISA5, URB2, and WASF2, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those 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 those genes' expression levels in corresponding synchronized cells derived from non-ADD patients. This invention also provides related diagnostic and therapeutic methods, including diagnostic methods based on NDS subject gene expression levels.

Description

GENE EXPRESSION-BASED TESTS FOR ALZHEIMER’S DISEASEThis application claims the benefit of U.S. Provisional Application No. 63 / 587,758, filed October 4, 2023, and U.S. Provisional Application No. 63 / 592,241 , filed October 23, 2023, the contents of both of which are incorporated herein by reference.Throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.Background of the InventionAlzheimer’s disease (“AD”) has long been the subject of considerable efforts to develop accurate diagnostic methods as well as therapeutic methods. Despite these efforts, there is an unmet need for methods of accurately diagnosing AD and differentiating it from non-Alzheimer’s dementia (“non-ADD”). There is also an unmet need for effective methods of treating AD.Summary of the InventionThis invention provides 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, wherein the suitable cells are cultured skin cell fibroblasts or cultured B lymphocytes; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1 , SHISA5, URB2, and WAS F2,whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those 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 those genes’ expression levels in corresponding synchronized cells derived from non-ADD patients.This invention also provides a method for treating a human subject afflicted with Alzheimer’s disease comprising administering to the subject a therapeutically effective amount of one or more agents known to favorably affect the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1 , SHISA5, URB2, and WASF2.This invention further provides a method for determining whether a human subject suspected of being afflicted with AD is afflicted with AD or is a non-demented subject (“NDS”), comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1 , SHISA5, URB2, and WAS F2, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients, and (ii) the subject is a NDS if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects.This invention further provides a method for determining whether a human subject not suspected of being afflicted with AD is afflicted with AD or is a NDS, comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1 , SHISA5, URB2, and WAS F2, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients, and (ii) the subject is a NDS if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects.This invention further provides a method for determining whether a human subject suspected of being afflicted with AD or non-ADD is afflicted with AD, is afflicted with non-ADD, or is a NDS, comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1 , SHISA5, URB2, and WAS F2, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients, (ii) the subject is afflicted with non-ADD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from non-ADD patients, and (iii) the subject is afflicted with neither AD not non-ADD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects.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(a) synchronizing a population of suitable cells derived from the subject, wherein the suitable cells are cultured skin cell fibroblasts or cultured B lymphocytes; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those 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 those genes’ expression levels in corresponding synchronized cells derived from non-ADD patients.This invention further provides a method for treating a human subject afflicted with Alzheimer’s disease comprising administering to the subject a therapeutically effective amount of one or more agents known to favorably affect the expression levels of two or more genes selected from the group consisting of a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family.This invention further provides a method for determining whether a human subject suspected of being afflicted with AD is afflicted with AD or is a NDS, comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients, and (ii) the subject is a NDS if the expression levelsmeasured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects.This invention further provides a method for determining whether a human subject not suspected of being afflicted with AD is afflicted with AD or is a NDS, comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients, and (ii) the subject is a NDS if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects.Finally, this invention provides a method for determining whether a human subject suspected of being afflicted with AD or non-ADD is afflicted with AD, is afflicted with non-ADD, or is a NDS, comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cellsderived from AD patients, (ii) the subject is afflicted with non-ADD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from non-ADD patients, and (iii) the subject is afflicted with neither AD not non-ADD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects.Brief Description of the FiguresFigure 1In this figure, five dysregulated genes show statistical significance in gold-standard autopsy-confirmed skin (N=17) and blood samples (N=18). Samples were measured by the overlap probability (P values) between Alzheimer's disease and non-Alzheimer’s disease dementia.Figure 2AIn this figure, eighteen families of dysregulated genes in the skin fibroblasts (N=17) of autopsy-confirmed AD patients are further validated in immortalized blood B- lymphocytes (N=18; P<0.05). The labels above the cylinders represent the number of members in the same gene family.Figure 2BThis figure shows an example of a radar plot for the statistically significant (P<=0.05) AD-dysregulated genes in blood corresponding to the skin-dysregulated gene IL18R1 (N=9).Figures 3A and 3BFigure 3A shows the conceptual relationship between early-onset AD (“EOAD”) and late-onset AD (“LOAD”). In Figure 3B, three families of genes (A Disintegrin and Metalloproteinase - ADAM, Interleukin - IL, and Interacting Protein - RAB) are well represented in blood B-lymphocytes (N=18) and skin fibroblasts (N=17), and bridge late-onset and early-onset Alzheimer's disease.Figure 4This figure shows that the comparison of Alzheimer's disease with non-Alzheimer's disease dementia samples has three validation levels.Figure 5This figure shows the number of samples analyzed with NGS RNA-seg and other biomarkers.Figures 6A-6CFigure 6A shows that differentially expressed genes identify AD vs. Non-ADD. Figure 6B shows that differentially expressed genes identify AD vs. HC. Figure 6C shows TPM values and cutoff values for six specific genes that are differentially expressed in skin fibroblasts (N=17) between AD and Non-ADD.Figure 7This figure shows ranked percent changes in TPM for AD / Non-ADD (n=26). Blue cylinders (darker) indicate the up-regulated genes, while the turguoise cylinders (lighter) indicate the down-regulated genes in the skin (N=17).Figures 8A-8CThese figures show certain measures of gene networks for the AD / non-ADD pair. Specifically, Figure 8A shows the number of edges as the y-axis, Figure 8B shows the average node degree as the y-axis, and Figure 8C shows the average clustering coefficient as the y-axis. One gene, MAPK1 , for AD / HC causally relates to two autopsy-validated biomarkers, PKC and AD index.Figure 9This figure shows the number of genes sorting into phenotype categories. All phenotypes (61) for the 36 genes were collected and sorted into 11 categories. The percentage for each phenotype category is represented on the top of each bar. For each of the 11 phenotype categories, the total number of genes was accounted for, and the percentage of the total was determined. The ratio was then ranked for the 11 phenotype categories.Detailed Description of the InventionDefinitionsIn 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 administration, intramuscular administration, oral administration, sublingual administration, transdermal administration, subcutaneous administration, intraperitoneal administration, and intrathecal administration.In addition, in this invention, 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 disease-afflicted subjects from whom samples were taken and studied, and for which data are presented herein, are autopsy-confirmed AD, non-ADD patients,and NDCs (who were hyper-validated because they were not demented at the time of biopsy collection).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 10 and its TPM measure is 100 in the same type of cells derived from non-ADD (or NDC) 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 50, below 40, below 30, below 20 or, ideally, 10 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 that preserve lymphocyte viability. In one embodiment, the temperature, salinity, and protein milieu permissive of cell growth is 37 °C, RPMI 1640 Medium with 10% fetal bovine serum (“FBS”) and 1 % 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 / pl. The high density is determined as the plateau that is typically more then 90% 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., nonimmortalized) 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 thatpreserve skin fibroblasts viability. In one embodiment, the temperature, humidity and protein milieu permissive of cell growth is 37 °C, DMEM Medium with 10% fetal bovine serum (“FBS”) and 1 % 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 100%, cells are passaged. Typically after two passages, fibroblasts are purified in a proportion greater than 95%.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 50% likelihood that the subject is afflicted with Alzheimer’s disease. Preferably, “diagnosing Alzheimer’s disease” means determining that there is greater than 60%, 70%, 80% or 90% 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 50% likelihood that the subject is afflicted with non-ADD. Preferably, “diagnosing non-ADD” means determining that there is greater than 60%, 70%, 80% or 90% 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, “expression level”, with respect to a gene, includes, without limitation, any of the following: (i) the rate and / or degree of transcription of the gene (i.e. , the rate at which, and / or degree to which, the gene is transcribed into RNA); (ii) the rate and / ordegree of processing of the RNA encoded by the gene; (iii) the rate and / or degree of maturation of non-protein-coding RNA encoded by the gene; (iv) the rate at which, and / or degree to which, the RNA encoded by the gene is exported; (v) the rate at which, and / or degree to which, the RNA encoded by the gene is translated (i.e. , the rate at which, and / or degree to which, the RNA is translated into protein); (vi) the rate at which, and / or degree to which, the protein encoded by the gene folds; (vii) the rate at which, and / or degree to which, the protein encoded by the gene is translocated; and (viii) the level of function (e.g., enzymatic activity or binding affinity) of the protein encoded by the gene.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 10 and its TPM measure were 100 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 (e.g., disease-free) 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 (20 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, e.g., 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 50 TPM. In another embodiment, measuring a gene’s expression level is done via protein quantification (e.g., via the known method of Western blotting). In a further embodiment, measuring a gene’s expression level is done via a quantitative assay for protein function (e.g., via known methods for measuring enzymatic activity and / or protein binding strength).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 1 ,000,000 cells. In another embodiment, the population of cells includes between 100,000 cells and 1 ,000,000 cells, between 10,000 cells and 100,000 cells, between 1 ,000 cells and 10,000 cells, between 100 cells and 1 ,000 cells, between 10 cells and 100 cells, and fewer than 10 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 50 years or older, 55 years or older, 60 years or older, 65 or older, 70 or older, 75 or older, 80 or older, 85 or older, or 90 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 (e.g., dementia) consistent with both AD and non-ADD.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 GO, G1 , G0 / G1 , S, G2 or M stage, and preferably in the GO stage. For example, the GO stage can be achieved after cell culturing to over-confluence followed by cell starvation. In one embodiment, synchronizing a population of cells means placing at least 60%, at least70%, at least 80%, at least 90%, at least 95% or preferably at least 99% 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 (also referred to as “double synchronization”). Cell confluence followed by serum starvation typically arrests the cells in the G0 / G1 stage [1-3],Doses, i.e. , “therapeutically effective amounts”, used in connection with this invention include, for example, a single administration, and two or more administrations (i.e., fractions). In one embodiment, a therapeutically effective amount of an agent is from 0.1 mg / kg to 100 mg / kg, from 1 mg / kg to 5 mg / kg, from 5 mg / kg to 10 mg / kg, from 10 mg / kg to 15 mg / kg, or from 15 mg / kg to 20 mg / kg.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.Embodiments of the InventionThis 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 of differential expression of certain genes between AD and non- ADD cells, thereby permitting accurately diagnosing the patient as having either AD ornon-ADD. This invention also provides methods for treating AD using certain gene expression-altering agents.Specifically, this invention provides 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, wherein the suitable cells are cultured skin cell fibroblasts or cultured B lymphocytes; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1 , SHISA5, URB2, and WAS F2, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those 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 those genes’ expression levels in corresponding synchronized cells derived from non-ADD patients.In one embodiment of this 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).In one embodiment of each of the present methods that comprise measuring the expression levels of two or more genes selected from the group consisting of FAM149B (also known as FAM149B1 ), NHLH1 , SHISA5, URB2, and WASF2, the method comprises measuring a combination of genes comprising one of the following combinations of genes: FAM149B and NHLH1 ; FAM149B and SHISA5; FAM149B and URB2; FAM149B and WASF2; NHLH1 and SHISA5; NHLH1 and URB2; NHLH1 and WASF2; SHISA5 and URB2; SHISA5 and WASF2; URB2 and WASF2; SHISA5, URB2, and WASF2; NHLH1 , URB2, and WAS F2; NHLH1 , SHISA5, and WAS F2;NHLH1 , SHISA5, and URB2; FAM149B, URB2, and WASF2; FAM149B, SHISA5, andWASF2; FAM149B, SHISA5, and URB2; FAM149B, NHLH1 , and WASF2; FAM149B, NHLH1 , and URB2; FAM149B, NHLH1 , and SHISA5; FAM149B, NHLH1 , SHISA5, and URB2; FAM149B, NHLH1 , SHISA5, and WASF2; FAM149B, NHLH1 , URB2, and WASF2; FAM149B, SHISA5, URB2, and WASF2;NHLH1 , SHISA5, URB2, and WASF2; and FAM149B, NHLH1 , SHISA5, URB2, and WASF2.This invention also provides a method for treating a human subject afflicted with Alzheimer’s disease comprising administering to the subject a therapeutically effective amount of one or more agents known to favorably affect the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1 , SHISA5, URB2, and WASF2. In one embodiment, the agent is D-tyrosine.This invention further provides a method for determining whether a human subject suspected of being afflicted with AD is afflicted with AD or is a non-demented subject (“NDS”, also referred to as “NDS patient”, “NDS subject”, “NDC” (i.e., non-demented control), “NDC patient”, and “NDC subject”), comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1 , SHISA5, URB2, and WAS F2, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients, and (ii) the subject is a NDS if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects.This invention further provides a method for determining whether a human subject not suspected of being afflicted with AD is afflicted with AD or is a NDS, comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1 , SHISA5, URB2, and WAS F2, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients, and (ii) the subject is a NDS if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects.This invention further provides a method for determining whether a human subject suspected of being afflicted with AD or non-ADD is afflicted with AD, is afflicted with non-ADD, or is a NDS, comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1 , SHISA5, URB2, and WAS F2, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients, (ii) the subject is afflicted with non-ADD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from non-ADD patients, and (iii) the subject is afflicted with neither AD not non-ADD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects. The various embodiments of the diagnostic methods herein for determining whether a human subject is afflicted with AD or non-ADD apply, mutatis mutandis, to this method and those below relating to NDS subjects.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(a) synchronizing a population of suitable cells derived from the subject, wherein the suitable cells are cultured skin cell fibroblasts or cultured B lymphocytes; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those 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 those genes’ expression levels in corresponding synchronized cells derived from non-ADD patients.In one embodiment of this 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).In one embodiment of each present method that comprises measuring the expression levels of two or more genes selected from the group consisting of a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family, the methods comprises measuring a combination of genes comprising one of the following combinations of genes: a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family; a gene from the ADAM gene family and a gene from the IL gene family; a gene from the ADAM gene family and a gene from the RAB gene family; a gene from the IL gene family and a gene from the RAB gene family; two or more genes from the ADAM gene family; two or more genes from the IL gene family; and two or more genes from the ADAM gene family.Genes from the ADAM gene family include, for example, ADAM20, ADAM21 , ADAM23, ADAMTS13, ADAMTS15, ADAMTS17, ADAMTS7, and ADAMTSL3 (which aredysregulated in skin cell fibroblasts). Genes from the ADAM gene family also include, for example, ADAM17 (which is dysregulated in B lymphocytes).Genes from the IL gene family include, for example, IL18R1 , IL12B, IL17RD, IL18R1 , IL1 RN, IL20RB, IL22RA1 , and IL23A (which are dysregulated in skin cell fibroblasts). Genes from the IL gene family also include, for example IL10RA, IL15RA, IL17D, IL17RB, IL20RA, IL2RG, IL21 R-AS1 , IL4R, and ILF2 (which are dysregulated in B lymphocytes).Genes from the RAB gene family include, for example, RAB3IP, RAB10, RAB11 FIP2, RAB11 FIP5, RAB14, RAB1A, RAB20, RAB2A, RAB32, RAB33B, RAB34, RAB35, RAB3GAP1 , RAB3IP, RAB40A, RAB43, RAB4B, RAB4B-EGLN2, RABEP1 , RABEPK, RABGAP1 L, RABGGTA, RABGGTB, RABL2A, and RABL3 (which are dysregulated in skin cell fibroblasts). Genes from the RAB gene family also include, for example, RAB20, RAB24, RAB33A, RAB33B, RAB39B, RAB6A, and RAB8A (which are dysregulated in B lymphocytes).This invention further provides a method for treating a human subject afflicted with Alzheimer’s disease comprising administering to the subject a therapeutically effective amount of one or more agents known to favorably affect the expression levels of two or more genes selected from the group consisting of a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family.This invention further provides a method for determining whether a human subject suspected of being afflicted with AD is afflicted with AD or is a NDS, comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family,whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients, and (ii) the subject is a NDS if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects.This invention further provides a method for determining whether a human subject not suspected of being afflicted with AD is afflicted with AD or is a NDS, comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients, and (ii) the subject is a NDS if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects.Finally, this invention provides a method for determining whether a human subject suspected of being afflicted with AD or non-ADD is afflicted with AD, is afflicted with non-ADD, or is a NDS, comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family,whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients, (ii) the subject is afflicted with non-ADD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from non-ADD patients, and (iii) the subject is afflicted with neither AD not non-ADD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects.In a preferred embodiment of each of the present diagnostic methods, step (b) further comprises measuring the expression level of RPL5.In one embodiment of each of the present methods that comprises measuring the expression level of RPL5, the method comprises measuring a combination of genes comprising one of the following combinations of genes: FAM149B and RPL5; NHLH1 and RPL5; SHISA5 and RPL5; URB2 and RPL5; WASF2 and RPL5; FAM149B, NHLH1 , and RPL5; FAM149B, SHISA5, and RPL5; FAM149B, URB2, and RPL5; FAM149B, WASF2, and RPL5; NHLH1 , SHISA5, and RPL5; NHLH1 , URB2, and RPL5; NHLH1 , WASF2, and RPL5; SHISA5, URB2, and RPL5; SHISA5, WASF2, and RPL5; URB2, WASF2, and RPL5; SHISA5, URB2, WASF2, and RPL5; NHLH1 , URB2, WASF2, and RPL5; NHLH1 , SHISA5, WASF2, and RPL5; NHLH1 , SHISA5, URB2, and RPL5; FAM149B, URB2, WASF2, and RPL5; FAM149B, SHISA5, WASF2, and RPL5;FAM149B, SHISA5, URB2, and RPL5; FAM149B, NHLH1 , WASF2, and RPL5; FAM149B, NHLH1 , URB2, and RPL5; FAM149B, NHLH1 , SHISA5, and RPL5; FAM149B, NHLH1 , SHISA5, URB2, and RPL5; FAM149B, NHLH1 , SHISA5, WASF2, and RPL5; FAM149B, NHLH1 , URB2, WASF2, and RPL5; FAM149B, SHISA5, URB2, WASF2, and RPL5; NHLH1 , SHISA5, URB2, WASF2, and RPL5; FAM149B, NHLH1 , SHISA5, URB2, WASF2, and RPL5; a gene from the ADAM gene family, and RPL5; a gene from the IL gene family, and RPL5; a gene from the RAB gene family, and RPL5; a gene from the ADAM gene family, a gene from the IL gene family, a gene from the RAB gene family, and RPL5; a gene from the ADAM gene family, a gene from the IL gene family, and RPL5; a gene from the ADAM gene family, a gene from the RAB gene family, and RPL5; a gene from the IL gene family, a gene from the RAB gene family, and RPL5; two or more genes from the ADAM gene family, and RPL5; two or moregenes from the IL gene family, and RPL5; and two or more genes from the ADAM gene family.Methods for synchronizing cell populations are known in the art. In one embodiment of the present methods, synchronizing the population of suitable cells comprises culturing the cells to over-confluence and then starving the resulting over-confluent cells.In the present methods, 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 or measuring the number of that gene’s RNA transcripts per one million transcripts (TPM).In the present methods, 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.The following are exemplary gene expression levels (in TPM) that are independently consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients (i.e., TPM values that are indicative of AD). In one embodiment of the present methods, an ADAM20 (an ADAM gene family member) expression level of < 0.16 TPM (in cultured skin cell fibroblasts) is indicative of AD. For example, in cultured skin cell fibroblasts, an ADAM20 expression level of between 0.09 and 0.11 TPM is indicative of AD, and an ADAM20 expression level of between 0.20 and 0.22 TPM is indicative of non-ADD. In another embodiment, a FAM149B expression level of < 22.60 TPM (in cultured skin cell fibroblasts) is indicative of AD. In a further embodiment, an IL18R1 (an IL gene family member) expression level of > 0.88 TPM (in cultured skin cell fibroblasts) is indicative of AD. In a further embodiment, a NHLH1 expression level of < 0.27 TPM (in cultured skin cell fibroblasts) is indicative of AD. In a further embodiment, a RAB3IP (a RAB gene family member) expression level of < 0.50 TPM (in cultured skin cell fibroblasts) is indicative of AD. In a further embodiment, an RPL5 expression level of > 794.88 TPM (in cultured skin cell fibroblasts) is indicative of AD. For example, in cultured skin cell fibroblasts, an RPL5expression level of between 850 and 880 TPM is indicative of AD, and an RPL5 expression level of between 280 and 600 TPM is indicative of non-ADD. In a further embodiment, a SHISA5 expression level of > 107.32 TPM (in cultured skin cell fibroblasts) is indicative of AD. For example, in cultured skin cell fibroblasts, a SHISA5 expression level of between 118 and 122 TPM is indicative of AD, and a SHISA5 expression level of between 67 and 89 TPM is indicative of non-ADD. In a further embodiment, an LIRB2 expression level of > 2.28 TPM (in cultured skin cell fibroblasts) is indicative of AD. In a further embodiment, a WASF2 expression level of > 23.74 TPM (in cultured skin cell fibroblasts) is indicative of AD. For example, in cultured skin cell fibroblasts, a WASF2 expression level of between 24.8 and 25.7 TPM is indicative of AD, and a WASF2 expression level of between 10.6 and 19.0 TPM is indicative of non-ADD.In another embodiment of each of the present methods that comprise measuring the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1 , SHISA5, URB2, and WASF2, the method comprises measuring a combination of genes comprising one of the following combinations of genes, wherein the expression levels shown (in TPM as measured in cultured skin cell fibroblasts) are indicative of AD: FAM149B (< 22.60 TPM) and NHLH1 (< 0.27 TPM); FAM149B (< 22.60 TPM) and SHISA5 (> 107.32 TPM); FAM149B (< 22.60 TPM) and URB2 (> 2.28 TPM); FAM149B (< 22.60 TPM) and WASF2 (> 23.74 TPM); NHLH1 (< 0.27 TPM) and SHISA5 (> 107.32 TPM); NHLH1 (< 0.27 TPM) and URB2 (> 2.28 TPM); NHLH1 (< 0.27 TPM) and WASF2 (> 23.74 TPM); SHISA5 (> 107.32 TPM) and URB2 (> 2.28 TPM); SHISA5 (> 107.32 TPM) and WASF2 (> 23.74 TPM); URB2 (> 2.28 TPM) and WASF2 (> 23.74 TPM); SHISA5 (> 107.32 TPM), URB2 (> 2.28 TPM), and WASF2 (> 23.74 TPM); NHLH1 (< 0.27 TPM), URB2 (> 2.28 TPM), and WASF2 (> 23.74 TPM); NHLH1 (< 0.27 TPM), SHISA5 (> 107.32 TPM), and WASF2 (> 23.74 TPM); NHLH1 (< 0.27 TPM), SHISA5 (> 107.32 TPM), and URB2 (> 2.28 TPM); FAM149B (< 22.60 TPM), URB2 (> 2.28 TPM), and WASF2 (> 23.74 TPM); FAM149B (< 22.60 TPM), SHISA5 (> 107.32 TPM), and WASF2 (> 23.74 TPM); FAM149B (< 22.60 TPM), SHISA5 (> 107.32 TPM), and URB2 (> 2.28 TPM); FAM149B (< 22.60 TPM), NHLH1 (< 0.27 TPM), and WASF2 (> 23.74 TPM); FAM149B (< 22.60 TPM), NHLH1 (< 0.27 TPM), and URB2 (> 2.28 TPM); FAM149B (< 22.60 TPM), NHLH1 (< 0.27 TPM), and SHISA5 (> 107.32 TPM); FAM149B (< 22.60 TPM), NHLH1 (< 0.27 TPM), SHISA5 (> 107.32 TPM), and URB2 (> 2.28 TPM); FAM149B (< 22.60 TPM), NHLH1 (< 0.27TPM), SHISA5 (> 107.32 TPM), and WASF2 (> 23.74 TPM); FAM149B (< 22.60 TPM), NHLH1 (< 0.27 TPM), URB2 (> 2.28 TPM), and WASF2 (> 23.74 TPM); FAM149B (<22.60 TPM), SHISA5 (> 107.32 TPM), URB2 (> 2.28 TPM), and WASF2 (> 23.74 TPM); NHLH1 (< 0.27 TPM), SHISA5 (> 107.32 TPM), URB2 (> 2.28 TPM), and WASF2 (> 23.74 TPM); and FAM149B (< 22.60 TPM), NHLH1 (< 0.27 TPM), SHISA5 (> 107.32 TPM), URB2 (> 2.28 TPM), and WASF2 (> 23.74 TPM).In another embodiment of each of the present methods that comprises measuring the expression level of RPL5, the method comprises measuring a combination of genes comprising one of the following combinations of genes, wherein the expression levels shown (in TPM as measured in cultured skin cell fibroblasts) are indicative of AD: FAM149B (< 22.60 TPM) and RPL5 (> 794.88 TPM); NHLH1 (< 0.27 TPM) and RPL5 (> 794.88 TPM); SHISA5 (> 107.32 TPM) and RPL5 (> 794.88 TPM); URB2 (> 2.28 TPM) and RPL5 (> 794.88 TPM); WASF2 (> 23.74 TPM) and RPL5 (> 794.88 TPM); FAM149B (< 22.60 TPM), NHLH1 (< 0.27 TPM), and RPL5 (> 794.88 TPM); FAM149B (< 22.60 TPM), SHISA5 (> 107.32 TPM), and RPL5 (> 794.88 TPM); FAM149B (<22.60 TPM), URB2 (> 2.28 TPM), and RPL5 (> 794.88 TPM); FAM149B (< 22.60 TPM), WASF2 (> 23.74 TPM), and RPL5 (> 794.88 TPM); NHLH1 (< 0.27 TPM), SHISA5 (>107.32 TPM), and RPL5 (> 794.88 TPM); NHLH1 (< 0.27 TPM), URB2 (> 2.28 TPM), and RPL5 (> 794.88 TPM); NHLH1 (< 0.27 TPM), WASF2 (> 23.74 TPM), and RPL5 (>794.88 TPM); SHISA5 (> 107.32 TPM), URB2 (> 2.28 TPM), and RPL5 (> 794.88 TPM); SHISA5 (> 107.32 TPM), WASF2 (> 23.74 TPM), and RPL5 (> 794.88 TPM); URB2 (> 2.28 TPM), WASF2 (> 23.74 TPM), and RPL5 (> 794.88 TPM); SHISA5 (>107.32 TPM), URB2 (> 2.28 TPM), WASF2 (> 23.74 TPM), and RPL5 (> 794.88 TPM); NHLH1 (< 0.27 TPM), URB2 (> 2.28 TPM), WASF2 (> 23.74 TPM), and RPL5 (>794.88 TPM); NHLH1 (< 0.27 TPM), SHISA5 (> 107.32 TPM), WASF2 (> 23.74 TPM), and RPL5 (> 794.88 TPM); NHLH1 (< 0.27 TPM), SHISA5 (> 107.32 TPM), URB2 (> 2.28 TPM), and RPL5 (> 794.88 TPM); FAM149B (< 22.60 TPM), URB2 (> 2.28 TPM), WASF2 (> 23.74 TPM), and RPL5 (> 794.88 TPM); FAM149B (< 22.60 TPM), SHISA5 (> 107.32 TPM), WASF2 (> 23.74 TPM), and RPL5 (> 794.88 TPM); FAM149B (<22.60 TPM), SHISA5 (> 107.32 TPM), URB2 (> 2.28 TPM), and RPL5 (> 794.88 TPM); FAM149B (< 22.60 TPM), NHLH1 (< 0.27 TPM), WASF2 (> 23.74 TPM), and RPL5 (>794.88 TPM); FAM149B (< 22.60 TPM), NHLH1 (< 0.27 TPM), URB2 (> 2.28 TPM), and RPL5 (> 794.88 TPM); FAM149B (< 22.60 TPM), NHLH1 (< 0.27 TPM), SHISA5 (>107.32 TPM), and RPL5 (> 794.88 TPM); FAM149B (< 22.60 TPM), NHLH1 (< 0.27TPM), SHISA5 (> 107.32 TPM), URB2 (> 2.28 TPM), and RPL5 (> 794.88 TPM); FAM149B (< 22.60 TPM), NHLH1 (< 0.27 TPM), SHISA5 (> 107.32 TPM), WASF2 (> 23.74 TPM), and RPL5 (> 794.88 TPM); FAM149B (< 22.60 TPM), NHLH1 (< 0.27 TPM), URB2 (> 2.28 TPM), WASF2 (> 23.74 TPM), and RPL5 (> 794.88 TPM); FAM149B (< 22.60 TPM), SHISA5 (> 107.32 TPM), URB2 (> 2.28 TPM), WASF2 (> 23.74 TPM), and RPL5 (> 794.88 TPM); NHLH1 (< 0.27 TPM), SHISA5 (> 107.32 TPM), URB2 (> 2.28 TPM), WASF2 (> 23.74 TPM), and RPL5 (> 794.88 TPM); FAM149B (< 22.60 TPM), NHLH1 (< 0.27 TPM), SHISA5 (> 107.32 TPM), URB2 (> 2.28 TPM), WASF2 (> 23.74 TPM), and RPL5 (> 794.88 TPM); a gene from the ADAM gene family (e.g., ADAM20 (< 0.16 TPM)), and RPL5 (> 794.88 TPM); a gene from the IL gene family (e.g., IL18R1 (> 0.88 TPM)), and RPL5 (> 794.88 TPM); a gene from the RAB gene family (e.g., RAB3IP (< 0.50 TPM)), and RPL5 (> 794.88 TPM); a gene from the ADAM gene family (e.g., ADAM20 (< 0.16 TPM)), a gene from the IL gene family (e.g., IL18R1 (> 0.88 TPM)), a gene from the RAB gene family (e.g., RAB3IP (< 0.50 TPM)), and RPL5 (> 794.88 TPM); a gene from the ADAM gene family (e.g., ADAM20 (< 0.16 TPM)), a gene from the IL gene family (e.g., IL18R1 (> 0.88 TPM)), and RPL5 (> 794.88 TPM); a gene from the ADAM gene family (e.g., ADAM20 (< 0.16 TPM)), a gene from the RAB gene family (e.g., RAB3IP (< 0.50 TPM)), and RPL5 (> 794.88 TPM); a gene from the IL gene family (e.g., IL18R1 (> 0.88 TPM)), a gene from the RAB gene family (e.g., RAB3IP (< 0.50 TPM)), and RPL5 (> 794.88 TPM); two or more genes from the ADAM gene family (e.g., ADAM20 (< 0.16 TPM)), and RPL5 (> 794.88 TPM); two or more genes from the IL gene family (e.g., one of the genes is IL18R1 (> 0.88 TPM)), and RPL5 (> 794.88 TPM); and two or more genes from the ADAM gene family (e.g., one of the genes is ADAM20 (< 0.16 TPM)).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.ExamplesExample 1 - Gene Expression Profile of Synchronized Cells Identifies Alzheimer'sDisease in Autopsy-Validated Skin and Blood SamplesAbstractA gene expression profile has never been identified for late-onset (sporadic) Alzheimer's disease (AD) (>95% cases), although genes with high penetrance are known for early onset (familial) AD. Further, the genetic bridges linking early with late- onset AD have yet to be identified despite the common neuropathology. Here, novel genetic analyses of living, synchronized skin fibroblasts and immortalized blood B- lymphocytes revealed an AD-specific gene expression profile for a core of five genes. These genes are as follows: (i) Family With Sequence Similarity 149 Member - FAM149B (P<0.0370 in training-set, P<0.0150 in validation-set in the skin, and P<0.0386 in the blood); (ii) Nescient Helix-Loop-Helix1 - NHLH1 (P<0.0319 in trainingset, P<0.0119 in validation-set in the skin, and P<0.0012 in the blood); (iii) Shisa Family Member 5 - SHISA5 (P<0.0188 in training-set, P<0.0169 in validation-set in the skin, and P<0.0121 in the blood); (iv) Ribosome Biogenesis Homolog - LIRB2 (P<0.0051 in training-set, P<0.0219 in validation-set in the skin, and P<0.0138 in blood); and (v) Wiskott-Aldrich Syndrome Protein Family Member 2 - WASF2 (P<0.0027 in training-set, P<0.0476 in validation-set in the skin, and P<0.0056 in blood). Experiments were performed using autopsy-confirmed, blinded samples for two cohorts: AD and non-AD dementia.The gene expression profiles of seventeen skin samples and eighteen blood samples converge with prior autopsy-validated biomarkers: Morphometric Imaging (N=73 in skin, N=9 in blood),1-4PKC epsilon (N=57 in skin, N=11 in blood),5AD index (N=88 in skin, N=0 in blood),67, cell size, growth rate, and protein production (N=33 in skin, N=9 in blood).89In Figure 1 , five dysregulated genes show statistical significance in gold-standard autopsy-confirmed skin (N=17) and blood samples (N=18). Samples were measured by the overlap probability (P values) between Alzheimer's disease and non-Alzheimer’s disease dementia.Eighteen Dysregulated Gene Families in Skin and BloodIn Figure 2A, eighteen families of dysregulated genes in the skin fibroblasts (N=17) of autopsy-confirmed AD patients are further validated in immortalized blood B- lymphocytes (N=18; P<0.05). The labels above the cylinders represent the number of members in the same gene family. Figure 2B shows an example of a radar plot for the statistically significant (P<=0.05) AD-dysregulated genes in blood corresponding to the skin-dysregulated gene IL18R1 (N=9).Bridges Between Late-Onset and Early-Onset Alzheimer's DiseaseThree well-represented families of late-onset dysregulated genes in skin fibroblasts and blood B-lymphocytes from autopsy-confirmed AD patients are also in the gene network of PSEN1 , 2, and APP. These families are as follows: (i) A Disintegrin and Metalloproteinase - ADAM (P<0.0321 in training-set, P<0.0082 in validation-set in the skin, and P<0.0016 in blood-one gene); (ii) Interleukin - IL (P<0.0298 in training-set, P<0.0399 in validation-set in the skin, and P<0.0484 in blood (nine genes)); and (iii) Interacting Protein - RAB (P<0.0137 in training-set, P<0.0186 in validation-set in the skin; and P<0.0357 in blood (seven genes)). This relationship suggests functional bridging between late-onset and early-onset AD.In Figure 3B, three families of genes (A Disintegrin and Metalloproteinase - ADAM, Interleukin - IL, and Interacting Protein - RAB) are well represented in blood B- lymphocytes (N=18) and skin fibroblasts (N=17), and bridge late-onset and early-onset Alzheimer's disease.Ribosomal Protein-RPL Bridges Protein Kinase C EpsilonThe family of genes (i.e. , ribosomal protein, RPL5 (P<0.0220 in training-set, P<0.00422 in validation-set in the skin (N=17), and P<0.0073 in blood (N=18)) found to be dysregulated in samples from both skin and blood from autopsy-confirmed AD has been linked to various human congenital disorders (termed ribosomopathies) and is in the interaction network of the PRKCE gene (which encodes protein kinase C epsilon).There are interaction networks between PRKCE gene (encoding protein kinase C epsilon) and ribosomal protein RPL5 (encoding ribosomal protein L5). PRKCE has 95 functional pathways, one of which contains the dysregulated gene RPL5 and the RPL family of genes found in this study. The RPL5 gene is also a hub with 26 nodes and 323 connections (edges), showing a 24.8 average node degree. There is interconnectivity between the PRKCE group and the RPL5 group.This Study has Three Validation LevelsFigure 4 shows a comparison of Alzheimer's disease with non-Alzheimer's disease dementia samples having three validation levels.Bridges to Five Prior Autopsy-Validated BiomarkersIn prior autopsy-validated clinical trials, we have developed three different but highly correlated diagnostic assays for AD using skin fibroblasts: Morphometric Imaging, 1 -4 PKC epsilon,5and AD index,67with extremely high sensitivity (86-100%) and specificity (83-100%).1-4These trials included 145 cases, of which > 80% were hyper-validated, i.e. , autopsy or genetically confirmed AD or non-ADD patients and non-demented healthy controls (HC).Figure 5 shows the number of samples analyzed with NGS RNA-seq and other biomarkers.Gene Expression Profile as a Biomarker for Alzheimer's Patients - Seven Genes Used as ExampleFigure 6A shows that differentially expressed genes identify AD vs. Non-ADD. Figure 6B shows that differentially expressed genes identify AD vs. HC. Figure 6C shows TPM values and cutoff values for six specific genes that are differentially expressed in skin fibroblasts (N=17) between AD and Non-ADD.Top Dysregulated GenesFigure 7 shows ranked percent changes in TPM for AD / Non-ADD (n=26). Blue cylinders (darker) indicate the up-regulated genes, while the turquoise cylinders (lighter) indicate the down-regulated genes in the skin (N=17).Gene Network MeasuresFigures 8A-8C show certain measures of gene networks for the AD / non-ADD pair. Specifically, Figure 8A shows the number of edges as the y-axis, Figure 8B shows the average node degree as the y-axis, and Figure 8C shows the average clustering coefficient as the y-axis. One gene, MAPK1 , for AD / HC causally relates to two autopsy-validated biomarkers, PKC and AD index.Top Functional Groups of Dysregulated GenesThe top four functional groups of dysregulated genes include vascular, cell dynamics, neurological, and inflammation. The AD-dysregulated genes found in this study are associated with growth, size, protein level differences in skin and blood cells, nine AD risk factors, and molecular pathways.Figure 9 shows the number of genes sorting into phenotype categories. All phenotypes (61 ) for the 36 genes were collected and sorted into 11 categories. The percentage for each phenotype category is represented on the top of each bar. For each of the 11 phenotype categories, the total number of genes was accounted for, and the percentage of the total was determined. The ratio was then ranked for the 11 phenotype categories.ReferencesI . Chirila, F.V., et al., Alzheimer’s & Dementia, 19(S2), Supplement Biomarkers, (2023). 2. Chirila, F.V., et al., Scientific Reports, 12, 17675 (2022).3. Chirila, F.V., et al., J. Alzheimer's Dis. 33, 165-176 (2013).4. Chirila, F. V., et al., D. L., J. Alzheimer's Dis. 42, 1279 - 1294 (2014).5. Khan, T. K., et al., J. Alzheimer's Dis., 43(2):491 -509 (2015).6. Zhao, W. Q. , et al. , Neurobiol. Dis 11 (1 ), 166-183 (2002). 7. Khan, T. K., Alkon D. L., Proc. Natl. Acad. Sci. U S A 103(35), 13203-7 (2006).8. Chirila, F.V., et al., Annual Meeting of the Society For Neuroscience, 2017.9. Chirila, F.V. and Alkon D.L., Annual Meeting of the Society For Neuroscience 201910. Spellman, P., et al., Mol. Biol. Cell. 9(12):3273-97(1998).I I . Chen, M., et al., PLoS ONE 7(4) (2012). 12. Baghdadchi, N., OSR Journal of Student Research, Vol. 1

[2013] ,13. Hayes, O., et al., Anim. Reprod. Sci. 87(3-4): 181 -92 (2005).

Claims

What is claimed is: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, wherein the suitable cells are cultured skin cell fibroblasts or cultured B lymphocytes; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1 , SHISA5, URB2, and WAS F2, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those 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 those genes’ expression levels in corresponding synchronized cells derived from non-ADD patients.

2. A method for treating a human subject afflicted with Alzheimer’s disease comprising administering to the subject a therapeutically effective amount of one or more agents known to favorably affect the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1 , SHISA5, URB2, and WASF2.

3. A method for determining whether a human subject suspected of being afflicted with AD is afflicted with AD or is a non-demented subject (“NDS”), comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1 , SHISA5, URB2, and WAS F2,whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients, and (ii) the subject is a NDS if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects.

4. A method for determining whether a human subject not suspected of being afflicted with AD is afflicted with AD or is a NDS, comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of FAM149B, NHLH1 , SHISA5, URB2, and WAS F2, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients, and (ii) the subject is a NDS if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects.

5. 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) synchronizing a population of suitable cells derived from the subject, wherein the suitable cells are cultured skin cell fibroblasts or cultured B lymphocytes; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family,whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those 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 those genes’ expression levels in corresponding synchronized cells derived from non-ADD patients.

6. A method for treating a human subject afflicted with Alzheimer’s disease comprising administering to the subject a therapeutically effective amount of one or more agents known to favorably affect the expression levels of two or more genes selected from the group consisting of a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family.

7. A method for determining whether a human subject suspected of being afflicted with AD is afflicted with AD or is a NDS, comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients, and (ii) the subject is a NDS if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects.

8. A method for determining whether a human subject not suspected of being afflicted with AD is afflicted with AD or is a NDS, comprising the steps of(a) synchronizing a population of cultured skin cell fibroblasts derived from the subject; and(b) in the resulting synchronized cell population, measuring the expression levels of two or more genes selected from the group consisting of a gene from the ADAM gene family, a gene from the IL gene family, and a gene from the RAB gene family, whereby (i) the subject is afflicted with AD if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from AD patients, and (ii) the subject is a NDS if the expression levels measured in step (b) are consistent with those genes’ expression levels in corresponding synchronized cells derived from NDS subjects.

9. The method of any of claims 1 , 3-5, 7, and 8, wherein step (b) further comprises measuring the expression level of RPL5.

10. The method of any of claims 1 , 3-5, and 7-9 wherein measuring the expression level of a gene comprises measuring the number of that gene’s RNA transcripts per number of total transcripts.

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