WO2019113277A1

WO2019113277A1 - Diagnosing disease via gene expression profile in synchronized cells

Info

Publication number: WO2019113277A1
Application number: PCT/US2018/064180
Authority: WO
Inventors: Florin Valentin Chirila; Daniel L. Alkon
Original assignee: Neurodiagnostics LLC
Current assignee: Neurodiagnostics LLC
Priority date: 2017-12-08
Filing date: 2018-12-06
Publication date: 2019-06-13
Anticipated expiration: 2020-06-08

Abstract

This invention provides a method for determining whether a human subject is afflicted with a predetermined disorder 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 patients afflicted with the disorder and those derived from non-afflicted patients, whereby the subject is afflicted with the disorder if the expression level measured in step (b) is consistent with that gene's expression level in corresponding synchronized cells derived from patients afflicted with the disorder, and the subject is not afflicted with the disorder if the expression level measured in step (b) is consistent with that gene's expression level in corresponding synchronized cells derived from non-afflicted patients.

Description

DIAGNOSING DISEASE VIA GENE EXPRESSION PROFILE IN

SYNCHRONIZED CELLS

This application claims the benefit of U.S. Provisional Application No.

62/596,613, filed December 8, 2017, the contents of which are incorporated herein by reference.

Throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference info this application to describe more fully the state of the art to which this invention pertains.

Summary of the invention

This invention provides a method for determining whether a human subject is afflicted with a predetermined disorder 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 patients afflicted with the disorder and those derived from non-affiicted patients, whereby the subject is afflicted with the disorder if the expression level measured in step (b) is consistent with that gene’s expression level in corresponding synchronized cells derived from patients afflicted with the disorder, and the subject is not afflicted with the disorder if the expression level measured in step (b) is consistent with that gene’s expression level in corresponding synchronized cells derived from non- afflicted patients.

Brief Description of the Figures

Figure 1

Statistically significant genes when comparing the Alzheimer’s Disease (AD) group with Non Alzheimer’s Disease Demented (Non-ADD) group. There are 2103 statistically significant genes for a P level less than 0.1 ; 1099 statistically significant genes for a P level less than 0.05; 285 statistically significant genes for a P level less than 0.01 ; and 6 statistically significant genes for a P level less or equal than 0.001.

Figure 2

Top 6 statistically significant genes (P<=0.001 ) for the 6 AD and 2 Non-ADD cases. Green squares represent the AD population while the purple circles represent the Non-ADD population.

Figure 3

Example of top 10 statistically significant genes (P<=0.01 ). A. Raw TPM data showing with green squares the AD population and with purple circles the Non- ADD population. B. Average TPM data showing with green squares the AD population and with purple circles the Non-ADD population. Error bars are standard deviations C Percent change (%Ch) in gene expression when comparing the AD with control (Non-ADD), i.e., 100*(AD-Non-ADD)/Non-ADD.

Figure 4

Genes ranked 11 to 20 at the statistical significance of 1 % overlap probability (P<=0.G1 ). A. Raw TPM data showing with green squares the AD population and with purple circles the Non-ADD population. B. Average TPM data showing with green squares the AD population and with purple circles the Non- ADD population. Error bars are standard deviations. C. Percent change (%Ch) in gene expression when comparing the AD with control (Non-ADD) i.e

1 G0*(AD-Non-ADD)/Non-ADD. Figure 5

Genes ranked 21 to 30 at the statistical significance of 1 % overlap probability (P<=0.01 ). A. Raw TPM data showing with green squares the AD population and with purple circles the Non-ADD population. B. Average TPM data showing with green squares the AD population and with purple circles the Non- ADD population. Error bars are standard deviations. C. Percent change (%Ch) in gene expression when comparing the AD with control (Non-ADD), i.e. ,

100*(AD-Non-ADD)/Non-ADD. Figure 6

Genes ranked 31 to 40 at the statistical significance of 1 % overlap probability (P<=0.01 ). A. Raw TPM data showing with green squares the AD population and with purple circles the Non-ADD population. B. Average TPM data showing with green squares the AD population and with purple circles the Non- ADD population. Error bars are standard deviations. C. Percent change (%Ch) in gene expression when comparing the AD with control (Non-ADD), i.e.,

100*(AD-Non-ADD)/Non-ADD.

Figure 7

Percent change (%Ch) in gene expression for top 40 genes.

Detailed Description of the Invention

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

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-Alzheimer’s dementia (‘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,“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, 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 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, the“human subject” can be of any age. In one embodiment, the subject is 40 years old or younger. In another embodiment, the subject is 50 years old or younger. In a further embodiment, the subject is over 40 years old. In yet a further embodiment, the subject is over 50 years old, over 60 years old, over 70 years old, over 80 years old, or over 90 years old.

As used herein, a“population” of cells includes any number of ceils permitting the manipulation and study required to assess gene expression. In one embodiment, the population of cells includes at least 1 ,000,000 ceils. In another embodiment, the population of cells includes between 100,000 cells and 1 ,000,000 cells, between 10,000 cells and 100,000 ceils, between 1 ,000 cells and 10,000 cells, between 100 ceils and 1 ,000 cells, between 10 ceils and 100 cells, and fewer than 10 cells (e.g., one cell).

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 ceils (e.g., contained in a punch biopsy) directly removed from the subject. As used herein,“synchronizing" a population of cells means placing at least a majority of cells in that population in the same ceil 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 60%, at least 70%, 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. Cell confluence followed by serum starvation typically arrests the cells in the G0/G1 stage [1 -3]

As used herein,“measuring" the expression level of a gene means

quantitatively determining the expression level via any means for doing so (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 FastG 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.

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 ceils 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, 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 again 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 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" skin fibroblasts is achieved, for example, by conducting the culturing at a temperature and in a growth factor milieu permissive of ceil 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 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,“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 below for proliferation) that preserve lymphocyte viability. In one embodiment, the temperature, salinity and protein milieu permissive of cel! growth is 37 °C, RPIVII 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/mI. 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. , 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”). mbodiments of the Invention

Generally

synchronized cells derived from patients afflicted with the disorder and those derived from non-afflicted patients, whereby the subject is afflicted with the disorder if the expression level measured in step (b) is consistent with that gene’s expression level in corresponding synchronized cells derived from patients afflicted with the disorder, and the subject is not afflicted with the disorder if the expression level measured in step (b) is consistent with that gene’s expression level in corresponding synchronized cells derived from non- afflicted patients.

In this invention, the predetermined disorder can be any disorder (e.g., cancer or a neurodegenerative disorder) wherein there is a defined gene known to be differentially expressed between corresponding synchronized cells derived from patients afflicted with the disorder and those derived from non-afflicted patients.

In the preferred embodiment, the disorder is AD, wherein a subject suspected of being afflicted with AD or non-ADD is diagnosed as being afflicted with one or the other. It is this preferred embodiment (also referred to herein as the “invention”) to which the remainder of this application is devoted.

AD and Non-ADD

This invention provides accurate gene-based methods 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. 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. 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; and

(b) in the resulting synchronized cell population, measuring the

expression level of a gene known to be differentially expressed between corresponding synchronized ceils derived from AD patients and those derived from non-ADD patients, 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 ceils 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. 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 ceil 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 50% between corresponding synchronized ceils derived from AD patients and those derived from non-ADD patients.

Preferably, the gene is known to be differentially expressed by at least 100% 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 [AD expression Nqh-ADDexpression / NqP-ADDexpression]·

In another preferred embodiment of the subject method, the gene is selected from the group consisting of PTCD2, ST20, AC090971.4, EIF4A2P1 , CFAP97, AL157871.3, AC00732.4, NR3C2, C60rf58 and LINC01393. In one embodiment, the following gene expression levels are indicative of AD: (^.) pjQp2 _ below 2 TPM; (ii) ST20 = above 10 TPM; (iii) AC090971_.4 = above 0.02 TPM; (iv) EIF4A2P1 = below 0.2 TPM; (v) CFAP97 = below 15 TPM; (vi) AL157871.3 = below 20 TPM; (vii) AC00732.4 = above 0 1 TPM; (viii) NR3C2 = below 1 TPM; (ix) C60rf58 = below 0.5 TPM; and (x) LINC01393 = above 0.7 TPM In 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, 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 20 genes, at least 100 genes, and at least 1 ,000 genes. Preferably, each gene of the plurality of genes is known to be differentially expressed by at least 50% (and more preferably by at least 100%) between corresponding synchronized ceils 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 two or more genes selected from the group consisting of PTCD2, ST20, AC090971.4, EIF4A2P1 , CFAP97, AL157871.3, AC00732.4, NR3C2, C60rf58 and LINC01393.

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 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 cultured skin cell fibroblasts derived from the subject, wherein the synchronizing comprises culturing the fibroblasts to over-confluence and then starving the resulting over-confluent fibroblasts; and (b) in the resulting synchronized fibroblast population, measuring the expression level of each of genes PTCD2, ST20, ACQ9Q971.4, EIF4A2P1 , CFAP97, AL157871.3, AC00732.4, NR3C2, C60rf58 and LINC01393, wherein measuring the expression level of each gene comprises measuring the number of its RNA transcripts per number of total transcripts, 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. In another preferred embodiment, the subject 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 cultured immortalized B

lymphocytes derived from the subject, wherein the synchronizing comprises culturing the lymphocytes to over-confluence and then starving the resulting over-confluent lymphocytes; and

(b) in the resulting synchronized lymphocyte population, measuring the expression level of each of genes PTCD2, ST20,

AC090971.4, EIF4A2P1 , CFAP97, AL157871.3, AC00732.4, NR3C2, C60rf58 and LINC01393, wherein measuring the expression level of each gene comprises measuring the number of its RNA transcripts per number of total transcripts, 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 ceils 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 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.

Examples

Table 1 - Top 285 statistically significant genes with less than 1 % overlap probability between AD and Non-ADD.

Table 2 - Genes with functional relevance to PKC and MARK

Table 3 - Genes with functional relevance for cell adhesion and cell division

References

1. Chen M et al.“Serum Starvation Induced Cell Cycle Synchronization Facilitates Human Somatic Ceils Reprogramming”, PLoS ONE 7(4) (2012)

2. Baghdadchi N “The Effects of Serum Starvation on Cell Cycle

Synchronization”, OSR Journal of Student Research (2013).

3. Hayes 0 et ai.“Cell confluency is as efficient as serum starvation for inducing arrest in the G0/G1 phase of the ceil cycle in granulosa and fibroblast cells of cattle”, 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 a predetermined disorder 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 ceils derived from patients afflicted with the disorder and those derived from non-afflicted patients, whereby the subject is afflicted with the disorder if the expression level measured in step (b) is consistent with that gene’s expression level in corresponding synchronized cells derived from patients afflicted with the disorder, and the subject is not afflicted with the disorder if the expression level measured in step (b) is consistent with that gene’s expression level in corresponding synchronized cells derived from non-afflicted patients.