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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCT 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 6 ; 
C12Q 1/68, G01N 33/50 



Al 



(11) Internati nal Publication Number: WO 99/23254 

(43) Internati nal Publication Date: 14 May 1999 (14.05.99) 



(21) International Application Number: PCT/U S98/22966 

(22) International Filing Date: 30 October 1998 (30.10.98) 



(30) Priority Data: 

60/063,857 



31 October 1997 (31.10.97) 



US 



(71) Applicant (for alt designated States except US): 

AFFYMETRIX, INC. [US/US]; 3380 Central Expressway, 
Santa Clara, CA 95051 (US). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only)i LOCKHART, David, J. 
[US/US]; 610 Mountain View Avenue, Mountain View, CA 
94041 (US). NAIR, Archana [IN/US]; 3589 Lochinvar Av- 
enue, No. 6, Santa Clara, CA 95051 (US). WARRINGTON, 
Janet, A. [US/US]; 325 Thompson Avenue, Mountain View, 
CA 94043 (US). 

(74) Agents: KAGAN, Sarah, A. et al.; Banner & Witcoff, Ltd., 1 1 th 
floor, 1001 G Street, N.W., Washington, DC 20001-4597 
(US). 



(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR, 
BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, 
GH, GM. HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, 
LC, LK, LR, LS, LT, LU, LV, MD, MG, MK t MN, MW, 
MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, 
TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW, ARIPO 
patent (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), Eurasian 
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM). European 
patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, 
IE, IT, LU, MC, NL, FT, SE), OAPI patent (BF, BJ, CF, 
CG, CI. CM, GA, GN, GW, ML, MR, NE, SN, TD, TG). 



Published 

With international search report. 

Before the expiration of the time limit for amending the 
claims and to be republished in the event of the receipt of 
amendments. 



(54) Title: EXPRESSION PROFILES IN ADULT AND FETAL ORGANS 



(57) Abstract 

Expression profiles have been constructed for liver and brain, in adult and fetal and adolescent tissue. These provide the art with 
probes which are highly differentially expressed among stages and organs. The profiles and probes can be used to prioritize potential drug 
targets, to monitor disease progression and remission, and to assess drug metabolism. Solid supports are also provided which facilitate 
these uses. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT 



AL 


Albania 


ES 


Spam 


LS 


Lesotho 


si 


Slovenia 


AM 


Armenia 


FI 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TO 


Chad 


BA 


Bosnia and Herzegovina 


GE 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


BG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy , 


MX 


Mexico 


uz 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan - 


NE 


Niger 


VN 


Viet Nam 


CC 


Congo 


KE 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzstan 


NO 


Norway 


. zw 


Zimbabwe 


CI 


C6te d'lvotre 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KJR 


Republic of Korea 


PT 


Portugal 






cv 


Cuba 


KZ 


Kazaksian 


RO 


Romania 






cz 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Germany 


U 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







WO 99/23254 PCT/US98/22966 



EXPRESSION PROFILES IN ADULT AND FETAL ORGANS 



R A nCGRQUN D OF THE INVENTION 

Expression profiles of genes in particular organs or disease states or 
developmental stages provide molecular tools for evaluating toxicity, drug efficacy, 
5 drug metabolism, development, and disease monitoring. Changes in the expression 

profile from a baseline profile can be used as an indication of such effects. 

SUMMARY O F THE INVENTION 

It is an object of the present invention to provide a method of screening a drug for 
deleterious side effects on a cell. 
10 It is another object of the present invention to provide a method of distinguishing 

between a fetal and an adult liver sample. 

It is an object of the present invention to provide a method of distinguishing 
between a fetal and adult brain tissue. 

Another object of the invention is to provide a method of determining the source 
15 of a tissue as adult brain or adult liver. 

Another object of the invention is to provide a method of distinguishing a tissue 
source as fetal brain or fetal liver. 

Another object of the invention is to provide a solid support for screening a drug 
for deleterious side effects on a cell. 
20 Another object of the invention is to provide a solid support for distinguishing 

between a fetal and an adult liver sample. 

Still another object of the invention is to provide a solid support for distinguishing 
between a fetal and adult brain tissue. 



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-2- 

Yet another object of the invention is to provide a solid support for determining 
the source of a tissue as adult brain or adult liver. 

Another object of the invention is to provide a solid support for distinguishing a 
tissue source as fetal brain or fetal liver. 

These and other objects of the invention are achieved by providing a method of 
screening a drug for deleterious side effects on a cell. The method comprises the step 
of: 

assaying for the amount of expression in the cell of two or more genes selected 
from the group consisting of: G6PD, calcium channel, synaptotagamin, neuromodulin, 
calmodulin, nicotinic acetylcholine receptor beta 2, VLDLR, 
udulinl/undulin/extracellular matrix glycoprotein, pyruvate dehydrogenase El, 
apolipoprotein B100, hepatocyte gf, IGF binding protein 1, ubiquitin, bone 
morphogenetic protein precursor, cytochrome p450-2El, and thymosin beta- 10, wherein 
the expression in the cell is assayed before and after the cell has been contacted with the 
drug, wherein alteration of the amount of expression of at least one of these genes by 
the drug is indicative of a deleterious side effect. 

According to another embodiment of the invention a method of distinguishing 
between a fetal and an adult liver sample is provided. The method comprises the step 
of: 

assaying for expression in the sample of two or more genes selected from the 
group consisting of: G6PD, calmodulin, VLDLR, udulinl/undulin/extracellular matrix 
glycoprotein, hepatocyte gf, IGF binding protein 1, ubiquitin, cytochrome p450-2El, 
and thymosin beta- 10, wherein expression of G6PD, VLDLR, 
udulinl/undulin/extracellular matrix glycoprotein, cytochrome p450-2El , and thymosin 
beta- 10 are indicative of an adult liver, and expression of calmodulin, hepatocyte gf, 
IGF binding protein 1, and ubiquitin are indicative of a fetal liver. 

According to still another embodiment of the invention a method of 
distinguishing between a fetal and adult brain tissue is provided. The method comprises 
the step of: 

assaying for expression in the tissue of two or more genes selected from the group 
consisting of: nicotinic acetylcholine receptor beta 2, ubiquitin, and thymosin beta- 10, 



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-3- 

wherein expression of nicotinic acetylcholine receptor beta 2 or ubiquitin indicates an 
adult brain. 

Another aspect of the invention is a method of determining the source of a tissue 
as adult brain or adult liven The method comprises the steps of: 
5 assaying for expression in the tissue of two or more genes selected from the group 

consisting of: calcium channel, synaptotagamin, neuromodulin, calmodulin, nicotinic 
acetylcholine receptor beta 2, VLDLR, udulinl/undulin/extracellular matrix 
glycoprotein, pyruvate dehydrogenase El, apoiipoprotein B100, hepatocyte gf, IGF 
binding protein 1, ubiquitin, bone morphogenetic protein precursor, and cytochrome 
10 p450-2El, wherein expression of calcium channel, synaptotagamin, neuromodulin, 

calmodulin , nicotinic acetylcholine receptor beta 2, ubiquitin, or bone morphogenetic 
protein precursor indicates a brain source for the tissue and wherein expression of 
pyruvate dehydrogenase El , VLDLR, udulinl/undulin/extracellular matrix 
glycoprotein, apoiipoprotein B100, thymosin beta- 10, hepatocyte gf, IGF binding 
1 5 protein 1 , or cytochrome p450-El indicates a liver source for the tissue. 

Still another aspect of the invention is a method of distinguishing a tissue source 
as fetal brain or fetal liver. The method comprises the step of : 

assaying for expression in the tissue of two or more genes selected from the group 
consisting of: G6PD, calcium channel, synaptotagamin, neuromodulin, pyruvate 
20 dehydrogenase El, apoiipoprotein B100, hepatocyte gf, IGF binding protein 1, 

ubiquitin, bone morphogenetic protein precursor, and thymosin beta- 10, wherein 
expression of G6PD, calcium channel, synaptotagamin, neuromodulin, thymosin beta- 
10 or bone morphogenetic protein precursor indicates a fetal brain source and 
expression of pyruvate dehydrogenase El, apoiipoprotein B100, hepatocyte gf, IGF 
25 binding protein 1, ubiquitin, indicates a fetal liver source. 

Another embodiment of the invention provides a solid support for screening a 
drug for deleterious side effects on a cell. The solid support comprises: at least two 
oligonucleotides for probing two or more genes selected from the group consisting of: 
G6PD, calcium channel, synaptotagamin, neuromodulin, calmodulin, nicotinic 
30 acetylcholine receptor beta 2, VLDLR, udulinl/undulin/extracellular matrix 

glycoprotein, pyruvate dehydrogenase El, apoiipoprotein B100, hepatocyte gf, IGF 



WO 99/23254 



PCT/US98/22966 



binding protein 1 , ubiquitin, bone morphogenetic protein precursor, cytochrome p450- 
2E 1 , and thymosin beta- 1 0, wherein each oligonucleotide comprises a sequence which 
is complementary to one of the two or more genes. 

Yet another aspect of the invention is a solid support for distinguishing between 
5 a fetal and an adult liver sample. The solid support comprises: two or more 

oligonucleotides for detecting two or more genes selected from the group consisting of: 
G6PD, calmodulin, VLDLR, udulinl/undulin/extracellular matrix glycoprotein, 
hepatocyte gf, IGF binding protein 1 , ubiquitin, cytochrome p450-2El , and thymosin 
beta- 10 wherein each oligonucleotide comprises a sequence which is complementary 

10 to one of the two or more genes. 

In another aspect of the invention a solid support for distinguishing between a 
fetal and adult brain tissue is provided. The solid support comprises: two more 
oligonucleotides for detecting two or more genes selected from the group consisting of: 
nicotinic acetylcholine receptor beta 2, ubiquitin, and thymosin beta-1 0, wherein each 

15 oligonucleotide comprises a sequence which is complementary to one of the two or 

more genes. 

In still another aspect of the invention a solid support for determining the source 
of a tissue as adult brain or adult liver is provided. The solid support comprises: 

two or more oligonucleotides for detecting two or more genes selected from the 
20 group consisting of: calcium channel, synaptotagamin, neuromodulin, calmodulin, 

nicotinic acetylcholine receptor beta 2, VLDLR, udulinl/undulin/extracellular matrix 
glycoprotein, pyruvate dehydrogenase El, apolipoprotein B100, hepatocyte gf, IGF 
binding protein 1, ubiquitin, bone morphogenetic protein precursor, and cytochrome 
p450-2El , wherein each oligonucleotide comprises a sequence which is complementary 
25 to one of the two or more genes. 

Another embodiment of the invention is a solid support for distinguishing a tissue 
source as fetal brain or fetal liver. The solid support comprises: two or more 
oligonucleotides for detecting two or more genes selected from the group consisting of: 
G6PD, calcium channel, synaptotagamin, neuromodulin, pyruvate dehydrogenase El, 
30 apolipoprotein B100, hepatocyte gf, IGF binding protein 1, ubiquitin, bone 



99/23254 



PCT/US98/22966 



-5- 

morphogenetic protein precursor, and thymosin beta- 10, wherein each oligonucleotide 
comprises a sequence which is complementary to one of the two or more genes. 
RRIEF DESC RIPTION OF THE DRAWINGS 

Figure 1 shows a plot of the fetal brain hybridization intensities, poly (A)* versus 
cDNA yielded a line with a slope of 1. 
DETAILED D ESCRIPTION 

Genes have been found whose expression is varied greatly (;> 10 fold) between 
different developmental stages and between different organs in the same developmental 
stages. Thus these genes can be used as interrogators (probes) to determine the 
expression pattern of unknown cells or samples to identify them as belonging to the 
appropriate developmental stage or organ source. The genes are listed in Table 4. The 
complete sequences of the genes are available from GenBank using the accession 
numbers shown in the table. 

Any selection of at least 2 through 16 of the genes can be used as interrogators. 
For a particular interrogation of two conditions or sources, it is desirable to select those 
genes which display a great deal of difference in the expression pattern between the two 
conditions or sources. At least a two-fold difference is desirable, but a five-fold or ten- 
fold difference is preferred. 

Interrogations of the genes or proteins can be performed to yield different 
information. Potential drugs can be screened to determine if the expression of these 
genes is inappropriately altered. This may be useful, for example, in determining 
whether a particular drug is prescribed to a pregnant woman. In the case where a fetally 
expressed gene's expression is affected by the potential drug, prohibition of the drug to 
pregnant woman is indicated. Similarly, a drug which causes expression of a gene 
which is not normally expressed by a fetus, should be prohibited to pregnant woman. 

Molecular expression markers for either brain or liver can be used to confirm 
tissue source identifications made on the basis of morphological criteria. In some 
situations, identifications of cell type or source is ambiguous based on classical criteria. 
In these situations the molecular expression markers of the present invention are useful. 

In addition, disease progression involving either the brain or the liver can be 
monitored by following the expression patterns of the involved organs using the 



WO 99/23254 



PCT/US98/22966 



-6- 

molecular expression markers of the present invention. Perturbed expression can be 
observed in the diseased state. Monitoring of the efficacy of certain drug regimens can 
also be accomplished by following the expression patterns of the molecular expression 
markers. 

Although only a few different developmental time points have been observed, as 
shown in the examples below, other developmental stages can be studied using these 
same molecular expression markers. The importance of these markers in development 
has been shown here, however, variations in their expression may occur at other times. 
For example, one could study the expression of these markers at other gestational 
stages, at birth, postnatally, and throughout the human life cycle. 

Solid supports containing oligonucleotide probes for differentially expressed 
genes can be filters, polyvinyl chloride dishes, etc. Any solid surface to which 
oligonucleotides can be bound, either directly or indirectly, either covalently or non- 
covalently, can be used. A preferred solid support is a high density array or DNA chip. 
These contain a particular oligonucleotide probe in a predetermined location on the 
array. Each predetermined location may contain more than one molecule of the probe, 
but each molecule within the predetermined location has an identical sequence. Such 
predetermined locations are termed features. There may be, for example, from 2, 10, 
100, 1000, to 10,000, 100,000, or 400,000 of such features on a single solid support. 
The solid support, or the area within which the probes are attached may be on the order 
r ' ^ of a square centimeter. 

Oligonucleotide probe arrays for expression monitoring can be made and used 
according to any techniques known in the art. See for example, Lockhart, D J. et al, 
Nature Biotechnology 74:1675-1680 (1996) and McGall, G. et al, Proc. Nat. Acad ScL 
USA 93:13555-13460 (1996). 

The genes which are assayed or interrogated according to the present invention 
are typically in the form of mRNA or reverse transcribed mRNA . The genes may be 
cloned or not The genes may be amplified or not. The cloning itself does not appear 
to bias the representation of genes within a population. However, it may be preferable 
to use polyA+ RNA as a source, as it can be used with less processing steps. The 
sequences of the expression marker genes are in the public databases. Table 4 provides 



WO 99/23254 



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-7 



the accession numbers for the sequences. The sequences of the genes in GenBank are 
expressly incorporated herein. Some of the genes are also the subject of scientific and 
learned journal articles. These include: Anand,R. and Lindstrom,J., Nucleotide 
sequence of the human nicotinic acetylcholine receptor beta 2 subunit gene, Nucleic 
5 Acids Res. 18 (14), 4272 (1990) (MEDLINE 90332444); Takizawa,T., HuangJ.Y., 

IkutaX and Yoshida^A., Human glucose-6-phosphate dehydrogenase: primary 
structure and cDNA cloning, Proc. Natl. Acad. Sci. U.S.A. 83 (12), 4157-4161 (1986) 
(MEDLINE 86233391); Karathanasis,S.K., Apolipoprotein multigene family: tandem 
organization of human apolipoprotein AI, CIII, and AIV genes, Proc. Natl. Acad. Sci. 

10 U.S.A. 82 (19), 6374-6378 (1985) (MEDLINE 86016704); SakaiJ., Hoshino,A., 

Takahashi,S., Miura,Y., Ishii,H., Suzuki,H., Kawarabayasi,Y. and Yamamoto,T., 
Structure, chromosome location, and expression of the human very low density 
lipoprotein receptor gene, J. Biol. Chem. 269 (3), 2173-2182 (1994) (MEDLINE 
94124575); Schuppan,D., CantaluppiJM., Becker J., Veit^A-, Bunte,T., Troyer,D., 

1 5 Schuppan,F., SchmidJvL, AckermannJL and HahnJL, Undulin, an Extracellular Matrix 

Glycoprotein Associated with Collagen Fibrils, J. Biol. Chem. 265, 8823-8832 (1990) 
(MEDLINE 90256812); Just,M., Herbst,H., Hummel,M., Durkop,H., TripierJ)., 
Stein,H. and SchuppanJ)., Undulin is a novel member of the fibronectin-tenascin 
family of extracellular matrix glycoproteins, J. Biol. Chem. 266, 17326-17332 

20 (1991)(MEDLINE 91373351); Ehrenborg3., Larsson,C, SternJL, Janson,M., 

PowellJXR. and LuthmanJL, Contiguous localization of the genes encoding human 
insulin-like growth factor binding proteins 1 (IGBP1) and 3 (IGBP3) on chromosome 
7, Genomics 12.(3), 497-502 (1992) (MEDLINE 92217971); Kosik,K.S., 
Orecchio,L.D M Bruns,GA., BenowitzJLL, MacDonald,G.P., Cox,D.R. and Neve,R.L., 

25 Human GAP-43: its deduced amino acid sequence and chromosomal localization in 

mouse and human, Neuron 1 (2), 127-132 (1988)(MEDLINE 90166498; Perin,M.S., 
Johnston,P.A., Ozcelik,T., Jahn,R., Francke,U. and Sudhof,T.C, Structural and 
functional conservation of synaptotagmin (p65) in Drosophila and humans, J. Biol. 
Chem. 266 (1), 615-622 (1991) (MEDLINE 91093190); Fischer,R., Koller,M., 

30 Flura,M., Mathews,S., Strehler-Page,M.A.,KrebsJ., PennistonJ.T., Carafoli,E. and 

Strehler,E.E,. Multiple divergent mRNAs code for a single human calmodulin, J. Biol. 



99/23254 



PCT/US98/22966 



-8- 

Chem. 263 (32), 17055-17062 (1988)(MEDLINE 89034207). Each of these articles 
is also expressly incorporated herein. 

Glucose-6-phosphate dehydrogenase deficiency can lead to significant 
hemolysis. Hemolytic crisis can be induced by exposure to an oxidant, producing 
profound drops in hematocrit and hemoglobin levels. Calmodulin is a calcium binding 
protein which controls the assembly of myosin molecules. Calcium channel is involved 
in ischemic heart disease, stroke and neuronal development and transmission. 
Neuromodulin is also known as GAP-43. It is involved in neuronal development. 
Prooncoprotein EWS binds to neuromodulin. VLDLR -is the very low density 
lipoprotein receptor. It is involved in hyper-cholesterolemia. Undulin is involved with 
kidney diseases, kidney transplantation and hemodialysis. (Clin Nephrol 44(3), 
178-184 (1995)). It is also involved in schistosomiasis and alcoholic liver cirrhosis. 
Hepatogastroenterology 42(1), 22-26 (1995). Pyruvate dehydrogenase El deficiency 
is associated with Leigh syndrome, microcephaly, and motor neuropathy. 
Apolipoprotein B100 is involved in atherosclerosis and hypercholesterolemia. 
Hepatocyte growth factor (gf) is involved in stimulation of the growth of hepatocytes. 
Insulin-like growth factor-binding protein (IGFBP-3) predisposes breast cancer cells to 
programmed ceil death, Ubiquitin is involved in dendrite outgrowth and differentiation. 
Cytochrome P450 2E1 is the principal catalyst of human oxidative halothane 
metabolism in vitro. (J Pharmacol Exp Ther 281(1), 400-411 (1997)). Thymosin 
beta- 10 is detected mainly in malignant breast tissue, particularly in the cancerous cells, 
whereas the normal cell population around the lesions shows very weak staining. Also, 
the intensity of staining in the cancerous cells was proportionally increased with the 
increasing grade of the lesions. (Br J Cancer 1996 Nov;74(9): 1441-1444). In addition, 
in the highly metastatic human melanoma cell line, BLM, thymosin beta- 10 correlated 
with the malignant phenotype. (Biochem Biophys Res Commun 1996 Aug 
23;225(3):808-816.) 

The protein product of the gene can be assayed to determine the amount of 
expression. Methods for assaying for a protein include Western blot, 
immunprecipitation, radioimmunoassay. However, it is preferred according to the 
invention that the mRNA be assayed as an indication of expression. Methods for 



WO 99/23254 



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-9 



assaying for mRNA include Northern blots, slot blots, dot blots, and hybridization to 
an ordered array of oligonucleotides. Any method for specifically and quantitatively 
measuring a specific protein or mRNA or DNA product can be used. 

Oligonucleotide probes for interrogating the tissue or cell sample are preferably 
5 of sufficient length to specifically hybridize only to appropriate, complementary genes 

or transcripts. Typically the oligonucleotide probes will be at least 10, 12, 14, 16, 18, 
20 or 25 nucleotides in length. In some cases longer probes of at least 30, 40, or 50 
nucleotides will be desirable. 

The above disclosure generally describes the present invention. A more complete 
1 0 understanding can be obtained by reference to the following specific examples which 

are provided herein for purposes of illustration only, and are not intended to limit the 
scope of the invention. 

EXAM PLE I 

Gene expression levels were quantitatively measured in human poly(A)+ and 
1 5 cDNA libraries using high density oligonucleotide arrays containing probes for more 

than 6500 human genes. Probe arrays (DNA chips) of this type have been shown to 
behave quantitatively with high specificity and sensitivity (Lockhart, D.J. et al., 1996, 
Nat. Biotech. 14:1657-1680). The arrays were designed and synthesized based on 
sequence information obtained from GenBank and dbEST. Half the genes were 
20 selected from full-length human sequences in Gen Bank and the remaining half were 

selected from clustered ESTs that have sequence similarity to genes of known function. 
Samples derived from adult brain, adult liver, fetal brain and fetal liver were hybridized 
to the probe arrays and the relative concentration of more than 6500 human genes were 
measured simultaneously. The RNAs were classified by relative abundance and 
25 differentially expressed genes were identified by direct comparison. The following 

comparisons were made; 1) fetal brain:adult brain, 2) fetal liver:adult liver, 3) adult 
brain:adult liver, 4) fetal brain: fetal liver. Using this method one can produce, in a 
relatively short period of time, a quantitative representation of gene expression for the 
major organs of the human body. 



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- 10- 



E XAMPLE 2 

The sources of the samples interrogated are shown in Table 1 . The samples were 
obtained commercially. 



WG99/23254 



-11 




- PCT/US98i/zi966 



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WO 99/23254 



PCT/US98/22966 



- 12- 
EX AMPIE3 

The profiles using cloned brain cDNA and brain poly(A) + mRNA (reverse 
transcribed) as samples were compared. As shown in Table 2, each sample type yielded 
a large number of low copy messages. The percentage of the unique messages which 
5 were low copy messages was about the same for each sample source. 

As shown in Figure 1 , a plot of the fetal brain hybridization intensities, poly(A) + 
versus cDNA yielded a line with a slope of 1. 



WO 99/23254 



- 13 - 



PCT/US98/22966 



TABLE 2 

cDNA and Poly (A) Brain Message Comparison 

cDNA - Poly (A) 
Common 1854 
Unique (cDNA) 680 
Low copy 500(0.74) 
Unique ( polyA) 329 
LOW Copy 239(0.73) 



SUBSTITUTE SHEET (RULE 26) 



WO 99/23254 



PCT/US98/22966 



- 14- 
EXAMPLE4 

The mRNA in brain and liver were compared (via their cDNA). Table 3 shows 
the results. The first column of data compares fetal brain and fetal liver. The second 
column of data compares fetal brain and adult brain. The third column of data 
5 compares the adolescent liver with the fetal liver. The percentage of low copy messages 

in fetal liver is very low. This indicates that the public databases (from which the 
probes were derived) contain an under representation of messages from fetal liver. 



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WO 99/23254 



PCT/US98/22966 



- 16- 
EXAMPLE 5 

The results of the comparison of expression levels for 16 particular genes in fetal 
and adult, lung and brain, are shown in Table 4. 



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WO 99/23254 



PCT/US98/22966 



-18- 

CLAHMS 

1. A method of screening a drug for deleterious side effects on a cell 
comprising the steps of: 

assaying for the amount of expression in the cell of two or more genes selected 
from the group consisting of: G6PD, calcium channel, synaptotagamin, neuromodulin, 
calmodulin, nicotinic acetylcholine receptor beta 2, VLDLR, 
uduiinl/undulin/extracellular matrix glycoprotein, pyruvate dehydrogenase El, 
apolipoprotein B100, hepatocyte gf, IGF binding protein 1, ubiquitin, bone 
morphogenetic protein precursor, cytochrome p450-2E 1 , and thymosin beta- 1 0, wherein 
the expression in the cell is assayed before and after the cell has been contacted with the 
drug, wherein alteration of the amount of expression of at least one of these genes by 
the drug is indicative of a deleterious side effect. 

2. A method of distinguishing between a fetal and an adult liver sample 
comprising the steps of: 

assaying for expression in the sample of two or more genes selected from the 
group consisting of: G6PD, calmodulin, VLDLR, uduiinl/undulin/extracellular matrix 
glycoprotein, hepatocyte gf, IGF binding protein 1, ubiquitin, cytochrome p450-2El, 
and thymosin beta- 10, wherein expression of G6PD, VLDLR, 
uduiinl/undulin/extracellular matrix glycoprotein, cytochrome p450-2El, and thymosin 
beta- 10 are indicative of an adult liver, and expression of calmodulin, hepatocyte gf, 
IGF binding protein 1, and ubiquitin are indicative of a fetal liver. 

3. A method of distinguishing between a fetal and adult brain tissue, 
comprising the steps of: 

assaying for expression in the tissue of two or more genes selected from the group 
consisting of: nicotinic acetylcholine receptor beta 2, ubiquitin, and thymosin beta- 10, 
wherein expression of nicotinic acetylcholine receptor beta 2 or ubiquitin indicates an 
adult brain. 

4. A method of determining the source of a tissue as adult brain or adult 
liver, comprising the steps of: 



WO 99/23254 



PCT/US98/22966 



- 19- 

assaying for expression in the tissue of two or more genes selected from the group 
consisting of: calcium channel, synaptotagamin, neuromodulin, calmodulin, nicotinic 
acetylcholine receptor beta 2, VLDLR, udulinl/undulin/extracellular matrix 
glycoprotein, pyruvate dehydrogenase El, apolipoprotein B100, hepatocyte gf, IGF 
5 binding protein 1, ubiquitin, bone morphogenetic protein precursor, and cytochrome 

p450-2El, wherein expression of calcium channel, synaptotagamin, neuromodulin, 
calmodulin , nicotinic acetylcholine receptor beta 2, ubiquitin, or bone morphogenetic 
protein precursor indicates a brain source for the tissue, and wherein expression of 
pyruvate dehydrogenase El, VLDLR, udulinl/undulin/extracellular matrix 
10 glycoprotein, apolipoprotein B100, thymosin beta- 10, hepatocyte gf, IGF binding 

protein 1, or cytochrome p450-El indicates a liver source for the tissue. 

5. A method of distinguishing a tissue source as fetal brain or fetal liver, 
comprising the steps of: 

assaying for expression in the tissue of two or more genes selected from the group 
IS consisting of: G6PD, calcium channel, synaptotagamin, neuromodulin, pyruvate 

dehydrogenase El, apolipoprotein B100, hepatocyte gf, IGF binding protein 1, 
ubiquitin, bone morphogenetic protein precursor, and thymosin beta* 10, wherein 
expression of G6PD, calcium channel, synaptotagamin, neuromodulin, thymosin beta- 
10 or bone morphogenetic protein precursor indicates a fetal brain source and 
20 expression of pyruvate dehydrogenase El, apolipoprotein B100, hepatocyte gf, IGF 

binding protein 1, ubiquitin, indicates a fetal liver source, 

6. The method of claim 1 , 2, 3, 4, or 5, wherein expression of at least 3 genes 
are assayed. 

7. The method of claim 1,2, 4, or 5, wherein expression of at least 4 genes 
25 are assayed. 

8. The method of claim 1,2, 4, or 5, wherein expression of at least 5 genes 
are assayed. 

9. The method of claim 1,2, 4, or 5, wherein expression of at least 6 genes 
are assayed. 

30 10. The method of claim 1,2, 4, or 5, wherein expression of at least 7 genes 

are assayed. 



WO 99/23254 



PCT/US98/22966 



-20- 



1 1 . The method of claim 1,2, 4, or 5, wherein expression of at least 8 genes 
are assayed. 

12. The method of claim 1,2, 4, or 5, wherein expression of at least 9 genes 
are assayed. 

5 13. The method of claim 1, 4, or 5, wherein expression of at least 10 genes 

are assayed. 

14. A solid support for screening a drug for deleterious side effects on a cell 
comprising: at least two oligonucleotides for probing two or more genes selected from 
the group consisting of: G6PD, calcium channel, synaptotagamin, neuromodulin, 

10 calmodulin, nicotinic acetylcholine receptor beta 2, VLDLR; 

udulinl/imdulin/extracellular matrix glycoprotein, pyruvate dehydrogenase El, 
apolipoprotein B100, hepatocyte gf, IGF binding protein 1, ubiquitin, bone 
morphogenetic protein precursor, cytochrome p450-2El , and thymosin beta- 1 0, wherein 
each oligonucleotide comprises a sequence which is complementary to one of the two 

15 or more genes. 

15. A solid support for distinguishing between a fetal and an adult liver 
sample comprising: two or more oligonucleotides for detecting two or more genes 
selected from the group consisting of: G6PD, calmodulin, VLDLR, 
udulinl/undulin/extracellular matrix glycoprotein, hepatocyte gf, IGF binding protein 

20 1 , ubiquitin, cytochrome p450-2El , and thymosin beta- 1 0 wherein each oligonucleotide 

comprises a sequence which is complementary to one of the two or more genes. 

16. A solid support for distinguishing between a fetal and adult brain tissue, 
comprising: two more oligonucleotides for detecting two or more genes selected from 
the group consisting of: nicotinic acetylcholine receptor beta 2, ubiquitin, and thymosin 

25 beta- 10, wherein each oligonucleotide comprises a sequence which is complementary 

to one of the two or more genes. 

17. A solid support for determining the source of a tissue as adult brain or 
adult liver, comprising: 

two or more oligonucleotides for detecting two or more genes selected from the 
30 group consisting of: calcium channel, synaptotagamin, neuromodulin, calmodulin, 

nicotinic acetylcholine receptor beta 2, VLDLR, udulinl/undulin/extracellular matrix 



WO 99/23254 



PCT7US98/22966 



-21- 

glycoprotein, pyruvate dehydrogenase El, apolipoprotein B100, hepatocyte gf, IGF 
binding protein 1, ubiquitin, bone morphogenetic protein precursor, and cytochrome 
p450-2El, wherein each oligonucleotide comprises a sequence which is complementary 
to one of the two or more genes. 

5 18. A solid support for distinguishing a tissue source as fetal brain or fetal 

liver, comprising: two or more oligonucleotides for detecting two or more genes 
selected from the group consisting of: G6PD, calcium channel, synaptotagamin, 
neuromodulin, pyruvate dehydrogenase El , apolipoprotein B 1 00, hepatocyte gf, IGF 
binding protein 1, ubiquitin, bone morphogenetic protein precursor, and thymosin beta- 

10 10, wherein each oligonucleotide comprises a sequence which is complementary to one 

of the two or more genes. 

19. The solid support of claim 14; 15, 16, 17, or 18 which is an array 
comprising at least 10 different oligonucleotides in discrete locations per cm 2 . 

20. The solid support of claim 14, 15, 16, 17, or 18 which is an array 
15 comprising at least 100 different oligonucleotides in discrete locations per cm 2 . 

21. The solid support of claim 14, 15, 16, 17, or 18 which is an array 
comprising at least 1,000 different oligonucleotides in discrete locations per cm 2 . 

22. The solid support of claim 14, 15, 16, 17, or 18 which is an array 
comprising at least 10,000 different oligonucleotides in discrete locations per cm 2 . 



20 



WO 99/23254 



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PCT/US98/22966 



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•WSOOCIO: <WO 9923254A1 I > 



SUBSTITUTE SHEET (RULE 26) 



INTERN ' TIO^K, SEARCH REPORT 



r Application No 

PCT/US 98/22966 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC 6 C12Q1/68 G01N33/50 



According to International Patent Classification (IPC) or to both national classification and IPC 



B. FIELDS SEARCHED 



Minimum documentation searched (classification system followed by classification symbols) 

IPC 6 C12Q 



Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched 



Electronic data base consulted during the international search (name of data base and. where practical, search terms used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category' 


Citation of document with indication, where appropriate, of the relevant passages 


Relevant to claim No. 


X 


WO 97 27317 A (CHEE MARK ;LAI CHAOOIANG 


1-22 




(US); LEE DANNY (US); AFFYMETRIX INC (US)) 






31 July 1997 






see the whole document 




X 


ZHAO ET AL.: "HIGH-DENSITY cDNA FILTER 


1-22 




ANALYSIS: NOVEL APPROACH FOR LARGE-SCALE, 






QUANTITATIVE ANALYSIS OF GENE EXPRESSION" 






GENE, 






vol. 156, 1995, pages 207-213, XP002094436 






see the whole document 






-/— 





m 



Further documents are listed in the continuation of box C. 



Patent family members are fisted In annex. 



* Special categories of cited documents : 

"A" document defining the general state of the art which Is not 

considered to be of particular relevance 
"E" earlier document but published on or after the international 

filing date 

*L" document which may throw doubts on priority daim(s) or 
which is cited to establish the publication date of another 
citation or other special reason (as specified) 

"O" document referring to an oral disclosure, use. exhibition or 
other means 

"P" document published prior to the international filing date but 
later than the priority date claimed 



*T" later document published after the international filing date 
or priority dale and not in conflict with the application but 
cited to understand the principle or theory underlying the 
invention 

"X" document of particular relevance; the claimed invention 
cannot be considered novel or cannot be considered to 
involve an inventive step when the document is taken alone 

"Y" document of particular relevance; the claimed Invention 

cannot be considered to Involve an inventive step when the 
document Is combined with one or more other such docu- 
ments, such combination being obvious to a person skilled 
in the art. 

document member of the same patent family 



Date of the actual completion of the international search 

23 February 1999 


Date of mailing of the international search report 

12/03/1999 


Name and mailing address of the ISA 

European Patent Office. P.B. 5616 Patentlaan 2 
NL-2280HVRijswi|k 
Tel. (+31-70) 340-2040, Tx. 31 651 epo nl. 
Fax: (+31-70) 340-3016 


Authorized officer 

Hagenmaler, S 



Form PCT/1SA/210 (Moond th*<rt) (July 1092) 



page 1 of 3 



INTE^^T 



^AL SEARCH REPORT 



I PCT 



tal Application No 

PCT/US 98/22966 



C.(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT 

Category ** Citation or document, with indication. where appropriate, of the relevant passages 



Relevant to daim No. 



TAKAHASHI ET AL. : "HIGH-DENSITY cDNA 
ANALYSIS OF THE EXPRESSION PROFILES OF THE 
GENES PREFERENTIALLY EXPRESSED IN HUMAN 
BRAIN" 
GENE, 

vol. 164, 1995, pages 219-227, XP002094437 
see the whole document 

SCHENA M ET AL: "PARALLEL HUMAN GENOME 
ANALYSIS: MICROARRAY-BASED EXPRESSION 
MONITORING OF 1000 GENES- 
PROCEEDINGS OF THE NATIONAL ACADEMY OF 
SCIENCES OF USA, 

vol. 93, no. 20, October 1996, pages 
10614-10619, XP002022507 
see the whole document 

B0RMAN S: "DNA CHIPS COME OF AGE AFTER 

PERIOD OF GESTATION, TECHNOLOGY FOR 

GENETIC ANALYSIS IS BLOSSOMING" 

CHEMICAL AND ENGINEERING NEWS, 

vol. 74, no. 50, 9 December 1996, page 

42/43 XP000633458 

see the whole document 

L0CKHART ET AL. : "EXPRESSION MONITORING 

BY HYBRIDIZATION TO HIGH-DENSITY 

OLIGONUCLEOTIDE ARRAYS" 

NATURE BIOTECHNOLOGY, 

vol. 14, December 1996, pages 1675-1680, 

XP002094438 

see the whole document 

SCHENA M ET AL: "QUANTITATIVE MONITORING 
OF GENE EXPRESSION PATTERNS WITH A 
COMPLEMENTARY DNA MICROARRAY" 
SCIENCE, 

vol. 270, no. 5235, 20 October 1995, pages 
467-470, XP000644675 
see the whole document 

KAUPPINEN ET AL.: "CONTRIBUTION OF 
CYTOPLASMIC POLYPEPTIDES TO THE PROTON NMR 
SPECTRUM OF DEVELOPING RAT CEREBRAL 
CORTEX" 

MAGN. RESON. MED., 

vol. 25, no. 2, 1992, pages 398-407, 

XP002094505 

See figure 3, page 404 

see the whole document 

WO 95 13374 A (BAYLOR COLLEGE MEDICINE) 

18 May 1995 

see the whole document 

-/— 



1-22 



1-22 



1-22 



1-22 



1-22 



1-22 



Fomi PCT/rSA/210 (continuation o< Mcond cheet) (Juty 1992) 
BNSOOCID: <WO 9923254A1 I > 



page 2 of 3 



INTERS TI 



SEARCH REPORT 



^ J^^ptppllcatlon No 

PCT/US 98/22966 



C.(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT 



Category " Citation of document, with indteatioawhere appropriate, of the relevant passages 



Relevant to claim No. 



EP 0 717 105 A ( PASTEUR INSTITUT) 

19 June 1996 

see the whole document 

WO 94 26905 A (US HEALTH ; NESTEC SA (CH)) 

24 November 1994 

see the whole document 



US 5 569 588 A (ASHBY MATTHEW 

29 October 1996 

see the whole document 



ET AL) 



P.X 



PATENT ABSTRACTS OF JAPAN 
vol. 096, no. 005, 31 May 1996 
& JP 08 009969 A (EISAI CO LTD), 
16 January 1996 
see abstract 

SEDLAK B J: "GENE CHIP TECHNOLOGY READY 

TO IMPACT DIAGNOSTIC MARKETS" 

GENETIC ENGINEERING NEWS, December 1997, 

page 1, 14, 34 XP000749581 

see the whole document 



1-22 



1-22 



1-22 



1-22 



1-22 



1 



Porni PCTrtSA/210 (continuation of second sheet) (Jtfy 1 WZ> 

«NSOOCI(><WO 0923254A1 1 > 



page 3 of 3 



tmormatlc 



NAL SEARCH REPORT 

tmSrmatto.. on patent family members 



at Application No 

PCT/US 98/22966 



Patent document 




1 Publication 




Patent family 




Publication 


cited in search report 




date 




member(s) 




dat 


wu y/^/oi/ 


A 

A 


31-07-1997 


AU 


2253397 


A 


20-08-1997 








EP 


0880598 


A 


02-12-1998 


WU 9b 133/4 


A 

A 


18-05-1995 


US 


5750367 


A 


12-05-1998 








AU 


1052695 


A 


29-05-1995 


- 






US 


5798209 


A 


25-08-1998 


EP 0717105 


A 


19-06-1996 


AU 


4037395 


A 


20-06-1996 








CA 


2165098 


A 


15-06-1996 








JP 


8242866 


A 


24-09-1996 








NZ 


280674 


A 


22-09-1997 


wu y^^oyub 


A 

A 


24-11-1994 


— — 

US 


5506131 


A 


09-04-1996 








AU 


697896 


B 


22-10-1998 








AU 


6914594 


A 


12-12-1994 


_ 






CA 


2163233 


A 


24-11-1994 








EP 


0700442 


A 


13-03-1996 








US 


5660986 A 


26-08-1997 




A 

A. 


Z9-10-1996 


AU 


6720996 


A 


05-03-1997 








CA 


2202154 


A 


20-02-1997 








EP 


0791078 A 


27-08-1997 








JP 


10507647 


T 


28-07-1998 








WO 


9706277 A 


20-02-1997 



Fom> PCT/TSA/210 (patent temBy anrwx) <JUy 1992) 
<WO 8S23264A 1 _l_> \