WO2014074942A1 - Risk variants of alzheimer's disease - Google Patents

Risk variants of alzheimer's disease Download PDF

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Publication number
WO2014074942A1
WO2014074942A1 PCT/US2013/069333 US2013069333W WO2014074942A1 WO 2014074942 A1 WO2014074942 A1 WO 2014074942A1 US 2013069333 W US2013069333 W US 2013069333W WO 2014074942 A1 WO2014074942 A1 WO 2014074942A1
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trem2
allele
disease
alzheimer
system
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PCT/US2013/069333
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French (fr)
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Hreinn Stefansson
Thorlakur JONSSON
Stacy STEINBERG
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Illumina, Inc.
Decode Genetics Ehf
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers

Abstract

The T allele of the single nucleotide polymorphism rs75932628 of the TREM2 gene encoding an arginine to histidine substitution at position 47 (R47H) of the TREM2 protein is associated with risk of Alzheimer's disease in humans. The present invention provides methods of determining susceptibility of Alzheimer's disease using the variant. Also provided are computer-implemented methods for determining susceptibility to Alzheimer's disease.

Description

RISK VARIANTS OF ALZHEIMER'S DISEASE

BACKGROUND

Alzheimer's disease (AD), the most common form of dementia in the elderly, is a

neurodegenerative disorder that is characterized by a slow but progressive loss of cognitive function, in particular memory. Extracellular amyloid plaques, intracellular neurofibrillary tangles, and loss of neurons and synapses resulting in brain atrophy are the main pathological hallmarks of AD. Onset of AD usually is after age 70, although the prevalence increases exponentially with age after the age of 65 and exceeds 25% in those over the age of 90.

The vast majority of variants in the sequence of the genome that have been shown to markedly affect the risk of AD are rare variants in APP, PSEN1 and PSEN2 (encoding amyloid precursor protein, presenilin-1 and presenilin-2, respectively) that appear to be fully penetrant and result in AD with an early onset, in most cases before age 60. These variants however do not shed light on the most common, late-onset form of AD. Although a number of common, low-risk variants have been reported to associate with late-onset AD, the ε4 allele of Apolipoprotein E, originally discovered as a risk factor for AD in 1993 remains by far the most important sequence variant affecting the risk of late-onset AD because of its prevalence and the size of its effect on risk.

The present invention provides variants in the human Triggering Receptor Expressed on Myeloid Cells-2 (TREM2) gene that confer risk of Alzheimer's disease.

SUMMARY

The present inventors have discovered that variants on chromosome 6p21 in the human TREM2 gene are predictive of risk of Alzheimer's disease. The present invention relates to the utilization of such variants in the risk management of Alzheimer's disease.

One aspect of the invention relates to a method of determining a susceptibility to Alzheimer's disease, the method comprising analyzing data representative of at least one allele of the TREM2 gene in a human subject, wherein different alleles of the human TREM2 gene are associated with different susceptibilities to Alzheimer's disease in humans, and determining a susceptibility to Alzheimer's disease for the human subject from the data . The analyzing may in certain embodiments comprise analyzing the data for the presence or absence of at least one mutant allele in TREM2 selected from the group consisting of a TREM2 missense allele, a TREM2 nonsense allele, a TREM2 promoter allele and a TREM2 3' UTR allele.

Another aspect relates to a method of determining whether an individual is at increased risk of developing Alzheimer's disease, the method comprising steps of (i) obtaining a biological sample containing nucleic acid from the individual; (ii) determining, in the biological sample, nucleic acid sequence about the human TREM2 gene; and (iii) comparing the sequence information to the wild-type sequence of human TREM2 (SEQ ID NO: 3); wherein an identification of a mutation in TREM2 in the individual is indicative that the individual is at increased risk of developing Alzheimer's disease.

The invention also provides a method of determining whether a human subject is at increased risk of developing Alzheimer's disease, the method comprising analyzing amino acid sequence data about an TREM2 polypeptide from the subject, wherein a determination of the presence of an altered TREM2 polypeptide compared with a wild-type TREM2 polypeptide with sequence as set forth in SEQ ID NO: 2 is indicative that the subject is at increased risk of developing Alzheimer's disease.

Also provided is a method of identifying a test sample having a TREM2 genotype that is predictive of risk of Alzheimer's disease in humans, comprising (a) obtaining a test sample containing nucleic acid from a human subject; (b) determining nucleic acid sequence about the human TREM2 gene in the test sample; and (c) determining whether a genotype of the TREM2 gene that correlates with increased risk of Alzheimer's disease in humans is present in the test sample; wherein determination of the presence of the genotype in the test sample identifies the test sample as having a TREM2 genotype that is predictive of Alzheimer's disease in humans.

The invention also provides methods of selecting individuals for therapy. Thus, the invention further provides a method of selecting a human subject with Alzheimer's disease, or a human subject experiencing symptoms characteristic for Alzheimer's disease, for treatment with a therapeutic regimen for treating Alzheimer's disease, the method comprising (i) obtaining a test sample comprising nucleic acid from the human subject; (ii) determine nucleic acid sequence about the human TREM2 gene in the nucleic acid; (iii) determine whether a genotype of the TREM2 gene that is predictive of increased risk of Alzheimer's disease in humans is present in the test sample; and (iv) selecting for treatment with the therapeutic regimen a subject identified as having a TREM2 genotype that is predictive of increased risk of Alzheimer's disease.

The invention also provides computer-implemented aspects for carrying out the methods described herein. In one such aspect, system for identifying susceptibility to Alzheimer's disease in a human subject, the system comprising (1) at least one processor; (2) at least one computer-readable medium; (3) a susceptibility database operatively coupled to a computer-readable medium of the system and containing population information correlating the presence or absence of one or more alleles of the human TREM2 gene and susceptibility to Alzheimer's disease in a population of humans; (4) a measurement tool that receives an input about the human subject and generates information from the input about the presence or absence of at least one mutant TREM2 allele in the human subject; and (5) an analysis tool that (i) is operatively coupled to the susceptibility database and the measurement tool; (ii) is stored on a computer-readable medium of the system; and (iii) is adapted to be executed on a processor of the system, to compare the information about the human subject with the population information in the susceptibility database and generate a conclusion with respect to susceptibility to Alzheimer's disease for the human subject.

Further details of these and other aspects of the inventions are described in the following detailed description of the invention.

It should be understood that all combinations of features described herein are contemplated, even if the combination of features is not specifically found in the same sentence or paragraph as set forth in the following detailed description. This refers for example to the variants described herein to be association with risk of Alzheimer's disease, which may all be useful in the various aspects of the invention, as described herein .

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention .

FIG 1 provides a diagram illustrating a system comprising computer implemented methods utilizing risk variants as described herein .

FIG 2 shows an exemplary system for determining risk of Alzheimer's disease as described further herein .

FIG 3 shows a system for selecting a treatment protocol for a subject diagnosed with Alzheimer's disease.

FIG 4 shows CPS scores of carriers (filled symbols) and non-carriers (open symbols) of rs75932628-T as a function of age. The graph shows data based on two year bins, i.e. the data point for 81 years of age contains data for ages 80 and 81, etc, except for the last bin which represents ages 98, 99 and 100. No CPS data was available for carriers in the last age bin . Each symbol represents the average CPS score of individuals in the respective age bin (in years) . Error bars represent ± 1 s.e. (standard error) . The graph is based on 307 data points from 53 carriers, and 24,152 data points from 3,699 non-carriers. Patients with AD diagnosis were not included in the analysis.

DETAILED DESCRIPTION

Definitions

Unless otherwise indicated, nucleic acid sequences are written left to right in a 5' to 3' orientation . Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by the ordinary person skilled in the art to which the invention pertains.

The following terms shall, in the present context, have the meaning as indicated :

A "polymorphic marker", sometime referred to as a "marker", as described herein, refers to a genomic polymorphic site. Each polymorphic marker has at least two sequence variations characteristic of particular alleles at the polymorphic site. Thus, genetic association to a polymorphic marker implies that there is association to at least one specific allele of that particular polymorphic marker. The marker can comprise any allele of any variant type found in the genome, including SNPs, mini- or microsatellites, insertion-deletions, translocations and copy number variations (insertions, deletions, duplications) . Polymorphic markers can be of any measurable frequency in the population. For mapping of disease genes, polymorphic markers with population frequency higher than 5-10% are in general most useful . However, polymorphic markers may also have lower population frequencies, such as 1-5% frequency, or even lower frequency, such as 0.1 to 1% frequency. The term shall, in the present context, be taken to include polymorphic markers with any population frequency.

An "allele" refers to the nucleotide sequence of a given locus (position) on a chromosome. A polymorphic marker allele thus refers to the composition (i .e., sequence) of the marker on a chromosome. Genomic DNA from an individual contains two alleles (e.g., allele-specific sequences) for any given polymorphic marker, representative of each copy of the marker on each chromosome. Sequence codes for nucleotides used herein are: A = 1, C = 2, G = 3, T = 4.

Sequence conucleotide ambiguity as described herein is according to WIPO ST.25 :

Figure imgf000005_0001
A nucleotide position at which more than one sequence is possible in a population (either a natural population or a synthetic population, e.g. , a library of synthetic molecules) is referred to herein as a "polymorphic site".

A "Single Nucleotide Polymorphism" or "SNP" is a DNA sequence variation occurring when a single nucleotide at a specific location in the genome differs between members of a species or between paired chromosomes in an individual. Most SNP polymorphisms have two alleles. Each individual is in this instance either homozygous for one allele of the polymorphism (i.e. both chromosomal copies of the individual have the same nucleotide at the SNP location), or the individual is heterozygous (i.e. the two sister chromosomes of the individual contain different nucleotides) . The SNP nomenclature as reported herein refers to the official Reference SNP (rs) ID identification tag as assigned to each unique SNP by the National Center for Biotechnological Information (NCBI) .

A "variant", as described herein, refers to a segment of DNA that comprises a polymorphic site. A "marker" or a "polymorphic marker", as defined herein, is a variant.

A "microsatellite" is a polymorphic marker that has multiple small repeats of bases that are 2-8 nucleotides in length (such as CA repeats) at a particular site, in which the number of repeat lengths varies in the general population.

An "indel" or an "insertion-deletion" is a common form of polymorphism comprising a small insertion or deletion that is typically only a few nucleotides long.

A "haplotype," as described herein, refers to a segment of genomic DNA that is characterized by a specific combination of alleles arranged along the segment. For diploid organisms such as humans, a haplotype comprises one member of the pair of alleles for two or more polymorphic markers or loci along the segment. In a certain embodiment, the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles.

Allelic identities are described herein in the context of the marker name and the particular allele of the marker, e.g., "rs75932628 allele T" refers to the T allele of marker rs75932628. Furthermore, allelic codes are as for individual markers, i.e. 1 = A, 2 = C, 3 = G and 4 = T.

The term "TREM2", as described herein, refers to the Triggering Receptor Expressed on Myeloid Cells-2 gene on chromosome 6p21.1. The gene is sometimes abbreviated as TREM2. The sequence of the TREM2 gene is set forth in SEQ ID NO: 3 herein, its cDNA is set forh in SEQ ID NO: l herein (corresponding to cDNA with NCBI Reference Sequence ID:

NM_018965.2), and the sequence of the encoded TREM2 protein is set forth in SEQ ID NO: 2 herein .

The term "susceptibility", as described herein, refers to the proneness of an individual towards the development of a certain state (e.g. , a certain trait, phenotype or disease, e.g., Alzheimer's disease), or towards being less able to resist a particular state than the average individual. The term encompasses both increased susceptibility and decreased susceptibility. Thus, particular alleles at polymorphic markers may be characteristic of increased susceptibility (i.e., increased risk) of Alzheimer's disease, as characterized by a relative risk (RR) or odds ratio (OR) of greater than one for the particular allele (e.g., allele T of rs75932628) .

The term "and/or" shall in the present context be understood to indicate that either or both of the items connected by it are involved. In other words, the term herein shall be taken to mean "one or the other or both".

The term "look-up table", as described herein, is a table that correlates one form of data to another form, or one or more forms of data to a predicted outcome to which the data is relevant, such as phenotype or trait. For example, a look-up table can comprise a correlation between allelic data for at least one polymorphic marker and a particular trait or phenotype, such as a diagnostic evaluation for Alzheimer's disease. Look-up tables can be

multidimensional, i.e. they can contain information about multiple alleles for single markers simultaneously, or they can contain information about multiple markers, and they may also comprise other factors, such as particulars about diseases diagnoses, racial information, biomarkers, biochemical measurements, therapeutic methods or drugs, etc.

The term "database" refers to a collection of data organized for one or more purposes. In the context of the invention, databases may be organized in a digital format for access, analysis, or processing by a computer. The data are typically organized to model features relevant to the invention . For instance, one component of data in a database may be information about variations in a population, such as genetic variation with respect to TREM2, but also variation with respect to other medically informative parameters, including other genetic loci, race, ethnicity, sex, age, behaviors and lifestyle (tobacco consumption (smoking), alcohol consumption (drinking), exercise, body mass indices), glucose tolerance/diabetes, blood pressure, lipid profile, cholesterol levels and any other factors that medical personnel may measure in the context of standard medical care or specific diagnoses. Other components of the database may include one or more sets of data relating to susceptibility to Alzheimer's disease in a population, and/or suitability or success of disease treatment, and/or suitability or success of a protocol for screening for Alzheimer's disease. Preferably the data is organized to permit analysis of how the biological variation in the population correlates with the susceptibility to the disease and/or the suitability or success of the treatment, protocol, etc. A look-up datable (or the information in a look-up table) may be stored in a database to facilitate aspects of the invention.

A "computer-readable medium", is an information storage medium that can be accessed by a computer using a commercially available or custom-made interface. Exemplary computer- readable media include memory (e.g., RAM, ROM, flash memory, etc.), optical storage media (e.g., CD-ROM), magnetic storage media (e.g., computer hard drives, floppy disks, etc.), punch cards, or other commercially available media. Information may be transferred between a system of interest and a medium, between computers, or between computers and the computer-readable medium for storage or access of stored information. Such transmission can be electrical, or by other available methods, such as IR links, wireless connections, etc.

The term "biological sample" refers to a sample obtained from an individual that contains nucleic acid and/or protein and/or fluid containing organic and/or inorganic metabolites and substances. In many variations of the invention, the biological sample comprises nucleic acid suitable for genetic analysis.

A "nucleic acid sample" as described herein, refers to a sample obtained from an individual that contains nucleic acid (DNA or RNA). In certain embodiments, i.e. the detection of specific polymorphic markers and/or haplotypes, the nucleic acid sample comprises genomic DNA. Such a nucleic acid sample can be obtained from any source that contains genomic DNA, including a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.

The term "antisense agent" or "antisense oligonucleotide" refers, as described herein, to molecules, or compositions comprising molecules, which include a sequence of purine an pyrimidine heterocyclic bases, supported by a backbone, which are effective to hydrogen bond to corresponding contiguous bases in a target nucleic acid sequence. The backbone is composed of subunit backbone moieties supporting the purine and pyrimidine heterocyclic bases at positions which allow such hydrogen bonding. These backbone moieties are cyclic moieties of 5 to 7 atoms in size, linked together by phosphorous-containing linkage units of one to three atoms in length. In certain preferred embodiments, the antisense agent comprises an oligonucleotide molecule.

TREM2 variation is predictive of Alzheimer's disease It has been discovered that genetic variation in the human TREM2 gene on chromosome 6p21 is predictive of risk of Alzheimer's disease. Missense variation in the TREM2 gene has been shown to confer greatly with increased risk of Alzheimer's disease.

In particular allele T of the marker rs75932628, which is located at position 3007 in the nucleic acid sequence of the TREM gene as set forth in SEQ ID NO:3 herein, encodes a argininge (R) to histidine (H) substitution at position 47 in the TREM protein as shown in SEQ ID NO: 2 herein. A C to T mutation at position 3007 in the TREM2 gene as set forth in SEQ ID NO:3 (position X in the cDNA sequence of TREM2 as set forth in SEQ ID NO:l herein) is found more commonly in individuals with Alzheimer's disease than in controls. This allele has been found to confer increased risk of Alzheimer's disease. The inventors have furthermore identified novel missense and nonsense varia nts at severa l additional positions in the human TREM2 gene, which are believed to represent fu rther variations that may confer risk of Alzheimer's disease . Identified varia nts a re sum marized in the va riant table.

Variant Table. Shown is identity of variant, its position in TREM2 cDNA (SEQ ID NO: l), TREM2 gene (SEQ ID NO: 3), flanking sequence of the variant, minor and major alleles, frequency of minor allele, and the encoded functional effect (with respect to SEQ ID NO: 2). Note that the cDNA sequence in SEQ ID NO: l is the reverse complement of the gene sequence in SEQ ID NO: 3

Figure imgf000009_0001

TREM2 was originally identified as a DAP12 associated receptor, expressed on macrophages and dendritic cells, and has also been shown to be expressed on osteoclasts and microglia . TREM2 is a tra nsmembra ne g lycoprotein, consisting of an extracellular im munog lobulin-like domain, a tra nsmem brane domain and a cytoplasmic tai l, which associates with DAP12 for its signalling fu nction . TREM2 has both exogenous liga nds on pathogens a nd endogenous ligands that remain largely u nknown, although a recent study has shown that Hsp60 is a n agonist of TREM2 in neu roblastoma cells and astrocytes, a nd a n endogenous ligand on dendritic cells (DCs) has been found .

In brai n, TREM2 is prima rily expressed on microg lia . Activation of microglia may lead to phagocytosis of cell debris and a myloid, but microglia can also be activated to promote production of pro-inflam matory cytokines, or they may differentiate into antigen-presenting cells. A recent study showed that TREM2 expression is induced concomitantly with the formation of a myloid plaques in APP transgenic mice expressing the Swedish (K670N/M671L) mutation in APP, a nd this expression was fou nd to correlate positively with amyloid phagocytosis by unactivated microglia . The expression of TREM2 a lso correlated positively with the a bility of microglia to stimulate CD4+ T-cell to proliferation, as well as their secretion of TNF and CCL2 but not IFNy into the extracel lula r milieu . TREM2+ microglia on plaques may thus captu re and present self-a ntigens to CNS-infiltrating lymphocytes without promoting pro-inflam matory responses. Fu rthermore, knockdown of TREM2 or DAP12 in microglia resu lts in reduced phagocytosis of apoptotic neurons, whereas overexpression of TREM2 increases the same, suggesting that microglia recognize and phagocytose apoptotic neurons via TREM2 ligation . TREM2 has an anti-inflammatory function; it inhibits macrophage response to Toll like receptor (TLR) ligation and it negatively regulates TLR-mediated maturation of dendritic cells, type I interferon responses and induction of antigen-specific T- cell proliferation . Furthermore, TREM2 stimulation of dendritic cells induces partial activation without any production of pro-inflammatory cytokines.

The R47H substitution encoded by rs75932628-T is located within the extracellular immunoglobulin-like domain of TREM2. It is postulated that the R47H amino acid substitution, or other amino acid substitution in TREM2 (i.e. missense variants or missense amino acid substitutions may result in decreased affinity of TREM2 for its natural ligands and affect its signalling. Alternatively, it is possible that rs75932628-T may affect proteolytic processing of TREM2 by γ-secretase. It has thus recently been proposed that TREM2 may represent a proteolytic substrate for γ-secretase.

The present inventors have shown that risk of Alzheimer's disease is influenced by rare mutations in the human TREM2 gene with large effects. These findings have significant implications for diagnostics of Alzheimer's disease, as described in the more detail herein .

Methods of determining susceptibility to Alzheimer's disease Accordingly, in one aspect, the invention provides a method of determining a susceptibility to Alzheimer's disease, the method comprising steps of (i) analyzing data representative of at least one allele of the TREM2 gene in a human subject, wherein different alleles of the human TREM2 gene are associated with different susceptibilities to Alzheimer's disease in humans, and

(ii) determining a susceptibility to Alzheimer's disease for the human subject from the data.

The data can be any type of data that is representative of polymorphic alleles in the TREM2 gene. In certain embodiments, the data is nucleic acid sequence data . The sequence data can be data that are sufficient to provide information about particular alleles in a nucleic acid sequence. In certain embodiments, the sequence data identifies the nucleotides at one specific position in a nucleic acid . In certain other embodiments, the sequence data identifies nucleotides at two or more sequential position in a nucleic acid. In certain embodiments, the nucleic acid sequence data is obtained from a biological sample comprising or containing nucleic acid from the human individual. The nucleic acids sequence may suitably be obtained using a method that comprises at least one procedure selected from (i) amplification of nucleic acid from the biological sample; (ii) hybridization assay using a nucleic acid probe and nucleic acid from the biological sample; (iii) hybridization assay using a nucleic acid probe and nucleic acid obtained by amplification of the biological sample, and (iv) sequencing, in particular high-throughput sequencing . The nucleic acid sequence data may also be obtained from a preexisting record. For example, the preexisting record may comprise a genotype dataset for at least one polymorphic marker. In certain embodiments, the determining comprises comparing the sequence data to a database containing correlation data between the at least one polymorphic marker and susceptibility to Alzheimer's disease. In certain embodiments, the sequence data is provided as genotype data, identifying the presence or absence of particular alleles at polymorphic locations.

In some embodiments, the analyzing comprises analyzing the data for the presence or absence of at least one mutant allele indicative of a defect in TREM2, such as a defect in an expressed TREM2 protein . The defect may for example be a missense mutation in the TREM2 gene relative to a wild-type TREM2 gene, such as the wild-type TREM2 gene with sequence as presented in SEQ ID NO: 3 herein. In one embodiment, anaysis of nucleic acid data includes analyzing the data for the presence or absence of at least one mutant allele in TREM2 selected from the group consisting of a TREM2 missense allele, a TREM2 nonsense allele, a TREM2 promoter allele and a TREM2 3' UTR allele. In one preferred embodiment, the missense allele is allele T of rs75932628, which encodes an arginine to histidine substitution at position 47 in TREM2 protein (SEQ ID NO: 2) .

Alternatively, the defect may be a premature truncation or frameshift of an encoded TREM2 protein, relative to a wild-type amino acid sequence, such as the wild-type amino acid sequence presented in SEQ ID NO: 2 herein . The TREM2 defect may also be expression of a TREM2 protein with reduced activity compared to a wild-type TREM2 protein. The defect can for example be a defect in the binding of a TREM2 ligand to TREM2, such as Hsp60. The defect can alternatively be a defect in the binding of TREM2 to DAP12. Alternatively, the defect is a defect in the proteolytic processing of TREM2 by γ-secretase. In one embodiment, the TREM2 defect is selected from one of these defects.

Determination of activities such as protein-protein binding, e.g. the interaction of TREM2 to its natural ligands, e.g. Hsp60, or to DAP12, can be performed using standard assays well known to the skilled person, some of which are described herein . Such assays can be used to confirm that a particular TREM2 mutation impairs or eliminates TREM2 activity and therefore would be expected to result in an increased susceptibility to Alzheimer's disease.

Alternatively, analyzing data to determine risk of Alzheimer's disease may comprise analyzing a biological sample from a human subject to obtain information selected from the group consisting of (a) nucleic acid sequence information, wherein the nucleic acid sequence information comprises sufficient sequence to identify the presence or absence of a TREM2 mutant allele in the subject; (b) nucleic acid sequence information, wherein the nucleic acid sequence information identifies at least one allele of a polymorphic marker in linkage disequilibrium (LD) with the mutant allele, wherein the LD is characterized by a value for r2 of at least 0.5; (c) measurement of the quantity or length of TREM2 mRNA, wherein the measurement is indicative of the presence or absence of the mutant a llele; (d) measurement of the quantity of TREM2 protein, wherein the measurement is indicative of the presence or absence of the mutant allele; and (e) measurement of TREM2 activity, wherein the measurement is indicative of the presence or absence of the mutant allele. In one embodiment, the TREM2 mutant allele is a mutant allele that results in a TREM2 defect, as described in the above.

Another aspect of the invention relates to a method of determining whether an individual is at increased risk of developing Alzheimer's disease, the method comprising steps of (i) obtaining a biological sample containing nucleic acid from the individual; (ii) determining, in the biological sample, nucleic acid sequence about the human TREM2 gene; (iii) analyzing the sequence information to determine the presence or absence of a TREM2 mutation that is indicative of increased risk of Alzheimer's disease in humans; and (iv) determining whether the human subject is at increased risk of Alzheimer's disease based on the presence of the

TREM2 mutation in the test sample. The mutation is in certain embodiments a mutation that results in a TREM2 defect selected from (a) premature truncation or frameshift of an encoded TREM2 protein, relative to the TREM2 amino acid sequence set forth in SEQ ID NO: 2; (b) expression of a TREM2 protein with reduced activity compared to a wild-type TREM2 protein; (c) reduced expression of TREM2 protein, compared to wild-type expression of TREM2; (d) reduced activity of TREM2 protein, compared with the activity of wild-type TREM2.

The data to be analyzed by the method of the invention is suitably obtained by analysis of a biological sample from a human subject to obtain information about particular alleles in the genome of the individual. In certain embodiments, the information is nucleic acid information which comprises sufficient sequence to identify the presence or absence of at least one allele in the subject (e.g. a mutant allele) . The information can also be nucleic acid information that identifies at least one allele of a polymorphic marker that is in linkage disequilibrium with a mutant allele.

Linkage disequilibrium may suitably be determined by the correlation coefficient between polymorphic sites. In one embodiment, the sites are correlated by values of the correlation coefficient r2 of greater than 0.5. Other suitable values of r2 that are also appropriate to characterize polymorphic sites in LD are however also contemplated, as discussed further herein .

The information may also be information about measurement of quantity of length of TREM2 mRNA, wherein the measurement is indicative of the presence or absence of the mutant allele. For example, mutant alleles may result in premature truncation of transcribed mRNA which can be detected by measuring the length of mRNA. The information may further be measurement of quantity of TREM2 protein, wherein the measurement of protein is indicative of the presence or absence of a mutant allele. Truncated transcripts will result in truncated forms of translated polypeptides, which can be measured using standard methods known in the art. For example, truncated proteins or proteins arising from a frameshift may have fewer or different epitopes from wild-type protein and can be distinguished with

immunoassays. Truncated proteins or proteins altered in other ways may migrate differently and be distinguished with electrophoresis. The information obtained may also be

measurement of TREM2 biological activity, wherein the measurement is indicative of the mutant allele. The activity is suitably selected from binding activity to DAP12, and binding activity to Hsp60. In one embodiment, the information is selected from any one of the above mentioned types of information .

Thus, in certain embodiments, analyzing data comprises analyzing a biological sample from the human subject to obtain information selected from the group consisting of (a) nucleic acid sequence information, wherein the nucleic acid sequence information comprises sequence sufficient to identify the presence or absence of the mutant allele in the subject; (b) nucleic acid sequence information, wherein the nucleic acid sequence information identifies at least one allele of a polymorphic marker in linkage disequilibrium (LD) with the mutant allele, wherein the LD is characterized by a value for r2 of at least 0.5;(c)

measurement of the quantity or length of TREM2 mRNA, wherein the measurement is indicative of the presence or absence of the mutant a llele; (d) measurement of the quantity of TREM2 protein, wherein the measurement is indicative of the presence or absence of the mutant allele; and (e) measurement of TREM2 activity, wherein the measurement is indicative of the presence or absence of the mutant allele.

In a further embodiment of the invention, a biological sample is obtained from the human subject prior to the analyzing steps. The analyzing may also suitably be performed by analyzing data from a preexisting record about the human subject. The preexisting record may for example include sequence information or genotype information about the individual, which can identify the presence or absence of mutant alleles.

In certain embodiments, information about risk for the human subject can be determined using methods known in the art. Some of these methods are described herein . For example, information about odds ratio (OR), relative risk (RR) or lifetime risk (LR) can be determined from information about the presence or absence of particular mutant alleles of TREM2.

In certain embodiments, the mutant allele of TREM2 is a missense mutation, a frameshift mutation, a splicing mutation or a nonsense mutation . In one preferred embodiment, the mutant allele is a missense mutation . In another preferred embodiment, the mutant allele is a nonsense mutation. In another preferred embodiment, the mutant allele is a frameshift mutation. In another preferred embodiment, the mutant allele is a splicing mutation. In certain embodiments, the mutation is rs75932628-T, which results in a C to T substitution in position 3007 a TREM2 gene with sequence as set forth in SEQ ID NO: 3. In another embodiment, the mutant allele is a missense mutation in TREM2 that results in expression of a TREM2 protein with reduced or no activity compared to a wild-type TREM2 protein . The activity may suitably be protein binding activity (e.g., binding to Hsp60, or binding to DAP12) . The mutant allele may also be a promoter polymorphism that leads to decreased expression of TREM2 compared with wild-type TREM2. In certain embodiments, the mutant allele in TREM2 is selected from the group consisting of rs79011726 allele T, chr6:41236977 allele C, rsl42232675 allele T, rsl43332484 allele T, rs75932628 allele T and chr6:41237236 allele T.

In certain embodiments, the mutant allele in TREM2 encodes one of the following amino acid substitutions in TREM2 : E151K, D87N, R62H, R47H and G45E. IN one preferred

embodiment, the mutant allele encodes an R47H amino acid substitution in TREM2.

It should be apparent from the foregoing that another aspect of the invention relates to a method of determining whether an individual is at increased risk of developing Alzheimer's disease, the method comprising steps of (a) obtaining a biological sample containing nucleic acid from the individual; (b) determining, in the biological sample, nucleic acid sequence about the TREM2 gene, and (c) comparing the sequence information to the wild-type sequence of TREM2, as set forth in SEQ ID NO: 3 herein, wherein the identification of a mutation in TREM2 in the individual is indicative that the individual is at increased risk of developing Alzheimer's disease.

A further aspect of the invention provides a method of determining a susceptibility to

Alzheimer's disease, the method comprising analyzing sequence data from a human subject for at least one variant in the human TREM2 gene, or in an encoded human TREM2 protein, wherein different alleles of the at least one variant are associated with different

susceptibilities to Alzheimer's disease in humans, and determining a susceptibility to

Alzheimer's disease for the human subject from the sequence data. In a preferred embodiment, the variant is a missense variant in the TREM2 gene. In another preferred embodiment, the variant is rs75932628, and wherein determination of the presence of allele T in rs75932628 is indicative of an increased susceptibility to Alzheimer's disease. In another embodiment, the variant is a variant that is correlated with rs75932628 by a value for the correlation coefficient r2 > 0.5.

The skilled person will appreciate that the present invention can be carried out by detecting either strand of a nucleic acid, i.e. a sense strand or an antisense strand. Thus, in certain embodiments of the invention, the allele that is detected is the allele of a complementary strand of DNA, such that the nucleic acid sequence data identifies an allele which is complement to the allele that would be detected on the alternate strand. For example, in one embodiment the allele that is detected may be the complementary allele of the at-risk T allele of rs75932628. It is well known to the skilled person that detecting either strand of DNA will produce quantitatively equal results, i.e. an equal estimate of risk based on the presence or absence of particular risk alleles of Alzheimer's disease.

It is contemplated that in certain embodiments of the invention, it may be convenient to prepare a report of results of risk assessment. Thus, certain embodiments of the methods of the invention comprise a further step of preparing a report containing results from the determination of risk, wherein said report is written in a computer readable medium, printed on paper, or displayed on a visual display. In certain embodiments, it may be convenient to report results of susceptibility to at least one entity selected from the group consisting of the individual, a guardian of the individual, a genetic service provider, a physician, a medical organization, and a medical insurer.

Obtaining nucleic acid sequence data

Sequence data can be nucleic acid sequence data, which may be obtained by means known in the art. Sequence data is suitably obtained from a biological sample of genomic DNA, RNA, or cDNA (a "test sample") from an individual ("test subject) . For example, nucleic acid sequence data may be obtained through direct analysis of the sequence of the polymorphic position (allele) of a polymorphic marker. Suitable methods, some of which are described herein, include, for instance, whole genome sequencing methods, whole genome analysis using SNP chips (e.g., Infinium HD BeadChip), cloning for polymorphisms, non-radioactive PCR-single strand conformation polymorphism analysis, denaturing high pressure liquid chromatography (DHPLC), DNA hybridization, computational analysis, single-stranded conformational polymorphism (SSCP), restriction fragment length polymorphism (RFLP), automated fluorescent sequencing; clamped denaturing gel electrophoresis (CDGE);

denaturing gradient gel electrophoresis (DGGE), mobility shift analysis, restriction enzyme analysis; heteroduplex analysis, chemical mismatch cleavage (CMC), RNase protection assays, use of polypeptides that recognize nucleotide mismatches, such as E. coli mutS protein, allele-specific PCR, and direct manual and automated sequencing. These and other methods are described in the art (see, for instance, Li et al., Nucleic Acids Research, 28(2) : el (i-v) (2000); Liu et al., Biochem Cell Bio 80 : 17-22 (2000); and Burczak et al.,

Polymorphism Detection and Analysis, Eaton Publishing, 2000; Sheffield et al., Proc. Natl. Acad. Sci. USA, 86: 232-236 (1989); Orita et al., Proc. Natl. Acad. Sci. USA, 86: 2766-2770 (1989); Flavell et al., Cell, 15 : 25-41 (1978); Geever et al., Proc. Natl. Acad. Sci. USA,

78: 5081-5085 (1981); Cotton et al., Proc. Natl. Acad. Sci. USA, 85:4397-4401 (1985); Myers et al., Science 230 : 1242-1246 (1985); Church and Gilbert, Proc. Natl. Acad. Sci. USA, 81 : 1991-1995 (1988); Sanger et al., Proc. Natl. Acad. Sci. USA, 74: 5463-5467 (1977); and Beavis et al., U.S. Patent No. 5,288,644) .

Recent technological advances have resulted in technologies that allow massive parallel sequencing to be performed in relatively condensed format. These technologies share sequencing-by-synthesis principle for generating sequence information, with different technological solutions implemented for extending, tagging and detecting sequences.

Exemplary technologies include 454 pyrosequencing technology (Nyren, P. et al. Anal Biochem 208: 171-75 (1993); http ://www.454.com), Illumina Solexa sequencing technology (Bentley, D.R. Curr Opin Genet Dev 16: 545-52 (2006); http://www.illumina .com), and the SOLiD technology developed by Applied Biosystems (ABI)

(http://www.appliedbiosystems.com; see also Strausberg, R.L., et al. Drug Disc Today 13 : 569-77 (2008)) . Other sequencing technologies include those developed by Pacific Biosciences (http://www.pacificbiosciences.com), Complete Genomics

(http://www.completegenomics.com), Intelligen Bio-Systems

(http://www.intelligentbiosystems.com), Genome Corp (http://www.genomecorp.com), ION Torrent Systems (http://www.iontorrent.com) and Helicos Biosciences

(http ://www. helicosbio.som). It is contemplated that sequence data useful for performing the present invention may be obtained by any such sequencing method, or other sequencing methods that are developed or made available. Thus, any sequence method that provides the allelic identity at particular polymorphic sites (e.g., the absence or presence of particular alleles at particular polymorphic sites) is useful in the methods described and claimed herein . Alternatively, hybridization methods may be used (see Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, including all supplements) . For example, a biological sample of genomic DNA, RNA, or cDNA (a "test sample") may be obtained from a test subject. The subject can be an adult, child, or fetus. The DNA, RNA, or cDNA sample is then examined . The presence of a specific marker allele can be indicated by sequence-specific hybridization of a nucleic acid probe specific for the particular allele. The presence of more than one specific marker allele or a specific haplotype can be indicated by using several sequence-specific nucleic acid probes, each being specific for a particular allele. A sequence- specific probe can be directed to hybridize to genomic DNA, RNA, or cDNA. A "nucleic acid probe", as used herein, can be a DNA probe or an RNA probe that hybridizes to a

complementary sequence. One of skill in the art would know how to design such a probe so that sequence specific hybridization will occur only if a particular allele is present in a genomic sequence from a test sample.

In certain embodiments, determination of a susceptibility to Alzheimer's disease comprises forming a hybridization sample by contacting a test sample, such as a genomic DNA sample, with at least one nucleic acid probe. A non-limiting example of a probe for detecting mRNA or genomic DNA is a labeled nucleic acid probe that is capable of hybridizing to mRNA or genomic DNA sequences described herein . The nucleic acid probe can be, for example, a full- length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 10, 15, 30, 50, 100, 250 or 500 nucleotides in length that is sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA. For example, the nucleic acid probe can comprise all or a portion of the nucleotide sequence of the TREM2 gene, or the probe can be the complementary sequence of such a sequence. Hybridization can be performed by methods well known to the person skilled in the art (see, e.g ., Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, including all supplements) . In one embodiment, hybridization refers to specific hybridization, i .e., hybridization with no mismatches (exact hybridization) . In one embodiment, the

hybridization conditions for specific hybridization are high stringency.

Specific hybridization, if present, is detected using standard methods. If specific

hybridization occurs between the nucleic acid probe and the nucleic acid in the test sample, then the sample contains the allele that is complementary to the nucleotide that is present in the nucleic acid probe.

Additionally, or alternatively, a peptide nucleic acid (PNA) probe can be used in addition to, or instead of, a nucleic acid probe in the hybridization methods described herein. A PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen et al., Bioconjug. Chem. 5 : 3-7 (1994)) . The PNA probe can be designed to specifically hybridize to a molecule in a sample suspected of containing one or more of the at-risk alleles (mutations) shown herein to be associated with risk of Alzheimer's disease.

Allele-specific oligonucleotides can also be used to detect the presence of a particular allele in a nucleic acid. An "allele-specific oligonucleotide" (also referred to herein as an "allele- specific oligonucleotide probe") is an oligonucleotide of any suitable size, for example an oligonucleotide of approximately 10-50 base pairs or approximately 15-30 base pairs, that specifically hybridizes to a nucleic acid which contains a specific allele at a polymorphic site (e.g., a polymorphic marker) . An allele-specific oligonucleotide probe that is specific for one or more particular alleles at polymorphic markers can be prepared using standard methods (see, e.g ., Current Protocols in Molecular Biology, supra) . PCR can be used to amplify the desired region. Specific hybridization of an allele-specific oligonucleotide probe to DNA from a subject is indicative of the presence of a specific allele at a polymorphic site (see, e.g ., Gibbs et al., Nucleic Acids Res. 17: 2437-2448 (1989) and WO 93/22456) .

In certain embodiments, arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from a subject can be used to identify particular alleles in a nucleic acid. For example, an oligonucleotide array can be used. Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods that incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods, or by other methods known to the person skilled in the art (see, e.g., Bier et al., Adv Biochem Eng Biotechnol 109 :433-53 (2008); Hoheisel, Nat Rev Genet 7 : 200-10 (2006); Fan et al., Methods Enzymol 410 : 57-73 (2006); Raqoussis & Elvidge, Expert Rev Mol Diagn 6: 145-52 (2006); Mockler et al., Genomics 85 : 1-15 (2005), and references cited therein, the entire teachings of each of which are incorporated by reference herein) . Many additional descriptions of the preparation and use of oligonucleotide arrays for detection of

polymorphisms can be found, for example, in US 6,858,394, US 6,429,027, US 5,445,934,

US 5,700,637, US 5,744,305, US 5,945,334, US 6,054,270, US 6,300,063, US 6,733,977, US 7,364,858, EP 619 321, and EP 373 203, the entire teachings of which are incorporated by reference herein . Also, standard techniques for genotyping can be used to detect particular marker alleles, such as fluorescence-based techniques (e.g., Chen et al., Genome Res. 9(5) : 492-98 (1999); Kutyavin et al., Nucleic Acid Res. 34: el28 (2006)), utilizing PCR, LCR, Nested PCR and other techniques for nucleic acid amplification. Specific commercial methodologies available for SNP genotyping include, but are not limited to, TaqMan genotyping assays and SNPlex platforms (Applied Biosystems), gel electrophoresis (Applied Biosystems), mass spectrometry (e.g ., MassARRAY system from Sequenom), minisequencing methods, real-time PCR, Bio-Plex system (BioRad), CEQ and SNPstream systems (Beckman), array hybridization

technology(e.g., Affymetrix GeneChip; Perlegen ), BeadArray Technologies (e.g ., Illumina GoldenGate and Infinium assays), array tag technology (e.g., Parallele), and endonuclease- based fluorescence hybridization technology (Invader; Third Wave) .

Suitable biological sample in the methods described herein can be any sample containing nucleic acid (e.g ., genomic DNA) and/or protein from the human individual. For example, the biological sample can be a blood sample, a serum sample, a leukapheresis sample, an amniotic fluid sample, a cerbrospinal fluid sample, a hair sample, a tissue sample from skin, muscle, buccal, or conjuctival mucosa, placenta, gastrointestinal tract, or other organs, a semen sample, a urine sample, a saliva sample, a nail sample, a tooth sample, and the like. Preferably, the sample is a blood sample, a saliva sample or a buccal swab.

Protein analysis

Missense, nonsense and frameshift nucleic acid variations may lead to an altered amino acid sequence, as compared to the non-variant (e.g., wild-type) protein, due to amino acid substitutions, deletions, or insertions, or truncations. Variations at splice sites may also lead to splice variation. In such instances, detection of an amino acid substitution or a truncated amino acid sequence of the variant protein may be useful. Thus, nucleic acid sequence data may be obtained through indirect analysis of the nucleic acid sequence of the allele of the polymorphic marker, i.e. by detecting a protein variation .

The variants described herein result in altered TREM2 protein. Accordingly, one aspect of the invention relates to a method of determining whether a human subject is at increased risk of developing Alzheimer's disease, the method comprising analyzing amino acid sequence data about a TREM2 polypeptide from the subject, wherein a determination of the presence of an altered TREM2 polypeptide compared with a wild-type TREM2 polypeptide with sequence as set forth in SEQ ID NO : 2 is indicative that the subject is at increased risk of developing Alzheimer's disease. In one embodiment, the amino acid sequence data is obtained from a biological sample comprising TREM2 polypeptide from the subject. In one embodiment, the amino acid sequence data is obtained from a biological sample from the human subject comprising human TREM2 polypeptide, using a method that comprises an antibody assay or protein sequencing. In a further embodiment, the amino acid sequence data is obtained from a preexisting record. In certain embodiment, the altered TREM2 polypeptide is a TREM2 polypeptide that contains a missense mutation or a nonsense mutation compared with wild-type TREM2. In certain embodiments, the altered TREM2 polypeptide has a reduced activity compared with wild-type TREM2, wherein the activity is protein binding activity. In one embodiment, the protein binding activity is Hsp60 binding activity or DAP12 binding activity. In another embodiment, the protein binding activity is binding to a TREM2 natural ligand.

Methods of detecting variant proteins are known in the art. For example, direct amino acid sequencing of the variant protein followed by comparison to a reference amino acid sequence can be used. Alternatively, SDS-PAGE followed by gel staining can be used to detect variant proteins of different molecular weights. Also, Immunoassays, e.g., antibody assays, e.g., immunofluorescent immunoassays, immunoprecipitations, radioimmunoasays, ELISA, and Western blotting, in which an antibody specific for an epitope comprising the variant sequence among the variant protein and non-variant or wild-type protein can be used. In certain embodiments, the amino acid sequence data about TREM2 protein is obtained or deduced from a preexisting record.

In certain embodiments of the present invention, an amino acid substitution in the human TREM2 protein is detected. In certain embodiments, the amino acid substitution is selected from the group consisting of E151K, D87N, R62H, R47H and G45E. In one preferred embodiment, the amino acid substitution is R47H. In another embodiment, a truncated polypeptide encoded by an altered TREM2 gene sequence is detected. The detection may be suitably performed, for example using any of the methods described in the above, or any other suitable method known to the skilled artisan.

Number of Polymorphic Markers/Genes Analyzed

With regard to the methods of determining a susceptibility to Alzheimer's disease described herein, the methods can comprise obtaining sequence data about any number of polymorphic markers and/or about any number of genes. For example, the method can comprise obtaining sequence data for about at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 500, 1000, 10,000 or more polymorphic markers. In certain embodiments, the sequence data is obtained from a microarray comprising probes for detecting a plurality of markers. In certain other embodiments, the sequence data is obtained through nucleic acid sequencing, for example by high-throughput nucleic acid sequencing. The sequence data may also be obtained by imputation of nucleic acid sequence using known methods, such as those described herein . The polymorphic markers can be the ones of the group specified herein or they can be different polymorphic markers that are not specified herein. In a specific embodiment, the method comprises obtaining sequence data about at least two polymorphic markers. In certain embodiments, each of the markers may be associated with a different gene. For example, in some instances, if the method comprises obtaining nucleic acid data about a human individual identifying at least one allele of a polymorphic marker, then the method comprises identifying at least one allele of at least one polymorphic marker. Also, for example, the method can comprise obtaining sequence data about a human individual identifying alleles of multiple, independent markers, which are not in linkage disequilibrium.

Linkage Disequilibrium

Linkage Disequilibrium (LD) refers to a non-random assortment of two genetic elements. For example, if a particular genetic element (e.g. , an allele of a polymorphic marker, or a haplotype) occurs in a population at a frequency of 0.50 (50%) and another element occurs at a frequency of 0.50 (50%), then the predicted occurrance of a person's having both elements is 0.25 (25%), assuming a random distribution of the elements. However, if it is discovered that the two elements occur together at a frequency higher than 0.25, then the elements are said to be in linkage disequilibrium, since they tend to be inherited together at a higher rate than what their independent frequencies of occurrence (e.g., allele or haplotype frequencies) would predict. Roughly speaking, LD is generally correlated with the frequency of recombination events between the two elements. Allele or haplotype frequencies can be determined in a population by genotyping individuals in a population and determining the frequency of the occurence of each allele or haplotype in the population. For populations of diploids, e.g., human populations, individuals will typically have two alleles for each genetic element (e.g., a marker, haplotype or gene) .

Many different measures have been proposed for assessing the strength of linkage disequilibrium (LD; reviewed in Devlin, B. & Risch, N., Genomics 29 : 311-22 (1995)) . Most capture the strength of association between pairs of biallelic sites. Two important pairwise measures of LD are r2 (sometimes denoted Δ2) and | D'| (Lewontin, R., Genetics 49:49-67 (1964); Hill, W.G. & Robertson, A. Theor. Appl. Genet. 22: 226-231 (1968)) . Both measures range from 0 (no disequilibrium) to 1 ('complete' disequilibrium), but their interpretation is slightly different. | D'| is defined in such a way that it is equal to 1 if just two or three of the possible haplotypes are present, and it is < 1 if all four possible haplotypes are present. Therefore, a value of | D'| that is < 1 indicates that historical recombination may have occurred between two sites (recurrent mutation can also cause | D'| to be < 1, but for single nucleotide polymorphisms (SNPs) this is usually regarded as being less likely than recombination) . The measure r2 represents the statistical correlation between two sites, and takes the value of 1 if only two haplotypes are present. Markers which are correlated by an r2 value of 1 are said to be perfectly correlated. In such an instance, the genotype of one marker perfectly predicts the genotype of the other.

The r2 measure is arguably the most relevant measure for association mapping, because there is a simple inverse relationship between r2 and the sample size required to detect association between susceptibility loci and SNPs. These measures are defined for pairs of sites, but for some applications a determination of how strong LD is across an entire region that contains many polymorphic sites might be desirable (e.g., testing whether the strength of LD differs significantly among loci or across populations, or whether there is more or less LD in a region than predicted under a particular model) . Measuring LD across a region is not straightforward, but one approach is to use the measure r, which was developed in population genetics. Roughly speaking, r measures how much recombination would be required under a particular population model to generate the LD that is seen in the data. This type of method can potentially also provide a statistically rigorous approach to the problem of determining whether LD data provide evidence for the presence of recombination hotspots.

A significant r2 indicative of markers being in linkage disequilibrium may be at least 0.1, such as at least 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or 1.0. A significant r2 indicates that the markers are highly correlated, and therefore in linkage disequilibrium. Highly correlated markers must, be definition, show highly comparable results in association mapping, since the genotypes for one marker predicts the genotype for another, correlated, marker. In one specific embodiment of invention, the significant r2 value can be at least 0.2. In another specific embodiment of invention, the significant r2 value can be at least 0.5. In one specific embodiment of invention, the significant r2 value can be at least 0.8. Alternatively, linkage disequilibrium as described herein, refers to linkage disequilibrium characterized by values of r2 of at least 0.2, such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.85, 0.9, 0.95, 0.96, 0.97, 0.98, 0.99. Thus, linkage disequilibrium represents a correlation between alleles of distinct markers. It is measured by correlation coefficient or | D'| (r2 up to 1.0 and | D'| up to 1.0) . Linkage disequilibrium can be determined in a single human population, as defined herein, or it can be determined in a collection of samples comprising individuals from more than one human population . In one embodiment of the invention, LD is determined in a sample from one or more of the HapMap populations. These include samples from the Yoruba people of Ibadan, Nigeria (YRI), samples from individuals from the Tokyo area in Japan (JPT), samples from individuals Beijing, China (CHB), and samples from U.S. residents with northern and western European ancestry (CEU), as described (The International HapMap Consortium, Nature 426: 789-796 (2003)) . In one such embodiment, LD is determined in the Caucasian CEU population of the HapMap samples. In another embodiment, LD is determined in the African YRI population . In another embodiment, LD is determined in samples from the lOOOgenomes project (http://www.1000genomes.org) . In yet another embodiment, LD is determined in samples from the Icelandic population .

If all polymorphisms in the genome were independent at the population level {i.e., no LD between polymorphisms), then every single one of them would need to be investigated in association studies, to assess all different polymorphic states. However, due to linkage disequilibrium between polymorphisms, tightly linked polymorphisms are strongly correlated, which reduces the number of polymorphisms that need to be investigated in an association study to observe a significant association. Another consequence of LD is that many polymorphisms may give an association signal due to the fact that these polymorphisms are strongly correlated.

Genomic LD maps have been generated across the genome, and such LD maps have been proposed to serve as framework for mapping disease-genes (Risch, N . & Merkiangas, K, Science 273 : 1516-1517 (1996); Maniatis, N ., et al., Proc Natl Acad Sci USA 99 : 2228-2233 (2002); Reich, DE et al, Nature 411 : 199-204 (2001)) .

It is now established that many portions of the human genome can be broken into series of discrete haplotype blocks containing a few common haplotypes; for these blocks, linkage disequilibrium data provides little evidence indicating recombination (see, e.g., Wall ., J.D. and Pritchard, J. K., Nature Reviews Genetics 4: 587-597 (2003); Daly, M . et al., Nature Genet. 29: 229-232 (2001); Gabriel, S.B. et al., Science 296: 2225-2229 (2002); Patil, N . et al., Science 294: 1719-1723 (2001); Dawson, E. et al., Nature 4.28: 544-548 (2002); Phillips, M .S. et al., Nature Genet. 33: 382-387 (2003)) .

Haplotype blocks (LD blocks) can be used to map associations between phenotype and haplotype status, using single markers or haplotypes comprising a plurality of markers. The main haplotypes can be identified in each haplotype block, and then a set of "tagging" SNPs or markers (the smallest set of SNPs or markers needed to distinguish among the haplotypes) can then be identified. These tagging SNPs or markers can then be used in assessment of samples from groups of individuals, in order to identify association between phenotype and haplotype. If desired, neighboring haplotype blocks can be assessed concurrently, as there may also exist linkage disequilibrium among the haplotype blocks.

It has thus become apparent that for any given observed association to a polymorphic marker in the genome, it is likely that additional markers in the genome also show association. This is a natural consequence of the uneven distribution of LD across the genome, as observed by the large variation in recombination rates. The markers used to detect association thus in a sense represent "tags" for a genomic region (i.e., a haplotype block or LD block) that is associating with a given disease or trait, and as such are useful for use in the methods and kits of the invention .

By way of example, the rs75932628 polymorphism may be detected directly to determine risk of Alzheimer's disease. Alternatively, any marker in linkage disequilibrium with the rs75932628 marker may be detected to determine risk. In other embodiments, markers in linkage disequilibrium with any one of the markers rs79011726, chr6:41236977, rsl42232675, rsl43332484, and chr6:41237236 may be used to determine risk.

Suitable surrogate markers may be selected using public information, such as from the International HapMap Consortium (http://www. hapmap.org) and the International lOOOgenomes Consortium (http://www.1000genomes.org) . The markers may also be suitably selected from results of whole-genome sequencing . The stronger the linkage disequilibrium (i.e., the higher the correlation) to the anchor marker, the better the surrogate, and thus the mores similar the association detected by the surrogate is expected to be to the association detected by the anchor marker. Markers with values of r2 equal to 1 (markers that are perfectly correlated) are perfect surrogates for the at-risk variants, i.e. genotypes for one marker perfectly predicts genotypes for the other. In other words, the surrogate will, by necessity, give exactly the same association data to any particular disease as the anchor marker. Markers with smaller values of r2 than 1 can also be surrogates for the at-risk anchor variant. For example, in certain embodiments, markers with r2 values greater than 0.5 are suitable surrogates for an at-risk variant. Association analysis

For single marker association to a disease, the Fisher exact test can be used to calculate two- sided p-values for each individual allele. Correcting for relatedness among patients can be done by extending a variance adjustment procedure previously described (Risch, N. &Teng, J. Genome Res., 8:1273-1288 (1998)) for sibships so that it can be applied to general familial relationships. The method of genomic controls (Devlin, B. & Roeder, K. Biometrics 55:997 (1999)) can also be used to adjust for the relatedness of the individuals and possible stratification.

For both single-marker and haplotype analyses, relative risk (RR) and the population attributable risk (PAR) can be calculated assuming a multiplicative model (haplotype relative risk model) (Terwilliger, J.D. & Ott, J., Hum. Hered.42:337-46 (1992) and Falk, C.T. & Rubinstein, P, Ann. Hum. Genet. 51 (Pt 3):227-33 (1987)), i.e., that the risks of the two alleles/haplotypes a person carries multiply. For example, if RR is the risk of A relative to a, then the risk of a person homozygote AA will be RR times that of a heterozygote Aa and RR2 times that of a homozygote aa. The multiplicative model has a nice property that simplifies analysis and computations— haplotypes are independent, i.e., in Hardy-Weinberg equilibrium, within the affected population as well as within the control population. As a consequence, haplotype counts of the affecteds and controls each have multinomial distributions, but with different haplotype frequencies under the alternative hypothesis.

Specifically, for two haplotypes, Λ, and h risk(ft)/risk(ftj) = (fi/Pi)/(fj/Pj), where fand p denote, respectively, frequencies in the affected population and in the control population. While there is some power loss if the true model is not multiplicative, the loss tends to be mild except for extreme cases. Most importantly, p-values are always valid since they are computed with respect to null hypothesis.

Risk assessment and Diagnostics

Within any given population, there is an absolute risk of developing a disease or trait, defined as the chance of a person developing the specific disease or trait over a specified time-period. For example, a woman's lifetime absolute risk of breast cancer is one in nine. That is to say, one woman in every nine will develop breast cancer at some point in their lives. Risk is typically measured by looking at very large numbers of people, rather than at a particular individual. Risk is often presented in terms of Absolute Risk (AR) and Relative Risk (RR) . Relative Risk is used to compare risks associating with two variants or the risks of two different groups of people. For example, it can be used to compare a group of people with a certain genotype with another group having a different genotype. For a disease, a relative risk of 2 means that one group has twice the chance of developing a disease as the other group. The risk presented is usually the relative risk for a person, or a specific genotype of a person, compared to the population with matched gender and ethnicity. Risks of two individuals of the same gender and ethnicity could be compared in a simple manner. For example, if, compared to the population, the first individual has relative risk 1.5 and the second has relative risk 0.5, then the risk of the first individual compared to the second individual is 1.5/0.5 = 3.

Risk Calculations

The creation of a model to calculate the overall genetic risk involves two steps: i) conversion of odds-ratios for a single genetic variant into relative risk and ii) combination of risk from multiple variants in different genetic loci into a single relative risk value.

Deriving risk from odds-ratios

Most gene discovery studies for complex diseases that have been published to date in authoritative journals have employed a case-control design because of their retrospective setup. These studies sample and genotype a selected set of cases (people who have the specified disease condition) and control individuals. The interest is in genetic variants (alleles) which frequency in cases and controls differ significantly.

The results are typically reported in odds ratios, that is the ratio between the fraction (probability) with the risk variant (carriers) versus the non-risk variant (non-carriers) in the groups of affected versus the controls, i.e. expressed in terms of probabilities conditional on the affection status:

OR = (Pr(c|A)/Pr(nc|A)) / (Pr(c| C)/Pr(nc|C))

Sometimes it is however the absolute risk for the disease that we are interested in, i .e. the fraction of those individuals carrying the risk variant who get the disease or in other words the probability of getting the disease. This number is typically not directly measured in case- control studies, in part, because the ratio of cases versus controls is typically not the same as that in the general population . However, under certain assumption, we can estimate the risk from the odds ratio.

It is well known that under the rare disease assumption, the relative risk of a disease can be approximated by the odds ratio. This assumption may however not hold for many common diseases. Still, it turns out that the risk of one genotype variant relative to another can be estimated from the odds ratio expressed above. The calculation is particularly simple under the assumption of random population controls where the controls are random samples from the same population as the cases, including affected people rather than being strictly unaffected individuals. To increase sample size and power, many of the large genome-wide association and replication studies use controls that were neither age-matched with the cases, nor were they carefully scrutinized to ensure that they did not have the disease at the time of the study. Hence, they often approximate a random sample from the general population . It is noted that this assumption is rarely expected to be satisfied exactly, but the risk estimates are usually robust to moderate deviations from this assumption .

Calculations show that for the dominant and the recessive models, where we have a risk variant carrier, "c", and a non-carrier, "nc", the odds ratio of individuals is the same as the risk ratio between these variants:

OR = Pr(A| c)/Pr(A| nc) = r

And likewise for the multiplicative model, where the risk is the product of the risk associated with the two allele copies, the allelic odds ratio equals the risk factor:

OR = Pr(A| aa)/Pr(A| ab) = Pr(A| ab)/Pr(A| bb) = r

Here "a" denotes the risk allele and "b" the non-risk allele. The factor "r" is therefore the relative risk between the allele types.

For many of the studies published in the last few years, reporting common variants associated with complex diseases, the multiplicative model has been found to summarize the effect adequately and most often provide a fit to the data superior to alternative models such as the dominant and recessive models.

Determining risk

In the present context, an individual who is at an increased susceptibility (i.e., increased risk) for Alzheimer's disease is an individual who is carrying at least one at-risk variant as described herein . In certain embodiments, the variant is within the human TREM2 gene, or a variant encoded by a variation in the human TREM2 gene. In one embodiment, significance associated with a marker is measured by a relative risk (RR) . In another embodiment, significance associated with a marker or haplotye is measured by an odds ratio (OR) . In a further embodiment, the significance is measured by a percentage. In one embodiment, a significant increased risk is measured as a risk (relative risk and/or odds ratio) of at least 1.5, including but not limited to at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3.0, at least 3.1, at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 4.0, at least 4.5, or at least 5.0. In a particular embodiment, a risk (relative risk and/or odds ratio) of at least 2.0 is significant. In another particular embodiment, a risk of at least 2.5 is significant.

An at-risk variant as described herein is one where at least one allele of at least one marker (e.g., allele T of marker rs75932628) is more frequently present in an individual at risk for Alzheimer's disease (affected), or diagnosed with Alzheimer's disease, compared to the frequency in a comparison group (control), such that the presence of the marker allele is indicative of susceptibility to Alzheimer's disease. The control group may in one embodiment be a population sample, i.e. a random sample from the general population . In another embodiment, the control group is represented by a group of individuals who are disease-free, i.e. individuals who have not been diagnosed with Alzheimer's disease. In another

embodiment, the control group is represented by a group of individuals who have lived to a certain age (for example, age 70, or age80) and remain free of cognitive decline.

The person skilled in the art will appreciate that for markers with two alleles present in the population being studied (such as SNPs), and wherein one allele is found in increased frequency in a group of individuals with a trait or disease in the population, compared with controls, the other allele of the marker will be found in decreased frequency in the group of individuals with the trait or disease, compared with controls. In such a case, one allele of the marker (the one found in increased frequency in individuals with the trait or disease) will be the at-risk allele, while the other allele will be a protective allele.

Database

Determining susceptibility can alternatively or additionally comprise comparing nucleic acid sequence data and/or protein sequence data (e.g., genotype data) to a database containing correlation data between polymorphic markers and susceptibility to Alzheimer's disease. The database can be part of a computer-readable medium described herein.

In a specific aspect of the invention, the database comprises at least one measure of susceptibility to Alzheimer's disease for the polymorphic markers. For example, the database may comprise risk values associated with particular genotypes at such markers. The database may also comprise risk values associated with particular genotype combinations for multiple such markers.

In another specific aspect of the invention, the database comprises a look-up table containing at least one measure of susceptibility to Alzheimer's disease for the polymorphic markers.

Further steps

The methods disclosed herein can comprise additional steps which may occur before, after, or simultaneously with one of the aforementioned steps of the method of the invention . In a specific embodiment of the invention, the method of determining a susceptibility to Alzheimer's disease further comprises reporting the susceptibility to at least one entity selected from the group consisting of the individual, a guardian of the individual, a genetic service provider, a physician, a medical organization, and a medical insurer. The reporting may be accomplished by any of several means. For example, the reporting can comprise sending a written report on physical media or electronically or providing an oral report to at least one entity of the group, which written or oral report comprises the susceptibility. Alternatively, the reporting can comprise providing the at least one entity of the group with a login and password, which provides access to a report comprising the susceptibility posted on a password-protected computer system.

Study population

In a general sense, the methods and kits described herein can be utilized from samples containing nucleic acid material (DNA or RNA) or protein material from any source and from any individual, or from genotype or sequence data derived from such samples. In preferred embodiments, the individual is a human individual. The individual can be an adult, child, or fetus. The individual can suitably be either male or female. The nucleic acid or protein source may be any sample comprising nucleic acid or protein material, including biological samples, or a sample comprising nucleic acid or protein material derived therefrom. The present invention also provides for assessing markers in individuals who are members of a target population . Such a target population is in one embodiment a population or group of individuals at risk of developing Alzheimer's disease, based on other genetic factors, biomarkers, biophysical parameters, or lifestyle factors. In certain embodiments, the group individuals comprises individuals above a certain age, e.g., age 60 years, age 65 years, age 70 years, age 75 years, or age 80 years.

The Icelandic population is a Caucasian population of Northern European ancestry. A large number of studies reporting results of genetic linkage and association in the Icelandic population have been published in the last few years. Many of those studies show replication of variants, originally identified in the Icelandic population as being associating with a particular disease, in other populations (Kiemeney, L.A. et al. Nat Genet 42 :415-19 (2010); Thorleifsson, G. et al. Nat Genet 41 : 926-30 (2009); Sulem, P., et al. Nat Genet 41 : 734-8 (2009); Rafnar, T., et al. Nat Genet 41 : 221-7 (2009); Greta rsdottir, S., et al. Ann Neurol 64:402-9 (2008); Stacey, S. N ., et al. Nat Genet 40 : 1313-18 (2008); Gudbjartsson, D.F., et al. Nat Genet 40 : 886-91 (2008); Styrkarsdottir, U., et al. N Engl J Med 358: 2355-65 (2008); Thorgeirsson, T., et al. Nature 452 :638-42 (2008); Gudmundsson, J., et al. Nat Genet. 40 : 281-3 (2008); Stacey, S.N ., et al., Nat Genet. 39 : 865-69 (2007); Helgadottir, A., et al., Science 316: 1491-93 (2007); Steinthorsdottir, V., et al., Nat Genet. 39 : 770-75 (2007); Gudmundsson, J., et al., Nat Genet. 39 :631-37 (2007); Frayling, TM, Nature Reviews Genet 8:657-662 (2007); Amundadottir, L.T., et al., Nat Genet. 38:652-58 (2006); Grant, S.F., et al., Nat Genet. 38: 320-23 (2006)) . Thus, genetic findings in the Icelandic population have in general been replicated in other populations, including populations from Africa and Asia .

It is thus believed that variants in TREM2 described herein to be associated with risk of Alzheimer's disease will show similar association in other human populations. It is further contemplated that additional variants in the human TREM2 gene may be conferring risk of Alzheimer's disease in other populations. Particular embodiments comprising individual human populations are thus also contemplated and within the scope of the invention. Such embodiments relate to human subjects that are from one or more human population including, but not limited to, White populations, Caucasian populations, European populations, American populations, Eurasian populations, Asian populations, Central/South Asian populations, East Asian populations, and African populations. In certain embodiments, the invention pertains to individuals from Caucasian populations. In certain embodiments, the invention pertains to Icelandic individuals. Thus certain embodiments relate to individual of an ancestry that includes any one, or a combination of, the human populations listed in the above.

In certain embodiments, the invention relates to markers identified in specific populations, as described in the above. The person skilled in the art will appreciate that measures of linkage disequilibrium (LD) may give different results when applied to different populations. This is due to different population history of different human populations as well as differential selective pressures that may have led to differences in LD in specific genomic regions. It is also well known to the person skilled in the art that certain markers, e.g. SNP markers, have different population frequency in different populations, or are polymorphic in one population but not in another. The person skilled in the art will however apply the methods available and as taught herein to practice the present invention in any given human population. This may include assessment of polymorphic markers in the LD region of the present invention, so as to identify those markers that give strongest association within the specific population. Thus, the at-risk variants of the present invention may reside on different haplotype background and in different frequencies in various human populations. However, utilizing methods known in the art and the markers of the present invention, the invention can be practiced in any given human population .

Diagnostic Methods

Polymorphic markers associated with increased susceptibility of Alzheimer's disease are useful in diagnostic methods. While methods of diagnosing Alzheimer's disease are known in the art, the detection risk markers for Alzheimer's disease advantageously may be useful for detection of Alzheimer's disease at its early stages and may also reduce the occurrence of misdiagnosis. In this regard, the invention further provides methods of diagnosing

Alzheimer's disease comprising obtaining sequence data identifying at least one risk allele as described herein, in conjunction with carrying out one or more clinical diagnostic steps for the identification of Alzheimer's disease. Alzheimer's disease has a slow onset, diagnoses not being established unless a progressive loss of function in one or multiple cognitive domains, in particular memory, is observed. Genetic variation that increases risk of Alzheimer's disease may thus be extremely useful for identification of disease in an individual who presents with mild symptoms which do not fulfill established diagnostic criteria but, when observed in the context of a particular genetic risk variant, such as those described herein, may be considered sufficient to establish a clinical diagnoses or at least be considered sufficient to apply therapeutic measures. The genetic variants described herein are thus useful to determine whether particular individuals are at high risk of developing Alzheimer's disease, such that onset of Alzheimer's disease may be monitored closely and appropriate

ameliorating or preventive treatment be undertaken at an early stage.

In certain embodiments, a sample containing genomic DNA or protein from an individual is collected. Such sample can for example be a buccal swab, a saliva sample, a blood sample, or other suitable samples containing genomic DNA or protein, as described further herein . In certain embodiments, the sample is obtained by non-invasive means (e.g., for obtaining a buccal sample, saliva sample, hair sample or skin sample) . In certain embodiments, the sample is obtained by non-surgical means, i.e. in the absence of a surgical intervention on the individual that puts the individual at substantial health risk. Such embodiments may, in addition to non-invasive means also include obtaining sample by extracting a blood sample (e.g., a venous blood sample) . The genomic DNA or protein obtained from the individual is then analyzed using any common technique available to the skilled person, such as high- throughput technologies for genotyping and/or sequencing . Results from such methods are stored in a convenient data storage unit, such as a data carrier, including computer databases, data storage disks, or by other convenient data storage means. In certain embodiments, the computer database is an object database, a relational database or a post- relational database. The genotype data is subsequently analyzed for the presence of certain variants known to be susceptibility variants for Alzheimer's disease, such as the variants described herein . Genotype and/or sequencing data can be retrieved from the data storage unit using any convenient data query method. Calculating risk conferred by a particular genotype for the individual can be based on comparing the genotype of the individual to previously determined risk (expressed as a relative risk (RR) or and odds ratio (OR), for example) for the genotype, for example for an heterozygous carrier of an at-risk variant. The calculated risk for the individual can be the relative risk for a person, or for a specific genotype of a person, compared to the average population with matched gender and ethnicity. The average population risk can be expressed as a weighted average of the risks of different genotypes, using results from a reference population, and the appropriate calculations to calculate the risk of a genotype group relative to the population can then be performed. Alternatively, the risk for an individual is based on a comparison of particular genotypes, for example heterozygous carriers of an at-risk allele of a marker compared with non-carriers of the at-risk allele. The calculated risk estimated can be made available to the customer via a website, preferably a secure website.

Prognostic methods

In addition to the utilities described above, the polymorphic markers of the invention are useful in determining a prognosis of a human individual with Alzheimer's disease. The variants described herein are indicative of risk of Alzheimer's disease. Individuals carrying mutant alleles that predispose to Alzheimer's disease are at increased risk of developing Alzheimer's disease. Such mutant alleles are useful in a prognostic context.

For example, the prognosis can relate to the progression of the deterioration of cognitive decline in individuals who present with memory deficits or other cognitive deficits that are consistent with early stages of Alzheimer's disease and that may develop, over time, leading to formal diagnosis of Alzheimer's disease. For example, in one embodiment an individual who presents with cognitive deficits and is a carrier of one of the risk alleles for Alzheimer's disease described herein is likely to have a worse prognosis than an individual who does not carry the risk allele. In other embodiments, the prognosis can relate to the severity of the symptoms, e.g. the rate of appearance or significance of the cognitive deficits, or how the disease will respond to therapeutic treatment.

In general, individuals who are diagnosed early with Alzheimer's disease are more likely to respond well to current therapeutic measures than those individuals who are diagnosed late, i.e. those individuals who have developed severe symptoms when diagnosed . It is therefore imperative that individuals that are likely to benefit early from treatment be identified at the early stages of disease. Such identification may include establishment of the presence of a genetic risk factor for Alzheimer's disease, such as one of the risk variants described herein, in the context of early cognitive deficits that may develop into Alzheimer's disease.

Accordingly, the invention provides a method of predicting prognosis of an individual at risk for, or diagnosed with, Alzheimer's disease. The method comprises analyzing data representative of at least one allele of a TREM2 gene in a human subject, wherein different alleles of the human TREM2 gene are associated with different susceptibilities to Alzheimer's disease in humans, and determining a prognosis of the human subject from the data. The analyzing may comprise analysis for a mutation in TREM that leads to reduced or altered activity of an encoded TREM2 protein. In certain embodiments, the analyzing comprises analyzing for the presence or absence of at least one mutant allele indicative of a TREM2 defect. The defect may in certain embodiments be a protein binding defect, e.g. binding of TREM2 to Hsp60 or binding to DAP12, relative to TREM2 protein with a wild-type amino acid sequence as set forth in SEQ ID NO: 2, expression of an TREM2 protein with reduced activity compared to a wild-type TREM2 protein, and reduced expression of TREM2 protein, compared to wild-type TREM2.

With regard to the prognostic methods described herein, the sequence data can be nucleic acid sequence data or amino acid sequence data. For example, in one embodiment, determination of the presence of a missense mutation, a frameshift mutation or a nonsense mutation in TREM2 is indicative of prognosis of the Alzheimer's disease. The determination of the presence of a mutation in TREM2 that leads to reduced or altered activity of TREM2 is in certain embodiments indicative of a worsened prognosis of Alzheimer's disease. In other words, the presence of such mutations is in certain embodiments indicative that the individual has a worse prognosis than do individuals with Alzheimer's disease who do not carry such mutations.

In certain embodiments, the allele is rs75932628 allele T, which encodes an arginine to histidine amino acid substitution in a TREM2 polypeptide with wild-type sequence as set forth in SEQ ID NO: 2.

In some variations, the prognostic method further includes one or more additional steps, such as a step relating to generating the data by analyzing a biological sample; and/or a step involving selecting or administering a medial protocol to the subject, as described elsewhere herein .

Kits

Kits useful in the methods of the invention comprise components useful in any of the methods described herein, including for example, primers for nucleic acid amplification, hybridization probes (e.g. probes for detecting particular mutant alleles), restriction enzymes (e.g., for RFLP analysis), allele-specific oligonucleotides, antibodies, e.g., antibodies that bind to an altered TREM2 polypeptide (e.g. a missense variant in TREM2 or a truncated TREM2 polypeptide) or to a non-altered (native) TREM2 polypeptide, means for amplification of nucleic acids, means for analyzing the nucleic acid sequence of nucleic acids, means for analyzing the amino acid sequence of polynucleotides, etc. The kits can for example include necessary buffers, nucleic acid primers for amplifying nucleic acids (e.g., a nucleic acid segment comprising one or more of the polymorphic markers as described herein), and reagents for allele-specific detection of the fragments amplified using such primers and necessary enzymes (e.g. , DNA polymerase) . Additionally, kits can provide reagents for assays to be used in combination with the methods of the present invention, e.g., reagents for use with other diagnostic assays for Alzheimer's disease.

In one embodiment, the invention pertains to a kit for assaying a sample from a subject to detect a susceptibility to Alzheimer's disease in the subject, wherein the kit comprises reagents necessary for selectively detecting at least one at-risk variant for Alzheimer's disease in the individual, wherein the at least one at-risk variant is a nucleic acid variant in the human TREM2 gene or an amino acid substitution in an encoded TREM2 protein . In certain embodiments, the markers encode an TREM2 protein with a reduced or altered activity compared with wild-type TREM2. In a particular embodiment, the reagents comprise at least one contiguous oligonucleotide that hybridizes to a fragment of the genome of the individual comprising at least one polymorphism of the present invention . In another embodiment, the reagents comprise at least one pair of oligonucleotides that hybridize to opposite strands of a genomic segment obtained from a subject, wherein each oligonucleotide primer pair is designed to selectively amplify a fragment of the genome of the individual that includes at least one variant associated with risk of Alzheimer's disease. In one embodiment, the variant is rs75932628 allele T. In yet another embodiment the fragment is at least 20 base pairs in size. Such oligonucleotides or nucleic acids (e.g. , oligonucleotide primers) can be designed using portions of the nucleic acid sequence flanking the polymorphism. In another embodiment, the kit comprises one or more labeled nucleic acids capable of allele- specific detection of one or more specific polymorphic markers or haplotypes, and reagents for detection of the label. Suitable labels include, e.g., a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.

In certain embodiments, determination of the presence of a particular variant (i.e., marker allele) is indicative of a increased susceptibility of Alzheimer's disease. In another embodiment, determination of the presence of a particular marker allele is indicative of prognosis of Alzheimer's disease, or selection of appropriate therapy for Alzheimer's disease. In another embodiment, the presence of the marker allele or haplotype is indicative of response to therapy for Alzheimer's disease. In yet another embodiment, the presence of the marker allele is indicative of progress of treatment of Alzheimer's disease.

In certain embodiments, the kit comprises reagents for detecting no more than 100 alleles in the genome of the individual. In certain other embodiments, the kit comprises reagents for detecting no more than 20 alleles in the genome of the individual.

In a further aspect of the present invention, a pharmaceutical pack (kit) is provided, the pack comprising a therapeutic agent and a set of instructions for administration of the therapeutic agent to humans diagnostically tested for an at-risk variant for Alzheimer's disease (e.g., a variant in TREM2, e.g., rs75932628 allele T) . The therapeutic agent can be a small molecule drug, an antibody, a peptide, an antisense or RNAi molecule, or other therapeutic molecules. In one embodiment, an individual identified as a carrier of at least one variant of the present invention is instructed to take a prescribed dose of the therapeutic agent. In one such embodiment, an individual identified as a carrier of at least one variant (e.g., an at-risk variant) is instructed to take a prescribed dose of the therapeutic agent.

The kit may additionally or alternatively comprise reagents for detecting an amino acid variation in a human TREM2 protein (e.g., an amino acid substitution, or a truncated or otherwise altered amino acid sequence of an encoded TREM2 protein) . In one embodiment, the kit comprises at least one antibody for selectively detecting a truncated or altered TREM2 polypeptide compared with wild-type TREM2 (SEQ ID NO : 2) . In one embodiment, the kit comprises at least one antibody for selectively detecting an arginine to histidine substitution at position 47 in a TREM2 polypeptide with sequence as set forth in SEQ ID NO: 2. Other reagents useful for detecting amino acid variations are known to the skilled person and are also contemplated.

In certain embodiments, the kit further comprises a set of instructions for using the reagents comprising the kit. In certain embodiments, the kit further comprises a collection of data comprising correlation data between the at least one at-risk variant and susceptibility to Alzheimer's disease. Antisense agents

The nucleic acids and/or variants described herein, or nucleic acids comprising their complementary sequence, may be used as antisense constructs to control gene expression in cells, tissues or organs. The methodology associated with antisense techniques is well known to the skilled artisan, and is for example described and reviewed in AntisenseDrug

Technology: Principles, Strategies, and Applications, Crooke, ed., Marcel Dekker Inc., New York (2001) . In general, antisense agents (antisense oligonucleotides) are comprised of single stranded oligonucleotides (RNA or DNA) that are capable of binding to a complimentary nucleotide segment. By binding the appropriate target sequence, an RNA-RNA, DNA-DNA or RNA-DNA duplex is formed . The antisense oligonucleotides are complementary to the sense or coding strand of a gene. It is also possible to form a triple helix, where the antisense oligonucleotide binds to duplex DNA.

Several classes of antisense oligonucleotide are known to those skilled in the art, including cleavers and blockers. The former bind to target RNA sites, activate intracellular nucleases (e.g., RnaseH or Rnase L), that cleave the target RNA. Blockers bind to target RNA, inhibit protein translation by steric hindrance of the ribosomes. Examples of blockers include nucleic acids, morpholino compounds, locked nucleic acids and methylphosphonates (Thompson, Drug Discovery Today, 7 :912-917 (2002)) . Antisense oligonucleotides are useful directly as therapeutic agents, and are also useful for determining and validating gene function, for example by gene knock-out or gene knock-down experiments. Antisense technology is further described in Lavery et al. , Curr. Opin. Drug Discov. Devel. 6: 561-569 (2003), Stephens et al., Curr. Opin. Mol. Then 5: 118-122 (2003), Kurreck, Eur. J. Biochem.

270 : 1628-44 (2003), Dias et a/., Mol. Cancer Ter. 1 : 347-55 (2002), Chen, Methods Mol. Med. 75 : 621-636 (2003), Wang et al., Curr. Cancer Drug Targets 1 : 177-96 (2001), and Bennett, Antisense Nucleic Acid Drug. Dev. 12 : 215-24 (2002) .

In certain embodiments, the antisense agent is an oligonucleotide that is capable of binding to a particular nucleotide segment. In certain embodiments, the nucleotide segment comprises the human TREM2 gene. In certain other embodiments, the antisense nucleotide is capable of binding to a nucleotide segment of a human TREM2 transcript. In one embodiment, the antisense nucleotide is capable of binding the a nucleotide segment of a human TREM2 transcript with an altered sequence, wherein the sequence is altered by the presence of at least one variant in the TREM2 gene selected from the group consisting of rs79011726 allele T, chr6:41236977 allele C, rsl42232675 allele T, rsl43332484 allele T, rs75932628 allele T and chr6:41237236 allele T. Antisense nucleotides can be from 5-400 nucleotides in length, including 5-200 nucleotides, 5-100 nucleotides, 10-50 nucleotides, and 10-30 nucleotides. In certain preferred embodiments, the antisense nucleotides is from 14- 50 nucleotides in length, including 14-40 nucleotides and 14-30 nucleotides.

The variants described herein can also be used for the selection and design of antisense reagents that are specific for particular variants. Using information about the variants described herein, antisense oligonucleotides or other antisense molecules that specifically target mRNA molecules that contain one or more variants of the invention can be designed. In this manner, expression of mRNA molecules that contain one or more variant of the present invention can be inhibited or blocked. In one embodiment, the antisense molecules are designed to specifically bind a particular allelic form of the target nucleic acid, thereby inhibiting translation of a product originating from this specific allele, but which do not bind other or alternate variants at the specific polymorphic sites of the target nucleic acid molecule. In one embodiment, the antisense molecule is designed to specifically bind to nucleic acids comprising a variant selected from rs79011726 allele T, chr6:41236977 allele C, rsl42232675 allele T, rsl43332484 allele T, rs75932628 allele T and chr6:41237236 allele T. As antisense molecules can be used to inactivate mRNA so as to inhibit gene expression, and thus protein expression, the molecules can be used for disease treatment. The methodology can involve cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Such mRNA regions include, for example, protein-coding regions, in particular protein-coding regions corresponding to catalytic activity, substrate and/or ligand binding sites, or other functional domains of a protein .

The phenomenon of RNA interference (RNAi) has been actively studied for the last decade, since its original discovery in C. elegans (Fire et al., Nature 391 : 806-11 (1998)), and in recent years its potential use in treatment of human disease has been actively pursued (reviewed in Kim & Rossi, Nature Rev. Genet. 8: 173-204 (2007)) . RNA interference (RNAi), also called gene silencing, is based on using double-stranded RNA molecules (dsRNA) to turn off specific genes. In the cell, cytoplasmic double-stranded RNA molecules (dsRNA) are processed by cellular complexes into small interfering RNA (siRNA). The siRNA guide the targeting of a protein-RNA complex to specific sites on a target mRNA, leading to cleavage of the mRNA (Thompson, Drug Discovery Today, 7 : 912-917 (2002)) . The siRNA molecules are typically about 20, 21, 22 or 23 nucleotides in length. Thus, one aspect of the invention relates to isolated nucleic acid molecules, and the use of those molecules for RNA interference, i.e. as small interfering RNA molecules (siRNA) . In one embodiment, the isolated nucleic acid molecules are 18-26 nucleotides in length, preferably 19-25 nucleotides in length, more preferably 20-24 nucleotides in length, and more preferably 21, 22 or 23 nucleotides in length.

Another pathway for RNAi-mediated gene silencing originates in endogenously encoded primary microRNA (pri-miRNA) transcripts, which are processed in the cell to generate precursor miRNA (pre-miRNA) . These miRNA molecules are exported from the nucleus to the cytoplasm, where they undergo processing to generate mature miRNA molecules (miRNA), which direct translational inhibition by recognizing target sites in the 3' untranslated regions of mRNAs, and subsequent mRNA degradation by processing P-bodies (reviewed in Kim & Rossi, Nature Rev. Genet. 8: 173-204 (2007)) . Clinical applications of RNAi include the incorporation of synthetic siRNA duplexes, which preferably are approximately 20-23 nucleotides in size, and preferably have 3' overlaps of 2 nucleotides. Knockdown of gene expression is established by sequence-specific design for the target mRNA. Several commercial sites for optimal design and synthesis of such molecules are known to those skilled in the art.

Other applications provide longer siRNA molecules (typically 25-30 nucleotides in length, preferably about 27 nucleotides), as well as small hairpin RNAs (shRNAs; typically about 29 nucleotides in length) . The latter are naturally expressed, as described in Amarzguioui et al. {FEBS Lett. 579 : 5974-81 (2005)) . Chemically synthetic siRNAs and shRNAs are substrates for in vivo processing, and in some cases provide more potent gene-silencing than shorter designs (Kim et ai., Nature Biotechnol. 23 : 222-226 (2005); Siolas et al., Nature Biotechnol. 23 : 227-231 (2005)) . In general siRNAs provide for transient silencing of gene expression, because their intracellular concentration is diluted by subsequent cell divisions. By contrast, expressed shRNAs mediate long-term, stable knockdown of target transcripts, for as long as transcription of the shRNA takes place (Marques et al., Nature Biotechnol. 23 : 559-565 (2006); Brummelkamp et al., Science 296: 550-553 (2002)) .

Since RNAi molecules, including siRNA, miRNA and shRNA, act in a sequence-dependent manner, the variants presented herein can be used to design RNAi reagents that recognize specific nucleic acid molecules comprising specific alleles and/or haplotypes (e.g., the alleles and/or haplotypes of the present invention), while not recognizing nucleic acid molecules comprising other alleles or haplotypes. These RNAi reagents can thus recognize and destroy the target nucleic acid molecules. As with antisense reagents, RNAi reagents can be useful as therapeutic agents (i .e., for turning off disease-associated genes or disease-associated gene variants), but may also be useful for characterizing and validating gene function (e.g., by gene knock-out or gene knock-down experiments) .

Delivery of RNAi may be performed by a range of methodologies known to those skilled in the art. Methods utilizing non-viral delivery include cholesterol, stable nucleic acid-lipid particle (SNALP), heavy-chain antibody fragment (Fab), aptamers and nanoparticles. Viral delivery methods include use of lentivirus, adenovirus and adeno-associated virus. The siRNA molecules are in some embodiments chemically modified to increase their stability. This can include modifications at the 2' position of the ribose, including 2'-0-methylpurines and 2'- fluoropyrimidines, which provide resistance to Rnase activity. Other chemical modifications are possible and known to those skilled in the art.

Nucleic acids and polypeptides

The nucleic acids and polypeptides described herein can be used in methods and kits of the present invention. An "isolated" nucleic acid molecule, as used herein, is one that is separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g ., as in an RNA library) . For example, an isolated nucleic acid of the invention can be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix. In other circumstances, the material can be purified to essential homogeneity, for example as determined by polyacrylamide gel electrophoresis (PAGE) or column chromatography (e.g ., HPLC) . An isolated nucleic acid molecule of the invention can comprise at least about 50%, at least about 80% or at least about 90% (on a molar basis) of all macromolecular species present. With regard to genomic DNA, the term "isolated" also can refer to nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. For example, the isolated nucleic acid molecule can contain less than about 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotides that flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived.

The invention also pertains to nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein (e.g. , nucleic acid molecules that specifically hybridize to a nucleotide sequence containing a polymorphic site associated with a marker or haplotype described herein) . Such nucleic acid molecules can be detected and/or isolated by allele- or sequence- specific hybridization (e.g. , under high stringency conditions) . Stringency conditions and methods for nucleic acid hybridizations are well known to the skilled person (see, e.g., Current Protocols in Molecular Biology, Ausubel, F. et al, John Wiley & Sons, (1998), and Kraus, M . and Aaronson, S., Methods Enzymol. , 200: 546-556 (1991), the entire teachings of which are incorporated by reference herein.

The percent identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence) . The nucleotides or amino acids at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions x 100) . In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, of the length of the reference sequence. The actual comparison of the two sequences can be accomplished by methods well known to the skilled person, for example, using the NBLAST and XBLAST programs, as described in Altschul, S. et al., Nucleic Acids Res., 25: 3389-3402 (1997) . Another example of an algorithm is BLAT (Kent, W.J. Genome Res. 12 : 656-64 (2002)) . The present invention also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleic acid that comprises, or consists of, the nucleotide sequence of the human TREM2 gene as set forth in SEQ ID NO: 3, or a nucleotide sequence comprising, or consisting of, the complement of the nucleotide sequence of SEQ ID NO: 3. In certain embodiments, the nucleotide sequence comprises at least one polymorphic allele as described herein (rs79011726 allele T, chr6:41236977 allele C, rsl42232675 allele T, rsl43332484 allele T, rs75932628 allele T or chr6:41237236 allele T) . The nucleic acid fragments of the invention may suitably be at least about 15, at least about 18, 20, 23 or 25 nucleotides, and can be up to 30, 40, 50, 100, 200, 300 or 400 nucleotides in length.

The nucleic acid fragments of the invention are used as probes or primers in assays such as those described herein . "Probes" or "primers" are oligonucleotides that hybridize in a base- specific manner to a complementary strand of a nucleic acid molecule. In addition to DNA and RNA, such probes and primers include polypeptide nucleic acids (PNA), as described in Nielsen, P. et a/., Science 254: 1497-1500 (1991) . A probe or primer comprises a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and in certain embodiments about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule. In one embodiment, the probe or primer comprises at least one allele of at least one polymorphic marker or at least one haplotype described herein, or the complement thereof. In particular embodiments, a probe or primer can comprise 100 or fewer nucleotides; for example, in certain embodiments from 6 to 50 nucleotides, or, for example, from 12 to 30 nucleotides. In other embodiments, the probe or primer is at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical, to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence. In another embodiment, the probe or primer is capable of selectively hybridizing to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence. Often, the probe or primer further comprises a label, e.g., a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.

Antibodies

The invention also provides antibodies which bind to an epitope comprising either an TREM2 variant amino acid sequence (e.g., a polypeptide comprising an amino acid substitution or a truncated polypeptide) encoded by a variant allele or the reference amino acid sequence encoded by the corresponding non-variant or wild-type allele of TREM2. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain antigen-binding sites that specifically bind an antigen . A molecule that specifically binds to a polypeptide of the invention is a molecule that binds to that polypeptide or a fragment thereof (e.g., a TREM2 polypeptide with sequence as set forth in SEQ ID NO: 2, or a fragment thereof), but does not substantially bind other molecules in a sample, e.g. , a biological sample, which naturally contains the polypeptide. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind to a polypeptide of the invention. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of a polypeptide of the invention. A monoclonal antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.

Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a desired immunogen, e.g. , polypeptide of the invention or a fragment thereof. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the antibody molecules directed against the polypeptide can be isolated from the mammal (e.g. , from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g. , when the antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein, Nature 256:495-497 (1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72 (1983)), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,1985, Inc., pp. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology (1994) Coligan et al., (eds.) John Wiley & Sons, Inc., New York, NY) . Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a polypeptide of the invention .

Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating a monoclonal antibody to a polypeptide of the invention (see, e.g. , Current Protocols in Immunology, supra; Galfre et al. , Nature 266: 55052 (1977); R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); and Lerner, Yale J. Biol. Med. 54: 387-402 (1981)) . Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods that also would be useful.

Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody to a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide. Kits for generating and screening phage display libraries are commercially available (e.g. , the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the

Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612) . Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U .S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al. , Bio/Technology 9 : 1370-1372 (1991); Hay et al., Hum. Antibod. Hybridomas 3 : 81-85 (1992); Huse et al. , Science 246: 1275-1281 (1989); and Griffiths et al. , EM BO J. 12: 725- 734 (1993) .

Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention . Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

In general, antibodies of the invention (e.g., a monoclonal antibody) can be used to isolate a polypeptide as described herein by standard techniques, such as affinity chromatography or immunoprecipitation. A polypeptide-specific antibody can facilitate the purification of natural polypeptide from cells and of recombinantly produced polypeptide expressed in host cells. Moreover, an antibody specific for a polypeptide of the invention can be used to detect the polypeptide (e.g., in a cellular lysate, cell supernatant, or tissue sample) in order to evaluate the abundance and pattern of expression of the polypeptide. Antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen . The antibody can be coupled to a detectable substance to facilitate its detection . Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include

streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

Antibodies can furthermore be useful for assessing expression of proteins, e.g. TREM2 expression . Antibodies specific TREM2, or variants or truncated forms of TREM2, may be used to determine the expression levels of TREM2 in a sample from an individual. Antibodies can be used in other methods. Thus, antibodies are useful as diagnostic tools for evaluating proteins, such as variant proteins of the invention, in conjunction with analysis by electrophoretic mobility, isoelectric point, tryptic or other protease digest, or for use in other physical assays known to those skilled in the art. Antibodies may also be used in tissue typing . In one such embodiment, a specific variant protein has been correlated with expression in a specific tissue type, and antibodies specific for the variant protein can then be used to identify the specific tissue type.

Subcellular localization of proteins, including variant proteins, can also be determined using antibodies, and can be applied to assess aberrant subcellular localization of the protein in cells in various tissues. Such use can be applied in genetic testing, but also in monitoring a particular treatment modality.

Antibodies are further useful for inhibiting variant protein function, for example by blocking the binding of a variant protein to a binding molecule or partner. Such uses can also be applied in a therapeutic context in which treatment involves inhibiting a variant protein's function. An antibody can be for example be used to block or competitively inhibit binding, thereby modulating (i.e., agonizing or antagonizing) the activity of the protein . Antibodies can be prepared against specific protein fragments containing sites required for specific function or against an intact protein that is associated with a cell or cell membrane. For administration in vivo, an antibody may be linked with an additional therapeutic payload, such as radionuclide, an enzyme, an immunogenic epitope, or a cytotoxic agent, including bacterial toxins (diphtheria or plant toxins, such as ricin) . The in vivo half-life of an antibody or a fragment thereof may be increased by pegylation through conjugation to polyethylene glycol.

The present invention further relates to kits for using antibodies in the methods described herein. This includes, but is not limited to, kits for detecting the presence or absence of a protein (e.g., TREM2, or variants or truncated forms thereof) in a test sample. One preferred embodiment comprises antibodies such as a labelled or labelable antibody and a compound or agent for detecting proteins in a biological sample, means for determining the amount or the presence and/or absence of protein (e.g., TREM2, or variants or truncated forms thereof) in the sample, and means for comparing the amount of variant protein in the sample with a standard, as well as instructions for use of the kit.

Computer-implemented aspects

Another aspect of the invention is a system that is capable of carrying out a part or all of the methods of the invention, or carrying out a variation of a method of the invention as described herein in greater detail . Exemplary systems include, as one or more components, computing systems, environments, and/or configurations that may be suitable for use with the methods and include, but are not limited to, personal computers, server computers, hand-held or laptop devices, multiprocessor systems, microprocessor-based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, distributed computing environments that include any of the above systems or devices, and the like. In some variations, a system of the invention includes one or more machines used for analysis of biological material (e.g ., genetic material), as described herein. In some variations, this analysis of the biological material involves a chemical analysis and/or a nucleic acid amplification .

With reference to Fig. 1, an exemplary system of the invention, which may be used to implement one or more steps of methods of the invention, includes a computing device in the form of a computer 110. Components shown in dashed outline are not technically part of the computer 110, but are used to illustrate the exemplary embodiment of Fig. 1. Components of computer 110 may include, but are not limited to, a processor 120, a system memory 130, a memory/graphics interface 121, also known as a Northbridge chip, and an I/O interface 122, also known as a Southbridge chip. The system memory 130 and a graphics processor 190 may be coupled to the memory/graphics interface 121. A monitor 191 or other graphic output device may be coupled to the graphics processor 190.

A series of system busses may couple various system components including a high speed system bus 123 between the processor 120, the memory/graphics interface 121 and the I/O interface 122, a front-side bus 124 between the memory/graphics interface 121 and the system memory 130, and an advanced graphics processing (AGP) bus 125 between the memory/graphics interface 121 and the graphics processor 190. The system bus 123 may be any of several types of bus structures including, by way of example, and not limitation, such architectures include Industry Standard Architecture (ISA) bus, Micro Channel Architecture (MCA) bus and Enhanced ISA (EISA) bus. As system architectures evolve, other bus architectures and chip sets may be used but often generally follow this pattern . For example, companies such as Intel and AMD support the Intel Hub Architecture (IHA) and the

Hypertransport™ architecture, respectively.

The computer 110 typically includes a variety of computer-readable media. Computer- readable media can be any available media that can be accessed by computer 110 and includes both volatile and nonvolatile media, removable and non-removable media . By way of example, and not limitation, computer readable media may comprise computer storage media . Computer storage media includes both volatile and nonvolatile, removable and nonremovable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data .

Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other physical medium which can be used to store the desired information and which can accessed by computer 110. The system memory 130 includes computer storage media in the form of volatile and/or nonvolatile memory such as read only memory (ROM) 131 and random access memory (RAM) 132. The system ROM 131 may contain permanent system data 143, such as identifying and manufacturing information. In some embodiments, a basic input/output system (BIOS) may also be stored in system ROM 131. RAM 132 typically contains data and/or program modules that are immediately accessible to and/or presently being operated on by processor 120. By way of example, and not limitation, Fig . 1 illustrates operating system 134, application programs 135, other program modules 136, and program data 137.

The I/O interface 122 may couple the system bus 123 with a number of other busses 126, 127 and 128 that couple a variety of internal and external devices to the computer 110. A serial peripheral interface (SPI) bus 126 may connect to a basic input/output system (BIOS) memory 133 containing the basic routines that help to transfer information between elements within computer 110, such as during start-up.

A super input/output chip 160 may be used to connect to a number of 'legacy' peripherals, such as floppy disk 152, keyboard/mouse 162, and printer 196, as examples. The super I/O chip 160 may be connected to the I/O interface 122 with a bus 127, such as a low pin count (LPC) bus, in some embodiments. Various embodiments of the super I/O chip 160 are widely available in the commercial marketplace.

In one embodiment, bus 128 may be a Peripheral Component Interconnect (PCI) bus, or a variation thereof, may be used to connect higher speed peripherals to the I/O interface 122. A PCI bus may also be known as a Mezzanine bus. Variations of the PCI bus include the Peripheral Component Interconnect-Express (PCI-E) and the Peripheral Component

Interconnect - Extended (PCI-X) busses, the former having a serial interface and the latter being a backward compatible parallel interface. In other embodiments, bus 128 may be an advanced technology attachment (ATA) bus, in the form of a serial ATA bus (SATA) or parallel ATA (PATA) .

The computer 110 may also include other removable/non-removable, volatile/nonvolatile computer storage media . By way of example only, Fig. 1 illustrates a hard disk drive 140 that reads from or writes to non-removable, nonvolatile magnetic media . The hard disk drive 140 may be a conventional hard disk drive.

Removable media, such as a universal serial bus (USB) memory 153, firewire (IEEE 1394), or CD/DVD drive 156 may be connected to the PCI bus 128 directly or through an interface 150. A storage media 154 may be coupled through interface 150. Other removable/nonremovable, volatile/nonvolatile computer storage media that can be used in the exemplary operating environment include, but are not limited to, magnetic tape cassettes, flash memory cards, digital versatile disks, digital video tape, solid state RAM, solid state ROM, and the like.

The drives and their associated computer storage media discussed above and illustrated in Fig. 1, provide storage of computer readable instructions, data structures, program modules and other data for the computer 110. In Fig. 1, for example, hard disk drive 140 is illustrated as storing operating system 144, application programs 145, other program modules 146, and program data 147. Note that these components can either be the same as or different from operating system 134, application programs 135, other program modules 136, and program data 137. Operating system 144, application programs 145, other program modules 146, and program data 147 are given different numbers here to illustrate that, at a minimum, they are different copies. A user may enter commands and information into the computer 20 through input devices such as a mouse/keyboard 162 or other input device combination. Other input devices (not shown) may include a microphone, joystick, game pad, satellite dish, scanner, or the like. These and other input devices are often connected to the processor 120 through one of the I/O interface busses, such as the SPI 126, the LPC 127, or the PCI 128, but other busses may be used . In some embodiments, other devices may be coupled to parallel ports, infrared interfaces, game ports, and the like (not depicted), via the super I/O chip 160.

The computer 110 may operate in a networked environment using logical connections to one or more remote computers, such as a remote computer 180 via a network interface controller (NIC) 170. The remote computer 180 may be a personal computer, a server, a router, a network PC, a peer device or other common network node, and typically includes many or all of the elements described above relative to the computer 110. The logical connection between the NIC 170 and the remote computer 180 depicted in Fig . 1 may include a local area network (LAN), a wide area network (WAN), or both, but may also include other networks. Such networking environments are commonplace in offices, enterprise-wide computer networks, intranets, and the Internet. The remote computer 180 may also represent a web server supporting interactive sessions with the computer 110, or in the specific case of location-based applications may be a location server or an application server.

In some embodiments, the network interface may use a modem (not depicted) when a broadband connection is not available or is not used. It will be appreciated that the network connection shown is exemplary and other means of establishing a communications link between the computers may be used.

In some variations, the invention is a system for identifying susceptibility to Alzheimer's disease in a human subject. For example, in one variation, the system includes tools for performing at least one step, preferably two or more steps, and in some aspects all steps of a method of the invention, where the tools are operably linked to each other. Operable linkage describes a linkage through which components can function with each other to perform their purpose.

In some variations, a system of the invention is a system for identifying susceptibility to Alzheimer's disease in a human subject, and comprises:

(a) at least one processor; (b) at least one computer-readable medium;

(c) a susceptibility database operatively coupled to a computer-readable medium of the system and containing population information correlating the presence or absence of one or more alleles of the human TREM2 gene and susceptibility to Alzheimer's disease in a population of humans;

(d) a measurement tool that receives an input about the human subject and generates information from the input about the presence or absence of at least one mutant TREM2 allele indicative of a TREM2 defect in the human subject; and

(e) an analysis tool or routine that:

(i) is operatively coupled to the susceptibility database and the information generated by the measurement tool,

(ii) is stored on a computer-readable medium of the system,

(iii) is adapted to be executed on a processor of the system, to compare the information about the human subject with the population information in the susceptibility database and generate a conclusion with respect to susceptibility to

Alzheimer's disease for the human subject.

Exemplary processors (processing units) include all variety of microprocessors and other processing units used in computing devices. Exemplary computer-readable media are described above. When two or more components of the system involve a processor or a computer-readable medium, the system generally can be created where a single processor and/or computer readable medium is dedicated to a single component of the system; or where two or more functions share a single processor and/or share a single computer readable medium, such that the system contains as few as one processor and/or one computer readable medium. In some variations, it is advantageous to use multiple processors or media, for example, where it is convenient to have components of the system at different locations. For instance, some components of a system may be located at a testing laboratory dedicated to laboratory or data analysis, whereas other components, including components (optional) for supplying input information or obtaining an output communication, may be located at a medical treatment or counseling facility (e.g ., doctor's office, health clinic, HMO, pharmacist, geneticist, hospital) and/or at the home or business of the human subject (patient) for whom the testing service is performed .

Referring to Figure 2, an exemplary system includes a susceptibility database 208 that is operatively coupled to a computer-readable medium of the system and that contains population information correlating the presence or absence of one or more alleles of the human TREM2 gene and susceptibility to Alzheimer's disease in a population of humans. For example, the one or more alleles of the TREM2 gene include mutant alleles that cause, or are indicative of, a TREM2 defect such as reduced or lost function, as described elsewhere herein . In a simple variation, the susceptibility database contains 208 data relating to the frequency that a particular allele of TREM2 has been observed in a population of humans with

Alzheimer's disease and a population of humans free of Alzheimer's disease. Such data provides an indication as to the relative risk or odds ratio of developing Alzheimer's disease for a human subject that is identified as having the allele in question. In another variation, the susceptibility database includes similar data with respect to two or more alleles of TREM2, thereby providing a useful reference if the human subject has any of the two or more alleles. In still another variation, the susceptibility database includes additional quantitative personal, medical, or genetic information about the individuals in the database diagnosed with

Alzheimer's disease or free of Alzheimer's disease. Such information includes, but is not limited to, information about parameters such as age, sex, ethnicity, race, medical history, weight, diabetes status, blood pressure, family history of Alzheimer's disease, smoking history, and alcohol use in humans and impact of the at least one parameter on susceptibility to Alzheimer's disease. The information also can include information about other genetic risk factors for Alzheimer's disease besides TREM2 variants. These more robust susceptibility databases can be used by an analysis routine 210 to calculate a combined score with respect to susceptibility or risk for developing Alzheimer's disease.

In addition to the susceptibility database 208, the system further includes a measurement tool 206 programmed to receive an input 204 from or about the human subject and generate an output that contains information about the presence or absence of the at least one TREM2 allele of interest. (The input 204 is not part of the system per se but is illustrated in the schematic Figure 2.) Thus, the input 204 will contain a specimen or contain data from which the presence or absence of the at least one TREM2 allele can be directly read, or analytically determined. In a simple variation, the input contains annotated information about genotypes or allele counts for TREM2 in the genome of the human subject, in which case no further processing by the measurement tool 206 is required, except possibly transformation of the relevant information about the presence/absence of the TREM2 allele into a format compatible for use by the analysis routine 210 of the system .

In another variation, the input 204 from the human subject contains data that is unannotated or insufficiently annotated with respect to TREM2, requiring analysis by the measurement tool 206. For example, the input can be genetic sequence of the chromosome or chromosomal region on which TREM2 resides, or whole genome sequence information, or unannotated information from a gene chip analysis of a variable loci in the human subject's genome. In such variations of the invention, the measurement tool 206 comprises a tool, preferably stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to receive a data input about a subject and determine information about the presence or absence of the at least one mutant TREM2 allele in a human subject from the data . For example, the measurement tool 206 contains instructions, preferably executable on a processor of the system, for analyzing the unannotated input data and determining the presence or absence of the TREM2 allele of interest in the human subject. Where the input data is genomic sequence information, and the measurement tool optionally comprises a sequence analysis tool stored on a computer readable medium of the system and executable by a processor of the system with instructions for determining the presence or absence of the at least one mutant TREM2 allele from the genomic sequence information.

In yet another variation, the input 204 from the human subject comprises a biological sample, such as a fluid (e.g ., blood) or tissue sample, that contains genetic material that can be analyzed to determine the presence or absence of the TREM2 allele of interest. In this variation, an exemplary measurement tool 206 includes laboratory equipment for processing and analyzing the sample to determine the presence or absence (or identity) of the TREM2 allele(s) in the human subject. For instance, in one variation, the measurement tool includes: an oligonucleotide microarray (e.g., "gene chip") containing a plurality of oligonucleotide probes attached to a solid support; a detector for measuring interaction between nucleic acid obtained from or amplified from the biological sample and one or more oligonucleotides on the oligonucleotide microarray to generate detection data; and an analysis tool stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to determine the presence or absence of the at least one TREM2 allele of interest based on the detection data.

To provide another example, in some variations the measurement tool 206 includes: a nucleotide sequencer (e.g., an automated DNA sequencer) that is capable of determining nucleotide sequence information from nucleic acid obtained from or amplified from the biological sample; and an analysis tool stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to determine the presence or absence of the at least one mutant TREM2 allele based on the nucleotide sequence information .

In some variations, the measurement tool 206 further includes additional equipment and/or chemical reagents for processing the biological sample to purify and/or amplify nucleic acid of the human subject for further analysis using a sequencer, gene chip, or other analytical equipment.

The exemplary system further includes an analysis tool or routine 210 that: is operatively coupled to the susceptibility database 208 and operatively coupled to the measurement tool 206, is stored on a computer-readable medium of the system, is adapted to be executed on a processor of the system to compare the information about the human subject with the population information in the susceptibility database 208 and generate a conclusion with respect to susceptibility to Alzheimer's disease for the human subject. In simple terms, the analysis tool 210 looks at the TREM2 alleles identified by the measurement tool 206 for the human subject, and compares this information to the susceptibility database 208, to determine a susceptibility to Alzheimer's disease for the subject. The susceptibility can be based on the single parameter (the identity of one or more TREM2 alleles), or can involve a calculation based on other genetic and non-genetic data, as described above, that is collected and included as part of the input 204 from the human subject, and that also is stored in the susceptibility database 208 with respect to a population of other humans. Generally speaking, each parameter of interest is weighted to provide a conclusion with respect to susceptibility to Alzheimer's disease. Such a conclusion is expressed in the conclusion in any statistically useful form, for example, as an odds ratio, a relative risk, or a lifetime risk for subject developing Alzheimer's disease.

In some variations of the invention, the system as just described further includes a communication tool 212. For example, the communication tool is operatively connected to the analysis routine 210 and comprises a routine stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to: generate a communication containing the conclusion; and to transmit the communication to the human subject 200 or the medical practitioner 202, and/or enable the subject or medical practitioner to access the communication. (The subject and medical practitioner are depicted in the schematic Fig. 2, but are not part of the system per se, though they may be considered users of the system . The communication tool 212 provides an interface for communicating to the subject, or to a medical practitioner for the subject (e.g ., doctor, nurse, genetic counselor), the conclusion generated by the analysis tool 210 with respect to susceptibility to Alzheimer's disease for the subject. Usually, if the communication is obtained by or delivered to the medical practitioner 202, the medical practitioner will share the communication with the human subject 200 and/or counsel the human subject about the medical significance of the communication. In some variations, the communication is provided in a tangible form, such as a printed report or report stored on a computer readable medium such as a flash drive or optical disk. In some variations, the communication is provided electronically with an output that is visible on a video display or audio output (e.g., speaker) . In some variations, the communication is transmitted to the subject or the medical practitioner, e.g., electronically or through the mail. In some variations, the system is designed to permit the subject or medical practitioner to access the communication, e.g ., by telephone or computer. For instance, the system may include software residing on a memory and executed by a processor of a computer used by the human subject or the medical practitioner, with which the subject or practitioner can access the communication, preferably securely, over the internet or other network connection. In some variations of the system, this computer will be located remotely from other components of the system, e.g., at a location of the human subject's or medical practitioner's choosing .

In some variations of the invention, the system as described (including embodiments with or without the communication tool) further includes components that add a treatment or prophylaxis utility to the system. For instance, value is added to a determination of susceptibility to Alzheimer's disease when a medical practitioner can prescribe or administer a standard of care that can reduce susceptibility to Alzheimer's disease; and/or delay onset of Alzheimer's disease; and/or increase the likelihood of detecting the disease at an early stage, to facilitate early treatment when the disease has not advanced and is most curable.

Exemplary lifestyle change protocols include increased mental activity, increase in exercise, cessation of unhealthy behaviors such as smoking, and change of diet. Exemplary medicinal intervention protocols include administration of pharmaceutical agents for prophylaxis.

For example, in some variations, the system further includes a medical protocol database 214 operatively connected to a computer-readable medium of the system and containing information correlating the presence or absence of the at least one TREM2 allele of interest and medical protocols for human subjects at risk for Alzheimer's disease. Such medical protocols include any variety of medicines, lifestyle changes, diagnostic tests, increased frequencies of diagnostic tests, and the like that are designed to achieve one of the aforementioned goals. The information correlating a TREM2 allele with protocols could include, for example, information about the success with which Alzheimer's disease is avoided or delayed, or success with which the disease is detected early and treated, if a subject has an TREM2 susceptibility allele and follows a protocol.

The system of this embodiment further includes a medical protocol tool or routine 216, operatively connected to the medical protocol database 214 and to the analysis tool or routine 210. The medical protocol tool or routine 216 preferably is stored on a computer- readable medium of the system, and adapted to be executed on a processor of the system, to: (i) compare (or correlate) the conclusion that is obtained from the analysis routine 210

(with respect to susceptibility to Alzheimer's disease for the subject) and the medical protocol database 214, and (ii) generate a protocol report with respect to the probability that one or more medical protocols in the medical protocol database will achieve one or more of the goals of reducing susceptibility to Alzheimer's disease; delaying onset of the disease; and increasing the likelihood of detecting the disease at an early stage to facilitate early treatment. The probability can be based on empirical evidence collected from a population of humans and expressed either in absolute terms (e.g ., compared to making no intervention), or expressed in relative terms, to highlight the comparative or additive benefits of two or more protocols.

Some variations of the system just described include the communication tool 212. In some examples, the communication tool generates a communication that includes the protocol report in addition to, or instead of, the conclusion with respect to susceptibility.

Information about TREM2 allele status not only can provide useful information about identifying or quantifying susceptibility to Alzheimer's disease; it can also provide useful information about possible causative factors for a human subject identified with Alzheimer's disease, and useful information about therapies for the patient with the disease. In some variations, systems of the invention are useful for these purposes. For instance, in some variations the invention is a system for assessing or selecting a treatment protocol for a subject diagnosed with Alzheimer's disease. An exemplary system, schematically depicted in Figure 3, comprises:

(a) at least one processor;

(b) at least one computer-readable medium;

(c) a medical treatment database 308 operatively connected to a computer-readable medium of the system and containing information correlating the presence or absence of at least one TREM2 allele and efficacy of treatment regimens for Alzheimer's disease;

(d) a measurement tool 306 to receive an input (304, depicted in Fig . 3 but not part of the system per se) about the human subject and generate information from the input 304 about the presence or absence of the at least one TREM2 allele indicative of an TREM2 defect in a human subject diagnosed with Alzheimer's disease; and

(e) a medical protocol routine or tool 310 operatively coupled to the medical treatment database 308 and the measurement tool 306, stored on a computer-readable medium of the system, and adapted to be executed on a processor of the system, to compare the information with respect to presence or absence of the at least one TREM2 allele for the subject and the medical treatment database, and generate a conclusion with respect to at least one of:

(i) the probability that one or more medical treatments will be efficacious for treatment of Alzheimer's disease for the patient; and

(ii) which of two or more medical treatments for Alzheimer's disease will be more efficacious for the patient.

Preferably, such a system further includes a communication tool 312 operatively connected to the medical protocol tool or routine 310 for communicating the conclusion to the subject 300, or to a medical practitioner for the subject 302 (both depicted in the schematic of Fig. 3, but not part of the system per se) . An exemplary communication tool comprises a routine stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to generate a communication containing the conclusion; and transmit the communication to the subject or the medical practitioner, or enable the subject or medical practitioner to access the communication .

The present invention will now be exemplified by the following non-limiting examples.

EXAMPLE 1

To search for sequence variants that influence the risk of AD, we performed a genome-wide association analysis with variants found by whole-genome sequencing of 2,261 Icelanders that are likely to affect protein function, followed by imputation of these variants, using long- range haplotype phasing information, in the chip-typed genomes of persons with AD and of controls.

We found a total of 191,777 nonsynonymous SNPs, frameshift variants, splicing variants and stop gain/loss variants, and imputed these in persons with AD and controls. A total of 3,550 persons with AD were included in the analysis. Our control group included individuals who have reached the age of 85 without the diagnosis of AD. Excluding the ApoE locus and the A673T variant in the Amyloid Protein Precursor (APP) gene, only one marker, rs75932628, showed genome-wide significant association, either using the Bonferroni-adjusted threshold of P < 0.5/191, 777=2.60xl0"7 or the conventional GWAS threshold of 5x10 s. The T allele of rs75932628, which encodes an arginine to histidine substitution at position 47 (R47H) in the triggering receptor expressed on myeloid cells 2 (TREM2) gene on chromosome 6p21.1, with an allelic frequency of 0.63% in Iceland, was found to confer significant risk of AD (OR = 2.92; 95% CI, 2.09 to 4.09; P-value=3.42xl010; Table 1). No other variant in TREM2 that is likely to affect protein function showed nominally significant association with AD. Risk variants of a late-onset disease such as AD are expected to be more common in the general population than in elderly controls without the disease. Thus, the use of elderly controls without a history of AD would in general be expected to result in increased power to detect risk variants for AD. We therefore investigated the association of rs75932628-T using cognitively intact elderly controls. We found an increased odds ratio (OR = 4.66; 95% CI, 2.38 to 9.14; P = 7.39xl0"6) when using controls who have remained cognitively intact at age 85, as determined by the cognitive performance scale (CPS). By contrast, a smaller OR was oberved when using general population controls of all ages (OR=2.26; 95% CI, 1.71 to 2.98, P=1.13xl0"8; Table 1). The larger P-value observed when using cognitively intact controls is due to the substantially smaller size of this control group (N = 1,236 vs 8,888 elderly controls and 110,050 population controls). Further, we found that the frequency of rs75932628-T in controls age 85 or greater without history of AD (0.46%) is significantly less than in controls below age 85 (0.64%; P=0.007). This observation is expected for alleles associated with common, late-onset diseases such as AD and thus provides further support for the association of rs75932628-T with AD. We identified four homozygous carriers of rs75932628-T in Iceland, two of whom were diagnosed with AD, and two of whom are currently living at age 51 and 52, respectively.

We investigated the effect of the ε4 allele of Apolipoprotein E on the association of rs7593262-T with AD. We found a somewhat higher odds ratio in ε4 non-carriers (4.03) than in ε4 carriers (2.38). The rs7593262-T frequency difference by ApoE status in cases was borderline significant (OR = 0.60; 95% CI, 0.37 to 0.98; P=0.04). However, in a logistic regression model, the interaction between rs7593262-T and ApoE ε4 was not significant (P=0.18)). Although the population frequency of rs75932628-T is low (0.63% in Iceland), it confers risk of AD (OR=2.92) which is comparable to that of the ε4 allele of ApoE, which has a population frequency of 17.3% in Iceland (OR=3.08 using controls age 85 or greater) . We also found that, in Iceland, each copy of rs75932628-T was associated with an age at onset of AD that is lower by 3.18 years (P=0.20) . While this effect is comparable to that of ApoE ε4 (3.22 years; P=4.1xl0"8), it is not even nominally significant due to the low frequency of the variant, which results in a reduced effective sample size and an elevated standard error (2.49 for rs75932628-T vs 0.58 for ApoE ε4) .

We also investigated how rs75932628-T affects cognitive function in the elderly without AD. As shown in Figure 4, cognitive function declines steadily with age in elderly individuals between the ages of 80 and 100. We found that carriers of rs75932628-T show on average 0.87 units worse cognition (higher CPS score) than do non-carriers of rs75932628-T

(P=0.0029) . Clinical determination of AD is, in part, based on progressive loss of cognitive function, in particular memory, with time. The decline in cognitive function we observe in rs75932628-T carriers may thus be due to early cognitive deficits that ultimately result in AD. Alternatively, the decline may (at least partially) be due to non-AD related loss of cognitive function at old age. The former explanation is in keeping with the hypothesis that AD may be the extreme of the cognitive decline of the elderly and caused by the same biochemical mechanism .

METHODS

Study Subjects

Icelandic study population. Approval for these studies was obtained from the National Bioethics Committee and the Icelandic Data Protection Authority. Written informed consent was obtained from all participants or their guardians before blood samples were drawn, and all sample identifiers were encrypted in accordance with the regulations of the Icelandic Data Protection Authority. Diagnosis of AD was established according to NINCDS-ADRDA criteria of definite, probable or possible AD12 (N = 1,062), or according to ICD-10 code F00 criteria (N = 2,697) . Cognitive function was assessed using data from the Resident Assessment Instrument (RAI). Assessment is performed on an individual basis and recorded in a Minimum Data Set (MDS 2.0) form . Data were primarily obtained through RAI 2.0 for Nursing Homes, which is a comprehensive and standardized instrument originally developed for residential facilities for the elderly (Morris et al., Gerontologist 1990; 30: 293-307), with additional information provided by the InterRAI assessment for Home Care (RAI-HC) (Morris et al., J Am Ger Soc 1997; 45 : 1017-24) . Cognitive function was assessed using the CPS, which combines selected MDS 2.0 items expressing different measures of cognitive function on a seven-category scale (score from 0 (intact) to 6 (severe impairment)) (Morris et al., J Gerontol 1994; 49 : M174-82) . The CPS is hierarchical and based on an assessment of several measures of cognitive function; one unit change is a reflection of distinct and measurable changes in at least one cognitive domain. Individuals with a score of 0 on the CPS scale (N = l,236) were used as cognitively intact controls. Population controls (N = 110,050) were selected from individuals participating in various research projects at Decode, excluding those diagnosed with AD. Data generation and analysis

Whole-genome sequencing, SNP calling and imputation. We performed whole-genome sequencing of 2,261 Icelandic samples, followed by SNP calling and genotype imputation, using methods as previously described (Jonsson et al., Nature 2012; 488: 96-9) . The chip- genotype imputation was based on chip genotypes from 95,085 individuals. Approximately 34 million markers (SNPs and indels), including the 191,777 functional variants identified through WGS analysis, were imputed in the Icelandic cases and controls. The information content for rs75932628 in the imputed data was 0.99918.

Single track assay SNP genotyping. Single SNP genotyping of rs75932628 was carried out using the Centaurus (Nanogen) platform (Kutyavin, et al., Nucl Acid Res 2006; 34: el28) . A comparison of genotypes determined through imputation and Centaurus genotyping of 964 individuals, including 30 individuals predicted to be heterozygous for the rare allele and 2 individuals predicted to be homozygous for the rare allele, resulted in no mismatches. Before analysis, samples with a genotype yield of less than 90% and one member of each pair of duplicate samples were excluded . Genotyping yield was at least 95% in both cases and controls in samples from all study locations. All genotypes were in Hardy-Weinberg equilibrium.

Association analysis

For the Icelandic data, case-control association testing of imputed genotypes was performed using methods as previously described (Jonsson et al., Nature 2012; 488: 96-9) . Odds Ratios (ORs) were calculated assuming a multiplicative mode! for the two chromosomes of each individual. The method of genomic control was used to correct for relatedness and potential population stratification . The relationship of age at onset and rs75932628-T was examined using a linear model with age at onset as the response and rs75932628-T and ApoE ε4 count as predictors. Analysis of effect of age on CPS scores: We analyzed the effect of age on CPS score, using determinations made at several ages for each individual . The Resident Assessment

Instrument for Nursing Homes (RAI-NH) on which the CPS score is based is applied on average three times per year in Icelandic Nursing Homes. Since the residency time in nursing homes in Iceland is on average 3-4 years, many determinations of CPS made at different times are available for most individuals. The difference in CPS score between rs75932628-T carriers and non-carriers in the 80 - 100 year age range was assessed using a mixed model with age and carrier status as fixed effects and individual as a random effect. Standard error bars for the CPS score versus age plot were calculated using bootstrapping .

Table 1. rs75932628-T associates with Alzheimer's disease, shown are results obtained using three different control groups; N represents number of controls, freq is the allelic frequency of rs75932628-T in controls, P-value is that for association with Alzheimer's disease using the control group, and OR represents odds ratio with 95% confidence intervals indicated.

freq

Control group N P-value OR (95% CI)

(%)

Population controls 110,050 0.63 1.13x10 s 2.26 (1.71-2.98)

Population controls age

8,888 0.46 3.42xl0"10 2.92 (2.09-4.09)

85 or greater

Cognitively intact controls

age 85 or greater 1,236 0.31 7.39xl0-6 4.66 (2.38-9.14)

Claims

1. A method of determining a susceptibility to Alzheimer's disease, the method comprising :
analyzing data representative of at least one allele of the TREM2 gene in a human subject, wherein different alleles of the human TREM2 gene are associated with different
susceptibilities to Alzheimer's disease in humans, and
determining a susceptibility to Alzheimer's disease for the human subject from the data .
2. The method according to claim 1, comprising analyzing the data for the presence or absence of at least one mutant allele selected from the group consisting of a TREM2 missense allele, a TREM2 nonsense allele, a TREM2 promoter allele and a TREM2 3' UTR allele.
3. The method of claim 1 or claim 2, wherein the analyzing data comprises analyzing a biological sample from the human subject to obtain information selected from the group consisting of:
(a) nucleic acid sequence information, wherein the nucleic acid sequence information comprises sufficient sequence to identify the presence or absence of a TREM2 mutant allele in the subject;
(b) nucleic acid sequence information, wherein the nucleic acid sequence information identifies at least one allele of a polymorphic marker in linkage disequilibrium (LD) with the mutant allele, wherein the LD is characterized by a value for r2 of at least 0.5;
(c) measurement of the quantity or length of TREM2 mRNA, wherein the measurement is indicative of the presence or absence of the mutant allele;
(d) measurement of the quantity of TREM2 protein, wherein the measurement is indicative of the presence or absence of the mutant allele; and
(e) measurement of TREM2 activity, wherein the measurement is indicative of the presence or absence of the mutant allele.
4. The method of claim 3, further comprising obtaining a biological sample comprising nucleic acid from the human subject.
5. The method of claim 3, wherein the analyzing data comprises analyzing data from a preexisting record about the human subject.
6. The method of any one of the preceding claims, wherein the presence of the mutant allele is indicative of increased susceptibility to Alzheimer's disease with a relative risk (RR) or odds ratio (OR) of at least 1.5, of at least 2.0, of at least 2.5, of at least 3.0, at least 3.5, of at least 4.0, at least 4.5, or of at least 5.0.
7. The method of any one of the preceding claims, wherein the allele encodes a TREM2 missense mutation .
8. The method of any one of the claims 1-7, wherein the allele is an allele encoding an arginine to histidine amino acid substitution in a TREM2 polypeptide with wild-type sequence as set forth in SEQ ID NO: 2.
9. The method of any one of the claims 1-7, wherein the allele is rs75932628 allele T.
10. A method of determining whether an individual is at increased risk of developing Alzheimer's disease, the method comprising steps of
obtaining a biological sample containing nucleic acid from the individual;
determining, in the biological sample, nucleic acid sequence about the human TREM2 gene; analyzing the sequence information to determine the presence or absence of a TREM2 mutation that is indicative of increased risk of Alzheimer's disease in humans; and determining whether the human subject is at increased risk of Alzheimer's disease based on the presence of the TREM2 mutation in the test sample.
11. The method of claim 10, wherein the mutation is a missense mutation, a nonsense mutation or a frameshift mutation in TREM2.
12. The method of claim 10 or claim 11, wherein the mutation results in a TREM2 defect selected from the group consisting of:
(a) premature truncation or frameshift of an encoded TREM2 protein, relative to the TREM2 amino acid sequence set forth in SEQ ID NO: 2;
(b) expression of a TREM2 protein with reduced activity compared to a wild-type TREM2 protein (SEQ ID NO: 2);
(c) reduced expression of TREM2 protein, compared to wild-type expression of TREM2, wherein mutant alleles indicative of the defect are associated with increased susceptibility to Alzheimer's disease.
13. The method of any one of the claims 10 to 12, wherein the mutation encodes an arginine to histidine substitution at position 47 in a TREM2 polypeptide with sequence as set forth in SEQ ID NO: 2.
14. The method of any of the claims 10 to 13, wherein the mutation is allele T in marker rs75932628.
15. A method of determining whether a human subject is at increased risk of developing Alzheimer's disease, the method comprising analyzing amino acid sequence data about a TREM2 polypeptide from the subject, wherein a determination of the presence of an altered TREM2 polypeptide compared with a wild-type TREM2 polypeptide with sequence as set forth in SEQ ID NO: 2 is indicative that the subject is at increased risk of developing Alzheimer's disease.
16. The method of claim 15, wherein the amino acid sequence data is obtained from a biological sample from the human subject comprising human TREM2 polypeptide, using a method that comprises at least one procedure selected from :
(i) an antibody assay; and
(ii) protein sequencing .
17. The method of claim 15, wherein the amino acid sequence data is obtained from a preexisting record .
18. A method of identifying a test sample having a TREM2 genotype that is predictive of risk of Alzheimer's disease in humans, comprising : obtaining a test sample containing nucleic acid from a human subject; determining nucleic acid sequence about the human TREM2 gene in the test sample; determining whether a genotype of the TREM2 gene that correlates with increased risk of Alzheimer's disease in humans is present in the test sample; wherein determination of the presence of the genotype in the test sample identifies the test sample as having a TREM2 genotype that is predictive of Alzheimer's disease in humans.
19. The method of claim 18, wherein the TREM2 genotype encodes a missense substitution in an encoded TREM2 protein with sequence as set forth in SEQ ID NO : 2.
20. The method of claim 18 or claim 19, wherein the TREM2 genotype encodes an arginine to histidine substitution at position 47 in a TREM2 polypeptide with sequence as set forth in SEQ ID NO: 2.
21. The method of any one of the claims 18 to 20, wherein the TREM2 genotype is a genotype about marker rs75932628, and wherein determination of the presence of allele T in rs75932628 in the test sample is predictive of increased risk of Alzheimer's disease.
22. A system for identifying susceptibility to Alzheimer's disease in a human subject, the system comprising :
at least one processor;
at least one computer-readable medium;
a susceptibility database operativeiy coupled to a computer-readable medium of the system and containing population information correlating the presence or absence of one or more alleles of the human TREM2 gene and susceptibility to Alzheimer's disease in a population of humans;
a measurement tool that receives an input about the human subject and generates information from the input about the presence or absence of at least one mutant TREM2 allele in the human subject; and
an analysis tool that:
is operativeiy coupled to the susceptibility database and the the measurement tool, is stored on a computer-readable medium of the system,
is adapted to be executed on a processor of the system, to compare the information about the human subject with the population information in the susceptibility database and generate a conclusion with respect to susceptibility to Alzheimer's disease for the human subject.
23. The system according to claims 22, further including :
a communication tool operativeiy coupled to the analysis tool, stored on a computer-readable medium of the system and adapted to be executed on a processor of the system to communicate to the subject, or to a medical practitioner for the subject, the conclusion with respect to susceptibility to Alzheimer's disease for the subject.
24. The system of claim 22 and claim 23, wherein the at least one mutant TREM2 allele is indicative of an TREM2 mutant allele selected from the group consisting of a missense allele, a nonsense allele, a promoter allele and a 3' UTR allele.
25. The system according to any one of claims 22 to 24, wherein the measurement tool comprises a tool stored on a computer-readable medium of the system and adapted to be executed by a processor of the system to receive a data input about a subject and determine information about the presence or absence of the at least one mutant TREM2 allele in a human subject from the data.
26. The system according to claim 25, wherein the data is genomic sequence information, and the measurement tool comprises a sequence analysis tool stored on a computer readable medium of the system and adapted to be executed by a processor of the system to determine the presence or absence of the at least one mutant TREM2 allele from the genomic sequence information.
27. The system according to any one of claims 22 to 26, wherein the input about the human subject is a biological sample from the human subject, and wherein the measurement tool comprises a tool to identify the presence or absence of the at least one mutant TREM2 allele in the biological sample, thereby generating information about the presence or absence of the at least one mutant TREM2 allele in a human subject.
28. The system according to claim 27, wherein the measurement tool includes:
an oligonucleotide microarray containing a plurality of oligonucleotide probes attached to a solid support;
a detector for measuring interaction between nucleic acid obtained from or amplified from the biological sample and one or more oligonucleotides on the oligonucleotide microarray to generate detection data; and
an analysis tool stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to determine the presence or absence of the at least one mutant TREM2 allele based on the detection data.
29. The system according to claim 28, wherein the measurement tool includes:
a nucleotide sequencer capable of determining nucleotide sequence information from nucleic acid obtained from or amplified from the biological sample; and
an analysis tool stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to determine the presence or absence of the at least one mutant TREM2 allele based on the nucleotide sequence information.
30. The system according to any one of claims 22 to 29, further comprising :
a medical protocol database operatively connected to a computer-readable medium of the system and containing information correlating the presence or absence of the at least one mutant TREM2 allele and medical protocols for human subjects at risk for Alzheimer's disease; and a medical protocol routine, operatively connected to the medical protocol database and the analysis routine, stored on a computer-readable medium of the system, and adapted to be executed on a processor of the system, to compare the conclusion from the analysis routine with respect to susceptibility to Alzheimer's disease for the subject and the medical protocol database, and generate a protocol report with respect to the probability that one or more medical protocols in the database will :
reduce susceptibility to Alzheimer's disease; or
delay onset of Alzheimer's disease; or
increase the likelihood of detecting Alzheimer's disease at an early stage to facilitate early treatment.
31. The system according to any one of claims 23 to 30, wherein the communication tool is operatively connected to the analysis routine and comprises a routine stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to:
generate a communication containing the conclusion; and
transmit the communication to the subject or the medical practitioner, or enable the subject or medical practitioner to access the communication.
32. The system according to claim 31, wherein the communication expresses the susceptibility to Alzheimer's disease in terms of odds ratio or relative risk or lifetime risk.
33. The system according to claim 31 or 32, wherein the communication further includes the protocol report.
34. The system according to any one of claims 22 to 33, wherein the susceptibility database further includes information about at least one parameter selected from the group consisting of age, sex, ethnicity, race, medical history, weight, diabetes status, blood pressure, family history of Alzheimer's disease, and smoking history in humans and impact of the at least one parameter on susceptibility to Alzheimer's disease.
35. A system for assessing or selecting a treatment protocol for a subject diagnosed with Alzheimer's disease, comprising :
at least one processor;
at least one computer-readable medium;
a medical treatment database operatively connected to a computer-readable medium of the system and containing information correlating the presence or absence of at least one mutant TREM2 allele and efficacy of treatment regimens for Alzheimer's disease; a measurement tool to receive an input about the human subject and generate information from the input about the presence or absence of the at least one mutant TREM2 allele indicative of a TREM2 defect in a human subject diagnosed with Alzheimer's disease; and a medical protocol tool operatively coupled to the medical treatment database and the measurement tool, stored on a computer-readable medium of the system, and adapted to be executed on a processor of the system, to compare the information with respect to presence or absence of the at least one mutant TREM2 allele for the subject and the medical treatment database, and generate a conclusion with respect to at least one of:
the probability that one or more medical treatments will be efficacious for treatment of Alzheimer's disease for the patient; and
which of two or more medical treatments for Alzheimer's disease will be more efficacious for the patient.
36. The system according to claim 35, wherein the measurement tool comprises a tool stored on a computer-readable medium of the system and adapted to be executed by a processor of the system to receive a data input about a subject and determine information about the presence or absence of the at least one mutant TREM2 allele in a human subject from the data .
37. The system according to claim 36, wherein the data is genomic sequence information, and the measurement tool comprises a sequence analysis tool stored on a computer readable medium of the system and adapted to be executed by a processor of the system to determine the presence or absence of the at least one mutant TREM2 allele from the genomic sequence information.
38. The system according to claim 36, wherein the input about the human subject is a biological sample from the human subject, and wherein the measurement tool comprises a tool to identify the presence or absence of the at least one mutant TREM2 allele in the biological sample, thereby generating information about the presence or absence of the at least one mutant TREM2 allele in a human subject.
39. The system according to any one of claims 36 to 38, further comprising a
communication tool operatively connected to the medical protocol routine for communicating the conclusion to the subject, or to a medical practitioner for the subject.
40. The system according to claim 39, wherein the communication tool comprises a routine stored on a computer-readable medium of the system and adapted to be executed on a processor of the system, to:
generate a communication containing the conclusion; and transmit the communication to the subject or the medical practitioner, or enable the subject or medical practitioner to access the communication.
41. The system according to any one of the claims 22 - 40, wherein the TREM2 allele is selected from the group consisting of an TREM2 missense allele, a TREM2 nonsense allele, a TREM2 promoter allele and a TREM2 3' UTR allele.
42. The system according to any one of the claims 22 - 41, wherein the TREM2 allele is rs75932628 allele T.
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