Patent Application
20220325347
Brain diseases such as neurodegenerative diseases and neuroinflammatory disorders are devastating conditions that affect a large subset of the population. Many are incurable, highly debilitating, and often result in progressive deterioration of brain structure and function over time. Disease prevalence is also increasing rapidly due to growing aging populations worldwide, since the elderly are at high risk for developing these conditions. Currently, many neurodegenerative diseases and neuroinflammatory disorders are difficult to diagnose due to limited understanding of the pathophysiology of these diseases. Meanwhile, current treatments are ineffective and do not meet market demand; demand that is significantly increasing each year due to aging populations. For example, Alzheimer's disease (AD) is marked by gradual but progressive decline in learning and memory, and a leading cause of mortality in the elderly. Increasing prevalence of AD is driving the need and demand for better diagnostics. According to Alzheimer's Disease International, the disease currently affects 46.8 million people globally, but the number of cases is projected to triple in the coming three decades. One of the countries with the fastest elderly population growth is China. Based on population projections, by 2030 1 in 4 individuals will be over the age of 60, which will place a vast proportion at risk of developing AD. In fact, the number of AD cases in China doubled from 3.7 million to 9.2 million from 1990-2010, and the country is projected to have 22.5 million cases by 2050. Hong Kong's population is also aging quickly. In 2012, the number of seniors aged 65+ made up 14.9% of the population. It is estimated that the elderly will make up 24% of the population by 2025, and 39.3% of the population by 2050. There were 103,433 cases of AD in 2009, but this number is projected to increase to 332,688 by 2039.
More worrisome is that, despite the increase in AD prevalence, many people fail to receive a correct AD diagnosis. According to Alzheimer's Disease International's World Alzheimer' Report 2015, in high-income countries only 20-50% of dementia cases are documented in primary care. The rest remain undiagnosed or incorrectly diagnosed. This ‘treatment gap’ is much more significant in low- and middle-income countries. Without a formal diagnosis, patients do not receive the treatment and care they need, nor do they or their care-givers qualify for critical support programs. Early diagnosis and early intervention are two important means of narrowing the treatment gap. Thus, early diagnostic tools that can determine disease risk both quickly and accurately have significant therapeutic value on many levels. Research has confirmed that AD affects the brain long before actual symptoms of memory loss or cognitive decline actually manifest. To this date, however, there are no diagnostic tools for early detection; by the time a patient is diagnosed with AD using methods currently available, which involves subjective clinical assessment, often the pathological symptoms are already at an advanced state. As such, for the purpose of improving AD treatment and long term management, there exists an urgent need for developing new and effective methods for early diagnosis of AD or for detecting an increased risk of developing AD in a patient at a later time. This invention addresses this and other related needs. The invention discloses compositions, methods, and kits related to the use of genetic variants or their combination, to assess individual risk of developing Alzheimer's disease (AD). Diseases such as AD may be associated with one or more genetic variations, where the presence of a genetic variation may increase the risk of developing AD or is indicative of AD, thus enabling genetic analysis as a tool for disease prediction.
Specifically, the inventors have determined that elevated soluble ST2 protein (sST2) in the plasma and cerebral spinal fluid (CSF), in conjunction with the presence of specific novel variants associated with sST2 protein in plasma, are associated with increased AD risk in Chinese populations. Moreover, the inventors have demonstrated that ST2 transcripts, eosinophil counts, and associated marker gene expression are altered in blood of AD patients. The inventors have further identified medication that can reduce plasma sST2 levels, which may be able to alleviate AD in clinical practice.
By leveraging these findings, the inventors have developed novel methods to determine if a subject has AD or is at increased risk of developing the disease by means of detecting specific variants in individuals' genomic DNA sequences. A polygenic risk scoring system based on genomic information, further optimized by including information on blood cell counts and plasma protein levels, has also been developed to predict the relative risk of AD. Taken together, this invention provides a significant breakthrough for clinical screening (e.g., diagnosis, prognosis) of the disease.
The invention relates to the discovery of novel genetic variants associated with the Alzheimer's Disease (AD). The invention thus provides methods and compositions useful for diagnosis or treating AD. Thus, in a first aspect, the present invention provides a method for detecting the presence of AD in a subject or an increased risk of developing AD at a later time by detecting in a biological sample taken from the subject the presence of one or more of the genetic variants in Table 3, for example, at least 5, 10, 15, 20, or 25 or more of the genetic variants in Table 3. In some embodiments, the subject is a Chinese, for example, a Hong Kong Chinese or mainland Chinese. In some embodiments, the subject has a family history of AD but does not exhibit symptoms of AD. In some embodiments, the subject has no family history of AD. In some embodiments, the sample is a blood sample, such as a whole blood sample or a sample of blood cells. In some embodiments, the sample contains cells or tissues from any part of the patient's body, such as saliva, oral swab, sweat, or urine. In some embodiments, the detecting step comprises an amplification reaction to amplify the genetic variant(s), such as polymerase chain reaction (PCR) including reverse transcription PCR (RT-PCR). In some embodiments, the detecting step comprises a hybridization-based method or a sequencing-based method. In some embodiments, the genetic variants comprise or consist of rs1921622, rs75687525, rs4499395, rs56238602, rs2140316, rs12052753, rs199977663, rs2141304, rs4103380, rs4703514, rs191531802, rs2300619, rs1229502, rs9886235, rs6587006, rs7047059, rs5006678, rs7849649, rs1907370, rs11615704, rs66994203, rs117523785, rs17641976, rs7208104, rs12600563, rs142226688, rs111246464, rs4583526, and rs56242654, or any combinations thereof. In some embodiments, the variants comprise or consist of rs1921622 and rs12052753. In some embodiments, the claimed method further comprises the step of determining the subject's APOE genotype or haplotype. In some embodiments, the claimed method further comprises the step of determining eosinophil count in a blood sample. In some embodiments, the method further comprises the step of determining sST2, full length ST2, or CCR3 expression level in a blood sample. In some embodiments, the expression level is protein level. In some embodiments, wherein the step of of determining sST2 or CCR3 expression level comprises an immunoassay or mass spectrometry. In other embodiments, the expression level is mRNA level. wherein the step of determining sST2, full length ST2, or CCR3 expression level comprises a reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization, or a sequencing-based method. In some embodiments, the claimed method further comprises a step of calculating a Polygenic Risk Score (PRS) as the sum of effect size-weighted (βi) genotype dosage of corresponding variants on sST2 level in the sample, wherein n is the number of genetic variants tested: PRS=β1 snp 1+β2snp2+ . . . +βnsnpn. In some embodiments, the number of genetic variants tested is 29. In some embodiments, the method is applied to determine the subject with a PRS no greater than 0 as not having AD or not having increased risk for AD and the subject with a PRS greater than 0 as having AD or having increased risk for AD. In some embodiments, the method further comprises a step of administering to the subject an agent effective for treating AD upon determining the subject as having AD or having an increased risk of developing AD. In some embodiments, the agent is trazodone.
In a second aspect, the invention provides a method for treating or reducing the risk of AD in a subject by administering to the subject a composition comprising (1) an effective amount of trazodone and (2) a pharmaceutically acceptable excipient. In some embodiments, the method further includes a step of administering to the subject an effective amount of a second therapeutically active agent for AD. In some embodiments, the composition being administered to the patient is formulated for subcutaneous, transdermal, intramuscular, intravenous, intraperitoneal, intracranial injection, topical, nasal, or oral administration. In some embodiments, the treatment method comprises these steps: (a) selecting a subject who has been determined as having AD or having increased risk for AD, according to any of the methods disclosed above or herein for detecting AD or an increased risk for later developing AD; and (b) administering to the subject a composition comprising an effective amount of trazodone and a pharmaceutically acceptable excipient.
In a third aspect, the invention provides a medicament for treating or reducing the risk of AD in a subject. The medicament comprises (1) an effective amount of trazodone; and (2) a pharmaceutically acceptable excipient. In some embodiments, the medicament further comprises a second therapeutically active agent for AD. In some embodiments, the medicament is formulated for subcutaneous, transdermal, intramuscular, intravenous, intraperitoneal, intracranial injection, topical, nasal, or oral administration.
In a four aspect, the present invention provides a kit for detecting the presence of Alzheimer's Disease (AD) or an increased risk of developing AD in a subject. The kit typically includes a set of oligonucleotide primers suitable for amplifying a genomic DNA sequence harboring at least one of the genetic variants in Table 3. The kit optionally also includes an agent capable of specifically identifying the at least one genetic variant. In some embodiments, the kit further comprises a primer suitable for directing reverse transcription of an mRNA transcribed from the genomic DNA seqeunce. In some embodiments, the kit further comprises an instruction manual for detecting the presence of AD or an increased risk of developing AD.
The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
The term “gene” means the segment of DNA involved in producing a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to chemical compounds having a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
There are various known methods in the art that permit the incorporation of an unnatural amino acid derivative or analog into a polypeptide chain in a site-specific manner, see, e.g., WO 02/086075.
Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
“Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
An “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed and/or expressed, operably linked to a promoter.
As used herein, the term “gene expression” is used to refer to the transcription of a DNA to form an RNA molecule encoding a particular protein or the translation of a protein encoded by a polynucleotide sequence. In other words, both mRNA level and protein level encoded by a gene of interest are encompassed by the term “gene expression level” in this disclosure.
In this disclosure the term “biological sample” or “sample” includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes, or processed forms of any of such samples. Biological samples include blood and blood fractions or products (e.g., whole blood, serum, plasma, platelets, blood cells such as eosinophils, and the like), sputum or saliva, lymph and tongue tissue, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, stomach biopsy tissue etc. A biological sample is typically obtained from a eukaryotic organism, which may be a mammal, may be a primate and may be a human subject.
The term “immunoglobulin” or “antibody” (used interchangeably herein) refers to an antigen-binding protein having a basic four-polypeptide chain structure consisting of two heavy and two light chains, said chains being stabilized, for example, by interchain disulfide bonds, which has the ability to specifically bind antigen. Both heavy and light chains are folded into domains.
The term “antibody” also refers to antigen- and epitope-binding fragments of antibodies, e.g., Fab fragments, that can be used in immunological affinity assays. There are a number of well characterized antibody fragments. Thus, for example, pepsin digests an antibody C-terminal to the disulfide linkages in the hinge region to produce F(ab)′2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)′2 can be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab′)2 dimer into an Fab′ monomer. The Fab′ monomer is essentially a Fab with part of the hinge region (see, e.g., Fundamental Immunology, Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody also includes antibody fragments either produced by the modification of whole antibodies or synthesized using recombinant DNA methodologies.
The phrase “specifically binds,” when used in the context of describing a binding relationship of a particular molecule to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated binding assay conditions, the specified binding agent (e.g., an antibody) binds to a particular protein at least two times the background and does not substantially bind in a significant amount to other proteins present in the sample. Specific binding of an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein or a protein but not its similar “sister” proteins. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or in a particular form. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective binding reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background. On the other hand, the term “specifically bind” when used in the context of referring to a polynucleotide sequence forming a double-stranded complex with another polynucleotide sequence describes “polynucleotide hybridization” based on the Watson-Crick base-pairing, as provided in the definition for the term “polynucleotide hybridization method.”
As used in this application, an “increase” or a “decrease” refers to a detectable positive or negative change in quantity from a comparison control, e.g., an established standard control (such as an average level/amount of sST2 or eosinophil found in samples from healthy non-AD subjects). An increase is a positive change that is typically at least 10%, or at least 20%, or 50%, or 100%, and can be as high as at least 2-fold or at least 5-fold or even 10-fold of the control value. Similarly, a decrease is a negative change that is typically at least 10%, or at least 20%, 30%, or 50%, or even as high as at least 80% or 90% of the control value. Other terms indicating quantitative changes or differences from a comparative basis, such as “more,” “less,” “higher,” and “lower,” are used in this application in the same fashion as described above. In contrast, the term “substantially the same” or “substantially lack of change” indicates little to no change in quantity from the standard control value, typically within ±10% of the standard control, or within ±5%, 2%, or even less variation from the standard control.
A “polynucleotide hybridization method” as used herein refers to a method for detecting the presence and/or quantity of a pre-determined polynucleotide sequence based on its ability to form Watson-Crick base-pairing, under appropriate hybridization conditions, with a polynucleotide probe of a known sequence. Examples of such hybridization methods include Southern blot, Northern blot, and in situ hybridization.
“Primers” as used herein refer to oligonucleotides that can be used in an amplification method, such as a polymerase chain reaction (PCR), to amplify a nucleotide sequence based on the polynucleotide sequence corresponding to a gene of interest, e.g., any one of the genetic variants described herein or a portion thereof. Typically at least one of the PCR primers for amplification of a polynucleotide sequence is sequence-specific for that polynucleotide sequence. The exact length of the primer will depend upon many factors, including temperature, source of the primer, and the method used. For example, for diagnostic and prognostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains at least 10, or 15, or 20, or 25 or more nucleotides, although it may contain fewer nucleotides or more nucleotides. The factors involved in determining the appropriate length of primer are readily known to one of ordinary skill in the art. In this disclosure the term “primer pair” means a pair of primers that hybridize to opposite strands a target DNA molecule or to regions of the target DNA which flank a nucleotide sequence to be amplified. In this disclosure the term “primer site,” means the area of the target DNA or other nucleic acid to which a primer hybridizes.
A “label,” “detectable label,” or “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins that can be made detectable, e.g., by incorporating a radioactive component into the peptide or used to detect antibodies specifically reactive with the peptide. Typically a detectable label is attached to a probe or a molecule with defined binding characteristics (e.g., a polypeptide with a known binding specificity or a polynucleotide), so as to allow the presence of the probe (and therefore its binding target) to be readily detectable.
The term “amount” as used in this application refers to the quantity of a substance of interest, such as a polynucleotide or a polypeptide of interest or a certain type of blood cells, present in a sample. Such quantity may be expressed in the absolute terms, i.e., the total quantity of the substance in the sample, or in the relative terms, i.e., the concentration of the substance in the sample.
The term “subject” or “subject in need of treatment,” as used herein, includes individuals who seek medical attention due to risk of (e.g., with family history), or having been diagnosed of, AD. Subjects also include individuals currently undergoing therapy that seek manipulation of the therapeutic regimen. Subjects or individuals in need of treatment include those that demonstrate symptoms of AD or are at risk of suffering from AD or its symptoms. For example, a subject in need of treatment includes individuals with a genetic predisposition or family history for AD, those that have suffered relevant symptoms in the past, those that have been exposed to a triggering substance or event, as well as those suffering from chronic or acute symptoms of the condition. A “subject in need of treatment” may be at any age of life.
“Inhibitors,” “activators,” and “modulators” of a target protein are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for the protein binding or signaling, e.g., ligands, agonists, antagonists, and their homologs and mimetics. The term “modulator” includes inhibitors and activators. Inhibitors are agents that, e.g., partially or totally block, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of the target protein. In some cases, the inhibitor directly or indirectly binds to the protein, such as a neutralizing antibody. Inhibitors, as used herein, are synonymous with inactivators and antagonists. Activators are agents that, e.g., stimulate, increase, facilitate, enhance activation, sensitize or up regulate the activity of the target protein. Modulators include the target protein's ligands or binding partners, including modifications of naturally-occurring ligands and synthetically-designed ligands, antibodies and antibody fragments, antagonists, agonists, small molecules including carbohydrate-containing molecules, siRNAs, RNA aptamers, and the like.
The term “treat” or “treating,” as used in this application, describes an act that leads to the elimination, reduction, alleviation, reversal, prevention and/or delay of onset or recurrence of any symptom of a predetermined medical condition. In other words, “treating” a condition encompasses both therapeutic and prophylactic intervention against the condition.
The term “effective amount,” as used herein, refers to an amount that produces therapeutic effects for which a substance is administered. The effects include the prevention, correction, or inhibition of progression of the symptoms of a disease/condition and related complications to any detectable extent. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).
The term “standard control,” as used herein, refers to a sample comprising an analyte of a predetermined amount to indicate the quantity or concentration of this analyte present in this type of sample (e.g., DNA/mRNA, protein, or blood cells such as eosinophils) taken from an average healthy subject not suffering from or at risk of developing a predetermined disease or condition (e.g., Alzheimer's Disease).
The term “average,” as used in the context of describing a healthy subject who does not suffer from and is not at risk of developing a relevant disease or disorders (e.g., AD) refers to certain characteristics, such as the level of a pertinent gene's mRNA or protein in the person's tissues (e.g., blood) or blood cell (e.g., eosinophil) counts that are representative of a randomly selected group of healthy humans who are not suffering from and is not at risk of developing the disease or disorder. This selected group should comprise a sufficient number of human subjects such that the average amount or concentration of the analyte of interest among these individuals reflects, with reasonable accuracy, the corresponding profile in the general population of healthy people. Optionally, the selected group of subjects may be chosen to have a similar background to that of a person whose is tested for indication or risk of the relevant disease or disorder, for example, matching or comparable age, gender, ethnicity, and medical history, etc.
The term “inhibiting” or “inhibition,” as used herein, refers to any detectable negative effect on a target biological process. Typically, an inhibition is reflected in a decrease of at least 10%, 20%, 30%, 40%, or 50% in one or more parameters indicative of the biological process or its downstream effect, e.g., plasma level of sST2 or eosinophil count or blood CCR3 level, when compared to a control where no such inhibition is present. The term “enhancing” or “enhancement” is defined in a similar manner, except for indicating a positive effect, i.e., the positive change is at least 10%, 20%, 30%, 40%, 50%, 80%, 100%, 200%, 300% or even more in comparison with a control. The terms “inhibitor” and “enhancer” are used to describe an agent that exhibits inhibiting or enhancing effects as described above, respectively. Also used in a similar fashion in this disclosure are the terms “increase,” “decrease,” “more,” and “less,” which are meant to indicate positive changes in one or more predetermined parameters by at least 10%, 20%, 30%, 40%, 50%, 80%, 100%, 200%, 300% or even more, or negative changes of at least 10%, 20%, 30%, 40%, 50%, 80% or even more in one or more predetermined parameters.
“Hong Kong Chinese” and “mainland Chinese” are terms used to refer to ethnic Chinese people who and whose ancestors have been residing in Hong Kong and mainland China, respectively, for a length of time, e.g., at least the last 3, 4, 5, 6, 7, or 8 generations or the last 100, 150, 200, 250, or 300 years.
AD patients face a grim prognosis due to the progressive and incurable nature of this disease. Early diagnosis of AD or detection of an increased risk for developing AD at a future time may provide the patients and their family more options in terms of treatment and long term management of the disease.
The present inventors discovered for the first time that certain genetic variants, especially those at the IL1RL1 locus, which directly influence the level of plasma sST2 protein, are correlated with the presence of AD in a patient or an increased risk of the patient developing the disease at a later time. This discovery thus provides novel methods and compositions useful for the early diagnosis and treatment of AD.
Practicing this invention utilizes routine techniques in the field of molecular biology. Basic texts disclosing the general methods of use in this invention include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).
For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Protein sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
Oligonucleotides that are not commercially available can be chemically synthesized, e.g., according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is performed using any art-recognized strategy, e.g., native acrylamide gel electrophoresis or anion-exchange high performance liquid chromatography (HPLC) as described in Pearson and Reanier, J. Chrom. 255: 137-149 (1983).
The sequence of interest used in this invention, e.g., the polynucleotide sequence of any one of the genetic variants shown in Tables 2 and 3, and synthetic oligonucleotides (e.g., primers useful for amplifying any one of the genetic variants) can be verified using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al., Gene 16: 21-26 (1981).
The present invention relates to detecting of AD-associated genetic variants, measuring the amount of a marker mRNA/protein found in a person's biological sample (for example, a blood sample), and/or measuring the number of a certain type of blood cells (for example, eosinophils), as a means to detect the presence, to assess the risk of developing, and/or to monitor the progression or treatment efficacy of AD. More specifically, the method involves (1) determining the presence of any one or more of the genetic variants in Table 2 or 3; (2) quantitatively determining the expression level of one or more marker gene(s), such as sST2 or CCR3, at the mRNA or protein level, and/or (3) determining eosinophil counts in a patient's biological sample and comparing with a standard control value in order to detect any quantitative changes, which in turn indicate the presence of AD or an elevated risk of later developing AD in the patient.
To practice this method one typically sequence the relevant genomic sequence to detect the AD-associated variants or analyzes the amount of the pertinent mRNA or protein found in a sample taken from a person being tested, e.g., a blood sample especially a whole blood sample or blood cell sample. Collection of blood from an individual is performed in accordance with the standard protocol hospitals or clinics generally follow. An appropriate amount of peripheral blood, e.g., typically between 5-50 ml, is collected in some cases and may be stored according to standard procedure prior to further preparation. Other biological samples taken from a broad range of anatomic sites containing somatic cells can also be used for the purpose of detecting genetic variants, for example, saliva, mouth swab, tissue biopsy, or cerebrospinal fluid (CSF).
To detect the presence of specific AD-associated genetic variants, e.g., those listing in Tables 2 and 3 or an APOE genotype such as APOE-ε4, polynucleotide sequence-based analysis is performed on a test subject's genomic DNA. In some cases, a polynucleotide hybridization method may be used in the analysis, e.g., by way of a chip containing a large number of immobilized polynucleotide probes allowing ready identification of various different genetic sequences of interest. In some cases, an amplification reaction is optional prior to the sequence analysis. A variety of polynucleotide amplification methods are well-established and frequently used in research. For instance, the general methods of polymerase chain reaction (PCR) for polynucleotide sequence amplification are well known in the art and are thus not described in detail herein. For a review of PCR methods, protocols, and principles in designing primers, see, e.g., Innis, et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, Inc. N.Y., 1990. PCR reagents and protocols are also available from commercial vendors, such as Roche Molecular Systems.
When the presence of specific AD-associated genetic variants, such as those listing in Tables 2 and 3 or an APOE genotype, is sought to be detected at the mRNA level, a step of reverse transcription (RT) is typically performed prior to the amplification reaction such as PCR. Once adequate DNA potentially containing the genetic variants has been obtained, sequencing can be performed to reveal the presence of such relevant variants, e.g., single-nucleotide polymorphisms (SNPs). Various high-through put, fully automated sequencing technologies are available through commerical suppliers (e.g., ThermoFisher Scientific) and can be used for practicing the present invention.
Although PCR amplification is often used in practicing the present invention, one of skill in the art will recognize that amplification of the relevant genomic sequence may be accomplished by any known method, such as the ligase chain reaction (LCR), transcription-mediated amplification, and self-sustained sequence replication or nucleic acid sequence-based amplification (NASBA), each of which provides sufficient amplification. Techniques for polynucleotide sequence determination are also well-established and widely practiced in the relevant research field. For instance, the basic principles and general techniques for polynucleotide sequencing are described in various research reports and treatises on molecular biology and recombinant genetics, such as Wallace et al., supra; Sambrook and Russell, supra, and Ausubel et al., supra. DNA sequencing methods routinely practiced in research laboratories, either manual or automated, can be used for practicing the present invention.
Similarly, the analysis of pertinent protein or mRNA (such as sST2 or CCR3) found in a patient's sample according to the present invention may be performed using, e.g., the whole blood, or a cellular sample such as all blood cells. Standard methods known in the research field can be used to isolate and analyze the protein or RNA level of a marker gene in the sample. See, e.g., Sambrook and Russell, supra).
In order to establish a standard control for practicing the method of this invention, a group of healthy persons free of any neurodegenerative disorders especially AD and not known to be at risk of developing the disorder are first selected. These individuals are within the appropriate parameters, if applicable, for the purpose of screening for and/or assessing future risk of such disorders using the methods of the present invention. Optionally, the individuals are of the same or comparable gender, age, ethnic background, and medical history. The healthy status of the selected individuals is confirmed by well established, routinely employed methods including but not limited to general physical examination of the individuals and general review of their medical records.
Furthermore, the selected group of healthy individuals must be of a reasonable size, such that the average amount/concentration of pertinent mRNA or protein or blood cells (e.g., eosinophils) in the samples obtained from the group can be reasonably regarded as representative of the normal or average level among the general population of healthy people without and not at risk of developing AD. Preferably, the selected group comprises at least 10 human subjects.
Once such an average value for the pertinent protein or mRNA or blood cells is established based on the individual values found in each subject of the selected healthy control group, this average or median or representative value or profile is considered a standard control. A standard deviation is also determined during the same process. In some cases, separate standard controls may be established for separately defined groups having distinct characteristics such as age, gender, ethnic background, or any distinct past event(s) in the medical history.
In a related aspect, the present invention also provides treatment methods for AD patients upon detection of AD or a heightened risk of later developing AD in a patient. For example, when the diagnostic method steps described above and herein are completed, optionally with additional diagnostic examination performed to provide further confirmatory information (for example, by brain imaging via CT scan or other imaging techniques to show excessive loss of brain volume, or by testing cognitive capability to show an accelerated decline), and a patient has been determined to either already have AD or is at a significantly increased risk of later developing AD, suitable therapeutic or prophylactic regimens may be ordered by physicians or other medical professionals to treat the patient, to manage/alleviate the ongoing symptoms, or to delay the future onset of the disease. The U.S. Food and Drug Administration (FDA) has approved a number of cholinesterase inhibitors, including donepezil (Aricept™, the only cholinesterase inhibitor approved to treat all stages of AD, including moderate to severe), rivastigmine (Exelon™, approved to treat mild to moderate AD), galantamine (Razadyne™, mild to moderate patients) and memantine (Namenda™) Donepezil is the only cholinesterase inhibitor approved to treat all stages of AD, including moderate to severe. Any one or more of these drugs can be prescribed for treating patients who have been diagnosed with AD in accordance with the methods of this invention. For patients having increased expression of plasma sST2, one possibility of treatment is administration of trazodone, which is currently approved for use as an antidepressant and has been shown by the present inventors as an effective agent to lower plasma sST2 level.
For patients who are deemed high risk for AD in a future time but do not yet exhibit any clinical symptoms, continuous monitoring is also appropriate, for example, the patients may be subject to regular testing (e.g., once a year or once every two years) to detect any change in their cognitive capabilities. Methods suitable for such regular monitoring include General Practitioner Assessment of Cognition (GPCOG), Mini-Cog, Eight-item Informant Interview to Differentiate Aging and Dementia (AD8), and Short Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE). Furthermore, prophylactic treatment with trazodone may also be recommended.
The present invention provides a kit for diagnosing or determining risk of AD in a subject. The kit typically comprises a first container containing an agent for amplifying a genomic sequence potentially harboring one or more genetic variants in Table 2 or 3. Optionally the kit includes a second container containing an agent for detecting the presence of one or more genetic variants in Table 2 or 3. For example, the agent in the first container may include a pair of primers or oligonucleotides useful for amplifying the genomic sequence, e.g., in a PCR. In the case of the kit intended for use in an RT-PCR, a further primer for reverse transcription is included as well. The second container may include a polynucleotide probe that specifically binds to a DNA sequence comprising the variant or variants so as to be able to indicate the presence or absence of the variant(s). In some embodiments, the primers and/or probe may be conjugated with a detectable label to faciliate the ready detection of the relevant DNA amplicons and/or the genetic variant(s) of interest.
Further optional components of the kit may include (a) one container containing an agent that is useful for determining the expression level of sST2 or CCR3 mRNA or protein in a biological sample taken from the subject, such as an antibody that specifically recognizes the protein or a polynucleotide probe that specifically binds a coding sequence for the mRNA; and (b) another container containing a standard control indicating the corresponding sST2 or CCR3 level (which may be protein or mRNA level) in the same type of biological sample taken from an average healthy subject not suffering from and not at risk of suffering from AD. Optionally, the polynucleotide probe or antibody is labeled with a detectable moiety. The antibody can be either a monoclonal antibody or a polyclonal antibody. In some cases, the kits may include at least two different antibodies, one for specific binding to the target protein (i.e., the primary antibody) and the other for detection of the primary antibody (i.e., the secondary antibody), which is often attached to a detectable moiety. In addition, the kits of this invention may provide instruction manuals to guide users in analyzing test samples and assessing the presence, risk, or state of gastric cancer in a test subject.
This invention also provides a kit for inhibiting or treating AD for therapy or prevention of the pertinent symptoms according to the method of this disclosure. The kits typically include a container that contains (1) a pharmaceutical composition having an effective amount of an active agent (for instance, trazodone) and (2) informational material containing instructions on how to dispense the pharmaceutical composition, including description of the type of patients who may be treated (e.g., patients suffering from AD), the schedule (e.g., dose and frequency) and route of administration, and the like. Optionally, an additional container may be included in the kit that provides a second therapeutic agent known to be effective for treating AD, for example, any one of cholinesterase inhibitors (Aricept, Exelon, Razadyne, Cognex) for early to moderate stages of AD or memantine (Namenda) to treat the cognitive symptoms (memory loss, confusion, and problems with thinking and reasoning) for moderate to severe stages of AD.
In a further aspect, the present invention can also be embodied in a device or a system comprising one or more such devices, which device is capable of carrying out all or some of the method steps described herein. For instance, in some cases, the device or system performs the following steps upon receiving a biological sample, e.g., a blood sample taken from a subject being tested for AD, assessing the risk of developing AD, or monitored for progression of the condition: (a) determining in sample the presence or absence of one or more genetic variants in Table 2 or 3; (b) generating a composite score (polygenic risk score, or PRS) from the information obtained in step (a); and (c) providing an output indicating whether AD is present in the subject or whether the subject is at risk of developing AD, or whether there is a change, i.e., worsening or improvement, in the subject's condition associated with AD.
In some cases, the device or system of the invention performs the task of steps (b) and (c), after step (a) has been performed and the presence or absence of relevant genetic variants from (a) has been entered into the device. Preferably, the device or system is partially or fully automated.
Optionally, the device or system may further perform, prior to step (c), the additional step(s) of assessing the quantity of additional marker(s), such as sST2 and/or CCR3 in either protein or mRNA form, present in the sample, or determining blood cell count such as eosinophil count in a blood sample, or determining a subject the APOE genotype especially APOE-ε4 genotype; in the alternative, the additional information, including the quantity/concentration of the marker(s) present in the sample, blood cell count, APOE genotype, or test subject's gender and/or ethnic background is obtained from one or more separate processes not performed with the device or system but is fed into the device or system in order to perform step (c) while taking into consideration of such additional information.
The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.
Alzheimer's disease (AD) is one of the most common forms of dementia worldwide, accounting for 60-70% of all dementia cases. It is an irreversible degenerative brain disease and a leading cause of mortality in the elderly. The hallmarks of AD are deposition of extracellular beta-amyloid (Aβ) plaques and intracellular neurofibrillary tangles, which result in progressively declining memory, reasoning, judgment, and locomotion abilities. An estimated 46.8 million people worldwide are afflicted with AD, but this figure is expected to rise significantly to 100 million by 2050 owing to longer life expectancies and aging populations. There is currently no cure for AD, and its pathophysiology is still relatively unknown.
There are only five drugs approved by the US Food and Drug Administration (FDA) to treat AD, but these only alleviate symptoms rather than alter disease pathology, i.e., they cannot reverse the condition or prevent further deterioration. Furthermore, AD affects the brain long before actual symptoms of memory loss or cognitive decline manifest. However, there are no diagnostic tools for early detection; by the time a patient is diagnosed with AD using current methods, which involves subjective clinical assessment, the pathological symptoms are already at an advanced stage. In addition, although several serum protein biomarkers such as serum APOE, Aβ, Tau, and p-Tau have been identified and brain imaging techniques such as positron emission tomography (PET) scan and functional magnetic resonance imaging (MRI) have been introduced to help predict AD progression, limitations such as high cost, high inter-individual variation, and low-throughput rates prevent their use for disease diagnosis. Thus, early therapeutic intervention is critical for the management of AD, and new and effective methods for the early diagnosis of AD and determining an individual's risk of developing the disease are urgently required.
The pathophysiological mechanisms of AD are complex, but genetic factors are believed to play critical roles. Genes can differ between individuals because of genomic variability—the most frequent of which can be due to single nucleotide polymorphisms (SNPs). Additional genetic polymorphisms in a human genome can be caused by duplication, insertion, deletion, translocation, and/or inversion of short and/or long stretches of DNA. Genetic variations may encode protein variants that can result in increased susceptibility to a disease or result in disease onset, including AD. AD may be associated with one or more genetic variations, wherein the presence of a genetic variation may increase the risk of developing AD or may be indicative of AD. Accordingly, genetic analysis can be used to determine the presence of such genetic variations.
AD is classified into two types based on the onset age: familial AD (also known as early-onset AD) with an onset age ≤45 years or late-onset AD with an onset age ≥65 years. Early-onset AD accounts for 10% of all cases and tends to be predominant in certain families because of the inheritance of specific and rare missense mutations in APP, PSEN1, or PSEN2. Meanwhile, late-onset AD accounts for 90% of all cases. Polymorphisms in APOE are the most relevant risk factors for late-onset AD. Genetics studies including, but not limited to, genome-wide association studies (GWAS), candidate gene sequencing, and whole-exome sequencing have identified several disease genes and risk alleles. However, most previous genetics studies on AD, including those on the APOE locus, have been conducted on individuals with Caucasian ancestry, whereas data from other ethnicities are limited. Given the diverse genomic content across different ethnic groups, the genetic risk factors for AD may be different between populations of Asian (e.g., Chinese) and Caucasian origins. Moreover, susceptibility to certain genetic risk factors varies among populations. Therefore, it is vital to systematically investigate the genetic risk factors in populations of different ethnicities.
Inflammatory disorders and cardiovascular diseases are potentially associated with AD risks (McGeer & McGeer, 2001; Newman et al., 2005). Individuals affected by inflammatory disorders and cardiovascular disease may harbor genetic predisposition to those disease (Grotenboer, Ketelaar, Koppelman, & Nawijn, 2013; Tu et al., 2013). Large-scale genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) in IL1RL1 (the gene that encodes full-length ST2 and sST2 protein) that associate with plasma protein levels of soluble ST2 (sST2), a decoy receptor for IL-33, in different diseases across various populations (Gudbjartsson et al., 2009; Zhu et al., 2018). Two of the IL1RL1 SNPs reported in asthma demonstrated a possible functional effect on airway inflammation (Gordon et al., 2016), whereas others were postulated to exert a protective function by influencing IL-33 activity (Ramirez-Carrozzi, Dressen, Lupardus, Yaspan, & Pappu, 2015). IL1RL1 SNPs associated with serum sST2 levels have also been identified in cardiovascular disease (Ho et al., 2013), while other studies have also demonstrated the utility of IL1RL1 SNPs as a predictor of mortality in coronary and peripheral artery disease (Lin et al., 2017). Thus, studies of the genetic variants of IL1RL1 in different diseases, and their regulation on sST2 levels in human blood may help to identify new biomarkers for different diseases and uncover new disease pathways.
Several studies have shown that IL-33/ST2 signaling mediates allergic asthma. IL-33 expression is elevated in airways of bronchial asthma, where it promotes airway remodeling. In a mouse model of pulmonary inflammation, pre-exposure to sST2 results in reduced production of TH2 cytokines, in keeping with the role of sST2 as a decoy receptor for IL-33, and, patients of bronchial asthma show elevated levels of serum sST2 (Kakkar & Lee, 2008). Meanwhile, IL-33/ST2 signaling exerts a protective role in cardiac fibroblasts and cardiomyocytes where the expression levels of IL-33, sST2, and ST2L increase in response to myocardial stress. In experimental models, IL-33/ST2 signaling prevents fibrosis and cardiomyocyte hypertrophy and reduces apoptosis, thus improving cardiac function. The antihypertrophic effect of IL-33 may be exerted via suppression of ROS (reactive oxygen species), regulation of NF-κB, or other mechanisms (Sanada et al., 2007). sST2 appears to antagonize the cardio-protective effects of IL-33/ST2L. In stimulated cardiomyocytes, pre-incubation with sST2 reversed the anti-hypertrophic effect of IL-33, and led to a decrease in free IL-33. High serum sST2 levels were also observed in patients with acute myocardial infraction and acute heart failure (Pascual-Figal & Januzzi, 2015). These results collectively suggest the potential usage of sST2 or associated genomic information as promising biomarkers for inflammatory diseases, cardiovascular disorders, as well as neurological disorders such as AD. Hence, the inventors undertook a comprehensive study of the genetic basis of AD in the Chinese population with a focus on sST2.
Levels of plasma sST2 were measured in a Hong Kong Chinese AD cohort comprising 427 participants by ELISA assay (Table 1). Female AD patients exhibited higher plasma sST2 levels compared to age- and gender-matched normal controls (NCs) (
Additionally, the inventors performed whole-genome sequencing (WGS) analysis on the Hong Kong Chinese AD cohort and conducted an association test between the genetic variants and plasma levels of sST2. The variants near the IL1RL1 (the gene that encodes full-length ST2 and sST2 protein) regions were associated with changes in plasma sST2 levels (
The elevated plasma levels of sST2 in AD (
Moreover, as levels of sST2 protein and full-length ST2 transcript were altered in the peripheral blood of AD patients, which implies dysregulation of IL-33/ST2 signaling in the peripheral circulatory system, the inventors examined whether this dysregulated IL-33/ST2 signaling leads to alteration of blood signatures, such as changes in the proportion of blood cells. Accordingly, the correlations between full-length ST2 transcript level and blood cell counts or transcript levels of specific blood cell-type markers were determined. The transcript levels of full-length ST2 were positively correlated with that of CCR3 (an eosinophil marker) in the blood as well as the absolute eosinophil count in the Hong Kong Chinese AD cohort (
Additionally, medications that may regulate the plasma sST2 protein level were investigated. The inventors conducted an association analysis of the plasma sST2 protein level and medical information collected in Hong Kong female AD patients. Trazodone may lead a decreased plasma sST2 level in AD patients (
Furthermore, the inventors developed a polygenic risk score (PRS) model, to calculate individual risk level, by summarizing the effects from a total of 29 sST2-associated variants as listed: rs75687525, rs4499395, rs56238602, rs1921622, rs2140316, rs12052753, rs199977663, rs2141304, rs4103380, rs4703514, rs191531802, rs2300619, rs1229502, rs9886235, rs6587006, rs7047059, rs5006678, rs7849649, rs1907370, rs11615704, rs66994203, rs117523785, rs17641976, rs7208104, rs12600563, rs142226688, rs111246464, rs4583526, rs56242654. Association analysis revealed significant association between the obtained PRS and AD for female participants harboring APOE-ε4 risk variants in the mainland Chinese AD cohort (effect size=0.344, P-value=0.046; Table 10), with concordant trend in the Hong Kong Chinese AD cohort.
The PRS model was further optimized by adding the information on APOE genotypes and plasma sST2 levels, to increase its accuracy in classifying AD and NC (AUC=0.6203 and 0.6678, for APOE alone and after adding PRS and plasma sST2, respectively;
Subject Recruitment for the Hong Kong Chinese AD Cohort: A cohort of Hong Kong Chinese was recruited from individuals who visited the Specialist Outpatient Department of the Prince of Wales Hospital, the Chinese University of Hong Kong. A total of 427 subjects were recruited: 233 with AD and 194 normal controls (NCs). All subjects were ≥60 years old. The clinical diagnosis of AD was established based on the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5). All subjects were given medical history assessment, cognitive and functional assessment through the Montreal Cognitive Assessment (MoCA) test, and neuroimaging assessment by MRI. Each individual's data, including age, sex, education, medical history, and cardiovascular disease history, were recorded. Individuals with any significant neurological disease or psychiatric disorder were excluded. The plasma of subjects was collected and stored at −80° C. as aliquots until use. This study was approved by the Prince of Wales Hospital, the Chinese University of Hong Kong, and the Hong Kong University of Science and Technology. All participants provided written informed consent for both study enrollment and sample collection. Absolute blood cell count was determined by complete blood count (CBC) at the Prince of Wales Hospital. The medication and disease records for the Hong Kong Chinese AD cohort were obtained from the Prince of Wales Hospital, as well.
Subject Recruitment for the Mainland Chinese AD Cohort: A cohort of mainland Chinese participants with AD, mild cognitive impairment (MCI), and age- and gender-matched normal controls were recruited by the Department of Neurology or Memory Clinic, Huashan Hospital, Fudan University, Shanghai, China, from 2007 to 2018. AD patients were diagnosed based on the recommendations of the National Institute on Aging and the Alzheimer's Association workgroup, and had an onset age of ≥50 years. Patients with MCI were diagnosed according to the Peterson criteria. Individuals with any significant neurological disease or psychiatric disorder were excluded. Part of the NCs without subjective memory complaints were recruited from the general community in Shanghai. A total of 1,696 participants (N=867 and 829, for AD and NC, respectively), with 911 female participants, were recruited. All participants were subjected to medical history assessment, neuropsychological assessment, and imaging assessment including computed tomography (CT) or magnetic resonance imaging (MRI). Some subjects also underwent positron emission tomography using Pittsburgh compound B. The study was approved by the Ethics Committee of Huashan Hospital, the Hong Kong University of Science and Technology (HKUST), and the HKUST Shenzhen Research Institute. All subjects provided written informed consent for both study enrollment and sample collection.
Human Cerebrospinal Fluid Samples: All human cerebrospinal fluid (CSF) samples were obtained from The MRC UK Brain Bank Network (UKBBN). The samples from AD and un-demented NCs were selected on the basis of clinical diagnosis, with post-mortem duration ≤30 h.
Detection of Soluble ST2 Protein by ELISA: Human plasma and CSF soluble ST2 (sST2) protein levels were measured using the Human ST2/IL-33 R Quantikine ELISA Kit (DST200).
Blood Transcriptome RNA Sequencing: Total RNA was extracted from blood samples collected in PAXgene tubes, followed by globin depletion using the GLOBINclear Kit (Thermo Fisher) to eliminate high-copy globin mRNA from the total RNA. The integrity, purity, and concentration were evaluated by Fragment Analyzer and Biodrop spectrophotometer. Library construction and RNA sequencing (RNA-seq) were performed by Novogene (Beijing). Briefly, mRNA was enriched from total RNA using oligo-dT beads and then subjected to chemical fragmentation. The fragmented mRNA was reverse-transcribed to cDNA, ligated to adapters, and amplified by PCR to generate cDNA libraries. All libraries were assessed for quality (i.e., concentration and library size distribution), and the ones that passed quality control requirements were subjected to the Illumina HiSeq X platform for transcriptome profiling, generating 12 million 150-bp paired-end reads per sample. Sequencing reads were mapped to the human reference genome (Grch37) using the splice-aware aligner, STAR, followed by Stringtie for transcript quantification.
Whole-genome sequencing and Variant Calling Method: Whole-genome sequencing (WGS, 5× coverage) was performed by Novogene. The genomic DNA libraries were sequenced on an Illumina Hiseq X Ten platform, which generated 150-bp paired-end reads. Variants from the sequencing data were detected by the Gotcloud pipeline, which were subsequently subjected to FastQC for quality checking and Trimmomatic to trim and filter low-quality reads. GRCh37, containing the decoy fragments, was adopted as the reference genome to map the filtered data by BWA-mem. After de-duplication and clipping of the overlapped paired-end reads, the BAM files were subjected to samtools to generate glf files, which contained the marginal likelihoods for genotypes, followed by glfFlex for population-based SNP calling. Hard filtering implemented in the Gotcloud pipeline as VcfCooker was applied to filter low-confidence variant calls based on distance with known insertion/deletion sites, allele balance, and mapping quality. Variants with high-confidence calls and a minor allele frequency (MAF) ≥5% were subjected to Beagle for phasing. The top five principal components were generated by PLINK software with the following parameters: -pca header tabs, -maf 0.05, -hwe 0.00001, and -not-chr x y.
Association Test and Data Visualization for GWAS: A genome-wide SNP association test with plasma sST2 level was performed using PLINK software, adjusting for covariates (including age, gender, and top five principal components), and with the following parameters: -keep-allele-order, -linear, -ci 0.95, -hwe 0.00001, and -maf 0.05. To visualize the data, Manhattan plots were generated using the R qqman package. Regional plots for IL1RL1 locus were generated using LocusZoom. Fine mapping analysis for the effects of the IL1RL1 locus on plasma sST2 expression was performed using CAVIAR software with association test results and pairwise linkage disequilibrium (LD) information generated from PLINK using the following parameters: -hwe 0.00001, -maf 0.05, -r, -matrix, -chr 2, -from-bp102000000, and -to-bp 104000000.
Sanger sequencing for the last exon of ILIRLI: Sanger sequencing was performed using the following primers for PCR with 10 ng extracted blood genomic DNA as input: forward primer: 5′-AGACTTTTAAATGTTCAGGATGTTT-3′ (SEQ ID NO:576); reverse primer: 5″-CCCAGAAGCAGGGAAATG-3′ (SEQ ID NO:577).
Genotyping of APOE by TaqMan assay: For APOE-ε4 (rs429358 and rs7412), genotyping was conducted by TaqMan assay with probe ordered from Thermo Scientific (assay ID: C_3084793_20 and C_904973_10). 10 ng genomic DNA were subjected to real-time PCR on the QuantStudio 7 Flex Real-Time PCR system (Applied Biosystems) and genotype calling was performed using QuanStudio Real-Time PCR software (Applied Biosystems).
Polygenic Risk Score for sST2 Level and Its Association with AD: sST2-associated variants with P-value lower than 1E-5 were subjected to SNP pruning (r2<0.2) to yield 29 independent signals. Polygenic risk scores (PRS) were calculated by weighting the genotype dosage by the effect size of each corresponding variants onto the sST2 level, to generate PRS at individual level. Lasso regression with cross-validation were performed for PRS alone, or in combination with plasma sST2 levels and eosinophil counts to estimate their effectiveness on classifying AD and NC. Logistic regression was performed using R programming with adjustment for age to test the association between PRS and AD.
Statistical Analyses and Data Visualization: All statistical plots were generated using GraphPad Prism version 6.0, Locuszoom or R programming. For sST2 level analysis, the investigators who performed the ELISA were blinded to the experimental groups and conditions. Linear regression was performed, adjusting for covariates including age, gender, disease history, and population structure (top principal components obtained from principal components analysis using whole-genome sequencing data). The level of significance was set at P<0.05. For blood transcriptome and blood cell count analysis, linear regression was performed, adjusting for age, gender, disease history, RNA integrity number (RIN), and population structure (top principal components obtained from principal components analysis using whole-genome sequencing data). The level of significance was set at P<0.05. For medication analysis, the linear regression analysis for plasma sST2 protein level, individual cognitive performance as indicated by MoCA score, and medication history in female AD patients were conducted using R, adjusting for age and population structure.
Polygenic risk derived sST2-assocaited variants in predicting AD risk: To investigate polygenic effects of IL1RL1 locus, which potentially influence the plasma sST2, on to the AD risk, a polygenic risk score (PRS) were calculated at individual level summarizing effects from multiple sST2-associated variants. Association analysis revealed the significant association between the obtained PRS and AD in mainland AD cohort for female participants harboring APOE-ε4 risk variants (effect size=0.344, P-value=0.046, with concordant trend can be observed in Hong Kong Chinese AD cohort). Particularly, a cutoff value of 0 for PRS is able to predict AD risks for individuals in mainland female APOE-ε4 carriers (Sensitivity=62.32%, specificity=57.74%; Table 11). Therefore, to predict the risk of AD for female APOE-ε4 carriers, genomic DNA can be extracted from blood for genotyping of 29 sST2-associated SNPs. The polygenic risk score (PRS) is calculated based on the genotype dosage of 29 SNPs as previously described and a PRS larger than 0 suggests the high risk of AD for the individual in testing (
Moreover, after adding the information of APOE genotypes and plasma sST2 level, the PRS model display more accuracy on classification of AD and NC (AUC=0.6203 and 0.6678, for APOE alone and after adding PRS and plasma sST2, respectively;
Polygenic Risk Score for sST2 Level and Its Association with AD: sST2-associated variants with P-value lower than 1E-5 were subjected to the SNP pruning (r2<0.2), yielding 29 independent signals. Polygenic risk scores (PRS) were calculated by the sum of effect size-weighted (βi) genotype dosage of corresponding variants (snpi) onto the sST2 level, to generate PRS at individual level:
Individual PRS=β1snp1+β2snp2+ . . . +β29snp29
The individual polygenic scores were further normalized by subtracting the mean and dividing by the standard deviation. Lasso regression with cross-validation were performed for PRS alone, or in combination with plasma sST2 level and eosinophil counts for estimation their effectiveness on classifying AD and NC. Logistic regression was performed using R programming with adjustment for age to test association between PRS and AD.
Trazodone treatment of mice: Mice were treated with Trazodone or vehicle control either by intraperitoneal (IP) injection or oral administration. For IP injection, mice were daily injected with 50 mg/kg Trazodone or vehicle (saline). For oral administration, mice were daily fed with 100 mg/kg Trazodone or vehicle (water). After the 2 days, 4 days, 7 days or 14 days of treatment, mouse blood was collected by intracardial bleed at time of sacrifice. The blood without anticoagulants was incubated at room temperature for 2 h and then centrifuged at 2,000×g for 20 min. The serum sST2 level was measured using the Mouse ST2/IL-33 R Quantikine ELISA Kit (MST200, R&D).
Medications that Modulate Human Plasma sST2 Level: To investigate medications that may regulate the plasma sST2 protein level, an association analysis was conducted of the plasma sST2 protein level and medications in Hong Kong female AD patients. Trazodone was associated with a decreased plasma sST2 level in AD patients (
All patents, patent applications, other publications, and GenBank Accession NOs or equivalents cited herein are incorporated by reference in the entirety for all purposes.
This application claims priority to U.S. Provisional Patent Application No. 62/893,502, filed Aug. 29, 2019, the contents of which are hereby incorpored by reference in the entirety for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/111095 | 8/25/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62893502 | Aug 2019 | US |
