Patent Application
20190033329
Schizophrenia is a heritable psychiatric disorder involving impairments in cognition, perception and motivation that usually manifest late in adolescence or early in adulthood. The pathogenic mechanisms underlying schizophrenia are unknown, but observers have repeatedly noted pathological features involving excessive loss of gray matter and reduced numbers of synaptic structures on neurons. While treatments exist for the psychotic symptoms of schizophrenia, there is no mechanistic understanding of, nor effective therapies to prevent or treat, the cognitive impairments and deficit symptoms of schizophrenia, its earliest and most constant features. New methods of identifying and treating patients having or at risk of developing schizophrenia are urgently needed.
As described below, the present invention features compositions and methods for (i) identifying a subject having or at risk of developing schizophrenia, (ii) monitoring treatment for schizophrenia, and (iii) treating or preventing schizophrenia in a subject.
In one aspect, the invention provides a method of treating schizophrenia in a subject. The method contains the step of administering to the subject an agent that inhibits the expression or activity of a complement component 4A (C4A) polypeptide or polynucleotide.
In another aspect, the invention provides a method of treating a subject having a neurodegenerative disease or disorder characterized by increased levels, activity, or expression of a complement component 4A (C4A) polypeptide or polynucleotide (e.g. Alzheimer's Disease, glaucoma, or age-related macular degeneration) by administering to the subject an agent that inhibits the expression or activity of a complement component 4A (C4A) polypeptide or polynucleotide.
In another aspect, the invention provides a method of reducing an interaction between a neuron and microglia and/or reducing synaptic elimination in a subject, the method involving the step of contacting a microglia or neuron (e.g., at a synapse) with an agent that inhibits the expression or activity of a complement component 4A (C4A) polypeptide or polynucleotide. In various embodiments, one or more of the microglia or neuron is contacted with the agent in vitro or in vivo (e.g., in a subject). In certain embodiments, engulfment of synapses by microglia is reduced. In some embodiments, the method involves administering an agent that inhibits the expression or activity of a complement component 4A (C4A) polypeptide or polynucleotide to the subject. In various embodiments, the agent is administered to the subject intrathecally.
In various embodiments, the agent inhibits the expression or activity of a complement component 4A (C4A) polypeptide or polynucleotide. In some embodiments, the agent inhibits the expression or activity of a complement component 4B (C4B) polypeptide or polynucleotide. In some other embodiments, the agent does not inhibit the expression or activity of a complement component 4B (C4B) polypeptide or polynucleotide. In some embodiments, the agent is an antibody or an inhibitory nucleic acid. In certain embodiments, the antibody specifically binds an epitope containing the amino acid sequence PCPVLD. In particular embodiments, the antibody does not bind an epitope containing the amino acid sequence LSPVIH. In various embodiments of any one of the aspects delineated herein, the subject is human.
In another aspect, the invention provides a method of treating schizophrenia in a pre-selected subject, the method containing the step of administering a schizophrenia treatment to the subject, where the subject is pre-selected by detecting an increase in a level of a complement component 4A (C4A) polynucleotide or polypeptide, an increase in a combined level of C4A and complement component 4B (C4B) polynucleotide or polypeptide, an increase in copy number of complement component 4A (C4A), and/or an alteration in a sequence of C4A or C4B polynucleotide relative to a reference in a biological sample obtained from the subject.
In yet another aspect, the invention provides a method of monitoring treatment progress in a subject having schizophrenia and administered with a schizophrenia treatment. The method contains the step of measuring a level of C4A polypeptide or polynucleotide or a combined level of C4A and C4B polypeptide or polynucleotide relative to a reference level in a biological sample obtained from the subject, where a decrease in the level or combined level indicates the subject is responsive to the schizophrenia treatment.
In still another aspect, the invention provides a method of determining efficacy of a schizophrenia treatment in a subject. The method contains the step of measuring a level of C4A polypeptide or polynucleotide or a combined level of C4A and C4B polypeptide or polynucleotide relative to a reference level in a biological sample obtained from the subject, where a decrease in the level or combined level indicates the the schizophrenia treatment is efficacious.
In another aspect, the invention provides method of characterizing a subject having a mental disorder. The method contains the step of measuring a level of a complement component 4A (C4A) polynucleotide or polypeptide, a combined level of C4A and complement component 4B (C4B) polynucleotide or polypeptide, a copy number of C4A polynucleotide, and/or a sequence of C4A and/or C4B polynucleotide relative to a reference in a biological sample obtained from the subject, where an increase in the level of C4A polynucleotide or polypeptide, an increase in the combined level of C4A and C4B polynucleotide or polypeptide, an increase in C4A copy number and/or an alteration in a sequence of C4A or C4B polynucleotide indicates the subject has schizophrenia or is at risk of developing schizophrenia.
In yet another aspect, the invention provides a method of identifying a subject having or at risk of developing schizophrenia, the method containing the step of measuring a level of a complement component 4A (C4A) polynucleotide or polypeptide, a combined level of C4A and complement component 4B (C4B) polynucleotide or polypeptide, a copy number of C4A polynucleotide, and/or a sequence of C4A and/or C4B polynucleotide relative to a reference in a biological sample obtained from the subject, where the subject is identified as having or at risk of developing schizophrenia if the level of C4A polynucleotide or polypeptide is increased, the combined level of C4A and C4B polynucleotide or polypeptide is increased, the copy number of C4A polynucleotide is increased, and/or the sequence of C4A or C4B polynucleotide is altered.
In another aspect, the invention provides a method of characterizing risk of schizophrenia in a subject, the method containing the step of measuring a level of a complement component 4A (C4A) polynucleotide or polypeptide, a combined level of C4A and complement component 4B (C4B) polynucleotide or polypeptide, a copy number of C4A polynucleotide, and/or a sequence of C4A and/or C4B polynucleotide relative to a reference in a biological sample obtained from the subject, where an increase in the level of C4A polynucleotide or polypeptide, an increase in the combined level of C4A and C4B polynucleotide or polypeptide, an increase in C4A copy number and/or an alteration in a sequence of C4A or C4B polynucleotide indicates the subject has schizophrenia or is at risk of developing schizophrenia.
In another aspect, the invention provides a transgenic mouse containing a polynucleotide sequence encoding a human complement component 4A (huC4A) or human complement component 4B (huC4B) polypeptide, where the polynucleotide sequence is operatively linked to a promoter sequence. In various embodiments, the transgenic mouse expresses the human complement component 4A (huC4A) or human complement component 4B (huC4B) polypeptide in the central nervous system. In various embodiments, the mouse complement component 4 (C4) gene is deleted or inactivated in the transgenic mouse.
In various embodiments, the method further contains the step of recommending the subject for schizophrenia treatment or for further evaluation for schizophrenia if the subject is identified as having or at risk of developing schizophrenia. In some other embodiments, the method further contains the step of administering a schizophrenia treatment to the subject if the subject is identified as having or at risk of developing schizophrenia. In some embodiments, the schizophrenia treatment involves inhibiting the expression or activity of a complement component 4A (C4A) polypeptide or polynucleotide, including for example, inhibiting the complement pathway with a complement inhibitor (e.g., anti-C1q, Eculizumab/Soliris and Cetor/Sanquin, etc.)
In some embodiments, the alteration in sequence is insertion of a human endogenous retrovirus (HERV) sequence. In some other embodiments, an increase in copy number of C4A polynucleotide and insertion of a human endogenous retrovirus (HERV) sequence in a C4A and/or C4B polynucleotide is detected. In still other embodiments, an increase in a level of C4A polynucleotide or polypeptide is detected. In some embodiments, an increase in a combined level of C4A and C4B polynucleotide or polypeptide is detected.
In various embodiments of any one of the aspects delineated herein, the biological sample is plasma, serum, or cerebrospinal fluid (CSF). In certains embodiments, schizophrenia or neurodegenerative disease is characterized by detecting changes in activated microglia/exosomes present in CSF. In various embodiments, the schizophrenia treatment is an antipsychotic agent or psychosocial therapy.
In another aspect, the invention provides a kit containing a capture reagent for detecting the sequence of complement component 4A (C4A) polynucleotide or complement component 4B (C4B), and an antipsychotic agent. In some embodiments, the kit further contains a capture reagent for detecting the sequence of a HERV. In some other embodiments embodiments, the capture reagent is a probe or a primer. In various embodiments, the level, copy number, and/or sequence of complement component 4A (C4A) polynucleotide or complement component 4B (C4B) is measured using the kit of any one of the aspects delineated herein.
In yet another aspect, the invention provides a method of identifying an agent that inhibits schizophrenia. The method contains the step of (a) contacting a cell or organism with a candidate agent, and (b) measuring a level of complement component 4A (C4A) polynucleotide or polypeptide in the cell or organism contacted with the candidate agent relative to a reference level, where a decrease in the level indicates the candidate agent inhibits schizophrenia.
In another aspect, the invention provides an expression vector contains an isolated polynucleotide encoding complement component 4A (C4A).
In still another aspect, the invention provides a host cell or host organism contains an expression vector that contains an isolated polynucleotide encoding complement component 4A (C4A). In various embodiments, the host cell or host organism is mammalian.
Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof. In some embodiments, the agent is a small molecule chemical compound. In particular embodiments, the agent is an antipsychotic agent. Exemplary antipsychotic agents include, but are not limited to, aripiprazole, asenapine, clozapine, iloperidone, lurasidone, olanzapine, paliperidone, quetiapine, risperidone, ziprasidone, chlorpromazine, fluphenazine, haloperidol, and perphenazine.
By “alteration” is meant a change (increase or decrease) in the expression levels, copy number, or sequence of a gene or polypeptide as detected by standard art known methods such as those described herein. In some embodiments, an alteration in expression level includes a 10% change in expression levels, a 25% change, a 40% change, and a 50% or greater change in expression levels. In some other embodiments, an alteration in copy number includes an increase or a decrease by at least 1, at least 2, at least 3, at least 4, or at least 5 copies of the gene in a genome. In some embodiments, the alteration in copy number is an increase by at least 1, at least 2, at least 3, at least 4, or at least 5 copies of the gene.
The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Methods of preparing antibodies are well known to those of ordinary skill in the science of immunology. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Tetramers may be naturally occurring or reconstructed from single chain antibodies or antibody fragments. Antibodies also include dimers that may be naturally occurring or constructed from single chain antibodies or antibody fragments. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab′) 2, as well as single chain antibodies (scFv), humanized antibodies, and human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antibody specifically binds to C4A polypeptide.
The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′) 2, and Fv fragments, linear antibodies, scFv antibodies, single-domain antibodies, such as camelid antibodies (Riechmann, 1999, Journal of Immunological Methods 231:25-38), composed of either a VL or a VH domain which exhibit sufficient affinity for the target, and multispecific antibodies formed from antibody fragments. The antibody fragment also includes a human antibody or a humanized antibody or a portion of a human antibody or a humanized antibody.
“Biological sample” as used herein means a biological material isolated from a subject, including any tissue, cell, fluid, or other material obtained or derived from the subject. In some embodiments, the subject is human. The biological sample may contain any biological material suitable for detecting the desired analytes, and may comprise cellular and/or non-cellular material obtained from the subject. In various embodiments, the biological sample may be obtained from the brain. In particular embodiments, the biological sample is blood. In certain embodiments, the biological sample is cerebrospinal fluid (CSF). Biological samples include tissue samples (e.g., cell samples, biopsy samples), such as tissue from the brain. Biological samples also include bodily fluids, including, but not limited to, cerebrospinal fluid, blood, blood serum, plasma, saliva, and urine.
By “capture reagent” is meant a reagent that specifically binds a nucleic acid molecule or polypeptide to select or isolate the nucleic acid molecule or polypeptide.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
A “complement component 4 polypeptide” or “C4 polypeptide” is a complement component 4A (C4A) polypeptide or a complement component 4B (C4B) polypeptide. By “complement component 4A polypeptide” or “C4A polypeptide” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to GenBank Accession No. AAA51855.1 and having activities that include binding to antigen-antibody complex and binding to other complement components. Human C4 exists as two paralogous genes (isotypes), C4A and C4B; the encoded polypeptides are distinguished at a key site that determines which molecular targets they bind. The sequence of C4A polypeptide provided at GenBank Accession No. AAA51855.1 is shown below:
By “complement component 4 polynucleotide” or “C4 polynucleotide” is meant a polynucleotide encoding a complement component 4A (C4A) polypeptide or a complement component 4B (C4B) polypeptide. By “complement component 4A polynucleotide” or “C4A polynucleotide” is meant a polynucleotide encoding a C4A polypeptide. An exemplary C4A polynucleotide sequence is provided at NCBI Accession No. NG_011638.1 (genomic sequence) and is reproduced below.
By “complement component 4B polypeptide” or “C4B polypeptide” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001002029.3 and having activities that include binding to antigen-antibody complex and binding to other complement components. The sequence at NCBI Accession No. NP_001002029.3 is shown below:
By “complement component 4B polynucleotide” or “C4B polynucleotide” is meant a polynucleotide encoding a C4B polypeptide. An exemplary C4B polynucleotide sequence is provided at NCBI Accession No. NG_011639.1 (genomic sequence) and is reproduced below.
“Detect” refers to identifying the presence, absence or amount of the analyte to be detected. In some embodiments, a copy number of complement component 4A (C4A) or complement component 4B (C4B) is detected. In other embodiments, presence of a human endogenous retrovirus (HERV) sequence is detected.
By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens. In some embodiments, the detectable label is a fluorescent polypeptide.
By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include schizophrenia, Alzheimer's Disease, glaucoma, and age-related macular degeneration. Such diseases are characterized by undesirably increased levels of complement component 4A (C4A) and/or synaptic pruning.
By “effective amount” is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. In particular embodiments, the disease is schizophrenia. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.
“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
As used herein, a “human endogenous retrovirus” or “HERV” polynucleotide sequence is a polynucleotide sequence that occurs in the human genome that is substantially identical to a sequence in a retrovirus or that was derived from a retrovirus. In some embodiments, the HERV sequence is a human endogenous retrovirus type K (HERV-K) sequence. In some other embodiments, the HERV sequence is a C4-HERV sequence. In certain embodiments, a retroviral (C4-HERV) sequence in intron 9 is inserted within a C4A polynucleotide sequence or a C4B polynucleotide sequence. An exemplary HERV sequence is provided at GenBank Accession No. AF164613.1, and is reproduced below.
“Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
By “inhibitory nucleic acid” is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.
The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. The preparation can be at least 75%, at least 90%, and at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By “marker” is meant any protein or polynucleotide having an alteration in expression level, copy number, sequence, or activity that is associated with a disease or disorder or risk of disease or disorder. In some embodiments, an alteration in the copy number and/or sequence of C4A polynucleotide and/or C4B polynucleotide is associated with risk of schizophrenia.
By “microglia” is meant an immune cell of myeloid lineage resident in the central nervous system.
As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
As used herein a “probe” or “nucleic acid or oligonucleotide probe” is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are preferably directly labeled with isotopes, for example, chromophores, lumiphores, chromogens, or indirectly labeled with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of a target gene of interest.
As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
By “reference” is meant a standard or control condition. In some embodiments, a “reference copy number” is a copy number of 0 or 1. In some other embodiments, a “reference level” is a level of C4A or C4B polynucleotide, such as C4A or C4B RNA, in a healthy, normal subject or in a subject that does not have schizophrenia.
A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, at least about 20 amino acids, or at least about 25 amino acids. The length of the reference polypeptide sequence can be about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, or at least about 75 nucleotides. The length of the reference nucleic acid sequence can be about 100 nucleotides, about 300 nucleotides or any integer thereabout or therebetween.
In some embodiments, the reference sequence is a sequence of a “short form” of complement component 4A (C4A) genomic polynucleotide. In some other embodiments, the reference sequence is the sequence of a short form of complement component 4B (C4B) genomic polynucleotide. As used herein, a “short form” of a C4A or C4B polynucleotide is a C4A or C4B polynucleotide that does not contain an insertion of a human endogenous retrovirus (HERV) sequence. As used herein, a “long form” of a C4A or C4B polynucleotide is a C4A or C4B polynucleotide that contains an insertion of a human endogenous retrovirus (HERV) sequence.
By “siRNA” is meant a double stranded RNA. Optimally, an siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3′ end. These dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream. Such siRNAs are used to downregulate mRNA levels or promoter activity.
By “specifically binds” is meant an agent that recognizes and binds a polypeptide or polynucleotide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polynucleotide of the invention. In some embodiments, the agent is a nucleic acid molecule. In some embodiments, the agent is an antibody that specifically binds C4A polypeptide.
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, or at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., at least about 37° C., and at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In one embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In yet another embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., at least about 42° C., and at least about 68° C. In one embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In yet another embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Such a sequence is at least 60%, at least 80%, at least 85%, at least 90%, at least 95% or even at least 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence.
By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. As used herein, “schizophrenia treatment” or “treatment for schizophrenia” includes, without limitation, antipsychotic agents and psychosocial therapy. Psychosocial therapy for schizophrenia includes individual therapy and family therapy.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
The invention features compositions and methods that are useful for determining risk of schizophrenia and treating schizophrenia in a subject. The invention is based, at least in part, on the discovery of a relationship between schizophrenia risk and structurally diverse alleles of the complement component 4 (C4) genes.
Schizophrenia is a heritable brain illness with unknown pathogenic mechanisms. Schizophrenia's strongest genetic association at a population level involves variation in the Major Histocompatibility Complex (MHC) locus, but the genes and molecular mechanisms accounting for this have been challenging to recognize. Studies described herein show that schizophrenia's association with the MHC locus arises in substantial part from many structurally diverse alleles of the complement component 4 (C4) genes. It was found that these alleles promoted widely varying levels of C4A and C4B expression and associated with schizophrenia in proportion to their tendency to promote greater expression of C4A in the brain. Human C4 protein localized at neuronal synapses, dendrites, axons, and cell bodies. In mice, C4 mediated synapse elimination during postnatal development. These results implicate excessive complement activity in the development of schizophrenia and may help explain the reduced numbers of synapses in the brains of individuals affected with schizophrenia.
Association of Loci with Schizophrenia Risk
Schizophrenia is a heritable psychiatric disorder involving impairments in cognition, perception and motivation that usually manifest late in adolescence or early in adulthood. The pathogenic mechanisms underlying schizophrenia are unknown, but observers have repeatedly noted pathological features involving excessive loss of gray matter1,2 and reduced numbers of synaptic structures on neurons3-5. While treatments exist for the psychotic symptoms of schizophrenia, there is no mechanistic understanding of, nor effective therapies to prevent or treat, the cognitive impairments and deficit symptoms of schizophrenia, its earliest and most constant features. An important goal in human genetics is to find the biological processes that underlie such disorders.
More than 100 loci in the human genome contain SNP haplotypes that associate with risk of schizophrenia6; the functional alleles and mechanisms at these loci remain to be discovered. By far the strongest such genetic relationship is schizophrenia's unexplained association with genetic markers across the Major Histocompatibility Complex (MHC) locus, which spans several megabases of chromosome 66-10. The MHC locus is best known for its role in immunity, containing 18 highly polymorphic human leukocyte antigen (HLA) genes that encode a vast suite of antigen-presenting molecules. In some autoimmune diseases, genetic associations at the MHC locus arise from alleles of HLA genes11,12; however, schizophrenia's association to the MHC is not yet explained.
Though the functional alleles that give rise to genetic associations have in general been challenging to find, the schizophrenia-MHC association has been particularly challenging, as schizophrenia's complex pattern of association to markers in the MHC locus spans hundreds of genes and does not correspond to the linkage disequilibrium (LD) around any known variant6,10. The most strongly associated markers in several large case/control cohorts were near a complex, multi-allelic, and only partially characterized form of genome variation that affects the C4 gene encoding complement component 4 (
In humans, adolescence and early adulthood bring extensive elimination of synapses in distributed association regions of cerebral cortex, such as the prefrontal cortex, that have greatly expanded in recent human evolution37-40. Synapse elimination in human association cortex appears to continue from adolescence into the third decade of life39. This late phase of cortical maturation, which may distinguish humans even from some other primates37, corresponds to the period during which schizophrenia most often becomes clinically apparent and patients' cognitive function declines, a temporal correspondence that others have also noted41. Principal pathological findings in schizophrenia brains involve loss of cortical gray matter without cell death: affected individuals exhibit abnormal cortical thinning12 and abnormally reduced numbers of synaptic structures on cortical pyramidal neurons3-5. The possibility that neuron-microglia interactions via the complement cascade contribute to schizophrenia pathogenesis—for example, that schizophrenia arises or intensifies from excessive or inappropriate synaptic pruning during adolescence and early adulthood—would offer a potential mechanism for these longstanding observations about age of onset and synapse loss. Many other genetic findings in schizophrenia involve genes that encode synaptic proteins6,42-44. Diverse synaptic abnormalities might interact with the complement system and other pathways45,46 to cause excessive stimulation of microglia and/or elimination of synapses.
The two human C4 genes (C4A and C4B) exhibited distinct relationships with schizophrenia risk, with increased risk associating most strongly with variation that increases expression of C4A. Human C4A and C4B proteins, whose functional specialization appears to be evolutionarily recent (
To date, few associations from genomewide association studies (GWAS) have been explained by specific functional alleles. An unexpected finding at C4 involves the large number of common, functionally distinct forms of the same locus that appear to contribute to schizophrenia risk. The human genome contains hundreds of other genes with complex, multi-allelic forms of structural variation49. It will be important to learn the extent to which such variation contributes to brain diseases and indeed to all human phenotypes.
Association of Risk of Schizophrenia with Structure of Complement 4 (C4) Alleles
In the studies described herein, allelic structure of complement 4 (C4) genes was found to be associated with risk of schizophrenia. In particular, increased expression of C4A mRNA in the brain was found to correlate with increased risk of schizophrenia. Increased C4A mRNA or C4B expression correlated with increased copy number of C4A or C4B genes. In addition, the presence of a human endogenous retrovirus (HERV) in C4A or C4B was found to increase expression of C4A relative to C4B.
Thus, information on allelic structure of C4 genes (e.g., copy number of C4A and/or C4B; presence or absence of HERV in C4A or C4B) may predict risk of schizophrenia in a subject. Accordingly, in one aspect, the invention provides a method of identifying a subject having or at risk of developing schizophrenia. The method contains the step of measuring copy number and/or sequence of C4A or C4B polynucleotide, where an alteration in copy number and/or sequence of C4A or C4B polynucleotide relative to a reference indicates the subject has or is at risk or developing schizophrenia. In some embodiments, the alteration in copy number is an increase in copy number. In some other embodiments, the alteration in sequence is insertion of a HERV sequence. In particular embodiments, the alteration is an increase in copy number of C4A polynucleotide. In some embodiments, the alteration is an increase in copy number of C4A polynucleotide containing a HERV sequence (i.e., long form of C4A polynucleotide). In certain embodiments, the alteration is any one of more of the following: an increase in copy number of C4A, increase in copy number of C4B, presence of HERV in one or more copies of C4A, and presence of HERV in one or more copies of C4B.
Early identification of risk of schizophrenia in a subject can be important in minimizing or preventing potentially irreversible deconstruction of a life that schizophrenia can bring to an individual and the individual's family and/or peers. If an individual is identified as having or at risk of developing schizophrenia at an early stage, proper treatment or therapy can be administered, which can help reduce symptoms of schizophrenia and/or help the individual (and family members and friends of the individual) cope with the individual's schizophrenia. Thus, in some embodiments, the methods contain the step of recommending an individual for further evaluation or for treatment of schizophrenia, if the individual is identified as having or at risk of developing schizophrenia. In some other embodiments, the methods contain the step of administering a schizophrenia treatment (e.g., antipsychotic agents and/or psychosocial therapy) to the individual if the individual is identified as having or at risk of developing schizophrenia.
In some aspects, the invention provides a method of treating schizophrenia in a pre-selected subject, where the subject is pre-selected for treatment by detecting an alteration in copy number and/or sequence of C4A or C4B polynucleotide relative to a reference. In some embodiments, the alteration in copy number is an increase in copy number. In some other embodiments, the alteration in sequence is insertion of a HERV sequence. In particular embodiments, the alteration is an increase in copy number of C4A polynucleotide. In some embodiments, the alteration is an increase in copy number of C4A polynucleotide containing a HERV sequence (i.e., long form of C4A polynucleotide). In certain embodiments, the alteration is any one of more of the following: an increase in copy number of C4A, increase in copy number of C4B, presence of HERV in one or more copies of C4A, and presence of HERV in one or more copies of C4B. For example, the subject can be diagnosed with schizophrenia and/or administered with schizophrenia treatment based on the results of the methods herein.
Further, studies herein have also found that increased level of C4A RNA, particularly in the brain, was associated with increased incidence of schizophrenia. Without being bound by theory, levels of C4 RNA associated with schizophrenia above and beyond what could be explained by effect of DNA variation at C4, indicate that dynamic biomarkers (that measure expression levels) might provide diagnostic information above and beyond that provided by DNA sequence and structure. Thus, in some aspects, the invention provides methods of identifying a subject having or at risk of developing schizophrenia, methods of treating schizophrenia in a subject, and methods of monitoring treatment progress in a subject, where the method contains the step of detecting an increased level of C4, or more specifically C4A RNA or C4A polypeptide, relative to a reference level.
In other aspects, the invention provides a method of treating schizophrenia in a pre-selected subject, where the subject is pre-selected by detecting an increased level of C4 or C4A protein or RNA relative to a reference level. Since C4 is a secreted protein, it can be detected in cerebrospinal fluid (CSF). Measuring levels of C4 in CSF could offer a way to dynamically measure C4 expression in a subject.
Analysis of C4A and C4B status can be performed in a variety of ways. In various embodiments of any of the aspects delineated herein, alterations in a polynucleotide or polypeptide of C4A and/or C4B (e.g, sequence, copy number, level) are analysed. In some embodiments, the method includes the step of measuring or detecting a level, copy number, or sequence of C4A and/or C4B polynucleotide in a biological sample obtained from the subject relative to a reference level, copy number, or sequence. In particular embodiments, DNA sequencing and copy number analysis are performed on C4A and/or C4B polynucleotide.
As described herein, an increase in copy number of C4A (particularly, the long form of C4A) and increased C4A expression were each associated with increased risk of schizophrenia. Thus, in some embodiments, an increase in copy number C4A is indicative of increased schizophrenia risk. Also, presence of a HERV sequence was found to increase C4A expression (particularly relative to C4B expression). Thus, increased copy number of a HERV sequence can be indicative of increased risk of schizophrenia, with risk increasing with increased numbers of copies. In certain embodiments, increased risk of schizophrenia can be indicated be any one of more of the following: an increase in copy number of C4A, presence of HERV in one or more copies of C4A, and presence of HERV in one or more copies of C4B.
In some embodiments, any one of the following combinations of C4A and C4B can be detected: one copy of C4B (short form), one copy of C4B (short form) and one copy of C4A (long form), one copy of C4B (long form) and one copy of C4A (long form), and two copies each of C4A (long form). In certain embodiments, the risk of schizophrenia associated with the combination of C4A and C4B is increased in the order in which the combination is listed as follows (from lowest to highest risk, respectively): one copy of C4B (short form), one copy of C4B (short form) and one copy of C4A (long form), one copy of C4B (long form) and one copy of C4A (long form), and two copies each of C4A (long form). As described elsewhere herein, the short form of either C4A or C4B does not contain a HERV sequence insertion in intron 9; the long form of either C4A or C4B contains a HERV sequence insertion in intron 9.
Alterations in polynucleotides or polypeptides of C4A and/or C4B (e.g, sequence, copy number, level) are detected in a biological sample obtained from an subject (e.g., a human). Biological samples include tissue samples (e.g., cell samples, biopsy samples), such as brain tissue. Biological samples that are used to evaluate the herein disclosed markers include without limitation brain tissue, blood, serum, plasma, and cerebrospinal fluid (CSF). In one embodiment, the biological sample is blood or serum. In another embodiment, the biological sample is brain tissue. In a particular embodiment, the biological sample is cerebrospinal fluid.
The sequence, level, or copy number of a polypeptide or polynucleotide of C4A and/or C4B detected in the method can be compared to a reference sequence, level, or copy number. The reference level of a C4A or C4B polynucleotide (e.g., a C4A or C4B RNA) can be level of C4A or C4B RNA in healthy normal controls. The reference copy number of C4A or C4B can be 0, 1, 2, or 3 copies. In some embodiments, the reference copy number is 0. The reference sequence of C4A or C4B can be C4A (short form) or C4B (short form) (i.e., C4A or C4B polynucleotide without an insertion of a HERV sequence in intron 9).
While the examples provided below describe specific methods of detecting levels of polynucleotides or polypeptides of the markers C4A and C4B, the skilled artisan appreciates that the invention is not limited to such methods. The biomarkers of this invention can be detected or quantified by any suitable method. For example, methods include, but are not limited to real-time PCR, Southern blot, PCR, mass spectroscopy, ELISA, and/or antibody binding. Methods for detecting a copy number and/or sequence of C4A or C4B or other polynucleotides of the invention include immunoassay, direct sequencing, and probe hybridization to a polynucleotide. In particular embodiments, a sequence and/or copy number of the markers is detected by DNA sequencing and/or copy number analysis.
The present invention provides methods of treating schizophrenia and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-schizophrenia agent (e.g., an antipsychotic agent) herein to a pre-selected subject (e.g., a mammal such as a human). In some embodiments, the subject is pre-selected by detecting an alteration in copy number and/or sequence of C4A and/or C4B polynucleotide relative to a reference. In other embodiments, the subject is pre-identified as having or at risk for schizophrenia, Thus, one embodiment is a method of treating a subject suffering from or susceptible to schizophrenia or disorder or symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of an agent (e.g., antipsychotic agent) herein sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.
The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of an agent described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method, such as the methods described herein).
The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the agents herein (such as an antipsychotic agent) to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a schizophrenia, disorder, or symptom thereof. In some embodiments, determination of those subjects “at risk” is made by an objective determination using the methods described herein.
In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (e.g., level of a polynucleotide or polypeptide of C4A and/or C4B) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a schizophrenia, or disorder or symptoms thereof, in which the subject has been administered a therapeutic or effective amount of a therapeutic agent described herein sufficient to treat the schizophrenia or symptoms thereof. The level of a polynucleotide or polypeptide of C4A and/or C4B determined in the method can be compared to known levels of a polynucleotide or polypeptide of C4A and/or C4B in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In some embodiments, a level of a polynucleotide or polypeptide of C4A and/or C4B in a cerebrospinal fluid (CSF) sample obtained from the subject is determined. In some embodiments, a second level of a polynucleotide or polypeptide of C4A and/or C4B in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain embodiments, a pre-treatment level, sequence, or copy number of a polynucleotide or polypeptide of C4A and/or C4B in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of a polynucleotide or polypeptide of C4A and/or C4B can then be compared to the level of a polynucleotide or polypeptide of C4A and/or C4B in the subject after the treatment commences, to determine the efficacy of the treatment.
In particular embodiments, the agent is an antipsychotic agent. Exemplary antipsychotic agents approved by the U.S. Food and Drug Administration for treatment of schizophrenia or symptoms thereof include, but are not limited to, aripiprazole, asenapine, clozapine, iloperidone, lurasidone, olanzapine, paliperidone, quetiapine, risperidone, ziprasidone, chlorpromazine, fluphenazine, haloperidol, and perphenazine. Commonly used first-line anti-psychotics for (first-episode) schizophrenia include quetiapine, risperidone, ziprasidone.
In some embodiments, the agent is a complement inhibitor. FDA-approved complement inhibitors that are currently in use for other indications include Eculizumab/Soliris and Cetor/Sanquin. In some embodiments, the complement inhibitor is an anti-C1q antibody or fragment thereof (see, e.g., U.S. Patent Publication No. 2016/0159890). In particular embodiments, the complement inhibitor inhibits synaptic pruning.
In some embodiments, the methods include administering psychosocial therapy or treatment to pre-selected subject. Psychosocial treatments for schizophrenia can include, for example, individual therapy, family therapy, social skills training, and vocational rehabilitation. Individual therapy is aimed at training an individual learn to cope with stress and identify early warning signs of relapse, which can help an individual with schizophrenia manage the illness. Family therapy provides support and education to families dealing with schizophrenia. Social skills training focuses on improving communication and social interactions of the individual with schizophrenia. Vocational rehabilitation focuses on helping individuals with schizophrenia prepare for, find and keep jobs. Most individuals with schizophrenia require some form of daily living support. Many communities have programs to help individuals with schizophrenia with jobs, housing, self-help groups and crisis situations. In some embodiments, a schizophrenia treatment can integrate antipsychotic agents, psychosocial therapies, case management, family involvement, and supported education and employment services, all aimed at reducing symptoms and improving quality of life of the individual with schizophrenia.
In other aspects, the invention provides a method of treating schizophrenia by selectively interfering with the function of C4A polypeptide. In some embodiments, the interference with C4A polypeptide function is achieved using an antibody binding to C4A polypeptide. In some embodiments, the antibody specifically binds to C4A polypeptide, and does not bind C4B polypeptide. In certain embodiments, the antibody binds to both C4A and C4B polypeptide.
In certain embodiments, the antibody disrupts or reduces interaction between a neuron and microglia. Without being bound by theory, it is believed that reduced interaction between a neuron and microglia decreases synaptic pruning. Accordingly, in some embodiments, the antibody reduces synaptic pruning.
Antibodies can be made by any of the methods known in the art utilizing a polypeptide of the invention (e.g., C4A and C4B polypeptide), or immunogenic fragments thereof, as an immunogen. One method of obtaining antibodies is to immunize suitable host animals with an immunogen and to follow standard procedures for polyclonal or monoclonal antibody production. The immunogen will facilitate presentation of the immunogen on the cell surface. Immunization of a suitable host can be carried out in a number of ways. Nucleic acid sequences encoding a polypeptide of the invention or immunogenic fragments thereof, can be provided to the host in a delivery vehicle that is taken up by immune cells of the host. The cells will in turn express the receptor on the cell surface generating an immunogenic response in the host. Alternatively, nucleic acid sequences encoding the polypeptide, or immunogenic fragments thereof, can be expressed in cells in vitro, followed by isolation of the polypeptide and administration of the polypeptide to a suitable host in which antibodies are raised.
Alternatively, antibodies against the polypeptide may, if desired, be derived from an antibody phage display library. A bacteriophage is capable of infecting and reproducing within bacteria, which can be engineered, when combined with human antibody genes, to display human antibody proteins. Phage display is the process by which the phage is made to ‘display’ the human antibody proteins on its surface. Genes from the human antibody gene libraries are inserted into a population of phage. Each phage carries the genes for a different antibody and thus displays a different antibody on its surface.
Antibodies made by any method known in the art can then be purified from the host. Antibody purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-immunoglobulin.
Antibodies can be conveniently produced from hybridoma cells engineered to express the antibody. Methods of making hybridomas are well known in the art. The hybridoma cells can be cultured in a suitable medium, and spent medium can be used as an antibody source. Polynucleotides encoding the antibody of interest can in turn be obtained from the hybridoma that produces the antibody, and then the antibody may be produced synthetically or recombinantly from these DNA sequences. For the production of large amounts of antibody, it is generally more convenient to obtain an ascites fluid. The method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse. The mammal may be primed for ascites production by prior administration of a suitable composition (e.g., Pristane).
Without intending to be bound by theory, results herein indicate that therapeutically it might be advantageous to selectively interfere with C4A while leaving C4B function intact. This could be important because ideally one would not want to entirely block complement function in the body, since complement is important for protection from immune assault and from auto-immunity. Thus, in some embodiments, therapeutic antibodies that selectively bind to C4A polypeptide and not to C4B polypeptide are generated by exploiting the amino-acid sequence differences between C4A and C4B to identify epitopes for isotope-specific antibodies. In some embodiments, the amino acid sequence difference between C4A and C4B is that shown in
The present invention features compositions useful for treating schizophrenia in a pre-selected subject. The administration of a composition comprising a therapeutic agent herein (e.g., an antipsychotic agent, an inhibitory nucleic acid inhibiting expression for C4A polypeptide, or an antibody specifically binding to C4A polypeptide) for the treatment of schizophrenia may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing schizophrenia in a subject. The composition may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Routes of administration include, for example, intrathecal, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the agent in the patient. In particular embodiments, the composition comprising a therapeutic agent herein is administered intrathecally to a subject. In some embodiments, the composition is injected into the spinal canal (in particular, subarachnoid space) of the subject such that the composition reaches the cerebrospinal fluid.
When the binding target is located in the brain, certain embodiments of the invention provide for the antibody or antigen-binding fragment thereof to traverse the blood-brain barrier. Certain neurodegenerative diseases are associated with an increase in permeability of the blood-brain barrier, such that the antibody or antigen-binding fragment can be readily introduced to the brain. When the blood-brain barrier remains intact, several art-known approaches exist for transporting molecules across it, including, but not limited to, physical methods, lipid-based methods, and receptor and channel-based methods.
In certain embodiments, a chimeric molecule is generated comprising a fusion of an antibody or other therapeutic polypeptide with a protein transduction domain which targets the antibody or therapeutic polypeptide for delivery to various tissues and more particularly across the brain blood barrier, using, for example, the protein transduction domain of human immunodeficiency virus TAT protein (Schwarze et al., 1999, Science 285: 1569-72) or BBB peptide (Brainpeps® database; http://brainpeps.ugent.be/; Van Dorpe et al., Brain Structure and Function, 2012, 217(3), 687-718). Other polypeptides facilitating transport across the blood-brain-barrier, include without limitation, transferrin receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low-density lipoprotein receptor related proteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptor, CRM197, a llama single domain antibody, TMEM 30(A), a protein transduction domain, Syn-B, penetratin, a poly-arginine peptide, an angiopep peptide, and ANG1005.
In certain embodiments, compositions disclosed herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that therapeutic compounds in compositions of the invention cross the BBB, they can be formulated, for example, in liposomes. Lipid-based methods of transporting an antibody or antigen-binding fragment across the blood-brain barrier include, but are not limited to, encapsulating the antibody or antigen-binding fragment in liposomes that are coupled to antibody binding fragments that bind to receptors on the vascular endothelium of the blood-brain barrier (see, e.g., U.S. Patent Application Publication No. 20020025313), and coating the antibody or antigen-binding fragment in low-density lipoprotein particles (see, e.g., U.S. Patent Application Publication No. 20040204354) or apolipoprotein E (see, e.g., U.S. Patent Application Publication No. 20040131692). For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134), different species of which may comprise the formulations of the invention, as well as components of the invented molecules (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.
Physical methods of transporting the antibody or antigen-binding fragment across the blood-brain barrier include, but are not limited to, circumventing the blood-brain barrier entirely, or by creating openings in the blood-brain barrier. Circumvention methods include, but are not limited to, direct injection into the brain (see, e.g., Papanastassiou et al., Gene Therapy 9: 398-406 (2002); interstitial infusion/convection-enhanced delivery (see, e.g., Bobo et al., Proc. Natl. Acad. Sci. USA 91: 2076-2080 (1994)), and implanting a delivery device in the brain (see, e.g., Gill et al., Nature Med. 9: 589-595 (2003); and Gliadel Wafers™, Guildford Pharmaceutical). Methods of creating openings in the barrier include, but are not limited to, ultrasound (see, e.g., U.S. Patent Publication No. 2002/0038086), osmotic pressure (e.g., by administration of hypertonic mannitol (Neuwelt, E. A., Implication of the Blood-Brain Barrier and its Manipulation, vols. 1 & 2, Plenum Press, N.Y. (1989))), permeabilization by, e.g., bradykinin or permeabilizer A-7 (see, e.g., U.S. Pat. Nos. 5,112,596, 5,268,164, 5,506,206, and 5,686,416), and transfection of neurons that straddle the blood-brain barrier with vectors containing genes encoding the antibody or antigen-binding fragment (see, e.g., U.S. Patent Publication No. 2003/0083299).
Receptor and channel-based methods of transporting the antibody or antigen-binding fragment across the blood-brain barrier include, but are not limited to, using glucocorticoid blockers to increase permeability of the blood-brain barrier (see, e.g., U.S. Patent Application Publication Nos. 2002/0065259, 2003/0162695, and 2005/0124533); activating potassium channels (see, e.g., U.S. Patent Application Publication No. 2005/0089473); inhibiting ABC drug transporters (see, e.g., U.S. Patent Application Publication No. 2003/0073713); coating antibodies with a transferrin and modulating activity of the one or more transferrin receptors (see, e.g., U.S. Patent Application Publication No. 2003/0129186), and cationizing the antibodies (see, e.g., U.S. Pat. No. 5,004,697).
The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of schizophrenia. Generally, amounts will be in the range of those used for other agents used in the treatment of schizophrenia, although in certain instances lower amounts will be needed because of the increased specificity of the agent. A composition is administered at a dosage that decreases effects or symptoms of schizophrenia as determined by a method known to one skilled in the art.
The therapeutic agent (e.g., an antipsychotic agent herein) may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
Pharmaceutical compositions according to the invention may be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with an organ, such as the liver; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target schizophrenia using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type (e.g., cells in the brain). For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.
Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the agent in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.
The pharmaceutical composition may be administered intrathecally or parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.
Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that reduces or ameliorates schizophrenia, the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) (e.g., antipsychotic agent) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.
In some embodiments, the composition comprising the active therapeutic (e.g., antipsychotic agent) is formulated for intravenous delivery. As indicated above, the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the agents is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.
Another therapeutic approach for treating or slowing progression of schizophrenia is polynucleotide therapy using an inhibitory nucleic acid that inhibits expression of a C4A and/or C4B polynucleotide (in particular, a C4A polynucleotide). Thus, provided herein are inhibitory nucleic acid molecules, such as siRNA, that target C4A and/or C4B polynucleotide. Such nucleic acid molecules can be delivered to cells of a subject having schizophrenia. The nucleic acid molecules are delivered to the cells of a subject in a form in which they can be taken up so that therapeutically effective levels of the inhibitory nucleic acid molecules are introduced.
Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). For example, an inhibitory nucleic acid as described can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. In some embodiments, the target cell type of interest is a neuron. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). In some embodiments, a viral vector is used to administer a polynucleotide encoding inhibitory nucleic acid molecules that inhibit C4A and/or C4B expression.
Non-viral approaches can also be employed for the introduction of the therapeutic to a cell of a patient requiring treatment of schizophrenia. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247:1465, 1990). Preferably the nucleic acids are administered in combination with a liposome and protamine.
Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of polynucleotide encoding inhibitory nucleic acid molecules into the affected tissues of a patient can also be accomplished by transferring a polynucleotide encoding the inhibitory nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue. cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.
In some embodiments, the inhibitory nucleic acid molecule is selectively expressed in a neuron. In some other embodiments, the inhibitory nucleic acid molecule is expressed in a neuron using a lentiviral vector. In still other embodiments, the inhibitory nucleic acid molecule is administered intrathecally. Selective targeting or expression of inhibitory nucleic acid molecules to a neuon is described in, for example, Nielsen et al., J Gene Med. 2009 July; 11(7):559-69. doi: 10.1002/jgm.1333.
The present invention further features methods of identifying modulators of a disease, particularly schizophrenia, comprising identifying candidate agents that interact with and/or alter the level or activity of a polynucleotide or polypeptide of C4A or C4B. As described elsewhere herein, increased expression of C4A was associated with increased risk of schizophrenia and increased synaptic elimination. Without being bound by theory, it is believed that interfering with C4A function or activity can decrease synaptic pruning and/or inhibit development or progression of schizophrenia in a subject.
Thus, in some aspects, the invention provides a method of identifying a modulator of schizophrenia, comprising (a) contacting a cell or organism with a candidate agent, and (b) measuring a level of polynucleotide or polypeptide of C4A or C4B in the cell relative to a control level. An alteration in the level of C4A or C4B polypeptide or polynucleotide indicates the candidate agent is a modulator of schizophrenia. In particular, a decrease in the level of C4A polynucleotide or polypeptide indicates the candidate agent is an inhibitor of schizophrenia. In some embodiments, the cell or organism is a recombinant cell or recombinant organism that overexpresses C4A polynucleotide or polypeptide.
Methods of measuring or detecting activity and/or levels of the polypeptide or polynucleotide are known to one skilled in the art. Polynucleotide levels may be measured by standard methods, such as quantitative PCR, Northern Blot, microarray, mass spectrometry, and in situ hybridization. Standard methods may be used to measure polypeptide levels, the methods including without limitation, immunoassay, ELISA, western blotting using an antibody that binds the polypeptide, and radioimmunoassay.
In some embodiments, the C4A polypeptide is fused to a detectable label (e.g., a fluorescent reporter polypeptide). Level(s) of C4A polypeptide in a cell contacted with a candidate agent can then be easily monitored by measuring fluorescence of the reporter polypeptide.
A recombinant cell or organism comprising an isolated C4A or C4B polynucleotide (in particular, a recombinant cell overexpressing C4A polynucleotide or polypeptide) can be useful in screening assays for identifying modulators (e.g., inhibitors) of schizophrenia. Accordingly, the invention provides a recombinant cell or organism heterologously expressing C4A polypeptide. In some embodiments, the cell is a mammalian cell. In some embodiments, the organism is a mouse.
Recombinant cells or organisms of the invention are produced using virtually any method known to the skilled artisan. Typically, recombinant cells are produced by transformation of a suitable host cell with all or part of a polypeptide-encoding nucleic acid molecule or fragment thereof in a suitable expression vehicle. Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to express (particularly, overexpress) C4A or C4B polypeptide in a host cell or organism. The precise host cell or organism used is not critical to the invention.
In some embodiments, the C4A or C4B polynucleotide or polypeptide is expressed in mammalian cells. Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al., Current Protocol in Molecular Biology, New York: John Wiley and Sons, 1997). The method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987).
A variety of expression systems exist for the expression of the polypeptides (e.g., C4A or C4B) of the invention in a host cell or organism. “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or organism. Expression vectors include all those known in the art, such as plasmids or viral vectors that incorporate the recombinant polynucleotide.
In some embodiments, the expression vector comprises an inducible or constitutive promoter operably linked to a C4A or C4B polynucleotide. Expression vectors useful for producing such polypeptides include, without limitation, chromosomal, episomal, and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof.
The invention provides kits for treating schizophrenia in a subject and/or identifying a subject having or at risk of developing schizophrenia. A kit of the invention provides a capture reagent (e.g., a primer or hybridization probe specifically binding to a C4A or C4B polynucleotide) for measuring relative expression level, copy number, and/or a sequence of a marker (e.g., C4A or C4B). In other embodiments, the kit further includes reagents suitable for DNA sequencing or copy number analysis of C4A and/or C4B.
In one embodiment, the kit includes a diagnostic composition comprising a capture reagent detecting at least one marker selected from the group consisting of a C4A polynucleotide and a C4B polynucleotide. In one embodiment, the capture reagent detecting a polynucleotide of C4A or C4B is a primer or hybridization probe that specifically binds to a C4A or C4B polynucleotide. The kits may further comprise a therapeutic composition comprising one or more antipsychotic agents. In some embodiments, the antipsychotic agent is aripiprazole, asenapine, clozapine, iloperidone, lurasidone, olanzapine, paliperidone, quetiapine, risperidone, ziprasidone, chlorpromazine, fluphenazine, haloperidol, and perphenazine.
In some embodiments, the kit comprises a sterile container which contains a therapeutic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
If desired, the kit further comprises instructions for using the diagnostic agents and/or administering the therapeutic agents of the invention. In particular embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for reducing schizophrenia symptoms; precautions; warnings; indications; counter-indications; over dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
Human C4 exists as two functionally distinct genes (isotypes), C4A and C4B; both vary in structure and copy number. One to three C4 genes (C4A and/or C4B) are commonly present as a tandem array within the MHC class III region (
A method (
Inheritance in father-mother-offspring trios were analyzed (
The series of many SNP alleles along a genomic segment (the SNP haplotype) can be used to identify chromosomal segments that come from shared common ancestors. The SNP haplotype(s) on which each C4 locus structure was present were identified (
Since C4A and C4B vary in both copy number and C4-HERV status (
Schizophrenia cases and controls from 22 countries have been analyzed genome-wide for SNPs, implicating the MHC locus as the strongest of more than 100 genome-wide-significant associations6. The analysis showed that long haplotypes defined by many SNPs carry characteristic C4 alleles (
SNP data from 28,799 schizophrenia cases and 35,986 controls, from 40 cohorts in 22 countries contributing to the Psychiatric Genomics Consortium (PGC)6 were analyzed. Association to 7,751 SNPs across the extended MHC locus (chr6: 25-34 Mb), to C4 structural alleles (
The association of schizophrenia to these genetic variants exhibited two prominent features (
Although the variation at C4 and in the distal extended MHC region associated to schizophrenia with similar strengths (p=3.6×10−24 and 5.5×10−28, respectively), their correlation with each other was low (r2=0.18,
In some autoimmune diseases with genetic associations in the MEW locus, alleles of HLA genes associate more strongly than do other variants in the MEW locus, appearing to explain the associations11,12. In contrast, in schizophrenia, classical HLA alleles associated to schizophrenia less strongly than other genetic variants in the MHC region did (
If each C4 allele affects schizophrenia risk via its effect on C4A expression, then this relationship should be visible across specific C4 alleles. Schizophrenia risk levels for the common C4 structural alleles (BS, AL-BS, AL-BL, and AL-AL) were measured; these alleles showed relative risks ranging from 1.00 to 1.27 (
These genetic findings (
To evaluate the extent to which levels of C4 protein in cerebrospinal fluid (CSF) are informative about disease status, levels of C4 protein were measured (by ELISA assay) in CSF samples derived from a group of 120 individuals who were either affected or unaffected with schizophrenia. CSF from affected individuals exhibited elevated levels of C4 protein (p<0.01;
C4 is a critical component of the classical complement cascade, an innate-immune-system pathway that rapidly recognizes and eliminates pathogens and cellular debris. In the brain, other genes in the classical complement cascade have been implicated in the elimination or “pruning” of synapses29-31.
To evaluate the distribution of C4 in human brain, immunohistochemistry on sections of the prefrontal cortex and hippocampus was performed. C4+ cells in the gray and white matter were observed, with the greatest number of C4+ cells detected in the hippocampus. Co-staining with cell-type-specific markers revealed C4 in subsets of NeuN+ neurons (
To further characterize neuronal C4, human primary cortical neurons were cultured and evaluated C4 expression, localization and secretion. Neurons expressed C4 mRNA and secreted C4 protein (
The association of increased C4 with schizophrenia (
In the immune system, C4 promotes C3 activation, allowing C3 to covalently attach onto its targets and promote their engulfment by phagocytic cells. In the developing mouse brain, C3 targets subsets of synapses and is required for synapse elimination by microglia, the principal CNS cells expressing receptors for complement29,30. It was found that in mice deficient in C436, C3 immunostaining in the dLGN was greatly reduced compared to WT littermates (
Whether mice deficient in C4 had defects in synaptic remodeling was then evaluated, as has been described for C3-deficient mice29. Mice lacking functional C4 exhibited greater overlap between RGC inputs from the two eyes (p<0.001) than wild-type littermate controls, suggesting reduced synaptic pruning (
In summary, described herein are methods to analyze a complex form of genome structural variation that were developed (
Microglia engulfed more synaptic particles in the presence of C4A in the frontal cortex of young adult mice (
Synapses in frontal cortex of P60 mice were quantified. Postnatal day 60 WT, C4−/−, hC4A/− and hC4B/− mice were perfused with 4% PFA and harvested brains were incubated in 4% PFA prior to cryopreservation in sucrose. Brain sections (12 μm) were stained with anti-SV2 (presynaptic marker) and anti-homer (post-synaptic marker) antibodies and layer of the frontal cortex was imaged using a confocal microscope (4 section/animal; 2 field of view/section). Staining for SV2 and homer identified synapses, defined as co-localized SV2 and Homer puncta (
In vitro C4 binding assay showed C4A preferential bound to synaptic membranes compared to C4B (
Changes in synapse number occurred during development in layer 2/3 of frontal cortex (
Results described herein were obtained using the following materials and methods.
Genomic DNA samples for the HapMap CEU population sample were obtained from Coriel Repositories (HapMap CEU plates 1 and 2). DNA samples for two groups of brain tissue donors were obtained from the Stanley Brain Resource of the Stanley Medical Research Institute (SMRI) and corresponded to the SMRI Array (SMRI-A) and SMRI Neuropathology (SMRI-N) collections. DNA samples for a third group of brain tissue donors, comprising 90 tissue donors for the NHGRI Gene and Tissue Expression Project (GTEx), were obtained from GTEx under an approved analysis proposal.
Copy number of each individual C4 structural element was first measured (C4A, C4B, C4L, and C4S) using droplet digital PCR (ddPCR)57. The following protocol for each genomic DNA sample in the study (including the HapMap CEU samples and the brain tissue donors) was used. First, genomic DNA was digested with AluI so that multiple tandem copies of C4 would then be on separate pieces of genomic DNA. (AluI cuts between structural features of C4 but not within any of the amplicons used for detection of them below.) For each genomic DNA sample, 50 ng of genomic DNA was digested in AluI (1 unit of enzyme in 10 ml of 1× reaction buffer, New England Biolabs) at 37° C. for 1 hour. The digested DNA was then diluted two-fold with water for subsequent analyses.
To measure the precise copy number of each structural element in each genomic DNA sample, digital PCR using nanoliter droplets (ddPCR) was performed, in which individual DNA molecules are dispersed into separate droplets, amplified with fluorescence detection probes (that detect with separate fluorescence colors the sequence of interest and a control, two-copy locus), and fluorescence-positive and -negative droplets of each color are then digitally counted57. 6.25 μl of the digested, diluted DNA from the above reaction was mixed with 1 ml of a 20× primer-probe mix (containing 18 μM of forward and reverse primers each and 5 μM of fluorescent probe) for C4 and a reference locus (RPP30) each, and 2×ddPCR Supermix for Probes (Bio-Rad Laboratories). The oligonucleotide sequences for the primers and probes used for assaying copy number of C4A, C4B, C4L, and C4S were from Wu et al58 and are listed in Table 1. For each sample, this reaction mixture was then emulsified into approximately 20,000 droplets in an oil/aqueous emulsion, using a microfluidic droplet generator (Bio-Rad). The droplets containing this reaction mixture were subjected to PCR using the following cycling conditions: 95° C. for 10 minutes, 40 cycles of 94° C. for 30 seconds and 60° C. (for C4A and C4L) or 59° C. (for C4B and C4S) for 1 minute, followed by 98° C. for 10 minutes. After PCR, the fluorescence (both colors) in each droplet was read using a QX100 droplet reader (Bio-Rad). Data were analyzed using the QuantaSoft software (Bio-Rad), which estimates absolute concentration of DNA templates by Poisson-correcting the fraction of droplets that are positive for each amplicon (C4 or RPP30). Since there are two copies of RPP30 (the control locus) in each diploid genome, the ratio of the concentration of the C4 amplicon to that of the reference (RPP30) amplicon is multiplied by two to yield the measurement of copy number of the C4 sequence per diploid genome (
The accuracy of copy number measurements from the above approach was evaluated in two ways. First, in every genome analyzed, the following relationship between the copy number of C4 structural elements is expected to hold because any given C4 gene is defined by its length (long or short) and its paralogous form (A or B):
C4A+C4B=C4L+C4S
Any deviation from this equality (for any sample) could flag a genotyping error for C4A, C4B, C4L, or C4S. Copy number measurements for all HapMap DNA samples and all brain donor DNA samples in this study satisfied this test in every case. In addition, copy number measurements for C4A and C4B from ddPCR were compared to those for 89 HapMap samples previously evaluated by Fernando et al.59 using Southern blot analysis of the same samples; measurements herein agreed with those of Fernando et al. for 89/89 samples.
The above analysis determines copy number of individual structural elements (A, B, L, S) but not of compound structural forms (AL, AS, BL, BS). Given that (for example) the numbers of copies of C4S are known, determining the ratio of the number of copies of C4AS and C4BS allows the copy number of these compound structural features to be readily calculated.
To determine how the known number of C4S copies (measured above) was composed of C4AS and C4BS copies, PCR was first performed to amplify 5.2-kilobase DNA molecules derived from C4S and spanning to the C4 A/B-defining molecular features (
Once C4A, C4B, C4L, C4S, C4AS, and C4BS copy numbers are calculated by the above methods, copy number of the remaining compound structural features (C4BL and C4AL) is easily calculated by the following formulas:
with the redundant calculation of C4AL copy number (by these two formulas) providing an additional checksum on the accuracy of measurements of copy number state.
For a multi-allelic CNV, multiple combinations of alleles can give rise to the same diploid copy number. For example, if a sample has 4 copies of the C4AL gene in a diploid genome, this could be a result of any of the following potential allelic combinations: 0+4, 1+3, or 2+2. To distinguish among these possibilities, we exploited allele frequency information that is implicit in the relative frequencies of the different diploid copy-number genotypes, together with additional constraints placed by inheritance in trios, as described below. An expectation-maximization (EM) algorithm that incorporated this information was applied to each C4 structural form (AL, AS, BL, and BS) separately. In this approach, each allelic configuration that could potentially give rise to each diploid copy number was enumerated. In certain trios only one configuration was possible under Mendelian inheritance (e.g., a trio in which father, mother, and offspring had a copy number of 0, 2, and 1, respectively). In the rest of the trios, allelic contributions were inferred using an EM algorithm with the following steps. First, probabilistic inferences of haploid copy number were made in each sample (with an “initial condition” that all possible combinations were equally likely). These inferences were then used to estimate frequencies of each copy-number allele in the population. The likelihood of each allelic combination in each trio was then re-calculated given these allele-frequency estimates. This allowed new estimates of allele frequency, which were then used to refine likelihoods of observing each allelic combination in each trio. This EM loop was repeated until the allele frequency estimates converged. In practice, these estimates converged very quickly to estimates that had low uncertainty in 45-55 of the 55 trios in the analysis (51 for AL, 55 for AS, 45 for BL, 49 for BS). In the remaining trios, the following further approach was used. First, a reference set of haplotypes was created from the trios in which inference of copy-number alleles had been unambiguous. This core set of haplotypes was then used as a reference to phase the remaining copy number alleles onto SNP haplotypes using Beagle genetic analysis software60.
C4 alleles were imputed from SNP genotypes using Beagle genetic analysis software61. To estimate the accuracy of inferences using our imputation approach, we performed leave-one-out trials. A different individual was removed from the reference panel in each trial, and the rest of the reference haplotypes were used to impute, using genetic analysis software61, the C4 structural form and haplogroup, with different subsets of SNPs in the extended MHC locus (chr6: 25-34 Mb): Illumina OmniExpress, Affymetrix 6.0, and Illumina Immunochip. The correlation (r2) between the probabilistic dosage from imputation and the experimentally-determined genotypes was calculated as a metric of imputation accuracy (Table 2). Note that these estimates of imputation efficacy will in many cases be lower bounds: (i) they will be exceeded by what it should be possible to do in the future (with larger reference panels derived from whole genome sequencing of many hundreds of families); and (ii) even in the current analysis, it was frequently observed that SNP haplotypes that were rare or unique in the reference panel (for example, the haplotypes grouped into the “-other” categories) were more common in the PGC cohorts and were presumably imputed with greater accuracy than a leave-one-out analysis would predict.
Expression of C4A and C4B was measured in eight panels of post mortem human brain RNA samples derived from three sets of donors. The first set (five brain-region-specific panels from one set of donors) was the Stanley Medical Research Institute Array Collection. This collection consists of 525 samples from 105 individuals. Five brain regions were sampled from each donor: anterior cingulate cortex, orbital frontal cortex, parietal cortex, cerebellum, and corpus callosum. The median age of the donors was 44 (range 19-64). Of the 105 individuals, 102 were of European ancestry and used in the analysis. The median post mortem interval (PMI) was 30 hours (range 9-84). 69 donors were male and 38 were female. Age, sex and PMI were evaluated as potential covariates in all analyses but were found to have insignificant regression coefficients in all analyses described. The second set (two tissue-specific panels) was obtained from the Stanley Medical Research Institute Neuropathology Consortium and contained 120 samples from 60 individuals. Two regions were sampled from each donor: anterior cingulate cortex and cerebellum. 36 donors were male and 24 were female. The median age was 47 (range: 30-68). The median PMI was 27 hours (range: 11-62). Age, sex and PMI were evaluated as potential covariates in all analyses but were found to have insignificant regression coefficients in all analyses described. The third set consisted of 93 samples (frontal cortex) from 93 individuals sampled by the Genotype-Tissue Expression (GTEx) Consortium. 67 donors were male and 26 were female. The median age was 53 (range: 22-59). Age, sex and BMI were evaluated as potential covariates in all analyses but were found to have insignificant regression coefficients in all analyses described. Copy number of C4 structural elements was measured using ddPCR in blood-derived genomic DNA samples from all individuals as described elsewhere herein.
Expression measurements were made using reverse-transcription ddPCR, in which total RNA is dispersed into thousands of nanodroplets; reverse transcription, PCR amplification, and fluorescence detection are then performed in droplets. Gene-expression measurements were normalized to the expression of a control gene (ZNF394) to account for variation in the amount of input RNA across samples; this gene was selected as a normalization control because in earlier brain transcriptomics data it showed uniform (low-variance) expression level across brain tissues sampled from many different individuals. In each reaction, the number of C4A-positive (or C4B-positive) and -negative droplets was counted, as well as the number of ZNF394-positive and -negative droplets. These numbers were then Poisson-corrected to yield an estimate of the underlying expression level, using the QuantaSoft software (Bio-Rad). ZNF394 was used as a normalization control and therefore calculate the ratio of C4A (or C4B) to ZNF394 expression.
For each brain donor in the two SMRI Brain Collection cohorts (each of which sampled multiple brain regions from each donor), a composite measure of expression across multiple brain regions was calculated in the following way. The calculation started with an i×j matrix (i individuals and j brain regions) of gene-expression measurements. A median normalization of the data was then performed for each region (more formally, the expression for ith individual in region j was re-calculated as a percentage of the median expression value across all the individuals for region j). To then obtain an overall summary value (across multiple brain regions) for an individual, the median (across regions) of these median-normalized values (more formally, a median value across the j columns was calculated for each row) was then calculated. Donors for whom measurements were available for at least 3 (of the 5) brain regions were carried into downstream analysis. Association between C4A (or C4B) expression and C4A (or C4B) copy number (
In the SMRI samples, the availability of genome-wide SNP data (together with our measurements of C4A, L, B, S copy number) allowed inference (by imputation) of the complex C4 structures present on each chromosome. To calculate the effect of each of the four common C4 structures on expression of C4A (
(C4A expression)i=Σjβj×(dose)ij+θ
where (dose)ij is the number of chromosomes in each diploid genome i that carry the structure j and θ is a constant (intercept).
To determine the C4 structural genotype for each individual in the SMRI array collection, copy number data for each C4 structural element (C4A, C4B, C4L, and C4S) from ddPCR were integrated together with SNP genotypes for these samples (from the Illumina Omni 2.5 SNP microarray). For each individual, the list of structural genotypes consistent with the set of copy numbers of C4 structural elements were enumerated, based on the 15 C4 structures that were identified in the HapMap CEU population sample (
To test association between gene expression and clinical diagnosis, the Mann-Whitney (nonparametric) test was used. The alternative hypothesis was specified based on the direction of effect of C4 structural variation on gene expression and on the risk of schizophrenia—given that C4 structural variants associating to increased risk of schizophrenia also associated to higher expression, it was hypothesized that the expression of C4 would be higher in patients with schizophrenia compared to unaffected controls. A Mann-Whitney test was performed to assess for differences in median normalized C4A expression values between patients with schizophrenia and unaffected controls. In order to test whether the expression of C4A associated with clinical diagnosis independently of structural variation in C4, the C4A expression-per-copy values were used and a Mann-Whitney test was again performed.
Expression of C4A and C4B was also tested for association to potential confounders, including age, sex, post mortem interval, preservation technique, and smoking. Parametric (Pearson) as well as non-parametric (Spearman) tests of correlation were used to evaluate correlation to continuous variables (age and post mortem interval), and association of expression to categorical variables (sex, preservation technique, and smoking) was tested using the Mann-Whitney test.
To derive a model for genetically predicting C4A and C4B expression to be used in association analysis of schizophrenia (in which it was expected that numerous genomes will have lower-frequency C4 structural haplotypes that are sparsely represented among the samples with measured expression values), C4A and C4B expression levels were sought to be predicted as a function of the dosage of each structural element (C4 AL, C4BL, C4AS, C4BS). All median-normalized expression data from samples across the SMRI array, SMRI Neuropathology, and GTEx cohorts was used to fit
(C4A or C4B expression)i=Σjβj×(dose)ij+θ
where (dose)ij is the number of structural elements j in sample i. From this model, samples with lower-frequency C4 haplotypes can have expected expression values computed by summing their structural element dosages multiplied by the corresponding coefficients. Regression coefficients that were significantly different from zero were included in the prediction models. The following prediction models were generated:
C4A expression=(0.47*AL)+(0.47*AS)+(0.20*BL)
C4B expression=(1.03*BL)+(0.88*BS)
Note that these are parameterized in internally normalized “expression units” that are not comparable between C4A and C4B, but are comparable across individuals for the same gene. These models explained 71% and 42% of inter-individual variation in measured C4A and C4B expression levels (respectively)—far more than explained by most known cis-eQTLs, but still consistent with a role for additional factors (beyond cis-acting variation at C4) in shaping C4 expression levels.
Case-Control Genotype Data from the Psychiatric Genomics Consortium (PGC)
Data from all 40 of the European-ancestry case-control cohorts for which individual level data could be made available by the PGC for such analyses was used (individual-level data from some cohorts could not be made available due to restricted level of patient consent). As described in the PGC manuscript62, all subjects provided written informed consent (or legal guardian consent and subject assent) with the exception of the CLOZUK sample, which obtained anonymous samples via a drug monitoring service under ethical approval and in accordance with the UK Human Tissue Act. The cohorts and array platforms used are listed in Table 3. These samples are further described in ref62 and in the individual studies referenced in Table 3.
Relatedness among samples and population structure was previously analyzed by the PGC Statistical Analysis Working Group, using a set of 19,551 autosomal SNPs across all cohorts, removing one member of each pair with π>0.2. The first ten principal components were included as covariates in all of the association analyses (as described below). All analyses were pursued in concordance with an analysis proposal approved by the PGC Schizophrenia Working Group. All analyses of individual-level genotype data were conducted on the PGC's computer server in the Netherlands.
The SNPs and individuals retained for association analysis were subject to the following quality control (QC) parameters previously applied by the PGC Statistical Analysis Group and including: (i) SNP missingness <0.05 (before sample removal); (ii) subject missingness <0. 02; (iii) autosomal heterozygosity deviation (|Fhet|<0.2); (iv) SNP missingness <0.02 (after sample removal); difference in SNP missingness between cases and controls <0.02; and SNP Hardy-Weinberg equilibrium (p>10−6 in controls or p>10−10 in cases).
In addition to the above parameters that were analyzed on a genome-wide scale, additional QC filters were applied to the SNP genotype data from the extended MHC locus in each of the 40 cohorts analyzed. SNPs that met the following criteria were removed: (i) those that were within the duplicated C4 locus (chromosome 6:31939608-32014384, hg 19); (ii) SNPs whose allele frequency differed by more than 0.15 from their frequency in our HapMap CEU reference panel for imputation; and (iii) transversion SNPs (A/T and G/C) whose minor allele frequency was greater than 0.35 (as it can be problematic to determine whether they have the same strand assignment as SNPs in the reference panel for imputation).
Imputation of C4 structural variation into the PGC data set was done with Beagle genetic analysis software5, using the HapMap CEU reference panel that we had supplemented with C4 structural alleles. C4 structural variation was imputed into each of the 40 cohorts in the PGC data set separately. Imputation was performed using two approaches, with highly similar results: (i) a “best guess” approach in which each genome is assigned the most likely pair of C4 structural alleles given the SNP data; and (ii) a “dosages” approach in which imputation uncertainty is advanced into subsequent stages of analysis by performing association analysis on the probabilistic “dosages” of each allele in each genome.
The reference panel used consisted of 222 haplotypes from 111 unrelated individuals, with C4 structural variants on haplotypes with HapMap phase III SNPs (see
This strategy enabled independent testing of association of each common combination of C4 structure and MHC SNP haplotype background. This strategy also allowed (i) inference of copy number of C4 structural elements (C4A, C4B, C4L, and C4S) based on the C4 alleles imputed in each individual (e.g., an individual with C4 alleles AL-AL-1 and AL-BL-2 has a diploid copy number of 3 for C4A, 1 for C4B, 4 for C4L and 0 for C4S); and (ii) inference of expected expression of C4A and C4B in the brain based on calculated copy number of C4 structural elements in each individual, using the linear model (described above) that was fit to the expression data from post mortem brain samples. A reference panel consisting of 9,956 haplotypes based on data collected by the Type 1 Diabetes Genetics Consortium (T1DGC)63 was used for imputation of HLA classical alleles from both class I and class II genes: HLA-A, B, C, DRB1, DQA1, DQB1, DPB1, DPA1. This reference panel enabled imputation of HLA classical alleles at four-digit resolution, HLA amino acids, intragenic SNPs in the MEW locus, and insertions/deletions.
A mega-analysis was performed that utilized individual-level genotype data from all 40 cohorts that were analyzed from the PGC data set. Association analysis was performed in a logistic regression framework that included study indicator variables to account for cohort-specific effects and principal components to control for population stratification:
log(oddsi)=βj×(dosei,j)+Σc=139βc×(chorti,c)+Σp=110βp×(PCi,p)+θ
where dosei,j is the number of chromosomes in each individual, i, that carried a C4 structural allele, j, and βj is the additive effect per copy of the C4 allele. 39 study indicator variables (the number of cohorts minus 1) were included, with cohorti,c equal to 1 if the ith individual belonged to the cth cohort and equal to 0 otherwise. In addition, ten principal components that associated to phenotype were included as covariates, with PC4 being the pth principal component for the ith individual. The same framework was used for testing association to (i) individual SNPs and HLA classical alleles, where dosei,j was the dosage of the minor allele, j, of the SNP or HLA classical allele in individual i; (ii) copy number of C4 structural features, where dosei,j was the diploid copy number of the C4 feature in individual i; (iii) genetically predicted expression of C4A and C4B, where dosei,j was calculated from the imputed C4 structures according to the above formulas (see the section, “Model for genetically predicting C4A and C4B expression”). To test association to C4 conditional on rs13194504 and rs210133 (representing the other two genome-wide significant associations within the extended MHC locus), the dosages of the minor alleles of those SNPs were used as additional covariates in the model.
The association of C4 alleles to schizophrenia was tested in multiple ways. The first test used aggregate genetic predictors (of C4A and C4B expression levels) as a composite genetic variable that combined information across many different alleles into an omnibus test; we started with this omnibus test (
Fresh frozen hippocampus and frontal cortex sections were obtained from the Stanley Medical Research Institute. Stained tissues were from schizophrenia patients aged 31-43. Sections were thawed on ice and then post-fixed for one hour at 4° C. in 4% paraformaldehyde in PBS. Sections were then washed three times in PBS and then permeabilized in 0.2% Triton X-100 in PBS on a shaker for one hour at room temperature. Sections were then blocked in 10% BSA with 0.2% Triton X-100 in PBS for one hour at room temperature on a shaker and then transferred into a carrier solution of 5% BSA in 0.2% Triton X-100 in PBS containing the primary antibody and were left to incubate overnight at 4° C. For pre-adsorption experiments, purified human C4 protein (Quidel) was pre-incubated with the C4c antibody at double the antibody concentration for 30 minutes at room temperature before being added to the slides for overnight incubation at 4° C. The following day sections were washed three times in PBS and incubated in carrier solution with Alexa-Flour conjugated secondary antibodies (1:500) and Hoechst (1:10,000) for one hour at room temperature on a shaker. The sections were then washed three times in PBS and then incubated in 0.5% Sudan Black dissolved in 70% ethanol to eliminate autoflourescence from lipofuscin vesicles. Sections were then washed 5-7 times in PBS to remove the excess Sudan Black. Coverslips were then added to the slides using 90% glycerol in PBS as the mounting media. Slides were imaged on an Ultraview Vox Spinning Disk Confocal microscope for images of cellular colocalization or Zeiss ELYRA PS1 structured illumination microscope (SIM) for synapse analysis. The following antibodies were used for staining; anti-C4c (Quidel, A211, 1:1000), anti-NeuN (Abcam, AB104225, 1:500), anti-Vglut1 (Millipore, AB5905, 1:1000), anti-Vglut2 (Millipore, AB2251, 1:2000), and anti-PSD95 (Invitrogen, 51-6900, 1:200). IHC was performed in brain tissue slices from 5 individuals affected with schizophrenia and 2 unaffected individuals. These were selected from the same brains as the RNA experiments (SMRI Neuropathology Consortium). Across different donors variable intensity of staining (down to almost no staining) was observed, but qualitatively different patterns were not observed. The level of RNA expression of C4 (in the corresponding RNA sample from the same donor) predicted the level of IHC staining—in tissue from donors with higher C4 RNA expression, the IHC staining was also stronger; in tissue from donors with little-to-no C4 RNA detected, little-to-no IHC staining was also observed.
The images in
Primary human cortical neurons were obtained from Sciencell Research Laboratories (catalog no. 1520). The neurons were characterized by Sciencell to be immunopositive for MAP2, neurafilament, and beta-tubulin III; are guaranteed to be negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi; and are not listed as a commonly misidentified cell line by ICLAC. Human cortical neurons were cultured in vitro on PLL-coated coverslips in neuronal media for up to 48 days. Coverslips were fixed with 4% paraformaldehyde at room temperature for 7 minutes. Non-specific binding sites were blocked with 5% BSA for 1 hour in PBST (0.1% Tween 20) followed by 4° C. overnight incubation with primary antibodies anti-MAP2 (EMD-Millipore, rabbit polyclonal, 1:10,000), anti-200 kD Neurofilament (Abcam, chicken polyclonal, 1:100,000), anti-Synaptotagmin (Synaptic Systems, rabbit polyclonal, 1:500), anti-PSD95 (Abcam, goat polyclonal, 1:500), and/or anti-C4c (Quidel, mouse monoclonal, 1:200). Coverslips were then washed with PBST and incubated for 1 hour at room temperature with secondary antibodies (Abcam, donkey or goat, 1:1000 in 5% BSA-PBST). Coverslips were mounted on slides using Vectashield with DAPI and visualized by fluorescent microscope (Zeiss Confocal).
Conditioned media was collected from in-vitro cultured human neurons at days 7 and 30 and frozen at −80° C. until quantification of C4 by western blot. Equal amounts of proteins (20 ug as determined by BCA Protein Assay) were diluted 1:1 with Native Sample Buffer (BioRad 161-0738) and separated on a 4-15% TGX precast polyacrylamide gel. Purified human C4 protein from Quidel (A402) was used as a positive control. Unconditioned neuronal media (Sciencell 1521) provided an appropriate negative control. Electrophoresis was performed using the Mini-PROTEAN Tetra Cell (BioRad). Proteins were then transferred onto polyvinylidene difluoride membranes (Immun-Blot PVDF, BioRad 162-0177) for Western Blot analysis. Membranes were blocked in a 5% milk solution in TBST (0.1% Tween 20) for 1 hour at room temperature and then incubated with anti-C4c (Dako, F016902-2, 1:1000) primary antibody overnight at 4° C. Following washes in TBST, secondary antibody goat-anti-rabbit HRP (Abcam, preadsorbed, 1:10,000) was hybridized for 1 hour at room temperature. Membranes were washed in TBST again and then reactivity was revealed by chemiluminescence reaction performed with ECL detection reagents (BioRad Clarity) and film exposure.
The generation of the C4−/− mice that were used to investigate synapse elimination in the retinogeniculate system is described in detail in earlier work64. In these mice, the sequence spanning part of exon 23 through exon 29 has been replaced with a PGK-Neo gene. Experiments involved litters created by crossing C4+/− heterozygous parents, so that all comparisons were among littermates of different C4 genotypes. Sample sizes were determined based on power calculations for each data set (to obtain >80% statistical power) and based on recommendations from IACUC to conserve animals. Mice from both sexes were analyzed in these experiments. Experiments were approved by the institutional animal use and care committee in accordance with NIH guidelines for the humane treatment of animals.
Human C4 transgenic mice were generated using BAC DNA transgenesis. BAC clones containing common human C4 alleles, i.e. C4A allele (MCF258G8), C4B (CH502) allele or C4A and C4B (CH501) were selected and purchased from Childrens Hospital Oakland Research Institute (CHORI) (http://bacpac.chori.org) (Horton et al. Immunogenetics. 2008 January; 60(1):1-18). The human C4 locus encodes two highly conserved isoforms, C4A (acidic) and C4B (basic), whose coding sequences differ by only four amino acids (Belt et al.). The structural differences between the two is conferred by the four amino-acid difference in the isotypic region that drive the efficient binding of C4A and C4B to different chemical targets (
In order to understand why increased C4A gene copies, but not C4B, confers schizophrenia risk and because mouse C4 is encoded by only one gene, transgenic mice were generated that express C4A and C4B. BAC DNAs were linearized prior to pronuclear injection into mouse zygotes. Offspring from injections were genotyped using digital droplet PCR (ddPCR) of genomic DNA using primers specific for the C4A or C4B isotypic region to confirm the number of copies of the BAC Tg. Mice were bred with C4−/− C57/B6 mice and backcrossed at least 10 generations (
Analysis of Dorsal Lateral Geniculate Nucleus (dLGN)
Visualization and analysis of RGC synaptic inputs in the mouse dLGN was performed as described9. Cholera toxin-β subunit (CTB) conjugated to Alexa 488 (green label) and CTB conjugated to Alexa 594 (red label) were intraocularly injected into the left and right eyes, respectively, of P9 mice, which were sacrificed the following day. Images were acquired using a Zeiss Axiocam microscope and quantified blind to experimental conditions and compared to age-matched littermate controls. The degree of left and right eye axon overlap in dLGN was quantified using an R-value analysis as described65 and by quantifying the percent overlap as previously described66. Pseudocolored images representing the R-value distribution were generated in ImageJ image analysis software.
For measurement of C4 expression in the retinal ganglion cells (RGCs) and LGN, RNA was isolated from tissue with the Qiagen RNeasy Lipid mini kit (cat. No 74804) with optional DNase digestion according to the manufacturer's protocol. RGCs were isolated, lysed, and DNase digested with Ambion Cells to Ct kit66. 15 ng of RNA was used as the input for the RT-ddPCR reaction with the primer-probe sets listed in Table 1.
Retinal ganglion cells were purified from p5 and p15 C57BL/6 mice through serial immunopanning as previously described67. To specifically isolate the lateral geniculate nucleus (LGN) from P5 C57BL/6 mice, LGN was first fluorescently labeled through bi-lateral intraorbital injection of flourophore-conjugated cholera toxin at P4 and then microdissected at P5 during visualization with a fluorescence dissecting microscope. Retinal tissue was harvested from separate P5 C57B16 mice. RNA was isolated from LGN and retinal tissue with the Qiagen RNeasy Lipid mini kit (cat. No 74804) with optional DNase digestion according to the manufacturer's protocol. RGCs were lysed, DNase digested with Ambion Cells to Ct kit, and RNA from the cell-free solution used in subsequent reactions. Mouse C4 expression was calculated as the average of two C4-specific reverse transcription-ddPCR assays, one with the primer-probe set spanning the junction of exons 23 and 24 and the other, the junction of exons 25 and 26, each normalized to the housekeeping mRNA, Eif4h.
Brains were harvested from mice after transcardial perfusion with 4% paraformaldehyde (PFA). Tissue was then immersed in 4% PFA for 2 hours following perfusion, cryoprotected in 30% sucrose, and embedded in a 2:1 mixture of OCT:20% sucrose PBS. Tissue was cryosectioned (12-14 microns), sections were dried, washed three times in PBS, and blocked with 2% BSA+0.2% Triton X in PBS for 1 hr. Primary antibodies were diluted in antibody buffer (+0.05% triton+0.5% BSA) as follows: anti-C3 (Cappel, 1:300), anti-vglut2 (Millipore, 1:2000) and incubated overnight at 4° C. Secondary Alexa-conjugated antibodies (Invitrogen) were added at 1:200 in antibody buffer for 2 hours at room temperature. Slides were mounted in Vectashield (+DAPI) and imaged using the Zeiss Axiocam microscope, Zeiss LSM700. In addition to the analysis of C3 localization, several commercial antibodies for mouse C4 were also tested and it was found that none were sufficiently specific.
Retinal flat mounts were prepared by dissecting out retinas whole from the eyecup and placing four cuts along the major axis, radial to the optic nerve. Each retina was stained with DAPI (Vector Laboratories, Burlingame, Calif.) to reveal cell nuclei. Measurements of RGC density based on Brn3a (goat anti-Brn3a, 1:200, Santa Cruz) immunohistochemistry were carried out blind to genotype from matched locations in the central and peripheral retina for all four retinal quadrants of each retina. Quantification was done on P10 retinas, which is the age at which eye specific segregation analysis was completed. For each retina (1 retina per animal; N=4 mice per treatment condition or genotype), 12 images of peripheral retina and 8 images of central retina were collected. For each field of view collected (20 per retina), Macbiophotonics ImageJ software (NIH) was used to quantify the total number of Brn3a-positive cells using the cell counter plugin. All analyses were performed blind to genotype.
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
This application claims priority to and the benefit of U.S. Provisional Application No. 62/286,867, filed Jan. 25, 2016, the disclosure of which is incorporated herein by reference in its entirety.
This invention was made with government support under Grant Nos. R01 HG006855, U01 MH105641, and R01 MH077139 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/014757 | 1/24/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62286867 | Jan 2016 | US |
