# Patent

## Title
Micro RNAs and their methods of use for the treatment and diagnosis of schizophrenia and schizophrenia spectrum disorders

## Abstract

A method of diagnosing, assessing susceptibility, and/or treating schizophrenia involving the identification and/or observation of microRNAs (miRNA) and variant miRNA are provided. Micro RNAs alleles associated with schizophrenia and schizophrenia spectrum disorders were identified and ultra-rare variants in the precursor or mature miRNA were identified. Functional analyses of ectopically expressed copies of the variant miRNA precursors demonstrate loss of function, gain of function and altered expression levels. The present invention also provides methods for selecting a preferred therapy for a particular subject or group of subjects or individuals at risk for or suffering from schizophrenia or psychosis by use of miRNAs.

## Background
PRIORITY CLAIM
This application claims the benefit of U.S. Provisional Application Ser. No. 61/060,892, filed Jun. 12, 2008, which is incorporated herein by reference.


GOVERNMENT INTEREST
The present invention was made with government support under National Institutes of Health grant numbers A129329 and HL07470. The government has certain rights in the present invention.


BACKGROUND
The present invention relates generally to neurological and psychiatric diseases diagnosed by and/or caused in whole or in part by microRNAs and, more specifically, to the use of individual microRNAs for the diagnosis and treatment of schizophrenia.
Schizophrenia is a chronic, severe and disabling brain disease. Approximately one percent of the population develops schizophrenia during their lifetime—more than two million Americans suffer from the illness in a given year. Schizophrenia typically presents in early adulthood or late adolescence. The illness is characterized by positive symptoms (delusions or hallucinations), negative symptoms (blunted emotions and lack of interest) and disorganized symptoms (confused thinking and speech or disorganized behavior and perception). Additionally, cognitive deficits are also frequently observed, particularly in elderly schizophrenia patients. For some patients, the disorder is life-long, while others may have periodic episodes of psychosis. Men have an earlier age of onset than women, and also tend to experience a more serious form of the illness with more negative symptoms, poorer chances of a full recovery, and a generally worse outcome [Jablensky, 2000]. Systematic reviews show that schizophrenia is 1.4 times more likely to occur in men than in women.
MicroRNAs (miRNAs) are a large family of small, non-coding RNAs that negatively regulate gene expression at the post-transcriptional level [Ambros, 2003; Lai, 2003; Bartel, 2004]. In animals, miRNAs bind to complementary sites in target mRNAs 3′ untranslated regions (UTRs) to create imperfectly paired RNA heteroduplexes that inhibit translation of the target RNAs. Many microRNAs are conserved in sequence and function between distantly related organisms.
miRNAs regulate various biological functions including developmental processes, developmental timing, cell proliferation, neuronal gene expression and cell fate [Klein et al., 2005], apoptosis [reviewed in [Mattick and Makunin, 2005; Croce and Calin, 2005], tissue growth, viral pathogenesis, brain morphogenesis [Giraldez et al., 2005], muscle differentiation [Naguibneva et al., 2006], stem cell division [Hatfield et al., 2005] and progression of human diseases [Ambros, 2003; Palatnik et al., 2003]. Condition-specific, time-specific, and individual-specific levels of gene expression may be due to the interactions of different miRNAs accounting for more accurate genetic expression of various traits [Ying and Lin, 2004].
The large number of miRNA genes, the diverse expression patterns and the abundance of potential miRNA targets suggest that miRNAs may be a significant but unrecognized source of human genetic disease, including neuropsychiatric disorders. A sequence variant in the binding site for the miRNA miR-189 in the SLITRK1 mRNA has been shown to be associated with Tourette's syndrome [Abelson et al., 2005]. In addition, components required for miRNA processing and/or function have also been implicated in fragile X mental retardation [Jin et al., 2004], DiGeorge syndrome [Landthaler et al., 2004] and cancer [Karube et al., 2005], pointing to the wide ranging involvement of miRNAs in disease.
A number of animal models have been developed for schizophrenia, utilizing both non-primate (rat) and primate (monkey) animals. In one commonly used animal model of schizophrenia, phencyclidene (PCP) is chronically administered to the animal subjects, resulting in dysfunctions similar to those seen in schizophrenic humans (Jentsch et al., 1997, Science 277:953 955; Piercey et al., 1988, Life Sci. 43(4):375 385).
The causes of schizophrenia are essentially unknown. Although it is believed to have a genetic component, environmental factors appear to influence the onset and severity of the disease. Neuropathological changes in schizophrenics may include enlargement of the lateral ventricles, cavities in the brain which are part of the cerebrospinal fluid system. Sometimes, there is a decrease in overall brain mass. Several different theories have been developed regarding the etiology of schizophrenia, including the dopaminergic, glutamatergic, and cholinergic theories of schizophrenia. The dopamine hypothesis posits that positive symptoms result from excess function of the neurotransmitter dopamine in the mesolimbic area of the brain. This hypothesis is based largely on indirect, pharmacological evidence that (1) dopamine-antagonizing drugs are effective antipsychotic agents; (2) dopamine-mimicking drug exacerbate schizophrenic symptoms and (3) certain symptoms of acute paranoid schizophrenia can be elicited in non-schizophrenics by amphetamine, a drug that activates dopamine systems. However, negative symptoms have been associated with regionally localized dopamine deficits in the prefrontal cortex. Thus, there is a need for a more effective diagnosis and treatment of this disease. Additionally, there is a need for an earlier detection method for schizophrenia, such as prior to the presentation or onset of noticeable symptoms. The present invention satisfies this need and provides related advantages as well.
SUMMARY
In accordance with one embodiment, there is provided a system and method for diagnosing and determining the susceptibility to schizophrenia.
In another embodiment, a method for determining the susceptibility to schizophrenia comprises obtaining a nucleic acid containing sample from a patient and detecting mutant miRNA alleles indicating schizophrenia.
In another embodiment, a method for diagnosing or identifying schizophrenia comprises obtaining a nucleic acid containing sample from a patient and detecting mutant miRNA alleles indicating schizophrenia.
A further embodiment includes a method for diagnosing schizophrenia which comprises determining the magnitude of expression of a miRNA gene or allele selected from the group consisting of those disclosed in Table 1 in a sample and comparing the magnitude of expression to a baseline magnitude of expression of the gene or allele in persons without schizophrenia, wherein elevated or depressed levels of the gene or allele (as compared to the baseline magnitude) indicate the presence of schizophrenia. This diagnostic assay may be performed examining one or a combination of more than one miRNA gene or allele associated with schizophrenia.
In another embodiment, a method for diagnosing schizophrenia comprises obtaining a nucleic acid containing sample from a patient and detecting one or more variant miRNA alleles.
Another aspect is a functional screen for testing new variants for likely contribution to schizophrenia. Variants in nucleic acid, such as in miRNA, include but are not limited to point mutations, deletions, insertions, and translocations.
In another embodiment, schizophrenia is treated or prevented by modifying the miRNA of a mammal affected or potentially affected by schizophrenia or a schizophrenia spectrum disorder using gene therapy techniques.
In another embodiment, schizophrenia is treated or prevented by administering agents to antagonize the function of the RNA, and particularly miRNA genes or alleles if such genes or alleles are upregulated, or to agonize the function of the miRNA genes or alleles if such genes or alleles are downregulated, which miRNA genes or alleles contribute to or cause schizophrenia.
Such miRNA genes or alleles referenced in these embodiments include let-7f-2, mir-18b, mir-505, mir-502, mir-188, mir-325, mir-660, mir-509-3, mir-510, mir-421, mir-934, and mir-450a-2



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the generation of 5p and 3p mature miRNA. This diagram was modeled after Han et al.
FIG. 2A-I shows stem-loop sequences of miRNA and its variants that are modeled after miRBase. FIG. 2A shows SEQ ID NOS: 1-4; FIG. 2B shows SEQ ID NOS: 5-9; FIG. 2C shows SEQ ID NOS: 10-14; FIG. 2D show SEQ ID NOS: 15-19; FIG. 2E shows SEQ ID NOS: 20-24; FIG. 2F shows SEQ ID NOS: 25-29; FIG. 2G shows SEQ ID NOS: 30-34; FIG. 2H shows SEQ ID NOS: 35-39; and FIG. 2I shows SEQ ID NOS: 40-43. 
FIG. 3 shows transfection data of miR-509-3 and its single nucleotide polymorphisms (SNPs). The repression of all reporters was the same for both the WT and the Mut (FIG. 3A: SEQ ID NOS: 44-47).
FIG. 4 shows co-transfection test results of miR-502. a. The expression of 5p-si, 5p-mi, and 3p-si reporters is reduced in the mutant whereas the expression of 3p-mi reporter is the same in the wild-type (“WT”) and mutant (“Mut”). Each bar represents the average of at least three independent transfections with duplicate determinations for each construct. Error bars represent the standard deviation (“S.D.”). b. Northern blot results. Top: Hybridization with a miR-502-3p probe; Middle: blot probed with a miR-502-5p probe: Bottom; hybridization with a U2 snoRNA probe and irrelevant HIV tat/rev siRNA probe. Lane 7 depicts RNAs from cells transfected with the miRNA expression vector fU1-miR; Lanes 1, 3, 5, and 6 contain RNAs from cells transfected with the WT miRNA construct; Lanes 2, 4, 8, and 9 contain samples from cells transfected with the Mut miRNA construct. U2 snoRNA was used as an RNA loading control and siRNA-1 (anti-tat/rev in HIV-1, synthetic annealed double-strand siRNA) was used as transfection control. c. Northern blot results. The left-hand panel is the RNA decade marker; the middle panel is blot probed with 502-5p; the right-hand panel is blot probed with 502-3p. For the two blots, fU1-miR-502 is run in lane 1; fU1 miR-502-C/G is run in lane 2; fU1-miR is run in lane 3. U2 and U6 are shown below. d. Nucleotide sequences of SiCheck-502-5p-si, SiCheck-502-5p-mi, SiCheck-502-3p-si, SiCheck-502-3p-mi (SEQ ID NOS: 48-55).
FIG. 5 shows a functional test of miR-510 and miR-510-G/A. a. Transfection test results. The expression of reporters for 5p-si, 5p-mi, 3p-si and 3p-m-si (G/A mutant form) from the mutant are all reduced. Each bar represents the average of at least three independent transfections with duplicate determinations for each construct. Error bars represent the S.D. b. Northern blot results. Top: hybridization with a miR-510-3p probe; Middle: hybridization with a miR-510-5p probe; U2 snoRNA was used as an RNA loading control. SiRNA-1 was used as a transfection control. Lane 9 is from cells transfected with fU1-miR; Lanes 1, 3, 5, and 6 are from cells transfected with fU1-miR-510; Lanes 2, 4, 7, and 8 are from cells transfected with fU1-miR-510-G/A. c. Northern blot results. The left-hand panel is a blot probed with 502-3p; the right-hand panel is a blot probed with 502-5p. For the two blots, fU1-miR is run in lane 1; fU1 miR-510 is run in lane 2; fU1-miR-G/A is run in lane; and fU1-miR-510-T/C is run in lane 4. Normalized signal ration of pre-510 and 510 is shown below.
FIG. 6 variant miR-510-m has a T (U) to C transition in the seed of the predicted miR-510* (3p). Functional test of miR-510 and miR-510-T/C. a. Transfection test results. The expression of reporters for 5p-si, 5p-mi, F3p-si, and 3p-m-si (mutant form) from the mutant form are reduced. The repression for 3p-mi is approximately the same for both the WT and the mutant. Each bar represents the average of at least three independent transfections with duplicate determinations for each construct. Error bars represent the S.D. b. Nucleotide sequences of miR-510, miR-510-G/A, SiCheck-510-5p-si, SiCheck-510-5p-mi, SiCheck-510-3p-si , SiCheck-510-3p-mi (SEQ ID NOS: 56-65).
FIG. 7 shows results of a Northern blot analysis in accordance with one embodiment of the present invention. Northern blot results. Top: blot that was probed with the miR-510-3p probe; Middle: blot probed with the miR-510-5p probe; Bottom: blot probed with a U2 snoRNA (RNA sample loading control) probe and a probe that was used to detect the siRNA-1 (transfection control). Lanes 1, 2, and 3 are samples transfected with miR-510 WT; Lanes 4 and 5 were transfected with the miR-510-T/C mutant.
FIG. 8 shows a functional test of miR-890 and miR-890-G/C. a. Transfection test results. The expression of reporters for 5p-si, 5p-mi, 3p-si, and 3pGC-si (mutant form) from the mutant form are reduced. The expression of the 3p-mi reporter is approximately the same for both the WT and mutant. Each bar represents the average of at least three independent transfections with duplicate determinations for each construct. Error bars represent the S.D. b. Northern blot results. Under lanes 0,1, and 2 were probed with miR-890-5p, 3pGC, and 3p for samples transfected with fU1-miR or fU1-miR plus siRNA-1. Lanes 7, 8, 9, and 10 were hybridized with probes for miR-890-5p, 3pGC, and 3p. Lanes 7 and 9 were transfected with fU1-miR-890; Lanes 8 and 10 were transfected with fU1-miR-890-G/C; Lanes 9 and 10 were also co-transfected with siRNA-1. U2 and U6 snoRNAs were used as RNA loading controls and siRNA-1 was used as transfection control. c. Nucleotide sequences of miR-890, mir-890-G/C, SiCheck-890-5p-si, SiCheck-890-5p-mi, SiCheck-890-3p-si, SiCheck-890-3p-GC-si, SiCheck-890-3p-mi (SEQ ID NOS: 66-77).
FIG. 9 shows a functional test of miR-892b and miR-892b-T/C. a. Transfection test results. The expression of reporters for 5p-si, 3p-si, 3pm-si (mutant form), and 3p-mi from the mutant form are reduced. Each bar represents the average of at least three independent transfections with duplicate determinations for each construct. Error bars represent the S.D. b. Northern blot results. Lanes 0, 1, and 2 were hybridized with probes for miR-892b-5p, 3p, and 3pTC (mutant form) using samples transfected with fU1-miR or fU1-miR plus siRNA-1. Lanes 7, 8, 9, and 10 were hybridized with probes for miR-892b-5p, 3p, and 3pTC. Lanes 7 and 9 were from cells transfected with fU1-miR-892b; Lanes 8 and 10 were from cells transfected with fU1-miR-892b-T/C; Lanes 9 and 10 were from cells also transfected with siRNA-1. U2 and U6 snoRNAs were used as RNA loading controls and siRNA-1 was used as transfection control. c. Nucleotide sequences of miR-892b, mir-892b-T/C, SiCheck-892b-5p-si, SiCheck-892b-3p-si, SiCheck-892b-3pm-si , SiCheck-892b-3p-mi (SEQ ID NOS: 78-87).
FIG. 10 shows a functional test of miR-934 and miR-934-T/G. a. Transfection test results. Expression of 5p-si and 5p-mi reporters are almost the same, but strong repression of the 3p-si and 3pm2-si reporters are observed from the mutant miRNA. Each bar represents the average of at least three independent transfections with duplicate determinations for each construct. Error bars represent the S.D. b. Northern blot results. Left: blot was probed with a miR-934 5p probe; right blot that was probed with a 3p probe. U2 and U6 snoRNAs were probed as RNA gel loading controls. SiRNA-1 was co-transfected in samples 2, 5, and 6 as a transfection control. Lanes 1 and 2 are from cells transfected with fU1-miR; Lanes 3, 5, and 7 are from cells transfected with fU1-miR-934; Lanes 4, 6, and 8 are from cells transfected with the mutant. c. Nucleotide sequences of miR-934, mir-934-T/C, SiCheck-934-5p-si, SiCheck-934-5p-mi, SiCheck-934-3p-si. (SEQ ID NOS: 88-95).
FIG. 11 shows variant miR-660 has a C to T (U) transition at the 15th position of the mature miRNA.
FIG. 12 shows Let7-f2 SNP si and mi target sequence test.
FIG. 13a shows miR-18b/18b* si and mi target sequence test. FIG. 13b is a stem-loop sequence of SEQ ID NO: 96.
FIG. 14 shows miR-188-5p/3p si and mi target sequence test.
FIG. 15 shows variant miR-505/505*-m has a C to T (U) transition at the 6th nt (relative to the 5′ end of the upper strand of the mature miR-505).
FIG. 16 shows dot blotting results. a. miR-510 verses 510-G/A. Blots 1, 2, and 3 are wild type and blots 4, 5, and 6 are mutant type. Left side blots were probed with 510-5p probe and right side blots were probed with 510-3p probe. b. miR-890 verses 890-G/C. Blots 1, 2, and 3 are wild type and blots 4, 5, and 6 are mutant type. Left side blots were probed with 890-5p probe and right side blots were probed with 890-3p probe. c. miR-934 verse 934-T/G. Blots 1, 2, and 3 are wild type and blots 4, 5, and 6 are mutant type. Left side blots were probed with 934-5p probe and right side blots were probed with 934-3p probe.



FIG. 17 (Table 1a) represents the results from an exemplary analysis of ultra-rare miRNA cohort-specific variants.
FIG. 18 (Table 1b) represents the results from an exemplary analysis of miRNA cohort-specific sequence variants in a gene pool analysis.
FIG. 19 (Table 2) lists exemplary miRNA primers useful in identifying X chromosome variants (SEQ ID NOS: 97-215).
FIG. 20 (Table 3) lists variants identified in cases and controls.
FIG. 21 (Table 4) lists variants with 509-2.
FIG. 22 (Table 5) lists variants without 509-2.
FIG. 23 (Table 6) lists target genes of miRNAs having ultra-rare cohort-specific variants.
FIG. 24 (Table 7) lists oligonucleotides useful for cloning pri-miRNA and probes for northern blots (SEQ ID NOS: 216-337).
FIG. 25 (Table 8) lists all the miRNA gene variants that were found in the control population or the patient samples.
DETAILED DESCRIPTION
The present invention relates to the involvement of microRNAs (miRNAs) in development, onset and expression of symptoms associated with schizophrenia and using the understanding of such miRNA expression and activity to diagnose and treat schizophrenia or to diagnose risk of developing schizophrenia and the development of functional assays to assist the search for schizophrenia therapies.
A first aspect is a method of diagnosing schizophrenia or a schizophrenia spectrum disorder in an individual by determining the presence of a variant of a miRNA gene or allele. The miRNA may be any miRNA known or found to affect or influence schizophrenia and such activity may be determined via the functional assay described herein, which was used to determine the function of miRNAs disclosed here, or by another method that allows determination of the function of miRNA as it relates to schizophrenia. The miRNAs may be one or a combination of more than one of the following: miRNA alleles consisting of let-7f-2, mir-18b, mir-505, mir-502, mir-188, mir-325, mir-660, mir-509-3, mir-510, mir-421, mir-934, and mir-450a-2.
Generally, the method, which can be used to either determine an increased risk for schizophrenia or propensity thereto in a subject or to diagnose schizophrenia existing in a subject, comprising the steps of analyzing the miRNA in a sample obtained from the subject and determining the presence of a variant of the miRNA wherein the presence of said variant is indicative of an increased risk for developing schizophrenia or propensity thereto in the subject. Certain variants that the present invention has determined influence on schizophrenia are let-7f-2, mir-18b, mir-505, mir-502, mir-188, mir-325, mir-660, mir-509-3, mir-510, mir-421, mir-934, and mir-450a-2. The biological samples may be any sample from a subject containing mRNA, including blood, serum, plasma, saliva, urine, or tissue samples. The mRNA is isolated using any method, including the method using RNA STAT-60 described herein. The presence of RNA may be determined via any reliable method, including the use of Northern blots.
The term “miRNA” includes miRNA molecules, variants, and analogs thereof, miRNA precursor molecules, and DNA molecules encoding miRNA or miRNA precursor molecules.
Further, therapeutic applications of the miRNA described herein may be used as modulators or targets of schizophrenia and related spectrum disorders. In general, the nucleic acid molecules may be used as a modulator of the expression of genes which are at least partially complementary to said nucleic acid and are sufficient to agonize or antagonize the activity as desired. miRNA may be used as raw materials for the manufacture of sequence-modified miRNA molecules to adjust target specificity or target binding efficacy. miRNA may also be used in an attempt to alter a differentiated cell line back into a stem cell or another cell type to avoid the onset or symptoms of schizophrenia. For therapeutic applications, miRNA may be administered alone or as part of a composition, which comprises the miRNA (or different miRNAs) and a pharmaceutically acceptable carrier. The therapeutic methods may be carried out by known methods, wherein a nucleic acid is introduced into a desired target cell in vitro or in vivo using gene transfer techniques include calcium phosphate, DEAE-dextran, electroporation, microinjection, viral vectors, and commercial liposomes. The composition may be in any suitable form such as an injectable solution, a cream, ointment, tablet, suspension, or pill and may be administered in any therapeutically effective manner, such as parenterally, intravenously, orally, or nasally.
Methods of screening and tests for the bioactivity of a miRNA are disclosed. The test determines altered functionality of a miRNA gene or allele variant. The test uses functionality activity comparisons. In one embodiment, polyadenylated RNA polymerase II transcripts, termed “primary miRNAs” (pri-miRNA) is and variant or putative variant miRNA is isolated. Then, the pri-miRNA and variant miRNA are co-transfected with an si target and an mi target. Preferably, each target has its own reporter. Then, the functionality of the variant is determined using miRNA and siRNA assays. Changes in the level of expected repression of the si and mi reporters (either reduced or enhanced repression) in transient transfection assays indicates altered functionality of the miRNA gene or allele variant. The variants may be the result of one or more of a SNP, point mutation, deletion, insertion, frameshift, or translocation. The screen may be used to test whether the miRNA variant indicates predisposition to or incidence of schizophrenia.
For the methods of diagnosing, assessing susceptibility, and/or treating schizophrenia involving the identification and/or observation of microRNAs (miRNA) and preferably, variant miRNA are provided and identified as follows: Individual microRNAs (miRNAs) effect moderate down-regulation of gene expression, typically by two-to four-fold. Micro RNAs alleles associated with schizophrenia and schizophrenia spectrum disorders were identified. The miRNA genes were amplified in patients and controls. Ultra-rare variants in the precursor or mature miRNA were identified in distinct miRNA genes in males with schizophrenia and one ultra-rare variant was identified in a control sample (8/193 versus 1/191, p=0.019). These variants were not found in an additional 7,197 control X-chromosomes. Functional analyses of ectopically expressed copies of the variant miRNA precursors demonstrate loss of function, gain of function and altered expression levels. The present invention also provides methods for selecting a preferred therapy for a particular subject or group of subjects or individuals at risk for or suffering from schizophrenia or psychosis by use of miRNAs.
miRNA genes are scattered among all the chromosomes in humans except for the Y chromosome. They primarily derive from intronic or exonic capped, polyadenylated RNA polymerase II transcripts, termed “primary miRNAs” (pri-miRNA). It is estimated that approximately one-third of human protein coding genes are post-transcriptionally controlled by miRNAs. The mature miRNAs are used to guide miRNA-induced silencing complex (“RISC”) to the complementary sequences in the 3′UTR of targeted transcripts. The result is site specific mRNA cleavage when the pairing is nearly complete (mostly in plants, rare in animals) or translational inhibition when imperfect base-pairing occurs (mostly in animals).
Translational suppression and mRNA degradation, modes by which mammalian miRNAs regulate gene expression, do not require complete complementarity between the miRNA and target. All that is required to reduce protein levels of the target is Watson-Crick base pairing between seven consecutive nucleotides in the target mRNAs 3′ UTR and nucleotides 2-7 or 2-8 (the “seed sequence”) at the miRNA's 5′ end. The critical role played by the “seed sequence” in the majority of miRNA/mRNA interactions implies that a single nucleotide change in the seed sequence, or shift of the processing sites during biogenesis of the miRNA/miRNA* duplex could result in a novel miRNA with alternated target-spectra. Therefore, both the 5′end of the mature miRNA that is generated from the 5′ arm of the pre-miRNA (5p) by Drosha, and the 5′ end of the mature miRNA that is produced by Dicer from the 3′ arm of the premiRNA (3p), will be under strong selective pressure to be highly conserved. (Drosha is an RNase III enzyme that initiates miRNA processing. Dicer is an RNase III endoribonuclease that cleaves miRNA into small interfering RNA (siRNA)). The sequence preceding the 5′ end or trailing the 3′ end of the pre-miRNAs form an  ̃11 bp long imperfect stem which is recognized by DGCR-8 as part the required structure for Drosha cutting. For most miRNAs/target combinations, a single nucleotide change in the seed sequence or any base shift during the Drosha/DGCR8 or Dicer/TRBP processing step can result in altered function or creation of a novel miRNA. The terminal loop is also important for Dicer/TRBP complex binding as well as for other protein binding. Sequences outside the seed in the mature miRNA sequence can also impact the strength of inhibition as well as the spectra of targeted transcripts.
Mature miRNAs are generated by a two-step processing mechanism (FIG. 1). PrimiRNAs are first processed to “hairpin-like” partially duplexed precursor miRNAs (premiRNA) in the nucleus. Aside from a small group of pre-miRNAs that are generated through mRNA splicing/debranching machinery termed the “miRtron pathway”, most pre-miRNAs are processed from pri-miRNAs by the nuclear RNase (ribonuclease) III Drosha which partners with the RNA binding protein DGCR8 (the DiGeorge syndrome critical region gene 8). Pre-miRNAs are typically 55-80 nt in length and are exported to the cytoplasm by exportin-5/RAN-GTP. The pre-miRNAs are processed into  ̃21-22 nt long miRNA/miRNA* duplexes by RNase III Dicer, which partners with the RNA binding protein TRBP (TAR RNA binding protein). The production of miRNA/miRNA* duplexes is an essential step in miRNA biogenesis and precisely defines the ends of the mature miRNAs for preferential loading of the guide strand. The choice of the guide strand is dependent in part on the thermodynamic end properties of the duplex, with the least thermodynamically stable 5′ end usually being chosen as the guide strand while the other strand, labeled miRNA*, is usually degraded. Most recently, the fates of the miRNA guide and miRNA* strands have been shown to be tissue dependent with both strands being functionally active under specific conditions. Argonaute-mediated loading into the processing complex can increase the bias of strand loading, and RNA binding proteins can selectively block the processing of primiRNAs.
The mature miRNAs are incorporated into a ribonucleoprotein complex, the RNA-induced silencing complex (RISC), which contains at least Dicer, TRBP and Ago2 [Hammond et al., 2000; Mourelatos et al., 2002; Hutvagner and Zamore, 2002]. In RISC, miRNAs can mediate down regulation of target gene activity by translational inhibition in animals [Zeng et al., 2002]. RISC can cleave, degrade or suppress translation of target mRNAs depending on the position and extent of the complementarity between miRNAs and target mRNAs. Given the complexity of protein interactions and the number of processing steps required to produce a functional miRNA, point mutations affecting any of these steps could dramatically impact the downstream function of these miRNAs.
The hairpin structure guided miRNA processing, the thermodynamic influences on strand loading, and the base pairing requirements for miRNA/mRNA interaction indicate that single nucleotide polymorphisms (“SNPs”) in miRNA genes affect miRNA biogenesis and function. Similarly, SNPs in the miRNA target also affect miRNA function. To clarify possible confusions in terminology with respect to SNPs, the terms “miR-SNP” refers to the variation that occurs in the miRNA gene sequence, and “miR-TS-SNP” refers to SNPs that occur in the miRNA target site (TS) or binding site. Since one miRNA can have multiple targets, miR-SNPs would exhibit more profound and broader biological effects than miR-TS-SNPs.
The roles that sequences flanking the pre-miRNA play in miRNA processing has been thoroughly studied. MiR-SNPs in miR-125a and Kaposi's sarcoma-associated herpes virus encoded miR-K5 impaired miRNA processing by the Drosha/DGCR8 complex. MiR-196a2-SNP (rs11614913) in the mature miR-196a2 was associated with a significantly decreased rate of survival in individuals with non-small cell lung cancer, and an association of rs11614913 with enhanced processing of mature miR-196a. MiR-146a-SNP (rs2910164) within the pre-miR-146a sequence reduced both the amount of pre-and mature miR-146a, and affected the Drosha/DGCR8 processing step. MiR-196a2-SNP, miR-146a-SNP, miR-149-SNP (rs2292832) and miR-499-SNP (rs3746444) are each associated with increased breast cancer risk. MiR-146a-SNP was associated with papillary thyroid carcinoma, breast/ovarian cancer, and hepatocellular carcinoma. Each of the above are examples of SNPs created by changes in DNA coding sequences, but miRNAs can also be post transcriptionally modified, such as by RNA editing via ADAR. A to I edited pre-miR-151 blocks its processing by Dicer/TRBP. ADAR edited pri-miR-142 was more easily degraded by Tudor-SN. Edited miR-376a-5p within the middle of the “seed” region alters the set of targets regulated by this miRNA. A survey of RNA editing of miRNAs from ten human tissues implies RNA editing of miRNA happens quite often and it is a mechanism to increase the diversity of miRNAs and their targets.
Mutant or post transcriptionally edited miRNAs can result in alterations of processing and function. Hence, SNPs that occur in sequences downstream or upstream of the pre-miRNA, sequences in the terminal loop of premiRNA, and sequences in the miRNA and miRNA* duplexes may also play important roles in miRNA biogenesis and function (FIG. 1).
In the present experiments, X-linked miRNA genes from patients with diagnosed schizophrenia or autism were analyzed and compared with a gene pool analysis consisting of over 7,000 chromosomes from normal individuals (i.e., individuals without schizophrenia or autism). Twenty-four different point mutations have been determined in either the mature miRNA sequences or the precursor regions for sixteen different X-linked miRNA genes. The present invention addresses the effects on miRNA generation and function generated by SNPs in X-linked miRNAs.
Of the tested miRNA-SNPs, one variant results in elevated levels of the mature miRNA sequence relative to the wild type, several variants result in reduced levels of the mature miRNA sequence relative to wild type, and another variant results in the generation of a novel miRNA due to an alteration in the Drosha and/or Dicer processing sites. This latter miRNA-SNP also has an alteration the strand loading bias relative to the wild type version. A single base alteration even outside of the mature miRNA sequence can have profound consequences on miRNA generation and function.
Most miR-SNPs are located in clusters and some of them, such as mir-510 and miR-509, rapidly evolved in primates. Table 4 shows variants with 509-2 and Table 5 shows variants without 509-2. One clear example is the generation of miR-509-3-5p by the deletion of an ‘A’ from miR-509-5p, which is processed from miR-509-1 or 2 (FIG. 2; SEQ ID NOS: 1-43). There are three copies of miR-509, miR-509-1 and miR-509-2 that produce the same mature miRNAs, while miR-509-3 produces a different 5p product. Most likely, the miR-509-3-5p was created by the deletion of an ‘A’ from miR-509-5p. A high percentage of an ATG insertion was also observed in the 5′ end of miR-509-1-3p. This insertion may affect both 5p and 3p processing. Eventually, under the selective pressure to target different mRNAs or to target with different specificities, the three copies of miR-509 may have developed into different family members with the same seed, like the let-7 family, or different miRNAs in the same cluster, like the miR-25-93-106b cluster. The miR-509 structure is more flexible, as there are no significant differences in the processing or function among three different miR-509-3 variants (FIG. 3, SEQ ID NOS: 44-47).
Many factors contribute to differences in miRNA expression profiles, including transcriptional regulation, post transcriptional miRNA processing, the stability of the pri-miRNA or pre-miRNA, and pre-miRNA export. The existence of miRNA targets may also result in miRNA stabilization because of engagement in miRISC. Different miRNA profiles are a consequence of subtle genetic changes in pre-miRNAs and their immediate flanking sequences.
Previous in silico studies from Bentwitck and Zhang show that miRNA family expansion during primate evolution may have occurred through tandem duplications. Copy number variations and high rates of gene conversion in the newly emerged miRNAs in primates may have resulted in production of novel miRNAs with more specialized functions. As a result, gene conversion may be a major mechanism in the biogenesis of miRNAs during evolution, especially in clusters of miRNAs, homologues, or miRNA families. Finally, some of the SNPs characterized by altered processing or abundance may play significant roles in disease development and progression.
The present invention is based on the novel finding that structural variants in the microRNA genes can predispose an individual to schizophrenia. In particular, microRNA mutant alleles associated with schizophrenia were identified.
Genomic sequence information of DNAs derived from patients diagnosed with schizophrenia was compared to that of large sets of normal patient samples. In certain experiments, the comparison focused on X-linked genes in male patients. From these analyses, several mutations in microRNA precursor and mature coding sequences were identified, some of which exhibit functional impairments in our cell culture based testing system. miRNA genes are transcribed by RNA polymerase II as long primary transcripts (pri-miRNA) containing stem-loop or “hairpin” structures  ̃60 nt in length.
The present invention identifies ultra-rare variants in the precursor or mature miRNA, each in distinct miRNA genes in males with schizophrenia and one ultra-rare variant was identified in a control (8/193 versus 1/191, p=0.019). These variants were not found in additional 7,197 control X-chromosome alleles using gene pool analyses. Functional analyses indicated that some of these variants display altered regulatory function consistent with dominant inheritance. The altered functions or defects in processing of the pre-miRNAs in the mutant alleles detected in our analyses indicate that these microRNAs may contribute to the development of schizophrenia. It is relevant to note that each microRNA can potentially regulate dozens, and perhaps even hundreds of different transcripts during development, so even subtle defects in activity can have profound effects on development of the nervous system.
Identified targets of the miRNAs in which ultra-rare variants were found are listed in Table 6. Such targets include neuregulin 1 (NGR1), Disrupted in schizophrenia 1 (DISC1) and Regulator of G-protein signaling 4 (RGS4). Defects in miRNAs altering the interactions between miRNAs and their mRNA targets thus likely contribute to schizophrenia.
To explore the role of miRNAs in schizophrenia, 59 microRNA genes on the X chromosome were analyzed in 193 males with schizophrenia spectrum disorders. The miRNA genes were amplified in patients and controls. Ultra-rare variants in the precursor or mature miRNA were identified in distinct miRNA genes in males with schizophrenia and one ultra-rare variant was identified in a control sample (8/193 versus 1/191, p=0.019). These variants were not found in an additional 7,197 control X-chromosomes. Functional analyses of ectopically expressed copies of the variant miRNA precursors demonstrate loss of function, gain of function and altered expression levels. These results provide the first statistically significant data linking microRNA gene dysfunction with schizophrenia.
EXAMPLES
Materials and Methods
Samples
All 193 male Caucasian schizophrenic patients met criteria for the disease as defined by the Diagnostic and Statistical Manual, Fourth Edition, Revised (DSM-IV-R). The majority of patients were ascertained through state mental institutions in Minnesota. The male controls were Midwest Caucasians with no known history of psychiatric illness.
PCR Amplification and Sequencing
The genomic sequence and adjacent flanking sequences of the precursors of 59 X-linked microRNA genes (miRBase 10.1, http://microrna.sanger.ac.uk/sequences/) were amplified and sequenced with the ABI model 3730 sequencer. Sequences of the primers are listed in Table 2 (SEQ ID NOS: 97-215). The nucleotide alterations were analyzed with Sequencher softwareTM (Gene Codes, Ann Arbor, Mich.). Mutations were confirmed by reamplifying from genomic DNA and sequencing in the opposite direction.
Gene Pool Analysis
Genomic DNA samples from 7,197 control X-chromosomes were ascertained from a Midwest population-based sample of overwhelmingly Caucasian individuals from Minnesota and a more ethnically and geographically diverse sample of hemophilia families.
The concentration of individual DNA samples was estimated by both UV spectrophotometry and agarose gel electrophoresis with diluted quantitation standards. Samples were diluted to 200 ng/μl and combined into pools of 10, 30 and 100 samples. The concentration of each sample per μl in the pool is 20 ng, 6.7 ng and 2 ng, respectively.
Allele-specific amplification assays were developed for each case or control cohort-specific variant. The specificity and selectivity of each assay were determined utilizing negative and positive controls spiked within gene pool samples.
Cell Lines and Plasmids
HEK293, Hela, NIH-3T3 cells were purchased from ATCC and maintained in high glucose (4.5 g/l) DMEM supplemented with 2 mM glutamine, 10% FBS, and 2 mM Penicillin/Streptomycin. Transfections to HEK293, NIH-3T3 and Hela cells were performed with Lipofectamine 2000 (Invitrogen) in duplicate in 24-well plate formats when cells are at 70-80% confluency.
Cell Based miRNA Processing Test
Primary miRNA expression plasmids and reporters bearing either fully complementary or seed sequence complements to the miRNAs were co-transfected into HEK293 cells. Dual-reporters (expressing both Firefly and Renilla luciferase) carrying the miRNA fully complementary sequences (si reporter) in the 3′ UTR of the Renilla transcript were used to validate the ability of cloned primary miRNA expression plasmids to produce functional, mature miRNAs. Dual-reporters carrying the partially complementary sequence (mi reporter: mis-matched at position 11 to 13 and the last two nts in miRNA/mRNA duplex) of a miRNA in the Renilla 3′UTR were used to measure the strength of translational repression from the corresponding miRNA.
In order to express the pri-miRNAs, the stem-loop sequences were retrieved from miRBase 10. The stem-loop sequence, plus flanking sequences extending over 100 bases in both directions, was PCR amplified from genomic DNA. A miRNA expression vector was constructed by first cloning the human Pol II U1 promoter upstream of a multiple cloning site in the Bluescript SK plasmid to create SK-U1. Secondly, the U1 transcriptional termination sequence was cloned downstream of the MCS of SK-UL to create the fU1-miR miRNA expression vector. The primary miRNA was cloned into the Xho I and BamH1 sites of fU1-miR. miRNA variants were cloned in the same manner as the wild type miRNAs from patient DNA when available. If samples were no longer available, the QuikChange II site-directed mutagenesis kit was used to create mutants within the wildtype expression constructs. All clones were sequenced to confirm the normal miRNA and mutant forms.
For si reporters, all miRNAs and their homologous mutant target sequences were designed as fully complementary to the mature miRNA sequence. The oligos for the two strands were inserted into the psiCHECK Xho I/Spe I or XhoI/Not I digested reporter 3′ UTR of the Renilla luciferase gene. All target clones were verified by sequencing. For mi reporters, all the inserted sequences in the reporter 3′ UTR of the Renilla luciferase gene were designed with bulges at positions 11 to 13 and were unpaired for two nucleotides at the 3′ end of the miRNA.
About 1×104 HEK293 cells per well in 500 microliters of growth media were plated in 24 well plates one day prior to transfection. The cells were at 70-80% confluency at the time of transfection. Each well was transfected with 5 ng reporter, 100 ng miRNA expression constructs (1:20 ratio, 1:5 ratio was used if the knockdown of the si target was >95%, then 25 ng of miRNA expression plasmid and 75 ng stuffer Blue-script SK were used) and 1 ul Lipofectomine 2000. Forty eight hours post transfection, the cells were lysed with 100 μl Passive lysis buffer (Promega) and luciferase levels were analyzed from 20 μl lysates using the Dual Luciferase reporter assay (50 μl of each substrate reagent, Promega) on a Veritas Microplate Luminometer (Turner Biosystems). Changes in expression of Renilla luciferase (target) were calculated relative to Firefly luciferase (internal control) and normalized to the miRNA expression vector control fU1-miR.
Point mutations were created with the QuikChange site-directed mutagenesis kit II (Stratagene) following the protocol included in the kit. Mutations were confirmed by sequencing.
RNA Isolation
RNA was isolated with RNA STAT-60 (Tel-Test Inc.) and 20 μg total RNA was loaded into a denaturing 12.5% SDS-PAGE gel. A DNA oligonucleotide probe complementary to the mature miRNA sequence was labeled with γ-32P-ATP.
Northern Blot
Two different transfections were performed in HEK 293 cells to detect processing of expressed pri-miRNA in vivo. One transfection contained pri-miRNA expression constructs alone, while the other was co-transfected with 25/27-mer synthesized siRNA duplex. Northern blots were performed with RNAs from both transfections. U2A or U6 snoRNA were used as the RNA loading controls and co-transfected S1 siRNA was used as the transfection control. 20 μg total RNA was loaded on a 12.5% PAGE-8M urea denatured gel. Gels were transferred to a Hybond-N+ (Amersham Pharmacia biotech, positive charged) membrane. DNA probes were used for all Northern blots, and hybridizations were carried out in PerfectHybTM Plus hybridization buffer (Sigma) for 16 hours (Table 7; SEQ ID NOS: 216-337). The blots were washed once for 10-30 minutes with 6×SSPE/0.1% SDS, followed by two washings with 6×SSC/0.1% SDS for 10-30 minutes each.
Mature miRNA Cloning
For small RNA cloning, small RNAs below 40 nt were fractionized by a flashPAGETM Fractionator System. Small RNAs were first polyadenylated, then ligated with a 5′ RNA adaptor. The 5′-adaptor-added polyadenylated small RNAs were RT-PCR amplified and the products cloned. Dot-blot hybridizations were used to identify positive clones. The positively hybridizing clones were sequenced to verify the processed mature miRNA sequences.
Dot-Blotting
Bio-Rad membranes were cut to the same size as the bottom of Petri-Dish plates. The membranes were laid on the colonies for 20 seconds or until they were wet, then lifted and washed twice in 0.5N NaOH for 5 minutes each (The plates were put back into the 37° C. incubator for 5-6 hours to preserve the colonies). Next, the membranes were washed twice in 0.5M Tris-HCl, pH 7.5 for 5 minutes each. Then, the membranes were washed twice in 6×SSC/0.1% SDS for 5 minutes each. Finally, the membranes were washed in 95% EtOH for 5 minutes and dried between two sheets of Whatman paper. All washings were performed at room temperature. Just before hybridization, membranes were soaked in 6×SSPE/0.1% SDS twice for 5 minutes each. The probe and the temperature of hybridization, and the washing condition were the same as those for the Northern blots above. The only difference was that the hybridization duration was one hour. Usually the signal is strong enough to detect after the blots are exposed to film for 5-6 hours. Positive colonies were located and plasmid DNAs were made for sequencing.
Results
Fifty-nine microRNA genes on the X chromosome were analyzed in 193 males with schizophrenia as well as 191 controls. Ultra-rare variants in the precursor or mature miRNA were identified, each in a distinct miRNA gene, in males with schizophrenia and one ultra-rare variant was identified in a control sample (8/193 versus 1/191, p=0.019, Fisher's exact test) (Table 1a). These variants were not found in an additional 7,197 control X-chromosomes using gene pool analyses.
Five cohort-specific variants, one in a patient and four in controls, were found in the gene pool analyses at a frequency greater than 0.02% (p=0.21, Fisher's exact test) (Table 1b). The schizophrenia patient's variant occurred in the mature miRNA mir509-3-5p. Of the four cohort-specific variants identified in control samples, three were found in the mature miRNAs, while one was found in the precursor.
Five common variants in miRNA precursors were found in both patients and controls with similar frequency (Table 3).
Twenty-four variants within pre-miRNAs and the immediate flanking regions were identified (Table 8). They are further characterized into four groups based on the location within the hairpin structure (FIG. 1): SNPs in the basal segment (miR-421-G/A, miR-888-A/C ), SNPs in the lower stem (miR-421-G/A, miR-502-C/G, miR-505-C/T, miR-509-2-G/T, miR-510-G/A), SNPs in the miRNA/miRNA* (let-7f-2-5p-G/A, miR-188-3p-C/T, miR-325-3p-G/A, miR-450a-2-T/C, an ‘A’ deletion in miR-509-2-5p, miR-509-3-5p-G/A, miR-509-3-5p-C/G, miR-509-3-3p-C/T, miR -510-T/C, miR-660-5p-C/T, miR-890-3p-G/C, miR-892b-3p-T/C, miR-934-T/G), SNPs in the terminal loop (miR-18b-A/G, -G/A, a TGA insertion in the terminal loop of miR-509-1, miR-891 b-C/G).
To assess the functional consequences of the point mutations, pri-miRNAs and mutant versions of each miRNA were co-transfected with their corresponding si and mi targets (using materials and methods described above). Three or more transfections were performed, with duplicates in each transfection. At least four of the variants identified, each having a point mutation in the mature coding region, have altered function (miRNA let-7f-2, miR-188, miR-660, and miR-509-3). The novel, ultra-rare variants in the mature miRNAs were predicted to have altered target specificity.
In order to evaluate the consequences of these point mutations, a novel assay was developed to monitor the processing and function of both strands of the miRNAs by employing both miRNA (seed sequence complementarity) and siRNA (fully complementary) assays. The functional assays were carried out in transient cotransfections of expressed pri-miRNA with target sequences in the 3′UTR of the Renilla luciferase encoding transcripts. At least six of the variants, miR-502-C/G (FIGS. 2 & 4, rare variant and associated with schizophrenia), miR-510-G/A (FIGS. 2 & 5), miR-510-T/C (FIGS. 2 & 6, rare variant and associated with schizophrenia), miR-890-C/G (FIGS. 2 & 8), miR-892b-T/C (FIGS. 2 & 9) and miR-934-T/G (FIGS. 2 & 10), showed reduced or enhanced repression of the ‘si’ and ‘mi’ reporters in transient transfection assays. For each of these variants, Northern blotting was performed to detect the effects of the SNP on processing of the pre and mature miRNAs. Mature miRNA cloning was performed on miR-510-G/A, miR-890-C/G and miR-934-T/G to study affect of the SNP on the maturation of the miRNAs. While all three SNPs apparently affect the miRNA processing, only the miR-934-T/G transversion also altered the Drosha or Dicer excision sites which also resulted in changing the strand bias for RISC loading relative to the wild type miRNA (FIG. 16).
Several examples in which miR-SNPs resulted in reduced processing were observed. Four of the six observed miR-SNPs, miR-502-C/G, miR-510-T/C, miR-890-C/G and miR-892b-T/C, produced less mature miRNA. While the 502-G/C SNP occurs two nts before the 5′end of 502-5p, the other three SNPs all occur in the mature 3p product.
Let-7f-2/7f-2*
A single base substitution G>A was identified in the mature miRNA of let-7f-2 at position 11. This site is highly conserved from C. elegans through human. This variant is not present in 7,197 control X-chromosomes (Table 1a). To examine the possible functional consequences of this mutation, the wild type and mutant variants were tested against its corresponding si and mi target sequence. The results obtained with these analyses demonstrate that the mutant sequence can down regulate its si sequence (FIG. 12, bar #6), but its knockdown of the let-7f si sequence was dramatically reduced (FIG. 12, bar #3). On the other hand, the let-7f knockdown of the mutant si-target remained unperturbed (FIG. 12, bar #5). These results demonstrate that the mutant produces a stronger siRNA phenotype than the wild type miRNA with the cognate complementary targets. On the other hand, the variant elicits a weaker miRNA phenotype than the wild type.
miR-18b/18b*
Variant miR-18b/18b*-m has an A to G mutation at the 5th nucleotide following the last base of the mature sequence, which is also in the predicted loop structure (FIG. 13, SEQ ID NO: 96). This sequence difference may affect processing and/or stability since there is a reduction in the level of target knockdown activity when compared to wild type in the si-target (FIG. 13, bar #2 vs #3) and the mi-target assays (FIG. 13, bar #5 vs #6). In contrast, the function of the miR-18* strand does not appear to be affected by this mutation (FIG. 13 bar #7 vs 8 and Bar #9 vs 10).
miR-502-5p/3p
Variant miR-502-5p/3p-m has a C to G transversion at the 3rd nucleotide (“nt”) upstream of the mature miR-502-5p sequence (FIG. 4, SEQ ID NOS: 44-47). This mutation will produce a bulge which changes the structure of the stem of the precursor miRNA (FIG. 2). Most likely, this structural change will affect the site of Drosha cleavage in producing pre-miR-502, therefore, both the 5p and 3p products should be affected. Reduced target knockdowns were observed in transfection assays (FIG. 4, bar #2 vs 3, #5 vs 6 and #8 vs 9). The impaired functional activity of the variant was supported by Northern blot analyses, as the production of pre-miR-502 and mature 502-5p/3p was both reduced. (FIG. 4).
miR-188-5p/3p
Variant miR-188-5p/3p-m has a C to T (U) transition at the 7th nt of the mature miR-188-3p within the seed sequence (FIG. 14). This variant results in a change of G:C to G:U pairing in the seed sequence. In our assay system, the effect of the variant is not dramatic (FIG. 14). Nevertheless, this variant will create a seed sequence where this position can pair with an A, thus potentially affecting the expression of new target sequences with a matched seed sequence.
miR-505/505*
Variant miR-505/505*-m has a C to T (U) transition at the 6th nt (relative to the 5′ end of the upper strand of the mature miR-505). This variant is distal to both Drosha and Dicer cleavage sites. Functional testing of this mutant (FIG. 15) revealed little difference when compared with the wild type miRNA.
miR-510
Variant miR-510-m has a T (U) to C transition in the seed of the predicted miR-510* (3p) (FIG. 6, SEQ ID NOS: 56-65). Transfection assays show processing of the miR-510-3p product and its ability to knockdown the corresponding si target sequence (FIG. 6, bar #8). The T (U)/C mutation produces a pre-miR-510 with much less activity for both 5p and 3p products (FIG. 6, Bar #2 vs 3, bar #5 vs 6 and bar #7 vs 8). This mutation most likely affects the structure of the pre-miR-510, as it affects the function of mature miR-510 on both strands. Northern blot analyses confirm that the production of both pre-miR510 and miR-510-5p/3p were reduced (FIG. 7).
miR-509-3
This miRNA variant has a C to T (U) transition at the 13th nucleotide of the mature miRNA (FIG. 3, SEQ ID NOS: 44-47). Our functional assays show that this mutation has a weak effect on the processing of the mature miRNA (FIG. 3). This base change could affect the endogenous targets down regulation as the 13th position is important for 3′end base pairing of miRNAs and mRNAs.
miR-660
Variant miR-660 has a C to T (U) transition at the 15th position of the mature miRNA (FIG. 11). The functional assay data shows it has little effect on the processing of the miRNA (FIG. 11). The base change could affect the natural targeting functions of the miRNA since this position affects the 3′ end base pairing of miRNA/mRNA.
miR-325
Variant miR-325-m has a G to A transition on the miR-325* strand. Interestingly, functional assays with the wild type and mutant variants did not generate knockdowns of the si target. This miRNA was originally cloned in a murine system and therefore there is no data demonstrating that the human homologue can be expressed and processed in HEK293 cells.
miR-890-C/G
miR-890 3p reporter transfection assays show its ability to knockdown the corresponding ‘si’ target sequence. Transfection and Northern blotting data show that the C/G transversion in miR-890 affects the production of the 3p strand, but not the 5p strand (FIG. 8, SEQ ID NOS: 66-77), with the production of 3p being reduced. Because the C/G transversion may be at the Drosha cleavage site, the exact sequence of its 3p products by miRNA cloning was defined. These data show that the cutting sites for both the 5p and 3p products were not altered by this miR-SNP. The 5p and 3p mature sequences were the same for both the wild type and mutant. Dot blotting analyses (FIG. 16) also show that more clones of the miR-890 than miR-890-C/G for the 3p probe were obtained, which is consistent with the transfection and Northern blot data (FIG. 8).
miR-892b-T/C
Like miR-510-T/C and miR-890-C/G, this SNP occurs in the 3p of miR-892b, but miR-892b-3p is listed in miRBase. Although there are currently no published data which support the existence of miR-892b 5p products, our 5p reporter transfection assays show its ability to knockdown the corresponding ‘si’ target sequence. Transfection and Northern blotting data show that the T/C transition in miR-892b affects the production of both 5p and 3p strands (FIG. 9, SEQ ID NOS: 78-87).
miR-510-G/A
A G/A transition in pri-miR-510 enhanced the production of miR-510-5p and -3p (-3p is miR-510*) (FIG. 5). The G to A transition occurs at the 4th nt upstream of the 5′ end of the mature miR-510-5p (FIG. 2). Variants at this position may affect Drosha processing of this substrate since it may provide a more stable stem preceding the mature miRNA sequence. The reporter transfection assay data show that the siRNA activity of the mutant is markedly higher than the wild type (FIG. 5A). Northern blot data show that the production of both pre-miR-510 and mature miR-510-5p/3p are increased (FIG. 5B). Dot blotting data also revealed more clones of the miR-510-G/A than miR-510 (FIG. 16A). The miRNA cloning data show that the generation of the 5p product is the same for both the wild type and mutant, and apparently this SNP does not affect the Drosha cutting sites. However, colonies hybridizing to the 3p probe were not observed in either the wild type or SNP blots.
miR-934-T/G
SNPs could alternate Drosha or Dicer excision sites since their cutting sites are structure based and not sequence based. Variant miR-934-T/G occurs at the first nucleotide of the miR-934-5p (FIG. 2), which is also the Drosha processing site. Because the variant occurs at the 5′ end and the base of the 5′end plays an important role in strand selection into miRISC, the T/G transversion of this variant is particularly significant. Transfection and Northern blot results show that this SNP affects the production of both strands (FIG. 10, SEQ ID NOS: 88-95).
First, the transfection assay shows that repression of the 5p reporter is reduced by the SNP, and Northern blots confirmed the reporter assay results (FIG. 10A). Second, the length of the 5p product seems to be increased in the Northern blot. The most dramatic changes are in the 3p product. Transfections show that repression of the 3p reporter by the SNP is increased, and Northern blots show that the variant produces more 3p than wild type. Thus, the guide strand and passenger strand in miRISC are inverted in the wild type versus mutant miRNAs. Cloning also yielded more 5p wild type clones and more mutant 3p clones. The cloning data also show that the production of 3p is altered, with both the Drosha and Dicer cutting sites being offset by one nucleotide from the wild type, resulting in a different 3p product. This not only produced a novel miRNA, but it also affected the strand selection in miR-934/miR-934*. The wild type miR-934-5p starts with a U and is most likely selected as the predominant guide strand due to the lower thermodynamic stability of the 5′ end. The U/G transversion changes the first nucleotide of the 5p product to ‘G’, which affected the Dicer cutting site, moving it back one nucleotide from the original ‘G’ to an ‘A’. Thus the 3p product in the mutant has a lower 5′ end thermodynamic stability and this is probably responsible for altered guide strand selectivity, which is consistent with the reported requirements for asymmetric strand loading.
The foregoing examples and methods of the invention are illustrative only and are not intended to be limiting of the invention in any way. Those of ordinary skill in the art will recognize that various modifications of the foregoing are within the intended scope of the invention.
All references cited are incorporated in their entirety as though fully set forth herein.
References


    1. Abelson, J. F. et al. Sequence variants in SLITRK1 are associated with Tourette's syndrome. Science 310, 317-20 (2005).
    2. Ambros V. 2003. MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell 113:673-676.
    3. Arisawa, T. et al. A polymorphism of microRNA 27a genome region is associated with the development of gastric mucosal atrophy in Japanese male subjects. Dig Dis Sci 52, 1691-7 (2007).
    4. Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281-97 (2004).
    5. Bentwich, I. et al. Identification of hundreds of conserved and nonconserved human microRNAs. Nat Genet 37, 766-70 (2005).
    6. Berezikov, E., Chung, W. J., Willis, J., Cuppen, E. & Lai, E. C. Mammalian mirtron genes. Mol Cell 28, 328-36 (2007).
    7. Blow, M. J. et al. RNA editing of human microRNAs. Genome Biol 7, R27 (2006).
    8. Bottema C D K, Sommer S S. 1993. PCR amplification of specific alleles: rapid detection of known mutations and polymorphisms. Mutat Res 288:93-102.
    9. Bushati, N. & Cohen, S. M. microRNA functions. Annu Rev Cell Dev Biol 23, 175-205 (2007).
    10. Cai, X., Hagedorn, C. H. & Cullen, B. R. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. Rna 10, 1957-66 (2004).
    11. Chen, W. et al. Mutation screening of brain-expressed X-chromosomal miRNA genes in 464 patients with nonsyndromic X-linked mental retardation. Eur J Hum Genet 15, 375-8 (2007).
    12. Chendrimada, T. P. et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436, 740-4 (2005).
    13. Chu, C. Y. & Rana, T. M. Translation Repression in Human Cells by MicroRNAinduced Gene Silencing Requires RCK/p54. PLOS Biol 4, e210 (2006).
    14. Clop, A. et al. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep. Nat Genet 38, 813-8 (2006).
    15. Croce C M, Calin G A. 2005. miRNAs, cancer, and stem cell division. Cell 122:6-7.
    16. Diederichs, S. & Haber, D. A. Sequence variations of microRNAs in human cancer: alterations in predicted secondary structure do not affect processing. Cancer Res 66, 6097-104 (2006).
    17. Du T, Zamore P D. 2005. microPrimer: the biogenesis and function of microRNA. Development 132:4645-4652.
    18. Duan, R., Pak, C. & Jin, P. Single nucleotide polymorphism associated with mature miR-125a alters the processing of pri-miRNA. Hum Mol Genet 16, 1124-31 (2007).
    19. Filipowicz, W., Bhattacharyya, S. N. & Sonenberg, N. Mechanisms of posttranscriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9, 102-14 (2008).
    20. Giraldez A J, Cinalli R M, Glasner M E, Enright A J, Thomson J M, Baskerville S, Hammond S M, Bartel D P, Schier A F. 2005. MicroRNAs regulate brain morphogenesis in zebrafish. Science 308:833-838.
    21. Gottwein, E., Cai, X. & Cullen, B. R. A novel assay for viral microRNA function identifies a single nucleotide polymorphism that affects Drosha processing. J Virol 80, 5321-6 (2006).
    22. Griffiths-Jones, S., Saini, H. K., van Dongen, S. & Enright, A. J. miRBase: tools for microRNA genomics. Nucleic Acids Res 36, D154-8 (2008).
    23. Haase, A. D. et al. TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing. EMBO Rep 6, 961-7 (2005).
    24. Hammond S M, Bernstein E, Beach D, Hannon G J. 2000. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293-296.
    25. Han, J. et al. Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125, 887-901 (2006).
    26. Han, J. et al. The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev 18, 3016-27 (2004).
    27. Harrison P J, Weinberger D R. 2005. Schizophrenia genes, gene expression, and neuropathology: on the matter of their convergence. Mol Psychiatry 10:40-68.
    28. Hatfield S D, Shcherbata H R, Fischer K A, Nakahara K, Carthew R W, Ruohola-Baker H. 2005. Stem cell division is regulated by the microRNA pathway. Nature 435:974-978.
    29. Hu, Z. et al. Common genetic variants in pre-microRNAs were associated with increased risk of breast cancer in Chinese women. Hum Mutat (2008).
    30. Hu, Z. et al. Genetic variants of miRNA sequences and non-small cell lung cancer survival. J Clin Invest 118, 2600-8 (2008).
    31. Hutvagner G, McLachlan J, Pasquinelli A E, Balint E, Tuschl T, Zamore P D. 2001. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293:834-838.
    32. Hutvagner G, Zamore P D. 2002. A microRNA in a multiple-turnover RNAi enzyme complex. Science 297:2056-2060.
    33. Jablensky A. 2000. Epidemiology of schizophrenia: the global burden of disease and disability. Eur Arch Psychiatry Clin Neurosci 250:274-285.
    34. Jazdzewski, K. et al. Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma. Proc Natl Acad Sci U S A 105, 7269-74 (2008).
    35. Jentsch et al., 1997, Science 277:953-955.
    36. Jin P, Zarnescu D C, Ceman S, Nakamoto M, Mowrey J, Jongens T A, Nelson D L, Moses K, Warren S T. 2004. Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway. Nat Neurosci 7:113-117.
    37. Karube Y, Tanaka H, Osada H, Tomida S, Tatematsu Y, Yanagisawa K, Yatabe Y, Takamizawa J, Miyoshi S, Mitsudomi T, Takahashi T. 2005. Reduced expression of Dicer associated with poor prognosis in lung cancer patients. Cancer Sci 96:111-115.
    38. Kawahara, Y. et al. Redirection of silencing targets by adenosine-to-inosine editing of miRNAs. Science 315, 1137-40 (2007).
    39. Kawahara, Y., Zinshteyn, B., Chendrimada, T. P., Shiekhattar, R. & Nishikura, K. RNA editing of the microRNA-151 precursor blocks cleavage by the Dicer-TRBP complex. EMBO Rep 8, 763-9 (2007).
    40. Khvorova, A., Reynolds, A. & Jayasena, S. D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209-16 (2003).
    41. Kim V N. 2005. MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol 6:376-385.
    42. Klein M E, Impey S, Goodman R H. 2005. Role reversal: the regulation of neuronal gene expression by microRNAs. Curr Opin Neurobiol 15:507-513.
    43. Lai E C. 2003. microRNAs: runts of the genome assert themselves. Curr Biol 13:R925-R936.
    44. Landthaler M, Yalcin A, Tuschl T. 2004. The human DiGeorge syndrome critical region gene 8 and Its D. melanogaster homolog are required for miRNA biogenesis. Curr Biol 14:2162-2167.
    45. Lee, Y., Jeon, K., Lee, J. T., Kim, S. & Kim, V. N. MicroRNA maturation: stepwise processing and subcellular localization. Embo J 21, 4663-70 (2002).
    46. Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415-9 (2003).
    47. Liu, J. et al. A role for the P-body component GW182 in microRNA function. Nat Cell Biol 7, 1261-6 (2005).
    48. Martin, M. M. et al. The human angiotensin II type 1 receptor +1166 A/C polymorphism attenuates microrna-155 binding. J Biol Chem 282, 24262-9
    49. (2007).
    50. Mattick J S, Makunin I V. 2005. Small regulatory RNAs in mammals. Hum Mol Genet 14 Spec No 1:R121-R132.
    51. Mishra, P. J., Humeniuk, R., Longo-Sorbello, G. S., Banerjee, D. & Bertino, J. R. A miR-24 microRNA binding-site polymorphism in dihydrofolate reductase gene leads to methotrexate resistance. Proc Natl Acad Sci USA 104, 13513-8 (2007).
    52. Mourelatos Z, Dostie J, Paushkin S, Sharma A, Charroux B, Abel L, Rappsilber J, Mann M, Dreyfuss G. 2002. miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev 16:720-728.
    53. Naguibneva I, Ameyar-Zazoua M, Polesskaya A, Ait-Si-Ali S, Groisman R, Souidi M, Cuvellier S, Harel-Bellan A. 2006. The microRNA miR-181 targets the homeobox protein Hox-A11 during mammalian myoblast differentiation. Nat Cell Biol 8:278-284.
    54. Okamura, K., Hagen, J. W., Duan, H., Tyler, D. M. & Lai, E. C. The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila. Cell 130, 89-100 (2007).
    55. Okamura, K. et al. The regulatory activity of microRNA* species has substantial influence on microRNA and 3′ UTR evolution. Nat Struct Mol Biol 15, 354-63 (2008).
    56. Palatnik J F, Allen E, Wu X, Schommer C, Schwab R, Carrington J C, Weigel D. 2003. Control of leaf morphogenesis by microRNAs. Nature 425:257-263.
    57. Pasquinelli A E, Reinhart B J, Slack F, Martindale M Q, Kuroda M I, Maller B, Hayward D C, Ball E E, Degnan B, Muller P, Spring J, Srinivasan A, Fishman M, Finnerty J, Corbo J, Levine M, Leahy P, Davidson E, Ruvkun G. 2000. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 408:86-89.
    58. Piercey et al., 1988, Life Sci. 43(4):375-385.
    59. Piskounova, E. et al. Determinants of microRNA processing inhibition by the developmentally regulated RNA-binding protein Lin28. J Biol Chem (2008).
    60. Purohit et al., 1993, Biol. Psychiatry 33(4):255-260.
    61. Rehwinkel, J., Behm-Ansmant, I., Gatfield, D. & Izaurralde, E. A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. Rna 11, 1640-7 (2005).
    62. Ro, S., Park, C., Young, D., Sanders, K. M. & Yan, W. Tissue-dependent paired expression of miRNAs. Nucleic Acids Res 35, 5944-53 (2007).
    63. Ruby, J. G., Jan, C. H. & Bartel, D. P. Intronic microRNA precursors that bypass Drosha processing. Nature 448, 83-6 (2007).
    64. Ruvkun, G. Molecular biology. Glimpses of a tiny RNA world. Science 294, 797-9 (2001).
    65. Saunders, M. A., Liang, H. & Li, W. H. Human polymorphism at microRNAs and microRNA target sites. Proc Natl Acad Sci USA 104, 3300-5 (2007).
    66. Schwarz, D. S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199-208 (2003).
    67. Seitz, H., Ghildiyal, M. & Zamore, P. D. Argonaute loading improves the 5′ precision of both MicroRNAs and their miRNA strands in flies. Curr Biol 18, 147-51 (2008).
    68. Sethupathy, P. et al. Human microRNA-155 on chromosome 21 differentially interacts with its polymorphic target in the AGTR1 3′ untranslated region: a mechanism for functional single-nucleotide polymorphisms related to phenotypes. Am J Hum Genet 81, 405-13 (2007).
    69. Shen, J. et al. A Functional Polymorphism in the miR-146a Gene and Age of Familial Breast/Ovarian Cancer Diagnosis. Carcinogenesis (2008).
    70. Sobell J L, Heston L L, Sommer S S. 1993. Novel association approach for determining the genetic predisposition to schizophrenia: case-control resource and testing of the first candidate gene. Am J Med Genet 48:28-35.
    71. Sommer S S, Cassady J D, Sobell J L, Bottema C D. 1989. A novel method for detecting point mutations or polymorphisms and its application to population screening for carriers of phenylketonuria. Mayo Clin Proc 64:1361-1372.
    72. Sommer S S, Groszbach A R, Bottema C D K. 1992. PCR amplification of specific alleles (PASA) is a general method for rapidly detecting known single-base changes. BioTechniques 12:82-87.
    73. Sun G, Li H, Rossi J J. 2007. Cloning and Detecting Signature MicroRNAs from Mammalian Cells. Methods Enzymol 427:123-138.
    74. Viswanathan, S. R., Daley, G. Q. & Gregory, R. I. Selective blockade of microRNA processing by Lin28. Science 320, 97-100 (2008).
    75. Xu, T. et al. A functional polymorphism in the miR-146a gene is associated with the risk for hepatocellular carcinoma. Carcinogenesis (2008).
    76. Yang, W. et al. Modulation of microRNA processing and expression through RNA editing by ADAR deaminases. Nat Struct Mol Biol 13, 13-21 (2006).
    77. Yi, R., Qin, Y., Macara, I. G. & Cullen, B. R. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17, 3011-6 (2003).
    78. Ying S Y, Lin S L. 2004. Intron-derived microRNAs—fine tuning of gene functions. Gene 342:25-28.
    79. Yu, Z. et al. Aberrant allele frequencies of the SNPs located in microRNA target sites are potentially associated with human cancers. Nucleic Acids Res 35, 4535-41 (2007).
    80. Zeng Y, Wagner E J, Cullen B R. 2002. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell 9:1327-1333.
    81. Zeng, Y. & Cullen, B. R. Efficient processing of primary microRNA hairpins by Drosha requires flanking nonstructured RNA sequences. J Biol Chem 280, 27595-603 (2005).
    82. Zeng, Y. Principles of micro-RNA production and maturation. Oncogene 25, 6156-62 (2006).
    83. Zeng, Y., Yi, R. & Cullen, B. R. Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha. Embo J 24, 138-48 (2005).
    84. Zeng, Y. & Cullen, B. R. Structural requirements for pre-microRNA binding and nuclear export by Exportin 5. Nucleic Acids Res 32, 4776-85 (2004).
    85. Zhang, R., Peng, Y., Wang, W. & Su, B. Rapid evolution of an X-linked microRNA cluster in primates. Genome Res 17, 612-7 (2007).









TABLE 1a







Ultra-rare miRNA cohort-specific variants



















Novel mature





Variant in mature
Variant in

miRNA


ID#
Disease
miRNA
miRNA
precursor
Gene pool
documenteda





S358
Schizophrenia
let-7f-2
11 G > A

0/7,197
yes


S418
Schizophrenia
mir-18b

32 A > G
0/7,197
?


S590
Schizophrenia
mir-505

 8 C > T
0/7,197
?


S356
Schizophrenia
mir-502

13 C > G
1/7,197b
yes


S014
Schizophrenia
mir-188
 7 C > T (188-3p)

0/7,197
yes


MC179
Psychosis
mir-325

66 G > A
0/7,197
?


S711
Schizophrenia
mir-660
15 C > T

0/7,197
yes


S596
Schizophrenia
mir-509-3
13 C > T (509-3p)

0/7,197
yes


MC149
Controlc
mir-510

48 T > C
0/7,197
?





aby functional analysis


bone otherwise healthy individual in the gene pool analysis, who has this variant, was found to have a history of depression; the extent of the depression requires further clarification


cthis individual was ascertained as a control sample, but upon examination of medical history was found to have a history of depression; the extent of the depression requires further clarification













TABLE 1b







miRNA cohort-specific sequence variants found


in the gene pooi analyses















Variant in







mature
Variant in
Gene


ID#
Disease
miRNA
miRNA
precursor
pool





S464
Schizophrenia
mir-509-3
22 G > A

 2/7,197





(509-3-5p)


MC527
Control
mir-509-3
19 C > G

10/4,962





(509-3-5p)


MC333
Control
mir-421

73 G > A
16/4,962


MC40
Control
mir-934
 1 T > G

 4/7,197


MC93
Control
mir-450-2
 5 T > C

 8/4,962
















TABLE 2







X Chromosome MicroRNA Primers (miRBase V10.1)




















Size

PCR size



#
miRNA
Oligo
SEQ ID NO.
Sequence
(bp)
Tm(° C.)
(bp)


















1
mir-221
mir-
SEQ ID NO. 97:
CAGTTATTCAGAAACATTATAGG
23
62
200





221D1




mir-
SEQ ID NO. 98:
AGGCAGTTGTGTTGAAATAGTA
22
60





221U1


 


2
mir-222
mir-
SEQ ID NO. 99:
TTATTAAAGACTGCCCAATAATC
23
60
195




222D1




mir-
SEQ ID NO. 100:
CTTCCACAGAGCCCCTCC
18
60





222U1


 


3
mir-188
mir-
SEQ ID NO. 101:
AGCATACCCATATGTCGTGC
20
60
182




188D1




mir-
SEQ ID NO. 102:
TGGTGAAGGAGTGCGTATGT
20
60





188U2


 


4
mir-98
mir-
SEQ ID NO. 103:
GAGGCAACACTGCTAAGACT
20
60
167




98D1




mir-
SEQ ID NO. 104:
CTTTTGCCTGCTGCCCTTAT
20
60





98U2


 


5
let-7f-2
let-7f-
SEQ ID NO. 105:
CCAGAACAAGAACCTGAACAG
21
60
184




2D1




let-7f-
SEQ ID NO. 106:
CCTGATAGTTCCGAGTAGCT
20
60





2U2


 


6
mir-223
mir-
SEQ ID NO. 107:
ACATCTCCCAGGAAGATCTC
20
60
192




223D1




mir
SEQ ID NO. 108:
GGCAGTCCATTCGTCATATC
20
62





223U1


 


7
mir-325
mir-
SEQ ID NO. 109:
ACCACTAGGCCTAAGTACCT
20
60
198




325D1




mir-
SEQ ID NO. 110:
GCTTAAATATAGGTTTTGAGATG
23
60





325U1


 


8
mir-361
mir-
SEQ ID NO: 111:
GATGCTTCTAAAGAAACGGTG
21
60
160




361D1




mir-
SEQ ID NO. 112:
TAGCAGTGGCACGCTTGAC
19
60





361U1


 


9
mir-224
mir-
SEQ ID NO. 113:
TCTGGTGCTTACCTACTGTC
20
60
170




224D1




mir-
SEQ ID NO. 114:
TGGGGACCCATCATCAAAAG
20
60





224U1


 


10
mir-374a
mir-
SEQ ID NO. 115:
AGGAGCTCACAGTCTAACTG
20
60
182




374D1




mir-
SEQ ID NO. 116:
GTTCCTCACCTCTCTTGATG
20
60





374U1


 


11
mir-384
mir-
SEQ ID NO. 117:
GCCAGTTAGCATCTTGAAGG
20
60
186




384D1




mir-
SEQ ID NO. 118:
GTTCCTTGCCTTTTAACTAGTAT
23
62





384U1


 


12
mir-220
mir-
SEQ ID NO. 119:
TCCAGCTGACGCACTTGCT
19
60
208




220D1




mir-
SEQ ID NO. 120:
GATGCAGTAGGTCTCATTCG
20
60





220U1


 


13
mir-92-2
mir-92-
SEQ ID NO. 121:
CTAAATATCAGAACTTACGGCT
22
60
177




2D1




mir-92-
SEQ ID NO. 122:
GTGAACACACCTTCATGCGT
20
60





2U1


 


14
mir-19b-
mir-
SEQ ID NO. 123:
TGAGTGCTGGAGATACGCAT
20
60
191



2
19b-2D1




mir-
SEQ ID NO. 124:
CTCTTGGATAACAAATCTCTTG
22
60





19b-2U1


 


15
mir-106a
mir
SEQ ID NO. 125:
TTATGCACAAACTACAGTTCTC
22
60
166




106aD1




mir-
SEQ ID NO. 126:
AGAAGAGCTCCTGGAAGACT
20
60




106aU1


 


16
mir-424
mir-
SEQ ID NO. 127:
GGGAGCGGGCACCTGGT
17
60
178




424D2




mir-
SEQ ID NO. 128:
GCTTCCTTCAGTCATCCAGT
20
60




424U3


 


17
mir-105-
mir-
SEQ ID NO. 129:
AATGGCTTTGGTCCAGAAGC
20
60
165



1
105-1D




mir-
SEQ ID NO. 130:
CTACTCCTATATATTGGATGTC
22
60





105-1U


 


18
mir-105-
mir-
SEQ ID NO. 131:
GAGTGGCTTTGTTCCAGAAG
20
60
170



2
105-2D




mir-
SEQ ID NO. 132:
GTCTACTCCCTATAACCTGG
20
60





105-2U


 


19
mir-651
mir-
SEQ ID NO. 133:
CTTGTGATGTAGATTAAATCGT
22
58
368




651D1




mir-
SEQ ID NO. 134:
CACTTTATTCATCCTGACTCT
21
58





651U1


 


20
mir-532
mir-
SEQ ID NO. 135:
TGTACACAAGCACACCTTCT
20
58
328




532D1




mir-
SEQ ID NO. 136:
GAAGCAGGACAGTCATCCA
19
58




532U1


 


21
mir-660
mir-
SEQ ID NO. 137:
GCACCTGACACTTTAATCTCA
21
60
365




660D1




mir-
SEQ ID NO. 138:
CTAATACCTCCACTAGATAGG
21
60





660U1


 


22
mir-652
mir-
SEQ ID NO. 139:
TGTTTGTGTGCTATGGCCAT
20
58
449




652D2




mir-
SEQ ID NO. 140:
GTTCTCCTATATGATGGCTC
20
58





652U2


 


23
mir-934
mir-
SEQ ID NO. 141:
TATGTATCTCGTGGATCTGC
20
58
259




934D1




mir-
SEQ ID NO. 142:
TTACAAGATAGGAAGTGCATAT
22
58





934U1


 


24
mir-421
mir-
SEQ ID NO. 143:
CATTGTCCGTGTCTATGGC
19
58
345




421D1




mir-
SEQ ID NO. 144:
AATTGGAGATGGTACTTGAGA
21
58





421U1


 


25
mir-766
mir-
SEQ ID NO. 145:
TATACACAGAGGATTGCTTAG
21
58
308




766D1




mir-
SEQ ID NO. 146:
CCTCATTACTCTCATTTCCTG
21
60





766U1


 


26
mir-450b
mir-
SEQ ID NO. 147:
ATCGCTGACTTGTGTCACTA
20
58
543




450bD3




mir-
SEQ ID NO. 148:
TATTCTAGGCCCACTGCTG
19
58





450bU2


 


27
mir-890
mir-
SEQ ID NO. 149:
TTCAGGGTTCAGGCATCCT
19
58
291




890D1




mir-
SEQ ID NO. 150:
ACACCTAAGGTTCAGGTGG
19
58





890U1


 


28
mir-888
mir-
SEQ ID NO. 151:
GACATCATGCTGTGCTCAG
19
58
279




888D1




mir-
SEQ ID NO. 152:
TGCCTGAATTCCAGGTGGT
19
58





888U1


 


29
mir-892a
mir-
SEQ ID NO. 153:
TCCAGATTCAGGCATCCTG
19
58
289




892aD1




mir-
SEQ ID NO. 154:
TTAAGGATGCCTGAGGTTCA
20
58





892aU1


 


30
mir-892b
mir-
SEQ ID NO. 155:
TCAAATTGTTCAGGCATCATG
21
58
279




892bD1




mir-
SEQ ID NO. 156:
ACATGGCCAGCTAGGTTCT
19
58





892bU1


 


31
mir-891b
mir-
SEQ ID NO. 157:
TAGCTACATAGGTCGTGAATA
21
58
315




891bD1




mir-
SEQ ID NO. 158:
CTACTACCAGTATCACTACAA
21
58





891bU1


 


32
mir-891a
mir-
SEQ ID NO. 159:
CATGCTGATAGTTACACAGG
20
58
319




891aD1




mir-
SEQ ID NO. 160:
ACTACCAGTGTCACTACAAG
20
58





891aU1


 


33
mir-509-
mir-
SEQ ID NO. 161:
ccaaattccaatggccacg
19
58
521



2
509-2D2




mir-
SEQ ID NO. 162:
atttggatgttggagtaggc
21
58





509-2U2


 


34
mir-509-
mir-
SEQ ID NO. 163:
TCTGTGAGTAACAGGACCTA
20
58
690



3
509-3D1




mir-
SEQ ID NO. 164:
TGAGAAAGGAAGCTAACCATT
21
58





509-3U1


 


35
mir-767
mir-
SEQ ID NO. 165:
TGATATCTCCTCCAGCATCA
20
58
331




767D1




mir-
SEQ ID NO. 166:
TGATCTAAGAGTAGAGAGTCA
21
58





767U1


 


36
mir-374b
mir-
SEQ ID NO. 167:
GTAAAGTGTTTGTCATAGGCA
21
58
329




374bD1




mir-
SEQ ID NO. 168:
CCTACAATGTGCACCGGAT
19
58





374bU1


 


37
mir-542
mir-
SEQ ID NO. 169:
GGTGGGATTAGAGCTGAAG
19
58
435




542D1




mir-
SEQ ID NO. 170:
GGCATTCCCATTACACTCC
19
58





542U1


 


38
mir-513-
mir-
SEQ ID NO. 171:
CAAGTTGCATTGTCCCTTGG
20
60
486



1
513-1D2




mir-
SEQ ID NO. 172:
TGGAGTAAAGCATTCCAGCT
20
58





513-1U2


 


39
mir-20b
mir-
SEQ ID NO. 173:
GTAGCAATGTCTTTGAATATTC
22
58
189




20bD1




mir-
SEQ ID NO. 174:
TGTTGGGAACAGATGGTGG
19
58





20bU1


 


40
mir-362
mir-
SEQ ID NO. 175:
ACATGCACACATACAAACACA
21
58
199




362D1




mir-
SEQ ID NO. 176:
ATAGCAAACACAAACATGTGAA
22
58





362U1


 


41
mir-18b
mir-
SEQ ID NO. 177:
ACCACTGAAATGTGTCCTATT
21
58
209




18bD1




mir-
SEQ ID NO. 178:
GAGAACTGTAGTTTGTGCATA
21
58





18bU1


 


42
mir-510
mir-
SEQ ID NO. 179:
ATGTGCTAAGAAAAGCATACC
21
58
219




510D1




mir-
SEQ ID NO. 180:
AGAGGTTGTTTGAAAAGTGTG
21
58





510U1


 


43
mir-363
mir-
SEQ ID NO. 181:
TAGCTTACTGTAGCGCTGAT
20
58
229




363D1




mir-
SEQ ID NO. 182:
ACTTGTCCCGGCCTGTGG
18
60





363U1


 


44
mir-503
mir-
SEQ ID NO. 183:
TGCAATCTTGGGTCTCGCT
19
58
239




503D1




mir-
SEQ ID NO. 184:
GGGCAGTATTCCTGGCTAG
19
60





503U1


 


45
mir-500
mir-
SEQ ID NO. 185:
AAGCTCAAGAACTGTTCTCTT
21
58
250




500D1




mir-
SEQ ID NO. 186:
ATAAATACTTGTGGAATGGATG
22
58





500U1


 


46
mir-501
mir-
SEQ ID NO. 187:
CAGAGATACTTTGCAGGAATG
21
60
260




501D1




mir-
SEQ ID NO. 188:
AAGAATGAATGAGGGTCAGAG
21
60





501U1


 


47
mir-505
mir-
SEQ ID NO. 189:
ATGATGCAAATGAGTATTAGGA
22
58
270




505D1




mir-
SEQ ID NO. 190:
TTCTAGATTATGGGTCATTCC
21
58





505U1


 


48
mir-452
mir-
SEQ ID NO. 191:
GCCAGCTGCACATTCCGA
18
58
278




452D1




mir-
SEQ ID NO. 192:
GTTGGTAAGCGTGCCAGG
18
58





452U1


 


49
mir-504
mir-
SEQ ID NO. 193:
AAGAGACTTATCAGGATTGAG
21
58
289




504D1




mir-
SEQ ID NO. 194:
CCATCCATTTCCCACATAGA
20
58





504U1


 


50
mir-502
mir-
SEQ ID NO. 195:
TCACCTAATATTCCTGCACG
20
58
299




502D1




mir-
SEQ ID NO. 196:
GGTGATGTCTAGGCTCTCT
19
58




502U1


 


51
mir-507
mir-
SEQ ID NO. 197:
TGATGGTGGTGGCACTGAC
19
60
310




507D1




mir-
SEQ ID NO. 198:
TCCTAGTGGACTGAGAGTGT
20
60





507U1


 


52
mir-545
mir-
SEQ ID NO. 199:
CAAAGAACTGTGTAGGAAGGA
21
60
320




545D1




mir-
SEQ ID NO. 200:
TCATCACTCGACAGTGATGG
20
60





545U1


 


53
mir-509-
mir-
SEQ ID NO. 201:
GTCCAGCATGGAAGTGGAG
19
60
330



1
509D1




mir-
SEQ ID NO. 202:
TGGATTGGATTCTGCAGAAGT
21
60





509U1




mir-
SEQ ID NO. 203:
TGGACAAAGGACTTCTGTAG
20
58
~920




509D2


 


54
mir-450-
mir-
SEQ ID NO. 204:
TAGTGCATATTAGGAACACATC
22
60
339



2
450-2D1




mir-
SEQ ID NO. 205:
ATAGGTATATAGGGAGCATTCT
22
60





450-2U1


 


55
mir-450-
mir-
SEQ ID NO. 206:
CACAGAAGTAAACCACAGATA
21
58
349



1
450-1D1




mir-
SEQ ID NO. 207:
TTGTGGTATAAAGGTGACCAA
21
58





450-1U1


 


56
mir-448
mir-
SEQ ID NO. 208:
CCAGGCCAGAAGAGGCTT
18
58
369




448D1




mir-
SEQ ID NO. 209:
AAGGTCATAGCAGTCAGTAC
20
58





448U1


 


57
mir-508
mir-
SEQ ID NO. 210:
AAGACCTGCCTATGGGAGT
19
58
379




508D1




mir-
SEQ ID NO. 211:
ACTGAAGAGAAGAAGTCCTC
20
58





508U1


 


58
mir-506
mir-
SEQ ID NO. 212:
CAGATTCTGGAGCAGATCTC
20
60
389




506D1




mir-
SEQ ID NO. 213:
CAGAACTACCCACTCAGTGA
20
60





506U1


 


59
mir-513-
mir-
SEQ ID NO. 214:
GAGTCCACAGTTCCTATGTA
20
58
399



2
513-2D1




mir-
SEQ ID NO. 215:
CTCACTTGGGGCAGGATG
18
58




513-2U1
















TABLE 3







Variants found in cases and controlsa















# of





# of
control





schizophrenia
patients




Variant
patients with
with the




in
the variant
variant


ID#
miRNA
Precursor
(n = 193)
(n = 191)














S329; MC124;
mir-
66 G > C
1
3


MC178; MC235
890


many
mir-
77 A > C
39
32



888


S014; S104;
mir-
35 C > G
4
4


S319; S599;
891b


MC73; MC207;


MC424; MC515


S211; S508;
mir-
54
2
4


MC129;
509-1
insTGA


MC162;


MC285; MC398


S345; S433;
mir-
9 G > T
2
2


MC348; MC370
509-2





aall 5 variants were found with similar frequencies in cases and controls













TABLE 4







With 509-2























# of
# of










schizophrenia
control









patients
patients







Variant

with the
with the







in mature
Variant in
variant
variant


ID#
Disease
Gender
Ethnicity
miRNA
miRNA
Precursor
(n = 288)
(n = 192)





S329;
schizophrenia
all male
all
mir-890

66 G < C
1
3



MC124;
& controls

Caucasian


MC178



schizophrenia
all male
all
mir-888

77 A < C
53 
32 



& controls

Caucasian


S599; S014;
schizophrenia
all male
all
mir-891b

35 C < G
4
4


S104; S319;
& controls

Caucasian


MC73;


MC207



schizophrenia
all male
all
mir-509-1

54 insTGA
3
4



& controls

Caucasian


6
509-2
g1461480
in
1/192
2/192
S433;
NA
1/94
yes




52t
precursor;


MC348;





11 bases


MC370





upstream of





the 5′ end





of the





mature





miRNA
















TABLE 5







Without 509-2























# of










control









# of
patients







Variant in

schizophrenia
with the







mature
Variant in
patients with the
variant


ID#
Disease
Gender
Ethnicity
miRNA
miRNA
Precursor
variant (n = 288)
(n = 192)


















S329; MC124;
schizophrenia
all male
all Caucasian
mir-890

66 G < C
1
3


MC178
& controls



schizophrenia
all male
all Caucasian
mir-888

77 A < C
53
32



& controls


S599; S014;
schizophrenia
all male
all Caucasian
mir-891b

35 C < G
4
4


S104; S319;
& controls


MC73; MC207



schizophrenia
all male
all Caucasian
mir-509-1

54 insTGA
3
4



& controls
















TABLE 6







Target genes of miRNAs in which we found ultra-rare cohort-specific variants.











miRNAs with ultra rare variants have binding


Genes
Function
site in 3'UTR





CLCN5
Chloride channel 5 (nephrolithiasis 2, X-linked, Dent disease)
Let-7f, miR-502, miR-18b, miR-660,



Mutations in this gene have been found in Dent disease and renal



tubular disorders complicated by nephrolithiasis


HMGA2
HMG proteins function as architectural factors and are essential
Let-7f, miR-505



components of the enhancesome. Identification of the deletion,



amplification, and rearrangement of this gene that are associated



with myxoid liposarcoma suggests a role in adipogenesis and



mesenchymal differentiation.


NRXN3
Neurexins are a family of proteins that function in the vertebrate
Let-7f



nervous system as cell adhesion molecules and receptors.


DISC1
Disrupted in schizophrenia 1: The protein is involved in neurite
Let-7f, miR-18b, miR-510, miR-188, miR-502



outgrowth and cortical development through its interaction with



other proteins. This gene is disrupted by a t(1; 11)(q42.1; q14.3)



translocation which segregates with schizophrenia and related



psychiatric disorders in a large Scottish family.


NRG1
Neuregulin 1: Interacts with the NEU/ERBB2 receptor tyrosine
miR-505



kinase to increase its phosphorylation on tyrosine residues. It induces



the growth and differentialtion of epithelial, neuronal, glial and other



types of cells.


MECP2
Methyl CpG binding protein 2: Mutations of MECP2 are the cause
Let-7f, miR-188 miR-325, miR-18b



of some cases of Rett syndrome, a progressive neurologic



developmental disorder, and are one of the most common causes of



mental retardation in females.


RGS4
Regulator of G-protein signaling 4: It negatively regulates signaling
miR-18b, miR-502



upstream or at the level of the heterotrimeric G protein and is



localized in the cytoplasm.


GRM3
Glutamate receptor, metabotropic 3: L-glutamate is the major
miR-325



excitatory neurotransmitter in the central nervous system and



activates both ionotropic and metabotropic glutamate receptors.



Glutamatergic neurotransmission is involved in most aspects of



normal brain function and can be perturbed in many neuropathologic



conditions.
















TABLE 7





Oligos used to clone pri-miRNA and probes for northern blots


















hsa-let-7f-2





5xho-let7f2
SEQ ID NO. 216:
attatCTCGAGaatctctcaggtaactctcc


3BamH-let7f2
SEQ ID NO. 217:
attatGGATCCAGAGTTGCAGTCAGGAAATG


 


5x-Let7f2-si
SEQ ID NO. 218:
TCGAAACTATACAATCTACTACCTCA


3s-Let7f2-si
SEQ ID NO. 219:
CTAGTGAGGTAGTAGATTGTATAGTT


 


5x-Let7f2-m-si
SEQ ID NO. 220:
TCGAAACTATACAATTTACTACCTCA


3s-Let7f2-m-si
SEQ ID NO. 221:
CTAGTGAGGTAGTAAATTGTATAGTT


 


Oligo for


rmutagenesis


5-let7f2G-A
SEQ ID NO. 222:
GGGATGAGGTAGTAAATTGTATAGTTTTAG


3-let7f2G-A
SEQ ID NO. 223:
CTAAAACTATACAATTTACTACCTCATCCC


 


5x-let7f-mi
SEQ ID NO. 224:
tcgaGGGTATACGGTCTACTACCTCA


3S-let7f-mi
SEQ ID NO. 225:
ctagTGAGGTAGTAGACCGTATACCC


 


5x-let7f-m-mi
SEQ ID NO. 226:
tcgaGGGTATACGGTTTACTACCTCA


3S-let7f-m-mi
SEQ ID NO. 227:
ctagTGAGGTAGTAAACCGTATACCC


 


let7f2 probe
SEQ ID NO. 228:
AACTATACAATCTACTACCTCA


 


hsa-mir-18b


5Xho-miR18b
SEQ ID NO. 229:
tatCTCGAGCTACTGAGAACTGTAGTTTGTGCA


3BamH-miR18b
SEQ ID NO. 230:
tatGGATCCACTGAAATGTGTCCTATTCAAATT


 


5x-18b-si
SEQ ID NO. 231:
tcgaCTAACTGCACTAGATGCACCTTA


3s-18b-si
SEQ ID NO. 232:
ctagTAAGGTGCATCTAGTGCAGTTAG


 


5x-18b-mi
SEQ ID NO. 233:
tcgagaAACTGCACatcATGCACCTTA


3s-18b-mi
SEQ ID NO. 234:
ctagTAAGGTGCATGATGTGCAGTTTC


 


5x-18bStar-si
SEQ ID NO. 235:
tcgaGCCAGAAGGGGCATTTAGGGCA


3s-18bStar-si
SEQ ID NO. 236:
ctagTGCCCTAAATGCCCCTTCTGGC


 


5x-18bStar-mi
SEQ ID NO. 237:
tcgacgCAGAAGGccgATTTAGGGCA


3s-18bStar-mi
SEQ ID NO. 238:
ctagTGCCCTAAATCGGCCTTCTGCG


 


hsa-mir-505


5Xho-miR505
SEQ ID NO. 239:
tatCTCGAGCATACTGTCATTGAAAGCACTTAC


3BamH-miR505
SEQ ID NO. 240:
tatGGATCCTGAGCAAATGAATGGGAGCAGTAA


 


5x-505-si
SEQ ID NO. 241:
tcgaAGGAAACCAGCAAGTGTTGACG


3s-505-si
SEQ ID NO. 242:
ctagCGTCAACACTTGCTGGTTTCCT


 


5x-505-mi
SEQ ID NO. 243:
tcgatcGAAACCAcgtAGTGTTGACG


3s-505-mi
SEQ ID NO. 244:
ctagCGTCAACACTACGTGGTTTCGA


 


5x-505Star-si
SEQ ID NO. 245:
tcgaACATCAATACTTCCTGGCTCCC


3s-505Star-si
SEQ ID NO. 246:
ctagGGGAGCCAGGAAGTATTGATGT


 


5x-505Star-mi
SEQ ID NO. 247:
tcgatgATCAATAgaaCCTGGCTCCC


3s-505STar-mi
SEQ ID NO. 248:
ctagGGGAGCCAGGTTCTATTGATCA


 


hsa-mir-502


5Xho-miR502
SEQ ID NO. 249:
tatCTCGAGAATATGTGTAGGAGACTTG


3BamH-miR502
SEQ ID NO. 250:
tatGGATCCTGTCTCACTCTGGATACCTG


 


5x-502-5p-si
SEQ ID NO. 251:
tcgaTAGCACCCAGATAGCAAGGAT


3s-502-5p-si
SEQ ID NO. 252:
ctagATCCTTGCTATCTGGGTGCTA


 


5x-502-5p-mi
SEQ ID NO. 253:
tcgaatGCACCCtctTAGCAAGGAT


3s-502-5p-mi
SEQ ID NO. 254:
ctagATCCTTGCTAAGAGGGTGCAT


 


5x-502-3p-si
SEQ ID NO. 255:
tcgaTGAATCCTTGCCCAGGTGCATT


3s-502-3p-si
SEQ ID NO. 256:
ctagAATGCACCTGGGCAAGGATTCA


 


5x-502-3p-mi
SEQ ID NO. 257:
tcgaacAATCCTTcggCAGGTGCATT


3s-502-3p-mi
SEQ ID NO. 258:
ctagAATGCACCTGCCGAAGGATTGT


 


miR-502-5p probe
SEQ ID NO. 259:
TAGCACCCAGATAGCAAGGAT


miR-502-3p probe
SEQ ID NO. 260:
AATCCTTGCCCAGGTGCATTGCATT


 


hsa-mir-188


5Xho-miR188
SEQ ID NO. 261:
tatCTCGAGCTGCCCACTTGCACCCCCCAGGCC


3BamH-miR188
SEQ ID NO. 262:
tatGGATCCCACCACATGGGTGTGTGTTTTTCT


 


5x-188-5p-si
SEQ ID NO. 263:
tcgaCCCTCCACCATGCAAGGGATG


3s-188-5p-si
SEQ ID NO. 264:
ctagCATCCCTTGCATGGTGGAGGG


 


5x-188-5p-mi
SEQ ID NO. 265:
tcgaggCTCCACgtaGCAAGGGATG


3s-188-5p-mi
SEQ ID NO. 266:
ctagCATCCCTTGCTACGTGGAGCC


 


5x-188-3p-si
SEQ ID NO. 267:
tcgaTGCAAACCCTGCATGTGGGAG


3s-188-3p-si
SEQ ID NO. 268:
ctagCTCCCACATGCAGGGTTTGCA


 


5x-188-3p-mi
SEQ ID NO. 269:
tcgaacCAAACCgacCATGTGGGAG


3s-188-3p-mi
SEQ ID NO. 270:
ctagCTCCCACATGGTCGGTTTGGT


 


5x-188-3pm-si
SEQ ID NO. 271:
tcgaTGCAAACCCTGCATATGGGAG


3s-188-3pm-si
SEQ ID NO. 272:
ctagCTCCCATATGCAGGGTTTGCA


 


5x-188-3pm-mi
SEQ ID NO. 273:
tcgaacCAAACCgacCATATGGGAG


3s-188-3pm-mi
SEQ ID NO. 274:
ctagCTCCCATATGGTCGGTTTGGT


 


hsa-mir-325


5Xho-miR325
SEQ ID NO. 275:
tatCTCGAGGTTCTGTGAGAAAAAGTTGCTTAA


3BamH-miR-325
SEQ ID NO. 276:
tatGGATCCTAACCACCACTAGGCCTAAGTACC


 


Oligo for


mutagensis


5-miR325-mut
SEQ ID NO. 277:
CATAATTTGTTTATTaAGGACCTCCTATCAA


3-miR325-mut
SEQ ID NO. 278:
TTGATAGGAGGTCCTTAATAAACAAATTATG


 


5Xho-miR325-L
SEQ ID NO. 279:
tatCTCGAgacagggattgtatggctta


3BamH-miR-325-L
SEQ ID NO. 280:
tatGGATcctcaacacactgaaatctg


 


5Xho-miR325-s
SEQ ID NO. 281:
tatCTCGAGATTCAAGTCCACAGAACCA


3BamH-miR-325-s
SEQ ID NO. 282:
tatGGATccTCAAAATGTAGGATGTTG


 


5x-325-5p-siL
SEQ ID NO. 283:
tcgaACAAACACTTACTGGACACCTACTAGGAA


3s-325-5p-siL
SEQ ID NO. 284:
ctagTTCCTAGTAGGTGTCCAGTAAGTGTTTGT


 


5x-325-3p-siL
SEQ ID NO. 285:
tcgaTTGATAGGAGGTCCTCAATAAACAAATT


3s-325-3p-siL
SEQ ID NO. 286:
ctagAATTTGTTTATTGAGGACCTCCTATCAA


 


5x-325-si
SEQ ID NO. 287:
tcgaACACTTACTGGACACCTACTAGG


3s-325-si
SEQ ID NO. 288:
ctagCCTAGTAGGTGTCCAGTAAGTGT


 


5x-325-mi
SEQ ID NO. 289:
tcgatgACTTACTGctgACCTACTAGG


3s-325-mi
SEQ ID NO. 290:
ctagCCTAGTAGGTCAGCAGTAAGTCA


 


hsa-mir-510


5Xho-miR510
SEQ ID NO. 291:
tatCTCGagtcctgaaaactGCCA


3BamH-miR510
SEQ ID NO. 292:
tatGGATCCTTGCAAGTTTGTAAAGAAGG


 


miR-510-5p probe
SEQ ID NO. 293:
GTGATTGCCACTCTCCTGAGTA


miR-510-3p (star)
SEQ ID NO. 294:
CCACTCTTAGAGGTTTCAATCA


probe


 


5x-510Star-si
SEQ ID NO. 295:
tcgaCCACTCTTAGAGGTTTCAATCA


3s-510Star-si
SEQ ID NO. 296:
ctagTGATTGAAACCTCTAAGAGTGG


 


5x-510Star-mi
SEQ ID NO. 297:
tcgaggACTCTTActcGTTTCAATCA


3s-510Star-mi
SEQ ID NO. 298:
ctagTGATTGAAACgagTAAGAGTcc


 


5x-510Star-m-si
SEQ ID NO. 299:
tcgaCCACTCTTAGAGGTTTCAgTCA


3s-510Star-m-si
SEQ ID NO. 300:
ctagTGAcTGAAACCTCTAAGAGTGG


 


5x-510Star-m-mi
SEQ ID NO. 301:
tcgaggACTCTTActcGTTTCAgTCA


3s-510Star-m-mi
SEQ ID NO. 302:
ctagTGAcTGAAACgagTAAGAGTcc


 


5x-510-si
SEQ ID NO. 303:
tcgaGTGATTGCCACTCTCCTGAGTA


3s-510-si
SEQ ID NO. 304:
ctagTACTCAGGAGAGTGGCAATCAC


 


5x-510-mi
SEQ ID NO. 305:
tcgacaGATTGCCtgaCTCCTGAGTA


3s-510-mi
SEQ ID NO. 306:
ctagTACTCAGGAGTCAGGCAATCTG


 


hsa-mir-660


5Xho-660
SEQ ID NO. 307:
tatCTCGAgcactgcttctccaggcgtg


3Bam-660
SEQ ID NO. 308:
tatGGATCCTGGGGAAGTCTAGGCACC


 


S660-5p-si
SEQ ID NO. 309:
tcgaCAACTCCGATATGCAATGGGTA


N660-5p-si
SEQ ID NO. 310:
ggccTACCCATTGCATATCGGAGTTG


 


S660-5p-si-m
SEQ ID NO. 311:
tcgaCAACTCCAATATGCAATGGGTA


N660-5p-si-m
SEQ ID NO. 312:
ggccTACCCATTGCATATTGGAGTTG


 


S660-3p-si
SEQ ID NO. 313:
tcgaCCTCCTGTAATCCATGCACACAGGAGGTG


N660-3p-si
SEQ ID NO. 314:
ggccCACCTCCTGTGTGCATGGATTACAGGAGG


 


S660-5p-mi
SEQ ID NO. 315:
tcgaGTACTCCGAGTCGCAATGGGTA


N660-5p-mi
SEQ ID NO. 316:
ggccTACCCATTGCgacTCGGAGTac


 


660-5p probe
SEQ ID NO. 317:
CAACTCCGATATGCAATGGGTA


 


hsa-miR-509-3


5Xho-509-2
SEQ ID NO. 318:
tatCTCgagtggacaggactcaaagc


3Bam-509-2
SEQ ID NO. 319:
tatGGATCCACGTGTCTGGTGGTCAGGC


 


S509-5p-si
SEQ ID NO. 320:
tcgaTGATTGCCACTGTCTGCAGTA


N509-5p-si
SEQ ID NO. 321:
ggccTACTGCAGACAGTGGCAATCA


 


S509-3p-si
SEQ ID NO. 322:
tcgaCTACCCACAGACGTACCAATCA


N509-3p-si
SEQ ID NO. 323:
ggccTGATTGGTACGTCTGTGGGTAG


 


5Xho-509-3
SEQ ID NO. 324:
tatCTCGAGtgggagtggacagcactcaa


3Bam-509-3
SEQ ID NO. 325:
tatGGATCCAAATTCCTAGACCATGTGTC


 


5S-509-3-5p-si
SEQ ID NO. 326:
tcgaCATGATTGCCACGTCTGCAGTA


3N-509-3-5p-si
SEQ ID NO. 327:
ggccTACTGCAGACGTGGCAATCATG


 


5S-509-3CT-3p-si
SEQ ID NO. 328:
tcgaCTACCCACAAACGTACCAATCA


3N-509-3CT-3p-si
SEQ ID NO. 329:
ggccTGATTGGTACGTTTGTGGGTAG


 


5S-509-3CG-5p-si
SEQ ID NO. 330:
tcgaCATCATTGCCACGTCTGCAGTA


3N-509-3CG-5p-si
SEQ ID NO. 331:
ggccTACTGCAGACGTGGCAATGATG


 


S509-3-5p-mi
SEQ ID NO. 332:
tcgaGTTGATTGCGTGGTCTGCAGTA


N509-3-5p-mi
SEQ ID NO. 333:
ggccTACTGCAGACcacGCAATCAac


 


U2A probe
SEQ ID NO. 334:
AGAACAGATACTACACTTGA


 


S1-27 mer siRNA


Sense
SEQ ID NO. 335:
GCGGAGACAGCGACGAAGAGCUCAUCA


Antisense
SEQ ID NO. 336:
UGAUGAGCUCUUCGUCGCUGUCUCCGC


Probe (Detect
SEQ ID NO. 337:
GCGGAGACAGCGACGAAGAGCTCATCA


antisense)
















TABLE 8







List of all miRNA gene variants that were found in the control


population or the patient samples.








miRNA
Variants





hsa-let-7f-2
5p 11: G > A


hsa-miR-18b
Stem-loop 32: A > G


hsa-miR-188
3p 60: C > T


hsa-miR-224
Stem-loop 41: G > A


hsa-miR-325
Stem-loop 66/3p 6: G > A


hsa-miR-421
Stem-loop 73: G > A


hsa-miR-421
4 nt downstream of the 3′ end of the stem-loop: G > A


hsa-miR-450a-2
5p 4: T > C


has-miR-502
Stem-loop 13: C > G


has-miR-505
Stem-loop 8: C > T


hsa-miR-509-1
Stem-loop 54: ins TGA


hsa-miR-509-2
Stem-loop 9: G > T


hsa-miR-509-2
5p 11: ‘A’ Deletion


hsa-miR-509-3
5p 22: G > A


hsa-miR-509-3
5p 19: C > G


hsa-miR-509-3
3p 13: C > T


hsa-miR-510
Stem-loop 48/3p 4: T > C


hsa-miR-510
Stem-loop 6: G > A


hsa-miR-660
5p 15: C > T


hsa-miR-888
Stem-loop 77: A > C


hsa-miR-890
Stem-loop 66: G > C


hsa-miR-891b
Stem-loop 35: C > G


hsa-miR-892b
3p 15: T > C


hsa-miR-934
5p 1: T > G

## Claims

1. A method of diagnosing certain cases of schizophrenia or a schizophrenia spectrum disorder in a human individual, the method comprising:
a. obtaining a nucleic acid sample from the individual; and
b. determining the presence of one or more variants of a microRNA (miRNA) gene or allele in the sample, the one or more variants selected from the group consisting of:
a G to A substitution at position 18 of let-7f-2 (SEQ ID NO:2);
an A to G substitution at position 32 of mir-18b (SEQ ID NO:4);
a C to T(U) substitution at position 8 of mir-505 (SEQ ID NO:19);
a C to G substitution at position 13 of mir-502 (SEQ ID NO:17);
a C to T(U) substitution at position 60 of mir-188 (SEQ ID NO:6);
a G to A substitution at position 60 of mir-325 (SEQ ID NO:10);
a C to T(U) substitution at position 30 of mir-660 (SEQ ID NO:33);
a C to T(U) substitution at position 56 of mir-509-3 (SEQ ID NO:26);
a G to A substitution at position 6 of mir-510 (SEQ ID NO:30);
a G to A substitution at position 73 of mir-421 (SEQ ID NO:12);
a T(U) to G substitution at position 15 of mir-934; (SEQ ID NO 43);
a G to C substitution at position 66 of mir-890 (SEQ ID NO:37);
a T(U) to C substitution at position 60 of mir-892 (SEQ ID NO:41);
a T(U) to C substitution at position 26 of mir-450a-2 (SEQ ID NO:15); and

diagnosing schizophrenia or a schizophrenia spectrum disorder in the individual when the one or more variants are present.

2. The method of claim 1, wherein determining the presence of one or more of the variants of an miRNA gene or allele comprises:
a) isolating miRNA from a sample taken from an individual; and
b) performing a Northern blot to detect the one or more variants of miRNA, which variants indicate schizophrenia or a schizophrenia spectrum disorder.

3. A method of determining an increased risk for developing schizophrenia or propensity thereto in a human subject comprising the steps of:
a) isolating the miRNA in a sample obtained from the subject; and
b) determining the presence of one or more variants of miRNA in the sample, the one or more variants selected from the group consisting of:
a G to A substitution at position 18 of let-7f-2 (SEQ ID NO:2);
an A to G substitution at position 32 of mir-18b (SEQ ID NO:4);
a C to T(U) substitution at position 8 of mir-505 (SEQ ID NO:19);
a C to G substitution at position 13 of mir-502 (SEQ ID NO:17);
a C to T(U) substitution at position 60 of mir-188 (SEQ ID NO:6);
a G to A substitution at position 60 of mir-325 (SEQ ID NO:10);
a C to T(U) substitution at position 30 of mir-660 (SEQ ID NO:33);
a C to T(U) substitution at position 56 of mir-509-3 (SEQ ID NO:26);
a G to A substitution at position 6 of mir-510 (SEQ ID NO:30);
a G to A substitution at position 73 of mir-421 (SEQ ID NO:12);
a T(U) to G substitution at position 15 of mir-934; (SEQ ID NO 43);
a G to C substitution at position 66 of mir-890 (SEQ ID NO:37);
a T(U) to C substitution at position 60 of mir-892 (SEQ ID NO:41);
a T(U) to C substitution at position 26 of mir-450a-2 (SEQ ID NO:15); and

wherein the presence of said variant is indicative of an increased risk for developing schizophrenia in the subject.