Patent Application
20180251834
Attention-Deficit/Hyperactivity Disorder (ADHD) is a common childhood and adolescent psychiatric disorder, characterized inattention, hyperactivity and impulsivity. It affects roughly 3% to 10% of school-age children worldwide, more than half of the ADHD children continue to struggle with symptoms through adulthood. Early and accurate diagnosis of ADHD is crucial to ameliorate patient's functional impairments throughout the lifecycle.
The diagnosis of ADHD is based upon the observation of behavioral signs and verbal reports of patients or their family, which are prone to bias. There is continuing controversy regarding the reliability and validity of ADHD diagnosis.
There is an unmet need for the identification of biomarkers, particularly measurable in vivo and noninvasive, to accurately diagnose ADHD and facilitate the development of novel therapeutic strategies. The present invention satisfy this and other needs.
In one embodiment, the present invention is directed to methods for detecting ADHD in a subject, comprising the step of measuring the expression level of at least one of the following miRNAs in the test sample of the subject: miR140-3p (SEQ ID NO:1), let-7g-5p (SEQ ID NO:2), miR-486-5p (SEQ ID NO:3), miR-151a-3p (SEQ ID NO:4), miR-151a-5p (SEQ ID NO:5), miR-126-5p (SEQ ID NO:6), miR-30e-5p (SEQ ID NO:7), miR-223-3p (SEQ ID NO:8), miR-142-5p (SEQ ID NO:9), miR-27a-3p (SEQ ID NO:10), miR-101-3p (SEQ ID NO:11), miR-150-5p (SEQ ID NO:12), and miR-92a-3p (SEQ ID NO:13) and compare with the expression level of the corresponding miRNA in an ADHD-free sample, wherein a higher expression level of the following miRNAs in the test sample, relative to the expression level of corresponding miRNA in an ADHD-free sample, is indicative of the subject having ADHD: let-7g-5p, miR-486-5p, miR-126-5p, miR-30e-5p, miR-223-3p, miR-27a-3p, and miR-92a-3p, and wherein a lower expression level of the following miRNAs in the test sample, relative to the expression level of corresponding miRNA in an ADHD-free sample, is indicative of the subject having ADHD: miR140-3p, miR-151a-3p, miR-151a-5p, miR-142-5p, miR-101-3p, and miR-150-5p.
In another embodiment, the present invention is directed to methods for detecting ADHD, comprising the step of measuring the expression level of at least two of the following miRNAs in the test sample of the subject: miR-151a-3p, miR-151a-5p, miR-126-5p, miR-140-3p, or miR-486-5p and compare with the expression level of corresponding miRNA in an ADHD-free sample, wherein a higher miRNA expression level of miR-126-5p and miR-486-5p in the test sample, relative to the expression level of corresponding miRNAs in the ADHD-free sample, is indicative of the subject having ADHD, and wherein a lower miRNA expression level of at least two of the following miRNAs in the test sample: miR-151a-3p, miR-151a-5p and miR-140-3p, relative to the expression level of corresponding miRNA in an ADHD-free sample, is indicative of the subject having ADHD.
The present invention provides kits for detecting ADHD in a subject, comprising an agent for sequencing or measuring the expression level of at least one of the following miRNAs in the test sample of the subject: miR140-3p, let-7g-5p, miR-486-5p, miR-151a-3p, miR-151a-5p, miR-126-5p, miR-30e-5p, miR-223-3p, miR-142-5p, miR-27a-3p, miR-101-3p, miR-150-5p, or miR-92a-3p. In one embodiment, the kit further comprises a label indicates that the agent is for measuring miRNA to diagnose ADHD, wherein a higher expression level of the following miRNAs in the test sample, relative to the expression level of corresponding miRNA in an ADHD-free sample, is indicative of the subject having ADHD: let-7g-5p, miR-486-5p, miR-126-5p, miR-30e-5p, miR-223-3p, miR-27a-3p, and miR-92a-3p, wherein a lower expression level of the following miRNAs in the test sample, relative to the expression level of corresponding miRNA in an ADHD-free sample, is indicative of the subject having ADHD: miR140-3p, miR-151a-3p, miR-151a-5p, miR-142-5p, miR-101-3p, and miR-150-5p.
Also provided are kits for detecting ADHD in a subject, comprising a first agent and a second agent, wherein the agents are for sequencing or measuring the expression level of at least two of the following miRNAs in the test sample of the subject: miR-151a-3p, miR-151a-5p, miR-126-5p, miR-140-3p, or miR-486-5p. In one embodiment, the kit further comprises a label indicates that the agent for measuring miRNA to diagnose ADHD, wherein a higher miRNA expression level of miR-126-5p and miR-486-5p in the test sample, relative to the expression level of corresponding miRNAs in an ADHD-free sample, is indicative of the subject having ADHD, wherein a lower miRNA expression level of at least two of the following miRNAs in the test sample: miR-151a-3p, miR-151a-5p and miR-140-3p, relative to the expression level of corresponding miRNAs in an ADHD-free sample, is indicative of the subject having ADHD.
Also provided are agents for sequencing or measuring the expression level of at least one of the following miRNAs in the manufacture of a kit for detecting ADHD in a subject: miR140-3p, let-7g-5p, miR-486-5p, miR-151a-3p, miR-151a-5p, miR-126-5p, miR-30e-5p, miR-223-3p, miR-142-5p, miR-27a-3p, miR-101-3p, miR-150-5p, and miR-92a-3p.
The terms “invention,” “the invention,” “this invention” and “the present invention” used in this patent are intended to refer broadly to all of the subject matter of this patent and the patent claims below. Statements containing these terms should be understood not to limit the subject matter described herein or to limit the meaning or scope of the patent claims below. Embodiments of the invention covered by this patent are defined by the claims below, not this summary. This summary is a high-level overview of various aspects of the invention and introduces some of the concepts that are further described in the Detailed Description section below. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all drawings and each claim.
The invention will become more apparent when read with the accompanying figures and detailed description which follow.
A more complete understanding of the invention may be obtained by reference to the accompanying drawings, when considered in conjunction with the subsequent detailed description. The embodiments illustrated in the drawings are intended only to exemplify the invention and should not be construed as limiting the invention to the illustrated embodiments.
As used herein, the articles “a” and “an” refer to one or more than one (i.e., at least one) of the grammatical object of the article.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
“Patient” or “subject” as used herein refers to a mammalian subject diagnosed with or suspected of having or developing ADHD. Exemplary subjects may be humans, apes, dogs, pigs, cattle, cats, horses, goats, sheep, rodents and other mammalians that can develop ADHD.
As used herein interchangeably, a “microRNA,” “miR,” or “miRNA” refers to the unprocessed (e.g., precursor) or processed (e.g., mature) RNA transcript from a miR gene. MicroRNAs are endogenous non-coding single-stranded RNAs of approximately 22 nucleotides in length that negatively regulate gene expression in eukaryotes and constitute a novel class of gene regulators (Chua, et al. (2009) Curr. Opin. Mol. Ther. 11:189-199). Individual miRNAs have been identified and sequenced in different organisms, and they have been given names. Names of miRNAs and their sequences are provided herein.
All numbers herein may be understood as modified by “about.” As used herein, the term “about,” when referring to a measurable value a temporal duration and the like or a range, is meant to encompass variations of ±10% from the specified value, as such variations are appropriate to expression of miRNA level unless otherwise specified.
The present invention is based, in part, on the identification of particular miRNA whose expression level is increased or decreased in a test sample of a subject, relative to the predetermined level of the corresponding miRNA in an ADHD-free sample. Some embodiments of the present invention are directed to methods of diagnosing whether a subject has, or is at risk for developing ADHD, comprising measuring the expression level of at least one miRNA in the test sample from the subject and comparing the expression level of the corresponding miRNA in the ADHD-free or control sample. In one embodiment, a higher miRNA expression level in the test sample, relative to that of the control sample, is indicative of the subject having ADHD. In another embodiment, a lower miRNA expression level in the test sample, relative to that of the control sample, is indicative of the subject having ADHD.
The predetermined miRNA expression level in an ADHD-free or control sample is from a representative pool of ADHD-free individuals, and are a mean, median or other statistically manipulated or otherwise summarized or aggregated representative miRNA expression level in the ADHD-free or control samples.
In one embodiment, the miRNA expression level is presented as the threshold cycles (Ct). Ct is defined as the polymerase chain reaction (PCR) cycle at which the fluorescent signal of the reporter dye crosses an arbitrarily placed threshold and is determined by known methods disclosed in Thomas D Schmittgen1 & Kenneth J Livak, “Analyzing real-time PCR data by the comparative CT method,” Nature Protocols, Vol. 3, No. 6 (2008), 1101-1108 and Caifu Chen et al. “Real-time quantification of microRNAs by stem-loop RT-PCR” Nucleic Acids Research, 2005, Vol. 33, No. 20 e179. In another embodiment, the miRNA expression level is the quantitative miRNA expression measured by methods described herein or any method known in the art. In another embodiment, the miRNA of the ADHD patient is compared with the predetermined miRNA expression level of an ADHD-free or control sample using the machine learning network described herein.
A “higher” or a “lower” miRNA expression is a relative term and can be determined by comparison of the miRNA expression level in the test sample to that from a referenced pool of subjects known to be ADHD-free (i.e., control subjects). In some embodiments, the expression of miRNA level in a test sample is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or any % thereinbetween higher than the miR expression level from a referenced pool of ADHD-free subjects. In other embodiments, the expression of miRNA level in a test sample is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or any % thereinbetween lower than that from a referenced pool of ADHD-free subject.
In one embodiment, the expression level of at least one of the following miRNAs is measured in the test sample of a subject to diagnose ADHD: let-7g-5p, miR-486-5p, miR-151a-5p, miR-140-3p, miR-126-5p, or miR-151a-3p.
In another embodiment, the expression level of one or more of the following miRNAs is measured in the test sample of subject to diagnose ADHD: miR140-3p, let-7g-5p, miR-486-5p, miR-151a-3p, miR-151a-5p, miR-126-5p, miR-30e-5p, miR-223-3p, or miR-142-5p.
In yet another embodiment, the expression level of one or more of the following miRNAs is measured in the test sample of subject to diagnose ADHD: miR140-3p, let-7g-5p, miR-486-5p, miR-151a-3p, miR-151a-5p, miR-126-5p, miR-30e-5p, miR-223-3p, miR-142-5p, miR-27a-3p, miR-101-3p, miR-150-5p, or miR-92a-3p.
In one exemplary embodiment, the expression level of miR-140-3p is measured in the test sample of a subject and a lower miR-140-3p level in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In another exemplary embodiment, the expression level of let-7g-5p is measured in the test sample of a subject and a higher let-7g-5p level in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In yet another exemplary embodiment, the expression level of miR-486-5p is measured in the test sample of a subject and a higher miR-486-5p level in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In a fourth embodiment, the expression level of miR-151a-3p is measured in the test sample of a subject and a lower miR-151a-3p level in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In a fifth embodiment, the expression level of miR-151a-5p is measured in the test sample of a subject and a lower miR-151a-5p level in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In a sixth embodiment, the expression level of miR-126-5p is measured in the test sample of a subject and a higher miR-126-5p level in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In some embodiments, the expression level of miR-30e-5p is measured in the test sample of a subject and a higher miR-30e-5p level in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In other embodiments, the expression level of miR-223-3p is measured in the test sample of a subject and a higher miR-223-3p level in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In other embodiments, the expression level of miR-142-5p is measured in the test sample of a subject and a lower miR-142-5p level in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In a tenth embodiment, the expression level of miR-27a-3p is measured in the test sample of a subject and a higher miR-27a-3p level in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In an eleventh embodiment, the expression level of miR-101-3p is measured in the test sample of a subject and a lower miR-101-3p level in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In some embodiments, the expression level of miR-150-5p is measured in the test sample of a subject and a lower miR-150-5p level in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In another embodiment, the expression level of miR-92a-3p is measured in the test sample of a subject and a higher miR-92a-3p level in the test sample relative to that of the control sample, is indicative of the subject having ADHD.
In yet another embodiment, the expression levels of at least two of the following miRNAs are measured in the test sample of a subject to diagnose ADHD: miR-151a-3p, miR-151a-5p, miR-126-5p, miR-140-3p, or miR-486-5p.
In one exemplary embodiment, the expression levels of miRNA-151a-3p and miR140-3p are measured in the test sample of a subject, wherein the expression levels of miRNA-151a-3p and miR140-3p are lower in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In another exemplary embodiment, the expression levels of miRNA-126-5p and miR140-3p are measured in the test sample of a subject, wherein the expression level of miRNA-126-5p is higher and the expression level of miR140-3p is lower in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In yet another exemplary embodiment, the expression levels of miR-151a-3p and miR-151a-5p are measured in the test sample of a subject, wherein the expression levels of miRNA-151a-3p and miR151a-5p are lower in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In a fourth embodiment, the expression levels of miR-151a-3p and miR-126-5p are measured in the test sample of a subject, wherein the expression level of miRNA-126-5p is higher and the expression level of miR151a-3p is lower in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In a fifth embodiment, the expression levels of miR-126-5p and miR-486-5p are measured in the test sample of a subject, wherein the expression levels of miRNA-126a-5p and miR486-5p are higher in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In a sixth embodiment, the expression levels of miR-151a-5p and miR-486-5p are measured in the test sample of a subject, wherein the expression level of miRNA-486-5p is higher and the expression level of miR151a-5p is lower in the test sample relative to that of the control sample, is indicative of the subject having ADHD. In some embodiments, the measurement of two or more miRNA expression levels offer a higher accuracy, sensitivity or specificity for ADHD diagnosis.
Measuring the level of miRNA expression refers to quantifying the amount of miRNA present in a sample. Measuring the expression level of any miRNA can be achieved using methods described herein, such as by real-time PCR, Northern blot analysis, or any technique known to those of skill in the art. Measuring the expression level of miRNA includes measuring the expression of either a mature form of miRNA or a precursor form that is correlated with miRNA expression.
In a particular embodiment, the level of at least one miRNA is detected using Northern blot analysis. For example, total cellular RNA can be purified from cells by homogenization in the presence of nucleic acid extraction buffer, followed by centrifugation. Nucleic acids are precipitated, and DNA is removed by treatment with DNase and precipitation. The RNA molecules are then separated by gel electrophoresis on agarose gels according to standard techniques, and transferred to nitrocellulose filters. The RNA is then immobilized on the filters by heating. Detection and quantification of specific RNA is accomplished using appropriately labeled DNA or RNA probes complementary to the RNA in question. See, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7, the entire disclosure of which is incorporated by reference.
In some embodiments, use of a microarray is desirable. A microarray is a microscopic, ordered array of nucleic acids, proteins, small molecules, cells or other substances that enables parallel analysis of complex biochemical samples. Microarrays can be fabricated using a variety of technologies, including printing with fine-pointed pins onto glass slides, photolithography using pre-made masks, photolithography using dynamic micromirror devices, ink-jet printing, or electrochemistry on microelectrode arrays.
Microarray analysis of miRNAs, for example, can be accomplished according to any method known in the art. In one embodiment, RNA is extracted from a cell such as white blood cell or a sample, the small RNAs (18-26-nucleotide RNAs) are size-selected from total RNA using denaturing polyacrylamide gel electrophoresis. Oligonucleotide linkers are attached to the 5′ and 3′ ends of the small RNAs and the resulting ligation products are used as templates for an RT-PCR reaction with 10 cycles of amplification. The sense strand PCR primer has a fluorophore attached to its 5′ end, thereby fluorescently labeling the sense strand of the PCR product. The PCR product is denatured and then hybridized to the microarray. An PCR product, referred to as the target nucleic acid that is complementary to the corresponding miRNA capture probe sequence on the array will hybridize, via base pairing, to the spot at which the capture probes are affixed. The spot will then fluoresce when excited using a microarray laser scanner. The fluorescence intensity of each spot is then evaluated in terms of the number of copies of a particular miRNA, using a number of positive and negative controls and array data normalization methods, which will result in assessment of the level of expression of a particular miRNA.
In one embodiment, the microarray comprises miRNA-specific probe oligonucleotides for sequencing or measuring the one or more of the following miRNAs: miR140-3p, let-7g-5p, miR-486-5p, miR-151a-3p, miR-151a-5p, miR-126-5p, miR-30e-5p, miR-223-3p, miR-142-5p, miR-27a-3p, miR-101-3p, miR-150-5p, miR-92a-3p, or the combination thereof.
In some embodiments, use of quantitative RT-PCR is desirable. Quantitative RT-PCR (qRT-PCR) is a modified PCR used to rapidly measure the quantity of a product of polymerase chain reaction. qRT-PCR is commonly used for the purpose of determining whether a genetic sequence, such as a miRNA, is present in a sample, and if it is present, the number of copies in the sample. Any method of PCR that can determine the expression of a nucleic acid molecule, including a miRNA, falls within the scope of the present disclosure. There are several variations of the qRT-PCR method known in the art, include, but are not limited to, via agarose gel electrophoresis, the use of SYBR Green (a double stranded DNA dye), and the use of a fluorescent reporter probe.
Some potential miRNAs biomarkers of ADHD, involved in unknown biological mechanisms can be identified using a global screening technology, such as microarray or next-generation sequencing (NGS). NGS is a technique of global screening for miRNA expression profile, showing high sensitivity and low background noise, and potentially discovers ADHD markers which have not been identified.
The identification of miRNAs that are differentially expressed in ADHD and non-ADHD subjects, allows the use of this information in a number of ways. For example, a particular treatment regime may be evaluated (e.g., to determine whether a therapy is effective in a subject with ADHD). Similarly, diagnosis may be done or confirmed by comparing the miRNA expression level in a test sample with known expression profiles from non-ADHD samples. Furthermore, these miRNA expression profiles allow screening of drug candidates that suppress miRNA expression in ADHD, or convert a poor prognosis profile to a better prognosis profile.
In one exemplary embodiment, methods for establishing ADHD-specific machine learning algorithms are provided, comprising: (a) determining the ΔCt value of at least one of the following miRNAs: miR140-3p, let-7g-5p, miR-486-5p, miR-151a-3p, miR-151a-5p, miR-126-5p, miR-30e-5p, miR-223-3p, miR-142-5p, miR-27a-3p, miR-101-3p, miR-150-5p, or miR-92a-3p from a reference pool of subjects without ADHD and a reference pool of subjects having ADHD; and (b) registering at least one ΔCt value of the miRNA from the reference pool of subjects without ADHD and at least one ΔCt value of the miRNA from the reference pool of subjects with ADHD from step (a) into a machine learning algorithm; and (c) establishing a binary classification model using the machine learning algorithm in step (b).
Non-limiting examples of machine learning network include Support Vector Machines (SVM), artificial neural networks, decision tree learning (e.g., CART, ID3, C4.5, CHAID, MARS, and Conditional Inference Trees), instance based learning (e.g., K-Nearest Neighbor), Bayesian networks, genetic algorithms and ensemble learning (e.g., Bagging and Boosting).
The present invention also provides kits for use in diagnosing ADHD. In one exemplary embodiment, the kit comprises at least one agent for sequencing or measuring the expression level of one or more miRNAs selected from the group consisting of: miR140-3p, let-7g-5p, miR-486-5p, miR-151a-3p, miR-151a-5p, miR-126-5p, miR-30e-5p, miR-223-3p, miR-142-5p, miR-27a-3p, miR-101-3p, miR-150-5p and miR-92a-3p, in a test sample of a subject in need of a diagnosis of ADHD. In another embodiment, the kit comprises at least two agents for sequencing or measuring the expression level of at least two of the following miRNAs: miR-151a-3p, miR-151a-5p, miR-126-5p, miR-140-3p or miR-486-5p in a test sample of a subject in need of a diagnosis of ADHD.
ADHD is diagnosed using the kit if the following miRNA expression level in the test sample is higher, relative to the expression level of corresponding miRNA in an ADHD-free sample: let-7g-5p, miR-486-5p, miR-126-5p, miR-30e-5p, miR-223-3p, miR-27a-3p or miR-92a-3p. ADHD is diagnosed using the kit if the following miRNA expression level in the test sample is lower, relative to the expression level of the corresponding miRNA in an ADHD-free sample: miR140-3p, miR-151a-3p, miR-151a-5p, miR-142-5p, miR-101-3p, or miR-150-5p. Non-limiting examples of the test sample include body fluid (e.g. serum, blood, and cerebrospinal fluid), cells (e.g., white blood cells) and tissue (brain biopsy).
In one embodiment, the kit further comprises an instruction for use of the kit to diagnose ADHD. In another embodiment, the agent is RT-PCR. In another embodiment, the agent is a probe oligonucleotide specific for sequencing or measuring any of the SEQ ID NO: 1 to SEQ ID NO: 13 miRNAs. The agent can be an agent known in the art for measuring the expression level of SEQ ID NO: 1 to SEQ ID NO: 13 miRNA.
Embodiments of the present invention are illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the invention. During the studies described in the following examples, conventional procedures were followed, unless otherwise stated. Some of the procedures are described below for illustrative purpose.
Study Participant: The research protocol was approved by the Institutional Review Board (IRB) at Kaohsiung Chang Gung Hospital in Taiwan. Eligible patients with ADHD were treated at the Child Psychiatry outpatient of Kaohsiung Chang Gung Children's Hospital in Taiwan and control (ADHD-free) subjects were recruited from the same hospital for this study.
The inclusion criteria for the study were (a) clinical diagnosis of ADHD by a senior pediatric psychiatrist based on the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) after structured interviews based on the Chinese version of the Schedule for Affective Disorders and Schizophrenia for School-Age Children, epidemiologic version (K-SADS-E); (b) age between 6 and 16 years; (c) Han Chinese ethnic origin; and (d) no prior ADHD medication. Patients were excluded if they had a major physical illness (such as genetic, metabolic, or infectious conditions) or a history of neuropsychiatric disease, such as intellectual disabilities, autism spectrum disorder, bipolar disorders, major depressive disorders, psychotic disorders, substance use disorders, epilepsy or severe head trauma.
The control subjects were children without ADHD of Han Chinese ethnic origin, between the age of 6 and 16 years old, and free of any major physical illnesses or aforementioned major neuropsychiatric diseases.
A Training Set based on the blood samples collected from 68 ADHD patients and 54 control subjects was used to establish a miRNA-based ADHD diagnostic model. A Testing Set based on the blood samples from 15 ADHD patients and 15 control subjects was used to further verify the miRNA-based ADHD diagnostic model identified in the Training Set.
Collecting Test Samples and Extracting RNA Samples
Approximately 5 ml of blood was collected from each subject. The whole blood sample was centrifuged at 3000 rpm for 10 min, followed by the addition of a red blood cell (RBC) lysis buffer (RBC Bioscience, Taiwan) to remove the RBCs. The RBC-free sample was processed with the mirVana miRNA isolation kit (Life technology, USA) to extract total RNAs from the white blood cells (WBC). The integrity of the RNAs was determined using Agilent 2100 (Agilent Technologies, USA). RNA sample with an RNA integrity number (RIN) less than 8 was excluded.
The Evaluation of miRNAs Profile in ADHD and ADHD-Free Samples
In the preliminary study, the RNA samples from five ADHD patients and five control subjects were evenly mixed to generate a pooled ADHD library and a pooled control library, respectively. The two pooled RNA libraries were prepared with TruSeq Small RNA preparation kit (Illumina, USA) and sequenced with MiSeq (Illumina), followed by miRSeq analysis to identify the 20 miRNA candidates for ADHD diagnosis.
Real-Time qPCR Validation of miRNA
The real-time quantitative reverse transcription PCR (qRT-PCR) was used to determine the expression profiles of the 20 miRNAs in the blood samples of all of the enrolled subjects. The TaqMan MicroRNA Transcription Kit (Applied Biosystems, USA) was used to prepare the RNA samples. Reverse-transcription reactions were carried out on a Veriti 96 well thermal cycler (Applied Biosystems) in accordance with the manufacturer's instructions. The real-time PCR cycling condition was as follows: 95° C. for 10 minutes, followed by 40 cycles at 95° C. for 15 s and 60° C. for 1 min. miRNAs expression abundances were determined by the ΔCt values with the small nucleolar RNA U44 as the endogenous control.
Clinical Measurements
All of the ADHD subjects and the control subjects were interviewed by a senior psychiatrist using the K-SADS-E diagnostic tool and ad child psychologist using the Wechsler Intelligence Scale for Children—Fourth Edition (WISC-IV), in a room designed to reduce variability in testing conditions. The Swanson, Nolan, and Pelham Version IV Scale (SNAP-IV) parent form and SNAP-IV teacher form were completed by the subject's parent(s) and the teacher, respectively. Subjects were interviewed by a clinician using the ADHD Rating Scale (ADHD-RS).
Statistical Analysis
Data were analyzed using the statistical software package SPSS, version 16.0 (SPSS Inc., Chicago, Ill., USA) and the MedCalc software Version 15.11.4. Variables were presented as either the mean (standard deviation) or frequency. Two-tailed p values of <0.05 were considered statistically significant.
Library for Support Vector Machine (LIBSVM), an integrated software for support vector classification, regression and distribution estimation, was used to develop an ADHD specific SVM alignment model to discriminate control and ADHD subjects based on the miRNA expression. Receiver operating characteristic (ROC) curves and the area under curve (AUC) were used to evaluate both the specificity and sensitivity of the probability score yielded by the LIBSVM. The optimal diagnostic point of the signature was evaluated at the cut-off values of the probability score at 0.5. Pearson correlation was performed to analyze the relationships between the probability score and ADHD symptoms in ADHD patients.
Results
miRNA Expression Profile by NGS
The miRNA expression profile by NGS shows 18 candidate miRNAs were differentially expressed between ADHD patients and the control. The candidate miRNAs was defined as: (1) TPM (transcript per million) >1,000 in either ADHD group or control group; (2) Ratios of miRNAs of ADHD patients/control subjects were approximately higher than 1.50 or lower than 0.67. Based on previous research data, miR-181a-5p and let-7d were also considered as potential biomarkers. A total of 20 candidate miRNAs served as diagnostic biomarkers for ADHD: miR-29a-3p, miR-142-3p, miR-140-3p, miR-423-3p, miR-192-5p, miR-27a-3p, miR-101-3p, miR-150-5p, let-7g-5p, miR-30e-5p, miR-223-3p, miR-142-5p, miR-143-3p, miR-92a-3p, miR-486-5p, miR-151a-3p, miR-151a-5p, miR-126-5p, miR-181a-5p, and let-7d.
qPCR Validation
qPCR validation on all samples from the Training Set shows the expression levels of following 9 miRNAs were significantly different between ADHD and non-ADHD subjects in the Training Set: miR140-3p, let-7g-5p, miR-486-5p, miR-151a-3p, miR-151a-5p, miR-126-5p, miR-30e-5p, miR-223-3p, and miR-142-5p (
The ADHD Diagnostic Model
To derive a more robust diagnosis model, the combination of the expression levels of the following 13 miRNAs was used to develop the ADHD diagnostic model using the SVM algorithm (hereafter “the ADHD diagnostic model”): miR-140-3p, miR-27a-3p, miR-101-3p, miR-150-5p, let-7g-5p, miR-30e-5p, miR-223-3p, miR-142-5p, miR-92a-3p, miR-486-5p, miR-151a-3p, miR-151a-5p, and miR-126-5p. The Multivariate analysis of covariance confirmed that the expression of these 13 miRNAs was not confounded by age, sex or intelligence quotient. To derive the best discriminative validity, 5-fold cross validations was used to derive the optimal parameters, gamma=0.015625 and cost=256. Using the default probability cutoff of 0.5, the ADHD diagnostic model has a sensitivity of 86.8% and a specificity of 88.9%, leading to an area under curve (AUC) value of 0.94 (p<0.001), when compare with the ADHD diagnosis by physician (the gold standard).
In addition, the combination of specific miRNAs lead to unexpected synergy in ADHD diagnostic accuracy (AUC), as shown in Table 1.
An independent blind test was conducted on 15 ADHD and 15 control subjects in the Testing Set. There was no significant difference between ADHD patients and control subjects in age, sex or intelligence quotient. The 13 miRNA ΔCt of the 30 subjects in the Testing Set was analysed using the ADHD diagnostic model. 13 of the 15 ADHD samples were correctly diagnosed with ADHD and 11 of the 15 control subjects were correctly classified as non-ADHD, demonstrating a sensitivity of 86.7%, a specificity of 73.3%, and an accuracy of 80%.
Further Validation of ADHD Diagnostic Model
The subjects in the Training Set and Testing Set were pooled together for additional sensitivity analysis. The subjects were stratified into a younger group (<108 months) and an older group (>108 months) and the ROC analysis showed the probability scores from the ADHD diagnostic model effectively discriminate ADHD and non-ADND subjects in both age groups (
Several miRNAs, such as miR124-1 and let-7d, have been reported to be potentially associated with ADHD. Theses miRNAs were identified as they play a role in the neurobiology mechanisms of ADHD, based on literature review or the miRNA database. The 13 miRNAs used to construct the ADHD diagnostic model in the present invention have not been reported to be involved in pathophysiology of ADHD and were identified by global screening technology (NGS).
This application claims the benefit of priority from U.S. Provisional Application No. 62/466,586, filed on 3 Mar. 2017, the entire content of which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62466586 | Mar 2017 | US |
